Name: solid-phase synthesis of [4.4] spirocyclic oximes
Uploaded: 2019-02-06T13:00:00
Description: Watch this Scientific Journal Video about Solid-phase Synthesis of [4.4] Spirocyclic Oximes at JoVE.com

Este trabajo fue financiado por una subvención del Consejo de investigación de Facultad a K.S. Huang (Azusa Pacific University - Estados Unidos). Drisko C.R. es un recipiente de la beca de Stauffer de John y la beca de investigación de pregrado de Gencarella. Griffin S.A. recibió una beca de investigación en pregrado de S2S del Departamento de biología y química.
<img alt="Image 1" src="/files/ftp_upload/58508/58508img1.jpg">
Autores (de izquierda a derecha) Cody Drisko, Dr. Kevin Huang y Silas Griffin llevado a cabo los experimentos y prepararon el manuscrito. Cody Drisko es John Stauffer Fellow y recibió la beca de investigación Gencarela. Silas es un S2S Azusa Pacific University Research Fellow. Dr. Kevin Huang proporcionó la orientación de la investigación y es un receptor de la Azusa Pacific University Facultad investigación Consejo concesión.

PRECAUCIÓN: Consulte todas las hojas de datos de seguridad del material (MSDS) antes de su uso. Varios de los productos químicos utilizados en estas síntesis son muy tóxicos y cancerígenos. Utilice todas las prácticas de seguridad apropiadas cuando se realizan las siguientes reacciones, incluyendo el uso de controles de ingeniería (campana y espectrómetros IR y RMN) y equipo de protección personal (gafas, guantes, bata, pantalones largos, y zapatos cerrados).
1. Michael además de Furfu...

En una típica REM vinculador/fase sólida sintética estrategia, antes de la liberación de una amina con el apoyo sólido, es fundamental para formar una sal de amonio cuaternario, como se describe en la sección 4 del protocolo39. Debido al impedimento estérico del sistema tricíclico y voluminosos R2 grupos (Haluros de bencilo y octilo), sólo pequeños reactivos alquilantes (metil y alil halides) pueden ser utilizados de esta reacción46. Con una simple mod...

A pesar de recientes descubrimientos en heterociclos funcionalizados altamente espirocícliclos de un número de sistemas biológicos1, una vía conveniente sigue siendo necesaria para su fabricación fácil. Tales sistemas y usos de estos heterociclos incluyen: MDM2 inhibición y otras actividades contra el cáncer2,3,4,5, enzima inhibición6,7,8 , actividad antibiótica9,10, fluorescente etiquetado10,11,12, enantioselectiva para ADN sondas13,14, 15 y ARN dirigida a16, junto con numerosas posibles aplicaciones a la terapéutica17,18,19. Con una demanda creciente de estos heterociclos, literatura actual sigue dividida sobre qué camino sintético es mejor. Enfoques sintéticos modernos este problema utilizan Isatina y isatin derivados a partir materiales para una variedad de heterociclos20,21, reordenamientos intramoleculares complicado22,23 ,24,25, Lewis ácido1,26,27 o metales de transición catálisis17,28,29, 30, o procesos asimétricos31. Aunque estos procedimientos han tenido éxito en la producción de oximas espirocícliclos específico con funcionalidad limitada, una estrategia sintética para producir una biblioteca de moléculas con alta diastereoselectividad ha sido explorada relativamente menos32.
La técnica presentada aquí muestra que estas moléculas de interés pueden ser generadas usando un número de técnicas sintéticas bien entendidos en tándem. A partir de la síntesis de la molécula sobre un soporte sólido mediante un vinculador de REM y cicloadición de nitruro-olefina de sililo intramolecular (ISOC), la vía propuesta implementa un recorrido no lineal, caracterizado por el enlace de cortar en un sistema tricíclico, dejando un ingredientes farmacéuticos activos altamente funcionalizados. Enlazadores de REM, conocidos por su comodidad y capacidad de reciclaje, utilizan un soporte sólido para sintetizar aminas terciarias33. Debido a la facilidad de purificación ante la REM vinculador por simple filtración, esta técnica de síntesis en fase sólida proporciona a los científicos con un enlazador reciclable y sin dejar huellas, que se ha utilizado aquí. Una vez completada la reacción, el vinculador de REM se regenera y puede ser reutilizado varias veces. El vinculador REM también es traceless porque, a diferencia de muchos elementos de conexión de fase sólida, el punto de conexión entre el producto y el polímero es indistinguible34,35. También bien estudiado y comprendido es la reacción de ISOC, útil en la síntesis de pirrolidina oximas36,37. Tal vez mejor conocido como un 1, 3-dipolar cycloaddition, estas reacciones forman un número de heterociclos con alta diastereoselectividad38,39,40,41,42 , 43 , 44 , 45. utilizando la técnica modificada de ISOC REM junto para la síntesis de moléculas de espirocícliclos rinde un producto altamente diastereoselectiva. Adjunto, Divulgamos sobre la producción eficiente de oximas espirocícliclos utilizando un enfoque sintético nuevo, combinando dos caminos bien entendidos y materias primas fácilmente disponibles.

<table>
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>REM Resin</td>
			<td>Nova Biochem</td>
			<td>8551010005</td>
			<td>Solid Polymer Support; 1.1 mmol/g loading</td>
		</tr>
		<tr>
			<td>Furfurylamine</td>
			<td>Acros Organics</td>
			<td>119800050</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Dimethylformamide (DMF)</td>
			<td>Sigma-Aldrich</td>
			<td>227056</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>trans-4-bromo-&#946;-nitrostyrene</td>
			<td>Sigma-Aldrich</td>
			<td>400017</td>
			<td>Nitro-olefin solid</td>
		</tr>
		<tr>
			<td>trans-3,4-dimethoxy-&#946;-nitrostyrene</td>
			<td>Sigma-Aldrich</td>
			<td>S752215</td>
			<td>Nitro-olefin solid</td>
		</tr>
		<tr>
			<td>trans-2,4-dichloro-&#946;-nitrostyrene</td>
			<td>Sigma-Aldrich</td>
			<td>642169</td>
			<td>Nitro-olefin solid</td>
		</tr>
		<tr>
			<td>trans-&#946;-nitrostyrene</td>
			<td>Sigma-Aldrich</td>
			<td>N26806</td>
			<td>Nitro-olefin solid</td>
		</tr>
		<tr>
			<td>Triethylamine (TEA)</td>
			<td>Sigma-Aldrich</td>
			<td>T0886</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Trimethylsilyl chloride (TMSCl)</td>
			<td>Sigma-Aldrich</td>
			<td>386529</td>
			<td>Reagent; CAUTION - highly volatile; creates HCl gas</td>
		</tr>
		<tr>
			<td>Tetra-n-butylammonium fluoride (TBAF) in Tetrahydrofuran (THF)</td>
			<td>Sigma-Aldrich</td>
			<td>216143</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Tetrahydrofuran (THF)</td>
			<td>Sigma-Aldrich</td>
			<td>401757</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>1-Bromooctane</td>
			<td>Sigma-Aldrich</td>
			<td>152951</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Iodomethane</td>
			<td>Sigma-Aldrich</td>
			<td>289566</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Allylbromide</td>
			<td>Sigma-Aldrich</td>
			<td>337528</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Benzylbromide</td>
			<td>Sigma-Aldrich</td>
			<td>B17905</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Glassware/Instrumentation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL solid-phase reaction vessel</td>
			<td>Chemglass</td>
			<td>CG-1861-02</td>
			<td>Glassware with filter</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nicole iS5</td>
			<td>Thermo Scientific</td>
			<td>IQLAADGAAGFAHDMAZA</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>AVANCE III NMR Spectrometer</td>
			<td>Bruker</td>
			<td>N/A</td>
			<td>Instrument; 300 MHz; Solvents: CDCl3 and CD3OH</td>
		</tr>
		<tr>
			<td>Wrist-Action Shaker Model 75</td>
			<td>Burrell Scientific</td>
			<td>757950819</td>
			<td>Instrument</td>
		</tr>
	</tbody>
</table>

solid-phase synthesis of [4.4] spirocyclic oximes

Una conveniente ruta sintética para heterociclos espirocícliclos es bien buscada debido al uso potencial de la molécula en los sistemas biológicos. Mediante síntesis en fase sólida, regenerar estrategias de vinculador de Michael (REM) y 1, 3-dipolar cycloaddition, una biblioteca de heterociclos estructuralmente similares, con o sin un centro de espirocícliclos, se puede construir. Las principales ventajas de la síntesis de sólido apoyo son los siguientes: en primer lugar, cada paso de la reacción puede ser conducido a la terminación con un exceso de reactivos, lo que resulta en altos rendimientos; siguiente, el uso de materias primas disponibles en el mercado y los reactivos de mantener los costos bajos; por último, las medidas de reacción son fáciles de purificar mediante filtración simple. La estrategia de vinculador de REM es atractiva debido a su reciclabilidad y la naturaleza sin dejar huellas. Una vez que un esquema de reacción se ha completado, el vinculador puede reutilizarse varias veces. En una síntesis típica de la fase sólida, el producto contiene una parte de o el vinculador entero, que puede resultar indeseable. El vinculador de REM es "traceless" y el punto de conexión entre el producto y el polímero es indistinguible. La alta diastereoselectividad de la cicloadición 1, 3-dipolar intramolecular está bien documentado. Limitada por la insolubilidad de la ayuda sólida, la progresión de reacción sólo controlables por un cambio en los grupos funcionales (si hubiera) mediante espectroscopia de infrarrojo (IR). Por lo tanto, la identificación estructural de productos intermedios no puede ser caracterizada por convencionales de resonancia magnética nuclear (NMR) espectroscopia. Otras limitaciones a este método se derivan de las compatibilidades de polímero/linker para el esquema de la reacción química deseada. Adjunto divulgamos un protocolo que permite para la producción conveniente de heterociclos espirocícliclos que, con simples modificaciones, se pueden automatizar con técnicas de alto rendimiento.

Como se indica en el procedimiento anterior, la ruta sintética para espirocícliclos oximas (ver figura 1) comienza con la adición de Michael de Furfurilamina para componer 1, el enlazador de REM, a pagar 2. Una posterior adición de Michael y 1, 3-dipolar cycloaddition del soporte 2 con producción de derivados de varios β-nitrostyrene los tricíclicos compuesto 3, un N- silyloxy...

Watch this Scientific Journal Video about Solid-phase Synthesis of [4.4] Spirocyclic Oximes at JoVE.com

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

Aquí presentamos un protocolo para demostrar un método eficiente para la síntesis de heterociclos espirocícliclos. El proceso de cinco pasos utiliza síntesis en fase sólida y regenerar estrategias de vinculador de Michael. Generalmente difícil de sintetizar, se presenta un método adaptable para la síntesis de moléculas de espirocícliclos otra manera inaccesibles a otros enfoques modernos.

Síntesis en fase sólida de oximas espirocícliclos [4,4]

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of ...

Esta técnica sintética orgánica de fase sólida puede encontrar aplicaciones en la construcción de una biblioteca combinatoria, mientras que la síntesis de heteciclos espirocíclicos het, que se pueden probar para actividades contra el cáncer utilizando bioensayos. Aunque este método se puede utilizar para la síntesis de compuestos espirocíclicos, también se puede aplicar a otras moléculas pequeñas, como isoxazolinas e isoxazoles. Para comenzar la síntesis, añadir a un recipiente de reacción de fase sólida de 25 mililitros un gramo de resina REM, 20 mililitros de dimetilformamida, y 2,4 mililitros de furfurilamina.Tapar el recipiente y agitarlo durante 24 horas en una coctelera a temperatura ambiente para cargar la resina con furfurilamina. A continuación, escurra la resina portadora de furfurylamina y agitar durante unos tres minutos en cinco mililitros de DMF para lavarla. Después de eso, lavar la resina cuatro veces, alternando entre cinco mililitros de diclorometano para hinchar la resina, y cinco mililitros de metanol para contraer la resina.A continuación, seque bien la resina con aire comprimido, que suele tardar unos 30 minutos. A continuación, en una campana de humo bien ventilada, añadir 1,48 mililitros de trietilamina, 10 mililitros de tolueno anhidro, y 637 gramos de beta nitrostireno a la resina de cojinete de furfurylamina seca. Añadir un mililitro de cloruro de trimetilo a la mezcla de reacción.Y deje el recipiente abierto hasta que la mezcla haya terminado de liberar gas clorhidrato. A continuación, tapar el recipiente y agitar la mezcla durante 48 horas a temperatura ambiente para formar el isoxazole intermedio. Después de eso, añadir cinco mililitros de metanol para apagar la reacción.Agitar la mezcla durante tres minutos, y luego dejar que se empape durante dos minutos. A continuación, escurra la resina y lávela cuatro veces, alternando entre cinco mililitros de DCM y cinco mililitros de metanol. Seque bien la resina lavada con aire comprimido.A continuación, añadir un mililitro de tetrahidrofurano anhidro a la resina seca. A continuación, añada 1,24 mililitros de una solución molar de tetra-butlammonio en el THF y agitar la mezcla a temperatura ambiente durante 12 horas para abrir el anillo de isoxazol. Después, colar la resina y lavarla con cinco mililitros de THF, seguido de cuatro lavados alternando entre cinco mililitros de DCM y cinco mililitros de metanol.Seque bien la resina con aire comprimido. A continuación, añadir cinco mililitros de DMF a la resina, seguido de un mililitro de un monlooctano. Agitar la mezcla durante 24 horas a temperatura ambiente para formar la amina cuaternaria.A continuación, escurra la resina y lávela con cinco mililitros de DMF. Lave la resina cuatro veces más, alternando entre DCM y metanol, y séquela con aire comprimido. A continuación, añadir tres mililitros de DCM a la resina seca, seguido de 1,5 mililitros de TEA.Agitar la mezcla durante 24 horas para cortar el oxime espirocíclico del soporte del polímero. A continuación, recoja el producto crudo en un vial o matraz. Lave la resina cuatro veces, alternando entre DCM y metanol, y recoja los lavados en el vial del producto.Retire los volátiles de la mezcla del producto en un evaporador rotatorio. A continuación, triturar el producto crudo con 5 mililitros de metanol hirviendo, y retirar cuidadosamente el líquido. Lavar la resina dos veces con cinco porciones de mililitro de DCM y secarla con aire comprimido para su reutilización en experimentos posteriores.Los oximes espiroclicólicos con varios sustitutos fueron sintetizados con excelente estereoselectividad. El rendimiento global indica un rendimiento medio de 80 a 88% para cada paso sintético. El progreso de la reacción a través de la abertura del anillo de isoxazol puede ser monitoreado por espectroscopia IR.Generalmente, los individuos nuevos en este método luchan con la selección del vinculador de soporte de polímero apropiado. Es esencial que el vinculador sea robusto para todas las condiciones de reacción durante la síntesis. Al intentar este procedimiento, recuerde lavar y secar la resina antes del siguiente paso de reacción.Los reactivos sobrantes del paso anterior complicarán la síntesis general y afectarán el rendimiento y la pureza del producto espirocíclico.

solid-phase-synthesis-of-44-spirocyclic-oximes

Research

JoVE Journal

Chemistry

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture&#160;and detection of circulating tumor cells (CTCs),&#160;and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.

Here, we describe a protocol for synthesis of magneto-plasmonic nanoparticles with a strong magnetic moment and a strong near-infrared (NIR) absorbance. The protocol also includes antibody conjugation to the nanoparticles through the Fc moiety for various biomedical applications which require molecular specific targeting.

Nanoclusters Síntesis de Immunotargeted Magneto-plasmónica

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications ...

Investigación

Química

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Metodología sintética para asimétrica Ferroceno derivados Sistemas de Bio-conjugadas vía sólida metodología a base de resina de fase

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zempl&#233;n.

Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

Síntesis de indoxil-glucósidos para la detección de glucosidasa Actividades

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

Hidroquinona basado Síntesis de oro Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

Synthesis of Hierarchical ZnO/CdSSe Heterostructure Nanotrees

A two-step chemical vapor deposition procedure is here employed to prepare tree-like hierarchical ZnO/CdSSe hetero-nanostructures. The structures are composed of CdSSe branches grown on ZnO nanowires that are vertically aligned on a transparent sapphire substrate. The morphology was measured via scanning electron microscopy. The crystal structure was determined by X-ray powder diffraction analysis. Both the ZnO stem and CdSSe branches have a predominantly wurtzite crystal structure. The mole ratio of S and Se in the CdSSe branches was measured by energy dispersive X-ray spectroscopy. The CdSSe branches result in strong visible light absorption. Photoluminescence (PL) spectroscopy showed that the stem and branches form a type-II heterojunction. PL lifetime measurements showed a decrease in the lifetime of emission from the trees when compared to emission from individual ZnO stems or CdSSe branches and indicate fast charge transfer between CdSSe and ZnO. The vertically aligned ZnO stems provide a direct electron transport pathway to the substrate and allow for efficient charge separation after photoexcitation by visible light. The combination of the abovementioned properties makes ZnO/CdSSe nanotrees promising candidates for applications in solar cells, photocatalysis, and opto-electronic devices.

Here, we prepare and characterize novel tree-like hierarchical ZnO/CdSSe nanostructures, where CdSSe branches are grown on vertically aligned ZnO nanowires. The resulting nanotrees are a potential material for solar energy conversion and other opto-electronic devices.

Síntesis del jerárquica ZnO / CdSSe heteroestructura Nanotrees

A two-step chemical vapor deposition procedure is here employed to prepare tree-like hierarchical ZnO/CdSSe hetero-nanostructures. The structures are ...

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

Integración de la Extracción de Fase Sólida Miniaturizada y la Detección de LC-MS / MS de 3-Nitrotirosina en Orina Humana para Aplicaciones Clínicas

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2&#039;-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

Protocolo para la síntesis en fase sólida de oligómeros de ARN que contiene un 2&#39;-<emO</em&gt; -tiofenilmetilo Modificación y caracterización mediante dicroísmo circular

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

Síntesis de nanocables de Au sustrato enlazado mediante un mecanismo activo crecimiento superficial

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a non-radioactive precursor in solution. This approach necessitates purification of the tracer by high performance liquid chromatography (HPLC) followed by reformulation in a biocompatible solvent for human administration. We recently developed a novel 11C-methylation approach for the highly efficient synthesis of carbon-11 labeled PET radiopharmaceuticals, taking advantage of solid phase cartridges as disposable &#34;3-in-1&#34; units for the synthesis, purification and reformulation of the tracers. This approach obviates the use of preparative HPLC and reduces the losses of the tracer in transfer lines and due to radioactive decay. Furthermore, the cartridge-based technique improves synthesis reliability, simplifies the automation process and facilitates compliance with the Good Manufacturing Practice (GMP) requirements. Here, we demonstrate this technique on the example of production of a PET tracer Pittsburgh compound B ([11C]PiB), a gold standard in vivo imaging agent for amyloid plaques in the human brains.

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

We report an efficient carbon-11 radiolabeling technique to produce clinically relevant tracers for Positron Emission Tomography (PET) using solid phase extraction cartridges. 11C-methylating agent is passed through a cartridge preloaded with precursor and successive elution with aqueous ethanol provides chemically and radiochemically pure PET tracers in high radiochemical yields.

Fase sólida 11C-Metilación, purificación y formulación para la producción de trazadores PET

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a ...

Gas Chromatography-Mass Spectrometry Paired with Total Vaporization Solid-Phase Microextraction as a Forensic Tool

Gas Chromatography &#8211; Mass Spectrometry (GC-MS) is a frequently used technique for the analysis of numerous analytes of forensic interest, including controlled substances, ignitable liquids, and explosives. GC-MS can be coupled with Solid-Phase Microextraction (SPME), in which a fiber with a sorptive coating is placed into the headspace above a sample or immersed in a liquid sample. Analytes are sorbed onto the fiber which is then placed inside the heated GC inlet for desorption. Total Vaporization Solid-Phase Microextraction (TV-SPME) utilizes the same technique as immersion SPME but immerses the fiber into a completely vaporized sample extract. This complete vaporization results in a partition between only the vapor phase and the SPME fiber without interference from a liquid phase or any insoluble materials. Depending upon the boiling point of the solvent used, TV-SPME allows for large sample volumes (e.g., up to hundreds of microliters). On-fiber derivatization may also be performed using TV-SPME. TV-SPME has been used to analyze drugs and their metabolites in hair, urine, and saliva. This simple technique has also been applied to street drugs, lipids, fuel samples, post-blast explosive residues, and pollutants in water. This paper highlights the use of TV-SPME to identify illegal adulterants in very small samples (microliter quantities) of alcoholic beverages. Both gamma-hydroxybutyrate (GHB) and gamma-butyrolactone (GBL) were identified at levels that would be found in spiked drinks. Derivatization by a trimethylsilyl agent allowed for conversion of the aqueous matrix and GHB into their TMS derivatives. Overall, TV-SPME is quick, easy, and requires no sample preparation aside from placing the sample into a headspace vial.

Total Vaporization Solid Phase Microextraction (TV-SPME) completely vaporizes a liquid sample whilst analytes are sorbed onto a SPME fiber. This allows for partitioning of the analyte between only the solvent vapor and the SPME fiber coating.

Cromatografía de Gases-Espectrometría de Masas Emparejada con La Vaporización Total De Microextracción En Fase Sólida Como Una Herramienta Forense

Gas Chromatography &#8211; Mass Spectrometry (GC-MS) is a frequently used technique for the analysis of numerous analytes of forensic interest, including ...