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Fig. 1. (A) Reduced-form amylin(1-12)-NH 2 (7) was released from the S-acyl isopeptide 6 inpH 7.6 phosphate buffer, and then oxidized to amylin(1-12)-NH 2 (1) by addition of DMSO tothe migration buffer. Process was monitored by analytical RP-HPLC: (i) 0 s, (ii) 1 min and(iii) 4 h (after the addition of DMSO). (B) HPLC profile of purified 1. Analytical HPLC wasperformed using a C18 reverse phase column (4.6 × 150 mm; YMC Pack ODS AM302) withbinary solvent system: a linear gradient of CH 3 CN ((A) 2%–32%/40 min, (B) 0–100%/40min) in 0.1% aqueous TFA at a flow rate of 0.9 mL min –1 (40 °C), detected at 230 nm.Cys 2 -Cys 7 , 1), which is known as a difficult sequence, was synthesized by the optimizedS-acyl isopeptide method (Scheme 1). Boc-Cys(Alloc-Thr(tBu))-OH was coupled to 2 bythe DIPCDI–HOAt method in CH 2 Cl 2 . After the Alloc group of resulting 3 was removedwith Pd, Alloc-Ala-OH was coupled by the HCTU–DIPEA method to give 4. Fmoc-Thr(tBu)-OH was coupled after the deprotection of the Alloc group of 4. Then, Fmoc-Asn(Trt)-OH was coupled after the removal of Fmoc group of 5 by a lower nucleophilicbase cocktail (1-methylpyrrolidine (25 v/v%)–hexamethyleneimine (2 v/v%)–HOBt(3 w/v%) in NMP-DMSO (1:1), also known as Reagent A [2]). After following residueswere coupled in a similar manner, isopeptide 6 was cleaved with TFA, and purified byRP-HPLC (Figure 1-A-i).The purified isopeptide 6 was dissolved in phosphate buffer (pH 7.6) and the mixturewas shaken at room temperature. After a 1 min reaction, quantitative S-to-N intramolecularacyl migration giving reduced amylin(1-12)-NH 2 (7) was observed (Figure 1-A-ii). Then,DMSO (final conc. 10%) was added to the solution, and the mixture was incubated at 37 ˚Cfor 4 h to form a disulfide bond at Cys 2 –Cys 7 (Figure 1-A-iii). After final purification, theyield of amylin(1-12)-NH 2 (1) was 30% from resin-bound Arg residue (Figure 1-B) [3].In this study, significant side reactions derived from thioester were not observed.Isopeptide 6 was rapidly and quantitatively converted to N-acyl peptide 7 in neutral buffer,and subsequent one-pot DMSO oxidation of 7 gave desired 1 cleanly. The total yield ofamylin(1-12)-NH 2 (1) by the S-acyl isopeptide method was ~1.5-fold higher than that ofconventional SPPS method (19%). Thus, the S-acyl isopeptide method would be a usefulmethod to prepare the difficult sequence-containing peptides in the future.AcknowledgmentsThis research was supported in part by the “Academic Frontier” Project for Private Universities:matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology)of the Japanese Government, and the 21st Century COE Program from MEXT. We thank Dr. J-T.Nguyen for his English correction. We thank Ms W. Takagi for technical assistance.References1. Yoshiya, T., Ito, N., Kimura, T., Kiso, Y. J. Pept. Sci. 14, 1203-1208 (2008).2. Li, X., Kawakami, T., Aimoto, S. Tetrahedron Lett. 39, 8669-8672 (1998).3. Yoshiya, T., Hasegawa, Y., Kawamura, W., Kawashima, H., Sohma, Y., Kimura, T., Kiso, Y.Biopolymers (Pept. Sci.) in press.113