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: All oxygen- and moisture-sensitive manipulations were carried out under an inert atmosphere (N2) using either standard air-free techniques or a glove box. THF (tetrahydrofuran), Et2O (diethyl ether), CH2Cl2 (dichloromethane), toluene, and pentane were purified by passing through a neutral alumina column under argon. Acetonitrile was dried over CaH2 (calcium hydride) and distilled under nitrogen atmosphere prior to use. Pd/C (palladium on carbon) was heated unde...

<ol>
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In the first part of this protocol, we described a modified synthesis of 1,2-azaborines based on previously reported methods12, 13. Triallylborane22 was used as a substitute for the routes using allyltriphenyl tin or potassium allyltrifluoroborate to prepare N-allyl-N-TBS-B-allyl chloride adduct (1). This method allows for a more atom-economical and environmentally friendly approach. For the synt...

Boron-nitrogen containing heterocycles (i.e. 1,2-azaborines) have recently drawn significant attention as isosteres of arenes. This isosterism can lead to diversification of existing structural motifs to expand the chemical space2,3,4. Azaborines have potential utility for application in biomedical research5,6,7,8, especially in the area of medicinal chemistry in which chemists carry out synthesis of libraries of structurally and functionally relevant molecules. Significantly, however, while there are numerous well-developed synthetic routes to available arene-containing molecules, only a limited number of methods for the synthesis of azaborines have been reported9,10,11,12,13. This is mainly due to a limited number of options for the boron source and the air- and moisture- sensitive nature of the molecule in the early stage of synthetic sequence.
In the first part of this article, we will describe a multi-gram scale synthesis of N-TBS-B-Cl-1,2-azaborine (3) using standard air-free techniques. This compound serves as a versatile intermediate that can be further functionalized to structurally more complex molecules14,15. Starting from 3, the synthesis and purification of N-H-B-ethyl-1,2-azaborine (5) for use in protein binding studies will be described. Due to the volatility of 5, its efficient isolation requires precise control of reaction temperature, time, and distillation conditions.
In the second part, protocols for protein expression and isolation of T4 lysozyme mutants (L99A and L99A/M102Q)17,18,19,20 will be presented, followed by protein crystallization and preparation of protein-ligand crystal complexes. T4 lysozyme mutants L99A and L99A/M102Q were chosen as biological model systems to examine the hydrogen bonding capability of N-H containing azaborine molecules17. Using a standard molecular biology protocol, the protein is expressed in Escherichia coli RR1 strain and induced with isopropyl-&#946;-D-1-thiogalactopyranoside (IPTG). Protein purification is carried out using ion-exchange column chromatography. Protein crystallization is performed with highly concentrated purified protein solution (&#62;95% purity by gel electrophoresis) using the hanging drop vapor diffusion method. Because of the sensitivity of this study&#39;s ligands to oxygen, the protein-ligand complexes are prepared under air-free conditions.

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	<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>
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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.

The schematic synthetic route for 1,2-azaborines is shown in Figure 1. This protocol applies to the synthesis of five different boron-nitrogen containing molecules. Figure 2 represents 11B NMR spectra measured during the course of step 1.3 to monitor the formation of the desired product (3). Protein purification was performed by using low-pressure chromatography system and a representative chromatogram is shown in Figur...

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.

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

The overall goal of this protocol is to describe a general method to synthesize 1, 2-Azaborines, and its potential application in biomedical research. The main advantage of this technique is that is shows a sure synthesis of 1, 2-azaborine compounds, which are a merging boron containing isosteres of the ubiquitous benzene motif. The precursor that is described can be further functionalized into potentially more complex molecules.The preparation of protein complexes with 1, 2-azaborine will give some insight into future application in biomedical research. To begin this procedure, prepare the ring-closed product as outlined in the text protocol. Add 14 grams of palladium on carbon to this prepared product in toluene.Then, remove the flask from the box. Fit the flask with an oven-dried 24/40 reflux condenser that has been purged with nitrogen and equipped with chilled water. Place the flask in an oil bath, and heat the reaction to a vigorous reflux at 135 degrees celsius while stirring.Maintain the reaction at reflux for 16 hours. After this, remove the flask from the oil bath to cool the reaction to room temperature. Once it's cooled, replace the reflux condenser with the septum on the reaction flask.Using a syringe equipped with an oven-dried Luer Lock needle, withdraw a 5 milliliter aliquot. Transfer the aliquot to a screw-top NMR tube. Analyze the sample via Boron-11 NMR.If the reaction is incomplete, transfer the reaction flask into a glove box. Then, add an additional three grams of palladium on carbon. Next, remove the flask from the glove box.Fit the flask with a 24/40 reflux condenser that has been purged with nitrogen and equipped with chilled water. Using an oil bath, heat the reaction to a vigorous reflux at 135 degrees celsius while stirring for 24 hours. Using Boron-11 NMR, analyze another aliquot to ensure the reaction is complete.After this, transfer the reaction flask back to the glove box. Pass the reaction mixture through a flash chromatography column packed with paper wipes. Then, collect the filtrate into a one liter round-bottom flask equipped with a stir bar.Wash the column with 50 milliliters of toluene. Remove the vessel of filtrate from the glove box and connect it to a vacuum gas manifold equipped with a nitrogen line. Place a water bath underneath the flask, and a dewar containing liquid nitrogen to trap solvent.Open the high vacuum valve to begin the removal of all volatiles from the filtrate. Then, return the reaction mixture to the glove box. Transfer the mixture into an oven-dried 24/40 250 milliliter round-bottom flask with a stir bar.Rinse the residual reaction mixture with dichloromethane, and transfer it into the flask. Cap the flask with a septum. Transfer the flask from the glove box to the vacuum manifold, and connect it.Open the high vacuum valve to remove the residual solvent. Next, quickly remove the septum and add calcium hydride using a scoopula. Connect the round-bottom flask to the distillation apparatus detailed in the text protocol, and set the oil bath temperature to 100 degrees Celsius.After distillation is complete, remove the heat source and cool the apparatus to room temperature. Re-pressurize the cooled apparatus with nitrogen, and tightly close the plug valve on the air-free storage flask. After transferring the isolated product from the flask to a vial in a glove box, store the vial in the glove box freezer.After purifying the protein, transfer the purified solution to dialysis membrane tubing. Place the dialysis tubing into a bin containing a four-liter solution of 100 millimolar sodium phosphate, 550 millimolar sodium chloride, and 02%sodium azide. Stir overnight at four degrees celsius.The next day, add 2-mercaptoethanol to the dialyzed sample, such that the final concentration is five millimolar. Then, use a centrifugal concentrator to concentrate the solution to 40 milligrams per milliliter. Spin the sample at 5, 000 RPM and six degrees celsius, and remove any precipitate after concentration.Next, in a 24-well plate, set up various conditions with the reservoir solution, as outlined in the text protocol. Use grease for tight sealing by applying it onto the edge of each well. Mix five microliters of the concentrated protein solution with five microliters of reservoir solution on a siliconized glass cover slide.Place the cover slide upside-down over the wells containing reservoir solution. Store the plate at 4 degrees celsius for crystal growth. Using a loop of cryogenic tubing, pick the grown crystals and place them in a 6 milliliter microcentrifuge tube.Add 50 microliters of mother liquor. Then, transfer the tube to a glove box. Transfer five microliters of the prepared azaborine ligand to the inside of the tube's snap cap.Cap the tube, and store in a refrigerator inside the glove box for two to seven days for equilibration by vapor diffusion. After this, deposit a droplet of N Paratone onto a glass slide. Using a micro pipette, transfer a crystal to the slide.Next, use a loop of cryogenic tubing to pick up the crystal and transfer it from the aqueous solution into the N Paratone. Plunge the crystal protected with N Paratone into a dewar containing liquid nitrogen. Using cryogenic tongs, mount the flash-frozen crystal on the loop.Then, analyze the collected crystal data as outlined in the text protocol. In this study, a modified synthesis of 1, 2-azaborines is presented based on previously reported methods. Tri-allele borine is used to prepare N-allele NTBSB-allele chloride adduct.A one pop two step sequence is employed for the synthesis of NTBS BCl 1, 2-azaborine, which results in higher isolated yield than previous methods. The synthesis of N-H-B-ethyl-1, 2-azaborine is accomplished in two steps, using ethyllithium in place of chromium complex used in previously reported methods. The protein is purified using a low-pressure chromatography system.A chromatogram run at 280 nanometers shows a peak consistent with the T4 Lysozyme mutant, L99am102q. Protein is then crystallized and complexed with ligands. While attempting this procedure, it's important to remember that the azaborine synthesis requires uses of air-free conditions, such as a glove box and a vacuum gas manifold, to prevent decomposition of compounds.After its development, this technique paved the way to explore the unique chemical and physical properties of azaborines compared to carbonaceous aromatics in classic biological areal reclamation pockets.

Research

JoVE Journal

Biochemistry

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