Metal-Peptide Bioconjugates for Targeted Anti-Cancer Therapy
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METAL-PEPTIDE BIOCONJUGATES FOR TARGETED ANTI-CANCER THERAPY Dissertation submitted to the Faculty of Chemistry and Biochemistry at the Ruhr-University Bochum, Germany to obtain the degree Doctor of Natural Sciences presented by Dariusz Śmiłowicz, M. Sc. Bochum, January 2020 This work has been carried out between December 2015 and January 2020 under the supervision of Prof. Dr. Nils Metzler-Nolte at the Chair of Inorganic Chemistry I – Bioinorganic Chemistry at the Faculty of Chemistry and Biochemistry, Ruhr-Universität Bochum. Date of oral examination: 08.05.2020 1st Referee: Prof. Dr. Nils Metzler-Nolte 2nd Referee: Prof. Dr. Gilles Gasser 2 Acknowledgements First of all, I would like to express my sincere gratitude to Prof. Dr. Nils Metzler-Nolte for giving me the opportunity to carry out my Ph.D. thesis under his supervision. In my opinion the most important is to work and learn from intimidating person and outstanding scientist. I was lucky to have both. Nils is a lively, enthusiastic, and energetic person, and is always in a good mood, eager to discuss any issue. This is particularly amazing. Moreover, I am also very grateful to Nils for his scientific advices, knowledge, many insightful discussions and suggestions, and for giving me the freedom to pursue various projects. I would like to gratefully acknowledge Prof. Dr. Gilles Gasser for being the second referee of my thesis. In particular I‘d like to thank Dr. Reece Miller for many scientific and life-related discussions. He belongs to the group of interesting people, which you may be lucky to encounter on your way. Dr. Jack Slootweg, my supervisor in the lab during my first practical time in this group, I also thank for his guidance and a highly inspiring time. To Dr. Matthew Reback, Sugina Thavalingam and Pina Eichert, I would like to say massive thanks for always advising on chemistry matters, the interesting discussions, countless advices, valuable time and for always being supportive. All the members of the AC1 group: Isabelle Daubit, Liudmila Janzen, Milena John, Nicole Lorenz, Daniel Obitz, Dr. Evgenia Olshvang, Dr. Sreedhar Kumar Vellas and Dr. Joan Soldevila-Barreda, I thank for the constant support and for their remarkable willingness to help. I would also like to thank the former Ph.D. students in this group, Dr. Martin Strack, Dr. Daniel Siegmund, Dr. Kathrin Klein, Dr. Anna Cordes and Dr. Yvonne Gothe. They have been helpful in providing advice many times during my research. And a special thanks to Nicole Ray, Dr. Christiane Klare, Carsten Lodwig, Marvin Heller, Annegret Knüfer and Dr. Klaus Merz. I could always ask them for advice and opinions on lab and on life related issues. And last but not least, I would like to especially thank my mom, sisters, and brothers. I know I can always rely on my family when times are rough. 3 ―Celui qui passe à coté de la plus belle histoire de sa vie n'aura que l'âge de ses regrets et tous les soupirs du monde ne sauraient bercer son âme.‖ - Yasmina Khadra, Ce que le jour doit à la nuit ―The man who let the love of his life pass him by will end up alone with his regrets and all the sighs in the world won't soothe his soul.‖ - Yasmina Khadra, What the day owes the night 4 Content 1 Introduction..............................................................................................................9 1.1 Metals in anti-cancer therapy..........................................................................9 1.2 Cell penetrating peptides................................................................................19 1.3 Cell targeting peptides....................................................................................22 1.4 Metal-peptide bioconjugates...........................................................................25 2 Objective....................................................................................................................29 3 Results........................................................................................................................30 3.1 Synthesis of monofunctional platinum(IV) carboxylate precursors for use in Pt(IV)–peptide bioconjugates..........................................................................30 3.1.1 Abstract...............................................................................................30 3.1.2 Introduction.........................................................................................31 3.1.3 Results and discussion.........................................................................33 3.1.4 Conclusion...........................................................................................44 3.1.5 Experimental section...........................................................................45 3.2 Bioconjugates of Co(III) Complexes with Schiff Base Ligands and Cell Penetrating Peptides: Solid Phase Synthesis, Characterization and Antiproliferative Activity...............................................................................51 3.2.1 Abstract..............................................................................................51 3.2.2 Introduction........................................................................................52 3.2.3 Results and discussion........................................................................55 3.2.4 Conclusion..........................................................................................65 3.2.5 Experimental section..........................................................................66 5 3.3 Bioconjugation of cyclometalated gold(III) lipoic acid fragment to linear and cyclic breast cancer targeting peptides...........................................................71 3.3.1 Abstract..............................................................................................71 3.3.2 Introduction........................................................................................72 3.3.3 Results and discussion........................................................................76 3.3.4 Conclusion..........................................................................................87 3.3.5 Experimental section..........................................................................88 4 Summary...................................................................................................................93 5 Literature..................................................................................................................95 6 List of contributions.................................................................................................121 7 Appendix (Supporting Information)......................................................................122 7.1 Appendix for chapter 3.1...............................................................................122 7.2 Appendix for chapter 3.2...............................................................................134 7.3 Appendix for chapter 3.3...............................................................................145 6 List of abbreviations AA amino acid ACN acetonitrile Boc tert.-butyloxycarbonyl Carboplatin [(diammine)(1,1-cyclobutanedicarboxylato)platinum(II)] Cisplatin cis-[PtCl2(NH3)2]) CBDC cyclobutane-1,1-dicarboxylate CDDP cis-[diamminedichloridoplatinum(II)] (cisplatin) DACH diaminocyclohexane DCM dichloromethane DIPEA N,N-diisopropylethylamine DMEM Dulbecco‘s Modified Eagle‘s Medium DMF dimethylformamide DMSO dimethyl sulfoxide EDTA ethylenediaminetetraacetic acid ESI electrospray ionisation Et2O diethyl ether FDA Food and Drug Administration Fmoc fluorenylmethoxycarbonyl Heptaplatin [(Propanedioato)(2-(1-methylethyl)-1,3-dioxolane-4,5- dimethanamine)platinum(II)] HOBt 1-hydroxybenzotriazole HPLC high performance liquid chromatography IC50 half maximal inhibitory concentration IR infrared lobaplatin [(2-Hydroxypropanoato)(1,2-cyclobutanedimethanamine)platinum(II)] 7 m/z mass to charge ratio MALDI matrix assisted laser desorption/ionization MeOH methanol MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide nedaplatin [diammine(hydroxyacetato)platinum(II)] NMR nuclear magnetic resonance ox oxalate oxaliplatin [1(R),2(R)-cyclohexane-1,2-diammine](oxalato)platinum(II) PBS phosphate buffered saline SPPS solid-phase peptide synthesis TBTU O-(benzotriazol-1-yl)-N,N,N‘,N‘-tetramethyluronium tetrafluoroborate TFA trifluoroacetic acid TIS triisopropylsilane For amino acids 1-letter codes as abbreviations are used. D-amino acids are shown in lower- case letters. 8 1 Introduction 1.1 Metals in anti-cancer therapy In medicinal bioinorganic chemistry, we can observe a clear shift from organic molecules to metal-containing complexes.1 Such choice is not surprising since metals possess advantages over conventional carbon-based compounds, such as partially filled d orbitals, a wide range of coordination numbers and geometries, accessible redox states, thermodynamic and kinetic characteristics, and the intrinsic properties of the cationic metal ion itself. 2 Different coordination numbers and modes provide a wide range of geometries, which eventually influence the pharmacological properties of metal complexes. 3 Furthermore, metal-based complexes exhibit one more major advantage over purely organic, carbon-based compounds, namely the ability to undergo ligand exchange reactions.4 This characteristic provides the capacity to interact reversibly as well as irreversibly with various biological molecules. 5 Of course such a shift and the wide use