Protein chemical synthesis is carried out by employing the ligation strategy, which has been developed by the use of peptide thioesters as building blocks. It is difficult to prepare the peptide thioester directly by the Fmoc solid phase peptide synthesis, because the thioester bond is decomposed during deprotection procedure using piperidine. Indirect methods, in which the thioester bond is constructed after peptide chain elongation, are being developed for preparing the peptide thioester. We have been focusing our efforts on an N to S acyl shift reaction to construct the thioester bond [1,2], and found that a peptide containing Cys-Pro ester (CPE) sequence at the C-terminus is spontaneously transformed into a peptide diketopiperazine (DKP) thioester under aqueous neutral buffer conditions [3,4]. In order to find a more reactive structure for the formation of peptide thioesters, we examined several different types of peptides, in which the proline residue of CPE moiety was replaced with N-substituted glycine derivatives (N-alkyl and benzyl substitutions). These peptides were transformed into peptide thioester more rapidly than the CPE peptide. In addition, when an amino acid residue was added at the C-teriminal of CPE structure, thus, Cys-Pro-OCH₂CO-Xaa-NH₂ (Xaa: amino acid residue), the conversion rate was faster than the original CPE peptide without an additional amino acid residue.