Дисертації з теми "Linear peptides"

The incorporation of unsymmetrical biaryl systems into peptide sequences is a strategy that can improve their biological activity. Due to the difficulty of arylating the 4(5}-position of the imidazole ring, this doctoral thesis was focused on the development of efficient methodologies for the solid-phase synthesis of 5-arylhistidine-containing antimicrobial undecapeptides through a Suzuki-Miyaura reaction under microwave irradiation. The extension of this protocol allowed the preparation of biaryl cyclic peptides of different ring sizes bearing a His-Phe or His-Tyr biaryl linkage. Then, it was developed a procedure for the total solid-phase synthesis of biaryl cyclic lipopeptides derived from arylomycins. These strategies were extended to the preparation of biaryl cyclic analogues of the marine bicyclic pptides aciculitins. In particular, it was achieved the synthesis of analogues of the northern and the southern hemispheres of aciculitins as well as biaryl bicyclic peptides incorporating a Phe-Phe, a Phe-Tyr, a His-Tyr or a Tyr-Tyr biaryl bridge
La incorporació de sistemes biarílics asimiètrics en seqüències peptídiques es considera un enfocament útil per a millorar l'activitat biològica de pèptids. Tenint en compte la dificultat d'arilar la posició 4 (5) de l'anell d'imidazole, aquesta tesi doctoral es centra en el desenvolupament de noves estratègies eficients per a la preparació en fase sòlida d'undecapèptids antimicrobians contenint una 5-arilhistidina a través d'una reacció de Suzuki-Miyaura sota irradiació microones. L'extensió d'aquesta metodologia ha permès la síntesi de pèptids biarílics cíclics de diferents mides que incorporen un enllaç His-Phe 0 His-Tyr. Posteriorment, s'ha desenvolupat un procediment per la síntesi total en fase sòlida de lipopèptids biarílics cíclics derivats de les arilomicines. Les estratègies anteriors s'han estès a la preparació de compostos biarílics anàlegs dels pèptids bicíclics marins aciculitines. Concretament, s'ha preparat anàlegs dels hemisferis nord i sud de las aciculitines així com pèptids biarílics bicíclics que incorporen un pont Phe-Phe, Phe-Tyr, Tyr-His 0 Tyr-Tyr.

This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin proteins in the context of bilayer interactions with model membranes and live cells. The studies involve spectroscopic techniques like fluorescence, fluorescence correlation spectroscopy (FCS), circular and linear dichroism (CD and LD), confocal fluorescence microscopy and NMR.

The peptides derived from the Prion and Doppel proteins combined with their subsequent nuclear localization-like sequences, makes them resemble cell-penetrating peptides (CPPs). mPrPp(1-28), corresponding to the first 28 amino acids of the mouse PrP, was shown to translocate across cell membranes, concomitantly causing cell toxicity. Its bovine counterpart bPrPp(1-30) was demonstrated to enter live cells, with and without cargo, mainly via macropinocytosis. The mPrPp(23-50) peptide sequence overlaps with mPrPp(1-28) sharing the KKRPKP sequence believed to encompass the driving force behind translocation. mPrPp(23-50) was however found unable to cross over cell membranes and had virtually no perturbing effects on membranes.

mDplp(1-30), corresponding of the first 30 N-terminal amino acids of the Doppel protein, was demonstrated to be almost as membrane perturbing as melittin. NMR experiments in bicelles implied a transmembrane configuration of its alpha-helix, which was corroborated by LD in vesicle bilayers. The positioning of the induced alpha-helix in transportan was found to be more parallel to the bilayer surface in the same model system.

Positioning of the native Influenza derived fusion peptide in bilayers showed no pH dependence. The glutamic acid enriched variant however, changed its insertion angle from 70 deg to a magic angle alignment relative the membrane normal upon a pH drop from 7.4 to 5.0. Concomitantly, the alpha-helical content dramatically rose from 18% to 52% in partly anionic membranes, while the native peptide’s helicity increased only from 39% to 44% in the same conditions.

A number of cyclic peptides containing a positively charged ring composed of arginine residues attached to hydrophobic tail made of tryptophan residues through a lysine linker namely [R5K]W5, [R6K]W5, [R5K]W6, [R7K]W5, [R5K]W7, [R6K]W6, and [R7K]W7 were synthesized and evaluated as molecular transporters. The peptides were evaluated for their ability to deliver, fluorescence-labeled cell-impermeable negatively charged phosphopeptide (F′-GpYEEI), and fluorescent labeled anti-HIV drugs (F′-FTC and F′-d4T). The results indicated that the presence of positively charged arginine residues on the ring and hydrophobic tryptophan residues in a sequential linear outside the ring was an optimal approach to improve the intracellular uptake of cargo molecules through non-covalent interactions. Some of these peptides were also evaluated for their efficiency for intracellular delivery of siRNA to triple-negative breast cancer cell lines in the presence and absence of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). [R6K]W6 and [R5K]W5 were found to be very efficient in the delivery of siRNA. Furthermore, co-formulation of peptides with lipid DOPE significantly enhanced the efficiency of siRNA delivery compared to peptide alone. Silencing of kinesin spindle protein (KSP) and Janus kinase 2 (JAK2) was evaluated in MDA-MB-231 cells in the presence of the peptides. The addition of DOPE significantly enhanced the silencing efficiency for all selected peptides. A chemotherapeutic drug, doxorubicin (Dox) was covalently conjugated to the cyclic peptide [R5K]W7A and linear peptide R5KW7A, and the biological activity was evaluated in cell-based assays. Comparative antiproliferative assays between covalently conjugated peptide-Dox and the corresponding noncovalent physical mixtures of the peptides and Dox were performed. The conjugation of Dox with cyclic [R5K]W7A-Dox exhibited similar antiproliferative activity compared to Dox alone after 72 h incubation time in all cancer cell lines, such as leukemia, ovarian and gastric cancer cells. However, [R5K]W7A-Dox significantly reduced the cell cytotoxicity in normal cell lines such as normal heart muscle and normal kidney cells after 72 h when compared with Dox alone. These results revealed that this cyclic peptide prodrug can be used as a potential candidate for the treatment of cancer cells with reduced side effects against normal cells in the body.

Im Rahmen der vorliegenden Arbeit gelang die Synthese einer Reihe von neuartigen linear oder tetragonal präorganisierten tektonischen Verbindungen mit peripheren Haftgruppen, bestehend aus den natürlich vorkommenden Aminosäuren Glycin und L-Alanin oder kurzen Peptiden. Neben der Synthese stand die strukturelle Charakterisierung der Zielsubstanzen und Vorstufen im Vordergrund, wobei die Aufklärung der Kristallstrukturen einer Zielverbindung und elf relevanter Vorstufen gelang, deren Packungsstrukturen überwiegend durch N–H•••O-Interaktionen determiniert sind. Ergänzend konnten Informationen bezüglich der von den Haftgruppen gebildeten Strukturmotive im Festkörper mittels FT-IR-Spektroskopie abgeleitet werden. Die Auswertung konformationssensitiver 1H- und 13C-NMR-Signale zeigt, dass Aminosäurereste innerhalb identischer Haftgruppen in Lösung jeweils in derselben Konformation vorliegen. Fluoreszenzspektroskopische Untersuchungen der hergestellten Zielverbindungen lassen interessante Anwendungen auf dem Gebiet der Sensorik erwarten.

The thesis consists of two parts targeting the characterization of chromatographic behavior of linear tryptic and cyclic peptides containing oxidized methionine (Met) in reversed-phased chromatography. The retention order of methionine-containing peptide analogues was observed to be the same in both studies: Met oxide < Met dioxide < Met. For linear tryptic peptides, the magnitude of the retention time shift may vary dramatically: from –9 % to +0.36 % acetonitrile. Particularly, large negative retention time shifts are found mostly associated with methionine being in the hydrophobic face of an amphipathic helix. Contrary to previously reported observations, I demonstrate for the first time that methionine oxidation may increase peptide hydrophobicity, this occurs only when methionine is in the N3 position of the N-capping stabilization motif preceding an amphipathic helix. In the second study, the effect of peak splitting was observed for some Met oxide-containing cyclolinopeptides, which most likely appear due to diastereomerization.

Several studies exist in the literature which utilise either dendritic (covalent) or self-assembling (non-covalent) strategies to achieve multivalent binding to a biological target, but rarely are the two explored together. Herein, we compare and contrast dendritic and self-assembling approaches to organise a multivalent array of ligands to bind the protein, integrin αvβ3. In the first instance, linear RGD (Arg-Gly-Asp) peptides were covalently attached to first and second generation dendritic frameworks, and a positive (monovalent) and negative control were synthesised. A fluorescence polarisation (FP) competition assay was used to quantify the binding. The first generation dendron (2.16) was the most effective binder, with an EC50 of 125 μM (375 μM per peptide unit); significantly better than the monovalent ligand (2.19), the binding of which could not be quantified in our assay, even at 1 mM concentration; whilst the second generation dendron (2.17) was somewhat less effective, indicating that there is an optimum number of ligands that can be displayed before the multiple ligand array becomes disadvantageous to the binding. To explore the non-covalent approach, the linear RGD peptide was conjugated to either a single hydrophobic C12 aliphatic chain (3.2), an aromatic pyrene (3.4), a C22 aliphatic chain (3.6), or two C12 aliphatic chains (3.18), which gave rise to amphiphilic peptides which were able to self-assemble to differing degrees and into markedly different morphologies, as shown by a Nile Red encapsulation study and TEM imaging. Spherical micelles were formed by amphiphiles 3.2 and 3.4, whereas 3.6 and 3.18 produced cylindrical, rod-like micelles. Compounds 3.2 and 3.4 were the most effective integrin binders at concentrations of 200 μM and 110 μM, respectively, whilst 3.6 and 3.18 failed to produce a quantifiable binding concentration. The results therefore show that not only does the micellar self-assembly approach yield multivalent ligand displays with improved efficiency of binding compared with the dendritic method, but the morphology of the self-assembled system can also be detrimental to the recognition of the protein, at least in our FP assay and using purified integrin in solution. Finally, we report on a family of linear RGD peptide conjugate hydrogelators. Of particular interest was the novel bolaamphiphile C12-[urea-RGD]2 (4.4), comprised of linear RGD peptide head groups connected to either end of a hydrophobic C12 aliphatic chain via urea linkages. The molecule undergoes thermoreversible chiral self-assembly in water and generates a sample-spanning nanofibrous gel network, as determined by circular dichroism spectroscopy and TEM/SEM imaging, respectively. Gels were formed at a minimum gel concentration (MGC) of 0.06 wt % (0.6 mg/ml, 0.5 mM), one of the lowest MGCs reported and represents a “super” hydrogel. We report on its responsiveness (breakdown) towards charge dense basic anions such as phosphate and acetate, but its stability in the presence of more charge diffuse halide and nitrate anions. Furthermore, we demonstrate the passive diffusion of encapsulated low MW additives out of the gel phase, whereas high MW, protein-sized molecules remain trapped within the fibrous network.

The evolution of drug resistant pathogens creates the need for the introduction of new antimicrobial drugs. Peptaibols, a class of naturally occurring peptides, contain large amounts of alpha aminoisobutyric acid (Aib). They are known to exhibit their antimicrobial activity by perturbing the membranes of pathogens. However, a comprehensive model of action for these peptides has not yet been identified. Aib residues support the formation of 310-helix conformation and it is thought that this secondary structure is important for their antimicrobial activity. It is possible to design small synthetic peptides, known as foldamers, endowed with specific properties; in particular, Aib-rich foldamers are used as a model for the understanding of the folding and membrane interaction of the naturally occurring species. The aim of this thesis is to investigate the conformational preference of monodisperse Aib-oligomers as well as understanding their interaction with bilayer membranes. A large set of spectroscopic techniques have been used to establish the conformation of Aib-rich foldamers both in solution and when bound to membranes. In particular: Raman, Raman Optical Activity (ROA), Infrared (IR), Vibrational Circular Dichroism (VCD), Linear Dichroism (LD) and Neutron Scattering (NR) were employed to provide new structural insights. These vibrational analysis (VA) and vibrational optical analysis (VOA) investigations in solution were focused on the identification of spectral features for 310-helix conformation, particularly with Raman and Raman Optical Activity spectroscopies. Spectroscopic markers for this conformation in the amide I region were successfully identified. Moreover, it is known that chiral Aib-rich peptides can show a right or left handed screw-preference based on the primary sequence. VOA studies successfully distinguished between peptides with opposite helicity. VCD, ROA, LD and NR of Aib-foldamers bound to membranes were shown to be useful for identification of conformational preferences of the peptides within the membrane as well as for determining their orientation in the bilayer, and ultimately the effect of the peptides on the membrane structure.

Computational methods such as linear free energy relationships (LFERs) offer a useful high-throughput solution to quickly evaluate drug developability, e.g. membrane permeability, organic solvent/water partition coefficients, and solubility. LFERs typically assume the contribution of structural components/functional groups to the overall properties of a given molecule to be constant and independent. This dissertation describes a series of studies in which linear free energy relationships were developed to predict solvation of small organic molecules in lipid formulations, specifically, triglyceride containing solvents and phospholipid-based liposomes. The formation of intermolecular HBs in triglyceride solvents (homogenous with H-bond accepting ability) and intramolecular HBs within the bilayer barrier domain (hydrocarbon-like) proved to be the major factors to consider in developing LFERs to account for the increased oil/water partition coefficients and enhanced bilayer permeability of small organic molecules. The triglyceride solvent/water partition coefficients of a series of model compounds varying in polarity and H-bond donating/accepting capability were used to establish a correlation between the solvent descriptors and the ester concentration in these solvents using the Abraham LFER approach. The LFER analyses showed that the descriptors representing the polarizability and H-bond basicity of the solvents vary systematically with the ester concentration. A fragment-based LFER to predict membrane permeability or 1,9- decadiene/water partition coefficients of small organic molecules including small peptides was systematically constructed using a total of 47 compounds. Significant nonadditivity was observed in peptides in that the contribution of the peptide backbone amide to the apparent transfer free energy from water into the bilayer barrier domain is considerably smaller than that of a “well-isolated” amide and greatly affected by adjacent polar substituents on the C-termini. In order to explain the phenomenon of nonadditivity, the formation of intramolecular HBs and inductive effects of neighboring polar groups on backbone amide, were investigated using FTIR and MD simulations. Both spectroscopic and computational results provided supportive evidence for the hypothesis that the formation of intramolecular HBs in peptides is the main reason for the observed nonadditivity of Δ(ΔG°)-CONH-. The MD simulation results showed that the inductive effect of neighboring groups is not as important as the effect of intramolecular HBs.

Au cours de ce travail, la génération de second harmonique optique des molécules biologiques a été étudiée. Cette technique optique non linéaire possède un caractère cohérent qui permet d'accéder aux propriétés de symétrie des molécules dont la taille est très inférieure aux longueurs d'onde optiques visibles utilisées et l'hyperpolarisabilité quadratique des acides aminés aromatiques a été mesurée en solution par diffusion non linéaire. L'hyperpolarisabilité des acides aminés non aromatiques a été obtenue de manière indirecte grâce à des mesures effectuées sur le collagène, une protéine ne contenant pas d'acide aminé aromatique. Le collagène possède en fait une très forte réponse non linéaire et fait l'objet d'études intenses en microscopie optique non linéaire. Par un modèle de sommation cohérente des champs harmoniques réémis, l'origine de la très forte efficacité non linéaire de cette protéine, liée à sa grande rigidité et à son organisation spatiale en triple hélice, a pu être obtenue. En parallèle, l'étude de plusieurs petits peptides synthétiques possédant de un à quatre tryptophanes a permis de comprendre la construction de la réponse optique non linéaire de ces objets à partir de motifs répétés. Par ailleurs, pour apporter des informations supplémentaires sur les nano systèmes, un nouveau montage expérimental a été développé. La grande sensibilité du montage a permis notamment d'atteindre la sensibilité d'une seule particule dans le volume sondé et de mettre en place un système de cartographie en trois dimensions par diffusion Hyper Rayleigh. La démonstration a été réalisée pour des nanoparticules métalliques uniques d'argent piégées dans une matrice de gélatine.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Porcine reproductive and respiratory syndrome virus (PRRSV) was isolated for the first time in 1991 and since then it has been associated with significant economic losses to the pig industry worldwide. Although vaccination against PRRSV is widely used, an important advance would be the development of marker vaccines allowing serologic discrimination between vaccinated and naturally infected animals. The present study aimed to identify immunogenic and conserved regions dispensable to viral replication in different PRRSV proteins, which could be used as negative serologic markers in a new generation of liveattenuated vaccines. A fine mapping of B-cell linear epitopes in different PRRSV proteins by Pepscan is presented in the first part of this thesis. The results indicated the presence of several B-cell linear epitopes in the non-structural protein 2 (Nsp2) and in all structural proteins encoded by PRRSV, which were consistently recognized by antibodies raised in pigs experimentally infected with a North American strain of the virus (NVSL97-7895). The Nsp2 was found to harbor the highest frequency of immunodominant epitopes (n=18) when compared to structural proteins. In the structural proteins, epitopes consistently recognized by immune sera were located in all studied proteins. Overall, the highest degree of immunogenicity and conservation was exhibited by two epitopes identified in the C-terminal end of the M protein (ORF6). The antibodies recognizing the immunodominant epitopes of each protein were detected as early as days 7 to 15 post-infection (p.i.) and remained detectable until the end of the experiment (day 90 p.i). Based on their immunodominance and level of amino acid (aa) conservation, two target epitopes were selected to serve as serological marker candidates in each of the following PRRSV proteins: Nsp2, GP3 and M. These epitopes were deleted in the wild-type cDNA infectious clone (FL-12) by site-directed mutagenesis. The results of this study are presented in the second part of this thesis. A Nsp2 mutant virus (FLdNsp2/44) was successfully rescued following RNA transfection in MARC 145 cells. This epitope deletion mutant fulfilled the requirements for a differential vaccine virus such as efficient growth in vitro and in vivo and induction of active seroconversion as measured by a commercial ELISA kit associated with the absence of a marker-specific peptide-ELISA response in 100% (n=15) of the vaccinated animals. In vitro and in vivo characterization of the mutant virus clearly showed that removal of a 15-mer Nsp2 epitope had no effect on the immunogenicity, growth properties or virulence when compared to the wild type virus. On the other hand, deletions of previously identified peptide marker candidates within GP3 and M genes were shown to be lethal for virus viability in vitro. Alternatively, by substitution of 5aa at a time within a M peptide marker candidate, a viable mutant virus could be recovered although it still resulted in a positive marker virus. In summary, our results provide proof of concept that PRRSV marker vaccines can be developed using such methodology. Taken together, these data indicate that the combination of a mutant virus carrying a deletion of an immunodominant epitope and the corresponding peptide ELISA represents an attractive approach for the development of PRRSV differential modified-live vaccines.
O vírus da síndrome respiratória e reprodutiva dos suínos (PRRSV) foi isolado pela primeira vez em 1991 e, desde então, tem sido associado a perdas significativas para a suinocultura mundial. Apesar da vacinação contra o PRRSV ser amplamente utilizada, um grande avanço seria alcançado com a elaboração de vacinas diferenciais que permitam a discriminação sorológica entre animais vacinados e naturalmente infectados. O presente estudo teve como objetivo a identificação de regiões imunogênicas, conservadas e dispensáveis a replicação viral, em diferentes proteínas do PRRSV, que pudessem ser utilizadas como marcadores sorológicos negativos em uma nova geração de vacinas atenuadas. Na primeira parte desta tese estão apresentados os resultados de um mapeamento de epítopos lineares de linfócitos B em diferentes proteínas do PRRSV, pelo uso da tecnologia de Pepscan. Os resultados indicam a presença de diversas regiões imunodominantes na proteína não estrutural 2 (Nsp2) e em todas as proteínas estruturais do vírus. Essas regiões foram consistentemente reconhecidas pelo soro de suínos experimentalmente infectados com uma cepa norte-americana do PRRSV (NVSL97-7895). A maior freqüência de epítopos imunodominantes foi identificada na Nsp2 (n=18) e o mais alto grau de imunogenicidade e nível de conservação de aminoácidos foi observado em dois epítopos identificados na extremidade carboxi-terminal da proteína M (ORF6). Anticorpos reagentes com epítopos imunodominantes de cada proteína foram detectados inicialmente entre os dias 7-15 pós-infecção (pi), permanecendo em altos títulos até o final do experimento (dia 90 pi). Com base na imunodominância e nível de conservação de amino ácidos (aa) das seqüências mapeadas, dois epítopos alvos foram selecionados como candidatos a marcadores sorológicos negativos em cada uma das proteínas Nsp2, Gp3 e M. Esses epítopos foram então deletados em um clone infeccioso de cDNA (FL12) por mutagênese sítio-direcionada. Os resultados desses experimentos encontram-se descritos na segunda parte da tese. Um vírus mutante carreando a deleção de um epítopo imunodominante da Nsp2 (FLdNsp2/44) foi obtido após transfeccção de RNA viral em células MARC145. A caracterização in vitro e in vivo do vírus mutante demonstrou que a remoção dos 15 aa da Nsp2 não produziu efeito sobre a imunogenicidade, replicação ou virulência quando comparado ao vírus parental. Além disso, observou-se indução de soroconversão contra o PRRSV em animais infectados, detectada pelo uso de um teste ELISA comercial. Por outro lado, não foi detectada resposta humoral específica contra a região deletada nos animais imunizados com o FLdNsp2/44, conforme resultados de um teste ELISA contendo como antígeno um peptídeo sintético correspondente a seqüência removida. Por outro lado, deleções dos epítopos previamente identificados na Gp3 e proteína M foram letais à viabilidade viral in vitro. Alternativamente, um outro vírus mutante foi gerado pela substituição de 5 aa do epítopo identificado na proteína M, embora a alteração de resíduos não tenha sido suficiente para eliminar a imunogenicidade da região. Em resumo, os resultados do presente estudo se constituem em uma prova de conceito no sentido do desenvolvimento de vacinas diferenciais contra o PRRSV. A utilização de um vírus mutante carreando a deleção de um epítopo imunodominante, associado com um teste de ELISA baseado no peptídeo sintético correspondente a região deletada, representam uma alternativa para o desenvolvimento de vacinas diferenciais atenuadas contra o PRRSV.

L’atome de fluor joue un rôle crucial dans la modulation des propriétés physico-chimiques des molécules organiques. Son incorporation au sein de la structure des médicaments a connu une expansion rapide et importante. En raison de sa forte électronégativité et de sa lipophilie, le motif trifluorométhyle est couramment employé par l’industrie pharmaceutique. De plus, parmi les composés fluorés, les alcènes substitués par du fluor sont également d’une grande importance en chimie médicinale. Dans ce contexte, nous nous sommes intéressés au développement de voies de synthèses pour incorporer des motifs trifluorométhyles et alcènes monofluorés au sein de molécules. Dans un premier temps, nous avons développé une méthode de chlorotrifluorométhylation d’alcènes internes, médiée par la lumière visible et catalysée par un catalyseur photoredox à base de cuivre. La réaction s’est déroulée avec une régiosélectivité totale dans des conditions de réaction douces en utilisant le CF3SO2Cl disponible dans le commerce comme source de trifluorométhyle et de chlore, conduisant à une synthèse de produits chimiques à haute valeur ajoutée avec une économie d'atomes. Les molécules obtenues ont été testés in vitro pour évaluer leurs cytotoxicités contre les lignées cellulaires cancéreuses ainsi que pour leurs activités antifongiques et antibactériennes. Dans un second temps, une synthèse sans solvant a été développée par mécanochimie afin d’accéder à des monofluoroalcènes. La réaction en une étape est très rapide, sans solvant et se déroule dans des conditions douces permettant de limiter l’impact environnemental de synthèse. Le protocole a montré une efficacité et une tolérance générales à un large panel de substrats carbonylés, y compris les aldéhydes et les cétones, et des dérivés de fluorophosphonoacétate.La douleur chronique touche 20 à 30% de la population mondiale et représente des milliards de dollars en coût annuel. Elle représente un véritable enjeu de santé publique rendant essentiel la poursuite des recherches scientifiques dans ce domaine afin de mieux comprendre la douleur chronique et de développer des solutions innovantes pour améliorer la qualité de vie des patients. Les peptides sont des cibles de choix dans la synthèse de nouveaux médicaments. Le peptide neurotensine (8-13) a démontré son efficacité dans le traitement de la douleur, par modulation des signaux nociceptifs, en se liant aux récepteurs neurotensinergiques, principalement NTS1 et NTS2. Néanmoins, la liaison au récepteur NTS1 provoque des effets secondaires néfastes, ce qui n’est pas le cas lors de la liaison à NTS2. Ainsi, une grande sélectivité envers le récepteur NTS2 est essentielle. Cependant, la forte sélectivité du peptide NT(8-13) envers le récepteur NTS1 ne fait pas de ce peptide un composé de choix dans le cas du traitement de la douleur. Ainsi, synthétiser des analogues peptidiques capables d'interagir de manière sélective avec NTS2, dans l'objectif d'améliorer leur efficacité et leur profil pharmacologique tout en minimisant les effets secondaires, représente un défi de choix. Dans ce contexte, ce dernier chapitre décrit la synthèse de composés capables de se lier aux récepteurs neurotensinergiques tout en maximisant la sélectivité envers NTS2. Pour cela, la synthèse peptidique de JMV 7323, JMV 7324, JMV 7325 et JMV 7326 sur support solide a été effectuée. De plus, les peptides obtenus doivent être capable de passer la barrière hémato-encéphalique. Pour cela, le peptide angiopep-2 a été couplé sur le peptide JMV 7324 pour être utilisé comme navette afin d’atteindre la barrière hémato-encéphalique. Les composés ont été testés in vivo dans des modèles de douleur induite par la formaline montrant des résultats prometteurs et les effets secondaires ont été étudiés
The fluorine atom plays a crucial role in modulating the physicochemical properties of organic molecules. Its incorporation into the structure of drugs has expanded rapidly and significantly. Due to its strong electronegativity and lipophilicity, the trifluoromethyl moiety is commonly used by the pharmaceutical industry. In addition, among fluorinated compounds, monofluoroalkenes are also of great importance in medicinal chemistry. In this context, we were interested in the development of synthetic pathways to incorporate trifluoromethyl and monofluoroalkene moieties into molecules. In a first project, we developed a method of chlorotrifluoromethylation of internal alkenes, mediated by visible light and catalyzed by a copper-based photoredox catalyst. The reaction took place with full regioselectivity under mild reaction conditions using commercially available CF3SO2Cl as the source of trifluoromethyl and chlorine, leading to a synthesis of value-added chemicals with atom economy. The molecules obtained were tested in vitro to evaluate their cytotoxicity against cancer cell lines as well as for their antifungal and antibacterial activities. In a second project, a solvent-free synthesis was developed by mechanochemistry to access monofluoroalkenes. The very fast, solvent-free one-step reaction under mild conditions has limited the environmental impact of synthesis. The protocol showed general efficacy and tolerance to a wide range of carbonyl substrates, including aldehydes and ketones, and fluorophosphonoacetate derivatives.Chronic pain affects 20 to 30% of the world's population and represents billions of dollars in annual costs. It represents a real public health issue that makes it essential to continue scientific research in this field to better understand chronic pain and develop innovative solutions to improve the quality of life of patients. Peptides are prime targets in the synthesis of new drugs. The neurotensin peptide (8-13) has demonstrated its efficacy in the treatment of pain, by modulating nociceptive signals, by binding to neurotensinergic receptors, mainly NTS1 and NTS2. Nevertheless, binding to the NTS1 receptor causes harmful side effects, which is not the case when binding to NTS2. Thus, a high selectivity towards the NTS2 receiver is essential. However, the high selectivity of the NT(8-13) peptide towards the NTS1 receptor does not make this peptide a compound of choice for pain treatment. Thus, synthesizing peptide analogues capable of selectively interacting with NTS2, with the aim of improving their efficacy and pharmacological profile while minimizing side effects, represents a challenge of choice. In this context, this chapter describes the synthesis of compounds capable of binding to neurotensinergic receptors while maximizing selectivity towards NTS2. For this, peptide synthesis of JMV 7323, JMV 7324, JMV 7325 and JMV 7326 on solid support was performed. In addition, the resulting peptides must be able to cross the blood-brain barrier. For this, the angiopep-2 peptide was coupled to the JMV 7324 peptide to be used as a shuttle to reach the blood-brain barrier. The compounds were tested in vivo in models of formalin-induced pain showing promising results and side effects were studied

This thesis describes the synthesis of a novel multimeric peptide ligand targeted to the human melanocortin 4 receptor. The synthesis of the peptide was attempted both by solid phase peptide synthesis and by solution phase peptide synthesis, leading to the conclusion that the necessary C- and N- terminal substituents were much easier to install via the solution phase route. The bifunctional peptide was purified and then multimerized in both protected and active amino acid forms using the copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction. The multimers were characterized using MS and UV-Vis spectroscopy. It was found that a large portion of the monomer cyclized under CuAAC conditions, though sufficient multimerization took place to form up to nonamers, as determined by mass spectrometry.

Nowadays, around a third of the agricultural production is lost due to the damage caused by pests, resulting in a significant economic impact. It is estimated that about a third of these losses are provoked by phytopathogens. Currently, the solution to these problems focuses on the use of pesticides, mainly copper derivatives, antibiotics or fungicides. These compounds are effective, but they are highly polluting. In addition, antibiotics are not allowed in many countries, because abusive use causes the rapid appearance of bacterial resistant strains. The lack of non-polluting and effective treatments for plant disease control has raised the search for new compounds. Antimicrobial peptides are one of the most promising alternatives to traditional pesticides. With this aim, in this doctoral thesis the design, synthesis and characterization of BP100 derivatives was considered
Actualment, es calcula que, aproximadament, un terç de la producció agrícola es perd a causa dels danys provocats per les plagues, resultant en un impacte econòmic molt important. Al voltant d’un terç d’aquestes pèrdues són provocades per fitopatògens. Avui en dia, la solució a aquests problemes es centra en l’ús de pesticides, fonamentalment derivats de coure, antibiòtics o fungicides; tanmateix, tot i ser eficaços, són altament contaminants. A més, els antibiòtics no estan permesos en molts països, perquè un ús abusiu provoca l’aparició ràpida de soques bacterianes resistents. La manca de tractaments no contaminants i efectius per al control de malalties de plantes ha motivat la recerca de nous compostos. Els pèptids antimicrobians són una de les alternatives més prometedores als pesticides tradicionals, perquè mostren una activitat biològica elevada. Per aquest motiu, en aquesta tesi doctoral es va plantejar el disseny, la síntesi i la caracterització de derivats de BP100

The Anterior Gradient 2 (AGR2) protein has been implicated in a variety of biological systems linked to cancer and metastasis, tamoxifen-induced drug resistance, pro-inflammatory diseases like IBD and asthma, and limb regeneration. The molecular mechanisms by which AGR2 mediates these various phenotypes in disease progression in both cancer and IBD are poorly understood, as is the biological function(s) of AGR2 under non-disease conditions. Here, we use a combination of biochemical techniques, organ culture, cell biology and mouse genetics to investigate the biological significance of AGR2 both in cell lines and in vivo. We present data based on phage-peptide inter-actomics screens suggesting a role for AGR2 in mediating the maturation and trafficking of a class of membrane and secretory proteins, and investigate a putative interaction between AGR2 and one member of this class of proteins. We also describe the construction of a universal vector for use in making a variety of transgenic animals, and then present data showing its use as a promoter reporter, and attempt to investigate the temporal and spatial expression of AGR2 in the developing and adult mouse. Further, we present data describing the localisation pattern of AGR2 in the developing murine kidney using a combination of organ culture and antibody staining, and suggest a role for AGR2 in the developing kidney based on this data that is in agreement with a chaperone function for membrane and secretory proteins. Together, these data suggest that AGR2 has an intrinsic consensus docking site for a subset of its client proteins, that AGR2 plays a role in protein maturation in ciliated cell types, and provides a novel biological model to dissect the role of AGR2 in ER-trafficking.

Proteins and protein interactions are involved in virtually all processes of life. Here we study interactions between globular domains and short linear motifs, which form a small interface ideal for transient interactions. Despite the small number of contacts involved, these domain-motif interactions (DMIs) are known to be highly specific in vivo. We have identified hundreds of instances of DMIs in high-resolution 3-dimensional (3D) structures to analyze the molecular basis of their high specificity. Furthermore, we have derived structural parameters to identify DMIs in 3D structures in a more general, motif-independent way. An important class of DMIs are kinase-substrate interactions. By combining the phosphorylation motif with different kinds of contextual information, we could predict substrates of the human kinase Aurora A. Lastly, we have incorporated DMIs into our database of 3D interacting domains (3did) to disseminate our results to the scientific community for future research.

Los procesos moleculares subyacentes a la mayoría de funciones biológicas implican la participación directa de una infinidad de proteínas y múltiples interacciones entre ellas. En esta tesis estudiamos un tipo particular de estas interacciones, de carácter transitorio y altamente específicas, dónde un dominio globular en una proteína reconoce un corto péptido lineal en otra (DMIs). En concreto, identificamos múltiples casos de DMIs en estructuras tridimensionales (3D) de alta resolución y analizamos las bases moleculares de su especificidad. Además, derivamos parámetros estructurales globales que nos permiten identificar nuevos casos de DMIs. Así mismo, y como caso práctico, combinamos el motivo de fosforilación propio de la quinasa humana Aurora A con diversas clases de información contextual para predecir y validar 90 nuevos substratos. Por último, incorporamos las caracterizadas DMIs en nuestra base de datos de interacciones en 3D (3did) con el fin de diseminar nuestros resultados entre la comunidad científica.

Labaditina é um decapeptídeo cíclico, hidrofóbico, extraído da Jatropha Multifida, uma planta da família Euphorbiaceae. É mais resistente à degradação proteolítica que seus respectivos isômeros lineares; e forma pontes de hidrogênio internamente, facilitando sua inserção em membrana biológica. Estudos tem mostrado que a restrição conformacional dos peptídeos cíclicos aumenta sua afinidade e especificidade à membrana. Devido à essas características físicas e às atividades biológicas apresentadas, tais como inibição da via clássica do sistema complemento humano in vitro e atividade antibacteriana para Streptococcus mutans, este peptídeo tem ganhado interesse biológico e farmacológico. Sobretudo, ainda não é conhecido seu mecanismo de ação. Devido à isso, os peptídeos Labaditina (Lo) e o análogo linear (L1), estruturalmente diferentes, foram estudados com o objetivo de obter informações quanto ao mecanismo de ação, interação e possíveis alterações estruturais frente a membranas biológicas. O comportamento do Lo e L1 foi avaliado na presença de diferentes composições de lipídios (DPPC, DPPC:Chol (9:1), DPPC:DPPS (8:2)) e de detergentes (SDS e LPC), utilizando sistemas miméticos de membrana: monocamada, micela e lipossomo. Em monocamada, sistema planar, foi observado um aumento da pressão superficial, provavelmente causado pela presença de peptídeo. Nos sistemas compostos por DPPC:Chol e DPPC:DPPS o efeito foi maior na presença do L1, sugerindo interação eletrostática entre o peptídeo e as monocamadas. Já o peptídeo Lo, por não possuir carga, apresentou maior interação com a monocamada de DPPC, por ser zwitteriônica. Resultados similares foram obtidos através do estudo com lipossomos constituídos por DPPC, DPPC:Chol (9:1) e DPPC:DPPS (8:2). Em todos os meios, através da espectrofotometria de fluorescência, foi observado um blue-shift, ou seja, migração do triptofano para um ambiente mais apolar. Para o Lo, isso foi maior na presença de DPPC; para o L1, na presença de DPPC:Chol e DPPC:DPPS. Através do DSC foi observado um aumento da entalpia e diminuição da cooperatividade (t1/2), causado pela presença de peptídeo na bicamada. Em DPPC:Chol (9:1) e DPPC:DPPS esse efeito foi maior na presença do L1; e em DPPC, na presença do Lo, confirmando os resultados anteriores. Essas interações peptídeo-mimético de membrana foram acompanhadas por mudanças conformacionais, observadas através do CD. O peptídeo Lo, tanto em meio aquoso, quanto na presença dos diferentes lipossomos está não-ordenado, entretanto, possui diferenças conformacionais em cada meio. O peptídeo L1 em meio aquoso apresenta estrutura ao acaso com interação entre os triptofanos, porém em DPPC e em DPPC:Chol (9:1) sofre alteração conformacional, distanciando os triptofanos; em DPPC:DPPS (8:2) sofreu alteração para -folha. Isso demonstra que a composição lipídica induz diferentes conformações nos peptídeos e pode afetar seu mecanismo de ação. No estudo com micelas também foi observado interação de ambos os peptídeos com SDS, e também com LPC. Em SDS os estudos sugerem que o L1 está mais inserido no meio apolar que o Lo; já em LPC, o Lo. Esses peptídeos também apresentaram alteração conformacional na presença das micelas. O peptídeo Lo, tanto em SDS, quanto em LPC, apresentou conformação não-ordenada, porém diferentes. Já o peptídeo L1 apresentou conformação -folha na presença de SDS e LPC, porém também com diferenças. Os resultados demonstram que o peptídeo com estrutura linear (L1) possui maior liberdade conformacional. Portanto, alguns fatores dirigem o processo de interação destes peptídeos: conformação e hidrofobicidade. Devido à diferença estrutural (cíclica e linear), esses peptídeos conferem diferentes hidrofobicidades, e isso interfere na conformação da molécula, além do meio lipídico. E finalizando o estudo, foi identificado através da DM que o resíduo de triptofano da posição 2 é o aminoácido mais inserido no meio apolar das micelas, após interação. Assim, um possível mecanismo de interação do peptídeo Lo é baseado, inicialmente, na adsorção do peptídeo na superfície lipídica. Em seguida ocorre a interação hidrofóbica membrana-peptídeo, acompanhada pela inserção do triptofano da posição 2 na região mais profunda da membrana, induzindo alterações conformacionais na molécula mediante a interação, dos outros resíduos, com a membrana.
Labaditin is a cyclic decapeptide with high hydrophobic character, extracted from Jatropha Multifida, a plant from Euphorbiaceae family. It is more resistant to proteolytic degradation than its corresponding linear isomers. Studies have been showed that conformational restriction of cyclic peptide increases its affinity and specificity to the membrane. Due to these physical characteristics and to the biological activities shown, such as inhibition of the classical pathway of human complement system in vitro and antibacterial activity for Streptococcus mutans, this peptide has attracted biological and pharmacological interest. However, neither the target nor the action mechanism are known yet. For this reason, the Labaditin (Lo) and the linear analogue (L1) peptides, different structures, were studied in an attempt to get information regarding the mechanism of action, interaction and possible conformational changes due to the interaction with biological membranes. The behavior of Lo and L1 was studied in the presence of different lipid compositions (DPPC, DPPC:Chol (9:1), DPPC:DPPS (8:2)) and of detergents (SDS and LPC), using membrane mimetic systems: monolayer, micelle and liposome. In monolayer, planar system, it was observed an increase of surface pressure, probably caused by the presence of peptide. In the systems composed by DPPC:Chol and DPPC:DPPS the effect was greater in the presence of L1, implying electrostatic interaction between the peptide and the monolayers. Lo peptide, on the other hand, due to the fact that it does not have charges, presented greater interaction with the DPPC monolayer, a zwitterionic molecule. Similar results were obtained through studies with liposome composed by DPPC, DPPC:Chol (9:1) and DPPC:DPPS (8:2). In all environments, through fluorescence spectroscopy, a blue-shift was observed, which means, migration of the tryptophan to a more non-polar environment. For Lo, it was higher in the presence of DPPC; for L1, in the presence of DPPC:Chol and DPPC:DPPS. Using the DSC technique an increase of enthalpy and a decrease of cooperativity was observed (t1/2), due to the presence of peptide in the bilayers. In DPPC:Chol (9:1) and DPPC:DPPS this effect was greater in the presence of L1; while in DPPC, in the presence of Lo, confirming the previous results. These peptide-membrane mimetic interaction was followed by conformational changes, observed through the CD. The Lo peptide has a unordered conformation in aqueous environment, and in the presence of liposomes also is unordered, although with differences. L1 peptide in aqueous environment presents random coil structure with interaction between tryptophan, but in DPPC and in DPPC:Chol (9:1) it suffers conformational changes, distancing tryptophan; in DPPC:DPPS (8:2) it changes to -sheet. This demonstrates that the lipidic composition induces conformational changes in peptides and it may affect their mechanism of action. In the study with micelles it was also observed interaction between peptides-SDS, and also with peptides-LPC. In SDS, the studies suggest that L1 is more inserted in the non-polar environment than Lo; in LPC, Lo is more inserted. These peptides also presented conformational changes in the presence of micelles. Lo peptide, both in SDS, and in LPC, presented unordered conformation, but differently. L1 peptide presented -sheet conformation in the presence of SDS and LPC, but also with differences. The results show that the peptide with linear structure (L1) has greater conformational liberty. Therefore, some factors are responsible to the interaction process of these peptides: conformation and hydrophobicity. Due to the structural difference (cyclic and linear), these peptides present different hydrophobicity, and it interferes in the conformation of the molecule, as well as the lipidic environment. On the last study it was identified through DM that the tryptophan residue from position 2 is the amino acid most inserted in the micelles, after interaction. Thus, a possible Lo peptide interaction mechanism is based, initially, on the adsorption of the peptide on the lipidic surface. Next, there is a hydrophobic interaction peptide-membrane followed by the tryptophan insertion of the position 2 in the deepest region of the membrane, inducing conformational changes in the molecule, through the interaction of the other residues with the membrane.

Background Obesity is an important risk factor for resistance to insulin-mediated glucose disposal, and is a precursor of type 2 diabetes and other disorders. Objectives To identify molecular pathways in adipose tissue and inflammatory cells that may result in obesity-associated insulin resistance, we exploited the fact that not all obese individuals are prone to insulin resistance. Thus the degree of obesity as a variable was removed by studying obese subjects of similar body mass index (BMI) who are insulin-sensitive (IS) versus insulin-resistant (IR). Methods Combining gene expression profiling with computational approaches, we determined the global gene expression signatures of omental and subcutaneous adipose tissue samples obtained from 10 obese-IR and 10 obese-IS patients undergoing gastric bypass surgery. In a secondary study, we isolated monocytes from 4 obese-IR, 3 obese-IS, and 4 nonobese-IS adolescent and young adult subjects for purposes of assessing differences in expression of inflammatory genes in monocytes using RT-PCR. Results Gene sets related to chemokine activity and chemokine receptor-binding were identified as most highly enriched in the omental tissue from obese-IR compared to obese-IS subjects, independent of BMI. Strikingly, insulin resistance, but not BMI, was associated with increased macrophage infiltration in the omental adipose tissue, as was adipocyte size. In the adolescent and young adult cohort, expression of two cytokine signaling molecules (IL8, SOCS3) and two downstream products of the JNK pathway (JunB, c-Fos) showed increased expression in the obese-IR subjects compared to the obese-IS and nonobese-IS subjects, suggesting the presence of a proinflammatory phenotype in monocytes in obesity, which is exacerbated in the insulin resistant state. Conclusions Our findings demonstrate that inflammation of omental adipose tissue and activation of proinflammatory monocytes is strongly associated with insulin resistance in human obesity. Manipulation of these pathways may result in the prevention of or delay in the onset of obesity-related co-morbidities.

Two photon absorption (TPA) is the near simultaneous absorption of two photons of light to achieve an electronically excited state. It has led to huge advances in microscopy and microfabrication due to its quadratic dependence on the local light intensity. This thesis describes the design, synthesis and application of dyes with strong TPA properties, and as such is divided into three chapters. The first introduces the theory and measurement of TPA as well as structure-property relationships known to maximise the efficiency of TPA. The subsequent chapters present explorations of the application of these dyes in biological applications; namely two-photon uncaging and two-photon photodynamic therapy. A recurring theme in my research is the discussion and evaluation of strategies for improving the compatibility of organic macromolecules with biological systems. Uncaging is the use of photolysis to achieve a rapid increase in the local concentration of a physiologically active species via a photoremovable protecting groups. Photoremovable protecting groups are covalently attached to the physiologically active species, thus rendering it inactive. At the desired time and location, a light dose releases the molecule in its active form. There are many compounds known to uncage following photoexcitation, but there are few examples of caging groups which exhibit both strong two-photon absorption properties and highly efficient uncaging. Chapter 2 discusses the rational design of such groups through the development of a new mechanism for uncaging, in which a photoinduced electron transfer (PeT) between a two-photon-excited electron donor and an electron acceptor/release group drives the uncaging event. Photodynamic therapy (PDT) is a treatment for neoplastic disorders such as cancer in which localised cell death is induced through photoexcitation of a sensitiser. Following light absorption, the photosensitiser enters a relatively long-lived excited state which reacts with cellular oxygen to produce its highly cytotoxic singlet form. The main challenges of the field are to achieve deep penetration of light into tissue and to reduce coincident damage to unaffected tissue by light scattering during irradiation. In 2008, the Anderson group reported the development of PDT photosensitisers with highly efficient two-photon absorption as well as high singlet oxygen quantum yields. Chapter 3 discusses strategies for improving the pharmacokinetics and defining the sub-cellular localisation of these photosensitisers.

碩士
東海大學
化學系
94
Osteoporosis is a silent disease after meridian age. Osteoporosis may cause the consequence that including the change of shape the body, hard walking、pain、even death. To know the situation of bone loss early, will get the great benefit to further awfully life. In clinical monitoring of osteoporosis, using bone turnover metabolic biomarkers in urine or blood can present the information of bone loss. Among the markers of bone resorption, type I collagen crosslinked N-telopeptides (NTx) are derived specifically from bone collagen degradation and not metabolized. Type I collagen is the major collagen product synthesized by bone cells and represents more than 90% by weight of the non-mineral component of bone. Thus, the rate of cross-linked N-telopeptides (NTx) excretion in urine is regarded as a highly specific index of bone resorption, and it is sensitively suppressed in response to antiresorptive therapies. Our previous study indicated that the designed linear peptides; pepide1, peptide2 and peptide3 had specific binding affinity with anti-NTx antibody. Here we designed a series of cyclic peptides which based on before results to explore the conformation reaction with the antibodies. Using UV-Vis. Spectrophotometer to observe the reaction between designed peptides anti-NTx antibodies then calculated the rate constants. The comparisons of linear and cyclic peptides in binding affinity difference show that cyclic peptides react higher affinity than linear peptides. Our further study is to produce anti-cyclic peptide monoclonal antibody to develop more specific and sensitive analysis for monitoring bone loss.

碩士
國立交通大學
應用化學系分子科學碩博士班
103
In cell signaling pathway, ubiquitin binding domains (UBDs) of various proteins specifically bind with different Ub linkages and trigger a vast array of cellular events including protein degradation, protein trafficking, transcription, DNA repair, cell-cycle progression and apoptosis…etc. We thus proposed that by creating Ub-linkage specific UBDs, these molecules may serve as novel recognition molecules for ubiquitination profiling tools and applications. A proof-of-principle study was performed, in which the coiled-coil 2 and leucine zipper (CoZi) region of NF  kappaB essential modulator (NEMO) has been employed as a specific linkage binder for linear polyubiquitin chains. Both quartz crystal microbalance (QCM) and the HaloTag-based assays have been applied to validate the specificity between the constructed binders and the corresponding linkage. This antibody-like molecular tool “A Novel Linear Ubiquitin peptide Binder Opted by tailored Tandem Iteration” (ANLUBOTI) enabled the recognition of highly ubiquitinated large molecules. The construction of these novel peptide binders shall facilitate the ubiquitinated proteome discovery, providing a tool readily for specific ubiquitin linkages profiling.

碩士
朝陽科技大學
應用化學系碩士班
100
Six somatostatin analogues, all containing the pharmaceutical active site F6-F7-W8-K9-T10-F11, were prepared by solid phase peptide synthesis protocol, including a hexapeptide, SRIF-6, an octpeptide, SRIF-8, and the alanine substitute of the octapeptide, SRIF-8 F6→A、SRIF-8 F7→A、SRIF-8 F11→A、SRIF-8 F6、7、11→A. The effect on the fluorescent property of tryptophan residue with the introduction of SDS micellar was investigated by fluorescence and nuclear magnetic resonance spectroscopy. The micellar environment caused quench effect in the tryptophan fluorescent signal for all the six peptides, while SRIF-8 F7→A displayed the least obvious quenching effect among the six peptides. NMR results showed that, under SDS micellar environment, a β-turn can be observed in all the peptides. When entering into SDS micelle environment, conformational change occurs in the fragement of somatostatin pharmaceutical active site and induce the F7 aromatic side-chain to move toward the fluorophore of W8 and results in static quench in tryptophan fluorescent intensity.

碩士
國立中山大學
化學系研究所
105
In recent years, it has been found that the histidine-containing peptide sequence has a very good effect on the chelation of cupric ion. It has been found that human neurodegeneration is associated with the generation of reactive oxygen species by cupric ion and β-amyloid protein. Such a histidine-containing peptide can effectively chelate cupric ion to prevent the production of reactive oxygen species. However, in the synthesis of histidine-containing peptide, the 1,3-diazole structure could react with the activated carboxylic acid to result in poor yields, making such sequences more difficult to synthesize. With reference to aminolysis of esters by using the anionic organic nucleophiles. Literatures shows that the azoles have good catalytic effect. Inspired by these work, our laboratory explored the potential of utilizing the imidazole moiety of histidine in peptide ligation, where the histidine imidazolate, generated by deprotonation. First undergoes a transesterification with a thioester followed by N to N acyl shift to result in the ligated product. In this thesis, the optimal yield of 84% was obtained in the case of alanine. The optimized conditions were applied on dipeptide synthesis of various amino acids, 2+1 tripeptide synthesis, 2+2 tetrapeptide synthesis and long range acyl transfer. All of the cases have good to excellent yield. This method is based on the absence of a protecting group on histidine and forms a native amide bond in the product. This novel methodology was achieved without using Cys/Ser/Tyr residues or an auxiliary group at the ligation site.

No
Stem cells are known to differentiate in response to the chemical and mechanical properties of the substrates on which they are cultured. Thus, supramolecular biomaterials with tunable properties are well suited for the study of stem cell differentiation. In this report, we exploited this phenomenon by combining stem cell differentiation in hydrogels with variable stiffness and metabolomics analysis to identify specific bioactive lipids that are uniquely used up during differentiation. To achieve this, we cultured perivascular stem cells on supramolecular peptide gels of different stiffness, and metabolite depletion followed. On soft (1 kPa), stiff (13 kPa), and rigid (32 kPa) gels, we observed neuronal, chondrogenic, and osteogenic differentiation, respectively, showing that these stem cells undergo stiffness-directed fate selection. By analyzing concentration variances of >600 metabolites during differentiation on the stiff and rigid gels (and focusing on chondrogenesis and osteogenesis as regenerative targets, respectively), we identified that specific lipids (lysophosphatidic acid and cholesterol sulfate, respectively), were significantly depleted. We propose that these metabolites are therefore involved in the differentiation process. In order to unequivocally demonstrate that the lipid metabolites that we identified play key roles in driving differentiation, we subsequently demonstrated that these individual lipids can, when fed to standard stem cell cultures, induce differentiation toward chondrocyte and osteoblast phenotypes. Our concept exploits the design of supramolecular biomaterials as a strategy for discovering cell-directing bioactive metabolites of therapeutic relevance.

碩士
東海大學
化學系
95
The type I collagen cross linked N-telopeptides (NTX) is a bone resorption maker by monitor bone loss. In this study, we synthesize the cyclic analogs of linear peptide of NTX fragments in order to enhance their specificity. Linear peptide fragments of NTX were synthesized by the solid phase method, followed by the chloroacetylation of the N-terminal amine on linear peptide-resin, the chloroacetylated linear peptides were cleaved from the resin and purified by RP-HPLC. The purified chloroacetylated linear peptides were cyclized in the solution phase, purified by RP-HPLC and identified by MALDI-TOF mass spectrophotometer. The yields of synthetic NTX peptide fragments were calculated using the integral area of RP-HPLC and summarized as the following: P3 (18.69%), chloroacetylated-P3 (11.38%), CP-3 (15.81%), P5 (94.87%), chloroacetylated-P5 (68.82%) and CP-5 (84.74%). Our results demonstrate that the amino acid sequence of P3 analogs is not easy to synthesize, thus, we increased the reaction time in order to enhance the yield of P3 analogs. Results of theses studies should provide important information for future work. Key words： NTX 、SPPS、Thioether bridge method、RP-HPLC、MALDI-TOF MS.

碩士
國立中正大學
分子生物研究所
93
In coronavirus, the spike proteins were known to locate on the surface of virus particle and to participate in the entrance of virus into cells. According to the previous reports, there was higher healing possibility within seven days after infection of SARS Co-V clinically. In order to increase the chance to be cured, we proposed to develop a fast and efficient diagnostics kit to detect SARS-CoV at early stage of infection. The LAE technique was used to generate DNA fragments encoding for repeated epitopes of spike protein fused with GST or PEIa proteins. These fusion proteins were purified with affinity chromatography and then used to immunized rabbits for producing antibodies. The ELISA assays showed the purified immunogens can induce antisera production and in the Western blot assays, the antisera can against each indicated epitope. From blockage test, an antiserum against epitope 8 was proved to block virus infection. In addition, other research also show that the monoclonal antibody against amino acid 130 to 150 of spike protein has neutralizing activity. Therefore, in order to obtain the stable antibodies for diagnosis or vaccination in the future, we prepared the monoclonal antibodies of epitope 1 and epitope 8. We obtained several hybridoma clones, but these clones could not produce the monoclonal antibodies specifically against epitope 1 and epitope 8.

This thesis is an investigation of the natural products deriving from marine algae and cyanobacteria and has resulted in the discovery of eleven new secondary metabolites. The structure elucidations of these new molecules were performed using a variety of spectroscopic techniques. Four new macrolides were isolated and characterized from the Madagascar marine cyanobacterium Geitlerinema sp. These ankaraholides are structurally similar to the potently cytotoxic swinholides and were found to have cytotoxicities ranging from 178 nM to 354 nM against human lung cancer (NCI-H460) and mouse neuro-2a cell lines. Since swinholide-type compounds were previously localized to the heterotrophic bacteria of sponges, these findings raise intriguing questions about their true metabolic source. Geitlerinema sp. was found to be particularly rich in chemistry, and also produced the new linear lipopeptide mitsoamide with unusual structural features including an aminal moiety, a homolysine residue and a polyketide unit (3,7- dimethoxy-5-methyl- nonanedioic acid) (DMNA). A collection of the red marine alga Portieria hornemannii from the south of Madagascar (Tolagniaro, Fort Dauphin), led to the isolation of the previously reported halogenated monoterpene, halomon, and the discovery of three new related metabolites. These molecules were found to inhibit DNA methyltransferase 1 (DNMT-1). As a result of efforts to identify bioactive agents from the marine cyanobacterium Lyngbya majuscula, tanikolide dimer, a novel SIRT2 inhibitor (IC50 = 176 nM), and tanikolide seco-acid were isolated. The depside molecular structure of tanikolide dimer, which is likely a meso compound, was established by NMR, MS and chiral HPLC analyses. The structure of tanikolide dimer raises a number of intriguing configurational and biosynthetic questions for further study. The bioassay guided fractionation of a collection of the brown marine alga Dictyota sp. from Netherland Antilles Playa Fort, led to the identification of a novel HDAC inhibitor with a dolastane carbon skeleton. The novel molecule was also found to possess antimalarial activity. Other known HDAC inhibitors with interesting antimalarial activity have been reported previously, and based on this efficacy against malaria, HDAC appears to be a viable target for the development of antiparasitic agents.
Graduation date: 2006

This thesis provides biophysical bases of the interactions between two proteins involved in microRNA (miRNA)-mediated silencing: CNOT1 and the silencing domain of GW182. The regulation of gene expression at the post-transcriptional level involves the crucial CCR4-NOT deadenylase complex, which deadenylates mRNA, and can also inhibit translation in an independent fashion. In miRNA-mediated silencing, the CCR4-NOT complex is brought into the vicinity of the target mRNA by the successive actions of the miRNA, the Argonaute protein and finally, the GW182 protein, which interacts directly with CCR4-NOT. In the case of silencing of mRNAs containing AU-rich elements, the same action is performed by the protein called tristetraprolin involved in the regulation of inflammatory processes. The interactions and interplay between all of these high molecular weight proteins are relatively poorly understood. In particular, the interaction sites between GW182 and the CCR4-NOT complex were previously unknown. Molecular biology experiments allowed the identification of CCR4-NOT interaction motifs on GW182. One of them is crucial for deadenylation, while the other is vital in mediating the interaction with CCR4-NOT via CNOT1, the scaffolding subunit of the CCR4-NOT complex. Biophysical experiments based on hydrogen-deuterium exchange mass spectrometry allowed the identification of the corresponding binding site on CNOT1(800-999). Surprisingly, the binding site of the GW182 silencing domain was found to be at the same CNOT1(800-999) surface region as the binding site of tristetraprolin. Biochemical experiments excluded their simultaneous binding to CNOT1. The GW182 and tristetraprolin proteins share a common motif, RLPXφ, that interacts with CNOT1 in a very similar, but not identical, manner. This sequence has been proposed to act as a short linear motif. Thus, the two different gene silencing pathways: miRNA-mediated silencing and ARE-mediated silencing intersect at CNOT1, which serves as a molecular hub. The structural dynamics of the GW182 silencing domain and the CNOT1(800-999) fragments were also studied. The GW182 silencing domain was experimentally proved to be natively unstructured except for an RNA-recognition motif (RRM) domain. The GW182 RRM domain was found to be a loose structure, contrary to the CNOT1(800-999) structure that was found to be very rigid. Experiments performed in this thesis have led to the discovery of the interaction sites between the natively disordered GW182 silencing domain and the helical CNOT1(800-999) protein fragment, contributing to the understanding of the molecular mechanisms of recognition within protein complexes involved in gene silencing in different physiological processes.
Niniejsza praca doktorska dotyczy biofizycznych podstaw oddziaływania między białkami zaangażowanymi w wyciszanie ekspresji genów przez mikro-RNA (miRNA), a mianowicie pomiędzy białkiem CNOT1 a domeną wyciszającą białka GW182. W procesie wyciszania ekspresji genów przez miRNA, cząsteczki te wiążą się z białkiem Argonaute i naprowadzają je na cząsteczkę mRNA, która ma ulec wyciszeniu. Z białkiem Argonaute oddziałuje białko GW182, które z kolei wiąże się z kompleksem deadenylaz CCR4-NOT. Kompleks ten deadenyluje mRNA oraz może także blokować jego translację, co łącznie prowadzi do wyciszenia ekspresji danego genu. Z kolei w wyciszaniu mRNA zawierających sekwencje bogate w adeninę i urydynę, rolę miRNA wraz z Argonaute i GW182 pełni białko o nazwie tristetraprolina, które odgrywa kluczową rolę w procesach odpowiedzi na stany zapalne. Oddziaływania pomiędzy składnikami tego skomplikowanego układu białek o wielkich masach cząsteczkowych są jeszcze stosunkowo słabo poznane. W szczególności, nieznane były miejsca odpowiedzialne za tworzenie kompleksu pomiędzy GW182 a CCR4-NOT. Doświadczenia z zakresu biologii molekularnej pozwoliły na identyfikację miejsc wiążących CCR4-NOT w sekwencji domeny wyciszającej białka GW182. Jedno z nich ma kluczowy wpływ na deadenylację, a drugie - kluczowy wpływ na oddziaływanie z kompleksem CCR4-NOT za pośrednictwem jego centralnej podjednostki CNOT1. Badania biofizyczne metodą wymiany wodór-deuter sprzężoną ze spektrometrią mas pozwoliły z kolei na identyfikację miejsca oddziaływania GW182 na białku CNOT1 (we fragmencie 800-999), które, niespodziewanie, okazało się bardzo dobrze pokrywać z miejscem oddziaływania CNOT1(800-999) z tristetraproliną. Eksperymenty biochemiczne wykazały, że białka te konkurują o miejsce oddziaływania na CNOT1(800-999). Białka GW182 i tristetraprolina oddziałują z CNOT1 wykorzystując ten sam motyw sekwencji, RLPXφ, w bardzo podobny, jednak nie identyczny sposób. Sekwencja ta prawdopodobnie działa jako tzw. krótki motyw liniowy (z ang. short linear motif, SLiM). Zatem te dwa szlaki kontroli nad ekspresją genów krzyżują się. W pracy zbadano także dynamikę strukturalną białka CNOT1(800-999) oraz domeny wyciszającej białka GW182. Wykazano eksperymentalnie, że białko GW182 ma nieustrukturyzowany charakter, oprócz domeny wiążącej RNA (RRM), która ma strukturę bardzo dynamiczną. Natomiast białko CNOT1(800-999) charakteryzuje się stabilną, ściśle upakowaną strukturą. Przeprowadzone badania doprowadziły do odkrycia miejsc oddziaływania pomiędzy natywnie nieustrukturyzowaną domeną wyciszającą GW182, a helikalnym fragmentem białka CNOT1(800 999), przyczyniając się do zrozumienia molekularnych mechanizmów rozpoznawania w kompleksach białkowych odpowiedzialnych za regulację ekspresji genów w różnych procesach komórkowych.