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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 Structure Confirmation of the Cysteine KnottedPeptide Gurmarin by Selective Disulphide FormationRasmus Eliasen 1 , Thomas Lars Andresen 2 , andKilian W. Conde-Frieboes 11 Novo Nordisk A/S, 2760, Måløv, Denmark; 2 Department of Micro- and Nanotechnology,Technical University of Denmark, 2800, Kgs. Lyngby, DenmarkIntroductionGurmarin is a 35-residue peptide from the plant Gymnema sylvestre [1]. The peptide hasbeen shown to function as a sweet taste inhibitor in mice [3]. The structure of Gurmarin(Figure 2) is defined by three disulphides in a common fold known as an inhibitor cysteineknot. Folding of such peptides is often achieved by equilibrium driven folding in a redoxbuffer [2]. In this study, the synthesis of Gurmarin was carried out in three different ways,where one of the three cysteine-pairs was trityl-protected, while the other two pairs wereacetamidomethyl-protected. This allowed for selective formation of the first disulphide byair oxidation, followed by iodine oxidation of the last two disulphides. The structures of thepeptides were confirmed by cleavage with thermolysin. In this way it was possible todetermine the disulphide connection.Results and DiscussionThe peptides were synthesized by standard Fmoc chemistry on a CEM Liberty peptidesynthesizer. HOBt was added to the piperidine deprotection to minimize aspartimideformation. The first disulphide was oxidized in 0.1 M Tris-HCl buffer (pH 7.8, 0.02mMpeptide), 2 vol% DMSO with air bubbled through until complete oxidation was confirmedby UPLC. The final two disulphides were oxidized using 20 eq. iodine in 1:4water:methanol (0.02 mM peptide) until complete oxidation was confirmed by MS. Thepeptides were cleaved with 1 mass eq. thermolysin in 0.2 M ammonium acetate (pH 6.5),10 mM CaCl 2 24h at 50°C.The synthesis of the peptide with tritylprotected Cys-3 and Cys-18 produced amixture of two products. One of these wasidentified as the native peptide by thefragment in Figure 1 and another fragmentcontaining two native disulphides (secondfragment, Table 1). In the latter fragmentthe disulphide between Cys-3 and Cys-18was oxidized selectively first in the airoxidation of the trityl protected cysteinesand consequently all three disulphides inthis peptide are native.The cleavage of the peptidesynthesized with trityl protected Cys-10 andFig. 1. Sequence and mass spectrum of anative disulphide fragment obtained bycleavage with thermolysin.Cys-23 produced one fragment with thenative disulphide between Cys-10 andCys-23, as expected. However no otherfragments containing native disulphideswere found from this cleavage.Fig. 2. The structure (PDB: 1C4E) of Gurmarin (left) and the primary sequence (right).144