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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, 2010S-Acyl Isopeptide Method: Preparation of Thioester-ContainingIsopeptides by Fmoc-Based SPPS with Alloc GroupTaku Yoshiya, Yuka Hasegawa, Wakana Kawamura, HiroyukiKawashima, Youhei Sohma, Tooru Kimura, and Yoshiaki KisoDepartment of Medicinal Chemistry, Center for Frontier Research in Medicinal Science,Kyoto Pharmaceutical University, Kyoto, 607-8412, JapanIntroductionWe developed the S-acyl isopeptide method [1] for the synthesis of Cys-containing difficultpeptides. In a study with a model peptide Ac-Val-Val-Cys-Val-Val-NH 2 , its S-acylisopeptide derivative had 1400-fold higher water-solubility than the native pentapeptide,and thus the isopeptide was readily purified by HPLC. The isopeptide could convert to thenative peptide via an S-to-N intramolecular acyl migration reaction. Additionally, thesynthesis of the isopeptide by Fmoc-based SPPS was improved when an allyl-typeprotective group was used. Side reactions at the thioester during base treatment for Fmocdeprotection (by aminolysis and/or diketopiperazine formation) were suppressed. Weherein report the application of the S-acyl isopeptide method to diabetes-related amylin.Results and DiscussionAmylin(1-12)-NH 2 (H–KCNTATCATQRL–NH 2 having a disulfide bond betweenBocHNOamylin(8-12) (protected)(a, b) x5H 2NH amylin(8-12) (protected)NovaSyn TGR resin 2cAlocHNSOO3BocHNOamylin(8-12) (protected)BocHNOamylin(8-12) (protected)d, e HSfAloc Ala NOOFmoc Thr(tBu) AlaHNOSO4 5g, h, g, i, g, jkTFA· H 2NNH 2 ·TFAHNOHSOAsnThr Ala6·TFATFA· H 2NHNHOSOOamylin(8-12)NH 2lH 2NNH 2HNOHSOAsnThr Ala7HONHHNOHSOamylin(8-12)NH 2mO Asn Thr Ala NH OHNH 2SHNOSNHONH amylin(8-12)2O1amylin(8-12): ATQRLNH 2Scheme 1. Reagents and conditions: (a) Fmoc-Xaa-OH, DIPCDI, HOBt, DMF, 2 h;(b) 20% piperidine/DMF, 20 min; (c) Boc-Cys(Alloc-Thr(tBu))-OH, DIPCDI, HOAt,CH 2 Cl 2 , 3 h; (d) Pd(PPh 3 ) 4 , PhSiH 3 , 0.1% TFA, toluene, Ar, 20 min (3 times); (e)Alloc-Ala-OH, HCTU, DIPEA, DMF, 2 h; (f) Pd(PPh 3 ) 4 , PhSiH 3 , 0.1% TFA, toluene,Ar, 20 min; then, Fmoc-Thr(tBu)-OH, HCTU, DIPEA, DMF, 40 min (3 cycles); (g)1-methylpyrrolidine (25 v/v%)–hexamethyleneimine (2 v/v%)–HOBt (3 w/v%) inNMP–DMSO (1:1) (also known as Reagent A [2]), 10 min; (h) Fmoc-Asn(Trt)-OH,DIPCDI, HOBt, DMF, 2 h; (i) Fmoc-Cys(Trt)-OH, DIPCDI, HOBt, DMF, 2 h; (j)Boc-Lys(Boc)-OH, DIPCDI, HOBt, DMF, 2 h; (k) TFA : ethanedithiol : H 2 O :triisopropylsilane (94:2.5:2.5:1), 90 min; (l) pH 7.6 phosphate buffer, 10 min, 25°C;(m) then, addition of DMSO (for the concentration of 10%), 4 h.112