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‣ Acanthoscurrin Fragment 101-132: Total Synthesis at 60 degrees C of a Novel Difficult Sequence

REMUZGO, Cesar; ANDRADE, Gustavo F. S.; TEMPERINI, Marcia L. A.; MIRANDA, M. Teresa M.
Fonte: JOHN WILEY & SONS INC Publicador: JOHN WILEY & SONS INC
Tipo: Artigo de Revista Científica
Português
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Glycine-rich proteins (GRP), serve a variety of biological functions. Acanthoscurrin is an antimicrobial GRP isolated front hemocytes-of the Brazilian spider Acanthoscurria gomesiana. Aiming to contribute to the knowledge of the secondary structure and stepwise solid-phase synthesis of GRPs` glycine-rich domains, we attempted to prepare G(101)GGLGGGRGGGYG(113) GGGGYGGGYG(123)GGy(126)GGGKYK(132)-NH(2), acanthoscurrin C-terminal amidated fragment. Although a theoretical prediction did not indicate high aggregation potential for this peptide, repetitive incomplete aminoacylations were observed after incorporating Tyr(126) to the growing peptide-MBHA resin (Boc chemistry) at 60 degrees C. The problem was not solved by varying the coupling reagents or solvents, adding chaotropic salts to the reaction media or changing the resin/chemistry (Rink amide resin/Fmoc chemistry). Some improvement was mode when CLEAR amide resin (Fmoc chemistry) was 32 used, as it allowed for obtaining fragment (G(113)-K(132) NIR-FT-Raman spectra collected for samples of the growing peptide-MBHA, -Rink amide resin and -CLEAR amide resin revealed the presence of beta-sheet structures. Only the combination of CLEAR-amide resin, 60 degrees C, Fmoc-(Fmoc-Hmb)Gly-OH and LiCl (the last two used alternately) was able to inhibit the phenomenon...

‣ Estudo da síntese convergente de peptídeos em fase sólida: abordagem clássica e uso de temperatura alta; Study of the convergent solid phase peptide synthesis: classical approach and use of high temperature

Ruiz, Cesar Manuel Remuzgo
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 12/12/2003 Português
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A síntese de peptídeos em fase sólida passo a passo (SPFS) tem sido aplicada com sucesso na preparação de peptídeos curtos, médios e de determinados sequências contendo mais de 30 resíduos. Entretanto, esta apresenta problemas e limitações que podem ser contornados pela síntese convergente de peptídeos em fase sólida (SCPFS) que se baseia na condensação entre fragmentos peptídicos Nα-acilados protegidos em suas cadeias laterais (doadores de acila) a fragmentos protegidos ligados a um suporte polimérico (receptores de acila). Além de desenvolver outros projetos enfocados na síntese de peptídeos ou no uso de sintéticos para o estudo de peptídeos biologicamente ativos ou proteinas, o nosso grupo de pesquisa tem se dedicado a estudar o emprego de temperaturas altas em SPFS. O objetivo final é propor protocolos ágeis alternativos aos empregados classicamente. Neste trabalho nos propusemos a investigar alguns aspectos da SCPFS e a explorar a possibilidade de agilizá-Ia a 60°C. Para tanto, empregamos como modelos a colecistocinina-33 humana (hCCK-33) não sulfatada e o análogo [Gln1]-gomesina. As seqüências destes peptídeos foram divididas em: doadores de acila (fragmentos central e N-terminal da hCCK-33 não sulfatada de 11 e de 5 resíduos...

‣ Novas observações e perspectivas do uso de lipases na síntese de peptídeos; New insights on the use of lipases as catalyst for peptide synthesis

Liria, Cleber Wanderlei
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 11/03/2004 Português
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Esta tese é composta de estudos realizados com a finalidade de encontrar condições experimentais e gerar informações confiáveis que possam ser utilizadas em síntese de peptídeos mediada por lipases. Inicialmente, foram caracterizadas por eletroforese do tipo PAGE-SDS e medida de atividade em óleo de oliva duas lipases purificadas de Candida cylindracea (CCL) e duas pancreáticas suínas, purificada (pPPL) e bruta (cPPL). As CCLs apresentaram atividades lipásicas e graus de purezas superiores às PPLs. Dentre os contaminantes das PPLs, foram identificadas a tripsina e a α-quimotripsina (α-QT). Em seguida, foi realizado um estudo sistemático da síntese do dipeptídeo modelo Ac-Tyr-Gly-NH2. A melhor condição experimental encontrada foi: mistura de n-hexano/tampão Tris-HCl, 0,5M, pH 8,0, (80/20,v/v), 50mg/mL de cPPL, 0,05M de Ac-Tyr-OEt, 0,5M de Gly¬NH2, 37°C e agitação de 300 rpm, que forneceu rendimento de ~90 % em 5 min de reação. Porém, nesta condição foi observada a hidrólise secundária do produto formado, a qual foi extinta pelo tratamento da cPPL com um inibidor específico de α-QT. A condição otimizada de síntese de Ac-Tyr-Gly-NH2 também se mostrou adequada à preparação de Z¬Asp-Gly-NH2 a partir de Z-Asp-OMe e de Gly-NH2. Quando se estudou a influência da percentagem de n-hexano na eficiência da formação de ligação peptídica...

‣ Solid-phase peptide synthesis in highly loaded conditions

Nakaie, Clovis R.; Oliveira, Eliandre; Vicente, Eduardo F.; Jubilut, Guita N.; Souza, Sinval E. G.; Marchetto, Reinaldo; Cilli, Eduardo Maffud
Fonte: Academic Press Inc. Elsevier B.V. Publicador: Academic Press Inc. Elsevier B.V.
Tipo: Artigo de Revista Científica Formato: 101-109
Português
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); The use of very highly substituted resins has been avoided for peptide synthesis due to the aggravation of chain-chain interactions within beads. To better evaluate this problem, a combined solvation-peptide synthesis approach was herein developed taking as models, several peptide-resins and with peptide contents values increasing up to near 85%. Influence of peptide sequence and loading to solvation characteristics of these compounds was observed. Moreover, chain-chain distance and chain concentration within the bead were also calculated in different loaded conditions. of note, a severe shrinking of beads occurred during the alpha-amine deprotonation step only when in heavily loaded resins, thus suggesting the need for the modification of the solvent system at this step. Finally, the yields of different syntheses in low and heavily loaded conditions were comparable, thus indicating the feasibility of applying this latter prohibitive chemical synthesis protocol. We thought these results might be basically credited to the possibility...

‣ Polystyrene-type resin used for peptide synthesis: application for anion-exchange and affinity chromatography

Carvalho, RSH; Ianzer, D. A.; Malavolta, L.; Rodrigues, M. M.; Cilli, Eduardo Maffud; Nakaie, C. R.
Fonte: Elsevier B.V. Publicador: Elsevier B.V.
Tipo: Artigo de Revista Científica Formato: 231-238
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This paper deals with an unusual application for a copolymer of styrene-1 % divinylbenzene bearing high amount of aminomethyl groups for anion-exchange and affinity chromatography. The so-called aminomethyl resin (AMR), to date only employed for peptide synthesis, swelled appreciably in water and was used successfully to purify negatively charged peptides. By correlating swelling degree of beads with pH of the media, it was possible to estimate that the AMR amino group pK(a) is approximately 5.5. In addition, the synthesized acetyl-(NANP)(3)-AMR succeeded in the affinity interaction with large antibody molecules related to malaria transmission and raised previously against this dodecapeptide sequence. (C) 2004 Elsevier B.V. All rights reserved.

‣ Comparative investigation of the cleavage step in the synthesis of model peptide resins: Implications for N-alpha-9-fluorenylmethyloxycarbonyl-solid phase peptide synthesis

Jubilut, Guita Nicolaewsky; Cilli, Eduardo Maffud; Crusca, Edson; Silva, Elias Horacio; Okada, Yoshio; Nakaie, Clovis Ryuichi
Fonte: Pharmaceutical Soc Japan Publicador: Pharmaceutical Soc Japan
Tipo: Artigo de Revista Científica Formato: 468-470
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Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the N-alpha-tert-butyloxycarbonyl (Boc)-peptide synthesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research but examines the N-alpha-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin II (All, DRVYIHPF) and its [Gly(8)]-All analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to establish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, depending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve complete removal of the peptide chains from the resin. Moreover...

‣ Use of commercial anion-exchange resins as solid support for peptide synthesis and affinity chromatography

Nakaie, C. R.; Lanzer, D. A.; Malavolta, L.; Cilli, Eduardo Maffud; Rodrigues, M. M.
Fonte: Elsevier B.V. Publicador: Elsevier B.V.
Tipo: Artigo de Revista Científica Formato: 39-46
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This report demonstrates that due to the presence of residual reactive sites in their matrices, classical diethylaminoethyl-attaching commercial anion-exchanger resins such as DEAE-MacroPrep and DEAE-Sephadex A50 supports can be used for peptide synthesis. Moreover, due to the high stability of the peptide-resin bond in the final cleavage treatments, desired peptidyl-resins free of side-chain protecting groups, which enables them to be further used as solid support for affinity chromatography, can be obtained. To demonstrate this potentiality, a fragment corresponding to the antigenic and immunodominant epitope of sporozoites of the Plasmodium falciparum malaria parasite was synthesized in these traditional resins and antibody molecules generated against the peptide sequence were successfully retained in these peptidyl supports. Due to the maintenance of their original anion-exchange capacities, the present findings open the unique possibility of applying, simultaneously, dual anion-exchange and affinity procedures for purification of a variety of macromolecules. (C) 2003 Elsevier B.V. (USA). All rights reserved.

‣ EPR investigation of the influence of side chain protecting groups on peptide-resin solvation of the Asx and Glx model containing peptides

Cilli, Eduardo Maffud; Vicente, Eduardo F.; Crusca, Edson; Nakaie, Clovis R.
Fonte: Elsevier B.V. Publicador: Elsevier B.V.
Tipo: Artigo de Revista Científica Formato: 5521-5524
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In spite of all progressive efforts aiming to optimize SPPS, serious problems mainly affecting the assembly of aggregating sequences have persisted. Following the study intended to unravel the complex solvation phenomenon of peptide-resin beads, the XING and XAAAA model aggregating segments were labeled with a paramagnetic probe and studied via EPR spectroscopy. Low and high substituted resins were also comparatively used, with the X residue being Asx or Glx containing the main protecting groups used in the SPPS. Notably, the cyclo-hexyl group used for Asp and Glu residues in Boc-chemistry induced greater chain immobilization than its tert-butyl partner-protecting group of the Fmoc strategy. Otherwise, the most impressive peptide chain immobilization occurred when the large trytil group was used for Asn and Gln protection in Fmoc-chemistry. These surprising results thus seem to stress the possibility of the relevant influence of the amino-acid side chain protecting groups in the overall peptide synthesis yield. (C) 2007 Elsevier Ltd. All rights reserved.

‣ Importance of the solvation degree of peptide-resin beads for amine groups determination by the picric acid method

Cilli,Eduardo M.; Jubilut,Guita N.; Ribeiro,Suely C. F.; Oliveira,Eliandre; Nakaie,Clovis R.
Fonte: Sociedade Brasileira de Química Publicador: Sociedade Brasileira de Química
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/10/2000 Português
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The classic and important picric acid method used in polymers biochemical and chemical fields of polymers for amine group quantification was chosen in this work as a model for evaluating the influence of the resin bead solvation during an analytical procedure. It was observed that this method, proposed almost three decades ago, failed to quantify amine groups of peptidyl-resin containing aggregating and polar sequence. This was due to inefficient solvation of resin beads when only CH2Cl2 was used for picrate anion binding and subsequent washing steps. It was demonstrated that the use of CH2Cl2/DMF (dimethylformamide) and CH2Cl2/EtOH solutions during these steps allows correct determination of peptidyl-resin amine groups. Besides the importance for the solid phase peptide synthesis methodology itself, these findings also represent the first quantitative demonstration of the relationship between solvation degree and the efficiency of a polymer-supported analytical method.

‣ Use of the same polymer for synthesis and purification of peptides

Silva,Elias H.; Etchegaray,Augusto; Carvalho,Regina S. H.; Jubilut,Guita N.; Miranda,Antonio; Nakaie,Clovis R.
Fonte: Sociedade Brasileira de Química Publicador: Sociedade Brasileira de Química
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/04/2005 Português
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This work reveals an uncommon but valuable biotechnological approach regarding the use of a same polymer (benzhydrylamine-resin, BHAR) for synthesis and anion exchange purification of peptides. Initially, the octapeptide DRVYIHPF-NH2 was synthesized in 1% and 3% cross-linked BHAR, attaching 2.5 mmol g-1 ammonium groups. Due certainly to its less rigid polymeric backbone, higher synthesis yield (about 80%) was achieved with the former resin. Next, the negatively charged peptides DEVYEHPF-NH2 and DEVYEDPF-NH2 (-1 and -3 in neutral pH, respectively), both synthesized in 1% BHAR were submitted to chromatographic separation test in this same type of resin (1% and 3%). Following comparative results of peptide synthesis and swelling data of resin beads obtained by microscopy, an improved separation of both peptides occurred with 1% BHAR batch. These findings demonstrated that BHAR applied so far for peptide synthesis, when containing high amount of positively charged ammonium groups, can be also used alternatively as a solid support for chromatographic purification of this type of biological molecule.

‣ P134-M Solid Phase Synthesis of C-Terminus Fluorescent Peptide: Comparison of Fmoc-Lys(5-FAM)-Resin and Fmoc-Lys[5-FAM(Trt)]-Resin

Tong, X.; Fu, W.; Sheng, L.
Fonte: The Association of Biomolecular Resource Facilities Publicador: The Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /02/2007 Português
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Many FRET (Fluorescent Resonance Energy Transfer) peptides require a C-terminus fluorescent label. In order to facilitate the synthesis of C-terminus fluorescent peptides, we prepared and compared two kinds of resins, Fmoc-Lys(5-FAM)-resin (I) and Fmoc-Lys[5-FAM(trt)]-resin (II).1 Resin (II) has a phenolic hydroxyl group protected with trityl group. The reason for introducing the protecting group was supposedly to prevent the phenolic hydroxyl groups from reacting with the activated amino acids during peptide synthesis.

‣ Cytophobic surface modification of microfluidic arrays for in situ parallel peptide synthesis and cell adhesion assays

Mandal, Suparna; Rouillard, Jean Marie; Srivannavit, Onnop; Gulari, Erdogan
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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A combination of PEG-based surface passivation techniques and spatially addressable SPPS (solid phase peptide synthesis) was used to demonstrate a highly specific cell-peptide adhesion assay on a microfluidic platform. The surface of a silicon-glass microchip was modified to form a mixed self-assembled monolayer that presented PEG moieties interspersed with reactive amino terminals. The PEG provided biomolecular inertness and the reactive amino groups were used for consequent peptide synthesis. The cytophobicity of the surface was characterized by on-chip fluorescent binding assays and was found to be resistant to non-specific attachment of cells and proteins. An integrated system for parallel peptide synthesis on this reactive amino surface was developed, using photogenerated acid chemistry and digital microlithography. A constant synthesis efficiency of >98% was observed for up to 7mer peptides. To demonstrate specific cell adhesion on these synthetic peptide arrays, variations of a 7mer cell binding peptide that binds to murine B lymphoma cells were synthesized. Sequence specific binding was observed on incubation with fluorescently labeled, intact murine B lymphoma cells and key residues for binding were identified by deletional analysis.

‣ Label-Free Optical Detection of Peptide Synthesis on a Porous Silicon Scaffold/Sensor

Furbert, Patrick; Lu, Caiyan; Winograd, Nicholas; DeLouise, Lisa
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Mesoporous porous silicon (PSi) microcavity sensors are used to conduct conventional solid-phase peptide synthesis. The sensor optical response provides a convenient means to monitor the synthesis reaction in a nondestructive manner. Measurements indicate that peptide synthesis occurs only when the PSi sensor/scaffold is amine-terminated using, for example, the amino silane or deprotected acid-labile Rink linker. Equivalent coupling efficiencies of the first amino acid to both amine terminations are observed. Kinetic studies indicate that coupling reactions are 90% complete in 1 h. Quantitative analysis of the optical response following the synthesis of homo-oligopeptides (4-mers) suggests that coupling efficiencies and/or optical thickness changes depend on the peptide length. The synthesis of the cell adhesive oligopeptide (RGD) was monitored by the optical sensor response and validated by the cell culture of primary dermal fibroblasts. Secondary ion mass spectrometry (SIMS) analysis successfully detected peptide on the silicon wafer adjacent to the PSi. Our findings suggest the potential to exploit the high surface area, efficient coupling, and intrinsic optical detection properties of PSi for label-free high-throughput screening.

‣ Building Blocks for the Construction of Bioorthogonally Reactive Peptides via Solid-Phase Peptide Synthesis

Zeglis, Brian M; Emmetiere, Fabien; Pillarsetty, Nagavarakishore; Weissleder, Ralph; Lewis, Jason S; Reiner, Thomas
Fonte: Wiley-VCH Verlag GmbH & Co Publicador: Wiley-VCH Verlag GmbH & Co
Tipo: Artigo de Revista Científica
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The need for post-synthetic modifications and reactive prosthetic groups has long been a limiting factor in the synthesis and study of peptidic and peptidomimetic imaging agents. In this regard, the application of biologically and chemically orthogonal reactions to the design and development of novel radiotracers has the potential to have far-reaching implications in both the laboratory and the clinic. Herein, we report the synthesis and development of a series of modular and versatile building blocks for inverse electron-demand Diels–Alder copper-free click chemistry: tetrazine-functionalized artificial amino acids. Following the development of a novel peptide coupling protocol for peptide synthesis in the presence of tetrazines, we successfully demonstrated its effectiveness and applicability. This versatile methodology has the potential to have a transformational impact, opening the door for the rapid, facile, and modular synthesis of bioorthogonally reactive peptide probes.

‣ Entwicklung neuer hochbeladener Trägermaterialien für die organische Festphasensynthese auf der Basis von vernetztem Polyethylenimin (Ultraharze); Development of new high loading supports for Solid-Phase Organic Synthesis on the base of cross-linked polyethylene imine (Ultraresins)

Barth, Michael
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
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Die vorliegende Dissertation befasst sich mit der Synthese, Charakterisierung, Evaluierung und Anwendungskonzepten von neuen hochbeladenen Trägermaterialien auf der Basis von vernetztem Polyethylenimin (Ultraharze). Im ersten Teil geht es um die Erarbeitung eines robusten Syntheseprotokolls für die Herstellung der Ultraharze. Durch Vernetzung von niedermolekularem Polyethylenimin (PEI) mit Terephthalaldehyd und anschließender Reduktion wurde ein hoch quellfähiges Trägermaterial erhalten, was eine Beladung von 15 mmol/g an Aminen aufweist. Durch verschiedene Derivatisierungsreaktionen konnte die ausgezeichnete Zugänglichkeit zu den Aminen des Polymers demonstriert werden. Es wurden Beladungen von bis zu 7.6 mmol/g erreicht, was einem Vielfachen der Kapazität herkömmlicher Trägermaterialien entspricht. Der Einsatz einer Reihe verschieden funktionalisierter Harze in der Festphasenpeptidsynthese zeigte das Potential dieser hochbeladenen Ultraharze. Der zweite Teil befasst sich mit der Evaluierung von Ultraharzen. Basierend auf einer genauen Charakterisierung der verwendeten PEIs, wurden verschiedene Ultraharze hergestellt, mit dem Ziel, ein optimiertes Harz zu finden. Sechs Ultraharze mit unterschiedlichen Crosslinkeranteilen und zwei verschiedenen PEI-Ausgangspolymeren wurden hergestellt...

‣ Total enzymatic synthesis of the cholecystokinin octapeptide (CCK 26-33); Vollenzymatische Synthese des Cholecystokininoctapeptids (CCK 26-33)

Meng, Li-Ping
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
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Enzymes can be used often favorably in organic syntheses, because they can be applied at room or slightly elevated temperature and in aqueous phase. Therefore these enzymatically catalyzed reactions are economically and environmentally superior to classical organic reactions. The objective of this thesis is to develop a synthetic path to the cholecystokinin octapeptide CCK-8 using exclusively enzymatic methods. The fragment CCK-8 has nearly the full biological activity of cholecystokinin and its therapeutic potential against type 2 diabetes, obesity and epilepsy is studied intensively. During the coupling process, many side reactions observed in the chemical peptide synthesis can be avoided in the enzymatic peptide synthesis. However, each coupling reaction has to be optimized. That means the optimal strategy, substrate structure and concentration, reaction media and temperature, type of the protease and its support for enzyme deposition. In the synthesis of the N-terminal tripeptide fragment Phac-Asp(OMe)-Tyr-Met-OAl, two strategies (Scheme 3.5, Scheme 3.6) with stepwise peptide chain elongation from the N-terminus to the C-terminus were investigated. Because of less reaction steps, higher overall yield and more economic starting materials...

‣ Application of Proteases for the Total Enzymatic Synthesis of the Cholecystokinin Octapeptide (CCK-8) using Benzoyl-Arginine as an Enzymatically Cleavable N-Terminal Protecting Group; Anwendung von Proteinen für die vollenzymatische Synthese des Cholecystokininoctapeptids (CCK-8) unter Verwendung von Benzoylarginin als enzymatisch spaltbare N-terminale Schutzgruppe

Joshi, Rajendra
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
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Enzymes can be used often favorably in organic syntheses, because they can be applied at room or slightly elevated temperature and in aqueous phase. Therefore, enzymatic reactions are economically and environmentally superior to classical organic reactions. Moreover, many side reactions, especially racemization occurring in the chemical peptide synthesis can be avoided and it is not necessary to protect all the side chains of amino acids involved in the coupling. However, there is no general protocol available for the enzymatic reactions therefore each step has to be optimized taking into account substrate structure, concentration, reaction media, temperature and the type of protease. The target octapeptide (CCK-8) is the minimum active sequence with the same biological activity as naturally occurring cholecystokinin and is a potential therapeutic agent in the control of gastrointestinal functions. The objective of this thesis is to synthesize N-terminal protected and deprotected CCK-8. In former investigations the enzymatically cleavable Phac group was used. But in our group it was found that the cleavability depends on the peptide sequence and sometimes can not be cleaved at all. As an alternative Bz-Arg was used as the N-terminal protecting group. This is possible because of the high specificity of trypsin towards basic amino acids in the P1 position and because there is no basic amino acid in the target peptide sequence. This protecting group could be introduced and removed easily with trypsin in test peptides and also in the octapeptide. The enzymatic removal of Bz-Arg from protected CCK 8 is a significant improvement because in the literature of CCK-8 syntheses...

‣ Solid-phase peptide synthesis and solid-state NMR spectroscopy of [Ala3-15N][Val1]gramicidin A.

Fields, G B; Fields, C G; Petefish, J; Van Wart, H E; Cross, T A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1988 Português
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[Ala3-15N][Val1]Gramicidin A has been prepared by solid-phase peptide synthesis and studied by solid-state 15N nuclear magnetic resonance spectroscopy. The synthesis of desformyl[Ala3-15N][Val1]gramicidin A employed N-hydroxysuccinimide esters of 9-fluorenylmethoxycarbonyl-N alpha-amino acids and completely avoided the use of acid. Since deblocking was done with piperidine and the peptide was removed from the resin by treatment with ethanolamine, this synthetic protocol prevented oxidation of the indole rings of this tryptophan-rich peptide and reduced truncations produced by acid hydrolysis. After formylation and purification by anion-exchange and high-pressure liquid chromatography, the peptide was obtained in an overall yield of 30%. Solid-state 15N nuclear magnetic resonance spectra of this peptide and uniformly labeled [15N]gramicidin A' oriented in hydrated lipid bilayers have been obtained, allowing unambiguous assignment of the [15N]Ala3 resonance in the latter. The solid-state 15N nuclear magnetic resonance experiments provide evidence that [Val1]gramicidin A is rotating about an axis that is perpendicular to the plane of the lipid bilayer and that the N--H axis is nearly parallel with the rotational axis. This study demonstrates that site-specifically labeled [15N]gramicidin A analogs prepared by solid-phase peptide synthesis are valuable tools in the study of the solid-state nuclear magnetic resonance spectra of samples in oriented lipid bilayers.

‣ Comparative behaviour of proteinases from the latex of Carica papaya and Funastrum clausum as catalysts for the synthesis of Z-Ala-Phe-OMe

Morcelle, Susana R.; Barberis, Sonia; Priolo, Nora; Caffini, Néstor O.; Clapés Saborit, Pere
Fonte: Elsevier Publicador: Elsevier
Tipo: Artículo Formato: 22195 bytes; application/pdf
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8 pages, 4 figures.-- Printed version published Aug 4, 2006.; The proteolytic extract obtained from the latex of Funastrum clausum (Jacq.) Schlechter (Asclepiadaceae), a South American climbing plant, was assayed as a novel catalyst for peptide synthesis and compared with commercial papain under the same conditions. After immobilization on polyamide, the synthesis of the bitter peptide precursor Z-Ala-Phe-OMe was performed and different conditions were tried. Acetonitrile and ethyl acetate with low water content were tested as organic solvents. Equilibrium- and kinetically-controlled synthesis were tried by using either Z-Ala-OH or Z-Ala-OMe as acyl donors, respectively. The best conditions for the synthesis of the desired product varied according to the catalyst used. For papain, thermodynamic control in acetonitrile (aw congruent with 0.12) in the presence of triethylamine (TEA) or boric acid–borate buffer (40 mM), and equilibrium- and kinetic-controlled synthesis in ethyl acetate (aw congruent with 0.75) proved to be the best conditions. The thermodynamic control in either acetonitrile with aw congruent with 0.12 (40 mM TEA or Na2CO3) or ethyl acetate (aw congruent with 0.75) were the best conditions found for funastrain. In all cases...

‣ Efficient Synthesis of Peptides with 4-Methylpiperidine as Fmoc Removal Reagent by Solid Phase Synthesis

Vergel Galeano,Cristian Francisco; Rivera Monroy,Zuly Jenny; Rosas Pérez,Jaiver Eduardo; García Castañeda,Javier Eduardo
Fonte: Sociedad Química de México A.C. Publicador: Sociedad Química de México A.C.
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2014 Português
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Solid phase peptide synthesis using the Fmoc/t-Bu strategy (SPPS-Fmoc/tBu) is the most widely used methodology for obtaining synthetic peptides. In this paper, we evaluate the viability of using 4-methylpiperidine as a reagent for deprotection of the amino acid alpha amino group in SPPS-Fmoc/tBu. For this purpose, the peptide (RRWQWRMKKLG) was simultaneously synthesized using 4-methylpiperidine or piperidine for Fmoc removal reagent. The obtained products had similar purities and yields. Finally, 21 peptides were synthesized using 4-methylpiperidine. Our results suggest that is possible to obtain synthetic peptides efficiently by the strategy SPPS-Fmoc/tBu when 4-methylpiperidine was used as reagent to remove Fmoc groups N-alpha protected amino acids.