Genetic Encoding of a Trifunctional Photo‐Cross‐Linker with a Cleavable Alkyl Ester Moiety

Genetically encoded photo‐cross‐linkable amino acids (PAAs) are powerful tools for analyzing direct protein–protein interactions (PPIs) in mammalian cells. Cleavable PAAs are particularly useful, enabling covalent capture and subsequent release of interacting partners, which facilitates the characterization of interaction interfaces using mass spectrometry. However, the limited options for cleavable linker structures have restricted the design of PAAs. In this study, we genetically encoded a novel trifunctional PAA, DiZAAsu, which contains three distinct chemical groups: diazirine, alkyne, and alkaline‐cleavable alkyl ester moieties. An archaeal pyrrolysyl‐tRNA synthetase was engineered to incorporate DiZAAsu efficiently into proteins in mammalian cells. We demonstrated the in‐cell photoreactive function of diazirine by cross‐linking the DiZAAsu‐introduced GRB2 protein to its binding partner, SHC. Using the alkyne group for biotinylation, we established a tandem affinity purification strategy that enabled efficient enrichment of the cross–linked complex, thereby reducing nonspecific protein contamination. The alkaline‐based cleavage of the ester group in DiZAAsu was also demonstrated, confirming its potential for the dissociation of covalently linked complexes. This system thus expands the design space of multifunctional PAAs and adds alkaline‐based dissociation to the limited repertoire of available cleavage strategies.