Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians Universität München Synthesis and Characterization of a Metal-Salen Base Pair for the Assembly of Programmed Metal Arrays inside the DNA Double Helix Synthese und Charakterisierung eines Metall-Salen Basenpaars für den Aufbau von programmierten Metallanordnungen im Inneren von DNA-Doppelhelices Guido Clever aus Bad Neuenahr 2006 1 2 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung der LMU München vom 29. Januar 1998 von Prof. Dr. T. Carell betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet. München, den 23.11.06 Guido Clever Dissertation eingereicht am 24.11.06 1. Gutachter: Prof. Dr. T. Carell 2. Gutachter: Prof. Dr. P. Klüfers Mündliche Prüfung am 19.12.06 3 4 Ltd. Doolittle: „Wenn Zusammenhänge logisch sind, dann sind sie das unabhängig von ihrem Ursprung.“ Bombe # 20: „Hmmm…“ Dark Star (John Carpenter, 1974) Meiner Familie und Michi gewidmet. 5 Parts of this work were published or presented on conferences G. H. Clever, K. Polborn, T. Carell, Angew. Chem. Int. Ed. 2005, 44, 7204 - 7208. “A Highly DNA-Duplex-Stabilizing Metal-Salen Base Pair” G. H. Clever, Y. Söltl, H. Burks, W. Spahl, T. Carell, Chem. Eu. J. 2006, 12, 8708 - 8718. “Metal-Salen-Base-Pair Complexes Inside DNA: Complexation Overrides Sequence Information“ G. H. Clever, T. Carell, Angew. Chem. 2006, in press (DOI: anie.200603099). “Controlled Stacking of 10 Transition Metal Ions inside a DNA Duplex” K. Tanaka*, G. H. Clever*, Y. Takezawa, Y. Yamada, C. Kaul, M. Shionoya, T. Carell, Nature Nanotech. 2006, in press (DOI: 10.1038/nnano.2006.141). „Programmable Self-Assembly of Metal Ions inside Artificial DNA Duplexes“ G. H. Clever, T. Carell, Coll. Symp. Ser. (M. Hocek, Ed), Vol 7, p. 389 - 391. Institute of Org. Chem and Biochem., Academy Of Sciences of the Czech Rep., Prague 2005. “Assembly of a Highly DNA-Duplex-Stabilizing Metal-Salen Base Pair” Poster presentations on JCF meeting, Heidelberg 2004 (poster prize); ORCHEM, Bad Nauheim 2004; Nanobionics III, Marburg 2005; NAC XIII, Prague 2005; Summer School FrontChem, Tokyo 2006; Int. COE Symposium, Tokyo 2006. Oral presentations on Volkswagen Foundation meeting, Aachen 2005; “Roche Symposium for Leading Scientists of the next decade”; Basel 2005, FCI stipend meeting, Munich 2006. 6 Table of contents 1 Summary ............................................................................................................. 9 2 Zusammenfassung............................................................................................ 13 3 Introduction........................................................................................................ 16 3.1 Bionanotechnology..................................................................................... 16 3.1.1 Structure and function of biopolymers................................................. 16 3.1.2 Nature as paragon for nanotechnology ............................................... 18 3.1.3 Examples of oligonucleotide-based nanoscale objects ....................... 20 3.2 Hypermodification of the DNA backbone and bases .................................. 27 3.3 Metal coordination inside/to nucleic acids .................................................. 28 3.3.1 Metal coordination to unmodified DNA................................................ 28 3.3.2 The metal-base pair concept and examples........................................ 30 3.4 Coordination chemistry: salen complexes and metal stacking ................... 37 3.4.1 Monomeric and oligomeric salen complexes....................................... 37 3.4.2 Interaction of metal-salen complexes with DNA .................................. 39 3.4.3 Metal stacks and arrays in solid state structures and in solution......... 39 4 Part I: Coordination of metals inside the DNA duplex........................................ 43 4.1 Aims of project (part I) ................................................................................ 43 4.2 Synthesis of ligand nucleosides ................................................................. 44 4.2.1 Synthesis of the salicylic aldehyde nucleobase................................... 44 4.2.2 Synthesis and X-ray structure of a monomeric Cu2+-salen-base pair.. 54 4.2.3 Attempts to prepare a hydroxyphenyl-oxazoline nucleoside ............... 58 4.2.4 Incorporation of an oxazolinylidene-indolone nucleoside into DNA..... 60 4.2.5 Synthesis of 3’-O-methylxylose-based C-nucleosides ........................ 64 4.3 Incorporation of the salicylic aldehyde nucleoside into oligonucleotides .... 70 4.3.1 Automated DNA synthesis .................................................................. 70 4.3.2 Deprotection of the incorporated nucleosides ..................................... 73 4.3.3 Chromatographic purification of aldehyde carrying oligonucleotides... 74 4.4 Assembly of the metal-salen base pair....................................................... 77 4.4.1 Hybridization and reaction with amines and metal ions....................... 77 4.4.2 Melting point studies ........................................................................... 80 4.4.3 UV-Vis and CD spectroscopy.............................................................. 92 4.4.4 ESI mass spectrometry ....................................................................... 98 4.4.5 Liquid chromatography-mass spectrometry (LC-MS).........................103 4.4.6 EPR spectroscopy..............................................................................104 7 4.5 Interplay of salen complex formation and DNA sequence.........................107 4.5.1 Complex formation in different sequence context ..............................107 4.5.2 Brick-wise elongation of overlapping sequences................................110 4.5.3 (Unspecific) hairpin formation and dynamics......................................111 4.5.4 Reaction of single strands..................................................................115 4.6 Metal stacks and arrays ............................................................................116 4.6.1 Two metal-salen complexes inside one duplex..................................116 4.6.2 Homo-polynuclear metal stacks inside DNA ......................................117 4.6.3 Hetero-polynuclear stacks: controlled mixing of metals inside DNA...121 4.7 Conclusion and outlook (part I) .................................................................130 5 Part II: Coordinating metals on the exterior of the DNA double helix................132 5.1 Aims of project (part II) ..............................................................................132 5.2 Synthesis of ligand-modified uridine compounds ......................................132 5.3 “Clicking” of gold clusters to DNA via glutathione-bisazide .......................139 5.4 Conclusion and outlook (part II).................................................................141 6 Experimental part .............................................................................................142 6.1 Materials and methods ..............................................................................142 6.2 DNA synthesis, cleavage and purification .................................................142 6.3 Melting point experiments .........................................................................143 6.4 UV and CD spectra and titrations..............................................................143 6.5 ESI mass spectrometry .............................................................................144 6.6 Synthesis of the salicylic aldehyde nucleobase.........................................145 6.7 Synthesis of a monomeric copper salen complex .....................................151 6.8 Synthesis of a 3-(2-oxazolidinylidene-)indol-2-one nucleoside..................154 6.9 Synthesis of 3’-O-methyl-xylopyranosyl nucleosides.................................159 6.10 Synthesis of a hydroxyphenyl-oxazoline-uridine nucleoside .....................165 6.11 Synthesis of a benzotriazole-uridine nucleoside .......................................169 6.12 Synthesis of a glutathione-bisazide...........................................................175 7 Appendix ..........................................................................................................177 7.1 Further selected ESI spectra.....................................................................177 7.2 Crystallographic data.................................................................................181 7.3 Abbreviations ............................................................................................186 8 References .......................................................................................................187 8 Chapter 1 Summary 1 Summary The controlled assembly of functional nanoscale materials from molecular entities is regarded as a key subject of future nanotechnology. Currently, the use of DNA, which features superior self organization properties, is heavily investigated. The aim of this work was the development of new systems for the controlled binding of multiple metal ions to the inside or outside of modified DNA double strands. In the “metal-base pair concept”, the natural DNA base pairs are replaced by flat metal complexes. The placement of metals inside the chiral DNA environment may result in enantioselective catalytic