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

• Applicant: Professor Dr. Oliver Seitz
• Subject Area: Biological and Biomimetic Chemistry
• Term: from 2017 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 367109134

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.

• DFG Programme: Research Grants