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Abstract a Practical Synthesis of N -Fmoc Protected L-Threo- -Hydroxyaspartic Acid Derivatives for Coupling

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Abstract a Practical Synthesis of N -Fmoc Protected L-Threo- -Hydroxyaspartic Acid Derivatives for Coupling CYCLIC LIPODEPSIPEPTIDES AS LEAD STRUCTURES FOR THE DISCOVERY OF NEW ANTIBIOTICS by Nina Bionda A Dissertation Submitted to the Faculty of The Charles E. Schmidt College of Science in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Florida Atlantic University Boca Raton, FL May 2013 ACKNOWLEDGEMENTS First and foremost, I want to thank my advisor, Dr. Predrag Cudic, for his guidance throughout my PhD endeavors, the endless discussions about science and life in general, and for teaching me to give my best every step of the way. You have been instrumental in my scientific education and for that I am forever in your debt. Immense gratitude goes to Dr. Lepore for embracing the difficult role of a co-advisor and fulfilling it truly beyond what I have expected. I will always appreciate your advice. I also wish to express my heartfelt thanks to my dissertation committee members, Dr. Laszlo Otvos Jr., Dr. Stefan Vetter and Dr. Andrew C. Terentis, for their valuable time and thoughtful suggestions, comments and critiques. I would also like to extend my appreciation to all of the faculty and students of the Department of Chemistry and Biochemistry that I have had contact with during my PhD studies. To Dr. Richard Houghten and everyone at the Torrey Pines Institute for Molecular Studies, thank you for providing me with a “home” for the last two and a half years of my PhD. And special thanks to Dr. Maré Cudic for sharing her scientific experience and the simple things of the everyday life. To Dr. Jasna Vorkapic-Furac and Dr. Andreja Jakas, thank you for introducing me into the world of science and inspiring me to pursue my scientific career. A very special thank you goes to my family, mom, dad and Ana, for believing in me and giving me their unconditional support and encouragement. Last, but certainly not least, big thanks to my friends for being there to share with me the ups and downs of my PhD experience, especially Sheldon’s crew from TPIMS, my dear friends from the Department of Chemistry at FAU, and most of all Patrick for helping me keep my mind together. iii ABSTRACT Author: Nina Bionda Title: Cyclic Lipodepsipeptides as Lead Structures for the Discovery of New Antibiotics Institution: Florida Atlantic University Dissertation Co-advisors: Dr. Salvatore D. Lepore Dr. Predrag Cudic Degree: Doctor of Philosophy Year: 2013 With antimicrobial resistance to current drugs steadily rising, the development of new antibiotics with novel mechanisms of action has become an imperative. The majority of life- threatening infections worldwide are caused by “ESKAPE” pathogens which are encountered in more than 40% of hospital-acquired infections, and are resistant to the majority of commonly used antibiotics. Naturally occurring cyclic depsipeptides, microbial secondary metabolites that contain one or more ester bonds in addition to amide bonds, have emerged as an important source of pharmacologically active compounds or lead structures for the development of novel antibiotics. Some of those peptides are either already marketed (daptomycin) or in advanced stages of clinical development (ramoplanin). Structurally simple, yet potent, fusaricidin/LI-F and lysobactin families of naturally occurring antibiotics represent particularly attractive candidates for the development of new antibacterial agents capable of overcoming infections caused by multidrug-resistant bacteria. These natural products exhibit potent antimicrobial activity against a variety of clinically relevant fungi and Gram-positive bacteria. Therefore, access to these classes of natural products and their synthetic iv analogs, combined with elucidation of their mode of action represent important initial steps toward full exploitation of their antmicrobial potential. This dissertation describes a general approach toward the solid-phase synthesis of fusaricidin/LI-F and lysobactin analogs and an extensive structure-activity relationship (SAR) study. We have devised a simple and robust preparation strategy based on standard Fmoc solid- phase peptide synthesis protocols. The SAR study revealed key structural requirements for fusaricidin/LI-F and related cyclic lipopeptides antibacterial activity, including the presence of the guanidino moietly at the end of the lipidic tail, hydrophobic amino acid residues, and peptide conformation. Moreover, substitution of the ester bond with an amide bond significantly improved stability under physiologically relevant conditions and reduced toxicity. In addition, we have shown that these antibacterial peptides exert their mode of action via a novel mechanism, which invloves bacterial membrane interactions, followed by peptide internalization. Altogether, the research described in this dissertation demonstrates that new antibiotics derived from fusaricidin/LI-F natural products, have the potential to meet the challenge of antibiotic resistance in Gram-positive bacteria. v Mojim roditeljima… “Science is a way of thinking much more than it is a body of knowledge” - Carl Sagan - CYCLIC LIPODEPSIPEPTIDES AS LEAD STRUCTURES FOR THE DISCOVERY OF NEW ANTIBIOTICS List of figures .............................................................................................................................. x List of tables............................................................................................................................... xii List of schemes ......................................................................................................................... xiii List of abbreviations .................................................................................................................. xiv 1. INTRODUCTION..................................................................................................................... 1 1.1 Overview– the challenges of new antibiotic discovery..................................................... 1 1.2 Antimicrobial resistance ................................................................................................. 6 1.2.1 Antibiotic resistance of bacteria in biofilms ........................................................... 8 1.3 Antimicrobial peptides (AMPs) ..................................................................................... 11 1.4 Cyclic lipodepsipeptides in novel antimicrobial drug discovery ...................................... 13 1.4.1 Overview ........................................................................................................... 13 1.4.2 Daptomycin ....................................................................................................... 15 1.4.3 Ramoplanins/enduracidins ................................................................................. 21 1.4.4 Katanosin A and lysobactin (katanosin B) .......................................................... 27 1.4.5 Fusaricidins/LI-Fs .............................................................................................. 30 1.4.6 Circulocins ......................................................................................................... 33 1.4.7 Heptadepsin ...................................................................................................... 34 1.5 Research goals ............................................................................................................ 35 2. STRUCTURAL MODIFIATION OF FUSARICIDIN/LI-F FAMILY OF CYCLIC LIPODEPSIPEPTIDES AND ITS EFFECT OF STRUCTURAL MODIFICATION ON STABILITY, ANTIMICROBIAL ACTIVITY AND HUMAN CELL TOXICITY .................................. 37 2.1 Overview ..................................................................................................................... 37 2.2 Solid-phase synthesis .................................................................................................. 38 2.2.1 Synthesis of depsipeptide analogs ..................................................................... 38 2.2.2 Synthesis of amide analogs ............................................................................... 41 2.2.3 Synthesis of the linear analog ............................................................................ 42 2.2.4 Synthesis of the N-methylamide analog ............................................................. 43 2.2.5 Guanidinylation of resin-bound amines .............................................................. 44 2.2.5.1 Introduction ............................................................................................. 44 vi 2.2.5.2 Results and discussion ............................................................................ 46 2.3 Conformational study ................................................................................................... 51 2.3.1 Circular dichroism (CD) spectroscopy ................................................................ 51 2.3.2 Molecular dynamics (MD) simulations ................................................................ 54 2.4 Structure-activity relationship study .............................................................................. 56 2.4.1 Antibacterial activity ........................................................................................... 56 2.4.2 Antifungal activity ............................................................................................... 58 2.5 Cytotoxicity .................................................................................................................. 59 2.5.1 Hemolytic activity ..............................................................................................
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