Peptide thioesters are used as key building blocks for contemporary protein synthesis, based on the ligation strategy such as thioester method and native chemical ligation. We found the methods for preparation of the peptide thioesters, in which the key reaction is an intramolecular N-S acyl shift reaction of thiol-containing peptides. These processes can be applied to Fmoc based solid phase peptide synthesis with minimum racemization at the thioester position. For the N-S acyl shift reaction, we use an N-2-mercapto-4,5-dimethoxybenzyl (Dmmb) group on the peptide bond, or a cysteine residue. The Dmmb-containing peptide is readily converted to the corresponding 2-sulfoethyl thioester via the N-S acyl shift reaction, followed by the intermolecular thiol-thioester exchange reaction. A peptide containing a Cys-Pro ester (CPE) moiety is spontaneously transformed into a diketopiperazine (DKP) thioester under neutral conditions. Furthermore, a peptide containing a Cys-Pro-Cys (CPC) sequence was also transformed into a peptide DKP thioester under acidic conditions via the N-S acyl shift reaction.