Phosphotyrosineresiduesare essential functional switches in health and disease. Thus, phosphotyrosine biomimeticsare crucial for the development of chemical tools and drug molecules. We report here the discovery and investigation of pentafluorophosphatoamino acids as novel phosphotyrosine biomimetics. A mild acidic pentafluorination protocol was developed and two PF5-amino acids were prepared and employed in peptide synthesis.Their structures, reactivities, and fluorine-specific interactions were studied by NMR and IR spectroscopy, X-ray diffraction, and in bioactivity assays. The mono-anionic PF5 motif displayed anamphiphilic character binding to hydrophobic surfaces, to water molecules, and to protein-bindingsites, exploiting charge and H-F-bonding interactions.The novel motifs bind 25- to 30-fold stronger to the phosphotyrosine binding site of the protein tyrosine phosphatase PTP1B than the best current biomimetics,as rationalized by computational methods, including molecular dynamics simulations.