Genetically Enabling Phosphorus Fluoride Exchange Click Chemistry in Proteins

Area of Science:

• Biochemistry
• Organic Chemistry
• Chemical Biology

Background:

• Click chemistry is crucial for bioconjugation and protein engineering.
• Phosphorus Fluoride Exchange (PFEx) is a novel click chemistry reaction.
• Adapting PFEx for biological systems requires specific reagents and methods.

Purpose of the Study:

• To explore the potential of PFEx in biological settings.
• To develop and incorporate novel amino acids for PFEx into proteins.
• To demonstrate PFEx reactivity within proteins in vitro and in living cells.

Main Methods:

• Genetic code expansion was used to incorporate PFY and PFK amino acids into proteins.
• PFY/PFK amino acids feature phosphoramidofluoridates for PFEx.
• Reactions were studied in vitro and in living cells, with pH and additive effects investigated.

Main Results:

• PFY/PFK selectively reacted with His, Tyr, Lys, or Cys residues in proteins.
• Proximity-driven PFEx occurred without external reagents in vitro and in cells.
• pH-dependent reactions with His and enhanced reactions with Tyr/Cys using Na2SiO3 were observed.
• Thermally sensitive linkages were formed with His.

Conclusions:

• PFEx is a versatile click chemistry applicable to proteins, forming covalent P-N/O linkages.
• This expands phosphorus-based linkages in proteins, mimicking natural phosphate connectors.
• PFEx offers advantages over SuFEx for protein engineering, broadening its use in biological and biomedical fields.