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

Detta arbete finansierades genom ett bidrag från fakulteten Research Council att K.S. Huang (Azusa Pacific University - USA). C.R. Drisko är mottagare av John Stauffer stipendiet och den Gencarella grundutbildning forskningsbidrag. S.A. Griffin fick en S2S Undergraduate Research Fellowship från Institutionen för biologi och kemi.
<img alt="Image 1" src="/files/ftp_upload/58508/58508img1.jpg">
Författarna (vänster till höger) Cody Drisko, Dr Kevin Huang och Silas Griffin genomfört experimenten och förberett manuskript. Cody Drisko är John Stauffer Fellow och en mottagare av bidraget som Gencarela forskning. Silas är en S2S Azusa Pacific University Research Fellow. Dr Kevin Huang den forskning som mentorskap och en mottagare av Azusa Pacific universitet fakulteten forskning rådet bidraget.

Varning: Läs alla relevanta säkerhetsdatablad (MSDS) före användning. Flera av de kemikalier som används i dessa synteser är akut giftigt och cancerframkallande. Använd alla lämpliga säkerhetsrutiner när du utför följande reaktioner, inklusive användning av tekniska kontrollåtgärder (spiskåpa och IR och NMR spectrometers) och personlig skyddsutrustning (handskar, labbrock, skyddsglasögon, hellånga byxor, och stängd tå skor).
1. Michael tillägg av Furfurylamine till REM länk...

I en typisk REM linker/fasta fasen syntetiska strategi, före utgivningen av en Amin från fasta stöd, är det viktigt att bilda ett kvaternära ammoniumsalter salt, som beskrivs i avsnitt 4 i det protokollet39. På grund av sterisk hindret tricykliska system och skrymmande R2 grupper (bensylbensoat och octyl halogenider), skulle endast små alkylerande reagenser (metyl och allyl halogenider) kunna utnyttjas i denna reaktion46. Med en enkel modifiering, vilket m...

Trots senaste upptäckter med högt-functionalized gröna föreningar i ett antal biologiska system1, är en bekväm väg fortfarande nödvändigt för deras lätt tillverkning. Sådana system och användningsområden för dessa föreningar inkluderar: MDM2 hämning och andra anticancer aktiviteter2,3,4,5, enzym hämning6,7,8 , antibiotiska aktivitet9,10, fluorescerande taggning10,11,12, enantioselective bindande för DNA-prober13,14, 15 och RNA inriktning16, tillsammans med många potentiella tillämpningar till therapeutics17,18,19. Med en ökande efterfrågan på dessa föreningar förblir aktuell litteratur splittrade om som syntetisk väg är bäst. Moderna syntetiska strategier på detta problem använder isatin och isatin derivat som utgångsmaterial för en mängd föreningar20,21, komplicerade intramolekylära rearrangements22,23 ,24,25, Lewis syra1,26,27 eller övergång metall katalys17,28,29, 30, eller asymmetrisk processer31. Medan dessa förfaranden har haft framgång i att producera specifika gröna oximes med begränsad funktionalitet, har en syntetisk strategi för att producera ett bibliotek av molekyler med hög diastereoselectivity varit utforskade relativt mindre32.
Den teknik som presenteras här visar att dessa molekyler av intresse kan genereras med ett antal väl förstått syntetiska tekniker i tandem. Den föreslagna vägen börjar med syntesen av molekylen på ett fast stöd med hjälp av en REM länkare och intramolekylära silyl nitronate-olefin cykloadditionen (ISOC), och distribuerar en ickelinjär rutt, kännetecknas av bond severing i ett tricykliska system, lämnar en Mycket functionalized heterocykel. REM linkers, känd för sin bekvämlighet och återvinningsbarhet, utnyttja ett fast stöd för att syntetisera tertiära aminer33. På grund av lätthet av rening ackrediterade till REM linker via enkel filtrering, ger denna fasta fasen syntes teknik forskare med en återvinningsbar och traceless länkare, som har använts här. När reaktionen är klar, REM länkaren regenereras och kan återanvändas flera gånger. REM länkaren beror också traceless, till skillnad från många fasta fasen linkers fästpunkten mellan produkten och polymeren är omöjlig att skilja34,35. Också väl studerat och förstås är ISOC reaktionen, användbar i syntesen av pyrrolidin oximes36,37. Kanske mer känd som en 1,3-tvåpolig cykloadditionen, dessa reaktioner bildar ett antal föreningar med hög diastereoselectivity38,39,40,41,42 , 43 , 44 , 45. med hjälp av modifierade REM-tillsammans-ISOC-tekniken för syntesen av gröna molekyler ger en mycket diastereoselective produkt. Häri, rapporterar vi om effektiv produktion av gröna oximes med en ny syntetisk metod, kombinera två väl förstått vägar och lättillgängliga utgångsmaterial.

<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 bekväm syntesväg för gröna föreningar är väl eftertraktade på grund av den molekylens potentiella användning i biologiska system. Med hjälp av fasta fasen syntes, kan regenererande Michael (REM) linker strategier och 1,3-tvåpolig cykloadditionen, ett bibliotek av strukturellt likartade föreningar, både med och utan en gröna center, konstrueras. De främsta fördelarna med solid-support syntesen är följande: för det första varje reaktion steg kan drivas till fullbordan med hjälp av ett stort överskott av reagenser som leder till hög avkastning; Nästa, användning av kommersiellt tillgängliga utgångsmaterial och reagenser hålla kostnaderna låga; Slutligen, reaktionen kliver är lätt att rena via enkel filtrering. REM linker strategin är attraktiva på grund av dess återvinningsbarhet och traceless natur. När en reaktionsformel är klar, kan länkare återanvändas flera gånger. I en typisk fasta fasen syntes innehåller produkten antingen en del av eller hela länkare, vilket kan vara önskvärt. REM länkaren är ”traceless” och fästpunkten mellan produkten och polymeren är omöjlig att skilja. Den höga diastereoselectivity av de intramolekylära 1,3-tvåpolig cykloadditionen är väl dokumenterat. Begränsas av olöslighet av fasta stöd, reaktion progression kan endast övervakas av en förändring i de funktionella grupperna (om någon) via infraröd (IR) spektroskopi. Således kan inte strukturella identifiering av intermediärer karakteriseras av konventionella kärnmagnetisk resonans (NMR) spektroskopi. Andra begränsningar till denna metod härstammar från kompatibla av polymer/länkaren till systemet med önskad kemisk reaktion. Häri rapportera vi ett protokoll som möjliggör bekväm produktionen av gröna föreningar som, med enkla ändringar, kan automatiseras med hög genomströmning tekniker.

Som beskrivs i proceduren ovan, börjar syntetiska vägen till gröna oximes (se figur 1) med Michael tillägg av furfurylamine till sammansatta 1, REM länkare, råd 2. En efterföljande Michael tillägg och 1,3-tvåpolig cykloadditionen av stöd 2 använder olika β-nitrostyrene derivat avkastning tricykliska förening 3, en N- silyloxy isoxazolidine med fyra unika stereogenic cent...

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

Här presenterar vi ett protokoll för att demonstrera en effektiv metod för syntesen av gröna föreningar. Den fem-stegs processen använder fasta fasen syntes och regenererande Michael linker strategier. Generellt svårt att syntetisera, presenterar vi en anpassningsbar metod för syntesen av gröna molekyler annars otillgängliga för andra moderna metoder.

Fasta fasen syntes av [4.4] gröna 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 ...

Denna fasta fas organiska syntetisk teknik kan hitta tillämpningar i byggandet av ett kombinatoriskt bibliotek, medan syntes av het spirocyclic heterocycles, som kan testas för anti-cancer aktiviteter med hjälp av bioassays. Även om denna metod kan användas för syntes av spirocykliska föreningar, det kan också tillämpas på andra små molekyler, såsom isoxazoliner och isoxazoler. För att påbörja syntesen, lägg till en 25 millileter solid-fas reaktionskärl ett gram REM harts, 20 millileters av dimetylformamid, och 2,4 milliliter av furfurylamin.Kapa kärlet och agitera det i 24 timmar på en shaker vid rumstemperatur för att ladda hartset med furfurylamin. Sedan dränera furfurylamin bärharts och agitera den i cirka tre minuter i fem milliliter DMF att tvätta den. Därefter tvätta hartset fyra gånger, omväxlande mellan fem milliliter av dichloromethane att svälla hartset, och fem milliliter metanol att kontraktera hartset.Torka sedan ordentligt hartset med tryckluft, som vanligtvis tar ca 30 minuter. Därefter, i en väl ventilerad rök huva, tillsätt 1,48 milliliter av trietylamin, 10 milliliter av vattenfri toluen, och 637 gram beta nitrostyren till den torra furfurylamin bärande harts. Tillsätt en milliliter trimetylsilylklorid till reaktionsblandningen.Och lämna kärlet öppet tills blandningen har släppt ut hydrokloridgas. Sedan locket kärlet och agitera blandningen i 48 timmar vid rumstemperatur för att bilda isoxazol mellanliggande. Därefter lägger du till fem milliliter metanol för att släcka reaktionen.Skaka blandningen i tre minuter, och låt den sedan dra i två minuter. Töm sedan hartset och tvätta den fyra gånger, omväxlande mellan fem milliliter DCM, och fem milliliter metanol. Torka noggrant den tvättade hartsen med tryckluft.Därefter tillsätt en milliliter av vattenfri tetrahydrofuran till den torra hartset. Tillsätt sedan 1,24 milliliter av en en molarlösning av tetra-n-butylammonium flouride i THF och agitera blandningen vid rumstemperatur i 12 timmar för att öppna isoxazolringen. Efteråt, sila hartset och tvätta den med fem milliliter av THF, följt av fyra tvättar omväxlande mellan fem milliliter DCM och fem milliliter metanol.Torka hartsen ordentligt med tryckluft. Därefter lägger du till fem milliliter DMF till hartset, följt av en milliliter av en-bromooctane. Agitera blandningen i 24 timmar vid rumstemperatur för att bilda kvartäramin.Töm sedan hartset och tvätta den med fem milliliter DMF. Tvätta hartsen ytterligare fyra gånger, växlande mellan DCM och metanol, och torka med tryckluft. Därefter lägger du till tre milliliter DCM till den torra hartsen, följt av 1,5 milliliter TEA.Agitera blandningen i 24 timmar för att klyva spirocykliska oxime från polymerstödet. Samla sedan upp råvaran i en injektionsflaska eller kolv. Tvätta hartset fyra gånger, omväxlande mellan DCM och metanol, och samla upp tvättarna i produktflaskan.Avlägsna flyktiga ämnen från produktblandningen på en roterande förångare. Sedan triturtera råvaran med 5 milliliter kokande metanol, och försiktigt ta bort vätskan. Tvätta hartset två gånger med fem milliliter portioner DCM och torka den med tryckluft för återanvändning i efterföljande experiment.Spirocyclic oximes med olika substituents var syntetiseras med utmärkta stereoselectivity. Den totala avkastningen indikerar i genomsnitt 80 till 88%avkastning för varje syntetiskt steg. Reaktionen framskrider genom isoxazolringens öppning kan övervakas genom IR-spektroskopi.Generellt individer nya till denna metod kämpar med att välja lämplig polymer stöd linker. Det är viktigt för länkaren att vara robust för alla reaktionsförhållanden under syntesen. Medan du försöker detta förfarande, kom ihåg att tvätta och torka hartset innan nästa reaktionssteg.Överblivna reagenser från föregående steg kommer att komplicera den totala syntesen och kommer att påverka avkastningen och renheten hos den spirocykliska produkten.

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.

Syntes av Immunotargeted Magneto-plasmoniska nanokluster

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 Metodik för Asymmetric Ferrocen Härledda Bio-konjugerade system via Solid Phase Harts-metodik

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.

Hydrokinon baserad syntes av guld nanostavar

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.

Syntes av Hierarkisk ZnO / CdSSe Heteronanoträd

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 av miniatyriserad fast fas extraktion och LC-MS / MS-detektion av 3-nitrotyrosin i human urin för kliniska tillämpningar

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.

Protokoll för fastfassyntesen av oligomerer av RNA innehållande en 2&#39;-<em&gt; O</em&gt; -tiofenylmetylmodifiering och karakterisering via cirkulär dikroism

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.

Syntesen av substrat-bundna Au nanotrådar Via en aktiv yta tillväxt mekanism

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 fas 11C-metylering, rening och formulering för produktion av 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-masspektrometri i kombination med total förångning Fast-Phase Microextraction som ett kriminaltekniskt verktyg

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