Lan enzyme-free construction of lanthionine-bridged macrocyclic phage libraries.

Phage display is a powerful technology for discovering macrocyclic peptide ligands. Many phage display-revealed peptide inhibitors comprise disulfide crosslinks, which unfortunately exhibit vulnerability to reduction and proteolysis. Lanthipeptides are a family of ribosomally synthesized peptides that harbor thioether cyclization instead of disulfides. Lanthipeptide libraries have been constructed on phage; however, only with the use of the Lan enzyme that demands specific recognition sequences. Here, we present an enzyme-free strategy for constructing lanthionine-bridged macrocyclic peptide libraries on M13 phage. Our strategy involves selective and reversible masking of the N-terminal cysteine (NCys), followed by Cys-to-Dha conversion and subsequent cyclization upon NCys deprotection. We have specifically optimized the chemistry for each step to allow easy preparation of lanthipeptide libraries. The utility of such libraries is demonstrated by panning against Keap1 as a model protein. To the best of our knowledge, this is the first demonstration of a Lan enzyme-free construction of lanthipeptide libraries on phage, which presents a significant addition to the increasing collection of cyclization chemistries that expand the chemical space of phage display.