Design of sensors for highly phosphorylated natural compounds Dissertation Submitted for the Degree Doctor rerum naturalium (Doctor rer. nat.) at the Faculty of Chemistry and Pharmacy University of Freiburg by Rahel Hinkelmann Rheinfelden (Baden) 2020 Vorsitzender des Promotionsausschusses: Prof. Dr. Stefan Weber Dekan: Prof. Dr. O. Einsle Referent/in: Prof. Dr. H. J. Jessen Korreferent/in: Prof. Dr. M. Müller Datum der mündlichen Prüfung: 30.10.2020 Abstract Highly phosphorylated metabolites, e.g. inositol polyphosphates (InsPs), diphospho- inositol phosphates (PP-InsPs), inorganic polyphosphate (polyP) and Magic spot nu- cleotides (MSN) regulate various biological processes. Their distinctive role in biology remain still elusive particularly concerning their dynamic turnover. The investigation of their contributions in metabolic processes asks for the development of, in vivo and in vitro, real time analytical detection. In this work the design, synthesis and evaluation of a set of new fluorescent chemosen- sors is presented, since inositol polyphosphates and polyP are lacking an intrinsic chro- mophore. The synthesized chemosensors can be divided into the following three cate- gories - Pyrene-excimer based, disassembly approach and DAPI derivatives. A 4’,6-diamidin-2-phenylindol (DAPI) synthesis was developed in this work. The photo- physical properties of the respective set of synthesized chemosensors in combination with the above mentioned phosphorylated metabolites was evaluated. Fe3+-Salen and PyDPA was successfully used as a polyacrylamide gel electrophoresis (PAGE) stain and the latter dye was applied as a post-column staining reagent for Ins(1,2,3,4,5,6)-hexakisphosphate (InsP6) on an ion chromatography system. In addition, the investigation and appli- cation of a new 31P-NMR based method using a chiral solvating agent revealed the stereoselectivity of the first described naturally occurring 1-phytase. Zusammenfassung Hoch phosphorylierte Metabolite, z.B InsPs, PP-InsPs, polyP und MSN regulieren ver- schiedene biologische Prozesse. Ihre besondere Rolle in der Biologie ist jedoch nach wie vor unklar, insbesondere was ihren dynamischen Umsatz betrifft. Die Untersuchung ihrer Beiträge zu Stoffwechselprozessen erfordert die Entwicklung einer in vivo und in vitro Echtzeitdetektion. In dieser Arbeit wird das Design, die Synthese und die Auswertung einer Reihe neuer fluoreszierender Chemosensoren vorgestellt. Die synthetisierten Chemosensoren können in die folgenden drei Kategorien unterteilt werden - Pyren-Excimer-basiert, «disassembly approach»und DAPI-Derivate. In dieser Arbeit wurde eine DAPI Synthese entwickelt. Die photophysikalischen Eigen- schaften des jeweiligen Sets der synthetisierten Chemosensoren in Kombination mit den oben genannten phosphorylierten Metabolite wurden diskutiert. Salen-Fe3+ und PyDPA wurden erfolgreich als PAGE-Färbung verwendet, und der letztgenannte Farbstoff konn- te als Nachsäulen-Färbereagenz für InsP6 auf einem Ionenchromatographiesystem ange- wendet werden. Darüber hinaus ergab die Anwendung eines neuen 31P-NMR basierten Verfahrens unter Verwendung eines chiralen Solvatisierungsmittels die Stereoselektivität der ersten beschriebenen natürlich vorkommenden 1-Phytase. Acknowledgements First of all, I want to thank Prof. Dr. Henning Jessen for the opportunity to be part of this exciting research from the very beginning in Freiburg. We started in empty labo- ratories and they were inspiring and productive years in which the group accomplished a great deal. I learned a lot from you about positive thinking, staying motivated and solving problems, in science and beyond. Thank you for your constant support, helpfull discussions and being such a great supervisor in the last years. I am grateful to Prof. Dr. M. Müller and Prof. Dr. P. Kurz for being part of my PhD committee. Furthermore, I want to thank the staff of the department of chemistry and pharmacy of the Albert-Ludwigs University. Especially Dr. M. Keller for his help regarding NMR and Dr. C. Warth for the HRMS measurements. Also, I want to thank Regine Schandera and Dr. Richard Krieger for the constant support during the last years. Especially to Regine for being the good soul of the group. Many thanks goes to all those who worked with me on this thesis: Larissa Pfennig and Paul Ebensperger for their support with the DAPI project. Ann-Kathrin Mündler for being the best Master student and the fun we had all the time. Alexander Ripp and Sophia Rauscher for their support in the Excimer project. Last but not least I have to express my deepest gratitude to Stephan Mundinger. Thank you for your invaluable help. v Special thanks go to Tamara Bittner, Verena Eisenbeis Nikolaus York, Dr. Jyoti Singh and Kathrin Hönerlage for proof-reading parts of my thesis. For the help in endless LaTeX questions I would like to say a big thank you to Kevin Ritter and Nicole Steck. A heartfelt thank you goes to all AK Jessen members, former and present. Dr. Jyoti Singh, Dr. Christopher Wittwer which whom I started this work years ago. Thomas Haas, Paul Ebensperger, Tobias Dürr, Isabel Pruker, Alexander Ripp, Dr. Danye Qui, Markus Häner, Sandra Moser for the good times in the lab. I enjoyed the time with you so much. I want to thank Paraskevi Fouka, Verena Eisenbeis, Jiahui Ma and Xuang Wang for the enjoyable company and conversations during lunch time. Also, I want to thank the friday evening group Tamara Bittner, Stephan Mundinger, Kevin Ritter, Nikolaus Jork and Dominik Bezold for the good times we had and the countless discussions about chemistry and more. Most importantly, I want to thank my friends and family for supporting me during the last years. Especially, my mother Andrea Hinkelmann and my father Horst Hinkelmann. Thank you for being with me for the whole journey and for always believing in me. A very special thanks goes to my love Claudio Werner. I cant find enough words to thank you for your support inside and outside the lab. And for all the great food! vi Contents Abbreviations vii 1. General Introduction 1 1.1. Biological importance of phosphates . 2 1.1.1. Myo-inositol phosphates . 5 1.1.1.1. Myo-inositol......................... 5 1.1.1.2. Inositol polyphosphates . 5 1.1.1.3. Biosynthesis of inositol polyphosphates . 7 1.1.1.4. Diphosphoinositol phosphate signalling . 9 1.1.1.5. IP6K1 as a drug target . 10 1.1.2. Inorganic polyphosphate (polyP) . 12 1.1.2.1. Biosynthesis of polyP . 12 1.1.2.2. PolyP signalling . 13 1.1.2.3. PPK as a drug target . 13 1.1.2.4. Phosphates in wastewater . 14 1.1.3. Magic Spot Nucleotides (MSN) . 15 1.1.3.1. Biosynthesis of (p)ppGpp . 16 1.1.3.2. (p)ppGpp signalling . 17 1.1.3.3. Rel as a drug target . 19 1.2. A brief history of fluorescence . 20 1.3. Supramolecular chemistry . 21 1.3.1. Fluorescent chemosensors . 22 i Contents 1.3.2. Anion sensing . 23 1.3.3. Ratiometric fluorescent probes . 23 1.3.3.1. Monomer/Excimer-based fluorescent probes . 24 1.3.3.2. ESIPT-based fluorescent probes . 25 1.3.4. Overview of chemosensors for myo-inositol polyphosphates, polyP and Magic Spot Nucleotides . 27 1.3.4.1. Chemosensor for myo-inositol polyphosphates . 27 1.3.4.2. Chemosensor for inorganic polyP . 32 1.3.4.3. Chemosensor for Magic Spot Nucleotides . 33 1.3.5. References............................... 35 2. State of the Art - Analytics 43 2.1. Radioactive labelling . 44 2.1.1. Inositol polyphosphates . 44 2.1.2. Magic Spot Nucleotides . 45 2.2. Nuclear Magnetic Resonance Spectroscopy (NMR) . 45 2.2.1. 31P-Nuclear Magnetic Resonance Spectroscopy . 46 2.2.1.1. Inorganic Polyphosphate (polyP) . 46 2.2.1.2. Inositol polyphosphates . 47 2.2.2. 13C-Nuclear Magnetic Resonance Spectroscopy . 48 2.2.2.1. Inositol polyphosphates . 48 2.3. EnzymeAssays ................................ 49 2.3.1. Inorganic Polyphosphate (polyP) . 49 2.4. Electrophoresis ................................ 50 2.4.1. Polyacrylamide Gel Electrophoresis (PAGE) . 51 2.4.2. Inorganic Polyphosphate (polyP) . 51 2.4.3. Inositol polyphosphates . 52 2.4.4. Capillary Electrophoresis (CE) . 54 2.4.5. Inositol polyphosphates . 55 2.4.6. Inorganic polyphosphate (polyP) . 55 ii Contents 2.5. Chromatography ............................... 55 2.5.1. Ionchromatography(IC). 55 2.5.2. Post column detection methods . 59 2.5.3. References............................... 62 3. Goals of the Thesis 67 4. Excimer based sensing 71 4.1. Background .................................. 72 4.1.1. Excimer fluorescence based chemosensors . 72 4.2. Results and Discussion . 74 4.3. Synthesis of excimer based chemosensors . 74 4.3.1. Tripodal chemosensors . 74 4.3.2. PyDPA chemosensor . 77 4.3.3. Linked chemosensor - Intramolecular excimer formation . 78 4.4. Sensor evaluation . 82 4.4.1. Photophysical properties . 82 4.4.1.1. Tripodal chemosensors . 83 4.4.1.2. Linked chemosensors . 86 4.4.2. PAGE-Gel staining . 89 4.4.3. Post column derivatisation - Ion Chromatography . 91 4.5. Summary & Outlook . 97 4.6. Experimental ................................. 101 4.6.1. Generalremarks ........................... 101 4.6.2. Synthesis ............................... 104 4.6.3. References............................... 123 5. Sensing via a disassembly approach 125 5.1. Background .................................. 126 5.1.1. Disassembly Approach . 126 iii Contents 5.2. Results and Discussion . 128 5.2.1. Synthesis of disassembly based fluorescent probes . 128 5.2.1.1. Salicylaldehyde-based fluorescent probes . 128 5.2.1.2. Naphthol-based fluorescent probes . 130 5.2.2. Excited state intramolecular proton transfer - ESIPT . 131 5.2.2.1. HBO-based fluorescent probes . 131 5.3. Sensor evaluation . 137 5.3.1. Photophysical properties . 138 5.3.1.1. Fe3+-Salen.......................... 138 5.3.1.2. HBO-based chemosensors . 139 5.3.1.3. Naphthol-based chemosensors . 144 5.3.1.4. PAGE-Gel staining . 145 5.4. Summary & Outlook . 148 5.5. Experimental ................................. 151 5.5.1. Generalremarks ........................... 151 5.5.2. Synthesis . 154 5.5.3. References............................... 167 6. Sensing via hydrogen bonding 169 6.1. Background .................................. 170 6.1.1.