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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, 2010Three Disulfide Bridged µ-Conopeptoids and Their MinimizedDisulfide-Depleted Selenopeptide DerivativesAleksandra Walewska 1 , Tiffany S. Han 2 , Min-Min Zhang 2 ,Doju Yoshikami 2 , Baldomero M. Olivera 2 ,Grzegorz Bulaj 3 , and Krzysztof Rolka 11 Faculty of Chemistry, University of Gdańsk, Poland; 2 Department of Biology and3 Medicinal Chemistry, University of Utah, Salt Lake City, UT, U.S.A.IntroductionPeptide neurotoxins from cone snails continue to provide compounds with a therapeuticpotential. The analgesic µ-conotoxin KIIIA is a three disulfide bridged peptide inhibitor ofvoltage-gated sodium channels, but more SAR studies are needed to optimize its subtypeselectivity. Recent studies suggested that Lys 7 in KIIIA might be an attractive target forengineering selectivity in µ-conotoxins toward neuronal sodium channel subtypes [1,2].Furthermore, converting this three disulfide bridged peptide into a one-disulfide-containingdisulfide-depleted selenopeptide analogue did not significantly affect its bioactivity, butdramatically simplified oxidative folding [2]. Here, we report chemical synthesis, oxidativefolding and bioactivity of two series of µ-conopeptoid analogues of KIIIA in which wereplaced Lys 7 with peptoid monomers of increasing side-chain size: N-methylglycine (Sar),N-butylglycine (Nnle) and N-octylglycine (Noct). Each of the µ-conopeptoid analoguesfrom the first series contained three disulfide bridges. To simplify oxidative folding, thesecond series of peptide-peptoid hybrid (peptomeric) analogues was introduced in thecontext of disulfide-depleted selenoconopeptides (Figure 1).AUNCSSXWARDHSRUC1 5 10 15Fig. 1. Primary structure of disulfide-depleted selenoconopeptomeric analogues, where inposition X= Sar, Nnle, or Noct.Results and DiscussionThe µ-conopeptoid analogues were synthesized using the Fmoc protocols. N-Substitutedglycine derivatives (peptoids) were introduced into the peptide chain by the submonomericapproach [3]. In the series of disulfide-depleted selenoconopeptoid analogues, the cysteinethiols were protected with trityl (Trt) groups, whereas the selenocysteine (Sec) residueswere protected with 4-methoxybenzyl (Mob) groups. The protected groups were removedduring the cleavage of the peptides from the resin with an enriched reagent K and the Mobgroups came off easily with 2,2’-dithiobis-(5-nitropyridine) (DTNP). The presence ofDTNP in the cleavage mixture was critical for the removal of the p-methoxybenzyl groupsand a simultaneous formation of a diselenide bridge. The oxidative folding was carried outin buffered solution (0.1 M Tris-HCl, pH 7.5) containing 1 mM reduced and 1 mMoxidized glutathione, followed by HPLC separation using reversed-phase C18 columns.The identity of each of the folded species was confirmed by MALDI-TOF or ESI massspectrometry.Electrophysiological experiments demonstrated that all µ-KIIIA analogues retainedactivity in blocking Na v 1.2, a neuronal subtype of sodium channel expressed in oocytes.Table 1 summarizes on- and off-rates of the block of Na v 1.2 as well as the percent block ofthe sodium currents by 10 µM peptide. Two analogues [Noct 7 ]KIIIA and [Ala 1,9 , Sec 2,15 ,Noct 7 ]KIIIA, with the long side-chain size of Noct, turned out to be the most potentpeptides.These results prove that the peptoid strategy can be effectively applied to conotoxinscontaining three disulfide bridges.250