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

Baumruck, Andreas (2020):
Chemical synthesis of membrane-associated peptides: A model study on influenza virus B protein BM2(1-51).
Darmstadt, Technische Universität, DOI: 10.25534/tuprints-00011731,
[Online-Edition: https://tuprints.ulb.tu-darmstadt.de/11731],
[Ph.D. Thesis]

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.

Item Type: Ph.D. Thesis
Erschienen: 2020
Creators: Baumruck, Andreas
Title: Chemical synthesis of membrane-associated peptides: A model study on influenza virus B protein BM2(1-51)
Language: English
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.

Place of Publication: Darmstadt
Divisions: 07 Department of Chemistry
07 Department of Chemistry > Fachgebiet Biochemie
07 Department of Chemistry > Fachgebiet Biochemie > Allgemeine Biochemie
Date Deposited: 18 May 2020 07:37
DOI: 10.25534/tuprints-00011731
Official URL: https://tuprints.ulb.tu-darmstadt.de/11731
URN: urn:nbn:de:tuda-tuprints-117312
Referees: Tietze, Ass. Prof. Alesia and Kolmar, Prof. Dr. Harald
Refereed / Verteidigung / mdl. Prüfung: 9 April 2020
Alternative Abstract:
Alternative abstract Language
Membranassoziierte Proteine haben oft eine Schlüsselrolle bei der Entstehung und Behandlung schwerer Krankheiten (z.B. Multiple Sklerose, Krebs). Zuverlässige Synthesemethoden für stark hydrophobe Peptide sind wesentlich für eine effiziente Herstellung membranassoziierter Peptide/Proteine. Insbesondere die chemische Peptidsynthese ermöglicht einen schnellen Einbau spezifischer Modifikationen, wie z.B. unnatürliche Aminosäuren, Schutzgruppen oder Isotopenmarkierungen für funktionelle oder strukturelle Studien. Die Herstellung ausreichender Peptidmengen durch Fmoc-basierte Festphasenpeptidsynthese (Fmoc-SPPS) und nativer chemische Ligation (NCL) ist jedoch häufig auf gut lösliche Peptidsequenzen beschränkt. Die Synthese von „schwierigen Sequenzen“ oder stark hydrophoben Peptiden ist oftmals mit schlechten Ausbeuten oder einem vollständigen Versagen der Synthese verbunden. Diese Arbeit konzentriert sich auf die Entwicklung zuverlässiger Synthesemethoden für stark hydrophobe Peptide, mit Hilfe temporär gebundener, C-terminaler Löslichkeits-Tags und löslichkeitsverbessernder Additive, wie fluorierten Alkoholen oder ionischen Flüssigkeiten. Als Modellpeptid für diese Studie wurde die Influenza B Protonenkanalsequenz BM2(1-51) verwendet. Diese Proteinsequenz spielt eine Schlüsselrolle bei der Vermehrung des Influenza B Virus und ist daher für die Entwicklung möglicher antigrippaler Arzneimittel von großer Bedeutung. Die entwickelte C-terminale Ligationsstrategie basiert auf einem abspaltbaren Löslichkeits-Tag, der an die Thioester-bildende Umlagerungsgruppe 2-Hydroxy-3-mercaptopropanamid (Hmp) gebunden ist. Durch Anbringen von Polylysin- oder PEG-Gruppen an Hmp konnte eine deutlich erhöhte Löslichkeit während der Aufreinigung und Ligation erreicht werden. Dieser Ansatz ermöglichte Ligationsausbeuten von bis zu 90% und erlaubte erstmals eine chemische Totalsynthese der BM2(1-51) Sequenz. Die in dieser Arbeit untersuchten externen Methoden zur Löslichkeitsverbesserung basieren auf der Zugabe von Hexafluorisopropanol (HFIP) oder der ionischen Flüssigkeit 1-Ethyl-3-methylimidazoliumacetat [C2mim][OAc] als Additive für die NCL und Cys-Desulfurierung. Dabei erwies sich HFIP als hocheffizientes Additiv für schwerlösliche Hmp-Peptide, was zu nahezu quantitativen Ausbeuten während der Ligation und Desulfurierung führte. Die Untersuchung der ionischen Flüssigkeit [C2mim][OAc] lieferte einen tiefen Einblick in die Modifizierung von Thiolverbindungen und cysteinhaltiger Peptide durch N-heterocyclische Carbene (NHC). Schließlich wurden Bedingungen gefunden, unter denen NHC-bedingte Nebenreaktionen vollständig unterdrückt werden konnten. Durch diese Erkenntnisse konnte ein neuer „IL-basierter Ligationspuffer“ entwickelt werden, der erfolgreich für die Herstellung von BM2(1-51) eingesetzt wurde.German
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