Furthermore, cyclization efficiency is largely affected by the composition of the target peptide sequence. (20-22) A limitation of this method however is the accessibility of a single type of cyclic peptide topology (i.e., N-to- C-end cyclic peptide). Enabling the synthesis of cyclic peptides in living cells, this approach has provided a powerful tool for the discovery of cyclopeptide inhibitors against a variety of protein targets upon integration with an intracellular selection or reporter system. (8, 9) As a notable exception, there is the split intein-mediated circular ligation method (SICLOPPS) introduced by Benkovic and co-workers, (19) in which head-to-tail cyclic peptides are generated via circularization of an internal peptide sequence upon a trans splicing reaction involving flanking domains from the natural split intein DnaE ( Supplementary Figure S1). Despite significant contributions in this area, (10-18) these approaches have yet remained largely limited to the production of macrocyclic peptides in vitro. (8, 9) Particularly attractive features of the latter are their high combinatorial potential and the possibility to interface the resulting libraries of constrained peptides with powerful display platforms. (3-7) Following an alternative approach, other groups, including ours, have focused on implementing strategies to enable the macrocyclization of ribosomally derived polypeptides of arbitrary sequence. (1, 2) To this end, one approach has involved the generation and manipulation of natural cyclopeptide scaffolds via the reconstruction and engineering of their respective biosynthetic pathways. Methods for generating macrocyclic peptides, and combinatorial libraries thereof, have attracted considerable interest owing to the peculiar conformational and molecular recognition properties of this structural class and their promise toward addressing challenging drug targets.

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