A Combinatorial Approach for the Synthesis of Multi-Phosphorylated Peptides: New tool for Studying Phosphorylation Patterns in Proteins

Phosphorylation of proteins at multiple sites creates different phosphorylation patterns that are essential for their biological activity. For example, such patterns contribute to the redirection of signalling to alternative pathways. Synthetic multi-phosphorylated peptides are excellent tools to systematically study the impact of unique phosphorylation patterns on signalling, but their synthesis is known to be extremely difficult. Here we present an efficient and general method for the synthesis of multi- phosphorylated peptides, using a combination of different tailor-made coupling conditions. The method was demonstrated for the synthesis of a library of Rhodopsin C terminal peptides with distinct phosphorylation patterns containing up to seven phosphorylated Ser (pSer) and Thr (pThr) residues in close proximity to one another. Our method can be used to synthesize peptides incorporating multiple phosphorylated amino acids at high efficiency using standard peptide laboratory techniques. This approach opens the way for quantitative mechanistic studies of phosphorylation patterns and their biological roles.