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Summary
Abstract
Introduction
Protocol
Results
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Acknowledgements
Materials
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Chemistry

寡 peptoids 的合成及质谱分析

Published: February 21, 2018
doi:
10.3791/56652
, , ,
1Department of Chemistry,University of the Pacific

Summary

本文介绍了用质谱法进行序列分析的 peptoids 的人工合成的一种协议。

Abstract

Peptoids 是由 n-烷基化甘氨酸单元组成的序列控制的多肽模仿寡聚物。在许多潜在的应用中, peptoids 被认为是一种分子信息存储类型。质谱分析已被认为是测序 peptoids 的选择方法。Peptoids 可以通过固体相化学合成, 使用重复的两步反应循环。本文提出了一种人工合成寡 peptoids 的方法, 并用串联质谱 (ms) 技术对 peptoids 序列进行了分析。样品 peptoid 是一个 nonamer 由交替 n-(2-methyloxyethyl) 甘氨酸 (Nme) 和 n-(2-苯) 甘氨酸 (北角), 以及 n-(2-氨基乙基) 甘氨酸 (Nae) 在 n 终点。peptoid 的序列公式为 ac Nae-(Nme)4-NH2, 其中 Ac 为乙酰基组。综合发生在一个商业上可利用的固相反应容器。溜冰场酰胺树脂作为固体支持, 以产生 peptoid 与酰胺组在 C 总站。所产生的 peptoid 产品是使用三极质谱仪耦合到电喷雾电离源进行序列分析。ms 和 ms 的测量产生了一系列的片段离子产生的分离的带电 peptoid。片段离子根据其质量-电荷比 (m-/z) 的值进行排序。根据 peptoid 碎片的方案, 将片段离子的 m-/z 值与理论上预测的片段离子的标称质量进行比较。分析生成带电 peptoid 的碎片模式。分裂模式与中性 peptoid 单体序列相关。在这方面, MS 分析宣读了 peptoids 的序列信息。

Introduction

Peptoids 是一类序列控制聚合物, 其骨干结构模仿肽的结构。Peptoids 可以从不同的胺合成, 这使 Peptoids 能够表现出高度可调谐的属性1,2。Peptoids 已被用作生物物理研究的分子模型, 被认为是治疗剂, 并被设计为蛋白质的配体3,4,5,6。Peptoids 已发展成为各种生物活性化合物, 如防污和抗体模拟材料, 抗菌剂, 和酶抑制剂7,8,9。具有高度有序和可调谐的性质, peptoids 也被认为是一种类型的分子信息存储10。这些不同应用的发现要求开发有效的分析方法来描述 peptoids 的序列和结构。串联质谱技术已表明, 作为分析序列控制聚合物序列特性的选择方法, 包括 peptoids11、12、13、 14,15。然而, 系统研究相关的 peptoid 离子碎片模式产生的质谱研究和结构信息的 peptoids 是非常有限的。

Peptoids 可以很容易地合成使用固相方法。成熟的方法包括两步单体加法循环的迭代16,17。在每个加法周期中, 树脂束缚胺是由卤乙酸酸 (通常溴乙酸酸, 乙酰), 这是其次是与主胺的位移反应。尽管自动合成协议已被例行地应用于 peptoid 合成, 但在标准化学实验室中, peptoids 可以人工合成, 并具有优异的产量16,18,19,20。

本实验室采用人工 peptoid 合成方法, 简化了现有方法中使用的仪器。我们以前研究了一系列 peptoids 的碎片模式, 使用 ms/ms 技术21,22,23。我们的结果表明, 当它们受到碰撞诱导的离解 (CID)21,23或电子捕获离解 (peptoids)22实验时, 产生特征碎裂。在本文中, 我们展示了如何在标准化学实验室中合成寡 peptoids, 如何使用三重四极质谱仪进行 CID 实验, 以及如何分析光谱数据。合成的 peptoid 和特征是 nonamer 与 n-末端乙酰化和 C 末端酰, Ac Nae-(北角 Nme)4-NH2。peptoid 的结构显示在图 1中。

Protocol

1. Peptoid 的合成 注: 合成始于激活树脂, 通过肿胀树脂和去除保护组。接着, 通过重复单体加法循环将 peptoid 链添加到树脂上。第一个与树脂耦合的单体是 C 端残渣。peptoid 从 C 总站拉长到 N 终点。一旦达到所需的 peptoid 序列, 树脂就会脱落, peptoid 产品被提纯。 试剂的制备注: 用微测量液体试剂, 用分析天平测量固体试剂。 混合6.2 毫升的 n, n-‘-diisopr…

Representative Results

图 1中显示了一个 9 peptoid 的结构, 具有 n-端乙酰化、交 Nae (Nme)4-NH2。peptoid 是通过固相法在烧结聚丙烯反应容器中人工合成的。溜冰场酰胺树脂 (0.047 毫摩尔, 84 毫克与装载0.56 毫摩尔/克) 被用来作为坚实的支持, 以产生 peptoid 与酰胺化的 C 总站。peptoid 链是由单体加法的多个循环所建立的。每个单体加法循环包括两个反应步骤, bromoacety…

Discussion

使用所介绍的协议合成了一个 nonamer peptoid、交流 Nae (北 Nme)4-NH2。该合成装置涉及一种类似于注射器的聚丙烯固相反应容器和机械振动筛。反应容器是商业上可利用和低成本。机械振动筛是化学实验室中常见的仪器。使用类似于注射器的反应容器, 可以通过手动移动柱塞将溶液抽出并推出容器。该技术允许单体添加和树脂裂解反应发生在一个单一的反应容器中, 并消除了将树脂绑定…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者要感谢 Zuckermann 先生和罗纳德博士 (分子铸造, 劳伦斯伯克利国家实验室) 在 peptoid 合成技术支持。我们感谢国家科学基金会 (CHE-1301505) 的支持。所有质谱实验都是在太平洋大学化学质谱仪的实验室进行的。

Materials

ESI-triple quadrupole mass spectrometer, Varian 320L Agilent Technologies Inc. The mass spectrometer was acquired from Varian, Inc.
Varian MS workstation, Version 6.9.2, a data acquisition and data review software Varian Inc. The software is a part of the Varian 320L package
Burrell Scientific Wrist-action shaker, Model 75 DD Fisher Scientific International Inc. 14-400-126
Hermle Centrifuge, Model Z 206 A Hermle Labortechnik GmbH
Solid phase reaction vessel, 10 mL Torviq SF-1000
Pressure caps for reaction vessels Torviq PC-SF
Syringe filters, pore size 0.2 μm Fisher Scientific Inc. 03-391-3B
Syringe filters, pore size 0.45 μm Fisher Scientific Inc. 03-391-3A
Polypropylene centrifuge tuges, 50 mL VWR International, LLC. 490001-626
Polypropylene centrifuge tuges, 15 mL VWR International, LLC. 490001-620
ChemBioDraw, Ultra, Version 12.0 CambridgeSoft Corporation CambridgeSoft is now part of PerkinElmer Inc.
Styrofoam cup, 12 Oz Common Supermarket
Rink amide resin Chem-Impex International, Inc. 10619
Piperidine Chem-Impex International, Inc. 02351 Highly toxic
N, N’-diisopropylcarbodiimide Chem-Impex International, Inc. 00110 Highly toxic
Bromoacetic acid Chem-Impex International, Inc. 26843 Highly toxic
2-Phenylethylamine VWR International, LLC. EM8.07334.0250
2-Methyoxyethylamine Sigma-Aldrich Co. LLC. 241067
N-Boc-ethylenediamine VWR International, LLC. AAAL19947-06
Acetic anhydride Sigma-Aldrich Co. LLC. 252845
N, N-dimethylformamide VWR International, LLC. BDH1117-4LG Further distillation before use
N, N-diisopropylethylamine Chem-Impex International, Inc. 00141
Triisopropylsilane Chem-Impex International, Inc. 01966
Trifluoroacetic acid Chem-Impex International, Inc. 00289 Highly toxic
Millipore MILLI-Q Academic Water Purification System Millipore Corporation ZMQP60001 For generating HPLC grade water
HPLC-grade Water Produced from Millipore MILLI-Q® Academic Water Purification System
Methanol Pharmco-Aaper 339USP/NF HPLC grade
Acetonitrile Fisher Scientific International, Inc. A998-4 HPLC grade
Diethyl ether VWR International, LLC. BDH1121-19L Further distillation before use
Dichloromethane VWR International, LLC. BDH1113-19L Further distillation before use
Nitrogen gas Fresno Oxygen/Barnes Supply NIT 50-C-F Ultra high purity, 99.9995%
Argon gas Fresno Oxygen/Barnes Supply ARG 50-C-F Ultra high purity, 99.9995%
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References

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Tags

Keywords: Oligo-peptoidsPeptoid SynthesisMass SpectrometryTandem Mass SpectrometryRink Amide ResinTMFPiperidineDMFBromoacetylation2-methoxyethylamineSolid-phase SynthesisCharacterizationMonomer Sequence
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Ren, J., Mann, Y. S., Zhang, Y., Browne, M. D. Synthesis and Mass Spectrometry Analysis of Oligo-peptoids. J. Vis. Exp. (132), e56652, doi:10.3791/56652 (2018).
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