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

<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>

All authors declare no competing financial interests in this manuscript.

Nella prima parte di questo protocollo, abbiamo descritto una sintesi modificata del 1,2-azaborines basate su metodi precedentemente riportati 12, 13. Triallylborane 22 è stato utilizzato come sostituto per le rotte che utilizzano allyltriphenyl stagno o di potassio allyltrifluoroborate per preparare N -allyl- N -TBS- B -allyl cloruro addotto (1). Questo metodo consente un approccio più atomi-...

Boro-contenenti azoto eterocicli (vale a dire 1,2-azaborines) hanno recentemente attirato notevole attenzione come isosteri di areni. Questo isosteria può portare alla diversificazione dei motivi strutturali esistenti per ampliare lo spazio chimica 2, 3, 4. Azaborines hanno potenziale utilità per l&#39;applicazione nella ricerca biomedica 5, 6, 7, 8, in particolare nel campo della chimica medicinale che i chimici svolgono sintesi di librerie di molecole strutturalmente e funzionalmente pertinenti. Significativamente, tuttavia, mentre ci sono numerose vie sintetiche ben sviluppati a molecole contenenti arene disponibili, sono stati riportati solo un numero limitato di metodi per la sintesi di azaborines 9, 10, </sup&gt; 11, 12, 13. Ciò è dovuto principalmente ad un numero limitato di opzioni per la sorgente di boro e l&#39;aria e la natura all&#39;umidità sensibile della molecola nella fase iniziale della sequenza sintetica. Nella prima parte di questo articolo, descriveremo una sintesi multi-scala grammo di N -TBS- B Cl-1,2-azaborine (3) utilizzando tecniche senza aria standard. Questo composto serve come intermedio versatile che può essere ulteriormente funzionalizzati al strutturalmente più complesse molecole 14, 15. Partendo da 3, verranno descritte la sintesi e la purificazione di N -H- B etil-1,2-azaborine (5) per l&#39;uso in studi di legame proteico. Data la volatilità del 5, il suo isolamento efficace richiede un controllo preciso della temperatura di reazione, tempo e distillation condizioni. Nella seconda parte, protocolli per l&#39;espressione della proteina e l&#39;isolamento di mutanti T4 lisozima (L99A L99A e / M102Q) 17, 18, 19, saranno presentati 20, seguita da cristallizzazione delle proteine e la preparazione di complessi di cristallo proteina-ligando. Mutanti T4 lisozima L99A L99A e / M102Q sono stati scelti come sistemi modello biologico per esaminare la capacità di idrogeno incollaggio di NH contenente molecole azaborine 17. Utilizzando un protocollo standard di biologia molecolare, la proteina è espressa in ceppo Escherichia coli RR1 e indotta con isopropil-β-D-1-thiogalactopyranoside (IPTG). Purificazione di proteine ​​viene effettuata utilizzando scambio ionico cromatografia su colonna. Protein cristallizzazione viene effettuata con soluzione altamente concentrata purificata proteine ​​(&gt; 95% di purezza mediante elettroforesi su gel) usando l&#39;attaccaturacadere metodo diffusione del vapore. A causa della sensibilità dei ligandi di questo studio all&#39;ossigeno, i complessi proteina-ligando sono preparati sotto condizioni di assenza di aria.

<table><tbody><tr><td>Tetrahydrofuran (THF), inhibitor-free, for HPLC, &amp;ge;99.9%</td><td>Sigma Aldrich</td><td>34865</td><td></td></tr><tr><td>Diethyl ether (Et&lt;sub&gt;2&lt;/sub&gt;O), for HPLC, &amp;ge;99.9%, inhibitor-free</td><td>Sigma Aldrich</td><td>309966</td><td></td></tr><tr><td>Methylene chloride&amp;nbsp; (CH&lt;sub&gt;2&lt;/sub&gt;Cl&lt;sub&gt;2&lt;/sub&gt;), (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 (CaH&lt;sub&gt;2&lt;/sub&gt;), 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, &amp;ge;99.5%</td><td>Sigma Aldrich</td><td>471283</td><td></td></tr><tr><td>Grubbs 1&lt;sup&gt;st&lt;/sup&gt; generation catalyst&amp;nbsp;</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&amp;nbsp;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 Et&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>455180</td><td></td></tr><tr><td>2-Methylbutane, anhydrous, &amp;ge;99%</td><td>Sigma Aldrich</td><td>277258</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt;, (Migula) Castellani and Chalmers (ATCC&amp;reg; 31343&amp;trade;)</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-&amp;beta;-D-1-thiogalactopyranoside (IPTG)&amp;nbsp;</td><td>Invitrogen</td><td>AM9464</td><td></td></tr><tr><td>Sodium phosphate monobasic 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&amp;nbsp;</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&amp;nbsp;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&amp;nbsp;</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&amp;nbsp;</td><td></td></tr><tr><td>Sartorius&amp;nbsp;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&amp;nbsp;</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&amp;nbsp;</td><td>Fisher</td><td>08-667E</td><td></td></tr><tr><td>&amp;nbsp;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 via sintetica schema per 1,2-azaborines è mostrato in Figura 1. Questo protocollo si applica alla sintesi di cinque diverse molecole boro contenenti azoto. Figura 2 rappresenta 11 B NMR spettri misurati nel corso del passo 1.3 per monitorare la formazione del prodotto desiderato (3). Purificazione di proteine è stata effettuata utilizzando il sistema di cromatografia a bassa pressione ed un cromatogramma rappresentativo ?...

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.

Sintesi di 1,2-Azaborines e la preparazione dei loro complessi proteici con T4 lisozima 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 ...

synthesis-12-azaborines-preparation-their-protein-complexes-with-t4

Universidad de Cartagena UDEC

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Biochemistry

7.5K Views.  Boston College. A protocol for the synthesis of 1,2-azaborines and the preparation of their protein complexes with T4 lysozyme mutants is presented.

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

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

Sintesi di proteine ​​bioconiugati<em&gt; via</em&gt; Chimica cisteina-maleimmide

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Ricerca

Chimica

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

L'analisi di complessi proteici dinamico montato su e rilasciato da Biolayer interferometria biosensore utilizzando la microscopia elettronica e spettrometria di massa

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Biochimica

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

Modifica Chemoselective delle superfici virali tramite Chimica Click Bioorthogonal

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

Immunologia e infezioni

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

Preparazione di stabile biciclico Aziridinium ioni e loro anello-apertura per la sintesi di Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

Sintesi enzimatica mediata OaAEP1 e immobilizzazione delle proteine polimerizzate per la spettroscopia a singola molecola

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

Nitrogen-containing heterocycle aziridines are synthetically very valuable for the preparation of azacyclic and acyclic molecules. However, it is very difficult and laborious to make aziridines in optically pure forms on a large scale to apply asymmetric synthesis of aza compounds. Fortunately, we successfully achieved both enantiomers (2R)- and (2S)-aziridine-2-carboxylates with the electron-donating &#945;-methylbenzyl group at the ring nitrogen as non-activated aziridines. These starting aziridines have two distinct functional groups-highly reactive three-membered ring and versatile carboxylate. They are applicable in ring-opening or ring-transformation with aziridine and in functional group transformation to others from carboxylate. Both of these enantiomers were utilized in the preparation of biologically important amino acyclic and/or aza-heterocyclic compounds in an asymmetric manner. Specifically, this report describes the first expedient asymmetric synthesis of both enantiomers of 5, 6-dihydrouracil-type marine natural products biemamide B and D as potential TGF-&#946; inhibitors. This synthesis consisted of regio- and the stereoselective ring-opening reaction of aziridine-2-carboxylate and subsequent formation of 4-aminoteterahydropyrimidine-2,4-dione. One more example in this protocol dealt a highly stereoselective Mukaiyama reaction of aziridine-2-carboxylate and silyl enol ether, following intramolecular aziridine ring-opening to provide easy and facile access to (-)-epiallo-isomuscarine.

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

In this study, we prepare both enantiomers of aziridine-2-carboxylate, which are used in the asymmetric synthesis of alkaloids, including biemamide B and D, and (-)-epiallo-isomuscarine.

Preparazione di aziridine non attivate enantiopure e sintesi di biemamide B, D ed epiallo-isomuscarina

Nitrogen-containing heterocycle aziridines are synthetically very valuable for the preparation of azacyclic and acyclic molecules. However, it is very ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Ingegneria fluorescenza 'D'oro' per incorporazione di pressione selettiva di aminoacidi Non canonico e l'analisi della proteina di fluorescenza e di spettrometria di massa

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Bioingegneria

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Specifici Sequenza Etichettatura degli acidi nucleici e proteine ​​con metiltransferasi e cofattore Analoghi

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Biologia

<meta charset="utf8"></head><body>Protocollo semplificato per la produzione di estratti traslazionali (LTE) di Leishmania

Production and Optimization of LTE, a Leishmania tarentolae Derived Cell-Free Protein Expression System for Recombinant Protein Production

This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate Leishmania tarentolae, referred to as Leishmania Translational Extract or LTE. Although this organism originally evolved as a parasite of geckos, it can be cultivated easily and inexpensively in flasks or bioreactors. Unlike Leishmania major, it is non-pathogenic to humans and does not require special laboratory precautions. Another advantage of using Leishmania for CFPS is that the addition of a single antisense oligonucleotide to the CFPS, targeting a conserved splice leader sequence on the 5'-end of all protein-coding RNAs, can suppress endogenous protein expression. We provide procedures for cell disruption and lysate processing, which have been simplified and improved compared to previous versions. These procedures start with simple flask cultures. Additionally, we explain how to introduce genetic information using vectors containing species-independent translation initiation sites (SITS) and how to perform straightforward batch optimization and quality control to ensure consistent protein expression quality.

<meta charset="utf8"></head><body>Autore in primo piano: Produzione di proteine utilizzando il sistema di espressione proteica libera da cellule eucariotiche LTE

Leishmania Translational Extract (LTE) is a eukaryotic cell-free protein expression system derived from the single-celled parasite, Leishmania tarentolae. This optimized protocol makes LTE simple and cost-effective to manufacture. It is suitable for various applications focused on the multiparallel expression and study of complex eukaryotic proteins and their interactions.

Produzione e ottimizzazione di LTE, un sistema di espressione proteica libera derivato da Leishmania tarantolae per la produzione di proteine ricombinanti

This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate ...

Sintesi di 1,2-Azaborines e la preparazione dei loro complessi proteici con T4 lisozima Mutants

Riepilogo

Esplora Altri Video

Capitoli in questo video

Sintesi di proteine bioconiugati

L'analisi di complessi proteici dinamico montato su e rilasciato da Biolayer interferometria biosensore utilizzando la microscopia elettronica e spettrometria di massa

Modifica Chemoselective delle superfici virali tramite Chimica Click Bioorthogonal

Preparazione di stabile biciclico Aziridinium ioni e loro anello-apertura per la sintesi di Azaheterocycles

Sintesi enzimatica mediata OaAEP1 e immobilizzazione delle proteine polimerizzate per la spettroscopia a singola molecola

Preparazione di aziridine non attivate enantiopure e sintesi di biemamide B, D ed epiallo-isomuscarina

Ingegneria fluorescenza 'D'oro' per incorporazione di pressione selettiva di aminoacidi Non canonico e l'analisi della proteina di fluorescenza e di spettrometria di massa

Specifici Sequenza Etichettatura degli acidi nucleici e proteine con metiltransferasi e cofattore Analoghi

Produzione e ottimizzazione di LTE, un sistema di espressione proteica libera derivato da Leishmania tarantolae per la produzione di proteine ricombinanti

Produzione e ottimizzazione di LTE, un sistema di espressione proteica libera derivato da Leishmania tarantolae per la produzione di proteine ricombinanti

Sintesi di 1,2-Azaborines e la preparazione dei loro complessi proteici con T4 lisozima Mutants

Riepilogo

Esplora Altri Video

Capitoli in questo video

Sintesi di proteine ​​bioconiugati

L'analisi di complessi proteici dinamico montato su e rilasciato da Biolayer interferometria biosensore utilizzando la microscopia elettronica e spettrometria di massa

Modifica Chemoselective delle superfici virali tramite Chimica Click Bioorthogonal

Preparazione di stabile biciclico Aziridinium ioni e loro anello-apertura per la sintesi di Azaheterocycles

Sintesi enzimatica mediata OaAEP1 e immobilizzazione delle proteine polimerizzate per la spettroscopia a singola molecola

Preparazione di aziridine non attivate enantiopure e sintesi di biemamide B, D ed epiallo-isomuscarina

Ingegneria fluorescenza 'D'oro' per incorporazione di pressione selettiva di aminoacidi Non canonico e l'analisi della proteina di fluorescenza e di spettrometria di massa

Specifici Sequenza Etichettatura degli acidi nucleici e proteine ​​con metiltransferasi e cofattore Analoghi

Produzione e ottimizzazione di LTE, un sistema di espressione proteica libera derivato da Leishmania tarantolae per la produzione di proteine ricombinanti

Produzione e ottimizzazione di LTE, un sistema di espressione proteica libera derivato da Leishmania tarantolae per la produzione di proteine ricombinanti

Sintesi di proteine bioconiugati

Specifici Sequenza Etichettatura degli acidi nucleici e proteine con metiltransferasi e cofattore Analoghi