Bioinspired Model Complexes For Vanadium Haloperoxidases DISSERTATION zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt dem Rat der Chemisch-Geowissenschaftlichen Fakult¨at der Friedrich-Schiller-Universit¨at Jena von M. Sc. Simona Filofteia Nica geborem am 19.12.1976 in Curtea de Arge¸s, Romania Gutachter 1. Winfried Plass 2. Matthias Westerhausen Tag der offen¨ tlichen Verteidigung: September 22nd 2005 "Most zealously I've studied matters great and small; Though I know much, I should like to know all." Goethe Din s^ınul vecinicului ieri Trˇaie¸ste azi ce moare, Un soare de s-ar stinge-n ceri S-aprinde iara¸si soare. Mihai Eminescu In memory of the most beloved father and to the memory of an extraordinary Professor, Dr. Ovidiu Maior. Acknowledgements I would like to thank my supervisor, Prof. Dr. Winfried Plass, for giving me the oppor- tunity to work in this interesting field and for support with new ideas and advises during my research. Special thanks to my partner, colleague and best friend, Adrian Ion, for moral sup- port, patience and especially for helping me in having new ideas through our multitude of scientific conversations. I also thank to my colleague, Dr. Axel Pohlmann, who helped a romanian student lost in the german bureaucracy. Thanks also for bringing me from Siegen to Jena and taking care of the new employment procedures. Without you, most likely, I would have been deported three years ago. Of course, I thank to him, more importantly, for helping me find the way through the german world of computers and teaching me how to use special programs. Lots of thanks to Dr. Andrew Hall for nice friendship and for language support. Only with your support my thesis reassembles now the English language. And because we are here, I say millions of thanks to Dr. Ioana Pera, my best romanian friend. Thank you very much for helping me to find my house in Siegen, for accompanying me shopping and for the very nice time spent together. Thanks to Masroor, my Pakistani colleague for nice and enjoyable evenings spent together and also for the nice talks about the structure of the enzyme and for correcting my English in the manuscripts. I also thank to my colleague Arne Roth for his patience in explaining me how the LATEXworks and especially for his help in the arrangement of the thesis. Many thanks to Ines Seidel for helping me with catalytic tests and some experiments in the last months while writing my manuscripts and my thesis. Also, lots of thanks to my student, Bj¨orn Kusebauch for bringing me so many results during his "Forschungsprak- tikum". I would like to thank to Dr. Manfred Rudolph, who measured the cyclic voltam- metry experiments of my complexes and also for helping me to interpret the spectropho- tometric titration data. I would like to thank to the NMR team, who measured my tons of samples and also for trying to understand my very bad german language. Many thanks to Lotte Neupert for the HPLC measurements. Lots of thanks to a special person, Dr. Helmar G¨orls for measuring my X-ray crystal structures and for his high sense of humor. He knew how to bring the smile on my face even when the measured structures were the by-products. Thank you very much. I also thank to my romanian friend, now Dr. Maria-Magadalena Titirici for her help and understanding me. Last but not least, I would like to thank to all my family for financial and moral support during all my studies. Thanks to my brother, Dr. Dorel-Daniel Nica for his kindness and financial support and also many thanks for the nice political and literature talks. Thanks to my parents for understanding and supporting me in all my life deci- sions. Unique thoughts of gratitude go to my wonderful father. I wish I would had the opportunity to share all this achievements with you. I miss you and I wish you were still here with me. Summary Vanadium haloperoxidases represent a class of non-heme peroxidases with a high stabil- ity to organic solvents and high temperatures. These enzymes proved also to be very stable in presence of excess of their product (HOCl) which will readily inactivate the heme-haloperoxidases. The active group of the enzymatic system consists in a vana- date moiety, solely covalently bonded to a histidine residue and embedded in the protein shell via a strong hydrogen bonding network. Structural similarities between the hydro- gen bonding site of vanadium haloperoxidases and some acid phosphatases are evident. An important role for the haloperoxidase activity has been attributed to the hydrogen bonding interactions which besides the fixation of the prosthetic group in the protein environment are also activating the peroxide-intermediate. The goal of this work was the synthesis of vanadium(v) complexes which model the active site and the catalytic function of these enzymes. Vanadium in the highest oxidation state proved to be a compulsory characteristic of model complexes, since the reduced form of vanadium haloperoxidase showed no activity. Therefore, vanadium(v) complexes with versatile N-salicylidene carbonic acid hydrazide were prepared. Both oxo and dioxovanadium complexes will be described in Chapter 2 and Chapter 3, with both types of vanadium coordination compounds regarded as model complexes for the natural system. Besides the crystallographic description of these complexes, reactivity in solution will be also discussed. Starting from a non-substituted aliphatic side chain of the N-salicylidene hydrazide ligand, new functionalities were introduced stepwise. This achievement had the role to probe the importance of the amino acid residues which are in hydrogen bonding inter- action with the equatorial oxygen atoms of the prosthetic group in vanadium containing haloperoxidases. The modification of the aliphatic side chain will be discussed in sepa- rate chapters, therefore additional groups such as hydroxyl, amine and guanidine afforded vanadium(v) complexes with relevant functionalized side chain for the vanadium haloper- oxidases, namely regarding the serine, lysine and arginine residues. The mechanism of the catalytic reaction performed by vanadium haloperoxidases was proposed to take place by addition first of the hydrogen peroxide at the vanadate 1 center to form a peroxo-intermediate which is subsequently able to oxidize the halides in slightly acidic media. In Chapter 2, the characterization and reactivity of an oxo- monoperoxovanadium(v) complex is presented. Further on, the cyclization on vanadium coordination of a short amino-functionalized aliphatic side chain of N-salicylidene hy- drazide ligand as well as the first example of guanilation reaction of a vanadium coordi- nation compound will be described in Chapter 4 and 5, respectively. The cis-dioxovanadium(v) complexes obtained with N-salicylidene carbonic acid hydrazides proved also to catalyze the biomimetic reaction. The bromide oxidation re- action by hydrogen peroxide was monitored by bromination of 1,3,5-trimetoxybenezene in acidified DMF solution. Apart from this specific enzymatic reaction, the model com- plexes were also able to catalyze the peroxide-mediated oxidation of phenyl methyl sulfide to the corresponding sulfoxide. The replacement of vanadate from vanadium haloperoxidase with molybdate or tungstate yielded an inactive enzyme. Therefore, cis-dioxomolybdenum(vi) complexes based on N-salicylidene amino-functionalized aliphatic acid hydrazide were also obtained in order to address the question whether a synthetic molybdenum complex can catalyze the bromoperoxidase reaction. This was also based on the reported high bromoperoxidase activity of the inorganic MoO3 (aq.) compound. But, however the cis-dioxomolybdenum complex based on N-salicylidene dihydropyrrole hydrazide showed no bromoperoxidase activity, but instead the complex was capable of catalyzing efficiently the oxidation of sulfide to sulfoxide. In the last chapter, bis-N-salicylidene hydrazide ligands were prepared by the con- densation of salicylaldehyde with bis-aliphatic hydrazides. The ligand H4Ln may be con- sidered as two N-salicylidene hydrazide cavities linked via a methylene chain of variable length. The ligand is able to bind two metal ions in a hexacoordinate fashion yielding bin- uclear cis-dioxovanadium(v) and cis-dioxomolybdenum(vi) complexes. The complexes showed a high efficient capacity in catalyzing the oxidation of sulfide when H2O2 was used as oxidant. Moreover, a chiral binuclear cis-dioxomolybdenum(vi) complex was isolated and used as catalyst of the sulfoxidation reaction. The complex proved to be an efficient catalyst of the reaction, but only 4% ee was obtained when the catalytic reaction was performed in dichlormethane. 2 Zusammenfassung Vanadium-Haloperoxidasen stellen eine Klasse von nicht-H¨am-Peroxidasen mit hoher Best¨andigkeitgegenub¨ er organischen L¨osungsmitteln und hohen Temperaturen dar. Diese Enzyme sind außerdem in Gegenwart von Ub¨ erschuss ihres Produktes (HOCl), was h¨am- Haloperoxidasen leicht inaktivieren wurde,¨ sehr stabil. Das aktive Zentrum des enzyma- tischen Systems besteht aus einer Vanadatumgebung, welche kovalent an nur einen Histid- inrest gebunden und im Proteinrumpf mittels eines starken Wasserstoffbruc¨ kennetzwerks verankert ist. Strukturelle Ahnlic¨ hkeiten zwischen der Wasserstoffbruc¨ kenumgebung von Vanadium-Haloperoxidasen und einigen sauren Phophatasen sind bewiesen. Eine wichtige Rolle fur¨ die Haloperoxidaseaktivit¨at wird den Wasserstoffbruc¨ kenwechselwirkungen ein- erseits bei der Fixierung der prosthetischen Gruppe in der Proteinumgebung