Summary

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

Published: March 25, 2017
doi:

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( 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- -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

注:所有氧和水分敏感的操作均在惰性气氛(N 2)使用标准无空气技术或在手套箱中进行。 THF(四氢呋喃),Et 2 O等(乙醚),CH 2 Cl 2中(二氯甲烷),甲苯和戊烷,通过中性氧化铝柱和氩气下经过纯化。乙腈经氢化钙2(氢化钙)干燥,并在使用前在氮气氛下蒸馏。的Pd / C(在碳上的钯)的混合物在100℃下在高真空下加热,使用前12小时。硅胶(230-400目?…

Representative Results

1,2- azaborines概略合成路线示于图1。该协议适用于五种不同的含硼氮分子的合成。 图2表示11步骤1.3的过程期间测量并监控所需产物(3)的生成乙NMR谱。蛋白纯化通过使用低压层析系统和有代表性的色谱如图3所示进行。所收集的级分的纯度通过SDS-PAGE测定。 T4溶菌酶突变体L99A的晶体在图4中被描绘。 <st…

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- 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- -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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Lee, H., Liu, S. Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants. J. Vis. Exp. (121), e55154, doi:10.3791/55154 (2017).

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