Due to the complex high-order structures and interactions of proteins within aqueous solution, majority of chemical functionalizations happen on the hydrophilic sites of protein external surfaces which are naturally exposed to the solution. However, hydrophobic pockets inside proteins are crucial for the ligand binding and function as catalytical centers and transporting tunnels. Herein, we describe a reagent pre-organization and in-situ photochemical trifluoromethylation strategy to profile the functional sites inside hydrophobic pockets of native proteins. Unbiased mass spectrometry profiling was applied for the characterization of trifluoromethylated sites with high sensitivity. Native proteins including myoglobin, trypsin, haloalkane dehalogenase, and human serum albumin have been engaged in this mild photochemical process and substantial hydrophobic site-specific and structure-selective trifluoromethylation substitutes are obtained without significant interference to their bioactivity and structures. Sodium triflinate is the only reagent required to functionalize the unprotected proteins with wide pH-range tolerance and high biocompatibility. This “in-pocket” activation model provides a general strategy to modify the potential binding pockets and gain essential structural insights of functional hotspots inside protein hydrophobic pockets.

This article is Open Access

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