Pentafluorophosphato‐Phenylalanines: Amphiphilic Phosphotyrosine Mimetics Displaying Fluorine‐Specific Protein Interactions

Abstract

Phosphotyrosine residues are essential functional switches in health and disease. Thus, phosphotyrosine biomimetics are crucial for the development of chemical tools and drug molecules. We report here the discovery and investigation of pentafluorophosphato amino acids as novel phosphotyrosine biomimetics. A mild acidic pentafluorination protocol was developed and two PF 5 -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 PF 5 motif displayed an amphiphilic character binding to hydrophobic surfaces, to water molecules, and to protein-binding sites, exploiting charge and HÀ 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.

A Formylglycine‐Peptide for the Site‐Directed Identification of Phosphotyrosine‐Mimetic Fragments**

“…95 nucleophilic amine or hydrazine fragments (<250 Da) were selected and tested as inhibitors of PTP1B alone and in combination with peptide 3 (Supporting Information Figures S23–28). Fragments were pre‐selected for library composition based upon representation of potential phosphate‐mimetic substructures including carboxylic acids, sulfonic acids, sulfonamides, and fluorine‐rich functional groups, which were to be investigated for fluorine‐specific interactions with phosphotyrosine binding sites [22,23] . Those fragments showing a significant enhancement of inhibition in the combination experiment were considered primary hits and tested in the same fragment ligation experiment with SHP2 (Figure 4).…”

“…Chemistry-A European Journal (22): Following the procedure for compound 21, using N-(4aminophenyl)-1,1,1-trifluoromethanesulfonamide (82 mg, 0.34 mmol, 1.1 equiv) and 2,5-bis(4-nitrophenyl)-2,4-dihydro-3Hpyrazol-3-one (100 mg, 0.31 mmol, 1 equiv), 22 (Z)-2-(4-(2-(1,3-Bis-(4-nitrophenyl)-5-oxo-1,5-dihydro-4H-pyrazol-4-ylidene)-hydrazinyl)-phenoxy)-acetic acid (23): Following the procedure for compound 21, using 2-(4-aminophenoxy)-acetic acid (28 mg, 0.17 mmol, 1.1 equiv) and 2,5-bis(4-nitrophenyl)-2,4-dihydro-3H-pyrazol-3-one (50 mg, 0.15 mmol, 1 equiv), 23 Protein Expression and purification: DNA encoding the catalytic domain of human PTP1B (amino acids 1-321) was subcloned into the pQLinkH vector. The gene encoding the N-terminal His 7 -tagged protein was over-expressed at 17 °C in E. coli Rosetta (DE3).…”

Fluorinated Protein–Ligand Complexes: A Computational Perspective

“…For example, fluorine can replace oxygen in a phosphate group, generating fluorinated analogues of phosphate groups. Accorsi et al 75 used this strategy to improve the phosphotyrosine mimetic 4-phosphono(difluoromethyl)phenylalanine, which inhibits the tyrosine phosphatase PTP1B ( Figure 6 ). The phosphate group in the original inhibitor is replaced by a pentafluorophosphato group (PF5), which improves the binding affinity from K I = 1555 μM to K I = 61 μM, where K I is the inhibition constant measured in an enzyme inhibition assays.…”

“…(b) Phosphotyrosine mimetic amino acid with the PF5 headgroup in the main binding pocket of PTP1B. Adapted from ref ( 75 ). Available under a CC BY-NC license.…”

A Formylglycine-Peptide for the Site-Directed Identification of Phosphotyrosine-Mimetic Fragments

“…Fragments were pre-selected for library composition based upon representation of potential phosphate-mimetic substructures including carboxylic acids, sulfonic acids, sulfona- Chemistry-A European Journal mides, and fluorine-rich functional groups, which were to be investigated for fluorine-specific interactions with phosphotyrosine binding sites. [22,23] Those fragments showing a significant enhancement of inhibition in the combination experiment were considered primary hits and tested in the same fragment ligation experiment with SHP2 (Figure 4).…”