Fmoc-based peptide thioester synthesis with self-purifying effect: heading to native chemical ligation in parallel...

The chemical synthesis of proteins has facilitated functional studies of proteins due to the site-specific incorporation of post-translational modifications, labels, and non-proteinogenic amino acids. Moreover, native chemical ligation provides facile access to proteins by chemical means. However, the application of the native chemical ligation reaction in the synthesis of parallel formats such as protein arrays has been complicated because of the often cumbersome and time-consuming synthesis of the required peptide thioesters. An Fmoc-based peptide thioester synthesis with self-purification on the sulfonamide 'safety-catch' linker widens this bottleneck because HPLC purification can be avoided. The method is based on an on-resin cyclization-thiolysis reaction sequence. A macrocyclization via the N-terminus of the full-length peptide followed by a thiolytic C-terminal ring opening allows selective detachment of the truncation products and the full-length peptide. A brief overview of the chemical aspects of this method is provided including the optimization steps and the automation process. Furthermore, the application of the cyclization-thiolysis approach combined with the native chemical ligation reaction in the parallel synthesis of a library of 16 SH3-domain variants of SHO1 in yeast is described, demonstrating the value of this new technique for the chemical synthesis of protein arrays.