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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, 2010Solid Phase and Ligation Approaches to DendrimericImmunogen SynthesisWioleta Kowalczyk, Marta Monsó, Beatriz G. de la Torre, andDavid AndreuDepartment of Experimental and Health Sciences, Pompeu Fabra University, BarcelonaBiomedical Research Park, Barcelona, SpainIntroductionDendrimeric immunogens such as multiple antigenic peptides (MAPs) were introduced byTam some 25 years ago [1] and have found numerous applications in vaccine anddiagnostics research. However, despite extensive and successful use, experimental reportson MAP and similar dendrimeric constructs are often lacking in chemical detail about theirpreparation and characterization.MAPs can be synthetically approached by direct or indirect (convergent) methods. Inthe former approach the branched poly-lysine core and the epitopes displayed on it areentirely built by stepwise SPPS while the convergent approach relies on the chemicalligation (e.g. thioether) of properly functionalized peptide epitopes onto the poly-lysinecore [2]. Theoretically, the use of pre-purified components in the ligation reactions canresult in chemically more unambiguous materials than in stepwise methods, where minutebut cumulative synthetic errors (deletions, truncations, etc.) become amplified bymultimerization and may predictably lead to relatively heterogeneous immunogens.In this study two methods of MAP preparation have been evaluated: fully stepwiseSPPS and thioether ligation in solution. The pros and cons of both approaches have beeninvestigated using a well-known epitope, the N-terminal ectodomain (M2e) of influenzatype A virus M2 protein [3], a transmembrane tetrameric protein on the virus surface actingas an ion channel and consisting ofthree regions: the ectodomain, atransmembrane region and thecytoplasmic tail (Figure 1). The 24-residue M2e, a rather challengingsequence [4], and a shorter (12-residue, sM2e), more manageableFig. 1. Schematic structure of protein M2.version have been used as testepitopes.Results and DiscussionThe all-SPPS (direct) approach was used to prepare two MAPs, each with four copies ofsM2e. MAP B (Figure 2) only differed from A in the presence of 6-aminohexanoic acid(Ahx) spacer units at every branching point, a modification aimed at enhancing the overallflexibility of the construct. Both A and B were readily assembled on a Rink amideChemMatrix resin using Fmoc chemistry and double couplings throughout the entire sM2esequence. A was obtained in a 9% isolated yield and well characterized by MALDI-TOFMS (C 281 H 432 N 80 O 102 S 1 , MW= 6595.05 Da, [M+H + ] = 6596.28). A parallel process for Bled to a more easily purifiable final product in significantly better yield (16.5%,C 317 H 498 N 86 O 108 S 1 , MW = 7273.99Da, [M+H + ] = 7274.34).Fig. 2. Analytical HPLC of tetravalent MAP constructs (crude product) obtained byall-SPPS approach (wavy lines denote flexibilizing Ahx residues).160