Active disulfide formed by 3-nitro-2-pyridinesulfenylation in the cysteine residue is an attractive reaction species to arise a selective disulfide exchange reaction, leading to the rapid disulfide bond formation when an unprotected thiol group coexists [1]. Taking advantage of this unique property of the 3-nitro-2-pyridinesulfenyl (Npys) group, we have recently reported a new synthetic methodology so-called "disulfide-driven cyclic peptide synthesis" [2]. Our strategy consists of two parts; 1) one-pot conjugation of two cysteine-containing peptide fragments by the Npys-mediated two-step solid-phase disulfide ligation (SPDSL) [2,3] and 2) its selective intramolecular cyclization through amide bond formation. To demonstrate this idea, we have synthesized oxytocin as a model toward more complex cyclic peptides [2].
In present study, our disulfide-driven method was applied to the synthesis of human endothelin-2, a 21-mer cyclic peptide with two disulfide bridges. As a synthetic strategy, the sequence of endothelin was divided into three fragments A, B and C from N-terminus, and central fragment B with a Cys(tBu) was firstly loaded onto Npys-Cl attached resin 1 as an active disulfide. Then, after this fragment B loaded resin 2 was washing with water, fragment A with both a Cys(tBu) and an unprotected Cys was mixed to the resin 2 in DMF to construct a disulfide peptide A-S-S-B (3). The resultant crude disulfide peptide 3 was cyclized via intramolecular amide bond formation to afford the cyclic peptide fragment c(A-B) (4). After HPLC purification, fragment 4 was again loaded onto the fresh resin 1 via Cys(tBu) originally derived from fragment A, captured by C-terminal fragment C with an unprotected Cys and cyclized by amide bond formation in a similar manner mentioned above. Final deprotection of the resultant fully constructed peptide 5 efficiently afforded human endothelin-2. In this presentation, the synthesis will be discussed in details.