Stabilization of peptides by site-specific incorporation of fluorinated amino acids: Model studies and the development of fluorinated, peptide-based HIV-1 fusion inhibitors Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by SUSANNE HUHMANN, M.Sc. from Goslar, Germany July 2018 1st Reviewer: Prof. Dr. Beate Koksch (Freie Universität Berlin) 2nd Reviewer: Prof. Dr. Nediljko Budisa (Technische Universität Berlin) date of defense: December 13, 2018 i ii Declaration The research presented in this doctoral thesis was performed under supervision of Prof. Dr. Beate Koksch from December 2013 until July 2018 at the Institute of Chemistry and Biochemistry in the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin. I hereby confirm that I have prepared this dissertation entitled “Stabilization of peptides by site- specific incorporation of fluorinated amino acids: Model studies and the development of fluorinated, peptide-based HIV-1 fusion inhibitors” autonomously and without impermissible help. All external sources and resources have been specified and properly cited or acknowledged. This thesis has not been submitted, accepted, rated as insufficient or rejected in any other doctorate degree procedure. Berlin, July 2018 Susanne Huhmann iii iv Publications, Oral and Poster Presentations Publications* (8) S. Huhmann, A. A. Berger, B. Koksch, Fluorinated protease substrates show position- dependent turnover. In Frontiers of Organofluorine Chemistry, I. Ojima, Eds., World Scientific, London, 2018, in revision. (7) S. Huhmann, B. Koksch, Fine-tuning the proteolytic stability of peptides with fluorinated amino acids. Eur. J. Org. Chem. 2018, DOI: 10.1002/ejoc.201800803. (6) S. Huhmann, A.-K. Stegemann, K. Folmert, D. Klemczak, J. Moschner, M. Kube, B. Koksch, Position-dependent impact of hexafluoroleucine and trifluoroisoleucine on protease digestion. Beilstein J. Org. Chem. 2017, 13, 2869-2882. (5) J. R. Robalo, S. Huhmann, B. Koksch, A. Vila Verde, The Multiple Origins of the Hydrophobicity of Fluorinated Apolar Amino Acids. Chem 2017, 3, 881-897. (4) S. Vukelić, J. Moschner, S. Huhmann, R. Fernandes, A. A. Berger, B. Koksch, Synthesis of Side Chain Fluorinated Amino Acids and their Effects on the Properties of Peptides and Proteins. In Modern Synthesis Processes and Reactivity of Fluorinated Compounds: Progress in Fluorine Science, H. Groult, F. Leroux, A. Tressaud, Eds., 1st Edition, Elsevier: London, 2016, pp 427-464. (3) S. Huhmann, E. K. Nyakatura, H. Erdbrink, U. I. M. Gerling, C. Czekelius, B. Koksch, Effects of single substitutions with hexafluoroleucine and trifluorovaline on the hydrophobic core formation of a heterodimeric coiled coil. J. Fluorine Chem. 2015, 175, 32-35. (2) J. Mortier, E. K. Nyakatura, O. Reimann, S. Huhmann, J. O. Daldrop, C. Baldauf, G. Wolber, M. S. Miettinen, B. Koksch, Coiled-Coils in Phage Display Screening: Insight into Exceptional Selectivity Provided by Molecular Dynamics. Journal of Chemical Information and Modeling 2015, 55, 495-500. (1) H. Erdbrink, E. K. Nyakatura, S. Huhmann, U. I. M. Gerling, D. Lentz, B. Koksch, C. Czekelius, Synthesis of enantiomerically pure (2S,3S)-5,5,5-trifluoroisoleucine and (2R,3S)- 5,5,5-trifluoro-allo-isoleucine. Beilstein J. Org. Chem. 2013, 9, 2009-2014. Oral Presentations at Conferences and Workshops (7) „Fluorine changes the proteolytic stability of peptides”; 58th Kollegseminar of the research training group 1582 “Fluorine as key element”, Berlin/Germany, 02/10/2017. (6) „Impact of fluorinated amino acids on the proteolytic stability of peptides”; 17. Deutscher Fluortag, Schmitten/Germany, 09/20/2016. * Publications (3) and (6) emerged from this doctoral thesis. v (5) „Towards fluorinated HIV-1 fusion inhibitors”; 7th PhD workshop of the research training group 1582 “Fluorine as key element”, Strausberg/Germany, 04/20/2016. (4) „Impact of fluorinated amino acids on the proteolytic stability of model peptides”; 6th PhD workshop of the research training group 1582 “Fluorine as key element”, Schwielowsee/Germany, 10/16/2015. (3) „Effects of Single Substitutions with Hexafluoroleucine and Trifluorovaline on the Hydrophobic Core Formation of a Heterodimeric Coiled Coil”; 5th PhD workshop of the research training group 1582 “Fluorine as key element”, Wandlitz/Germany, 11/20/2014. (2) „Effects of Single Substitutions with Hexafluoroleucine and Trifluorovaline on the Hydrophobic Core Formation of a Heterodimeric Coiled Coil”; 16. Deutscher Fluortag, Schmitten/Germany, 09/23/2014. (1) „Effects of Single Substitutions with Hexafluoroleucine and Trifluorovaline on the Hydrophobic Core Formation of a Heterodimeric Coiled Coil”; 39th Kollegseminar of the research training group 1582 “Fluorine as key element”, Berlin/Germany, 07/03/2014. Posters (9) „Impact of fluorinated amino acids on the proteolytic stability of peptides“; GDCh- Wissenschaftsforum Chemie, Berlin/Germany, 09/10 - 09/14/2017. (8) „Fluorine changes the proteolytic stability of peptides“; 25th American Peptide Symposium and 9th International Peptide Symposium, Whistler B.C./Canada, 06/17 - 06/22/2017. (Travel Award Winner) (7) „Impact of fluorinated amino acids on the proteolytic stability of peptides“; 13th German Peptide Symposium, Erlangen/Germany, 03/20 - 03/23/2017. (6) „Towards fluorinated peptidic HIV-1 fusion inhibitors“; 34th European Peptide Symposium and 8th International Peptide Symposium, Leipzig/Germany, 09/04 - 09/09/2016. (5) „Impact of fluorinated amino acids on the proteolytic stability of peptides“; Bremen Fluorine Days (5th International Symposium on Organofluorine Compounds in Biomedical, Materials and Agriculture Sciences), Bremen/Germany, 07/03 - 07/07/2016. (4) „Effects of Single Substitutions with Hexafluoroleucine and Trifluorovaline on the Hydrophobic Core Formation of a Heterodimeric Coiled Coil”; 5. Berliner Chemie Symposim, Berlin/Germany, 04/12/2016. (Poster Price Winner) (3) „Effects of Single Substitutions with Hexafluoroleucine and Trifluorovaline on the Hydrophobic Core Formation of a Heterodimeric Coiled Coil”; 21st International Symposium on Fluorine Chemistry, Como/Italy, 08/23 - 08/28/2015. vi (2) „Impact of trifluoroisoleucine on the proteolytic stability of model peptides“; 29. Tag der Chemie, Berlin/Germany, 06/18/2015. (1) „Hydrophobicity and Helix-propensity of Fluorinated Isoleucine Variants”; 17th European Symposium on Fluorine Chemistry, Paris/France, 07/21 - 07/25/2013. vii viii Acknowledgements Above all, I would like to thank Prof. Dr. Beate Koksch for the confidence she has shown in me, and her support and guidance throughout my studies. She gave me encouragement and freedom to pursue my research interests. I am deeply grateful for her continuing promotion to present the results of my work at several national and international academic conferences. Besides benefiting for my research, I could thereby develop myself further. Many thanks! I also wish to thank Prof. Dr. Nediljko Budisa for being the second supervisor of this dissertation. Additionally, I am very grateful for the excellent research environment, and the pleasant and motivating working atmosphere in the Koksch group. Everyday lab work, discussions, lunch and coffee breaks as well as the many cake times would not have been so inspiring, enjoyable and relaxing without all the current and former group members. All have broadened my scientific horizon and promoted this thesis tremendously. Especially, I would like to extend my sincere thanks to Dr. Elisabeth Nyakatura and Dr. Ulla Gerling-Driessen for the kind welcoming in the group and introduction to several methods and techniques; to Dr. Allison Berger for proofreading English manuscripts and this doctoral thesis; to Dr. Johann Moschner for his kind advice and willingness to discuss questions regarding organic synthesis or joint projects. I also thank him, Dipl.-Chem. Valentina Stulberg and Dipl.-Chem. Katharina Hellmund for carefully proofreading this thesis. My deepest gratitude goes to M. Sc. Kristin Folmert for introducing me to the wonders of HPLCs, her understanding and endearing character, and for being the best labmate one could wish for. Without her support it would not have been possible to achieve some of the results. Thank you for our great friendship and always being there. I would like to thank my students Anne, Damian, Jakob, Michelle, and Ayşe for their experimental support and contribution to my research in the course of internships or master and bachelor theses. I am also greatly indebted to Prof. Dr. Czekelius, Dr. Holger Erdbrink and Dr. Johann Moschner for providing the fluorinated amino acids. Moreover, I thank Dr. Bernhard Loll, Claudia Alings and Nicole Holton from the Wahl group for their advice and assistance in crystallization experiments and ITC measurements. Thanks are due to all the staff members of the Core Facility BioSupraMol and Material Management for their friendly service and provision of the necessary infrastructure. I wish to acknowledge the Deutsche Forschungsgemeinschaft in the context of the Research Training Group 1582 “Fluorine as Key Element” for financial support. ix Finally, I am especially grateful to my friends and most of all to my family and Mone, the best “little” sister one can wish for. Their emotional and splendid support, as well as their unconditional love always gave me strength and motivation. Without them I would never have managed