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 arbeidet ble finansiert av en bevilgning fra Forskningsrådet fakultetet til K.S. Huang (i Azusa Pacific University - USA). CR Drisko er en mottaker av John Stauffer stipend og Gencarella Undergraduate Research Grant. S.A. Griffin mottok en S2S Undergraduate Research Fellowship fra Institutt for biologi og kjemi.
<img alt="Image 1" src="/files/ftp_upload/58508/58508img1.jpg">
Forfatterne (venstre) Cody Drisko, Dr. Kevin Huang og Silas Griffin utført eksperimenter og forberedt manuskriptet. Cody Drisko er John Stauffer Fellow og en mottaker av Gencarela Research Grant. Silas er en S2S i Azusa Pacific University Research Fellow. Dr. Kevin Huang gitt forskning veiledning og er en mottaker av i Azusa Pacific University fakultet Research Council Grant.

FORSIKTIG: Se alle relevante sikkerhetsdatablader (MSDS) før bruk. Flere av kjemikaliene som brukes i disse synteser er akutt giftige og kreftfremkallende. Kan du bruke alle nødvendige sikkerhets praksis når du utfører følgende reaksjoner, inkludert bruk av engineering kontroller (avtrekksvifte og IR og NMR spektrometre) og personlig verneutstyr (vernebriller, hansker, laboratoriefrakk, full lengde bukser, og lukket tå sko).
1. Michael tillegg av Furfurylamine til REM Linker
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I en typisk REM linker/solid-fase syntetiske strategi, før utgivelsen av et Amin fra solid støtte, er det avgjørende å danne kvartær ammonium salt, som beskrevet i del 4 av protokollen39. På grunn av den steric hindring av trisykliske systemet og omfangsrik R2 grupper (benzyl og octyl halides), kan bare små alkylating reagenser (metyl og allyl halides) benyttes i denne reaksjonen46. Med en enkel endring, slik at for å bruk av større, steric reagenser, st...

Til tross for nyere funn med svært functionalized spirocyclic heterocycles i en rekke biologiske systemer1, en praktisk sti er fortsatt nødvendig for deres lett produsere. Slike systemer og bruker for disse heterocycles inkluderer: MDM2 hemming og andre anticancer aktiviteter2,3,4,5, enzym hemming6,7,8 , antibiotika aktivitet9,10, fluorescerende merking10,11,12, enantioselective bindende for DNA sonder13,14, 15 og RNA målretting16, sammen med mange bruksmuligheter til therapeutics17,18,19. Etterspørselen for disse heterocycles forblir gjeldende litteratur delte om hvilke syntetiske veien er best. Moderne syntetiske tilnærminger til problemet bruke isatin og isatin derivater som starter materiale for en rekke heterocycles20,21, komplisert intramolekylære rearrangements22,23 ,24,25, Lewis syre1,26,27 eller transisjonsmetall katalyse17,28,29, 30, eller asymmetrisk prosesser31. Mens disse prosedyrene har hatt suksess i å produsere bestemte spirocyclic oximes med begrenset funksjonalitet, har en syntetisk strategi for å produsere et bibliotek av molekyler med høye diastereoselectivity vært utforsket relativt mindre32.
Teknikken presenteres her viser at disse molekylene rundt kan genereres ved hjelp av en rekke godt forstått syntetiske teknikker i tandem. Starter med syntese av molekylet på en solid støtte bruker REM linker og intramolekylære silyl nitronate-olefiner cycloaddition (ISOC), den foreslåtte veien distribuerer en ikke-lineær rute, preget av bond kuttet i en tricyclic system, forlate en svært functionalized heterocycle. REM linkers, kjent for sin bekvemmelighet og resirkulerbarhet, utnytte en solid støtte for å syntetisere tertiær aminer33. På grunn av brukervennlighet for rensing akkreditert til REM koblingsfunksjonalitet via enkle filtrering, gir denne solid-fase syntese teknikken forskere med et resirkulerbare og traceless linker, som er brukt her. Når reaksjonen er fullført, REM linker genereres og kan brukes på nytt flere ganger. REM linker er også traceless fordi, i motsetning til mange solid-fase linkers, poenget med vedlegg mellom produktet og polymer er utvisket34,35. Også godt studert og forstått er ISOC reaksjonen, nyttig for syntese av pyrrolidine oximes36,37. Kanskje bedre kjent som en 1,3-dipolar cycloaddition, disse reaksjonene danne en rekke heterocycles med høy diastereoselectivity,38,,39,,40,,41,,42 , 43 , 44 , 45. med endrede REM-sammen-ISOC teknikk for syntese av spirocyclic molekyler gir et svært diastereoselective produkt. Her, rapportere vi om en effektiv produksjon av spirocyclic oximes med en ny syntetisk tilnærming, kombinere to godt forstått stier og tilgjengelige starter materialer.

<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 praktisk syntetiske rute for spirocyclic heterocycles er også ettertraktet molekylets potensiell bruk i biologiske systemer. Med solid-fase syntese, kan regenererer Michael (REM) koblingsfunksjonalitet strategier og 1,3-dipolar cycloaddition, et bibliotek med samme heterocycles, både med og uten en spirocyclic sentrum, konstrueres. De viktigste fordelene med solid-støtte syntese er som følger: første steg reaksjonen kan bli drevet til ferdigstillelse med en stor overskudd av reagenser som resulterer i høy avkastning; neste, bruk av kommersielt tilgjengelige starter materialer og reagenser holde kostnadene lave; Endelig er reaksjon trinnene enkle å rense via enkle filtrering. REM koblingsfunksjonalitet strategien er attraktive på grunn av sin resirkulering og traceless natur. Når en reaksjon ordningen er fullført, linker kan brukes på nytt flere ganger. I en typisk solid-fase syntese inneholder produktet en del av eller hele linker, som kan være uønsket. REM linker er "traceless" poenget med vedlegg mellom produktet og polymer er utvisket. Den høye diastereoselectivity av intramolekylære 1,3-dipolar cycloaddition er godt dokumentert. Begrenset av insolubility av solid støtte, reaksjon progresjon kan bare bli overvåket av en endring i de funksjonelle gruppene (hvis noen) via infrarød (IR) spektroskopi. Dermed kan strukturell identifikasjon av mellomprodukter være preget av konvensjonelle kjernefysiske magnetisk resonans (NRM) spektroskopi. Andre begrensninger til denne metoden stammer fra av kompatibiliteten til polymer/linker ønsket kjemisk reaksjon ordningen. Her rapporterer vi en protokoll som tillater for praktisk produksjon av spirocyclic heterocycles som, med enkle modifikasjoner, kan automatiseres med høy gjennomstrømming teknikker.

Som beskrevet i fremgangsmåten ovenfor, begynner syntetiske ruten til spirocyclic oximes (se figur 1) med Michael tillegg av furfurylamine til sammensatte 1, REM linker, råd til 2. En etterfølgende Michael tillegg og 1,3-dipolar cycloaddition av støtte 2 ulike β-nitrostyrene derivater avkastning trisykliske sammensatte 3, en N- silyloxy isoxazolidine med fire unike stereogenic s...

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 presenterer vi en protokoll for å demonstrere en effektiv metode for syntese av spirocyclic heterocycles. Fem-trinns prosess benytter solid-fase syntese og regenererende Michael koblingsfunksjonalitet strategier. Generelt vanskelig å syntetisere, presenterer vi en passelig metode for syntese av spirocyclic molekyler ellers utilgjengelige for andre moderne tilnærminger.

Solid-fase syntese av [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 solidfasede organiske syntetiske teknikken kan finne anvendelser i byggingen av et kombinatorisk bibliotek, mens syntese av het spirosykliske heterocycles, som kan testes for anti-kreftaktiviteter ved hjelp av bioanalyser. Selv om denne metoden kan brukes til syntese av spirosykliske forbindelser, det kan også brukes på andre små molekyler, som isoksazoliner og isoksazoler. For å starte syntesen, legg til en 25 millileter solid-fase reaksjonsbeholder ett gram REM harpiks, 20 millileter dimetylformamid og 2,4 milliliter furfurylamin.Cap fartøyet og agiter det i 24 timer på en shaker ved romtemperatur for å laste harpiksen med furfurylamin. Tøm deretter furfurylaminbærerharpiksen og agiterer den i omtrent tre minutter i fem milliliter dmf for å vaske den. Deretter vasker du harpiksen fire ganger, vekslende mellom fem milliliter diklormetan for å hovne opp harpiksen, og fem milliliter metanol for å trekke harpiksen sammen.Tørk deretter harpiksen grundig med trykkluft, noe som vanligvis tar ca. 30 minutter. Deretter, i en godt ventilert røykhette, tilsett 1,48 milliliter trietyamin, 10 milliliter vannfri toluen og 637 gram betanitrostyren til den tørre furfurylaminbærende harpiksen. Tilsett en milliliter trimetylsilylklorid til reaksjonsblandingen.Og la fartøyet stå åpent til blandingen er ferdig med å slippe ut hydrokloridgass. Deretter cap fartøyet og agiter blandingen i 48 timer ved romtemperatur for å danne isoxazole mellomliggende. Etter det, legg til fem milliliter metanol for å slukke reaksjonen.Rist blandingen i tre minutter, og la den suge i to minutter. Tøm deretter harpiksen og vask den fire ganger, vekslende mellom fem milliliter DCM og fem milliliter metanol. Tørk den vaskede harpiksen grundig med trykkluft.Deretter legger du til en milliliter vannfri tetrahydrofuran til den tørre harpiksen. Tilsett deretter 1,24 milliliter av en molarløsning av tetra-n-butylammoniummelid i THF og agiterer blandingen ved romtemperatur i 12 timer for å åpne isoksazolringen. Etterpå, spenne harpiks og vaske den med fem milliliter av THF, etterfulgt av fire vasker vekslende mellom fem milliliter DCM og fem milliliter metanol.Tørk harpiksen grundig med trykkluft. Deretter legger du til fem milliliter DMF til harpiksen, etterfulgt av en milliliter en bromooctan. Agg om blandingen i 24 timer ved romtemperatur for å danne det kvarternære aminet.Tøm deretter harpiksen og vask den med fem milliliter DMF. Vask harpiksen fire ganger til, vekslende mellom DCM og metanol, og tørk med trykkluft. Deretter legger du til tre milliliter DCM til den tørre harpiksen, etterfulgt av 1,5 milliliter TE.Agiter blandingen i 24 timer for å holde spirosyklisk oksym fra polymerstøtten. Deretter samler du råoljeproduktet i et hetteglass eller kolbe. Vask harpiksen fire ganger, vekslende mellom DCM og metanol, og samle vaskene i hetteglasset med produktet.Fjern flyktige fra produktblandingen på en roterende fordamper. Deretter triturate råproduktet med 5 milliliter kokende metanol, og fjern forsiktig væsken. Vask harpiksen to ganger med fem milliliter deler av DCM og tørk den med trykkluft for gjenbruk i påfølgende eksperimenter.Spirocykliske oksymer med ulike substitutents ble syntetisert med utmerket stereovalg. Den totale avkastningen indikerer et gjennomsnitt på 80 til 88% utbytte for hvert syntetiske trinn. Reaksjonen utvikler seg opp gjennom isoksazolringåpningen kan overvåkes av IR-spektroskopi.Vanligvis sliter personer som er nye på denne metoden med å velge riktig polymerstøttekobling. Det er viktig at linkeren skal være robust for alle reaksjonsforhold under syntesen. Mens du prøver denne prosedyren, husk å vaske og tørke harpiksen før neste reaksjonstrinn.Rester av reagenser fra forrige trinn vil komplisere den generelle syntesen og vil påvirke utbyttet og renheten til spirosyklisk 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 av 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 ...

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 Methodology for Asymmetric Ferrocene Avledet Bio-konjugerte systemer via Solid Phase Resin-basert 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 av indoksyl-glykosider for deteksjon av glykosidase 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.

Hydrokinon Basert Syntese av gull 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 av hierarkisk ZnO / CdSSe hetrostruktur 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.

Integrasjon av miniatyrisert Solid Phase Extraction og LC-MS / MS Detection av 3-Nitrotyrosin i Human Urin for Kliniske 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 ...

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 for fastfasesyntese av oligomerer av RNA inneholdende en 2&#39;-<em&gt; O</em&gt; -tiofenylmetylmodifikasjon og karakterisering via sirkulær 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 av underlaget-bundet Au Nanowires Via en aktiv overflate vekst 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 Phase 11C-metylering, rensing og formulering for produksjon av pet Bevegelsesuskarphet

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.

Gasskromatografi-massespektrometri sammen med total fordampning fastfase mikroekstraksjon som rettsmedisinsk verktøy

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