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Summary
Abstract
Introduction
Protocol
Results
Discussion
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Acknowledgements
Materials
References
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생화학

马萨霉素的合成，一种革兰氏阳性细菌生长的小分子抑制剂

Published: January 07, 2022
doi:
10.3791/63191
, ,
1Department of Science and Technology,Bryant University, 2Center for Health and Behavioral Sciences,Bryant University

Summary

提出了制备抑菌二酰胺马萨霉素的详细方案，马萨里霉素是一种小分子探针，通过靶向细胞壁降解来抑制 枯草芽孢杆菌 和 肺炎链球菌 的生长。其作为化学探针的应用在与 枯草芽孢杆菌 和 肺炎链球菌的协同作用/拮抗作用测定和形态学研究中得到证明。

Abstract

细菌细胞壁中的肽聚糖（PG）是一种独特的大分子结构，赋予形状和保护周围环境。了解细胞生长和分裂的核心是了解PG降解如何影响生物合成和细胞壁组装。最近，已经报道了通过引入改性糖或氨基酸来标记PG的代谢标记。虽然用小分子抑制剂对生物合成步骤进行化学询问是可能的，但研究自身溶酶降解PG的化学生物学工具尚不发达。细菌自溶素是一类广泛的酶，参与PG的紧密协调降解。这里提出了制备小分子探针masarimycin的详细方案，masarimycin是 枯草芽孢杆菌 中N-乙酰葡萄糖苷酶LytG的抑制剂，以及 肺炎链球菌中的细胞壁代谢。 提供通过 微波辅助和经典有机合成制备抑制剂的方法。介绍了其作为研究生物测定中革兰氏阳性生理学的工具的适用性。

Introduction

肽聚糖（PG）是一种网状聚合物，可描绘革兰氏阳性和革兰氏阴性细菌1，2中的细胞形状和结构。这种杂聚物是由短肽3，4，5，6与由β-（1，4）连接的交替的N-乙酰葡糖胺（GlcNAc）和N-乙酰基壁氨酸（MurNAc）残基组成的主链的氨基糖基质（图1）1。附着在MurNAc的C-3乳酰基部分的是茎肽。PG的代谢涉及生物合成和降解酶的紧密协调系统，以将新材料掺入细胞壁7，8。PG的降解由统称为自溶蛋白9的酶进行，并根据切割的键的特异性进一步分类。自溶蛋白参与许多细胞过程，包括细胞生长，细胞分裂，运动，PG成熟，趋化性，蛋白质分泌，遗传能力，分化和致病性10，11。解开单个自溶蛋白的特定生物学功能可能令人生畏，部分原因是功能冗余。然而，最近的生物物理8，12，13和计算研究12为它们在PG代谢中的作用提供了新的见解。此外，最近的报告提供了对PG代谢中合成14和膜介导的15，16，17步骤的进一步了解。对PG代谢的降解和合成途径之间关系的透彻理解可能会产生以前未开发的抗生素靶标。

虽然在研究真核生物糖生物学的方法上取得了重大进展，但细菌糖生物学，特别是PG代谢并没有以类似的速度发展。目前研究PG代谢的化学方法包括荧光标记的抗生素18，荧光探针19，20和代谢标记21，22，23，24。这些新方法为询问细菌细胞壁代谢提供了新的方法。虽然其中一些策略能够在体内标记PG ， 但它们可以是物种特异性的19，或者仅在缺乏特定自溶素25的菌株中起作用。许多PG标记策略旨在用于分离的细胞壁26 或与 体外 重建的PG生物合成途径20，27，28。荧光标记抗生素的使用目前仅限于生物合成步骤和转吞18。

目前细菌自身溶解素及其在细胞壁代谢中的作用的知识来自遗传和 体外 生化分析11，29，30，31，32。虽然这些方法提供了有关这种重要酶类的大量信息，但破译它们的生物学作用可能具有挑战性。例如，由于功能冗余33，在大多数情况下，自动溶血素的缺失不会导致细菌生长停止。尽管它们在细胞生长和分裂中隐含着作用7，12。另一个并发症是细菌自身溶解素的遗传缺失可引起元表型34。元表型源于受遗传缺失影响的途径与其他相互关联的途径之间的复杂相互作用。例如，元表型可以通过直接 效应（例如 缺乏酶）或间接效应（例如调节剂的破坏）而产生。

目前，只有少数糖苷酶自溶蛋白抑制剂，如N-乙酰氨基葡萄糖酶（GlcNAcase）和N-乙酰基muramidases，可用作化学探针来研究PG的降解。为了解决这个问题，已经鉴定并表征了35 作为 枯草芽孢杆菌 生长的抑菌抑制剂，其靶向GlcNAcase LytG32 （图1）。LytG是一种 外源作用的GlcNAcase36，是糖基水解酶家族73（GH73）中簇2的成员。它是营养生长过程中的主要活性GlcNAcase32。据我们所知，马沙霉素是抑制细胞生长的PG作用GlcNAcase的第一种抑制剂。马沙霉素与 肺炎链球菌 的进一步研究发现，马沙霉素可能抑制该生物体中的细胞壁代谢37。在这里，据报道，马沙霉素的制备被用作化学生物学探针，以研究革兰氏阳性生物 枯草芽孢 杆菌和 肺炎链球菌 的生理学。本文介绍了用马沙霉素进行亚最小抑制浓度治疗的形态学分析实例，以及协同/拮抗作用测定。使用具有明确定义的作用模式的抗生素进行协同作用和拮抗作用测定可以是探索细胞过程38，39，40之间连接的有用方法。

Protocol

1. 一般方法 注意：所有化合物均从标准供应商处购买，无需进一步纯化即可使用。 在预涂有硅胶XG F254的铝板上进行薄层色谱（TLC）。通过浸入对茴香醛污渍或暴露于I2蒸气来检测紫外灯下的斑点。 在 400 MHz 光谱仪上记录所有核磁共振 （NMR） 光谱。注意： 1H-NMR和 13C-NMR光谱参考残留溶剂峰。耦合常数以 [Hz] 为单?…

Representative Results

马萨里霉素是枯草芽孢杆菌和肺炎链球菌的小分子抑菌抑制剂，已被证明可抑制枯草芽孢杆菌35，37中的外效GlcNAcase LytG，并靶向肺炎链球菌37中的细胞壁。马沙霉素可以通过经典或微波辅助有机合成有效制备，收率在55%-70%范围内。微波辅助合成的优点是可以显著缩短合成化合物的时间。微波辅助合成将合成?…

Discussion

马沙霉素是 枯草芽孢杆菌35 和肺炎链球菌37 生长的单微摩尔抑菌抑制剂。在 枯草芽孢杆菌中， 马萨利霉素已被证明可以抑制GlcNAcase LytG35，而 肺炎链球菌 细胞壁中的精确分子靶标尚未确定37。使用经典的有机合成或微波程序合成马萨霉素为抑制剂提供了良好的产量和高纯度。马沙霉素的低产率通常可归?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

研究得到了美国国家科学基金会的支持，拨款编号为2009522。马沙霉素的NMR分析得到了美国国家科学基金会重大研究仪器计划奖的支持，授予号为1919644。本材料中表达的任何意见，发现和结论或建议均为作者的观点，并不一定反映美国国家科学基金会的观点。

Materials

2-Iodobenzoic acid SIGMA-ALDRICH I7675-25G corrosive, irritant, light yellow to orange-brown powder
2-Propanol SIGMA-ALDRICH 109827-4L flammable, irritant, colorless liquid
Acetonitrile SIGMA-ALDRICH 34851-4L flammable, irritant, colorless liquid
Aluminum backed silica plates Sorbtech 4434126 silica gel XG F254 on aluminum backed plates
chloroform-d SIGMA-ALDRICH 151823-50G solvent for NMR
Compact Mass Spectrometer Advion-Interchim Advion CMS compact mass spectrometer equiped with APCI source and atmospheric solids analysis probe
Corning Costar 96 well flat bottom plates-sterile fisher chemical 07-200-90 for synergy/antagonism assays
cover slips fisher chemical 12-547 for microscopy
Cyclohexanecarboxaldehyde CHEM-IMPEX INT'L INC. 24451 flammable, irritant, colorless to pink liquid
Cyclohexyl isocyanide SIGMA-ALDRICH 133302-5G irritant, colorless liquid, extremly unpleasant odor
Cyclohexylamine SIGMA-ALDRICH 240648-100ML corrosive, flammable, irritant, colorless liquid unless contaminated
Ethyl acetate SIGMA-ALDRICH 537446-4L flammable, irritant, colorless liquid
flash silica cartridge (12g) Advion-Interchim PF-50SIHP-F0012 pack of flash silica columns (12g) for purification of masarimycin
formaldehyde SIGMA-ALDRICH F8775-25ML fixing agent for microscopy
HEPES SIGMA-ALDRICH H8651-25G buffer for microscopy fixing solution
Hexane, mixture of isomers SIGMA-ALDRICH 178918-4L environmentally damaging, flammable, irritant, health hazard, colorless liquid
High performance compact mass spectrometer Advion expression Atmospheric Solids Analysis Probe (ASAP), low resolution
High Vac eppendorf Vacufuge plus vacuum aided by centrifugal force and temperature
Hydrochloric acid SIGMA-ALDRICH 258148-2.5L corrosive, irritant, colorless liquid
hydrochloric acid SIGMA-ALDRICH 320331-2.5L strong acid
immersion oil fisher chemical 12-365-19 for microscopy
Iodine, resublimed crystals Alfa Aesar 41955 environmentally damaging, irritant, health hazard, dark grey/purple crystals
Mestre Mnova MestreLab Research software for processing NMR spectra
Methanol SIGMA-ALDRICH 439193-4L flammable, toxic, health hazard, colorless liquid
methylene blue SIGMA-ALDRICH M9140-25G microscopy stain for staining cell walls
meuller-hinton agar plates + 5% sheep blood fisher chemical B21176X growth media for Streptococcus pneumoniae
meuller-hinton broth fisher chemical DF0757-17-6 growth media for Streptococcus pneumoniae
microscope slides fisher chemical 22-310397 for microscopy
Microwave Synthesis Labstation MILESTONE START SYNTH device that requires the ventilation of a fume hood, equipped with synthesis carousel
NMR tubes SIGMA-ALDRICH Z562769-5EA 5mm NMR tubes 600 MHz
Nuclear Magnetic Resonance (NMR) Bruker Ascend 400 large superconducting magnet (400MHz)
optochin fisher chemical AAB21627MC ethylhydrocupreine hydrochloride
petrie plates Celltreat 229695 for preparing agar plates for bacterial growth
Primo Star Bright field/Phase contrast Microscope with ERc5s camera Zeiss for morphology studies
puriFlash interchim XS520plus flash chromatography purification system
resazurin SIGMA-ALDRICH R7017-1G for synergy/antagonism assays
Rotary Evaporator Heidolph Hei-VAP Value "The Collegiate" solvent evaporator
Sodium bicarbonate SIGMA-ALDRICH S6014-500G irritant, white powder
Sodium chloride fisher chemical S271-1 crystalline, colorless
Sodium chloride SIGMA-ALDRICH S5886-500G for growth of B.subtilis and preparation of LB media
Sodium sulfate SIGMA-ALDRICH 7985592-500G anhydrous, granular, white
tryptone fisher chemical BP1421-500 for growth of B.subtilis and preparation of LB media
Whitney DG250 Workstation Microbiology International DG250 anaerobic workstation. Anaerobic gas mixture used: 5% hydrogen, 10% carbon dioxide, balance nitrogen
yeast extract fisher chemical BP1422-500 for growth of B.subtilis and preparation of LB media
Zen Lite (blue) software Zeiss for acquiring micrographs
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Tags

MasarimycinGram-positive BacteriaAutolysin InhibitorBacterial Growth InhibitorOrganic SynthesisCyclohexylamineCyclohexyl CarboxaldehydeOrtho-iodobenzoic AcidCyclohexyl IsocyanideMethanolThin-layer ChromatographyEthyl AcetateHydrochloric AcidSodium BicarbonateSodium ChlorideSodium Sulfate
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Gallati, M., Point, B., Reid, C. W. Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth. J. Vis. Exp. (179), e63191, doi:10.3791/63191 (2022).
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