View of the Zif268–DNA Complex, Showing the Side Chains That Make 7 Direct Base Contacts
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UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ Creation and Investigation of Protein Core Mimetics and DNA Binding Molecules A dissertation submitted to the Division of Research and Advance Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY in the Department of Chemistry of the College of Arts and Sciences 2005 by Juris Fotins B. S., University of Latvia, 1999 M. S., University of Cincinnati, 2002 Committee Chair: David B. Smithrud, Ph. D. Abstract The goal of this research is to design and synthesize small molecules that mimic structural and functional elements of the zinc finger containing proteins. Two fairly independent areas have been explored to determine stable minimal structure and minimal DNA binding motif of zinc fingers. Mimetic protein cores were created that align a set of L-Phe, D-Phe, or L-Leu residues in a parallel or an antiparallel arrangement in chloroform. Not all cores show a single conformation at room temperature. Stable structures require a synergistic relationship between the H-bonding groups and the residues within the core. The spatial arrangement of the side chains dictates whether a zippered or a crossed pattern of H-bonds is observed for these cores. Variable-temperature 1H NMR experiments were used to determine the strengths of the H-bonds. The existence of H-bonds was verified through FTIR spectroscopic analysis. Large temperature coefficients exist for some protons of aromatic rings that are held in a T-shaped arrangement. A comparison of these temperature coefficients shows that a more stable core is obtained by combining benzenoid and nitrobenzenoid rings as compared to benzenoid rings. Structures were determined using a combination of 2D NMR analysis and molecular modeling. Detailed results of this studies have been published in “Creation and Investigation of Protein - Core Mimetics with Parallel and Antiparallel Aligned Amino Acids”, Fotins, J., Smithrud, D. B. J. Org. Chem, Vol. 70, No. 11, 2005. Based on previously studied DNA binding molecules, we have designed second generation mimetic. In addition to a major groove binder already present in the molecule, the mimetic can be derivatized with an intercalator, such as acridine. ii Functionalization of the aromatic ring provides another attachment point for oligopeptides containing lysine residues, which are known to bind to DNA at the phosphate backbone. Docking experiments performed with HyperChem supported that the overall geometry of the newly designed mimetic is favorable for multiple mode interactions. iii Acknowledgements I would first like to thank my research adviser professor David B. Smithrud for his support, guidance and patience that he extended throughout my graduate career. I also want to extend my appreciation to my research committee members: professors George P. Kreishman, Apryll Stalcup and R. Marshall Wilson for many useful discussions and their encouragement. I would like to thank Dr. Elwood Brooks for his advice and guidance with NMR spectroscopy. It is my pleasure to acknowledge my research colleagues: Dr. Shawn Dickess, Michael Herr, Brian House and Dr. Inese Smukste for numerous discussions that significantly contributed to my academic and experimental knowledge. I would especially like to acknowledge Dr. Jeff Turk for his pioneering work; without his results, it would have been much harder to develop my project. I would also like to thank Vadim Dvornikov and Sergei Berdnikov for being my best friends for all these years. I am indebted to my undergraduate adviser Dr. Valerjans Kauss for his contribution to my professional career. Without his knowledge and passion for chemistry, I would probably never have become an organic chemist. Above all, I am grateful to my parents, Vilma and Sergei, for giving endless support and unconditional love. iv Table of Contents Abstract ii Acknowledgements iv Table of Contents v List of Figures vi List of Schemes viii Abbreviations ix 1. Introduction to Protein Mimicry 1 Protein Mimicry 2 Zinc Finger Proteins 5 Protein Core Mimetics 11 2. Synthesis and Physical Properties of Protein-Core Mimetics with Parallel and Antiparallel Aligned Amino Acids 15 Introduction 16 Mimetics with Parallel Aligned Amino Acids 19 Design and Synthesis 19 Experimental Methods 22 Physical Properties 24 Mimetics with Antiparallel Aligned Amino Acids 30 Design and Synthesis 30 Physical Properties 33 Discussion 36 Conclusion 44 3. Design and Synthesis of Small Molecules that Bind DNA 45 Introduction 46 Design and Synthetic Efforts 49 References 56 Experimental Section 68 Spectral Data 93 v List of Figures Figure 1. Structure diagram of the classical C2H2 zinc-binding motif 6 Figure 2. Overview of the Zif268–DNA complex, showing the side chains that make 7 direct base contacts Figure 3. Cartoon representation of typical zinc finger protein containing a 9 hydrophobic core, three-stranded structural domain and the DNA binding domain Figure 4. Schematic representation of parallel β-sheet and antiparallel β-sheet 12 Figure 5. Artificial β–structures that have stable hydrophobic cores around 13 dibenzofuran and dipyridine scaffold Figure 6. The “molecular torsion balance” has two gently restricted conformational 14 states Figure 7. The number of aromatic rings held in a T-shaped arrangement determines 16 whether a zippered or a crossed pattern of H-bonds is formed. A nonsynergistic relationship between the core and H-bonding residues, however, results in multiple structures Figure 8. (A) The original PCM that displays interactions between both aromatic side 17 chains and one of the scaffold s aromatic rings and two isoenergetic H-bonds. (B) The new PCM also contains a scaffold and H-bonding plane, but its internal amines are not methylated Figure 9. General design of mimetics with parallel aligned amino acids 19 Figure 10. Typical variable temperature 1H NMR spectrum. With an increase in 23 temperature NH proton signals move upfield and aromatic signals move downfield 1 Figure 11. An overlay of the H NMR spectra of (L, D)-Pheparallel and (L, L)-Pheparallel, 25 showing that a correct matching of core with H-bonding residues will lead to a stable structure Figure 12. Schematic drawings of the compounds showing key NOE cross-peaks 26 (double-headed arrows). Temperature coefficients (standard deviations are less than (0.1) for amide N-H and aromatic Ar-H protons are given. Highlighted in (L, L)- PheNO2parallel are the observed diastereomeric differences Figure 13. FTIR spectral data from the N-H stretching region of the PCMs and N- 27 Ac-phenylalanine methyl amide. All samples were 3 mM in CHCl3 at 298 K. Each spectrum had its baseline corrected, and the absorbance of CHCl3 was subtracted Figure 14. Amino acid that induces β-turn to aligned strands of mimetic in 30 antiparallel fashion vi List of Figures (continued) Figure 15. Key NOE cross-peaks (double-headed arrows), temperature coefficients 33 for amide N-H and aromatic Ar-H protons, and FTIR spectral data from the N-H stretching region are given for the antiparallel compounds Figure 16. The diastereomeric sets of the antiparallel compounds, a cartoon depiction 35 of their calculated stable structures highlighting the orientation of the aromatic rings and whether a single conformer is observed Figure 17. A zippered or a crossed pattern of H-bonds exists between the amino 37 acids of the parallel aligned compounds and the antiparallel aligned compounds Figure 18. Low-energy structures obtained from molecular modeling studies that are 38 consistent with the observed properties of the compounds Figure 19. First generation DNA binder containing hydrophobic core, Arg-His 47 recognition strand and fluorescence tag DAE (dansyl group with ethylene diamine linker) Figure 20. Representative fluorescence quenching assays of mimetic IV binding to 47 mDd (A) and d(A9T9)2 (B) in 0.1 M phosphate buffer pH 7.0 at 25 ºC Figure 21. Proposed design of the second generation mimetic that will bind DNA 49 vii List of Schemes Scheme 1 20 Scheme 2 21 Scheme 3 31 Scheme 4 32 Scheme 5 50 Scheme 6 51 Scheme 7 52 Scheme 8 53 Scheme 9 54 Scheme 10 55 viii Abbreviations Ac2O acetic anhydride AcOH acetic acid Arg arginine Bn benzyl Boc t-butoxycarbonyl BOM benzyloxymethyl CDI N,N’-carbonyldiimidazole DCM dichloromethane DIEA diisopropylethylamine DMF dimethylformamide DMSO dimethyl sulfoxide EtOAc ethyl acetate His histidine HOBt 1-hydroxybenzotriazole NMR nuclear magnetic resonance Phe phenylalanine PCM protein core mimetic PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate RT room temperature TC temperature coefficient TFA trifluoroacetic acid TFAA trifluoroacetic acid anhydride THF tetrahydrofuran ix Chapter I Introduction to Protein Mimicry 1 Protein Mimicry Peptides are short, sequence- and length-specific oligomers composed of amino acids. These familiar biomolecules are ubiquitous in living cells and assume myriad roles. Each role assumed by a bioactive peptide will typically correspond to a unique three-dimensional structure. Protein – protein interactions are crucial events in most biological processes and are therefore important