Name: synthesis and characterization of 1,2-dithiolane modified self-assembling peptides
Uploaded: 2018-08-20T13:00:00.000+00:00
Duration: 9 min 54 s
Description: Watch this Scientific Journal Video about Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides at JoVE.com

Os autores gostaria de agradecer o Dr. B. Ellen Scanley para sua formação técnica e ajudar usando a temperatura no centro da Universidade (CSCU) e faculdades do estado de Connecticut para nanotecnologia e Dr. Ishita Mukerji na Universidade wesleyana para acesso ao seu CD Espectrofotómetro. O trabalho relatado foi apoiado em parte pelo Instituto de Ciências da Universidade de Fairfield, a NASA Connecticut Space Grant Consortium e pela National Science Foundation sob Grant número-1624774.

1. síntese e purificação de 1,2-Ditiolano modificado do Peptide
<ol><li>Síntese do precursor de Ditiolano, 3-(acetylthio) -2-(acetylthiomethyl) ácido propanoico19.
	<ol>
<li>Adicionar 1 g de 3-bromo - 2-(bromomethyl) ácido propiônico (1 equiv) dissolvido em uma quantidade mínima de 1 M NaOH (aproximadamente 4 mL) para um balão de fundo redondo de 25 mL reação agitando a 55 ° C. Selar o balão de reação com um septos e coloque sob atmosfera de nitrogênio.</li>
		<li>Preparar uma solução contendo 1,49 g de tioacetato de potássio (3.2 equiv) em 4 mL de água desionizada e 3 mL de ácido sulfúrico de 2 M (H2SO4) para criar tioacético ácido em situ.</li>
		<li>Puxe a solução ácida tioacético em uma seringa de plástico descartável 10 mL e coloque uma agulha na seringa. Adicione a mistura de gota a gota no balão de reação por perfuração através de septos com a agulha. Continuar a reação durante a noite a 55 ° C.</li>
		<li>Monitore a reação por cromatografia em camada fina (TLC) em placas de sílica gel 60 F254 usando uma mistura de metanol e diclorometano (1:9). Visualize o progresso de reação pela mancha verde de bromocresol. O produto tem um Rf = 0,57.</li>
		<li>Depois que a reação é completa e refrigeração à temperatura ambiente, acidificar a mistura de pH de 1 com 2 M de H2então4. Um óleo amarelo separa fora da solução.</li>
		<li>Extrair o produto com clorofórmio frio (40 mL x 3). Combine as camadas orgânicas e secar sobre sulfato de magnésio. Remova o clorofórmio sob pressão reduzida.</li>
		<li>Confirme a identidade do produto isolado, 1, por espectroscopia de ressonância magnética nuclear (NMR), como mostrado nas Figuras 1B e C. Esperar os seguintes resultados: 1H NMR, CDCl3, 300 MHz: d = 10.1 (b, 1 H) 3.2 (m, 4h), 2.9 (m, 1 H), 2.4 (s, H 6); 13 C NMR, CDCl3, 75 MHz: d = 195.1 (CH3COS-), 177.6 (-COOH), 45,1 (-CH2S), 30.5 (C. H), 29,2 (-SCOCH3). Nota: O produto é um óleo amarelo e tem um rendimento total de 83%. Utilize o produto sem mais purificação.</li>
	</ol></li>
	<li>SPPS e acoplamento na resina de Ditiolano Precursor Nota: A síntese do peptide de fase sólida descrita abaixo foi realizada em um sintetizador de peptídeo automatizado, seguindo o protocolo recomendado do fabricante. Configurações e reagentes podem ser adaptados para outros instrumentos comerciais ou ao usar aminoácidos especializados.<ol>
<li>Pesar 0,156 g de resina de 4-methylbenzhydrylamine (MBHA) de amida de Rink (0,1 mmol) e coloque em um recipiente de reação. Inche a resina em dimetilformamida (DMF) durante pelo menos 15 minutos antes do início da síntese.</li>
		<li>Pesagem para fora 4 equivalentes de cada fluorenylmethyloxycarbonyl (Fmoc) protegido aminoácido (0,4 mmol) na sequência e 0,152 g de N, N, N', N'- tetrametil-O-(1H- benotriazol-1-yl) Hexafluorofosfato de urânio (0,4 mmol, HBTU) para cada aminoácido na sequência. Cada cartucho contém o aminoácido Fmoc-protegido e HBTU.</li>
		<li>Depois de executar todos os pre-síntese verifica para o sintetizador (reencher os reagentes, liberadas linhas de reagente e pressurizado todos os frascos de reagente), coloque os cartuchos de aminoácido no carrossel no C' n ' direção do terminal. Coloque um cartucho vazio após a posição final de aminoácidos para a etapa final de desproteção de Fmoc N-terminal.</li>
		<li>Sintetiza o peptídeo usando as configurações padrão e recomendadas.<ol>
<li>DePROTECT o grupo de Fmoc da resina com 5 mL de piperidina de 20% em DMF (5 min x 2).</li>
			<li>Lave a resina com DMF (6 x 5 mL) antes da etapa de acoplamento.</li>
			<li>Para um passo único acoplamento, adicione 4 mL de 0,4 M N-methylmorpholine em DMF para o aminoácido Fmoc-protegido e HBTU. Ativar a solução de aminoácido Fmoc-protegido por 30 s antes de transferir a solução para o recipiente de reação. Nota: O sintetizador de peptídeo automatizado mistura da resina e a solução pela subida N2 gás cada 30 s por 20 min enquanto a reação de acoplamento leva a lugares. Para a síntese do peptide manual, coloque o recipiente de reação em um agitador orbital a baixa velocidade para a duração da etapa de acoplamento.</li>
			<li>Escorra a solução e lavar a resina com DMF (3 x 5 mL).</li>
			<li>Repita as etapas 1.2.4.1 através de 1.2.4.4 para cada aminoácido Fmoc-protegido no C' n '-direção de terminal para sintetizar o peptídeo de interesse.</li>
		</ol>
</li>
		<li>Após a etapa final de desproteção N-terminal, transferi a resina uma seringa descartável fritada. Lave a resina com DMF (3 x 5 mL) e diclorometano (DCM, 3 x 5 mL). Nota: A resina pode ser armazenada após DCM lavagem num exsicador de vácuo. Se a resina foi armazenada antes do engate, certifique-se para inchar a resina em DMF antes da reação de acoplamento.</li>
		<li>Alguns o precursor de Ditiolano (1) para N-terminal do peptide na resina, adicionando 4 equivalentes de 1, 5ml de DMF, 4 equivalentes de HBTU e 10 equivalentes de N, N-diisopropylethylamine (DIPEA). Pre-ative a mistura de acoplamento por 10 min antes de adicionar à resina contendo seringa fritada.</li>
		<li>Agite a reação de acoplamento para 2 h. Após 2 h, lave a resina com DMF (3 x 5 mL) e repetir a reação de acoplamento com agitação durante a noite.</li>
		<li>Após o segundo acoplamento, lave a resina com DMF (3 x 5 mL) e DCM (3 x 5 mL). Nota: A resina pode ser armazenada neste momento sob vácuo, até o decote.</li>
	</ol></li>
	<li>Tioacetato desproteção e clivagem de peptídeo de resina
	<ol>
<li>Para deprotect o grupo de tioacetato do precursor do Ditiolano N-terminal, transferir a resina seca para um tubo de reação de microondas de 10 mL e adicione 2 mL de DMF. Permitir que a resina a inchar, adicionar uma barra de agitação magnética pequena para o navio e re-suspender com baixa velocidade de agitação magnética por 15 min.</li>
		<li>Adicionar 2 mL de hidróxido de amônio concentrado, a embarcação da reação com os septos do silicone do tampão e coloque o recipiente de reação em um reator de microondas usando as configurações de microondas de 75 ° C por 45 min com agitação.</li>
		<li>Após a reação de microondas completa, transferi a resina uma seringa limpa descartável fritada. Lave com DMF (2 x 5 mL) e metanol (MeOH, 2 x 5 mL).</li>
		<li>Adicione uma solução de hidróxido de amônio concentrado em metanol (1:4), para um volume total de 5 mL. Deixe a tremer durante a noite para aumentar a oxidação intramolecular de ligação dissulfeto no anel Ditiolano.</li>
		<li>Lave a resina com MeOH (2 x 5 mL) e DCM (3 x 5 mL). Nota: A resina seca pode ser armazenada num exsicador de vácuo neste ponto.</li>
		<li>Adicione o cocktail de clivagem para a resina contendo seringa agitando suavemente para 1,5 h. O cocktail de clivagem usado é 95% o ácido trifluoroacético (TFA), 2,5% triisopropylsilane (dicas) e 2,5% de água em um volume total de 5 mL. Cuidado: Trabalho sob coifa química. TFA é volátil e corrosivo. Nota: Para a maioria de sequências peptídicas e aminoácido sidechain proteger grupos, a solução acima de cocktail clivagem é suficiente; no entanto, coquetéis de clivagem alternativos podem ser necessários para certa cadeia lateral de ácido aminado proteger grupos (em particulares peptídeos contendo Cys, Met, Trp e Arg) ou outra resina química20.</li>
		<li>Precipitar o peptídeo bruto em 25 mL de frio em um tubo cónico de 50 mL de éter dietílico por adição gota a gota da seringa fritada. O peptídeo precipita-se como um sólido branco. Pelota do peptide por centrifugação a 1300 x g durante 10 min. decantar cuidadosamente o éter etílico em um recipiente separado para recolha de resíduos.</li>
		<li>Adicionar mais 25 mL de éter dietílico para o tubo cônico e ressuspender o precipitado pela utilização do vortex. Repetir a centrifugação a 1300 x g por 10 min e decantar o éter dietílico novamente. Seque a pelota sob vácuo.</li>
	</ol></li>
	<li>Purificação de 1,2-Ditiolano modificado do Peptide Nota: Purifica o peptídeo bruto por HPLC-fase reversa. Coletar e combinar os picos do peptide e confirmar a massa por espectrometria de massa MALDI-TOF.<ol>
<li>Dissolva a pelota crua peptídeo em quantidade mínima de acetonitrilo com 0,1% TFA. Devido a hidrofobicidade do peptide e propensão de agregação, aquece lentamente a amostra a 40 ° C, para auxiliar na solubilidade. Nota: Evite maiores temperaturas e sonication do peptide para evitar potenciais bissulfeto troca reações21,22,23.</li>
		<li>Para preparar 1 mL de peptídeo bruto para a purificação de HPLC, adicione 400 μL de estoque concentrado peptídeo em acetonitrila para 600 μL de H2O com 0,1% TFA e filtro através de um filtro de seringa 22 μm dentro de um frasco de HPLC. Um isopropanol 5% adicionais pode ser adicionado para evitar a precipitação e a agregação de peptídeo.</li>
		<li>Purificar o peptídeo com C-18 semipreparativa coluna com uma vazão de 3 mL/min, ao longo de um gradiente linear de 15-55% acetonitrila em 20 min. conjunto dos detectores de UV para 222 nm (espinha dorsal Amida) e 330 nm (ligação dissulfeto). Coletar e combinar os picos de interesse (Figura 2A).</li>
		<li>Confirme a massa de produto de peptídeo pelo espectrômetro de massa MALDI-TOF no modo reflectron (Figura 2B). Para análise, misturar 0,5 μL do pico coletado na placa MALDI com 0,5 μL de matriz de ácido (DHB) 2,5-benzoico (10 mg/mL em 50% acetonitrila, 0,1% DHB TFA). Nota: Comum adutos em MALDI-TOF espectrometria de massa incluem o sódio e o sal de potássio aduto ([M + nd]+ e [M + K]+) picos. Dessalinização da amostra antes da análise recomenda-se a picos de sal aduto suprimem o sinal do principal pico [M + H]+ . Além disso, um pico de oxidado de [M + O]+ também é detectado no peptídeo de 1,2-Ditiolano modificado. Um relatório sobre laser induzida por oxidação da ionização de MALDI usando DHB matrix sugere que fatores incluindo a concentração da amostra, solvente e intensidade do laser podem ser modificados para limitar o MALDI induzida por oxidação artefato24.</li>
		<li>Após a confirmação da correta massa MALDI-TOF lyophilize o peptídeo após congelamento flash. Manter o pó liofilizado peptídeo sob vácuo até montagem.</li>
	</ol></li>
</ol>2. caracterização de Supramolecular auto-montagem estruturas
<ol><li>
Formação de fibras amiloides
	<ol>
<li>Para preparar a auto-montagem solução, pese 1 mg do pó do peptide usando uma balança analítica. Dissolver em uma mistura (pH 7,5) de 20% acetonitrila e 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) em um tubo de microcentrifugadora de 1,5 mL, para uma concentração final da mistura de montagem de peptídeo de 1 mg/mL. Vórtice a solução de montagem e deixar de reunir-se na temperatura ambiente.</li>
	</ol>
</li>
	<li>
Caracterização espectroscópica de fibras amiloides
	<ol>
<li>Siga o processo de montagem do peptídeo via transformada de Fourier espectroscopia de infravermelho (FTIR) todos os dias. Um pico amplo, centrado em torno de 1670 cm-1 é a assinatura de IR decorrentes de peptídeos desmontados em amostra17. As amostras de montagem de peptídeo costumam levam uma ou duas semanas para o amplo pico desmontado desaparecer e alcançar a maturação.<ol>
<li>Seco, uma alíquota de 8-10 μL da solução de montagem como um filme fino sobre o cristal de diamante ATR. Monitore o desaparecimento de um pico de água grande e amplo de 1640 a 1630 cm-1 , como as formas de película seca.</li>
			<li>Aquisição de espectros de IR de 1500-1800 cm-1 , com média de 50 digitalizações com resolução de 2 cm-1 . Adquirir e subtrair as varreduras em segundo plano antes de cada verificação de amostra. A assinatura de IR para a montagem de β-folha é um pico afiado entre 1625 e 1635 cm-1 (Figura 3A)25,26.</li>
		</ol>
</li>
		<li>Caracteriza o assembly de peptídeo em estruturas supramoleculares ricos de β-folha por Dicroísmo circular (CD). Grave os espectros usando spectropolarimeter um CD com um sistema de controle de temperatura Peltier.<ol>
<li>Adicionar 30 μL da solução de montagem em um microcuvette de comprimento de caminho de 0,1 mm. Nota: O titular de uma célula é necessária para fixar e posicionar a célula de comprimento de caminho mais curto no instrumento.</li>
			<li>Para cada espectro, definir o instrumento de CD para os seguintes parâmetros: digitalização de comprimentos de onda de 300 nm a 180 nm, taxa de exploração de 100/min, largura de banda de 1 nm, 25 ° C, média de três exames.</li>
			<li>Coletar um espectro do buffer (20% acetonitrila/10 mM HEPES, pH 7,5) e subtrair de cada amostra de verificação como um controle. A assinatura de CD para β-folhas é que uma mínimo de elipticidade centrado em torno de 220 nm (Figura 3B)27.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Microscopia de fibras amiloides
	<ol>
<li>Permitir que duas a três semanas para as amostras de peptídeo amadurecer-se em estruturas supramoleculares ricas de β-folha. Nota: Os módulos (assemblies) pode ser fotografada usando microscopia eletrônica de transmissão (TEM) em fases anteriores do processo de montagem também.<ol>
<li>Adicionar 10 μL da solução de montagem de peptídeo para a superfície da grade de carbono TEM. Nota: tenha cuidado para não tocar a ponta da pipeta para a superfície da grade. Alta precisão, de fecho automático pinças são usadas para segurar a grade TEM durante a preparação.</li>
			<li>Espere 1-2 min para permitir que os assemblies de adsorção na superfície da grade. Retire o excesso amostra, tocando o papel de filtro para a borda da grade.</li>
			<li>Prepare uma mancha de acetato de uranilo 2% adicionando 100 μL de água desionizada para solução de acetato de uranilo 4% comercialmente disponível. Adicionar 10 μL da mancha de acetato de uranilo de 2% para a superfície da grade e incubar durante 2-3 min. Após a incubação, remova mancha em excesso, tocando o papel de filtro para a borda da grade.</li>
			<li>Coloca as grades TEM num exsicador de vácuo durante a noite. Armazenar sob vácuo até imagens.</li>
			<li>Imagem as amostras preparadas com temperatura (Figura 3). Parâmetros típicos para microscopia são como segue: imagens em ampliações variando de 9.300 X 23, 000 X, filamento de tungstênio com uma tensão de aceleração de 120 kV. Nota: ImageJ pode ser usado para medir a largura média da fibra das estruturas supramoleculares de de imagens adquiridas TEM28. Atenção: Por favor consulte todas as folhas de dados relevantes de segurança (SDS) antes do uso. Vários produtos químicos usados na síntese, purificação, caracterização dos peptides auto-montagem descrita 1,2-Ditiolano modificado são corrosivos ou tóxicos e só deve ser usado sob uma coifa de química. Use sempre equipamento de protecção adequado (incluindo óculos de segurança, jaleco, calça comprida, sapatos fechados) quando se trabalha no laboratório.</li>
		</ol>
</li>
	</ol>
</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disulfide+exchange:+Exposing+supramolecular+reactivity+through+dynamic+covalent+chemistry.">Disulfide exchange: Exposing supramolecular reactivity through dynamic covalent chemistry.</a> Chemical Society Reviews. 43, 1861-1872 (2014).</li><li>Vendetti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dihydroasparagusic+acid:+Antioxidant+and+tyrosinase+inhibitory+activities+and+improved+synthesis.">Dihydroasparagusic acid: Antioxidant and tyrosinase inhibitory activities and improved synthesis.</a> Journal of Agricultural and Food Chemistry. 61, 6848-6855 (2013).</li><li>Stawikowski, M., Fields, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+peptide+synthesis.">Introduction to peptide synthesis.</a> Current Protocols in Protein Science. 26, (2002).</li><li>Canadell, J., Goossens, H., Klumperman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-healing+materials+based+on+disulfide+links.">Self-healing materials based on disulfide links.</a> Macromolecules. 44, 2536-2541 (2011).</li><li>Lafont, U., van Zeijl, H., van der Zwaag, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+cross-linkers+on+the+cohesive+and+adhesive+self-healing+ability+of+polydisulfide-based+thermosets.">Influence of cross-linkers on the cohesive and adhesive self-healing ability of polydisulfide-based thermosets.</a> ACS Applied Materials and Interfaces. 4, 6280-6288 (2012).</li><li>Komaromy, D., Stuart, M. C. A., Santiago, G. M., Tezcan, M., Krasnikov, V. V., Otto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+can+direct+dynamic+covalent+bond+formation+toward+diversity+or+specificity.">Self-assembly can direct dynamic covalent bond formation toward diversity or specificity.</a> Journal of the American Chemical Society. 139, 6234-6241 (2017).</li><li>McAvery, K. M., Guan, B., Fortier, C. A., Tarr, M. A., Cole, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+oxidation+of+cholesterol+observed+during+MALDI-TOF+mass+spectrometry.">Laser-induced oxidation of cholesterol observed during MALDI-TOF mass spectrometry.</a> Journal of the American Society for Mass Spectrometry. 22, 659-669 (2011).</li><li>Krimm, S., Bandekar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrational+spectroscopy+and+conformation+of+peptides,+polypeptides,+and+proteins.">Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins.</a> Advances in Protein Chemistry. 38, 181-364 (1986).</li><li>Halverson, K. J., Sucholeiki, I., Ashburn, T. T., Lansbury, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+of+&#946;-sheet-forming+sequences+in+amyloid+proteins+by+FTIR.">Location of &#946;-sheet-forming sequences in amyloid proteins by FTIR.</a> Journal of the American Chemical Society. 113, 6701-6703 (1991).</li><li>Greenfield, N., Fasman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computed+circular+dichroism+spectra+for+the+evaluation+of+protein+confirmation.">Computed circular dichroism spectra for the evaluation of protein confirmation.</a> Biochemistry. 8, 4108-4116 (1969).</li><li>. ImageJ Available from: <a target="_blank" href="https://imagej.nih.gov/ij">https://imagej.nih.gov/ij</a> (2016)</li><li>Roy, S., Shinde, S., Hamilton, G. A., Hartnett, H. E., Jones, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+[FeFe]-hydrogenase:+On+resin+modification+of+an+amino+acid+to+anchor+a+hexacarbonyldiiron+cluster+in+a+peptide+framework.">Artificial [FeFe]-hydrogenase: On resin modification of an amino acid to anchor a hexacarbonyldiiron cluster in a peptide framework.</a> European Journal of Inorganic Chemistry. 2011, 1050-1055 (2011).</li><li>Van Duinen, S. G., Castano, E. M., Prelli, F., Bots, G. T. A. B., Luyendijk, W., Frangione, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hereditary+cerebral+hemorrhage+with+amyloidosis+in+patients+of+Dutch+origin+is+related+to+Alzheimer+disease.">Hereditary cerebral hemorrhage with amyloidosis in patients of Dutch origin is related to Alzheimer disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 84, 5991-5994 (1987).</li><li>Barth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+infrared+absorption+of+amino+acid+sidechains.">The infrared absorption of amino acid sidechains.</a> Progress in Biophysics and Molecular Biology. 74, 141-173 (2000).</li><li>Jayaraman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+amyloid+nucleation+via+&#945;-helix-rich+oligomeric+intermediates+in+short+polyglutamine-containing+Huntingtin+fragments.">Slow amyloid nucleation via &#945;-helix-rich oligomeric intermediates in short polyglutamine-containing Huntingtin fragments.</a> Journal of Molecular Biology. 415, 881-899 (2012).</li></ol>

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Este artigo discute os detalhes de tanto a síntese e a purificação de um peptídeo de auto-montagem de 1,2-Ditiolano modificado N-terminal e a caracterização das estruturas supramoleculares resultantes. A síntese do peptide 1,2-Ditiolano relatado aqui tem as vantagens, incluindo a síntese de uma etapa para produzir o precursor de Ditiolano, 3-(acetylthio) -2-ácido (acetylthiomethyl) e a resina no microondas reação de desproteção do precursor tioacetato grupo para render o moiety oxidado 1,2-Ditiolano, utiliza...

Robusta peptídica formando a química envolvida na síntese do peptide de fase sólida e a capacidade de controlar a composição e o comprimento da sequência fazer os peptídeos que auto-montagem em estruturas supramoleculares um campo fortemente pesquisado. Os fatores que controlam e estabilizar a estrutura Self montada do peptide, incluindo cadeia lateral estérica e interações eletrostáticas, ligações de hidrogênio e efeitos hidrofóbicos1, servem como um conjunto de regras de design. Como a investigação sobre essas regras de design fundamental continua a progredir, o próximo passo lógico na peptídeo auto-montagem envolve expandir a diversidade de funções e estruturas do peptide-baseado. Ao montar auto peptídeos são um biomaterial versátil que foram usados para muitas aplicações biomédicas ajustando o peptídeo sequência ou conjunto condições2,3,4, o desenvolvimento de estratégias para modificações pós-montagem do peptide nanofibras5,6,7,8,9 continua a ser uma área relativamente inexplorada.
Bissulfeto dinâmico intercâmbio e tiol química na superfície de estruturas supramoleculares é uma área que tem potencial para produzir biomateriais novos e funcionais. A incorporação de partes de 1,2-Ditiolano (comumente um derivado do ácido lipoico (la) ou asparagúsico (aa)) têm sido relatados em lipossomas sistemas10,11,12,de copolímeros bloco13e como organizando as âncoras às superfícies14,15. Neste documento, nós relatamos a síntese e caracterização de um peptídeo de auto-montagem derivado do núcleo de nucleação do peptide Aβ associado com a doença de Alzheimer que é modificada no N-terminal com um grupo funcional de 1,2-Ditiolano16, 17. As fibras supramoleculares resultantes agora servem como uma plataforma experimental para estudar a reatividade de bissulfeto-troca e tiol na superfície supramolecular de fibras amiloides18.

<table><tbody><tr><td>Rink amide MBHA resin, high load</td><td>Gyros Protein Technologies</td><td>RAM-5-HL</td><td>Avoid contact with skin and eyes; do not inhale</td></tr><tr><td>N,N-Dimethylformamide</td><td>Fisher Scientific</td><td>D119-4</td><td>Flammable liquid and vapor; irritating to eyes and skin; Use personal protective equipment; keep away from open flame</td></tr><tr><td>Fmoc-L-Val-OH</td><td>Gyros Protein Technologies</td><td>FLA-25-V</td><td>Wear personal protective equipment; do not inhale</td></tr><tr><td>Fmoc-L-Leu-OH</td><td>Gyros Protein Technologies</td><td>FLA-25-L</td><td>Wear personal protective equipment; do not inhale</td></tr><tr><td>Fmoc-L-Lys(Boc)-OH</td><td>Gyros Protein Technologies</td><td>FLA-25-KBC</td><td>Wear personal protective equipment; do not inhale</td></tr><tr><td>Fmoc-L-Phe-OH</td><td>Gyros Protein Technologies</td><td>FLA-25-F</td><td>Wear personal protective equipment; do not inhale</td></tr><tr><td>Fmoc-L-Ala-OH</td><td>Gyros Protein Technologies</td><td>FLA-25-A</td><td>Wear personal protective equipment; do not inhale</td></tr><tr><td>Fmoc-L-Gln(Trt)-OH</td><td>Gyros Protein Technologies</td><td>FLA-25-QT</td><td>Wear personal protective equipment; do not inhale</td></tr><tr><td>N,N,N&amp;prime;,N&amp;prime;-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate</td><td>Gyros Protein Technologies</td><td>26432</td><td>Causes skin, eye and respiratory irritation; do not inhale; use under hood or in well ventilated area</td></tr><tr><td>0.4 M N-methylmorpholine in DMF</td><td>Gyros Protein Technologies</td><td>PS3-MM-L</td><td>highly flammable; wear personal protective equipment; keep away from heat and keep container tightly closed; do not inhale or swallow; wash skin thoroughly after handling</td></tr><tr><td>20% piperidine in DMF</td><td>Gyros Protein Technologies</td><td>PS3-PPR-L</td><td>Causes severe eye and skin burns; Flammable Liquid and vapor; Do not inhale</td></tr><tr><td>dichloromethane</td><td>Fisher Scientific</td><td>D37-4</td><td>May cause cancer; Do not inhale; Wear personal protective equipment; use under hood only; if contacted rise with water for at least 15 minutes and obtain medical attention</td></tr><tr><td>acetonitrile</td><td>Fisher Scientific</td><td>A998-4</td><td>Flammable; irritating to eyes; Use personal protective equipment; Use only under a fume hood; keep away from open flame or hot surface; if contacted rinse wiith water for at least 15 minutes and obtain medical attention</td></tr><tr><td>trifluoroacetic acid</td><td>Fisher Scientific</td><td>A116-50</td><td>Causes severe burns; do not inhale; harmful to aquatic life; use personal protective equipment; use only under fume hood; if contacted rinse with water for at least 15 minutes and obain immediate medical attention</td></tr><tr><td>4% uranyl acetate</td><td>Electron Microscopy Sciences</td><td>22400-4</td><td>Do not inhale; harmful to aquatic life</td></tr><tr><td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid</td><td>Acros Organics</td><td>AC172571000</td><td>Do not inhale; use outdoors or in well-ventilated area</td></tr><tr><td>nitrogen Gas</td><td>TechAir</td><td></td><td>Contents under pressure, may explode if heated</td></tr><tr><td>3-bromo-2-(bromomethyl)propionic acid</td><td>Alfa Aesar</td><td>AAA1963014</td><td>Do not inhale; causes irritation to skin and eyes; corrosive</td></tr><tr><td>sodium hydroxide</td><td>Fisher Scientific</td><td>S318-100</td><td>Use personal protective equipment; use only under fume hood; if contact rinse area for at least 15 minutes and obtain medical attention</td></tr><tr><td>potassium thioacetate</td><td>Acros Organics</td><td>AC221300250</td><td>Causes skin and eye irritation; do not inhale; use personal protective equipment</td></tr><tr><td>sulfuric acid</td><td>Fisher Scientific</td><td>SA213</td><td>Causes burns; keep away from water; keep away from combustible material; do not inhale; use personal protective equipment; if contact rinse area for at least 15 minutes and obtain medical attention</td></tr><tr><td>chloroform-d</td><td>Acros Organics</td><td>AC320690075</td><td>Possible cancer hazard; irritating to skin and eyes; do not inhale; Use personal protective equipment; use only under fume hood; If contact rinse area for at least 15 minutes and obtain medical attention</td></tr><tr><td>chloroform</td><td>Fisher Scientific</td><td>C298-4</td><td>Possible cancer hazard; irritating to skin and eyes; do not inhale; Use personal protective equipment; use only under fume hood; If contact rinse area for at least 15 minutes and obtain medical attention</td></tr><tr><td>N,N-diisopropylethylamine</td><td>Acros Organics</td><td>AC367841000</td><td>Highly flammable; harmful to aquatic life; wear personal protective equipment; do not swallow</td></tr><tr><td>ammonium hydroxide</td><td>Fisher Scientific</td><td>A669S-500</td><td>Corrosive; do not inhale</td></tr><tr><td>methanol</td><td>Fisher Scientific</td><td>A452-4</td><td>Flammable liquid and vapor; use personal protective equipment; do not inhale; If contact rinse area for at least 15 minutes and obtain medical attention</td></tr><tr><td>triisopropylsilane</td><td>Sigma Aldrich</td><td>233781</td><td>Flammable; use personal proctective safety equipment; keep container tightly closed</td></tr><tr><td>diethyl ether</td><td>Fisher Scientific</td><td>E138-1</td><td>Extremely flammable; Irritating to skin and eyes; Use personal protective equipment</td></tr><tr><td>2,5-dihydroxybenzoic acid</td><td>Sigma Aldrich</td><td>39319-10x10MG-F</td><td>do not inhale; irritating to skin and eyes</td></tr><tr><td>alpha-cyano-4-hydroxycinnamic acid</td><td>Alfa Aesar</td><td>AAJ67635EXK</td><td></td></tr><tr><td>c18 zip-tip</td><td>Millipore</td><td>ZTC18S096</td><td></td></tr><tr><td>tris(2-carboxyethyl) phospine hydrochloride</td><td>Thermo Scientific</td><td>PI20490</td><td></td></tr><tr><td>silica gel 60 F254 coated aluminum-backed TLC sheets</td><td>EMD Millipore</td><td>1.05549.0001</td><td></td></tr><tr><td>Thin walled Precision NMR tubes</td><td>Bel-Art</td><td>663000585</td><td>5mm O.D.</td></tr><tr><td>All-plastic Norm-Ject syringes</td><td>Air Tite</td><td>AL10</td><td></td></tr><tr><td>single-use needle</td><td>BD PrecisionGlide</td><td>BD 305185</td><td>used needles get disposed on in sharps waste container</td></tr><tr><td>disposable fritted syringe</td><td>Torviq</td><td>SF1000LL</td><td>10mL fritted syringes were used in the report, but larger syringes are avaibale if needed for larger scale synthesis.</td></tr><tr><td>carbon grid</td><td>Ted Pella, Inc.</td><td>CF200-CU</td><td>Make sure to prepare samples and staining on the carbon grid side, not the shiny copper side of grid</td></tr><tr><td>self-closing tweezers</td><td>Electron Microscopy Sciences</td><td>78318-3X</td><td>very sharp tips, length: 120 mm</td></tr><tr><td>0.1 mm short path length cell</td><td>Starna Cells, Inc.</td><td>20/C-Q-0.1</td><td>Fragile</td></tr><tr><td>10mL Vessel Caps</td><td>CEM</td><td>909210</td><td></td></tr><tr><td>10mL Pressure Vessels</td><td>CEM</td><td>908035</td><td></td></tr><tr><td>Aeris Semi-Prep HPLC column</td><td>Phenomenex</td><td>00F-4632-N0</td><td>150 x 10mm</td></tr><tr><td>cell holder</td><td>Starna Cells, Inc.</td><td>CH-2049</td><td>Needed when using short pathlength cells</td></tr><tr><td>PS3 peptide synthesizer</td><td>Gyros Protein Technologies</td><td></td><td></td></tr><tr><td>DiscoverSP Microwave Reactor</td><td>CEM</td><td></td><td></td></tr><tr><td>centrifuge</td><td>HERMLE</td><td>Z 206 A</td><td>used a fixed 6x50 mL rotor</td></tr><tr><td>HPLC</td><td>Shimadzu</td><td></td><td>UV Detector</td></tr><tr><td>nuclear magnetic resonance spectrometer</td><td>Avance, Bruker</td><td></td><td>300 MHz</td></tr><tr><td>MALDI-TOF mass spectrometer</td><td>Axima Confidence, Shimadzu</td><td></td><td></td></tr><tr><td>lyophilizer</td><td>Millrock Technology</td><td>BT85A</td><td></td></tr><tr><td>Fourier-Transform Infrared Spectrometer</td><td>Alpha Tensor, Bruker</td><td></td><td></td></tr><tr><td>Transmission Electron Microscope</td><td>Tecnai Spirit, FEI</td><td></td><td>Used with Gatan Orius Fiberoptic CCD digital camera. Accessed at CSCU Center for Nanotechnology</td></tr><tr><td>Circular Dichroism Spectropolarimeter</td><td>J-810, JASCO</td><td></td><td>Used with a six-cell Peltier temperature controller. Accessed at Wesleyan University.</td></tr></tbody></table>

synthesis and characterization of 1,2-dithiolane modified self-assembling peptides

Este relatório centra-se na síntese de um N-terminal 1,2-Ditiolano modificado auto-montagem de peptídeo e a caracterização dos resultante Self montadas estruturas supramoleculares. A rota sintética tira proveito da síntese do peptide de fase sólida com o engate em resina Ditiolano precursor da molécula de, 3-(acetylthio) -2-ácido (acetylthiomethyl) e o tioacetato assistida por microondas deprotection do peptide N-terminal antes de clivagem final da resina para produzir o 1,2-Ditiolano modificado do peptide. Após a purificação de cromatografia líquida de alto desempenho (HPLC) do peptide 1,2-Ditiolano, derivado do núcleo de nucleação do peptide Aβ associado com a doença de Alzheimer, o peptídeo é mostrado a auto-montagem em fibras de amiloides Cruz-β. Protocolos para caracterizar as fibras amiloides por espectroscopia de infravermelho-transformada de Fourier (FT-IR), espectroscopia de Dicroísmo circular (CD) e microscopia eletrônica de transmissão (TEM) são apresentados. Os métodos de modificação do N-terminal com um grupo de 1,2-Ditiolano bem caracterizadas péptidos auto-montagem agora podem ser explorados como sistemas modelo para desenvolver estratégias de modificação pós-montagem e explorar o dinâmica química covalente superfícies de nanofibras de peptídeo supramolecular.

Afora a síntese de uma etapa inicial da molécula precursora de Ditiolano, o resto da síntese do peptide 1,2-Ditiolano modificado ocorre na sustentação contínua (figura 1A). A conversão da 3-bromo - 2-(bromomethyl) ácido propiônico para 3-(acetylthio) -2-ácido (acetylthiomethyl), o precursor de Ditiolano, é confirmado por 1H e 13C NMR (figura 1B e C) antes está acoplada à livre N...

Watch this Scientific Journal Video about Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides at JoVE.com

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

Um protocolo para a síntese de um 1,2-Ditiolano modificado de peptídeo e a caracterização das estruturas supramoleculares resultantes do peptide auto-montagem.

Síntese e caracterização de 1,2-Ditiolano modificado peptídeos auto-montagem

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

synthesis-characterization-12-dithiolane-modified-self-assembling

Universidad San Sebastian

Research

JoVE Journal

Chemistry

7.2K visualizações.  Fairfield University. Um protocolo para a síntese de um 1,2-Ditiolano modificado de peptídeo e a caracterização das estruturas supramoleculares resultantes do peptide auto-montagem.

Vídeo: Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

A formação de estruturas biomoleculares ordenada pelo auto-montagem de pequenos peptídeos

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Química

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Split-and-piscina Síntese e Caracterização de Peptídeos Terciário Amide Biblioteca

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

Nucleosídeos Trisfosfatos - Da síntese à Caracterização Bioquímica

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

Síntese em fase sólida Submonomer de Polímeros Peptoid e seus Automontagem em altamente ordenada nanofolhas

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

Bioengenharia

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

Construindo Tioéter/sulfeto de vinil-amarrados induzida por peptídeos helicoidais Via foto Thiol-ene/ino Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

Síntese de Peptoides portadores de informações e sua Auto-montagem Dinâmica Covalent e dirigida por sequência

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as a consequence, they are now used clinically. It is, therefore, critical to seek effective strategies for synthesizing cyclic peptides to promote their application in the field of drug discovery. This paper reports a detailed protocol for the efficient synthesis of cyclic peptides using on-resin or intramolecular (intermolecular) bisalkylation. Using this protocol, linear peptides were synthesized by taking advantage of solid-phase peptide synthesis with cysteine (Cys) and methionine (Met) coupled simultaneously on the resin. Further, cyclic peptides were synthesized via bisalkylation between Met and Cys using a tunable tether and an on-tether sulfonium center. The whole synthetic route can be divided into three major processes: the deprotection of Cys on the resin, the coupling of the linker, and the cyclization between Cys and Met in a trifluoroacetic acid (TFA) cleavage solution. Furthermore, inspired by the reactivity of the sulfonium center, a propargyl group was attached to the Met to trigger thiol-yne addition and form a cyclic peptide. After that, the crude peptides were dried and dissolved in acetonitrile, separated, and then purified by high-performance liquid chromatography (HPLC). The molecular weight of the cyclic peptide was confirmed by liquid chromatography-mass spectrometry (LC-MS), and the stability of the cyclic peptide combination with the reductant was further confirmed using HPLC. In addition, the chemical shift in the cyclic peptide was analyzed by 1H nuclear magnetic resonance (1H NMR) spectra. Overall, this protocol aimed to establish an effective strategy for synthesizing cyclic peptides.

This protocol presents the synthesis of cyclic peptides via bisalkylation between cysteine and methionine and the facile thiol-yne reaction triggered by the propargyl sulfonium center.

Construindo peptídeos cíclicos usando um centro de sulfônio on-tether

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as ...

<meta charset="utf8"></head><body> Hidrogéis peptídicos: métodos para gelificação ambientalmente responsiva de ECF-5

Preparation of Mechanically Stable Self-Assembled Peptides Hydrogels

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking networks. Their morphology closely resembles that of the extracellular matrix, allowing them to exhibit both the biological functions of peptides and responsive gelation properties. These unique characteristics have led to their extensive application in tissue engineering, three-dimensional cell culture, cancer therapy, regenerative medicine, and other biomedical fields. This article describes three methods for preparing ECF-5 peptide hydrogels using self-assembling peptides with environmentally responsive gelation processes: (1) pH-responsive gelation: varying pH levels induce the protonation or deprotonation of amino acid residues, altering electrostatic interactions between peptide molecules and promoting their self-assembly into hydrogels; (2) Metal ion addition: polyvalent metal ions chelate with negatively charged amino acid residues, acting as bridges between peptides to form a network hydrogel; (3) Solvent exchange: hydrophobic peptides are initially dissolved in non-polar organic solvents and subsequently induce self-assembly into hydrogels upon transitioning to a polar aqueous environment. These methods utilize conventional experimental procedures to facilitate peptide self-assembly into hydrogels. By designing peptide sequences to align with specific gelation-inducing conditions, it is possible to achieve finely tuned micro/nanostructures and biological functions, highlighting the significant potential of peptide hydrogels in the biomedical domain.

<meta charset="utf8"></head><body>Autor em destaque: Melhorando a produção de fibras automontáveis e hidrogéis peptídicos para biocompatibilidade superior

This protocol presents three fast and simple preparation methods that use environmental conditions to trigger the self-assembly of peptides into hydrogels. Additionally, the characterization of peptide hydrogels is described, demonstrating that mechanically stable peptide hydrogels can be formed under these straightforward conditions.

Preparação de hidrogéis de peptídeos automontados mecanicamente estáveis

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking ...

Síntese e caracterização de 1,2-Ditiolano modificado peptídeos auto-montagem

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Preparação de hidrogéis de peptídeos automontados mecanicamente estáveis