Este estudio fue apoyado por el Departamento de Energía de EE.UU. (DE-FG02-09ER09-08 y DE-PS02-09ER09 18), el Centro Comprensivo del Cáncer Jonsson de la UCLA, y el Programa de Industria-Universidad de Investigación Cooperativa (UC Discovery Grant, bio07 -10.665). Agradecemos al Dr. Nagichettiar Satyamurthy y el personal en las instalaciones de UCLA Biomédica ciclotrón para la prestación del F-18 de radioisótopos y muchas discusiones profundas. Agradecemos a los Dres. Michael Collins, Greg Leblanc, José Lambert, y Keller Barnhardt de CEM para su asesoramiento y apoyo técnico. Damos las gracias a Dirk Williams, Williams Darin, los Dres. Joseph Hong Lin Dun y Michael van Dam para el diseño y mecanizado de piezas para modificar el reactor de microondas CEM y para los módulos de depuración de SPE.

1. Los preparativos iniciales <ol><li> Un V-vial (5 ml) RV1 (con barra de agitación) se utiliza como recipiente de la reacción principal para llevar a cabo la síntesis de microondas. Se conecta a un adaptador de PEEK con siete de entrada / salida conecta los puertos y dentro de la cavidad de microondas (ver Figura 2). RV2 está conectado al cartucho de SPE (I) para recoger el crudo [18 F] SFB. RV3 está conectado al cartucho de SPE (II) para la recogida de final [18 F] solución de SFB. Se puede colocar en un baño de agua tibia (40 ° C) para concentrar la solución correspondiente antes de la reconstitución en tampón PBS, especialmente para el radiomarcado aguas abajo de las biomoléculas. </li><li> Configuración para la recolección de crudo [18 F] SFB: Llene MeCN / H 2 O [6 mL, 1:4 (v: v)] solución acuosa al 5% AcOH (8 ml), MeCN (2 ml) para un depósito, B y C, respectivamente. A continuación, activar un cartucho de SPE (I) (poliestireno, Merck LiCholut EN) con etanol (10 ml), seguido por acuosa al 5% AcOH (10 ml) de lavado. </li><li> Configuración para la recogida purificado [18 F] SFB: Prepare depósito D y E se llenó con 10 ml de H 2 O y 3 ml de éter dietílico, respectivamente. El segundo cartucho de SPE (II) (poliestireno, Merck LiCholut EN) es activada por el mismo procedimiento antes mencionado. </li><li> Inicie el HPLC (tampón de elución: MeCN / H 2 O, 1:1 (v / v) que contenía 0,2% TFA, caudal: 3 ml / min) para pre-acondicionamiento de la columna HPLC [a fase inversa semi-prep columna (Luna, 5 micras C18 (2) 100 A, 250 x 10 mm), Phenomenex, Torrance, CA, EE.UU.]. </li></ol> 2. Preparación de secado [es decir, no portador añadido, (NCA)] [18 F] fluoruro <ol><li> [18 F] una solución de fluoruro en [18 O] H 2 O (100 l) se añadió a una mezcla de Kryptofix 222 (20 mg), 1M acuosa K 2 CO 3 (26 l) y MeCN (0,8 ml) en un Eppendorf tubo. Toda la solución se mezcla bien antes de transferir a la RV1 a través de la línea de entrada 1. El [18 F] una solución de fluoruro también puede ser pasado a través de un cartucho de intercambio aniónico (por ejemplo, CMA-luz Sep-Pak de Waters) para atrapar el flúor-18 y luego se eluye con una mezcla de K 2 CO 3 y Kryptofix en MeCN. </li><li> Ejecutar la secuencia de secado (20 W, 3 min), bajo el programa de control de microondas para eliminar el agua residual en RV1 [al vacío]. Después de que el enfriamiento de la temperatura del sistema es inferior a 50 ° C, más MeCN (1,0 ml) se introduce en el reactor y la secuencia se repite una vez. </li></ol> 3. Síntesis de 4 - [18 F] fluorobenzoato <ol><li> A una solución de DMSO (0,4 ml) contiene 4 - (N, N, N-trimetilamonio) benzoato de triflato (1,5 mg) en RV1 través de la línea de entrada 2. </li><li> Ejecutar la secuencia de etiquetado (50 W, 1 min), bajo el programa de control de microondas con el enfriamiento de agitación buque, y todas las válvulas cerradas para pagar 4 - [18 F] fluorobenzoato ([18 F] 2). </li></ol> 4. Síntesis de potasio 4 - [18 F] fluorobenzoato <ol><li> A una solución de DMSO (0,5 ml) contiene KOtBu (13 mg) en RV1 a través de la línea de entrada 3. </li><li> Ejecutar programa de desproteger (40 W, 1 min), bajo el programa de control de microondas con el enfriamiento de agitación buque, y todas las válvulas cerradas para permitir el 4 - [18 F] fluorobenzoato sal ([18 F] 3). </li></ol> 5. Síntesis de crudo [18 F] SFB <ol><li> Para una solución de acetonitrilo (2,5 ml) contiene TSTU (30 mg) se añadió a la línea de entrada a través de RV1 6. TSTU es la humedad y sensibles a la luz. Cabe alícuotas en frasco pequeño y se almacenan a 4 ° C en un recipiente cerrado, cubiertas con papel de aluminio. </li><li> Ejecutar la secuencia de acoplamiento (30 W, 2 min), bajo el programa de control de microondas con el enfriamiento de agitación buque, y todas las válvulas cerradas para permitir el crudo [18 F] SFB. </li></ol> 6. La preparación de los SPE-purificado [18 F] SFB <ol><li> Acuosa al 5% AcOH (1,0 ml) se añadió a través de RV1 línea de entrada de 7 a neutralizar la mezcla de reacción. La solución había sido trasladado en B vial que contiene 8 ml de una solución acuosa al 5% AcOH (Figura 2). </li><li> Pase la mezcla de reacción se diluyó a través del cartucho SPE (I) para atrapar crudo [18 F] SFB utilizando nitrógeno (10 psi). </li><li> LAVADO SPE cartucho (I) con una mezcla de MeCN y H 2 O [10 mL, 1:4 (v: v)] desde el embalse de A. </li><li> [18 F] SFB se eluyó a cabo en RV2 con MeCN (2 ml) desde el embalse de C. </li></ol> 7. Purificación del crudo [18 F] SFB con Radio-HPLC <ol><li> Diluir bien crudo [18 F] SFB o purificada-SPE [18 F] SFB con H 2 O (2 ml) en RV2 y la transferencia de la mezcla en el circuito de HPLC (5 ml). La solución se inyecta en la radio-HPLC [O MeCN / H 2, 1:1 (v / v) que contenía 0,2% TFA, caudal: 3 ml / min]. </li><li> Recoger la fracción que contiene purificado [18 F] SFB (retención de time: 8-10 minutos) en el vial D (precargada con 10 ml de H 2 O) (Figura 2). Paso crítico: Si se realiza correctamente, el volumen de fracción recogida aquí debe ser 4-5 ml. </li><li> Pase la mezcla de reacción se diluyó a través del cartucho SPE (II) para atrapar purificado [18 F] SFB utilizando nitrógeno (10 psi). Secar el cartucho con una corriente de nitrógeno durante 2-3 minutos. </li><li> [18 F] SFB se eluyó a cabo en RV3 con éter dietílico (3 ml) desde el embalse de E.. </li><li> Se evapora el solvente en RV3 a sequedad mediante una suave corriente de gas nitrógeno (10 psi) con un baño de agua (40 ° C). El secado final [18 F] SFB se puede reconstituir en tampón PBS para la aplicación posterior. </li></ol> 8. Los resultados representativos: Hemos desarrollado un sistema simplificado y rápido, una olla de método para la síntesis de [18 F] SFB utilizando una estrategia de desprotección en condiciones anhidras y calentamiento con microondas en cada transformación radioquímica / químicos. La figura 1 presenta los detalles de nuestro radiosíntesis. La identidad del producto final se confirmó mediante la comparación del tiempo de retención de HPLC con una referencia no radiactivo SFB. El purificado [18 F] SFB también fue analizado a través de la radio-TLC y HPLC para determinar la pureza radioquímica y química. El RCY de [18 F] SFB fue de 35 ± 5% dentro de los 60 minutos después de la purificación por HPLC (n&gt; 30), de alta pureza radioquímica (&gt; 99%) y buena pureza química (ver la huella UV en el perfil de HPLC, la Figura 3 ). La actividad específica fue de aprox. 67-330 GBq / mmol (1.8 a 9.0 Ci / mmol), dependiendo de la radiactividad inicial. <img src="/files/ftp_upload/2755/2755fig1.jpg" alt="Figura 1" /> Figura 1. Asistida por microondas en un único recipiente radiosíntesis de [18 F] SFB. En primer lugar, la radiofluorination de 4 - (N, N, N-trimetilamonio) benzoato de triflato (1) se realizó en el calentamiento por microondas (50 W, 1 min) en presencia de [K ⊃ 2.2.2] [18 F] F - complejo en dimetilsulfóxido (DMSO) para dar 4 - [18 F] fluorobenzoato ([18 F] 2). Sin purificación, una solución de DMSO de terc-butóxido (t BuOK) y se añadió el recipiente de reacción se irradia microondas (40 W, 1 min) para completar la desprotección anhidro. La conversión final de [18 F] 3 en [18 F] SFB se logró mediante O-(N-succinimidil) - N, N, N &#39;, N&#39;-tetramethyluronium tetrafluoroborato (TSTU) de activación. TSTU en acetonitrilo se añadió a la mezcla de reacción que contiene el 4 - [18 F] fluorobenzoato ([18 F] 3) la sal, este paso dado crudo sintético pasado [18 F] SFB después de la calefacción (30 W, 2 min). <img src="/files/ftp_upload/2755/2755fig2.jpg" alt="Figura 2" /> Figura 2. El esquema de configuración de microondas con ayuda de una olla de [18 F] síntesis de SFB. <img src="/files/ftp_upload/2755/2755fig3.jpg" alt="Figura 3" /> Figura 3. HPL Radio-cromatogramas de final [18 F] SFB. Arriba: La señal de UV a 254 nm, en la planta: la señal radiactiva; recuadro: UV señal a 254 nm (x 33,3). <img src="/files/ftp_upload/2755/2755table1.jpg" alt="Tabla 1" /> . Tabla 1 Resumen de [18 F] radiosyntheses SFB en la literatura con alquilo 4 - (trimetilamonio) triflato benzoato como precursores.

Este método se ha presentado para la aplicación de patente de los EE.UU..

Esto simplifica en tres pasos, en un único recipiente radiosíntesis de los 18 F-acilación reactivo [18 F] SFB se desarrolla sobre la base de la química no acuosos. Este proceso tiene una excelente reproducibilidad y podría ser utilizado de forma fiable para la producción de [18 F] SFB en los módulos de radioquímica automatizado, debido a dos modificaciones principales que se describen como de seguidores: 1. Contamos con una etapa de desprotección / saponificación en anhidro KOt...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> ácido acético en solución acuosa (5% v / v) </td><td> Pescador </td><td> A38-500 </td><td> Preparado en nuestro laboratorio </td></tr><tr><td> Acetonitrilo </td><td> Sigma-Aldrich </td><td> 75-05-8 </td><td></td></tr><tr><td> Éter dietílico </td><td> Sigma-Aldrich </td><td> 14775 </td><td></td></tr><tr><td> Dimetilsulfóxido (DMSO) </td><td> Sigma-Aldrich </td><td> 472301 </td><td></td></tr><tr><td> 4 - (N, N, N-trimetilamonio) benzoato de triflato </td><td> Preparados en el laboratorio </td><td></td><td></td></tr><tr><td> 4,7,13,16,21,24-Hexaoxa-1 ,10-diazabiciclo [8.8.8] hexacosane (K222) </td><td> Sigma-Aldrich </td><td> 29,111-0 </td><td></td></tr><tr><td> O - (N-succinimidil) - N, N, N &#39;, N&#39;-tetramethyluronium tetrafluoroborato (TSTU) </td><td> Sigma-Aldrich </td><td> 105832-38-0 </td><td></td></tr><tr><td> El carbonato de potasio en solución acuosa (1M) </td><td> Sigma-Aldrich </td><td> 209619 </td><td> Preparado en nuestro laboratorio </td></tr><tr><td> Terc-butóxido </td><td> Sigma-Aldrich </td><td> 156671 </td><td></td></tr></tbody></table>

microwave-assisted one-pot synthesis of n-succinimidyl-4-[18f]fluorobenzoate ([18f]sfb)

Biomoléculas, como péptidos, proteínas 01.09, 10,11 y anticuerpos y sus fragmentos de ingeniería, 12-14 están ganando importancia como agentes terapéuticos potenciales y de imagen molecular. En particular, cuando se han etiquetado con radioisótopos emisores de positrones (por ejemplo, Cu-64, Ga-68 o F-18), que pueden ser utilizados como sondas para la imagen objetivo de muchos procesos fisiológicos y patológicos. 15-18 Por lo tanto, un esfuerzo significativo ha dedicado a la síntesis y la exploración de 18 F-etiquetados biomoléculas. Aunque hay ejemplos de la elegante directa 18 F-etiquetado de los péptidos, 19-22 de las condiciones de reacción dura (es decir, un disolvente orgánico pH, extrema, alta temperatura) asociados con radiofluorination directos suelen ser incompatibles con las muestras de proteínas frágiles. Hasta la fecha, por lo tanto, la incorporación de grupos prostéticos radiomarcado en biomoléculas sigue siendo el método de elección 23,24. N-succinimidil-4-[18 F] fluorobenzoato ([18 F] SFB), 25-37 de un reactivo tipo de Bolton-Hunter que reacciona con los grupos amino primarios de las biomoléculas, es un grupo prostético muy versátil para los 18 F-etiquetado de un amplio espectro de entidades biológicas, en términos de su evidente en la estabilidad y el rendimiento in vivo de alta radiomarcado. Tras el marcaje con [18 F] SFB, el resultado [18 F] biomoléculas fluorobenzoylated podrían explorarse como trazadores de PET potencial de los estudios in vivo de imágenes. La mayoría de 1 [18 F] radiosyntheses SFB se describe en la literatura actual requieren dos o hasta tres reactores y purificaciones múltiples mediante el uso de cualquiera de extracción en fase sólida (SPE) o de alto rendimiento de cromatografía líquida (HPLC). Tales procesos largos dificultan la producción de rutina y amplias aplicaciones en el radiomarcado de biomoléculas. A pesar de varios módulos con ayuda de [18 F] síntesis SFB se ha informado, 29-32, 41-42 que se basan principalmente en procedimientos complicados y largos con costosos disponibles en el mercado cajas de radioquímica (Tabla 1). Por lo tanto, una mayor simplificación de la radiosíntesis de [18 F] SFB utilizando una configuración de bajo costo sería muy beneficioso para su adaptación a un proceso automatizado. Presentamos una preparación concisa de [18 F] SFB, basado en una versión simplificada de una olla asistida por microondas de síntesis (Figura 1). Nuestro enfoque no requiere la purificación entre los pasos o los reactivos acuosos. Además, la irradiación de microondas, que se ha utilizado en la síntesis de varios trazadores de PET, 38-41 se da un mayor RCYs y una mejor selectividad de la reacción térmica correspondiente o que proporcionan rendimientos similares en los tiempos de reacción más cortos. 38 más importante, cuando el etiquetado biomoléculas , el tiempo ahorrado podría ser desviado a bioconjugation paso posterior o PET 28,43 La novedad de nuestro mejor [18 F] síntesis de SFB es doble: (1). la estrategia de desprotección anhidro no requiere la purificación de intermedios (s) entre cada paso y (2) el horno de microondas con ayuda de las transformaciones radioquímica permitir la producción rápida y fiable de [18 F] SFB.

Watch this Scientific Journal Video about Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB at JoVE.com

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB

Una fácil, one-pot síntesis de N-succinimidil-4-[<sup&gt; 18</sup&gt; F] fluorobenzoato ([<sup&gt; 18</sup&gt; F] SFB) fue desarrollado sobre la base de un no-acuoso, proceso radioquímico de tres pasos. Usando calentamiento por microondas, el procedimiento se puede completar en menos de 30 minutos o 60 minutos con posterior purificación por HPLC preparativa. La decadencia corregida rendimiento radioquímico (RCYs) fueron 35-5% (n&gt; 30).

Asistida por microondas de un bote de síntesis de N-Succinimidil-4-[ 18 F] fluorobenzoato ([ 18 F] SFB)

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential ...

microwave-assisted-one-pot-synthesis-n-succinimidyl-4

Research

JoVE Journal

Biology

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)

12.9K vistas.  University of California at Los Angeles. Una fácil, one-pot síntesis de N-succinimidil-4-[ 18 F] fluorobenzoato ([ 18 F] SFB) fue desarrollado sobre la base de un no-acuoso, proceso radioquímico de tres pasos. Usando calentamiento por microondas, el procedimiento se puede completar en menos de 30 minutos o 60 minutos con posterior purificación por HPLC preparativa. La decadencia corregida rendimiento radioquímico (RCYs) fueron 35-5% (n> 30).

Video: Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described. Triplicate runs of 48 parallel experiments are reported, varying concentration, solvent and the excess of some of the reagents. The isolation of the product was achieved by a simple filtration and wash procedure. The highest yield obtained was 66% from 0.4 M methanol with 1.2 eq. of imine. This is significantly above the 49% yield obtained from the initial reaction under equimolar concentration at 0.4 M in methanol. Methanol solutions with reagent concentrations of 0.4M or 0.2M gave superior yields while all solvent systems at 0.07M performed poorly. At 0.2M, methanol and ethanol/methanol (60/40) mixtures were statistically equally good while THF/methanol (60/40) was poor and acetonitrile/methanol (60/40) was intermediate. Good reproducibility of the precipitate yields was obtained in these replicate experiments, allowing for subtle interaction effects to be positively identified.

The Ugi reaction has proved to be a convenient way to quickly create diverse libraries of compounds. It involves the reaction of an amine, an aldehyde, a carboxylic acid and an isonitrile typically in methanol at room temperature. In this video, we utilize a 48-slot Mettler-Toledo MiniBlock equipped with filtration tubes and a Mettler-Toledo MiniMapper automated liquid handler was used to deliver the reagents and solvent. The parameters of interest were the concentration, the solvent composition and the excess of some of the reagents.

Optimización de la reacción de Ugi mediante la síntesis en paralelo y manejo de líquidos automatizados

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described.  Triplicate runs ...

Investigación

Biología

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

Síntesis y caracterización de nanosondas fosforescentes de imágenes de oxígeno en sistemas biológicos

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

Síntesis de un<em&gt; En vivo</em&gt; RM-detectable apoptosis de la sonda

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides1.
Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides2, which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield1, but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation.
Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an &alpha;-amine)3,4. Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.

Solid Phase Synthesis of a Functionalized Bis-Peptide Using &quot;Safety Catch&quot; Methodology

The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.

Síntesis en fase sólida de un péptido funcionalizado Bis-El uso de &quot;Cierre de seguridad&quot; Metodología

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Análisis de RNA Global Síntesis en el nivel de células individuales siguiente Hipoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Medición de niveles hemo síntesis en células de mamífero

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

Síntesis in vitro de ARNm modificado para la inducción de la expresión proteica en las células humanas

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

Síntesis de ADN sondas de hibridación de longitud de onda de desplazamiento mediante fotoestable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

Preparación de muestras biológicas por congelación de alta presión, realce del contraste asistida por microondas y mínima inclusión de proyección de imagen de volumen

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Here, we introduce and describe a nonradioactive and noninvasive method to assess de novo protein synthesis in vivo, utilizing the nematode Caenorhabditis elegans and fluorescence recovery after photobleaching (FRAP). This method can be combined with genetic and/or pharmacological screens to identify novel modulators of protein synthesis.

Evaluación de las tasas de síntesis de proteínas de novo en Caenorhabditis elegans

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and ...