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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Synthesis of (Glyco)protein by Ligation MethodsHironobu Hojo, Hidekazu Katayama, Akiharu Ueki, Yuko Nakahara,and Yoshiaki NakaharaDepartment of Applied Biochemistry, Institute of Glycoscience, Tokai University, 4-1-1Kitakaname, Hiratsuka, Kanagawa, 259-1292, JapanIntroductionProteins and glycoproteins have been mainly synthesized by the ligation methods, such asthe native chemical ligation (NCL) [1] and the thioester method [2]. However, bothmethods still require further modifications to overcome their intrinsic drawbacks. In thispaper, we report our recent approaches to overcome these problems.Results and DiscussionNCL reaction at Xaa-Ser/Thr siteIn the NCL method, the ligation site is limited to the Xaa-Cys site. As the naturalabundance of cysteine in proteins is quite low, this problem severely limits the selection ofthe ligation site. We have developed an extended ligation reaction at the Xaa-Ser/Thr site,which is far more abundant in natural proteins than the Xaa-Cys site as shown in Scheme 1[3]. In this method, a mercaptomethyl group on the hydroxyl group of Ser and Thr wasused as a thiol auxiliary group. The advantage of this method is that this group is a labilethiohemiacetal and thus, an additional deprotection step for this auxiliary group is notrequired, as it is spontaneously hydrolyzed after the ligation. The efficiency of the methodwas demonstrated by the synthesis of glycopeptide, contulakin-G, and human calcitonin.Fmoc-Ser/Thr(CH 2 SSBu t )-OH used for ligation were prepared in 5 steps starting fromFmoc-Ser/Thr-OBu t . The obtained serine unit was used for the synthesis of contulakin-G 1by NCL at Gly 6 -Ser 7 . N-terminal peptide thioester, pGlu-Ser-Glu-Glu-Gly-Gly-SPh 2, wasprepared by the solid-phase peptide synthesis (SPPS) using the N-alkylcysteine (NAC)-assisted thioesterification method [4]. C-Terminal glycopeptide, H-Ser(CH 2 SSBu t )-Asn-Ala-Thr(GalNAcBn)-Lys-Lys-Pro-Tyr-Ile-Leu-OH 3, was prepared by the Fmoc methodusing thiol-free TFA cocktail for final cleavage. Then two peptides were ligated in sodiumphosphate buffer pH 7.0 containing triscarboxyethylphosphine (TCEP). The thioesterintermediate 4 was obtained in good yield within 10 min. After overnight, however, thedesired product 6 was obtained in a yield of only 42%. As the S- to N-acyl shift in thisligation proceeds through 7-membered ring, the hydrolysis (Path B) significantly competedwith the reaction. To avoid the hydrolysis, the reaction mixture was diluted with DMFcontaining 5% acetic acid after a 10 min ligation, when most of the starting peptides wereconverted to the thioester intermediate 4. As a result, the desired product 6 was successfullyOOPeptide 1 HSPh + 2 NPeptide 2R 2 SORR 2 = SBu tR 2 = HH(1-6) SPhO2R 2 S+OH 2 NOGalNAc(Bn)(8-16)OH3a: R 2=SBu t3b: R 2 =HTCEPPeptide 1OSH 2 NOORPeptide 2H(1-6)SOOH 2 NOGalNAc(Bn)(8-16) OH4+ HSR 1R O SHOPeptide 1 Peptide 2NHOH(1-6)HSOONHOPath AGalNAc(Bn)(8-16) OH5HPath B(1-6) OH7O+R OHOPeptide 1 NPeptide 2HOH(1-6)OHONHOGalNAc(Bn)(8-16) OH6HOH 2 NOGalNAc(Bn)(8-16) OH8Scheme 1. Novel ligation at Xaa-Ser/Thr site. Scheme 2. Pathway of the ligation for 6.110