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

未受保护的肽硫酯和未受保护的半胱氨酰肽之间的天然化学连接（NCL）提供了以限定的翻译后修饰状态获得蛋白质的途径。然而，对半胱氨酸的要求限制了NCL反应的范围。连接-脱硫方法扩展了NCL化学的所有组成部分。不幸的是，制备巯基官能化的氨基酸结构单元所需的努力太高，限制了在任意连接位点的应用。此外，其缺点是远程半胱氨酸残基（其不参与NCL）需要保护。依赖于辅助基团的NCL方法仅需要单个构件，在理想情况下，其可以提供对任何连接位点的访问。然而，在这方面，现有的国家，在本研究项目中，我们将开发第一种普遍适用的方法，该方法能够在几乎任何给定的连接位点进行快速的天然化学连接反应，但在没有甘氨酸的情况下耐受未保护的半胱氨酸残基的存在。远程站点。我们将开发高性能连接助剂，其i）可以在固相肽合成的最后一步中在任意选择的氨基酸处引入; ii）即使在空间挑战性NCL反应中也提供高反应性，以及iii）允许在温和条件下无副产物的切割，iv）对未保护的半胱氨酸侧链没有损害。为了实现这些目标，我们将建立2-硒基-2-苯乙基-和2-巯基-2-芳基乙基-支架作为新的辅助类别。助剂的位阻需求很小。因此，NCL反应将通过5元过渡态快速进行。自由基条件将用于选择性地触发片段化反应，该片段化反应使靶蛋白变性而不损害远程位点的半胱氨酸残基。在蛋白质全合成的现实场景中，我们将建立Leu-Thr-、Gln-Asn-、Leu-His-和Ile-Met-连接。值得注意的是，这样的连接点将无法通过使用现有的天然化学连接方法来访问。为了评估新方法的有用性，我们将通过化学全合成来制备衔接蛋白p130 Cas和NEDD 9的SH 3结构域。我们将通过与已知SH 3结构域配体的蛋白结合实验来检查合成蛋白的功能。位点特异性磷酸化的p130 Cas和NEDD 9蛋白的全合成将允许研究，在该研究中，我们探索了一种假定的磷酸开关，其通过改变所涉及的SH 3结构域的识别库来精细调节细胞信号转导。

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.