Dissertations / Theses on the topic 'Histidine peptide'

Die Arbeit behandelt die Ergebnisse von Experimenten über die Wirkung des Dipeptides Carnosin (β Alanyl L Histidin) und der Aminosäuren L Histidin und β-Alanin auf Kulturen der humanen Zellreihen U87, T98G und LN405, welche von Zellen des malignen Hirntumors Glioblastoma multiforme abgeleitet sind. Die Vitalität der Zellen nach Inkubation mit Carnosin oder L Histidin wurde anhand der Adenosintriphosphatproduktion und der Dehydrohenaseaktivität für Inkubationszeiträume von 24, 48 und 72 Stunden bestimmt. Dabei zeigte sich eine signifikant niedrigere Vitalität der mit Carnosin oder L Histidin inkubierten Zellen gegenüber der unbehandelten Kontrolle. Dieser Effekt war bei L Histidin stärker ausgeprägt. Bei Messungen der Lakatdehydrogenaseaktivität im Medium der Zellen, welche als Indikator für Zellnekrosen diente, zeigten nur die mit L Histidin inkubierte Zellen Zeichen von Nekrose. Die gleichen Messungen wurden auch an humanen embryonalen Nierenzellen durchgeführt (HEK 293), wobei sich ein ähnliches Ergebnis feststellen ließ. In den drei Zellreihen wurde zudem mittels qRT-PCR die mRNA-Expression für die beiden Enzyme Carnosinase 1 und Carnosinase 2 bestimmt, welche L Histidin von Carnosin abspalten. Im Vergleich mit Proben aus normalem Hirngewebe war die Expression beider Enzyme in den Glioblastomzellen deutlich geringer, wenngleich nachweisbar. Nachdem vorhergehende Studien [8] einen Anstieg der Expression von mRNA der Pyruvatdehydrogenasekinase 4 (PDK4) in mit Carnosin inkubierten Glioblastomzellen gezeigt hatten, wurde dieser Effekt hier auch mittels qRT-PCR in mit L Histidin inkubierten Zellen nachgewiesen. Eine Wirkung von Carnosin oder L Histidin auf ein Reportergen des PDK4-Promoters wurde ebenfalls untersucht, wobei sich kein signifikanter Effekt nachweisen ließ.

The Amyloid Cascade Hypothesis states that fibrillation of the amyloid beta (Aβ) peptide is the primary cause of Alzheimer’s pathology. The trigger for the fibrillation is a subject of much debate, although it is clear, oxidative stress is a key feature of Alzheimer’s aetiology. This thesis explores a possible role of oxidation of Aβ, in particular the effect of histidine and methionine side-chain oxidation, on Aβ fibril growth rates. Within chapters 2 and 3 of this thesis is a discussion of various approaches to chemical synthesis of 2-oxo-histidine with a view to the incorporation of the oxidised amino acids into Aβ peptide using Fmoc approaches. Chapter 2 describes attempted chemical transformation of (protected) L-histidine into L-oxohistidine. Dimethyldioxirane oxidised Boc-His-OMe yielded products containing isopropylidene groups, while oxidation using a Cu(II)/ascorbate generated 2-oxo-histidine but gave very low yields. Within chapter 3, a successful synthesis of protected 2-oxo-histidine is described, via the known imidazolin-2-one-4-carboxylic. Chapter 4 analyses Aβ(1-40) fibrillation kinetics by treating the intact peptide with various oxidants. Contrary to previous reports, hydrogen peroxide alone did not slow fibrillation rates. Cu(II)/Cu(I)- catalysed oxidation increased the likelihood of amorphous aggregation over fibrillation. This thesis shows oxidation of Aβ has a profound influence on fibril growth and that incorporation of a stable oxidised histidine into Aβ is a realisable goal.

β-alanil-L-istidina (carnosina) è un peptide endogeno che possiede innumerevoli proprietà (chelante dei metalli, antiossidante, sequestrante delle specie reattive carboniliche). Diversi studi clinici hanno dimostrato un’attività farmacologica della carnosina in malattie su base ossidative, tuttavia il meccanismo dell’attività in vivo non è ancora noto. Questo progetto ha come scopo quello di comprendere il meccanismo in vivo della carnosina. Per far ciò, sono stati sviluppati nuovi metodi analitici di cromatografia liquida accoppiata a spettrometria di massa per la quantificazione in campioni biologici dei peptidi istidinici, loro derivati e gli addotti con le specie reattive carboniliche. Come prima cosa, un metodo analitico basato su una colonna ad interazione idrofiliche è stato sviluppato per l’analisi del carnosinolo in matrici biologiche di modelli animali di sindrome metabolica. La concentrazione di carnosinolo è stata determinata in diversi tessuti e, per la prima volta, l’addotto carnosinolo-acroleina è stato identificato in omogenato di fegato. Questo conferma l’attività del carnosinolo e dei peptidi istidinici come sequestranti delle specie reattive carboniliche in vivo. Tuttavia, è stata identificata l’instabilità metabolica dell’addotto carnosinolo-HNE in diversi tessuti. Saranno quindi necessari ulteriori studi per la caratterizzazione del metabolismo di questi addotti e l’identificazione della corretta entità chimica da ricercare nelle matrici biologiche come indice dell’attività sequestrante di carnosina e derivati. Il metodo basato su colonne ad interazioni idrofiliche è stato anche utilizzato per sviluppare un metodo a rivelazione diretta per determinare l’attività idrolitica del siero umano della carnosina. La carnosinasi serica è stata identificata come principale enzima impiegato nel metabolismo della carnosina. Rispetto ad altri metodi pubblicati in letteratura, quello sviluppato in questo elaborato si basa su una determinazione diretta della carnosina, senza dover effettuare processi complessi di preparazione del campione. I dati ottenuti sono stati convalidati con dati presenti in letteratura, dimostrando che il nostro metodo risulta essere affidabile ed accurato. È stato possibile anche condurre esperimenti di competizione fra substrati naturali e alcune molecole per valutare le principali interazioni substrato/enzima, con l’obiettivo di identificare inibitori della carnosinasi. I dati ottenuti sono stati condivisi con colleghi chimici computazionali che attraverso esperimenti di docking, virtual screening e dinamica molecolare hanno identificato dei possibili inibitori naturali della carnosinasi serica umana. Un nuovo meccanismo d’azione della carnosina è stato approfondito, in quanto recenti pubblicazioni hanno evidenziato un ruolo della carnosina nella prevenzione della formazione di addotti fra la 3,4-diidrofenilglicolaldeide (DOPEGAL), un metabolita intermedio del catabolismo della noradrenalina, e le proteine. La capacità della carnosina di legare covalentemente la DOPEGAL tramite la formazione di un prodotto di Amadori è stata determinata in vitro e in lisato cellulare dove la DOPEGAL è stata formata aggiungendo noradrenalina al lisato enzimaticamente attivo. Studi futuri dovranno caratterizzare la stabilità metabolica di quest’addotto e le caratteristiche della sua formazione in matrici biologiche in quanto risulta essere un interessante biomarcatore di tossicità noradrenalinergica. In fine è stata valutato l’impatto della carnosina e del carnosinolo sul proteoma di cellule endoteliali umane derivanti dalla vena ombelicale. È ormai noto che i farmaci non agiscono unicamente col meccanismo d’azione per il quale sono stati sviluppati, ma possono interferire con l’espressione delle proteine cellulari, aumentandone, o diminuendone l’espressione e di conseguenza attivando o disattivando vie biologiche. Carnosina e carnosinolo non inducono una variazione nell’espressione delle proteine in cellule sane. Questo conferma la sicurezza delle molecole, soprattutto prevedendone un uso come terapia cronica. In futuro l’effetto del trattamento andrà valutato su cellule in condizioni patologiche, per comprendere se, in queste condizioni, carnosina o carnosinolo riescono a influenzare vie metaboliche e risposte cellulari. Sebbene ci siano ancora diverse domande che sono rimaste senza risposta, i dati ottenuti in questo elaborato hanno portato all’aumento della conoscenza del meccanismo d’azione di carnosina e derivati e all’identificazione di composti inibitori della carnosinasi.
β-alanil-L-histidine (i.e. carnosine) is an endogenous peptide that have been extensively characterized for a number of in vitro properties (i.e. metal chelating, antioxidant, reactive carbonyl species quenching). Several clinical trials highlighted the potential benefits of carnosine in the treatment of oxidative stress-based diseases, although the in vivo mechanism of action is not known, yet. The research project herein tries to expand upon the in vivo mechanism of action of carnosine. New analytical methods have been developed by means of liquid chromatography – tandem mass spectrometry for the quantification of histidine dipeptides, their derivatives, and the adducts formed with reactive carbonyl species into biospecimens. A first step was the implementation of hydrophilic interaction chromatography to skip some sample preparation steps and to reduce the chance of systematic errors. The method allowed the quantification of carnosine and carnosinol (a carnosine derivative stable to carnosinase) in biospecimens. Carnosinol tissue distribution in animal models of metabolic syndrome was determined and carnosinol-acrolein adduct was detected for the first time in liver matrices. This finding experimentally confirmed the reactive carbonyl species (RCS quenching activity of histidine dipeptides and derivatives in vivo. However, the metabolic instability of carnosinolHNE adduct was proved and such an evidence requires further studies aiming at understanding the metabolic fate of RCS-adducts to characterize their disposal. Subsequently, a new method for the measurement of carnosine hydrolysis in serum was developed as well. Human serum carnosinase has been identified as the enzyme responsible for such an activity. Compared to other published assays, the method employs a direct detection of the substrate and the use of less sample. Competition experiments with either natural derivatives or other molecules were set to identify hit compounds acting as carnosinase inhibitors. The collected data were shared with computational chemists who identified putative hit compounds via docking, virtual screening, and molecular dynamic approaches. Furthermore, a novel carnosine mechanism of action was studied starting from the evidence that carnosine can prevent the formation of protein adducts with 3,4- dihydroxyphenylglycolaldehyde (DOPEGAL) (i.e. an aldehyde intermediate of norepinephrine metabolism). This could be relevant for the in vivo mode of action of carnosine since DOPEGAL can accumulate in cells because of oxidative stress and as it covalently binds proteins, it can alter their structures and functions. Carnosine quenching activity via the formation of an Amadori product with DOPEGAL was determined in vitro and in cell lysates producing DOPEGAL from enzymatic transformation of norepinephrine. Future studies should be done to characterize the metabolic stability of the adduct and its formation in biospecimens as potential biomarker of norepinephrine toxicity. Finally, the project included proteomics studies on human umbilical vein cells (HUVECs) to assess the impact of carnosine and carnosinol on protein expression. It is widely recognized that drugs exert their pharmacological effects also by an alteration of biological pathways by modifying protein expression. Carnosine and carnosinol have little or no impact on protein expression as detectable on proteome or secretome of healthy endothelial cells. In the future the impact on pathological cells should be carried out as well. These data support the hypothesis of a low toxicity for these molecules, making them suitable candidates for a chronic administration. Although a lot of questions are still unanswered, these data have given new insights in the mechanism of action of carnosine and in the discovery of molecules acting either as carnosine-like compounds or as carnosinase inhibitors.

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.

Orientador: Marisa Masumi Beppu
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química
Made available in DSpace on 2018-08-22T23:38:34Z (GMT). No. of bitstreams: 1 Mahl_CynthiaReginaAlbrecht_M.pdf: 2998402 bytes, checksum: 1db8236b0532958615debe390fa28bc7 (MD5) Previous issue date: 2013
Resumo: A doença de Alzheimer está relacionada à ligação anômala que ocorre entre o peptídeo ?- amilóide (?A) e o íon cobre. Segundo a literatura, utilizando-se espectroscopia Raman, observou-se forte evidência de que o cobre se liga ao núcleo da ?A através de anéis imidazólicos de histidina. Agentes quelantes, em princípio, podem ser usados farmacologicamente no tratamento da intoxicação com metais pesados. Neste trabalho, verificou-se a ação quelante da quitosana atuando na remoção de íons cobre, na presença de ?-amilóide ou de um composto equivalente que contenha histidinas (conhecidos como os responsáveis pela ligação com o íon cobre). Devido a limitações no uso da ?A, decidiu-se por usar histidina, para um melhor entendimento da interação que ocorre no sistema. Inicialmente, esferas porosas de quitosana foram preparadas para utilização na adsorção de íons cobre, com e sem a presença de histidina. Estudos de equilíbrio, envolvendo isotermas e cinéticas de adsorção foram realizadas, e a partir desses constatou-se, que a histidina compete com a quitosana pelos íons cobre, levando a uma diminuição na capacidade de adsorção. Em contrapartida, observa-se uma diminuição não tão brusca da capacidade de adsorção, mostrando que mesmo com a competição ocorrendo a quitosana continua adsorvendo cobre. Com a aplicação dos modelos isotérmicos, foi possível prever que a adsorção ocorre em monocamadas e também em sítios heterogêneos e que não existe somente um grupamento responsável pela quelação dos íons cobre. Em relação aos modelos cinéticos foi proposto que a etapa limitante no processo é adsorção química. Empregando Espectroscopia de Infravermelho por Transformada de Fourier (FTIR-ATR), observou-se que a histidina atua principalmente nos grupos amino e hidroxil da quitosana, afetando os átomos de nitrogênio e oxigênio do mesmo. Foram também realizadas análises de espalhamento de Raios-X a baixo ângulo (SAXS) revelando que a adição de histidina ou ?A no sistema proporciona provavelmente um desenovelamento da quitosana. Através de análises de Estrutura Fina Estendida de Absorção de Raios-X (EXAFS) verificou-se que após adição de histidina ocorre uma mudança qualitativa relacionada à primeira esfera de coordenação referente à ligação de Cu-O, o que pode indicar novas ligações, agora com os átomos de nitrogênio. Para finalizar, uma isoterma de adsorção empregando-se o peptídeo ?A foi obtida, os resultados foram semelhantes aos obtidos com a histidina, mantendo-se uma de adsorção de cobre por parte da quitosana
Resumo: A hidrogenólise da sacarose é uma reação química de interesse industrial, uma vez que os produtos obtidos, sob a forma de glicóis e polióis, podem ser empregados em diversos setores produtivos, tais como indústrias farmacêuticas e de alimentos. Nesse contexto, o presente trabalho tem como objetivo estudar a preparação de catalisadores de Ni e Ru suportados em carvão ativado, destinados à hidrogenólise da sacarose em meio aquoso. Para tanto, catalisadores com uma fração mássica total de 5 % de metal foram preparados através dos métodos de impregnação incipiente e úmida, a partir de soluções aquosas de precursores clorados. Antes das impregnações, o suporte de carvão ativado comercial foi submetido a um tratamento químico com solução aquosa de KOH. Os sólidos preparados por meio da impregnação incipiente foram reduzidos a 473 K (200 ºC), sob fluxo de H2. Por sua vez, a impregnação úmida foi conduzida a 353 K (80 ºC), para diversos valores de pH, sendo a redução dos catalisadores realizada com formaldeído ou NaBH4. Os sólidos foram caracterizados através das técnicas de adsorção de N2 (método de B.E.T.), titulação de Boehm, titulação potenciométrica, espectroscopia de fotoelétrons excitados por raios X, microscopias eletrônicas de varredura e de transmissão, difratometria de raios X e redução à temperatura programada. Os desempenhos catalíticos na reação de hidrogenólise da sacarose foram conduzidos num reator Parr do tipo "slurry", à temperatura de 523 K (250 ºC) e sob pressão de H2 de 5,0 MPa (50 atm), utilizando-se uma massa de 5 g do catalisador. Os resultados revelam que os catalisadores de Ni/C preparados por impregnação úmida são mais ativos e seletivos a 1,2 propanodiol que os preparados via impregnação incipiente. A impregnação úmida também leva a catalisadores de Ru/C seletivos, que se mostram mais ativos que os preparados por impregnação incipiente. Contudo, a impregnação incipiente permite obter catalisadores igualmente seletivos, sendo que, em ambos os casos, o tratamento do suporte com KOH tem um efeito marcante no aumento do rendimento de 1,2 propanodiol
Abstract: Alzheimer's disease is related to the anomalous binding that occurs between the protein ?-amyloid (?A) and copper metal ion. By using Raman spectroscopy, literature has shown strong evidence that copper and zinc bind to A? peptide core through the imidazole ring of histidine. Chelating agents, in principle, can be used pharmacologically in the treatment of heavy metal poisoning. In this study, we verified the chelating action of chitosan acting in the removal of copper ions in the presence of amyloid-ß or an equivalent compound structural that presents histidine (known as the responsible for binding with copper ion). Due to limitations in using ?A, it was decided to use just histidine, aiming a better understanding of the interaction that occurs in the system. Initially, porous chitosan beads were prepared for the use in the adsorption of copper ions, with and without the presence of histidine. Equilibrium studies involving adsorption isotherms and kinetics were carried out, and from them it was observed that histidine competes with chitosan for the copper ions, leading to a decrease in the adsorption capacity. On the other hand, there is no sharp decrease of the adsorption capacity showing that even with a competition taking place, chitosan remains adsorbing copper. By applying the isothermal models, it was possible to predict that an adsorption monolayer occurs and that there is not only one group responsible for chelation of copper ions. With regard to kinetic models it was proposed that the limiting step in the adsorption process is the chemical adsorption. By using Fourier Transform Infrared Spectroscopy (FTIR-ATR) it was observed that histidine acts mainly in the amino and hydroxyl groups of chitosan, affecting the nitrogen and oxygen atoms of the same. Analyses were performed for Small Angle X-Ray Scattering (SAXS) revealing that the addition of histidine or ?A in the system provides probably one unfolding of chitosan. Through the analyses of Extended X-Ray absorption fine structure (EXAFS), it was found that after addition of histidine a qualitative change occurs in the first coordination sphere attributed to Cu-O binding, which may indicate new bindings, now with nitrogen atoms. Finally, the adsorption isotherm employing ?A protein was similar to those obtained with histidine; chich shows that chitosan kept the ability to absorb copper
Abstract: The sucrose hydrogenolysis is a chemical reaction of industrial interest, since the obtained products, under the form of glycols and polyols, can be employed in different production sectors such as pharmaceutical and food industries. The present work aims to study the preparation of Ni and Ru catalysts supported on activated carbon, for the sucrose hydrogenolysis in aqueous media. For this purpose, catalysts with a total fraction of 5 wt % of metal were prepared by the methods of incipient and wet impregnation from aqueous solutions of chlorinated precursors. Before impregnation, the support of activated carbon was subjected to a chemical treatment in an aqueous solution of KOH. The solids prepared by incipient impregnation were reduced at 473 K (200 °C) under H2 flow. In its turn, the wet impregnation was conducted at 353 K (80 °C), for various pH values, being the catalysts reduction performed with formaldehyde or NaBH4. The solids were characterized by the techniques of N2 adsorption (B.E.T. method), Boehm titration, potentiometric titration, X ray photoelectron spectroscopy, scanning and transmission electronic microscopies, X ray diffraction and temperature programmed reduction. The catalytic performances for the sucrose hydrogenolysis were conducted in a Parr reactor of slurry type at temperature of 523 K (250 ° C) under H2 pressure of 5.0 MPa (50 atm), employing a catalyst mass of 5 g. The results reveal that the Ni/C catalysts prepared by wet impregnation are more active and selective for 1,2 propanediol than those prepared via incipient impregnation. The wet impregnation also leads to selective Ru/C catalysts, which are more active than those prepared by incipient impregnation. However, the incipient impregnation achieves also to selective catalysts, being that in both cases, the support treatment with KOH has a marked effect on increasing the yield of 1,2 propanediol
Mestrado
Engenharia de Processos
Mestra em Engenharia Química

Ce travail concerne la synthèse, les propriétés et l’étude du potentiel de liquides ioniques chiraux dérivés d’aminoacides. Des sels de thiazolinium substitués en position 2 ont été préparés avec de très bons rendements à partir d’aminoalcool énantiopurs commerciaux et de dithioesters. Les propriétés physico-chimiques de ces sels ont été modulées en jouant sur la structure du cation ou la nature de l’anion. Les sels de thiazolinium synthétisés présentent une bonne stabilité thermique et chimique. Leur potentiel en reconnaissance chirale a été démontré en mettant en évidence des intéractions diastéréomériques avec un sel racémique de l’acide de Mosher. Les sels de thiazolinium sont des milieux de choix pour la réaction d’aldolisation de Mukaiyama entre le benzaldéhyde et le diène de Danishefsky, permettant de s’affranchir de l’utilisation d’un catalyseur. Néanmoins, aucun transfert de chiralité du liquide ionique vers le produit n’a été observé. Afin d’avoir de meilleures interactions entre le liquide ionique et le substrat, nous nous sommes orientés vers la synthèse de liquides ioniques fonctionnalisés. Dans cette optique, deux nouvelles familles de liquides ioniques ont été préparées. Les thiazolinium portant un hydrogène en position 2 catalysent efficacement la réaction d’aldolisation de Mukaiyama, mais ils ne permettent pas de transférer la chiralité. Ils peuvent aussi être utilisés pour générer des carbènes de type thiazolinylidènes. Bien que ces carbènes soient trop réactifs pour être isolés, les dimères correspondants ont été isolés et parfaitement caractérisés. Les liquides ioniques chiraux à tâche spécifique de type histidinium, dont l’originalité est de posséder une partie liquide ionique et une partie chirale fonctionnalisée ont été préparés simplement à partir de l’histidine. Ces composés présentent la même réactivité que les aminoacides simples et le couplage peptidique avec ces espèces originales s’est montré efficace
This work concerns the synthesis, the properties and the application of original chiral ionic liquids derived from chiral aminoacids. Starting from commercially available aminoalcohols, a novel family of chiral ionic liquids having a 2-substituted thiazolinium cation was prepared by a simple and straightforward procedure in good overall yield. The properties of the new salts can be fine tuned by careful selection of the anion and the cation. This new salts are thermally and chemically stable under acidic or basic conditions. Their potential in chiral recognition was highlighted by the formation of diastereomeric interactions between several thiazolinium salts and racemic Mosher’s acid salt. Their use as solvent in the Mukaiyama aldol reaction between benzaldehyde and Danishefsky’s diene allowed the reaction to proceed under mild conditions (rt) in the absence of a catalyst. However no chirality transfer was observed. In order to favour better interactions between the ionic liquid and the substrate, we considered the synthesis of functionalized ionic liquids. Accordingly, two new families of chiral ionic liquids were designed, 2H-thiazolinium salts and histidinium salts ; Thiazolinium salts, bearing an hydrogen in the 2-position, efficiently catalyse the Mukaiyama aldol reaction, but like their substituted analogues, no chirality transfer occurs. Nevetheless, these new thiazolinium salts can be used to produce rather unknow thiazolylidene carbene derivatives. If the free carbenes proved to be too reactives to be isolated, the corresponding dimers were isolated and fully characterized. Task-specific chiral ionic liquids, whose originality is to have an ionic liquid part and a functionalized chiral part, were easily prepared from histidine. These compounds showed similar reactivity than conventional amino acids and peptide coupling with these original species was as effective as with free amino-acids

Résumé : Grâce à des caractéristiques physiques particulières, le [indice supérieur 64]Cu (T[indice inférieur 1/2]= 12.7 h; β[indice supérieur+], 0.65 MeV [17.8 %]; β[indice supérieur −], 0.58 MeV [38.4 %]) est un candidat idéal pour l’imagerie TEP et la radiothérapie ciblée du cancer. Son utilisation est actuellement limitée par la disponibilité de chélateurs bi-fonctionnels (CBFs) offrant une résistance élevée aux réactions de transmétallation in vivo. Récemment nous avons développés deux nouveaux CBFs cycliques, DOTHA[indice inférieur 2] et NOTHA[indice inférieur 2], portant des ligands hydroxamates pour la complexation au [indice supérieur 64]Cu. Ces CBFs possèdent une cinétique de marquage rapide dans des conditions très douces, une stabilité élevée in vivo et un profil de biodistribution favorable avec une clairance rapide. Nous proposons maintenant d’étendre notre approche à la préparation de CBFs acycliques plus flexibles et compacts afin de moduler les propriétés biologiques et la pharmacocinétique des traceurs peptidiques. Le but de mon projet de maîtrise est de développer une série de chélateurs acycliques dérivés de l'histidine et de l'acide glutamique et fonctionalisés avec des groupements hydroxamates pour identifier un CBF offrant un complexe stable in vivo avec le [indice supérieur 64]Cu(II). Les CBFs ont été préparés en solution pour faciliter l’optimisation de chaque étape réactionnelle. Les groupements chélatants hydroxamates ont été sélectionnés pour leur habilité à former des complexes stables avec différents métaux et ils ont été liés en position N-terminale et sur la chaîne latérale des acides aminés grâce à des réactions de substitution nucléophile. Les groupements para-methoxy-benzyles ont été judicieusement sélectionnés pour la protection des groupements hydroxamates afin de faciliter, au besoin, une déprotection sélective sous des conditions très douces. L’optimisation du marquage a été effectuée avec l’isotope stable du cuivre et ensuite avec le [indice supérieur 64]Cu en faisant varier le contre ion métallique, le pH, la concentration, et la température. Le CBF offrant la plus grande stabilité, soit celui dérivé de l’histidine, a été conjugué à un peptide, le H[indice inférieur 2]N-PEG-[D-Tyr[indice supérieur 6],βAla[indice supérieur 11],Thi[indice supérieur 13],Nle[indice supérieur 14]]bombesin(6-14) (BBN), se liant fortement aux récepteurs de la relâche de la gastrine surexprimés dans les cancers du sein et de la prostate. La stabilité et l’activité spécifique du CBF-histidine et du radiotraceur marqués au [indice supérieur 64]Cu s’est avérée faible in vitro. Il est connu que l’activité antibactérienne de ligands hydroxamates est associée à leur capacité à complexer le fer. En perspective, comme nos chélateurs complexent très fortement le Fe(III), une alternative pour ces composés serait d’évaluer leur capacité à inhiber la croissance et la prolifération des bactéries. || Abstract : Thanks to its particular physical characteristics, [superscript 64]Cu (T[subscript ½= 12.7 h; β[superscript +], 0.65MeV [17.8 %]; β[superscript −], 0.58MeV [38.4 %]) is an ideal candidate for PET imaging and targeted cancer radiotherapy. Currently, its use is limited by the availability of bi-functional chelators (BFCs) which give high resistance to in vivo transmetallation reactions. Recently, we developed two new cyclic BFCs, DOTHA[subscript 2] and NOTHA[subscript 2], bearing hydroxamate pendant arms for the complexation with [superscript 64]Cu. Those BFCs have fast labeling kinetics under very mild conditions, a high in vivo stability and a biodistribution profile which is favorable with a fast clearance. Now, we propose to expand our approach to the preparation of acyclic BFCs, which are more flexible and compact, in order to better modulate biological properties and the pharmacokinetics of the peptidic tracers. The goal of my Master’s degree project is to develop a series of acyclic chelators derived from histidine and glutamic acid and functionalized with hydroxamate pendant arms to identify a BFC that shows highly stable in vivo complexes with [superscript 64]Cu(II). BFCs have been prepared in solution to facilitate the optimization of each reactive step. Hydroxamate chelating groups have been selected for their ability to form stable complexes with different metals and they have been conjugated in N-terminal position and on the lateral chain of amino acids via nucleophilic substitution reactions. Para-methoxy-benzyl groups have been judiciously selected for the protection of the hydroxamate groups to facilitate, if needed, a selective deprotection under mild conditions. The labeling optimization has been performed with a stable copper isotope, and then with [superscript 64]Cu varying the metallic counter-ion, pH, concentration and temperature. The BFC having the highest stability, the one derived from histidine, was conjugated to a peptide, H[subscript 2]N-PEG-[D-Tyr[superscript 6],βAla[superscript 11],Thi[superscript 13],Nle[superscript 14]]bombesin(6-14) (BBN), strongly bounding the gastrin releasing peptide receptor, which is overexpressed in breast and prostate cancers. Both the stability and specific activity of BFC-histidine of the radiotracer labeled with [superscript 64]Cu were low in vitro. It is known that the antibacterial activity of hydroxamate ligands is associated with their ability to complex iron. In perspective, because our hydroxamate ligands strongly complex Fe(III), an alternative for these compounds would be to assess their ability to inhibit the growth and proliferation of bacteria.

Orientadora: Profa Dra Fernanda Dias da Silva
Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2016.
A microplusina e um peptideo antimicrobiano (PAM), quelante de cobre e ferro, presente no carrapato bovino Riphicephalus microplus. Possui estrutura globular, arranjada em cinco ¿¿-helices e e rica em residuos de cisteina e histidina. As histidinas His-2 e His-74 estao entre os possiveis residuos de aminoacidos envolvidos na formacao do sitio de ligacao ao cobre, de acordo com dados de RMN. Seu amplo espectro de atividade contra bacterias Gram-positivas e fungos pode estar associado a sua capacidade de quelar cobre e estudos com Micrococcus luteus e Cryptococcus neoformans, mostraram que a adicao de cobre ao meio anula o seu efeito bacteriostatico e fungistatico, respectivamente. A microplusina ao ligar cobre, possivelmente, diminui a disponibilidade deste metal, o que pode afetar a atividade de enzimas dependentes de cobre. Culturas de M. luteus e C. neoformans tratadas com o peptideo apresentaram uma reducao no seu consumo de oxigenio e dados sugerem que a deplecao de cobre pode ter afetado a atividade de heme cobre oxidases pertencentes a uma das vias da cadeia transportadora de eletrons. A microplusina tambem interfere na sintese de melanina de C. neoformans, o que pode estar relacionado a uma diminuicao da atividade da lacase, uma enzima cobre dependente. Esses dados indicam o enorme potencial da microplusina como farmaco e estudos sobre quais aminoacidos de sua sequencia sao importantes para manutencao de sua estrutura e atividades tornam-se essenciais. Sendo assim, o presente projeto propos como objetivo principal avaliar o efeito da substituicao das histidinas amino-terminais His-1 e His-2 por residuos de alanina, utilizando-se para isso, a expressao recombinante de duas variantes da microplusina (MPmH1A e MPmH2A). Os resultados demonstraram o surgimento inesperado de mais de uma molecula com atividade antimicrobiana para a variante MPmH1A, indicando que mais estudos serao necessarios para avaliar a atividade destes peptideos. Por sua vez a variante MPmH2A teve sua atividade antimicrobiana mantida mesmo sem a presenca do residuo de histidina 2 (His- 2). Porem em ambas nao foi possivel detectar a atividade quelante de cobre, indicando assim que atividade antimicrobiana pode ter ocorrido por outras vias, bem como que o residuo de histidina 2 pode ser essencial para a atividade quelante de cobre.
Microplusin is an antimicrobial and chelating peptide (AMP) for both copper II and iron II, present in the cattle tick Riphicephalus microplus. Its structure is globular, with five alpha helices and rich in cysteine and histidine residues. The histidines His-2 and His-74 are the possible amino acids involved in the copper-binding site. Its broad activity spectrum against Gram-positive bacteria and fungi may be associated with its ability to chelate copper and studies with Micrococcus luteus and Cryptococcus meoformans have shown the copper addition in the media abrogate its bacteriostatic and fungistatic effect, respectively. The binding of microplusin to copper, possibly decreases the metal availability in the media, which may affect the activity of copper dependent enzymes. In fact, M. luteus and C. neoformans cultures treated with microplusin, shown a reduction in their oxygen consumption and data suggest the copper depletion may have affected the activity of heme copper oxidases, belonging to one of the transport chain of electrons.This peptide also affects the melanization of C. neoformans. The melanization reduction is related to a lacase activity decrease, a copper dependent enzyme. Microplusin activities indicate its huge potential as a promising therapeutic and studies about which amino acids in its sequence are important to its structure maintenance and activities become essential. Therefore, the main objective of this project was evaluate the effect of amino-terminal histidines His-1 e His-2 substitution for alanine residues, using, for that, a recombinant expression of two microplusin variants (MPmH1A e MPmH2A). The results showed the unexpected onset of more than one molecule with antimicrobial activity for MPmH1A variant, indicating that more studies are necessary to evaluate the activity of these peptides. In turn, the MPmH2A variant had its antimicrobial activity maintained even without the presence of histidine residue 2 (His-2). However in both has not been possible detect the chelating activity of copper, indicating that antimicrobial activity may have happened by other means well as the residue of histidine 2 can be essential for the chelating activity of copper.

Bien que présents dans l'organisme en quantités infinitésimales, les métaux de transition jouent un rôle physiologique essentiel. On les trouve généralement sous forme de chélates avec des coordinats biologiques de masse molaire élevée dont l'étude est particulièrement difficile. Il est cependant possible de modéliser les sites actifs de ces macromolécules à l'aide de petites molécules plus faciles à étudier. Nous avons synthétisé des pseudo-peptides à base d'histamine pour simuler les sites de coordination de protéines telles que la sérum albumine. Ces peptidoamines sont également intéressantes du fait de leur activité antioxydante. L'étude des propriétés complexantes de ces molécules vis-à-vis du Cu(II) et du Ni(II) a permis de mettre en évidence une variété de complexes parfois très différents par leur nature et leur stabilité. À partir de ces résultats, nous avons proposé la synthèse de nouveaux tensioactifs possédant des propriétés complexantes par greffage d'un chaîne hydrophobe sur des peptides contenant l'histidine. Les têtes polaires correspondent aux pseudo-peptides peptides ß-alanyl-histidine et glycyl-glycyl-histidine. Les concentrations micellaires critiques ont été déterminées par tensiométrie et fluorimétrie et les diagrammes de phase binaires ont été tracés. Les propriétés complexantes ont été démontrées et les diagrammes de répartition de espèces ont été déterminés à partir de molécules modèles à courte chaîne hydrophobe. Enfin des tensioactifs trimodulaires ont été préparés en greffant sur les molécules précédentes une partie de type polyoxyéthylène, pour augmenter l'hydrophilie.

Previous nutritional physiology research using L-histidine and zinc in American lobster intestine (Homarus americanus) has suggested that these solutes can be co-transported as complexes (Histidine-Zinc-Histidine) across the intestine using a peptide transporter. Furthermore, transport of L-leucine was shown to be inhibited by high calcium concentrations. Dipeptide and bis-complex transport and the role of calcium were investigated in the perfused intestines of lobster and Atlantic white shrimp (Litopenaeus setiferus). Following trans-intestinal transport, serosal medium was analyzed for amino acid composition by gas chromatography. In lobster, the transport of glycylsarcosine (Gly-Sar) from mucosa to serosa was stimulated two-fold with luminal pH 8.5, compared to the pH 5.5 control. Mucosa to serosa and serosa to mucosa fluxes of Gly-Sar were measured; the dipeptide was transported intact in both directions, but the net flux was from mucosa to serosa. The use of 0.5mM calcium chloride stimulated Gly-Sar transport two-fold, compared to 25 mM. In shrimp, the addition of 50 µM zinc chloride increased the rate of L-histidine transport, while Gly-Sar inhibited histidine transport in the presence of zinc. The rate of histidine transport was significantly higher with 1mM calcium chloride than with 25mM. These results suggest that shrimp transport bis-complexes in a manner similar to lobster. High calcium concentration had an inhibitory effect on both amino acid and dipeptide transport. Proposed mechanisms accounting for the effects of metals and calcium on trans-intestinal transports of both amino acids and dipeptides by lobster and shrimp digestive tracts are discussed.

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.

Antimicrobial (cationic) proteins play an important role in innate imunity. Such proteins can possess antibacterial, antiendotoxic or fungicidal abilities. The rising resistence of microbes to common antibiotics evokes acute need of studying more endogenous proteins to reveal new potential antibiotics. Ticks, the blood-feeding ectoparasites with effectual defense system, present an endless source of newly described and unknown antimicrobial peptides/proteins with significant theurapeutic potential. This study represents identification of histidine-rich proteins detected in Ixodes ricinus and Ixodes scapularis, that are related to recently described new family of proteins isolated from Rhipicephalus microplus (protein microplusin) and Amblyomma hebraeum (protein heraein). Analysis and characterization of newly identified histidine rich proteins, study of their antimicrobial and protease inhibitory effect are the main goals of this study.

Lysozyme is a natural antimicrobial agent that is effective against many food spoilage and pathogenic microorganisms by disintegrating their cell walls. Immobilization of lysozyme has attractive applications for use in the food industry: (1) The enzyme could be readily separated from treated foods and beverages and re-used while the foods could still be claimed additive-free; (2) It could impart stable antimicrobial capability to the surface of food packaging polymers. In this study, a novel method is described for the preparation of a highly active immobilized lysozyme system. The method addressed three key issues in the covalent attachment of a biological active protein to an insoluble support: 1.) The protein should be attached to the matrix by the fewest possible bonds to minimize conformational change; 2.) The binding site(s) on the enzyme to the supports should be located as far as practical from its active center and be nonessential for its tertiary structure; 3.) The binding method should minimize the steric interference between the support and the immobilized enzyme. Using polystyrene resin beads as support matrix, peptide spacers of various lengths composed of 6-aminocaproic acid were synthesized with the solid phase peptide synthesis method. Then the amino terminals of the spacers were derivatized with bromoacetyl bromide and coupled to the protein's only histidine residue (His-15) that is nonessential for its lytic activity. Immobilized lysozyme with a spacer composed of three 6-aminocaproic acid units displayed the best lytic result against lyophilized M. lysodeikticus cells: 2736 U/g resin with a protein load of 2.21 mg/ resin. Retained activity was 14.2% of that of the free enzyme. Preparations with longer spacers yielded higher protein load yet the retained activity remained at about 14% level. A control consisted of random coupling of lysozyme to polystyrene beads without spacer gave an activity of 158 U/G with a protein load of 1.24 mg/g resin and 1.4% of retained activity, Properties of the immobilized lysozyme system were studied, including stability, effect of pH, surface characteristics of the support. A kinetics study of the system using Eadie-Hofstee plot demonstrated strong external diffusion effects, which resulted in deviation from classic Michaelis-Menton kinetic behavior.
Graduation date: 2004

碩士
國立交通大學
應用化學系分子科學碩博士班
103
In recent years, protein microparticles have received much attention in the drug delivery systems due to the improved protein stability and prolonged release in vivo. However, most of current preparation processes involved harsh conditions, introducing chemical crosslinkers or high salt, which often led to protein inactivation and failed to apply in drug delivery systems. In this study, we employed the well-established hexahistidine (His)-tag recombinant protein technology as well as a metal-triggerable peptide to enhance the binding strength between protein and metal ion and to fine-tune the protein drug release. The His-tagged proteins could self-assemble to form microparticles (~ 2 μm) upon zinc chloride (1 mM) treatment and an eight-hour sustained protein drug release has been achieved in physiological saline. The experimental results also indicated that by adjusting the peptide concentration and the N- and C-terminal hexahistidine-tags, the protein release could be controlled. Moreover, no protein denaturation has been observed. We have developed a universal strategy to enable facile protein microparticles fabrication under mild conditions, and their potential in release-tunable protein drug delivery systems has been successfully demonstrated.

碩士
高雄醫學大學
醫藥暨應用化學研究所
97
There are many surface groups on the dendrimers surfaces. Histidine accesses the good binding affinity of metal ions, such as copper and nickel ions. In this thesis, we modified the surface groups of PAMAM dendrimer with different number of histidine peptide. The designed peptides were prepared by solid phase peptide synthesis (SPPS). Meanwhile, we modified the PAMAM dendrimer with histidine residue successfully, and the ability to bind copper was confirmed through the electrochemistry. In the future, these compounds may be a good metal binder. Meanwhile, prostate cancer is one of the top ten cancer incidence. The most important way to improve the drugs solubility and diminish side effect is to modify the drugs with specific peptides. For the prostate cancer, we choose the Bombesin (BBN) as our target peptides, the sequences are QWAVGHLM. At first, we synthesized the final compounds on solid phase, but the drug was decomposed in TFA which is necessary to collect peptide from resin. Thereafter, we have to synthesize and purify the peptide first then to modify the drug with the peptide. With this, we compared the solubility before and after the peptide modification by UV detection.

Die Arbeit behandelt die Ergebnisse von Experimenten über die Wirkung des Dipeptides Carnosin (β Alanyl L Histidin) und der Aminosäuren L Histidin und β-Alanin auf Kulturen der humanen Zellreihen U87, T98G und LN405, welche von Zellen des malignen Hirntumors Glioblastoma multiforme abgeleitet sind. Die Vitalität der Zellen nach Inkubation mit Carnosin oder L Histidin wurde anhand der Adenosintriphosphatproduktion und der Dehydrohenaseaktivität für Inkubationszeiträume von 24, 48 und 72 Stunden bestimmt. Dabei zeigte sich eine signifikant niedrigere Vitalität der mit Carnosin oder L Histidin inkubierten Zellen gegenüber der unbehandelten Kontrolle. Dieser Effekt war bei L Histidin stärker ausgeprägt. Bei Messungen der Lakatdehydrogenaseaktivität im Medium der Zellen, welche als Indikator für Zellnekrosen diente, zeigten nur die mit L Histidin inkubierte Zellen Zeichen von Nekrose. Die gleichen Messungen wurden auch an humanen embryonalen Nierenzellen durchgeführt (HEK 293), wobei sich ein ähnliches Ergebnis feststellen ließ. In den drei Zellreihen wurde zudem mittels qRT-PCR die mRNA-Expression für die beiden Enzyme Carnosinase 1 und Carnosinase 2 bestimmt, welche L Histidin von Carnosin abspalten. Im Vergleich mit Proben aus normalem Hirngewebe war die Expression beider Enzyme in den Glioblastomzellen deutlich geringer, wenngleich nachweisbar. Nachdem vorhergehende Studien [8] einen Anstieg der Expression von mRNA der Pyruvatdehydrogenasekinase 4 (PDK4) in mit Carnosin inkubierten Glioblastomzellen gezeigt hatten, wurde dieser Effekt hier auch mittels qRT-PCR in mit L Histidin inkubierten Zellen nachgewiesen. Eine Wirkung von Carnosin oder L Histidin auf ein Reportergen des PDK4-Promoters wurde ebenfalls untersucht, wobei sich kein signifikanter Effekt nachweisen ließ.:Table of contents Bibliographische Beschreibung I List of Abbreviations II List of Figures IV List of Tables V 1 Introduction 1 1.1 Overview 1 1.2 Glioblastoma 1 1.3 Carnosine 4 1.4 Histidine 8 1.5 Human pyruvate dehydrogenase kinase 4 gene and enzyme 9 2 Objectives of the study 12 3 Materials and Methods 13 3.1 Materials 13 3.1.1 Cell lines 13 3.1.2 Primers 13 3.1.3 Plasmids 14 3.1.4 cDNA of normal brain tissue 14 3.1.5 Solutions and buffers 14 3.1.6 Enzymes and kits 15 3.1.7 Media 16 3.1.8 Ready-made chemicals 17 3.1.9 Instruments 18 3.1.10 Software 19 3.1.11 Consumables 20 3.2 General microbiological and cytological methods 21 3.2.1 Production of competent E. coli 21 3.2.2 Transformation of RbCl-competent E. coli 21 3.2.3 Preparation of plasmid DNA from cultures of transformed E. coli 22 3.2.4 Cell culture conditions for human cell lines 22 3.3 Assay methods and protocols 24 3.3.1 Transfections and reporter gene assays 24 3.3.2 mRNA-isolation and qRT-PCR 25 3.3.3 Cell viability assays 26 3.4 Construction of the reporter gene (-3968/+319)_PDK4_GauIII 27 3.5 Statistical analysis 28 4 Results 29 4.1 Cell viability of glioblastoma cells under the influence of carnosine, L histidine and β-alanine 29 4.1.1 ATP production under the influence of carnosine, L histidine and β alanine 29 4.1.2 Dehydrogenase activity under the influence of carnosine, L histidine and β alanine 34 4.1.3 Lactate dehydrogenase activity and necrotic cell death under the influence of carnosine, L histidine and β alanine 38 4.1.4 Concentration dependence of viability decrease under the influence of carnosine and L histidine 42 4.1.5 Effect of carnosine and β-alanine on viability of HEK 293 cells 44 4.2 Carnosinase mRNA expression 46 4.3 PDK4-mRNA expression under the influence of L-histidine 48 4.3.1 Enhancement of PDK4-mRNA-expression under the influence of L histidine 48 4.3.2 Development of L-histidine-mediated PDK4-mRNA increase over time 51 4.4 Reporter gene assays 54 5 Discussion 60 5.1 Conclusions 60 5.2 Outlook and suggestions for further research 63 6 Summary 65 7 Literature 68 8 Appendix - Optimization of transfection conditions for U87 cells 74

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Lenka Javorská Supervisor: RNDr. Lucie Škarydová, Ph.D Title of diploma thesis: Detection of His-tag and FLAG-tag recombinant proteins Recombinant proteins are proteins produced in genetically modified organism by recombinant DNA technology that enable insert DNA sequence of target protein in expression cell. This technology allows preparation of actually any protein in any quantity without difficulties associated with demanding purification of native protein from tissues. Recombinant proteins are produced often in form of fusion proteins with suitable peptide tag (e.g. His-tag, FLAG- tag) that can help with their detection and enable simple purification. The aim of this diploma thesis was to introduce and optimize universal method for the detection of recombinant proteins labelled with His-tag (6x histidine) and FLAG-tag. The model recombinant protein with His-tag was enzyme carbonylreductase 1 (CBR1) produced by expression system Escherichia coli. For FLAG-tag labelled protein were chosen enzymes DHRS12, HSD11β1, RODH4, DHRS3, DHRS7, DHRS8 produced by insect cells (strain Sf9). SDS polyacrylamide electrophoresis for protein separation and immunochemical detection after Western...

碩士
國立清華大學
化學系
101
Collagen is a biodegradable and biocompatible material, and has been applied in medical uses for decades. However, animal-derived collagens have several drawbacks, such as low thermal stability, nonspecific cell adhesion, and antigenicity. To solve these problems, preparing collagen-related biomaterial from short mimetic collagen peptides has received many attentions and become an emerging research topic. Our previous studies have shown that His-metal coordination can induce unstable short mimetic collagen peptides to assemble into a higher order structure. In this work, we prepared three collagen related peptides (CRPs): HG(POG)9GH, HG(POG)4PHG(POG)4GH, and GG(POG)9GG, of which two peptides contain His residues, to study their assembled structures. The size and topology of results show that His-metal coordination can promote mimetic collagen peptides to form macro-scale structures, and the topologies depend on metals and the time of adding metal ions into peptide solutions. Circular dichroism spectroscopy was used to examine the structure and the thermal stability of collagen mimetic peptides. Dynamic light scattering (DLS), SEM, and TEM were used to assess the size and the topology of the assembled structures. The CRPs in this work can form microstructures without the assistance of metal ions. Thus, pH dependent assembly of these CRPs was also examined. Although we are not able to clarify the process of self-assembly at the present stage, we did find the impact of the rate of self-assembly, His-metal coordination, and the His-His interaction on the assembly of CRPs. Our results may be useful and helpful for the future development of collagen-related materials.

碩士
國立清華大學
化學系
99
Collagen, the most abundant protein in mammals, has been widely used in biomedical materials. Searching for an effective way to assemble short mimetic collagen peptides into a higher order structure has been an emerging topic for the preparation of collagen-related biomaterials. In this work, we have incorporated histidine residue into two mimetic collagen peptides to promote the self-assembly of short collagen triple helices into supermolecular structure via His-metal coordination. Our results indicate that His-metal coordination can serve as an effective force to assemble mimetic collagen peptides into large scale structures and their topology depends on metal ions and His-metal coordination sites. Furthermore, the process of self-assembly can be reversed upon adding the cation chelator, EDTA, in solution. In addition, we have introduced a cationic residue into the N-terminus and an aromatic residue into the C-terminus of a collagen-related peptide which can generate favorable cation-π interactions between the termini of collagen triple helices. The experimental results demonstrate that cation-π interactions can promote the self-assembly of collagen triple helices into higher-order fibril structures in a head-to-tail manner. The work shows that cation-π interactions can serve as an effective force in preparing collagen-related biomaterials.

碩士
國立清華大學
化學系所
105
Recently, a number of simple oligopeptides have been developed as artificial enzymes for mimicking the activity and selectivity of natural hydrolases. Although the arrangement of amino acid residues in natural enzymes has been reported and their composition of active sites could provide a strategy for designing artificial enzymes, creating catalysts with both efficient binding and catalytic activity is still challenging. In this study, we designed a series of peptides with polyproline scaffold as artificial enzymes and also utilized the concept of catalytic dyad or triad and the co-assembly strategy. Their secondary structures were characterized by CD spectroscopy and their catalytic activities on ester hydrolysis were measured by UV-Vis spectroscopy using a series of p-nitrophenyl ester assay. In the first part, the results indicate that a well formed polyproline II (PPII) structure could result in a much higher catalytic efficiency. In addition, deprotonation steps in artificial enzymes are an important factor for obtaining a higher catalytic efficiency. In the second part, the results show that the triple helical stability of collagen-mimetic peptides could dramatically affect their catalytic efficiency. Furthermore, introducing zinc ions into collagen-mimetic peptides could mimic the active site in metalloenzymes and result in a higher catalytic efficiency. This is the first report of a functional dyad or triad engineered into a polyproline helix framework. Despite the fact that our designs are not as efficient as natural enzymes, the activity of our designs is still greater than some reported nanostructures. Our investigation has also revealed the necessity of maintaining a stable three-dimensional structure and a well-organized catalytic site for effective biocatalysts.

Title: Raman optical activity and conformational flexibility of peptides in solution Author: Jana Hrudíková Department: Institute of Physics of Charles University Supervisor: Doc. RNDr. Vladimír Baumruk, DrSc. Supervisor's e-mail address: baumruk@karlov.mff.cuni.cz Abstract: Molecular flexibility can significantly modify Raman and ROA spectral intensities, band positions and the ROA signs. Taking into account dynamic aspects of behavior of studied molecules in solution via conformational averaging therefore seems to be crucial for spectral interpretation. The first of studied models, histidine, plays an important role in metallo-enzymatic reactions and peptide folding, due to its imidazole ring. ROA spectra of His at different pH, His complexed with Cu2+ and dipeptides His- Gly and Gly-His were recorded on the spectrometer built at the Institute of Physics of the Charles University as a first step of the subsequent study. The second studied system, a cyclic hexapeptide c-(Phe-D-Pro-Gly-Arg-Gly-Asp), serves as a convenient model for β- hairpin and anti-parallel β-sheet. It was previously studied by means of VCD and IR. From molecular dynamics simulations 10 peptide geometries were selected for spectral modeling. The Raman and ROA spectra were calculated ab initio. For a model fragment Phe-D-Pro, which...

碩士
國立交通大學
應用化學系所
94
On the basis of the high surface area to volume ratio and the superparamagnetic property, iron oxide magnetic nanoparticles have been widely used as the affinity probes for specific analytes. After trapping process, the affinity probes-target species conjugates can be readily isolated from the sample solution by employing an external magnetic field. The whole analysis time used in concentration and isolation is therefore dramatically reduced. In this dissertation, two types of metal ions immobilized iron oxide magnetic nanoparticles for enriching histidine (his)-tagged proteins and phosphorylated proteins and peptides were generated. Nitriacetic acid (NTA) was first immobilized on the surfaces of magnetic nanoparticles. NTA immobilized magnetic nanoparticles were capable of chelating metal ions onto their surfaces. Ni(II) and Zr(IV) ions were selected to be chelated by the NTA immobilized magnetic nanoparticles. Ni(II) immobilized magnetic nanoparticles were successfully employed to selectively enrich his-tagged proteins and peptides from complex samples such as cell lysates. Pipeting the sample solution in and out of a tip in a sample vial for only 30 sec could enrich sufficient target species for matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) analysis. The detection limit for a his-tagged peptide (6×his) is 10-9 M (50 μL), while the detection limit for a his-tagged protein (C192S) is 10-7 M (50 μL). Additionally, Zr(IV) ions immobilized magnetic nanoparticles have been demonstrated to have the capacity of enriching phosphoproteins and phosphopeptides such as α&β-caseins, milk samples, and their tryptic digestion products. The sample solution was enriched by pipeting the sample solution in and out of a tip in a sample vial for 30 sec. The results indicated that the detection limit for α&β-caseins is 10-9 M (50 μL).