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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, 2010Analogs of Contulakin-G, an Analgetically Active Glycopeptidefrom Conus GeographusSamson Afewerki 1 , Ola Blixt 2 , Henrik Clausen 2 , and Thomas Norberg 11 Department of Biochemistry and Organic Chemistry, Uppsala University, PO Box 576,751 23, Uppsala, Sweden; 2 Department of Cellular and Molecular Medicine, Faculty ofHealth Sciences, University of Copenhagen, 2200, Copenhagen N, DenmarkIntroductionCone snails are marine predators who use immobilizing venoms for catching prey.Chemical analysis of the venoms has revealed a variety of biologically active small andintermediate size peptides rich in post-translational modifications (modified amino acids,glycosylation). Contulakin-G (structure, see 2 in Scheme 1) is a potent analgesic fromConus geographus venom. The in vivo activity of synthetic Contulakin-G was previouslyfound [1] to be significantly higher compared to that of a peptide lacking the glycan. Theseobservations touch on the general question of the function of glycans of glycopeptides andglycoproteins in Nature. We believe that Conus glycopeptides are among the best modelcompounds available to address this question, since they are comparatively small moleculeswhich can be readily prepared and modified by chemical synthesis. In order to furtherinvestigate the importance of the glycan of Contulakin-G, we have now synthesizedglycopeptide analogs where the glycan chain has been altered. The glycopeptides wereprepared by a combination of solid-phase peptide synthesis and enzymatic synthesis.Fig. 1. A typical cone snail hunting session.Results and DiscussionThe glycopeptide 1 (see Scheme 1) was prepared by Fmoc-type solid-phase peptidesynthesis on Fmoc-Leu-NovaSyn TGA resin using commercial Fmoc-protected amino acidderivatives and in-house prepared [2] N-Fmoc-O-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxyβ-D-galactopyranosyl)-L-threonine.After resin cleavage and de-O-acetylation withmethanolic sodium methoxide the glycan was enzymatically elongated, first with1,3-GalT/UDP-Gal to produce 2 (native Contulakin-G) and then with 2,3-ST/CMP-NeuActo produce glycopeptide 3, which was purified with HPLC and characterized by MALDI-TOF mass spectrometry. To our knowledge, this is the first time that a cone snailglycopeptide with a sialic acid residue has been prepared. Evaluations of biological activityof 1, 2 and 3 will be carried out in various test systems.420