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

Quest'opera è stata finanziata da una sovvenzione dal Consiglio di ricerca di facoltà K.S. Huang (Azusa Pacific University - Stati Uniti d'America). C.R. Drisko ha ricevuto la borsa di studio di John Stauffer e il Gencarella Undergraduate Research Grant. S.A. Griffin ha ricevuto un S2S Undergraduate Research Fellowship dal dipartimento di biologia e chimica.
<img alt="Image 1" src="/files/ftp_upload/58508/58508img1.jpg">
Autori (da sinistra a destra) Cody Drisko, Dr. Kevin Huang e Silas Griffin condotti gli esperimenti e preparato il manoscritto. Cody Drisko è John Stauffer Fellow e un destinatario del Gencarela Research Grant. Silas è un S2S Azusa Pacific University Research Fellow. Dr. Kevin Huang fornito il mentoring di ricerca ed è un destinatario di Azusa Pacific University Facoltà Research Council Grant.

Attenzione: Si prega di consultare tutte le schede di dati di sicurezza (MSDS) prima dell'uso. Molte delle sostanze chimiche utilizzate in queste sintesi sono acutamente tossici e cancerogeni. Si prega di utilizzare tutte le pratiche di sicurezza appropriate quando si eseguono le seguenti reazioni, compreso l'uso di controlli tecnici (cappa e spettrometri IR e NMR) e dispositivi di protezione individuale (occhiali di sicurezza, guanti, camice, pantaloni lunghi, e Scarpe chiuse).
1. Michael aggiu...

In una tipica REM del linker/fase solida strategia sintetica, prima del rilascio di un'ammina dal supporto solido, è fondamentale per formare un sale di ammonio quaternario, come descritto nella sezione 4 del protocollo39. A causa di impedimento sterico del sistema triciclico e gruppi di ingombranti R2 (alogenuri benzilici e ottilico), unico piccoli reagenti alchilanti (Alogenuri metilici e allile) potrebbero essere utilizzati in questa reazione46. Con una sempl...

Nonostante le recenti scoperte utilizzando composti eterociclici funzionalizzati altamente spirocyclic in un certo numero di sistemi biologici1, un comodo sentiero è ancora necessario per la loro fabbricazione facile. Tali sistemi e la usi per questi eterocicli includono: MDM2 inibizione e altre attività anticancro2,3,4,5, enzima inibizione6,7,8 , attività antibiotica9,10, tagging10,11,12, enantioselettiva vincolante per DNA fluorescente sonde13,14, 15 e16, insieme a numerose applicazioni potenziali a terapeutica17,18,19di targeting RNA. Con una crescente domanda per questi eterocicli, letteratura corrente rimane divisa su quale via sintetica è migliore. Sintetici moderni approcci a questo problema utilizzano isatin e isatin derivati come materie prime per una varietà di eterocicli20,21, riarrangiamenti intramolecolare complicati22,23 ,24,25, Lewis acido1,26,27 o metalli di transizione catalisi17,28,29, 30, o processi asimmetrica31. Mentre queste procedure hanno avuto successo nella produzione di specifici spirocyclic ossime con funzionalità limitate, una strategia sintetica per la produzione di una libreria di molecole con alta diastereoselettività è stato esplorato relativamente meno32.
La tecnica presentata qui dimostra che queste molecole di interesse possono essere generate utilizzando un numero di tecniche di sintesi ben capiti in tandem. A partire con la sintesi della molecola su un supporto solido utilizzando un linker di REM e intramolecolari Silile nitronate-olefina cicloaddizione (ISOC), il percorso proposto consente di distribuire un percorso non lineare, caratterizzato da bond dividente in un sistema triciclico, lasciando un heterocycle altamente funzionalizzati. Linker di REM, noti per la loro convenienza e riciclabilità, utilizzare un supporto solido per sintetizzare ammine terziarie33. A causa della facilità di purificazione accreditati alla REM del linker tramite semplice filtrazione, questa tecnica di sintesi in fase solida offre ai ricercatori con un linker riciclabile e traceless, che è stato utilizzato qui. Una volta che la reazione è completa, il linker di REM viene rigenerato e può essere riutilizzato più volte. Il linker di REM è traceless anche perché, a differenza di molti linker di fase solida, il punto di attacco tra il prodotto e il polimero è indistinguibile34,35. Anche ben studiato e capito è la reazione di ISOC, utile nella sintesi di pyrrolidine ossime36,37. Forse meglio conosciuto come una cicloaddizione 1,3-dipolare, queste reazioni formano un numero di eterocicli con alta diastereoselettività38,39,40,41,42 , 43 , 44 , 45. utilizzando la tecnica di REM-accoppiato-ISOC modificata per la sintesi di molecole spirocyclic produce un prodotto altamente diastereoselettiva. Qui, segnaliamo su una produzione efficiente di ossime spirocyclic utilizzando un nuovo approccio sintetico, che unisce due vie ben comprese e prontamente disponibili materiali di partenza.

<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 comoda rotta sintetica per spirocyclic eterocicli è ben ricercata a causa di uso potenziale della molecola nei sistemi biologici. Per mezzo di sintesi in fase solida, rigenerante strategie del linker Michael (REM) e la cicloaddizione 1,3-dipolare, una libreria di eterocicli strutturalmente simili sia con che senza un centro di spirocyclic, può essere costruito. I principali vantaggi della sintesi solido-supporto sono come segue: in primo luogo, ogni passaggio di reazione può essere guidato a completamento utilizzando un grande eccesso di reagenti conseguente rese elevate; avanti, l'uso di materie prime disponibili in commercio e reagenti mantenere i costi bassi; Infine, i punti di reazione sono facili da purificare tramite semplice filtrazione. La strategia del linker di REM è attraente a causa della sua natura traceless e riciclabilità. Una volta completato uno schema di reazione, il linker può essere riutilizzato più volte. In una tipica sintesi in fase solida, il prodotto contiene una parte di o il linker intero, che può risultare indesiderato. Il linker di REM è "traceless" ed il punto di attacco tra il prodotto e il polimero è indistinguibile. L'alta diastereoselettività della cicloaddizione 1,3-dipolare intramolecolare è ben documentato. Limitata dall'insolubilità del supporto solido, la progressione di reazione possa solo essere monitorata da un cambiamento in gruppi funzionali (se presente) tramite spettroscopia nell'infrarosso (IR). Così, l'identificazione strutturale di intermedi non può essere caratterizzata da convenzionale risonanza magnetica nucleare (NMR). Altre limitazioni di questo metodo derivano dalle compatibilità del polimero/linker al regime di reazione chimica desiderata. Qui segnaliamo un protocollo che consente la produzione conveniente di eterocicli spirocyclic che, con semplici modifiche, possono essere automatizzato con tecniche di alto-rendimento.

Come descritto nella procedura precedente, il percorso sintetico per spirocyclic ossime (Vedi Figura 1) inizia con l'aggiunta di Michael di furfurylamine Compound 1, il linker di REM, permettersi 2. Una successiva addizione di Michael e la cicloaddizione 1,3-dipolare del supporto 2 utilizzando vari β-nitrostirene derivati resa il triciclico composto 3, un N- silyloxy isoxazolidine c...

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

Qui presentiamo un protocollo per dimostrare un metodo efficiente per la sintesi di eterocicli spirocyclic. Sintesi in fase solida e rigenerante strategie del linker Michael utilizza il processo in cinque fasi. Generalmente difficili da sintetizzare, presentiamo un metodo personalizzabile per la sintesi di molecole spirocyclic altrimenti inaccessibili ad altri approcci moderni.

Sintesi in fase solida di [4,4] Spirocyclic ossime

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 ...

Questa tecnica sintetica organica in fase solida può trovare applicazioni nella costruzione di una biblioteca combinatoria, mentre la sintesi di eterocicli eterociclici et spirociclici, che possono essere testati per attività antitumorali utilizzando test biologici. Sebbene questo metodo possa essere utilizzato per la sintesi di composti spirociclici, può anche essere applicato ad altre piccole molecole, come isoxazoline e isoxazoli. Per iniziare la sintesi, aggiungere a un recipiente di reazione in fase solida da 25 millilitri un grammo di resina REM, 20 millileter di dimetilformamide e 2,4 millilitri di furfurilammina.Limitare il recipiente e agitarlo per 24 ore su uno shaker a temperatura ambiente per caricare la resina con furfurilammina. Quindi scolare la resina portante furfurilammina e agitarla per circa tre minuti in cinque millilitri di DMF per lavarla. Successivamente, lavare la resina quattro volte, alternando cinque millilitri di diclorometano per gonfiare la resina e cinque millilitri di metanolo per contrarre la resina.Quindi asciugare accuratamente la resina con aria compressa, che di solito richiede circa 30 minuti. Successivamente, in una cappa fumi ben ventilata, aggiungere 1,48 millilitri di trietilammina, 10 millilitri di toluene anidro e 637 grammi di beta nitrostirolo alla resina portante furfurilammina secca. Aggiungere un millilitro di cloruro di trimetilsilil alla miscela di reazione.E lasciare il recipiente aperto fino a quando la miscela non ha finito di rilasciare gas cloridrato. Quindi limitare il vaso e agitare la miscela per 48 ore a temperatura ambiente per formare l'intermedio isoxazolo. Successivamente, aggiungere cinque millilitri di metanolo per spegnere la reazione.Agitare il composto per tre minuti, quindi lasciarlo in ammollo per due minuti. Quindi scolare la resina e lavarla quattro volte, alternando cinque millilitri di DCM e cinque millilitri di metanolo. Asciugare accuratamente la resina lavata con aria compressa.Quindi, aggiungere un millilitro di tetraidrofurano anidrofurano alla resina secca. Quindi aggiungere 1,24 millilitri di una soluzione molare di farineuro di tetra-n-butilammonio nel THF e agitare la miscela a temperatura ambiente per 12 ore per aprire l'anello di isoxazolo. Successivamente, filtrare la resina e lavarla con cinque millilitri di THF, seguiti da quattro lavaggi alternati tra cinque millilitri di DCM e cinque millilitri di metanolo.Asciugare accuratamente la resina con aria compressa. Successivamente, aggiungere cinque millilitri di DMF alla resina, seguiti da un millilitro di un bromoottano. Agitare la miscela per 24 ore a temperatura ambiente per formare l'ammina quaternaria.Quindi scolare la resina e lavarla con cinque millilitri di DMF. Lavare la resina altre quattro volte, alternando DCM e metanolo, e asciugare con aria compressa. Successivamente, aggiungere tre millilitri di DCM alla resina secca, seguiti da 1,5 millilitri di TEA.Agitare la miscela per 24 ore per scindere l'ossima spirociclica dal supporto del polimero. Quindi, raccogliere il prodotto grezzo in una fiala o in un pallone. Lavare la resina quattro volte, alternandosi tra DCM e metanolo, e raccogliere i lavaggi nel flaconcino del prodotto.Rimuovere i volatili dalla miscela di prodotto su un evaporatore rotante. Quindi, triturare il prodotto grezzo con 5 millilitri di metanolo bollente e rimuovere con cura il liquido. Lavare la resina due volte con cinque porzioni millilitri di DCM e asciugarla con aria compressa per il riutilizzo in esperimenti successivi.Le ossima spirocicliche con vari sostituenti sono state sintetizzati con un'eccellente stereoseletività. La resa complessiva indica una resa media dall'80 all'88% per ogni fase sintetica. La reazione che avanza attraverso l'apertura dell'anello isoxazolo può essere monitorata mediante spettroscopia IR.Generalmente, gli individui nuovi a questo metodo hanno difficoltà a selezionare il linker di supporto polimerico appropriato. È essenziale che il linker sia robusto a tutte le condizioni di reazione durante la sintesi. Durante il tentativo di questa procedura, ricordarsi di lavare e asciugare la resina prima della fase di reazione successiva.I reagenti rimanenti del passo precedente complicheranno la sintesi complessiva e influenzeranno la resa e la purezza del prodotto spirociclico.

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.

Sintesi di Immunotargeted magneto-plasmonica nanocluster

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

Ricerca

Chimica

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 sintetico per Asymmetric Ferrocene derivati ​​sistemi bio-coniugati attraverso Solid Metodologia Resina a base di 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.

Sintesi di indoxyl-glicosidi per il rilevamento di glicosidasi Attività

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.

Idrochinone Based Sintesi di 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.

Sintesi di 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 ...

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.

Integrazione di estrazione miniaturizzata di fase solida e rilevazione LC-MS / MS di 3-Nitrotirosina nell&#39;urina umana per applicazioni cliniche

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.

Protocollo per la sintesi in fase solida di oligomeri di RNA contenente un 2&#39;-<em&gt; O</em&gt; -thiophenylmethyl Modifica e caratterizzazione attraverso il Dicroroo circolare

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.

Sintesi di nanofili Au associato a substrato tramite un meccanismo di crescita superficiale attiva

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 solida 11C-Metilazione, purificazione e formulazione per la produzione di traccianti 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.

Gas cromatografia-spettrometria di massa abbinata alla microestrazione a fase solida di vaporizzazione totale come strumento forense

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