Installation of Site-Specific Inorganic and Organic Phosphate to Peptides and Proteins via Vinylaryl Sulfonium Reagents

Abstract

Protein phosphorylation is a very important post-translational modification that regulates diverse cellular activities. In addition to classic monophosphorylation, there is also oligophosphorylation from pyrophosphorylation to polyphosphorylation. Moreover, organophosphates may modify residues such as via AMPylation and ADPylation. Although plenty of new types of protein phosphorylation have been revealed, molecular mechanisms of the biological functions are still challenging to study due to the lack of a good method to prepare proteins of interest with such site-specific PTMs. Here we report a facile method to install inorganic and organic phosphates on peptides and proteins. Vinylaryl sulfonium with an electron-withdrawing group was applied to cysteine alkylation and subsequent cyclization by γ-sulfur yielding episulfonium. This highly electrophilic intermediate was later attacked by a phosphate reagent to yield site-specifically phosphorylated cysteine peptides and proteins. As a result, this method does not require a special precursor residue on peptides/proteins or activation of phosphate reagents. In addition, this method is applicable to diverse inorganic phosphates and organophosphate. Therefore, we believe that this method will accelerate protein phosphorylation research by simple preparation of site-specific modified proteins. We also believe it offers a simple bioconjugation strategy via a phosphate linker.