Polyarene dendrimers are composed entirely of aromatic rings and are of interest as they possess rigid and shape-persistent structures, which have potential applications in materials chemistry and as scaffold for functional molecules. This thesis describes the synthesis and characterisation of a number of new polyarene dendrimers containing pentaphenylbenzene-like (pentaaryl dendrimers) and hexaphenylbenzene-like (hexaaryl dendrimers) branching units which are capable of focal and peripheral expansions. These structures are based on terminal and internal 1,3-dialkyne cores, onto which two polyaryl sections were introduced via Diels-Alder reaction. These compounds possess inherent axial chirality as a result of the dialkyne position on the core, and a range of differently substituted polyaryl units which result in further chiral axes. Several crystal structures of pentaryl dendrimers were obtained, with these crystallising either as meso conformers or conformers with C2 symmetry. Introduction of a chiral auxiliary onto the central aromatic ring of these dendrimers provided evidence of the atropisomers in solution. These were studied via VT 13C NMR, revealing fast rotation and an energy barrier of about 66 kJ mol-1. The presence of additional aromatic rings on a hexaaryl dendrimer enabled observation of atropisomerism in solution owing to the greater steric hindrance of rotation of its branching units about the chiral axes. Fluorine-containing analogues of these dendrimers were synthesised and subjected to conformational studies through 19F-19F Exchange Spectroscopy (EXSY). Qualitative and quantitative analysis of the spectra revealed the kinetic and thermodynamic parameters, showing that the energy barrier of rotation of these dendrimers was within the range of 75-80 kJ mol-1, which was sufficient for spectroscopic observation but not for physical separation. Polyaryls with symmetry or lacking symmetry have been synthesised. The lack of symmetry was achieved by stepwise introduction of the polyaryl fragments. This demonstrates a route to polyaryls with sections possessing different structural features and thus potentially engineered properties and future chemical differentiations.

A deeper understanding of the diversity of carbohydrates and the many applications of oligo- and polysaccharides found in nature are of high interest. Many of the processes involving carbohydrates affect our everyday life. This thesis is based on six papers all contributing to an extended perspective of carbohydrate property and functionality. An introduction to carbohydrate chemistry together with a presentation of selected carbohydrate synthesis and analysis methods introduces the reader to the research field. The first paper is an NMR spectroscopy reinvestigation of the structures of the O-antigens from the lipopolysaccharides (LPS) of Shigella dysenteriae type 3 and Escherichia coli O124. The repeating units were concluded to be built of identical branched pentasaccharides now with the correct anomeric configurations. Paper II is a structural investigation of the O-antigen from the LPS of E. coli O74 which is built of branched tetrasaccharide repeating units including the uncommon monosaccharide d-Fuc3NAc. Paper III is a conformational study of a rhamnose derivative, using NMR spectroscopy and X-ray crystallography. The benzoyl ester group positioned at C4 prefers an “eclipsed” conformation in the crystal as well as in solution. The use of site-specifically 13C-labeled compounds in conformational studies is discussed in Papers IV and V. The disaccharide α-L-Rhap-(1→2)-α-L-Rhap-OMe was synthesized together with two 13C-isotopologues and studied with NMR spectroscopy to give seven J-couplings related to torsion angles φ and ψ. The trisaccharide α-L-Rhap-(1→2)[α-L-Rhap-(1→3)]-α-L-Rhap-OMe was synthesized with 13C-labeling at two positions which presented a solution to a problem of overlapping signals in the 1H NMR spectrum. The site-specific labeling also facilitated the measurement of two 3JCC and two 2JCH coupling constants. Finally, chapter 6 gives a short introduction to glycosynthase chemistry and discusses the synthesis of α-glycosyl fluorides. A novel cyclic heptasaccharide was synthesized from α-laminariheptaosyl fluoride using a mutant of the enzyme laminarase 16A and subsequently analyzed by NMR spectroscopy. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

Made available in DSpace on 2015-07-22T20:50:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-01-27. Added 1 bitstream(s) on 2015-08-11T03:25:32Z : No. of bitstreams: 1 Publico-00399a.pdf: 1787027 bytes, checksum: 6cb684251c1894e3685b611ace5c9056 (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:32Z : No. of bitstreams: 2 Publico-00399a.pdf: 1787027 bytes, checksum: 6cb684251c1894e3685b611ace5c9056 (MD5) Publico-00399b.pdf: 1285455 bytes, checksum: 52050426f68cebcd4b7e00081ce49a38 (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:32Z : No. of bitstreams: 3 Publico-00399a.pdf: 1787027 bytes, checksum: 6cb684251c1894e3685b611ace5c9056 (MD5) Publico-00399b.pdf: 1285455 bytes, checksum: 52050426f68cebcd4b7e00081ce49a38 (MD5) Publico-00399c.pdf: 2029206 bytes, checksum: 480bbfb2deea834d9cc92a52def4805e (MD5) / A gomesina (Gm) é um potente peptídeo antimicrobiano catiônico. Este peptídeo e seu análogo linear [Ser2,6,11,15]-Gm, com menor atividade lítica, foram sintetizados pela metodologia da fase sólida, empregando-se a estratégia t-Boc. Neste estudo, investigamos a interação da Gm e da [Ser2,6,11,15]-Gm com vesículas unilamelares gigantes (GUVs), compostas por membranas lipídicas de POPC ou POPC/POPG, através de microscopias óticas de contraste de fase e fluorescência. As análises se dividiram em duas diferentes partes. Na primeira, observou-se o efeito da injeção de uma solução peptídica, com micropipeta, na vizinhança das GUVs. Como resultado da interação peptídeo/lipídio, as GUVs foram desestabilizadas e romperam-se rapidamente, sem observação de formação de poros estáveis durante todo o experimento. Nos estudos controles, em ausência de peptídeo, as GUVs não romperam de forma espontânea. Peptídeos marcados com a sonda fluorescente rodamina (Rh) foram injetados e mostram que a Gm primeiramente acumula-se na superfície da vesícula, na qual, então, pequenas partículas de alto-contraste são formadas e ocorre, eventualmente, a destruição das GUVs. Este fato leva-nos a especular que a Gm rompe as membranas via modo carpete. Na segunda parte, uma solução contendo GUVs foi misturada a crescentes concentrações peptídicas para quantificação da ação lítica destes peptídeos em vesículas de diferentes composições lipídicas. O efeito de atividade lítica observado foi maior que 90% em baixas concentrações tanto de Gm quanto de [Ser2,6,11,15]-Gm em GUVs compostas de POPC, lipídio eletricamente neutro, e de uma mistura de POPG, negativamente carregados, a POPC em diferentes proporções. Estudos conformacionais foram feitos por duas técnicas espectroscópicas distintas: dicroísmo circular (CD) e fluorescência. A Gm e seu análogo linear aparentemente interagiram com as cargas negativas de SDS nas concentrações acima e abaixo da CMC. Os espectros de CD da [Ser2,6,11,15]-Gm em água apresentaram uma banda fortemente negativa em 198 nm, indicando tratar-se de uma estrutura desordenada, como esperado para peptídeos livres em solução, e não de uma dobra beta como apresentada pela Gm. A partir dos resultados obtidos, foi possível concluir que ambos os peptídeos analisados interagem com vesículas compostas por fosfolipídios e induzem o vazamento de seu conteúdo interno dependentemente da carga de membrana destas, o que corrobora estudos prévios que sugerem que as interações eletrostáticas com a bicamada lipídica dos micro-organismos representam um importante papel no mecanismo de ação da Gm. / TEDE / BV UNIFESP: Teses e dissertações

Ce travail de thèse porte sur l’analyse conformationelle de biomolécules en phase gazeuse. L’étude d’objets complexes en phase gazeuse permet de connaître les facteurs intrinsèques stabilisant leur structure. La première étape de mes travaux a été le couplage entre un appareil de mobilité ionique (IMS) et un appareil de spectrométrie de masse (MS). Nous avons simulé (SimIon) et développé différentes optiques ioniques fonctionnant à haute pression (entonnoirs à ions, piège ionique). Ces modifications expérimentales nous ont permis de commencer des études conformationnelles de biomolécules en phase gazeuse.Les premières expériences avaient pour objectif d’observer les facteurs stabilisants une structure secondaire sur des séries de peptides analogues. La première étude a été réalisée sur des séries de polyalalanines et de polyglycines de formules Arg(Ala)4XxxAla4Lys et Arg(Gly)4Xxx(Gly)4Lys, où Xxx est l’un des 20 acides aminés naturels. Nous nous sommes intéressés à l’influence de l’acide aminé central sur la conformation globale. Puis nous avons observés la stabilité de l’hélice de la partie transmembranaire de la protéine M2 du virus de la grippe A et de différents mutants. Ces études ont permit de montrer l’importance de la solvatation des charges en phase gazeuse.Enfin nous avons initié une étude sur le repliement des protéines en utilisant une protéine modèle, le lysozyme. Nous nous sommes plus particulièrement intéressés aux mécanismes de repliements en fonction du degré d’oxydation de cette dernière / This work deals with the conformational studies of biomolecules in gas phase. The gas phase allows observing the intrinsic factors that stabilize the structure.The first step of this work consisted in coupling an ion mobility tube and a mass spectrometer (IM-MS). Especially, ion optics working at high pressure had to be developed (ion funnels, cylindrical ion trap). These experimental modifications allowed us to realising conformational studies in vacuo.This work is build around two major axes. The first one deals with the understanding of the factors that stabilize the secondary structure in gas phase. We have studied series of peptides with similar sequences. We studied a series of polyalanines and polyglycines who have the following formula Arg(Ala)4XxxAla4Lys et Arg(Gly)4Xxx(Gly)4Lys, where Xxx is one of the 20 natural amino acids. We are looking the influence of central amino acid on the global structure of peptides. After that we have observed the stability of the helix of the transmembran domain of the M2 protein of virus Influenza A and some mutants. These studies allow showing the importance of the charge solvatation in the gas phase.Finally, we are initiated a study on the refolding of proteins and more particularly on a model protein: the Lysozyme. We are studied the refolding in function of oxide degrees

Le travail de cette thèse a permis tout d’abord d’aboutir à un protocole simplifié pour la synthèse des p-tert-butylcalix[7] et [9]arènes, composés jusqu’à lors difficilement préparables. Ces macrocycles ont été obtenus en grosse quantité, de l’ordre de la dizaine de gramme. Il faut également noter que cette synthèse sur grande échelle permet d’obtenir les p-tert-butylcalix[10] et [12]arènes avec des rendements modestes mais une simplicité remarquable. A partir de ces calixarènes, deux voies principales de per-fonctionnalisation ont été mises en oeuvre : La réaction de Williamson, consistant à former des éthers, et l’estérification via l’utilisation d’anhydrides. Au total, ce sont 10 nouveaux calixarènes fonctionnalisés qui ont été obtenus. Le comportement dynamique et conformationnel des calix[9]arènes originaux a été évalué en solution et à l’état solide. Dans le cas où cela a été possible, la détermination des constantes de vitesse et d’énergie libre des mouvements conformationnels a montré que ces calix[9]arènes fonctionnalisés étaient très mobiles. En outre, trois nouvelles structures RX ont été obtenues et ont confirmé que ces macrocycles adoptaient une conformation désordonnée. En l’état, ces nouveaux calixarènes ne sont pas des candidats de choix pour devenir des hôtes moléculaires efficaces. Toutefois, ce travail a permis d’améliorer nos connaissances sur des structures dérivées de p-tert-butylcalix[n]arènes (n = 7, 9, 10 et 12) peu étudiés jusqu’à présent / The work of this thesis allowed to propose a simplified procedure for the synthesis of p-tertbutylcalix[ 7] and [9]arenes, which were hardly synthesizable until now. Those macrocycles were obtained in a large amount, about a dozen of grams. It’s important to emphasize that this large scale synthesis allow to obtain the p-tert-butylcalix[10] and [12]arenes with moderate yields but in a straight and simple procedure. From these calixarenes, two main ways of per-functionalization were attempted: Williamson reaction, consisting in forming ethers and esterification by using anhydrides. Totally, 10 new functionalized calixarenes were obtained. Their dynamic and conformational behaviors in solution as well as in the solid state were explored. The determination of the rate constants and of the free energy barrier of the conformational motion has shown that those compounds were mobile. Moreover, three original X-Ray structures were obtained and have revealed that those macrocycles adopted a disordered conformation. At this point, those new calixarenes are not well designed to act as efficient molecular host. However, this work allowed to improve our knowledges on structures made from the p-tert-butylcalix[n]arene (n = 7, 9, 10 and 12), a skeleton rarely described and studied up to now