One-pot chemical pyro- and tri-phosphorylation of peptides by using diamidophosphate in water

Abstract

Protein (pyro)phosphorylation is emerging as a post-translational modification (PTM) in signalling pathways involved in many cellular processes. However, access to synthetic pyrophosphopeptides that can serve as tools for understanding protein pyrophosphorylation is quite limited. Herein, we report a chemical phosphorylation method that enables the synthesis of pyrophosphopeptides in aqueous medium without the need for protecting groups. The strategy employs diamidophosphate (DAP) in a one-pot sequential phosphorylation-hydrolysis of mono-phosphorylated peptide precursors. This operationally simple method exploits the intrinsic nucleophilicity of a phosphate moiety installed on serine, threonine or tyrosine residues in complex peptides with excellent chemoselectivity and good yields under mild conditions. We demonstrate the installation of the pyrophosphate group within a wide range of model peptides and showcase the potential of this methodology by selectively pyrophosphorylating the highly functionalized Nopp140 peptide fragment. The potential to produce higher (poly)phosphorylated peptides was demonstrated as a proof-of-principle experiment where we synthesized the triphosphorylated peptides using this one-pot strategy.