Summary

1,2- Azaborines의 합성과 T4 리소자임 돌연변이와 그들의 단백질 복합체의 제조

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

헤테로 사이클 (즉, 1,2- azaborines)를 포함하는 붕소 – 질소는 최근 아렌의 isosteres로 크게 주목 받고있다. 이 isosterism 화학 공간 2, 3, 4를 확장 기존 구조 모티프의 다변화로 이어질 수있다. Azaborines 특히 화학자 라이브러리 구조적 기능적으로 중요한 분자의 합성을 수행하는 의약 화학 분야에서 생물 의학 연구 5, 6, 7, 8에서의 응용 가능성 유용성을 갖는다. 현저하게, 그러나 동시에 사용할 아렌 – 함유 분자에 다수의 잘 개발 된 합성 경로가, azaborines의 합성 방법에 한정 수가보고되었다 (9), (10) </s> 11, 12, 13입니다. 이는 합성 순서의 초기 단계에서의 붕소 원과 공기와 분자의 습기 민감성 옵션의 수가 제한을 주로한다.

이 문서의 첫 부분에서, 우리는 (3)의 표준 공기없는 기술을 사용하여 N -TBS- B -Cl -1,2- azaborine의 멀티 그램 규모의 합성을 설명한다. 이 화합물은 더 이상의 구조적으로 복잡한 분자 (14) (15)에 작용 화 될 수있는 다목적 중간체로서 작용한다. 3부터 단백질 결합 연구에 사용하기위한 N -H- B의 에틸 -1,2- azaborine (5)의 합성 및 정제를 설명한다. (5)의 변동성으로 인해 효율적인 분리는 반응 온도, 시간, DIST의 정밀한 제어를 필요추리 조건.

두 번째 부분에서, 단백질 발현 및 T4 라이소자임 돌연변이 (L99A 및 L99A / M102Q) (17), (18, 19)의 분리를위한 프로토콜은 20 단백질 결정화 단백질 – 리간드 결정 복합체의 제조 다음으로 제시한다. T4 라이소자임 돌연변이 L99A 및 L99A / M102Q는 NH가 azaborine 분자 (17)를 함유 한 수소 결합 능력을 조사하는 생물학적 시스템 모델로 선택했다. 표준 분자 생물학 프로토콜을 사용하여 단백질을 대장균 RR1 균주에서 발현 및 이소 프로필 β-D-1- 티오 갈 락토 피 라노 시드 (IPTG)에 유도된다. 단백질 정제는 이온 교환 컬럼 크로마토 그래피를 이용하여 수행된다. 단백질 결정화 매달려 사용 (겔 전기 영동으로> 95 % 순도) 정제 된 단백질의 고농도 용액으로 수행증기 확산 방법을 놓습니다. 때문에 산소 본 연구의 리간드의 민감성, 단백질 – 리간드 복합체는 공기가없는 조건에서 제조된다.

Protocol

주 : 모든 산소 및 습기에 민감한 조작 표준 공기없는 기술 또는 글로브 박스를 이용하여 불활성 분위기 (N 2)에 따라 수행 하였다. THF (테트라 히드로 푸란), 등이 O (디 에틸 에테르)을 CH2Cl2 (디클로로 메탄), 톨루엔, 펜탄 아르곤 하에서 중성 알루미나 칼럼을 통과시킴으로써 정제 하였다. 아세토 니트릴이 CAH (칼슘 하이드 라이드)상에서 건조시키고, 사용하…

Representative Results

1,2-azaborines에 대한 개략적 인 합성 경로는 그림 1과 같다. 이 프로토콜은 다섯 가지 붕소 – 질소 함유 분자의 합성에 적용됩니다. 도 2는 목적하는 생성물 (3)의 형성을 모니터링하는 단계 130의 과정 동안 측정 된 11 B NMR 스펙트럼을 나타낸다. 단백질 정제는 저압 크로마토 그래피 시스템 및 대표적인 크로마토 그램은도 3?…

Discussion

이 프로토콜의 첫 번째 부분에서는 이전에보고 된 방법 (12, 13)에 기초하여 1,2- azaborines의 변형 합성을 설명했다. Triallylborane 22 제조 allyltriphenyl 주석 칼륨 allyltrifluoroborate를 사용 경로에 대한 대체물로서 사용 하였다 N 알릴 N -TBS- B 알릴 클로라이드의 부가 물 (1). 이 방법은 더 많은 원자를 경제적이고 ?…

Disclosures

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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Cite This Article
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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