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obtained in 80% yield (Scheme 2). The contulakin G 1 was easily obtained by the lowacidityTfOH treatment of glycopeptide 6. In the same manner, human calcitonin wassuccessfully prepared. These results demonstrates that this extended ligation method isuseful, when cysteine residue does not exist at an appropriate position.Azido group for use as an amino protecting group in the thioester methodThioester method has the advantage that there is no restriction on the selection of theligation site. Instead, the side chain amino and thiol groups have to be protected. To realizethis, the Boc groups have to be reintroduced to the side chain amino groups, which weremade free during the deprotection after SPPS. We examined the direct synthesis of the sidechain-N-protected peptides by introducing azido-protected Fmoc-Lys during SPPS toovercome the inconvenience. The method was used for the synthesis of N- andO-glycosylated pro-opiomelanocortin (POMC) (1-74) 9 shown in Figure 1 [5]. The peptidechain was divided at Gly 37 -Asn 38 and both segments were prepared by the Fmoc strategy.To apply the NAC method for the synthesis of N-terminal thioester 10, peptide chain waselongated starting from Fmoc-Gly-(Et)Cys(Trt)-[Arg(Pbf)] 2 -NH-resin. Lys 25 was introducedusing Fmoc-Lys(N 3 )-OH by DCC-HOBt method. After the completion of the chainassembly, peptide was deprotected by TFA and then thioesterified by 4-mercaptophenylaceticacid to obtain Fmoc-[Cys(Acm) 2,8,20,24 , Lys(N 3 ) 25 ]-POMC(1-37)-SC 6 H 4 CH 2 COOH 10. C-Terminal segment 11 was also prepared by the Fmoc strategy. Forthe introduction of Thr 45 , Lys 50 , Asn 65 , Fmoc-Thr(3-O-benzyl-4,6-O-benzylidene-GalNAc)-OH, Fmoc-Lys(N 3 )-OH, Fmoc-Asn(GlcNAcBn 3 )-OH, were used, respectively.After the protected-peptide resin was treated with Reagent K for 1 hr to deprotect thepeptide portion, the obtained peptide was further treated with 20% thioanisole-TFA at 30ºC for 14 h to remove benzyl groups to obtain the desired C-terminal glycopeptide[Asn(GlcNAc) 65 , Lys(N 3 ) 50 , Thr(GalNAc) 45 ]-POMC(38-74) 11. Then the peptides 10 and11 were condensed by the Ag + -free thioester method in DMSO containing HOOBt andDIEA. After the Fmoc removal by piperidine, followed by the reduction of azido groupwith Zn in aq AcOH and by the removal of Acm groups by AgNO 3 treatment, the reducedform of the product 9 was obtained. Finally, disulfide bond was formed in the redox buffercomposed of oxidized and reduced form of glutathione to successfully obtain the desiredproduct 9 in 15% overall yield from peptide 10 and 11. These results demonstrate that theuse of azido group for amino group protection facilitates the peptide ligation by thethioester method.WCLESSQCQD LTTESNLLAC IRACKLDLSLHOOHOHOAcHNETPVFPGNGD EQPLTENPRK YVMGHFRWDRHOHOOHONHAcHNFGPRNSSSAG SAAQFig. 1. Structure of POMC. An arrow indicates the site of segment coupling.AcknowledgmentsThis work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry ofEducation, Sport, Science and Technology of Japan. We thank Tokai University for a Grant-in-Aid forhigh-technology research.References1. Hojo, H., Aimoto, S. Bull. Chem. Soc. Jpn. 64, 111-117 (1991).2. Dawson, P.E., Muir, T.W., Clark-Lewis, I., Kent, S.B.H. Science 266, 776-779 (1994).3. Hojo, H., Ozawa, C., Katayama, H., Ueki, A., Nakahara, Y., Nakahara, Y. Angew. Chem. Int. Ed.49, 5318-5321 (2010).4. Hojo, H., Onuma, Y., Akimoto, Y., Nakahara, Y., Nakahara, Y. Tetrahedron Lett. 48, 25-28 (2007).5. Katayama, H., Hojo, H., Shimizu, I., Nakahara, Y., Nakahara, Y. Org. Biomol. Chem. 8, 1966- 1972(2010).5025111