JoVE Journal > Chemistry
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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화학

分裂和池合成及肽叔酰胺图书馆表征

Published: June 20, 2014
doi:
10.3791/51299
,
1Scripps Florida,The Scripps Research Institute

Summary

肽叔酰胺（贸易协定）的肽模拟物，包括但不限于肽，拟肽和N-甲基化的肽的一个家族。在这里，我们描述它结合了两个分裂和池和子策略单体合成家教会的一珠一化合物库的合成方法。

Abstract

肽是蛋白质配体的巨大来源。这些化合物的低聚性质使我们利用组合化学访问的固相合成大库。一个模拟肽的研究最多也类是拟肽。拟肽是易于合成，并已被证明是蛋白酶解抗性和细胞渗透性。在过去的十年中，许多有用的蛋白质配体已通过拟肽库的筛选鉴定。然而，大多数从拟肽库确定的配体不显示高亲和力，除了极少数例外。这可能是由于，在某种程度上，缺乏手性中心的构象和限制在类肽分子。最近，我们描述了一种新的合成路线来访问肽叔酰胺（家教）。贸易协定是肽模拟物，包括但不限于肽，拟肽和N-甲基化的肽的一个家族。与在两个α-碳和主链氮原子的侧链这些分子的构象可得到很大的空间位阻和烯丙基1,3应变的限制。 （ 图1）我们的研究表明，这些PTA分子是高度结构化的溶液中，并且可以被用来确定蛋白质的配体。我们相信，这些分子可以是高亲和力的蛋白配体未来的源泉。在这里，我们描述的合成方法结合两种分裂和池和子单体策略的力量来合成样品家教会的一珠一化合物（OBOC）库。

Introduction

肽模拟物是模拟天然肽的结构的化合物。它们被设计为保留的生物活性，同时克服了与天然肽，包括细胞的渗透性和稳定性对蛋白水解1-3有关的问题。由于这些化合物的低聚物的性质，大合成文库可以容易地通过单体或子单体的合成路线4-7访问。一个模拟肽的研究最多的类别是拟肽。拟肽是，可以很容易地使用子单体策略8,9来合成N-烷基化甘氨酸的低聚物。许多有用的蛋白质配体已成功识别从筛选对蛋白指标1，10-14大合成拟肽库。不过，从拟肽库确定了“命中”很少归档非常高的亲和力对蛋白质靶1,10-14,22。一马约旦拟肽和天然肽之间的区别是，大部分的拟肽的普遍缺乏，以形成二级结构，由于缺乏手性中心和构象约束的能力。为了解决这一问题，开发了在过去的十年多种策略，主要着眼于包含在主链氮原子的15-22侧链的修饰。最近，我们已经开发出一种新的合成路线，以引进天然氨基酸侧链到拟肽骨干创造肽叔酰胺23。

肽叔酰胺（贸易协定）的肽模拟物，包括但不限于肽（R 2 = H），拟肽（R 1 = H）和N-甲基化的肽的超家族（R 1≠H，R 2 = Me）的。 （ 见图1）本合成路线采用天然存在的氨基酸为手性和侧链上的源45; – 碳，和市售的伯胺，得到N-取代。因此，比单纯肽，拟肽或N-甲基化多肽的化学较大的空间可以探索。圆二色光谱表明，PTA分子是高度结构化的解决方案。在PTA-蛋白质复合物的一个表征清楚地表明，PTA的构象限制所需要的具有约束力。最近，我们还发现，一些PTA分子具有改善细胞通透性比他们的拟肽和肽同行。我们相信，这些PTA库可以是高亲和力配体蛋白指标的良好来源。在本文中，我们将讨论的样本中以及这些化合物的耦合和裂解一些改善条件细节一珠一化合物（OBOC）PTA库的合成。

Protocol

1，斯普利特和池合成基础知识为了有效地产生在固相上的大量的化合物，分裂和池合成经常被用作一个一般的策略。 如图4的TentaGel珠是第一分割成三个部分。各部分进行反应以不同的试剂，生成的小珠的第一个残基。在第一反应后，所有的三个部分被汇集在一起​​，混合，然后再分成三个部分。每个部分将再次用不同的试剂反应，生成对珠子的第二个残基。经…

Representative Results

在这里，我们将展示从PTA三聚体与接头三个有代表性的电离光谱。 如图6A所示 ，当用50％TFA / DCM溶液在室温下裂解，显著降解是观察。在图6A中 ，峰593和484分别对应于连接体和三聚体的PTA，表明整个分子被成功地合成了在胎圈，但裂解过程中降解。当如上述那样低的温度条件下裂解，三氟乙酸诱导的降解的量被大大地抑制， 如图6B所示 。这样的裂解的机制已…

Discussion

肽叔酰胺（自由贸易协定）是肽低聚物的超家族。除了充分研究的肽，拟肽和N-甲基化肽，这个家庭中的化合物有很大一部分仍然是充分研究，majorly由于缺乏接入通用N-烷基化肽的合成方法。这里我们描述了一种有效的方法来合成贸易协定与从氨基酸衍生的手性结构单元。此前，我们曾报道使用一个新的子单体路线的PTA分子23的合成库。我们已经表明，自由贸易协定是其通过骨干具有构象?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者要感谢JUMPEI森本博士和托德多伦博士宝贵的援助。这项工作是由来自NHLBI（NO1-HV-00242）的合同支持。

Materials

2,4,6 trimethylpyridine ACROS 161950010 CAS:108-75-8
2-morpholinoethanamine Sigma-Aldrich 06680  CAS:2038-03-1  
48% HBr Water solution ALFA AESAR AA14036AT CAS:10035-10-6
Acetaldehyde Sigma-Aldrich 402788 CAS:75-07-0  
Acetonitrile Fisher SR015AA-19PS CAS:75-05-8
Anhydrous Tetrahydrofuran (THF) EMD EM-TX0277-6  CAS:109-99-9
Benzylamine Sigma-Aldrich 185701 CAS:100-46-9
bis(trichloromethyl) carbonate (BTC) ACROS 258950050 CAS:32315-10-9
Bromoacetic acid ACROS 106570010 CAS:79-08-3
Chloranil Sigma-Aldrich 23290 CAS:118-75-2
Cyclohexanemethylamine Sigma-Aldrich 101842 CAS:3218-02-8
D2O Cambridge Isotope DLM-4-99.8-1000 CAS:7789-20-0
D-alanine Anaspec 61387-100 CAS:338-69-2  
Dichloromethane (DCM) Fisher BJ-NS300-20 CAS:75-09-2
Dimethylformamide (DMF) Fisher BJ-076-4 CAS:68-12-2
Ethylene glycol Oakwood 44710 CAS:107-21-1
Isopentylamine Sigma-Aldrich W321907 CAS:107-85-7
KBr ACROS 424070025 CAS:7758-02-3
L-alanine Anaspec 61385-100 CAS:56-41-7 
3-Methoxypropylamine Sigma-Aldrich M25007 CAS:5332-73-0
2-Methoxyethylamine Sigma-Aldrich 143693 CAS:109-85-3
N-(3-Aminopropyl)-2-pyrrolidinone Sigma-Aldrich 136565  CAS:7663-77-6 
N,N'-Diisopropylcarbodiimide (DIC) ACROS 115211000 CAS:693-13-0
N,N-Diisopropylethylamine (DIPEA) Sigma-Aldrich D125806 CAS:7087-68-5
NaNO2 ACROS 424340010 CAS:7631-99-4
NAOD 40% solution in water ACROS 200058-506 CAS:7732-18-5
Piperidine ALFA AESAR A12442-AE CAS:110-89-4
Piperonylamine Sigma-Aldrich P49503  CAS:2620-50-0
Propylamine Sigma-Aldrich 240958 CAS:107-10-8
Trifluoroacetic acid Sigma-Aldrich 299537 CAS:76-05-1
α-Cyano-4-hydroxycinnamic acid  Sigma-Aldrich 39468 CAS:28166-41-8  
α-ketoglutarate ALFA AESAR AAA10256-22 CAS:328-50-7
Tentagel Resin with RINK linker Rapp-Polymere S30023
Alanine transaminase Roche 10105589001 AKA: Glutamate-Pyruvate Transaminase (GPT)
Incubator New Brunswick Scientific Innova44
NMR Bruker 400MHz
MALDI mass spectrometer Applied Biosystems  4800 MALDI-TOF/TOF
Lyophilizer SP Scientific VirTis benchtop K
Syringe reactor INTAVIS  Reaction Column 3ml, 5ml, 10ml, 20ml
Vacuum manifold  Promega A7231 Vac-Man
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Tags

Peptide Tertiary AmidesPeptidomimeticsPeptoidsProtein LigandsSplit-and-pool SynthesisCombinatorial ChemistryOne-bead One-compound LibraryConformational Constraints

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Gao, Y., Kodadek, T. Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library. J. Vis. Exp. (88), e51299, doi:10.3791/51299 (2014).
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