Enzymatic Peptide and Protein Bromination: The BromoTrp Tag

Bio-orthogonal reactions for modification of proteins and unprotected peptides are of high value in chemical biology. The combination of enzymatic halogenation with transition metal-catalyzed cross-coupling provides a feasible approach for the modification of proteins and unprotected peptides. By a semirational protein engineering approach, variants of the tryptophan 6-halogenase Thal were identified that enable efficient bromination of peptides with a C-terminal tryptophan residue. The substrate scope was explored using di-, tri-, and tetrapeptide arrays, leading to the identification of an optimized peptide tag we named BromoTrp tag. This tag was introduced into three model proteins. Preparative scale post-translational bromination was possible with only a single cultivation and purification step using the brominating E. coli coexpression system Brocoli. Palladium-catalyzed Suzuki-Miyaura cross-coupling of the bromoarene was achieved with Pd nanoparticle catalysts at 37 degrees C, highlighting the rich potential of this strategy for bio-orthogonal functionalization and conjugation. An engineered variant of the Trp 6-halogenase Thal allows post-translational bromination of a tetrapeptide tag with C-terminal tryptophan in proteins on a preparative scale using the brominating E. coli coexpression strain Brocoli. The bromoarene can be derivatized by palladium-catalyzed Suzuki-Miyaura cross-coupling at 37 degrees C. This opens new avenues for bio-orthogonal modification of proteins by biocatalytic bromination of minimally tagged proteins.+image