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 ...... 59 2.5.2 Toxicity towards human normal and cancer cell lines ...... 61 2.6 Serum stability ...... 63 2.7 Discussion and conclusions ...... 65 2.8 Materials and methods ...... 72 2.8.1 Chemicals and instrumentation ...... 72 2.8.2 General procedure for peptide synthesis and purification ...... 73 2.8.3 Methods for guanidinylation of resin-bound amines ...... 75 2.8.3.1 Synthesis of model cyclic lipopeptide ...... 75 2.8.3.2 Guanidinylation using N,N’-di-Boc-N’’-triflylguanidine ...... 75 2.8.3.3 Guanidinylation using 1H-pyrazole-1-carboxamidine hydrochloride ...... 76 2.8.3.4 Guanidinylation using N,N’-di-Boc-thiourea and Mukaiyama’s reagent as an activator ...... 76 2.8.3.5 Guanidinylation using N,N’-di-Boc-thiourea and NIS as an activator ...... 76 2.8.4 Circular dichroism (CD) spectroscopy ...... 77 2.8.5 Molecular dynamics (MD) simulations ...... 77 2.8.6 Antibacterial activity ...... 78 2.8.7 Antifungal activity ...... 79 2.8.8 Hemolytic activity ...... 79 2.8.9 Cytotoxicity ...... 80 2.8.10 Proteolytic stability asssays ...... 80 3. TOWARDS DECIPHERING THE MODE OF ACTION OF THE FUSARICIDIN/LI-F CLASS OF ANTIMICROBIAL PEPTIDES ...... 81 3.1 Introduction ...... 81 3.2 Membrane depolarization ...... 82 3.3 Bacterial survival following membrane depolarization ...... 86 3.4 Bactericidal vs bacteriostatic activity ...... 88 3.5 Disruption of S. aureus biofilm...... 89 3.6 Assessment of of fusaricidin/LI-F class of antibacterial peptides ability to enter the cells of Gram-positive bacteria ...... 90

vii 3.7 Assessment of interaction with the known target in cell wall biosynthesis, the D-Ala-D-Ala dipeptide ...... 91 3.8 Influence of human serum and physiological salt concentrations on peptides’ activity ... 91 3.9 Effect of 12-GDA substitution with cell-penetrating peptide (CPP) sequences ...... 92 3.10 Discussion and conclusions ...... 92 3.11 Materials and methods ...... 94 3.11.1 Bacterial strains and reagents ...... 94 3.11.2 Peptide synthesis ...... 95 3.11.3 Antibacterial activity ...... 95 3.11.4 Membrane depolarization assay ...... 96 3.11.5 Time-kill assays ...... 96 3.11.6 Biofilm-susceptibility assay ...... 97 3.11.7 Fluorescence microscopy ...... 98 4. LYSOBACTIN (KATANOSIN B) – SYNTHESIS AND EVALUATION OF ACTIVITY ...... 99 4.1 Introduction ...... 99 4.2 Synthesis of N-Fmoc protected L-threo--hydroxyaspartic acid derivatives ...... 101 4.2.1 Background ...... 101 4.2.2 Enantioresolution of D,L-threo--hydroxyaspartic acid...... 102 4.2.3 Design of a protection scheme...... 105 4.2.4 Discussion and conclusions...... 108 4.2.5 Materials and methods ...... 108 4.3 Synthesis of Fmoc-SPPS-compatible L-threo-phenylserine (L-tPhSer) and L- hydroxyleucine (L-HyLeu) building blocks ...... 113 4.3.1 Synthesis of Fmoc-L-threo-PhSer-OH ...... 113

4.3.2 Synthesis of Fmoc-L-HyLeu(TBDMS)-OH ...... 114 4.4 Optimization of Fmoc-SPPS approach towards lysobactin ...... 115 4.4.1 Synthesis of lysobactin analog 49 and antimicrobial activity assessment ...... 115 4.4.2 Synthesis of lysobactin analog 51 and antimicrobial activity assessment ...... 119 4.4.3 The challenges of the on-resin ester bond formation ...... 121 4.5 Discussion and conclusions ...... 123 5. APPENDIX ...... 125 5.1 RP HPLC and MALDI-TOF MS spectra of fusaricidin/LI-F analogs ...... 125 5.2 Hemolytic activity of all tested fusaricidin/LI-F analogs ...... 147 5.3 Additional membrane depolarization data ...... 148 5.4 Enantioresolution of D,L-tHyAsp ...... 151 5.5 General method for derivatization with Marfey’s reagent...... 151 5.6 Specific optical rotation calculation ...... 152 5.7 Selected NMR spectra of L-tHyAsp derivatives ...... 153

viii 5.8 Assessment of epimerization upon introduction of Dmab protecting group ...... 156 REFERENCES ...... 158

ix LIST OF FIGURES

Figure 1 New systemic (i.e., nontopical) antibacterial agents approved by the US FDA ...... 2 Figure 2 Timeline of introduction of novel drug classes on the market ...... 3 Figure 3 Structures of linezolid and daptomycin, members of the novel antibiotic classes, oxazolidinones and lipopeptides, respectively...... 4 Figure 4 Inactivation of penicillins with β-lactamase hydrolytic activity ...... 7 Figure 5 Schematic representation of the biofilm life cycle ...... 8 Figure 6 Models explaining the mechanisms of membrane permeabilization by AMPs ...... 12

Figure 7 Structures of daptomycin and A21978C1-C3...... 16 Figure 8 Proposed mechanism of action of daptomycin ...... 17 Figure 9 Representative member of the A54145 family, A54145A ...... 21

Figure 10 Structures of ramoplanins A1-A3 and ramoplanose ...... 22 Figure 11 Structures of enduracidins A and B ...... 23 Figure 12 Peptidoglycan biosynthesis and potential targets of ramoplanins ...... 26 Figure 13 Structures of katanosin A and lysobactin (katanosin B) ...... 28 Figure 14 Structures of fusaricidin/LI-F family of compounds and the synthetic analog...... 31 Figure 15 Structures of circulocins α-δ...... 34 Figure 16 Structure of heptadepsin...... 35 Figure 17 Sequences of synthetic fusaricidin/LI-F analogs ...... 39 Figure 18 Structure of linear fusaricidin/LI-F analog ...... 43 Figure 19 Structures of guanidinylating agents used in the study ...... 45 Figure 20 RP HPLC analysis of guanidinylation reactions ...... 48 Figure 21 Structures of compounds isolated in the guanidinylation study ...... 49 Figure 22 CD spectra of fusaricidin/LI-F analogs: a) 6, b) 14 and c) 18 ...... 52 Figure 23 CD spectra of N-methylated fusaricidin/LI-F analog 19 (a) and comparison with the CD spectra of analogs 6 and 14 in 0.5 % AcOH (b) and TFE (c) ...... 53 Figure 24 Representative structures of low energy conformers of depsipeptide 6, amide 14 and NMe-amide 19 in water and TFE and their calculated amphiphilic moments, μ ...... 55 Figure 25 Anifungal activity of fusaricidin/LI-F analog 6 against A. niger ...... 59 Figure 26 Hemolytic activity of fusaricidin/LI-F analogs expressed as percent of hemolysis caused by Triton X-100 ...... 60 Figure 27 Structures of fusaricidin/LI-F analogs used in the cytotoxicity study ...... 61

x Figure 28 Cell survival measured by MTT assay after 24 h treatment with fusaricidin/LI-F analogs. Normal cell line WRL 68 (upper panel), cancer cell line HepG2 (lower panel)...... 62 Figure 29 RP HPLC trace of analog 6 in 50% human serum at time 0 (up) and after 24 h incubation at 37C (down) ...... 63 Figure 30 RP HPLC trace of analog 6 in the EMEM media at time 0 (up) and after 24 h incubation at 37C (down) ...... 64 Figure 31 Stability of fusaricidin/LI-F analog 6 in the EMEM media containing 10% FBS (left) and analogs 6 and 14 in 50% human serum (right)...... 65 Figure 32 MALDI-TOF MS analysis of RP HPLC fractions obtained after 24 h incubation of 6 in the EMEM media ...... 65 Figure 33 Structures of analogs used in the study towards deciphering the mode of action of fusaricidin/LI-F class of antimicrobial peptides ...... 82 Figure 34 Time- and dose-dependent membrane depolarization at r.t...... 85 Figure 35 Maximal membrane depolarization at r.t. for depsipeptide 6 (left) and amide 14 (right) ...... 86 Figure 36 Time-kill following membrane depolarization at; A) room temperature, B) 37C ...... 87 Figure 37 Time-kill studies for three Gram-positive strains ...... 88 Figure 38 Effect of synthetic fusaricidin/LI-F analogs on S. aureus biofilm ...... 89 Figure 39 Confocal fluorescence microscopic images of B. subtilis ATCC 82 upon incubation with fluorescein-labeled analog 29...... 90 Figure 40 Retrosynthetic analysis of lysobactin ...... 100 Figure 41 RP HPLC traces after derivatization with Marfey’s reagent ...... 104 Figure 42 Graphical representation of HyAsp derivatives compatible with Fmoc-SPPS ...... 108 10 10 Figure 43 Structure of lysobactin analog 49 with D,L-tHyAsn instead of L-tHyAs ...... 116 Figure 44 Proposed structure of the side product 50 obtained during the synthesis of lysobactin 49 ...... 117 Figure 45 Isolation and characterization of products obtained from synthesis of 49 ...... 118 10 10 Figure 46 Structure of lysobactin analog 51 containing Asn instead of L-tHyAsn ...... 120 Figure 47 RP HPLC analysis of crude lysobactin analog 51 ...... 120 Figure 48 Magnified RP HPLC traces obtained after coupling of Aloc-Ser(tBu)-OH to tetrapeptide D-Leu-L-tPhSer-Leu-Leu on different resins ...... 122 Figure 49-70 RP HPLC and MALDI-TOF MS spectra of fusaricidin/LI-F analogs 1-19 and analogs 29-31 ...... 125-146 Figure 71 Hemolytic activity of all tested fusaricidin/LI-F analogs ...... 147 Figure 72 membrane depolarization of S. aureus with fusaricidin/LI-F analogs 11 and 16 ...... 148 Figure 73 Comparison of membrane depolarization data against S. aureus for gramicidin S at 20 μg/mL and fusaricidin/LI-F analogs 6, 14 and 18 at 48 μg/mL ...... 149 Figure 74 Time- and dose-dependent membrane depolarization at 37C...... 150 Figure 75-77 NMR spectra of 36, 40 and 42 ...... 153-155 Figure 78 RP HPLC analysis of L-tHyAsp derivative 42 after removal of Fmoc and Dmab protecting groups ...... 156 Figure 79 COSY 2D NMR spectrum of 41 ...... 157 xi LIST OF TABLES

Table 1 Mechanisms of action of antibacterial agents ...... 7 Table 2 In vivo efficacy of katanosin A and lysobactin (katanosin B) against bacterial infection in mice ...... 29 Table 3 In vivo toxicity of lysobactin (katanosin B) and vancomycin ...... 30 Table 4 Antibacterial activity of fusaricidin/LI-F analog ...... 57 Table 5 Membrane composition of different bacterial strains ...... 83 Table 6 Specific optical rotations for resolved tHyAsp enantiomers ...... 104 Table 7 Tested conditions for selective removal of benzyl protecting group from 37 ...... 107 Table 8 Antimicrobial activity of lysobactin analogs 49 and 50 ...... 119

xii LIST OF SCHEMES

Scheme 1 Synthesis of fusaricidin/LI-F depsipeptide analogs 1-12 ...... 40 Scheme 2 O→N acyl shift ...... 41 Scheme 3 Synthesis of fusaricidin/LI-F amide analogs 13-17 ...... 42 Scheme 4 Synthesis of N-methylated fusaricidin/LI-F analog 19 ...... 44 Scheme 5 Solid-phase guanidinylation ...... 46 Scheme 6 General scheme of D,L-tHyAsp enantioresolution ...... 103 Scheme 7 General protection scheme for HyAsp...... 106 Scheme 8 Enantioresolution and Nα-Fmoc protection of tPhSer ...... 114

Scheme 9 Orthogonal protection of L-HyLeu ...... 115 Scheme 10 Synthetic approach toward lysobactin analog 49 ...... 116 Scheme 11 Synthesis of a model tetrapeptide for testing of on-resin ester bond formation ...... 121

xiii LIST OF ABBREVIATIONS

12-ADA/ADA 12-Aminododecanoic acid 12-GDA/GDA 12-Guanidino dodecanoic acid ACN Acetonitrile AcOH Acetic acid Aloc Allyloxycarbonyl AMP Antimicrobial peptide Bn-OH Benzyl alcohol CAMP Cationic antimicrobial peptide CD Circular dichroism CL Cardiolipin Cl-TrtCl 2-Chlorotrytyl chloride CPP Cell-penetrating sequence Dab 2,4-Diaminobutyric acid Dap 2,3-Diaminopropionic acid DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCM Dichloromethane DHP Dihydropyran DIAD Diisopropyl azodicarboxylate DIC Diisopropylcarbodiimide DIEA Diisopropylethyl amine Dmab-OH 4{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino} benzyl alcohol DMAP 4-Dimethylaminopyridine DMF N,N’-Dimethylformamide DMSO Dimethyl sulfoxide EMEM Eagle’s minimal essential medium EtOAc Ethyl acetate EtOH Ethanol FBS Fetal bovine serum Fmoc-OSu N-(9-Fluorenylmethoxycarbonyloxy) succinimide hRBCs Human red blood cells

xiv HATU (Dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3- yloxy)methaniminium hexafluorophosphate HBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HOAt N-Hydroxy-7-azabenzotriazole HOBt N-Hydroxybenzotriazole IPA Isopropanol KPC Klebsiella pneumoniae carbapenemase LPS Lipopolysaccharide MALDI-TOF MS Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy MeOH Methanol MHB Müller-Hinton broth MIC Minimum inhibitory concentration MRSA Methicillin-resistant Staphylococcus aureus MRSE Methicillin-resistant Staphylococcus epidermidis MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Mtt 4-Methyltrityl chloride NMM N-Methylmorpholine NMR Nuclear magnetic resonance o-NBS-Cl 2-Nitrobenzenesulfonyl chloride PBS Phosphate buffered saline PE Phosphatidylethanolamine PEG Polyethylene glycol PG Phosphatidylglycerol PPTS Pyridinium p-toluenesulfonate PS Polystyrene PyBOP Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate RND Resistance-nodulation-cell division RP-HPLC Reversed-phase high performance liquid chromatography SPPS Solid-phase peptide synthesis TBAF Tetrabutylammonium fluoride TBDMS-Cl tert-Butyldimethylsilyl chloride TCA Trichloroacetic acid TFA Trifluoroacetic acid TFE Trifluoroethanol THF Tetrahydrofurane VRE Vancomycin resistant Enterococci VRSA Vancomycin resistant Staphylococcus aureus

xv CHAPTER ONE

INTRODUCTION

1.1 Overview – the challenges of new antibiotic discovery

For almost half of the century, since the first discovery of penicillin in the 1940s up until the

1990s, we have been able to fairly quickly respond to the emergence of bacterial resistance by developing new antibiotics. The “golden era” of antibiotics (1945-1965) was mainly a result of our advances in the ability to solve molecular structures of antibacterial agents which lead to further development of (semi)synthetic derivatives of already known antibiotics. Until 1985 well over 100 antibiotics were clinically tested and about 60 of them were released on the market.1

In the last 20 years, however, we have been loosing the battle. The occurrence of resistance is in constant increase making multi-drug resistant microorganisms currently a global public health problem.2-6 The development of novel antiobitics, on another hand, does not follow that trend and is in constant decline, Figure 1.

The majority of life-threatening infections worldwide are caused by the ESKAPE pathogens

(Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species).7,8 This group of bacteria is encountered in more than 40% of hospital-acquired infections, and is resistant to the majority of commonly used antibiotics. Even antibiotics we consider to be our “last resort” are under the threat of loosing potency due to the development of bacterial resistance.9

The danger of antibiotic resistance is not only observed in poor or underdeveloped countries but all over the world. Alarmingly, in US hospitals today more people die of infections caused by methicillin-resistant S. aureus (MRSA) than of HIV/AIDS and tuberculosis combined.10,11

1 Almost 2 million Americans develop hospital-acquired infections (HAIs) per year, the vast majority of which caused by antibiotic-resistant pathogens, resulting in about 99,000 deaths annually.11

Figure 1. New systemic (i.e., nontopical) antibacterial agents approved by the US FDA (modified from Spellberg 2008)5

In the last decade, there have been various initiatives from the World Health Organization

(WHO), Center for Disease Control and Prevention (CDC), National Institute of Health (NIH),

Infectious Diseases Society of America (IDSA) and other agencies urging the development of new antibiotics.6,8,9,12 Furthermore, just this year the IDSA has proposed, in adition to the Gain act approved by the Food and Drug Administration in 2011, a Special Population Limited Medical

Use (SPLMU) mechanism which should instigate more flexibility in the creation of new antibiotics and foster prudent use of novel anti-infective drugs to slow the rate at which resistance to the drugs develops.12 This paradox of keeping a potent drug on the shelf, though necessary, is unseen with any other diseases and speaks of the gravity of the situation in the antibiotic field.

There are particular challenges in the development of novel antimicrobial agents that are not present in the development of drugs for other diseases. Antibiotics need to be active against multiple bacterial species which are constantly changing, mutating, developing resistance in

2 different ways and thus “hiding” from the effectiveness of the antibiotics. Even if we consider targeting a specific enzyme present in the bacteria, rather than the bacteria itself, a few years down the road the same target may prove to be redundant. Comparing to cancer, where we face a similar problem of drug resistance, this resistance is not transmissible among the patients as it is in the case of infectious diseases.13 Moreover, due to their daily dosages, antibiotics have to have very low toxicity compared to other drugs.2 There are also pharmacoeconomical reasons that influence development of new antibiotics which include short treatment period, resulting in poor investment return and development of resistance can make the newly released drug nearly obsolete. All of these factors contribute significantly to the lack of interest from the pharmaceutical industry for investing in new antibiotics.

A majority of the antibiotics currently in use belong to classes that have been discovered before the 1970s, Figure 2. Since then, the only truly new classes of antibiotics were oxazolidinones (linezolid)14 discovered in 1978 and released on the market in 2000, and lipopeptides (daptomycin)15 discovered in 1986 and released on the market in 2003, Figure 3.

Both of these classes of antibiotics exhibit novel modes of action and are active only against

Gram-positive bacteria.16,17

Figure 2. Timeline of introduction of novel drug classes on the market (modified from Walsh 20094 and IDSA report 20046) 3 The situation is even worse in the case of Gram-negative bacteria where no new classes of antibiotics have been introduced since trimethoprim in the 1970s, and the clinicians are turning to recycling old classes of compounds such as polymyxins.18,19 The rest of the development involved modifications/improvements of existing drugs with more or less significant success in increasing their potency and ability to evade already existing resistance. Some of the examples are fluoroquinolones versus nalidixic acid,20,21 amikacin versus kanamycin,22 and glycylcyclines versus tetracycline.13,23 Unfortunately, this approach became obsolete with the emergence of mechanisms affording resistance to whole classes of compounds.

Figure 3. Structures of linezolid and daptomycin, members of the novel antibiotic classes, oxazolidinones and lipopeptides, respectively

Most notable examples are multiple topoisomerase mutations, which compromise efficacy of all fluoroquinolones;24 metallo- and KPC -lactamases, compromising nearly all -lactams;25 16S rRNA methylases, compromising nearly all aminoglycosides;26 and up-regulation of resistance, nodulation and division (RND) efflux pumps,27 compromising multiple drug classes.13 More importantly, resistance to our latest classes of antibiotics is occurring. In the case of daptomycin some cross-resistance with vancomycin was observed in the changes in the membrane and alterations in the cell wall structure and/or function, mainly thickening of the cell wall,28 as well as

4 alteration in the cellular metabolism and differences in the cell wall turnover.29 Linezolid resistance has been reported, though seldom, for methicillin-resistant Staphylococci. However, more importantly, resistance of vancomycin-resistant E. faecium to linezolid has been widely reported.30-32 Almost all known resistance mechanisms to linezolid involve small alterations to the linezolid binding site on the ribosome.

In the 1990s, fueled by the lack of new leads from the screening of natural products, and turning to the newly developed technologies, the world of antibiotic discovery adopted genomics- based approach. The idea was to identify essential conserved genes encoding targets that do not have counterparts in mammalian cells through sequencing genomes of multiple pathogens. This would then be used for the development of highthroughput screens of existing compound libraries to identify “druggable” molecules that bind to the newly identified targets. However, 20 years later, not a single antibiotic reached the market as a direct result of this approach.2 There are many potential explanations for the failure of the genomics in discovery of antibiotics. One major example being the compatibility of the libraries used for screening,2,33 the selection of which was mainly guided by the Lipinski’s “rule of five”34 which is not applicable to antibiotics. Furthermore, finding compounds that bind the target of interest does not equate to finding antibiotics. Drugs that bind a certain intracellular target may fail to penetrate the bacteria or may be removed by efflux mechanisms.2,33 Also, drugs with single targets are more susceptible to mutational resistance, as identified in the case of rifampicin, streptomycin and fosfomycin,33 as opposed to drugs with multiple targets, such as -lactams, quinolones and aminoglycosides.33 The disappointment with the results, or lack thereof, of the genomic strategy had led to the abandonment of antibiotic research from many of the major pharmaceutical companies.35

Currently, novel approaches for discovering new antibiotics include finding new classes of compounds that interfere with already established targets, such as various non--lactam inhibitors of -lactamases, including NXL104,36 MK-765537 and ME1071,38 and non-quinolone topoisomerase inhibitors39 for broad spectrum use, as wells as inhibitors that interfere with multidrug RND pumps thereby restoring quinolone activity against P. aeruginosa.40 Advances are

5 also seen with known antibiotic classes, including novel tetracycline analogues. The lead compound, TP434 has MICs 4-fold lower than tigecycline against both Gram-positive and Gram- negative strains and is now in Phase II clinical trials.41 We also see a comeback of polymyxins and the development of analogs with lower toxicities and greater potencies.18,19 Furthermore, learning from the lessons of the genomics approach, screening of the libraries now includes unconventional classes of molecules, which has already led to the discovery of several compounds that have successfully completed Phase I trials.42 The natural product screening is also coming back, though to a much lesser degree and the focus has broadened to encompass not only traditional antibiotic producing soil streptomycetes43-45 but also plants,46 deep-sea bacteria,47 actinomycetes,48 etc.

One of the most promising classes of compounds for the discovery of new antibiotics are antimicrobial peptides (AMPs), which can be found in the majority of living organisms, from prokaryotes to humans.49-51 Although AMPs exhibit diverse biological activities, their antibacterial and immunomodulatory properties have gained the most attention.51-55 This class of compounds will be discussed to further detail in section 1.3.

1.2 Antimicrobial resistance

Most antimicrobial agents can be categorized according to their principal mode of action,

Table 1. Modes of action include interference with the synthesis of bacterial cell wall, inhibition of protein synthesis, interference with the synthesis of nucleic acids, and inhibition of metabolic pathways.56-58 Disruption of membrane structure and function may comprise the fifth category, though this mechanism of action is not yet well understood and more evidence is coming to light indicating that some of the compounds believed to act purely by membrane disruption have in fact (multiple) internal targets.51,56,58

Whenever a new antibiotic is introduced to the market, the development of antimicrobial resistance follows. In some instances, such as in the case of penicillins, the resistance was found

6 within a few months. On the other hand, it took almost 30 years for the resistance to vancomycin to be reported.59 However, this is mainly due to the limited use of vancomycin in the early years.

Table 1. Mechanisms of action of antibacterial agents56-58

Interference Inhibition of protein Interference with Inhibition of Membrane with cell wall synthesis nucleic acid metabolic disruption synthesis synthesis pathways

β-Lactams Macrolides Fluoroquonolones Sulfonamides Polymyxins Glycopeptides Aminoglycosides Rifamycins Trimethoprim Daptomycin Tetracyclines Chloramphenicol Oxazolidinones Streptogramins Lincosamides

Some bacterial species are inherently resistant to one or more classes of antibiotics, for example bacteria that are antibiotic producers,58,60 and Gram-negative bacteria which have reduced permeability of antibiotics due to presence of an additional, outer membrane, not found in Gram-positive bacteria.61 Acquired resistance occurs when initially susceptible subpopulation of bacteria becomes resistant either through mutation and selection (vertical evolution), or by acquiring resistance encoding genetic information from other bacteria through mobile genetic elements (transposones) or plasmid-sized DNA (horizontal evolution).58,62

Resistance mechanisms vary but can be grouped into three major categories:56-58,62

a) Enzymatic destruction or modification of the antibiotic itself. For example enzymes β-

lactamases hydrolyze the four-membered β-lactam ring of β-lactam antibiotics

(penicillins, cephalosporins, etc.) rendering them inactive, Figure 4.

Figure 4. Inactivation of penicillins with β-lactamase hydrolytic activity

7 b) Active export or efflux of antibiotics. This mode of resistance can be clinically relevant for

several antibiotic classes but is most notable for tetracyclines and fluoroquinolones.

c) Replacement or modification of the antibiotic target. For example cell wall reprogramming

discovered in vancomycin-resistant enterococci (VRE) where loss of one hydrogen bond

between vancomycin and D-Ala-D-lactate results in 1000-fold drop in binding affinity.

To complicate things further, the same kind of resistance mechanism can be a result of many different genes and for the same antibiotic there can be more than one resistance mechanism.57

1.2.1 Antibiotic resistance of bacteria in biofilms

The National Institutes of Health estimate that biofilms account for over 80% of microbial infections in the body.63,64 A biofilm is a community of bacteria encased in a matrix composed of exopolysaccharides, proteins and extracellular DNA and adhered to a biotic or abiotic surface.

Biofilms can be made up of single or multiple bacterial species and the process of formation, maturation, maintenance and dissemination of biofilm is very dynamic with different factors involved at different stages, Figure 5.65-69

Figure 5. Schematic representation of the biofilm life cycle65-68

8 Various nosocomial infections related to implants such as catheters, heart valves, prostetic implants, as well as opportunistic lung infections in patients with cystic fibrosis, are caused by biofilm-forming bacteria. The most notable biofilm-forming pathogens are S. aureus and

Staphylococcus epidermidis from Gram-positive and E. coli and P. aeruginosa from Gram- negative strains.70-72 Infections that involve biofilm are generally chronic, difficult to treat because they exhibit inherent resistance to antibiotics, and very often persist until the colonized surface

(tissue) is surgically removed.73,74

Although these infections occur at different sites and are caused by different microbial hosts, they share certain common characteristics. Most importantly, they evade host defense systems and withstand antimicrobial treatment.63,75 The conventional mechanisms of resistance (described in the previous section) do not seem to be responsible for resistance of biofilm-growing bacteria, although they may be present. This is supported by the evidence showing that strains that are normally susceptible to certain antibiotics, when growing in biofilm can become 10–1000 times more resistant to the effects of the same antibiotics.63,76-78 However, when the bacteria are dispersed from the biofilm, the antibiotics are active suggesting that this resistance is not acquired via mutations or mobile genetic elements.79,80

It is believed that there are several modes of resistance inherent to biofilms. The first of which is the poor penetration of antibiotics into the biofilm, (e.g. positively charged aminoglycosides).81,82 However, some antibiotics are able to penetrate, but they are still not active against biofilms.83 The second factor that may be involved is the altered chemical microenvironment within the biofilm. For example, oxygen can be completely consumed in the surface layers creating anaerobic niches,84 while local accumulation of acidic waste products leads to local pH differences.85 Both of these effects may antagonize the influence of an antibiotic.86 Also, in the state of nutrient deprivation and/or accumulation of waste products, some of the bacteria in the biofilm enter the stationary phase in which they are protected from the effect of antibiotics that target mainly processes occurring in growing, dividing bacteria.87 In addition, the osmotic microenvironment within the biofilms may be altered, inducing an osmotic stress

9 response in bacteria rendering the cell envelope less permeable to antibiotics.88 The third effect believed to be occurring in biofilms is the phenotypic change in the subpopulation of bacteria residing in biofilm, similar to spore formation. This has been supported by the fact that even bacteria in the young, newly formed biofilms which are not thick enough to pose a barrier either for antibiotics or nutrients and waste products, exhibit resistance to antibiotics.89,90 All of these mechanisms of antimicrobial resistance depend on the multicellular nature of biofilms which explains why bacteria, once released from the biofilm, rapidly reverts to the susceptible phenotype. We know that very specific gene regulation and bacterial cross-talk (quorum sensing) are involved both in the early stages of biofilm formation, as well as biofilm maturation and dissemination73 and that multiple resistance mechanisms act together and contribute to the biofilm persistence.

A successful antibiotic has to overcome the multiple challenges found in biofilms, including heterogeneity of the bacterial population, the microenvironment containing a decreased concentrations of nutrients and increased concentrations of microbial waste, and gradient of antibiotic concentration throughout biofilm. Very important progress is being done in identifying genes (and gene products) that mediate biofilm resistance to antibiotics, because these have the potential to become targets for chemotherapeutics or chemotherapeutic adjuvants that could be used to enhance the effectiveness of existing antibiotics against biofilm infections. Future strategies for biofilm treatment may involve therapies that disrupt the multicellular structure of the biofilm which is crucial for biofilm existence. If the multicellularity of the biofilm is disintegrated, the host defenses might be able to resolve the infection, and the efficacy of antibiotics may be restored. This includes enzymes that dissolve the matrix polymers of the biofilm,91 chemical reactions that block biofilm matrix synthesis,92 and analogues of microbial signalling molecules that interfere with cell-to-cell communication, required for normal biofilm formation.93 It seems that the main approach is targeting primarily the biofilm structure, rather than essential functions of individual cells.73

10 1.3 Antimicrobial peptides (AMPs)

Antimicrobial peptides (AMPs) are evolutionary conserved components of the innate immune response and are found in virtually all living organisms, from prokaryotes to humans.49-51 The expression of AMPs can be constitutive or inducible by infections and/or inflammatory stimuli, such as proinflammatory cytokines, bacteria, or bacterial toxins, for example lipopolysaccharides

(LPS).53 AMPs may also possess immunomodulatory activities, act as chemokines and/or induce chemokine production, inhibiting lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production, promoting wound healing, etc. Hence, they appear to be broadly involved in the coordination of the innate immune and inflammatory responses.53-55

AMPs in general have up to 100 amino acid residues and a net positive charge between +2 and +9. They are very diverse both in structure and origin, hence can only be broadly categorized. For example on the basis of their secondary structure there are four major classes:

β-sheet, α-helical, loop and extended peptides.94 In addition to the naturally occurring peptides, a vast number of synthetic peptides have been reported as well. The common feature is the adaptation of an amphiphatic conformation upon membrane interaction.95 AMPs have a broad spectrum of activity, though by far the best studied cationic antimicrobial peptides are those with antibacterial activity.51,52

The mechanism of AMPs antibacterial activity depends on several factors, most notably net positive charge (interaction with lipids in bacterial membrane and other targets), overall peptide hydrophobicity (often driving force for peptide internalization) and flexibility (transition between conformations).96 The activity and selectivity of AMPs can be modulated by balancing these factors and there is no universal empirical solution applicable to all AMPs.51 The exact mode of action of AMPs has been a topic of many debates and it was long believed that their main mode of action is compromising the bacterial membrane. There is an increasing body of evidence indicating that AMPs may in fact have multiple cellular targets. However, regardless of their exact target of action, all antibacterial cationic peptides must interact with the bacterial cytoplasmic membrane, if only to get to their site of action.96,97 Several models can explain the mechanisms of

11 membrane permeabilization by AMPs, Figure 6, and it is not excluded that the same peptide under different concentrations interacts with the membrane through different mechanisms.

The final result of A-D is that some peptide monomers get translocated into the cytoplasm, dissociate from the membrane and bind to cellular targets such as DNA and RNA, inhibit enzymatic activity such as protein synthesis or chaperone assisted protein folding.98 For example, lantibiotics such as nisin can inhibit the transglycosylation of lipid II, which is necessary for the synthesis of peptidoglycan, buforins bind to bacterial DNA99 while members of another, short, proline-rich peptide family kill bacteria by selectively binding to the 70 kDa bacterial heat shock protein DnaK.100

Figure 6. Models explaining the mechanisms of membrane permeabilization by AMPs:98,101,102 (A) carpet, (B) barrelstave, (C) wormhole or toroidal, and (D) aggregate channel

Among AMPs, cyclic lipodepsipeptides are particularly attractive as new sources of antimicrobial agents due to their potent antimicrobial activity, and presence of non-proteinogenic amino acids and one or more ester bonds, rendering them less prone to proteolytic hydrolysis.

Moreover, daptomycin, a representative cyclic lipodepsipeptide is already on the market for

12 treatment of infections caused by Gram-positive bacteria putting this group of natural products in the spotlight for development of new antibiotics.

1.4 Cyclic lipodepsipeptides in novel antimicrobial drug discovery

1.4.1 Overview

Historically, natural products have served as important sources of pharmacologically active compounds or lead structures for the development of new drugs.103-105 Although natural-product research efforts have recently lost popularity in the industry,106 a large number (over 25%) of new drugs approved between 1981 and 2006 are of natural origin.107-109 Among natural products, peptides are particularly interesting because of the key roles they play in biological processes. In

2004 more than 40 peptides were in the world market for clinical applications, and more than 400 were in advanced preclinical phases worldwide.110 Peptides’ potential for high efficacy and their minimal side effects combined with advances in solid-phase synthetic chemistry, purification technology and new strategies for peptide drug delivery made them widely considered as lead compounds in drug development.111-113 At present, peptide-based therapeutics exist for a wide variety of human diseases and conditions, including osteoporosis (calcitonin), diabetes (insulin), infertility (gonadorelin), carcinoid tumors and acromegaly (octreotide), hypothyroidism

(thyrotropin-releasing hormone [TRH]), HIV (enfuvirtide) and lipodystrophy realted to HIV treatment (egrifta), pain (ziconotide), and bacterial infections (vancomycin, daptomycin).114

Despite the great potential, there are still some limitations for peptides as drugs per se. Major disadvantages are short half-life, rapid metabolism, and poor oral bioavailability.115,116

Nevertheless, pharmacokinetic properties of peptides can be improved by various modifications.114 Peptidomimetic modifications or cyclization of linear peptides are frequently used as an attractive method to provide more conformationally constrained and thus more stable and bioactive peptides.117-122 In addition, replacement of the amide groups that undergo proteolytic hydrolysis with ester groups may lead to longer-acting compounds not so prone to proteolysis.123-126 Considering all these modifications that can potentially improve peptide

13 metabolic stability, naturally occurring cyclic depsipeptides that contain one or more ester bonds in addition to the amide bonds have emerged as promising lead compounds for drug discovery.

Cyclic depsipeptides belong to a large and structurally very diverse family of naturally occurring peptides, with a wide variety of important biological activities.127,128 The biosynthesis of these peptides proceeds nonribosomally and is catalyzed by complex multi-functional enzymes; termed non-ribosomal peptide synthases (NRPSs). NRPSs have unique modular structure in which each module contains the requisite domains for the recognition and activation of a single amino acid, generating huge structural and functional diversity of nonribosomal peptides.129 It is very well documented that cyclic depsipeptides and their lipidated derivatives exhibit a broad spectrum of biological activities including insecticidal, antiviral, antimicrobial, antitumor, tumorpromotive, anti-inflammatory, and immunosuppressive actions. Our interest for these compounds stems from the observation that they act as potent antimicrobial agents. There are numerous literature reports describing isolation, characterization, and antimicrobial activities of cyclic lipodepsipeptides. To illustrate the clinical potential of cyclic lipopeptides as novel antibacterial agents, in this chapter selected families of cyclic lipodepsipeptides will be discussed.

One of these compounds, such as cyclic lipodepsipeptide daptomycin, Figure 7, (Cubicin, Cubist

Pharmaceuticals, Inc.) has already been approved in the USA, European Union and Canada for the treatment of bloodstream and complicated skin infections caused by multidrug-resistant bacterial strains. Ramoplanin A2 (Nanotherapeutics, Inc.) is another good example of cyclic depsipeptide potential in the development of new antibiotics, Figure 10. Ramoplanin is currently in a Phase III trial for the treatment of C. difficile-associated diarrhea (CDAD) and prevention of vancomycin-resistant enterococci bloodstream infections.

The following sections will give an overview of the selected families of cyclic lipodepsipeptides that are of greater interest for the development of new antibiotics. The discovery, structural characterization, synthesis and mode of action (where applicable) are described. Besides already mentioned daptomycin and ramoplanins, the emphasis is placed on novel cyclic lipodepsipeptides with a unique structural feature, such as fusaricidins/LI-Fs,

14 circulocins and heptadepsin which, beside the ester bond and non-proteinogenic amino acid residues, contain a lipidic tail with a guanidino group at the end.

For a broader overview of the cyclic lipodepsipeptides refer to our review in Croatica

Chemica Acta 130 and references within.

1.4.2 Daptomycin

In the early 1980s, a group at Lilly Research Laboratories isolated a series of novel antibiotics designated as A21978A, B, C, D and E from Streptomyces roseosporus NRRL 11379 culture broth, Figure 7.15 The major component, A21978C, was highly active against Gram-

131 positive bacteria. RP HPLC analysis of A21978C showed the presence of six compounds (C0-

C5), out of which three compounds were most abundant, A21978C1-C3. Structural analysis revealed all compounds to be cyclic lipodepsipeptides comprised of 13 amino acid residues, 5 of which were unusual amino acids, including L-ornithine (L-Orn), L-threo-3-methylglutamic acid (L-3-

2 MeGlu), L-kynurenine (L-Kyn) and two D-amino acid residues. Initially, Asn configuration was

132 assigned as L, however, this assignation was later corrected to a D-configuration. Ten amino

13 acids form the depsipeptide macrocyclic core with a lactone linkage between the L-Kyn carboxyl group and the Thr4 hydroxyl group. The remaining tripeptide, Asp3-Asn2-Trp1, is attached to the

4 Thr carboxyl group. The amino acid sequence is conserved in all six C0-C5 daptomycins. The structural differences lie in the fatty acid chain attached to the Trp1 amino group. In the compounds C1-C3, the fatty acid chain was found to be anteiso-undecanoyl, iso-dodecanoyl and anteiso-tridecanoyl.

A minor compound of the complex C0, was found to have a mixture of both linear and

133 branched decanoyl, while C4 and C5 both contain dodecanoyl fatty acids. Daptomycin, a member of the A21978C complex, contains a straight C10 lipid side-chain, Figure 7. Comparison of A21978C1 activity with those of vancomycin, teichomycin and several -lactam antibiotics, showed that A21978C1 was at least as active as vancomycin against all streptococci and

15 staphylococci tested, including MRSA and penicillin-resistant pneumococci.134 It was also found to be bactericidal against enterococci at concentrations close to MICs (2 μg/mL).

Figure 7. Structures of daptomycin and A21978C1-C3

When combined with gentamycin, a bactericidal synergism was observed by time-kill

134 methods. A21978C1 did not interact with penicillin-binding proteins of bacterial cell membranes nor did it interfere with the DNA, RNA or protein synthesis. However, it inhibited peptidoglycan biosynthesis of both S. faecalis and S. aureus. Even more interesting was the finding that activity of this family of naturally occurring antibiotics strongly depends on the concentration of Ca2+ in the culture medium, whereas no increase in activity was observed if medium is supplemented with Mg2+, Zn2+ or Ba2+.134 A21978C’s maximum antibacterial potency is reached at the Ca2+ concentration of 50 mg/L (1.25 mM); the concentration of ionized calcium normally found in human serum.135 It has also been shown that these antibiotics cause potassium release from S. aureus, suggesting that their bacterial target is membrane and/or cell wall, Figure 8.136,137 The activity of A21978C antibiotics was proposed to be very complex, depending on incorporation of the antibiotic into bacterial membrane. Studies performed by Lakey et al. and recently by Jung et al. on daptomycins’ interaction with phospholipid vesicles and Ca2+, suggested that charge neutralization through Ca2+ complexation facilitates penetration of the peptide into the 16 membrane.138,139 It was found that the extent of penetration into the lipid bilayer is inversely proportional to the side chain length, indicating that Ca2+ dependent interaction is a specific interaction involving the polar head groups of the phospholipids and the daptomycins, with the role of the lipidic side chain being most important for the initial antibiotic attachment to the membrane.

Figure 8. Proposed mechanism of action of daptomycin; insertion of daptomycin into bacterial cytoplasmic membrane in a calcium-dependent fashion (1), oligomerization and disruption of the functional integrity of the cytoplasmic membrane (2), release of intracellular ions and rapid cell death (3)137

However, the length of the side chain was shown to influence the peptide’s toxicity. Peptides bearing longer side-chain fatty acids exhibited higher toxicity. The optimal balance between toxicity and antimicrobial activity was obtained with daptomycin, an A21978C derivative containing a decanoyl chain.140,141

In 1988 Huber et al. reported large quantity production of desired daptomycin by supplying decanoic acid during S. roseosporus fermentation.142 The lack of suitable solution or solid-phase synthetic approaches toward daptomycin and its analogs makes chemoenzymatic methods important alternatives to generate and scale up daptomycin derivatives for antibacterial screening and drug development. Chemoenzymatic methods usually combine standard solid-phase peptide synthesis (SPPS) with enzymatic peptide cyclization mediated through excised thioesterase (TE) domains from NRPSs.143-145 Antibacterial activity studies of chemoenzymatically synthesized 17 7 9 12 13 daptomycin derivatives revealed that four amino acids, Asp , Asp , and L-3-MeGlu and L-Kyn , are important for the peptide’s antibacterial potency. Furthermore, derivatives containing different macrocyclic core size were prepared by shifting the Thr4 residue to position 3, 5 or 6 in the linear precursor generating 8, 9 and 11-membered rings.145 Genetic engineering of the NRPS in the daptomycin biosynthesis was further exploited for combinatorial biosynthesis of novel antibiotics.

Baltz et al. combined NRPS module exchanges, NRPS subunit exchanges, inactivation of the tailoring enzyme glutamic acid 3-methyltransferase, and natural variation of the lipid tail to generate a library of daptomycin analogs.132,146,147 These bioengineered daptomycin analogs

13 include modifications at the L-Kyn position, and modifications of the daptomycin macrocyclic core at residues that are not conserved among structurally related lipopeptides. Some of these analogs were as active as daptomycin, demonstrating that the combinatorial biosynthesis is an effective tool to generate daptomycin analogs with significant structural diversity for further clinical evaluation.

A semisynthetic method was also successfully applied to modify the daptomycin amino acid core. In an attempt to enhance the potency of daptomycin, Parr et al. synthesized a series of N-

6 148 acylated L-Orn analogs using activated esters, anhydrides, and guanidinylating reagents.

Based on the MIC data, it was shown that the Orn6 amino group is not essential for daptomycin’s antibacterial activity. Some analogs maintaining a free amine in the Orn region showed similar or slightly better antibacterial activity, and significantly different pharmacokinetic profiles than daptomycin.

Although it is well established that daptomycin exerts its mode of action on the bacterial cell membrane in Gram-positive bacteria, its precise mode of action has not been completely clarified.

Initially, Allen et al. have shown that daptomycin has a disruptive effect on membrane permeability as demonstrated by the loss of intracellular potassium upon bacteria exposure to the peptide. The fact that this antibiotic inhibited incorporation of [14C] alanine and [14C]DAP into the peptidoglycan of S. aureus and Bacillus megaterium, and the lack of any significant inhibition of protein, RNA, DNA or lipid biosynthesis indicated that the membrane is not likely a lethal target

18 for this antibiotic. Instead, they suggested that daptomycin inhibits formation of peptidoglycan precursor, UDP-MurNAc-pentapeptide.149 Silverman et al. proposed a multistep mode of action for daptomycin.137 According to the proposed mechanism, in the first step, daptomycin binds weakly to the cytoplasmic membrane and complex Ca2+. This causes conformational change, and leads to daptomycin’s insertion into the plasma membrane and its subsequent oligomerization. In the second step, daptomycin oligomers form a channel through which intracellular potassium ion is lost, leading to membrane depolarization and bacterial cell death. Later, a more complex mode of action was proposed for daptomycin. In 2004 Hancock et al. suggested that the bactericidal action of daptomycin is not exclusively a result of the membrane depolarization, but rather daptomycin’s interaction with several bacterial components such as cell wall, various enzymes,

RNA and DNA.139 Chopra et al. in 2008 re-examined the daptomycin mode of action proposed by

Silverman et al. by performing more detailed experiments on kinetics of membrane depolarization and loss of K+, Mg2+, and ATP.150 Obtained experimental data showed that daptomycin-induced efflux of Mg2+, and ATP occurs in conjunction with K+ leakage. Based on these findings, Chopra et al. proposed that the bactericidal activity of daptomycin is not simply a consequence of K+ efflux and probably involves more general disruption of the membrane.

Eli Lilly and Co. began development of daptomycin in 1985, however, they later abandoned further pursuit due to Phase II clinical trial results which showed the occurrence of potential drug- induced myopathic events.151 In 1997 Cubist Pharmaceuticals Inc. licensed daptomycin and re- instigated clinical development of the drug. To determine the dosing regimen with potentially lower muscle toxicity two studies were conducted with dogs. Repeated intravenous administration every 24 h versus every 8 h for 20 days indicated that once-daily administration appeared to have minimized potential for daptomycin-induced skeletal-muscle effects.152 Further clinical trials were conducted using a single daily dose of the antibiotic. Daptomycin was approved in the United

States in 2003 for treatment of complicated skin and skin structure infections associated with methicillin-susceptible S. aureus (MSSA), MRSA, S. pyogenes, Streptococcus agalactiae,

Streptococcus dysgalactiae subsp. equisimilis and E. faecalis (vancomycin-susceptible only) and

19 in 2006 for treatment of bacteraemia and right-sided endocarditis caused by MSSA and

MRSA.153,154 Also, it was approved in Europe in 2006 for treatment of complicated skin and soft tissue infections. Daptomycin is marketed under the trade name Cubicin.

Although daptomycin exhibits strong activity against variety of Gram-positive bacteria including S. pneumoniae, the experimental and clinical data for mice and humans showed that daptomycin lacks efficacy against Gram-positive bacterial infections of the lung.155 In phase III clinical trial for the treatment of patients with community-acquired pneumonia (CAP) daptomycin

(applied at a dose of 4 mg/kg) failed to achieve superiority over ceftriaxone, a drug of choice for the treatment of CAP. Reported efficacies for daptomycin and ceftriaxone are 79% and 87%, respectively.156 Daptomycin's poor efficacy against CAP may be attributed to its interaction with pulmonary surfactant components.155 Recent attempts to generate more potent daptomycin derivatives in the presence of bovine surfactant utilize combinatorial biosynthesis approach.157

A5415 is a complex of eight cyclic lipodepsipeptide antibiotics produced by Streptomyces fradiae and structurally similar to daptomycin, Figure 9.158-161

These antibiotics exhibited potent activity against S. aureus and S. pyogenes infections in mice. However, acute mouse toxicities hampered their further development as a novel drug.162

Some of these antibiotics were shown to be much less inhibited by bovine surfactant than daptomycin, providing therefore a lead structure for modifications to explore structure-activity relationship (SAR) around A5415.

Very recently, Baltz et al. reported preparation of several A5415 analogs by modifying the

A5415 NRPS using segments of the daptomycin NRPS genes.163-165 Three mutants defective in

IptJ, IptK or IptL genes encoding the enzymes involved in biosynthesis of hydroxy-Asn3 and methoxy-Asp9 were generated. These deletions were combined with deletion of the IptI gene involved in biosynthesis of 3-methyl-Glu12 and with the plasmid containing combination of Ipt genes to produce a series of novel A5415/daptomycin analogs. For all the compounds, removal of the methoxy group from Asp9 and the hydroxy group from Asn3 yielded compound

A5415(Asn3Asp9) with comparable antibacterial activity to the parent A5415 or daptomycin, and

20 without loss in activity in the presence of bovine surfactant.164 The structure of A5415(Asn3Asp9) predicted from the genetic changes and bioinformatics studies was confirmed by chemical transformations, amino acid quantitation by enantiomer labeling, LC-MS/MS and 2D NMR techniques.165 This approach creates new possibilities for daptomycin SAR, and discovery of new and more potent antibiotics of this class.

13 O Ile HO 2 Glu12 D-Gln

11 O O NH D-Asn HN 2 O 1 HN O Trp H N O O 2 O H H O O N N 4 N N R NH Thr H H 10 O NH N O Gly 2 H2N Sar5 OH HN O HN O HO HN O O HyAsn3 O O 9 mOAsp O HN NH Ala6 N H O O 8 D-Lys OH Asp7 R

O A54145A

Figure 9. Representative member of the A54145 family, A54145A

1.4.3 Ramoplanins/enduracidins

In 1984 a research group from Gruppo Lepetit (Biosearch Italia) isolated from a culture broth of Actinoplanes sp. ATCC 33076 an antibiotic complex named A-16686, later renamed to ramoplanin, Figure 10. The isolated antibiotic complex was a mixture of three structurally similar

166 compounds designated as A1-A3, out of which A2 represented the major component. Initial studies, including chemical degradation, mass spectrometry and 2D NMR experiments, revealed the structural complexity of these antibiotics.167,168 Isolated compounds were identified to be cyclic lipoglycodepsipeptides containing 17 amino acids, including several nonproteinogenic amino acids such as L-hydroxyphenylglycine (L-Hpg), D- and L-aThr, L-threo--hydroxyasparagine (L-

21 tHyAsn), D-ornithine (D-Orn) and L-3-chloro-4-hydroxyphenylglycine (L-Chp). The macrocycle is

17 2 formed via lactone linkage between the L-Chp carboxyl group and the L-tHyAsn -hydroxyl group. The latter one is also linked to Asn1 bearing an unusual unsaturated acyl chain on its N-

11 terminus. In addition, L-Hpg residue carries a disaccharide moiety, -1,2-dimannose, attached to its hydroxyl group. The amino acid sequence within the ramoplanin family of natural products is conserved; the only difference lies in the unsaturated acyl chain, Figure 9. The stereochemistry of ramoplanin’s acyl chain was initially assigned to be (2Z,4Z).167 However, this was subsequently reexamined and corrected to (2Z,4E).169,170 Ramoplanins are structurally similar to ramoplanose, a trimannosylated ramoplanin isolated also from Actinoplanes sp.171 and to enduracidins.

Figure 10. Structures of ramoplanins A1-A3 and ramoplanose

Enduracidin A and B, Figure 11, were isolated in 1968 from Streptomyces fungicides B5477 culture broth.172,173 Enduracidins A and B differ only in the length of the lipid tail. Enduracidins are

17-mer peptides forming a macrocyclic core composed of 16-amino acids cyclized via lactone

2 17 bridge between Thr and L-Hpg residues, Figure 6. The characteristic features of enduracidins are the amino acids D- and L-enduracididine (D-, L-End), L-citrulline (L-Cit), and L-3,5-dichloro-4-

22 hydroxyphenylglycine (L-Dpg). Enduracidin and ramoplanin share a number of structural similarities; however, enduracidins do not have a sugar moiety. These common structural features indicate a possible similarity in mode of action and a common active pharmacophore.174

Figure 11. Structures of enduracidins A and B

The 3D NMR structure of ramoplanose revealed a double-stranded antiparallel -sheet with seven intramolecular hydrogen bonds and two reverse turns.171 Almost identical structural characteristics were found in ramoplanin A2. In 1996 Kurtz and Guba determined the 3D structure of ramoplanin A2 in aqueous solution from multidimensional NMR experiments. The structure is characterized by two antiparallel -sheets connected with six intramolecular hydrogen bonds and

169 175 one reverse -turn. Quite interestingly, Walker et al. showed that ramoplanin A2 in methanol forms a symmetric dimer at the interface of amino acid residues 10-14, and is stabilized with four intermolecular hydrogen bonds.

In 2009 McCafferty et al. reported the first X-ray structure of ramoplanin A2, showing that ramoplanin A2 in the crystal forms an amphiphatic dimer with C2 symmetry. This mutual orientation of monomers allows formation of a four-stranded antiparallel -sheet, whose interface consists of amino acid residues 9-15 and six intermolecular hydrogen bonds.176 The fact that ramoplanin A2 forms dimers in a hydrophobic solvent that mimics the environment at the bacterial

23 cell surface and in the crystal may indicate the manner in which this antibiotic interacts with its bacterial target.

As expected, due to partial amino acid sequence similarity, the overall backbone structure of enduracidin and ramoplanin share a high degree of similarity.177 Enduracidin’s structure is characterized by two antiparallel -sheets that include residues 5-7 and 10-12 connected by a turn composed of the residues 8 and 9. Three hydrogen-bonding interactions stabilizing this -

5 12 7 10 7 hairpin arrangement are between residues D-aThr and D-Ser , D-Hpg and D-End , and D-Hpg

9 177 and L-Cit .

Ramoplanins A1-A3 possess identical antibiotic activities, and are active against Gram- positive bacteria, including resistant strains such as VRE and MRSA with MICs below 1

μg/mL.178-182 They have a similar spectrum of antibacterial activities as vancomycin, but they are

183 4-8 times more potent. In addition, ramoplanin A2 exhibits bactericidal activity at concentrations close to MICs for most Gram-positive bacteria, and at concentrations twice the MIC value against

181,184 VRE, while vancomycin only displays bacteriostatic activity at its MICs. Ramoplanins A1-A3 exhibit no activity against Gram-negative bacteria.183,185 Soon afterward, three other ramoplanin analogs were isolated from the culture broth of the producing microorganism and designated as

A1’-A3’, differing from A1-A3 in the -glycosidic moiety. Ramoplanins A1’-A3’ contain only a

186 monosaccharide unit. Interestingly, A2’ showed better activity against certain bacterial strains

186 than A2; otherwise, their antimicrobial activities are similar. Enduracidins exhibited a similar spectrum of antibacterial activities to ramoplanins, whereas no data for ramoplanose antibacterial activity was reported.

SAR studies were performed by several groups in order to determine important structural characteristics modulating antibacterial activity. Ciabatti et al. prepared deglycosylated ramoplanin analogs, and analogs containing saturated N-terminal fatty acid chains.167 These modifications had no significant effect on ramoplanin’s antibacterial activity. McCafferty et al. had also reported synthesis of several semi-synthetic ramoplanin analogs with modifications on the D-

4 10 174 Orn and D-Orn side chains. Guanidylation or reductive amination of the Orn residues had

24 minimal to moderate effects on ramoplanin’s antimicrobial activity. In contrast, loss of the cationic charge by Orn acetylation, and hydrolysis of the lactone linkage severely diminished or fully

174,187,188 deleted depsipeptide’s activity. Total solution-phase syntheses of ramoplanin A2, ramoplanose aglycon, and ramoplanin A1-A3 aglycons, were accomplished by Boger et

170,187,189 al. The retrosynthetic analysis was designed around ramoplanin A2 3D structure in solution. Based on this analysis, three linear peptide precursors (heptapeptide composed of residues 3-9, pentadepsipeptide composed of residues 1, 2 and 15-17, and pentapeptide composed of residues 10-14) were prepared, sequentially coupled and cyclized to yield the ramoplanin aglycon macrocyclic core. The coupling sites were chosen to maximize the efficacy of the synthesis, prevent potential racemization that may occur during the final macrocyclization step, and to promote formation of a -sheet secondary structure of the linear 17-amino-acid precursor that would facilitate the final macrocyclization step. Ramoplanin A1 and A3 aglycons were prepared by acylation introduction of the lipid side-chains onto the synthetic ramoplanin macrocyclic core.190 In order to provide a more chemically stable alternative to natural ramoplanin

2 A2, two amide aglycon analogs have been synthesized where L-tHyAsn has been replaced by L-

191 2,3-diaminopropionic acid (L-Dap) or L-2,4-diaminobutyric acid (L-Dab). The L-Dap-analog maintained the same activity as ramoplanin A2, whereas the ring extension by one methylene group, L-Dab-analog, led to a loss of antibiotic activity. Recently, Boger et al. reported an alanine- scan on ramoplanin’s L-Dap-analogue providing insight into the potential role and importance of each amino acid residue for its antibacterial activity.192 In contrast, there are no reports on the total synthesis of enduracidins. However, biosynthesis of enduracidin A analogs with altered halogenation patterns has been reported in 2010, and initial in vitro antibacterial evaluation showed that these analogs retained activity against S. aureus.193

In 1990 Somner and Reynolds showed that ramoplanin blocks bacterial cell wall biosynthesis at the step catalyzed by membrane-associated glycosyltrensferase MurG.194 They proposed a mechanism of action that involves ramoplanin binding to MurG substrate Lipid I and inhibition of

Lipid II formation. Eight years later, Brötz, et al. showed that ramoplanin, as well as some

25 lantibiotics, interacted with Lipid II, and raised a question of a possible second mode of action for ramoplanin.195 Recently Walker et al. demonstrated that ramoplanin has higher affinity for Lipid II, and proposed the transglycosylation step as the primary target. Inhibition kinetic experiments revealed that ramoplanin binds Lipid II with a stoichiometry of 2:1, Figure 12.196

Figure 12. Peptidoglycan biosynthesis and potential targets of ramoplanins (circled in red)196-198

Interestingly, fibril formation of Lipid II analogs was observed upon titration with ramoplanin.174,175 Walker’s group also found that ramoplanin inhibits MurG by the mechanism that does not include binding to Lipid I, but rather via direct interaction with the enzyme.197 However, since Lipid I and MurG are intracellular, and therefore not accessible to ramoplanin,175,198 and considering ramoplanin’s higher affinity toward Lipid II, it is not clear whether inhibition of MurG is of physiological relevance, Figure 12. Enduracidin has an almost identical mode of action as 26 ramoplanin.196 The main difference is in the MurG inhibition. Ramoplanin displayed noncompetitive inhibition of MurG, whereas enduracidin produced an inhibition curve consistent with substrate binding.196 Ramoplanin is currently in Phase III trial for the treatment of C. difficile associated diseases (CDAD), and in Phase III development for the prevention of vancomycin- resistant enterococci bloodstream infections. Enduracidin has been shown to be effective in humans for treating urinary tract and skin infections caused by methicillin-resistant S. aureus.199

No toxicity or side effects for enduracidin in these trials were reported.

1.4.4 Katanosin A and lysobactin (katanosin B)

Katanosin A and lysobactin (also known as katanosin B) are lipophilic, cyclic depsipeptides isolated in 1988 independently from two sources. Shionogi & Co., Japan, isolated the two natural products from a strain related to the genus Cytophaga, whereas the Squibb Institute, USA, isolated lysobactin from Lysobacter sp. SC 14067.200-203 These two natural products have a macrocyclic core composed of nine amino acids connected via lactone bridge between L-threo--

11 phenylserine (L-tPhSer) and C-terminal L-Ser . A hydrophobic D-Leu-Leu side chain is attached to the N-terminus of L-tPhSer3, Figure 1. Out of eleven amino acids, six are nonproteinogenic, including L-tPhSer, L-threo--hydroxyaspartic acid (L-tHyAsp), L-hydroxyleucine (L-HyLeu), L-allo- threonine (L-aThr) and two D-amino acids, D-Arg and D-Leu. The main structural difference between katanosins A and lysobactin (katanosin B) lies in the amino acid residue at position 7,

Figure 12. Katanosin A contains a Val residue, whereas lysobactin contains Ile residue.200-

202,204,205 Several groups have reported synthesis of lysobactin fragments,206-211 and an Fmoc solid-phase synthesis of katanosin A and lysobactin analogs containing Asp10, Thr8, Leu4 instead

10 8 4 212 of L-tHyAsp , L-aThr , and L-HyLeu residues has been reported by Egner and Bradley. The first total solution-phase syntheses of lysobactin have been reported recently. In 2007 von

Nussbaum et al. at Bayer AG reported a synthesis of lysobactin rationalized by its crystal structure.213 A key step in this synthesis, peptide’s macrocyclic core assembly, was achieved via conformation–directed cyclization in which H-bonding involving unprotected hydroxyl side chains

27 of amino acids plays a crucial role. Consequently, no side chain hydroxyl group protections are required, simplifying somewhat lysobactin’s total synthesis. Shortly afterward, Van Nieuwenhze et al. reported peptide-chemistry based solution phase total synthesis of lysobactin214 and few years later solid-phase approach215 where the linear peptidyl precursor was prepared on solid support and cyclized in solution. The site of macrolactamization was the same as in the case of solution phase synthesis reported previously214 and involved a glycine-derived activated ester.216 Besides

3 the natural product, the same group prepared an analog in which L-tPhSer was substituted with

3 6 3 Thr in order to probe the role of the cation-π interaction between D-Arg and L-tPhSer which was observed in the crystal structure.213

Figure 13. Structures of katanosin A and lysobactin (katanosin B)

Both katanosin A and lysobactin (katanosin B) show quite potent in vitro activity against

Gram-positive bacteria such as antibiotic-resistant S. aureus, E. faecium, Streptococcus pyogenes, Streptococcus pneumoniae and Enterococcus faecalis. Reported antibacterial activity, as indicated by the minimum inhibitory concentrations (MIC), ranged from 0.1 to 0.8 μg/mL.200,203

Katanosin A and lysobactin exhibit promising in vivo activity as well, as demonstrated by their curative effects when administered subcutaneously to mice infected with Gram-positive pathogens, Table 2.200

28

Table 2. In vivo efficacy of katanosin A and lysobactin (katanosin B) against bacterial infection in mice200

Antibiotica Katanosin A Lysobactin (Katanosin B)

Bacteria ED50 mg/kgx2 ED50 mg/kgx2

S. aureus Smith 1.20 0.67 S. aureus SR 2030 1.90 0.77 S. pyogenes C-203 2.86 1.55 S. faecalis SR 700 2.10 1.80 a Compounds were administered subcutaneously at 1 and 5 h after infection

Modest to no activity against Gram-negative bacteria has been reported.203 The synthetic analog with substituted L-tPhSer3 retained its antimicrobial activity and showed significantly higher levels of Bacillus subtilis membrane permeabilization than the parent compound, even at concentrations 4 times lower than MIC.215

The spectrum of antibacterial activity for lysobactin parallels that of vancomycin, and although lysobactin shows higher potency (up to four times lower MICs), it is also slightly more toxic as demonstrated by the in vivo toxicity in mice, Table 3.203

In 1989 Tymiak et al. reported structure determination of lysobactin and structural requirements for its biological activity.204 Based on lysobactin’s semisynthetic modifications, it

1 was demonstrated that the macrocyclic lactone bridge and N-terminal D-Leu are crucial structural elements contributing to its antibacterial activity.

Table 3. In vivo toxicity of lysobactin (katanosin B) and vancomycin in mice203

LD50 (mg/kg) Antibiotic iva ipb Lysobactin 77 132 (Katanosin B) Vancomycin >400 >1000 a iv=intravenously, b ip=intraperitoneally

29 Experiments performed by Bonner et al.203 have suggested that lysobactin is primarily a cell wall acting agent probably affecting a step prior to UDP-N-acetylglucosamine formation.

According to recent data published by Maki et al.,217 lysobactin inhibits the transglycosylation and transpeptidation steps of bacterial cell wall biosynthesis by an unidentified mechanism that differs from the D-Ala-D-Ala binding characteristic for vancomycin, a drug of choice for treatments of infections caused by resistant bacterial strains. It was shown that lysobactin inhibits nascent peptidoglycan biosynthesis much like vancomycin; but unlike vancomycin, it also inhibits lipid intermediate formation. Interestingly, the MIC of lysobactin against S. aureus SRM133 was not affected by addition of acetyl-Lys-D-Ala-D-Ala, while the MIC of vancomycin increased significantly.217 These data strongly suggest that the inhibition of bacterial cell wall biosynthesis is a result of binding to lipid intermediates, substrates of several successive enzymes, rather than a result of the direct effect on one enzyme. However, lysobactin’s mode of action is not yet precisely understood.

1.4.5 Fusaricidins/LI-Fs

Fusaricidins or LI-Fs are a family of cyclic lipodepsipeptide antifungal antibiotics isolated from

Paenibacillus sp, Figure 14.218-220 Their common structural feature is the macrocyclic portion

1 4 6 consisting of six amino acid residues, three of which, Thr , D-aThr , D-Ala are conserved throughout the family, and a 15-guanidino-3-hydroxypentadecanoic acid attached via amide bond to the N-terminal Thr1. Fusaricidins/LI-Fs are cyclized by a lactone bridge between N-terminal

1 6 Thr hydroxyl group and C-terminal D-Ala . In 1987 Kurusu and Ohba reported isolation of LI-Fs from Paenibacillus polymyxa L-1129 strain.218,221 Although RP HPLC analysis of the antifungal antibiotics mixture isolated from the fermentation broth showed the presence of at least ten compounds, only five were isolated, which were subsequently named LI-F03, F04, F05, F07 and

F08. It was also noticed that each isolated antifungal antibiotic was, in fact, a mixture of two homologous components that were not successfully separated at the time.218 In 2000 Kuroda et al. reported sequences of all isolated members of LI-F family.221 The (R)-configuration of fatty

30 acid for LI-F04a was determined just recently upon total synthesis of both fatty acid enantiomers and their comparison with the natural LI-F04a.222 However, it is not yet known whether this stereochemistry is conserved within the family. All isolated antimicrobials showed high activity against a variety of fungi and Gram-positive bacteria, whereas no activity was observed against

Gram-negative bacteria.218 Approximately ten years after the discovery of the LI-F family of antifungal antibiotics, Kajimura and Kaneda isolated a series of compounds named fusaricidins from the culture broth of P. polymyxa KT-8 strain.219,220 Fusaricidins A-D, Figure 14, are analogous to the LI-F family of antimicrobial antibiotics. In fact, LI-F peptides were later determined to be mixtures containing previously structurally characterized and new fusaricidins.221,223

Analog R2 R3 R5

LI-F03a = Fusaricidin C D-Val Tyr D-Asn

LI-F03b = Fusaricidin D D-Val Tyr D-Gln

LI-F04a = Fusaricidin A D-Val Val D-Asn

LI-F04b = Fusaricidin B D-Val Val D-Gln

LI-F05a D-Val Ile D-Asn

LI-F05b D-Val Ile D-Gln

LI-F06a D-aIle Val D-Asn

LI-F06b D-aIle Val D-Gln

LI-F07a D-Val Phe D-Asn

LI-F07b D-Val Phe D-Gln

LI-F08a D-Ile D-Ile D-Asn

LI-F08b D-Ile D-Ile D-Gln Fusaricidin analog 23 D-Val Val D-Asn

Figure 14. Structures of fusaricidin/LI-F family of compounds and the synthetic analog

Out of four isolated fusaricidin antibiotics, fusaricidin A (LI-F04a),221 showed the most promising antimicrobial activity against a variety of fungi, including clinically important Candida albicans and Cryptococcus neoformans. Fusaricidin A (LI-F04a) also exhibits potent activity against Gram-positive bacteria such as S. aureus (MICs ranging from 0.78-3.12 g/mL for most of the tested strains). Fusaricidins/LI-Fs did not, however, show activity against Gram-negative bacteria.219,220 Low acute toxicity in mice have been reported for LI-F03, LI-F04, LI-F05, LI-F07,

218 and LI-F08 (LD50 150-200 mg/kg). Fusaricidins/LI-Fs’ mode of action is still unknown. Two 31 solid-phase synthetic approaches toward the most active fusaricidin A (LI-F04a) have been reported recently. Our laboratory reported in 2006 Fmoc solid-phase synthesis of a fusaricidin A

(LI-F04a) analog containing 12-guanidino dodecanoic acid instead of naturally occurring 15- guanidino-3-hydroxyhexadecanoic acid.224 One reported synthetic approach includes resin

5 attachment of the first amino acid, D-Asp , via side chain, successful combination of four quasiorthogonal temporary protecting groups, stepwise Fmoc solid-phase synthesis of a linear precursor peptide, lipid tail attachment followed by last amino acid coupling via ester bond and on-resin head-to-tail macrolactamization. This strategy allows the complete suppression of the undesired ON acyl shift, and an efficient automated solid-phase synthesis of cyclic lipodepsipeptides.224 Since the entire synthesis was accomplished on the solid support, this opens a possibility for combinatorial modification of fusaricidin A (LI-F04a) natural products and their analogs. Total synthesis of this natural product was reported in 2010 by Cochrane et al. In this approach, a linear peptide sequence was assembled on a solid-support using Fmoc- chemistry, followed by peptide cleavage, in solution macrolactonization, and final attachment of

15-guanidino-3-hydroxyhexadecanoic acid.222 Both enantiomers of this fatty acid were assembled by Yamaguchi-Hiaro alkylation of corresponding chiral epoxide precursors. By comparison of the optical rotations of both synthetic fusaricidin A (LI-F04a) analogs with the natural product, it was determined that the natural product has fatty acid with (R)-absolute configuration. Two groups,

Jensen et al.225 and Park et al.,226 have recently reported identification and isolation of putative LI-

F/fusaricidin synthetase gene, fusA, from P. polymyxa, opening the possibility for the development of biosynthetic approaches toward this family of naturally occurring cyclic lipodepsipeptides and their analogs.

1.4.6 Circulocins

The strains of Bacillus are known to produce diverse structures of bioactive cyclic lipopeptides227 which can be classified into two groups based on common structural features. The members of the first group have the peptide N-terminus connected via an amide bond to a β-

32 hydroxy or β-amino fatty acid and the C-terminus carboxyl group forms either a lactone with the

β-hydroxy group of the fatty acid, as seen in surfactin228 and lichenysins,229 or a lactam with the β- amino group, as seen in iturins230 and bacillomycins.231 In this case the fatty acid chain is incorporated in the into the ring system. The second group, in which the fatty acid is attached to the N-terminus of the cyclic core via amide bond and lies outside the ring system, includes members of fusaricidin/LI-F family,218-220 plipastatins,232,233 colistins234 and octapeptins.235

In 2001 He et al. reported isolation and structural characterization of novel cyclic lipopeptides isolated from Bacillus circulans J2154 named circulocins α-δ, Figure 15.236 Circulocins belong to the second group and contain a depsi-pentapeptide, α and β, or depsi-hexapeptide core, γ and δ, to which is attached a saturated fatty acid with a terminal guanidino function. The amino acid

1 2 3 4 5 sequence found in circulocins α and β is Thr -D-Phe -Ile -L-Dab -Asp while circulocins γ and δ

1 2 3 4 5 6 contain Thr -D-Leu -Ile -D-allo-Thr -D-Asp -D-Ala .

Circulocin β differs from α only in the structure of the fatty acid by lacking the 3-hydroxy group in the 19-guanidino-3-hydroxy-nonadecanoic acid, while circulocin δ has the longest fatty acid tail with two additional methylene groups. The presence of fatty acids is very common in the natural products; however this length has thus far not been reported.

Circulocins α-γ showed potent activity against Gram-positive bacteria, including multiple-drug resistant Staphylococci and vancomycin-resistant Enterococci, and yeast Candida albicans, but poor activity against Gram-negative bacteria. Among the circulocins tested, circulocin γ showed the highest activity against all tested organisms with MICs in the range of 0.5-2 μg/mL.236 The presence of both hydrophilic and hydrophobic features indicates potential surface-active properties which have been observed for other lipopeptides produced by Bacillus.

33

Figure 15. Structures of circulocins α-δ

1.4.7 Heptadepsin

In 2004, Umezawa et al. search for bacterial metabolites in the culture broth of Paenibacillus sp. BML771-113F9 for compounds that inhibit adhesion of human myelocytic cell line HL-60 cells to LPS-stimulated human umbilical vein endothelial cells (HUVEC) led to a discovery of a cyclic lipodepsipeptide named heptadepsin.237 Structural features of heptadepsin, cyclic depsipeptide core and long fatty acid tail with a guanidine group at its end, Figure 16, are reminiscent of those belonging to fusaricidin/LI-F221 and circulocin236 families of compounds and produced by Bacillus.

1 2 3 4 5 However, the cyclic core is made of seven amino acid residues, Ser -D-Val -Ile -D-Ala -Ser -

6 7 D-Asn -D-Ala , instead of five or six which was found in fusaricidins/LI-Fs and circulocins. The tail on the other hand is identical to the one found in fusaricidin/LI-F family. Stereochemistry of the 3- hydroxy position, however, is still unknown.

Interestingly, although Bacilli produce a plethora of cyclic lipodepsipeptides, none of those have been reported to inhibit LPS-induced cell adhesion. Fusaricidin A and B were tested in the same experiments as heptadepsin and they showed no inhibitory activity.237

34

Figure 16. Structure of heptadepsin

1.5 Research goals

The development and spreading of multi-drug bacterial resistance can not be circumvented and ultimately our most efficient way to combat resistance remains the discovery of new classes of antibiotics. Natural products are a very important source of biologically active compounds or lead structures for the development of new drugs. Among them, cyclic depsipeptides are particularly interesting because of their broad spectrum of biological activity, including antimicrobial activity, enhanced metabolic stability, and in general decreased potential for resistance induction. However, difficult isolation and purification required for larger quantities of naturally occurring depsipeptides, and especially limited access to their synthetic analogs hampers their full utilization.

We are particularly interested in fusaricidin/LI-F and the katanosin family of natural products because of their antibacterial potency and unique structure. The positive charge on these peptides is separated from the amino acid sequence, differentiating them from the rest of the

AMPs, suggesting a potentially novel mode of action. The main research goals of these studies were the preparation of Fmoc-SPPS compatible building blocks and the development of Fmoc solid-phase syntheses for preparation of cyclic lipodepsipeptides belonging to fusaricidin/LI-F and lysobactin families of natural products, as well as the evaluation of ther bioactivity profiles. In addition, we performed a structure-activity relationship study (SAR), to determine the minimal

35 structural requirements for antibacterial activity, and nonselective toxicity of the fusaricidin/LI-F class of antibacterial peptides.

The SAR involves evaluation of the role of the lipidic tail and its charge, as well as the roles of each of the residues in the amino acid sequence on the antimicrobial activity of the peptides.

Furthermore, these synthetic peptides were evaluated for stability in human serum and toxicity towards human cells. Conformational studies were performed to assess the effect of peptide conformational flexibility on their bioactivity profile. Since biofilms are in general major medical concern, our most potent analogs were evaluated for anti-biofilm activity as well.

The preparation of lysobactin analogs was more challenging due to the lack of Fmoc-SPPS compatible building blocks, in particular orthogonaly protected L-threo-hydroxyaspartic acid. We have developed a simple and efficient synthetic approach toward this unusual amino acid that involves enantioresolution of the commercially available D,L-threo-hydroxyaspartic acid racemic mixture followed by its orthogonal protection. The prepared building blocks were used in synthesis of lysobactin analogs which were also evaluated for antimicrobial activity.

36 CHAPTER 2

STRUCTURAL MODIFICATION OF FUSARICIDIN/LI-F FAMILY OF CYCLIC

LIPODEPSIPEPTIDES AND ITS EFFECT ON STABILITY,

ANTIMICROBIAL ACTIVITY AND HUMAN CELL TOXICITY

2.1 Overview

With the antibiotic resistance in continuous rise, the development of new antibiotics, in particular those with novel mode of action became an imperative.2-5 Unique simple structure of fusaricidin/LI-F family of naturally occurring antibiotics, Figure 14, and their promising activity against a wide range of microorganisms including Gram-positive bacteria and fungi, have encouraged us to further investigate the antimicrobial potential of this family of peptides.

Fusaricidins/LI-Fs common structural feature is the macrocyclic ring consisting of six amino acid

1 4 6 residues, three of which, Thr , D-aThr , and D-Ala , are conserved throughout the family, as well as the 15-guanidino-3-hydroxypentadecanoic acid tail attached to the N-terminal Thr1 residue by an amide bond. Fusaricidins/LI-Fs are cyclized by a lactone bridge between the N-terminal Thr1

6 hydroxy group and the C-terminal D-Ala . Taking into consideration fusaricidins/LI-Fs unique structural characteristics and their promising antibacterial activity, we hypothesize that this class of antibacterial peptides exert their antibacterial effects through a novel mode of action.

Therefore, synthesis of fusaricidins/LI-Fs analogs and better understanding of their structure- activity relationship represent first steps toward full exploitation of their antibacterial potentials.

To gain better insight into fusaricidin’s structural requirements for antibacterial activity, their stability under physiologically relevant conditions and nonselective toxicity, we have synthesized

19 fusaricidin/LI-F analogs varying in the amino acid sequences, the macrocyclic ring closure, and the lipidic tail structure.

37 We have found that substituting the ester bond in the natural sequence with an amide bond significantly improved peptide stability and minimized toxicity toward human cells, whereas having no or minimal effect on antibacterial activity. The positive charge of the lipidic tail in a form of guanidinium group also showed to be crucial for their activity. More stable and less cytotoxic amide analogues may have significant advantages over natural products and their depsipeptide derivatives as lead structures for the development of new antibacterial agents. In addition, amide analogues are synthetically more accessible than the parent depsipeptides, allowing for further structural optimization including a combinatorial chemistry approach.

2.2 Solid-phase synthesis

Amino acid sequences and lipid tails of synthesized fusaricidin/LI-F analogs 1-19 are shown

1 6 in Figure 17. Analogs 1-12 are depsipeptides containing an ester bond between Thr and D-Ala

6 1 6 or Gly residues. In analog 13, naturally occurring amino acid residues Thr and D-Ala are replaced with Lys thus substituting the ester moiety, as well as two chiral centers, while keeping the same number of the atoms in the ring. Four cyclic analogs, 14-17, have an ester bond replaced with an amide bond by substituting while analog 18 is a linear version of 6 and was prepared as a control. Analog 19 also has Thr1 substituted for Dap1, however the amide bond

1 6 between Dap and D-Ala is methylated.

2.2.1 Synthesis of depsipeptide analogs

224 Synthesis of depsipeptide analogs 1-12 is shown in Scheme 1. In the first step, the C- terminal amino acid Fmoc-D-Asp-OAllyl was attached to a PEG-PS based amide resin (TentaGel

S RAM) via side chain using standard HBTU/HOBt/NMM synthetic protocol. Standard Fmoc

SPPS strategy was used throughout. Ester bond was formed between Thr1 side chain hydroxyl group and Aloc-D-Ala-OH or Aloc-Gly-OH carboxyl group using DIC/DMAP coupling conditions in

DCM. In order to avoid potential epimerization during Aloc-D-Ala-OH coupling, a catalytic amount

38 of DMAP (0.2 eq.) was used.238 Under applied experimental conditions no epimerization was observed as indicated by analytical RP HPLC (data not shown).

LIPID TAIL R2 O H O O H LIPID TAIL N C N C C Ac CH H NH R3 R1 CH HC O C O H N X 2 12-ADA 5 O C NH HC H O CH 4 H N N R HN H C R 2 12-GDA 6 C C N 5 O NH H O R5 X=O,NH,NMe

[a] [b] Analog Lipid Tail Sequence Rt (min)

1 Ac Thr1-D-Val2-Val3-D-Thr4-D-Asn5-D-Ala6 11.89 2 Ac Thr1-D-Val2-Val3-D-aThr4-D-Asn5-D-Ala6 11.96 3 12-ADA Thr1-D-Val2-Val3-D-Thr4-D-Asn5-D-Ala6 14.78 4 12-ADA Thr1-D-Val2-Val3-D-aThr4-D-Asn5-D-Ala6 14.87 5 12-GDA Thr1-D-Val2-Val3-D-Thr4-D-Asn5-D-Ala6 15.76 6 12-GDA Thr1-D-Val2-Val3-D-aThr4-D-Asn5-D-Ala6 15.81 7 12-GDA Thr1-D-Val2-Val3-D-Ala4-D-Asn5-D-Ala6 15.16 8 12-GDA Thr1-D-Val2-Ala3-D-aThr4-D-Asn5-D-Ala6 14.95 9 12-GDA Thr1-D-Ala2-Val3-D-aThr4-D-Asn5-D-Ala6 14.7 10 12-GDA Thr1-D-Val2-Val3-D-aThr4-D-Asn5-Gly6 15.48 11 12-GDA Thr1-D-Val2-Phe3-D-aThr4-D-Asn5-D-Ala6 16.82 12 12-GDA Thr1-D-Val2-Tyr3-D-aThr4-D-Asn5-D-Ala6 15.18 13 12-GDA Lys1-D-Val2-Val3-D-aThr4-D-Asn5 15.44 14 12-GDA Dap1-D-Val2-Val3-D-aThr4-D-Asn5-D-Ala6 15.12 15 12-GDA Dap1-D-Val2-Ala3-D-aThr4-D-Asn5-D-Ala6 14.17 16 12-GDA Dap1-D-Val2-Phe3-D-aThr4-D-Asn5-D-Ala6 16.27 17 12-GDA Dap1-D-Val2-Tyr3-D-aThr4-D-Asn5-D-Ala6 14.58 18 12-GDA Thr1-D-Val2-Val3-D-aThr4-D-Asn5-D-Ala6 15.01 Linear 1 2 3 4 5 6 19 12-GDA NMe-Dap -D-Val -Val -D-aThr -D-Asn -D-Ala 15.25

Figure 17. Sequences of synthetic fusaricidin/LI-F analogs. [a] Differences among the sequences of naturally occurring fusaricidin/LI-F and synthetic analogs are highlighted in bold. [b] Rt=retention time obtained by analytical RP HPLC. Method: 2% B for 0.5 min followed by linear gradient 2→98% B over 30 min where A is 0.1% TFA in water and B is 0.08%TFA in ACN

The lipid tail, Fmoc-12-aminododecanoic acid (Fmoc-ADA-OH), was incorporated into the

6 6 linear peptide precursor prior to D-Ala /Gly coupling via ester bond and on-resin cyclization in

39 order to avoid the undesired ON acyl shift known to occur under basic conditions required for

Fmoc removal, Scheme 2.224,239,240 After selective removal of Aloc and Allyl protective groups by

241 treatment with Pd(Ph3P)4 and non-basic borane dimethylamine complex as scavenger, the

6 6 5 linear peptide was cyclized through an amide bond between D-Ala or Gly and D-Asn residues using PyBOP.

Scheme 1. Synthesis of fusaricidin/LI-F depsipeptide analogs 1-12. Reagents and conditions: a)

Fmoc-D-Asp-OAllyl, and Fmoc-AA-OH, standard Fmoc-SPPS deprotection and coupling

protocols; b) Ac-Thr-OH, standard Fmoc-SPPS deprotection and coupling protocols; c) Aloc-D-

Ala-OH or Aloc-Gly-OH (4 eq), DIC (4 eq), DMAP (0.2 eq), DCM, r.t., 18 h; d) Pd(Ph3P)4 (0.1 eq),

HN(CH3)2•BH3 (4 eq), DCM, r.t. 2x10 min; e) PyBOP (2 eq), DIEA (6 eq), DMF, r.t. 18 h; f)

TFA:thioanisole:H2O=95:2.5:2.5 (v/v/v), r.t. 3 h; g) Fmoc-Thr-OH, standard Fmoc-SPPS deprotection and coupling protocols; h) Fmoc-ADA-OH, standard Fmoc-SPPS deprotection and coupling protocols; i) 20% piperidine/DMF (v/v), r.t., 25 min); j) N,N’-di-Boc-N’’-triflylguanidine (5 eq), TEA (5 eq), DCM, r.t. or N,N’-bis(tert- butoxycarbonyl)thiourea (3 eq), 2-chloro-1-methylpyridinium iodide (3 eq), TEA (4 eq), DMF, r.t. 18 h

40 The conversion of the lipid tail’s amino into the desired guanidino group was achieved by the removal of the Fmoc-protecting group using standard piperidine deprotection protocol and subsequent treatment of the peptidyl-resin with N,N’-bis(tert-butoxycarbonyl)thiourea followed by

Mukaiyama’s reagent, 2-chloro-1-methylpyridinium iodide242 or N’,N’’-di-Boc-N’’-triflyl guanidine243,244 (see section 2.2.5). Final deprotection and cleavage from the resins was carried out using a cleavage cocktail of TFA:thioanisole:water (95:2.5:2.5 v/v/v). All analogs were purified using preparative RP HPLC, and purity (95%) was confirmed with analytical RP HPLC and

MALDI-TOF MS.

Scheme 2. O→N acyl shift

2.2.2 Synthesis of amide analogs

Amide analogs 13-17 were prepared by replacing the Thr1 residue with Lys 13 or Dap, 14-17,

Scheme 3. For this purpose, an identical Fmoc SPPS strategy was employed. In the case of analog 13, upon synthesis of linear precursor, Allyl and Aloc protecting groups were removed and

1 5 the peptide was cyclized through Lys ε-amino group and D-Asn α-carboxyl group. In the case of analogs 14-17, selective removal of Mtt protecting group from Dap1 with 2% TFA in DCM allowed coupling of Aloc-D-Ala-OH using standard coupling protocols. Final synthetic steps were performed as described above.

41

Scheme 3. Synthesis of fusaricidin/LI-F amide analogs 13-17. Reagents and conditions: a) Fmoc-

D-Asp-OAllyl, and Fmoc-AA-OH, standard Fmoc-SPPS deprotection and coupling protocols; b) Fmoc-Lys(Aloc)-OH, standard Fmoc-SPPS deprotection and coupling protocols; c) Fmoc-ADA-

OH, standard Fmoc-SPPS deprotection and coupling protocols; d) Pd(Ph3P)4 (0.1 eq),

HN(CH3)2•BH3 (4 eq), DCM, r.t. 2x10 min; e) PyBOP (2 eq), DIEA (6 eq), DMF, r.t. 18 h; f) 20% piperidine/DMF (v/v), r.t., 25 min); g) N,N’-di-Boc-N’’-triflylguanidine (5 eq), TEA (5 eq), DCM, r.t. or N,N-bis(tert-butoxycarbonyl)thiourea (3 eq), 2-chloro-1-methylpyridinium iodide (3 eq), TEA (4

eq), DMF, r.t. 18 h; h) TFA:thioanisole:H2O=95:2.5:2.5 (v/v/v), r.t. 3 h; i) Fmoc-Dap(Mtt)-OH, standard Fmoc-SPPS deprotection and coupling protocols; j) TFA:thioanisole:DCM=2:3:95, r.t.,

30 min; k) Aloc-D-Ala-OH, standard Fmoc-SPPS coupling protocol

2.2.3 Synthesis of the linear analog

Linear analog 18, Figure 18, was prepared on 2-chlorotrityl chloride resin (Cl-TrtCl)245 using standard Fmoc SPPS chemistry, starting with attachment of Fmoc-D-Ala-OH via carboxyl group.

Loading of the resin was determined to be 0.5 mmol/g.246 Use of Cl-TrtCl resin in this case afforded a linear peptide with a C-terminal carboxyl group identical to the hydrolysis product of 6.

42 NH O

H2N NH N Thr-D-Val-Val-D-allo-Thr-D-Asn-D-Ala-OH 18 10 H

Figure 18. Structure of linear fusaricidin/LI-F analog

2.2.4 Synthesis of the N-methylated analog

In order to further probe the effect of substituting the ester bond for an isosteric equivalent,247 we synthesized an N-methylated analog containing NMe-Dap1 19 (NMe-amide) instead of Thr1 found in the natural product. The synthesis of this analog begins with formation of a linear peptidyl precursor in the same fashion as described above with the exception that the amino group at the end of the lipidic tail is converted into the guanidine moiety before methylation,

Scheme 4. This was necessary because the Fmoc protecting group at the lipidic tail can be cleaved during removal of nosyl group (2-nitrobenzenesulfonyl, o-NBS) which is used as a temporary protecting group to ensure monomethylation of the Dap β-amino group.248 The o-NBS group was introduced using Fukuyama’s method249 followed by racemization-free N-methylation using Mitsunobu conditions. The reaction progress was monitored by RP HPLC and MALDI-TOF

MS. The nosyl group removal was achieved by a nucleophilic addition reaction using DBU and 2- mercaptoethanol.250 Although the N-methylation changes the stereoelectronic property of an amino group and subsequent coupling to that amino group is considered to be difficult, with use of HOAt/HATU coupling reagents the coupling of Aloc-D-Ala-OH was completed (> 95% based on

RP HPLC) within 1 h. The removal of the Allyl/Aloc protecting groups and subsequent cyclization and cleavage from the resin proceeded using the same protocols as above without any difficulties observed.

43

Scheme 4. Synthesis of N-methylated fusaricidin/LI-F analog 19. Reagents and conditions: a) o-

NBS-Cl (4 eq), collidine (10 eq), NMP, 15 then 10min; b) PPh3, MeOH, DIAD, THF, portionwise over 1 h; c) HSCH2CH2OH (10 eq), DBU (5 eq), NMP, 30 min; d) Aloc-D-Ala-OH (3 eq), HOAt (3 eq), HATU (3 eq), DIEA (6 eq), NMP, 1h; e) Pd(PPh3)4 (0.1 eq), NMe2·BH3 (4 eq), DCM, 30 min; f)

PyBOP (2 eq), DIEA (6 eq), DMF, overnight; g) TFA:thioanisole:H2O=95:2.5:2.5 (v/v/v), r.t. 3 h

2.2.5 Guanidinylation of resin-bound amines

2.2.5.1 Introduction

Gunidino functionality is often found in natural products and is considered as an important pharmacophoric element due to its strong basic property and capabilities of forming intermolecular H-bonds, charge pairing and cation- interactions.251,252 Many synthetic and natural products possessing guanidinium functionality exhibit a wide range of biological

44 activities253 including antimicrobial activity,51,218-220 Na-H exchange (NHE) inhibition,254,255 thrombin inhibition,256 gene delivery,257,258 and cell-penetrating properties.259,260

In the case of fusaricidin/LI-F family of natural products, the guanidinium group is located at the end of the lipidic tail and showed to be important for their antibacterial activity. Since appropriately protected guanidino fatty acid building blocks are not commercially available, our strategy toward guanidinylated fusaricidin/LI-F analogs include solid-phase synthesis of a cyclic lipopeptide precursor possessing a lipidic tail with a terminal amino group followed by its conversion into corresponding guanidine. To find the optimal method for this conversion, we have examined commonly used guanidinylation reagents under the conditions compatible with standard solid-phase peptide synthesis.

Although a number of methods for guanidinylation of resin-bound primary amines have been reported,251,252 only a few of the described protocols are fully compatible with the standard solid- phase peptide chemistry.242-244,261-265 The most commonly used guanidinylating reagents are diprotected triflylguanidines 20,243,244 reagents based on 1H-pyrazole-1-carboxamidine 21262 and di-urethane-protected thiourea 22 most frequently in combination with an activating agent such as

Mukaiyama’s reagent 23, N-iodosuccinimide (NIS) 24, or one of the carbodiimides, Figure

19.242,251,265-267

Tf N N S N Boc Boc Boc Boc O N O N N N N N Cl . - H H HN NH2 HCl H H I I 20 21 22 23 24

Figure 19. Structures of guanidinylating agents used in the study

However, these reagents are not without shortcomings. For example, it was observed by us and others that 1H-pyrazole-1-carboxamidine 21 fails to completely guanidinylate resin-bound amines even after prolonged reaction time,224,242,265 although it is used in great excess. Self- condensation of 21 under guanidinulation reaction conditions has been reported as well.262 In the

45 case of guanidinylation of resin-bound amines using di-Boc-protected thiourea 22 and

Mukaiyama’s reagent 23, the reaction efficacy depends on the steric hindrance of the amino group and the solvent.242,266 In adition, reaction of resin-bound amine with 23 has also been reported,265 as well as the reaction with thiourea 22 via S-atom resulting in corresponding S- aminoisothiourea.268

Taking these reports into consideration, we have explored the reaction conditions for efficient solid-phase guanidinylation of fusaricidin/LI-F class of cyclic lipopeptides. For this purpose three different guanidinylation reagents (a) N,N’-di-Boc-N”-triflylguanidine 20,243,244 (b) 1H-pyrazole-1- carboxamidine hydrochloride 21262 and (c) di-Boc thiourea 22 activated with the Mukaiyama’s reagent 23242,265 or N-iodosuccinimide (NIS) 24263,268 were tested, Figure 19.

O NH O

H2N NH 1) Guanidinylation H2N N NH 10 H H 10 N 2) Cleavage NH2 Dap-D-Val-Val-D-Thr-D-Asp Dap-D-Val-Val-D-Thr-D-Asp O Resin O N D-Ala 25 N D-Ala H O H O

Scheme 5. Solid-phase guanidinylation

2.2.5.2 Results and discussion

Since it is well known that the efficiency of SPPS depends on the properties and the quality of the solid-support,269,270 the effect of resin on guanidinylation was assessed as well. For this purpose polystyrene (PS) based Rink amide MBHA and polyethylene glycol (PEG) based

TentaGel S RAM resins were employed. Both resins are commonly used in Fmoc-SPPS and, according to manufacturer’s specifications, have similar physical properties (see section 2.8.1).

However, as demonstrated recently by Krchnak et al.,271 even the resins with identical specifications from different or the same sources often behave differently in solid-phase synthesis. The model compound for testing guanidinylation conditions was fusaricidin/LI-F analog

1 1 4 4 with Dap instead of Thr and D-Thr instead of D-aThr 25, Scheme 5. The solid-phase synthesis

46 followed previously described protocol (see section 2.2.2). Upon removal of Fmoc-protection, the lipidic tail primary amino group was subjected to guanidinylation conditions. Peptides were cleaved from the solid support with TFA:thioanisole:H2O (90:5:5 v/v/v) cleavage cocktail, and the progress of the reactions was monitored by RP HPLC and MALDI-TOF MS analysis, Figure 20.

As Figure 20 clearly shows, the best results were obtained using N,N’-di-Boc-N”- triflylguanidine 20, independently of the resin used, Figure 20A. Diprotected triflylguanidines were for the first time described by Goodman et al.243,244 and used in direct guanidinylation of primary and secondary amines under mild conditions in solution and on solid support. In our case, the solid-phase guanidinylation of resin-bound peptidyl-amine with 20 was completed within 5 hours on both resins, resulting in desired product 25 in high yields with no or insignificant amount of byproducts detected. On the other hand, guanidinylation reagents 21-24 gave unsatisfactory results on both resins. Guanidinylation with 1H-pyrazole-1-carboxamidine 21 did not go to completion even after prolonged reaction times. After 8 h roughly 91% of the starting material 26 was recovered on TentaGel S RAM resin and about 82% was recovered using polystyrene based

Rink amide MBHA resin. Further extension of the reaction time to 18 hours had a modest impact improving the yield of the desired product 25 by 10 % on TentaGel S RAM and 15 % on Rink amide MBHA resins, Figure 20B. On another hand, side products were not observed. This is in agreement with previous reports which indicate the necessity of prolonged reaction time using this guanidinylating reagent.

The conversion of amines into guanidines using thioureas requires initial activation.251 Among various activators that can be used for this purpose251,266 Mukaiyama’s reagent 23 and NIS 24 are fully compatible with Fmoc SPPS. It has been suggested that both Mukaiyama’s reagent 23 and

NIS 24 activate the sulfur-leaving group leading to a highly electrophilic carbodiimide intermediate,242,272 and consequently conversion of the amino group into the desired protected guanidine. In the case of activation with the Mukaiyama’s reagent 23, consumption of the starting resin-bound peptidyl amine was completed within 3 h on both resins, resulting in multiple reaction products, Figure 20C.

47

Figure 20. RP HPLC analysis of the guanidinylation reactions; A) N,N’-di-Boc-N’’-triflylguanidine; B) 1H-pyrazole carboxamidine; C) di-Boc-thiourea and Mukaiyama’s reagent; D) di-Boc-thiourea and NIS, reaction mixture analysis with 3 eq (solid line) and 1.1 eq (dashed line) of guanidinylating reagents. Analytical RP HPLC; method: 2% solvent B for 0.5 min followed by linear gradient 2→98% solvent B over 30 min, for which solvent A is 0.1% TFA in H2O, and B is 0.08% TFA in ACN

48 The byproduct 27 has been identified as the main product of this reaction(Rt= 15.9 min, m/z expected 851.5341, found [M+Na]+ 873.4537), Figure 21. The increase in molecular weight in the case of 27 over the starting resin-bound peptidyl-amine is compatible with the addition of two formimidamide groups. Other reaction products include the desired monoguanidine 25 (Rt= 15.1 min, m/z expected 809.5123, found [M+H]+ 810.5260), and the Mukaiyama adduct 28, a side

+ product reported in the literature (Rt= 16.2 min, m/z expected 859.5405, found [M] 859.6324).

However, as illustrated in Figure 20C, the relative distribution of (by)products depends on the type of resin. TentaGel S RAM resin gave 39 % of the desired product 25, 54 % of 27 and 7.3 % of the Mukaiyama’s adduct 28, whereas use of Rink amide MBHA resin resulted in only 14 % of the desired product 25, and 79 % and 8 % of the byproducts 27 and 28, respectively. Somewhat better results were obtained using NIS 24 as an activator, Figure 20D.

R R'

H NH O 25 N OH R' NH2 R NH 10 NH2 26 NH2 OH Dap-D-Val-Val-D-Thr-D-Asp NH O H NH N O N D-Ala 27 NH H O NH2 2 H N N 28 OH

Figure 21. Structures of compounds isolated in the guanidinylation study

In the case of TentaGel S RAM resin the starting material was completely consumed within 2 h, yielding almost identical amounts of guanidine 25 (51%) and byproduct 27 (49 %).

Guanidinylation on Rink amide MBHA resin under the same reaction conditions was less efficient as evidenced by 14 % recovery of non-guanidinylated cyclic lipopeptide 26 in addition to formation of 39 % of 25 and 48% of 27. Although reported in the literature,268 formation of S- aminoisothiourea byproduct was not observed under applied guanidinylation conditions. 49 Considering that thiourea 22 activation with Mukaiyama’s reagent 23 and NIS 24 proceeds through a common carbodiimide intermediate, formation of the byproduct 27 in both cases is not surprising. The excess (3 eq) of thiourea and the activator is required due to carbodiimide intermediate low stability.242 However, the excess of guanidinylation reagents may facilitate formation of byproducts as well. To assess the effect of reagents excess on guanidinylation, resin-bound peptidyl amine guanidinylation was performed in the presence of 1.1 equivalents of reagents. In this model reaction NIS 24 was chosen as an activator since activation of thiourea with NIS resulted in better yields of monoguanidine product 25 compared to thiourea activation with Mukaiyama’s reagent 23. As shown in Figure 20D, lowering amounts of guanidinylation reagents to 1.1 eq. resulted in incomplete consumption of starting resin-bound peptidyl amine, and formation of 25 and 27 within 2 h, with more 27 formed on Rink amide MBHA resin. The fact that lower amounts of the guanidinylation reagents did not suppress formation of byproducts indicates that the formation of 27 is relatively fast and occurs in parallel with formation of desired product 25. These results indicate the possibility of carbodiimide intermediate reaction with other functional groups present in the cyclic lipopeptide structure. Taking into consideration the amino acid sequence of cyclic lipopeptide, such possibility could include partial deprotection of Thr3 side-chain hydroxyl group under Mukaiyama’s and NIS guanidinylation conditions, allowing therefore for a competitive nucleophilic attack of the hydroxyl group on the carbodiimide intermediate and formation of an urea adduct 27.273-275 The feasibility of forming byproduct 27 was assessed by guanidinylation of 12-aminododecanoic acid attached to Rink amide MBHA resin. Standard Fmoc SPPS chemistry was applied in preparation of this control compound.

Guanidinylation of resin bound 12-aminododecanoic acid using Mukaiyama’s reaction conditions resulted in exclusive formation of corresponding monoguanidine product as evidenced by MALDI-

TOF MS analysis of the reaction mixture (12-aminododecanamide, m/z calculated 214.2045; 12- guanidinododecanamide, m/z expected 256.2263; found 257.3374 [M]+, 279.3320 [M+Na]+).

Obtained results eliminate the possibility of guanidine group dimerization and strongly support our

50 initial assumption of byproduct 27 formation in parallel with desired product 25 under the experimental conditions required for guanidinylation using thiourea and activators 23 or 24.

Our experimental results demonstrated superiority of N,N’-di-Boc-N”-triflylguanidine 20 in solid-phase guanidinylation of cyclic lipopeptide 25. Guanidinylation with 20 resulted in a sole monoguanidinylated product regardless of the type of resin used. On the other hand, use of pyrazole based reagent or activated thiourea gave unsatisfactory results. In both of these approaches the reaction kinetics and the product distribution depends on guanidinylation reagents as well as on the type of the resin. Selection of the appropriate method for the guanidinylation of resin-bound amines is critical in order to minimize formation of the undesired byproducts and increase guanidinylation reagents efficacy.

The efficacy of guanidinylating reagents depends strongly on the substrate, hence, we can not claim that these results can be extrapolated to other systems but they can at the very least serve as a guide when choosing reagents for solid phase guanidinylation.

2.3 Conformational study

Since small cyclic peptides still have considerable mobility of their backbones,276-279 it is reasonable to expect that the ester bond substitution with isosteric amide280 or NMe amide247 will also lead to significant conformational changes. Therefore, we should expect different CD spectra of depsipeptide 6, amide analog 14 and NMe-analog 19 in water and less polar TFE, due to their different conformational flexibility and the ability of TFE to promote intramolecular H-bonds and stabilize preferential conformation.281 In order to get a better insight into conformational changes induced by these ester-bond substitution analogs, we have also conducted molecular dynamics

(MD) simulations.

2.3.1 Circular dichroism (CD) spectroscopy

The structural features of representative fusaricidin/LI-F analogs; depsipeptide 6, amide 14, linear peptide 18 and NMe-amide analog 19 were monitored by circular dichroism (CD)

51 spectroscopy, Figures 22 and 23. The CD spectra were recorded in an aqueous medium and different concentrations of trifluoroethanol (TFE).282 Besides membrane mimicking properties,

TFE is also known to induce formation of the stable conformations in peptides which are otherwise unstructured in aqueous solutions.281,283 In order to increase solubility of peptides in aqueous media and to inhibit potential peptide aggregation at the concentrations required for CD experiments, the analyzed analogs were dissolved in 0.5% aqueous AcOH.284,285

The interpretation of these CD spectra is rather difficult, due to the fact that none of the spectra can be attributed to a single conformation. Moreover, the contribution of D-amino acid, residues, which predominate in the sequence, Figure 17, to peptide's conformation also has to be taken into account.

Figure 22. CD spectra of fusaricidin/LI-F analogs: a) 6, b) 14 and c) 18

The CD spectrum of the depsipeptide 6 in 0.5% AcOH exhibits a minimum at approximately

197 nm and a weak maximum around 220 nm, resembling the type C spectra and suggesting presence of type I (III) β-turns, Figure 22a.286,287 -Sheet/-turn containing small cyclic peptides are not unusual, and examples can be found in gramicidin S and its analogs.288-290 Water replacement with less polar 50% TFE/water mixture and 100% TFE resulted in a drastic change of depsipeptide 6 CD spectra. The CD spectrum in 100% TFE shows a double minimum at 192 nm and 201 nm and maximum at 225 nm. In addition to these changes, a marked decrease in the intensity of the CD spectrum in TFE was observed as well.

52 The CD spectra of the amide analog 14 are markedly different from the parent depsipeptide 6 in both aqueous and TFE solutions, indicating significant differences in structural flexibility and conformations induced by ester-to-amide substitution. The CD spectrum of amide analog 14 in

0.5% AcOH is characterized by a maximum at 205 nm and minimum at 188 nm, whereas in TFE the CD maximum at 205 nm is completely lost and the intensity of the spectral minimum at 188 nm is decreased and slightly shifted toward shorter wavelengths, Figure 22b.

As expected, due to lack of the structural constraints, linear peptide 18 shows different CD spectra in aqueous medium and TFE. In 0.5% AcOH, the CD spectrum of peptide 18 has a weak maximum at 195 nm, whereas in TFE a weak minimum at 200 nm was observed, Figure

22c.287,291

The CD spectra of the NMe-amide analog 19 in the aqueous media has a maximum around

200 nm and a small positive band around 225 nm which is similar to the inverted antiparallel β- sheet, Figure 23a. The spectra in 50 % TFE is very similar to the spectra in the aqueous media with a very slight shift of the maxima. On another hand, the CD spectra in 100% TFE is has a weak minimum at about 210 nm indicating a presence of a β-turn.

a) b) c)

Figure 23. CD spectra of N-methylated fusaricidin/LI-F analog 19 (a) and comparison with the CD spectra of analogs 6 and 14 in 0.5 % AcOH (b) and TFE (c)

When we compare the CD spectra of the NMe-amide analog 19 with those of the depsipeptide 6 and amide 14, the differences are again remarkable, Figure 23b and c, showing clearly that small changes introduced in a constrained system such as a small cyclic hexapeptide, have a big impact on the conformation of the peptide. 53 2.3.2 Molecular dynamics (MD) simulations

MD calculations were performed to further explore the conformational differences caused by ester-bond substitution with an amide and NMe-amide in fusaricidin/LI-F analogues and to assess the possibility of forming more organized structures under conditions that mimic the membrane environment. Low energy conformations were generated by conformational analysis in Molecular

Operating Environment (MOE)292 using a Monte Carlo search with the Generalized Born solvation model implemented in MOE.293 The amphiphilic moment descriptor, which is an established measure of balance between hydrophilic and lipophilic moieties, was computed in MOE for the lowest energy conformations.294

As shown in Figure 24, MD studies are in qualitative agreement with the experimental CD spectroscopy data indicating that the depsipeptide 6 can adopt different conformations in polar and nonpolar environments, whereas conformational differences of the amide analog 14, and the

NMe-amide analog 19 under the same conditions are less pronounced suggesting more rigid peptide backbone structure. In addition, calculated amphiphilic moments for depsipeptide 6 and amide 14 are markedly different with depsipeptide being more amphiphilic in both solvent systems, Figure 24. The NMe-amide analog 19, however, shows an opposite trend and greater amphiphilic moment in the aqueous conditions.

54

Figure 24. Representative structures of low energy conformers of depsipeptide 6, amide 14 and NMe-amide 19 in water and TFE and their calculated amphiphilic moments,  (see section 2.8.5)

55 2.4 Structure-activity relationship study

2.4.1 Antibacterial activity

To identify the structural determinants for the antibacterial activity and cytotoxicity, a total of nineteen fusaricidin/LI-F analogs were prepared, Figure 17. The structural variations of the natural product include modification of the lipid tail, substitution of amino acid residues including alanine-scan analogs (Ala-scan), ester-to-amide substitution, and a linear control peptide for comparison. The antibacterial in vitro activity of synthesized fusaricidin/LI-F analogs 1-19, expressed as the minimum inhibitory concentration (MIC, μg/mL), was determined for Gram- positive and Gram-negative bacteria using standard micro dilution broth method in 96-well plates, Table 4.295,296

Similar to the natural products, fusaricidin/LI-F analogs are active against Gram-positive bacteria including antibiotic resistant strains, and did not show activity against Gram-negative bacteria (data not shown). To probe the role of the lipid tail in antibacterial activity, depsipeptides containing an acetyl group 1, 2, 12-ADA 3, 4, and 12-GDA 5-12 attached to Thr1 were synthesized. No antibacterial activity was observed for the analogs containing an acetyl group or

12-ADA, even at the highest concentration tested. On the other hand, depsipeptide analog containing 12-GDA 6 showed potent antibacterial activity (MIC 8 g/mL) against Gram-positive bacteria, Table 1. Ala-scan and several other substitutions at key amino acid residues were performed to reveal the role of each individual amino acid in the antimicrobial activity of synthetic fusaricidin/LI-F analogs.

However, requirements of our solid-phase synthetic approach,224 Schemes 1 and 3, permitted

2 3 4 6 replacement of four (D-Val , Val , D-aThr , and D-Ala ) out of six amino acids. Obtained MIC values, Table 4, showed that the substitution of Val3 with Ala, analog 8, preserved the

4 2 antibacterial activity of the parent depsipeptide 6, whereas substitutions of D-aThr and D-Val with D-Ala, analogs 7 and 9, respectively, resulted in significant reduction of antimicrobial activity.

6 1 6 1 In contrast, replacement of both D-Ala with Gly, analog 10, or both Thr and D-Ala with Lys , analog 13, led to a complete loss of antibacterial activity.

56 Table 4. Antibacterial activity of fusaricidin/LI-F analogs

Compd MIC (μg/mL)

S. aureus S. aureus S. aureus S. epidermidis S. pyogenes

ATCC 29213 ATCC 33591 ATCC 700699 ATCC 27626 ATCC 19615

1 -a > 128 > 128 - - 2 - > 128 > 128 - - 3 - > 128 > 128 - - 4 - > 128 > 128 - - 5 16 16 16 16 32 6 8 8 16 8 16 7 - 64 64 32 64 8 16 16 16 8 16 9 - 32 64 16 64 10 - 64 64 64 64 11 8 8 16 8 8 12 > 128 > 128 - 16 64 13 - 64 64 64 64 14 8 16 16 16 16 15 - 64 - 64 > 64 16 8 8 - 16 16 17 - 32 - 64 64 18 > 128 > 128 - > 128 > 128 19 > 128 > 128 - - - a Not determined.

4 No significant reduction in antimicrobial activity was observed in the case where D-aThr was

4 replaced with D-Thr , analog 5. Quite interestingly, despite significant changes in hydrophobicity, amphiphilicity and conformation caused by ester-to-amide substitution, antibacterial activities of amide analogs 14-17 parallel those of the parent depsipeptides 6, 8, 11 and 12. As mentioned earlier, fusaricidins/LI-F is a family of naturally occurring cyclic lipodepsipeptide antibiotics

1 4 6 130 consisting of 12 compounds in total, with conserved Thr , D-aThr , D-Ala amino acid residues.

Aliphatic nonpolar amino acid residues are present at the position 2 (D-Val, D-Ile and D-aIle), and polar amino acids D-Asn and D-Gln are present at position 6 of the peptide sequence. Most diverse changes occur in position 3 where Tyr, Val, Ile, Phe, and D-Ile were found. Taking this into consideration, we have synthesized fusaricidin/LI-F analogs possessing Phe, analogs 11 and

57 16, and Tyr, analogs 12 and 17, residues in position 3 in the sequence. Replacement of Val3 with polar Tyr resulted in reduction of antimicrobial activity, whereas substitution with less polar Phe did not affect the antibacterial activity, Table 3. In addition, the MIC data obtained for depsipeptides 6, 8, 11 and 12 showed that an increase of overall hydrophobicity, as indicated by the HPLC retention times, Figure 17, did not correlate with their antibacterial activities. Analogs 6,

8 and 11 showed almost identical, within experimental error, antibacterial activities for tested bacterial strains while analog 12 showed reduced activity. In contrast, similar comparison of amide analogs 14-16 showed different results. Amide analogs possessing hydrophobic amino acids in position 3 of the peptide sequence such as Val and Phe (analogs 14 and 16) exhibited better antibacterial activity than analogs with more polar Ala and Tyr (analogs 15 and 17), indicating in this case correlation between the peptides’ overall hydrophobicity and antibacterial activity. Despite a much higher degree of conformational flexibility and complete sequence homology with biologically active depsipeptide 6, control linear peptide 18 did not show any biological activities in described assays.

Interestingly, the NMe-amide analog 19 was not active at the conditions tested, up to 128

μg/mL. Considering the enormous differences seen in the CD spectra of depsipeptide 6, amide

14 and NMe-amide 19, Figure 23b and c, predicted by the MD simulations as well, Figure 24, it is obvious that, what seem to be minor changes in the peptide scaffold, result in big differences in the activity of the peptides which may explain the differences observed in the antimicrobial activity.

2.4.2 Antifungal activity

The depsipeptide analog 6 was tested for antifungal activity against three strains, Aspergillus niger, Fusarium oxysporum and Rhyzopus spp. The fungi were treated with 2-fold serial dilutions of peptide and their growth was monitored after 24, 48 and 72 h. The peptide was active against

A. niger and F. oxysporum at 32 μg/mL even after 24 h and maintained its activity until the end of the experiment. In the case of Rhyzopus spp. the peptide showed activity at 64 μg/mL after 24 h incubation. Some regrowth was observed after 48 h and after 72 h only the highest concentration

58 tested, 128 μg/mL, showed significant decrease in the fungal population. Figure 25 shows a representative graph of the antifungal activity.

Figure 25. Antifungal activity of fusaricidin/LI-F analog 6 against A. niger

2.5 Cytotoxicity

2.5.1 Hemolytic activity

Hemolytic activity was determined against human erythrocytes (0.5% in PBS buffer), as described in the Materials and methods section 2.8.8, PBS and 0.5% Triton X-100 were used as a reference for 0 and 100% hemolysis, respectively. Peptides that were not active in the antimicrobial assays did not give noticeable hemolytic activity. None of the active depsipeptides

6, 8, 11 and their amide counterparts, peptides 14-16, showed hemolytic activity at MIC concentrations (see Figure 26 and Appendix). However, at higher concentrations, depsipeptides

6, 8 and 11 showed considerable hemolysis relative to reference Triton X-100, Figure 26.

The degree of hemolysis appears to correlate with the increase in hydrophobicity of depsipeptides. For example, at 64 μg/mL (8 x MIC) least hydrophobic depsipeptide 8 with Ala3 exhibited the lowest hemolytic activity (30%) followed by analog 6 with Val3 causing 45% 59 hemolysis, whereas most hydrophobic analog 11 with Phe3 showed the highest level of hemolysis, 65%. Interestingly, none of the amide analogs 14-16 exhibited appreciable hemolytic activity at this concentration. At much higher concentrations, 128 and 256 μg/mL (16 and 32 x

MIC), depsipeptide analogs 6 and 11 showed hemolysis comparable to that of Triton X-100, whereas amide analogs 14 and 16 at 256 μg/mL reached 15 % and 50% hemolysis, respectively,

Figure 26. Control peptide 18 did not exhibit appreciable hemolytic activity even at the highest tested concentration of 64 μg/ml.

Figure 26. Hemolytic activity of fusaricidin/LI-F analogs expressed as percent of hemolysis

caused by Triton X-100

It is important to mention that in this assay up to 5% v/v final concentration of DMSO was added to increase tested fusaricidin/LI-F analog solubility. Therefore, potential aggregation due to lower solubility of amide analogs at higher concentrations may contribute to the observed hemolytic activity.297-299 Although DMSO is hemolytic,300,301 our control experiments showed that in the conditions used DMSO did not cause appreciable hemolysis.

60 2.5.2 Toxicity towards human normal and cancer cell lines

To further assess the therapeutic potential of synthetic fusaricidin/LI-F analogs, we tested the in vitro toxicity of analogs 1, 4, 6 and 14 (Figure 27) on the human liver embryonic cell line WRL

68 and the HepG2 cancer cell line. A known cytotoxic drug Adriamycin (Doxorubicin)302 was used as a positive control in these assays.

1 4 6 14

Figure 27. Structures of fusaricidin/LI-F analogs used in the cytotoxicity study

Depsipeptide analog 1, lacking a lipid tail and inactive in the antimicrobial assays, did not show appreciable cytotoxicity toward WRL 68 or HepG2 cells within the tested concentration range. Interestingly, lipidated depsipeptide analogs 4 and 6, regardless of their antibacterial or hemolytic activities, were cytotoxic at higher concentrations to both tested cell lines. Analog 4 was highly cytotoxic at 150 μg/mL, whereas more active depsipeptide analog 6 showed somewhat similar trend in cytotoxicity to doxorubicin and was highly cytotoxic at 90 μg/mL concentration. In comparison, the cytotoxicity of less hydrophobic and amphiphylic amide analog 14 was much lower at the same concentrations, Figure 28.

61

Figure 28. Cell survival measured by MTT assay after 24 h treatment with fusaricidin/LI-F analogs. Normal cell line WRL 68 (upper panel), cancer cell line HepG2 (lower panel)

62 2.6 Serum stability

To investigate the effect of ester-to-amide substitution on cyclic peptide proteolytic stability, the disappearance of the intact peptide incubated in 50% human serum and EMEM media containing 10% FBS for 24 h at 37C was followed by RP HPLC,303 Figures 29 and 30, respectively. Depsipeptide 6 and amide 14 were used in this study as representative examples of each group of synthetic fusaricidin/LI-F analogs. Approximately 35% change in the concentration of depsipeptide 6 was observed within an hour of incubation in 50% human serum. However, prolonged incubation of 6 did not result in complete depsipeptide degradation, and after 24 h, about 35% of 6 was still detected in the serum, Figures 29 and 31.

*

Figure 29. RP HPLC trace of analog 6 in 50% human serum at time 0 (up) and after 24 h

incubation at 37C (down). Peak at Rt=15.8 min corresponds to unchanged 6. (*) Marks

approximate elution time (Rt= 15-15.2 min) of the major degradation product

63 Stability of 6 in EMEM buffer containing 10% FBS used for cytotoxicity assays was significantly better. In this case approximately 23% loss in concentration of 6 was observed after

24 h. Analysis of depsipeptide 6 degradation using RP HPLC and MALDI-TOF mass spectrometry revealed that the main degradation product is a result of this depsipeptide's ring

+ opening via ester bond hydrolysis (depsipeptide 6: Rt=15.8 min, [M+H] m/z=825.9709; hydrolysis

+ product: Rt=15.04 min, found [M+H] m/z=843.9988, Figure 32). Obtained identical MALDI-TOF m/z and RP HPLC Rt for the depsipeptide 6 degradation product and control peptide 18 (Rt=15.05 min, [M+H]+ m/z=844.1160, calculated [M+H]+ m/z=843.0228) additionally confirmed this finding.

In contrast, no degradation was observed for amide analog 14 under the same experimental conditions, Figure 31.

*

Figure 30. RP HPLC trace of analog 6 in the EMEM media at time 0 (up) and after 24 h

incubation at 37C (down). Peak at Rt=15.7 min corresponds to unchanged 6. (*) Marks elution

time (Rt=15.1 min) of the major degradation product

64

Figure 31. Stability of fusaricidin/LI-F analog 6 in the EMEM media containing 10% FBS (left), and analogs 6 and 14 in 50% human serum (right)

Voyager Spec #1[BP = 844.1, 55982] Voyager Spec #1[BP = 826.2, 30648] 826.2208 844.1015 100 100 3.1E+4 5.6E+4 90 90 80 80 70 70 60 60 50 50

40 40 %Intensity % Intensity 30 30 848.2219 866.0889 20 20 882.0575 10 864.1920 10 0 0 500 640 780 920 1060 1200 500 640 780 920 1060 1200 Mass (m/z) Mass (m/z)

Figure 32. MALDI-TOF MS analysis of RP HPLC fractions obtained after 24 h incubation of 6 in the EMEM media. Left: fraction at 15.7 min, right: fraction at 15.1 min

2.7 Discussion and conclusions

Due to their structural diversity and high potency, naturally occurring cyclic depsipeptides have attracted a great deal of attention for discovery of new antibiotics with superior activity against multidrug-resistant bacteria 98,130. However, full exploitation of the antimicrobial potentials of this class of natural products strongly depends on the synthetic access to their structures, in particular their analogs, and understanding important details of their mode of action. It has been shown in the literature that the antibacterial and hemolytic activities of antimicrobial peptides depend on different structural components such as charge, size, secondary structure,

65 hydrophobicity and amphipathicity.289,304-306 Among naturally occurring antibacterial peptides, fusaricidin/LI-F family of cyclic lipodepsipeptides represent attractive lead compounds for the development of new antibiotics because of their unique structure in which peptide sequences are composed of six, mostly hydrophobic, amino acids and a positive charge located at the end of the lipid tail.130 In addition, there is substantial evidence in the literature showing that substitution of the ester bond in depsipeptides with an amide bond may afford derivatives with comparable activities and markedly increased serum stabilities.190,280,307 With this in mind, we synthesized nineteen fusaricidin/LI-F analogs and investigated the effects of structural modification on their overall hydrophobicity/amphiphilicity, conformation, stability and biological activity. All analogs were synthesized on a solid support using Fmoc methodology. However, solid-phase synthesis of depsipeptides 1-12 turned out to be particularly challenging. Our strategy for the solid-phase

5 synthesis of cyclic lipodepsipeptides involved attachment of D-Asp via side-chain, stepwise

Fmoc-synthesis of a linear peptide precursor, and on-resin head-to-tail cyclization, Scheme 1.

The key step in the synthesis of cyclic depsipeptides is the ring closure. Considering the greater reactivity of the amino group, and therefore minimal possibility of side reactions, we have chosen macrolactamization for depsipeptide ring closure, as opposed to macrolactonization. Although this synthetic strategy appears to be a better choice for the solid-phase depsipeptide synthesis, an undesired intramolecular ON acyl shift may occur if basic conditions are used.239,308

Reversible intramolecular ON or NO acyl shifts are well described side reactions in peptide chemistry.239,308 Typically, peptides containing Ser or Thr residues undergo NO acyl shift under acidic conditions, whereas the exposure of corresponding depsipeptides to basic conditions leads to opposite ON acyl shift. Incorporation of the lipid tail or acetyl group into the linear peptide

6 precursor prior to D-Ala coupling via ester bond and on-resin cyclization completely suppressed the ON acyl shift resulting in the desired cyclic lipodepsipeptide, Scheme 1. In addition, we have found that the efficacy of the ester bond formation strongly depends on the type of solid support and the solvent used. Optimal results were obtained using PEG-based resins, DIC/DMAP coupling method, and DCM.224 Superiority of PEG-PS over PS-based resins in ester bond

66 formation could be explained by a better solvation of peptide-PEG-PS resins, 309,310 rapid DIC activation of the carboxyl group,311 and significant suppression of N-acylurea byproduct formation312 in a non-polar solvent such as DCM. Crucial steps in synthesis of fusaricidins, including esterification, are monitored using RP HPLC and MALDI-TOF MS and using our conditions racemization of Ala was not observed. Synthesis of amide analogs 13-17 using the same approach was straightforward, Scheme 3. The desired cyclic lipo(depsi)peptides were obtained in satisfactory yields.

Several studies have pointed out the impact of amide-to-ester substitution on cyclic peptide's conformation and proteolytic stability.276,313-315 In general, replacement of a hydrogen-donating

NH-group by the oxygen atom in depsipeptides results in removal of the backbone H-bond, affecting therefore conformation of peptides. As long as the side chains are not altered, intramolecular hydrogen bonds affect the equilibrium distribution of peptide conformers. The effect of ester-to-amide substitution on the conformation of fusaridicin A/LI-F analogs was assessed by CD spectroscopy, Figures 22 and 23. Although the conformations of the peptides can not be attributed to a single structure, rather a combination of structures, there are drastic differences between the CD spectra of depsipeptide 6, its amide analog 14, as well as the linear peptide 18. MD were performed to further explore the conformational differences caused by ester-to-amide substitution in fusaricidin/LI-F analogs and to assess the possibility of forming more organized structures under conditions that mimic the membrane environment. The results are shown in Figure 24. Our experimental CD data fully supports the MD simulations indicating higher conformational flexibility of depsipeptide analogs. MD calculations showed no significant conformational differences between depsipeptide 6 and amide analog 14 in water, whereas the differences become markedly more pronounced in a less polar environment such as TFE.

To further elucidate the influence of conformation on peptides activity and evaluate the influence of the hydrogen-donating NH-group which was introduced in the place of ester bond, we prepared NMe-amide analog 19. The conformational differences predicted by the MD simulations

67 were confirmed by CD spectroscopy showing again how significant effect the changes in the peptide ring can cause.

Besides conformational changes, ester-to-amide substitution altered the biochemical properties of the fusaricidin/LI-F analogs. The assessments of synthesized fusaricidin/LI-F analogs’ stability in human serum and cytotoxicity are important secondary screening assays, mainly because these assays eliminate cytotoxic analogs and analogs with short half-lives.316 Our experimental data suggest that low serum stability of depsipeptide 6 can be attributed mainly to the hydrolysis of its ester bond. This finding is in accordance with the literature data showing that cyclic depsipeptides can be degraded through competition between the protease action and esterase action. However, the depsipeptides were more easily subjected to hydrolysis by

317-319 esterase-type enzymes. Cyclic structure, the presence of D-amino acids, and greater stability of an amide bond all together contribute to the enhanced proteolytic stability of the amide analogs.117,120,122,320-324 Quite importantly, ester-to-amide substitution did not affect the antibacterial potency of these peptides. A positive charge positioned at the end of the lipid tail, hydrophobicity and amphiphilicity appeared to be crucial for fusaricidin/LI-F analogs’ antibacterial activities and separation of their antibacterial and human cell toxicity. Antibacterial assays showed that only analogs bearing a guanylated lipid tail are effective against Gram-positive bacteria, indicating the importance of the lipid tail and positively charged guanidino functionality for their antimicrobial activity, Table 4. Based on the role of the lipid tail in other lipopeptide antibiotics,132,141,145,162,325 and our experimental data showing that nonlipidated analog 2 is not active against tested bacterial strains, we can assume that the lipid moiety helps target fusaricidin/LI-F analogs to bacterial membrane. The antibacterial activity of peptides having a guanidylated lipid moiety could possibly be explained by the strong ability of the guanidinium group to bind the anionic phosphate of the bacterial phospholipid membrane through a combination of H-bonding and charge-charge interactions. In general, high pKa value, diffused charge density, and the geometry of the guanidinium group which allows better alignment of H-

68 bonds, all contribute to better interactions with the phosphate anion compared to the ammonium group.326

2 The results of the antibacterial assays of the Ala-scan analogs revealed that residues D-Val ,

4 6 D-aThr and D-Ala are crucial for antibacterial activity. Replacement of any of these amino acids with corresponding Ala, analogs 7, 9 and 10, resulted in complete loss of depsipeptide antibacterial activity, Table 4. On the other hand, position 3 in the depsipeptide sequence is more tolerable to changes. Depsipeptide analogs containing neutral Ala3, 8, and bulky hydrophobic

Val3, 6, or Phe3, 11, exhibited almost identical antibacterial activities, whereas analog 12 with more polar Tyr3 was not active. Equally potent in vitro antibacterial activity was determined for their amide counterparts, analogs 14 and 16 possessing hydrophobic Val3 and Phe3, respectively.

However, differences in the antibacterial activity were observed between depsipeptide 8 and its amide counterpart 15, both containing Ala in position 3. Depsipeptide 8 exhibited antibacterial activity comparable to the most potent analogs, whereas the amide 15 showed significant decrease in activity, Table 4. Since the relative overall hydrophobicity order of Ala < Val < Phe is the same for both amide and depsipeptide analogs, greater change in amphiphilicity upon substitution of lipophylic Val3 with neutral Ala3 in structurally constrained amide 15 may explain the loss of its antibacterial activity.

Substitution of the ester bond with an N-methylamide bond, analog 19, was detrimental for the activity of the peptide. No inhibition of bacterial growth was observed in the conditions tested, up to 128 μg/mL. This may be surprising on the first hand, however, it is not uncommon that small structural changes, particlarly in such small cyclic peptides, have significant, in this case deleterious impact on the activity.

Comparison of the overall hydrophobicity of depsipeptides 6, 8, 11 and 12 based on RP

HPLC retention times, Figure 17, shows no correlation with their antibacterial activities, Table 4.

In contrast, an increase in depsipeptide hydrophobicity can be associated with the enhanced hemolytic activities, Figure 26. More hydrophobic depsipeptides 6 and 11 were also more hemolytic. Similarly, depsipeptides 6, 8, 11 and 12 exhibited higher hemolytic activities than their

69 less hydrophobic amide counterparts 14-17. At concentrations 8 x MIC (64 μg/mL) none of the amide analogs 14-17 were hemolytic. The fact that linear peptide 18 is not hemolytic (see

Appendix), suggests that the hydrolysis of the ester bond which leads to opening of the depsipeptide ring is not a cause of hemolysis.

Liver toxicity is one of the most critical issues in drug development, often leading to the delay or failure of drug candidates.327-329 Therefore, peptides that show no in vitro liver cell toxicity may prove to be better candidates for further preclinical or clinical studies. HepG2 and WRL 68 cell lines were used to assess the potential toxicity of fusaricidin/LI-F analogs. In both cell lines, depsipeptide 6 exhibited much higher in vitro cytotoxicity than amide counterpart 14. However, absence of cytotoxicity to human cells observed for amide analog 14 cannot be solely explained by lower hydrophobicity. Conformational differences among these fusaricidin/LI-F analogs, as illustrated by the MD calculations and the differences in the CD spectra between analogs 6 and

14, Figures 22-24, should be taken into consideration as well. While hemolytic depsipeptide 6 has the ability to form more ordered structures in a membrane mimicking environment, non-hemolytic amide analog 14 fails to undergo any significant conformational changes regardless of the solvent system. Conformational changes and hence changes in the amphipathicity has been ascribed to the low hemolytic activity of some synthetic and naturally occurring cyclic cationic antimicrobial peptides.289,304,306,330 Typically, in these cyclic antimicrobial peptides, presence of cationic amino acids in the peptide sequence, defined secondary structure (-sheet and hairpin loop), and amphiphilic character are common structural characteristics that determine their biological activities.304,330 In the case of fusaricidin/LI-F and its analogs, a positive charge is positioned at the terminus of the 12-carbon-atom lipid tail, and peptide sequences are composed mostly of hydrophobic amino acids, Figure 17. Nevertheless, experimental CD data and molecular modeling studies showed that the peptide ring in both depsipeptide 6 and amide 14 analogs can adopt different amphiphilic conformations depending on the environment, with depsipeptides being more amphiphilic. Relatively lower amphiphilicity of the amide analogs may contribute to dissociation of antibacterial activity and human cell cytotoxicity.

70 In conclusion, structure-activity relationship studies of fusaricidin/LIF depsipeptide analogs reported in this work reveal key structural requirements for antibacterial activity and decreasing cytotoxicity. The positively charged guanidinium group at the end of the 12-carbon-atom lipid tail, and the presence of hydrophobic amino acids in the depsipeptide sequence are crucial for antibacterial activity. Ala-scan results and comparison of the fusaricidin/LI-F natural product sequences suggest that position 3 in the depsipeptide sequence is more tolerable to amino acid change. By introduction of neutral and hydrophobic amino acids into this position, we were able to manipulate the depsipeptide’s overall hydrophobicity and amphiphilicity without losing antibacterial potency. However, structural changes leading to an increase in the depsipeptide’s overall hydrophobicity and amphiphilicity resulted in an increase of their cytotoxicity. On the other hand, substitution of an ester bond in depsipeptides with an amide bond gave more stable cyclic lipopeptide analogs with preserved in vitro antibacterial activities, yet greatly improved serum stabilities and minimized human cell toxicity. Lower overall hydrophobicity/amphiphilicity of amide analogs in comparison to their parent depsipeptides may explain dissociation of antibacterial activity and human cell cytotoxicity. More stable and less cytotoxic amide analogs may have significant advantages over naturally occurring fusaricidin/LI-F and its depsipeptide analogs as lead structures for the development of new antibacterial agents. In addition, amide analogs are synthetically more accessible than the parent depsipeptides, allowing for further structural optimization using a combinatorial chemistry approach. Interestingly, N-methylamide analog completely lost antibacterial activity.

Synthesis of a focused combinatorial library based on fusaricidin/LI-F amide analogs, and elucidation of the mode of action of both the depsipeptide and amide analogs are currently underway.

71 2.8 Materials and methods

2.8.1 Chemicals and instrumentation

All solvents were purchased from Fisher Scientific (Atlanta, GA) or Sigma Aldrich (St. Louis,

MO) and were were high-performance liquid chromatography (HPLC) grade. TentaGel S RAM resin was obtained from Advanced ChemTech (Louisville, KY; substitution level: 0.25 mmol/g; mesh size: 90 m; swelling: 3.9 mL/g in DMF, > 5 mL/g in DCM). Rink amide MBHA resin was purchased from Novabiochem (Gibbstown, NJ; substitution level: 0.66 mmol/g; mesh size: 75-150

m; swelling: 3.5 mL/g in DMF, 5.2 mL/g in DCM). 2-chlorotrityl chloride was obtained from

Novabiochem (Gibbstown, NJ; substitution level: 1.3 mmol/g). Fmoc-protected amino acids and coupling reagents (HBTU, HOBt, PyBOP) were purchased from Chem-Impex (Wood Dale, IL) or

Novabiochem (Gibbstown, NJ). DIC was purchased from Acros Organics (Thermo Fisher

Scientific, Waltham, MA). DMAP was purchased from Sigma Aldrich (St. Louis, MO). Kaiser ninhydrin kit was purchased from AnaSpec (Fremont, CA). All other reagents used were purchased from Sigma-Aldrich, Fisher Scientific or Alfa Aesar (Ward Hill, MA).

Microbial strains and human cells were purchased from American Type Culture Collection

(ATCC, Manassas, VA). Dehydrated culture media and agar, and polystyrene plates used for antimicrobial assays were purchased from BD (Franklin Lakes, NJ). Control antibiotics were purchased from Sigma Aldrich (St. Louis, MO). Antimicrobial activity assays were carried out in standard, sterile 96-well plates, and MIC values were determined by measuring turbidity at 600 nm using a Stat Fax 2100 Microplate reader (Awareness Technology Inc., Palm City, FL). Human red blood cells (hRBCs) were purchased from Innovative Research (Novi, MI). Human serum was purchased from Sigma Aldrich (St. Louis, MO).

Linear peptidyl-resin precursors were synthesized on a PS3 automated peptide synthesizer

(Protein Technologies Inc., Tucson, AZ). Mass spectrometry was performed on MALDI-TOF

Voyager-DETM STR (Applied Biosystems, Foster City, CA) in reflector mode using -cyano-4- hydroxycinnamic acid as a matrix and positive mode. Analytical RP HPLC analyses and peptide purifications were performed on 1260 Infinity (Agilent Technologies, Santa Clara, CA) liquid

72 chromatography systems equipped with a UV/Vis detector. For analytical RP HPLC analysis, a

C18 monomeric column (Grace Vydac, 250 x 4.6 mm, 5 μm, 120 Å), 1 mL/min flow rate, and elution method with a linear gradient of 2→98% B over 30 min, where A was 0.1% TFA in H2O and B was 0.08% TFA in ACN was used. For peptide purification, a preparative C18 monomeric column (Grace Vydac, 250 x 22 mm, 10 μm, 120 Å) was used. Elution method was identical to the analytical method except for the flow rate, which was 19 mL/min.

CD spectra were recorded on a JASCO 810 spectropolarimeter (Easton, MD). Cytotoxicity assays were analyzed on a Synergy H4 microplate reader (BioTek, Winooski, VT).

2.8.2 General procedure for peptide synthesis and purification

Linear peptidyl-resin precursors for cyclic lipopeptides 1-17 were synthesized on amide

TentaGel S RAM resin (substitution 0.26 mmol/g, 0.25 mmol scale) using automated peptide synthesizer. The solid-phase synthesis of cyclic peptides 1-17 started by attaching C-terminal

Fmoc-D-Asp-OAllyl via side chain to the resin using HBTU/HOBt/NMM protocol (4 eq of Fmoc- amino acid, HOBt and HBTU, 0.4 M NMM in DMF, 1 h). The same coupling protocol was used throughout, including coupling of the lipid tail (Fmoc-ADA-OH, 1.5 eq each). Deprotection of the

Nα-Fmoc protecting group was achieved using 20% piperidine/DMF (3 x 6 min on the automated synthesizer or 1 x 10 min plus 1 x 20 min when done manually).

In the case of depsipeptide analogs 1-12, Aloc-D-Ala-OH (4 eq) or Aloc-Gly-OH (4 eq) was coupled manually via ester bond to the hydroxyl group of Fmoc-Thr-OH using DIC (4 eq) and

DMAP (0.2 eq) coupling reagents in DCM. Amide analogs 13-17 were prepared by coupling

Fmoc-Dap(Mtt)-OH instead of Fmoc-Thr-OH using the same coupling protocol as above.

Selective removal of Allyl and Aloc protecting groups was performed by treatment of peptidyl- resin precursors with borane dimethylamine complex (4 eq), followed by addition of Pd(PPh3)4

(0.1 eq) in DCM under argon.241 Mtt was selectively removed under mild acidic conditions (1%

TFA in DCM, 30 min). Solid-phase cyclization of linear precursors was carried out in a manual reaction vessel overnight using PyBOP and DIEA (2 and 6 eq, respectively) in DMF. The

73 conversion of the lipid tail’s amino into the desired guanidino group was achieved by the removal of the Fmoc-protecting group using standard piperidine deprotection protocol and the treatment of the peptidyl-resin with N,N-bis(tert-butoxycarbonyl)thiourea (3 eq) followed by Mukaiyama’s reagent 2-chloro-1-methylpyridinium iodide/TEA (3:4 eq) in DMF242 or using N,N’-di-Boc-N’’- triflylguanidine 7 (5 eq) and TEA (5 eq) in DCM for 5 h at r.t.243,244

Control peptide 18 was synthesized on 2-chlorotrityl chloride resin (substitution 1.3 mmol/g).

The synthesis started by attaching Fmoc-D-Ala-OH (4 eq) to the resin using an equimolar amount of DIEA in DCM, followed by resin endcapping with MeOH,280 and the chain elongation using standard Fmoc-chemistry. Quantitative Fmoc substitution of the resin (0.5 mmol/g) was determined by Fmoc cleavage and absorption measurement at 304 nm.246 In all cases, the reaction progress was monitored by RP HPLC, MALDI-TOF MS, and where applicable ninhydrin colorimetric test.331

N-methylated analog 19 was prepared using the same approach as described above for depsipeptide and amide analogs. After attachment of the protected lipidic tail Fmoc-ADA-OH,

Fmoc-protecting group was removed using 20% piperidine/DMF and the amino group converted into guanidinium moiety using the same conditions as described above. Mtt protecting group was selectively removed under mild acidic conditions (1% TFA in DCM, 30 min). The o-NBS was introduced using by treating the peptidyl-resin with o-NBS-Cl (4 eq) and collidine (10 eq) in NMP, first 15 min. The resin was washed and treated with the same reaction mixture for another 10 min. For the N-methylation mixture of PPh3 (5 eq), MeOH (10 eq) and DIAD (5 eq) in THF (1 mL) was added portionwise, 200 μL aliquots every 10 min. The o-NBS protection was removed using excess of 2-mercaptoethanol (10 eq) and DBU (5 eq) in DBU and was monitored by visual observation of the release of a yellow chromophore.248 Although the protocol stated 5 min reaction time, the resin was treated several times with the reaction mixture until the release of yellow color was no longer observed. Total time necessary for the complete removal was 30 min.

The next amino acid Fmoc-D-Ala-OH was coupled using 3 eq of the amino acid and each of the

74 coupling reagents, HOAt and HATU, and 6 eq of DIEA, in NMP for 1 h. The removal of Allyl/Aloc protecting groups and cyclization were performed as described above.

Peptides were removed from the resin using TFA:thioanisole:H2O (95:2.5:2.5 v/v/v) for 3 h.

The crude peptides were precipitated with cold methyl tert-butyl ether, and purified using preparative RP HPLC. HPLC fractions were analyzed for purity, combined and lyophilized to give white powder. Final purity of synthesized peptides was confirmed by analytical RP HPLC, and was  95% in all cases. The yields for all peptides were 40-60% after RP HPLC purification or

>70% if crude peptides were purified using dialysis.

2.8.3 Methods for guanidinylation of resin-bound amines

2.8.3.1 Synthesis of model cyclic lipopeptide

Synthesis of the linear precursor of the model cyclic lipopeptide was performed as described above. Upon cyclization of the linear precursor, Fmoc protecting group was removed from the lipidic tail terminal amino group using standard 20% piperidine in DMF deprotection protocol generating a free amine suitable for subsequent guanidinylation. Four different guanidinylation conditions were used for this purpose. The guanidinylation reactions were carried out under the reaction conditions described in sections 2.8.3.2-2.8.3.5. Progress of guanidinylation reaction was monitored every hour by Kaiser test.331 Guanidinylated cyclic peptide was cleaved from the resin using TFA;thioanisole;H2O (95:2.5:2.5 v/v) cleavage cocktail and crude peptide was analyzed by

RP HPLC (Grace Vydac C18 monomeric column 250 x 4.6 mm, 5 μm, 120 Å, 1 mL/min flow rate, elution method with a linear gradient of 2→98% B over 30 min, where A was 0.1% TFA in H2O and B was 0.08% TFA in ACN) and MALDI-TOF MS. Relative quantification is based on the integrated areas of the reaction products RP HPLC peaks.

2.8.3.2 Guanidinylation using N,N’-di-Boc-N’’-triflylguanidine

The peptidyl-resin was swollen in DCM for 20 min followed by solvent removal. The solution of N,N’-di-Boc-N’’-triflylguanidine 20 (5 eq) and TEA (5 eq) in DCM (5 mL) was added to the

75 peptidyl-resin and reaction mixture was allowed to agitate at room temperature. Kaiser test indicated complete consumption of resin-bound amine within 5 h on both TentaGel S RAM and

Rink amide MBHA resins. Cyclic lipopeptide was cleaved from the solid support and crude product analyzed as described above.

2.8.3.3 Guanidinylation using 1H-pyrazole-1-carboxamidine hydrochloride

Peptidyl-resin was swollen in DMF for 20 min followed by solvent removal. The solution of

1H-pyrazole-1-carboxamidine 21 (3 eq) and DIEA (3 eq) in DMF (5 mL) was added to the peptidyl-resin and allowed to agitate at room temperature. Peptidyl-resin samples (cca 20 mg) were taken after 8 h and 18 h. Cyclic lipopeptide was cleaved from the solid support and crude product analyzed as described above.

2.8.3.4 Guanidinylation using N,N’-di-Boc-thiourea and Mukaiyama’s reagent as an activator

Peptidyl-resin was swollen in DCM for 20 min followed by solvent removal. The solution of

N,N’-di-Boc-thiourea 22 (3 eq) and TEA (4 eq) in DCM (4 mL) was added to the resin and reaction mixture was allowed to agitate at room temperature for 15 min. Solution of Mukaiyama’s reagent 23 (3 eq) in DCM (1 mL) was then added and agitation was continued at room temperature. Kaiser test indicated complete consumption of free resin-bound amine within 3 h on both TentaGel S RAM and Rink amide MBHA resins. Cyclic lipopeptide was cleaved from the solid support and crude product analyzed as described above.

2.8.3.5 Guanidinylation with N,N’-di-Boc-thiourea and NIS as an activator

Peptidyl-resin was swollen in DCM for 20 min followed by solvent removal. The solution of

N,N’-di-Boc-thiourea 22 (3 eq), NIS 24 (3 eq) and TEA (4 eq) in DCM (5 mL) was added to the peptidyl-resin and allowed to agitate at room temperature. Kaiser test indicated complete consumption of resin-bound amine within 2 h on TentaGel S RAM, whereas on Rink amide

MBHA resin guanidinylation reaction did not go to completion even after prolonged reaction time

76 of 8 h. In both cases, the peptidyl-resin samples (cca 20 mg) were taken after 2 h, cyclic lipopeptide was cleaved from the solid support and crude product 3 analyzed as described above.

The guanidinylation reaction was repeated with 1.1 eq of di-Boc-thiourea, 1.1 eq of NIS and 1.5 eq of TEA.

2.8.4 Circular dichroism (CD) spectroscopy

All CD spectra were recorded on JASCO 810 spectropolarimeter at 25C using a 0.1 cm path length cell. The spectra were acquired in the range 180-250 nm, 1 nm band width, 4 accumulations and 200 nm/min scanning speed. All spectra were obtained using 0.1-0.2 mM concentrations in 0.5% or 1% AcOH, 25-100% TFE/water (v/v) solution. Spectra at 0.5 and 1%

AcOH were virtually identical for each peptide. Each experiment was repeated at least once.

2.8.5 Molecular dynamics (MD) simulations

The MD simulations were performed by Dr. Franco Medina’s group at Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL.

First low energy conformations were generated by conformational analysis in Molecular

Operating Environment (MOE)70 using a Monte Carlo search with the Generalized Born solvation model implemented in MOE.293 The dielectric constant was set up to 26.14 or 80 to simulate the search in TFE or water, respectively. The search was conducted with the MMFF94x force field using default parameters. The lowest energy conformers found for each compound in the Monte

Carlo search were the starting point of 40 ns of molecular dynamics simulation using the module

MacroModel 9.9 from Maestro software.321 In brief, the initial structures were equilibrated by 2 ps.

The “Bonds to Hydrogens” option from the Shake procedure was selected in order to use 2 fs as time step. The simulation temperature was set to 300.0 K as default. The Optimized Potential for

Liquid Simulation (OPLS)-2005 force field332 was used to calculate the potential energy.

Conformations were sampled every 20 ps and optimized using the same force field. The dielectric

77 constant was set up to 26.14, to simulate the search in TFE. The GB/SA solvation model implemented in MacroModel333 was used for water simulation.

The amphiphilic moment descriptor,  which is an established measure of the balance between hydrophilic and lipophilic,54 was computed in MOE for the lowest energy conformations obtained in the dynamics within 5 kcal/mol of the lowest minimum.

2.8.6 Antibacterial activity

Total of six Gram-positive and two Gram-negative bacterial strains were used, including multiple-drug resistant bacterial strains; Staphylococcus aureus ATCC 29213, Staphylococcus aureus (MRSA) ATCC 33591, Staphylococcus aureus Mu50 (VRSA) ATCC 700699,

Staphylococcus epidermidis (MRSE) ATCC 27626, Streptococcus pyogenes ATCC 19615,

Escherichia coli K-12 ATCC 29181 and Klebsiella pneumoniae K6 ATCC 700603. Quantification of the antibacterial activity of synthesized analogs 1-18 was performed in sterile 96-well flat- bottomed polystyrene plates by the standard micro dilution broth method.295,296 Tests were performed using Müller-Hinton broth (MHB) without dilution. Controls on each plate were media without bacteria, bacterial inoculum without antimicrobials added, and bacterial inoculum containing methicillin or vancomycin. Assay setup: Stocks of microorganisms maintained at -80C in 30% glycerol were thawed and grown in media recommended by ATCC protocols for each particular microorganism. The following day, an aliquot of the bacterial suspension (100 L) was transferred into 10 mL of fresh media and incubated for 4-5 h, until OD600 of the suspension was

0.3-0.4. Bacterial suspension (100 μL) was then transferred into sterile Eppendorf tubes and centrifuged for 5 min at 4000 rpm. Supernatant was discarded and cell pellet resuspended in

MHB. Upon measuring OD600 of the suspension, appropriate dilution was made so that the final

OD of the suspension was approximately 0.001 (based on calculation). Concentrations of the analogs 1-18, as well as control antibiotics, were in the range 1-128 μg/mL. All samples were loaded in duplicate, and the average OD value was taken for calculating MIC. Each assay was repeated twice. Stock solutions of synthesized analogs 1-18, as well as control antibiotics were

78 prepared in 5-10% of DMSO/water (v/v) solvent mixture, depending on the analog solubility. After dilutions with MHB medium, final concentration of DMSO in wells was 0.5-1%. Plates were loaded with 90 μL of bacterial suspension (with initial OD600 of 0.001) of the tested microorganism, and 10 μL aliquots of two-fold serial dilutions of the analogs 1-18 or control antibiotics. Plates were then incubated at 37C overnight with gentle shaking. In the case of S. pyogenes, plates were incubated in 5% CO2 atmosphere. The inhibition of the bacterial growth was determined by measuring OD600; reduction in OD600 indicates inhibition of bacterial growth.

2.8.7 Antifungal activity

The testing of antifungal activity was done by Dr. Tim Gottwald’s group Agricultural Research

Services, USDA facility in Fort Pierce, FL.

Antifungal activity of fusaricidin/LI-F analog 6 was determined using broth dilution method. As controls chlorothalonil (negative control) and fluconazole (positive control) were used.

2.8.8 Hemolytic activity

Human red blood cells (hRBCs) were diluted with PBS to 1%. Depending on solubility, analogs 1-18 were dissolved in 5-10% DMSO/water (v/v) solvent mixture to concentrations of 16-

512 μg/mL. Into each well of the clear, flat-bottom 96-well plate, 50 μL of the hRBCs were placed followed by addition of 50 μL of analog solution to final peptide concentrations of 8-256 μg/mL.

Assays were performed in triplicate, and each experiment was repeated twice. To determine the potential effect of DMSO on hemolytic activity, controls containing 5-10% DMSO in water (v/v) were added to the assay setup. As a positive control, 50 μL of Triton X-100 in water was used at a final concentration of 0.5% (v/v). As a negative control, 50 μL of water and PBS was used.

Plates were incubated for 1h at 37 C. To each well 100 μL of PBS was added, and the plates were centrifuged for 10 min at 1000 g. Supernatants (150 μL) were transferred into a new plate, and absorbance at 405 nm was measured. Within the tested concentration range, the effect of

DMSO on hemolysis was minimal, and was deducted to obtain solely hemolytic activity of the

79 peptides. The degree of hemolysis was expressed in percent relative to total hemolysis caused by Triton-X.

2.8.9 Cytotoxicity

Cytotoxicity of analogs 1-18 against human normal (WRL-68) and cancer (HepG2) cell line was determined using MTT colorimetric assay. Assay was set up in flat-bottom polystyrene 96- well plates with 10,000 cells/well grown in EMEM media containing 10% FBS, 5% penicillin/streptomycin and 5% of L-glutamine (v/v). After an overnight incubation at 37C in humidified atmosphere with 5% CO2, media was removed, and fresh media containing analogs 1-

18 in the concentration range of 1-250 μM was added. Plates were again incubated at 37C in humidified atmosphere with 5% CO2. As a control, doxorubicin was used in the same concentration range. After 24 or 48 h incubation, media was removed, and 100 μL of MTT dissolved in serum free medium (1 mg/mL) was added to each well. Plates were again incubated for 3 h under the same conditions. Media containing MTT was removed and 100 μL of DMSO was added to each well. Plates were shaken for 5 min before reading absorbance at 540 nm.

2.8.10 Proteolytic stability assays

Stabilities of selected fusaricidin/LI-F analogs in 50% human serum, and as well as in EMEM medium (used in cell toxicity assays) were determined. For stability in 50% human serum, peptides 6 and 14 (0.5 mg each) were dissolved in 200 μL of DMSO to which 800 μL of water, and 1 mL of human serum was added. The solution was incubated at 37C. After 0 min, 45 min, 2 h, 4 h, 8 h and 24 h, three samples (3 x 100 μL) of each peptide were taken and precipitated by addition of 20 μL of 15 % aqueous TCA. Samples were quickly vortexed and then centrifuged at

10,000 rpm for 10 min. Supernatant was analyzed by analytical RP HPLC and MALDI-TOF MS.

For stability of depsipeptide 6 in the EMEM medium containing 10% FBS, 0.25 mg of the peptide was dissolved in 10 μL of DMSO, followed by addition of 990 μL of media and incubation at 37C. Peptide samples were treated and analyzed in the same way as described above.

80 CHAPTER 3

TOWARDS DECIPHERING THE MODE OF ACTION OF THE

FUSARICIDIN/LI-F CLASS OF ANTIMICROBIAL PEPTIDES

3.1 Introduction

Bacterial resistance is a pressing matter and the world of antibiotics is in need of new drugs with novel modes of action. Furthermore, biofilms, surface-attached communities of bacteria, represent a particularly serious threat due to their inherent resistance to antibiotics and host defense systems.69,74 Biofilms are the main cause of over 80% of the infections occurring in the body.63,64 Among Gram-positive bacteria S. aureus and S. epidermidis are the most commonly associated with complications caused by biofilm-related infections.334 More importantly, sepsis caused by the biofilm dissemination of S. epidermidis infected catheters is one of the most severe forms of S. epidermidis infection with a very high rate of morbidity and mortality, especially in neonates.335

Fusaricidin/LI-F is a family of potent antimicrobial agents with unique structure. Although we can assume some parallels with the mode of action of cationic antimicrobial peptides, due to their amphipilic structure and the positive charge present at the end of the lipidic tail, there is no actual data to prove or disprove this. And since the interactions of antimicrobial peptides with the membranes are very specific, and can not be easily predicted, we evaluated selected fusaricidin/LI-F analogs for membrane depolarization activity against three Gram-positive bacterial strains that differ in membrane composition and/or cell wall thickness. Also, the internalization of the peptides was monitored using fluorescence microscopy. Both depsipeptide and amide analogs were evaluated for bactericidal activity and activity against biofilm-forming S. aureus.

81 Moreover, the lipidic tail was substituted with cell-penetrating sequences (CPPs) to determine whether the role of the lipidic tail is merely peptide internalization. The structures of the analogs used in this study is shown in Figure 33.

Figure 33. Structures of analogs used in the study towards deciphering the mode of action of fusaricidin/LI-F class of antimicrobial peptides

3.2 Membrane depolarization

Cationic antimicrobial peptides (CAMPs) have the ability to electrostatically interact with the negatively charged phospholipidis in the bacterial membranes. This initial interaction allows insertion of CAMPs into the membrane bilayers to form pores or channels by different mechanisms, carpet, barrel-stave, toroidal pore.98,101,102 This causes leakages of intracellular components and ultimately cell death. However, for some peptides, the interaction with the bacterial membrane is only an intermediate step in the uptake into the cytoplasm where they reach their target. The extent of the interactions with the membrane depends on multiple factors, such as overall charge, hydrophobicity and amphiphylic character of the peptide. 51,96 Considering 82 the different structures of the synthesized fusaricidin/LI-F analogs 1-19, in comparison to typical

CAMP, we hypothesize that these cyclic lipopeptides exert their antibacterial activity via a novel mode of action. Therefore, assessment of the synthesized fusaricidin/LI-F analogs interaction with bacterial membranes represents the first step toward understanding their antibacterial mode of action.

The effect of synthetic fusaricidin/LI-F analogs on the membrane depolarization of Gram- positive bacteria was determined by using a membrane potential sensitive dye DiSC3(5). This fluorescent dye distributes between cells and media depending on the cytoplasmic membrane potential. Once inside the cells it self-quenches, most likely due to aggregates formed, which results in a decrease of the fluorescence signal. If a peptide forms channels or in other way causes dissipation of the membrane potential, the dye leaks out of the cells and the fluorescence signal increases.336 In these experiments 0.1 M KCl was added to the buffer to balance the chemical potential of K+ ions outside and inside the cells.337 The effect of the presence of 0.1 M

KCl on antimicrobial activity was tested and the activity of peptides was not affected.

The membrane depolarization experiments were performed using three strains, S. aureus

ATCC 29213, vancomycin-resistant S. aureus Mu50 (VRSA) ATCC 700699 and methicillin resistant S. epidermidis (MRSE) ATCC 27626.

Table 5. Membrane composition of different bacterial strains338,339

Strain PE (%) PG (%) CL (%)

S. aureus 0 57 19 ATCC 29213

S. aureus VRSA a 0 57 19 ATCC 700699

S. epidermidis MRSE 0 90 1 ATCC 27626 a Thicker peptidoglycan layer. PE= phosphatidylethanolamine, PG=phosphatidylglycerol, CL=cardiolipin

These strains differ in the membrane lipid composition and the thickness of the peptidoglycan layer, Table 5. For example, S. epidermidis contains much higher levels of negatively charged

83 phospholipids (PG) compared to S. aureus.338 On another hand, the vancomycin-resistant S. aureus strain has a significantly thicker peptidoglycan layer which confers its resistance to vancomycin, as well as daptomycin.29,339-341

In these experiments two of the depsipeptide analogs were used, 6 and 11, differing in position 3 in the peptide sequence (Val and Phe, respectively), and their amide analogs 14 and

16, Figure 33. Linear lipopeptide 19 and gramicidin S were used as negative and positive controls, respectively. Lipopeptide 19 did not show antibacterial activity in our assays, whereas gramicidin S is known to depolarize the cytoplasmic membrane of both Gram-positive and Gram- negative bacteria and makes pores in the membranes.342,343

Both depsipeptide 6 and amide 14 depolarized the membranes of tested Gram-positive bacteria in a concentration-dependent manner, Figure 34. Overall, the depsipeptide analog 6 consistently showed higher levels of depolarization compared to the amide analog 14 with all three bacterial strains tested. The level of membrane depolarization was comparable for the two

S. aureus strains (see Figure 34A and B), suggesting that the thickness of the peptidoglycan layer does not have an effect on the membrane depolarization capability of these peptides. These results are in good agreement with the MIC assays, Table 4, showing that the thicknes of the bacterial cell wall, a common feature of vancomycin-resistance in S. aureus,339 does not affect antibacterial activity of peptides 6 and 14. The levels of depolarization were in general higher for

S. epidermidis than for S. aureus strains (Figure 34C), which was not unexpected since S. epidermidis has higher level of negatively charged phospholipids in the membrane.

At a concentration of 48 g/mL of depsipeptide 6 and amide 14, the levels of membrane depolarization of all tested strains reached their maximum very quickly, within the first 5-10 min.

However, only in the case of S. epidermidis the depsipeptide 6 showed significant levels of depolarization at the MIC concentrations (8-16 g/mL) with a maximum of about 60% while amide analog 14 at the same concentrations only reached about 10% of depolarization, Figure 35.

84 Depsipeptide 6 Amide 14

A

B

C

Figure 34. Time- and dose-dependent membrane depolarization of A) S. aureus ATCC 29213; B) S. aureus Mu50 ATCC 700699 (VRSA); C) S. epidermidis ATCC 27626 (MRSE) with synthetic fusaricidin/LI-F analogs (performed at r.t.). Gramicidin S (20 g/mL) was used as internal reference

85 Similar results were obtained with depsipeptide 11 and amide 16 against S. aureus 29213, see Appendix, Figure 72. The depsipeptide analog 11 showed much higher levels of depolarization compared to its amide counterpart 16 at concentrations close to MIC. On another hand, the linear analog 19 did not elicit detectable depolarization even at the highest concentration tested (48 μg/mL), see Appendix, Figure 73, indicating the importance of peptide macrocyclic structure for bacterial membrane permeabilization and thus antibacterial activity.

Altogether, obtained results strongly suggest that the bacterial membrane is not the target for this family of antimicrobial peptides.

The temperature at which the experiment is performed does not affect the levels of depolarization. The same trends of concentration-dependent depolarization were observed for experiments done at room temperature (Figure 34) and at 37C (see Appendix, Figure 74) and the levels of depolarization were comparable.

Figure 35. Maximal membrane depolarization at r.t. for depsipeptide 6 (left) and amide 14 (right)

3.3 Bacterial survival following membrane depolarization

To evaluate whether the depolarization of the membranes is directly linked to the antimicrobial activity of peptides,137 we monitored bacterial survival under membrane depolarization experimental conditions. In these experiments bacteria was treated with 48 μg/mL

86 (3-6 x MIC) of either depsipeptide 6 or amide 14 and non-treated bacteria was used as control. In

10 minute intervals, aliquots of the bacterial suspension were taken to determine bacterial survival and the values are expressed as Log(CFU/mL).

If the membrane depolarization experiments were done at room temperature, neither the depsipeptide 6 nor the amide analog 14 showed bactericidal activity over the course of the experiment (1 h) against either susceptible or vancomycin-resistant S. aureus. In the case of S. epidermidis decrease of 3 log units in the number of bacterial colonies was oberved when bacteria was treated with depsipeptide 6, whereas the number of bacteria treated with the amide analog 14 remained unchanged, Figure 36A. In the case when membrane depolarization was performed at 37C, the depsipeptide analog 6 showed very fast bactericidal activity against all three Gram-positive strains with a decrease of > 3 log-units in the number of bacterial survivors.

Under the same experimental conditions, the amide analog 14 showed bacteriostatic activity,

Figure 36B.

S. aureus VRSA MRSE

A

B

Figure 36. Time-kill following membrane depolarization at: A) room temperature; B) 37C. Concentration of peptides was 48 μg/mL

87 Observed bacteriostatic activity at room temperature and fast bacterial killing at 37C for 6 suggest bacterial metabolism rather than bacterial membrane as potential target for this depsipeptide. The fact that the amide analog 14 exhibit bacteriostatic activity at both room temperature and 37°C indicates that the mechanism of action of 14 is different from that of 6.

However, further mechanistic studies are nesessary to precisely elucidate mechanisms of actions for both cyclic lipopeptides.

3.4 Bactericidal vs bacteriostatic activity

The real time-kill studies of the depsipeptide 6 and amide analog 14 against the three bacterial strains was assessed over the timeline of 18 h in MH broth at 37°C and 220 rpm.344

Similar to the results observed above, the depsipeptide analog 6 showed bactericidal activity against all three strains, while the amide analog 14 showed only bacteriostatic activity. The kinetics of the killing caused by the depsipeptide analog is similar for all three strains; very fast killing within the first hour where the number of bacteria drops 3-log units.345 The time-kill studies were performed at diffeent pressure of bacteria and peptides were able to tolerate even high inoculum, up to 109 CFU/mL.

A B C

Figure 37. Time-kill studies for three Gram-positive strains: A) S. aureus ATCC 29213; B) S. aureus Mu50 (VRSA) ATCC 700699; C) S. epidermidis MRSE ATCC 27626. Concentration of peptides was 48 μg/mL

88 3.5 Disruption of S. aureus biofilm

The ability of the synthetic fusaricidin/LI-F analogs to disrupt biofilm of Gram-positive bacteria was tested using model strain S. aureus ATCC 29213 and Minimal Biofilm Eradication

Concentration (MBEC) Assay™ (formerly Calgary Biofilm Device, Innovotech, Edmonton,

Canada) that allows simultaneous testing of a larger number of compounds in a 96-well plate format using a lid with pegs on which the biofilm is formed.346

In this assay S. aureus biofilm was treated with serial dilutions of both depsipeptide 6 and amide 14 (8-64 μg/mL). Daptomycin and vancomycin (100 μg/mL) were used a controls. Both peptides, 6 and 14, showed remarkable activity against S. aureus biofilm at very low concentrations, Figure 38.

Figure 38. Effect of synthetic fusaricidin/LI-F analogs on S. aureus biofilm

At the concentration corresponding to MIC (8 g/mL), the depsipeptide 6 reduced the number of bacteria in the biofilm for more than 3 log-units while at 4 x MIC concentration (24 g/mL) the biofilm was completely eradicated. The amide 14 was slightly less potent, at 8 g/mL it removed about 2 log-units of bacteria and complete biofilm eradication was observed at 6 x MIC (32 89 g/mL). On another hand, vancomycin was able to reduce 4 log-units of bacteria at much higher concentration, 100 g/mL. At the same concentration daptomycin was a bit more successful and removed approx. 6 log-units. However, it is important to note that these values are far greater than the MIC values vancomycin and daptomycin have against S. aureus which are in the range

0.5-1 μg/mL.

Similar to the results obtained from the killing curves, Figure 37, depsipeptide 6 was bactericidal against planktonic bacteria, even at 16 μg/mL, while amide 14 showed bacteriostatic activity (data not shown).

3.6 Assessment of of fusaricidin/LI-F class of antibacterial peptides ability to enter the cells of

Gram-positive bacteria

To assess the ability of fusaricidin/LI-F class of antibacterial cyclic lipopeptides to enter the cells of Gram-positive bacteria, we have prepared fluorescently labeled 29, Figure 33. To determine peptide localization, confocal fluorescence microscopy was performed after treatment of B. subtilis ATCC 82 strain 29 for 30 min. The data clearly shows that the peptide translocated into the cells and seems to be homogeneously distributed throughout the cells, Figure 39.

10 μm

Figure 39. Confocal fluorescence microscopic images of B. subtilis ATCC 82 cells upon incubation with fluorescein-labeled analog 29. Three images represent slices through the preparation

90 3.7 Assessment of interaction with the known target in cell wall biosynthesis, the D-Ala-D-Ala dipeptide

In order to assess whether fusaricidin/LI-F family of antibacterial peptides can interrupt bacterial cell wall biosynthesis, in particular the transpeptidation step, we have evaluated antibacterial activity of depsipeptide 6 in the presence of D-Ala-D-Ala dipeptide. The transpeptidation is the last major step in the biosynthesis of the peptidoglycan that results in a crosslink between differnet pentapeptide chains of the peptidoglycan precursor which is crucial for the three-dimensional structure and the rigidity of the peptidoglycan. The specific amino acid sequence and molecular structure of the peptidoglycan vary with the bacterial species.56 One of the main mechanisms of vancomycin-resistance in staphylococci is the replacement of the terminal D-Ala-D-Ala on the pentapeptide of the peptidoglycan precursor into D-Ala-D-lactate, resulting in loss of one hydrogen bond between vancomycin and its binding partner rendering the bacteria resistant to vancomycin.

During the screening of antimicrobial activity, we added up to 50 μg/mL of D-Ala-D-Ala in the media in order to see if the MIC values will change. In fact, addition of vancomycin’s binding partner did not have an effect on the activity of fusaricidin/LI-F analogs meaning that this particular resistance observed for vancomycin will affect the activity of our peptides.

3.8 Influence of human serum and physiological salt concentrations on peptides’ activity

To evaluate the influence of more physiologically relevant conditions on the activity of the fusaricidin/LI-F family of antibiotics, the antimicrobial activity of the peptides was performed in the presence of physiological concentrations of salts; 2 mM Ca2+, 1 mM Mg2+ and 150 mM NaCl, and in the presence of 25% human serum.347 A decrease in antimicrobial activity was not observed for neither of the peptides tested, depsipeptide 6 and amide 14. Even though we observed partial hydrolysis of the ester bond of 6 in the presence of human serum (see section 2.6), since the peptide acts rapidly, within 1 h (see section 3.4), the potential hydrolysis does not affect its activity.

91 3.9 Effect of 12-GDA substitution with cell-penetrating peptide (CPP) sequences

Bacterial membrane depolarization assays and the fluorescence microscopy study strongly suggest that the fusaricidin/LI-F class of antimicrobial peptides exert their antibacterial effect after translocation into the cytoplasm. Our antibacterial assays showed that the lipidic tail and presence of guanidinium functionality is essential for fusaricidins/LI-Fs and related cyclic lipopeptides antibacterial activity, Table 4. However, these experiments do not indicate if the positively charged lipidic tail’s role is to solely deliver the peptide into bacterial cytoplasm or it also participates in the mechanism of action. To better understand the role of the lipidic tail in activity of fusaricidin/LI-F class of antimicrobial peptides, we have prepared analogs containing short

IPMIK 30 and TAT 31 cell penetrating peptides (CPPs) instead of the lipidic tail, 12-GDA. IPMIK is the shortest cell penetrating peptide derived from Ku70,348 whereas the HIV-1 virus nuclear transcription activating protein TAT-derived peptide (GRKKRRQRRRPQ, residues 46–58) is a basic peptide that has been successfully shown to deliver a large variety of cargoes, from small particles to proteins, peptides and nucleic acids. It was shown recently that TAT-derived peptides exhibits antibacterial activity349 by disrupting bacterial membrane via a surface-active (carpet-like) mechanism.350

In our case substitution of the lipidic tail with either IPMIK or TAT CPPs resulted in complete loss of fusaricidin/LI-F cyclic lipopeptides antibacterial activity (MIC > 128 μg/mL). This data suggest that the lipidic tail plays an important role in this class of antibacterial peptide’s mode of action that exceeds antibacterial peptide translocation through the membrane.

3.10 Discussion and conclusions

With the experiments described in this chapter our goal was to obtain more insight into the mode of action of the fusaricidin/LI-F family of antimicrobial cyclic lipopeptides. Throughout SAR and conformational studies we have designed analogs of the natural products that exhibit comparable antimicrobial activity, but significantly improved stability under phisiologically relevant conditions and reduced nonselective toxicity. Since the structure of fusaricidin/LI-F class of

92 antibacterial peptides differ from those of the classical AMPs, we hypothesize that this new class of antibacterial peptided exerts their mode of action via a novel mechanism.

The activity of the synthetic analogs was assessed through examination of bacteriostatic/bactericidal properties, membrane depolarization activity and fluorescence microscopy study. Our experiments clearly showed that depolarization of bacterial membrane by depsipeptide 6 and amide 14 is not necessarily a lethal event. Unlike the peptides that form pores in the bacterial membrane, such as gramicidin S, our peptides showed significant depolarization at concentrations far above MIC level for the two S. aureus strains. Only in the case of S. epidermidis was notable depolarization found at MIC level. However, this can be rationalized simply through electrostatic interactions since the membrane of S. epidermidis contains significantly higher levels of negatively charged phospholipids (PG) than the membrane of S. aureus (Table 5). The level of depolarization was in all cases both concentration and time dependent but independent of the temperature. The confocal fluorescence microscopy with fluorescently labeled amide analog 29 clearly showed internalization of the peptide and its distribution throughout the cells.

In the case of the experiments performed at room temperature, no significant decrease in the number of bacteria was detected for peptide 6 and 14. At 37C, however, the killing of all three tested strains was rapid with the depsipeptide 6, whereas amide 14 showed bacteriostatic activity. Obtained results are somewhat surprising considering that the MIC values for depsipeptide 6 and amide 14 against these three strains are comparable (see section 2.3.1).

However, the differences in overall mechanism of action between 6 and 14 may be explained by the conformational differences between these two cyclic lipopeptides (see section 2.2).

Based on our SAR study (Chapter 2) 351 and the data reported in this chapter, the presence of the lipid tail and the positive charge on its terminus are crucial for the activity. This was established by replacing the 12-GDA acid tail with non-charged lipid versions and by lipid tail elimination. Peptide possessing 12-ADA or acetyl group instead of lipidic tail did not show

93 antimicrobial activity. Moreover, replacement of the lipidic tail with CPP sequences completely abolished the activity.

The problem of biofilms and complications caused by biofilms, including chronic infections and resistance to multiple antibiotics, make treatment of biofilms particularly challenging. The minimum biofilm eradication concentration (MBEC) is defined as the minimal concentration necessary to remove ≥ 3 log-units of bacteria from biofilm. By definition, our depsipeptide analog

6 has MBEC value equal to its MIC value, 8 μg/mL. For amide 14 the MBEC value is between 24 and 32 μg/mL. To put these numbers in an even more important perspective, the MBEC of daptomycin is > 100 x MIC,71 a similar case was also observed for vancomycin and linezolid.71,352

Daptomycin is also a cyclic lipodepsipeptide and it has been approved for the treatment of treatment of complicated skin and skin structure infections as well as treatment of bacteraemia and right-sided endocarditis caused by MSSA and MRSA.153,154 The problem of biofilm resistance affects all of the antibiotic classes currently on the market, however, fusaricidin/LI-F analogs reported here show very high efficacy against biofilm encapsulated bacteria, even at MIC level.

Furthermore, fusaricidins/LI-Fs seem to have an internal target and mode of action that does not depend on membrane depolarization, as seen in the case of daptomycin and many AMPs.

The antimicrobial activity is unaffected by the addition of D-Ala-D-Ala indicating that the resistance observed for vancomycin will not affect fusaricidins/LI-Fs.

The results reported in this and previous chapter show that new antibiotics derived from fusaricidin/LI-F natural products have the potential to meet the challenge of antibiotic resistance in Gram-positive bacteria, both planktonic, and biofilm growing.

3.11 Materials and methods

3.11.1 Bacterial strains and reagents

Bacterial strains used in this study; B. subtilis ATCC 82, S. aureus ATCC 29213, S. aureus

Mu50 ATCC 700699 and S. epidermidis ATCC 27626, were obtained from the American Type

Culture Collection (ATCC, Manassas, VA). Media and agar were purchased from BD (Franklin

94 Lakes, NJ). The dye 3,3'-dipropylthiadicarbocyanine iodide (DiSC3(5)) was purchased from

AnaSpec (Fremont, CA). Antibiotics (gramicidin S, methicillin, daptomycin and vancomycin) were purchased from Sigma Aldrich (St. Louis, MO). Vectashield H-1000 mounting media for fluorescence was purchased from Vector Laboratories (Burlingame, CA). Fmoc-protected amino acids and coupling reagents were purchased from Chem-Impex (Wood Dale, IL) or Novabiochem

(Gibbstown, NJ). TentaGel S RAM Fmoc resin for peptide synthesis was obtained from Advanced

ChemTech (Louisville, KY). All other chemicals, solvents and buffers were from Sigma-Aldrich or

Fisher Scientific (Atlanta, GA) and were HPLC grade or better. Plates for biofilm susceptibility assays were purchased from Innovotech Inc., Edmonton, Canada.

3.11.2 Peptide synthesis

Fusaricidin/LI-F analogs, Figure 33, were synthesized using standard Fmoc-SPPS methodology as reported previously (see Chapter 2).351 Fluorescently labeled analog 29 was prepared using standard protocols as described above, except that Val3 was replaced with Lys3 by using Fmoc-Lys(ivDde)-OH. Upon synthesis of the peptide and conversion of the amino moiety on the lipidic tail into guanidinium (as described above), the ivDde protecting group was removed using 2% hydrazine in DMF, 3 x 10 min. Coupling of fluorescein (5,6-FAM) was achieved using DIC/HOBt (10 eq of both 5,6-FAM and coupling reagents) in DMF overnight.353

For the preparation of peptides with CPP sequences instead of the lipid tail the cyclic amide core was synthesized first, leaving Fmoc protection on Dap1 which was subsequently removed and the peptide sequence continued using standard Fmoc methodology to obtain the desired CPP sequence.

3.11.3 Antibacterial activity

Minimal inhibitory concentrations (MICs) were determined using 96-well flat-bottomed polystyrene plates and the standard micro dilution broth method following CLSI guidelines as described in section 2.8.6.296 Each assay was performed at least twice.

95 3.11.4 Membrane depolarization assay

Cytoplasmic membrane depolarization was assessed using membrane potential sensitive

354 336,337,354 cyanine dye DiSC3(5) as described in the literature. Stocks of microorganisms maintained at -80C in 15% glycerol were thawed and grown at 37C and 220 rpm in media recommended by ATCC. The following day, an aliquot of the bacterial suspension was transferred into fresh media and incubated for 4-5 h, until OD600 of the suspension was 0.3-0.4.

After centrifugation at 2800 rpm for 10 min and washing once with a HEPES buffer containing 5 mM glucose (pH 7.2), the culture was resuspended in a HEPES buffer containing 5 mM glucose

8 and 100 mM KCl (pH 7.2) to final OD600=0.05 (approx. 10 CFU/mL). KCl was added to equilibrate cytoplasmic and external K+ concentrations to prevent any effect of internal K+ on the

336 assay. To the bacterial suspension DiSC3(5) dye was added to a final concentration of 0.4 μM.

Aliquots (100 L) of the bacterial suspension were placed in a 96-well non-culture treated white flat-bottomed plate and incubated until the fluorescence signal stabilized due to dye internalization (20-30 min at r.t. or 37C, depending on the experiment). The fluorescence signal was monitored using Synergy H4 microplate reader (BioTek, Winooski, VT) with excitation wavelength of 622 nm and emission wavelength of 670 nm. After the fluorescence signal stopped decreasing, 100 μL of solutions containing different concentrations of peptides (8, 12, 16, 24, 32 and 48 μg/mL) were added to the wells and the fluorescence was monitored in 2 min intervals over 1 h. As positive controls 20 μg/mL gramicidin S and 1% Triton X-100 were used and negative control was untreated bacteria. Values are expressed as percentage of maximum fluorescence caused by Triton X-100. All samples were done in triplicate. To evaluate potential influence of the temperature on the outcome of the experiments, membrane depolarization assays were performed both at r.t. and at 37C. Each experiment was performed at least twice.

3.11.5 Time-kill assays

During the membrane depolarization assay, in 10 min intervals, aliquots of the bacterial suspension treated with the peptides (48 μg/mL) were taken out of the wells and, following proper

96 dilutions, plated on nutrient agar. After 24 h incubation at 37C colonies were counted to determine the CFU/mL. For control untreated bacteria were used. The survival of bacteria was determined by enumerating CFUs from treated samples and comparing with control samples.

Real time-kill study was performed using the same starting bacterial inoculum as in the MIC

6 assays; OD600=0.001 (approx. 10 CFU/mL). Bacteria were incubated for 18 h in the presence of

48 g/mL of a fusaricidin/LI-F analog. At certain time points (0, 0.5, 1, 2, 4, 8 and 18 h) aliquots of the bacterial suspension were taken and, following proper dilutions, plated on nutrient agar and incubated for 24 h at 37C. Control was bacteria without antibiotic added. The survival of bacteria was determined by enumerating CFUs from treated samples and comparing with control samples.

3.11.6 Biofilm susceptibility assay

Biofilm formation and susceptibility testing was performed according to previously reported protocols.346,355,356 The bacterial culture of S. aureus ATCC 29213 was grown overnight in trypticase soy broth (TSB) and the stationary phase culture was diluted in TSB to OD600 of approx. 0.001 which corresponds to about 106 CFU/mL. Biofilm formation was carried out in

MBEC® P&G plates with a peg lid with a final volume of bacterial suspension 200 L. For sterility control TSB only was used. After incubation in a humidified incubator with gyrorotary shaking at

35C and 110 rpm for 9 h the pegs were rinsed in PBS to remove any loosely adhered bacteria.

To determine the starting number of bacteria in the biofilm several pegs were clipped off, sonicated to disrupt the biofilm and after proper dilutions plated on nutrient agar plates. The lid was then placed on the challenge plate containing different concentrations of our synthetic peptides 6 and 14 and control antibiotics. Controls were vancomycin and daptomycin, used at single concentration of 100 μg/mL, as well as media by itself. Both synthetic and control peptides were prepared in media. The challenge plate was incubated in the same conditions as described above for 18 h after which the pegs were rinsed twice in PBS and then placed on a recovery plate

97 containing 200 μL of TSB media/well and sonicated for 45 min. To determine bacterial survival, both planktonic and bacteria from biofilm were, after proper dilutions, plated on NA.

3.11.7 Fluorescence microscopy

Microscopy studies were performed using fluorescently labeled analog 29, Figure 33, and B. subtilis ATCC 82. The bacteria was grown as described above, harvested during the exponential growth and resuspended in MHB to OD600 of approx. 0.1. Fluorescently labeled peptide was added to the bacterial suspension in final concentration of 64 μg/mL and the suspension was incubated at 37C for 1 h. Cells were washed twice with PBS, resuspended in PBS with 1% paraformaldehyde and 10 μL of the suspension was placed on a slide. To prevent photo bleaching 5 μL of Vectashield mounting media was added and the samples were analyzed the same day using Zeiss Axio Imager.Z2 LSM 780 (Carl Zeiss MicroImaging GmbH, Jena,

Germany).

98 CHAPTER 4

LYSOBACTIN (KATANOSIN B) – SYNTHESIS AND EVALUATION OF ACTIVITY

4.1 Introduction

Lysobactin (katanosin B) 32, Figure 40, belongs to a large and diverse family of the non- ribosomally synthesized peptides, a family of microbial natural products that contain, in addition to the known 20 proteinogenic amino acids, unusual residues that are not present in proteins. These include D-amino acids, -amino acids, and hydroxy-amino acids and the peptide is further modified by heterocyclic ring formation. However, recombinant techniques have not yet been routinely applied to the synthesis of peptides containing non-proteinogenic amino acids.

Therefore, total solution and/or solid–phase peptide synthesis combined with the combinatorial chemistry approach still remain the most efficient way for the preparation of these important natural products, including lysobactin, and its structural modifications.

Several groups have thus far reported synthesis of lysobactin, both in solution213,214 and on solid phase.212,215 Von Nussbaum et al. developed elegant in solution approach guided by the crystal structure of lysobactin where they utilized conformation-directed cyclization starting with three peptide fragments previously prepared. Although the macrolactamization proceeded with high yields (72 %), the overall synthesis allows very little manipulations which are necessary for

SAR studies. On another hand, utilizing the solid phase approach several groups obtained lysobactin and its analogs. However, only Egner and Bradley212 reported total solid phase synthesis while Van Nieuwehnze et al.215 reported a combined solid-phase and solution phase strategy. In this case, the lysobactin linear precursor was synthesized using SPPS approach, whereas peptide cyclization was performed in solution.

99 We have devised a synthetic strategy that offers the advantages of the complete lysobactin synthesis on solid-support. Based on the retrosynthetic analysis of lysobactin 32, Figure 40, our synthetic approach toward this molecule and related analogs includes attachment of β- hydroxyaspartic acid to the amine resin via side chain, linear peptide precursor assembly using standard Fmoc-SPPS, coupling of the Ser to L-threo-phenylserine via ester bond, and on-resin macrolactamization.

Pepride sequence of lysobactin 32, Figure 40, contains three unusual amino acids, L- hydroxyleucine (L-HyLeu), L-threo-phenylserine (L-tPhSer), and L-threo-β-hydroxyaspartic acid (L- tHyAsp), which are not commercially available as Fmoc SPPS compatible building blocks and therefore needed to be prepared as such. Among them, perhaps, the most challenging is the synthesis of orthogonally protected and enantiomerically pure L-tHyAsp.

Figure 40. Retrosynthetic analysis of lysobactin

100  4.2 Synthesis of N -Fmoc protected L-threo--hydroxyaspartic acid derivatives

4.2.1 Background

Stereoisomers of -hydroxyaspartic acid (HyAsp) as well as its analog -hydroxyasparagine

(HyAsn) are found in both the free form and as peptide constituents in many natural products. L-

357 threo--hydroxyaspartic acid (L-tHyAsp) alone inhibits growth of various fungi, and its derivatives exhibit a series of biological activities such as inhibition of glutamate/aspartate transporters,358,359 inhibition of tumor cell growth,360,361 and antiretroviral activity.362 HyAsp and

HyAsn were found in sequences of antibacterial peptides such as ramoplanin,166,179 katanosins,201,202 lanthiopeptin,363 alterobactins,364 plusbacins,200,217 and cormycin A.365 Among them, ramoplanin shows the most promising clinical potential and is currently in Phase III trials for treatment of Clostridium difficile-associated disease (CDAD) and for the prevention of vancomycin-resistant Enterococci bloodstream infections.366,367 Other examples of naturally occurring peptides containing HyAsp/HyAsn are theonellamide368 and cepacidine,369,370 exhibiting strong antifungal activity, and microviridins371 that act as protease inhibitors.

Since Fmoc solid-phase peptide synthesis (Fmoc SPPS) represents a standard approach for the routine synthesis of peptides, access to orthogonally protected HyAsp and HyAsn fully compatible with this methodology is of practical importance. Asymmetric syntheses of L-tHyAsn bearing standard protecting groups for Fmoc- solution or solid-phase peptide synthesis have been reported in the literature.372-374 In contrast, a few synthetic protocols are reported for preparation of HyAsp, but none of the reported derivatives are compatible with the standard Fmoc

375 SPPS methodology. Hanessian and Vanasse reported the synthesis of L-threo-HyAsp (L- tHyAsp) and L-erythro-HyAsp (L-eHyAsp) mixtures of diesters by hydroxylation of L-aspartic acid

(L-Asp) diesters. Starting with trans-ethyl cinnamate, Sharpless asymmetric aminohydroxylation

376 was used by Khalaf and Datta to prepare D-tHyAsp. In all cases reported, HyAsp derivatives possess unprotected -amino and -carboxyl groups. Boc-protecting strategy was chosen to synthesize N-protected HyAsp derivatives. Hansson and Kihlberg377 used tartaric acid to prepare

101  378 protected L-eHyAsp, whereas N -Boc-L-Ser was used by Wagner and Tilley to prepare

 protected L-tHyAsp fully compatible with Boc peptide synthetic methodology. N -Boc-L-tHyAsp was also prepared by Deng et al.379 starting from methyl cinnamate and using Sharpless dihydroxylation reaction. In this case, synthesized L-tHyAsp derivative underwent additional

  deprotection and reprotection to obtain the desired N -Boc protected L-tHyAsp. All reported N -

Boc-HyAsp derivatives possess a free α-carboxyl group, limiting somewhat their synthetic applicability.

Reported here is a practical and efficient synthetic methodology towards orthogonally protected D- and L-tHyAsp, fully compatible with the standard Fmoc SPPS methodology. Besides complete orthogonal protection, our approach allows synthetic exploitation of both HyAsp carboxyl groups expanding the versatility of these peptide building blocks.

4.2.2 Enantioresolution of D,L-threo--hydroxyaspartic acid

The enantioselective enzyme-catalyzed hydrolysis of N-acyl amino acids had been a widely used method in the resolution of amino acid racemic mixtures. In particular, acylase I (N- acetylamino-acid amidohydrolase) was shown to be very efficient in the preparation of large

380-382 quantities of enantiopure L-amino acids. However, several studies demonstrated that acyl derivatives of aspartic acid are not substrates for acylase I due to unfavorable charge

383,384 interactions, thereby eliminating this method as an option for enantioresolution of D,L- tHyAsp. Instead, we have focused on their chemical resolution. Attempts to separate the D,L- mixture of compound 3 via co-crystallization with (-)-ephedrine hydrochloride,385,386 a method

 traditionally used for resolution of N -benzyloxycarbonyl-D,L-amino acids, were unsuccessful.

Limited solubility of D,L-33 in a variety of solvents including CHCl3, MeOH, EtOH, ACN, acetone,

THF, EtOAc, EtOAc:MeOH 1:1, EtOAc:petroleum ether 2:3, EtOAc:diethyl ether 1:1,

EtOAc:CHCl3 1:1, EtOAc:DCM 2:3, EtOAc:ACN 1:3 resulted in poor resolution of the corresponding diastereomeric ephedrine salts. The D,L-tHyAsp 33 was successfully enantioresolved by modified method previously described by Okai et al.387 via co-crystallization 102 with L-Lys followed by ion-exchange chromatography. Both enantiomers were obtained with satisfactory yield (75%) and enantiomeric purity (e.e. >99%).

D,L-tHyAsp racemic mixture 33 was successfully separated by co-crystallization with L-Lys

(equimolar ratio) followed by ion exchange chromatography,387 Scheme 6. Enantiomerically pure

L-Lys • L-tHyAsp salt crystallized from the H2O:MeOH solvent mixture overnight at 4°C, whereas

L-Lys • D-tHyAsp remained in the mother liquor (see Appendix). In both cases L-Lys was separated by ion exchange chromatography, and pure L- and D-tHyAsp, 33a and 33b, were obtained in 75% yields.

NH2 O NH2 HO + HO OH NH2 O OH O D,L-threo-HyAsp L-Lys

33 1) Co-crystallization water/MeOH 1:1 2) Filtration

Crystals Mother liquor L-threo-HyAsp L-Lys D-threo-HyAsp L-Lys

Ion-exchange chromatography

NH2 O NH2 O HO HO OH OH O OH O OH

L-threo-HyAsp D-threo-HyAsp 33a 33b

Scheme 6. General scheme of D,L-tHyAsp enantioresolution

103 2200 UV2000-340nm 2200 UV2000-340nm HyAsp Marfey 2 DHyAsp 2000 a) 2000 b) 1800 Marfey’s 1800 Rt-21.2 min 1600 reagent 1600 1400 1400

1200 1200 mAU mAU 1000 1000 800 800 600 600 400 400 200 200 0 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Minutes Minutes

2200 UV2000-340nm L-HyAsp run2-1 120908 2000 c) 1800 1600 1400 Rt-21.6 min 1200

mAU 1000 800 600 400 200 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Minutes Figure 41. RP HPLC traces after derivatization with Marfey’s reagent: a) D,L-tHyAsp; b) D-tHyAsp; c) L-tHyAsp. RP HPLC conditions: 100% A 5 min, 0-100% B, 45 min; A - 0.1% TFA in water, B - 0.1% TFA in ACN

388 RP HPLC analysis of D- and L-tHyAsp after derivatization with Marfey’s reagent, Figure 41, shows that in both cases enantiomeric purity was >99%. Specific optical rotations ([]D) for the

387 obtained D- and L-tHyAsp are in very good agreement with the literature data, further confirming the enantiomeric resolution efficiency, Table 1.

Table 6. Specific optical rotations for resolved tHyAsp enantiomers

Literature values387 Observed values

[]D

5 N HCl H2O 5 N HCl H2O

L-tHyAsp (33a) +6.4° -8.5° +6.0° -8.2°

D-tHyAsp (33b) -6.5° +8.9° -6.0° +8.4°

104 4.2.3 Design of a protection scheme

Appropriate selection of orthogonal protecting groups and their selective manipulation is an essential requirement in peptide and amino acid synthesis. In order to find the optimal combination of orthogonal protecting groups for tHyAsp, we have tested benzyl (Bn), allyl (Allyl), and 4{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino} benzyl (Dmab) for protection of carboxyl groups, 9-fluorenylmethylmethoxycarbonyl (Fmoc) for protection of amino, and tert-butyldimethylsilyl (TBDMS) and tetrahydropyranyl (THP) for protection of the hydroxyl group. Taking into consideration reaction conditions for the standard Fmoc SPPS and the requirement for the tHyAsp coupling via either - or -carboxyl group, combination of the Fmoc,

Dmab, TBDMS and Bn protecting groups gave the best results. The general protection scheme for L-tHyAsp 33a is shown in Scheme 7. Selective protection of -carboxyl group was achieved by

Fischer-type esterification, resulting in compound 34 (87% yield). Due to the presence of acid catalyst, protonation of the -amino group determined the esterification selectivity.389 In order to prepare tHyAsp derivative with a free -carboxyl group, we have devised two protecting approaches. One includes Fmoc protection of the -amino group resulting in compound 35, 96% yield,390 followed by -hydroxyl group protection with THP391 with a 65% yield to obtain compound

36.

In another approach, due to the basic reaction conditions requirements for TBDMS introduction, the order of protections was reversed. The -hydroxyl group was protected first with

TBDMS 392 (39, 82% yield), followed by -amino group protection with Fmoc (40, 96% yield).

Although both THP and TBDMS protecting groups fulfill the requirement for orthogonal protection, we gave preference to the TBDMS due to better reaction yields, the lack of need for chromatographic purification, less complex reaction monitoring and final product characterization.

Introduction of the THP group added an additional stereocenter to the tHyAsp molecule, resulting in two diastereomers of 36 with distinguishable Rf values on the silica-gel TLC plates (0.35, 0.33; toluene:EtOAc:AcOH 10:1:1), and complex NMR spectra.393 On the other hand, preparation of

105 the orthogonally protected L-tHyAsp derivate with a free -carboxyl group turned out to be more challenging.

NH2 O NH2 O a HO HO OH OBn O OH O OH 33a 34

b f

Fmoc

NH O NH2 O HO HO OBn OBn O OH 35 39 O OTBDMS

c g

Fmoc Fmoc NH O NH O HO HO OBn OBn O OTHP 36 40 O OTBDMS

d h

Fmoc Fmoc NH O NH O AllylO DmabO OBn OBn O OTHP 37 41 O OTBDMS

e i

Fmoc Fmoc NH O NH O AllylO DmabO OH OH O OTHP 38 42 O OTBDMS

Scheme 7. General protection scheme for HyAsp. Reaction conditions: a) Bn-OH, HCl, 4 h, 87%; b) Fmoc-OSu (1.1 eq), NaHCO3 (2 eq), acetone/H2O, 2 h, 96%; c) DHP (2.5 eq), PPTS (0.3 eq),

CH2Cl2, 72 h, 65%; d) NaHCO3 (1 eq), Allyl-Br (2.5 eq), Aliquat 336 (1 eq), DCM, 72 h, 85%; e) see Table 2; f) TBDMS-Cl (2 eq), DBU (3.5 eq), ACN, 3-5 h, 82%; g) same as b); h) Dmab-OH

(1.2 eq), DIC (1.2 eq), DMAP (0.2 eq), DCM, 4-6 h, 70%; i) H2, 5% Pd/C, EtOAc, 50 min, 75%

After successful introduction of an allyl protecting group (37, 85% yield, 394), selective removal of benzyl ester using various approaches was unsuccessful. We have tested several reaction conditions including mild base, Lewis acids, catalytic hydrogenation, neutral reagents, or lipase C from Candida antarctica, and in all cases either no Bn removal or more than one protecting group 106 removal was observed. The reaction conditions used for benzyl ester removal are summarized in

Table 7.

Table 7. Tested conditions for selective removal of benzyl protecting group from 37

Fmoc Fmoc NH O NH O AllylO AllylO OBn OH O OTHP O OTHP 37 38 Conditions Observeda

395 20% K2CO3/H2O, EtOH, 1 h Removal of Fmoc, Bn and Allyl

396 15% K2CO3/H2O, THF, 4 h No deprotection

397 4 mM NaOH, 0.5 M CaCl2, IPA/H20 (7:3), 2 h Removal of Fmoc and Bn 1 N LiOH, THF, 2 h 398 Removal of Fmoc and Bn

399 1 N BCl3, DCM, -10 °C to r.t., 1 h Removal of THP first

400 Short time-no deprotection, longer time-all protecting Pd(OAc) , Et SiH, Et N, DCM, 1 h-overnight 2 3 3 groups removed except THP

401 H2, 10% Pd/C, EtOAc, 1 h Removal of Bn and Allyl or Allyl reduction TBAF, THF, 1 h 402 Removal of Fmoc first

403 HCOONH4/Mg, MeOH, 3 h Removal of Bn and Allyl Enzymatic removal, 24 h 404 No deprotection aBased on 1H NMR analysis and TLC comparison of reaction mixtures with compounds 33a-37.

Replacement of the allyl protecting group with the Dmab, compound 41, allowed preparation of the desired L-tHyAsp derivative 42 over five reaction steps in a satisfactory yield of 36%.

Among several previously published methods for coupling of Dmab-OH to amino acid's carboxyl group, the most satisfactory results were obtained using DIC/DMAP coupling reagents 405.

However, this method had to be modified for optimal Dmab-OH coupling to compound 40. If catalytic amounts of DMAP were to be used, the required reaction time is significantly prolonged resulting in a poor yield for compound 41. On the other hand, use of equimolar amount of DMAP causes Fmoc deprotection. The best results were obtained with 0.2 eq of DMAP. In this case compound 41 was obtained in 70% yield.

Final benzyl protecting group removal was successfully achieved by hydrogenation of compound 9 with 5% Pd/C in ethyl-acetate.401 Yield for this final step was 75%. The outlined protection scheme is also expected to be useful for the preparation of orthogonally protected D- tHyAsp, as well as D- and L-eHyAsp derivatives. 107 4.2.4 Discussion and conclusions

We have successfully devised a simple and practical general synthetic protocol towards orthogonally protected HyAsp derivatives, fully compatible with Fmoc solid-phase peptide synthetic methodology, Figure 42. This approach allows utilization of the commercially available

 D,L-tHyAsp racemic mixture and preparation of multi-gram quantities of enantiomerically pure N -

Fmoc protected tHyAsp derivatives for coupling via either - or -carboxyl group onto the resins or the growing peptide chain. In addition, the coupling of HyAsp via free -carboxyl group onto amino resins enables the preparation of peptides containing HyAsn sequences, further increasing the utility of the designed protection scheme.

Considering the increasing demand for peptides containing unusual sequences due to their interesting biological activities, the described synthesis of N-Fmoc protected tHyAsp building blocks is of particular practical value allowing multiple possibilities for their incorporation into a peptide backbone.

Fmoc NH O HO O PG1 O O NH2 O NH2 O PG2 HO HO OH OH Fmoc O OH O OH NH O

PG3 O OH O O PG2

Figure 42. Graphical representation of HyAsp derivatives compatible with Fmoc-SPPS

4.2.5 Materials and methods

Synthesis of (2S,3S)-2-amino-4-(benzyloxy)-3-hydroxy-4-oxobutanoic acid (34)

To benzyl alcohol (10 mL) and concentrated HCl (1.2 mL) L-tHyAsp 33a (1 g, 6.71 mmol) was added, and the resulting mixture was stirred at 70°C for 4.5 h. Upon reaction completion, the pH was adjusted to 6 with NH4OH, and cold EtOH was added to precipitate the product. Precipitate was recrystallized from hot water, and pure (2S,3S)-2-amino-4-(benzyloxy)-3-hydroxy-4- 108 oxobutanoic acid (34) (1.4 g, 5.86 mmol, 87%) was obtained as white solid. 1H NMR (400 MHz,

DMSO-d6) δ 7.33-7.43 (m, 5H, Bn-ArH), 5.11 (dd, 2H, Bn-CH2), 4.52 (d, 1H, Hβ), 3.47 (d, 1H, Hα)

13 ppm; C NMR (100 MHz, DMSO-d6) δ171.7, 167.7, 136.0, 128.4, 128.0, 69.7, 66.1, 55.9 ppm.

Synthesis of (2S,3S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(benzyloxy)-3-hydroxy-

4-oxobutanoic acid (35)

A solution of Fmoc-OSu (0.141 g, 0.42 mmol) in acetone was added dropwise to a solution of compound 34 (0.1 g, 0.42 mmol) and NaHCO3 (0.07 g, 0.84 mmol) in water:acetone mixture (1:2,

15 mL). The reaction was completed within 2 h, as determined by TLC (solvent A/solvent B= ratio). The reaction mixture was concentrated via rotary evaporation, and the residual water was acidified with 2N HCl until pH 4 and extracted two times with EtOAc. EtOAc was evaporated and the crude residue was loaded on a silica gel column (solvent system toluene:EtOAc:HOAc, 5:5:1) yielding product (0.185 g, 0.4 mmol, 96%) as a white solid. HRMS calculated for C26H23NO7 m/z

+ 1 (M + Na) 484.1372; found 484.4560. H NMR (400 MHz, CD3OD) δ 7.12-7.82 (m, 13H, Fmoc-

ArH, Bn-ArH), 5.16 (dd, 2H, Bn-CH2), 4.83 (d, 1H, Hα), 4.76 (d, 1H, Hβ), 4.27 (m, 2H, Fmoc-CH2),

13 4.19 (t, 1H, Fmoc-CH) ppm; C NMR (100 MHz, CD3OD) δ 173.0, 172.8, 158.6, 145.4, 145.3,

142.7, 137.1, 129.6, 129.6, 129.5, 128.9, 128.4, 128.3, 126.53, 126.49, 121.1, 72.5, 68.5, 68.5,

58.5 ppm.

Synthesis of (2S,3S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(benzyloxy)-4-oxo-3-

(tetrahydro-2H-pyran-2-yloxy)butanoic acid (36)

PPTS was added (0.008 g, 0.0325 mmol) to a solution of 3 (0.15 g, 0.325 mmol) in dry DCM

(15 mL).To the stirred reaction mixture, DHP was added dropwise over 10 min (0.045 mL, 0.487 mmol). Stirring was continued at room temperature for 72 h with occasional addition of PPTS and

DHP (total 0.3 eq PPTS and 2.5 eq DHP). The reaction mixture was then extracted with water.

DCM was evaporated and crude product purified using silica gel chromatography (solvent system toluene:EtOAc:HOAc, 10:1:1) yielding 0.115 g of product (0.211 mmol, 65%). HRMS calculated

109 + 1 for C31H31NO8 m/z (M + Na) 568.1947; found 568.4458. H NMR (400 MHz, CDCl3) δ 7.28-7.77

(m, 13H, Fmoc-ArH, Bn-ArH), 5.69 (d, 1H, Hα), 5.17 (s, 2H, Bn-CH2), 5.01 (m, 1H, Hβ), 4.86 (t,

1H, THP), 4.33 (m, 2H, Fmoc-CH2), 4.22 (t, 1H, Fmoc-CH), 3.49-3.75 (m, 2H, THP), 1.50-1.77

13 (m, 6H, THP) ppm; C NMR (100 MHz, CDCl3) δ 173.0, 169.1, 156.5, 143.9, 141.4, 135.2, 128.8,

128.7, 128.6, 127.9, 127.3, 125.4, 120.2, 97.6, 73.5, 67.8, 67.7, 62.6, 56.2, 47.1, 30.0, 25.2, 18.9 ppm.

Synthesis of (2S,3S)-1-allyl 4-benzyl 2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-

(tetrahydro-2H-pyran-2-yloxy)succinate (37)

Phase transfer catalyst aliquat 336 (0.084 mL, 0.183 mmol) and allyl-Br (0.017 mL, 0.183 mmol) were dissolved in DCM (15 mL). This mixture was added to a stirred solution of 4 (0.1 g,

0.183 mmol) and NaHCO3 (0.016 g, 0.183 mmol) in water (15 mL). Reaction was stirred at room temperature for 72 h with occasional addition of 0.5 eq of allyl-Br (total added was 2.5 eq). DCM was separated from the aqueous layer and extracted one more time with water, evaporated, and crude product was purified on a silica column (solvent system toluene:EtOAc:HOAc, 10:1:1)

+ yielding 0.109 g (85%) of pure product 5. HRMS calculated for C34H35NO8 m/z (M + Na)

1 608.2260; found 608.6071. H NMR (400 MHz, CDCl3) δ 7.25-7.77 (m, 13H, Fmoc-ArH, Bn-ArH),

5.91 (m, 1H, Allyl), 5.65 (d, 1H, Hα), 5.36 (dd, 1H, Allyl), 5.27 (dd, 1H, Allyl), 5.16 (s, 2H, Bn-CH2),

5.02 (m, 1H, Hβ), 4.87 (t, 1H, THP), 4.67 (m, 2H, Allyl-CH2), 4.33 (m, 2H, Fmoc-CH2), 4.22 (t, 1H,

13 Fmoc-CH), 3.43-3.62 (m, 2H, THP), 1.46-1.79 (m, 6H, THP) ppm; C NMR (100 MHz, CDCl3) δ

169.2, 169.1, 156.3, 144.0, 143.9, 141.4, 135.2, 131.5, 128.8, 128.7, 128.7, 127.9, 127.3, 125.5,

120.2, 119. 5, 101.0, 96.9, 73.2, 67.7, 67.7, 67.5, 66.8, 66.8, 61.9, 56.4, 47.2, 30.2, 29.9, 25.3,

25.2, 19.0, 18.4 ppm.

110 Synthesis of (2S,3S)-2-amino-4-(benzyloxy)-3-(tert-butyldimethylsilyloxy)-4-oxobutanoic acid

(39)

To a stirred solution of 34 (0.56 g, 2.33 mmol) in dry ACN (300 mL), DBU was added (0.351 mL, 2.33 mmol) to obtain pH approximately 9 after which TBDMS-Cl was added in half equimolar portions (0.175 g, 1.17 mmol). Occasionally DBU was added to correct the pH of the solution.

Reaction mixture was refluxed at 50°C. Total 2 eq of TBDMS-Cl and 3.5 eq of DBU were used.

The reaction was monitored by TLC and was completed within 3-5 h. The solvent was evaporated; residue redissolved in water, acidified with 2N HCl until pH 4 and extracted two times with EtOAc. Crude product (yellow oil, 0.674 g, 82% yield) was used in the next step without further purification.

Synthesis of (2S,3S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(benzyloxy)-3-(tert- butyldimethylsilyloxy)-4-oxobutanoic acid (40)

To a solution of 39 (0.674 g, 1.91 mmol) and NaHCO3 (0.321 g, 3.82 mmol) in water:acetone mixture (1:2, 100 mL), a solution of Fmoc-OSu (0.644 g, 1.91 mmol) in acetone was added dropwise. The reaction was monitored by TLC and was completed within 2 h. Reaction mixture was concentrated, residual water acidified with 2N HCl until pH 4 and extracted two times with

EtOAc. EtOAc was evaporated and crude residue was loaded on a silica gel column and purified

(solvent system toluene:EtOAc:HOAc, 10:1:1) yielding pure product 8 (1.06 g, 1.84 mmol, 96%

+ yield) as a white solid. HRMS calculated for C32H37NO7Si m/z (M + Na) 598.2237; found

1 598.2075. H NMR (400 MHz, CDCl3) δ 7.3-7.76 (m, 13H, Fmoc-ArH, Bn-ArH), 5.58 (d, 1H, Hα),

5.16 (dd, 2H, Bn-CH2), 4.93 (d, 1H, Hβ), 4.33 (m,2H, Fmoc-CH2), 4,23 (t, 1H, Fmoc-CH), 0.89 (s,

13 9H, TBDMS-tBu), 0.11 (s, 3H, TBDMS-CH3), 0.03 (s, 3H, TBDMS-CH3) ppm; C NMR (100 MHz,

CDCl3) δ 174.3, 169.9, 156.1, 143.7, 141.2, 141.2, 135.0, 128.57, 128.55, 128.50, 127.7, 127.1,

125.2, 119.9, 72.2, 67.6, 67.5, 57.3, 46.9, 25.5, 25.4, 18.7, -4.9, -5.8 ppm.

111 Synthesis of (2S,3S)-1-(4-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3- methylbutylamino)benzyl) 4-benzyl 2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(tert- butyldimethylsilyloxy)succinate (41)

To a solution of 40 (0.3 g, 0.52 mmol) in DCM (30 mL), DMAP (0.013 g, 0.1 mmol) and

Dmab-OH (0.205 g, 0.624 mmol) were added. DIC (0.097 mL, 0.624 mmol) was added dropwise over 15 min. Reaction was stirred at room temperature for 4-6 hours with monitoring using TLC.

The solvent was removed and silica gel chromatography (solvent system toluene:EtOAc:HOAc,

10:5:1) afforded product 9 (0.319 g, 0.36 mmol, 70%) as a yellowish solid. HRMS calculated for

+ 1 C52H62N2O9Si m/z (M + Na) 909.4122; found 909.1248. H NMR (400 MHz, CDCl3) δ 7.07-7.78

(m, 17H, Fmoc-ArH, Bn-ArH, Dmab-ArH), 5.63 (d, 1H, Hα), 5.10-5.15 (m, 4 H, Bn-CH2,Dmab-

CH2), 4.97 (d, 1H, Hβ), 4.31 (m, 2H, Fmoc-CH2), 4.23 (t, 1H, Fmoc-CH), 2.49 (s, 2H, Dmab-CH2),

2.40 (s, 2H, Dmab-CH2), 2.09 (d, 2H, Dmab-CH2 isopropyl), 1.82 (m, 1H, Dmab-CH isopropyl),

1.08 (d, 6H, 2 Dmab-CH3), 0.88 (s, 9H, TBDMS-tBu), 0.76 (s, 3H, Dmab-CH3 isopropyl), 0.74 (s,

13 3H, Dmab-CH3 isopropyl), 0.09 (s, 3H, TBDMS-CH3), -0.04 (s, 3H, TBDMS-CH3) ppm; C NMR

(100 MHz, CDCl3) δ 200.2, 196.5, 176.5, 170.0, 169.8, 169.2, 169.5, 156.0, 155.5, 143.74,

143.71, 143.6, 141.2, 137.1, 136. 9, 135.1, 135.1, 134.4, 134.3, 129.2, 129.2, 128.64, 128.58,

128.53, 127.7, 127.1, 126.7, 126. 6, 120.0, 107.8, 72.4, 67.6, 67.4, 67.0, 66.8, 66.8, 57.7, 53.7,

52.3, 47.0, 46.9, 38.4, 30.0, 29.6, 28.3, 25.6, 22.6, 18.2, -4.8, -5.8 ppm.

Synthesis of (2S,3S)-4-(4-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3- methylbutylamino)benzyloxy)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-(tert- butyldimethylsilyloxy)-4-oxobutanoic acid (42)

Fully protected amino acid 41 (0.27 g, 0.3 mmol) was dissolved in EtOAc, and 5% Pd/C (270 mg) was added. The reaction vessel was set up at 1 atm of H2 and shaken at room temperature for 50 min. Reaction mixture was filtered through celite and evaporated. Product was precipitated with petroleum ether yielding pure 10 (0.184 g, 0.23 mmol, 75 %). HRMS calculated for

+ 1 C45H56N2O9Si m/z (M) 795.3677; found 795.6108. H NMR (400 MHz, CDCl3) δ 7.04-7.76 (m,

112 12H, Fmoc-ArH, Dmab-ArH), 5.71 (d, 1H, Hα), 5.10-5.13 (dd, 2 H, Dmab-CH2), 4.98 (d, 1H, Hβ),

4.33 (m, 2H, Fmoc-CH2), 4.23 (t, 1H, Fmoc-CH), 2.49 (s, 2H, Dmab-CH2), 2.41 (s, 2H, Dmab-

CH2), 2.06 (d, 2H, Dmab-CH2 isopropyl), 1.81 (m, 1H, Dmab-CH isopropyl), 1.06 (d, 6H, 2 Dmab-

CH3), 0.90 (s, 9H, TBDMS-tBu), 0.75 (s, 3H, Dmab-CH3 isopropyl), 0.73 (s, 3H, Dmab-CH3

13 isopropyl), 0.14 (s, 3H, TBDMS-CH3), 0.01 (s, 3H, TBDMS-CH3) ppm; C NMR (100 MHz,

CDCl3) δ 203.2, 197.0, 176.8, 176.5, 173.3, 169.2, 167.4, 156.3, 144.3, 143.7, 143.6, 141.5,

141.2, 136.9, 134.4, 129.2, 127.7, 127.6, 127.1, 126.7, 125.2, 125.2, 124.7, 120.0, 107.6, 71.9,

67.7, 66.8, 61.4, 57.6, 53.5, 49.3, 46. 9, 39.0, 38.5, 30.0, 29.6, 28.2, 25.6, 22.7, 22.6, 20.7, 18.2, -

4.8, -5.7 ppm.

4.3 Synthesis of Fmoc-SPPS-compatible L-threo-phenylserine and L-hydroxyleucine building blocks

4.3.1 Synthesis of Fmoc-L-tPhSer-OH

General procedure for D,L-threo-PhSer enantioresolution: Commercially available threo-

PhSer racemic mixture (D,L-tPhSer, 2 g, 11 mmol) 43 was treated with 2 eq of acetic anhydride in acetic acid and refluxed for 4-5 h. Solvent was evaporated and the remaining crude residue 44 redissolved in 1mM CoCl2·6H2O in water, final concentration of amino acid was 0.1 M. pH of the solution was adjusted to 8 with LiOH and enzyme was added so that the final concentration of enzyme was 16.6 U/mmol of L-tPhSer. Reaction mixture was stirred at room temperature overnight and monitored via TLC. Occasionally, LiOH was added to maintain pH 8. Upon reaction completion the reaction mixture was acidified to pH 5, activated carbon was added and the reaction was heated to 60C. After 15 min the reaction mixture was filtered, filtrate acidified to pH

1.5 and extracted with EtOAc. Aqueous layer, containing deacetylated L-tPhSer 43a was evaporated and subjected to ion-exchange chromatography using Dowex 50, fractions were combined and evaporated (0.3 g, 1.65 mmol, 30%).212 Sample of the remaining residue was

388 derivatized with Marfey’s reagent, injected on RP HPLC and presence of only L-tPhSer enantiomer was confirmed (data not shown).

113 L-tPhSer 43a was redissolved in acetone:water mixture (2:1) to which 1.1 eq of Fmoc-OSu

390 (0.612 g, 1.815 mmol) and up to 4 eq of NaHCO3 (0.554 g, 6.6 mmol) were added. Reaction mixture was stirred at room temperature for 3-5 h and monitored via TLC. Upon completion, the reaction mixture was acidified to pH 3 and extracted several times with EtOAc. Organic layer was evaporated and the residue purified using column chromatography and toluene:EtOAc:AcOH

10:5:1 solvent system to obtain product, Fmoc-L-tPhSer-OH 45 (0.554 g, 1.375 mmol), as white solid in total yield of approx. 25% starting from the racemic mixture, Scheme 8 (MALDI-TOF MS calculated [M+Na]+=425.1239, observed [M+Na]+=425.9919).

O O HN OH

OH O O O H N HN 2 OH OH (CH3CO)2O Acylase I OH OH O AcOH H2N O O OH O O O O N O OH HN 43 44 O OH

NaHCO OH 43a 3 45

Scheme 8. Enantioresolution and Nα-Fmoc protection of tPhSer

4.3.2 Synthesis of Fmoc-L-HyLeu(TBDMS)-OH

Commercially available L-HyLeu 46 (0.5 g,, 3.4 mmol) was suspended in acetonitrile and 1 eq of 1,8-diazabicycloundec-7-ene (DBU, 0.508 mL, 3.4 mmol) was added to obtain pH 9. Reaction was heated to 50C and both tert-butyldimethylsilyl chloride (TBDMS-Cl, 0.256g, 1.7 mmol) and

DBU were added in 0.5 eq portions until the reaction went to completion (approx. 4 h) as observed by TLC.392 Upon reaction completion the solvent was evaporated, residue redissolved in water, acidified to pH 3 and extracted several times with EtOAc. Organic layers were combined and evaporated. Crude product 47 was used for the next step, protection of Nα with Fmoc protecting group. Crude product 47 was dissolved in acetone:water mixture (2:1 v/v) to which 1.1 eq (based on 100% theoretical yield) of Fmoc-OSu (1.26 g, 3.74 mmol) and up to 4 eq of

NaHCO3 (1.142 g, 13.6 mmol) were added. Reaction mixture was stirred at room temperature for 114 3-5 h and monitored via TLC.390 Upon completion the reaction mixture was acidified to pH 3, extracted several times with ethyl-acetate. Organic layer was evaporated and the residue purified using column chromatography and toluene:EtOAc:AcOH 10:5:1 solvent system to obtain product,

Fmoc-L-HyLeu(TBDMS)-OH 48, as yellow solid in total yield of 77% over two steps (1.265 g, 2.62 mmol), Scheme 9 (MALDI-TOF MS calculated [M+Na]+=505.2260, observed [M+H]+=505.9231).

O O O O O O N Si Cl H N H N O O 2 OH 2 OH O O HN OH DBU O NaHCO3 OH Si O 46 47 48 Si

Scheme 9. Orthogonal protection of L-HyLeu

4.4 Optimization of Fmoc-SPPS approach towards lysobactin

Our synthetic approach towards lysobactin 32 and its analogs is based on the utilization of orthogonally protected tHyAsp,238 and Fmoc methodology for solid-phase synthesis of cyclic lipodepsipeptides, previously developed in our laboratory.224

4.4.1 Synthesis of lysobactin analog 49 and antimicrobial activity assessment

In order to optimize the SPPS, we initially used the racemic mixture of orthogonally protected tHyAsp instead of L-tHyAsp, Figure 43.

The synthetic approach towards 49 is depicted in Scheme 10. Free -carboxyl group of D,L-

42 (from Scheme 6) had to first be activated as pentafluorophenyl ester (Pfp) to facilitate the coupling to the resin. The efficiency of the coupling (90%) was determined by a

246 spectrophotometric Fmoc-quantitation test. The linear precursor peptide (Boc-D-Leu-Leu-L- tPhSer-L-HyLeu-Leu-D-Arg-Ile-L-aThr-Gly-D,L-tHyAsp) was assembled using standard Fmoc- chemistry.

115 H2N NH HN D-Arg6

L-Leu5 L-HyLeu4 O L-Ile7 HN NH HN O O L-Leu2 HO O HN NH L-a Thr8 O O OH O H H2N N HO N NH H O O H 1 O N Gly9 D-Leu O O N H NH2 HO O L-t PhSer3 49 D,L-t HyAsn10 L-Ser11

10 10 Figure 43. Structure of lysobactin analog 49 with D,L-tHyAsn instead of L-tHyAsn

Scheme 10. Synthetic approach toward lysobactin analog 49 116 The ester bond was formed between Fmoc-Ser(tBu)-OH and resin bound L-tPhSer3 using

N,N'-diisopropylcarbodiimide/4-dimethylaminopyridine (DIC/DMAP) in DCM as previously described.224 Subsequently, the Dmab protecting group was removed from the α-carboxyl group of HyAsp10 with the use of 2% hydrazine in DMF. Hydrazine also partially removed Fmoc from

Ser11. Removal of Fmoc was completed using 20% piperidine in DMF. Linear peptide was cyclized using PyBOP coupling reagent and cleaved from the resin using TFA:thioanisole:H2O

95:2.5:2.5 (v/v/v) cleavage cocktails. The crude product was purified by RP HPLC.

In addition to the desired product 49 (MALDI-TOF MS calculated [M+H]+=1275.72, observed

[M+H]+=1274.47), a significant amount of a side product was observed (MALDI-TOF MS

[M+H]+=1257.17) which most likely corresponds to the dehydration of HyAsp 50, Figures 44 and

45. Since this side product is not observed during monitoring progress of the synthesis, we assume that the dehydration occurs during the cleavage of the cyclized peptide from the solid support under acidic conditions (TFA:thioanisole:H2O=95:2.5:2.5 v/v/v).

H2N NH HN

O HN NH HN O HO O O HN NH O O OH O H H2N N HO N NH O H H O O N O O N H NH2 O 50

Figure 44. Proposed structure of the side product 50 obtained during synthesis of lysobactin 49

117 3000 UV2000-220nm P4000 - Solvent: B KB2 final 5%DMSO in MeOH 100ul 05152009 100 2800 a)

90 2600

2400 80

2200 70 2000

1800 60

1600

50 %

mAU 1400 40 1200

1000 30

800 20 600

10 400

200 0

0 -10 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 Minutes

1500 1500 3500 3500 UV2000-220nm UV2000-220nm KB3 run 2 fr 5 100ul of solution used for CD H2O 06032009 KB2 run1 fr6 0.59mg in 200ul det conc for assay 5ul 05192009

1400 1400 3250 3250

1300 1300 b) 3000 c) 3000

1200 1200 2750 2750

1100 1100

2500 2500

1000 1000

2250 2250

900 900

Rt-25.2 min 2000 2000 800 800

1750 1750

700 700

mAU mAU

mAU mAU

1500 1500 600 600 Rt-26.4 min 500 500 1250 1250

400 400 1000 1000

300 300 750 750

200 200 500 500

100 100

250 250

0 0

0 0

-100 -100 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 Minutes Minutes Voyager Spec #1[BP = 1274.5, 30162] Voyager Spec #1[BP = 1257.1, 44650] 1274.4678 3.0E+4 100 1257.1739 100 4.5E+4

90 d) 1274.47 90 e) 1257.17 80 80

70 70

60 60

50 50

% Intensity % % Intensity % 40 40 1259.1641 1276.4547

30 30

1240.7509 20 550.7269 20 1296.4254 1176.2306 10 1056.3928 10 1312.3499 1279.1316 551.1125 971.9560 1172.1987 1172.3990 1314.3930 1483.4636 0 0 0 0 499.0 899.4 1299.8 1700.2 2100.6 2501.0 499.0 899.4 1299.8 1700.2 2100.6 2501.0 Mass (m/z) Mass (m/z)

Figure 45. Isolation and characterization of products obtained from synthesis of 49; a) RP HPLC trace of the crude product; b) RP HPLC trace of isolated lysobactin analog 1; c) RP HPLC trace of isolated side product; RP HPLC conditions: 100% A 5 min, 0-100% B, 45 min; A - 0.1% TFA in water, B - 0.1% TFA in ACN; d) MALDI-TOF MS of lysobactin analog; e) MALDI-TOF MS of the side product 118 The assessment of antibacterial activity was done in 96 well microtiter plates using a standard micro dilution broth method as described above (see section 2.8.6).296 The lysobactin analog 49 showed MIC of 8 μg/mL against all tested Gram-positive bacterial strains, Table 8. On another hand, the side product 50 had significantly higher MIC values.

Table 8. Antimicrobial activity of lysobactin analogs 49 and 50

MIC* (μg/mL) Microorganism 49 50

S. aureus ATCC 33591 8 64

S. aureus Mu50 ATCC 700699 8 > 80

S. epidermidis ATCC 27626 8 > 32

S. aureus ATCC 6538P 8 > 32 *MIC=minimum inhibitory concentration

4.4.2 Synthesis of lysobactin analog 51 and antimicrobial activity assesment

In order to optimize the synthetic methodology prior to using valuable L-tHyAsp derivative, and to also reveal whether L-tHyAsp is necessary for the activity of the peptide, we decided to

10 10 use lysobactin analog which contains Asn instead of L-tHyAsn as a model, Figure 46.

The synthetic methodology followed the approach outlined in Scheme 10 with several modifications that significantly improved the purity and the yield of the final product 51. During the synthesis of 49 we observed that the partial loss of the ester bond occurs upon treatment of peptidyl-resin with 2% hydrazine necessary for the removal of Dmab protecting group from Asp10

α-carboxyl group. Hence, we optimized the amount of hydrazine to achieve complete removal of

Dmab protecting group without losing the ester moiety. Treatment of the peptidyl-resin 2 x 1 min with only 0.1% of hydrazine in DMF gave us the best results. Furthermore, to form the ester bond

3 with resin bound L-tPhSer , Fmoc-Ser(tBu)-OH was substituted with Aloc-Ser(tBu)-OH thus avoiding the treatment of peptidyl-resin with 20% piperidine in DMF subsequent to ester bond

119 formation. The cyclization of the linear precursor and subsequent cleavage from the solid support were performed as described above and the target peptide 51 was identified as the major product, Figure 47.

10 10 Figure 46. Structure of lysobactin analog 51 with Asn instead of L-tHyAsn

*

Figure 47. RP HPLC analysis of crude lysobactin analog 51. Asterisk denotes the peak corresponding to the desired product. Inserted figure: MALDI-TOF MS analysis of the main product (m/z calculated: 1259.7238; found: [M+H]+ 1260.5865, [M+Na]+ 1282.5497, [M+K]+ 1298.5214)

120 Lysobactin analog 51 was tested against methicillin-resistant S. aureus (MRSA) ATCC 33591 and showed MICs of 2 μg/mL. For comparison, MICs of the natural product against a panel of

Gram-positive clinical isolates was reported to be in the range 0.1 to 0.8 μg/mL.200,203 The difference in the observed MIC values may be rationalized by the fact that a different method was used. The method reported in the literature was disk diffusion method while we used a standardized CLSI approved broth microdilution method. Also, the effect of substituting L- tHyAsn10 with Asn10 can have an effect as well.

4.4.3 The challenges of the on-resin ester bond formation

The data previously obtained in our laboratory suggested that Tenta Gel S RAM resin from

Advanced ChemTech gives the best results for ester coupling.224 However, we have observed that the batch-to-batch properties of the solid support may differ. In order to evaluate the suitability of different resins for ester bond formation, several polyethylene glycol (PEG) based resins were used, including NovaPEG Rink Amide (Novabiochem), Tenta Gel S RAM (Chem-

Impex), Tenta Gel S RAM (Acros), Novagel Rink Amide (Novabiochem) and Rink Amide PEGA

(Novabiochem). We chose to test only PEG-based resins because of our previous observation that polystyrene based resins do not support ester bond formation.224

1 2 3 4 For testing of ester bond formation the tetrapeptide D-Leu -L-tPhSer -Leu -Leu was used as a model due to the similarity to the original lysobactin 32 sequence (L-HyLeu is substituted for

Leu3), Scheme 11.

Scheme 11. Synthesis of a model pentapeptide for testing of on-resin ester bond formation

121 Synthesis on all resins were performed using the same conditions; 4 eq of DIC and Aloc-

Ser(tBu)-OH and 0.2 eq of DMAP, in DCM, shaking for 24 h, followed by cleavage from the resin and RP HPLC and MALDI-TOF MS analysis, Figure 48. Out of five resins tested, only Rink Amide

PEGA gave satisfactory results. The remaining four resins showed only partial formation of the ester bond. Interestingly, the Rink Amide PEGA resin comes from the manufacturer swollen in

EtOH, however, if the resin is washed with with DCM and dried, it seems to change its properties and the re-swelling does not give satisfactory results.

900 UV2000-220nm 900 UV2000-220nm 900 ACROS 4 h ester coupling 10122009 900 NovaPEG Alloc-Ser coupling 09212009

800 a) Tenta Gel S RAM (Acros) 800 800 b) NovaPEG Rink Amide 800

700 700 700 (Novabiochem) 700 Ester not formed 600 600 600 600

500 Ester formed 500 500 Ester formed 500

Ester not formed

mAU

mAU mAU 400 400 mAU 400 400

300 300 300 300

200 200 200 200

100 100 100 100

0 0 0 0

23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 29.0 23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 29.0 Minutes Minutes

UV2000-220nm UV2000-220nm CHEM-IMPEX Alloc-Ser coupling 09222009001 900 900 NOVAGEL ester formation 10212009001 900 900

800 800 d) Novagel Rink Amide (Novabiochem) c) Tenta Gel S RAM (Chem-Impex) 800 800

700 700 Ester not formed 700 Ester not formed 700 600 600 600 600

500 500

500 Ester formed 500

mAU

mAU mAU 400 Ester formed 400 mAU 400 400

300 300 300 300

200 200 200 200

100 100 100 100

0 0 0 0

23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 29.0 23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 29.0 Minutes Minutes UV2000-220nm 900 PEGA ester formation 10262009 900

800 e) Rink Amide PEGA (Novabiochem) 800

700 700

600 Ester formed 600

500 500 mAU mAU 400 400

300 300

200 200

100 100

0 0

23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 29.0 Minutes

Figure 48. Magnified RP HPLC traces obtained after coupling of Aloc-Ser(tBu)-OH to tetrapeptide

D-Leu-L-tPhSer-Leu-Leu on different resins; RP-HPLC conditions: 0→100% B over 45 min; A - 0.1% TFA in water, B - 0.1% TFA in ACN 122 4.5 Discussion and conclusions

Egner and Bradley212 were the first to report the synthesis of a lysobactin analog on solid support. Their approach involved side-chain attachment of the first residue (Asp10), followed by

N→C coupling of H2N-Ser-OAllyl to the α-carboxyl group of the resin-bound Asp. Standard C→N extension of the linear peptide through the Nα-amino group of Asp gave the linear peptidyl precursor which was cyclized in the final step through the ester bond. Recently, VanNieuwenhze reported another approach where the linear peptidyl precursor was assembeled on the solid support with final two steps, macrolactamization and cleavage of protecting groups, performed in solution.215

We have used a synthetic approach for the synthesis of lysobactin analogs, similar to the one from Egner and Bradley, that utilizes the side-chain attachment of the first residue, followed by the formation of the linear peptidyl precursor in standard C→N fashion, and on-resin ester bond

3 11 formation between L-tPhSer and Ser . In the final step, the peptide was cyclized through amide bond between α-carboxyl group of Ser11 and Nα–amino group of resin bound (Hy)Asp10 followed by cleavage from the solid support.

A crucial step in the synthesis is attachment of the first amino acid, especially when HyAsp was used. If the α-carboxyl group of HyAsp was not activated as a Pfp ester, the reaction proceeded with low yields (< 20% after 24 h). Furthermore, treatment of the resin-bound depsipeptide precursor with hydrazine can result in complete hydrolysis of the ester bond unless very low concentrations of hydrazine are used (≤ 0.2% hydrazine in DMF). Also, during peptide cleavage from the solid support, we observed significant amount of dehydration of HyAsp indicating that the time of the cleavage reaction should be decreased and potentially the protecting group for the side hydroxyl of the HyAsp revisited. Interestingly, the byproduct 50 showed significantly lower antimicrobial activity compared to compound 49 which contains intact

10 10 HyAsp (D,L-mixture). We also prepared an analog containing Asp instead of HyAsp which showed to be very potent against Gram-positive bacteria.

123 Furthermore, during the course of synthesis optimization, we found that the choice of the solid support plays a significant role in the final outcome of the synthesis. Hovewer, very few commercially available resins support the on-resin esterification reaction. Among many resins tested, TentaGer S RAM resin was proven to produce tha highest yield and purity of desired cyclic depsipeptide. Despite the difficulties, we have established a succesful solid-phase synthetic protocol for preparation of lysobactin analogs, oppening a possibility for their diversification using a combinatorial chemistry approach.

124 CHAPTER 5

APPENDIX

5.1 RP HPLC and MALDI-TOF MS spectra of synthetic fusaricidin/LI-F analogs

Voyager Spec #1=>BC=>NF0.7[BP = 650.7, 23466] 650.6558 100 2.3E+4 90 80 70 60

50 666.6310

40 % Intensity % 30 628.6735 20 668.6559 10 0 500 560 620 680 740 800 Mass (m/z)

Figure 49. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 1

125

Voyager Spec #1=>BC=>SM 9[BP = 650.7, 4297] 650.6790 100 4297.2 90 80

70 650.4244 60 50 40

% Intensity % 666.3824 30 628.7007 20 10 0 500 560 620 680 740 800 Mass (m/z)

Figure 50. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 2

126

Voyager Spec #1[BP = 783.8, 6641] 783.7851 100 6641.2 90 80 70 60 50

40 % Intensity % 30 20 10 805.8465 0 600 720 840 960 1080 1200 Mass (m/z)

Figure 51. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 3

127

Voyager Spec #1=>BC=>SM 5=>SM 5=>SM 9[BP = 783.2, 20234] 783.2229 100 2.0E+4 90 80 70 805.1652 60 50 821.1249

40 % Intensity % 30 20 10 0 600 720 840 960 1080 1200 Mass (m/z)

Figure 52. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 4

128

Voyager Spec #1[BP = 825.6, 14131] 825.5641 100 1.4E+4 90 80 70 60 50

40 847.1996 % Intensity % 30 20 863.1735 10 0 600 720 840 960 1080 1200 Mass (m/z)

Figure 53. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 5

129

Voyager Spec #1[BP = 826.0, 13230] 825.9749 100 1.3E+4 90 80 70 60 50

40 % Intensity % 30 20 847.9485 10 0 600 720 840 960 1080 1200 Mass (m/z)

Figure 54. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 6

130 Voyager Spec #1[BP = 795.8, 16200] 795.8084 100 1.6E+4 90 80 70 817.8027 60 50

40 % Intensity % 30 20 10 0 500 640 780 920 1060 1200 Mass (m/z)

Figure 55. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 7

131

Voyager Spec #1[BP = 798.1, 6047] 798.0692 100 6047.0 90 80 70 60 50

40 % Intensity % 30 820.0621 20 10 0 500 640 780 920 1060 1200 Mass (m/z)

Figure 56. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 8

132 Voyager Spec #1[BP = 797.6, 10675] 797.5884 100 1.1E+4 90 80 70 819.5709 60 50

40 % Intensity % 30 20 10 835.5355 0 500 640 780 920 1060 1200 Mass (m/z)

Figure 57. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 9

133 Voyager Spec #1[BP = 812.3, 23818] 812.3234 100 2.4E+4 90 80 70 60 50

40 % Intensity % 30 20 10 834.3278 0 600 720 840 960 1080 1200 Mass (m/z)

Figure 58. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 10

134

Voyager Spec #1[BP = 874.1, 37023] 874.1084 100 3.7E+4 90 80 70 60 50

40 % Intensity % 30 20 896.0902 10 0 499.0 699.4 899.8 1100.2 1300.6 1501.0 Mass (m/z)

Figure 59. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 11

135

Voyager Spec #1[BP = 890.3, 48222] 890.3375 100 4.8E+4 90 80 70 60 50

40 % Intensity % 30 912.3322 20 928.2991 10 0 600 720 840 960 1080 1200 Mass (m/z)

Figure 60. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 12

136

Voyager Spec #1[BP = 781.9, 24893] 781.8370 100 2.5E+4 90 80 70 803.8141 60 50

40 % Intensity % 30 20 819.7741 10 0 500 640 780 920 1060 1200 Mass (m/z)

Figure 61. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 13

137

Voyager Spec #1[BP = 810.8, 30623] 810.7949 100 3.1E+4 90 80 70 60 50

40 % Intensity % 30 832.7360 20 848.7080 10 0 600 740 880 1020 1160 1300 Mass (m/z)

Figure 62. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 14

138

Voyager Spec #1[BP = 782.9, 23700] 782.9501 100 2.4E+4 90 80 70 60 50

40 % Intensity % 30

20 804.9542 10 820.9247 0 500 640 780 920 1060 1200 Mass (m/z)

Figure 63. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 15

139

Voyager Spec #1[BP = 859.0, 31903] 859.0195 100 3.2E+4 90 80 70 60 50

40 881.0028 % Intensity % 30 896.9771 20 10 0 600 720 840 960 1080 1200 Mass (m/z)

Figure 64. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 16

140

Voyager Spec #1[BP = 875.1, 20521] 875.0820 100 2.1E+4 90 80 70 60 50

40 % Intensity % 30 20 10 897.0670 0 600 720 840 960 1080 1200 Mass (m/z)

Figure 65. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 17

141 Voyager Spec #1=>BC=>SM 5[BP = 844.1, 6328] 844.1160 100 6328.1 90 80 70 60 50

40 % Intensity % 30 20 10 866.0974 0 600 720 840 960 1080 1200 Mass (m/z)

Figure 66. RP HPLC trace (up) and MALDI-TOF spectrum (down) of control lipopeptide 18

142

Figure 67. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 19

143

Figure 68. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 29

144

Figure 69. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 30

145

Figure 70. RP HPLC trace (up) and MALDI-TOF spectrum (down) of analog 31

146 5.2 Hemolytic activity of fusaricidin/LI-F analogs

Figure 71. Hemolytic activity of all fusaricidin/LI-F analogs tested

147 5.3 Additional membrane depolarization data

Figure 72. Membrane depolarization of S. aureus with fusaricidin/LI-F analogs 11 and 16

148

Figure 73. Comparison of membrane depolarization data against S. aureus for gramicidin S at 20 μg/mL and fusaricidin/LI-F analogs 6, 14 and 18 at 48 μg/mL

149

Figure 74. Time- and dose-dependent membrane depolarization of A) S. aureus ATCC 29213; B) S. aureus Mu50 ATCC 700699 (VRSA); C) S. epidermidis ATCC 27626 (MRSE) with synthetic fusaricidin/LI-F analogs (performed at 37C)

150 5.4 Enantioresolution of D,L-tHyAsp

D,L-tHyAsp mixture (purchased from TCI America, Portland, OR) derivatizatized with Marfey’s reagent (Bhushan and Brückner 2004) showed presence of these two stereoisomers in 1:1 ratio,

Figure S1a. Enantioresolution of D,L-tHyAsp was performed using previously described method by Okai et al. (1967). L-Lys•HCl (4.57 g, 25 mmol) was dissolved in H2O (5-10 mL) and passed through previously activated Dowex 50 (H+ form) ion exchange column to obtain Lys in a salt-free form (eluent 2M NH4OH). D,L-tHyAsp (3.72 g, 25 mmol) was dissolved in water (30 mL) with gentle heating and Lys residue was added to the amino acid. Once everything was in the solution, MeOH (25 mL) was added in small portions. The solution was left at room temperature to cool down and then left at 4°C overnight to crystallize. Precipitated crystals were filtered, washed with a small amount of cold methanol and recrystallized from water (20 mL) with addition of methanol (16 mL). Crystals were filtered off (3 g) and applied on a previously activated Dowex

1 (acetate form) ion exchange column. Once Lys is eluted from the column using 0.5 M AcOH, column is treated with 2 M AcOH to elute pure L-tHyAsp. Fractions containing pure L-tHyAsp were combined and eluent evaporated yielding 1.4 g (75%) of the final product. The original mother liquor was again left at 4°C overnight and formed crystals containing both D- and L-tHyAsp were removed by filtration. MeOH from the mother liquor was evaporated and the aqueous part was treated as described above in order to obtain pure D-tHyAsp (1.4 g, 75%). Enantiomeric composition of each sample was determined by derivatization with Marfey’s reagent followed by

RP HPLC analysis, Figure 41.

5.5 General method for derivatization with Marfey’s reagent

Sample of amino acid (5 μmol) is dissolved in 100 μL of water. 2 mg of FDAA (Marfey’s reagent) is dissolved in 200 μL of acetone (1% solution). Solution of Marfey’s reagent is added to the dissolved amino acid followed by 40 μL of 1 M NaHCO3. Reaction is set up at 40°C for 1 h after which is quenched by addition of 20 μL of 2 M HCl. Small aliquot is taken, diluted with water and injected on RP HPLC. Elution method 100% A for 5 min followed by linear gradient of 0-

151 100% B over 40 min where A is 0.1% TFA in H2O and B is 0.1% TFA in ACN. Eluting products were detected by UV at 220 nm and 340 nm.

5.6 Specific optical rotation calculation

Optical rotations α were measured at 25ºC and 589 nm (Na-D). Specific optical rotations [α]D were calculated using equation below.

[] = specific optical 25 = optical rotation 25  []  rotationc = concentration in  cl l = optical pathlength in g/mL  = wavelength dm

152 5.7 Selected NMR spectra of L-tHyAsp derivatives

O OH O O

O N O H O O

Figure 75. 1H (up) and 13C (down) NMR spectra of 36

153 O OH O O

O N O H O Si

Figure 76. 1H (up) and 13C (down) NMR spectra of 40

154 O O O NH O O HN O O O Si OH

Figure 77. 1H (up) and 13C (down) NMR spectra of 42

155 5.8 Assessment of epimerization upon introduction of Dmab protecting group

In order to assess the level of epimerization upon introduction of Dmab protecting group by

RP HPLC analysis, both Fmoc and Dmab protecting groups were removed from 42 using 4% hydrazine in DMF for 20 min. Figure S9. RP HPLC fractions were analyzed by MALDI-TOF MS.

Molecular ion for the peak designated with asterisk corresponds to the expected HyAsp derivative. None of the other isolated peaks showed correct molecular ion mass.

*

Figure 78. RP HPLC trace linear gradient 0-45 min 2→98% ACN with 0.08% TFA; + + molecular mass C17H28NO5Si calculated m/z (M + H) 354.1731, found (M + H) 354.6248.

COSY 2D NMR of compound 41, Figure 79, shows presence of only one Hand

Hindicating no detectable epimerization during introduction of Dmab protecting group.

156

Figure 79. COSY 2D NMR spectrum of 41

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