High performance auxiliaries for a cysteine-tolerant native chemical ligation at arbitrary sites

用于在任意位点进行半胱氨酸耐受的天然化学连接的高性能辅助剂

基本信息

批准号：
367109134
负责人：
Professor Dr. Oliver Seitz
金额：
--
依托单位：
Institut für Chemie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2017
资助国家：
德国
起止时间：
2016-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/367109134?language=en
关键词：
performance auxiliaries cysteine tolerant native

项目摘要

The Native Chemical Ligation (NCL) between unprotected peptide thioesters and unprotected cysteinyl peptides provides access to proteins in defined posttranslational modification states. However, the requirement for cysteine limits the scope of NCL reactions. The ligation-desulfurization method extends the repertoire of NCL chemistry. Unfortunately, the efforts required for the preparation of the mercapto-functionalized amino acid building blocks is too high and limits applications at arbitrary ligation sites. Furthermore, it is a drawback that remote cysteine residues (which are not involved in the NCL) need protection. NCL methods that rely on auxiliary groups require only a single building block, which in the ideal case may provide access to any ligation site. However, the existing state-of-the-art auxiliaries are limited either by their steric demand which prevents ligations to succeed in absence of glycine or by side reactions at unprotected cysteine residues during auxiliary removal.In this research project we will develop the first generally applicable method that enables rapid native chemical ligation reactions at virtually any given ligation site and yet tolerates the presence of unprotected cysteine residues at remote sites. We will develop high performance ligation auxiliaries, which i) can be introduced at arbitrarily chosen amino acids in the last step of solid phase peptide synthesis; ii) provide high reactivity even in sterically challenging NCL reactions and iii) allow by-product-free cleavage under mild conditions, iv) without harm to unprotected cysteine side chains. To achieve these aims, we will establish 2-seleno-2-phenethyl- and 2-mercapto-2-arylethyl-scaffolds as new auxiliary categories. The auxiliaries have little steric demand. As a result, NCL reactions will proceed rapidly via 5-membered transition states. Radical conditions will be used to selectively trigger a fragmentation reaction which furnishes the target proteins without detriment to cysteine residues at remote sites. In a realistic scenario of protein total synthesis, we will establish Leu-Thr-, Gln-Asn-, Leu-His- and Ile-Met-ligations. Of note, such ligation junctions would not be accessible by using existing native chemical ligation methodology.To assess the usefulness of the new method we will prepare SH3 domains of the adapter proteins p130Cas and NEDD9 by chemical total synthesis. We will examine the functionality of the synthetic proteins by means of protein binding experiments with known SH3 domain ligands. The total synthesis of site specifically phosphorylated p130Cas and NEDD9 proteins will allow studies, in which we explore a putative phospho switch that fine regulates cellular signal transduction by altering the recognition repertoire of the SH3 domains involved.

未受保护的肽硫代酯和未受保护的白细胞蛋白酶肽之间的天然化学连接（NCL）为定义的翻译后修饰状态提供了蛋白质的访问。但是，半胱氨酸的需求限制了NCL反应的范围。结扎硫化方法扩展了NCL化学的曲目。不幸的是，制备墨托官能化的氨基酸构建块所需的努力太高，并且限制了在任意连接部位的应用。此外，远程半胱氨酸残基（不参与NCL）需要保护是一个缺点。依靠辅助组的NCL方法仅需要一个单个构建块，在理想情况下，这可以提供对任何连接站点的访问。但是，现有的最新辅助因素受其空间需求的限制，这可以防止辅助因甘氨酸缺乏辅助或在辅助清除过程中未受保护的半胱氨酸残基处于未受保护的半胱氨酸残基处而取得成功。在这项研究项目中，我们将开发出第一个普遍适用的方法，可在任何给定的位置上均能在任何远处均能耐受地位，但可耐受地位的频率，但可耐受地位，但可耐受地点的差异。我们将开发高性能连接辅助机，我可以在固相肽合成的最后一步中以任意选择的氨基酸引入； ii）即使在空间挑战性的NCL反应中也具有高反应性，iii）允许在轻度条件下允许无副产物的裂解，iv），而不会损害未保护的半胱氨酸侧链。为了实现这些目标，我们将建立2-塞勒诺-2-苯基苯基和2-甲基-2-芳基-2-芳基甲基甲基体系作为新的辅助类别。辅助机构的需求很小。结果，NCL反应将通过5元过渡状态迅速进行。根本条件将用于选择性触发碎裂反应，该反应可提供靶蛋白，而不会损害偏远部位的半胱氨酸残基。在蛋白质总合成的现实情况下，我们将建立leu-thr-，gln-asn-，leu-his-和ile-met-ligations。值得注意的是，使用现有的本机化学连接方法无法访问这种连接连接。要评估新方法的有用性，我们将通过化学总合成来制备适配器蛋白P130CAS和NEDD9的SH3域。我们将通过具有已知SH3结构域配体的蛋白结合实验来检查合成蛋白的功能。位点的总合成，特异性磷酸化的P130CA和NEDD9蛋白将允许研究，其中我们探索了假定的磷酸开关，该开关通过改变涉及的SH3域的识别库来调节细胞信号转导。