In modern procedures for total chemical protein synthesis, the concept of chemical ligation plays an essential role in the assembly of target protein polypeptide chains.[1] The peptide a-thioester is the key component for chemical ligation such as native chemical ligation (NCL), direct segment coupling methods, or traceless Staudinger ligation.[2] Thus, substantial effort has been expended on the establishment of peptide-athioester synthesis based on conventional Boc or Fmoc solidphase peptide synthesis (SPPS, Boc = tert-butoxycarbonyl, Fmoc = 9-fluorenylmethyloxycarbonyl).[3] However, because of the inherent limitations of SPPS, synthesis of peptide athioesters with more than 50 amino acids (aa) is still challenging. For the preparation of such a polypeptide athioester, an expression method using the intein system has come to be recognized as a robust technology with the capacity to provide polypeptide a-thioesters of more than 50 aa.[4] Inspired by the biological system, we explored an inteinlike chemical methodology for preparing a long peptide athioester by using a native (unprotected) peptide as the starting material. Some groups recently reported thioesterification of E. coli-expressed peptides using acid treatment, but these intriguing methods lead to epimerization of the Cterminal amino acid residue and still have sequence limitations.[5] Thus we set out to find a widely usable new methodology.The key point was the manipulation of an unprotected peptide to install a C-terminal a-thioester. This task appears to require the selective activation of a native amide bond and subsequent thiolysis. Recently, several groups reported elegant methods of activating the peptide backbone to install an a-thioester at the peptide C terminus.[6] In these methods, the activation of the peptide bond was performed by a selective acylation strategy of an Na-amide nitrogen atom at a specific amino acid residue. To examine the thioesterification of unprotected peptides, we focused on the cysteine (Cys) residue, because Cys possesses a thiol group, which might be more easily modified than the other amino acid side chains and thus selectively induce N-acylation. In fact, selective peptide-cleavage methods at the Cys residue employing an Nacylation strategy have been reported for protein sequencing (Scheme 1 a). [7] In these methods, N-acylation is induced by an electrophilic auxiliary group introduced on a Cys side chain (e.g. S-carbonyl group), and this auxiliary group provides the activation of the peptide bond and subsequent hydrolysis. These reports encouraged us to hypothesize the feasibility of the selective activation of a native amide bond at the Cys residue and subsequent thiolysis, instead of hydrolysis, to obtain a peptide a-thioester. However, our preliminary examination of thiolysis for the activated Cys residue did not proceed and regenerated unprotected peptide substrate or hydrolyzed product instead of the desired peptide athioester (Scheme 1 a). Therefore, we have explored a new nucleophile as an alternative to a...

In modern procedures for total chemical protein synthesis, the concept of chemical ligation plays an essential role in the assembly of target protein polypeptide chains. [1] The peptide a-thioester is the key component for chemical ligation such as native chemical ligation (NCL), direct segment coupling methods, or traceless Staudinger ligation. [2] Thus, substantial effort has been expended on the establishment of peptide-athioester synthesis based on conventional Boc or Fmoc solidphase peptide synthesis (SPPS, Boc = tert-butoxycarbonyl, Fmoc = 9-fluorenylmethyloxycarbonyl). [3] However, because of the inherent limitations of SPPS, synthesis of peptide athioesters with more than 50 amino acids (aa) is still challenging. For the preparation of such a polypeptide athioester, an expression method using the intein system has come to be recognized as a robust technology with the capacity to provide polypeptide a-thioesters of more than 50 aa. [4] Inspired by the biological system, we explored an inteinlike chemical methodology for preparing a long peptide athioester by using a native (unprotected) peptide as the starting material. Some groups recently reported thioesterification of E. coli-expressed peptides using acid treatment, but these intriguing methods lead to epimerization of the Cterminal amino acid residue and still have sequence limitations. [5] Thus we set out to find a widely usable new methodology.The key point was the manipulation of an unprotected peptide to install a C-terminal a-thioester. This task appears to require the selective activation of a native amide bond and subsequent thiolysis. Recently, several groups reported elegant methods of activating the peptide backbone to install an a-thioester at the peptide C terminus. [6] In these methods, the activation of the peptide bond was performed by a selective acylation strategy of an Na-amide nitrogen atom at a specific amino acid residue. To examine the thioesterification of unprotected peptides, we focused on the cysteine (Cys) residue, because Cys possesses a thiol group, which might be more easily modified than the other amino acid side chains and thus selectively induce N-acylation. In fact, selective peptide-cleavage methods at the Cys residue employing an N-acylation strategy have been reported for protein sequencing (Scheme 1 a). [7] In these methods, N-acylation is induced by an electrophilic auxiliary group introduced on a Cys side chain (e.g. S-carbonyl group), and this auxiliary group provides the activation of the peptide bond and subsequent hydrolysis. These reports encouraged us to hypothesize the feasibility of the selective activation of a native amide bond at the Cys residue and subsequent thiolysis, instead of hydrolysis, to obtain a peptide a-thioester. However, our preliminary examination of thiolysis for the activated Cys residue did not proceed and regenerated unprotected peptide substrate or hydrolyzed product instead of the desired peptide athioester (Scheme 1 a). Therefore, we have explored a new nucleophile as an alternat...