On-Resin P(V) Polyphosphorylation of Serine, Threonine, and Tyrosine

Abstract Protein phosphorylation is the most abundant post-translational modification (PTM) and plays a central role in regulating biological processes. Attaining discretely polyphosphorylated peptides is an open challenge. Their chemical synthesis relies on coupling of phosphorylated amino acid building blocks, which suffer from base-mediated elimination and incomplete couplings, or postsynthesis functionalization via a global phosphoramidite approach, which is prone to side reactions during oxidation. Herein, we developed an on-resin phosphorylation that utilizes a protected and activated phosphorus(V) species. This method gave ≥95% conversion for monophosphorylation of serine, threonine, and tyrosine and tolerated oxidation-sensitive methionine, cysteine, and tryptophan residues. Up to five phosphorylations were installed site-selectively, with superior performance to building block approaches, and it remained effective on sequences up to 83 residues. With an efficient method for site-selective polyphosphorylation established, the specificity of antibodies directed against phosphorylated variants of the oncoprotein MYC was evaluated. These antibodies are often used for evaluating the role of phosphorylation sites (phosphosites) in regulating MYC in cancer. Although all antibodies maintained selectivity for their specific phosphosite, binding varied upon additional phosphorylation sites around the epitope, underscoring the need for careful validation of antibodies to prevent misinterpretation. Overall, this strategy enables efficient access to complex, defined polyphosphorylated peptides for future investigations into the biological roles of phosphorylation sites on MYC, and provides a broadly applicable method for the synthesis of precisely (poly)phosphorylated peptides and proteins.