Rsc_cc_c1cc13533c 1..3

Macrocyclic peptides and peptide-based structures have attracted significant interest as a source of chemical probes and therapeutic agents. While peptides and peptidomimetics in rigidified configurations can be prepared synthetically, genetic encoding offers the advantage to couple the creation of vast chemical libraries (10–10) with ultrahigh-throughput screeningmethods. Notable approaches involve the introduction of disulfide bridges within randomized peptide sequences or formation of cyclic peptides via split intein-mediated cyclization, but the range of building blocks available to assemble these structures remains inherently limited compared to synthetic methods. Alternatively, ribosomal peptides have been constrained through the use of cysteineor amine-reactive cross-linking agents but these methods rely on non-directional and non-bioorthogonal reactions which limits the choice of the cross-linking scaffolds and it may lead to multiple undesired products. To overcome these major limitations, we have undertaken efforts toward implementing general methods for chemoselectively embedding variable synthetic scaffolds within ribosomal peptides to generate macrocycles with a hybrid peptidic/non-peptidic backbone, referred to as Macrocyclic Organo-Peptide Hybrids or MOrPHs. Here, we report an efficient strategy for MOrPH synthesis which exploits a highly chemoselective, bioorthogonal, and catalyst-free tandem reaction between a trifunctional oxyamino/amino-thiol synthetic precursor (SP) and genetically encoded biosynthetic precursors (BPs) incorporating a keto group (Fig. 1A). Based on our recent investigations, we envisioned that a suitable biosynthetic precursor for MOrPH construction could be generated by framing a target peptide sequence (‘TS’) between the unnatural amino acid para-acetylphenylalanine (pAcF) and an intein (species ‘a’ in Fig. 1A). This protein would display two functional groups with orthogonal reactivity, namely the keto group of pAcF at the N-terminus of the target sequence and the reactive thioester bond transiently formed at its C-terminus via intein-catalyzed N,S-acyl transfer (species ‘b’). Macrocyclization could then be achieved via a synthetic precursor equipped with (i) an oxyamino group to form a stable oxime linkage with a pAcF side chain, and (ii) an amino-thiol moiety to coordinate an intein-mediated ligation and concomitant excision of the intein from the biosynthetic precursor. To test this approach, we prepared a first set of six biosynthetic precursors with target sequences spanning 4, 5, 6, 8, 10, or 12 amino acids (CBD4(pAcF) to CBD12(pAcF), Table S1 (ESIz)).