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

Summary

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

Abstract

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.

Introduction

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 space^2,3,4. Azaborines have potential utility for application in biomedical research^5,6,7,8, especially in the area of medicinal chemistry in which chemists carry out synthesis of libraries of structurally and functional....

Protocol

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 chromatograph....

Results

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.......

Discussion

In the first part of this protocol, we described a modified synthesis of 1,2-azaborines based on previously reported methods^{12, 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.......

Materials

Name	Company	Catalog Number	Comments
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