Fig. 1. Design of PABK as a multifunctional UAA for versatile protein manipulations. (A) An evolved PylRS mutant can recognize and site-specifically incorporate PABK into the growing polypeptide chain with its cognitive tRNA in response to an amber codon (UAG). The p-azidobenzyl group on PABK possesses a panel of unique properties listed on the right (1–4), while coupling with a carbamate linker further renders it susceptible to the bioorthogonal cleavage reaction (5). The background shape of PylRS is drawn according to its crystal structure (PDB 2ZIN). (B) The mechanism of the protein decaging strategy based on the TCO and p-azidobenzyl pair: (I) strain-promoted 1,3-dipolar cycloaddition; (II) rearrangement and N2 release; (III) hydrolysis and (IV) 1,6-elimination. The caged primary amine can be restored by the TCO activator.
