Investigations on 2,7-diamino-9-fluorenol photochemistry INAUGURALDISSERTATION zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von DRAGANA ZIVKOVIC aus Pirot, Serbien Basel, 2007 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. J. Wirz und Prof. Dr. H. Huber. Basel, den 24. 04. 2007. Prof. Dr. Hans Peter Hauri Dekan Dedicated to my family and to Ian Acknowledgments First of all, I would like to thank Prof. Dr. Jakob Wirz for giving me the opportunity to join his research group, for guiding and supporting my work. I thank Prof. Dr. Hanspeter Huber for agreeing to act as co-referee. I thank Prof. Dr. Martin Jungen for agreeing to act as chairman of the thesis committee. A special thanks to the members of the Wirz group: Hassen Boudebous, Martin Gaplovsky, Yavor Kamdzhilov, Gaby Persy, Bruno Hellrung, Jürgen Wintner, Pavel Müller and Dominik Heger. Thanks for making the atmosphere in the lab so enjoyable, for the useful discussions and for always being ready to help. I thank my family and friends, especially Mat and Janni, for their affection and constant encouragement. I thank piggy, for being there for me, and for cheering me up when experiments didn’t work. Finally, I would also like to thank the Swiss National Science Foundation and the University of Basel for their financial support. Table of Contents 1. Introduction.................................................................................................................1 1.1. Major photoremovable protecting groups........................................................... 3 1.1.1. The 2-nitrobenzyl group ............................................................................. 3 1.1.2. The benzoin group ...................................................................................... 5 1.1.3. The p-hydroxyphenacyl group.................................................................... 7 1.1.4. The coumarinyl group................................................................................. 9 1.1.5. The other groups....................................................................................... 10 1.2. Applications of the photoremovable protecting groups.................................... 11 1.2.1. Photorelease of neurotransmitters............................................................. 11 1.2.2. Photorelease of second messengers .......................................................... 14 1.2.3. Photorelease of peptides and proteins....................................................... 17 1.2.4. Photoactivatable fluorophores .................................................................. 19 1.2.5. Two-photon excitation.............................................................................. 20 1.3. Photochemistry of 9-fluorenol .......................................................................... 21 1.3.1. The polyfluorinated alcohols case ............................................................ 25 2. Problem statement..................................................................................................... 27 3. Synthesis ................................................................................................................... 29 3.1. Synthesis of 2,7-diamino-9-fluorenol ............................................................... 29 3.2. Synthesis of 2,7-diamino-9-fluorenol derivatives............................................. 31 4. Photochemical Studies.............................................................................................. 42 4.1. Photorelease from 2,7-diamino-9-fluorenol...................................................... 43 4.1.1. Product studies.......................................................................................... 43 4.1.1.1. In polyfluorinated alcohols ............................................................... 43 4.1.1.2. In methanol ....................................................................................... 44 4.1.2. Laser Flash Photolysis .............................................................................. 47 4.1.2.1. In polyfluorinated alcohols ............................................................... 47 4.1.2.2. In methanol and water....................................................................... 56 4.1.3. Discussion................................................................................................. 58 4.2. Photorelease from 2,7-diamino-9-fluorenyl acetate ......................................... 61 4.2.1. Product studies.......................................................................................... 61 4.2.2. Discussion................................................................................................. 63 4.3. Photorelease from 2,7-diamino-9-fluorenyl phenyl ether................................. 64 4.3.1. Product studies.......................................................................................... 64 4.3.2. Discussion................................................................................................. 66 5. Conclusions............................................................................................................... 67 6. Experimental............................................................................................................. 68 6.1. Irradiation.......................................................................................................... 68 6.2. Flash photolysis ................................................................................................ 68 6.3. Analytical equipment........................................................................................ 69 6.4. Materials ........................................................................................................... 71 7. References................................................................................................................. 72 8. Summary................................................................................................................... 79 9. Curriculum Vitae ...................................................................................................... 81 10. Appendix................................................................................................................... 83 1. Introduction Protecting groups have been long known as useful synthetic tools, especially in multistep organic synthesis. They are introduced into a molecule by chemical modification of a functional group to obtain maximum control and selectivity in the subsequent reaction. There are numerous considerations which define how effectively a protecting group will fulfill its assigned role of shielding a functional group from destruction1,2. The protecting group should be easily and efficiently introduced, as well as cheap and readily available. It should be stable to the widest possible range of reaction conditions and removed selectively and efficiently under highly specific conditions, leaving intact all the other groups (the so-called “orthogonality”)3. The removal of the protecting group can be achieved by chemical, electrolytic or photolytic methods. When it is achieved photolytically, i.e. no reagent for its cleavage is required - just light, we are talking about photoremovable protecting groups. This category of protecting groups offers the possibility of dealing with extremely sensitive molecules, otherwise incompatible with acids or bases4. Also, the release of the substrate can be strictly controlled, both the concentration and the spatial distribution. Very good general reviews have been published in this field2,5-8. Photoremovable protecting groups are used for a broad range of applications in organic synthesis, biophysics and biology. They are commonly linked to a substrate of interest to make them unable to react, forming in this manner a “caged compound”6-11. The term “caged compound” was coined in 1978 for photolabile derivatives of natural substrates such as ATP, first reported by Kaplan, Forbush and Hoffman12. More recently, the term phototrigger has become more broadly accepted as a more accurate of the photoactive protective group. Caged compounds are widely used to start biological reactions by the photolytic release of an effector molecule from a biologically inactive precursor. Upon irradiation, typically breaking the photolabile bond with a pulse of intense light in the near ultraviolet (UV; 350–360 nm) range, the protecting group undergoes a photochemical reaction that results in release of the substrate and restores its activity. In this fashion it is possible to deliver bioactive materials such as neurotransmitters, ATP, L-glutamate, γ-aminobutyric acid (GABA) or Ca2+ ions 1 rapidly to small target sites and therefore enable following ensuing physiological events in real time. Amplified chemical effects may be achieved by controlling enzyme activity, gene expression or ion channel permeability with light (phototriggers). Typical procedure for caging and uncaging the protein is shown as an example below: Several potential chromophores have been examined for cage applications, but only very few have met the demands or been successfully developed. The constraints include both mechanistic requirements and changes in physical properties that accompany the attachment of a chromophore to a substrate. An important limiting factor is an ease of synthesis