Genetic Encoding of Pentafluorophosphato‐Phenylalanine Provides PF ₅ ‐Proteins as Phosphoprotein Mimetics

Protein phosphorylation is one of the most important posttranslational modifications altering the structure, stability, and activity of more than 13 000 human proteins. In this work, the phosphotyrosine mimetic pentafluorophosphato-difluoromethyl-phenylalanine (PF₅CF₂Phe) was genetically encoded and incorporated into three different proteins. Screening two libraries of orthogonal aminoacyl-tRNA synthetases identified enzymes enabling the efficient and specific incorporation of PF₅CF₂Phe into red fluorescent protein (RFP) via amber stop codon suppression. Two model proteins, human ubiquitin (Ubq) and the B1 immunoglobulin-binding domain of streptococcal protein G (GB1), were prepared with PF₅CF₂Phe mutations and investigated for potential interaction partners. While native GB1 showed no binding to protein tyrosine phosphatases (PTP), PF₅-GB1, with PF₅CF₂Phe at position 17, was a strong inhibitor of the phosphatases PTP1B and SHP2. PF₅-Ubq was produced and converted into the first example of a protein carrying the most prominent phosphotyrosine mimetic, phosphono-difluoromethyl phenylalanine (PO₃CF₂Phe). With increasing need in the biosciences to delineate the functions of complex phosphorylation patterns, genetic encoding of PF₅CF₂Phe yielding phosphoprotein mimetics opens unique opportunities for precise functional studies where site-specific and homogeneous protein modifications are required.