Discovery and Application of a Lysine 5-Hydroxylase for Bioorthogonal Chemistry

The selective functionalization of unactivated C(sp3)–H bonds remains an ongoing challenge in synthetic organic chemistry. In this context, biocatalysis provides an attractive strategy to perform such chemistry under mild reaction conditions. We now report the discovery of K5H, the first enzyme that catalyzes the one-step conversion of free L-lysine into enantiopure (2S, 5R)-5-hydroxylysine (5-Hyl), producing a β-amino alcohol motif on the lysine side-chain. As chiral β-amino alcohols are versatile synthetic motifs found in natural products, pharmaceuticals, ligands, and other complex molecules, we demonstrate that this noncanonical amino acid can be incorporated into several pharmaceutically-relevant peptides in place of lysine through a tandem one-pot biocatalytic cascade using in vitro transcription/translation. Indeed, we show that the introduction of a single hydroxyl group adjacent to the ε-amine on lysine serves as a selective handle for downstream bioorthogonal chemistry such as heterocyclization, ligation to various payloads, and formation of branched peptides. Taken together, the discovery and characterization of K5H provides a modular genetically-encoded platform to tune the structure and properties of diverse bioactive peptides via biocatalytic transformations of unactivated C(sp3)–H bonds.