Name: synthesis of 1,2-azaborines and the preparation of their protein complexes with t4 lysozyme mutants
Uploaded: 2017-03-25T13:00:00
Description: Watch this Scientific Journal Video about Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants at JoVE.com

This research was supported by the National Institutes of Health NIGMS (R01-GM094541) and Boston College.

NOTE: Toutes les manipulations oxygène et sensibles à l&#39; humidité ont été effectuées sous atmosphère inerte (N 2) en utilisant soit des techniques libres d&#39;air standard ou une boîte à gants. THF (tétrahydrofuranne), Et2O (éther diéthylique), un groupe CH 2 Cl 2 (dichlorométhane), le toluène et le pentane ont été purifiés par passage à travers une colonne d&#39;alumine neutre sous argon. L&#39; acétonitrile a été séché sur CaH2 (hydrure...

<ol>
	<li>Ducruix, A., Gi&#233;ge, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Crystallization of Nucleic Acids and Proteins, A Practical Approach. , (1992).</li><li>Bosdet, M. J. D., Piers, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-N+as+a+C-C+substitute+in+aromatic+systems.">B-N as a C-C substitute in aromatic systems.</a> Can. J. Chem. 87 (1), 8-29 (2009).</li><li>Campbell, P. G., Marwitz, A. J., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Advances+in+Azaborine+Chemistry.">Recent Advances in Azaborine Chemistry.</a> Angew. Chem. Int. Ed. 51 (25), 6074-6092 (2012).</li><li>Wang, X. -. Y., Wang, J. -. Y., Pei, J. B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterosuperbenzenes:+Synthesis+and+Properties.">Heterosuperbenzenes: Synthesis and Properties.</a> Chem. Eur. J. 21 (9), 3528-3539 (2015).</li><li>Zhou, H. -. B., 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=Elemental+isomerism:+a+boron-nitrogen+surrogate+for+a+carbon-carbon+double+bond+increases+the+chemical+diversity+of+estrogen+receptor+ligands.">Elemental isomerism: a boron-nitrogen surrogate for a carbon-carbon double bond increases the chemical diversity of estrogen receptor ligands.</a> Chem. Biol. 14 (6), 659-669 (2007).</li><li>Ito, H., Yumura, K., Saigo, K. <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+binding+property+of+isoelectronic+analogues+of+nucleobases,+B(6)-substituted+5-aza-6-borauracils+and+-thymines.">Synthesis, characterization, and binding property of isoelectronic analogues of nucleobases, B(6)-substituted 5-aza-6-borauracils and -thymines.</a> Org. Lett. 12 (15), 3386-3389 (2010).</li><li>Vlasceanu, A., Jessing, M., Kilburn, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BN/CC+isosterism+in+borazaronaphthalenes+towards+phosphodiesterase+10A+(PDE10A)+inhibitors.">BN/CC isosterism in borazaronaphthalenes towards phosphodiesterase 10A (PDE10A) inhibitors.</a> Bioorg. Med. Chem. 23 (15), 4453-4461 (2015).</li><li>Rombouts, F. J., Tovar, F., Austin, N., Tresadern, G., Trabanco, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzazaborinines+as+Novel+Bioisosteric+Replacements+of+Naphthalene:+Propranolol+as+an+Example.">Benzazaborinines as Novel Bioisosteric Replacements of Naphthalene: Propranolol as an Example.</a> J. Med. Chem. 58 (23), 9287-9295 (2015).</li><li>Culling, G. C., Dewar, M. J. S., Marr, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Heteroaromatic+Compounds.+XXIII.+Two+Analogs+of+Triphenylene+and+a+Possible+Route+to+Borazarene.">New Heteroaromatic Compounds. XXIII. Two Analogs of Triphenylene and a Possible Route to Borazarene.</a> J. Am. Chem. Soc. 86 (6), 1125-1127 (1964).</li><li>White, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-Phenyl-2,1-borazarene+and+Derivatives+of+1,2-Azaboracycloalkanes.">2-Phenyl-2,1-borazarene and Derivatives of 1,2-Azaboracycloalkanes.</a> J. Am. Chem. Soc. 85 (22), 3634-3636 (1963).</li><li>Ashe III, A. J., Fang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Synthesis+of+Aromatic+Five-+and+Six-Membered+B&#8722;N+Heterocycles+via+Ring+Closing+Metathesis.">A Synthesis of Aromatic Five- and Six-Membered B&#8722;N Heterocycles via Ring Closing Metathesis.</a> Org. Lett. 2 (14), 2089-2091 (2000).</li><li>Marwitz, A. J. V., Matus, M. H., Zakharov, L. N., Dixon, D. A., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Hybrid+Organic/Inorganic+Benzene.">A Hybrid Organic/Inorganic Benzene.</a> Angew. Chem. Int. Ed. 48 (5), 973-977 (2009).</li><li>Abbey, E. R., Lamm, A. N., Baggett, A. W., Zakharov, L. N., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+Group-Free+Synthesis+of+1,2-Azaborines:+A+Simple+Approach+to+the+Construction+of+BN-Benzenoids.">Protecting Group-Free Synthesis of 1,2-Azaborines: A Simple Approach to the Construction of BN-Benzenoids.</a> J. Am. Chem. Soc. 135 (34), 12908-12913 (2013).</li><li>Rudebusch, G. E., Zakharov, L. N., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodium-catalyzed+boron+arylation+of+1,2-azaborines.">Rhodium-catalyzed boron arylation of 1,2-azaborines.</a> Angew. Chem. Int. Ed. 52 (35), 9316-9319 (2013).</li><li>Brown, A. N., Li, B., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negishi+Cross-Coupling+Is+Compatible+with+a+Reactive+B-Cl+Bond:+Development+of+a+Versatile+Late-Stage+Functionalization+of+1,2-Azaborines+and+Its+Application+to+the+Synthesis+of+New+BN+Isosteres+of+Naphthalene+and+Indenyl.">Negishi Cross-Coupling Is Compatible with a Reactive B-Cl Bond: Development of a Versatile Late-Stage Functionalization of 1,2-Azaborines and Its Application to the Synthesis of New BN Isosteres of Naphthalene and Indenyl.</a> J. Am. Chem. Soc. 137 (28), 8932-8935 (2015).</li><li>Knack, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BN/CC+Isosteric+Compounds+as+Enzyme+Inhibitors:+N-+and+B-Ethyl-1,2-azaborine+Inhibit+Ethylbenzene+Hydroxylation+as+Non-Convertible+Substrate+Analogs.">BN/CC Isosteric Compounds as Enzyme Inhibitors: N- and B-Ethyl-1,2-azaborine Inhibit Ethylbenzene Hydroxylation as Non-Convertible Substrate Analogs.</a> Angew. Chem. Int. Ed. 52 (9), 2599-2601 (2013).</li><li>Eriksson, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+a+Protein+Structure+to+Cavity-Creating+Mutations+and+Its+Relation+to+the+Hydrophobic+Effect.">Response of a Protein Structure to Cavity-Creating Mutations and Its Relation to the Hydrophobic Effect.</a> Science. 255 (5041), 178-183 (1992).</li><li>Eriksson, A. E., Baase, W. A., Wozniak, J. A., Matthews, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cavity-containing+mutant+of+T4+lysozyme+is+stabilized+by+buried+benzene.">A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene.</a> Nature. 355 (6358), 371-373 (1992).</li><li>Morton, A., Baase, W. A., Matthews, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetic+Origins+of+Specificity+of+Ligand+Binding+in+an+Interior+Nonpolar+Cavity+of+T4+Lysozyme.">Energetic Origins of Specificity of Ligand Binding in an Interior Nonpolar Cavity of T4 Lysozyme.</a> Biochemistry. 34 (27), 8564-8575 (1995).</li><li>Wei, B. Q., Baase, W. A., Weaver, L. H., Matthews, B. W., Shoichet, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Model+Binding+Site+for+Testing+Scoring+Functions+in+Molecular+Docking.">A Model Binding Site for Testing Scoring Functions in Molecular Docking.</a> J. Mol. Biol. 322 (2), 339-355 (2002).</li><li>Lee, H., Fischer, M., Shoichet, B. K., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+Bonding+of+1,2-Azaborines+in+the+Binding+Cavity+of+T4+Lysozyme+Mutants:+Structures+and+Thermodynamics.">Hydrogen Bonding of 1,2-Azaborines in the Binding Cavity of T4 Lysozyme Mutants: Structures and Thermodynamics.</a> J. Am. Chem. Soc. 138 (37), 12021-12024 (2016).</li><li>Brown, H. C., Racherla, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoboranes.+43.+A+Convenient,+Highly+Efficient+Synthesis+of+Triorganylboranes+via+a+Modified+Organometallic+Route.">Organoboranes. 43. A Convenient, Highly Efficient Synthesis of Triorganylboranes via a Modified Organometallic Route.</a> J. Org. Chem. 51 (4), 427-432 (1986).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Liu, L., Baase, W. A., Matthews, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Halogenated+Benzenes+Bound+within+a+Non-polar+Cavity+in+T4+Lysozyme+Provide+Examples+of+I.S+and+I.Se+Halogen-bonding.">Halogenated Benzenes Bound within a Non-polar Cavity in T4 Lysozyme Provide Examples of I.S and I.Se Halogen-bonding.</a> J. Mol. Biol. 385 (2), 595-605 (2009).</li></ol>

Dans la première partie de ce protocole, nous avons décrit une synthèse modifiée de 1,2-azaborines basés sur des méthodes précédemment rapportées 12, 13. Triallylborane 22 a été utilisé comme un substitut pour les routes à l&#39; aide de allyltriphenyl étain ou potassium allyltrifluoroborate pour préparer N -allyl- N -TBS- B chlorure de produit d&#39; addition (1). Cette méthode ...

Le bore-azote contenant des hétérocycles (ie 1,2-azaborines) ont récemment attiré l&#39; attention significative en tant isostères de arenes. Cette isosterism peut conduire à la diversification des motifs structurels existants pour élargir l&#39;espace chimique 2, 3, 4. Azaborines ont une utilité potentielle pour les applications en recherche biomédicale , 5, 6, 7, 8, en particulier dans le domaine de la chimie médicinale dans laquelle les chimistes effectuent la synthèse de banques de molécules structuralement et fonctionnellement pertinents. De manière significative, cependant, alors qu&#39;il existe de nombreuses voies de synthèse bien développés à des molécules disponibles contenant arene, seul un nombre limité de méthodes pour la synthèse de azaborines ont été rapportés 9, 10, </sup&gt; 11, 12, 13. Ceci est principalement dû à un nombre limité d&#39;options pour la source de bore et de l&#39;air et de la nature sensible à l&#39;humidité de la molécule dans le stade précoce de la séquence synthétique. Dans la première partie de cet article, nous allons décrire une synthèse à l&#39;échelle multi-gramme de N -TBS- B -Cl-1,2-azaborine (3) en utilisant des techniques de libre-air standard. Ce composé sert comme intermédiaire polyvalent qui peut encore être fonctionnalisé pour structurellement plus complexes molécules 14, 15. A partir de 3, dont la synthèse et la purification du N -H- B -éthyl-1,2-azaborine (5) destiné à être utilisé dans des études de liaison de protéine seront décrits. En raison de la volatilité de 5, son isolement efficace nécessite un contrôle précis de la température de réaction, le temps, et distillation conditions. Dans la deuxième partie, les protocoles pour l&#39; expression de la protéine et l&#39; isolement des mutants T4 lysozyme (L99A et L99A / M102Q) 17, 18, 19, 20 seront présentés, suivie d&#39; une cristallisation de la protéine et la préparation de complexes cristallins protéine-ligand. Les mutants T4 lysozyme L99A et L99A / M102Q ont été choisis en tant que systèmes modèles biologiques pour examiner la capacité de liaison hydrogène de NH contenant des molécules azaborine 17. En utilisant un protocole de biologie moléculaire standard, la protéine est exprimée dans la souche Escherichia coli RR1 et induite par l&#39; isopropyl-β-D-thiogalactopyranoside 1 (IPTG). La purification des protéines est réalisée en utilisant une Chromatographie sur colonne échangeuse d&#39;ions. La cristallisation des protéines est effectuée avec une solution fortement concentrée et purifiée de protéine (&gt; 95% de pureté par électrophorèse sur gel) en utilisant la tenturedéposer méthode de diffusion de la vapeur. En raison de la sensibilité des ligands de la présente étude à l&#39;oxygène, les complexes protéine-ligand sont préparés dans des conditions exemptes d&#39;air.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tetrahydrofuran (THF), inhibitor-free, for HPLC, &#8805;99.9%</td>
			<td>Sigma Aldrich</td>
			<td>34865</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether (Et2O), for HPLC, &#8805;99.9%, inhibitor-free</td>
			<td>Sigma Aldrich</td>
			<td>309966</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene chloride&#160; (CH2Cl2), (Stabilized/Certified ACS)</td>
			<td>Fisher</td>
			<td>D37-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Fisher</td>
			<td>T290-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentane, HPLC</td>
			<td>Fisher</td>
			<td>P399-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher</td>
			<td>A21-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium hydride (CaH2), reagent grade, 95%</td>
			<td>Sigma Aldrich</td>
			<td>208027</td>
			<td>Pyrophoric</td>
		</tr>
		<tr>
			<td>Palladium on activated carbon (Pd/C), 10 wt% Pd</td>
			<td>Strem</td>
			<td>46-1900</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 M Boron trichloride solution in hexane</td>
			<td>Sigma Aldrich</td>
			<td>211249</td>
			<td>Highly toxic/ Pyrophoric</td>
		</tr>
		<tr>
			<td>Triethylamine, &#8805;99.5%</td>
			<td>Sigma Aldrich</td>
			<td>471283</td>
			<td></td>
		</tr>
		<tr>
			<td>Grubbs 1st generation catalyst&#160;</td>
			<td>materia</td>
			<td>C823</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetamide</td>
			<td>Sigma Aldrich</td>
			<td>A0500</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Butanol, anhydrous, 99.8%</td>
			<td>Sigma Aldrich</td>
			<td>281549</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyllithium solution, 0.5 M in benzene/cyclohexane</td>
			<td>Sigma Aldrich</td>
			<td>561452</td>
			<td>Highly toxic/ Pyrophoric</td>
		</tr>
		<tr>
			<td>HCl solution, 2.0 M in Et2O</td>
			<td>Sigma Aldrich</td>
			<td>455180</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Methylbutane, anhydrous, &#8805;99%</td>
			<td>Sigma Aldrich</td>
			<td>277258</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli, (Migula) Castellani and Chalmers (ATCC&#174; 31343&#8482;)</td>
			<td>ATCC</td>
			<td>31343</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 lysozyme WT* (L99A)</td>
			<td>Addgene</td>
			<td>18476</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 lysozyme mutant (S38D L99A M102Q N144D)</td>
			<td>Addgene</td>
			<td>18477</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>A0166</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropyl-&#946;-D-1-thiogalactopyranoside (IPTG)&#160;</td>
			<td>Invitrogen</td>
			<td>AM9464</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic&#160; anhydrous</td>
			<td>Fisher</td>
			<td>BP329</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate dibasic anhydrous</td>
			<td>Fisher</td>
			<td>BP332</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher</td>
			<td>S642212&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Fisher</td>
			<td>BP118</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma Aldrich</td>
			<td>M4880</td>
			<td>Corrosive</td>
		</tr>
		<tr>
			<td>Thermo scientific pierce DNaseI</td>
			<td>Fisher</td>
			<td>PI-90083</td>
			<td></td>
		</tr>
		<tr>
			<td>GE Healthcare&#160;Sepharose Fast Flow Cation Exchange Media</td>
			<td>Fisher</td>
			<td>45-002-931</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Fisher</td>
			<td>BP152-500&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>TCI</td>
			<td>S0489</td>
			<td>Highly toxic</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Fisher</td>
			<td>ICN806443&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sartorius&#160;Vivaspin 20 Centrifugal Concentrators</td>
			<td>Fisher</td>
			<td>14-558-501</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>P5379</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Hydroxyethyl disulfide</td>
			<td>Sigma Aldrich</td>
			<td>380474</td>
			<td></td>
		</tr>
		<tr>
			<td>N-paratone&#160;</td>
			<td>Hampton Research</td>
			<td>HR2-643</td>
			<td></td>
		</tr>
		<tr>
			<td>4 RC Dialysis Membrane Tubing 12,000 to 14,000 Dalton MWCO&#160;</td>
			<td>Fisher</td>
			<td>08-667E</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;CryoLoop</td>
			<td>Hampton Research</td>
			<td></td>
			<td>cryogenic tubing shaped into a loop</td>
		</tr>
		<tr>
			<td>CryoTong</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td>cryogenic tong</td>
		</tr>
		<tr>
			<td>Coot</td>
			<td></td>
			<td></td>
			<td>Electron density images are generated from the software</td>
		</tr>
	</tbody>
</table>

synthesis of 1,2-azaborines and the preparation of their protein complexes with t4 lysozyme mutants

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor diffusion. Oxygen- and moisture-sensitive compounds are prepared and isolated under an inert atmosphere (N2) using either a vacuum gas manifold or a glove box. As an example of azaborine synthesis, we demonstrate the synthesis and purification of the volatile N-H-B-ethyl-1,2-azaborine by a five-step sequence involving distillation and column chromatography for the isolation of products. T4 lysozyme mutants L99A and L99A/M102Q are expressed with Escherichia coli RR1 strain. Standard protocols for chemical cell lysis followed by purification using carboxymethyl ion exchange column affords protein of sufficiently high purity for crystallization. Protein crystallization is performed in various concentrations of precipitant at different pH ranges using the hanging drop vapor diffusion method. Complex preparation with the small molecules is carried out by vapor diffusion method under an inert atmosphere. X-ray diffraction analysis of the crystal complex provides unambiguous structural evidence of binding interactions between the protein binding site and 1,2-azaborines.

La voie de synthèse schématique pour le 1,2-azaborines est représenté sur la figure 1. Ce protocole applicable à la synthèse de cinq molécules du bore contenant de l&#39;azote différentes. La figure 2 représente les spectres 11 RMN B mesuré au cours de l&#39; étape 1.3 pour suivre la formation du produit désiré (3). La purification des protéines a été réalisée en utilisant un système de Chromatographie à ba...

Watch this Scientific Journal Video about Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants at JoVE.com

Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants

A protocol for the synthesis of 1,2-azaborines and the preparation of their protein complexes with T4 lysozyme mutants is presented.

Synthèse de 1,2-Azaborines et la préparation de leurs complexes protéiques avec T4 lysozyme Mutants

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor ...

Research

JoVE Journal

Biochemistry

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