Rozprawy doktorskie na temat „Γ Amino Acid”

L’élaboration de nouveaux oligomères capables de mimer les propriétés des protéines naturelles est devenue un domaine de recherche très important, non seulement du point de vue structural mais aussi médicinal. Les peptides comportant des acides β-aminés ont été extensivement étudiés tandis que les recherches dans le domaine des γ-peptides sont plus récentes. Néanmoins, ces derniers montrent déjà des propriétés structurales uniques ainsi que des activités biologiques prometteuses.Dans notre laboratoire nous nous intéressons au développement d’une synthèse générale de nouveaux acides β,γ-diaminés à partir d’acides ∝-aminés naturels, notamment à la 3-déoxyaminostatine, et à leur utilisation dans la synthèse peptidique pour obtenir des peptides non-naturels afin d’étudier leurs propriétés conformationnelles. La stratégie de synthèse élaborée dans notre laboratoire et améliorée au cours de ces travaux a permis d’élargir la gamme des acides β,γ-diaminés. Deux diastéréomères cis et trans issus de la L-leucine, de la L- et D-phénylalanine ont été obtenus et engagés dans la synthèse de nouveaux peptides hybrides α/γ. Deux séries de peptides hybrides α/γ ont été étudiées. Les analyses structurales de la série comportant des acides ∝-aminés de configuration L montrent une capacité à adopter des structures secondaires bien définies, stabilisées par des liaisons hydrogènes intramoléculaires, impliquant notamment l’azote situé en β. De plus, une voie de synthèse vers un analogue d’un antibiotique naturel, le gramicidine S, a été proposée. Dans cet analogue, le motif D-Phe-L-Pro est remplacé par l’acide β,γ-diaminé issu de la D-phénylalanine
The design of a new oligopeptides, capable to mimic the properties of natural proteins, is an important field not only for structural studies but also in the developpement of new efficient drugs. The peptides featuring β-amino acids have been extensively explored, whereas the research in γ-peptides is more recent. The studies of γ-peptides show their ability to adopt stable secondary structures and also to have a promising biological activity. Our laboratory is interested in the synthesis of new β,γ-diaminoacids.The aim of this work is a developpement of new synthetic route starting from different natural α -amino acids and to use obtained β,γ-diaminoacids to access novel unnatural peptides having specific conformational properties.The synthetic strategy, developed and optimized in our laboratory during this work, gives access to diastereomers cis and trans from L-leucine, L- and D-phenylalanine which were used in the synthesis of a new hybrid α/γ-peptides. The structural studies were performed on two series of hybrid α/γ-peptides consisting of β,γ-aminoacid from L-leucine and L- or D-α-amino acids. In the first case, the peptides are able to adopt stable secondary structures stabilized by intramolecular hydrogen bonds involving the nitrogen on the β-position. In addition, we present the synthesis of an analogue of gramicidine S, a naturally occuring antibiotic cyclic peptide. The dipeptide pattern D-Phe-L-Pro has been replaced with the β,γ-diaminoacid synthesized from D-phenylalanine

Les travaux décrits dans ce manuscrit concernent la synthèse, l’étude structurale et l’évaluation biologique d’oligopeptides abiotiques incorporant le gamma-aminoacide hétérocyclique nommé ATC (acide-4-Aminométhyl-1,3-Thiazole-5-Carboxylique). Les ATCs sont construits autour d’un noyau thiazole et présentent deux points de diversité structurale. De précédents travaux ont déterminé que la présence du noyau thiazole entre les positions alpha et béta bloquait l'angle zéta autour de 0°, structurant les homo-oligomères de poly-(S)-ATCs en une hélice 9 droite et les faisant ainsi entrer dans le domaine des foldamères. Dans une première partie, nous avons entrepris de développer une voie de synthèse simple, flexible et énantiosélective permettant d’obtenir les ATCs stéréochimiquement purs sur une échelle de plusieurs grammes à partir d'alpha-aminoacides commerciaux. L’introduction de la diversité chimique est réalisée via deux étapes-clés que sont la condensation croisée de Claisen et la réaction de cyclisation de Hantzsch. Puis l’identification des marqueurs de structuration RMN et IR-TF des oligomères d'ATCs a été mise à profit pour caractériser le repliement d’hétéro-oligomères combinant ATCs et alpha-aminoacides. Ainsi, une étude structurale par RMN, IR-TF, cristallographie RX et dichroïsme circulaire a démontré que l’enchaînement 1:1 (L)-alpha:(S)-ATC se structurait en un ruban, stabilisé par un réseau intramoléculaire de liaisons hydrogène bifides formant des pseudocycles à 9 et 12 chaînons. La distribution des chaînes latérales le long du squelette principal présente une forte analogie avec l’hélice alpha, ce qui pourrait constituer un atout majeur pour le développement de composés à finalité thérapeutique. La dernière partie de ce travail a porté sur la conception de pseudo-peptides amphipatiques pour des applications en temps qu'antimicrobiens
This manuscript describes the synthesis, the structural study and the biological evaluation of abiotic oligopeptides incorporating the heterocyclic gamma-amino acid ATC (4-Aminomethyl-1,3-Thiazole-5 Carboxylic acid). This original block is built around a thiazole ring and displays two lateral chains. Previous work in our laboratory highlighted that the presence of the thiazole ring between the positions alpha and beta implied that zeta angle was blocked around 0°, thus structuring poly-(S)-ATCs homo-oligomers in a right-handed 9-helix foldamer. First, development of a simple, flexible and enantioselective synthesis on a few grams scale has allowed to get access of a highly diverse ATC library from commercial alpha-amino acids. Introduction of the chemical diversity occurs via two key steps implying a cross-Claisen condensation and a Hantzsch cyclization. Then identification of NMR and FT-IR structural markers of ATC-containing oligomers was used to characterize the folding propensity of hybrid α:ATC oligomers. We demonstrated that 1:1 (L)-alpha:(S)-ATC heterochiral oligomers are structured in solution in a new ribbon-like shape stabilized by a bidentate intramolecular hydrogen bond network forming 9- and 12-membered pseudorings. The distribution of lateral chains along the main skeleton shows a high analogy with alpha-helix thus constituting a major advantage for potential medicinal applications. The last part of this work has focused on the design of amphipathic ATC-containing pseudo-peptides as antimicrobial agents

Ce travail visait à étudier la neurotransmission dans la première synapse de la rétine, ou l’inhibition latérale permet un renforcement du contraste spatial et de la perception des bords des objets. Or, la nature de son mécanisme n’est pas connue à ce jour. L’utilisation de techniques d’électrophysiologie, d’immunohistochimie et de biologie moléculaire nous a permis de démontrer, à partir d’une étude comparative sur quatre souches de souris, qu’une forte proportion de cellules horizontales expriment la GAD65, mais contiennent du GABA uniquement chez la souris Balb/c. Notre approche pour mettre en évidence une transmission GABA, a permis de révéler la présence du transporteur du glutamate EAAT5 dans les cellules bipolaires à bâtonnet. Son activation permet un rétrocontrôle rapide renforçant le contraste dans la voie des bâtonnets. Enfin, nous avons apporté de nouveaux éléments pour comprendre le rôle des dystrophines dans la première synapse, et dont l’absence conduit à un défaut de transmission.

Lactacystin is a microbial metabolite isolated by Omura that exhibits neurotrophic activity in neuroblastoma cell lines. Lactacystin and especially its β-lactone analog are the first examples of non-polypeptide small molecules capable of specifically inhibiting the 20S proteasome. Various asymmetric total syntheses of lactacystin and its analogs have been reported. The total synthesis of clasto -lactacystin β-lactone is achieved using L-serine methyl ester as the starting material and the sole source of stereochemical induction. The success of this synthesis hinges on two featured transformations. The first key step involves formation of the γ -lactam core via rhodium (II) catalyzed intramolecular C-H insertion of the α-diazo-α-(phenylsulfonyl)acetamide intermediate. The methodology for this transformation has been developed and applied to the synthesis of highly functionalized stereogenic γ-lactams from natural α-amino acids. Three control elements that govern γ-lactam formation are described. This step is highlighted by the xvi simultaneous creation of two stereogenic centers of the γ-lactam core. The second key step involves the late stage aldol coupling for quaternary carbon formation and installation of the hydroxyisobutyl group. In all previously reported syntheses, this is the very first aspect which is addressed. The stereochemical outcome of this step is directed by the chiral environment of the enolate itself. Various attempts to achieve selectivity are explored and reported. Completion of the synthesis of clasto-lactacystin β-lactone requires 17 steps with an overall yield of 10%. Some general attempts for optimizing the synthetic scheme are discussed as well as the future direction of this research.

Dans notre groupe, nous nous intéressons au développement de peptides contenant des acides γ-aminés. Comme d’autres peptides contenant des acides aminés non naturels, ils ont montré leur capacité à posséder des conformations stables et/ou des propriétés biologiques intéressantes. De plus, ces peptides sont généralement résistant à la protéolyse. Dans l’objectif de synthétiser des acides -diaminés sous la forme d’un seul stéréoisomère, nous avons développé une voie de synthèse reposant sur une réaction de Blaise suivie d’une réduction diastéréosélective. En appliquant cette méthode, nous avons synthétisé des acides β,γ-diaminés dérivés de la D-phénylalanine et de l’acide L-glutamique. Le premier a été utilisé pour concevoir des analogues d’un peptide antimicrobien, la gramicidine S. Comparé à la molécule parent, les analogues ont montré une cytotoxicité beaucoup moins importante pour les cellules hôtes tout en conservant une activité antibactérienne intéressante. Cette étude nous a donné de meilleures connaissances pour développer d’autres analogues de la gramicidine S ainsi que d’autres peptides antimicrobiens. Nous avons également effectué de nombreuses optimisations pour synthétiser de façon efficace des acides β,γ-diaminés cycliques à partir de l’acide L-glutamique. Les oligomères incorporant ces acides β,γ-diaminés et des acides α-aminés ont montré un fort potentiel pour l’adoption de conformations stables. Ces études vont être poursuivies
In our group, we are interested in developing peptides containing β,γ-diamino acids . Along with many other peptides containing unnatural amino acids, they have shown the ability to possess stable conformations and/or interesting biological activities. Moreover, those peptides are usually more resistant to proteolysis. In order to synthesize stereopure γ-amino acids, we have developed a synthetic route using Blaise reaction and subsequent diastereoselective reduction as key reactions. Through applying this method, we have synthesized β,γ-diamino acids derived from D-phenylalanine and L-glutamic acid. The former β,γ-diamino acid was used for designing antimicrobial peptide gramicidin S analogues. Compared with mother molecule, the analogues exerted much less host cell cytotoxicity while remaining interesting antibacterial activity. Meanwhile, it gave us more knowledge for further developing analogues of gramicidin S as well as other antimicrobial peptides. We also paid lots of effort to efficiently synthesize cyclic β,γ-diamino acids starting from L-glutamic acid. Interestingly, when oligomers incorporating this β,γ-diamino acids and α-amino acids, they have shown the potential to adopt stable conformations. The following studies will be continuously investigated

This thesis is concerned with the development of new synthetic routes for the asymmetric syntheses of a range of β- and γ-amino acids. Chapter 1 introduces the various biological activities displayed by cyclic β-amino acid containing compounds together with their occurrence in pharmaceutical molecules and β-peptides. Some of the most commonly used synthetic strategies for the preparation of carbocyclic β-amino acids are briefly described, with the focus on the formation and functionalisation of a five-membered carbocyclic ring. Chapter 2 describes a full investigation into a highly diastereoselective Ireland-Claisen rearrangement of stereodefined allyl β-amino esters to access enantiopure α-substituted β-amino acid products. The synthetic utility of this methodology is highlighted by its application in the asymmetric syntheses of five previously inaccessible C(5)-substituted 1,2-anti-1,5-syn-transpentacins. Chapter 3 delineates investigations into a highly diastereoselective conjugate additionelimination protocol for the preparation of a cyclic β'-amino-α,β-unsaturated ester. Subsequent chemo- and diastereoselective conjugate addition reactions of Grignard reagents and lithium amides to this substrate enabled the asymmetric syntheses of four C(5)-substituted 1,2-anti-1,5-syn-transpentacins and two five-membered β,β'-diamines. Chapter 4 details the extension of the protocol developed in Chapter 3 for the conjugate addition of Grignard reagents to a range of acyclic γ-(N,N-dibenzylamino)-substituted α,β-unsaturated esters. Elaboration of the β,γ-disubstituted γ-amino ester products culminated in the asymmetric syntheses of six β,γ-disubstituted γ-amino acids. Chapter 5 chronicles the preparation of an azabicyclic α,β-unsaturated ester, following which attempts towards the asymmetric synthesis of various substituted pyrrolizidines using a conjugate addition protocol are subsequently described. Chapter 6 contains full experimental procedures and characterisation data for all compounds synthesised in Chapters 2, 3, 4 and 5.

(1) Chapter three describes the attempted investigation of the mechanism of cepham formation catalysed by the enzyme isopenicillin N synthase of Acremonium cephalosporium. The stereochemistry of this cyclisation was to be investigated with a specifically labelled butyrate residue in the unnatural substrate. 8-(a-amino) adipoyl-cysteinyl- a-aminobutyric acid. The chiral methyl and methylene groups were to be synthesised by the tritiation of specifically deuterated vinylglycine. This, in turn, was to be prepared from phenyl (2-lrimcthylsilylethynyl) sulphone. Scrambling of the deuterium labelling occurred in this synthesis of vinylglycine and so forced the abandonment of the experiment without concluding the investigation. (2) Chapter four describes the preparation of vinylglycine in a simple, inexpensive, three step synthesis. The synthetic pathway involved a Pinner reaction, sodium hypochlorite treatment and an aqueous Neber rearrangement. The overall yield was 52% from the vinylcyanide. This method was applied to the production of other b.y-unsaturated a-amino acids with mixed success. The resolution of the r-butyloxycarbonyI derivatives of the amino acids so prepared was investigated with the thiol protease, papain. Ethyl L-N-t-butyloxycarbonylvinylglycine prepared in this manner was transformed by epoxidation, dihydroxylation and cyclopropylation. An attempt to prepare a fluorothreonine derivative from the epoxide gave only the 2.3-dehydrohomoserine analogue. (3) Chapter five describes the development of methyl (R)-(2-2H)- N-4-mcthoxyphcnylglycinatc (I) as a potential reagent for determining enantiomeric excess in chiral acids. The diastereomera formed differ only in isotopic stereochemistry, thus the possibility of kinetic resolution is reduced to a minimum. The ee is measured by the proton nmr spectrum of the diastereomera. In coupling (I) to a range of racemic chiral carboxylic acids the expected 1:1 ratio was observed. However during one of the steps in the preparation of (I) racemisation of the proton label occurred thus a single diastcrcomcr has yet to be prepared.

L’acide γ-aminobutyrique ou GABA est le principal neurotransmetteur inhibiteur présent dans le système nerveux central (CNS). Afin d’obtenir un nouveau dérivé cyclobutanique du GABA, le cis-3,4CB-GABA, sous forme énantiomériquement pure, deux stratégies de synthèses efficaces et reproductibles ont été mises au point. Ces deux voies de synthèse impliquent toutes les deux une étape-clé de photocycloaddition [2+2] qui permet de créer le cycle à 4 chaînons. La première consiste en une homologation de l’acide cis-2-aminocyclobutanique (cis-ACBC), et la deuxième est une synthèse multi-étape qui utilise le caprolactame comme composé de départ.D’autre part, grâce à une synthèse stéréosélective du (1R,2S)-cis-2,3CB-GABA, quelques oligomères C- et N-protégés – di, tri, et tétra-peptides – de cet aminoacide ont été préparés. Ceux-ci ont été caractérisés par les techniques de RMN 1D et 2D, IR, RX. Les analyses ont montré qu’il n’existe pas d’interactions non-covalentes (liaisons hydrogène) inter-résidu au sein de ces structures moléculaires. En revanche, la propriété de gélification de ces oligomères dans différents solvants organiques a été mise en évidence. Des solutions et des gels formés à partir de ces peptides ont été analysés par microscope électronique à balayage et des clichés ont été obtenus montrant une organisation du dipeptide et du tetrapeptide en fibrilles. Le tripeptide lui n’a présenté aucun assemblage intermoléculaire régulier
The γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). In order to obtain new enantiomerically pure cyclobutanic derivative of GABA, the cis-3,4CB-GABA, two efficient synthetic strategies have been established. Both synthetic routes employed a photocycloaddition [2 +2] protocol, which provided the cyclobutanic ring. The first route involved the homolgation of the cis-2-aminocyclobutanecarboxylic acid (cis-ACBC), whereas the second route is a multi-step synthesis using caprolactam as starting material.On the other hand, the (1R,2S)-cis-GABA-2,3CB was synthetized, and a series of N- and C-protected oligomers of di, tri, and tetrapeptides of this amino acid were prepared. These oligomers were characterized by NMR (1D and 2D) techniques, IR, and X-ray. The analyses have shown that there are no non-covalent interactions (hydrogen bonds) between the residues of each oligomers. However, the gelation property of these oligomers in various organic solvents was demonstrated. Solutions and gels formed from these peptides were analyzed by scanning electron microscopy, and the obtained images showed a fibrous organization of the di- and tetrapeptide, while the tripeptide showed no regular intermolecular assembly

Pericentrin is a molecular scaffold protein. It anchors protein kinases, (PKB, (Purohit, personal communication), PKC, (Chen et al., 2004), PKA Diviani et al., 2000), the γ tubulin ring complex, (γ TuRC) (Zimmerman et al., 2004), and possibly dynein (Purohit et al., 1999) to the spindle pole. The γ TuRC is a ~ 2 MDa complex which binds the minus ends of microtubules and nucleates microtubules in vitro, (Zheng et al., 1995). Prior to this work, nothing was known about the association of the γTuRC with pericentrin. Herein I report the biochemical identification of a large protein complex in Xenopus extracts containing pericentrin, the γ TuRC, and other as yet unidentified proteins. Immunodepletion of γ tubulin results in co-depletion of pericentrin, indicating that virtually all the pericentrin in a Xenopus extract is associated with γ tubulin. However, pericentrin is not a member of the, γ TuRC, since isolated γ TuRCs do not contain pericentrin. The association of pericentrin with the γ TuRC is readily disrupted, resulting in two separable complexes, a small pericentrin containing complex of approximately 740 KDa and the the γ TuRC, 1.9 MDa in Xenopus. Co overexpression/ coimmunoprecipitation and yeast two hybrid studies demonstrate that pericentrin binds the γTuRC through interactions with both GCP2 and GCP3. When added to Xenopus mitotic extracts, the GCP2/3 binding domain uncoupled γ TuRCs from centrosomes, inhibited microtubule aster assembly and induced rapid disassembly of pre-assembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding, and were specific for mitotic centro somal asters as I observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Overexpression of the GCP2/3 binding domain of pericentrin in somatic cells perturbed mitotic astral microtubules and spindle bipolarity. Likewise pericentrin silencing by small interfering RNAs in somatic cells disrupted γ tubulin localization and spindle organization in mitosis but had no effect on γ tubulin localization or microtubule organization in interphase cells. Pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. I conclude that pericentrin anchoring of γ tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death. Additionally, I provide functional and in vitro evidence to suggest that the larger pericentrin isoform (pericentrin B/ Kendrin) is not functionally homologous to pericentrin/pericentrin A in regard to it's interaction with the γ TuRC.

Deux souches de Lactobacillus pentosus, Ind1 et Ind3, ont été isolées à partir de " Naigeda ", un fromage traditionnel Chinois produit dans la région du Xinjiang. Les propriétés probiotiques de L. pentosus étant peu connues, la présente étude a été conduite afin de déterminer si ces 2 souches, Ind1 et Ind3, sont susceptibles d'être utilisées comme probiotiques. Les propriétés physiologiques de L. pentosus Ind1 et Ind3 ont fait l'objet d'essais in vitro afin de déterminer leur tolérance à l'environnement gastro-intestinal et leur adhérence à l'épithélium intestinal. Leurs propriétés de dégradation de 3 substances carcinogènes (phénol, p-crésol et indole ; concentrations comprises entre 50 et 150 µg/mL) ainsi que leur inhibition éventuelle par ces mêmes substances ont été étudiées. Les effets des 2 souches de L. pentosus sur la microflore intestinale de souris, après administration orale de 109cfu/mL dans 0.5mL de lait écrémé, ont été analysés. A cet effet, les populations de Lactobacilles, Bifidobactéries, Entérobacilles, Entérocoques et Clostridium perfringens, contenues dans les fèces des souris, avant, pendant et après leur alimentation en probiotiques, ont été considérées. Enfin, les capacités des 2 souches de L. pentosus à produire de l'acide -amino butyrique ont été étudiées, et les conditions de milieu et de culture assurant la meilleure production définies. Les résultats montrent que les 2 souches de L. pentosus, Ind1 et Ind3, présentent des taux de survie élevés : plus de 90 % en milieu acide et de 80% dans une solution de bile. Les aptitudes à l'adhérence sont souches dépendantes, avec pour Ind3 un potentiel similaire à celui de souches probiotiques reconnues (NCFM et Lp115). Ind1 et Ind3 ont également montré une bonne résistance aux substances carcinogènes (phénol, p-crésol, indole à 150 μg/mL). Enfin, ces 2 souches permettent un accroissement des concentrations de Lactobacilles et de bifidobactéries, dans le tractus intestinal des souris, tout en inhibant la croissance des Entérobacilles et de C. perfringens. Ces résultats démontrent les aptitudes potentielles des deux souches de L. pentosus étudiées pour une utilisation comme souches probiotiques au sein de régimes diététiques ou pour l'élaboration de produits laitiers fermentés.

Les travaux décrits dans cette thèse s'articulent autour de la réactivité et de la synthèse de composés bicycliques chiraux, les 2,3-aziridino-gamma-Iactones et leur utilisation pour 1âpréparation d'acides beta-aminés polysubstitués. Nous avons d'abord étudié la réactivité des 2,3-aziridino-gamma-Iactones vis-à-vis de nucléophiles mous carbonés tel que des organocuprates. Pour cela, différentes aziridines ont été préparées à partir de la D-ribonolactone puis mises en réaction avec différents nucléophiles carbonés. Ainsi nous avons pu introduire de manière régiosélective un groupement méthyle en position C-2, le composé obtenu étant néanmoins accompagné de produits secondaires. Nous avons ensuite utilisé ces composés bicycliques chiraux dans le cadre de la synthèse de l'APTO, acide beta-aminé polysubstitué composant des microsclérodermines C et D. Ainsi, nous avons développé une synthèse énantiospécifique d'un précurseur cyclique de l'APTO au départ du L-gulose. Cette stratégie est basée sur la préparation de l'aziridineelactone appropriée suivie de l'ouverture régiosélective en C-2 de l'aziridine et d'une réaction de Heck. Ce composé a pu être ouvert par différents nucléophiles aminés montrant ainsi son potentiel synthétique. Enfin, nous avons étudié une nouvelle voie d'accès aux 2,3-aziridino-gamma-Iactones à partir d'un sulfamate insaturé. Cette aziridination intramoléculaire par transfert de nitrène se fait par catalyse au rhodium, médiée par le diacétate d'iodosylbenzène. Ce procédé permet d'obtenir des 2,3-aziridino-gamma-Iactones de stéréochimie différente de celles préparées par la méthode précédente, et donc d'accéder à de nouveaux acides beta-aminés
The work described in this thesis concems the synthesis and reactiviy of chiral bicyclic compounds, 2,3gamma-Iactones, and their use in the preparation ofpolysubstituted beta-amino acids. The reactivity of 2,3-aziridino-gamma-Iactones with soft carbon nucleophiles such as organocuprates was first studied. Different aziridines were prepared and reacted with various carbon nucleophiles. A methyl group was successfully introduced at the C-2 position but this was accompanied by the formation of many by-products. The 2,3-aziridino-gamma-Iactones were then used to synthesize APTO, a polysubstituted beta-amino acid fragment of microsclerodermines C and D. An enantiospecific synthesis of a cyclic precursor of APTO was developed starting from L-gulose. This strategy is based upon the preparation of the appropriate aziridine-Iactone, followed by regioselective CC2 ring-opening by an acetate and a Heck reaction. This intermediate was successfully opened by various amines, demonstrating its synthetic potential. Lastly, a new process for the preparation of 2,3-aziridino-gamma-Iactones starting from an unsaturated sulfamate was studied. This intramolecular nitrene transfer aziridination was catalyzed by rhodium salts and mediated by iodosylbenzene diacetate. This new methodology allowed the preparation of 2,3-aziridino-gamma-Iactones with a different stereochemistry than those synthesized previously, potentially leading to new beta-amino acids

T lymphocytes are essential components of the immune system and as such are continually regulated by a variety of factors. Every step of their development, survival and function is tightly monitored to ensure their ability to recognize most foreign agents and mount adaptive immune responses during pathogenic infections, while remaining tolerant to self-antigens. Among the many factors that participate in the regulation of T cell development and function are the cytokines. Cytokines that signal through the common gamma (γс) chain and the Janus kinase 3 (Jak3) include IL-2, -4, -7, -9, -15, and -21 and have been implicated in the regulation of every stage in the life of a T cell. Therefore, it is not surprising that mutations in the γс chain or Jak3 lead to a SCID condition in humans and mice. Specifically, Jak3-deficient mice are characterized by a reduction in thymic cellularity and dysregulated T cell homeostasis. They have an expansion of memory-like CD4+ mature T cells and an almost complete absence of mature CD8+ T cells. By investigating the TCR repertoire of CD4+ T cells in the thymus and spleen of Jak3-/- mice, I deduced that the CD4+ T cell activation and expansion is TCR-specific and takes place in the periphery of the mice. After crossing Jak3-deficient mice to Bcl-2 transgenic mice I showed that the developmental block observed in Jak3-/- mice could not be rescued by the anti-apoptotic factor, despite the fact that its expression did increase, slightly, the total numbers of developing thymocytes. The enforced expression of Bcl-2 was also not sufficient to revert the dysregulation of T cell homeostasis in Jak3-/- mice. Finally, in order to further understand the role played by γс cytokines during T cell function, I investigated the ability of mature Jak3-/- CD8+ T cells to become activated and differentiate into effector cells in response to a viral infection. My results indicate that CD8+ T cells are activated and proliferate in response to a viral infection, but their survival, as well as their ability to proliferate and differentiate into effector cells are greatly impaired, resulting in the inability of Jak3-deficient mice to mount a protective response.

Ce travail porte sur l'étude de la régiosélectivité d'addition de carbanions en alpha de nitrile sur des composés carbonylés alpha-bêta éthyléniques dans le but de réaliser la synthèse de composés carbonylés gamma fonctionnalisés. Dans le premier chapitre, nous avons montré que l'addition du dérivé lithié du pyridyl-3 acétonitrile sur diverses énones conduit dans le tétrahydrofuranne après protonation exclusivement aux adduits 1,4. Les cétones bicycliques obtenues présentent une jonction de cycle cis tandis que la stéréochimie cis ou trans des cyclanones-2,3 disubstituées est orientée par le contrôle cinétique ou thermodynamique de la protonation des énolates. L'addition 1,2 de ce carbanion sur le méthyl-3 butène-2 al est réalisée dans le tétrahydrofuranne tandis que l'addition 1,4 est observée sous contrôle thermodynamique en présence d'hexaméthylphosphorotriamide ou d'éthérate de trifluorure de bore. Dans le second chapitre, nous avons réalisé les additions régiosélectives des anions lithié et potassé du N-diméthylammophénylacétonitrile et lithié de l'(éthoxy-éthoxy) phénylacétonitrile (équivalents de benzoyle) sur divers aldéhydes insaturés. Notre étude a porté sur 1'tnfluence de la nature des réactifs, du solvant, du cation, des acides de Lewis et des conditions réactionnelles sur la régiosélectivité et la stéréosélectivité de cette addition. Nous avons mis au point une méthodologie permettant d'accéder aux cétoaldéhydes et aux cétoalcools allyliques, d'obtention généralement délicate, que nous avions appliquée à la synthèse de composés optiquement actifs dont la formation est discutée. Dans le troisième chapitre, l'étude de la décyanation réductrice d'un aminonitrile modèle par divers borohydrures a été réalisée afin d'accéder aux aminoaldéhydes et ammoalcools allyliques. Suivant l'agent réducteur, l'amine attendue est accompagnée du complexe stable amine-borane en proportions variables. L'application de cette méthodologie aux adduits 1,4 a conduit à l'aminoalcool correspondant ; l'adduit 1,2 subit une rétrocondensation
This work deals with the regioselectivity of addition of alpha nitrile carbanions on unsaturated carbonyl compounds in the aim of synthesizing gamma functionalized carbonyl compounds. In the first chapter, we have shawn that the addiion of lithiated 3-pyridylacetonitrile to various enones in tetrahydrofurane, followed by protonation, leads exclusively to 1,4 adducts, the bicylcic ketones are Cis ring fused. The cis or trans stereochemistry of the 2,3-disubstituted cyclanones depends on the protonation of enolates under kinetic or thermodynamic control. The 1,2 addition of this anion on 3-methyl 2-butene 1-al is performed in tetrahydrofurane whereas 1,4 addition is observed under thermodynamic control in the presence of hexamethylphosphoramide or trifluoroboron etherate. In the second chapter, we have realized regioselective additions of lithiated and potassiated dimethylaminophenylacetonitrile and lithiated (ethoxy-ethoxy) phenylacetonitrile (latent benzoyl equivalents) on various unsaturated aldehydes. Our study centers on the influence of the nature of the reactants, solvents, cations, Lewis acids, and experimental conditions on the regio and stereoselectivities of these additions. A methodology leading to ketoaldehydes and keto allylic alcohols, usually difficult to obtain, was improved and applied to the synthesis of optically active compounds. In the third chapter, the reductive decyanation by different borohydrides was studied on an aminonitrile model with the aim of accessing to aminoaldehydes and aminoallylic alcohols. The desired free amine is accompanied by various amounts of stable amine-borane complexe according to the reducing agent. This methodology, applied to 1,4 adducts, leads to the corresponding aminoalcohols while retrocondensation occurs from the1,2 adducts

L'allodynie mécanique est un symptôme cardinal des douleurs persistantes. Elle est due à l’activation de circuits, habituellement bloqués, des couches superficielles de la corne dorsale spinale ou du sous-noyau caudal du trijumeau (Sp5C), par lesquels les afférences mécaniques à bas seuil peuvent accéder aux neurones nociceptifs de projection de la couche I. Un élément déterminant de ces circuits est une classe d’interneurones excitateurs de la couche II interne (IIi) exprimant l'isoforme gamma de la protéine kinase C (PKCγ), et recevant des afférences des mécanorecepteurs à bas seuil. La modulation de l’inhibition tonique de ces interneurones PKCγ contribue à l’apparition de l’allodynie mécanique. Cependant la morphologie, les propriétés électrophysiologiques et les caractéristiques des afférences excitatrices et inhibitrices de ces interneurones PKCγ ne sont toujours pas connues. Utilisant des techniques d’électrophysiologie (enregistrements patch-clamp) et d'immunohistochimie sur tranches de Sp5C, nous avons caractérisé les propriétés des interneurones PKCγ de la couche IIi du Sp5C chez le rat adulte et comparé ces propriétés avec celles d’interneurones voisins n’exprimant pas la PKCγ.Cette étude révèle que l’arborisation neuritique des interneurones PKCγ s’étend largement au sein de la couche IIi, et peut se prolonger du coté dorsal jusqu’à la couche II externe, sans jamais atteindre la couche I. En outre, en fonction de cette extension neuritique, au moins deux sous-populations d'interneurones PKCγ peuvent être dissociées – centrales et radiales – qui s’avèrent être aussi fonctionnellement différentes. Comparés aux autres neurones non-PKCγ de la conche IIi, les interneurones PKCγ, dans leur ensemble, présentent un seuil de déclenchement des potentiels d’action plus bas et, souvent associée, plus fréquemment une réponse tonique à un courant dépolarisant, indiquant ainsi qu’ils sont plus facilement excitables. Cependant, ils reçoivent inversement une excitation synaptique plus faible. Quant aux afférences inhibitrices, la plupart des interneurones PKCγ expriment des synapses mixtes associant récepteurs GABAAergiques (GABAAR) et récepteurs glycinergiques (GlyR). Seul un petit nombre d’entre eux exprime des synapses uniquement GABAAR ou GlyR. Pourtant, tous les interneurones PKCγ reçoivent non seulement des mIPSCs mixtes GABAAR-GlyR, mais aussi des mIPSCs uniquement GABAAR ou uniquement GlyR
Mechanical allodynia, a cardinal symptom of persistent pain, is associated with the unmasking of usually blocked local circuits within the superficial spinal or medullary dorsal horn (MDH), through which low-threshold mechanical inputs can gain access to the lamina I nociceptive output neurons. Key determinants of these circuits are lamina II (IIi) excitatory interneurons that selectively concentrate the gamma isoform of protein kinase C (PKCγ) and receive low-threshold mechanical receptor (LTMR) inputs. Tonic inhibition of PKCγ interneurons is thought to gate circuits underlying mechanical allodynia. However, the morphology, electrophysiological properties and excitatory and inhibitory synaptic inputs on these PKCγ interneurons are still unknown. Using whole-cell patch-clamp recordings and immunohistochemical techniques in slices of adult rat MDH, we characterized these lamina IIi PKCγ interneurons and compared them with neighboring non-PKCγ interneurons. Our results reveal that the neurites of PKCγ interneurons arborize extensively within lamina IIi, can spread dorsally into lamina IIo, but never reach lamina I. In addition, according to cell bodies and the orientation and extent of dendritic arbors, at least two morphologically different classes of PKCγ interneurons can be identified – central and radial – which appear to be also functionally different. Compared with neighboring lamina IIi non-PKCγ interneurons, PKCγ interneurons exhibit a lower threshold for action potentials, consistent with a more frequent tonic spike discharge to depolarizing step current, indicating that they are more excitable than other lamina IIi neurons. On the other hand, they receive a weaker excitatory synaptic drive. According to inhibitory inputs, most PKCγ interneurons display mixed-GABAA (GABAAR) and glycine (GlyR) receptor synapses with only very few of them displaying also GABAAR-alone or GlyR-alone synapses. Interestingly, all PKCγ interneurons exhibit mixed GABAAR–GlyR as well as GABAAR-only and GlyR-only mIPSCs. Altogether, this study indicates that PKCγ interneurons within lamina IIi of MDH are different from other lamina IIi neighboring neurons according to morphology, electrophysiological properties and synaptic inputs. This is consistent with their specific role in the gating of dorsally directed circuits within the MDH underlying mechanical allodynia. Moreover, we have identified two morphological and functional subclasses of PKCγ interneurons which might thus differently contribute to this gating

碩士
中華醫事科技大學
生物醫學研究所
100
GABA is the chief inhibitory neurotransmitter in the mammalian central nervous system. It plays a role in regulating neuronal excitability throughout the nervous system. Many studies have showed that GABA was involved in many physiological functions. (1) Activated GABA receptor induces vasodilation and angiotensin converting enzyme (ACE) inhibition. The blood vessel relaxation leads to decrease blood pressure. (2) It is well established that activation of GABAA receptors favors sleep. The GABAA receptor-mediated inhibitory processes decrease waking and increase slow-wave sleep and the paradoxical sleep is decreased by agonistic modulators of GABAA receptors. (3) Benzodiazepine-induced conformational changes increase GABA's receptor affinity, and thus increase the frequency of ion channel openings. Chloride ion influx hyperpolarizes GABA neurons and produces GABA's inhibitory interneuronal effects. (4) Adding GABA and GABA agonists improve the function of cells in the visual cortex (area V1) and facilitate visual function in old animals. (5) The activation of GABAA receptor leading to Ca2+ channels opening are involved in progesterone-induced acrosomal exocytosis in mouse spermatozoa. (6)To inhibit the inflammatory response and stimulate the epidermal revive. (7)GABA receptors control gastrointestinal motility. (8) Be supplement feed to make domestic birds and animail health. Many Lactic acid bacterium (LAB) studies found, these beneficial effects not only came from bacterium itself but also metabolism products, including lactic acid, bacteriocin, polysaccharide and GABA. The thesis review and summarize these publications and according to these studies, we believed that GABA is worthy to develop and apply in functional food.

碩士
國立臺灣海洋大學
食品科學系
98
Rice bran (RB), due to contain considerable glutamate decarboxylase activity, could be used to incubate with monosodium L-glutamate (MSG) substrate to produce -aminobutyric acid (GABA). After water-soluble portion was removed, large amounts of active components such as phytic acid (PA) and oryzanol remained and is worthy of recovery. This study investigated to use RB not only for the production of GABA but also to to extract PA under appropriate processing conditions. PA amounts recovered from acetone-defatted rice bran (DRB) were 25.1~47.8% higher than that from raw RB. But it might be lose of the activity of GAD, the production of GABA using DRB decreased by 32.8%. After the incubation of raw RB and MSG at 35oC for 6 hours, the whole reactant was called rice bran cake (RBC). Among raw RB, DRB and RBC as starting material, RBC without a further centrifugation (RBC before centrifugalizing) was the best source for the recovery of GABA and PA. Factors that affecting the recovery of PA including hydrochloric acid concentration (0.1~2.0 M), extraction temperature (25~45oC) and extraction time (0~5 hr) were compared. To extract RBC before centrifugal by 2.0 M HCl for 1 hr at ambient temperature had the highest yield of PA (100.01 mg/g RB). Also yield of GABA from the subsequent recovery producedure was not affected, amounting in a range of 3,579 ± 252.10 ~ 4,377 ± 15.57 g/g and showing no significant difference with initial content. For separation of PA from acid extract, acid extract was neutralized by adding NaOH solution and this resulted in a precipitation of phytate salt. The recovery of PA, however, was only 44.7%. On the other hand, when acid extract was firstly titrated with NaOH solution to pH 3.0 and then mixed with acetate buffer (pH 3.0), the solution was applied into a Dowex 50WX8 ion-exchange column to elute GABA and PA. It was found that the PA recovery rose to 70%. Also GABA and PA were completely separated from each other. The heating of PA sample thus obtained at 120oC for 24 hr showed the lowest PA remained, suggesting that the production of inositol might be the largest.

碩士
國防醫學院
藥學研究所
92
The activity of γ-secretase controls the amount of amyloid β-peptide (Aβ) formation, which is central in the pathogenesis of Alzheimer’s disease (AD). γ-Secretase has been considered central to understanding the etiology of AD because it determines the proportion of the highly fibrillogenic Aβ42 peptide. The fact that Aβ42 is increase in all forms of early-onset AD is very intriguing because this form of Aβ fibrils is toxic to cultured neurons. Therefore, γ-secretase has been one of the major goals of drug discovery for AD. In this study, we started the development of potential γ-secretase inhibitors based on two lead compounds (i) Boc-FΨF-Leu-Val-OMe, a peptidomimetic with hydroxyethyl moiety mimicking the transition-state of aspartyl protease catalysis, (ii) 3,5-difluorophenylacetyl- alanyl-(S)-phenylglycine t-butyl ester (DAPT), an amino acid derivative. Moieties on P1’ and P3’ positions of Boc-FΨF-Leu-Val-OMe and difluorophenylacetyl and alanyl groups of DAPT were the focus of the series of chemical optimizations, respectively. An assay that expresses a Gal4-VP16 (GVP) tagged APP or C99 and a Gal4-promoter luciferase reporter gene were established to monitor the inhibitory potencies for the series of compounds against γ-secretase activity. The results indicated that hydroxyethylureas 23c, 23d, 23e, 23f, 24a, 24b and 24c and amino acid derivative 47 possess the similar or higher activity as compared with Compound E, whereas compounds derived from modification of difluorophenylacetyl and alanyl groups at DAPT dramatically decreased the inhibitory potency against γ-secretase.

The work reported in this thesis is focused on understanding the conformational properties of peptide foldamers containing β or γ amino acid residues. Chapter 1 includes a brief state-of-the-art literature review on peptide foldamers containing β and/or γ amino acids. Chapter 2 describes the effect of insertion of an Aib residue in a β3(R) peptide sequence with the help of two model peptides of comparable length- Boc-[β3(R)Val]9-OMe and Boc-[(β3(R)Val)3-Aib-(β3(R)Val)4]-OMe. Results in chapter 2 demonstrate the influence of the gem-dimethyl effect of Aib residues on the conformation of Aib/β3(S) peptides. In principle, the nature and chirality of the component residues also might affect the conformation of the heterogeneous peptides. Chapter 3 attempts to demonstrate a few of such effects with the help of NMR studies on few model peptides- (Boc-(Val)2-Ala-(Leu)2-OMe, Boc-(Val)2-β3(S)Ala-(Leu)2-OMe, Boc-(Val)2-γ4(S)Ala-(Leu)2-OMe, Boc-Val-DVal-β3(S)Ala-(Leu)2-OMe & Boc-Val-DVal-γ4(S)Ala-(Leu)2-OMe) and with a crystal structure database (CSD) analysis. Chapter 4 reports the first solution state structural characterization of C12/C14/C12 helices in peptides of the type- [γ4(R)-Aib-γ4(R)]2 and [γ4(R)-α(L)-γ4(R)]2. Δδ values of amide NHs (from DMSO-d6 titration in CDCl3) in the hexapeptides used in this study, as well as the hydrogen bond parameters and some other features of the crystal structures, reported in a previous study consistently and conclusively points to the existence of the hydrogen bond heterogeneity in C12/C14/C12 helix. Chapter 5 demonstrates the effect of insertion of a DPro-Gly segment in a γ4(R) peptide sequence using three model peptides- Boc-γ4(R)Leu-γ4(R)Leu-γ4(R)Leu-γ4(R)Leu-DPro-Gly-γ4(R)Leu-γ4(R)Leu-γ4(R)Leu-γ4(R)Leu-OMe, Boc-γ4(R)Ile-γ4(R)Ile-γ4(R)Ile-γ4(R)Ile -DPro-Gly-γ4(R)Ile-γ4(R)Ile-γ4(R)Ile-γ4(R)Ile-OMe and Boc-γ4(R)Val-γ4(R)Val-γ4(R)Val-γ4(R)Val-DPro-Gly-γ4(R)Val-γ4(R)Val-γ4(R)Val-γ4(R)Val-OMe.
CSIR

碩士
朝陽大學
應用化學研究所
87
We build our lab's method to synthesize β-amino-α,β-unsaturated,γ-thiolactone 1 (achiral)、2 (chiral), and have good yield. From thiolactone 1、2, we have to synthesize thiophene 3、4, and study thiophene's Diels-alder reaction and Lewis acid catalyze Michael addition

碩士
朝陽大學
應用化學系碩士班
88
A novel procedure for the synthesis of achiral thiopenone from Methyl 3-pyrrolidino(E)-2-butenoate and it’s application on the studies of Aldol reactions under basic(including the temperature,counter ion , base reagent effect) and Lewis acid conditions (Lewis such as BF3.Et2 , SnCl4 , TiCl4) to generate syn - product or anti-product will be discussed.

碩士
東海大學
食品科學系
105
The aim of this study was to screen soybeans cultured in Taiwan with highest glutamate decarboxylase (GAD) in their sprouts. The optimum conditions for the production of Gamma-amino butyric acid (GABA) were also studied by extracting GAD crude enzyme from the selected soybean sprouts. First of all, sprouts of black soybean Tainan 3, black soybean Tainan 5, Jinzhu soybean and Kaohsiung 10, were prepared under 27℃, lightless and every 2 hrs automatic sprinkler culture. The results showed that GAD activity in all cultivars increased with the germination time. GAD enzyme in each breed of bean sprouts can reach maximum activity within 10 days. Black soybean Tainan 3 had its best enzyme activity on the 3rd day and 7-9th days. Both black soybean Tainan 5 and Jinzhu soybean showed their best enzyme activity on the 7th day. Kaohsiung 10 revealed its best enzyme activity during the 3-10th days, The maximum GAD activity in various varieties was between 2.20 and 3.41 U. But there was no significant difference between them (p <0.01). Therefore, sprouts of Kaohsiung 10 were selected for further experiments, because of its shortest required germination time and relative higher GAD activity. Furthermore, response surface methodology was applied to optimize the independent variables including reaction temperature 28.6~75.2℃, pH 5.32~8.68 and glutamate concentration 3.2~36.8 mM for GABA production. Based on response surface regression analysis (RSREG) procedure, the optimum reaction conditions for maximizing GABA production of 601 mg/L were 57.5 ℃, pH 7.75 and 27.5 mM glutamate. The results showed that the highest yield of GABA production could be up to 1027.49 mg/L when the reaction time was extended to 6 hrs. Finally, it were verified by the conversion rate. The highest conversion of glutamate to GABA by GAD enzyme was also carried out in 6 hrs.

碩士
國立屏東科技大學
生物機電工程系所
95
The general populace starts to take care of their health in recent years. Therefore, the acceptation regarding to high nutritional value food gradually become popular. However, the soybean is the Chinese peoples' often edible food. Soybean itself has includes the extremely high GABA(γ－Amino butyric acid), GABA was proved at many experiments that soybean has many effects. The physiology function like stable spirit, the improvement of arteriosclerosis, the improvement of losing sleep, the suppression of blood pressure rise, to promotes the brain metabolism, to promote the memory, the prevention arteriosclerosis, the activation kidney function and so on. Germinates is an important process which the plant grows, when the new bud sprouts it can activate the sleep enzyme, as increase growth needs nutrient. According to the literature pointed out germinates truly can cause GAD(glutamate decarboxylase) to escape. From the activation it has increase GABA, but this research is then urges using the soybean natural growth way to stimulates soybean's GABA increase, in order to use it to manufacture the soybean milk. Firstly, to germinate the soybean under the following environment, by the temperature 30℃ and humidities 75%. Then, uses HPLC(high peformance liquid chromatograph) to analysis. Meter to examines the soybean sample's quantity contained for GABA and with bud length between relations. The experimental result showed, using the way which germinates may cause the originally not germinated soybean's GABA content to enhance from 1289 mg/100g to rise to the germinates 24 hour after 1729 mg/100g, its content increases 34.1%. When the bud length is 16.1 ~ 23.3 mm, GABA all presents condition of the increase. Nevertheless, after makes the soybean milk, the not germinated soybean milk and which the soybean does with to germinate 24 hours's soybean milk its GABA content, respectively is 70 mg/100ml and 83 mg/100ml. However, when it is in the use of N-hexane in degreasing experiment, by 100℃ steams the soybean milk which has not germinated, Its increase normal hexane may make the GABA content to increase from 70 mg/100g to 84 mg/100g. However, by 100℃ steams germinates 24 hours soybean milk, Its increase normal hexane may make the GABA content to increase from 83 mg/100g to 96 mg/100g. In conclusion, the use of germinates way truly may cause to increase GABA in the soybean. However, when manufactures under the soybean milk ,it is also to be allowed to retain more than 70% above GABA not to be able to destroy during the soybean milk manufacture process. Therefore, uses of germinates the soybean in order to use in to manufacture the soybean milk, it does have the high feasibility.

碩士
東海大學
食品科學系
100
Abstract The aim of this study was to investigate the optimum production conditions of γ-amino butyric acid (GABA) by glutamate decarboxylase (GAD) from germinated Adzuki bean. Response surface methodology was applied to evaluate the independent variables including reaction temperature 28.6~45.5℃, pH 6.32~9.68 and PLP concentration 0.032~0.368 mM under fixed enzyme to substrate ratio of 1：75, 0.25 mM CaCl2 and reaction time 1h. Based on response surface regression analysis (RSREG) procedure, the optimum reaction conditions for maximizing GABA production of 326.7 mg/L were 37.2 ℃, pH 7.6 and 0.208 mM PLP. The maximum predicted value could be achieved whin 95% confidence interval of experimental values. Furthermore, bioreactor was employed to convert glutamate with GAD into GABA and to recycle the GAD. The productivity of bioreactor was 1.663 mg GABA/mg protein, which was three fold than that of batch reactor after reusing the GAD for three times. The molecular weight of the GAD from germinated Adzuki bean was about 101 kDa (tetramer). Based on enzymatic kinetic study, the parameters of km and Vmax were 16.66 mM and 555 mg /L, respectively. The result of storage stability test indicated that 70% GAD activity was maintained after a half year storage under both 4 ℃and -20 ℃.

Yes
A reliable description of ion pair interactions for biological systems, particularly those involving polyatomic ions such as carboxylate and divalent ions such as Ca2+, using biomolecular force-fields is essential for making useful predictions for a range of protein functions. In particular, the interaction of divalent ions with the double carboxylate group present in γ-carboxyglutamic acid (Gla), relevant to the function of many proteins, is relatively understudied using biomolecular force-fields. Using force-field based metadynamics simulations to predict the free energy of binding between Ca2+ and the carboxylate group in liquid water, we show that a widely-used biomolecular force-field, CHARMM22*, substantially over-estimates the binding strength between Ca2+ and the side-chains of both glutamic acid (Glu) and Gla, compared with experimental data obtained for the analogous systems of aqueous calcium–acetate and calcium–malonate. To correct for this, we propose and test a range of modifications to the σ value of the heteroatomic Lennard–Jones interaction between Ca2+ and the oxygen of the carboxylate group. Our revised parameter set can recover the same three association modes of this aqueous ion pair as the standard parameter set, and yields free energies of binding for the carboxylate–Ca2+ interaction in good agreement with experimental data. The revised parameter set recovers other structural properties of the ion pair in agreement with the standard CHARMM22* parameter set.

X-ray crystallographic studies of designed peptides provide definitive proof of the success of a design strategy and yield essential structural information that can be utilized in the future design of biologically and structurally important polypeptides. The ability to create locally folded, hydrogen bonded structures in short peptide oligomers is important for peptide design strategies, which rely on the use of folding nuclei in the construction of protein secondary structure modules like helices and β-hairpins. The realization that backbone homologated amino acids, specifically  and  residues can be incorporated into folded polypeptide structures, has stimulated considerable recent interest in the areas of peptide mimetic and foldamer design. Polypeptides with unnatural, modified backbones provide an entry to novel classes of intramolecularly hydrogen bonded helical structures, which are without precedent in conventional peptides composed of  amino acid residues. The design of hybrid sequences, which contain ,  and  amino acid residues, greatly expands the diversity of peptide structure space. The insertion of additional backbone atoms into amino acid residues enhances the number of torsional variables that describe local residue conformation. For example, in the case of  residues, four torsional variables describe the conformational space,  = (Ci-1-Ni-C i-C i), 1 = (Ni-C i-C i-C i), 2 = (C i-C i-C i-Ci), and  = (C i-C i-Ci-Ni+1), as compared to two Ramachandran angles , , for an  residue. In developing the structural chemistry of  residues, it has proved fruitful to explore the conformational properties of residues which are conformationally constrained, either by backbone cyclisation or by the use of gem-dialkyl substitution. In these approaches, conformational choices at selected positions are biased, using local stereochemical constraints that limit the range of accessible backbone torsion angles. Recent trends in peptide research focus on the incorporation of β-, γ- and higher homologues of the α-amino acid residues in designed peptides as they confer significant proteolytic stability. X-ray crystallographic studies of such modified peptides containing non-protein residues are essential, since information on the geometric and stereochemical properties of modified amino acids can only be gathered from systematic structural studies of synthetic peptides incorporating them. This thesis reports a study of the structures and conformations of designed peptides containing stereo chemically unconstrained γ-amino acid residues, derived from naturally occurring proteinogenic α-amino acid residues by backbone homologation. The structures described in this thesis contain the backbone homologated, unconstrained γ-amino acid residues, 4Val, 4Leu and 4Ile and the 3-monosubstituted γ-amino acid residue pregabalin (Pgn). The crystal structure determination of peptides permitted the characterization of intramolecularly hydrogen bonded helices in hybrid sequences and homooligomeric γ- peptides. The studies enabled the precise determination of conformational and geometric parameters of three backbone homologated γ-amino acid residues, 4Val, 4Leu and 4Ile and one monosubstituted unconstrained γ-amino acid residue, pregabalin (Pgn). A detailed analysis of the backbone conformations and intramolecular hydrogen bond parameters of γ- amino acid residues in different hetero- and homooligomeric helices is also provided. This thesis is divided into 7 chapters. Chapter 1 provides a brief introduction to the stereochemistry of polypeptide chains, description of backbone torsion angles of α-, β- and γ- amino acid residues and the major secondary structures of α-, β-, γ- and hybrid peptides. Some previous studies on unconstrained backbone homologated γ-residues are briefly reviewed, followed by a concise introduction to X-ray diffraction and the methods of structure solution. Chapter 2 describes the characterization of the  C12 helix in oligopeptides containing the unconstrained  residue, 4(R)Val. The crystal structure determinations of [Aib-4(R)Val]n oligomers viz. Boc-[Aib-4(R)Val]2-OMe, Boc-[Aib-4(R)Val]4-OMe, Boc-[Aib-4(R)Val]5- OMe and Boc-[Aib-4(R)Val]8-OMe reveal the formation of C12 helical structures in the solid state. The structures of ()n peptides ranging in length from 4 to 16 residues permit characterization of the conformational parameters for -residues. A comparison of the structures of 4 and 8 residue peptides containing repeat ()n, (αβ)n and ()n sequences is also described. The structure determination of the ()n sequences, Boc-[Aib-(S)Val]n-OMe (n = 2 and 4) and the (αβ)n sequences, Boc-[Aib-β3(R)Val]n-OMe (n = 2 and 4), permitted a comparison of the intramolecular hydrogen bonding patterns in peptide helices. The ()n sequences formed 310 helical structures stabilized by C10 hydrogen bonding rings, while the corresponding ()n sequence yielded the backbone expanded C12 helix. In the case of the (αβ)n series the tetrapeptide yielded two consecutive C11 hydrogen bonds corresponding to a C11 helix. In contrast, the (αβ)4 octapeptide yielded a mixed C14/C15 helical structure generated by successive three residue turns of the type αβα and βαβ. Chapter 3 presents the crystallographic characterization of an ()4 dodecapeptide Boc-[Aib-γ4(R)Val-γ4(R)Val]4-OMe. This study was intended to establish whether helical folding could be maintained in sequences containing contiguous γ4(R)Val residues. Specifically, an (αγγ)n sequence composed of successive two residue, 4→1 hydrogen bonded turn can result in a helical structure in which the αγ and γα sequences form C12 hydrogen bonded turns while the γγ segment forms a C14 turn. The crystal structure of the 12-residue peptide Boc-[Aib-γ4(R)Val-γ4(R)Val]4-OMe described in this chapter does indeed demonstrate formation of a mixed C12/C14/C12 helix. Three independent molecules are observed in the crystallographic asymmetric unit permitting an analysis of the conformational and hydrogen bond parameters in this hybrid helical structure. Chapter 4 addresses the question whether ()n helices can be characterized in crystals in sequences composed of unconstrained  and  residues. The crystal structures of 4  hybrid peptides, Boc-Leu-γ4(R)Val-Val-OH, Boc-[Leu-γ4(R)Val-Val]2-OH, Boc-[Leu- γ4(R)Leu]2-OMe and Boc-[Leu-γ4(R)Val]5-OMe are presented. In all four cases folded intramolecularly hydrogen bonded structures are obtained. C12 intramolecular hydrogen bonding is observed in all 4 peptides, suggesting that helical folding is readily obtained in()n and ()n sequences even in the absence of local backbone constraints. Preorganization of  residues does not appear to be a necessary condition for initiating helical folding in hybrid peptides containing -residues. Chapter 5 follows up the results obtained in the preceding chapters and examines the conformational properties of homooligomeric ()n sequences. The structures of the - peptides Boc-[4Val]n-OMe (n = 2-6, 8), Boc-[4Leu]n-OMe (n = 4) and Boc-[4Ile]n-OMe (n = 6, 10) determined in crystals by X-ray diffraction, are described. The tetrapeptides Boc- [4(R)Leu]4-OMe and Boc-[4(S)Leu]4-OMe reveal incipient C14 helical structures of opposite handedness. The hexapeptide Boc-[4(R)Ile]6-OMe and decapeptide Boc-[4(R)Ile]10-OMe fold into C14 helices stabilized by 4 and 8 intramolecular hydrogen bonds respectively, which are backbone expanded analogues of the -peptide 310 helices. The foldability of unconstrained homooligomeric -peptides, is in sharp contrast to the tendency of their - counterparts to form extended, sheet like structures. The structures presented in this chapter constitute the first reported characterization of C14 helix in homooligomers of unconstrained -residues. Chapter 6 describes the crystal structures of two pregabalin zwitterions, (S) Pgn and (R, S) Pgn, five derivatives, one γγ dipeptide, Boc-Gpn-(R, S) Pgn-OH (Gpn, gabapentin) and two hybrid isomeric pentapeptides with the template Boc-Aib-Xxx-Leu-Phe-Val-OMe [where, Xxx = (S)Pgn and γ4(R)Leu]. Five derivatives of pregabalin, Boc-(R, S) Pgn-OH, Boc-(R, S) Pgn-Cyclohexylamide, Boc-(R, S) Pgn-NHMe, Piv-(S) Pgn-NHMe and Piv-(R, S) Pgn-NHMe, reported in this chapter do not possess any intramolecular hydrogen bonds. The γγ dipeptide, Boc-Gpn-(R, S) Pgn-OH forms a C9 hydrogen bond, involving the Gpn residue. Pregabalin (Pgn) is a γ3 substituted analogue and is isomeric with γ4Leu, described in Chapters 4 and 5. Two model pentapeptide sequences were examined to compare the conformational characteristics of γ3 and γ4 monosubstituted γ-residues. The crystal structures of two isomeric pentapeptides, Boc-Aib-(S)Pgn-Leu-Phe-Val-OMe and Boc-Aib- γ4(R)Leu-Leu-Phe-Val-OMe are reported. In the case of the Pgn containing pentapeptide two polymorphic crystal forms are obtained, containing three independent peptide molecules. All three independent molecules fold into short helical segments, with C12 hydrogen bonds at αγ and γα segments and C10 at αα segments. The γ4Leu pentapeptide yielded a conformation almost identical to that observed in the γ3 (Pgn) pentapeptides. Chapter 7 presents a summary of the results obtained and highlights the major, conclusions. Stimulated by the structural results obtained in this study a detailed analysis of γ residue containing helical turns was also carried out by extracting the structures from the CCDC (Cambridge Crystallographic Data Centre) database. Appendix presents a series of tables listing the backbone torsion angles and hydrogen bond parameters of the peptides relevant for the present study. The structures reported in this thesis are listed below (for the structures deposited in the Cambridge Crystallographic Data Centre, the respective CCDC numbers are indicated in parentheses): 1. Boc-[Aib-α(S)Val]2-OMe [C24 H44 N4 O7] (881183) 2. Boc-[Aib-α(S)Val]4-OMe [C42 H76 N8 O11] (882909) 3. Boc-[Aib-β3(R)Val]2-OMe [C26 H48 N4 O7. 2H2O (2O)] (881179) 4. Boc-[Aib-β3(R)Val]4-OMe [C46 H84 N8 O11] (881180) 5. Boc-[Aib-4(R)Val]2-OMe [C28H52N4O7] (881181) 6. Boc-[Aib-4(R)Val]4-OMe [C50 H92 N8 O11] (881182) 7. Boc-[Aib-4(R)Val]5-OMe [C61 H112 N10 O13] (881177) 8. Boc-[Aib-4(R)Val]8-OMe [C78H144N12O15. 2H2O] (881178) 9. Boc-[Aib-4(R)Val-4(R)Val]4-OMe [C94 H172 N16 O19] (909490) 10. Boc-Leu-4(R)Val-Val-OH [C23H43N3O6] (881185) 11. Boc-[Leu-4(R)Val-Val]2-OH [C41H76N6O9] (881186) 12. Boc-[Leu-4(R)Leu]2-OMe [C34H64N4O7. 2H2O (O)] (930729) 13. Boc-[Leu-4(R)Val]5-OMe [C71H132N10O13. 2H2O (2O). 2CH3OH (CO)] (922799) 14. Boc-[4(S)Leu]4-OMe [C38H72N4O7 . 0.5 H2O (0.5 O)] (922802) 15. Boc-[4(R)Leu]4-OMe [C38H72N4O7 . 0.5 H2O (0.5 O)] (922800) 16. Boc-[4(R)Ile]6-OMe [C54H102N6O9] (930731) 17. Boc-[4(R)Ile]10-OMe [C86H162N10O13] (930730) 18. Pregabalin Zwitterion (S) Pgn [C8H17NO2] 19. Pregabalin Zwitterion (R, S) Pgn [C8H17NO2] 20. Boc-(R,S) Pgn-OH [C13H25NO4] 21. Boc-(R, S) Pgn-Cyclohexylamide [C19H36N2O3] 22. Boc-(R, S) Pgn-NHMe [C14H28N2O3] 23. Piv-(S) Pgn-NHMe [C14H28N2O2] 24. Piv-(R, S) Pgn-NHMe [C14H28N2O2] 25. Boc-Gpn-(R, S) Pgn-OH [C22H40N2O5] 26. Boc-Aib-(S) Pgn-Leu-Phe-Val-OMe (Form-I) [C38H63N5O8. H2O] (971416) 27. Boc-Aib-(S) Pgn-Leu-Phe-Val-OMe (Form-II) [C38H63N5O8. H2O (O)] (971417) 28. Boc-Aib-4(R)Leu-Leu-Phe-Val-OMe (Form-I) [C38H63N5O8] (971418)

Στην παρούσα διατριβή παρουσιάζεται μια νέα μεθοδολογία σύνθεσης μη-πρωτεϊνικών αμινοξέων (ΜΠΑ) κατάλληλων για εφαρμογές στην πεπτιδική σύνθεση με χρήση του ασπαραγινικού οξέος ως χειρόμορφου εκμαγείου. Η μεθοδολογία αυτή βασίζεται στην επιτυχή και σε καλές αποδόσεις σύνθεση της β-αλδεΰδης του Ν-τριτυλοασπαραγινικού τριτ-βουτυλεστέρα, η οποία αποτελεί την ένωση «κλειδί». Αυτή στη συνέχεια έδωσε μια ποικιλία νέων ΜΠΑ μέσω αντιδράσεων Wittig, Horner-Emmons ή με επίδραση οργανοψευδραργυρικών αντιδραστηρίων. Ανάλογες μελέτες με σκοπό την σύνθεση των αντίστοιχων ομολόγων τους με έναν άνθρακα λιγότερο ή περισσότερο στην πλευρική αλυσίδα, ξεκινώντας από σερίνη ή γλουταμινικό οξύ δεν απέδωσαν τα αναμενόμενα αποτελέσματα ή ήταν ανεπιτυχείς.
In the present dissertation a new methodology is reported for the synthesis of novel non-proteinogenic amino acids (NPAAs) suitable for applications in solid phase peptide synthesis. This methodology involves aspartic acid as chiral template and relies on the successful and in good yields pepraration of the key N-tritylaspartic tert-butyl ester’s β-aldehyde, which when followed by Wittig, Horner-Emmons or Grignard type reactions may result in a variety of new NPAAs. Furthermore unsuccessful attempts towards the application of the present methodology for the synthesis of NPAA homologues bearing one carbon atom less or more in the side chain by using as chiral templates serine or glutamic acid, respectively, are also reported.

Synopsis The thesis entitled “Synthesis of Glyco-amino-acids, Glycosyl-amino-acids, and α-Amino γ-Lactams from Carbohydrate Derived Donor-Acceptor Cyclopropanes” is divided into five chapters. Chapter 1: Introduction and Background: Carbohydrate Derived Cyclopropanes and Glycoconjugates of Amino Acids and Peptides In this chapter, introduction and background on cyclopropanes, carbohydrate derived DA-cyclopropanes, glycopeptides and its mimetics is discussed Chapter 2: Efficient Synthesis of Glycosyl Esters of Amino Acids from Carbohydrate Derived Cyclopropanecarboxylates In this chapter, the N-iodosuccinimide (NIS) mediated ring opening of carbohydrate derived donor-acceptor (DA) cyclopropanes with carboxylic group of various N-protected amino acids is discussed. Under mild conditions, glucosyl esters of amino acids have been synthesized in moderate to good yields. This methodology has also been applied to galactose derived DA-cyclopropanes for the synthesis of galactosyl-amino-acid derivative. Among three N-protected valine derivatives (–Fmoc, –Boc, and –Cbz), the reaction of N-Fmoc protected valine derivative of glycosyl-amino-acid has not been successful due to the steric hindrance of bulky Fmoc group. Chapter 3: Synthesis of O–Linked Glycosyl-amino-acids & C–Linked Glyco-amino-acids. In this chapter, the synthesis of glycosyl-amino-acids and glyco-amino-acids by the NIS mediated ring opening of carbohydrate derived DA-cyclopropanes is reported. To synthesize the precursors of glycopeptides, deprotection of NHBoc has been performed with trifluoroacetic acid (TFA) and trimethylsilyl chloride (TMSCl). Trimethylsilyl chloride is found to be a better reagent than trifluoroacetic acid for this reaction. The synthesis of both O–linked glycosyl-amino acids and C–linked glycopeptides from single starting material using the orthogonal strategy at amine groups has been achieved. In these glycoconjugates of amino acids, the azide group (–N3) has been used as a masked amine (–NH2) which circumvents the protection and deprotection steps. Chapter 4: Synthesis of Carbohydrate Fused α-Amino γ-Lactams. In this chapter a flexible protocol for the synthesis of carbohydrate fused α-Amino γ-Lactams from carbohydrate derived cyclopropanecarboxylates has been disclosed. Also, the synthesis of carbohydrate fused γ-Lactams in a single-step from the iodo-azide by reductive cyclization has been reported. The formation of -lactam is achieved in low yield using both methods (A & B). The utility of the carbohydrate fused α-Amino γ-Lactams in the synthesis of Agl-bridged glycopeptide conjugates in a single-step with high efficiency has been demonstrated. Chapter 5: Studies on the Synthesis of Septanosides from Carbohydrate Derived DA-Cyclopropanes In carbohydrate derived DA 1,2-cyclopropanes, generally, the electron withdrawing group is attached at C-7 (type-I), C-2 (type-II), or C-3 (type-III). In this chapter, studies on the synthesis and use of carbohydrate derived DA-cyclopropanes of type-II & -III to form the the septanoside derivatives have been described. Attempts at the synthesis of 3,4,6-tri-O-methyl-D-glucal derived cyclopropanecarboxylates of type-II have not been successful. The failure of the cyclopropanation reactions might be due to presence of the carbmethoxy group at C-2 causing steric hindrance on the olefinic bond of 3,4,6-tri-O-tri-methyl-D-glucal methyl ester. It was then speculated that replacement of the of the carboxylate group in tri-O-methyl-glucal with hydroxymethyl group can promote the cyclopropanation reaction. Cyclopropanation of benzyl protected corresponding alcohol furnished the desired cyclopropane derivative in moderate yield (48%) as an inseparable mixture of diastereomers (1:1). Hence the synthesis of cyclopropanecarboxylates of type-II has not been achieved using this methodology. Glucose derived 3-oxo-1,2-cyclopropanes is synthesized from 3,4,6-tri-O-acetyl-D-glucal in good yield. This glucose derived cyclopropane of type-III did not furnish the septanoside derivative under different conditions. The synthesis of 3-oxo-1,2-cyclopropanated galactose derivative has been achieved in reasonably good yield from D-galactal with in three steps. When the galactose derived DA-cyclopropane is reacted with NIS and MeOH in the presence of catalytic amount of TMSOTf, it furnished the desired septanoside along with many side-products. The attempts at separation and identification of the septanoside in pure form have not been successful.

Synopsis The thesis entitled “Synthesis of Glyco-amino-acids, Glycosyl-amino-acids, and α-Amino γ-Lactams from Carbohydrate Derived Donor-Acceptor Cyclopropanes” is divided into five chapters. Chapter 1: Introduction and Background: Carbohydrate Derived Cyclopropanes and Glycoconjugates of Amino Acids and Peptides In this chapter, introduction and background on cyclopropanes, carbohydrate derived DA-cyclopropanes, glycopeptides and its mimetics is discussed Chapter 2: Efficient Synthesis of Glycosyl Esters of Amino Acids from Carbohydrate Derived Cyclopropanecarboxylates In this chapter, the N-iodosuccinimide (NIS) mediated ring opening of carbohydrate derived donor-acceptor (DA) cyclopropanes with carboxylic group of various N-protected amino acids is discussed. Under mild conditions, glucosyl esters of amino acids have been synthesized in moderate to good yields. This methodology has also been applied to galactose derived DA-cyclopropanes for the synthesis of galactosyl-amino-acid derivative. Among three N-protected valine derivatives (–Fmoc, –Boc, and –Cbz), the reaction of N-Fmoc protected valine derivative of glycosyl-amino-acid has not been successful due to the steric hindrance of bulky Fmoc group. Chapter 3: Synthesis of O–Linked Glycosyl-amino-acids & C–Linked Glyco-amino-acids. In this chapter, the synthesis of glycosyl-amino-acids and glyco-amino-acids by the NIS mediated ring opening of carbohydrate derived DA-cyclopropanes is reported. To synthesize the precursors of glycopeptides, deprotection of NHBoc has been performed with trifluoroacetic acid (TFA) and trimethylsilyl chloride (TMSCl). Trimethylsilyl chloride is found to be a better reagent than trifluoroacetic acid for this reaction. The synthesis of both O–linked glycosyl-amino acids and C–linked glycopeptides from single starting material using the orthogonal strategy at amine groups has been achieved. In these glycoconjugates of amino acids, the azide group (–N3) has been used as a masked amine (–NH2) which circumvents the protection and deprotection steps. Chapter 4: Synthesis of Carbohydrate Fused α-Amino γ-Lactams. In this chapter a flexible protocol for the synthesis of carbohydrate fused α-Amino γ-Lactams from carbohydrate derived cyclopropanecarboxylates has been disclosed. Also, the synthesis of carbohydrate fused γ-Lactams in a single-step from the iodo-azide by reductive cyclization has been reported. The formation of -lactam is achieved in low yield using both methods (A & B). The utility of the carbohydrate fused α-Amino γ-Lactams in the synthesis of Agl-bridged glycopeptide conjugates in a single-step with high efficiency has been demonstrated. Chapter 5: Studies on the Synthesis of Septanosides from Carbohydrate Derived DA-Cyclopropanes In carbohydrate derived DA 1,2-cyclopropanes, generally, the electron withdrawing group is attached at C-7 (type-I), C-2 (type-II), or C-3 (type-III). In this chapter, studies on the synthesis and use of carbohydrate derived DA-cyclopropanes of type-II & -III to form the the septanoside derivatives have been described. Attempts at the synthesis of 3,4,6-tri-O-methyl-D-glucal derived cyclopropanecarboxylates of type-II have not been successful. The failure of the cyclopropanation reactions might be due to presence of the carbmethoxy group at C-2 causing steric hindrance on the olefinic bond of 3,4,6-tri-O-tri-methyl-D-glucal methyl ester. It was then speculated that replacement of the of the carboxylate group in tri-O-methyl-glucal with hydroxymethyl group can promote the cyclopropanation reaction. Cyclopropanation of benzyl protected corresponding alcohol furnished the desired cyclopropane derivative in moderate yield (48%) as an inseparable mixture of diastereomers (1:1). Hence the synthesis of cyclopropanecarboxylates of type-II has not been achieved using this methodology. Glucose derived 3-oxo-1,2-cyclopropanes is synthesized from 3,4,6-tri-O-acetyl-D-glucal in good yield. This glucose derived cyclopropane of type-III did not furnish the septanoside derivative under different conditions. The synthesis of 3-oxo-1,2-cyclopropanated galactose derivative has been achieved in reasonably good yield from D-galactal with in three steps. When the galactose derived DA-cyclopropane is reacted with NIS and MeOH in the presence of catalytic amount of TMSOTf, it furnished the desired septanoside along with many side-products. The attempts at separation and identification of the septanoside in pure form have not been successful.

Les composés carbonylés y, δ - insaturés constituent des groupes fonctionnels important présents dans plusieurs produits naturels possédant des activités biologiques diverses. Quelques méthodes de préparation de composés carbonylés y, δ - insaturés obtenues par des réactions type réarrangement [3,3] sont rapportées dans la littérature. Cependant, à notre connaissance il n'existe aucun exemple de réarrangement de Claisen [3,3] sur des composés type B-allyloxyénamides. Pourquoi s'intéresser aux composés carbonylés y, δ - insaturés? Parce que l'on peut obtenir des acides aminés non naturels par oxydation et hydrolyse de ces derniers. Les composés carbonylés y, δ - insaturés sont obtenus par un réarrangement de Claisen [3,3], de composés type B-allyloxyénamides. Ces derniers ont pu être synthétisés dans le cadre de travaux préliminaires dans notre laboratoire, par couplage cuivré entre des alcools allyliques et des B-iodoénamides. En outre, les B-iodoénamides ont été synthétisés grâce à une méthodologie de couplage cuivré, entre un amide et du (E)-diiodoéthène. Cette méthodologie a été développée et optimisée dans notre laboratoire, et cela dans le cadre d'une étude précédant celle-ci. Nous avons optimisé la synthèse des B-allyloxyénamides, et ainsi nous avons pu décrire une méthode générale permettant l'obtention de ces derniers, avec des rendements allants de bons à excellents. Nous avons également développé une méthodologie permettant la synthèse de composés carbonylés y, δ - insaturés, en effectuant un réarrangement de Claisen [3,3] des B-allyloxyénamides. Nous avons montré via cette étude, que l'on pouvait obtenir de manière diastéréosélective, des composés carbonylés y, δ - insaturés, avec de bons rendements, et cela notamment par l'emploi des micro-ondes. Ce travail a permis de confirmer la viabilité globale de la stratégie de synthèse conçue pour préparer de façon diastéréosélective des composés très prisés par l'industrie pharmaceutique, tels que les acides aminés. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : acides aminés, couplage, cuivre, réarrangement de Claisen, micro-ondes

Structural studies of peptides are of great importance in developing novel and effective biomaterials ranging from drugs and vaccines to nano materials with industrial applications. In addition, they provide model systems to study and mimic the protein conformations. The ability to generate folded intramolecularly hydrogen bonded structures in short peptides is essential for peptide design strategies, which rely on the use of folding nuclei in the construction of secondary structure modules like helices and β-hairpins. In these approaches, conformational choices at selected positions are biased, using local stereochemical constraints, that limit the range of accessible backbone torsion angles. X-ray crystallographic studies of designed peptides provide definitive proof of the success of a design strategy, and provide essential structural information that can be utilized in the future design of biologically and structurally important polypeptides. Recent trends in peptide research focus on the incorporation of β-, γ- and higher homologs of the α-amino acid residues in designed peptides as they confer more proteolytic stability to the polypeptides. X-ray crystallographic studies of such modified peptides containing non-protein residues are essential, since information on the geometric and stereochemical properties of modified amino acids can only be gathered from the systematic structural studies of synthetic peptides incorporating them. This thesis reports a systematic study of the structures and conformations of amino acid derivatives and designed peptides containing stereochemically constrained α-, β- and γ-amino acid residues and the structural studies of polymorphic peptide helices. The structures described in thesis contain the Cα,α-dialkyalted α-residues α-aminoisobutyric acid (Aib) and 1-aminocyclohexane-1-carboxylic acid (Ac6c), the β-amino acid residue 1-aminocyclohexane acetic acid (β3,3Ac6c) and the γ-amino acid residue 1-aminomethylcyclohexaneacetic acid (gabapentin, Gpn). The crystal structure determination of peptides incorporating conformationally constrained α-, β- and γ- amino acid residues permitted the characterization of new types of hydrogen bonded turns and polymorphs. The studies enabled the precise determination of conformational and geometric parameters of two ω-amino acid residues, gabapentin and β 3,3Ac6c and provided detailed information about the conformational excursions possible for peptide molecules. This thesis is divided into 10 chapters. Chapter 1 gives a general introduction to the stereochemistry of the polypeptide chain, description of backbone torsion angles of α- and ω- amino acid residues and the major secondary structures of α-peptides, β-peptides, γ-peptides and hybrid peptides. A brief introduction to polymorphism and weak interactions, in particular aromatic interactions, is also provided, followed by a discussion on X-ray diffraction and solution to the phase problem. Chapter 2 describes the crystal structures of gabapentin zwitterion and its eight derivatives (Ananda, Aravinda, Vasudev et al., 2003). The crystal structure of the gabapentin zwitterions determined in this study is identical to that previously reported (Ibers, J. A. Acta Crystallogr. 2001, C57, 641-643). Eight of the nine achiral compounds crystallized in centrosymmetric space groups P21/c, C2/c or Pbca, while one derivative (Tos-Gpn-OH) crystallized in non-centrosymmetric space group Pna21 with four independent molecules in the asymmetric unit.The structural studies presented in this chapter reveal that the geminal substituents on the Cβ atom limits the values of dihedral angles θ1 and θ2 to ±60°, resulting in folded backbone conformations in all the examples. Intramolecular hydrogen bonds with 7-atoms in the hydrogen bond turn (C7) are observed in three derivatives, gabapentin hydrochloride (GPNCL), Boc-Gpn-OH (BGPNH) and Piv-Gpn-OH (PIVGPN), while a 9-atom hydrogen bonded turn (C9) is observed in Ac-Gpn-OH (ACGPH). Unique structural features, such as an unusual anti conformation of the COOH group (in ACGPH) and positional disorder of the cyclohexane ring (in BGPNN), indicating the co-existence of both the interconvertible chair conformations, are revealed by the crystal structure analyses. Chapter 3 describes the structural characterization of novel hydrogen bonded conformations of homo oligomers of Gpn. The crystal structures of three peptides, Boc-Gpn-Gpn-NHMe (GPN2), Boc-Gpn-Gpn-Leu-OMe (GPN2L) and Boc-Gpn-Gpn-Gpn-Gpn-NHMe (GPN4) provide the first crystallographic characterization of two new families of polypeptide structures, the C9 helices and C9 ribbons (Vasudev et al., 2005, 2007), in which the molecular conformations are stabilized by contiguous C9 turns formed by the hydrogen bonding between the CO group of residue (i) and the NH group of residue (i+2). The C9 hydrogen bond is characterized by a specific combination of the four torsion angles for the Gpn backbone, with the torsion angles θ1 and θ2 adopting g+/g+ or g /g- conformations. The structural analysis also permits precise determination of hydrogen bond geometry for the C9 structures, which is highly linear in contrast to the analogous γ-turn hydrogen bonds in α-peptides. A comparison of the backbone conformations in the three peptides reveals two classes of C9 hydrogen bonded secondary structures, namely C9 helices and C9 ribbons. The packing arrangement in these γ-peptides follows the same patterns as the helix packing in crystals of α-peptides. Chapter 4 describes ten crystal structures of short hybrid peptides containing the Gpn residue (Vasudev et al., 2007). In addition to the C7 and C9 hydrogen bonded turns which are defined by the backbone conformations at the Gpn residue, hybrid turns defined by a combination of backbone conformations at the α and γ-residues or at the β and γ-residues have been determined. Peptides Boc-Ac6c-Gpn-OH (ACGPH), Piv-Pro-Gpn-Val-OMe (PPGPV) and Boc-Val-Pro-Gpn-OH (VPGPH) reveal molecular conformation stabilized by intramolecular C9 hydrogen bonds, while Boc-Ac6c-Gpn-OMe (ACGPO) and Boc-Gpn-Aib-OH (GPUH) are stabilized by a C7 hydrogen bonded turn at the Gpn residue. An αγ hybrid turn with 12 atoms in the intramolecular hydrogen bonded rings (C12 turns) has been observed in the tripeptide Boc-Ac6c-Gpn-Ac6c-OMe (ACGP3), while βγ hybrid turns with 13 atoms in the hydrogen bonded ring (C13 turns) have been characterized in the tripeptides Boc-βLeu-Gpn-Val-OMe (BLGPV) and Boc- βPhe-Gpn-Phe-OMe (BFGPF). The two βγ C13 turns belong to two different categories and are characterized by different sets of backbone torsion angles for the β and γ residues. A γα C10 hydrogen bond, which is formed in the N→C direction (NHi ••• COi+2), as opposed to the regular hydrogen bonded helices of α-peptides, has also been observed in BFGPF. The Chapter provides a comparison of the backbone torsion angles of the Gpn residue in various hydrogen bonded turns and a brief comparison of the observed hydrogen bonded turns with those of the α-peptides. Chapter 5 describes the crystal structures of three αγ hybrid peptides which show C12/C10 mixed hydrogen bond patterns (Vasudev et al., 2007, 2008a; Chatterjee, Vasudev et al.,2008a). The insertion of gabapentin in the predominantly α-amino acid sequences in Boc-Ala-Aib-Gpn-Aib-Ala-OMe (AUGP5) and Boc-Leu-Gpn-Aib-Leu-Gpn-Aib-OMe results in the observation of helices stabilized by αα C10 (310-turn) and αγ C12 turns. The tetrapeptide Boc-Leu-Gpn-Leu-Aib-OMe reveals a novel conformation, stabilized by C12 (αγ) and C10 (γα) hydrogen bonds of opposite hydrogen bond directionalities. The conformations observed in crystals have been extended to generate C12 helix and C12/C10 helix with alternating hydrogen bond polarities in ( αγ)n sequences. The structure determination of three crystals, providing five molecular conformations, presented in this chapter provides the first crystallographic characterization of two types of helices predicted for the regular αγ hybrid peptides from theoretical calculations. The crystal structure of Boc-Ala-Aib-Gpn-Aib-Ala-OMe also provides an example for the co-existence of left-handed and right-handed helix in the asymmetric unit. Chapter 6 describes the structural studies of αγ hybrid peptides containing Aib and Gpn residues, and is divided into two parts. The first part presents the crystal structure analysis of peptides of sequence length 2 to 4, with alternating Aib and Gpn residues, and illustrates the conformational variability in αγ hybrid sequences as evidenced by the observation of conformational polymorphs (Chatterjee, Vasudev et al., 2008b; Vasudev et al., 2007; Ananda, Vasudev et al., 2005). The peptide Boc-Gpn-Aib-NHMe (GUN), Boc-Aib-Gpn-Aib-OMe (UGU), Boc-Gpn-Aib-Gpn-Aib-OMe (GU4O), Boc-Aib-Gpn-Aib-Gpn-OMe (UG4O) and Boc-Aib-Gpn-Aib-Gpn-NHMe (UG4N), all of which are potential candidates for exhibiting αγ C12 hydrogen bonds, reveal molecular conformations stabilized by diverse hydrogen bonded turns such as C7, C9, C12 and C17 in crystals. The conformational heterogeneity in this class of hybrid peptides is further evidenced by the observation of three polymorphs in the monoclinic space group P21/c for the tetrapeptide Boc-Aib-Gpn-Aib-Gpn-NHMe (UG4N), providing four independent peptide molecules adopting two distinct backbone conformations. In one polymorph, C12 helices terminated with an unusual three residue ( γαγ) C17 turn is observed, while the unfolding of helical conformation by solvent insertion into the backbone is observed in the other two polymorphs. The studies indicate the possible utility of Gpn residue in stabilizing locally folded conformations in the folding pathway, thus permitting their crystallographic characterization in multiple crystal forms. A discussion of the structural and conformational features of Gpn residues determined from all the crystal structures is presented in the Chapter, along with a φ-ψ plot for the Gpn residue. Part 2 of Chapter 6 describes the crystal structures of two octapeptides, Boc-Gpn-Aib-Gpn-Aib-Gpn-Aib-Gpn-Aib-OMe (GU8) and Boc-Leu-Phe-Val-Aib-Gpn-Leu-Phe-Val-OMe (LFVUG8), featuring C12 turns at the Aib-Gpn segments (Chatterjee, Vasudev et al., 2009). GU8 folds into a C12 helix flanked by C9 hydrogen bonds at both the termini, while LFVUG8 adopts β-hairpin conformation with a chain-reversing C12 turn at the central Aib-Gpn segment. A remarkable feature of the Aib-Gpn turn in the β-hairpin structure is the anti conformation about the Cβ-Cα (θ2) bond, which is the only example of a Gpn residue not adopting gauche conformation for both θ1 and θ2. The crystal structures of the two peptides, mimicking the two major secondary structural elements of α-peptides in hybrid polypeptides, permits a comparative study of the mode of molecular packing in crystals of α-peptides and hybrid peptides. The chapter also discusses theoretical calculations on αγ hybrid sequences, which reveal new types of C12 hydrogen bonded turns. Chapter 7 describes the crystal structures of conformationally biased tert-butyl derivatives of Gpn. The crystallographic characterization of the E (trans) and Z (cis) isomers of the residue,three protected derivatives and a tripeptide provides examples of C7 and C9 hydrogen bonded conformations, suggesting that the C7 and C9 hydrogen bonds can be formed by Gpn residues with both the chair conformations of the cyclohexane ring. Chapter 8 describes the systematic structural studies of the derivatives and peptides of the stereochemically constrained β- amino acid residue, β3,3Ac6c (Vasudev et al., 2008c). The backbone torsion angles φ and θ adopt gauche conformation in majority of the examples, owing to the presence of a cyclohexane ring on the Cβ atom. In contrast to Gpn, β3,3Ac6c does not show strong preference for adopting intramolecularly hydrogen bonded conformations. Of the 16 crystal structures determined, intramolecular hydrogen bonds involving the β-residue are observed only in 4 cases. The amino acid zwitterion (BAC6C), the hydrochloride (BACHCL) and the dipeptide Boc-β3,3Ac6c-β3,3Ac6c-NHMe (BAC62N) form N-H•••O hydrogen bonds with 6-atoms in the hydrogen bond ring (C6 turns). An αβ hybrid C11 hydrogen bonded turn is characterized in the dipeptide Piv-Pro-β3,3Ac6c-NHMe, which is distinctly different from the C11 hydrogen bonds observed in αβ hybrid peptide helices. Several unique structural features such as a dynamic disorder of the hydrogen atom of the carboxylic acid group (in BBAC) and cis geometry of the urethane bond (in BBAC, BAC62N and BPBAC) have been observed in this study. A comparison of the backbone conformations of β3,3Ac6c with other β- amino acid residues is also provided. Chapter 9 describes the crystallographic characterization of a new polymorph of gabapentin monohydrate and crystal structures of the zwitterions of E and Z isomers of tert-butylgabapentin and its hydrochloride and hydrobromide (Vasudev et al., 2009). A comparison of the crystal structures of the monoclinic form (Ibers, J. A. Acta Crystallogr. 2001, C57, 641-643) of gabapentin monohydrate and the newly characterized orthorhombic form reveals identical molecular conformations and intermolecular hydrogen bond patterns in both the polymorphs. The two polymorphs show differences in the orientation of molecules constituting a layer of hydrophobic interactions between the cyclohexyl side chains. A comparison of the packing arrangements of the zwitterionic amino acid molecules in the crystal structures of gabapentin monohydrate, the tert-butyl derivatives and other co-crystals of gabapentin that had been characterized so far, is provided which would facilitate prediction of new polymorphs of the widely used drug molecule, Gpn. Chapter 10 describes the crystallization of α-peptide helices in multiple crystal forms (Vasudev et al., 2008b). Crystal structures of two peptides, Boc-Leu-Aib-Phe-Phe-Leu-Aib-Ala-Ala-Leu-Aib-OMe (LFF), Boc-Leu-Aib-Phe-Ala-Leu-Ala-Leu-Aib-OMe (D1) in two crystal forms and the crystal structure of a related sequence, Boc-Leu-Aib-Phe-Ala-Phe-Aib-Leu-Ala-Leu-Aib-OMe (D10) permit an analysis of the molecular conformation and packing patterns of peptide helices in crystals. The two polymorphs of LFF, crystallized in the space groups P21 and P22121, reveal very similar molecular conformation (α/310-helix) in both the polymorphic crystals; the two forms differ significantly in the pattern of solvation. The crystal structure determination of a monoclinic (P21) and an orthorhombic polymorph (P21212) of D1 provides five different peptide conformations, four of which are α-helical and one is a mixed 310/α-helix. The crystal structure determination of the three peptides provide an opportunity to compare the nature and role of aromatic interactions in stabilizing molecular conformation and packing and its significance in the observation of polymorphism. An analysis of the Cambridge Structural Database and a model for nucleation of crystals in hydrophobic peptide helices are also discussed.

Structural studies of peptides are of great importance in developing novel and effective biomaterials ranging from drugs and vaccines to nano materials with industrial applications. In addition, they provide model systems to study and mimic the protein conformations. The ability to generate folded intramolecularly hydrogen bonded structures in short peptides is essential for peptide design strategies, which rely on the use of folding nuclei in the construction of secondary structure modules like helices and β-hairpins. In these approaches, conformational choices at selected positions are biased, using local stereochemical constraints, that limit the range of accessible backbone torsion angles. X-ray crystallographic studies of designed peptides provide definitive proof of the success of a design strategy, and provide essential structural information that can be utilized in the future design of biologically and structurally important polypeptides. Recent trends in peptide research focus on the incorporation of β-, γ- and higher homologs of the α-amino acid residues in designed peptides as they confer more proteolytic stability to the polypeptides. X-ray crystallographic studies of such modified peptides containing non-protein residues are essential, since information on the geometric and stereochemical properties of modified amino acids can only be gathered from the systematic structural studies of synthetic peptides incorporating them. This thesis reports a systematic study of the structures and conformations of amino acid derivatives and designed peptides containing stereochemically constrained α-, β- and γ-amino acid residues and the structural studies of polymorphic peptide helices. The structures described in thesis contain the Cα,α-dialkyalted α-residues α-aminoisobutyric acid (Aib) and 1-aminocyclohexane-1-carboxylic acid (Ac6c), the β-amino acid residue 1-aminocyclohexane acetic acid (β3,3Ac6c) and the γ-amino acid residue 1-aminomethylcyclohexaneacetic acid (gabapentin, Gpn). The crystal structure determination of peptides incorporating conformationally constrained α-, β- and γ- amino acid residues permitted the characterization of new types of hydrogen bonded turns and polymorphs. The studies enabled the precise determination of conformational and geometric parameters of two ω-amino acid residues, gabapentin and β 3,3Ac6c and provided detailed information about the conformational excursions possible for peptide molecules. This thesis is divided into 10 chapters. Chapter 1 gives a general introduction to the stereochemistry of the polypeptide chain, description of backbone torsion angles of α- and ω- amino acid residues and the major secondary structures of α-peptides, β-peptides, γ-peptides and hybrid peptides. A brief introduction to polymorphism and weak interactions, in particular aromatic interactions, is also provided, followed by a discussion on X-ray diffraction and solution to the phase problem. Chapter 2 describes the crystal structures of gabapentin zwitterion and its eight derivatives (Ananda, Aravinda, Vasudev et al., 2003). The crystal structure of the gabapentin zwitterions determined in this study is identical to that previously reported (Ibers, J. A. Acta Crystallogr. 2001, C57, 641-643). Eight of the nine achiral compounds crystallized in centrosymmetric space groups P21/c, C2/c or Pbca, while one derivative (Tos-Gpn-OH) crystallized in non-centrosymmetric space group Pna21 with four independent molecules in the asymmetric unit.The structural studies presented in this chapter reveal that the geminal substituents on the Cβ atom limits the values of dihedral angles θ1 and θ2 to ±60°, resulting in folded backbone conformations in all the examples. Intramolecular hydrogen bonds with 7-atoms in the hydrogen bond turn (C7) are observed in three derivatives, gabapentin hydrochloride (GPNCL), Boc-Gpn-OH (BGPNH) and Piv-Gpn-OH (PIVGPN), while a 9-atom hydrogen bonded turn (C9) is observed in Ac-Gpn-OH (ACGPH). Unique structural features, such as an unusual anti conformation of the COOH group (in ACGPH) and positional disorder of the cyclohexane ring (in BGPNN), indicating the co-existence of both the interconvertible chair conformations, are revealed by the crystal structure analyses. Chapter 3 describes the structural characterization of novel hydrogen bonded conformations of homo oligomers of Gpn. The crystal structures of three peptides, Boc-Gpn-Gpn-NHMe (GPN2), Boc-Gpn-Gpn-Leu-OMe (GPN2L) and Boc-Gpn-Gpn-Gpn-Gpn-NHMe (GPN4) provide the first crystallographic characterization of two new families of polypeptide structures, the C9 helices and C9 ribbons (Vasudev et al., 2005, 2007), in which the molecular conformations are stabilized by contiguous C9 turns formed by the hydrogen bonding between the CO group of residue (i) and the NH group of residue (i+2). The C9 hydrogen bond is characterized by a specific combination of the four torsion angles for the Gpn backbone, with the torsion angles θ1 and θ2 adopting g+/g+ or g /g- conformations. The structural analysis also permits precise determination of hydrogen bond geometry for the C9 structures, which is highly linear in contrast to the analogous γ-turn hydrogen bonds in α-peptides. A comparison of the backbone conformations in the three peptides reveals two classes of C9 hydrogen bonded secondary structures, namely C9 helices and C9 ribbons. The packing arrangement in these γ-peptides follows the same patterns as the helix packing in crystals of α-peptides. Chapter 4 describes ten crystal structures of short hybrid peptides containing the Gpn residue (Vasudev et al., 2007). In addition to the C7 and C9 hydrogen bonded turns which are defined by the backbone conformations at the Gpn residue, hybrid turns defined by a combination of backbone conformations at the α and γ-residues or at the β and γ-residues have been determined. Peptides Boc-Ac6c-Gpn-OH (ACGPH), Piv-Pro-Gpn-Val-OMe (PPGPV) and Boc-Val-Pro-Gpn-OH (VPGPH) reveal molecular conformation stabilized by intramolecular C9 hydrogen bonds, while Boc-Ac6c-Gpn-OMe (ACGPO) and Boc-Gpn-Aib-OH (GPUH) are stabilized by a C7 hydrogen bonded turn at the Gpn residue. An αγ hybrid turn with 12 atoms in the intramolecular hydrogen bonded rings (C12 turns) has been observed in the tripeptide Boc-Ac6c-Gpn-Ac6c-OMe (ACGP3), while βγ hybrid turns with 13 atoms in the hydrogen bonded ring (C13 turns) have been characterized in the tripeptides Boc-βLeu-Gpn-Val-OMe (BLGPV) and Boc- βPhe-Gpn-Phe-OMe (BFGPF). The two βγ C13 turns belong to two different categories and are characterized by different sets of backbone torsion angles for the β and γ residues. A γα C10 hydrogen bond, which is formed in the N→C direction (NHi ••• COi+2), as opposed to the regular hydrogen bonded helices of α-peptides, has also been observed in BFGPF. The Chapter provides a comparison of the backbone torsion angles of the Gpn residue in various hydrogen bonded turns and a brief comparison of the observed hydrogen bonded turns with those of the α-peptides. Chapter 5 describes the crystal structures of three αγ hybrid peptides which show C12/C10 mixed hydrogen bond patterns (Vasudev et al., 2007, 2008a; Chatterjee, Vasudev et al.,2008a). The insertion of gabapentin in the predominantly α-amino acid sequences in Boc-Ala-Aib-Gpn-Aib-Ala-OMe (AUGP5) and Boc-Leu-Gpn-Aib-Leu-Gpn-Aib-OMe results in the observation of helices stabilized by αα C10 (310-turn) and αγ C12 turns. The tetrapeptide Boc-Leu-Gpn-Leu-Aib-OMe reveals a novel conformation, stabilized by C12 (αγ) and C10 (γα) hydrogen bonds of opposite hydrogen bond directionalities. The conformations observed in crystals have been extended to generate C12 helix and C12/C10 helix with alternating hydrogen bond polarities in ( αγ)n sequences. The structure determination of three crystals, providing five molecular conformations, presented in this chapter provides the first crystallographic characterization of two types of helices predicted for the regular αγ hybrid peptides from theoretical calculations. The crystal structure of Boc-Ala-Aib-Gpn-Aib-Ala-OMe also provides an example for the co-existence of left-handed and right-handed helix in the asymmetric unit. Chapter 6 describes the structural studies of αγ hybrid peptides containing Aib and Gpn residues, and is divided into two parts. The first part presents the crystal structure analysis of peptides of sequence length 2 to 4, with alternating Aib and Gpn residues, and illustrates the conformational variability in αγ hybrid sequences as evidenced by the observation of conformational polymorphs (Chatterjee, Vasudev et al., 2008b; Vasudev et al., 2007; Ananda, Vasudev et al., 2005). The peptide Boc-Gpn-Aib-NHMe (GUN), Boc-Aib-Gpn-Aib-OMe (UGU), Boc-Gpn-Aib-Gpn-Aib-OMe (GU4O), Boc-Aib-Gpn-Aib-Gpn-OMe (UG4O) and Boc-Aib-Gpn-Aib-Gpn-NHMe (UG4N), all of which are potential candidates for exhibiting αγ C12 hydrogen bonds, reveal molecular conformations stabilized by diverse hydrogen bonded turns such as C7, C9, C12 and C17 in crystals. The conformational heterogeneity in this class of hybrid peptides is further evidenced by the observation of three polymorphs in the monoclinic space group P21/c for the tetrapeptide Boc-Aib-Gpn-Aib-Gpn-NHMe (UG4N), providing four independent peptide molecules adopting two distinct backbone conformations. In one polymorph, C12 helices terminated with an unusual three residue ( γαγ) C17 turn is observed, while the unfolding of helical conformation by solvent insertion into the backbone is observed in the other two polymorphs. The studies indicate the possible utility of Gpn residue in stabilizing locally folded conformations in the folding pathway, thus permitting their crystallographic characterization in multiple crystal forms. A discussion of the structural and conformational features of Gpn residues determined from all the crystal structures is presented in the Chapter, along with a φ-ψ plot for the Gpn residue. Part 2 of Chapter 6 describes the crystal structures of two octapeptides, Boc-Gpn-Aib-Gpn-Aib-Gpn-Aib-Gpn-Aib-OMe (GU8) and Boc-Leu-Phe-Val-Aib-Gpn-Leu-Phe-Val-OMe (LFVUG8), featuring C12 turns at the Aib-Gpn segments (Chatterjee, Vasudev et al., 2009). GU8 folds into a C12 helix flanked by C9 hydrogen bonds at both the termini, while LFVUG8 adopts β-hairpin conformation with a chain-reversing C12 turn at the central Aib-Gpn segment. A remarkable feature of the Aib-Gpn turn in the β-hairpin structure is the anti conformation about the Cβ-Cα (θ2) bond, which is the only example of a Gpn residue not adopting gauche conformation for both θ1 and θ2. The crystal structures of the two peptides, mimicking the two major secondary structural elements of α-peptides in hybrid polypeptides, permits a comparative study of the mode of molecular packing in crystals of α-peptides and hybrid peptides. The chapter also discusses theoretical calculations on αγ hybrid sequences, which reveal new types of C12 hydrogen bonded turns. Chapter 7 describes the crystal structures of conformationally biased tert-butyl derivatives of Gpn. The crystallographic characterization of the E (trans) and Z (cis) isomers of the residue,three protected derivatives and a tripeptide provides examples of C7 and C9 hydrogen bonded conformations, suggesting that the C7 and C9 hydrogen bonds can be formed by Gpn residues with both the chair conformations of the cyclohexane ring. Chapter 8 describes the systematic structural studies of the derivatives and peptides of the stereochemically constrained β- amino acid residue, β3,3Ac6c (Vasudev et al., 2008c). The backbone torsion angles φ and θ adopt gauche conformation in majority of the examples, owing to the presence of a cyclohexane ring on the Cβ atom. In contrast to Gpn, β3,3Ac6c does not show strong preference for adopting intramolecularly hydrogen bonded conformations. Of the 16 crystal structures determined, intramolecular hydrogen bonds involving the β-residue are observed only in 4 cases. The amino acid zwitterion (BAC6C), the hydrochloride (BACHCL) and the dipeptide Boc-β3,3Ac6c-β3,3Ac6c-NHMe (BAC62N) form N-H•••O hydrogen bonds with 6-atoms in the hydrogen bond ring (C6 turns). An αβ hybrid C11 hydrogen bonded turn is characterized in the dipeptide Piv-Pro-β3,3Ac6c-NHMe, which is distinctly different from the C11 hydrogen bonds observed in αβ hybrid peptide helices. Several unique structural features such as a dynamic disorder of the hydrogen atom of the carboxylic acid group (in BBAC) and cis geometry of the urethane bond (in BBAC, BAC62N and BPBAC) have been observed in this study. A comparison of the backbone conformations of β3,3Ac6c with other β- amino acid residues is also provided. Chapter 9 describes the crystallographic characterization of a new polymorph of gabapentin monohydrate and crystal structures of the zwitterions of E and Z isomers of tert-butylgabapentin and its hydrochloride and hydrobromide (Vasudev et al., 2009). A comparison of the crystal structures of the monoclinic form (Ibers, J. A. Acta Crystallogr. 2001, C57, 641-643) of gabapentin monohydrate and the newly characterized orthorhombic form reveals identical molecular conformations and intermolecular hydrogen bond patterns in both the polymorphs. The two polymorphs show differences in the orientation of molecules constituting a layer of hydrophobic interactions between the cyclohexyl side chains. A comparison of the packing arrangements of the zwitterionic amino acid molecules in the crystal structures of gabapentin monohydrate, the tert-butyl derivatives and other co-crystals of gabapentin that had been characterized so far, is provided which would facilitate prediction of new polymorphs of the widely used drug molecule, Gpn. Chapter 10 describes the crystallization of α-peptide helices in multiple crystal forms (Vasudev et al., 2008b). Crystal structures of two peptides, Boc-Leu-Aib-Phe-Phe-Leu-Aib-Ala-Ala-Leu-Aib-OMe (LFF), Boc-Leu-Aib-Phe-Ala-Leu-Ala-Leu-Aib-OMe (D1) in two crystal forms and the crystal structure of a related sequence, Boc-Leu-Aib-Phe-Ala-Phe-Aib-Leu-Ala-Leu-Aib-OMe (D10) permit an analysis of the molecular conformation and packing patterns of peptide helices in crystals. The two polymorphs of LFF, crystallized in the space groups P21 and P22121, reveal very similar molecular conformation (α/310-helix) in both the polymorphic crystals; the two forms differ significantly in the pattern of solvation. The crystal structure determination of a monoclinic (P21) and an orthorhombic polymorph (P21212) of D1 provides five different peptide conformations, four of which are α-helical and one is a mixed 310/α-helix. The crystal structure determination of the three peptides provide an opportunity to compare the nature and role of aromatic interactions in stabilizing molecular conformation and packing and its significance in the observation of polymorphism. An analysis of the Cambridge Structural Database and a model for nucleation of crystals in hydrophobic peptide helices are also discussed.