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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, 2010Synthesis and Oxidative Folding of Cyclic Cystine KnotPeptides: Towards Backbone EngineeringTeshome Leta Aboye 1 , Richard J. Clark 2 , Robert Burman 1 ,David J. Craik 2 , and Ulf Göransson 11 Department of Medicinal Chemistry, Div. of Pharmacognosy, Uppsala University,Biomedical Centre, Box 574, SE-751 23, Uppsala, Sweden; 2 Institute for MolecularBioscience, The University of Queensland, Brisbane, QLD 4072, AustraliaIntroductionCyclotides are a family of plant miniproteins with a range of biological activities ofpotential therapeutic interest, including antimicrobial, anti-HIV, antitumour and cardiotoxicactivities [1]. They display a head-to-tail cyclized peptide backbone interconnected by threedisulfide bonds (I-VI) forming a cyclic cystine knot (CCK) motif (Figure 1). The cystineknot in combination with the cyclic backbone appears to be a highly efficient motif forstructure stabilization, resulting in exceptional conformational rigidity, together withstability against denaturing conditions, as well as against proteolytic degradation [2].Möbius and bracelets are the two main subfamilies of cyclotides [1]. Bracelets displaymore sequence diversity, and higher potency in some assays, but has largely beenintractable for chemical synthesis and oxidative folding.Due to exceptional stability, there has been significant interest to exploit cyclotides asa scaffold in drug design; for example by grafting new bioactivities onto the CCKframework [3,4]. Using this kind of biomolecular engineering it may be possible toovercome the main deficiencies of peptide-based drugs' susceptibility to proteolyticdegradation and poor bioavailability. However for this potential to be realized efficientsynthetic and oxidative folding methods need to be developed.Here we present a successful strategy for the cyclization and oxidative folding ofbracelet cyclotides. In addition we present the first systematic quantification andcomparison of heterogenous intermediates with different numbers and/or connectivities ofdisulfide bond(s) (1SS, 2SS, and 3SS) species in the process of oxidative refolding tonative 3SS form (N) for selected subfamilies of cyclotides in four different buffers.Fig. 1. Structures and sequences of selected cyclotides. Panel A. 3 D structures of threesubfamilies: bracelet, cycloviolacin O2 (cyO2); hybrid, kalata B8 (KB8); and Möbius,kalata B1 (KB1) (from left to right). Panel B. shows the sequences of the four cyclotidesused in the current study.142