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Table 1. Integration results of RP-HPLC chromatograms obtained after solid phasepeptide synthesis of HBVpreS/2-78A 214 nmPeptide sequencespectrometric analysis of the crude peptide after solid phase synthesis showed that none ofthese strategies could enhance the efficiency of the peptide synthesis – an almost identical2-80 (product)25-80 Ac36-80 Ac42-80 Ac46-80 Ac18 19 20 21222324Time (min)Fig. 2. RP-HPLC of the crude peptideafter solid phase synthesis at roomtemperature (grey) and at elevatedtemperature (black).Area [%]Room temp. 50 °C2-80 23.8 31.225-80 Ac 6.2 3.936-80 Ac 7.9 4.942-80 Ac 4.5 2.646-80 Ac 3.3 2.5pattern of side products was detected.By comparing the crude product obtainedafter heating (50 °C) with the synthesis at roomtemperature, it could be demonstrated that theformation of almost all side products wasdecreased (Table 1, Figure 2). After purificationthe HPLC chromatogram consists of a singleproduct peak. The analysis of the synthesizedpeptide by LC-MS confirmed formation of thedesired product. The resulting purity ofMyr-HBVpreS/2-78 defined after RP-HPLCanalysis was found to be 98%. The overall yieldwas 7.3%.Although the classical stepwise chainassembly of large peptides is complicated, weshowed that the solid phase peptide synthesisdescribed provides a suitable access to the 77-merlipopeptide Myr-HBVpreS/2-78. The preliminaryidentification and assessment of difficult regionswithin the peptide chain by a predictive method helped to consider individual solutions.Attempts were undertaken to optimize the synthesis by heating, double coupling or the useof pseudoproline dipeptides. The efficiency of the synthesis could be increased best byapplying elevated temperature resulting in a higher purity of the crude product after solidphase synthesis.AcknowledgmentsThis work was funded by the Bundesministerium für Bildung und Forschung (BMBF), InnovativeTherapieverfahren, Grant Number 01GU0702.References1. Gripon, P., Cannie, I., Urban, S. Journal of Virology 79, 1613-1622 (2005).2. Petersen, J., Dandri, M., Mier, W., Lutgehetmann, M., Volz, T., von Weizsacker, F., Haberkorn, U.,Fischer, L., Pollock, J.M., Erbes, B., Seitz, S., Urban, S., Braun, W., Wider, G., Lee, K.H.,Wüthrich, K. Nature Biotechnology 26, 335-341 (2008).3. Gripon, P, Rumin, S., Urban, S., Le Seyec, J., Glaise, D., Cannie, I., Guyomard, C., Lucas, J.,Trepo, C., Guguen-Guillouzo, C. PNAS 99, 15655-15660 (2002).4. Le Seyec, J., Chouteau, P., Cannie, I., Guguen-Guillouzo, C., Gripon, P. Journal of Virology 73,2052-2057 (1999).5. Schieck, A., Müller, T., Schulze, A., Haberkorn, U., Urban, S., Mier, W. Molecules 15, 4773-4783(2010).173