Este trabajo fue apoyado por la Fundación Nacional Suiza para la Ciencia (Becas n ° PZ00P2_126430 / 1 y PZ00P2_144595). Prof. C. Leumann se agradece para proporcionar el espacio y equipo de laboratorio, así como por su constante apoyo. La Sra. Sue Knecht se reconoce para un debate fructífero.

1. Síntesis de los trifosfatos de nucleósido modificado El enfoque sintético elegido sigue el procedimiento desarrollado por Ludwig y Eckstein ya que este método es generalmente fiable y conduce a muy pocos subproductos secundarios (Figura 1A) 39. <ol><li> Coevaporate el nucleósido protegido adecuadamente-3&#39;-OAc (típicamente 0,1 mmol) dos veces con piridina anhidra (2 ml) y después se seca a vacío durante la noche. Al mismo tiempo, pirofosfato de tributilamonio seca (0,13 mmol) bajo vacío durante la noche. </li><li> Se disuelve el nucleósido en un mínimo de piridina seca (0,2 ml) y añadir dioxano seco (0,4 ml) como codisolvente. Por último, añadir 2-cloro-1, 3,2-benzodioxaphosphorin-4-ona (0,11 mmoles) y dejar reaccionar a temperatura ambiente durante 45 min. Nota: Es de destacar que en particular se debe tener cuidado con la 2-cloro-1, 3,2-benzodioxaphosphorin-4-ona reactivo. De hecho, incluso el almacenamiento de este reactivo en virtud de un ambi gas inerteaquí no es suficiente para evitar su descomposición. Por lo tanto, el sólido blanco que se forma en esta descomposición puede y debe ser levantada antes de su uso. </li><li> Preparar una solución de pirofosfato de tributilamonio en DMF seca (0,17 ml) y tributilamina recién destilado (58 l; nunca añadir tamices moleculares). Añadir la solución resultante a la mezcla de reacción (un precipitado blanco aparece pero desaparece rápidamente) y dejar reaccionar a temperatura ambiente durante 45 min. </li><li> Preparar una solución de yodo (0,16 mmol) en piridina (0,98 ml) y H2O (20 l) y añadir a la mezcla de reacción con el fin de oxidar el Pα (III) central. Deje que la solución oscura resultante en agitación a temperatura ambiente durante 30 min. </li><li> Utilice una solución acuosa al 10% de NaS 2 O 3 para inactivar el exceso de I 2. Eliminar el disolvente en vacío (la temperatura del baño de agua del evaporador rotatorio debe mantenerse por debajo de 30 ° C). Añadir H 2 O (5 ml) y permitir que el millasxture en reposo a temperatura ambiente durante 30 min para hidrolizar el resto trifosfato cíclico. </li><li> En esta etapa, los grupos protectores se eliminan normalmente (es decir, el 3&#39;-OAc y los grupos en las cadenas laterales de la nucleobase). En consecuencia, añadir NH4OH (30% en H2O, 10 ml) a la crudo y se agita durante 1,5 horas a temperatura ambiente, y eliminar el disolvente bajo vacío. </li><li> Añadir 2 ml de H 2 O y se dividió en 2 tubos. Añadir 12 ml de una solución 2% de NaClO 4 en acetona y se centrifuga (1.000 xg) durante 30 min. Este procedimiento se repitió una vez más. Esta precipitación permite la separación de los disolventes y reactivos utilizados en la síntesis de la trifosfato (que precipitará), simplificando de este modo la subsiguiente purificación por RP-HPLC sustancialmente. </li><li> Después de aire de secado del residuo aceitoso, grabar un espectro de RMN P-31 de la crudo (siguiendo procedimientos estándar, ver también Lista de Materiales y Figura 3) ydisolver en 4 ml de H2O Nota: Este procedimiento de síntesis se centra principalmente en la generación de trifosfatos de desoxinucleósidos modificados, pero un procedimiento muy similar se puede aplicar para sus contrapartes de ARN (por el simple uso de 2 &#39;, 3&#39;-bis-O-acetilado precursores). </li></ol> 2. Purificación por HPLC de los trifosfatos de nucleósido modificado <ol><li> Preparación de una solución 1 M de stock de bicarbonato de trietilamonio (TEAB): 40 <ol><li> Preparar una solución de 1 mol de trietilamina (139 ml) en ≈ 600 ml de agua destilada y se filtró H 2 O. </li><li> Burbuja de CO 2 (a través de hielo seco y un burbujeador de gas) en la solución con agitación vigorosa hasta que se alcanza un pH de 7,6 (esto llevará al menos 10 horas). Esta solución madre se puede almacenar en el refrigerador hasta por un mes. </li></ol></li><li> Purificación: <ol><li> Prepare dos soluciones tampón de la acción 1 M: 2 L de 50 mM TEAB en agua ultrapura (eluyente A) unND 1 L de 50 mM de TEAB en 50% de MeCN (eluyente B). Degas ambos eluyentes bajo vacío y agitación durante 20 min (se requiere una cuidadosa atención a fin de no alterar el pH mediante la eliminación de CO 2 durante la desgasificación). </li><li> Preparar una muestra analítica por disolución de 10 l de la trifosfato de crudo en 300 l de H 2 O destilada y se inyecta en un sistema de HPLC equipado con una columna RP semi-preparativa (C18) y usando un gradiente que varía de 0-100% de B en 40 min (Figura 4). Ajuste el programa de HPLC de acuerdo con el Rt del trifosfato (que normalmente es el pico principal en el cromatograma) y el difosfato (que tiene un R ligeramente inferior t). Se purifica la mezcla bruta mediante estas condiciones y removiendo primeras fracciones que puedan contener algunos difosfato. </li><li> Combinar todas las fracciones que contienen el producto y liofilizar (que se prefiere a la evaporación en el evaporador rotatorio con el fin de minimizar la hidrólisis a la diphosph no deseadaate). Coevaporate varias veces con agua ultrapura (para eliminar trietilamina remanente de los eluyentes). Evaluar la pureza del trifosfato por RMN (tanto 1 H y 31 P RMN) y MALDI-TOF por la aplicación de los protocolos estándar (ver Figura 5). Los rendimientos típicos obtenidos mediante la aplicación de este protocolo típicamente se encuentran en el intervalo de 30-70%, dependiendo del sustrato. </li></ol></li></ol> 3. Primer extensión reacciones y TdT polimerización <ol><li> Etiquetado extremo 5 &#39;del cebador <ol><li> Mezclar 30 pmol del cebador apropiado (por ejemplo P1, Lista de Materiales) con 4 l de 10x polinucleótido quinasa tampón (PNK), 3 l de γ-[32 P]-ATP, 1 l de PNK, y ddH2O (por un volumen de reacción total de 40 l). Incubar la mezcla de reacción a 37 ° C durante 30 min y después de calor-desactivar la quinasa (10 min a 70 ° C). </li><li> Durante la reacción de marcaje prepararuna columna de Sephadex G-10 conectando una punta de pipeta de 1 ml con lana de vidrio silanizada y llenando la punta con una solución de G-10 (10% en ddH2O, en autoclave). Decantar y lavar tres veces con 500 l ddH 2 O y un lavado final con 50 l ddH 2 O. Ejecute la mezcla de reacción a través del G-10 (para eliminar el marcador radioactivo gratis). </li><li> Gel de purificar (PAGE 20%) el cebador radiomarcado y recuperarse mediante la aplicación de la aglomeración y remojo método (es decir, la elución con 500 l de una solución acuosa que contiene 1% LiClO 4 y 1 mM de NEt3 (pH 8) a 72 ° C durante 15 min). El etanol y el precipitado G10 desalar el oligonucleótido eluido. </li></ol></li><li> Reacción de extensión del cebador <ol><li> Recocido 1 pmol de cebador radiomarcado P1, 10 pmol de cebador P1, y 10 pmol de plantilla T1 (Lista de Materiales) en tampón de reacción 10x (suministrado por el proveedor de la polimerasa para ser utilizado) mediante la colocación de la tUbe en (90-95 ° C) de agua caliente y al permitir que se enfríe gradualmente hasta la temperatura ambiente (durante 45 min). </li><li> Poner el tubo en hielo y añadir (a su vez) el cóctel de dNTP (que contiene tanto el modificado y los trifosfatos naturales, 100 mM concentración final) y 1 U de la ADN polimerasa (Vent (exo -), de Klenow, de Pwo o 9 ° N m) y completar con agua (para un volumen total de reacción de 20 l). Incubar a la temperatura de trabajo óptima de la enzima (por ejemplo, 60 ° C durante 30 min cuando Vent (exo - se está utilizando)). </li><li> Añadir 20 l de solución de parada (formamida (70%), ácido etilendiaminotetraacético (EDTA; 50 mM), azul de bromofenol (0,1%), cianol xileno (0,1%)), calentar las muestras (95 º C, 5 min), fresco hacia abajo (0 ° C), y resolver por electroforesis en gel desnaturalizante de poliacrilamida. Visualizar phosphorimager. </li></ol></li><li> Polimerización TdT <ol><li> Diluir 7 pmol de una sola hebra, Cebador P2 sin marcar (Lista de Materiales), 1 pmol de cebador radiomarcado en 1 l de tampón TdT 10x. </li><li> Poner el tubo en hielo y añadir (a su vez) el cóctel de dNTP (a una concentración adecuada comprendida entre 10 y 200 mM) y la TDT de la polimerasa (4 U). Incubar a 37 ° C durante 1 hora. Añadir 10 l de la solución de parada, calentar las muestras (95 ° C, 5 min), enfriamiento (0 º C), y resolver mediante la desnaturalización electroforesis en gel de poliacrilamida (PAGE 15%). Visualizar phosphorimager. </li></ol></li></ol> 4. PCR con trifosfatos de nucleósidos modificados <ol><li> Mezclar 8 pmol de tanto la cebadores directo e inverso con 0,5 pmol de la plantilla a amplificar. Añadir 10x tampón de reacción y el cóctel de dNTP (para una concentración final 200 mM) y poner el tubo en hielo. Añadir 1 U de la ADN polimerasa y completa con H 2 O para llegar a un volumen final de 20 l. Transferir la mezcla a un vial de PCR y llevar a cabo 30 ciclos de PCR.</li><li> Diluir 6 l con 6 l de tampón de carga 2x sacarosa y resolver por agarosa al 2% (teñido con bromuro de etidio) electroforesis. Visualizar phosphorimager. </li></ol>

No hay conflictos de interés declarado.

La inclusión de las modificaciones en los ácidos nucleicos es de interés para numerosas aplicaciones prácticas, incluyendo el desarrollo de agentes antisentido y antigenes 42,43, el etiquetado y el marcado funcional de oligonucleótidos 41, y en los esfuerzos para ampliar el alfabeto genético 44-46. Las alteraciones químicas y grupos funcionales son por lo general introducidos en ácidos nucleicos mediante la aplicación de protocolos de síntesis en fase sólida estándar y automa...

<html> Trifosfatos de nucleósidos 5&#39;-((d) PNT) representan una clase de biomoléculas vitales que están implicadas en incontables procesos y funciones que van desde ser la moneda universal de energía a los reguladores del metabolismo celular. Además de su papel en estas transformaciones biológicas fundamentales, sus homólogos modificados han avanzado como una plataforma versátil y suave para la introducción de grupos funcionales en oligonucleótidos, una metodología que complementa muy bien la síntesis en fase sólida automatizada que por lo general se aplica 1,2. De hecho, siempre que las (d) los PNT pueden actuar como sustratos para ARN y ADN polimerasas 3, una gran cantidad de grupos funcionales que incluyen aminoácidos 4-13, ácidos bórico 14,15, nornbornene 16, residuos diamondoid-17, como parte de las cadenas de organocatálisis 18, los ácidos biliares 19, e incluso oligonucleótidos 20 se puede introducir en oligonucleótidos. _content &quot;&gt; Más allá de lo que representa un vector conveniente para la funcionalización de los ácidos nucleicos, dNTPs modificados pueden acoplarse en SELEX y otros métodos combinatorios relacionados de selección in vitro para la generación de ácidos nucleicos catalíticos modificados 21-30 y aptámeros para diversas aplicaciones prácticas 10, 31-36. Las cadenas laterales adicionales que se introducen por la polimerización de los dNTPs modificados se cree que aumentar el espacio químico que puede ser explorado durante un experimento de selección y complementar el bien pobre arsenal funcional de ácidos nucleicos 37. Sin embargo, a pesar de estos rasgos atractivos y los recientes progresos realizados en el desarrollo de tanto sintéticos como los métodos de análisis, universalmente aplicable y el procedimiento de alto rendimiento existe para la elaboración de nucleósidos trifosfato modificados 2,38. El objetivo del presente protocolo es arrojar luz en la (a veces) los procedimientos intrincados líder to la síntesis y caracterización bioquímica de estos bloques de construcción activados (Figura 1B). Se hará especial hincapié en todos los detalles sintéticos que a menudo son difíciles de encontrar o están ausentes en tramos experimentales, pero que son, pero crucial para el buen fin de la ruta sintética que conduce al aislamiento de (d) los PNT puros (Figura 1).

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		<tr height="21">
			<td height="21" style="height:21px;width:333px;">tributylammonium pyrophosphate&#160;</td>
			<td style="width:109px;">Sigma Aldrich</td>
			<td style="width:143px;">P8533</td>
			<td style="width:215px;">Hygroscopic solid, keep under Ar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-chloro-1,3,2-benzodioxaphosphorin-4-one&#160;</td>
			<td style="width:109px;">Sigma Aldrich</td>
			<td align="right" style="width:143px;">324124</td>
			<td>Moisture sensitive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Pyridine</td>
			<td style="width:109px;">Sigma Aldrich</td>
			<td align="right" style="width:143px;">82704</td>
			<td>Under molecular sieves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Dioxane</td>
			<td style="width:109px;">Sigma Aldrich</td>
			<td align="right" style="width:143px;">296309</td>
			<td>Under molecular sieves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">dimethylformamide (DMF)</td>
			<td style="width:109px;">Sigma Aldrich</td>
			<td align="right" style="width:143px;">40248</td>
			<td>Under molecular sieves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:333px;">Acetonitrile&#160;</td>
			<td style="width:109px;">Fisher Scientific</td>
			<td style="width:143px;"></td>
			<td>HPLC grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Triethylamine</td>
			<td style="width:109px;">Sigma Aldrich</td>
			<td align="right" style="width:143px;">90342</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Tributylamine</td>
			<td style="width:109px;">Sigma Aldrich</td>
			<td align="right" style="width:143px;">90781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:333px;">ddH2O</td>
			<td style="width:109px;">Milli-Q</td>
			<td style="width:143px;"></td>
			<td>deionized and purified water, autoclaved in the presence of Diethylpyrocarbonate (DEPC)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diethylpyrocarbonate (DEPC)</td>
			<td style="width:109px;">Sigma Aldrich</td>
			<td align="right" style="width:143px;">159220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:333px;">D2O</td>
			<td style="width:109px;">Cambridge Isotope Laboratories, Inc.</td>
			<td style="width:143px;">DLM-4-25</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:333px;">Biochemical reagents</td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">g-[32P]-ATP</td>
			<td style="width:109px;">Hartmann Analytics</td>
			<td style="width:143px;">FP-301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Natural dNTPs</td>
			<td style="width:109px;">Promega</td>
			<td style="width:143px;">U1420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:333px;">Vent (exo-) DNA polymerase</td>
			<td style="width:109px;">NEB</td>
			<td style="width:143px;">M0257S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">DNA polymerase I, Large (Klenow) Fragment</td>
			<td style="width:109px;">NEB</td>
			<td style="width:143px;">MO210S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:333px;">9&#176;Nm DNA polymerase</td>
			<td style="width:109px;">NEB</td>
			<td style="width:143px;">MO260S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Terminal deoxynucleotidyl Transferase (TdT)</td>
			<td style="width:109px;">Promega</td>
			<td style="width:143px;">M828A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Pwo DNA polymerase</td>
			<td style="width:109px;">Peqlab</td>
			<td style="width:143px;">01 01 5010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">T4 PNK</td>
			<td style="width:109px;">Thermo Scientific</td>
			<td>EK0032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Acrylamide/bisacrylamide (19:1, 40%)</td>
			<td style="width:109px;">Serva</td>
			<td align="right" style="width:143px;">10679.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">Agarose</td>
			<td style="width:109px;">Apollo Scientific</td>
			<td style="width:143px;">BIA1177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">G10 Sephadex</td>
			<td style="width:109px;">Sigma</td>
			<td style="width:143px;">G10120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">Urea</td>
			<td style="width:109px;">Apollo Scientific</td>
			<td style="width:143px;">BIU4110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Equipment</td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">Jupiter semi-preparative RP-HPLC column (5m C18 300&#197;)</td>
			<td style="width:109px;">Phenomenex</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Gene Q Thermal Cycler</td>
			<td style="width:109px;">Bioconcept</td>
			<td style="width:143px;">BYQ6042E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">PCR vials</td>
			<td style="width:109px;">Bioconcept</td>
			<td style="width:143px;">3220-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:333px;">HPLC system</td>
			<td style="width:109px;">Amersham Pharmacia Biotech</td>
			<td style="width:143px;"></td>
			<td>&#196;kta basic 10/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Oligonucleotides</td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">All oligonucleotides were purchased from Microsynth and purified by PAGE</td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">5&#39;-CAAGGACAAAATACCTGTATTCCTT P1</td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:333px;">5&#39;-GACATCATGAGAGACATCGCCTCTGGGCTAAT-AGGACTACTTCTAATCTGTAAGAGCAGATCCCTGG-ACAGGCAAGGAATACAGGTATTTTGTCCTTG T1</td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">5&#39;-GAATTCGATATCAAG P2</td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"></td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:333px;">More information on experimental procedures and equipment can be found in the following articles:</td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Chem. Eur. J. 2012, 18, 13320 &#8211; 13330</td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">Org. Biomol. Chem. 2013, DOI: 10.1039/C3OB40842F.</td>
			<td style="width:109px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

nucleoside triphosphates - from synthesis to biochemical characterization

La estrategia tradicional para la introducción de funcionalidades químicas es el uso de la síntesis en fase sólida añadiendo precursores de fosforamidita adecuadamente modificados a la cadena naciente. Sin embargo, las condiciones utilizadas durante la síntesis y la restricción de las secuencias más cortas dificultan la aplicación de esta metodología. Por otro lado, trifosfatos de nucleósidos modificados son bloques de construcción que se han empleado para la introducción suave de numerosos grupos funcionales en los ácidos nucleicos, una estrategia que allana el camino para el uso de ácidos nucleicos modificados en una gama de colores amplia de aplicaciones prácticas activan tales como etiquetado funcional y generación de ribozimas y ADNzimas. Uno de los principales desafíos reside en la complejidad de la metodología que conduce al aislamiento y caracterización de estos análogos de nucleósidos. En este artículo de vídeo, se presenta un protocolo detallado para la síntesis de these análogos modificados utilizando fósforo reactivos (III) con sede en. Además, el procedimiento para su caracterización bioquímica es divulgada, con un énfasis especial en las reacciones de extensión de cebador y polimerización del tizón TdT. Este protocolo detallado será de utilidad para la elaboración de dNTPs modificados y su uso posterior en la biología química.

Trifosfatos de nucleósidos modificados son atractivos objetivos sintéticos ya que permiten la introducción fácil de una gran variedad de grupos funcionales en los ácidos nucleicos 41. Sin embargo, el aislamiento y la caracterización de estos bloques de construcción activados es frecuentemente revelado a ser ardua. En consecuencia, se cree que los resultados que se muestran en este documento para ofrecer una mano de ayuda para seguir los diferentes pasos dentro de los procedimientos sintéticos y bioqu?...

Watch this Scientific Journal Video about Nucleoside Triphosphates - From Synthesis to Biochemical Characterization at JoVE.com

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

El protocolo descrito en el presente documento tiene como objetivo explicar y limite los numerosos obstáculos en el camino de la ruta intrincada que conduce a trifosfatos de nucleósidos modificados. En consecuencia, este protocolo facilita tanto la síntesis de estos bloques de construcción activados y su disponibilidad para las aplicaciones prácticas.

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 ...

nucleoside-triphosphates-from-synthesis-to-biochemical

Research

JoVE Journal

Chemistry

17.0K vistas.  University of Bern.  El protocolo descrito en el presente documento tiene como objetivo explicar y limite los numerosos obstáculos en el camino de la ruta intrincada que conduce a trifosfatos de nucleósidos modificados. En consecuencia, este protocolo facilita tanto la síntesis de estos bloques de construcción activados y su disponibilidad para las aplicaciones prácticas. 

Video: Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

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 ...

Investigación

Química

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

Marcos de síntesis y caracterización de funcionalizado metal-orgánico

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Metal-free Synthesis of Ynones from Acyl Chlorides and Potassium Alkynyltrifluoroborate Salts

Ynones are a valuable functional group and building block in organic synthesis. Ynones serve as a precursor to many important organic functional groups and scaffolds. Traditional methods for the preparation of ynones are associated with drawbacks including harsh conditions, multiple purification steps, and the presence of unwanted byproducts. An alternative method for the straightforward preparation of ynones from acyl chlorides and potassium alkynyltrifluoroborate salts is described herein. The adoption of organotrifluoroborate salts as an alternative to organometallic reagents for the formation of new carbon-carbon bonds has a number of advantages. Potassium organotrifluoroborate salts are shelf stable, have good functional group tolerance, low toxicity, and a wide variety are straightforward to prepare. The title reaction proceeds rapidly at ambient temperature in the presence of a Lewis acid without the exclusion of air and moisture. Fair to excellent yields may be obtained via reaction of various aryl and alkyl acid chlorides with alkynyltrifluoroborate salts in the presence of boron trichloride.

The goal of this manuscript is to demonstrate a straightforward method for the preparation of ynones from acyl chloride and potassium alkynyltrifluoroborate salt starting materials. The one-pot reaction proceeds rapidly in the presence of boron trichloride without exclusion of air and moisture.

Sin metal Síntesis de Ynones de Acil cloruros y potasio Alkynyltrifluoroborate Sales

Ynones are a valuable functional group and building block in organic synthesis. Ynones serve as a precursor to many important organic functional groups ...

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the hydroxylmethylene phosphine precursor with ammonia in methanol under a nitrogen atmosphere. The N-triphosPh ligand precipitates from the solution after approximately 1 hr of reflux and can be isolated analytically pure via simple cannula filtration procedure under nitrogen. Reaction of the N-triphosPh ligand with [Ru3(CO)12] under reflux affords a deep red solution that show evolution of CO gas on ligand complexation. Orange crystals of the complex [Ru(CO)2{N(CH2PPh2)3}-&#954;3P] (2) were isolated on cooling to RT. The 31P{1H} NMR spectrum showed a characteristic single peak at lower frequency compared to the free ligand. Reaction of a toluene solution of complex 2 with oxygen resulted in the instantaneous precipitation of the carbonate complex [Ru(CO3)(CO){N(CH2PPh2)3}-&#954;3P] (3) as an air stable orange solid. Subsequent hydrogenation of 3 under 15 bar of hydrogen in a high-pressure reactor gave the dihydride complex [RuH2(CO){N(CH2PPh2)3}-&#954;3P] (4), which was fully characterized by X-ray crystallography and NMR spectroscopy. Complexes 3 and 4 are potentially useful catalyst precursors for a range of hydrogenation reactions, including biomass-derived products such as levulinic acid (LA). Complex 4 was found to cleanly react with LA in the presence of the proton source additive NH4PF6 to give [Ru(CO){N(CH2PPh2)3}-&#954;3P{CH3CO(CH2)2CO2H}-&#954;2O](PF6) (6).

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Ruthenium phosphine complexes are widely used for homogeneous catalytic reactions such as hydrogenations. The synthesis of a series of novel tridentate ruthenium complexes bearing the N-triphos ligand N(CH2PPh2)3 is reported. Additionally, the stoichiometric reaction of a dihydride Ru&#8211;N-triphos complex with levulinic acid is described.

La síntesis, caracterización y reactividad de una serie de rutenio<em&gt; N</em&gt; -triphos<sup&gt; Ph</sup&gt; Complejos

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the ...

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large extinction coefficients which can be tuned across the visible spectrum. Research into the plasmonic enhancement of optical transitions has become popular, due to the possibility of altering and in some cases improving photo-absorption or emission properties of nearby chromophores such as molecular dyes or quantum dots. The electric field of the plasmon can couple with the excitation dipole of a chromophore, perturbing the electronic states involved in the transition and leading to increased absorption and emission rates. These enhancements can also be negated at close distances by energy transfer mechanism, making the spatial arrangement of the two species critical. Ultimately, enhancement of light harvesting efficiency in plasmonic solar cells could lead to thinner and, therefore, lower cost devices. The development of hybrid core/shell particles could offer a solution to this issue. The addition of a dielectric spacer between a gold nanoparticles and a chromophore is the proposed method to control the exciton plasmon coupling strength and thereby balance losses with the plasmonic gains. A detailed procedure for the coating of gold nanoparticles with CdS and ZnS semiconductor shells is presented. The nanoparticles show high uniformity with size control in both the core gold particles and shell species allowing for a more accurate investigation into the plasmonic enhancement of external chromophores.

The synthesis of uniform gold nanoparticles coated with semiconductor shells of CdS or ZnS is performed. The semiconductor coating is conducted by first depositing a silver sulfide shell and exchanging the silver cations for zinc or cadmium cations.

Síntesis, caracterización y funcionalización de híbridos Au / CdS y Au / ZnS Core / Shell nanopartículas

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Síntesis y caracterización de supramoleculares Coloides

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

Ensayo basado en HPLC para supervisar extracelular de nucleótidos / nucleósidos de metabolismo en las células de la leucemia crónica humana linfocítica

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis. The cyclic polymer exhibits multiple-mode diffusion which is distinct from its linear counterpart. The results demonstrate that the diffusional heterogeneity of polymers that is often hidden behind ensemble averaging can be revealed by the efficient synthesis of the cyclic polymers using the ESA-CF process and the quantitative analysis of the diffusive motion at the single molecule level using the MSD and CDF analyses.

A protocol for the synthesis and characterization of diffusive motion of cyclic polymers at the single molecule level is presented.

Síntesis de polímeros cíclicos y caracterización de su movimiento difusivo en el estado fundido en la molécula de un solo nivel

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single ...

Synthesis and Characterization of Fe-doped Aluminosilicate Nanotubes with Enhanced Electron Conductive Properties

The goal of the protocol is to synthesize Fe-doped aluminosilicate nanotubes of the imogolite type with the formula (OH)3Al2-xFexO3SiOH. Doping with Fe aims at lowering the band gap of imogolite, an insulator with the chemical formula (OH)3Al2O3SiOH, and at modifying its adsorption properties towards azo-dyes, an important class of organic pollutants of both wastewater and groundwater.
Fe-doped nanotubes are obtained in two ways: by direct synthesis, where FeCl3 is added to an aqueous mixture of the Si and Al precursors, and by post-synthesis loading, where preformed nanotubes are put in contact with a FeCl3&#8226;6H2O aqueous solution. In both synthesis methods, isomorphic substitution of Al3+ by Fe3+ occurs, preserving the nanotube structure. Isomorphic substitution is indeed limited to a mass fraction of ~1.0% Fe, since at a higher Fe content (i.e., a mass fraction of 1.4% Fe), Fe2O3 clusters form, especially when the loading procedure is adopted. The physicochemical properties of the materials are studied by means of X-ray powder diffraction (XRD), N2 sorption isotherms at -196 &#176;C, high resolution transmission electron microscopy (HRTEM), diffuse reflectance (DR) UV-Vis spectroscopy, and &#950;-potential measurements. The most relevant result is the possibility to replace Al3+ ions (located on the outer surface of the nanotubes) by post-synthesis loading on preformed imogolite without perturbing the delicate hydrolysis equilibria occurring during nanotube formation. During the loading procedure, an anionic exchange occurs, where Al3+ ions on the outer surface of the nanotubes are replaced by Fe3+ ions. In Fe-doped aluminosilicate nanotubes, isomorphic substitution of Al3+ by Fe3+ is found to affect the band gap of doped imogolite. Nonetheless, Fe3+ sites on the outer surface of nanotubes are able to coordinate organic moieties, like the azo-dye Acid Orange 7, through a ligand-displacement mechanism occurring in an aqueous solution.

Here, we present a protocol to synthesize and characterize Fe-doped aluminosilicate nanotubes. The materials are obtained by either sol-gel synthesis upon addition of FeCl3&#8226;6H2O to the mixture containing the Si and Al precursors or by post-synthesis ionic exchange of preformed aluminosilicate nanotubes.

Síntesis y caracterización de dopado con Fe aluminosilicato nanotubos con propiedades conductoras mejoradas Electron

The goal of the protocol is to synthesize Fe-doped aluminosilicate nanotubes of the imogolite type with the formula (OH)3Al2-xFexO3SiOH. Doping with Fe ...

Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. Their synthesis by conventional organic chemistry often requires tedious multi-step synthesis processes, with difficult control of the stereochemical outcome. We present a protocol to enzymatically synthetize amino alcohols starting from the readily available L-lysine in 48 h. This protocol combines two chemical reactions that are very difficult to conduct by conventional organic synthesis. In the first step, the regio- and diastereoselective oxidation of an unactivated C-H bond of the lysine side-chain is catalyzed by a dioxygenase; a second regio- and diastereoselective oxidation catalyzed by a regiodivergent dioxygenase can lead to the formation of the 1,2-diols. In the last step, the carboxylic group of the alpha amino acid is cleaved by a pyridoxal-phosphate (PLP) decarboxylase (DC). This decarboxylative step only affects the alpha carbon of the amino acid, retaining the hydroxy-substituted stereogenic center in a beta/gamma position. The resulting amino alcohols are therefore optically enriched. The protocol was successfully applied to the semipreparative-scale synthesis of four amino alcohols. Monitoring of the reactions was conducted by high performance liquid chromatography (HPLC) after derivatization by 1-fluoro-2,4-dinitrobenzene. Straightforward purification by solid-phase extraction (SPE) afforded the amino alcohols with excellent yields (93% to &#62;95%).

Chiral amino alcohols are versatile molecules for use as scaffolds in organic synthesis. Starting from L-lysine, we synthesize amino alcohols by an enzymatic cascade reaction combining diastereoselective C-H oxidation catalyzed by dioxygenase followed by cleavage of the carboxylic acid moiety of the corresponding hydroxyl amino acid by a decarboxylase.

Reacciones de la cascada enzimática para la síntesis de aminoalcoholes quirales de L-lisina

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. ...