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 fikk økonomisk støtte fra følgende for dette prosjektet: Connecticut NASA Space Grant Alliance (Award Number P-1168), Fairfield University Science Institute, College of Arts and Sciences Publication Fund, Fairfield University Fakultet Summer Research Stipend, og National Science Foundation-Major Research Instrumentation Program (Grant Number CHE-1827854) for midler til å kjøpe en 400 MHz NMR spektrometer. Han takker også Terence Wu (Yale University) for hjelp til å skaffe elektrospray masse spektra. Jerry Jasinski anerkjenner National Science Foundation-Major Research Instrumentation Program (Grant Number CHE-1039027) for midler til å kjøpe en røntgendiffractometer. Sheila Bonitatibus, Emilse Almanza, Rami Kharbouch og Samantha Zygmont anerkjenner Hardiman Scholars Program for å gi sitt sommerforskningsstipend.

1. Syntese av klor-(n 3-S,S,N)-[2,6-bis(N-isopropyl-N'-metylenimidazol-2-tione)pyridin]kobolt(II)tetraklorkoökoökoösetter [4]3
<ol><li>For å forberede kompleks 4,legg til 0,121 g (3,12 x 10-4 mol) på 2,6-bis(N-isopropyl-N'-methyleneimidazole-2-thione)pyridin (C19H25N5S2)6 til 15 ml acetonitrile i en 100 ml rund nedre kolbe. Deretter tilsetter du 0,0851 g (3,58 x 10-4 mol) koboltklorid (II) heksahyd...

<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>

Utarbeidelsen av komplekser 4 og 5 er facile. Hovedtrinnet er å legge til den solide CoCl2·6H2O til en acetonitrile løsning som inneholder den respektive ligandforløperen. Løsningen blir mørk grønn i løpet av sekunder etter tillegg av CoCl2·6H2O for å danne kompleks 4. Løsningen blir lys blå etter tillegg av CoCl2·6H2O for å danne kompleks 5. For å sikre fullstendig reaksjon...

Formålet med den presenterte metoden er å forberede småmolekylanaloger av LADH for å forstå katalytisk aktivitet av metalloenzymer ytterligere. LADH er et dimeric enzym som inneholder et kofaktorbindende domene og sink (II) metallholdig katalytisk domene1. LADH, i nærvær av co-faktor NADH, kan redusere ketoner og aldehyder til sine respektive alkoholderivater2. I nærvær av NAD+kan LADH utføre omvendt katalysere av oksidasjon av alkoholer til ketoner og aldehyder2. Krystallstrukturen til LADh sin aktive side viser at dets sink (II) metallsenter er bundet til ett nitrogenatom, levert av en histidinsidekjede og to svovelatomer og tilbys av to cysteinligander3. Videre forskning har vist at sink metallsenteret er ligated med et labile vannmolekyl, noe som resulterer i pseudo-tetrahedral geometri rundt metallsenteret4.
Vi har tidligere rapportert og utnyttet SNS pincer ligand forløpere samt metallert ligand forløperne med ZnCl2 å danne Zn (II) komplekser som inneholder tridentate ligand forløperen5,6,7. Disse ligandforløperne er vist i figur 1. Disse sink (II) komplekser utstilt aktivitet for stoichiometric reduksjon av elektron-fattige aldehyder og er dermed modell komplekser for LADH. Deretter har syntesen og karakteriseringen av en rekke kobber(I) og kobber (II) komplekser som inneholder SNS ligand forløpere blitt rapportert8,9,10.
Selv om LADH er et sink(II) enzym, er vi interessert i å forberede kobolt(II) modellkomplekser av LADH for å få mer spektroskopisk informasjon om kobolt (II) analoger av LADH. Kobolt(II) komplekser er farget, mens sink (II) komplekser er off-white. Siden kobolt(II) komplekser er farget, ultrafiolett synlig spektra av kompleksene kan oppnås, der informasjon om styrken av ligand feltet i kobolt (II) komplekser kan også samles. Ved å bruke informasjon fra gaussiske beregninger og eksperimentelt innhentet ultrafiolett synlig spektra, kan informasjon om styrken i ligandfeltet utledes. Kobolt(II) er en god erstatning for sink(II), siden begge ionene har lignende ioniske radier og lignende Lewis-syrer11,,12.
Den presenterte metoden innebærer å syntetisere og karakterisere modellkomplekser for å forsøke å etterligne den naturlige katalytiske oppførselen til LADH5,6. Vi har tidligere metallert en familie av ligand forløpere med ZnCl2 for å danne sink (II) modellkomplekser av LADH, som modellerte strukturen og reaktiviteten til sink aktive området i LADH4. Gjennom flere eksperimenter har disse pincer ligandene vist seg å være robuste under ulike miljøforhold og har holdt seg stabile med en mangfoldig samling av vedlagte R-grupper. 5,6(andre kan være på)
Tridentate ligands er å foretrekke i forhold til monodentate ligands, fordi de har blitt funnet å være mer vellykket med metallering på grunn av de sterke chelate effektene av tridentate ligands. Denne observasjonen skyldes en mer favorisert entropi av tridentatpincer ligand dannelse i forhold til en monodentate ligand13. Videre er tridentate pincer ligands sannsynlig å hindre dimerization av metallkomplekser, som favoriseres fordi dimerization er sannsynlig å bremse katalytisk aktivitet av et kompleks14. Dermed, ved hjelp av tridentate pincer ligands har vist seg vellykket i organometallisk kjemi i utarbeidelsen av katalytiske aktive og robuste komplekser. SNS pincer komplekser har blitt mindre studert enn andre pincer systemer, som pincer komplekser vanligvis inneholder andre og tredje rad overgang metaller15.
Denne forskningen på metalloenzymer kan bidra til å fremme forståelsen av deres enzymatiske aktivitet, som kan brukes på andre områder i biologi. Denne metoden for å syntetisere modellkomplekser sammenlignet med den alternative metoden (syntetisere hele proteinet til LADH) er gunstig av flere grunner. Den første fordelen er at modellkomplekser er lave i molekylær masse og fortsatt er i stand til nøyaktig å representere katalytisk aktivitet og miljøforhold i det naturlige enzymets aktive område. For det andre er modellkomplekser enklere å arbeide med og produsere pålitelige og relatable data.
Dette manuskriptet beskriver syntetisk forberedelse og karakterisering av to kobolt (II) pincer modell komplekser av LADH. Begge kompleksene har en pincer ligand som inneholder svovel, nitrogen og svoveldonoratomer. Det første komplekset (4) er basert på en imidazol forløper, og den andre (5) er basert på en triazol forløper. Kompleksene viser reaktivitet for stoichiometry reduksjon av elektron fattige aldehyder i nærvær av en hydrogendonor. Disse reaktivitetsresultatene vil bli rapportert i et påfølgende manuskript.

<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

Kjemiske modellkomplekser er forberedt på å representere det aktive stedet for et enzym. I denne protokollen, en familie av tridentate pincer ligand forløpere (hver besitter to svovel og en nitrogen donor atom funksjonalitet (SNS) og basert på bis-imidazol eller bis-triazole forbindelser) er metallated med CoCl2·6H2O å råd tridentate SNS pincer kobolt (II) komplekser. Tilberedning av kobolt(II) modellkomplekser for leveralkohol dehydrogenase er facile. Basert på en rask fargeendring ved å legge til CoCl2·6H2O til acetonitrile løsning som inneholder ligand forløperen, danner komplekset raskt. Dannelsen av metallkomplekset er fullført etter at løsningen kan bli refluks over natten. Disse kobolt(II) komplekser tjene som modeller for sink aktive stedet i leveren alkohol dehydrogenase (LADH). Kompleksene er karakterisert ved hjelp av enkelt krystall røntgendiffraksjon, elektrospraymassespektromi, ultrafiolett synlig spektroskopi og elementær analyse. For å nøyaktig bestemme strukturen av komplekset, må den seneste krystallstrukturen bestemmes. Enkeltkrystaller av kompleksene som er egnet for røntgendiffraksjon dyrkes deretter via langsom dampdiffusjon av diettyleter til en acetonitrileløsning som inneholder kobolt(II) komplekset. For krystaller av høy kvalitet foregår rekrystallisering vanligvis over en 1 ukes periode, eller lenger. Metoden kan brukes på utarbeidelse av andre modell koordineringskomplekser og kan brukes i lavere undervisninglaboratorier. Til slutt, Det antas at andre kan finne denne rekrystallisering metoden for å få enkeltkrystaller gunstig for sin forskning.

Syntese Syntsavrene av komplekser 4 og 5 ble utført ved å reagere på en acetonitrile oppløsning som inneholder en bis-tione ligand forløper med kobolt (II) klorid heksahydrat (Figur 2). Denne reaksjonen skjedde ved en reflukstemperatur i nærvær av luft. Generelt ble komplekser 4 og 5 observert å være løselig i acetonitrile, dimetylsulfoksid, diklormetan og metanol. Kompleks 4 va...

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

Utarbeidelsen av SNS pincer kobolt(II) modellkomplekser av leveralkohol dehydrogenase presenteres her. Kompleksene kan tilberedes ved å reagere på ligandforløperen med CoCl2·6H2O og kan deretter rekrystalliseres ved å la dietyeher sakte spre seg til en acetonitrileløsning som inneholder koboltkomplekset.

Utarbeidelse av SNS Kobolt (II) Pincer Modell Komplekser av Leveralkohol 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 ...

Denne metoden kan brukes til å forberede et kjemisk modellkompleks av et enzym leveralkohol dehydrogenase og å vokse enkeltkrystaller. Fordelene med denne teknikken er at den komplekse formasjonen er facile og at rekrystalliseringsmetoden kan brukes til fremstilling av andre koordineringskomplekser. Denne metoden kan gi innsikt i forberedelse og rekrystallisering av uorganiske koordineringsforbindelser og kan brukes på enhver forbindelse av interesse for å vokse enkeltkrystaller.Det tar praksis å vokse enkeltkrystaller. Nøkkelen er å gjøre acetonitrilløsningen som inneholder metallkomplekset så konsentrert som mulig. For å forberede komplekse fire, tilsett 0,121 gram 2, 6-BIS-N-isopropyl-N'metylenimidzole-2-tione-pyridin og 0,0851 gram kobolt II kloridheksahydrat til en 100 milliliter rund bunnkolbe som inneholder 15 milliliter acetonitril.Reaksjonen bør endres fra en lys gul farge til smaragdgrønn umiddelbart etter at kobolt II kloridheksahydrat er tilsatt. Refluks og rør deretter reaksjonen i 20 timer for å sikre fullstendig reaksjon før du bruker en rotovap under redusert trykk for å fjerne oppløsningsvæsken. For klor-N3-S, S, N-2, 6-BIS-N-isopropyl-N'metylenimidzole-2-tione-pyridin kobolt II tetrachlorocobaltate rekrystallisering ved langsom dampdiffusjon, oppløs solute i 10 milliliter acetonitril, filer løsningen, deretter aliquot løsningen jevnt mellom en dram hetteglass.Tilsett 1,5 ml fersk acetonitriloppløsning til hvert hetteglass og tetthet av hetteglassene med bomull. Legg hetteglassene i en 240 ml krukke som inneholder 50 milliliter dietyleter og lukk krukken med en hette. La deretter krystallene vokse i opptil en uke.For å forberede komplekse fem, tilsett 0,183 gram 2, 6-BIS-N-isopropyl-N'metylentriazole-2-tione-pyridin og 0,223 gram kobolt II kloridheksahydrat til 15 milliliter acetonitril i en 100 milliliter rund bunnkolbe. Reaksjonen bør endre farge fra en lys gul farge til kongeblå umiddelbart etter kobolt II klorid heksahydrat er lagt til. Refluks og rør deretter reaksjonen i 20 timer for å sikre fullstendig reaksjon før du fjerner oppløsningsvæsken ved hjelp av en rotovap under redusert trykk.For klor-N3-S, S, N-2, 6-BIS-N-isopropyl-N'metylentriazole-2-tione-pyridin kobolt II tetraklorocobaltate rekrystallisering ved langsom dampdiffusjon, oppløs solute i 10 milliliter acetonitril og filtrere oppløsningen før du aliquoting løsningen jevnt i ett dram hetteglass. Fyll hvert hetteglass med 1,5 milliliter acetonitriloppløsning og tett hette hetteglassene med bomull. For å vokse enkeltkrystaller av høy kvalitet er det avgjørende at bomullspluggen sitter godt i ett dram hetteglass.Vi bruker litt mindre enn én bomullsdott til hvert hetteglass. Legg deretter hetteglassene i en krukke som inneholder 50 milliliter dietyleter, lukk krukken med en hette, og la krystallene vokse i opptil en uke. Syntesen av komplekser fire og fem kan utføres med hell ved å reagere på en acetonitrilløsning som inneholder en bis-thione ligand forløper med kobolt II kloridheksahydrat.Oppkjøpet av enkeltkrystaller av komplekser fire og fem via langsom dampdiffusjon er en utmerket metode for dyrking av enkeltkrystaller for vanskelig å krystallisere prøver. Elementanalyse av de rekrystalliserte kompleksene tyder på at komplekser fire og fem er rene fordi de beregnede prosentandelene av karbon, hydrogen og nitrogen er i utmerket enighet med de funnet prosentandelene av karbon, hydrogen og nitrogen. I tillegg viser ultrafiolett synlig spektroskopi av komplekser fire og fem at komplekse fire viser tre topper i den synlige regionen på 680, 632 og 589 nanometer, og at komplekse fem viser fire topper i den synlige regionen på 682, 613, 588 og 573 nanometer.Det viktigste å huske er at når du forbereder komplekset, må du legge koboltklorid heksahydrat til en acetonitrilløsning som inneholder en ligand forløper. Rekrystalliseringsmetoden kan brukes på organisk eller uorganisk forbindelse som er vanskelig å rekrystallisere. Vi forbereder for tiden kobolt II-komplekser som ikke inneholder kobolt i motangrepet og screening kompleksene for deres reaktivitet.Sørg for å utføre alle reaksjonene i en røykhette.

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

Transition Metals and Coordination Complexes

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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.

Produksjon av disulfide-stabilisert transmembrane Peptid Komplekser for Strukturelle studier

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

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.

Anticancer Metal Komplekser: Syntese og Cytotoksisitet Evaluering av MTT analysen

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.

Bruk av gasskromatografi å analysere kompositorisk Endringer av fettsyrer i Rat levervev under graviditet

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.

The Synthesis, Characterization og reaktivitet av en serie av Ruthenium<em&gt; N</em&gt; -triphos<sup&gt; Ph</sup&gt; Komplekser

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.

Utarbeidelse av Carbon Nanosheets ved romtemperatur

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.

Facile Fremstilling av 4-Substituerte kinazolinderivater

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.

Utarbeidelse av Biopolymer Aerogels Bruke Grønne Løsemidler

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.

Kontrollert Photoredox Ring-åpning polymerisering av O- Carboxyanhydrides formidlet av Ni/Zn komplekser

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.

Microfluidic utarbeidelse av flytende krystallinsk Elastomer aktuatorer

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