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 trabalho foi financiado por uma bolsa do Conselho de pesquisa da faculdade de Huang Konzen (Azusa Pacific University - Estados Unidos). C.R. Drisko é um receptor da John Stauffer bolsa e a bolsa de pesquisa de graduação Gencarella. Griffin S.A. recebeu uma bolsa de pesquisa S2S graduação do departamento de biologia e química.
<img alt="Image 1" src="/files/ftp_upload/58508/58508img1.jpg">
Autores (da esquerda para a direita) Cody Drisko, Dr. Kevin Huang e Silas Griffin conduziram os experimentos e preparado o manuscrito. Cody Drisko é um companheiro de Stauffer de John e um destinatário da subvenção Gencarela pesquisa. Silas é um S2S Azusa Pacific University Research Fellow. Dr. Kevin Huang desde a pesquisa de tutoria e é um receptor da Azusa Pacific University faculdade pesquisa Conselho subvenção.

Atenção: Por favor consulte todas as fichas de dados de segurança (MSDS) antes do uso. Vários produtos químicos utilizados nestas sínteses são extremamente tóxico e cancerígeno. Por favor, use todas as práticas de segurança adequadas ao executar as seguintes reações, incluindo o uso de controles de engenharia (coifa e espectrômetros de IR e NMR) e equipamentos de proteção individual (óculos de segurança, luvas, jaleco, calça longa-metragem, e sapatos fechados).
1. Michael adi?...

Em uma típico REM vinculador/fase sólida sintética estratégia, antes do lançamento de uma amina do suporte sólido, é fundamental para formar um sal quaternário de amônio, conforme descrito na secção 4 do protocolo39. Devido o estérico do sistema tricíclico e volumosos R2 grupos (haletos de benzila e octilo), apenas pequenos reagentes alquilantes (haletos de metilo e de alilo) poderiam ser utilizados nesta reação46. Com uma modificação simples, pe...

Apesar das recentes descobertas usando heterocíclicos spirocyclic altamente acrescida em um número de sistemas biológicos1, um caminho conveniente ainda é necessário para o seu fabrico fácil. Tais sistemas e usos para estes heterocíclicos incluem: MDM2 inibição e outras atividades anticancer2,3,4,5, enzima inibição6,7,8 ,13,14, sondas de actividade antibiótica9,10, fluorescente de10,11,12, enantioselective vinculativas para DNA de marcação 15 e RNA alvo16, juntamente com numerosas aplicações potenciais para terapêutica17,18,19. Com uma crescente demanda por estes heterocíclicos, literatura atual continua dividida sobre qual caminho sintético é melhor. Abordagens sintéticas modernas para este problema usam Isatina e Isatina derivados como partida materiais para uma variedade de heterocíclicos20,21, rearranjos intramolecular complicado22,23 ,24,25, Lewis ácido1,26,27 ou metais de transição catálise17,28,29, 30, ou assimétrica processos31. Enquanto estes procedimentos tiveram sucesso em produzindo oximas spirocyclic específicos com funcionalidade limitada, uma estratégia sintética para produzir uma biblioteca de moléculas com alta diastereoseletividade tem sido explorada relativamente menos32.
A técnica aqui apresentada mostra que estas moléculas de interesse podem ser geradas usando um número de técnicas sintéticas bem compreendidas em tandem. Começando com a síntese da molécula sobre um suporte sólido usando um vinculador de REM e intramolecular silil nitronate-olefin cicloadição (ISOC), o percurso proposto implanta uma rota não-linear, caracterizada por bond, separando-se em um sistema tricíclico, deixando um anel de altamente funcionalizado. Linkers REM, conhecidos por sua conveniência e reciclabilidade, utilizam um suporte sólido para sintetizar aminas terciárias33. Devido à facilidade de purificação credenciada para a REM vinculador através de simples filtração, esta técnica de síntese de fase sólida fornece os cientistas com um vinculador traceless e reciclável, que tem sido usada aqui. Uma vez que a reação é completa, o vinculador REM é regenerado e pode ser reutilizado várias vezes. O vinculador REM também é traceless porque, ao contrário de muitos linkers de fase sólida, o ponto de ligação entre o produto e o polímero é indistinguível34,35. Também estudada e compreendida é a reação de ISOC, útil na síntese de pirrolidina oximas36,37. Talvez mais conhecido como uma cicloadição 1,3-dipolar, essas reações formam um número de heterocíclicos com alta diastereoseletividade38,39,40,41,42 , 43 , 44 , 45. usando a técnica modificada de REM-ISOC-acoplado para a síntese de moléculas de spirocyclic produz um produto altamente diastereoselective. Neste documento, nós relatamos sobre a produção eficiente de oximas spirocyclic usando uma nova abordagem sintética, combinando duas vias bem compreendidas e matérias-primas disponíveis.

<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

Uma rota sintética conveniente para spirocyclic heterocíclicos é bem procurada devido ao uso potencial da molécula em sistemas biológicos. Por meio da síntese de fase sólida, regenerar estratégias de vinculador de Michael (REM) e a cicloadição 1,3-dipolar, uma biblioteca de heterocíclicos estruturalmente semelhantes, com e sem um centro de spirocyclic, pode ser construído. As principais vantagens da síntese sólido suporte são os seguintes: primeiro, a cada etapa de reação pode ser conduzida à conclusão usando um grande excesso de reagentes, resultando em rendimentos elevados; próximo, o uso de matérias-primas disponíveis comercialmente e reagentes manter os custos baixos; Finalmente, as etapas de reação são fáceis purificar através de filtração simples. A estratégia de vinculador de REM é atraente devido à sua capacidade de reciclagem e natureza traceless. Uma vez que um esquema de reação é concluído, o vinculador pode ser reutilizado várias vezes. Em uma típica síntese de fase sólida, o produto contém uma parte de ou o vinculador inteiro, que pode ser indesejável. O vinculador REM é "traceless" e o ponto de ligação entre o produto e o polímero é indistinguível. A alta diastereoseletividade da cicloadição 1,3-dipolar intramolecular é bem documentada. Limitado pela insolubilidade do suporte sólido, a progressão da reação apenas pode ser acompanhada por uma mudança nos grupos funcionais (se houver) através de espectroscopia infravermelha (IR). Assim, a identificação estrutural de intermediários não pode ser caracterizada por espectroscopia convencional da ressonância magnética nuclear (NMR). Outras limitações para este método decorrem as compatibilidades do polímero/vinculador ao regime de reação química desejada. Aqui nós relatamos um protocolo que permite a produção conveniente de heterocíclicos spirocyclic que, com simples modificações, podem ser automatizados com técnicas de alta produtividade.

Conforme descrito no procedimento acima, a rota sintética para spirocyclic oximas (ver Figura 1) começa com a adição de Michael de furfurylamine para compostos 1, o vinculador REM, a pagar 2. Um subsequente adição de Michael e a cicloadição 1,3-dipolar do suporte 2 usando vários rendimento de derivados de β-nitrostyrene os tricíclicos composto 3, uma N- silyloxy isoxazolid...

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

Aqui nós apresentamos um protocolo para demonstrar um método eficiente para a síntese de heterocíclicos spirocyclic. O processo de cinco etapas utiliza síntese de fase sólida e regenerando estratégias de vinculador de Michael. Geralmente é difícil sintetizar, apresentamos um método personalizável para a síntese de moléculas de spirocyclic inacessíveis para outras abordagens modernas.

Fase sólida síntese de [4,4] Spirocyclic oximas

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 orgânica de fase sólida pode encontrar aplicações na construção de uma biblioteca combinatória, enquanto a síntese de het spirocyclic het sterocicclic het heterocicics, que pode ser testado para atividades anticâneas usando bioensa. Embora este método possa ser usado para a síntese de compostos espirráclicos, ele também pode ser aplicado a outras pequenas moléculas, como isoxazolinas e isoxazoles. Para começar a síntese, adicione a um vaso de reação de fase sólida de 25 mililitros um grama de resina REM, 20 mililitros de dimetilformamida e 2,4 mililitros de furfurilamina.Tampe o recipiente e agitar-o por 24 horas em um shaker à temperatura ambiente para carregar a resina com furfurylamina. Em seguida, escorra a resina de rolamento de furfurylamina e agitar-a por cerca de três minutos em cinco mililitros de DMF para lavá-la. Depois disso, lave a resina quatro vezes, alternando entre cinco mililitros de diclorometano para inchar a resina, e cinco mililitros de metanol para contrair a resina.Em seguida, seque completamente a resina com ar comprimido, que geralmente leva cerca de 30 minutos. Em seguida, em um capô de fumaça bem ventilado, adicione 1,48 mililitros de trietilamina, 10 mililitros de tolueno anidro e 637 gramas de beta nitrostyrena à resina seca de rolamento de furfurilamina. Adicione um mililitro de cloreto de trimetilsil à mistura de reação.E deixe o vaso aberto até que a mistura termine de liberar gás cloridrato. Em seguida, tampe o recipiente e agitar a mistura por 48 horas em temperatura ambiente para formar o intermediário isoxazole. Depois disso, adicione cinco mililitros de metanol para saciar a reação.Agite a mistura por três minutos e deixe-a de molho por dois minutos. Em seguida, escorra a resina e lave-a quatro vezes, alternando entre cinco mililitros de DCM, e cinco mililitros de metanol. Seque completamente a resina lavada com ar comprimido.Em seguida, adicione um mililitro de tetrahidrofurano anidrofurano à resina seca. Em seguida, adicione 1,24 mililitros de uma solução molar de farinha de tetra-n-butylammonium no THF e agitar a mistura em temperatura ambiente por 12 horas para abrir o anel de isoxazole. Depois, coe a resina e lave-a com cinco mililitros de THF, seguida por quatro lavagens alternando entre cinco mililitros de DCM e cinco mililitros de metanol.Seque a resina completamente com ar comprimido. Em seguida, adicione cinco mililitros de DMF à resina, seguido por um mililitro de um bromooctano. Agitar a mistura por 24 horas em temperatura ambiente para formar a amina quaternária.Em seguida, escorra a resina e lave-a com cinco mililitros de DMF. Lave a resina mais quatro vezes, alternando entre DCM e metanol, e seque com ar comprimido. Em seguida, adicione três mililitros de DCM à resina seca, seguido por 1,5 mililitros de TEA.Agitar a mistura por 24 horas para cortar o oxime spirocyclic do suporte do polímero. Em seguida, colete o produto bruto em um frasco ou frasco. Lave a resina quatro vezes, alternando entre DCM e metanol, e colete as lavagens no frasco do produto.Remova os voláteis da mistura do produto em um evaporador rotativo. Em seguida, triturar o produto bruto com 5 mililitros de metanol fervente, e remover cuidadosamente o líquido. Lave a resina duas vezes com cinco porções mililitros de DCM e seque-a com ar comprimido para reutilização em experimentos subsequentes.Oximes spirocyclic com vários substituintes foram sintetizados com excelente estereoseletividade. O rendimento geral indica um rendimento médio de 80 a 88% para cada etapa sintética. O progresso da reação através da abertura do anel isoxazole pode ser monitorado por espectroscopia de RI.Geralmente, indivíduos novos neste método lutam com a seleção do linker de suporte de polímero apropriado. É essencial que o linker seja robusto a todas as condições de reação durante a síntese. Ao tentar este procedimento, lembre-se de lavar e secar a resina antes da próxima etapa de reação.Os reagentes remanescentes da etapa anterior complicarão a síntese geral e afetarão o rendimento e a pureza do produto spirocyclic.

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

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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.

Síntese de Immunotargeted magneto-plasmónico Nanoclusters

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

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.

Metodologia sintética para Asymmetric Ferroceno Derivados Sistemas Bio-conjugadas via Metodologia base de resina em Fase Sólida

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.

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 baseada Síntese de ouro 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.

Síntese de hierárquicos 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 ...

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.

Integração da Extração de Fase Sólida Miniaturizada e Detecção de LC-MS / MS de 3-Nitrotirosina em Urina Humana para Aplicações 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 síntese de fase sólida de oligómeros de ARN contendo um 2&#39;-<em&gt; O</em&gt; -tiofenilmetilo Modificação e Caracterização através do 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.

Síntese de nanofios de Au substrato ligados através de um mecanismo de crescimento ativo de superfície

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-metilação, purificação e formulação para a produção de traçadores 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.

Espectrometria de massa de cromatografia a gás emparelhada com microextração de fase sólida de vaporização total como ferramenta forense

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