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 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.
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-β-D-1-thiogalactopyranoside (IPTG). Protein purification is carried out using ion-exchange column chromatography. Protein crystallization is performed with highly concentrated purified protein solution (>95% purity by gel electrophoresis) using the hanging drop vapor diffusion method. Because of the sensitivity of this study's ligands to oxygen, the protein-ligand complexes are prepared under air-free conditions.
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 under high vacuum at 100 °C for 12 h prior to use. Silica gel (230-400 mesh) was dried for 12 h at 180 °C under high vacuum. Flash chromatography was performed with this silica gel under an inert atmosphere. All other chemicals and solvents were purchased and used as received.
NOTE: Caution, please consult all relevant material safety data sheets (MSDS) before use. Several of the chemicals used in the synthesis are acutely toxic and carcinogenic.
1. Preparation of 1,2-Azaborines
NOTE: Characterization data of all compounds in this study have been previously reported12, 13, 16.
Figure 1: Synthetic scheme of 1,2-azaborines. Detailed protocols for the synthesis of each compound (1-5) are described in the protocol section. Please click here to view a larger version of this figure.
Figure 2: 11B NMR spectra for monitoring formation of N-TBS-B-Cl-1,2-azaborine (3). A) N-Allyl-N-TBS-B-allyl chloride adduct (1), starting material for ring closing metathesis. B) Oxidation after 16 h. (Unreacted N-TBS-B-Cl ring-closed product (2) and isomerized B-vinyl ring-closed product (2') along with N-TBS-B-Cl-1,2-azaborine (3) are shown.) C) Oxidation after additional 24 h. D) Isolated product (3) after purification by vacuum distillation. Please click here to view a larger version of this figure.
2. Protein Preparation and Crystallization of T4 Lysozyme Mutants
Figure 3: Representative results for the protein purification of T4 lysozyme mutant L99A/M102Q. A) Chromatogram showing the measured UV absorbance in A. U. at 280 nm (blue line) and conductivity in mS/cm (red line). Fractions 20–23 were combined and the purity was determined by SDS-PAGE. B) 15% SDS-PAGE gel showing the presence of purified protein of fractions 20–23 at 18.6 kDa. Please click here to view a larger version of this figure.
Figure 4: Representative picture of crystals of T4 lysozyme mutant L99A prior to complexation with ligands. Crystals are in the mother liquor (2.1 M sodium/potassium phosphate, pH 6.9, 50 mM 2-mercaptoethanol, 50 mM 2-hydroxyethyl disulfide). Please click here to view a larger version of this figure.
Figure 5: Atomic models of T4 lysozyme mutant L99A binding pocket with electron density. A) L99A cavity with an un-modeled electron density blob in the binding site. B) L99A cavity with the modeled azaborine ligand 5 in two alternative conformers. Please click here to view a larger version of this figure.
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 Figure 3. The purity of the collected fractions was determined by SDS-PAGE. Crystals of T4 lysozyme mutant L99A are depicted in Figure 4. Figure 5 shows the L99A binding pocket with un-modeled electron density and the binding cavity after refinement with the ligand 5 bound.
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 synthesis of N-TBS-B-Cl-1,2-azaborine (3), a one-pot, two-step sequence (ring closing metathesis followed by oxidation) was employed, which resulted in higher isolated yield than the previous method involving isolation of the intermediate (2) (52% vs. 29% over two steps). (However, depending on the purity of the adduct (1), isolation of the ring-closed product before oxidation step might be necessary using vacuum distillation. In this case, using dichloromethane as solvent in place of toluene reduces time for removal of volatiles prior to distillation.) Monitoring 11B NMR (Figure 2) during the oxidation step also benefits in reduction in the total amounts of Pd/C used in this reaction (7 mol% vs 15 mol%).
Synthesis of N-H-B-ethyl-1,2-azaborine (5) was accomplished in two steps from 4 using ethyllithium in place of the previously reported multistep sequence16 that required use of a chromium complex. The isolation required precise temperature control and attenuated-vacuum manipulation due to the volatility of the product. The techniques presented in this protocol can be also applied to purification of other volatile compounds.
In the second part, we demonstrated protein purification and crystal complexation of T4 lysozyme mutants L99A and L99A/M102Q. A standard protein expression in E. coli RR1 strain and induction using IPTG, followed by chemical cell lysis afforded isolation of the desired protein. It is advised to run SDS-PAGE after protein expression and cell lysis to confirm the success of each step or identify the failed process. For the L99A T4 lysozyme, the expressed protein can be found in both the supernatant and pellet due to its activity to lyse bacterial cells. In this case, protein in the supernatant can be dialyzed against 20 mM sodium phosphate buffer and combined with lysate from cell lysis for column purification. Induction conditions can be adjusted to higher temperature and shorter time (from 21 h at 25 °C to 2-3 h at 37 °C). Cell lysis can be also performed by using sonication with an ice bath to prevent overheating. Carboxymethyl ion exchange column with a 300 mM NaCl linear gradient was used for protein purification. Only >95% pure (based on SDS-PAGE) protein fractions were collected and used in protein crystallization.
Prior to concentrating the protein solution, addition of 2-mercaptoethanol (final concentration of 5 mM) (step 2.2.2) to the dialyzed protein was necessary to prevent its spontaneous precipitation, especially for L99A/M102Q protein which tends to precipitate readily at high concentration. As protein complexation with 5 required an air-free atmosphere, the protein-ligand complex was prepared in a glove box. The materials prepared in this protocol were used in binding studies of 1,2-azaborines in a biological model system: cavity-bearingT4 lysozyme mutants.
The authors have nothing to disclose.
This research was supported by the National Institutes of Health NIGMS (R01-GM094541) and Boston College.
Tetrahydrofuran (THF), inhibitor-free, for HPLC, ≥99.9% | Sigma Aldrich | 34865 | |
Diethyl ether (Et2O), for HPLC, ≥99.9%, inhibitor-free | Sigma Aldrich | 309966 | |
Methylene chloride (CH2Cl2), (Stabilized/Certified ACS) | Fisher | D37-20 | |
Toluene | Fisher | T290-4 | |
Pentane, HPLC | Fisher | P399-4 | |
Acetonitrile | Fisher | A21-4 | |
Calcium hydride (CaH2), reagent grade, 95% | Sigma Aldrich | 208027 | Pyrophoric |
Palladium on activated carbon (Pd/C), 10 wt% Pd | Strem | 46-1900 | |
1.0 M Boron trichloride solution in hexane | Sigma Aldrich | 211249 | Highly toxic/ Pyrophoric |
Triethylamine, ≥99.5% | Sigma Aldrich | 471283 | |
Grubbs 1st generation catalyst | materia | C823 | |
Acetamide | Sigma Aldrich | A0500 | |
n-Butanol, anhydrous, 99.8% | Sigma Aldrich | 281549 | |
Ethyllithium solution, 0.5 M in benzene/cyclohexane | Sigma Aldrich | 561452 | Highly toxic/ Pyrophoric |
HCl solution, 2.0 M in Et2O | Sigma Aldrich | 455180 | |
2-Methylbutane, anhydrous, ≥99% | Sigma Aldrich | 277258 | |
Escherichia coli, (Migula) Castellani and Chalmers (ATCC® 31343™) | ATCC | 31343 | |
T4 lysozyme WT* (L99A) | Addgene | 18476 | |
T4 lysozyme mutant (S38D L99A M102Q N144D) | Addgene | 18477 | |
Ampicillin sodium salt | Sigma Aldrich | A0166 | |
isopropyl-β-D-1-thiogalactopyranoside (IPTG) | Invitrogen | AM9464 | |
Sodium phosphate monobasic anhydrous | Fisher | BP329 | |
Sodium Phosphate dibasic anhydrous | Fisher | BP332 | |
Sodium chloride | Fisher | S642212 | |
Ethylenediaminetetraacetic acid | Fisher | BP118 | |
Magnesium chloride | Sigma Aldrich | M4880 | Corrosive |
Thermo scientific pierce DNaseI | Fisher | PI-90083 | |
GE Healthcare Sepharose Fast Flow Cation Exchange Media | Fisher | 45-002-931 | |
Tris-base | Fisher | BP152-500 | |
Sodium azide | TCI | S0489 | Highly toxic |
2-Mercaptoethanol | Fisher | ICN806443 | |
Sartorius Vivaspin 20 Centrifugal Concentrators | Fisher | 14-558-501 | |
Potassium phosphate monobasic | Sigma Aldrich | P5379 | |
2-Hydroxyethyl disulfide | Sigma Aldrich | 380474 | |
N-paratone | Hampton Research | HR2-643 | |
4 RC Dialysis Membrane Tubing 12,000 to 14,000 Dalton MWCO | Fisher | 08-667E | |
CryoLoop | Hampton Research | cryogenic tubing shaped into a loop | |
CryoTong | Thermo Fisher | cryogenic tong | |
Coot | Electron density images are generated from the software |