1,2- Azaborinesの合成とT4リゾチーム変異体とのタンパク質複合体の調製
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
複素環（ すなわち 、1,2- azaborines）を含むホウ素-窒素は、最近、アレーンのアイソスターとして大きな注目を集めています。この等電子性化学空間2、3、4を展開するために、既存の構造モチーフの多様化につながる可能性があります。 Azaborines特に化学が構造のライブラリーと機能的に関連する分子の合成を行った医薬品化学の分野で、生物医学研究5、6、7、8で使用するための潜在的な有用性を有します。重要なことに、しかし、一方、利用可能なアレーン含有分子に多数のよく発達した合成経路があり、azaborinesの合成のための方法の限られた数が報告されている9、10、 </s> 11、12、13アップ。これは、ホウ素源と空気と合成配列の初期段階における分子の湿気に敏感な性質のためにオプションの限られた数の主です。
この記事の最初の部分では、（3）標準エアフリー技術を用いて、N -TBS- B -Cl-1,2- azaborineのマルチグラム規模の合成を説明します。この化合物は、さらに、構造的に、より複雑な分子14、15に官能化することができる汎用性の高い中間体として機能します。 3から出発して、タンパク質結合研究に使用するためのN -H- Bエチル-1,2- azaborineの合成および精製（5）について説明します。 5の揮発性のために、その効率的な分離は、反応温度、時間、およびDISTの正確な制御を必要とします推理条件。
第二部では、タンパク質発現およびT4リゾチーム変異体（L99A及びL99A / M102Q）17、18、19の単離のためのプロトコルは、20は、タンパク質結晶化およびタンパク質-リガンドの結晶複合体の調製に続いて、提示されます。 T4リゾチーム変異体L99A及びL99A / M102Qがazaborine分子17を含むNHの水素結合能を調べるために、生物学的モデル系として選択しました。標準的な分子生物学プロトコルを用いて、タンパク質を大腸菌 RR1株で発現させ、イソプロピルβ-D-1-チオガラクトピラノシド（IPTG）で誘導されます。タンパク質精製は、イオン交換カラムクロマトグラフィーを用いて行われます。タンパク質の結晶化は、吊り下げを使用し、高度に濃縮された精製されたタンパク質溶液（ゲル電気泳動によって純度> 95％）を用いて行われます蒸気拡散法をドロップします。そのため、酸素に対する本研究のリガンドの感度が、タンパク質 – リガンド複合体は、空気を含まない条件下で製造されます。
Protocol
Representative Results
Discussion
このプロトコルの最初の部分では、我々は以前に報告された方法12、13に基づいて、1,2- azaborinesの修正された合成を説明しました。 Triallylborane 22を準備するallyltriphenylスズやカリウムallyltrifluoroborateを使用して、ルートの代替として使用されたN -allyl- N -TBS- B -アリル塩化付加物（1）。この方法は、よ…
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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