A Synthetic Approach to a Peptide α‐Thioester from an Unprotected Peptide through Cleavage and Activation of a Specific Peptide Bond by N‐Acetylguanidine

Abstract

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...

An efficient solid‐phase synthesis of peptidyl‐N‐acetylguanidines for use in native chemical ligation

“…We also tested the same NCL using the alkylthiol sodium 2‐mercaptoethanesulfonate (MESNa) as a thiol additive. In our previous model NCL, MESNa unexpectedly gave higher conversion yield of a ligated product than that of MPAA . However, in the work reported here, NCL in the presence of 200 mM MESNa afforded only 9% conversion yield (Figure e), which was lower than the case of 20 mM MPAA condition (Figure d).…”

“…Uniquely, this peptide derivative can be converted into a corresponding peptide‐α‐thioester only by treatment with an excess amount of thiol in aqueous buffer at around neutral pH. We have tested model NCL between a 4‐residue peptidyl‐ N ‐acetylguanidine and a 4‐residue Cys‐peptide in a previous work . This preliminary experiment demonstrated the utility of a peptidyl‐ N ‐acetylguanidine as a peptide‐α‐thioester precursor.…”

“…Recently our group found a peptidyl‐ N ‐acetylguanidine as a new type of peptide‐α‐thioester precursor . Peptidyl‐ N ‐acetylguanidines have an amide bond between a peptide C ‐terminal carboxylic acid and an N ‐acetylguanidine (Figure ).…”

Site-Selective Peptide/Protein Cleavage

“…In 2012, Kajihara and Okamoto reported a method for cleavage of the N-terminus side peptide bond of a Cys residue and generation of a thioester (Fig. 17b) [114]. This method involved three steps: (1) selective thiocarbonylation of the thiol group of Cys by using O-phenyl chlorothionoformate [PhOC(S)Cl], (2) cyclization to generate thiazolidine-2-thione moiety, followed by amidolysis with N-acetylguanidine, and (3) thiolysis of the obtained peptidyl-Nacetylguanidine to give the peptide thioester.…”

Semisynthesis of a Post‐Translationally Modified Protein by Using Chemical Cleavage and Activation of an Expressed Fusion Polypeptide

“…We have already developed a method for the conversion of a unprotected peptide into a peptide‐α‐thioester (Figure 1 B). 6 This method, called the “guanidine method” hereafter, consists of four chemical‐reaction steps (Figure 1 B): 1) Boc protection of the side‐chain amino group of lysine (Lys) residues and an N‐terminal amino group, 2) S ‐thiocarbonylation of the internal Cys residue, 3) treatment with N ‐acetylguanidine to provide a peptidyl N ‐acetylguanidine derivative, and 4) trans ‐thioesterification to yield a peptide‐α‐thioester. By applying this method to the recombinant polypeptide, which is an unprotected polypeptide, we expected to obtain the peptide‐α‐thioester segment that corresponds to the N‐terminal sequence.…”