Characterization and Synthesis of Selected Secondary Metabolites produced by Xenorhabdus and Photorhabdus spp Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt dem Fachbereich der Biowissenschaften (15) der Johann Wolfgang von Goethe Universität, Frankfurt a. M. von Friederike Inga Nollmann geboren am 30. 11. 1985 in Stade D30 vom Fachbereich für Biowissenschaften (15) der Johann-Wolfgan-von-Goethe-Universität als Dissertation angenommen. Dekanin: Prof. Dr. Meike Piepenbring Gutachter: Prof. Dr. Helge B. Bode Jun. Prof. Dr. Martin Grininger Datum der Disputation: 3 4 There is no answer as big as the question, there is no victory as big as the lesson, you go on and you see where your detours will take you to, there is no power like understanding. Tina Dico To my friends and my family who were neglected now and then in the process of this work but nevertheless helped to make it happen. 5 6 Acknowledgement Anybody who has been seriously engaged in scientific work of any kind realizes that over the entrance to the gates of the temple of science are written the words: “Ye must have faith.” Max Planck With these words I would like to thank all the people who were involved in this work and had to restore my faith in sciences from time to time, namely Prof. Dr. Helge B. Bode, my mentor, who gave me the opportunity to work on a quite diverse topic which never stopped being challenging. I also appreciate it that he always had the confidence in me to make it click. Jun. Prof. Dr. Martin Grininger, my second reviewer, who was willing to survey this work without hesitation. Thanks a lot for your time! Christina Dauth, my tutor, who showed me that organic chemistry can be fun and never grew tired of supplying me with new ideas or solutions. Thanks for everything, animation coach! Max Kronenwerth, my optimist, who talked me into this thesis and hopefully never regretted it. Daniela Reimer, my mom in the bio-lab, who always shared her pipettes, media, antibiotics, working space and knowledge with me without a second thought. Carsten Kegler, my constant supplier of critical remarks and the daily weather forecast, who had to put up with me in the office and nevertheless, always was happy to help and educate me. Samina Atri, my sun shine, who brought colors into my every day and never lost her high spirtis. 7 Arne Kittler, my voice of reason, who picked me up when I shattered into pieces and turned out to be solid as a rock. Peter Grün, Olivia Schimming, Yvonne Engel and Hélène Adihou, my amazon crew, who put up with me and the occasionally HPLC break down. Katharina Scholz, Lena Kalinowsky, Jens Frindert, Sylvester Größl and Aljoscha Joppe, my students, we learned and grew together – thanks for the fun with you, guys! Vivien Schulz, Carsten Kegler, Martina Blum, Gesine Hübner and Barbara Fleischer, my proofreading crew, who merciless eliminated every mistake they could find. And last but not least, the whole Bode lab, who always guaranteed a constant supply of cakes and sweets as well as the badly needed cup of coffee. 8 Table of Contents Abstract .......................................................................................................................... 13 Zusammenfassung ............................................................................................................ 15 List of Selected Publications ............................................................................................ 21 List of Selected Contribution to Conferences ................................................................. 22 1 Introduction ........................................................................................................ 27 1.1 Xenorhabdus and Photorhabdus spp. .................................................................. 28 1.2 Secondary metabolites ......................................................................................... 29 1.2.1 Nonribosomal peptide synthetase-derived secondary metabolites ........................ 32 1.3 Organic Peptide Synthesis and its Limitations ...................................................... 34 1.3.1 Solution Phase Synthesis ..................................................................................... 35 1.3.2 Solid Phase Synthesis .......................................................................................... 35 1.3.3 Limitations and how to Circumvent them .............................................................. 36 2 Objectives ........................................................................................................... 47 3 Small Molecules ................................................................................................. 49 3.1 A Photorhabdus natural product inhibits insect juvenile hormone epoxide hydrolase ............................................................................................................. 51 4 Cyclic Peptides ................................................................................................... 53 4.1 Synthesis of the Xenotetrapeptide produced by Xenorhabdus nematophila ......... 57 4.2 Insect-specific production of novel GameXPeptides, widespread natural products in entomopathogenic Photorhabdus and Xenorhabdus bacteria .............................. 63 4.3 Synthesis of Ambactin, a cyclic peptide from Xenorhabdus miraniensis ............... 67 5 Cyclic Depsipeptides ......................................................................................... 71 5.1 Synthesis of szentiamide, a depsipeptide from entomopathogenic Xenorhabdus szentirmaii with activity against Plasmodium falciparum ...................................... 73 5.2 Synthesis of Selected Xentrivalpeptides ............................................................... 77 6 Methylated peptides ........................................................................................... 85 6.1 Synthesis of short partly methylated sequences in solution .................................. 86 6.2 Synthesis of partly methylated sequences on resin .............................................. 91 6.3 Synthesis of permethylated sequences ................................................................ 92 6.4 Synthesis of highly methylated sequences ........................................................... 95 6.5 Fragment condensation approach ........................................................................ 96 7 Target Identification ......................................................................................... 103 9 Table of Contents 7.1 Biotinylated GameXPeptides .............................................................................. 103 7.2 Intrinsically labelled Xenephematides ................................................................. 106 8 Discussion ........................................................................................................ 115 8.1 Small Molecules ................................................................................................. 115 8.1.1 Phurealipids ..................................................................................................... 115 8.2 Cyclic Peptides ................................................................................................... 116 8.2.1 Xenotetrapeptide ................................................................................................ 116 8.2.2 GameXPeptides ................................................................................................. 117 8.2.3 Ambactin ..................................................................................................... 118 8.3 Cyclic Depsipeptides .......................................................................................... 119 8.3.1 Szentiamide ..................................................................................................... 119 8.3.2 Xentrivalpeptides ................................................................................................ 120 8.3.3 Xenephematide .................................................................................................. 120 8.4 Methylated Peptides ........................................................................................... 121 APPENDIX ........................................................................................................................ 129 General Procedures ......................................................................................................... 131 I HPLC Coupled to ESI-MS .................................................................................. 131 II NMR ............................................................................................................ 131 III Solid Phase Synthesis ..................................................................................... 131 III.I Immobilization of the First Amino Acid on the Solid Support ....................... 131 III.I.I Wang resin ......................................................................................................... 131 III.I.II 2-Chloro Trityl Chloride resin .............................................................................. 132 III.I.III Rink Acid Resin .................................................................................................. 132 III.II Deprotection ..................................................................................................... 132 III.II.I Orthogonal Deprotection of Fmoc-protected Amines .........................................