A síntese de peptídeos em fase sólida passo a passo (SPFS) tem sido aplicada com sucesso na preparação de peptídeos curtos, médios e de determinados sequências contendo mais de 30 resíduos. Entretanto, esta apresenta problemas e limitações que podem ser contornados pela síntese convergente de peptídeos em fase sólida (SCPFS) que se baseia na condensação entre fragmentos peptídicos Nα-acilados protegidos em suas cadeias laterais (doadores de acila) a fragmentos protegidos ligados a um suporte polimérico (receptores de acila). Além de desenvolver outros projetos enfocados na síntese de peptídeos ou no uso de sintéticos para o estudo de peptídeos biologicamente ativos ou proteinas, o nosso grupo de pesquisa tem se dedicado a estudar o emprego de temperaturas altas em SPFS. O objetivo final é propor protocolos ágeis alternativos aos empregados classicamente. Neste trabalho nos propusemos a investigar alguns aspectos da SCPFS e a explorar a possibilidade de agilizá-Ia a 60°C. Para tanto, empregamos como modelos a colecistocinina-33 humana (hCCK-33) não sulfatada e o análogo [Gln1]-gomesina. As seqüências destes peptídeos foram divididas em: doadores de acila (fragmentos central e N-terminal da hCCK-33 não sulfatada de 11 e de 5 resíduos, respectivamente, e N-terminal de 8 resíduos do [Gln1]-gomesina) e receptores de acila (fragmentos C-terminais da hCCK-33 não sulfatada de 17 resíduos e do [Gln1]-gomesina de 10 resíduos). As peptidil-resinas correspondentes foram sintetizadas manualmente por SPFS passo a passo e caracterizadas por análise de aminoácidos para determinação de seus graus de substituição. Suas propriedades de solvatação em diferentes sistemas de solventes foram também examinadas. Análises por RP-HPLC e LC/ESI-MS dos peptídeos brutos obtidos após clivagem das resinas e desproteção total permitiram avaliar as sínteses realizadas. Os doadores de acila foram então gerados a partir das peptidil-resinas empregando procedimentos conhecidos (catálise por DBU ou NaOH) e um proposto por nós (assistência por íons metálicos). Finalmente, os acoplamentos entre os doadores e receptores de acila foram realizados a 37 e 60°C empregando sistemas de solventes adequados à solvatação dos receptores de acila e diferentes reagentes acopladores. As peptidil-resinas alongadas foram analisadas em seu conteúdo de aminoácidos e submetidas à clivagem e desproteção total para liberação dos peptídeos brutos correspondentes. Estes foram submetidos à análise comparativa usando RP-HPLC e LC/ESI-MS. Os resultados obtidos neste primeiro trabalho que emprega alta temperatura na SCPFS demonstraram que: 1) o conhecimento do grau de solvatação das peptidil-resinas auxilia na escolha do sistema de solventes a ser empregado na geração dos doadores de acila (desligamento dos peptídeos protegidos correspondentes) e nos seus acoplamentos aos receptores de acila; 2) a geração de doadores de acila a partir de peptidil-KOR não é trivial como sugere a literatura (o fragmerto 6-19 da hCCK-33 na forma protegida não foi obtido) e deve ser melhor estudada. O tamanho e a seqüência peptídica parecem estar diretamente relacionados à eficiência do processo; 3) o uso de alta temperatura agiliza o processo; 4) a geração de doadores de acila a partir de peptidil-2-CI-Trt empregando 1%TFA em DCM e misturas AcOH:TFE:DCM é simples e eficiente; 5) o uso combinado de DMF, do agente acoplador TBTU e de 60°C levou à agilização dos acoplamentos convergentes realizados, fornecendo os peptídeos desejados com boa qualidade em tempos relativamente menores; 6) vantagens e desvantagens da SCPFS em alta temperatura devem ser melhor avaliadas; 7) a SCPFS também apresenta limitações e problemas a serem contornados, o que demanda exploração sistemática empregando seqüências peptídicas e resinas variadas. / Stepwise solid-phase peptide synthesis (SSPPS) has been applied successfully for the preparation of most peptides containing up to 30 residues. However, it presents problems and limitations. Convergent soIid-phase peptide synthesis (CSPPS) can overcome part of them. This methodology is based on the synthesis of peptide segments by SSPPS followed by their condensations: the Nα- acylated protected segments act as acyl donors and the protected segments bound to the resin as acyl receptors. When the sequence is completed this is detached from the resin and fully deprotected to give the crude peptide. Besides developing other projectsfocused on peptide synthesis itself or on the use of synthetics to study biologically active peptides or proteins, we have systematically examined solid-phase peptide synthesis at elevated temperature. The aim of the present work was to investigate different aspects of convergent solid-phase peptide synthesis and examine the possibility to improve it at 60°C. Thus, unsulfated human cholecystokinin-33 and [Gln1]-gomesin were divided in three and two fragments, respectively. In the first case, the acyl donors were built-up by SSPPS on Kaiser oxime resin while in the second peptide elongation was done on 2-CI-Trt resin. The acyl receptor was synthesized and kept on the MBHA resin. The synthetic process was evaluated through characterization of every peptidyl-resin by amino acid analysis and of each crude peptide obtained from resin cleavage/full deprotection by RP-HPLC and LC/ESI-MS. The swelling degrees of the peptidyl-resins were determined in various solvents or mixtures of high boiling points. The peptidyl-KOR and peptidyl-2-CI-Trt were then submitted to peptide detachment under a few experimental conditions in order to release the Nα-acyl protected peptides (the acyl donors). Finally, their couplings with the acyl receptors were carried-out at 37 and 60°C in solvents or mixtures suitable for peptidyl-resin solvation containing specific coupling agents. The resulting peptidyl-resins were isolated, dried and submitted to HF treatment to release the corresponding unprotected crude peptides, which were analysed by RP-HPLC and LC/ESI-MS. The results found indicated that: 1) the knowledge of the swelling properties of the peptidyl-resins in different solvents systems is very useful in SCPFS. Indeed, it may guide the selection of experimental conditions to be used in peptide detachment from KOR and 2-CI-Trt and in segment condensation at elevated temperature, 2) peptide detachment from KOR is not as trivial as described (it was impossible to release hCCK-33 fragment 6-19 in its protected form). Further studies are certainly required to improve it. It seems that the size and the sequence are strictly related to the process efficiency, 3) high temperature can improve it, 4) peptide detachment from 2-CI-Trt is as simple as described, 5) combination of DMF as solvent, TBTU as coupling agent and 60°C was suited for the segment couplings studied: the reactions were completed in relatively short times and crude peptides of good quality were obtained, 6) this is the first attempt to carry-out CSPFS at elevated temperature, thus its advantages and disadvantages must be studied, 7) CSPPS also presents problems and limitations to overcome. Such task requires further investigation using various resins and peptide sequences.

<p>This study addresses the issue of how to convert peptides into drug-like non- peptides with retained biological activities at peptide receptors. Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Ang II) was used as a model peptide. </p><p>Knowledge of the bioactive conformations of endogenous peptides is invaluable for the conversion of peptides into less peptidic analogues. Effectively constrained cyclic analogues, with retained pharmacological activities, may provide valuable information about the bioactive conformations of the peptide in question. </p><p>This thesis describes the development of synthesis for a number of protected, aldehyde-functionalized building blocks for standard solid phase peptide synthesis, and their use for the preparation of cyclic peptide analogues. The effect of variations in the side-chain lengths of the building blocks, on the outcome of the cyclizations was studied. Incorporation of a building block derived from L-aspartic acid afforded bicyclization towards the C-terininal end of the peptide, while for the corresponding L-glutamic acid derived building block, N-terminal directed bicyclization was achieved. A building block derived from L-2-aminoadipic acid was exploited for monocyclization furnishing <i>cis-</i> and <i>trans-</i> vinyl sulfide bridged peptide analogues. </p><p>The described cyclization methods have been applied to the synthesis of a number of conformationally constrained Ang II analogues, for which the pharmacological properties have been evaluated. Two of the Ang II analogues synthesized displayed high affinities and full agonist activities at the AT₁ angiotensin receptor, and have proven to be useful tools in the search for the bioactive conformation of Ang II.</p>

This study addresses the issue of how to convert peptides into drug-like non- peptides with retained biological activities at peptide receptors. Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Ang II) was used as a model peptide. Knowledge of the bioactive conformations of endogenous peptides is invaluable for the conversion of peptides into less peptidic analogues. Effectively constrained cyclic analogues, with retained pharmacological activities, may provide valuable information about the bioactive conformations of the peptide in question. This thesis describes the development of synthesis for a number of protected, aldehyde-functionalized building blocks for standard solid phase peptide synthesis, and their use for the preparation of cyclic peptide analogues. The effect of variations in the side-chain lengths of the building blocks, on the outcome of the cyclizations was studied. Incorporation of a building block derived from L-aspartic acid afforded bicyclization towards the C-terininal end of the peptide, while for the corresponding L-glutamic acid derived building block, N-terminal directed bicyclization was achieved. A building block derived from L-2-aminoadipic acid was exploited for monocyclization furnishing cis- and trans- vinyl sulfide bridged peptide analogues. The described cyclization methods have been applied to the synthesis of a number of conformationally constrained Ang II analogues, for which the pharmacological properties have been evaluated. Two of the Ang II analogues synthesized displayed high affinities and full agonist activities at the AT1 angiotensin receptor, and have proven to be useful tools in the search for the bioactive conformation of Ang II.

The immune system often recognises tumour cells and infectious agents from the unique peptides found on their surfaces therefore, synthetic peptides of similar structure can be used as vaccines to stimulate the immune system. Despite the problems associated with proteolysis and delivery to the immune system, peptide-based vaccines have enormous potential due to their ease of synthesis and purification. The aim of this research was to synthesise ligands for mannose receptors (MRs) that are found on human Antigen Presenting Cells (APCs), for use in synthetic vaccines. Carbohydrate bearing antigens are recognised by MRs which play an important role in binding antigens, migration of dendritic cells (DCs) and interaction of DCs with lymphocytes. Hence, incorporation of a sugar residue into a peptide chain can be used to enhance antigen presentation. This thesis describes the synthesis of fluorescein labelled O-mannosylated peptides using either manual or microwave assisted solid phase glycopeptide synthesis (SPGS) on pre-loaded WANG resin. The mannosylated peptides thus prepared can be tested for their ability to bind mannose receptors on human APCs in vitro. In order to prepare compounds that could be analysed in biological screens, a fluorescent label (5(6)-carboxyfluorescein) was introduced into the glycopeptides via the Nα- or the Nε-amino group of the lysine residue. It was found that preparation of the glycopeptide was more facile when the peptide chain was built onto the Nε of Lys (label into Nα) rather than onto the Nα of Lys (label into Nε). In order to overcome problems experienced when introducing more than one glycosylated building block into the peptide chain, a polyethylene glycol (PEG) linker was employed as a sugar carrier. It was found that mono- and dimannosylated building blocks attached to PEG carrier were incorporated more easily into the peptide chain compared to mono- and dimannosylated serine units. Importantly, microwave technology (CEM Liberty microwave peptide synthesiser) was used for SPGS which resulted in improved purity and yields of the glycopeptides thus prepared with a significant reduction in reaction times. The first fifteen glycopeptides prepared in the present study were tested for binding to mannose receptors. Several compounds have shown improved binding to monocytes (bear MRs) in comparison to lymphocytes (do not bear MRs), in the presence of calcium ions. Calcium dependent binding is specific for C type lectin receptor family that MRs belong to. Five remaining glycopeptides are currently undergoing biological evaluation.

The immune system often recognises tumour cells and infectious agents from the unique peptides found on their surfaces therefore, synthetic peptides of similar structure can be used as vaccines to stimulate the immune system. Despite the problems associated with proteolysis and delivery to the immune system, peptide-based vaccines have enormous potential due to their ease of synthesis and purification. The aim of this research was to synthesise ligands for mannose receptors (MRs) that are found on human Antigen Presenting Cells (APCs), for use in synthetic vaccines. Carbohydrate bearing antigens are recognised by MRs which play an important role in binding antigens, migration of dendritic cells (DCs) and interaction of DCs with lymphocytes. Hence, incorporation of a sugar residue into a peptide chain can be used to enhance antigen presentation. This thesis describes the synthesis of fluorescein labelled O-mannosylated peptides using either manual or microwave assisted solid phase glycopeptide synthesis (SPGS) on pre-loaded WANG resin. The mannosylated peptides thus prepared can be tested for their ability to bind mannose receptors on human APCs in vitro. In order to prepare compounds that could be analysed in biological screens, a fluorescent label (5(6)-carboxyfluorescein) was introduced into the glycopeptides via the Nα- or the Nε-amino group of the lysine residue. It was found that preparation of the glycopeptide was more facile when the peptide chain was built onto the Nε of Lys (label into Nα) rather than onto the Nα of Lys (label into Nε). In order to overcome problems experienced when introducing more than one glycosylated building block into the peptide chain, a polyethylene glycol (PEG) linker was employed as a sugar carrier. It was found that mono- and dimannosylated building blocks attached to PEG carrier were incorporated more easily into the peptide chain compared to mono- and dimannosylated serine units. Importantly, microwave technology (CEM Liberty microwave peptide synthesiser) was used for SPGS which resulted in improved purity and yields of the glycopeptides thus prepared with a significant reduction in reaction times. The first fifteen glycopeptides prepared in the present study were tested for binding to mannose receptors. Several compounds have shown improved binding to monocytes (bear MRs) in comparison to lymphocytes (do not bear MRs), in the presence of calcium ions. Calcium dependent binding is specific for C type lectin receptor family that MRs belong to. Five remaining glycopeptides are currently undergoing biological evaluation.

The immune system often recognises tumour cells and infectious agents from the unique peptides found on their surfaces therefore, synthetic peptides of similar structure can be used as vaccines to stimulate the immune system. Despite the problems associated with proteolysis and delivery to the immune system, peptide-based vaccines have enormous potential due to their ease of synthesis and purification. The aim of this research was to synthesise ligands for mannose receptors (MRs) that are found on human Antigen Presenting Cells (APCs), for use in synthetic vaccines. Carbohydrate bearing antigens are recognised by MRs which play an important role in binding antigens, migration of dendritic cells (DCs) and interaction of DCs with lymphocytes. Hence, incorporation of a sugar residue into a peptide chain can be used to enhance antigen presentation. This thesis describes the synthesis of fluorescein labelled O-mannosylated peptides using either manual or microwave assisted solid phase glycopeptide synthesis (SPGS) on pre-loaded WANG resin. The mannosylated peptides thus prepared can be tested for their ability to bind mannose receptors on human APCs in vitro. In order to prepare compounds that could be analysed in biological screens, a fluorescent label (5(6)-carboxyfluorescein) was introduced into the glycopeptides via the Nα- or the Nε-amino group of the lysine residue. It was found that preparation of the glycopeptide was more facile when the peptide chain was built onto the Nε of Lys (label into Nα) rather than onto the Nα of Lys (label into Nε). In order to overcome problems experienced when introducing more than one glycosylated building block into the peptide chain, a polyethylene glycol (PEG) linker was employed as a sugar carrier. It was found that mono- and dimannosylated building blocks attached to PEG carrier were incorporated more easily into the peptide chain compared to mono- and dimannosylated serine units. Importantly, microwave technology (CEM Liberty microwave peptide synthesiser) was used for SPGS which resulted in improved purity and yields of the glycopeptides thus prepared with a significant reduction in reaction times. The first fifteen glycopeptides prepared in the present study were tested for binding to mannose receptors. Several compounds have shown improved binding to monocytes (bear MRs) in comparison to lymphocytes (do not bear MRs), in the presence of calcium ions. Calcium dependent binding is specific for C type lectin receptor family that MRs belong to. Five remaining glycopeptides are currently undergoing biological evaluation.

The immune system often recognises tumour cells and infectious agents from the unique peptides found on their surfaces therefore, synthetic peptides of similar structure can be used as vaccines to stimulate the immune system. Despite the problems associated with proteolysis and delivery to the immune system, peptide-based vaccines have enormous potential due to their ease of synthesis and purification. The aim of this research was to synthesise ligands for mannose receptors (MRs) that are found on human Antigen Presenting Cells (APCs), for use in synthetic vaccines. Carbohydrate bearing antigens are recognised by MRs which play an important role in binding antigens, migration of dendritic cells (DCs) and interaction of DCs with lymphocytes. Hence, incorporation of a sugar residue into a peptide chain can be used to enhance antigen presentation. This thesis describes the synthesis of fluorescein labelled O-mannosylated peptides using either manual or microwave assisted solid phase glycopeptide synthesis (SPGS) on pre-loaded WANG resin. The mannosylated peptides thus prepared can be tested for their ability to bind mannose receptors on human APCs in vitro. In order to prepare compounds that could be analysed in biological screens, a fluorescent label (5(6)-carboxyfluorescein) was introduced into the glycopeptides via the Nα- or the Nε-amino group of the lysine residue. It was found that preparation of the glycopeptide was more facile when the peptide chain was built onto the Nε of Lys (label into Nα) rather than onto the Nα of Lys (label into Nε). In order to overcome problems experienced when introducing more than one glycosylated building block into the peptide chain, a polyethylene glycol (PEG) linker was employed as a sugar carrier. It was found that mono- and dimannosylated building blocks attached to PEG carrier were incorporated more easily into the peptide chain compared to mono- and dimannosylated serine units. Importantly, microwave technology (CEM Liberty microwave peptide synthesiser) was used for SPGS which resulted in improved purity and yields of the glycopeptides thus prepared with a significant reduction in reaction times. The first fifteen glycopeptides prepared in the present study were tested for binding to mannose receptors. Several compounds have shown improved binding to monocytes (bear MRs) in comparison to lymphocytes (do not bear MRs), in the presence of calcium ions. Calcium dependent binding is specific for C type lectin receptor family that MRs belong to. Five remaining glycopeptides are currently undergoing biological evaluation.

A síntese de peptídeos em fase sólida passo a passo (SPFS) tem sido aplicada com sucesso na preparação de peptídeos curtos, médios e de determinados sequências contendo mais de 30 resíduos. Entretanto, esta apresenta problemas e limitações que podem ser contornados pela síntese convergente de peptídeos em fase sólida (SCPFS) que se baseia na condensação entre fragmentos peptídicos N&#945;-acilados protegidos em suas cadeias laterais (doadores de acila) a fragmentos protegidos ligados a um suporte polimérico (receptores de acila). Além de desenvolver outros projetos enfocados na síntese de peptídeos ou no uso de sintéticos para o estudo de peptídeos biologicamente ativos ou proteinas, o nosso grupo de pesquisa tem se dedicado a estudar o emprego de temperaturas altas em SPFS. O objetivo final é propor protocolos ágeis alternativos aos empregados classicamente. Neste trabalho nos propusemos a investigar alguns aspectos da SCPFS e a explorar a possibilidade de agilizá-Ia a 60°C. Para tanto, empregamos como modelos a colecistocinina-33 humana (hCCK-33) não sulfatada e o análogo [Gln1]-gomesina. As seqüências destes peptídeos foram divididas em: doadores de acila (fragmentos central e N-terminal da hCCK-33 não sulfatada de 11 e de 5 resíduos, respectivamente, e N-terminal de 8 resíduos do [Gln1]-gomesina) e receptores de acila (fragmentos C-terminais da hCCK-33 não sulfatada de 17 resíduos e do [Gln1]-gomesina de 10 resíduos). As peptidil-resinas correspondentes foram sintetizadas manualmente por SPFS passo a passo e caracterizadas por análise de aminoácidos para determinação de seus graus de substituição. Suas propriedades de solvatação em diferentes sistemas de solventes foram também examinadas. Análises por RP-HPLC e LC/ESI-MS dos peptídeos brutos obtidos após clivagem das resinas e desproteção total permitiram avaliar as sínteses realizadas. Os doadores de acila foram então gerados a partir das peptidil-resinas empregando procedimentos conhecidos (catálise por DBU ou NaOH) e um proposto por nós (assistência por íons metálicos). Finalmente, os acoplamentos entre os doadores e receptores de acila foram realizados a 37 e 60°C empregando sistemas de solventes adequados à solvatação dos receptores de acila e diferentes reagentes acopladores. As peptidil-resinas alongadas foram analisadas em seu conteúdo de aminoácidos e submetidas à clivagem e desproteção total para liberação dos peptídeos brutos correspondentes. Estes foram submetidos à análise comparativa usando RP-HPLC e LC/ESI-MS. Os resultados obtidos neste primeiro trabalho que emprega alta temperatura na SCPFS demonstraram que: 1) o conhecimento do grau de solvatação das peptidil-resinas auxilia na escolha do sistema de solventes a ser empregado na geração dos doadores de acila (desligamento dos peptídeos protegidos correspondentes) e nos seus acoplamentos aos receptores de acila; 2) a geração de doadores de acila a partir de peptidil-KOR não é trivial como sugere a literatura (o fragmerto 6-19 da hCCK-33 na forma protegida não foi obtido) e deve ser melhor estudada. O tamanho e a seqüência peptídica parecem estar diretamente relacionados à eficiência do processo; 3) o uso de alta temperatura agiliza o processo; 4) a geração de doadores de acila a partir de peptidil-2-CI-Trt empregando 1%TFA em DCM e misturas AcOH:TFE:DCM é simples e eficiente; 5) o uso combinado de DMF, do agente acoplador TBTU e de 60°C levou à agilização dos acoplamentos convergentes realizados, fornecendo os peptídeos desejados com boa qualidade em tempos relativamente menores; 6) vantagens e desvantagens da SCPFS em alta temperatura devem ser melhor avaliadas; 7) a SCPFS também apresenta limitações e problemas a serem contornados, o que demanda exploração sistemática empregando seqüências peptídicas e resinas variadas. / Stepwise solid-phase peptide synthesis (SSPPS) has been applied successfully for the preparation of most peptides containing up to 30 residues. However, it presents problems and limitations. Convergent soIid-phase peptide synthesis (CSPPS) can overcome part of them. This methodology is based on the synthesis of peptide segments by SSPPS followed by their condensations: the N&#945;- acylated protected segments act as acyl donors and the protected segments bound to the resin as acyl receptors. When the sequence is completed this is detached from the resin and fully deprotected to give the crude peptide. Besides developing other projectsfocused on peptide synthesis itself or on the use of synthetics to study biologically active peptides or proteins, we have systematically examined solid-phase peptide synthesis at elevated temperature. The aim of the present work was to investigate different aspects of convergent solid-phase peptide synthesis and examine the possibility to improve it at 60°C. Thus, unsulfated human cholecystokinin-33 and [Gln1]-gomesin were divided in three and two fragments, respectively. In the first case, the acyl donors were built-up by SSPPS on Kaiser oxime resin while in the second peptide elongation was done on 2-CI-Trt resin. The acyl receptor was synthesized and kept on the MBHA resin. The synthetic process was evaluated through characterization of every peptidyl-resin by amino acid analysis and of each crude peptide obtained from resin cleavage/full deprotection by RP-HPLC and LC/ESI-MS. The swelling degrees of the peptidyl-resins were determined in various solvents or mixtures of high boiling points. The peptidyl-KOR and peptidyl-2-CI-Trt were then submitted to peptide detachment under a few experimental conditions in order to release the N&#945;-acyl protected peptides (the acyl donors). Finally, their couplings with the acyl receptors were carried-out at 37 and 60°C in solvents or mixtures suitable for peptidyl-resin solvation containing specific coupling agents. The resulting peptidyl-resins were isolated, dried and submitted to HF treatment to release the corresponding unprotected crude peptides, which were analysed by RP-HPLC and LC/ESI-MS. The results found indicated that: 1) the knowledge of the swelling properties of the peptidyl-resins in different solvents systems is very useful in SCPFS. Indeed, it may guide the selection of experimental conditions to be used in peptide detachment from KOR and 2-CI-Trt and in segment condensation at elevated temperature, 2) peptide detachment from KOR is not as trivial as described (it was impossible to release hCCK-33 fragment 6-19 in its protected form). Further studies are certainly required to improve it. It seems that the size and the sequence are strictly related to the process efficiency, 3) high temperature can improve it, 4) peptide detachment from 2-CI-Trt is as simple as described, 5) combination of DMF as solvent, TBTU as coupling agent and 60°C was suited for the segment couplings studied: the reactions were completed in relatively short times and crude peptides of good quality were obtained, 6) this is the first attempt to carry-out CSPFS at elevated temperature, thus its advantages and disadvantages must be studied, 7) CSPPS also presents problems and limitations to overcome. Such task requires further investigation using various resins and peptide sequences.

Le but de cette thèse est de montrer la réponse de la membrane mitochondriale au cours la régulation de l’apoptose en étudiant l’effet de domaines clés sur la dynamique membranaire et l’importance de la composition phospholipidiques des modèles utilisés. Le domaine BH4 est la partie spécifique anti-apoptotique de la famille Bcl-2. La première étape a été de synthétiser le peptide par voie chimique en utilisant la synthèse peptidique en phase solide. Un protocole décrivant les étapes de purification par chromatographie liquide et de caractérisation par spectroscopie de masse, garantissant une pureté indispensable pour des études biophysiques, a été établi. La modification de la structure secondaire du peptide interagissant avec des vésicules a été étudiée par spectroscopie infrarouge ainsi que par dichroïsme circulaire. Le peptide s’agrège à la surface et s’insère peu profondément dans la partie hydrophobe de la membrane. En utilisant la résonance magnétique nucléaire (RMN) et la calorimétrie, il a été montré que le peptide BH4 modifie l’organisation et la dynamique des liposomes mimant la surface mitochondriale. La deuxième étude a porté sur la première hélice de la protéine pro-apoptotique Bax (Bax-a1) qui a la propriété de diriger la protéine cytosolique vers la mitochondrie. Un protocole de synthèse et purification a été à nouveau établi. Le but de cette étude est de démontrer le rôle de l’interaction spécifique entre la cardiolipine, un phospholipide uniquement présent dans la mitochondrie et le peptide Bax-a1. Les études RMN ont montré que Bax-a1 n’interagissait uniquement que si la cardiolipine était présente, produisant un fort effet électrostatique piégeant le peptide à la surface de la membrane. Enfin, un nouveau protocole permettant d’étudier la réponse des lipides de mitochondries isolées toujours actives par RMN est présenté. Le but est de pouvoir directement observer les modifications subies par chaque phospholipide de la mitochondrie. . / The aim of this thesis was the investigation of the mitochondrial response mechanisms upon apoptotic stimuli. The specific objectives were the biophysical characterization of membrane dynamics and the specific roles of lipids in the context of apoptotic regulation occurring at the mitochondrion and its complex membrane systems. The BH4 domain is an anti-apoptotic specific domain of the Bcl-2 protein. Solid phase peptide synthesis was used to produce large amount of the peptide for biophysical studies. A protocol has been established and optimized, guarantying the required purity for biophysical studies. In detail the purification by high performance liquid chromatography and the characterisation via mass spectroscopy are described. The secondary structure of BH4 changes significantly in the presence of lipid vesicles as observed by infrared spectroscopy and circular dichroism. The BH4 peptide aggregates at the membrane surface and inserts slightly into the hydrophobic part of the membrane. Using nuclear magnetic resonance (NMR) and calorimetry techniques, it could even be shown that the BH4 domain modifies the dynamic and organization of the liposomes which mimic a mitochondrial surface. The second study was on the first helix of the pro-apoptotic protein Bax. This sequence called Bax-a1 has the function to address the cytosolic Bax protein to the mitochondrial membrane upon activation. Once again a protocol has been established for the synthesis and purification of this peptide. The aim was to elucidate the key role of cardiolipin, a mitochondria-specific phospholipid, in the interaction of Bax-a1 with the mitochondrial membrane system. The NMR and circular dichroism studies showed that Bax-a1 interacts with the membrane models only if they contain the cardiolipin, producing a strong electrostatic lock effect which is located at the membrane surface. Finally, a new NMR approach was developed which allows the investigation of the lipid response of isolated active mitochondria upon the presence of apoptotic stimuli. The goal was there to directly monitor lipid specific the occurring changes during these physiological activities.

No description available.