A two-fold symmetric linchpin (TSL) converts readily available phage-displayed disulfide peptide libraries to proteolytically stable bicyclic peptides. The bicyclic phage library was screened to discover an antagonist of NODAL morphogen.

Diversity‐oriented approach to large artificial macrocycle libraries with a ring size of 13—18 atoms relying on the “build‐couple‐pair” strategy is disclosed. The “couple” phase included three one‐pot steps including consequent amide coupling of N‐Boc‐monoprotected vicinal diamines with two alkenyl carboxylic acids, followed by ring‐closing metathesis as the key “pair” step. The scope and limitations of the method were established for all three reagents. In particular, various acyclic, mono‐ and bicyclic aliphatic diamine derivatives with the N−C−C−N dihedral angle less than ca. 130° appeared to be suitable substrates. The proposed approach was used to construct a virtual library of 1.8 ⋅ 105 macrocycles derived from 12,283 different scaffolds. More than 40 % of members of this library contained a protected amino function and hence can be suitable for the post‐pairing modification, thus giving rise to at least a billion‐size chemical space based on the REAL‐type synthetic methodology. Validation of the approach under parallel synthesis conditions on a 383‐member subset showed a 61 % success rate over the whole 4–5‐step reaction sequence. Finally, the synthetic approach also worked on a gram scale (up to 8.0 g).

In this manuscript, we developed a Two-fold Symmetric Linchpin (<b>TSL</b>) that converts readily available phage display peptides libraries made of 20 common amino acids to genetically-encoded libraries of bicyclic peptides displayed on phage. <b>TSL</b> combines an aldehyde-reactive group and two thiol-reactive groups; it bridges two side chains of cysteine [C] with an N-terminal aldehyde group derived from the N-terminal serine [S], yielding a novel bicyclic topology that lacks a free N-terminus. Phage display libraries of SX₁CX₂X₃X₄X₅X₆X₇C sequences, where X<i>_i</i> is any amino acids but Cys, were converted to a library of bicyclic <b>TSL</b>-[<u>S</u>]X₁<u>[C]</u>X₂X₃X₄X₅X₆X₇[<u>C]</u> peptides in 45 ± 15% yield. Using this library and protein morphogen NODAL as a target, we discovered bicyclic macrocycles that specifically antagonize NODAL-induced signaling in cancer cells. At a 10 µM concentration, two discovered bicyclic peptides completely suppressed NODAL-induced phosphorylation of SMAD2 in P19 embryonic carcinoma. The <b>TSL</b>-[<u>S</u>]Y<u>[C]</u>KRAHKN[<u>C]</u> bicycle inhibited NODAL-induced proliferation of NODAL-Tky-nu ovarian carcinoma cells with apparent IC50 1 µM. The same bicycle at 10 µM concentration did not affect the growth of the control Tky-nu cells. <b>TSL</b>-bicycles remained stable over the course of the 72 hour-long assays in a serum-rich cell-culture medium. We further observed general stability in mouse serum and in a mixture of proteases (Pronase^TM) for 33 diverse bicyclic macrocycles of different ring sizes, amino acid sequences, and cross-linker geometries. <b>TSL</b>-constrained peptides expand the previously reported repertoire of phage display bicyclic architectures formed by cross-linking Cys side chains. We anticipate that it will aid the discovery of proteolytically stable bicyclic inhibitors for a variety of protein targets.