Genetically enabling phosphorus fluoride exchange click chemistry in proteins

Phosphorus fluoride exchange (PFEx), recently debuted in small molecules, represents the forefront of click chemistry. To explore PFEx’s potential in biological settings, we developed amino acids phosphoramidofluoridate tyrosine (PFY) and phosphoramidofluoridate lysine (PFK) featuring phosphoramidofluoridates and incorporated them into proteins through genetic code expansion. PFY/PFK selectively reacted with nearby His, Tyr, Lys, or Cys in proteins, both in vitro and in living cells, demonstrating that proximity enabled PFEx reactivity without external reagents. The reaction with His showed unique pH-dependent properties and created thermally sensitive linkages. Additionally, Na2SiO3 enhanced PFEx reactions with Tyr and Cys. PFEx, by generating defined covalent P-N/O linkages, extends the utility of phosphorus linkages in proteins, aligning with nature’s use of phosphate connectors in other biomolecules. More versatile and durable than sulfur fluoride exchange (SuFEx), PFEx in proteins expands the latent bioreactive arsenal for covalent protein engineering and will facilitate the broad application of this potent click chemistry in biological and biomedical fields.