Chemical synthesis of membrane-associated peptides: A model study on influenza virus B protein BM2(1-51)

Abstract

Membrane-associated proteins play often a key role in the development and treatment of heavy diseases (i.g. multiple sclerosis, cancer). Reliable synthetic methods for highly hydrophobic peptides are therefore essential for an efficient production of membrane-associated peptides/proteins. Especially, chemical peptide synthesis allows a fast incorporation of specific modifications including unnatural amino acids, protecting groups or precise isotopic labelling for functional or structural studies. However, providing enough material by Fmoc-based solid phase peptide synthesis (Fmoc-SPPS) and native chemical ligation (NCL) is often limited to good soluble peptide sequences. The synthesis of “difficult sequences” or highly hydrophobic peptides is usually associated with poor yields or a complete failure of the synthesis. This thesis is focused on the development of reliable synthetic methods for highly hydrophobic peptides including the incorporation of temporary bound, C-terminal solubilizing tags and the addition of solubility improving additives such as fluorinated alcohols or ionic liquids. As a model peptide for these studies, the influenza B proton channel sequence BM2(1-51) was chosen. This protein sequence plays an important role in the multiplication of the influenza B virus and is therefore of great relevance for the development of an anti-flu drug. The C-terminal solubilizing tag strategy is based on a removable solubilizing tag which is attached to the thioester-forming rearrangement group 2-hydroxy-3-mercaptopropanamide (Hmp). By attaching polylysine or PEG tags to Hmp moiety an increased solubility was achieved for purification and NCL. With this approach ligation yields of approximately 90% were obtained and the total chemical synthesis of BM2(1-51) was achieved for the first time. The investigated external conditions, presented in this work, represent the addition of hexafluoroisopropanol (HFIP) or the ionic liquid 1-ethyl-3-methylimidazolium acetate [C2mim][OAc] as efficient additives for NCL or Cys desulfurization. Thereby HFIP turned out as highly efficient additive for poorly-soluble Hmp-peptides leading to nearly quantitative ligation and desulfurization yields. The investigation of the ionic liquid [C2mim][OAc] as alternative ligation media provided deeper insight into N-heterocyclic carbene (NHC) induced modifications of thiol-containing compounds and Cys-containing peptides. Finally, conditions were found in which NHC-related side reactions could be entirely suppressed. Applying these findings a new “ionic liquid-buffer” was developed which was successfully applied for BM2(1-51) synthesis.