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

Dette arbejde blev finansieret af et tilskud fra Fakultet Research Council til K.S. Huang (Azusa Pacific University - USA). C.R. Drisko er en modtager af John Stauffer legat og Gencarella Undergraduate Research Grant. S.A. Griffin modtaget en S2S Undergraduate Research Fellowship fra Institut for biologi og kemi.
<img alt="Image 1" src="/files/ftp_upload/58508/58508img1.jpg">
Forfattere (fra venstre mod højre) Cody Drisko, Dr. Kevin Huang og Silas Griffin udført eksperimenter og udarbejdet manuskriptet. Cody Drisko er en John Stauffer fyr og en modtager af Gencarela forskning tilskud. Silas er en S2S Azusa Pacific University Kandidatstipendiat. Dr. Kevin Huang forskning mentorordninger og er modtager af Azusa Pacific University Faculty forskning Rådet Grant.

Forsigtig: Kontakt venligst alle relevante materiale sikkerhedsdatablade (MSDS) før brug. Flere af kemikalier, der anvendes i disse sammenstillinger er akut giftige og kræftfremkaldende. Brug venligst alle relevante sikkerhedspraksis, når du udfører de følgende reaktioner, herunder brugen af tekniske kontrol (stinkskab og IR og NMR spektrometre) og personlige værnemidler (beskyttelsesbriller, handsker, laboratoriekittel, fuld længde bukser, og lukket tå sko).
1. Michael tilsætning af Fu...

I en typisk REM linker/fast-fase syntetiske strategi, forud for frigivelsen af en amine fra solid støtte, er det kritisk at danne en kvaternære ammoniumsalt, som beskrevet i punkt 4 i protokollen39. På grund af sterisk hindring af tricykliske system og voluminøse Rasmussen2 grupper (benzyl og octyl halogenider), kunne kun små alkylerende reagenser (methyl og allylisothiocyanat halogenider) udnyttes i denne reaktion46. Med en simpel ændring, giver mulighed f...

Trods de seneste opdagelser ved hjælp af stærkt functionalized spirocyclic heterocycles i en række biologiske systemer1, er en praktisk vej stadig nødvendige for deres let fremstilling. Sådanne systemer og anvendelser for disse heterocycles omfatter: MDM2 hæmning og andre anticancer aktiviteter2,3,4,5, enzym hæmning6,7,8 , antibiotiske aktivitet9,10, fluorescerende tagging10,11,12, enantioselective binding til DNA sonder13,14, 15 og RNA målretning16, sammen med mange potentielle applikationer til therapeutics17,18,19. Med en stigende efterspørgsel efter disse heterocycles, aktuelle litteratur er stadig splittet om hvilke syntetisk vej er bedst. Moderne syntetiske tilgange til dette problem bruge isatin og isatin derivater som råvarer til en bred vifte af heterocycles20,21, kompliceret intramolekylære rearrangementer22,23 ,24,25, Lewis syre1,26,27 eller overgangen metal katalyse17,28,29, 30, eller asymmetrisk processer31. Mens disse procedurer har haft succes i at producere specifikke spirocyclic oximes med begrænset funktionalitet, har en syntetisk strategi for at producere et bibliotek af molekyler med høje diastereoselectivity været udforsket relativt mindre32.
Teknikken præsenteret her viser, at disse molekyler af interesse kan genereres ved hjælp af en række velforståede syntetiske teknikker i tandem. Startende med syntesen af molekylet på en solid støtte ved hjælp af en REM linker og intramolekylære silyl nitronate-olefin cycloaddition (ISOC), den foreslåede vej udsender en ikke-lineær rute, karakteriseret ved bond severing i et tricyklisk system, forlader en meget functionalized heterocycle. REM linkers, kendt for deres bekvemmelighed og genvindelighed, udnytte en solid støtte til at syntetisere tertiære aminer33. På grund af lethed, hvormed rensning akkrediteret til REM linker via simpel filtrering, giver denne fast-fase syntese teknik forskere med et genanvendeligt og traceless linker, som har været brugt her. Når reaktionen er færdig, REM linker er regenereret og kan genbruges flere gange. REM linker er også traceless, fordi i modsætning til mange fast-fase linkers, stopgarnets mellem produktet og polymeren er umulig at skelne34,35. Også godt undersøgt og forstået er ISOC reaktion, nyttige i syntesen af pyrrolidine oximes36,37. Måske bedre kendt som en 1,3-dipolære cycloaddition, disse reaktioner udgør en række heterocycles med høj diastereoselectivity38,39,40,41,42 , 43 , 44 , 45. ved hjælp af modificerede REM-kombineret-ISOC teknik til syntese af spirocyclic molekyler giver et meget diastereoselective produkt. Heri, rapport vi om effektiv produktion af spirocyclic oximes ved hjælp af en ny syntetisk tilgang, kombinerer to velforståede veje og let tilgængelige råvarer.

<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

En bekvem syntesevejen for spirocyclic heterocycles er godt eftertragtede molekyles potentielle anvendelse i biologiske systemer. Ved hjælp af solid-fase syntese, kan regenererende Michael (REM) linker strategier og 1,3-dipolære cycloaddition, et bibliotek af strukturelt lignende heterocycles, både med og uden en spirocyclic center, være konstrueret. De vigtigste fordele ved solid-støtte syntesen er som følger: først, hver reaktion skridt kan være drevet til afslutning ved hjælp af et stort overskud af reagenser medfører høje udbytter; Næste, brug af kommercielt tilgængelige råvarer og reagenser holde omkostningerne lave; Endelig, reaktion foranstaltninger er let at rense via simpel filtrering. REM linker strategi er attraktivt på grund af dets genvindelighed og traceless karakter. Når en reaktion ordningen er afsluttet, kan linker genbruges flere gange. I en typisk solid-fase syntese indeholder produktet enten en del af eller hele linker, der kan være uønsket. REM linker er "traceless" og stopgarnets mellem produktet og polymeren er umulig at skelne. Den høje diastereoselectivity på den intramolekylære 1,3-dipolære cycloaddition er veldokumenteret. Begrænset af kan den faste støtte, reaktion progression kan kun overvåges af en ændring i funktionelle grupper (hvis nogen) via infrarød (IR) spektroskopi. Således, den strukturelle identifikation af mellemprodukter kan karakteriseres ved konventionelle Kernemagnetisk resonans (NMR) spektroskopi. Andre begrænsninger denne metode stammer fra kompatibilitet af polymer/linker til ordningen ønskede kemisk reaktion. Heri rapportere vi en protokol, der giver mulighed for bekvem produktion af spirocyclic heterocycles, der med enkle ændringer, kan automatiseres med høj overførselshastighed teknikker.

Som beskrevet i ovenstående fremgangsmåde, begynder syntesevejen til spirocyclic oximes (Se figur 1) med Michael tilsætning af furfurylamine til sammensatte 1, REM linker til at råd til 2. En efterfølgende Michael tilsætning og 1,3-dipolære cycloaddition støtte 2 ved hjælp af forskellige β-nitrostyrene derivater udbytte de tricykliske sammensatte 3, en N- silyloxy isoxazoli...

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

Her præsenterer vi en protokol for at påvise en effektiv metode til syntese af spirocyclic heterocycles. Den fem-trins proces udnytter fast-fase syntese og regenererende Michael linker strategier. Generelt vanskeligt at syntetisere, vi præsenterer en tilpasselig metode til syntese af spirocyclic molekyler ellers utilgængelige for andre moderne tilgange.

Fast-fase syntese af [4.4] Spirocyclic Oximes

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 ...

Denne organiske syntetiske teknik i fast fase kan finde anvendelser i konstruktionen af et kombinatorisk bibliotek, mens syntese af het spirocyklic heterocycles, som kan testes for anti-cancer aktiviteter ved hjælp af bioassays. Selv om denne metode kan anvendes til syntese af spirocykliske forbindelser, Det kan også anvendes på andre små molekyler, såsom isoxazolines og isoxazoler. For at begynde syntesen, tilføje til en 25 millileter solidfase reaktion fartøj et gram REM harpiks, 20 millileters dimethylformamid, og 2,4 milliliter furfurylamin.Beholderen må bæres under, og den omrøres i 24 timer på en shaker ved stuetemperatur for at laste harpiksen med furfurylamin. Derefter drænes furfurylaminbærende harpiks og omrørs i ca. tre minutter i fem milliliter DMF for at vaske den. Herefter vaskes harpiksen fire gange, skiftevis mellem fem milliliter dichlormethan for at svulme harpiksen op, og fem milliliter methanol for at få en kontrakt med harpiksen.Tør derefter harpiksen grundigt med trykluft, som normalt tager ca. 30 minutter. Dernæst i et godt ventileret røghætte, tilsættes 1,48 milliliter triethylamin, 10 milliliter vandfri toluen og 637 gram beta nitrostyren til den tørre furfurylaminbærende harpiks. Der tilsættes en milliliter trimethylsilylchlorid til reaktionsblandingen.Og lad fartøjet stå åbent, indtil blandingen er færdig med at frigive hydrochloridgas. Derefter hætten beholderen og omrør blandingen i 48 timer ved stuetemperatur for at danne isoxazol mellemprodukt. Derefter tilsættes fem milliliter methanol for at slukke reaktionen.Ryst blandingen i tre minutter, og lad det derefter suge i to minutter. Derefter dræne harpiks og vaske det fire gange, skiftevis mellem fem milliliter DCM, og fem milliliter methanol. Tør den vaskede harpiks grundigt med trykluft.Dernæst tilsættes en milliliter vandfri tetrahydrofuran til den tørre harpiks. Derefter tilsættes 1,24 milliliter af en en molar opløsning af tetra-n-butylammonium flourid i THF og omrøre blandingen ved stuetemperatur i 12 timer for at åbne isoxazol ring. Derefter stammes harpiksen og vaskes med fem milliliter THF, efterfulgt af fire vasker skiftevis mellem fem milliliter DCM og fem milliliter methanol.Reling harpiksen grundigt med trykluft. Dernæst tilsættes fem milliliter DMF til harpiksen, efterfulgt af en milliliter en bromooctan. Omrør blandingen i 24 timer ved stuetemperatur for at danne kvatnær amin.Derefter tømmes harpiksen og vaskes med fem milliliter DMF. Vask harpiksen fire gange mere, skiftevis mellem DCM og methanol, og tør med trykluft. Dernæst tilsættes tre milliliter DCM til den tørre harpiks, efterfulgt af 1,5 milliliter TE.Omgit blandingen i 24 timer for at kløve spirocyclic oxime fra polymerstøtten. Råproduktet opsamles derefter i et hætteglas eller en kolbe. Vask harpiksen fire gange, skiftevis mellem DCM og methanol, og opsaml vaskene i produkthætteglasset.Flygtige stoffer fjernes fra produktblandingen på en roterende fordamper. Derefter triturere rå produkt med 5 milliliter kogende methanol, og forsigtigt fjerne væsken. Harpiksen vaskes to gange med fem milliliterportler af DCM, og den tørres med trykluft til genbrug i efterfølgende forsøg.Spirocyklic oximes med forskellige substituents blev syntetiseret med fremragende stereoselectivity. Det samlede udbytte angiver et gennemsnit på 80 til 88% udbytte for hvert syntetisk trin. Reaktionen skrider op gennem isoxazol ring åbning kan overvåges af IR spektroskopi.Generelt, enkeltpersoner nye til denne metode kamp med at vælge den relevante polymer støtte linker. Det er vigtigt, at linkeren er robust over for alle reaktionsforhold under syntesen. Under forsøg på denne procedure, skal du huske at vaske og tørre harpiksen før næste reaktionstrin.Rester reagenser fra det foregående trin vil komplicere den samlede syntese og vil påvirke udbyttet og renheden af spirocyclic produkt.

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.

Syntese af Immunotargeted Magneto-plasmoniske nanoklynger

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

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.

Syntetisk Metode til Asymmetrisk Ferrocen Afledte Bio-konjugat systemer via Solid Phase Resin-metode

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.

Syntese af indoxyl-glycosider til påvisning af glycosidase Aktiviteter

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.

Hydroquinon Based Syntese af guld 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.

Syntese af Hierarkisk ZnO / CdSSe heterostruktur 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.

Integration af miniaturiseret fastfaseekstraktion og LC-MS / MS-detektion af 3-nitrotyrosin i human urin til kliniske applikationer

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.

Protokol til fastfasesyntese af oligomerer af RNA indeholdende en 2&#39;-<em&gt; O</em&gt; -thiophenylmethyl-modifikation og karakterisering via cirkulær dikroisme

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.

Syntese af substrat-bundet Au Nanowires Via en aktiv overflade vækst mekanisme

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.

Solid fase 11C-methylering, oprensning og formulering til fremstilling af PET-Tracers

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.

Gaskromatografi-massespektrometri parret med total fordampning Solid-Phase Microextraction som et retsmedicinsk værktøj

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