Name: preparation of sns cobalt(ii) pincer model complexes of liver alcohol dehydrogenase
Uploaded: 2020-03-19T13:00:00
Description: Watch this Scientific Journal Video about Preparation of SNS CobaltII Pincer Model Complexes of Liver Alcohol Dehydrogenase at JoVE.com

John Miecznikowski recibió apoyo financiero de lo siguiente para este proyecto: Connecticut NASA Space Grant Alliance (Número de Premio P-1168), el Fairfield University Science Institute, College of Arts and Sciences Publication Fund, Fairfield Estipendio de Investigación de Verano de la Facultad universitaria, y National Science Foundation-Major Research Instrumentation Program (Número de subvención CHE-1827854) para fondos para adquirir un espectrómetro NMR de 400 MHz. También agradece a Terence Wu (Universidad de Yale) por su ayuda en la adquisición de espectros de masas electrospray. Jerry Jasinski reconoce el Programa de Instrumentación de Investigación Mayor de la Fundación Nacional de Ciencias (Número de Subvención CHE-1039027) para fondos para comprar un difractómetro de rayos X. Sheila Bonitatibus, Emilse Almanza, Rami Kharbouch y Samantha Zygmont reconocen el Programa de Académicos Hardiman por proporcionar su estipendio de investigación de verano.

1. Síntesis de cloro-(n3-S,S,N)-[2,6-bis(N-isopropyl-N'-metileimidazol-2-thione)piridina]cobalto(II)tetraclorocobaltato [4]
<ol><li>Para preparar el complejo 4, añadir 0.121 g (3.12 x 10-4 mol) de 2,6-bis(N-isopropyl-N'-metilemidazol-2-thione)piridina (C19H25N5S2)6 a 15 mL de acetonitrilo en un matraz inferior redondo de 100 ml. A continuación, a esta solución, añadir 0,0851 g (3,58 x 10-4 mol) de ...

<ol>
	<li>Holm, R. H., Kennepohl, P., Solomon, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+Functional+Aspects+of+Metal+Sites+in+Biology.">Structural and Functional Aspects of Metal Sites in Biology.</a> Chemical Reviews. 96 (7), 2239-2314 (1996).</li><li>Ibers, J. A., Holm, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+coordination+sites+in+metallobiomolecules.">Modeling coordination sites in metallobiomolecules.</a> Science. 209 (4453), 223-235 (1980).</li><li>Kannan, K. K., 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=Crystal+structure+of+human+erythrocyte+carbonic+anhydrase+B.+Three-dimensional+structure+at+a+nominal+2.2-A+resolution.">Crystal structure of human erythrocyte carbonic anhydrase B. Three-dimensional structure at a nominal 2.2-A resolution.</a> Proceedings of the National Academy of Sciences USA. 72 (1), 51-55 (1975).</li><li>Eklund, H., Br&#228;nd&#233;n, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+differences+between+apo-+and+holoenzyme+of+horse+liver+alcohol+dehydrogenase.">Structural differences between apo- and holoenzyme of horse liver alcohol dehydrogenase.</a> Journal of Biological Chemistry. 254, 3458-3461 (1979).</li><li>Miecznikowski, J. R., 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=Syntheses,+Characterization,+Density+Functional+Theory+Calculations+and+Activity+of+Tridentate+SNS+Zinc+Pincer+Complexes.">Syntheses, Characterization, Density Functional Theory Calculations and Activity of Tridentate SNS Zinc Pincer Complexes.</a> Inorganica Chimica Acta. 376, 515-524 (2011).</li><li>Miecznikowski, J. R., 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=Syntheses,+Characterization,+Density+Functional+Theory+Calculations,+and+Activity+of+Tridentate+SNS+Zinc+Pincer+Complexes+Based+on+Bis-Imidazole+or+Bis-Triazole+Precursors.">Syntheses, Characterization, Density Functional Theory Calculations, and Activity of Tridentate SNS Zinc Pincer Complexes Based on Bis-Imidazole or Bis-Triazole Precursors.</a> Inorganica Chimica Acta. 387, 25-36 (2012).</li><li>Sunderland, J. R., 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=Investigation+of+liver+alcohol+dehydrogenase+catalysis+using+an+NADH+biomimetic+and+comparison+with+a+synthetic+zinc+model+complex.">Investigation of liver alcohol dehydrogenase catalysis using an NADH biomimetic and comparison with a synthetic zinc model complex.</a> Polyhedron. 114, 145-151 (2016).</li><li>Miecznikowski, J. R., 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=Synthesis+and+characterization+of+three-+and+five-coordinate+copper(II)+complexes+based+SNS+ligand+precursors.">Synthesis and characterization of three- and five-coordinate copper(II) complexes based SNS ligand precursors.</a> Polyhedron. 80, 157-165 (2014).</li><li>Miecznikowski, J. R., 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=Synthesis,+Characterization,+and+Computational+Study+of+Three-Coordinate+SNS+Copper(I)+Complexes+based+on+Bis-Thione+Ligand+Precursors.">Synthesis, Characterization, and Computational Study of Three-Coordinate SNS Copper(I) Complexes based on Bis-Thione Ligand Precursors.</a> Journal of Coordination Chemistry. 67, 29-44 (2014).</li><li>Lynn, M. A., 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=Copper(I)+SNS+Pincer+Complexes:+Impact+of+Ligand+Design+and+Solvent+Coordination+on+Conformer+Interconversion+from+Spectroscopic+and+Computational+Studies.">Copper(I) SNS Pincer Complexes: Impact of Ligand Design and Solvent Coordination on Conformer Interconversion from Spectroscopic and Computational Studies.</a> Inorganica Chimica Acta. 495, (2019).</li><li>. Web Elements Available from: <a target="_blank" href="https://www.webelements.com/zinc/atom_sizes.html">https://www.webelements.com/zinc/atom_sizes.html</a> (2019)</li><li>. Web Elements Available from: <a target="_blank" href="https://www.webelements.com/cobalt/atom_sizes.html">https://www.webelements.com/cobalt/atom_sizes.html</a> (2019)</li><li>Caballero, A., D&#237;ez-Barra, E., Jal&#243;n, F. A., Merino, S., Tejeda, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1,1'-(pyridine-2,6-diyl)bis(3-benzyl-2,3-dihydro-1H-imidazol-2-ylidine),+a+new+multidentate+N-heterocyclic+bis-carbene+and+its+silver(I)+complex+derivative.">1,1'-(pyridine-2,6-diyl)bis(3-benzyl-2,3-dihydro-1H-imidazol-2-ylidine), a new multidentate N-heterocyclic bis-carbene and its silver(I) complex derivative.</a> Journal of Organometallic Chemistry. 617-618, 395-398 (2001).</li><li>Albrecht, M., van Koten, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platinum+Group+Organometallics+Based+on+"Pincer"+Complexes:+Sensors,+Switches,+and+Catalysis.">Platinum Group Organometallics Based on "Pincer" Complexes: Sensors, Switches, and Catalysis.</a> Angewandte Chemie International Edition. 40 (20), 3750-3781 (2001).</li><li>Peris, E., Crabtree, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+factors+in+pincer+ligand+design.">Key factors in pincer ligand design.</a> Chemistry Society Reviews. 47, 1959-1968 (2018).</li><li>Dolomanov, O. V., Bourhis, L. J., Gildea, R. J., Howard, J. A. K., Puschmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complete+structure,+solution,+refinement,+and+analysis+program.">A complete structure, solution, refinement, and analysis program.</a> Journal of Applied Crystallography. 42, 339-341 (2009).</li><li>Sheldrick, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+Space+Group+and+Crystal+Structure+Determination.">Integrated Space Group and Crystal Structure Determination.</a> Acta Crystallography. 71, 3-8 (2015).</li><li>Sheldrick, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+Structure+Refinement+with+SHELXL.">Crystal Structure Refinement with SHELXL.</a> Acta Crystallography. 71, 3-8 (2015).</li><li>Pauling, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal-metal+bond+lengths+in+complexes+of+transition+metals.">Metal-metal bond lengths in complexes of transition metals.</a> Proceedings of the National Academies of the Sciences of the United States of America. 73, 4290-4293 (1976).</li><li>Trzhtsinskaya, B. V., Abramova, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imidazole-2-Thiones:+Synthesis,+Structure,+Properties.">Imidazole-2-Thiones: Synthesis, Structure, Properties.</a> Sulfur Reports. 10 (4), 389 (1991).</li><li>Schneider, G., Eklund, H., Cedergren-Zeppezauer, E., Zeppezauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+active+site+in+specifically+metal-depleted+and+cobalt+substituted+horse+liver+alcohol+dehydrogenase+derivatives.">Crystal structure of the active site in specifically metal-depleted and cobalt substituted horse liver alcohol dehydrogenase derivatives.</a> Proceedings of the National Academies of the Sciences of the United States of America. 80, 5289-5293 (1983).</li><li>Yang, L., Powell, D. R., Houser, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+variation+in+copper(I)+complexes+with+pyridylmethylamide+ligands:+structural+analysis+with+a+new+four-coordinate+geometry+index,+&#964;4.">Structural variation in copper(I) complexes with pyridylmethylamide ligands: structural analysis with a new four-coordinate geometry index, &#964;4.</a> Dalton Transactions. , 955-964 (2007).</li></ol>

La preparación de los complejos 4 y 5 es fácil. El paso clave es añadir el sólido CoCl2s 6H2O a una solución de acetonitrilo que contiene el precursor de ligando respectivo. La solución se vuelve verde oscuro en cuestión de segundos después de la adición de CoCl2s 6H2O para formar complejo 4. La solución se vuelve azul brillante después de la adición de CoCl2s 6H2O para formar complejo ...

El propósito del método presentado es preparar análogos de moléculas pequeñas de LADH para comprender mejor la actividad catalítica de las metalózimas. LADH es una enzima dimerica que contiene un dominio de unión a cofactores y undominiocatalítico que contiene zinc(II) 1 . LADH, en presencia de cofactor NADH, puede reducir cetonas y aldehídos a sus respectivos derivados del alcohol2. En presencia de NAD+, LADH puede realizar catálisis inversa de oxidación de alcoholes a cetonas y aldehídos2. La estructura cristalina del sitio activo de LADH muestra que su centro metálico de zinc (II) está unido a un átomo de nitrógeno, proporcionado por una cadena lateral de histidina y dos átomos de azufre y ofrecido por dos ligandos de cisteína3. Investigaciones posteriores han demostrado que el centro de metal de zinc está ligado con una molécula de agua lábil, lo que resulta en geometría pseudo-tetraédrica alrededor del centro metálico4.
Hemos informado y utilizado previamente precursores de ligando pinza SNS, así como metalizado los precursores de ligando con ZnCl2 para formar complejos Zn(II) que contienen el precursor de ligando tridentato5,6,7. Estos precursores de ligandose se muestran en la Figura 1. Estos complejos de zinc(II) mostraron actividad para la reducción estequiométrica de los aldehídos pobres en electrones y, por lo tanto, son complejos modelo para LADH. Posteriormente, se han notificado la síntesis y caracterización de una serie de complejos de cobre(I) y cobre(II) que contienen precursores de ligando SNS8,9,10.
Aunque LADH es una enzima zinc (II), estamos interesados en preparar complejos modelo de cobalto (II) de LADH con el fin de obtener más información espectroscópica sobre los análogos de cobalto (II) de LADH. Los complejos cobalto(II) son de color, mientras que los complejos de zinc(II) son blanquecinos. Dado que los complejos de cobalto (II) están coloreados, también se pueden obtener espectros ultravioleta visibles de los complejos, en los que también se puede recopilar información sobre la fuerza del campo de ligandoen en los complejos de cobalto(II). Mediante el uso de información de cálculos gaussianos y los espectros visibles ultravioletas obtenidos experimentalmente, se puede deducir información sobre la fuerza del campo de ligando. El cobalto (II) es un buen sustituto del zinc (II), ya que ambos iones tienen radios iónicos similares y acidezes Lewis similares11,12.
El método presentado consiste en sintetizar y caracterizar complejos de modelos para intentar imitar el comportamiento catalítico natural de LADH5,,6. Anteriormente hemos metalizado una familia de ligandos precursores con ZnCl2 para formar complejos modelo de zinc (II) de LADH, que modeló la estructura y reactividad del sitio activo de zinc en LADH4. A través de múltiples experimentos, estos ligandos de pinza han demostrado ser robustos bajo diferentes condiciones ambientales y se han mantenido estables con una colección diversa de grupos R adjuntos. 5,6
Los ligandos tridentatos son preferibles en comparación con los ligandos monóditados, porque se ha encontrado que tienen más éxito con la metalización debido a los fuertes efectos quelatos de los ligandos tridentitatos. Esta observación se debe a una entropía más favorecida de la formación de ligando pinza tridentita en comparación con un ligando monodentate13. Además, es probable que los ligandos de pinza de tridente eviten la dimerización de los complejos metálicos, lo que se favorece porque es probable que la dimerización difiacere la actividad catalítica de un complejo14. Por lo tanto, el uso de ligandos de pinza tridente se ha demostrado exitoso en la química organometálica en la preparación de complejos catalíticos activos y robustos. Los complejos de pinzas SNS han sido menos estudiados que otros sistemas de pinzas, ya que los complejos de pinzas suelen contener metales de transición de segunda y tercera fila15.
Esta investigación sobre metaloenzimas puede ayudar a comprender aún más su actividad enzimática, que se puede aplicar a otras áreas de la biología. Este método de síntesis de complejos de modelos en comparación con el método alternativo (sintetizar toda la proteína de LADH) es favorable por una serie de razones. La primera ventaja es que los complejos modelo son bajos en masa molecular y todavía son capaces de representar con precisión la actividad catalítica y las condiciones ambientales del sitio activo de la enzima natural. En segundo lugar, los complejos de modelos son más sencillos de trabajar y producir datos fiables y relacionables.
Este manuscrito describe la preparación sintética y caracterización de dos complejos modelo de pinza de cobalto (II) de LADH. Ambos complejos cuentan con un ligando de pinza que contiene átomos de donantes de azufre, nitrógeno y azufre. El primer complejo (4) se basa en un precursor de imidazol, y el segundo (5) se basa en un precursor de triazol. Los complejos muestran reactividad para la reducción de estequiometría de aldehídos pobres en electrones en presencia de un donante de hidrógeno. Estos resultados de reactividad se notificarán en un manuscrito posterior.

<table>
	<tbody>
		<tr>
			<td>100 mL Round Bottomed Flask</td>
			<td>Chem Glass</td>
			<td>CG150691</td>
			<td>100mL Single Neck Round Bottomed Flask, 19/22 Outer Joint</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher</td>
			<td>HB9823-4</td>
			<td>HPLC Grade</td>
		</tr>
		<tr>
			<td>Chiller for roto-vap</td>
			<td>Lauda</td>
			<td>L000638</td>
			<td>Alpha RA 8</td>
		</tr>
		<tr>
			<td>Cobalt Chloride hexahydrate</td>
			<td>Acros Organics</td>
			<td>AC423571000</td>
			<td>Acros Organics</td>
		</tr>
		<tr>
			<td>Diethyl Ether</td>
			<td>Fisher</td>
			<td>E-138-1</td>
			<td>Diethyl Ether Anhydorus</td>
		</tr>
		<tr>
			<td>graduated cylinder</td>
			<td>Fisher</td>
			<td>S63456</td>
			<td>25 mL graduated cylinder</td>
		</tr>
		<tr>
			<td>hotplate</td>
			<td>Fisher</td>
			<td>11-100-49SH</td>
			<td>Isotemp Basic Stirring Hotplate</td>
		</tr>
		<tr>
			<td>jars</td>
			<td>Fisher</td>
			<td>05-719-481</td>
			<td>250 mL jars</td>
		</tr>
		<tr>
			<td>Ligand</td>
			<td>-----</td>
			<td>-----</td>
			<td>Synthezied previously by Professor Miecznikowski</td>
		</tr>
		<tr>
			<td>medium cotton balls</td>
			<td>Fisher</td>
			<td>22-456-80</td>
			<td>medium cotton balls</td>
		</tr>
		<tr>
			<td>one dram vials</td>
			<td>Fisher</td>
			<td>03-339</td>
			<td>one dram vials with TFE Lined Cap</td>
		</tr>
		<tr>
			<td>pipet</td>
			<td>Fisher</td>
			<td>13-678-20B</td>
			<td>5.75 inch pipets</td>
		</tr>
		<tr>
			<td>pipet bulbs</td>
			<td>Fisher</td>
			<td>03-448-21</td>
			<td>Fisher Brand Latex Bulb for pipet</td>
		</tr>
		<tr>
			<td>recrystallizing dish for sand bath</td>
			<td>Fisher</td>
			<td>08-741 D</td>
			<td>325 mL recrystallizing dish for sand bath</td>
		</tr>
		<tr>
			<td>reflux condensor</td>
			<td>Chem Glass</td>
			<td>CG-1218-A-22</td>
			<td>Condenser with 19/22 inner joint</td>
		</tr>
		<tr>
			<td>Rotovap</td>
			<td>Heidolph Collegiate</td>
			<td>36000090</td>
			<td>Brinkmann; Heidolph Collegiate Rotary Evaporator with Heidolph WB eco bath Heidolph Rotary Evaporator</td>
		</tr>
		<tr>
			<td>sea sand for sandbath</td>
			<td>Acros Organics</td>
			<td>612355000</td>
			<td>washed sea sand for sand bath</td>
		</tr>
		<tr>
			<td>Stir bar</td>
			<td>Fisher</td>
			<td>07-910-23</td>
			<td>Egg-Shaped Magnetic Stir Bar</td>
		</tr>
		<tr>
			<td>Vacum grease</td>
			<td>Fisher</td>
			<td>14-635-5D</td>
			<td>Dow Corning High Vacuum Grease</td>
		</tr>
		<tr>
			<td>vacuum pump for rotovap</td>
			<td>Heidolph Collegiate</td>
			<td>36302830</td>
			<td>Heidolph Rotovac Valve Control</td>
		</tr>
	</tbody>
</table>

preparation of sns cobalt(ii) pincer model complexes of liver alcohol dehydrogenase

Los complejos de modelos químicos están preparados para representar el sitio activo de una enzima. En este protocolo, una familia de precursores de ligando de pinza tridentato (cada uno posee dos funcionalidades de átomo de donante de azufre y un nitrógeno (SNS) y se basa en compuestos de bis-imidazol o bis-triazol) se metalizan con CoCl2s 6H2O para permitir complejos tridentatos de cobalto pinza SNS(II). La preparación de los complejos modelo cobalto(II) para el alcohol hepático deshidrogenasa es fácil. Basado en un cambio de color rápido al agregar el CoCl2s 6H2O a la solución de acetonitrilo que contiene el precursor de ligando, las formas complejas rápidamente. La formación del complejo metálico se completa después de permitir que la solución se refluya durante la noche. Estos complejos de cobalto (II) sirven como modelos para el sitio activo de zinc en alcohol deshidrogenasa hepática (LADH). Los complejos se caracterizan utilizando difracción de rayos X de cristal único, espectrometría de masas de electrospray, espectroscopia visible ultravioleta y análisis elemental. Para determinar con precisión la estructura del complejo, se debe determinar su estructura cristalina única. Los cristales individuales de los complejos que son adecuados para la difracción de rayos X se cultivan a través de la difusión lenta de vapor de éter dietílico en una solución de acetonitrilo que contiene el complejo cobalto(II). Para cristales de alta calidad, la recristalización normalmente tiene lugar durante un período de 1 semana, o más. El método se puede aplicar a la preparación de otros complejos de coordinación modelo y se puede utilizar en laboratorios de enseñanza de grado. Finalmente, se cree que otros pueden encontrar este método de recristalización para obtener cristales únicos beneficiosos para su investigación.

Síntesis Las síntesis de los complejos 4 y 5 se llevaron a cabo con éxito reaccionando una solución de acetonitrilo que contiene un precursor de ligando bis-tione con cloruro de cobalto (II) hexahidrato(Figura 2). Esta reacción se produjo a una temperatura de reflujo en presencia de aire. En general, se observó que los complejos 4 y 5 eran solubles en acetonitrilo, dimetilsulfóxido, diclorometano y...

Watch this Scientific Journal Video about Preparation of SNS CobaltII Pincer Model Complexes of Liver Alcohol Dehydrogenase at JoVE.com

Preparation of SNS CobaltII Pincer Model Complexes of Liver Alcohol Dehydrogenase

Aquí se presenta la preparación de los complejos modelo de pinza SNS(II) de alcohol deshidrogenasa hepática. Los complejos se pueden preparar reaccionando el precursor de ligando con CoCl2s 6H2O y luego se pueden recristalizar permitiendo que el éter de dietil se difunda lentamente en una solución de acetonitrilo que contiene el complejo de cobalto.

Preparación de SNS Cobalt(II) Pincer Model Complexes of Liver Alcohol Dehydrogenase

Chemical model complexes are prepared to represent the active site of an enzyme. In this protocol, a family of tridentate pincer ligand precursors (each ...

Este método se puede utilizar para preparar un complejo de modelos químicos de una enzima alcohol hepático deshidrogenasa y para crecer cristales individuales. Las ventajas de esta técnica son que la formación compleja es fácil y que el método de recristalización se puede aplicar a la preparación de otros complejos de coordinación. Este método puede proporcionar información sobre la preparación y recristalización de compuestos de coordinación inorgánicos y se puede aplicar a cualquier compuesto de interés para cultivar cristales individuales.Se necesita práctica para cultivar los cristales individuales. La clave es hacer que la solución de acetonitrilo que contiene el complejo metálico esté lo más concentrado posible. Para preparar cuatro complejos, añadir 0.121 gramos de 2, 6-BIS-N-isopropyl-N'metilenoimidzol-2-thione-piridina y 0.0851 gramos de cloruro de cobalto II hexahidrato a un frasco inferior redondo de 100 mililitros que contiene 15 mililitros de acetonitrilo.La reacción debe cambiar de un color amarillo claro a verde esmeralda inmediatamente después de añadir el cloruro de cobalto II hexahidrato. A continuación, revuelva y remueva la reacción durante 20 horas para garantizar una reacción completa antes de usar un rotovap bajo presión reducida para eliminar el disolvente. Para cloro-N3-S, S, N-2, 6-BIS-N-isopropyl-N'metileenemilzol-2-thione-pyridina cobalto II tetracloocaltate recrystallization por difusión lenta de vapor, disolver el soluto en 10 mililitros de acetonitrile, filete la solución, luego alícuota la solución uniformemente entre un vial de dram.Añadir 1,5 mililitros de solución fresca de acetonitrilo a cada vial y tapar perfectamente los viales con algodón. Coloque los viales en un frasco de 240 mililitros que contenga 50 mililitros de éter dietílico y cierre el frasco con una tapa. Luego deja que los cristales crezcan hasta por una semana.Para preparar cinco complejos, añadir 0.183 gramos de 2, 6-BIS-N-isopropyl-N'metilenotriazol-2-thione-pyridina y 0.223 gramos de cloruro de cobalto II hexahidrato a 15 mililitros de acetonitrilo en un matraz inferior redondo de 100 mililitros. La reacción debe cambiar de color de un color amarillo claro a azul real inmediatamente después de añadir el cloruro de cobalto II hexahidrato. A continuación, revuelva y remueva la reacción durante 20 horas para garantizar una reacción completa antes de retirar el disolvente utilizando un rotovap bajo presión reducida.Para cloro-N3-S, S, N-2, 6-BIS-N-isopropyl-N'metilenotriazol-2-thione-pyridina cobalto II tetracloroocaltate recrystallization por difusión lenta de vapor, disolver el soluto en 10 mililitros de acetonitrilo y filtrar la solución antes de alícuota de la solución uniformemente en un vial de dram. Llene cada vial con 1,5 mililitros de solución de acetonitrilo y tapone perfectamente los viales con algodón. Para cultivar cristales individuales de alta calidad, es fundamental que el tapón de algodón encaje perfectamente dentro del vial de un dram.Usamos un poco menos de una bola de algodón para cada vial. A continuación, coloque los viales en un frasco que contenga 50 mililitros de éter dietílico, cierre el frasco con una tapa y deje que los cristales crezcan hasta por una semana. La síntesis de los complejos cuatro y cinco se puede llevar a cabo con éxito reaccionando una solución de acetonitrilo que contiene un precursor de ligando de bis-tione con hexahidrato de cloruro de cobalto II.La adquisición de cristales individuales de complejos cuatro y cinco a través de la difusión lenta de vapor es un excelente método para el cultivo de cristales individuales para muestras difíciles de cristalizar. El análisis elemental de los complejos recristalizados sugiere que los complejos cuatro y cinco son puros porque los porcentajes calculados de carbono, hidrógeno y nitrógeno están en excelente acuerdo con los porcentajes encontrados de carbono, hidrógeno y nitrógeno. Además, la espectroscopia ultravioleta visible de los complejos cuatro y cinco revela que los cuatro complejos exhiben tres picos en la región visible con 680, 632 y 589 nanómetros, y que los complejos cinco exhiben cuatro picos en la región visible a 682, 613, 588 y 573 nanómetros.Lo más importante a recordar es que cuando se prepara el complejo, debe añadir cloruro de cobalto hexahidrato a una solución de acetonitrilo que contiene un precursor de ligando. El método de recristalización se puede aplicar a cualquier compuesto orgánico o inorgánico que sea difícil de recristalizar. Actualmente estamos preparando complejos de cobalto II que no contienen cobalto en el contraanión y cribado de los complejos para su reactividad.Asegúrese de llevar a cabo todas las reacciones en una campana de humos.

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Preparation of SNS Cobalt(II) Pincer Model Complexes of Liver Alcohol Dehydrogenase

Transition Metals and Coordination Complexes

SCIENTISTS IN ACTION

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.

La producción de disulfuro estabilizados complejos transmembrana de péptidos de Estudios Estructurales

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

Investigación

Química

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

Anticancerosos complejos metálicos: Síntesis y Evaluación de la citotoxicidad por el ensayo MTT

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

El uso de la cromatografía de gases para analizar los cambios de composición de ácidos grasos en el tejido de hígado de rata durante el embarazo

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the hydroxylmethylene phosphine precursor with ammonia in methanol under a nitrogen atmosphere. The N-triphosPh ligand precipitates from the solution after approximately 1 hr of reflux and can be isolated analytically pure via simple cannula filtration procedure under nitrogen. Reaction of the N-triphosPh ligand with [Ru3(CO)12] under reflux affords a deep red solution that show evolution of CO gas on ligand complexation. Orange crystals of the complex [Ru(CO)2{N(CH2PPh2)3}-&#954;3P] (2) were isolated on cooling to RT. The 31P{1H} NMR spectrum showed a characteristic single peak at lower frequency compared to the free ligand. Reaction of a toluene solution of complex 2 with oxygen resulted in the instantaneous precipitation of the carbonate complex [Ru(CO3)(CO){N(CH2PPh2)3}-&#954;3P] (3) as an air stable orange solid. Subsequent hydrogenation of 3 under 15 bar of hydrogen in a high-pressure reactor gave the dihydride complex [RuH2(CO){N(CH2PPh2)3}-&#954;3P] (4), which was fully characterized by X-ray crystallography and NMR spectroscopy. Complexes 3 and 4 are potentially useful catalyst precursors for a range of hydrogenation reactions, including biomass-derived products such as levulinic acid (LA). Complex 4 was found to cleanly react with LA in the presence of the proton source additive NH4PF6 to give [Ru(CO){N(CH2PPh2)3}-&#954;3P{CH3CO(CH2)2CO2H}-&#954;2O](PF6) (6).

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Ruthenium phosphine complexes are widely used for homogeneous catalytic reactions such as hydrogenations. The synthesis of a series of novel tridentate ruthenium complexes bearing the N-triphos ligand N(CH2PPh2)3 is reported. Additionally, the stoichiometric reaction of a dihydride Ru&#8211;N-triphos complex with levulinic acid is described.

La síntesis, caracterización y reactividad de una serie de rutenio<em&gt; N</em&gt; -triphos<sup&gt; Ph</sup&gt; Complejos

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the ...

Preparation of Carbon Nanosheets at Room Temperature

Amphiphilic molecules equipped with a reactive, carbon-rich "oligoyne" segment consisting of conjugated carbon-carbon triple bonds self-assemble into defined aggregates in aqueous media and at the air-water interface. In the aggregated state, the oligoynes can then be carbonized under mild conditions while preserving the morphology and the embedded chemical functionalization. This novel approach provides direct access to functionalized carbon nanomaterials. In this article, we present a synthetic approach that allows us to prepare hexayne carboxylate amphiphiles as carbon-rich siblings of typical fatty acid esters through a series of repeated bromination and Negishi-type cross-coupling reactions. The obtained compounds are designed to self-assemble into monolayers at the air-water interface, and we show how this can be achieved in a Langmuir trough. Thus, compression of the molecules at the air-water interface triggers the film formation and leads to a densely packed layer of the molecules. The complete carbonization of the films at the air-water interface is then accomplished by cross-linking of the hexayne layer at room temperature, using UV irradiation as a mild external stimulus. The changes in the layer during this process can be monitored with the help of infrared reflection-absorption spectroscopy and Brewster angle microscopy. Moreover, a transfer of the carbonized films onto solid substrates by the Langmuir-Blodgett technique has enabled us to prove that they were carbon nanosheets with lateral dimensions on the order of centimeters.

We present the synthesis of an amphiphilic hexayne and its use in the preparation of carbon nanosheets at the air-water interface from a self-assembled monolayer of these reactive, carbon-rich molecular precursors.

Preparación de nanoláminas de carbono a temperatura ambiente

Amphiphilic molecules equipped with a reactive, carbon-rich "oligoyne" segment consisting of conjugated carbon-carbon triple bonds self-assemble into ...

Facile Preparation of 4-Substituted Quinazoline Derivatives

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted 2-aminobenzophenones and thiourea in the presence of dimethyl sulfoxide (DMSO). This is a unique complementary reaction system in which thiourea undergoes thermal decomposition to form carbodiimide and hydrogen sulfide, where the former reacts with 2-aminobenzophenone to form 4-phenylquinazolin-2(1H)-imine intermediate, whilst hydrogen sulfide reacts with DMSO to give methanethiol or other sulfur-containing molecule which then functions as a complementary reducing agent to reduce 4-phenylquinazolin-2(1H)-imine intermediate into 4-phenyl-1,2-dihydroquinazolin-2-amine. Subsequently, the elimination of ammonia from 4-phenyl-1,2-dihydroquinazolin-2-amine affords substituted quinazoline derivative. This reaction usually gives quinazoline derivative as a single product arising from 2-aminobenzophenone as monitored by GC/MS analysis, along with small amount of sulfur-containing molecules such as dimethyl disulfide, dimethyl trisulfide, etc. The reaction usually completes in 4-6 hr at 160 &#186;C in small scale but may last over 24 hr when carried out in large scale. The reaction product can be easily purified by means of washing off DMSO with water followed by column chromatography or thin layer chromatography.

A protocol for facile preparation of 4-substituted quinazoline derivatives from 2-aminobenzophenones, thiourea and dimethyl sulfoxide is presented.

Preparación Facile de derivados de quinazolina 4-sustituidos

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted ...

Preparation of Biopolymer Aerogels Using Green Solvents

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers (gelatin, agar, cellulose), only recently have biomasses been recognized as an abundant source of chemically diverse macromolecules for functional aerogel materials. Biopolymer aerogels (pectin, alginate, chitosan, cellulose, etc.) exhibit both specific inheritable functions of starting biopolymers and distinctive features of aerogels (80-99% porosity and specific surface up to 800 m2/g). This synergy of properties makes biopolymer aerogels promising candidates for a wide gamut of applications such as thermal insulation, tissue engineering and regenerative medicine, drug delivery systems, functional foods, catalysts, adsorbents and sensors. This work demonstrates the use of pressurized carbon dioxide (5 MPa) for the ionic cross linking of amidated pectin into hydrogels. Initially a biopolymer/salt dispersion is prepared in water. Under pressurized CO2 conditions, the pH of the biopolymer solution is lowered to 3 which releases the crosslinking cations from the salt to bind with the biopolymer yielding hydrogels. Solvent exchange to ethanol and further supercritical CO2 drying (10 - 12 MPa) yield aerogels. Obtained aerogels are ultra-porous with low density (as low as 0.02 g/cm3), high specific surface area (350 - 500 m2/g) and pore volume (3 - 7 cm3/g for pore sizes less than 150 nm).

A new way for the production of biopolymer-based aerogels by carbon dioxide (CO2) induced gelation is shown. The technique utilizes pressurized carbon dioxide (5 MPa) for the production of biopolymer hydrogels and supercritical CO2 (12 MPa) to convert gels into aerogels. The only solvents needed besides CO2 are water and ethanol.

Preparación de biopolímeros aerogeles el uso de disolventes verdes

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers ...

Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes

Here, we describe an effective protocol that combines photoredox Ni/Ir catalysis with the use of a Zn-alkoxide for efficient ring-opening polymerization, allowing for the synthesis of isotactic poly(&#945;-hydroxy acids) with expected molecular weights (&#62;140 kDa) and narrow molecular weight distributions (Mw/Mn &#60; 1.1). This ring-opening polymerization is mediated by Ni and Zn complexes in the presence of an alcohol initiator and a photoredox Ir catalyst, irradiated by a blue LED (400 - 500 nm). The polymerization is performed at a low temperature (-15 &#176;C) to avoid undesired side reactions. The complete monomer consumption can be achieved within 4 - 8 hours, providing a polymer close to the expected molecular weight with narrow molecular weight distribution. The resulted number-average molecular weight shows a linear correlation with the degree of polymerization up to 1000. The homodecoupling 1H NMR study confirms that the obtained polymer is isotactic without epimerization. This polymerization reported herein offers a strategy for achieving rapid, controlled O-carboxyanhydrides polymerization to prepare stereoregular poly(&#945;-hydroxy acids) and its copolymers bearing various functional side-chain groups.

Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes

A protocol for the controlled photoredox ring-opening polymerization of O-carboxyanhydrides mediated by Ni/Zn complexes is presented.

Controla la polimerización de apertura de Photoredox de O- Carboxyanhydrides mediada por complejos de Ni/Zn

Here, we describe an effective protocol that combines photoredox Ni/Ir catalysis with the use of a Zn-alkoxide for efficient ring-opening polymerization, ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation usually consists in the formation of droplets containing low molar mass liquid crystals at elevated temperatures. Subsequently, these particle precursors are oriented in the flow field of the capillary and solidified by a crosslinking polymerization, which produces the final actuating particles. The optimization of the process is necessary to obtain the actuating particles and the proper variation of the process parameters (temperature and flow rate) and allows variations of size and shape (from oblate to strongly prolate morphologies) as well as the magnitude of actuation. In addition, it is possible to vary the type of actuation from elongation to contraction depending on the director profile induced to the droplets during the flow in the capillary, which again depends on the microfluidic process and its parameters. Furthermore, particles of more complex shapes, like core-shell structures or Janus particles, can be prepared by adjusting the setup. By the variation of the chemical structure and the mode of crosslinking (solidification) of the liquid crystalline elastomer, it is also possible to prepare actuating particles triggered by heat or UV-vis irradiation.

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

Preparación de microfluídica de elastómero cristalino líquido actuadores

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...