Latent Thioesters in Protein Chemistry and Chemical Biology

蛋白质化学和化学生物学中的潜在硫酯

• 批准号: EP/J007560/1
• 负责人: Derek MacMillan
• 金额: $ 42.23万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FJ007560%2F1
• 关键词: Latent; Thioesters; Protein; Chemistry; Chemical

The peptide thioester is an important tool in modern protein chemistry and chemical biology, particularly for the synthesis of proteins using a chemical reaction called Native Chemical Ligation (NCL). The synthetic thioester, which may contain unnatural amino acids or molecular probes, combines with additional peptide components to form a full length protein. NCL has enabled detailed studies of how proteins work and can provide access to pure samples of therapeutic peptides and proteins.The driving force for NCL is the formation of the amide linkage. However we recently described a new transformation where peptides and proteins fragment to afford thioesters in a reverse-NCL type reaction, a process that occurs via Nitrogen to Sulfur (N to S) acyl transfer. This reaction yields the important thioester tools for NCL which can be extremely challenging to prepare. Peptides of synthetic or biological origin can serve as substrates simply by introduction of a C-terminal cysteine residue, which functions as a latent thioester. When peptides are additionally furnished with an N-terminal cysteine, head to tail cyclic peptides are produced through a retro NCL/NCL reaction sequence.We have successfully applied the thioesters from our reaction to assembly of several synthetic bioactive peptides and semi-synthetic proteins. However, only through a detailed understanding of this fascinating transformation will we deliver a general, scalable, enabling methodology for peptide and protein synthesis, labelling, and chemical biology. Furthermore, this transformation is of fundamental interest since amide bonds, usually considered the more stable carboxylic acid derivatives, are broken under near physiological conditions in the absence of any enzymes, and formed in water, in the absence of typical peptide coupling reagents.Our first goal is to improve the process through a detailed study in model peptides, exploring the ability of new terminal functional groups to expedite thioester formation. These experiments will ultimately tell us how reactions can be performed in shorter times, at room temperature or below. N to S acyl transfer can also be employed to remove a single amino acid from a proteins' N-terminus allowing access to N-terminal cysteine containing proteins which can also be very challenging to produce by other means. We will investigate optimal procedures for N-cysteinyl peptide production using fluorescence-based detection and apply an optimised protocol to an expressed protein. An interesting feature of our process is the ease with which head-to-tail cyclic peptides can be prepared. We propose that the cyclic product accumulates, in part, because solvent is excluded from the reaction site upon cyclisation and can explore this hypothesis using mass spectrometry in a model system. This process also highlights the dominance of NCL under our reaction conditions and additives that may temper competing NCL during thioester formation will also be explored.An observed weakness is competing peptide hydrolysis at aspartate residues and so developing conditions, protecting groups, or aspartate "surrogates" that circumvent this problem will also be explored.The second goal is to apply optimized protocols in more challenging contexts. First we will prepare an analogue of the HIV fusion inhibitor enfuvertide. Completion of the synthesis, in three sections, will provide a valuable proof of concept, employing two thioesters derived from our new methodology. The second application explores the synthesis of cyclic mirror image peptides derived from the naturally occurring beta defensin family of antimicrobials. The stability of these analogues is far superior to their L-peptide counterparts. To better understand the molecular basis for their antimicrobial activity we will conduct the synthesis of further analogues and prepare sufficient material for characterisation by NMR spectroscopy and X-ray crystallography.

肽硫酯是现代蛋白质化学和化学生物学的重要工具，特别是用于利用天然化学结扎（NCL）化学反应合成蛋白质。合成的硫酯可能含有非天然氨基酸或分子探针，与额外的肽成分结合形成全长蛋白质。NCL使蛋白质如何工作的详细研究成为可能，并可以提供治疗肽和蛋白质的纯样品。NCL的驱动力是酰胺键的形成。然而，我们最近描述了一种新的转化，其中肽和蛋白质片段在反ncl型反应中提供硫酯，这一过程通过氮到硫（N到S）酰基转移发生。这个反应产生了NCL的重要的硫酯工具，这是极具挑战性的制备。合成或生物来源的多肽可以简单地通过引入c端半胱氨酸残基作为底物，其功能是潜在的硫酯。当多肽额外提供n端半胱氨酸时，通过反向NCL/NCL反应序列产生从头到尾的环状肽。我们已经成功地将从我们的反应中获得的硫酯应用于几种合成的生物活性肽和半合成蛋白质的组装。然而，只有通过对这一迷人转变的详细了解，我们才能为肽和蛋白质合成、标记和化学生物学提供一种通用的、可扩展的、可行的方法。此外，这种转化具有根本性的意义，因为酰胺键通常被认为是更稳定的羧酸衍生物，在没有任何酶的接近生理条件下断裂，并在没有典型肽偶联试剂的情况下在水中形成。我们的第一个目标是通过对模型肽的详细研究来改进这一过程，探索新的末端官能团加速硫酯形成的能力。这些实验最终将告诉我们如何在更短的时间内，在室温或更低的温度下进行反应。N到S酰基转移也可以用于从蛋白质的N端去除单个氨基酸，从而使其能够进入含有N端半胱氨酸的蛋白质，这也非常具有挑战性通过其他方式产生。我们将使用基于荧光的检测来研究n -半胱氨酸肽生产的最佳程序，并将优化的方案应用于表达的蛋白质。我们的工艺的一个有趣的特点是易于与头到尾的环状肽可以制备。我们提出循环产物的积累，部分原因是溶剂在环化时被排除在反应位点之外，并且可以在模型系统中使用质谱法来探索这一假设。这一过程也突出了NCL在我们的反应条件下的主导地位，并且还将探索在硫酯形成过程中可能缓和竞争NCL的添加剂。观察到的一个弱点是在天冬氨酸残基上竞争性肽水解，因此开发条件、保护基团或天冬氨酸“替代品”来规避这个问题也将被探索。第二个目标是在更具挑战性的环境中应用优化的协议。首先，我们将制备HIV融合抑制剂恩弗肽的类似物。合成的完成，分三个部分，将提供一个有价值的概念证明，使用从我们的新方法衍生的两种硫酯。第二个应用程序探索了从自然发生的抗微生物防御素家族衍生的环状镜像肽的合成。这些类似物的稳定性远远优于它们的l肽对应物。为了更好地了解其抗菌活性的分子基础，我们将进行进一步的类似物的合成，并准备足够的材料通过核磁共振波谱和x射线晶体学进行表征。

项目成果

Examination of mercaptobenzyl sulfonates as catalysts for native chemical ligation: application to the assembly of a glycosylated Glucagon-Like Peptide 1 (GLP-1) analogue.

• DOI: 10.1039/c4cc09502b
• 发表时间: 2015-02
• 期刊: Chemical communications
• 影响因子: 4.9
• 作者: B. Cowper;Tsz Mei Sze;B. Premdjee;Aileen F Bongat White;A. Hacking;D. Macmillan

Cysteine promoted C-terminal hydrazinolysis of native peptides and proteins.

• DOI: 10.1002/anie.201304997
• 发表时间: 2013-12-02
• 期刊: ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
• 影响因子: 16.6
• 作者: Adams, Anna L.;Cowper, Ben;Morgan, Rachel E.;Premdjee, Bhavesh;Caddick, Stephen;Macmillan, Derek
• 通讯作者: Macmillan, Derek