Name: preparation of dmmtav and dmdtav using dmav for environmental applications: synthesis, purification, and confirmation
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Description: Watch this Scientific Journal Video about Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation at JoVE.com

Questa ricerca è stata sostenuta dal programma di ricerca di scienza di base (numero di progetto: 2016R1A2B4013467) attraverso la nazionale Ricerca Fondazione della Corea (NRF) finanziato dal Ministero della scienza, ICT & futuro pianificazione 2016 e supportato anche da scienza di base di Corea Istituto di ricerca programma (numero di progetto: C36707).

1. sintesi di MAURIZIOBANDIERIV
<ol><li>
Preparazioni chimiche e rapporto molare di miscelazione di DMAV, Na2S e H2SO4 Nota: DMAV: Na2S:H2così4 = 1:1.6:1.6<ol>
<li>Sciogliere g 5,24 di DMAV in 40 mL deionizzata e N2-eliminato l'inceppo (epurato per almeno 30 min) acqua in un tubo di centrifuga da 50 mL.</li>
		<li>Preparazione del reagente2S Na sciogliendo 14,41 g di Na2S·9H2O in 50 mL deionizzata e N2-eliminate l'acqua in un pallone da 250 mL.</li>
		<li>Preparazione H2così4 reagente aggiungendo 3,3 mL di acido solforico concentrato (96%) a 40 mL di deionizzata e N2-eliminate l'acqua in un tubo di centrifuga da 50 mL. Nota: Finale rapporto molare di DMAV: Na2S:H2SO4 = 1:1.6:1.6 1,3,4,8,15.</li>
		<li>Aggiungere il preparato 40 mL di soluzione DMAV (punto 1.1.1) 50 mL della soluzione di Na2S contenuta nella beuta di 250 mL (punto 1.1.2). Sciacquare il tubo contenente DMAV con 10 mL di acqua eliminata l'inceppo e aggiungere questo al pallone da 250 mL pure.</li>
		<li>Tappare il recipiente con un tappo di gomma tre fori con tubi di vetro. Utilizzare il vetro tubi per H2, deflusso e afflusso di gas di N2 entrata di soluzione SO4 , rispettivamente. Immediatamente dopo aver chiuso il pallone, permettere il flusso di gas di2 N nel pallone. Nota: Pressione del Gas dovrebbe essere effettuate per flusso sopra la superficie della soluzione di reazione senza schizzi.</li>
		<li>Collegare la tubazione resistente all'acido per il tubo di vetro di H2dell'ingresso SO4 soluzione con una siringa da 50 mL aggiungere il preparato 40 mL di H2così4 soluzione (punto 1.1.3). Aggiungere 40 mL di H2così4 soluzione, lentamente e in modo graduale. Attenzione: Al momento l'aggiunta di acido solforico, fumi bianchi saranno generati; utilizzare una cappa ben ventilata.</li>
		<li>Monitorare il cambiamento di colore della miscela di reazione nel pallone durante l'aggiunta di acido solforico a intervalli regolari. Mantenere un intervallo di 4-5 mL gocce di H2così4; la miscela deve essere una soluzione bianca nuvolosa. Nota: Precipitazione giallo istantanea potrebbe apparire a causa della rapida aggiunta di acido solforico concentrato.</li>
		<li>Garantire che la soluzione di reazione è stato in piedi per 1 h dall'inizio del passaggio 1.1.4.</li>
	</ol>
</li>
	<li>
Estrazione diV MAURIZIOBANDIERI utilizzando il metodo di estrazione liquido-liquido
	<ol>
<li>Dopo 1 h, versare la soluzione di reazione in un imbuto separatore contenente circa 200 mL di etere etilico.</li>
		<li>Agitare l'imbuto per 5-10 min, liberando il gas più volte passando il rubinetto. Nota: Sintetizzato MAURIZIOBANDIERIV verrà trasferito allo strato superiore di etere etilico (0.713 g·mL-1). Attenzione: Etere etilico gas potrebbe essere nocivo; utilizzare una cappa ben ventilata.</li>
		<li>Raccogliere la soluzione di reazione in un becher e raccogliere separatamente l'etere etilico contenente MAURIZIOBANDIERIV in una bottiglia. Rimettere la soluzione di reazione nell'imbuto separatore stessa e aggiungere circa 200 mL di etere etilico fresco per riscuotendo. Ripetere i passaggi 1.2.2 - 1.2.3 tre volte.</li>
		<li>Versare l'etere etilico raccolto dal punto 1.2.3 nell'imbuto separatore stesso nuovamente e aggiungere circa 100 mL N2-eliminato l'inceppo acqua deionizzata. Agitare per 5-10 min e scartare il N2-eliminato l'inceppo acqua deionizzata e pochi mL di etere etilico per scopi di purezza. Raccogliere l'etere etilico rimanente in un piatto di petri di vetro (diametro interno minimo di 160 mm) e altezza minima di 50 mm.</li>
		<li>Trasferimento di Petri di vetro in una scatola di guanto N2 atmosfera per impedire la trasformazione di specie. Attenzione: Garantire che il solvente non è tirato nella pompa del vuoto attraverso l'uscita della finestra di passaggio.</li>
		<li>Asciutto fino ad un precipitato bianco di dimethylmonothioarsinate (cristallizzato MAURIZIOBANDIERIV) è formata su di Petri di vetro. Nota: Il protocollo può essere messo in pausa qui.</li>
	</ol>
</li>
	<li>
Verifica di sintetizzato MAURIZIOBANDIERIV e deposito
	<ol>
<li>Prendere il precipitato bianco cristallizzato MAURIZIOBANDIERIVe misurare e registrare il suo peso totale. Attenzione: Utilizzare una cappa ben ventilata o portaoggetti per impedire l'inalazione di gas di idrogeno solforato.</li>
		<li>Sciogliere cristallizzato MAURIZIOBANDIERIV in 50 mL di N2-eliminato l'inceppo acqua deionizzata e filtrare il precipitato giallo attraverso un filtro per siringa da 0,2 µm.</li>
		<li>Si supponga che il totale a partire dal usate DMAV viene convertito in MAURIZIOBANDIERIV, cioè≈9, 649 mg As· L-1. Diluire la soluzione di riserva MAURIZIOBANDIERIV a ≈ 1 magnesio · L-1 e ≈40 µg· L-1 per l'analisi di verifica tramite ionizzazione Electrospray Spectromtery di massa (ESI-MS) e HPLC-ICP-MS, rispettivamente.</li>
		<li>Analizzare m/z di MAURIZIOBANDIERIV mediante ESI-MS11,16 e frammenti a m/z 155 in modalità ioni positivi o a m/z 153 in modalità negativo-ione (tabella 1). Nota: Vedi i valori di riferimento di m/z (tabella 1).</li>
		<li>Analizzare il cromatogramma di MAURIZIOBANDIERIV in soluzione di riserva mediante HPLC-ICP-MS11,16,17 con condizioni di eluente appropriato e un picco maggiore è stato trovato presso il tempo di ritenzione descritto nella conferma la letteratura. Nota: Purezza di sintetizzato MAURIZIOBANDIERIV deve essere calcolato utilizzando i risultati dell'analisi dal punto 1.3.5.</li>
		<li>Analizzare la concentrazione di arsenico totale mediante ICP-MS dopo digestione acida11 e calcolare la concentrazione effettiva di MAURIZIOBANDIERIV in soluzione di riserva di MAURIZIOBANDIERIV sintetizzati utilizzando fattori di diluizione e purezza, come nella seguente equazione: Totale analizzato come concentrazione (µg· L-1) · Fattore di diluizione · Purezza (%) = True MAURIZIOBANDIERIV concentrazione in soluzione di riserva MAURIZIOBANDIERIV (µg· L-1)</li>
		<li>Conservare al buio per ulteriori quantitativi speciazione analisi18MAURIZIOBANDIERIV soluzione a 4 ° C.</li>
	</ol>
</li>
</ol>2. sintesi di DMDTAV
<ol><li>
Preparazioni chimiche e rapporto molare di miscelazione di DMAV , Na2S e H2SO4 Nota: DMAV: Na2S:H2così4 = 1:7.5:7.5<ol>
<li>Sciogliere 1,38 g di DMAV in 40 mL di acqua deionizzata in un tubo di centrifuga da 50 mL.</li>
		<li>Preparazione del reagente2S Na sciogliendo 18,01 g di Na2S·9H2O in un 50 mL di acqua deionizzata in un pallone da 250 mL.</li>
		<li>Preparare, H2così4 reagente con l'aggiunta di 4 mL di acido solforico concentrato (96%) a 40 mL di acqua deionizzata contenuta in un tubo di centrifuga da 50 mL. Nota: Finale rapporto molare di DMAV: Na2S:H2SO4 = 1:7.5:7.51,3,4,8,15.</li>
		<li>Aggiungere il preparato 40 mL di soluzione DMAV (punto 2.1.1) 50 mL della soluzione di Na2S contenuta nella beuta di 250 mL (punto 2.1.2). Sciacquare il tubo contenente DMAV con 10 mL di acqua deionizzata e aggiungere questo al pallone da 250 mL pure.</li>
		<li>Aggiungere il preparato 40 mL di H2così4 soluzione (punto 2.1.3), lentamente e in modo graduale, nel pallone.</li>
		<li>Monitorare il cambiamento di colore della miscela di reazione nel pallone durante l'aggiunta di acido solforico a intervalli regolari. Mantenere un intervallo di 4-5 mL gocce di H2così4; la miscela deve essere una soluzione nuvolosa bianco/giallo. NOTE: Precipitazione giallo istantaneo può essere visualizzati a causa di una rapida aggiunta di acido solforico concentrato. Attenzione: Al momento l'aggiunta di acido solforico, fumi bianchi saranno generati; utilizzare una cappa ben ventilata.</li>
		<li>Mantenere la soluzione di reazione nel pallone durante la notte senza copertura. Nota: Il protocollo può essere messo in pausa qui.</li>
	</ol>
</li>
	<li>
Estrazione diV DMDTA utilizzando il metodo di estrazione in fase solida (SPE)
	<ol>
<li>Dopo la reazione in piedi durante la notte, filtro, la soluzione di reazione usando una siringa C18 digitare SPE a base di silice per intrappolare sintetizzato DMDTAV sulla resina. Attenzione: Utilizzare una cappa ben ventilata.</li>
		<li>Preparate l'acetato di ammonio 10 mM (pH 6,3) sciogliendo 0,77 g di ammonio acetato in 1 L di acqua deionizzata. Eluire un volume sufficiente di acetato di ammonio 10 mM tramite la siringa C18 (punto 2.2.1) per estrarre l'adsorbiti DMDTAV. Raccogliere l'acetato di ammonio filtrato in un bicchiere di Petri (diametro interno minimo di 160 mm) e altezza minima di 50 mm.</li>
		<li>Trasferimento di Petri di vetro in una scatola di guanto N2 atmosfera per impedire la trasformazione di specie. Attenzione: Garantire il solvente non è tirato nella pompa del vuoto attraverso l'uscita della finestra di passaggio.</li>
		<li>Asciugare fino a quando un precipitato bianco di dimethyldithioarsinate è formata (cristallizzato DMDTAV) del piatto di petri di vetro. Nota: Il protocollo può essere messo in pausa qui.</li>
	</ol>
</li>
	<li>
Verifica di sintetizzato DMDTAV e deposito
	<ol>
<li>Prendere il precipitato bianco cristallizzato DMDTAVe misurare il suo peso totale, la misura di registrazione. Attenzione: Utilizzare una cappa ben ventilata o portaoggetti per impedire l'inalazione di gas di idrogeno solforato.</li>
		<li>Sciogliere cristallizzato DMDTAV in 50 mL di N2-eliminato l'inceppo acqua deionizzata in un 50 mL Centrifuga tubo e filtrare qualsiasi precipitato attraverso un filtro per siringa da 0,2 µm.</li>
		<li>Si supponga che il totale a partire dal usate DMAV viene convertito in DMDTAV, cioè≈2, 539 mg As· L-1. Diluire la soluzione di riserva DMDTAV a ≈ 1 magnesio · L-1 e ≈40 µg· L-1 per l'analisi di verifica mediante ESI-MS e HPLC-ICP-MS, rispettivamente.</li>
		<li>Analizzare m/z di DMDTAV mediante ESI-MS11,16 e frammenti a m/z 171 in modalità ioni positivi o a m/z 169 nella modalità negativo-ione (tabella 1). Nota: Vedi i valori di riferimento di m/z (tabella 1).</li>
		<li>Analizzare il cromatogramma di DMDTAV della soluzione stock mediante HPLC-ICP-MS11,16,17 con condizioni di eluente appropriato e che è stato trovato un picco al tempo di ritenzione descritto nella conferma la letteratura. Nota: Purezza di sintetizzato DMDTAV deve essere calcolato utilizzando i risultati dell'analisi dal punto 2.3.5.</li>
		<li>Analizzare la concentrazione di arsenico totale mediante ICP-MS dopo digestione acida11 e calcolare la concentrazione effettiva di DMDTAV DMDTAV sintetizzato soluzione stock utilizzando fattori di diluizione e purezza come la seguente equazione: Totale analizzato come concentrazione (µg· L-1) · Fattore di diluizione · Purezza (%) = concentrazione di vero DMDTAV DMDTAV in soluzione di riserva (µg· L-1)</li>
		<li>Conservare la soluzione stock di DMDTAV a 4 ° C al buio per ulteriori quantitativi speciazione analisi18.</li>
	</ol>
</li>
</ol>

<ol>
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Toxicol. 26, 952-962 (2013).</li><li>Alava, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPLC-ICP-MS+method+development+to+monitor+arsenic+speciation+changes+by+human+gut+microbiota.">HPLC-ICP-MS method development to monitor arsenic speciation changes by human gut microbiota.</a> Biomed. Chromatogr. 26, 524-533 (2012).</li><li>Kurosawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+metabolic+activation+associated+with+glutathione+in+dimethylmonoarsinic+acid+(DMMTAV)-induced+toxicity+obtained+from+in+vitro+reaction+of+DMMTAV+with+glutathione.">A novel metabolic activation associated with glutathione in dimethylmonoarsinic acid (DMMTAV)-induced toxicity obtained from in vitro reaction of DMMTAV with glutathione.</a> J. trace Elem. Med. Biol. 33, 87-94 (2016).</li></ol>

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Il protocollo sviluppato ha chiarito i passaggi critici che precedenti studi1,3,4,8,15 omesso o abbreviato, che potrebbero aver condotto a difficoltà con o errore durante SintesiV MAURIZIOBANDIERIV e DMDTA. Come MAURIZIOBANDIERIV è sensibili all'ossidazione1,5, reagen...

Poiché l'acido dimethylarsinic (DMAV) è stata dimostrata ad esporre sia la tossicità acuta e genotossicità dovuto in fase di metilazione e thiolation su ingestione1,2, la via metabolica di arsenico thiolation ha stato intensamente studiato sia in vitro che in vivo3,4 , nonché come in media ambientali (ad es., percolato di discarica)5,6. Gli studi precedenti hanno trovato entrambi ridotti e chitosani analoghi di DMAV nella vita delle cellule, per esempio, dimethylarsinous acido (DMAIII), acido dimethylmonothioarsinic (MAURIZIOBANDIERIV) e dimethyldithioarsinic (acido DMDTAV)7,8,9, con dimetilata thioarsenicals come MAURIZIOBANDIERIV esibendo una maggiore tossicità rispetto altri noti arsenicali organici od inorganici10. L'abbondanza di thioarsenicals altamente tossico ha gravi implicazioni ambientali, dal momento che potrebbero presentare un rischio per gli esseri umani e all'ambiente in condizioni altamente solo nei confronti di11. Tuttavia, i meccanismi di MAURIZIOBANDIERIV e DMDTAV (trans) formazione e i loro destini in media ambientali richiedono ulteriore studio. Così, l'analisi quantitativa di thioarsenicals è necessaria per migliorare la comprensione degli effetti ambientali di MAURIZIOBANDIERIV e DMDTAV.
Anche se i prodotti chimici standard sono il requisito fondamentale per l'analisi quantitativa, gli standard di MAURIZIOBANDIERIV e DMDTAV sono difficili da ottenere replicando gli studi precedenti, a causa dell'elevato rischio di trasformazione della specie in altre specie e procedure di sintesi Unstandardized12. Inoltre, i metodi di riferimento hanno limitazioni che possono portare a difficoltà pratiche nel sintetizzare le sostanze chimiche standard e l'esecuzione di analisi quantitativa. MAURIZIOBANDIERIV e DMDTAV sono comunemente preparato mescolando DMAV, Na2S e H2così4 in un certo rapporto molare1 o bolle H2S gas attraverso una soluzione di DMAV 13,14. La sostituzione di caratteristiche metodo ribollimento di ossigeno da zolfo utilizzando una fornitura diretta di gas di H2S, che è altamente tossico e difficile da controllare per un utente inesperto. Al contrario, il sopra miscelazione metodo1, ampiamente usato per l'analisi qualitativa di MAURIZIOBANDIERIV e DMDTAV in studi ambientali5,6,12, dispone il thiolation di DMAV con H2S generato mescolando Na2S e H2così4 e produce MAURIZIOBANDIERIV e DMDTAV, consentendo un più facile controllo di stechiometrico per produrre sostanze chimiche di destinazione, come rispetto al diretto uso di H2S gas.
Il riferimento di miscelazione metodo procedure1,3,4,8,15 citato in questo studio mostra limitazioni in alcuni dei loro passaggi critici sperimentale, che potrebbero condurre ad errore sperimentale. Ad esempio, i dettagli della preparazione di solvente specifico (cioè, deionizzata acqua) e l'estrazione e la cristallizzazione l'arsenicali sintetizzati sono sovra-abbreviati o non descritto in modo sufficientemente dettagliato. Tale dispersi e informazioni limitate su passaggi procedurali potrebbero condurre alla formazione incoerente di thioarsenicals e l'analisi di quantificazione inaffidabile. Pertanto, il protocollo modificato sviluppato questo documento descrive la sintesi di MAURIZIOBANDIERIV e DMDTAV soluzioni di riserva con analisi di separazione quantitativa specie.

<table><tbody><tr><td>Cacodylic acid</td><td>Sigma-Aldrich</td><td>20835-10G-F</td><td></td></tr><tr><td>Sodium sulfide nonahydrate</td><td>Sigma-Aldrich</td><td>S2006-500G</td><td></td></tr><tr><td>Sulfuric acid 96%</td><td>J.T.Baker</td><td>0000011478</td><td></td></tr><tr><td>Ammonium acetate</td><td>Sigma-Aldrich</td><td>A7262-500G</td><td></td></tr><tr><td>Formic acid 98%</td><td>Wako Pure Chemical Industries, Ltd.</td><td>066-00461</td><td></td></tr><tr><td>Diethyl ether (Extra Pure)</td><td>Junsei Chemical</td><td>33475-0380</td><td></td></tr><tr><td>Adapter cap for 60 mL Bond Elut catridges</td><td>Agilent Technologies</td><td>12131004</td><td>Syringe type of SPE</td></tr><tr><td>Bond Elut C18 cartridge</td><td>Agilent Technologies</td><td>14256031</td><td>Syringe type of SPE</td></tr><tr><td>HyPURITY C-18</td><td>Thermo Scientific</td><td>22105-254630</td><td>5 um, 125 x 4.6 mm</td></tr><tr><td>Glovebox</td><td>Chungae-chun, Rep. of Korea</td><td></td><td>Customized&amp;nbsp;</td></tr><tr><td>Agilent 1260 Infinity Bio-inert LC</td><td>Agilent Technologies</td><td>DEAB600252, DEACH00245</td><td></td></tr><tr><td>Agilent Technologies 7700 Series ICP-MS</td><td>Agilent Technologies</td><td>JP12031510</td><td></td></tr><tr><td>Finnigan LCQ Deca XP MAX Mass Spectrometer System</td><td>Thermo Electron Corporation</td><td>LDM10627</td><td></td></tr></tbody></table>

preparation of dmmtav and dmdtav using dmav for environmental applications: synthesis, purification, and confirmation

Dimetilata thioarsenicals come l'acido dimethylmonothioarsinic (MAURIZIOBANDIERIV) e l'acido dimethyldithioarsinic (DMDTAV), che sono prodotti dalla via metabolica del dimethylarsinic acido thiolation (DMAV), sono stati recentemente trovate l'ambiente, nonché di organi umani. MAURIZIOBANDIERIV e DMDTAV può essere quantificata per determinare gli effetti ecologici di dimetilata thioarsenicals e la loro stabilità in matrici ambientali. Il metodo di sintesi per questi composti è unstandardized, rendendo la replica precedenti studi impegnativi. Inoltre, c'è una mancanza di informazioni sulle tecniche di conservazione, tra cui deposito di composti senza trasformazione di specie. Inoltre, poiché soltanto le informazioni limitate sui metodi di sintesi sono disponibile, ci possono essere difficoltà sperimentali nella sintesi di prodotti chimici standard e l'esecuzione di analisi quantitativa. Il protocollo presentato qui fornisce un metodo di sintesi praticamente modificate per il thioarsenicals dimetilata, MAURIZIOBANDIERIV e DMDTAVe vi aiuterà nella quantificazione dell'analisi di separazione specie utilizzando liquido ad alte prestazioni cromatografia in combinazione con spettrometria di massa di plasma accoppiato induttivamente (HPLC-ICP-MS). La procedura sperimentale di questa procedura sono stata modificata concentrandosi sulla preparazione di reagenti chimici, i metodi di filtrazione e stoccaggio.

Poiché MAURIZIOBANDIERIV è stato erroneamente preparato da DMAIII sintesi metodo19, verifica di sintetizzato MAURIZIOBANDIERIV e DMDTAV è un passo fondamentale per la sintesi e di estrazione e di determinare lo standard ideale materiali chimici. Sostanze chimiche sintetizzate possono essere verificate dal picco di MAURIZIOBANDIERIV (154 MW g · mol-1) e DMDTAV (MW 170 g · mol-1</s...

Watch this Scientific Journal Video about Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation at JoVE.com

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Preparazione di MAURIZIOBANDIERIV e DMDTAV mediante DMAV per applicazioni ambientali: sintesi, purificazione e conferma

Questo articolo presenta modificati protocolli sperimentali per l'acido dimethylmonothioarsinic (MAURIZIOBANDIERIV) e la sintesi di acido (DMDTAV) dimethyldithioarsinic, inducendo dimethylarsinic acido (DMAV) thiolation attraverso la miscelazione di DMAV , Na2S e H2SO4. Il protocollo modificato fornisce una guida di riferimento sperimentale, superando così le limitazioni i passaggi di sintesi che hanno causato errori sperimentali in analisi quantitativa.

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

preparation-dmmtav-dmdtav-using-dmav-for-environmental-applications

Research

JoVE Journal

Environment

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

5.5K Views.  Korea Basic Science Institute, Seoul Center. Questo articolo presenta modificati protocolli sperimentali per l'acido dimethylmonothioarsinic (MAURIZIOBANDIERIV) e la sintesi di acido (DMDTAV) dimethyldithioarsinic, inducendo dimethylarsinic acido (DMAV) thiolation attraverso la miscelazione di DMAV , Na2S e H2SO4. Il protocollo modificato fornisce una guida di riferimento sperimentale, superando così le limitazioni i passaggi di sintesi ch...

Video: Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do so is highly important in view of a number of applications in functional soft matter including electronics, biomedical engineering, and sensors. In the past, successful strategies to control the size and shape of supramolecular polymers typically focused on the use of templates2,3, end cappers4 or selective solvent techniques5. 
Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing electrostatic repulsive interactions on the hydrophilic rim and attractive non-covalent forces within the hydrophobic core of the polymerizing building block, we manage to create small and discrete spherical objects6,7. Increasing the salt concentration to screen the charges induces a sphere-to-rod transition. Intriguingly, this transition is expressed in an increase of cooperativity in the temperature-dependent self-assembly mechanism, and more stable aggregates are obtained. 
For our study we select a benzene-1,3,5-tricarboxamide (BTA) core connected to a hydrophilic metal chelate via a hydrophobic, fluorinated L-phenylalanine based spacer (Scheme 1). The metal chelate selected is a Gd(III)-DTPA complex that contains two overall remaining charges per complex and necessarily two counter ions. The one-dimensional growth of the aggregate is directed by &pi;-&pi; stacking and intermolecular hydrogen bonding. However, the electrostatic, repulsive forces that arise from the charges on the Gd(III)-DTPA complex start limiting the one-dimensional growth of the BTA-based discotic once a certain size is reached. At millimolar concentrations the formed aggregate has a spherical shape and a diameter of around 5 nm as inferred from 1H-NMR spectroscopy, small angle X-ray scattering, and cryogenic transmission electron microscopy (cryo-TEM). The strength of the electrostatic repulsive interactions between molecules can be reduced by increasing the salt concentration of the buffered solutions. This screening of the charges induces a transition from spherical aggregates into elongated rods with a length &gt; 25 nm. Cryo-TEM allows to visualise the changes in shape and size. In addition, CD spectroscopy permits to derive the mechanistic details of the self-assembly processes before and after the addition of salt. Importantly, the cooperativity -a key feature that dictates the physical properties of the produced supramolecular polymers- increases dramatically upon screening the electrostatic interactions. This increase in cooperativity results in a significant increase in the molecular weight of the formed supramolecular polymers in water.

The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.

Controllo della dimensione, forma e stabilità dei polimeri supramolecolari in acqua

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do ...

Ricerca

Ingegneria

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Produzione di disolfuro-stabilizzati peptide transmembrana per gli studi strutturali

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Chimica

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

Compact Quantum Dots per singola molecola Imaging

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

Misure biochimiche di ipossia neonatale

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

Medicina

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

Preparazione e<em&gt; In Vitro</em&gt; Caratterizzazione del dendrimero a base di agenti di contrasto per risonanza magnetica

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and cost savings through the &#34;scaling out&#34; strategy as opposed to the traditional &#34;scaling up&#34;. Herein, we report the reaction of diphenyldiazomethane with p-nitrobenzoic acid in both batch and flow modes. To effectively transfer the reaction from batch to flow mode, it is essential to first conduct the reaction in batch. As a consequence, the reaction of diphenyldiazomethane was first studied in batch as a function of temperature, reaction time, and concentration to obtain kinetic information and process parameters. The glass flow reactor set-up is described and combines two types of reaction modules with &#34;mixing&#34; and &#34;linear&#34; microstructures. Finally, the reaction of diphenyldiazomethane with p-nitrobenzoic acid was successfully conducted in the flow reactor, with up to 95% conversion of the diphenyldiazomethane in 11 min. This proof of concept reaction aims to provide insight for scientists to consider flow technology&#39;s competitiveness, sustainability, and versatility in their research.

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Flow chemistry carries environmental and economic advantages by leveraging superior mixing, heat transfer and cost benefits. Herein, we provide a blueprint to transfer chemical processes from batch to flow mode. The reaction of diphenyldiazomethane (DDM) with p-nitrobenzoic acid, conducted in batch and flow, was chosen for proof of concept.

Chimica di flusso continuo: Reazione di Diphenyldiazomethane con l'acido p- nitrobenzoico

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and ...

Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl dimethyl azlactone) (PGMA-b-PVDMA), are presented. Due to the high reactivity of azlactone groups towards amine, thiol, and hydroxyl groups, PGMA-b-PVDMA surfaces can be modified with secondary molecules to create chemically or biologically functionalized interfaces for a variety of applications. Previous reports of patterned PGMA-b-PVDMA interfaces have used traditional top-down patterning techniques that generate non-uniform films and poorly controlled background chemistries. Here, we describe customized patterning techniques that enable precise deposition of highly uniform PGMA-b-PVDMA films in backgrounds that are chemically inert or that have biomolecule-repellent properties. Importantly, these methods are designed to deposit PGMA-b-PVDMA films in a manner that completely preserves azlactone functionality through each processing step. Patterned films show well-controlled thicknesses that correspond to polymer brushes (~90 nm) or to highly crosslinked structures (~1-10 &#956;m). Brush patterns are generated using either the parylene lift-off or interface directed assembly methods described and are useful for precise modulation of overall chemical surface reactivity by adjusting either the PGMA-b-PVDMA pattern density or the length of the VDMA block. In contrast, the thick, crosslinked PGMA-b-PVDMA patterns are obtained using a customized micro-contact printing technique and offer the benefit of higher loading or capture of secondary material due to higher surface area to volume ratios. Detailed experimental steps, critical film characterizations, and trouble-shooting guides for each fabrication method are discussed.

Surface fabrication methods for patterned deposition of nanometer thick brushes or micron thick, crosslinked films of an azlactone block co-polymer are reported. Critical experimental steps, representative results, and limitations of each method are discussed. These methods are useful for creating functional interfaces with tailored physical features and tunable surface reactivity.

Fabbricazione di superfici reattive con spazzola e film reticolato di co-polimeri funzionalizzati Azlactone blocco

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

Esplorazione del metiloma dell'arginina mediante spettroscopia di risonanza magnetica nucleare

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

Ricerca sul cancro

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of the levels of oxidative stress within a biological sample, provided that the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), which leads to the formation of MDA-TBA2 adducts called TBARS. TBARS yields a red-pink color that can be measured spectrophotometrically at 532 nm. The TBARS assay is performed under acidic conditions (pH = 4) and at 95 &#176;C. Pure MDA is unstable, but these conditions allow the release of MDA from MDA bis(dimethyl acetal), which is used as the analytical standard in this method. The TBARS assay is a straightforward method that can be completed in about 2 h. Preparation of assay reagents are described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at a low cost rather than buying an expensive TBARS assay kit that only permits construction of a single standard curve (and thus can only be used for one experiment). The applicability of this TBARS assay is shown in human serum, low density lipoproteins, and cell lysates. The assay is consistent and reproducible, and limits of detection of 1.1 &#956;M can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.

The goal of the thiobarbituric acid reactive substances assay is to assess oxidative stress in biological samples by measuring the production of lipid peroxidation products, primarily malondialdehyde, using visible wavelength spectrophotometry at 532 nm. The method described here can be applied to human serum, cell lysates, and low density lipoproteins.

Valutazione dello stress ossidativo in campioni biologici utilizzando il test delle sostanze reattive dell'acido tiobarbiturico

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic ...

Rilevamento della trimetilammina nella medicina di origine animale mediante Headspace GC-MS

An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the poor compliance of clinical patients. Trimethylamine (TMA) is one of the key fishy odor components in animal-derived medicine. It is difficult to identify TMA accurately using the existing detection method due to the increased pressure in the headspace vial caused by the rapid acid-base reaction after the addition of lye, which causes TMA to escape from the headspace vial, stalling the research progress of the fishy odor of animal-derived medicine. In this study, we proposed a controlled detection method that introduced a paraffin layer as an isolation layer between acid and lye. The rate of TMA production could be effectively controlled by slowly liquefying the paraffin layer through thermostatic furnace heating. This method showed satisfactory linearity, precision experiments, and recoveries with good reproducibility and high sensitivity. It provided technical support for the deodorization of animal-derived medicine.

Autore in primo piano: Una tecnica migliorata per la rilevazione della trimetilammina nella medicina di origine animale mediante gascromatografia nello spazio di testa-spettrometria di massa a quadrup

Here, a headspace gas chromatography-tandem quadrupole mass spectrometry (HS-GC-MS/MS) method suitable for the determination of trimethylamine (TMA) in animal-derived medicines is described. The protocol includes sample pretreatment, headspace treatment, analysis conditions, methodological validation, and the determination of TMA in animal-derived medicines.

Una tecnica migliorata per il rilevamento della trimetilammina nella medicina di derivazione animale mediante gascromatografia dello spazio di testa-spettrometria di massa a quadrupolo tandem

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the ...

Preparazione di MAURIZIOBANDIERI^V e DMDTA^V mediante DMA^V per applicazioni ambientali: sintesi, purificazione e conferma

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