Monodisperse Chemical Oligophosphorylation of Peptides via Protected Oligophosphorimidazolide Reagents

Abstract

Protein poly‐ and oligophosphorylation are recently discovered post‐translational modifications that remain poorly characterized due to (1) the difficulty of extracting endogenously polyphosphorylated species without degradation and (2) the absence of synthetic and analytical tools to prepare and characterize poly‐ and oligophosphorylated species in biochemical contexts. Herein, we report a methodology for the selective oligophosphorylation of peptides with monodisperse phosphate chain lengths (Pn = 3–6). A library of oligophosphorimidazolide (oligoP‐imidazolide) reagents featuring benzyl and o‐nitrophenylethyl protecting groups were synthesized in moderate‐to‐good yields (65–93%). These oligoP‐imidazolide reagents enabled the selective and simultaneous conjugation of multiple phosphate units to phosphoryl nucleophiles, circumventing tedious iterative processes. The generalizability of this approach is illustrated by a substrate scope study that includes several biologically relevant phosphopeptide sequences, culminating in the synthesis of >60 examples of peptide oligophosphates (Pn = 2–6). Moreover, we report the preparation of oligoP‐diimidazolides (Pn = 3–5) and discuss their application in generating unique condensed phosphate‐peptide conjugates. We also demonstrate that human phospho‐ubiquitin (pS65‐Ub) is amenable to functionalization by our reagents. Overall, we envision the methods described here will enable future studies that characterize these newly discovered but poorly understood phosphorylation modes.