Los autores desean agradecer al Dr. B. Ellen Scanley su capacitación técnica y ayuda usar la temperatura en el centro de la Universidad (CSCU) y universidades del estado de Connecticut para nanotecnología y Dr. Ishita Mukerji en Universidad de Wesleyan para tener acceso a su CD Espectrofotómetro. El trabajo divulgado fue apoyado en parte por el Instituto de Ciencias en la Universidad de Fairfield, Connecticut NASA espacio Grant consorcio y por la National Science Foundation bajo la beca número-1624774.

1. síntesis y purificación de 1, 2-Dithiolane péptido modificado
<ol><li>Síntesis del precursor dithiolane, 3-(acetylthio) -2-(acetylthiomethyl) propanoico ácido19.
	<ol>
<li>Añadir 1 g de 3-bromo - 2-(bromometil) ácido propiónico (1 equivalente) disuelto en una cantidad mínima de 1 M NaOH (aproximadamente 4 mL) a un matraz de reacción de fondo redondo de 25 mL con agitación a 55 ° C. Tapar el matraz de reacción con un septo y bajo atmósfera de nitrógeno.</li>
		<li>Preparar una solución que contiene 1,49 g de potasio thioacetate (3.2 equiv) en 4 mL de agua desionizada y 3 mL de ácido sulfúrico de 2 M (H2SO4) crear thioacetic ácido en situ.</li>
		<li>Tire la solución ácido thioacetic en una jeringa de plástico desechable de 10 mL y colocar una aguja en la jeringa. Añadir gota a gota la mezcla en el matraz de reacción por la perforación a través de los tabiques con la aguja. Continuar la reacción durante la noche a 55 º C.</li>
		<li>Vigilar la reacción por cromatografía en capa fina (CCF) en gel de sílice 60 F254 placas usando una mezcla de metanol y diclorometano (1:9). Visualizar el progreso de la reacción por la mancha verde de bromocresol. El producto tiene un Rf = 0.57.</li>
		<li>Después de la reacción es completa y enfriados a la temperatura ambiente, acidificar la mezcla a pH 1 con 2 M H2hasta4. Un aceite amarillo se separa de la solución.</li>
		<li>Extraer el producto con cloroformo frío (40 mL x 3). Combinar las capas orgánicas y seca sobre sulfato de magnesio. Retire el cloroformo a presión reducida.</li>
		<li>Para confirmar la identidad del producto aislado, 1, espectroscopia de resonancia magnética nuclear (RMN) como se muestra en las figuras 1B y C. Esperar los resultados siguientes: 1H NMR, CDCl3, 300 MHz: d = 10.1 (b, 1 H), 3.2 (m, 4 H), 2,9 (m, 1 H), 2.4 (s, 6 H); 13 C RMN CDCl3, 75 MHz: d = 195,1 (CH3COS-), 177.6 (-OOHC), 45.1 (-CH2S-), 30.5 (CH) 29.2 (-SCOCH3). Nota: El producto es un aceite amarillo y tiene un rendimiento global del 83%. Utilizar el producto sin una posterior purificación.</li>
	</ol></li>
	<li>SPPS y acoplamiento en resina de Dithiolane Precursor Nota: La síntesis del peptide de la fase sólida que se describe a continuación se llevó a cabo en un sintetizador de péptidos automatizada, siguiendo los protocolos recomendados del fabricante. Configuración y reactivos pueden ser adaptados para otros instrumentos comerciales o al usar aminoácidos especializados.<ol>
<li>Pesar 0,156 g de resina de 4-methylbenzhydrylamine (MBHA) pista amida (0.1 mmol) y colocar en un recipiente de la reacción. Hincharse la resina en dimetilformamida (DMF) para por lo menos 15 minutos antes del inicio de la síntesis.</li>
		<li>Peso a 4 equivalentes de cada fluorenylmethyloxycarbonyl (Fmoc) protegido del aminoácido (0.4 mmol) en la secuencia y 0,152 g de N, N, N', N'- tetrametil-O-(1H- benotriazol-1-yl) hexafluorofosfato de uranio (0.4 mmol, HBTU) para cada aminoácido en la secuencia. Cada cartucho contiene los aminoácidos Fmoc-protegido y HBTU.</li>
		<li>Después de ejecutar todas la síntesis comprueba en el sintetizador (rellenar reactivos, lavado de líneas de reactivo y a presión todas las botellas de reactivos), coloque los cartuchos del aminoácido en el carrusel en el C' n ' Dirección terminal. Coloque un cartucho vacío después de la posición final del aminoácido para el paso final de la desprotección de N-terminal Fmoc.</li>
		<li>Sintetizar el péptido usando la configuración recomendada estándar.<ol>
<li>Desprotejera el grupo Fmoc de la resina con 5 mL de 20% piperidina en DMF (5 min x 2).</li>
			<li>Lavar la resina con DMF (6 x 5 mL) antes de la etapa de acoplamiento.</li>
			<li>Para un acoplamiento único paso, añadir 4 mL de 0,4 M de N-methylmorpholine en DMF y el protegido de Fmoc Aminoácidos HBTU. Activar la solución de aminoácidos Fmoc-protegido por 30 s antes de transferir la solución al recipiente de reacción. Nota: El sintetizador de péptidos automatizada mezcla la resina y la solución por los burbujeantes N2 gas cada 30 s por 20 min mientras que la reacción de acoplamiento toma lugares. Para síntesis de péptidos manual, coloque el recipiente de la reacción en un agitador orbital a una velocidad baja para la duración de la etapa de acoplamiento.</li>
			<li>Vaciar la solución y lavar la resina con DMF (3 x 5 mL).</li>
			<li>Repita los pasos 1.2.4.1 a través 1.2.4.4 para cada aminoácidos Fmoc-protegidos en el C' n '-dirección de terminus para sintetizar el péptido de interés.</li>
		</ol>
</li>
		<li>Después de la etapa final de la desprotección de N-terminal, transferencia de la resina en una jeringa desechable de sinterizado. Lavar la resina con el DMF (3 x 5 mL) y diclorometano (DCM, 3 x 5 mL). Nota: La resina se puede almacenar después de DCM en un desecador de vacío. Si la resina ha sido seleccionada antes de acoplamiento, asegúrese de hincharse la resina en DMF antes de la reacción de acoplamiento.</li>
		<li>Pareja dithiolane precursor (1) a N-terminal del péptido de la resina mediante la adición de 4 equivalentes de 1, 5 mL de DMF, 4 equivalentes de HBTU y 10 equivalentes de N, N-diisopropylethylamine (DIPEA). Activar previamente la mezcla de acoplamiento por 10 min antes de agregar a la resina que contiene la jeringa sinterizado.</li>
		<li>Agite la reacción de acoplamiento de 2 h. Después de 2 h, lavar la resina con DMF (3 x 5 mL) y repetir la reacción de acoplamiento con agitación durante la noche.</li>
		<li>Después el segundo acoplamiento, lavar la resina con el DMF (3 x 5 mL) y DCM (3 x 5 mL). Nota: La resina se puede almacenar en este punto bajo vacío hasta el escote.</li>
	</ol></li>
	<li>Thioacetate desprotección y escote de péptido de la resina
	<ol>
<li>Para desprotejera del grupo thioacetate el precursor de dithiolane N-terminal, transferir la resina seca a un tubo de reacción de microondas de 10 mL y añadir 2 mL de DMF. Permita que la resina se hinchan, añadir una barra de agitación magnética pequeña al recipiente y resuspender con baja velocidad de agitación magnética durante 15 minutos.</li>
		<li>Añadir 2 mL de hidróxido de amonio concentrado, tapa el recipiente de la reacción con los tabiques de silicona y coloque el recipiente de la reacción en un reactor de microondas utilizando la configuración de microondas de 75 ° C por 45 min con agitación.</li>
		<li>Una vez finalizada la reacción de la microonda, transferencia de la resina en una jeringa desechable limpias de sinterizado. Lave con DMF (2 x 5 mL) y metanol (MeOH, 2 x 5 mL).</li>
		<li>Agregar una solución de hidróxido de amonio concentrado en metanol (1:4), para un volumen total de 5 mL. Deje a temblar durante la noche para aumentar la oxidación intramolecular del enlace de disulfuro en dithiolane anillo.</li>
		<li>Lavar la resina con MeOH (2 x 5 mL) y DCM (3 x 5 mL). Nota: La resina seca se puede almacenar en un desecador de vacío en este punto.</li>
		<li>Añadir el cóctel de escote a la resina que contiene jeringa con agitándolo suavemente durante 1,5 h. El escote cóctel utilizado es de 95% de ácido trifluoroacético (TFA), Triisopropilsilano (TIPS) de 2,5% y 2.5% de agua en un volumen total de 5 mL. Atención: Trabajar bajo campana química solamente. TFA es volátil y corrosivo. Nota: Para la mayoría de secuencias de péptidos y aminoácidos cadena protección de los grupos, la solución cóctel escote anterior es suficiente; sin embargo, cocteles escote alternativos pueden ser necesaria para cierta cadena lateral del aminoácido protección grupos (en particular péptidos que contienen Cys, Met, Trp y Arg) u otra resina química20.</li>
		<li>Precipitar el péptido crudo en 25 mL de frío éter etílico en un tubo cónico de 50 mL por adición gota a gota desde la jeringa sinterizada. El péptido se precipita como un sólido blanco. Pelotilla del péptido por centrifugación a 1300 x g durante 10 minutos decantar con cuidado el éter dietílico en un contenedor separado para recogida de residuos.</li>
		<li>Agregar otros 25 mL de éter etílico en el tubo cónico y Resuspenda el precipitado con un vórtex. Repetir la centrifugación 1300 x g durante 10 minutos y decantar el éter dietílico otra vez. Secar el precipitado al vacío.</li>
	</ol></li>
	<li>Purificación de 1, 2-Dithiolane modificado péptido Nota: Purificar el péptido crudo por HPLC de fase reversa. Recoger y combinar los picos del péptido y confirmar la masa por espectrometría de masas MALDI-TOF.<ol>
<li>Disolver los pellets de peptide crudo en cantidad mínima de acetonitrilo con 0,1% TFA. Debido a la hidrofobicidad del péptido y propensión de agregación, calentar suavemente la muestra a 40 º C para facilitar la solubilidad. Nota: Evite mayores temperaturas y sonicación del péptido para evitar potencial intercambio de disulfuro reacciones21,de22,23.</li>
		<li>Para preparar 1 mL de crudo péptido para la purificación del HPLC, añadir 400 μL de caldo concentrado de péptidos en acetonitrilo a 600 μL de H2O con 0,1% TFA y el filtro a través de un filtro de jeringa de 22 μm en un frasco HPLC. Puede añadirse un isopropanol al 5% adicional para prevenir la precipitación y la agregación del péptido.</li>
		<li>Purificar el péptido utilizando columna semi-preparativa C-18 con un caudal de 3 mL/min en un gradiente lineal de acetonitrilo de 15-55% en conjunto de 20 minutos los detectores UV a 222 nm (espina dorsal amida) y 330 nm (Enlace disulfuro). Reunir y combinar los picos de interés (figura 2A).</li>
		<li>Confirmar la masa de producto péptido por espectrómetro de masas MALDI-TOF en el modo reflectron (figura 2B). Para el análisis, mezclar 0,5 μL de pico recogido en la placa MALDI con 0,5 μL de la matriz de ácido (DHB) 2,5-dihydroxybenzoic (10 mg/mL DHB en 50% acetonitrilo, 0.1% TFA). Nota: Común aductos en MALDI-TOF espectrometría de masas son el sodio y potasio aducción ([M + Na]+ y [M + K]+) picos. La muestra antes del análisis de la desalación se recomienda si los picos de aducto de sal suprimen la señal del pico [M + H]+ principal. Además, un pico oxidado [M + o]+ se detecta también en el péptido de 1, 2-dithiolane modificado. Un informe sobre la oxidación inducida por láser de la ionización MALDI utilizando DHB matriz sugiere que factores como la concentración de muestra, solvente y la intensidad de láser pueden ser modificados para limitar el MALDI inducido por oxidación artefacto24.</li>
		<li>Después de la confirmación de MALDI-TOF de la masa correcta, liofilizar el péptido después de congelación flash. Mantenga el polvo liofilizado péptido bajo vacío hasta asamblea.</li>
	</ol></li>
</ol>2. Caracterización de Supramolecular uno mismo-montaje de estructuras
<ol><li>
Formación de fibras amiloides
	<ol>
<li>Para preparar uno mismo-Asamblea solución, pesar 1 mg de polvo péptido utilizando una balanza analítica. Disolver en una mezcla (pH 7,5) de 20% de acetonitrilo y 10 mM (4-(2-hydroxyethyl)-1--1-ácido ácido (HEPES) en un tubo de microcentrífuga de 1,5 mL, a una concentración final de 1 mg/mL péptido ensamblado de una mezcla. Mezclar la solución de montaje y dejar montar a temperatura ambiente.</li>
	</ol>
</li>
	<li>
Caracterización espectroscópica de fibras amiloides
	<ol>
<li>Seguir el proceso de ensamblaje del péptido mediante la espectroscopia infrarrojo de Fourier (FTIR) cada pocos días. Un amplio pico centrado alrededor de 1670 cm-1 es la firma IR derivados de péptidos sin montar en la muestra17. Las muestras de ensamblaje del péptido suelen tener una o dos semanas para el amplio pico sin montar desaparecer y llegar a maduración.<ol>
<li>Una alícuota de 8-10 μL de la solución de montaje en seco como una película fina sobre el cristal de diamante ATR. Seguimiento de la desaparición de un pico de agua grande y amplio de 1640 a 1630 cm-1 como las formas de la película seca.</li>
			<li>Adquisición de espectros IR de 1500-1800 cm-1 con un promedio de 50 escaneos con una resolución de 2 cm-1 . Adquirir y resta el análisis de fondo antes de cada exploración de muestra. La firma de IR para el montaje de la β-hoja es un pico agudo entre 1625 y 1635 cm-1 (Figura 3A)25,26.</li>
		</ol>
</li>
		<li>Caracterizar el conjunto péptido β-hoja ricas estructuras supramoleculares por dicroísmo circular (CD). Registrar los espectros usando un spectropolarimeter de CD con un sistema de control de temperatura Peltier.<ol>
<li>Tomar con pipeta 30 μL de la solución de montaje en un camino 0,1 mm longitud microcubeta. Nota: Es necesario un soporte de celular a la abrazadera y coloque la celda de longitud de ruta corta en el instrumento.</li>
			<li>Para cada espectro, establece el instrumento de CD en los siguientes parámetros: Análisis de longitudes de onda de 300 nm a 180 nm, velocidad de barrido de 100/min, ancho de banda de 1 nm, 25 ° C, promedio de tres análisis.</li>
			<li>Recoge un espectro del búfer (20% acetonitrilo/10 mM HEPES, pH 7,5) y restar cada scan muestra como control. La firma de CD para las β-hojas es que una elipticidad mínima centrado alrededor 220 nm (figura 3B)27.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Microscopia de fibras amiloides
	<ol>
<li>Permiten dos o tres semanas para las muestras del péptido madurar en β-hoja ricas estructuras supramoleculares. Nota: Las Asambleas pueden ser reflejadas mediante microscopía electrónica de transmisión (TEM) en las etapas anteriores del proceso de montaje.<ol>
<li>Pipetear 10 μL de la solución de montaje del péptido en la superficie de la rejilla del carbón TEM. Nota: tenga cuidado para no tocar la punta de la pipeta a la superficie de la rejilla. Alta precisión, cierre pinzas se utilizan para sostener la rejilla TEM durante la preparación.</li>
			<li>Esperar 1-2 minutos para permitir que las Asambleas fijar por adsorción sobre la superficie de la rejilla. Quitar exceso muestra por contacto con papel de filtro en el borde de la cuadrícula.</li>
			<li>Preparar una tinción de acetato de uranilo 2% mediante la adición de 100 μL de agua desionizada para la solución de acetato de uranilo 4% disponible en el mercado. Pipetee 10 μL de la mancha de acetato de uranilo 2% sobre la superficie de la rejilla e incubar durante 2-3 minutos. Después de la incubación, retirar exceso mancha por contacto con papel de filtro en el borde de la cuadrícula.</li>
			<li>Coloque las rejillas TEM en un desecador de vacío durante la noche. Almacenar bajo vacío hasta la proyección de imagen.</li>
			<li>Imagen de las muestras preparadas con TEM (figura 3). Parámetros típicos para microscopía son las siguientes: imágenes con aumentos que van desde 9.300 X 23, 000 X, filamento de tungsteno con un voltaje de aceleración de 120 kV. Nota: ImageJ puede utilizarse para medir el ancho de la fibra media de las estructuras supramoleculares de adquirido TEM imágenes28. PRECAUCIÓN: Por favor consulte todas hojas de datos de seguridad (SDS) antes de su uso. Varios productos químicos utilizan en la síntesis, purificación, y caracterización de los péptidos de uno mismo-montaje descrito 1, 2-dithiolane modificado son tóxicos o corrosivos y debe utilizarse sólo bajo una campana de humos químicos. Utilice siempre equipos de protección personal (incluyendo gafas de seguridad, bata de laboratorio, pantalón largo, zapatos cerrados) cuando se trabaja en el laboratorio.</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 artículo discute los detalles de la síntesis y la purificación de un peptide uno mismo-montaje N-terminal 1, 2-dithiolane modificado y la caracterización de las estructuras supramoleculares resultantes. La síntesis del péptido 1, 2-dithiolane divulgado aquí tiene las ventajas, incluyendo una síntesis de un solo paso para producir el precursor dithiolane, 3-(acetylthio) -2-(acetylthiomethyl) propanoico ácido y la resina en microondas reacción de desprotección de la precursor thioacetate protección de grup...

El enlace peptídico sólido formación química implicada en síntesis del peptide de la fase sólida y la capacidad para controlar la composición y la longitud de la secuencia hacen los péptidos que uno mismo-montan en estructuras supramoleculares un campo muy investigado. Los factores que controlan y estabilizan péptido uno mismo-montado estructuras, incluyendo cadena lateral estérico e interacciones electrostáticas, hidrógeno y efectos hidrofóbicos1, servir como un conjunto de reglas de diseño. Como la investigación en estas reglas de diseño fundamental continúa progresando, el siguiente paso lógico en péptido autoensamblaje implica ampliar la diversidad de funciones y estructuras de péptidos. Mientras que uno mismo-montaje de péptidos es un biomaterial versátil que se han utilizado para muchas aplicaciones biomédicas templando el péptido secuencia o conjunto de condiciones2,3,4, el desarrollo de estrategias para la Asamblea modificaciones a péptido nanofibras en5,6,7,8,9 sigue siendo un área relativamente inexplorada.
Química intercambio y tiol disulfuro dinámicos en la superficie de estructuras supramoleculares es un área que tiene el potencial para producir biomateriales nuevos y funcionales. La incorporación de moléculas de 1, 2-dithiolane (comúnmente un derivado de ácido lipoico (la) o ácido asparagusic (aa)) se han divulgado en liposomas sistemas10,11, bloque copolímeros12,13y como Organización de anclajes en superficies14,15. Adjunto, divulgamos la síntesis y caracterización de un peptide uno mismo-montaje derivado del núcleo nucleating del péptido Aβ asociado con la enfermedad de Alzheimer que es modificada en el N-terminal con 1, 2-dithiolane grupo funcional16, 17. Las fibras supramoleculares que ahora sirven como una plataforma experimental para el estudio de la reactividad en la superficie supramolecular de fibras amiloides18intercambio de disulfuro y tiol.

<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 informe se centra en la síntesis de un N-terminal 1, 2-dithiolane modificado peptide uno mismo-montaje y la caracterización de la resultante uno mismo-montar estructuras supramoleculares. La ruta sintética aprovecha la síntesis de péptidos de fase sólida con el acoplamiento en la resina de la molécula del precursor dithiolane, 3-(acetylthio) -2-(acetylthiomethyl) propanoico ácido y la thioacetate asistida por microondas deprotection del péptido N-terminal antes de la división final de la resina para dar 1, 2-dithiolane modificado péptido. Después de la purificación de la cromatografía líquida de alto rendimiento (HPLC) del péptido 1, 2-dithiolane, derivado de la base nucleating del péptido Aβ asociado con la enfermedad de Alzheimer, el péptido aparece uno mismo-montar en fibras amiloideas Cruz-β. Protocolos para la caracterización de las fibras amiloides por espectroscopia infrarroja de Fourier (FT-IR), espectroscopía de dicroísmo circular (CD) y microscopía electrónica de transmisión (TEM) se presentan. Ahora pueden explorar los métodos de modificación de la N-terminal con una molécula de 1, 2-dithiolane a péptidos uno mismo-montaje bien caracterizados como sistemas modelo para desarrollar estrategias de modificación de la Asamblea y explorar la química covalente dinámica en superficies de nanofibras de péptido supramolecular.

Aparte de la síntesis de un paso inicial de la molécula del precursor dithiolane, el resto de la síntesis del péptido 1, 2-dithiolane modificado se realiza en soporte sólido (figura 1A). La conversión de 3-bromo - 2-(bromometil) ácido propiónico para 3-(acetylthio) -2-(acetylthiomethyl) propanoico ácido dithiolane precursor, es confirmado por 1H y 13C NMR (figura 1B y C) antes de qu...

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

Un protocolo para la síntesis de un 1, 2-dithiolane modificada de péptidos y la caracterización de las estructuras supramoleculares resultantes del peptide uno mismo-Asamblea.

Síntesis y caracterización de 1, 2-Dithiolane modifican uno mismo-montaje de péptidos

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

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7.1K vistas.  Fairfield University. Un protocolo para la síntesis de un 1, 2-dithiolane modificada de péptidos y la caracterización de las estructuras supramoleculares resultantes del peptide uno mismo-Asamblea.

Video: 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.

Formación de estructuras biomoleculares ordenada por el auto-ensamblaje de péptidos cortos

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

Investigación

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.

Síntesis Split-y-piscina y caracterización del péptido Terciario Amida 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.

Nucleósidos Trifosfatos - De la síntesis a la Caracterización 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.

En fase sólida Submonomer Síntesis de Polímeros peptoide y su auto-ensamblaje en altamente ordenadas nanoláminas

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

Bioingeniería

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.

Construcción de Thioether/sulfuro de vinilo-atado Hydrothiolation de péptidos helicoidales mediante foto-inducida tiol-eno/Ino

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.

Síntesis de peptoides que llevan información y su autoensamblaje covalente dinámico dirigido por secuencia

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.

Construcción de péptidos cíclicos usando un centro de sulfonio en la correa

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> Hidrogeles peptídicos: métodos para la gelificación ambientalmente sensible 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 destacado: Mejora de la producción de fibras autoensamblables e hidrogeles peptídicos para una biocompatibilidad 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.

Preparación de hidrogeles de péptidos autoensamblados mecánicamente estables

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

Síntesis y caracterización de 1, 2-Dithiolane modifican uno mismo-montaje de péptidos

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Formación de estructuras biomoleculares ordenada por el auto-ensamblaje de péptidos cortos

Síntesis Split-y-piscina y caracterización del péptido Terciario Amida Biblioteca

Nucleósidos Trifosfatos - De la síntesis a la Caracterización bioquímica

En fase sólida Submonomer Síntesis de Polímeros peptoide y su auto-ensamblaje en altamente ordenadas nanoláminas

Construcción de Thioether/sulfuro de vinilo-atado Hydrothiolation de péptidos helicoidales mediante foto-inducida tiol-eno/Ino

Síntesis de peptoides que llevan información y su autoensamblaje covalente dinámico dirigido por secuencia

Construcción de péptidos cíclicos usando un centro de sulfonio en la correa

Preparación de hidrogeles de péptidos autoensamblados mecánicamente estables

Preparación de hidrogeles de péptidos autoensamblados mecánicamente estables

Preparación de hidrogeles de péptidos autoensamblados mecánicamente estables