1,2- Azaborines的合成及其蛋白复合物的用T4溶菌酶突变体的制备
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
含杂环（ 即 1,2- azaborines）硼-氮最近已经引起显著关注，芳烃等排。此isosterism可导致现有的结构图案的多样化，扩大化学空间2，3，4。 Azaborines具有在生物医学研究5，6，7，8为应用潜在效用，尤其是在药物化学中化学家进行的结构库和功能相关的分子的合成的区域。显著，然而，尽管有无数发达合成路线可用含芳烃分子，只对azaborines的合成方法有限数量已报道9，10， </s达> 11，12，13。这主要是由于对于硼源和空气和该分子的湿气敏感性质在合成序列的早期阶段的选择是有限的。
在本文的第一部分，我们将介绍使用标准无空气技术（3）N -TBS- 乙 -Cl-1,2-azaborine的多克规模的合成。该化合物作为可被进一步官能化以在结构上更复杂的分子14,15一种多用途的中间体。从3开始，N- -H- 乙 -乙基-1,2- azaborine（5）在蛋白结合研究使用的合成和纯化将描述。由于5的波动，其高效分离需要的反应温度，时间，和DIST的精确控制推理的条件。
在第二部分中，用于蛋白质表达和T4溶菌酶突变体（L99A和L99A / M102Q）17，18，19的分离方案，20将呈现，接着蛋白质结晶和制备蛋白质-配体晶体复合物。 T4溶菌酶突变体L99A和L99A / M102Q被选为生物模型系统来检查包含azaborine分子17 NH的氢键能力。使用标准的分子生物学协议，该蛋白在大肠杆菌 RR1菌株表达并用异丙基β-D-1-硫代半乳糖苷（IPTG）诱导。蛋白质纯化进行使用离子交换柱层析。蛋白质结晶使用悬高度浓缩纯化的蛋白质溶液（> 95％纯度通过凝胶电泳）上进行滴气相扩散法。由于本研究的配位体对氧的敏感性，蛋白质 – 配体配合物是无空气的条件下制备。
Protocol
Representative Results
Discussion
在这个协议中的第一部分中，我们描述的基于先前报道的方法12,13 1,2- azaborines修饰合成。 Triallylborane 22被用作使用allyltriphenyl锡或钾allyltrifluoroborate制备路线的替代Ñ-allyl-Ñ-TBS- 乙烯丙基氯化物加合物（1）。这种方法允许更原子经济和环保的做法。对于N -TBS- 乙 -Cl -1,2- azaborine（3）</st…
Divulgations
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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