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

无保护肽硫酯和无保护半胱氨酸肽之间的天然化学连接（NCL）提供了进入特定翻译后修饰状态的蛋白质的途径。然而，对半胱氨酸的需求限制了NCL反应的范围。结扎-脱硫法扩展了NCL化学的范围。不幸的是，制备巯基功能化氨基酸构建块所需的努力太高，限制了在任意连接位点的应用。此外，它的缺点是远端半胱氨酸残基（不涉及NCL）需要保护。依赖辅助组的NCL方法只需要一个构建块，在理想情况下，它可以提供任何连接部位的访问。然而，现有的最先进的助剂受到空间需求的限制，这使得在没有甘氨酸的情况下无法成功结联，或者在辅助去除过程中在无保护的半胱氨酸残基处发生副反应。在这个研究项目中，我们将开发第一种普遍适用的方法，使快速的天然化学连接反应在几乎任何给定的连接位点，但在遥远的位置耐受无保护的半胱氨酸残基的存在。我们将开发高性能的连接助剂，i)可以在固相肽合成的最后一步任意选择的氨基酸上引入；ii)即使在具有空间挑战性的NCL反应中也能提供高反应活性；iii)在温和条件下允许无副产物裂解；iv)不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-硒-2-苯基-和2-巯基-2-芳基-支架作为新的辅助类别。助剂几乎没有空间需求。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碎片反应，该反应提供目标蛋白而不损害远程位置的半胱氨酸残基。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，使用现有的天然化学连接方法无法获得这种连接连接。为了评估新方法的有效性，我们将通过化学全合成制备适配器蛋白p130Cas和NEDD9的SH3结构域。我们将通过与已知SH3结构域配体的蛋白质结合实验来检查合成蛋白质的功能。位点特异性磷酸化p130Cas和NEDD9蛋白的完全合成将允许研究，我们将探索一种假定的磷酸化开关，该开关通过改变相关SH3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.