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 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...

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

“…As a result, this synthetic protocol successfully afforded [GIP1‐27]‐NAcGu in 13% isolated yield from the 20 µmol resin used (Figure (C) and (D)). As mentioned in our previous work , peptidyl‐ N ‐acetylguanidines are slightly labile under 0.1% TFA solution as the common eluent for RP‐HPLC purification; therefore, it is essential to keep the fractions containing the desired material in an ice bath during HPLC purification until lyophilization to avoid their decomposition.…”

“…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.…”

“…In our previous study, peptidyl‐ N ‐acetylguanidines were synthesized from fully unprotected peptides through solution‐phase reactions (Figure (A)). We found that S ‐thiocarbonylation of a Cys side‐chain followed by treatment with N ‐acetylguanidine selectively activated and cleaved a native peptide bond at the Cys residue (Figure A) . Intriguingly, the activation of the peptide bond by the side‐chain S ‐thiocarbonyl group and a nucleophilic attack of an N ‐acetylguanidine seem to occur as a concerted reaction; consequently, this reaction affords the peptidyl‐ N ‐acetylguanidines from the unprotected peptides.…”

“…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 ).…”

Design and synthesis of cysteine-rich peptides

“…Various methods employing similar strategy have been developed to prepare peptide thioesters containing up to 35 amino acids. [220][221][222][223][224][225] (Figure 1.14) Since the C-terminus of the peptide thioester is highly activated during the conversion process, the steric hindrance of C-terminal residue is not a problem in these methods. Various peptide thioesters with C-terminal Phe, Tyr, Val or Pro can be synthesized by safety catch methods.…”