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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, 2010The Synthesis of Peptide DualAGZhiguo Liu, Shengwu Wang, Qian Qiu, Shawn Lee, and Xiaohe TongCPC Scientific Inc., 1245 Reamwood Ave., Sunnyvale, CA, 94089, U.S.A.IntroductionRecently, it was reported that peptide DualAG, H-s-QGTFTSDYSKYLDSRRAQDFVQW-LMNTKRNRNNIAC(CH 2 CO-PEG4-Cholesterol), exhibits superior weight loss, lipidlowering activity, and antihyperglycemic efficacy [1]. The preparation and conjugation ofcholesterol with PEG 4 linker to the C-terminus of native oxyntomodulin via a Cyssidechain improve pharmacokinetics and are also key steps for synthesis of peptideDualAG which incorporate a D-Ser 2 substitution that confers resistance to dipeptidylpeptidase 4(DPP-4). Here we report the synthesis of DualAG with Fmoc solid phasesynthesis to obtain peptide precursors and solution phase work to make and react cholest-5-en-3-yl 1-iodo-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oate(3) with the free thiolcontainingpeptide to obtain peptide DualAG.HH-s-QGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-Cys-NH 2Fig. 1. The structure of DualAG.SONHOO4OHHResults and DiscussionCholesterol (1) was reacted with Fmoc-PEG 4 -OH in dry DCM solution containing DIC andDMAP to give the Fmoc-PEG 4 -cholesterol with 85% yield. The Fmoc-PEG 4 -cholesterolwas treated with diethylamine in THF and the reaction mixture was purified by silica gel toobtain the NH 2 -PEG 4 -cholesterol (2) with 45% yield. The NH 2 -PEG 4 –cholesterol (2) wasiodoacetylated with iodoacetic anhydride in anhydrous DCM and the reaction mixture waspurified by silica gel to give a key intermediate cholest-5-en-3-yl 1-iodo-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oate(3) with 90% yield.HOH1. Fmoc-PEG 4 -OH/DIC/DMAP/DCMHH H2. DEA / THFH HH 2 NO O14O 2(ICH 2 CO) 2 ODCMFig. 2. Synthesis of DualAG.IONHOO4O3HH HPeptide-Cys precursorsDMF/ACN/buffer(pH7.8)DualAGThe free thiol-containing peptide precursor of DualAG, H-s-QGTFTSDYSKYLDSRRA-QDFVQWLMNTKRNRNNIAC, was synthesized by standard solid-phase peptidesynthesis using Fmoc/t-Bu chemistry and purified by RP HPLC using water/acetonitrile(0.1% TFA) gradients with >95% purity. Cholesterol-peptide conjugate (DualAG) wassynthesized by reaction of the free thiol-containing peptide precursors with 3 in mixturesolvent of DMF/acetonitrile/PBS buffer (pH 7.8) at room temperature. After the reactionwas completed, the peptide conjugates was purified by RP HPLC using water/acetonitrile(0.1% TFA) gradients. Purified DualAG was characterized by electrospray MS.258