Figure 1.

Design and synthesis of linaclotide and its analogues. (A) Linaclotide is a hybrid design of bacterial heat-stable enterotoxin (STa) and endogenous peptide hormones guanylin and uroguanylin. (B) Sequence of linaclotide and its analogues. Modifications are highlighted in blue; c/- backbone cyclization; y: d-Tyr; v: d-Val; U: selenocysteine (C) Peptides were obtained by Fmoc-SPPS followed by oxidative folding. (D) Fresh 2-chlorotrityl hydrazine resin was prepared to synthesize the peptides via Fmoc-SPPS followed by one-pot cyclization via intramolecular hydrazide-based native chemical ligation (NCL) and oxidative folding. The dashed line indicates the ligation site. (E) [Sec^1,6; d-Tyr¹⁴]-Linaclotide was synthesized via Boc-SPPS using Boc-Sec(Meb)-OH to introduce the two selenocysteine residues followed by HF cleavage and oxidative folding.