Síntesis de 1,2-Azaborines y la preparación de sus complejos de proteínas con lisozima T4 Mutantes
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
El boro-heterociclos que contienen nitrógeno (es decir, 1,2-azaborines) recientemente han llamado la atención significativa como isosteras de arenos. Este isosterism puede conducir a la diversificación de los motivos estructurales existentes para ampliar el espacio químico 2, 3, 4. Azaborines tienen utilidad potencial para la aplicación en la investigación biomédica 5, 6, 7, 8, especialmente en el área de la química médica en la que los químicos llevar a cabo la síntesis de bibliotecas de moléculas estructuralmente y funcionalmente relevantes. Significativamente, sin embargo, si bien hay numerosas rutas sintéticas bien desarrollados a moléculas que contienen areno-disponibles, se han reportado sólo un número limitado de métodos para la síntesis de azaborines 9, 10, </shasta> 11, 12, 13. Esto se debe principalmente a un número limitado de opciones para la fuente de boro y el aire y la naturaleza sensible a la humedad de la molécula en la fase inicial de la secuencia sintética.
En la primera parte de este artículo, describiremos una síntesis escala multi-gramo de N -TBS- B -Cl-1,2-azaborine (3) usando técnicas sin aire estándar. Este compuesto sirve como un intermedio versátil que puede ser además funcionalizado para estructuralmente más complejas moléculas 14, 15. A partir de 3, se describirán la síntesis y purificación de N -H- B etil-1,2-azaborine (5) para su uso en estudios de unión a proteínas. Debido a la volatilidad de 5, su aislamiento eficiente requiere un control preciso de la temperatura de reacción, tiempo, y distillation condiciones.
En la segunda parte, los protocolos para la expresión de proteínas y el aislamiento de mutantes T4 lisozima (L99A y L99A / M102Q) 17, 18, 19, se presentarán 20, seguido de la cristalización de proteínas y la preparación de los complejos de cristal de proteína-ligando. Mutantes de lisozima T4 L99A y L99A / M102Q fueron elegidos como modelo de los sistemas biológicos para examinar la capacidad de enlace de hidrógeno de las moléculas que contienen NH azaborine 17. El uso de un protocolo de biología molecular estándar, la proteína se expresa en la cepa Escherichia coli RR1 y indujo con isopropil-β-D-1-tiogalactopiranósido (IPTG). Purificación de proteínas se lleva a cabo usando cromatografía en columna de intercambio iónico. cristalización de proteínas se lleva a cabo con una solución altamente concentrada y purificada de proteínas (> 95% de pureza por electroforesis en gel) usando el colgantedeje caer método de difusión de vapor. Debido a la sensibilidad de los ligandos de este estudio para el oxígeno, los complejos proteína-ligando se preparan en condiciones libre de aire.
Protocol
Representative Results
Discussion
En la primera parte de este protocolo, se describe una síntesis modificada de 1,2-azaborines basadas en métodos reportados previamente 12, 13. Triallylborane 22 fue utilizado como un sustituto de las rutas usando allyltriphenyl estaño o de potasio allyltrifluoroborate para preparar N -allyl- N -TBS- B alil aducto de cloruro de (1). Este método permite un enfoque más átomos-económica y res…
Declarações
The authors have nothing to disclose.
Acknowledgements
This research was supported by the National Institutes of Health NIGMS (R01-GM094541) and Boston College.
Materials
| 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 |
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