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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, 2010Microwave-Assisted Total Synthesis of MacrocyclicCystine Knot MiniproteinsS. Park, S. Gunasekera, T. Aboye Teshome, and U. GöranssonDiv. of Pharmacognosy, Dept. of Medicinal Chemistry, Uppsala University,BMC Box 574, SE-751 23, Uppsala, SwedenIntroductionCyclotides are mini-proteins of approximately 30 amino acids residues that have a uniquestructure consisting of a head-to-tail cyclic backbone with a knotted arrangement of threedisulfide bonds [1] (Figure 1). This unique structure provides exceptional stability tochemical, enzymatic and thermal treatments [2] and has been implicated as an ideal drugscaffold for the development into agricultural and biotechnological agents [3]. Wedeveloped the first method for microwave assisted Fmoc-SPPS of cyclotides [4]. Weovercame the difficulties that previous studies [5-7] encountered for synthesis of cyclicpeptides which involves numerous and time consuming reaction steps.Results and DiscussionThe protocol adopts a strategy that combines the optimized microwave assisted chemicalreactions for Fmoc-SPPS of peptide backbone synthesis, thioesterification of theC-terminal carboxylic acid of the peptide and a one pot reaction that promotes cyclisationthrough native chemical ligation and oxidative folding (Figure 1). The application of thisprotocol was exemplified for the synthesis of three prototypic cyclotides; kalata B1,MCoTI-II and cycloviolacin O2.By utilization of current protocol, the results are as follows. For peptide chainelongation, microwave assisted coupling reaction showed the yield higher than 99.3%.With microwave application, the cleavage time was effectively shortened from 3 hours atroom temperature into 45 minutes. Thioester was efficiently introduced at C-terminalprotected peptide; LC-MS analysis confirmed full conversion into thioester withPyBOP/p-acetamidothiophenol/DIPEA (5/5/10 equiv) in DMF under microwave radiation.190Fig. 1. Synthetic strategy forsynthesis of cyclotides. (a) Assemblyof the protected peptide backbone byMW assisted Fmoc SPPS (couplingreaction for non-Cys: 5 min, 35W,75 o C for Cys: 7min, 30W, 55 o C); (b)AcOH/TFE/DCM (1/1/8) undermicrowave radiation (45 min, 10W,40 o C); (c) (1) PyBOP/p-acetamidothiophenol/DIPEA (5/5/10 equiv) inDMF under microwave radiation(5 min, 35W, 75 o C), (2) TFA/TIPS/water (95/2.5/2.5), rt, 2 hr; (d) 1:1(v/v) mixture of 0.25 M Tris buffer(pH 8.0) and isopropanol, containingreduced and oxidised glutathione (8respective 2 mM), rt, 24 hr.