Proceedings book download - 5Z.com

Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Peptide Diketopiperazine Thioester Formation at theCys-Pro-Cys PositionToru Kawakami, Sakiko Shimizu, and Saburo AimotoInstitute for Protein Research, Osaka University, Suita, 565-0871, JapanIntroductionThe peptide thioester is one of the key intermediates in protein synthesis by the ligationstrategies, such as the thioester method [1,2] and native chemical ligation [3,4]. The focusof our research has been on the use of the N to S acyl shift reaction for preparing peptidethioesters. In previous studies, we reported on the thiol-auxiliary mediated thioesterformation [5,6] and on the diketopiperazine (DKP)-thioester formation by the use of aCys-Pro ester (CPE) unit [7,8]. The peptide containing the CPE unit can be directly utilizedin a ligation reaction with a peptide containing a cysteine residue at the N-terminus(Cys-peptide) in a one pot reaction. In this reaction, the ester group played a crucial role asa leaving group at the DKP-forming step. The next step is to design a general sequence thatleads to thioester production without an ester moiety, which would make it possible to usegenetically engineered proteins as peptide thioester precursors. We report herein on theformation of a peptide DKP thioester using a peptide containing a Cys-Pro-Cys sequence(CPC peptide) [9].Results and DiscussionWe initially prepared a peptide library, H-Ala-Lys-Leu-Arg-Phe-Gly-Cys-Pro-Xaa-Xbb-Xcc-Arg-NH 2 (1), in which the Cys-Pro sequence was fixed and the Xaa-Xbb-Xcc regioncontained random sequences of 3 amino acid residues consisting of Asn, Asp, Cys, Gln,Gly, His, Leu, or Ser. The library was treated with an excess amount of H-Cys-Tyr-NH 2 (2)in a carbonate buffer at pH 8.2. A ligated product, H-Ala-Lys-Leu-Arg-Phe-Gly-Cys-Tyr-NH 2 (3), would be expected to be produced, if the active sequence is contained in thelibrary. A library 1a, in which the Xaa residue was fixed with 8 different amino acidresidues respectively, and Xbb and Xcc contained random residues, was treated withpeptide 2. When a Ser or Cys residue was located at the Xaa position, the ligated product 3was observed as a small but distinct peak by RP-HPLC and by mass spectroscopy. When aSer residue was located at the Xaa position and the Xbb or Xcc position was fixed with the8 different amino acid residues respectively, the results were similar; a signalcorresponding to peptide 3 was observed in the majority of cases. These findings suggestthat only Ser or Cys residues at the Xaa position are essential and that the ligation reactionproceeds via the following steps (Figure 1): An N-S/O acyl shift reaction at the second Seror Cys residue of a peptide 4 containing Cys-Pro-Ser/Cys sequence (path b) would producea (thio)ester structure at the C-terminus, as shown in structure 5b which contains a similarstructure to the CPE unit. Once the CPE structure is constructed, the DKP thioesterformation (structure 7) might occur via an N-S acyl shift reaction at the first Cys residue(path a), followed by DKP formation (path c). The order of the acyl shift reactions a and bwould not be critically important. Finally, the native chemical ligation reaction of thioester7 proceeds with a Cys-peptide 9 to give the ligation product 10.A Ser or Cys residue at the Xaa position in the library was required for the ligation,whereas the reaction proceeded in low yield under neutral conditions. Next, some of theselected peptides were treated in an acid solution since the equilibrium between a thioesterand an amide forms at a Cys site shifts in favor of the thioester under the acidic conditions[10]. As a result, when a peptide, H-Ala-Lys-Leu-Arg-Phe-Gly-Cys-Pro-Cys-NH 2 (4a) wasincubated in a 0.1 M HCl solution at 110 ºC in an evacuated sealed tube for 2 h, the massnumber corresponding to peptide DKP thioester 7a was observed and the isolated yield was12%. In this condition a hydrolysis product, H-Ala-Lys-Leu-Arg-Phe-Gly-OH (11) wasobserved, while the CPC peptide 4a was remained. When peptide 4a was treated withheptafluorobutyric acid vapors at 110 ºC in an evacuated sealed tube for 4 h [11], the DKPthioester product 7a was isolated (9 %) with its epimer in the DKP moiety (12%), in whichCPC peptide 4a disappeared and the production of hydrolysis peptide 11 was reduced.When this crude mixture was reacted with an excess amount of Cys-peptide 2 to give asingle ligated isomer 3 in a yield of 17% based on CPC peptide 4a.52