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Structural Characterization, Biological Evaluation and Synthesis of Novel Secondary Metabolites from Guamanian Marine Cyanobacteria Lyngbya Spp.

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Title:
Structural Characterization, Biological Evaluation and Synthesis of Novel Secondary Metabolites from Guamanian Marine Cyanobacteria Lyngbya Spp.
Creator:
Montaser, Rana A
Place of Publication:
[Gainesville, Fla.]
Publisher:
University of Florida
Publication Date:
Language:
english

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Pharmaceutical Sciences
Medicinal Chemistry
Committee Chair:
Luesch, Hendrik
Committee Members:
James, Margaret O
Sloan, Kenneth B
Edison, Arthur Scott
Graduation Date:
5/4/2013

Subjects

Subjects / Keywords:
Amides ( jstor )
Esters ( jstor )
Fatty acids ( jstor )
Infrared spectrum ( jstor )
Magnetic resonance spectroscopy ( jstor )
Mass spectroscopy ( jstor )
Metabolites ( jstor )
Rotational spectra ( jstor )
Solvents ( jstor )
Spectral correlation ( jstor )
Medicinal Chemistry -- Dissertations, Academic -- UF
Genre:
Electronic Thesis or Dissertation
bibliography ( marcgt )
theses ( marcgt )
Pharmaceutical Sciences thesis, Ph.D.

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 2013.
Local:
Adviser: Luesch, Hendrik.
Electronic Access:
INACCESSIBLE UNTIL 2015-05-31
Statement of Responsibility:
by Rana A Montaser.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
5/31/2015
Classification:
LD1780 2013 ( lcc )

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1 STRUCTURAL CHARACTERIZATION, BIOLOGICAL EVALUATION AND SYNTHESIS OF NOVEL SECONDARY METABOLITES FROM GUAMANIAN MARINE CYANOBACTERIA LYNGBYA SPP By RANA MONTASER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013

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2 2013 Rana Montaser

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3 To my parents, my husband and my daughter.

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4 ACKNOWLEDGM ENTS First, I would like to express my greatest gratitude to my advisor Dr. Hendrik Luesch for giving me the opportunity to learn and explore the field of marine natural products in his reputable lab, and for his continuous guidance, teaching, and support throughout my PhD years My experience in his lab has been a lifetime experience that fostered my interest in this research field and helped me clarify my career goals For the rest of my career, I will always be indebted to him for his mentorship. I would also like to thank my committee members Dr. Kenn eth Sloan, Dr. Margaret James and Dr. Arthur Edison for their help and their precious time and comments I would like to thank our collaborator Dr. Valerie Paul from the Smithsonian Marine Station, for pro vid ing us with the cyanobacterial samples and for her valuable insights and discussions which enriched the projects I would also like to thank Dr. Khalil Abboud, the director of the X ray facilities at the Department of Chemistry at the University of Flor ida for his helpful remarks regarding crystallization and for the analysis of the crystal structures. In addition, I would like to thank James Rocca from the AMRIS facility at the McKnight Brain Institute, for his assistance with the NMR experiments and for all the helpful discussions. I am also grateful for all the present and former members and colleagues in the Luesch group, Dr. Jason Kwan, Dr. Susan Matthew, Dr. Ranjala Ratnayake, Dr. Qi Yin Chen, Dr. Yanxia Liu, Dr. Wei Zhang, Dr. Rui Wang, Lilibeth Salvador Kamolrat Metavarayuth, Michelle Bousquet Fatma Al Awadhi and Weijing Cai for their generous assistance during the past years. Their help and guidance have been invaluable. Special thanks go to all my family for their continuous encouragement and support, specially my parents and my brothers. My parents have been a constant

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5 source of support that was always pushing me forward and giving me the motivation and strength to achieve my goals Above all, my deepest expression of appreciation goes to my wonderful husband Mohamed for his indispensable love, support and encouragement, and for continuously believing in me. Without him by my side, I wouldn t have been able to make it this far and face the challenges of multitasking as a graduate student, a w ife and a mother. He is the most amazing husband and friend, and his incredible support is one of the major pillars of my success Finally, I would like to thank my little angel my daughter Malak for allowing me to share her world of fairy tales which help ed to relieve the graduate school stress and get me back on track Her smile s and giggles have always been and will always be my sunshine and the best remedy for all my worries

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 16 ABSTRACT ................................................................................................................... 20 CHAPTER 1 GENERAL INTRODUCTION .................................................................................. 22 Oceans Harbor Unique Bioactive Chemical Entities ............................................... 22 Marine Drugs on the Market ................................................................................... 24 Marine Cyanobacteria: A Prolific Source of Bioactive Compounds ......................... 26 Lyngbya spp. Have a Characteristic Chemical Profile ............................................ 27 Drug Discovery from Lyngbya Samples from Guam ............................................... 28 Isolation of Marine Cyanobacterial Secondar y Metabolites .............................. 28 Chemical Characterization of Marine Cyanobacterial Secondary Metabolites 2 9 Biological Characterization of Mari ne Cyanobacterial Secondary Metabolites .................................................................................................... 30 Synthesis of Marine Cyanobacterial Secondary Metabolites ............................ 32 2 PITIPROLAMIDE, A PROLIN E RICH DOLASTATIN 16 ANALOGUE FROM THE MARINE CYANOBACTERIUM LYNGBYA MAJUSCULA FROM GUAM ....... 39 Introduction ............................................................................................................. 39 Isolation and Struct ure Determination ..................................................................... 40 X ray Crystallography and Stereochemical Assignment ......................................... 41 Related Analogues .................................................................................................. 42 Biological Activity Evaluation ................................................................................... 43 Experimental Section .............................................................................................. 44 General Experimental Procedures ................................................................... 44 Extraction and Isolation .................................................................................... 45 Acid Hydrolysis and Enantioselective Amino Acid Analysis by HPLC/MS ........ 46 X ray Crystallography ....................................................................................... 46 Cell Viability Assays ......................................................................................... 47 Antibacterial Assays ......................................................................................... 47 3 PITIPEPTOLIDES C F, ANTIMYCOBACTERIAL CYCLODEPSIPEPTIDES FROM THE MARINE CYANOBACTERIUM LYNGBYA MAJUSCULA FROM GUAM ..................................................................................................................... 65

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7 Introduction ............................................................................................................. 65 Isolation and Structure Determination ..................................................................... 65 Related Analogues ................................................................................................. 67 Biological A ctivity Evaluation .................................................................................. 69 Towards Target Identification of Pitipeptolide A ...................................................... 71 Design of Synthetic Probes to Investigate Mechanism of Action ...................... 72 Bioactivity Evaluation of the Synthetic Probes .................................................. 73 Conclusion .............................................................................................................. 74 Experimental Section .............................................................................................. 75 General Experimental Procedures ................................................................... 75 Marine Cyanobacterial Sample ........................................................................ 75 Extraction and Isolation .................................................................................... 76 Acid Hydrolysis and Enantioselective Analysis ................................................. 78 Cell Viabilit y Assay ........................................................................................... 79 Disc Diffusion Assay ......................................................................................... 79 Synthetic Procedures ....................................................................................... 80 4 MAR INE CYANOBACTERIAL FATTY ACID AMIDES ACTING ON CANNABINOID RECEPTORS ................................................................................ 99 Introduction ............................................................................................................. 99 Isolation and Structure Determination ................................................................... 101 Biological Activity Evaluation ................................................................................ 103 Conclusion ............................................................................................................ 106 Experimental Procedures ...................................................................................... 107 General Experimental Procedures ................................................................. 107 Extraction and Isolation .................................................................................. 107 Jones Oxidation and Enantioselective Amino Acid Analysis by HPLC/MS ..... 108 Cannabinoid CB1/CB2 Receptor Binding Assays ............................................ 109 cAMP Functional Assay .................................................................................. 110 FAAH Inhibitor Enzyme Assay ....................................................................... 110 NO Assay ....................................................................................................... 110 Cell Viability Assays ....................................................................................... 111 5 CHARACTERIZATION AND SYNTHESIS OF BIOACTIVE LIPIDS FROM A GUAMANIAN MARINE CYANOBACTERIUM ...................................................... 117 Introduction ........................................................................................................... 117 Isolation and Structure Determination ................................................................... 118 Synthesis of the Chlorinated Ester ........................................................................ 120 Biological Activity Evaluation ................................................................................ 124 Experimental Section ............................................................................................ 126 General Experimental Procedures ................................................................. 126 Extraction and Isolation .................................................................................. 127 Enantioselective Analysis ............................................................................... 128 Synthetic Procedures ..................................................................................... 128 Pyocyanin and Elastase Quantitation in Pseudomonas aeruginosa ............... 138

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8 RT qPCR in Pseudomonas aeruginosa ......................................................... 139 THP 1 Cell Culture and RT qPCR .................................................................. 141 6 CONCLUSIONS AND FUTURE DIRECTIONS .................................................... 151 APPENDIX A NMR DATA OF ISOLATED MARINE CYANOBACTERIAL SECONDARY METABOLITES ..................................................................................................... 154 B NMR DATA OF SYNTHETIC COMPOUNDS ....................................................... 206 LIST OF REFERENCES ............................................................................................. 249 BIOGRAPHICAL SKETCH .......................................................................................... 258

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9 LIST OF TABLES Table page 1 1 Marine drugs on the market ................................................................................ 33 2 1 NMR spectroscopic data for pitiprolamide ( 2 1 ) in benzened6 ........................... 50 2 2 Crystal data and struct ure refinement for pitiprolamide ( 2 1 ). ............................. 53 2 3 Atomic coordinates and equivalent is otropic displacement parameters for pitiprolamide ( 2 1 ). .............................................................................................. 54 2 4 Hydrogen bonds for pitiprolamide ( 2 1 ) .............................................................. 57 3 1 NMR spectroscopic data for pitipeptolides C ( 3 3 ) and D ( 3 4 ) in CDCl3 ............ 87 3 2 NMR spectroscopic data for pitipeptolides E ( 3 5 ) and F ( 3 6 ) in CDCl3 ............ 89 3 3 Cytotoxicity in cancer cells and antimycobacterial activities of pitipeptolides A F ( 3 1 3 6 ) ..................................................................................................... 91 3 4 Antimycobacterial activities of the semi synthetic probes piti A biotin ( 3 7 ) and piti A fluor ( 3 8 ) ............................................................................................ 92 4 1 NMR spectroscopic d ata for s erinolamide B ( 4 1 ) in CDCl3 .............................. 112 4 2 Cannabinoid receptors affinities ( Ki) and consequent functional effects on cAMP accumulation (EC50) by serinolamide B ( 4 1 ) and malyngamide B ( 4 2 ) 113 5 1 NMR spectroscopic data for 5methylene decanoic acid ( 5 1 ) and its chlorinated ester ( 5 2 ) in CDCl3 ........................................................................ 142

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10 LIST OF FIGURES Figure page 1 1 Marine drugs on the market. ............................................................................... 34 1 2 Examples for bioactive marine natural products used as pharmacological probes. ............................................................................................................... 35 1 3 Examples for bioactive secondary metabolites reported from marine cyanobacteria. .................................................................................................... 36 1 4 Schematic summary of the chemical and biologic al characterization of bioactive marine secondary metabolites through NMR guided fractionation. ..... 37 2 1 Structures of pitiprolamide ( 2 1 ) and its closely related analogues dolastatin 16 and ho modolastatin 16. ................................................................................. 59 2 2 Important HMBC correlations for sequence identification for pitiprolamide ( 2 1 ). ....................................................................................................................... 60 2 3 MS/MS fragmentat ion pattern for pitiprolamide ( 2 1 ). ......................................... 61 2 4 1H NMR spectrum for pitiprolamide ( 2 1 ) in benzened6 at 600 MHz at 25 C shows conformational changes over time. .......................................................... 62 2 5 Crystal structure of pitiprolamide ( 2 1 ) shows intraand intermolecular hydrogen bonding. .............................................................................................. 63 2 6 Residue sequences for dolastatin 16, homodolastatin 16, k ulokekahilide1 and pitiprolamide ( 2 1 ). ....................................................................................... 64 3 1 Structures of pitipeptolides A F ( 3 1 3 6 ). ......................................................... 93 3 2 ESI MS/MS fragmentation patterns of pitipeptolides C F ( 3 3 3 6 ). .................. 94 3 3 Structural comparison between pitipeptolide A ( 3 1 ) and antanapeptin A. ......... 95 3 4 Struc tures of the probes piti A biotin ( 3 7 ) and piti A fluor ( 3 8 ). ........................ 96 3 5 Synthetic scheme to obtain piti A biotin ( 3 7 ). .................................................... 97 3 6 Sy nthetic reaction to obtain piti A fluor ( 3 8 ). ..................................................... 98 4 1 Structures of the endocannabinoid anandamide and fatty acid amides from marine cyanobacteria with binding affinities to the cannabinoid receptors. ...... 114 4 2 General structural scaffold of malyngamides and the structures of three malyngamides. ................................................................................................. 115

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11 4 3 Cannabimimetic ef fects of serinolamide B ( 4 1 ) and malyngamide B ( 4 2 ). ..... 116 5 1 Chemical structure of the 5methylene decanoic acid ( 5 1 ) and its chlorinated ester ( 5 2 ). ........................................................................................................ 143 5 2 LRMS data for compound 5 2 ( ve mode) ........................................................ 144 5 3 Retrosynthetic strategies for the semi synthesis of the chlorinated ester ( 5 2 ). 145 5 4 Schemes showing the initial trials to obtain the alcohol fragment for coupling with the acid 5 1 to obtain the chlorinated ester 5 2 ........................................ 146 5 5 Scheme for semi synthesis of the chlorinated ester 5 2 ................................... 147 5 6 Comparison of 1H NMR Spectra of the natural product ( 5 2 ) and the synthetic compound. ........................................................................................................ 148 5 7 Quorum sensing inhibition by the fatty acid 5methylene decanoic acid ( 5 1 ) in P. aeruginosa (PAO1) ................................................................................... 149 5 8 Effect of the chlorinated ester ( 5 2 ) on the transcript levels o f pro inflammatory cytokines IL 6 IL IL 8 and TNF in differentiated THP 1 cells. ................................................................................................................. 150 A 1 1H NMR Spectrum of Pitiprolamide ( 2 1 ) in Benzened6 (600 MHz) at 20 C. .. 155 A 2 13C NMR Spectrum of Pitiprolamide ( 2 1 ) in Benzened6 (125 MHz) at 20 C. 156 A 3 COSY Spectrum of Pitiprolamide ( 2 1 ) in Benzene d6 (600 MHz) at 20 C ...... 157 A 4 TOCSY Spectrum of Pitiprolamide ( 2 1 ) in Benzened6 (600 MHz) at 20 C .... 158 A 5 ROESY Spectrum of Pitiprolamide ( 2 1 ) in Benzened6 (600 MHz) at 20 C ... 159 A 6 HSQC Spectrum of Pitiprolamide ( 2 1 ) in Benzene d6 (600 MHz) at 20 C ...... 160 A 7 HMB C Spectrum of Pitiprolamide ( 2 1 ) in Benzened6 (600 MHz) at 20 C ..... 161 A 8 1H NMR Spectrum Pitiprolamide ( 2 1 ) in Acetoned6 (400 MHz) at 25 C ........ 162 A 9 1H NMR Spectrum of Pitipeptolide C ( 3 3 ) in CDCl3 (600 MHz). ...................... 163 A 10 13C NMR Spectrum of Pitipeptolide C ( 3 3 ) in CDCl3 (125 MHz) ...................... 164 A 11 COSY Spectrum of Pitipeptolide C ( 3 3 ) in CDCl3 (600 MHz). ......................... 165 A 12 TOCSY Spectrum of Pitipeptolide C ( 3 3 ) in CDCl3 (600 MHz). ....................... 166 A 13 ROESY Spectrum of Pitipeptolide C ( 3 3 ) in CDCl3 (600 MHz). ....................... 167

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12 A 14 HSQC Spectrum of Pitipeptolide C ( 3 3 ) in CDCl3 (600 MHz). ......................... 168 A 15 HMBC Spectrum of Pitipeptolide C ( 3 3 ) in CDCl3 (600 MHz). ......................... 169 A 16 1H NMR Spectrum of Pitipeptolide D ( 3 4 ) in CDCl3 (600 MHz). ....................... 170 A 17 COSY Spectrum of Pitipeptolide D ( 3 4 ) in CDCl3 (600 MHz). ......................... 171 A 18 TOCSY Spectrum of Pitipeptolide D ( 3 4 ) in CDCl3 (600 MHz). ....................... 172 A 19 ROESY Spectrum of Pitipeptolide D ( 3 4 ) in CDCl3 (600 MHz). ....................... 173 A 20 HSQC Spectrum of Pitipeptolide D ( 3 4 ) in CDCl3 (6 00 MHz). ......................... 174 A 21 HMBC Spectrum of Pitipeptolide D ( 3 4 ) in CDCl3 (600 MHz). ......................... 175 A 22 1H NMR Spectrum of Pitipeptolide E ( 3 5 ) i n CDCl3 (600 MHz). ....................... 176 A 23 COSY Spectrum of Pitipeptolide E ( 3 5 ) in CDCl3 (600 MHz). ......................... 177 A 24 TOCSY Spectrum of Pitipeptolide E ( 3 5 ) in CDCl3 (600 MHz). ....................... 178 A 25 ROESY Spectrum of Pitipeptolide E ( 3 5 ) in CDCl3 (600 MHz). ....................... 179 A 26 HSQC Spectrum of Pitipeptolide E ( 3 5 ) in CDCl3 (600 MHz). ......................... 180 A 27 HMBC Spectrum of Pitipeptolide E ( 3 5 ) in CDCl3 (600 MHz). ......................... 181 A 28 1H NMR Spectr um of Pitipeptolide F ( 3 6 ) in CDCl3 (600 MHz). ....................... 182 A 29 COSY Spectrum of Pitipeptolide F ( 3 6 ) in CDCl3 (600 MHz). .......................... 183 A 30 TOC SY Spectrum of Pitipeptolide F ( 3 6 ) in CDCl3 (600 MHz). ....................... 184 A 31 ROESY Spectrum of Pitipeptolide F ( 3 6 ) in CDCl3 (600 MHz). ....................... 185 A 32 HSQC Spectrum of Pitipeptolide F ( 3 6 ) in CDCl3 (600 MHz). ......................... 186 A 33 HMBC Spectrum of Pitipeptolide F ( 3 6 ) in CDCl3 (600 MHz). ......................... 187 A 34 1H NMR Spectrum of Serinolamide B ( 4 1 ) in CDCl3 (600 MHz). ..................... 188 A 35 COSY Spectrum of Serinolamide B ( 4 1 ) in CDCl3 (600 MHz). ........................ 189 A 36 TOCSY Spectrum of Serinolamide B ( 4 1 ) in CDCl3 (600 MHz). ...................... 190 A 37 NOESY Spectrum of Serinolamide B ( 4 1 ) in CDCl3 (600 MHz). ...................... 191 A 38 HSQC Spectrum of Serinolamide B ( 4 1 ) in CDCl3 (600 MHz). ........................ 192

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13 A 39 HMBC Spectrum of Serinolamide B ( 4 1 ) in CDCl3 (600 MHz). ........................ 193 A 40 1H NMR Spectrum of 5 Methylene Decanoic Acid ( 5 1 ) in CDCl3 (600 MHz). .. 194 A 41 COSY Spectrum of 5 Methylene Decanoic Acid ( 5 1 ) in CDCl3 (600 MHz). .... 195 A 42 TOCSY Spectrum of 5Methylene Decanoic Acid ( 5 1 ) in CDCl3 (600 MHz). ... 196 A 43 NOESY Spectrum of 5 Methylene Decanoic Acid ( 5 1 ) in CDCl3 (600 MHz). .. 197 A 44 HSQC Spectrum of 5Methylene Decanoic Acid ( 5 1 ) in CDCl3 (600 MHz). ..... 198 A 45 HMBC Spectrum of 5 Methylene Decanoic Acid ( 5 1 ) in CDCl3 (600 MHz). .... 199 A 46 1H NMR Spectrum of Chlorinated Ester ( 5 2 ) in CDCl3 (600 MHz). .................. 200 A 47 COSY Spectrum of Chlorinated Ester ( 5 2 ) in CDCl3 (600 MHz). ..................... 201 A 48 TOCSY Spectrum of Chlorinated Ester ( 5 2 ) in CDCl3 (600 MHz). .................. 202 A 49 NOESY Spectrum of Chlorinated Ester ( 5 2 ) in CDCl3 (600 MHz). .................. 203 A 50 HSQC Spectrum of Chlorinated Ester ( 5 2 ) in CDCl3 (600 MHz). .................... 204 A 51 HMBC Spectrum of Chlorinated Ester ( 5 2 ) in CDCl3 (600 MHz). .................... 205 B 1 1H NMR Spectrum of Intermediate 3 9 in CDCl3 (400 MHz). ............................ 207 B 2 13C NMR Spectrum of Intermediate 3 9 in CDCl3 (100 MHz). .......................... 208 B 3 1H NMR Spectrum of Intermediate 3 10 in CDCl3 (400 MHz) ........................... 209 B 4 1H NMR Spectrum of Intermediate 3 11 in CDCl3 (400 MHz). .......................... 210 B 5 1H NMR Spectrum of Intermediate 3 12 in CDCl3 (400 MHz). .......................... 211 B 6 13C NMR Spectrum of intermediate 3 12 in CDCl3 (100 MHz). ......................... 212 B 7 1H NMR Spectrum of Intermediate 3 13 in CDCl3 (400 MHz). .......................... 213 B 8 1H NMR Spectrum of Intermediate 3 14 in CDCl3 (400 MHz). .......................... 214 B 9 1H NMR Spectrum of Intermediate 3 15 in CDCl3 (400 MHz). .......................... 215 B 10 13C NMR Spectrum of Intermediate 3 15 in CDCl3 (100 MHz). ......................... 216 B 11 1H NMR Spectrum of PitiA Biotin ( 3 7 ) in CDCl3 (400 MHz). ........................... 217 B 12 APT Spectrum of Piti A Biotin 3 7 in CDCl3 (100 MHz). ................................... 218

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14 B 13 1H NMR Spectrum of Intermediate 5 3 in CDCl3 (400 MHz) ............................. 219 B 14 13C NMR Spectrum of Intermediate 5 3 in CDCl3 (100 MHz). .......................... 220 B 15 1H NMR Spectrum of Intermediate 5 4 in CDCl3 (400 MHz). ............................ 221 B 16 13C NMR Spectrum of Intermediate 5 4 in CDCl3 (100 MHz) ........................... 222 B 17 1H NMR Spectrum of Intermediate 5 6 in CDCl3 (400 MHz) showing mixture o f isomers. ........................................................................................................ 223 B 18 1H NMR Spectrum of Intermediate 5 7 in CDCl3 (400 MHz). ............................ 224 B 19 13C NMR Spectrum of Intermediate 5 7 in CDCl3 (100 MHz). .......................... 225 B 20 1H NMR Spectrum of Intermediate 5 8 in CDCl3 (400 MHz). ............................ 226 B 21 13C NMR Spectrum of Intermediate 5 8 in CDCl3 (100 MHz). .......................... 227 B 22 1H NMR Spectrum of Intermediate 5 9 in CDCl3 (400 MHz). ............................ 228 B 23 13C NMR Spectrum of Intermediate 5 9 in CDCl3 (100 MHz). .......................... 229 B 24 1H NMR Spectrum of Intermediate 5 10 in CDCl3 (400 MHz). .......................... 230 B 25 13C NMR Spectrum of Intermediate 5 10 in CDCl3 (100 MHz). ......................... 231 B 26 1H NMR Spectrum of Intermediate 5 11 in CDCl3 (400 MHz). .......................... 232 B 27 13C NMR Spectrum of Intermediate 5 11 in CDCl3 (100 MHz). ......................... 233 B 28 1H NMR Spectrum of Intermediate 5 12 in CDCl3 (400 MHz). .......................... 234 B 30 13C NMR Spectrum of Int ermediate 5 13 in CDCl3 (100 MHz). ......................... 236 B 31 1H NMR Spectrum of Intermediate 5 14 in CDCl3 (400 MHz). .......................... 237 B 33 1H NMR Spectrum of Intermediate 5 15 in CDCl3 (400 MHz). .......................... 239 B 35 1H NMR Spectrum of Intermediate 5 16 in CDCl3 (400 MHz). .......................... 241 B 36 13C NMR Spectr um of Intermediate 5 16 in CDCl3 (100 MHz). ......................... 242 B 37 1H NMR Spectrum of Intermediate 5 17 in CDCl3 (400 MHz) .......................... 243 B 38 13C NMR S pectrum of Intermediate 5 17 in CDCl3 (100 MHz). ......................... 244 B 39 1H NMR Spectrum of Intermediate 5 18 in CDCl3 (400 MHz).. ......................... 245 B 40 13C NMR Spectrum of Intermediate 5 18 in CDCl3 (100 MHz). ......................... 246

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15 B 41 1H NMR Spectrum of the Synthesized Chlorinated Ester 5 2 in CDCl3 (400 MHz). ................................................................................................................ 247 B 42 13C NMR Spectrum of the Synthesized Chlorinated Ester 5 2 in CDCl3 (100 MHz).. ............................................................................................................... 248

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16 LIST OF ABBREVIATIONS AcOH Acetic acid (Boc)2O Ditert butyl dicarbonate CDCl3 Chloroform COSY Correlation spec troscopy CrCl2 Chromous chloride CrO3 Chromium trioxide CuSO4 C upric sulfate DCC N N Dicyclohexylcarbodiimide DCM Dichloro methane DIBAL Diisobutylaluminium hydride DMAP 4 Dimethylaminopyridine DMF Dimethylformamide DMSO Dimethyl sulfoxide EC50 Half maxim al effective concentration EDC 1 Ethyl 3 (3 dimethylaminopropyl)carbodiimide ESIMS Electrospray ionization mass spectrometry EtOAc Ethyl acetate EtOH Ethanol Et2O Diethyl ether Et3N Triethyl amine Fmoc Cl Fluorenylmethyloxycarbonyl chloride g Gram h Hour H2O Water

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17 H2O2 Hydrogen peroxide H2SO4 Sulfuric acid HCl Hydrochloric acid HCOOH Formic acid HMBC Heteronuclear multiple bond correlation spectroscopy HMQC Heteronuclear multiple quantum correlation spectroscopy HOAt 1 Hydroxy7 azabenzotriazole HOBt Hydro xybenzotriazole HPLC High performance liquid chromatography HRESIMS High resolution electrospray ionization mass spectroscopy HT 29 HT 29 colorectal adenocarcinoma Hz Hertz IC50 Half maximal inhibitory concentration i PrOH Isopropanol Ki Binding affinity IR Infrared LB LuriaBertani LiOH Lithium hydroxide MCF7 MCF7 breast adenocarcinoma MeCN Acetonitrile MeOH Methanol MgSO4 Magnesium sulfate MHz Megahertz MRM Multiple Reaction Monitoring MS Mass spectrometry

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18 m Multiplet M Molar mm Millimeter mM Millimolar NaBr Sodium bromide NaHCO3 Sodium bicarbonate NaN3 Sodium azide NaNO2 Sodium nitrite NaOH Sodium hydroxide n Bu OH 1 Butanol NMM N methyl morpholine NMR Nuclear magnetic resonance PCC Pyridinium chlorochromate Ph3P Triphenylphosphine ppm Parts per million R OESY Rotating frame nuclear Overhauser effect spectroscopy RT qPCR Quantitative Polymerase Chain Reaction after Reverse Transcription SAR Structure activity relationship TBAF Tetra n butylammonium fluoride TBDPS Cl t ert Butylchlorodiphenylsilane TBS Cl te rt Butyldimethylsilyl chloride THF Tetrahydrofuran THP 1 Human acute monocytic leukemia cell line TOCSY Total correlation spectroscopy TsCl 4 Toluenesulfonyl chloride

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19 UV Ultraviolet g Microgram m Micrometer M Micromolar

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20 Abstract of Dissertation Prese nted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy STRUCTURAL CHARACTERIZATION, BIOLOGICAL EVALUATION AND SYNTHESIS OF NOVEL SECONDARY METABOLITES FROM GUAMANIAN MARINE CYANOBACTERIA LYNGBYA SPP By Rana Montaser May 2013 Chair: Hendrik Luesch Major: Pharmaceutical Sciences Medicinal Chemistry The marine environment is one spectacular unexplored resource that is characterized by its huge biodivers ity. During our quest for novel drug leads from the marine environment, we explored Guamanian varieties of the marine cyanobacterium Lyngbya spp. Cyanobacteria of the genus Lyngbya are known for the production of peptides, polyketides or peptide/polyketide hybrids. E xploring two samples collected from the Piti Bay area led to the characterization of a group of compounds which are chemically classified as peptides and fatty acid derivatives. Exploring the samples involved extraction, isolation, structural characterization as well as biological evaluation of pure secondary metabolites, aided by synthetic studies. Seven cyclic peptides were isolated from a Lyngbya sample collected from Piti Bomb Holes from Guam; a novel proline rich cyclic peptide named pitipr olamide ( 2 1 ) and the six cyclic peptide analogues pitipeptolides A F ( 3 1 3 6 ). The structure of 2 1 was deduced using NMR, MS, X ray crystallography and enantioselective HPLC MS techniques Pitiprolamide ( 2 1 ) has distinctive structural features with proline representing half of the structural resid u es. The structures of pitipeptolides were

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21 elucidated using 2D NMR, MS and enantioselective HPLC experiments. Biological activities of the seven cyclic peptides were investigated highlighting their cytotoxi c and antimycobacterial activities. Additionally, the biological activities of the six analogues 3 1 3 6 suggested some structureactivity relationship features. Accordingly, two probes of pitipeptolide A were semi synthesized and proved to retain the an timycobacterial bioactivity, and therefore could be used for further target identification studies. The same sample also yielded a new fatty acid amide named serinolamide B ( 4 1 ). Serinolamide B ( 4 1 ) and malyngamide B ( 4 2 ) a representative member of a l arge class of cyanobacterial fatty acid amides were evaluated for cannabimimetic activities where they proved to act as cannabinoid receptor agonists Another sample from Piti Bay was rich in a simple fatty acid 5methylene decanoic acid ( 5 1 ). The fatty acid 5 1 can modulate quorum sensing in bacteria. Additionally, a chlorinated ester ( 5 2 ) of this abundant fatty acid was isolated but in minute amounts. Therefore, more material was obtained through semi synthetic efforts, and further biological evaluation showed compound 5 2 to possess anti inflammatory properties .

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22 CHAPTER 1 GENERAL INTRODUCTIONa Oceans Harbor Unique Bioactive Chemical Entities The discovery of new drug leads is a constant biomedical need, especially with the existence of difficult to cur e diseases, the rapidly growing incidence of drug resistant infectious diseases and the presence of neglected diseases. While n ature is an ancient pharmacy that used to be the solitary source of therapeutics for the early eras, y et, the renaissance of science and the deeper understanding of the pathology of several diseases enabled the design and synthesis of drug molecules for specific molecular targets, and thus shifted the attention from the natural pharmacy to the purely synthetic drugs.1, 2 This attention was further biased towards synthetic drugs with the development of highthroughput screening (HTS) technologies. The ability to test a large number of chemical entities in a certain assay at the same time required a larger and faster supply of compound libraries. Combinatorial chemistry fulfilled this latter requirement and preceded natural products in this specific obligation and thus was mor e tempting for drug discovery. However, relying on those faster drug discovery approaches that are irrelevant to our chiral world led to a concomitant 20year low number of New Chemical Entities in 2001.3 Natural products are often structurally complex compounds that possess a well defined spatial orientation. Those chemical compounds evolved to interact efficiently with their biological targets; therefore they occupy a biologically relevant chemical space a Reproduced in part with permission from Montaser & Luesch, Future Med. Chem 2011 3 1475 1489 Copyright (2011) Future Science Ltd.

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23 and represent validated starting points for drug discovery. Accordingly, it is not surprising that 40% of the chemical scaffolds in the Dictionary of Natural Products occupy unprecedented chemical space that was not represented by synthetic compounds.4 Moreover, the ingenuity of natural products allowed them to occupy a significant market share as well as a special preference by the consumers. About half of all new drugs in the time frame reported are of natural product origin or designed on the basi s of natural product structures5 and about half of the 20 best selling nonprotein drugs are related to natural products.6 Almost all of the current natural product derived therapeutics have terrestrial origins. However, mining novel sources, such as the marine environment, will open the way for chemical and biological novelties as well The marine environment is a largely unexplored resource that is characterized by its huge biodiversity. The first Census of Marine Life (CoML 20002010) has completed a decade inventory that revealed an ast ounding level of biodiversity.7 From a pharmaceutical perspective, this wealthy resource yields a plethora of chemically novel bioactive secondary metabolites, which sometimes possess unprecedented mechanisms of action.8 15 A comparative analysis by Kong and coworkers showed that marine natural products are superior to terrestrial natural products in terms of chemical novelty.16 The analysis, which compared molecular scaffolds reported in the Dictionary of Natural Products to those in the Dictionary of Marine Natural Products, showed that approximately 71% of the molecular scaffolds in the Dictionary of Marine Natural Products were exclusively utilized by marine organisms. In addit ion, marine organisms show higher incidence of significant bioactivity compared to terrestrial organisms. For example, in a National Cancer

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24 Institute preclinical cytotoxicity screen, approximately 1% of the tested marine samples showed anti tumor potential versus 0.1% of the tested terrestrial samples.15 Marine Drugs on the Market As a result of th e chemical novelty associated with marine natural products and its concu rrent biochemical significance, the market has already welcomed the first marinederived drugs (Table 11 ), and there is more yet to come.11, 12, 17, 18 Currently, there are six FDA approved marine or marine derived drugs besides one additional drug registered in the EU.12, 1720 Cytarabine (AraC) and vidarabine (AraA) (Figure 1 1 ), the first FDA approved marine derived drugs, are synthetic pyrimidine and purine nucleosides, respectively, developed from naturally occurring nucleosides originally isolated from the Caribbean sponge Tethya crypta.12 Cytarabine was approved by FDA in 1969 as an anticancer drug, while vidarabine was approved in 1976 as an antiviral agent. More than twenty years later, ziconotide (Prialt) (Figure 1 1 ) gained FDA approval in 2004 for the management of severe chronic pain. It is a synthetic equivalent of a naturally occurring peptide isolated from the venom of the cone snail Conus magus .12, 21 Also, FDA granted approval for the marine omega3 acid ethyl esters (Lovaza) (Figure 1 1) product in 2004 for the treatment of hypertriglyceridemia. This fatty acid mixture is composed mainly of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) esters, and is reported to be sourc ed from South Pacific fish including anchovies, herring, salmon, mackerel, smelts and jacks.18, 20 Next came trabectedin (Yondelis) (Figure 1 1 ), the first marine anticancer agent to gain approval by the European U nion for the treatment of soft tissue sarcoma and relapsed ovarian cancer, and it is now awaiting FDA approval.12, 18 Trabectedin is a marine alkaloid isolated from the marine tunicate Ecteinascidia turbinata 22 that was registered in 2007

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25 Eribulin mesylate (Halaven) (Figure 11 ) is another marine derived drug which gained FDA approval in November 2010 f or metastatic breast cancer .19 This totally synthesized drug is a structurally simplified macrocyclic ketone analogue of the potent cytotoxic compound halichondrin B, which was first isolated from the marine sponge Halichondria okadai in 1986.23, 24 The latest addition to the market is the antitumor drug Brentuximab vedotin (Adcetris) (Figure 1 1) which was developed as an antibody drug conjugate and has gained FDA approval in August 2011 for the treatment of Hodgkin lymphoma (HL) and another rare lymphoma known as systemic anaplastic large cell lymphoma (ALCL).17 This drug is a derivative of the marine cytotoxic cyanobacterial metabolite dolastatin 10 (Figure 1 3 ).25 Notably, the approval of this marine derived drug represents the first new FDA approval for the management of HL since 1977 and the first indicated to treat ALCL.17 In addition, there is a continuous supply of auspicious candidates in the preclinical pipeline which is continuously feeding the clinical pipeline.12, 18 Promisingly, the number of new marine compounds reported each year is increasing, and more than 1000 new compounds with different potencies and biological activities have been reported each year for the past couple of years .9, 10, 26, 27 Even if a discovered marine secondary metabolite was not effective in directly treating diseases, it could still possess unique biochemical properties that will enable it to be developed in to a pharmacological probe, and thus can indirectly contribute to disease treatment by helping us better understand the underlying biochemistry of a disease.8 For example, manoalide (Figure 1 2) a sesterterpenoid first isolated from the sponge Luffariella variabilis ,28 is the first selective phospholipase A2 inhibitor29, 30, and it is now commercially available for

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26 biochemical research.31 Another example for a marine natural product that is widely used in pharmacological research is kainic acid (Figure 1 2) which is used as a tool in neurobiology research.32, 33 This metabolite was isolated from the red algae Digenia simplex and has a characteristic biological activity as a selective kainate receptor agonist where it is commonly used to distinguish those receptor types .34, 35 As a conclusion, t he worlds oceans contain a seemingly endless number of promising unique chemical scaffolds awaiting discovery. Marine Cyanobacteria: A Prolific Source of Bioactive Compounds In particular, marine cyanobacteria are one of the most prolific sources of secondary metabolites with promising biomedical potential.3638 Although bioactive marine compounds that reached the market or are currently in clinical trials have been discovered from different marine organisms including mollusks, tunicates and sponges, there have been doubts about the true sources of those metabolites.18 Oftentimes, the true producers of those bioactive compounds have been suspected or even proved to be symbiotic microorganisms associated with the collected source. Significantly, up to 80% of those pharmaceutical agents in the clinical pipeline and on the market might be metabolically produced by symbiotic marine bacteria and cyanobacyteria.18 For example, recent studies showed that the bacterial sym biont Candidatus Endobugula sertula is the source of the cytotoxic bryostatins that were discovered from the marine bryozoan B. neritina .39, 40 It was shown that the bacterial aggregates produce a coating of bryostatins around the bryozoan larvae for protection against predators. Similarly, dolastatin 10 (Figure 1 3), from which the recently approved antitumor drug brentuximab vedotin was developed, was initially reported from the sea hare Dolabella auricularia.41

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27 Later, it was found to originate from the marine c yanobacterium Symploca sp. dietary source.25 Lyngbya spp. H ave a C haracteristic C hemical P rofile Among the marine cyanobacteria, Lyngbya sp p are well known for their enormous potential to mix nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) pathways for the production of peptidepolyketide hybrids with a wide range of biological activities .37, 42 Some of those secondary metabolites will have a major modif ied peptide core with one or more fatty acid derived moieties, such as the potent cytotoxins apratoxin A43 and lagunamides A C4446 (Figure 1 3 ) Others will have a major fatty acid portion with few attached amino acid or amino ac id derived moieties, such as the neurotoxic dimeric lipopeptide somocystinamide A47 and the potent cyt otoxin curacin A48, 49 (Figure 1 3 ). Malyngamides represent another group of peptidepolyketide hybrids that are largely related to the marine cyanobacterium L. majuscula and possess different biological activ ities including cytotoxic50, 51, antibacterial52 and anti inflammatory5355 activities. To date, there are more than 30 malyngamide analogues reported, most of which enclose a characteristic fatty acid side chain which is mostly 7( S ) methoxytetradec 4( E ) enoic ac id (lyngbic acid)56 (Figure 1 3 ) or a derivative ther eof. The significantly high yield of bioactive secondary metabolites from the genus Lyngbya, and particularly from a single species Lyngbya majuscula has recently triggered intensive investigations of the phylogenetic classification of this genus to unc over the true biodiversity of this cyanobacterial group.5759 Taxonomic identification of cyanobacterial samples commonly follow morphology based methods, and little genomic information is usually available. However a recent genomic investigation of

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28 several cyanobacterial samples that were previously classified as Lyngbya members showed that those are morphologically similar but phylogenetically distant lineages, and accordingly re classified several Lyngbya majuscula species .58 However, since the investigated Lyngbya samples here were not clearly reclassified yet,59 we will continue to re fer to those samples as Lyngbya majuscula in the coming studies Drug Discovery from Lyngbya S amples from Guam Isolation of Marine Cyanobacterial Secondary Metabolites In our quest for novel drug leads from marine cyanobacteria, samples of the marine cyanobacterium Lyngbya majuscula collected from Piti Bay area from Guam were subjected to chemical and biological investigations ( Figure 1 4 ). Chemical analysis followed an NMR guided fractionation approach targeting peptides and fatty acid structural classes which represent the characteristic biosynthetic signatures of this organism In 1D NMR spectra, the presence of peptides i s usually marked by some characteristic peaks, including peaks for alphaprotons ( H 5 ppm) and secondary amide protons ( H 8 ppm) in the proton NMR spectrum and peaks for carbonyls ( C 175 ppm) in the carbon NMR spectrum. In case of fatty acids, characteristic peaks in 1D NMR spectra include terminal methyl groups ( H C methylene groups ( H .3 ppm and C 35 ppm) and typical resonances for overlapping methylene groups ( H C fatty acids show distinctive olefinic methine peaks ( H C Isolation and purification of secondary metabolites followed a typical scheme (Figure 1 4), which started by extracting the freeze dried samples using polar and nonpolar solvents. The nonpolar extract (EtOAc MeOH, 1:1) was further partitioned between hexanes, n BuOH and water, to separ ate the components between nonpolar,

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29 semi polar and polar fractions, respectively. T he semi polar n BuOH fraction was further fractionated using silica gel column chromatography to generate fractions of different polarities using a gradient solvent system that shows the best separation, such as DCM/ i PrOH or DCM/MeOH gradients. Based on NMR profiles, silica gel column fractions were prioritized for HPLC purification to isolate pure secondary metabolites Once pure compounds were obtained, structure elucida tion techniques were tackled Chemical C haracterization of M arine C yanobacterial S econdary M etabolites Isolated secondary metabolites were obtained in the highest pure form using HPLC techniques. Once a pure compound is obtained, subsequent experiments fo cused in part on its chemical characterization, where the planar structure is first identified followed by stereochemical assignments. The identification of the planar structure usually involves a set of NMR experiments and MS analysis First, a complete set of 1D and 2D NMR spectral data are collected, including 1H NMR, 13C NMR, COSY, TOCSY, ROESY, HSQC and HMBC data. 1H and 13C 1DNMR experiments provide insight into the number and type of protons, carbons and possible functional groups present in the mol ecule which are usually collected in an inventory. Then, 2D HSQC experiments provide connectivity information relating specific carbon atoms and the protons bound to them. Partial structures could then be formed based on data from 2D COSY, TOCSY and HMBC e xperiments. HMBC experimental data will also aid in providing final information about some critical connectivities, such as bonds that will cyclize a linear planar structure or final connections between partial structures to form a complete molecular struc ture. Once the planar structure is known, stereogenic centers will be clear, and experiments to identify their configurations will follow. In the work presented here, functional groups with stereo genic centers were alphaprotons in amino

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30 acids, modified am ino acids and in alphahydroxy acids as well as olefinic geometry. The methods used to establish the configurations were hydrolysis to liberate single amino acid or modified amino acid moieties followed by LC MS enantioselective analysis using a chiral HPLC column, where the retention times on the chiral HPLC column and the MRM ion pairs from MS analysis for the samples were compared to commercial or synthetic standards. For the olefinic geometry, ozonolysis with oxidative workup yielded a known moiety that has available commercial standards, and enantioselective HPLC analysis with UV detection was done using a chiral column to compare retention times of the chemically modified sample to known standards. In addition, w henever crystallization was successful, X ray crystallography aided in confirming the proposed chemical structure of a cyanobacterial metabolite and in solving its relative stereochemistry. Biological C haracterization of M arine C yanobacterial S econdary M etabolites Natural products evolved to int eract with biological targets, and therefore they are considered as privileged structures and optimum starting points for drug discovery. In other words, natural products could be viewed as bioactive compounds which often times produce physiological outco mes in the human body, but the challenge is to find the correct assay/method to discover this bioactivity. In order to screen for bioactivity of a certain molecule, there are two major approaches to follow; target based screens or phenotypic screens. Target based screens are bioassays where the molecule is tested for binding to or modulating the activity of a certain preidentified target. The endpoint of this approach is a molecule with an already known molecular target and mechanism of action. However, this approach has major disadvantages; first, it is characterized by a lower hit rate since it only screens for one specific interaction.

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31 S econd, very often the hit compound fails to reproduce its bioactivity in vivo T hird sometimes the desired compoundta rget interaction could be masked by unexpected off target interactions, leading to undesired outcomes.60, 61 On the other hand, phenotypic screens are bioassays where the molecule is screened for producing specific phenotypic changes in treated cells or organisms. Although this approach does not offer mechanistic knowledge about the hit compounds detected bioactivity, it offers major advantages including a higher hit rate, since several available targets are simulta neously screened in phenotypic assays, and a higher chance of reproducing the desired bioactivity in in vivo models.60, 61 Additionally, the challenge of identifying the molecular target of the hit compound and its mechanism of action has been tackled by the development of technologies for this latter purpose and therefore increased the focus on cellphenotypebased screening .61 In the work presented here, phenotypic screens were always pursued, where isolated secondary metabolites were routinely screened for certain phenotypic outcomes in mammalian or bacterial cell s. First line assays include d cytotoxicity assays in mammalian cancer cell lines, ant ibacterial assays against Gram +ve and Gram ve bacteria and anti inflammatory assays in murine monocyte cell line RAW 264.7 and human macrophages THP 1 cells Compounds that show significant biological activities could be further characterized in more specialized assays according to the detected phenotype, and more attempts could be directed towards deciphering their mechanisms of action. For the bioactive compounds identified in this work, a good candidate for follow up mechanism of action studies will be a compound with interesting bioactivity

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32 ( moderate to strong potency ) and abundant yield that allows for extensive mechanis tic exploration. Synthesis of Marine Cyanobacterial Secondary Metabolites O ne of the major challenges that faces drug discovery from natural samples is the isolation of minute amounts of a bioactive compound. Although recent advanced technologies have provided solutions for successful chemical characterization of bioactive natural products in minute amounts62, 63, this scarcity hurdles biological evaluation of those oftentimes structurally interesting mol ecules. This issue could be addressed through chemical synthesis to provide a resupply for further assays Also, structural modifications through chemical synthesis/semi synthesis add the advantage of gaining insight into some st ructure activity relationship (SAR) features. In the work presented here, chemical synthesis was pursued for two reasons: the modification of a bioactive compound to develop a useful derivative that can be used to probe its mechanism of action, and the semi synthesis of a bioactive secondary metabolite that was isolated in micro gram quantities to allow for its biological characterization.

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33 Table 11 Marine drugs on the market Marketed Drug (Year Approved) Compound Name Parent Compound Marine O rganis m Chemical Class Indicated Disease Cytosar U (1969) Cytarabine, Ara C Spongothymidine Sponge Nucleoside Cancer Vira A (1976) Vidarabine, Ara A Spongouridine Sponge Nucleoside Antiviral Prialt (2004) Ziconotide conotoxi n MVIIA Cone Snail Peptide Pain Lovaza (2004) Omega 3 acid ethyl esters Docosahexaenoic/ Eicosapentaenoic acid s Fish Omega 3 fatty acids Hypertri glyceridemia Yondelis (2007) a Trabectedin Ecteinascidin 743 Tunicate Alkaloid Cancer Halaven TM (2010) Eribulin mesylate Halichondrin B Sponge Macrolide Cancer Adcetris TM (2011) b Brentuximab vedotin Dolastatin 10 Bacterium Peptide b Cancer a EU registered only, and now awaiting FDA approval. b Developed as a drug antibody conjugate.

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34 Figure 11. Marine drugs on the market

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35 Figure 12. Examples for bioactive marine natural products used as pharmacological probes.

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36 Figure 13 Examples for bioactive secondary metabolites reported from marine cyanobacteria.

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37 Figure 14 Schematic summary of the chemical and biological characterization of bioactive marine secondary metabolites through NMR guided fractionation.* Interesting fractions: fractions where NMR profiles indicate the presence of a certain class of co mpounds (e.g.: peptides, modified peptides, fatty acids, polyketides). **Significant biological activity: biological activity (e.g.: IC50, MIC ) in the low/submicromolar range.

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38 Further fractionation and purification (HPLC) Solvent extraction and solvent solv ent partitioning Freeze dried m arine sample NMR guided fractionation Biological characterization Structure Elucidation (NMR, MS, X ray crystallography etc ) Yes No No further action Interesting NMR profiles? Yes No No further action Significant biological activity ? ** Target identification and mechanism of action studies Yes No Enough isolated compound? Total chemical synthesis or semi synthesis SAR Compound derivatization

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39 CHAPTER 2 PITIPROLAMIDE, A PROLINE RICH DOLASTATIN 16 ANALOGUE FROM THE MARINE CYANOBACTERIUM LYNGBYA MAJUSCULA FROM GUAMa Introduction The marine cyanobacterium Lyngbya majuscula is a prolific source of structurally diverse secondary metabolites. One of its multifarious structural themes is the establishment of cyclic peptides by utilizing a diversity of standard as well as modified amino acids.38 Among these, prolinerich cyclic peptides re present an intriguing class. The interest in this class arises from the distinctively low activation barrier for the prolyl amide bond to switch between cis and trans conformations readily compared to other amide linkages, which would impact the three dime nsional structure of the peptide.64 The rate of this interconversion is solvent dependent, being higher in nonpol ar solvents, where the charge separation and resonance stabilization of the ground state of both isomers are lost, lowering the cis to trans torsion barrier.6567 Recently, further attention has been directed toward this prolyl isomerization, as it has been suggested to be involved in the ratelimiting steps for protein folding68 and to act as a molecular timer in several biological and pathological processes.69 In our qu e st for novel drug leads from marine cyanobacteria, we isolated a novel prolinerich cyclodepsipeptide, pitiprolamide ( 2 1 ), from a collection of the cyanobacterium L. majuscula from Piti Bomb Holes, Guam; the same population that previously yielded pitipeptolides A and B.70 Compound 2 1 is structu rally related to a Reproduced with permission from Montaser, R., Abboud, K. A., Paul, V. J., Luesch, H J. Nat. Prod 2011 74 109 1 11. Copyright (2011) American Chemical Society.

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40 dolastatin 1671 and other marine secondary metabolites isolated from marine invertebrates and cyanobacteria. Isolation and Structure Determination The non polar extract of this sample was subjected to solvent partitioning followed by silica chromatography. NMR guided fractionation led to the identification of a pitipeptolidecontaining fraction, which was further subjected to purification by reversedphase HPLC to furnish pitiprolamide ( 2 1 ) (Figure 2 1 ) as a colorless, amorphous solid. The structure of 2 1 was elucidated by interpreting the 1H NMR, 13C NMR, COSY, TOCSY, HSQC, HMBC, and ROESY spectra recorded in benzened6. HRESIMS for 2 1 suggested a molecular formula of C49H72N6O10 ( m/z 927.5209 for [M + Na]+ and m/z 905.5390 for [M + H]+). The low field signals at H 7.73 and 9.1 7 ppm suggested that these arise from amide protons. The presence of amide bonds was protons ( H 5 ppm) and detecting their HMBC correlations with neighboring carbonyls ( C 175 ppm). However, the chemical shifts of two carbons (C28, C 77.7 and C44, C 79.3 ppm) suggested their oxygenation and the presence of hydroxy acids. This further indicated that 2 1 could be a depsipeptide. Aromatic carbons ( C 126.3, 2 128.7, 2 129.8, 141.1) present were characteristic of a mono substituted benzene. Further analysis of the 2D NMR spectra led to the assignment of eight moieties: five proteinogenic amino acids (four proline units (Pro1, C1 C5; Pro2, C17 C21; Pro3, C22 C26; Pro4, C32 C36) and a valine unit (Val, C37C41)), the modified amino acid dolaphenvaline (Dpv, C6C16), and two hydroxy acids, namely, 2hydroxy isovaleric acid (Hiva, C27 C31) and 2,2dimethyl 3 hydroxyhexanoic acid (Dmhha, C42C49) (Figure 2 1 Table 21 ). To satisfy the molecular requirements fr om the MS analysis,

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41 compound 2 1 had to be a cyclic depsipeptide. The sequence of the residues forming 2 1 was determined by HMBC correlations (Figure 2 2 ) along with MS MS fragmentation data (Figure 2 3 ). X ray Crystallography and Stereochemical Assignme nt The 1H NMR spectrum of 2 1 in benzened6 revealed the presence of two isomers showing a slow interconversion over time (Figure 2 4 ) due to prolyl amide isomerization, which complicated the structure elucidation process. However, as the compound crystall ized out of an aqueous MeOH solution, the implied sequence was confirmed by X ray crystallography (Figure 2 5 ). The availability of an X ray crystal structure for 2 1 simplified its stereochemical assignment by establishing the relative configuration. Enantioselective amino acid analysis of the acid hydrolysate by HPLC MS established the Lconfiguration for all Pro units and for Val unit. Consequently, the Dmhha unit had a 3R and Hiva had an S configuration. Notably, the Dpv unit was first reported in dolastatin 16 (Figure 2 1) ,71 but its stereochemistry remained unassigned. In pitiprolamide ( 2 1 ), the X ray crystal stru cture showed that the Dpv moiety had a 2 S 3 R configuration. This is the first crystal structure of a Dpv containing compound. Shortly thereafter, the X ray crystal structure of dolastatin 16 was reported72 and its stereochemistry was confirmed to be the same as in 2 1 Th e difference in chemical shifts of the and carbon signals in Pro2 = 10.2 ppm) suggested a cis orientation for the Pro2Pro3 linkage for the major conformer in the NMR solvent benzened6. The bonds preceding the three other Pro units appeared to be trans concluded from the lower values (less than 6 ppm).64 This

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42 matches the X ray crystal structure and the ROESY correlations, pointing out the similarity between the solution and the solidstate structure s. Because pitiprolamide ( 2 1 ) crystallized from an aqueous solvent, hydration was detected in the crystal structure (Figure 2 5 ) Four water molecules formed H bonds with the four carbonyls in Dmhha, Hiva, Pro2, and Pro3 units. Moreover, two intramolecular hydrogen bonds were observed. The first was between the amide proton in Val and the hydroxy derived oxygen in the Dmhha unit. The second one occurred between the other amide proton in the Dpv unit and the carbonyl in the Hiva unit. This latter 4 1 type H bond granted the cyclic peptide a turn, enclosing the cis amide linked Pro2Pro3 residues ( Figure 2 5A ) This matches what is known about the contribution of proline to various turn types, as well as the induction of a turn by cis Pro Pro sequences.73 Related Analog ue s Pitiprolamide ( 2 1 ) is structurally related to dolastatin 16 (Figure 2 1 ) which was r eported more than 10 years ago from the sea hare Dolabella auricularia.71 To the best of our knowledge, compound 2 1 is the second dolastatin 16 analogue from a marine cyanobacterium. Homodolastatin 16 (Figure 2 1 ) has been isolated from a different collection of L. majuscula from Kenya.74 The isolation of 2 1 from L. majuscula adds to the growing body of evidence regarding the cyanobacterial biosynthetic origin of dolastatin 16 and other dolastatins as well. Kulokekahilide1 is another dolastatin 16 analogue from the marine mollusk Philinopsis speciosa assumed to be of dietary cyanobacterial origin.75

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43 The building blocks of dolastatin 16 and its three analogues share several features (Figure 2 6 ). First, they are all octabidepsipeptides. Second, five conserved residues could be noted in the four structural analogues: ProDpv Pro and HivaPro where the stereochemistry is also conserved for those units (the stereochemistry of Dpv unit in homodolastatin 16 was not reported though). Third, the four compounds share derivatives of hexanoic and isohexanoic acids at the same position. Nonetheless, there are some distinctive differences characterizing 2 1 Pitiprolamide ( 2 1 ) has four Pro residues, which corresponds to 50% of all the residues. Also, it has a hydroxy hexanoic acid derivative versus amino hexanoic and isohexanoic acid derivatives in the other three analogues. This difference shifts the position of the second ester linkage in 2 1 (Figure 2 6) Moreover, a configurational change is prominent, where Pro3 in 2 1 which replaces the N Me -DVal/Ile in the other analogues, has an Lconfiguration. Compound 2 1 also shares some structural features with other marine cyanobacterial secondary metabolites. For example, the rare Dmhha residue in 2 1 was f irst reported in guineamides E and F76 and was recently illustrated in palmyramide A.77 Also, the prolinerich aspect in 2 1 has been characterized in some other secondary metabolites reported from different marine sponges; examples include phakellistatins,78 stylisins,79 and stylissamides.80 However, 2 1 contains all of these rare structural characteristics in one molecule. Biological Activity E valuation Because natural products emerged from evolutionary selection as biologically active compounds,81 bioactivity assessment was initiated for 2 1 Pitiprolamide ( 2 1 ) showed weak cytotoxic activity against HCT116 colorectal carcinoma and MCF7 breast adenocarcinoma cell lines (IC50 33 M for both). While dolastatin 16, homodolastatin 16,

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44 and kulokekahilide1 showed more potent cytotoxic acti vities against different cancer cell lines,71, 74, 75 the structural differences in the four analogues could signify the relationship betw een some residues and cytotoxicity. Compound 2 1 showed weak antibacterial activity against Mycobacterium tuberculosis starting at 50 g in a dis c diffusion assay (Table 2 5 ) and against Bacillus cereus starting at 1 M in a microtiter platebased assay with an approximate IC50 value of 70 M. No antibacterial activities were detected against either Staphylococcus aureus or Pseudomonas aeruginosa. Experimental Section General Experimental Procedures The optical rotation was measured on a PerkinElmer 341 polarimeter. UV and optical density were measured on a SpectraMax M5 (Molecular Devices), and IR data were obtained on a PerkinElmer Sp ectrum One FT IR Spectrometer. The 13C NMR spectrum was recorded on a Bruker 500 MHz spectrometer operating at 125 MHz. 1H and 2D NMR spectra were recorded on a Bruker Avance II 600 MHz spectrometer. All spectra were obtained in benzened6 using residual s olvent signals ( H 7.16, C 128.06 ppm) as internal standards. HSQC and HMBC experiments were optimized for 1JCH= 145 and 1JCH =7 Hz, respectively. The 1H NMR spectrum in acetone d6 was recorded on a Varian Mercury 400 MHz spectrometer using residual solvent signal ( H 2.05 ppm) as a reference. HRMS data was recorded on an Agilent LC TOF mass spectrometer equipped with an APCI/ESI multimode ion source detector in positive ion mode. LC MS data were obtained using an API 3200 triple quadrupole MS (Applied Biosystems) equipped with a Shimadzu LC system. ESIMS fragmentation data were recorded on an API 3200 by direct injection with a syringe driver.

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45 Extraction and Isolation Lyngbya majuscula was collected from Piti Bomb Holes, Guam in February 2000 by V. Paul The freezedried organism was extracted three times with EtOAc MeOH (1:1) to afford an organic extract (35.5 g). The resulting extract was partitioned between hexanes and 8 0% aqueous MeOH, the methanolic phase was evaporated to dryness, and the residue was further partitioned between n BuOH and H2O. After concentrating the n BuOH extract in vacuo, the resulting residue (7.2 g) was subjected to flash chromatography over silica gel, eluting with CH2Cl2 followed by increasing gradients of i PrOH in CH2Cl2, and finally with MeOH. The fraction eluting with 4% i PrOH/ CH2Cl2 was fractionated on a semipreparative reversedphase HPLC column (YMC Pack ODS AQ, 250 10 mm, 5 m, 2 mL/min; UV detection at 220/254 nm) using a MeOH/H2O linear gradient (75% to 100 % over 30 min, and then 100% MeOH for 10 min). The fraction eluting between tR 19.2 to 20.2 min was then repurified using semipreparative reversedphase HPLC (Luna C18, 250 10 mm, 5 m, 2.0 mL/min; UV detection at 200/220 nm) using a MeOH/H2O linear gradient (75% to 100 % over 20 minutes followed by 100% MeOH for 10 min) to afford compound 2 1 (22 mg) at tR 19 min. Pitiprolamide ( 2 1 ) 20 D 65 ( c 0.3, MeOH); UV (MeOH) max (log ) 202 (4.41) nm; IR (film) max 3749, 2964, 2876, 1734, 1643, 1546, 1522, 1502, 1436, 1282, 1190, 1092 cm 1 ; 1H NMR, 13C NMR and HMBC data, see Table 2 1 ; HRESI/APCIMS m/z 927.5209 [M + Na]+ (calcd C49H72N6O10Na 927.5201) and m/z 905.5390 [M+H]+ (calcd C49H73N6O10 905.5383).

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46 Acid Hydrolysis and Enantioselective Amino Acid Analysis by HPLC/MS A sample of 2 1 (0.1 mg) was treated with 6N HCl (0.5 mL) at 110 C for 24 h. The hydrolysate was concentrated to dryness, resuspended in 100 L H2O, and then analyzed by enantioselective HPLC [column: Chirobiotic TAG (250 4.6 mm), Supelco; solvent: MeOH/10 mM NH4OAc (40:60, pH 5.6); flow rate 0.5 mL/min; detection by ESIMS in positive ion mode (MRM scan)]. The retention times ( tR min; MRM ion pair, LDPro (41.4), LDVal (16.0). The hydrolysate of 2 1 showed peaks corresponding to LPro a nd LVal at tR 14.8 and 8.5 respectively. The MS parameters used were as follows: DP 32 EP 4 CE 21.8 CXP 2.8 CUR 50, CAD medium, IS 4500 TEM 750 GS1 65 and GS2 65. X ray Crystallographyb C49H79N6O13.5, Mr = 968.18, Orthorhombic P 212121, a = 10.534(2) b = 14.952(3) c = 33.511(7) V = 5278.0(19) 3, Z = 0, Dcalc. = 1.218 g cm3, Mo K ( = 0.71073 ), T = 100 K (Table 22 Table 23 ) Data were collected at 100 K on a Bruker DUO system equipped with an APEX II area detector and a graphite monochromator utilizing Mo K radiation ( = 0.71073 ). Cell parameters were refined using 3973 reflections. A hemisphere of data was collected using the scan method (0.5 frame width). Absorption corrections by integration were applied based on measured indexed crystal faces. The structure was solved by the Direct Methods in SHELXTL6 and refined using fullmatrix least squares. The non H atoms were treated b Crystallo graphic data for pitiprolamide have been deposited w ith the Cambridge Crystallographic Data Centre (CCDC accession no. 801619). Copies of the data can be obtained, free of charge, on application to the director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44(0)1223 336033 or email: deposit@ ccdc.cam.ac.uk).

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47 anisotropically, whereas the hydrogen atoms were calculated in ideal positions and were riding on their respective carbon atoms. In addition to the molecule, there are three and a half water molecules in the asymmetric unit (one water refined with 50% occupancy). The water protons were obtained from a Difference Fourier map and were treated riding on their parent oxygen atoms. The amino protons were also obtained from a Difference Fourier map and were refined freely. All of those protons are involved in a network of hydrogen bonded (Table 24) A total of 622 parameters were refined in the final cycle of refinement using 3819 reflections with I > 2 ( I ) to yield R1 and wR2 of 4.99% and 6.26%, respec tively. X ray crystals were analyzed by Dr. Khalil A. Abboud (Department of Chemistry, University of Florida) Cell Viability Assays Cell culture medium was purchased from Invitrogen and fetal bovine serum (FBS) from Hyclone. Cells were propagated and maintained in DMEM supplemented with 10% FBS at 37 C humidified air and 5% CO2. Cells were seeded in 96well plates (MCF7 10,500 cells/well; HCT116 10,000 cells/well). After 24 h, cells were treated with various concentrations of compound 2 1 or solvent cont rol (1% EtOH). After 48 h of incubation, cell viability was measured using MTT according to the manufacturers instructions (Promega). Antibacterial Assay s Pitiprolamide ( 2 1 ) was tested against Mycobacterium tuberculosis [ATCC # 25177] in a disc diffusion assay. The compound was dissolved in EtOH and three different amounts were loaded on sterile filter paper discs. The dry discs where applied to an inoculated agar plate and incubated for 12 days at 37 C, after which the diameter of the zone of inhibition was measured. The microtiter platebased assay method82 was

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48 used to test the antibacterial activity of 2 1 against three human pathogenic bacteria; Bacillus cereus [ATCC # 10987], Staphylococcus aureus [ATCC # 25923] and Pseudomonas aeruginosa [AT CC # BAA 47]. The former organism was grown in LB broth medium at 30 C, while the two latter strains were grown in the same medium at 37 C. For each organism, a standard curve was constructed and used to interpret the assay data. Solvents were used as negative controls, while the positive controls used were ciprofloxacin for Bacillus cereus chloramphenicol for Staphylococcus aureus and carbenicillin for Pseudomonas aeruginosa. To generate the standard curve, an overnight liquid shake culture was serially diluted for each organism to give a range of samples. For each sample, the turbidity was measured at 600 nm in triplicates and the rest of the sample was centrifuged at 13, 200 rpm for 10 min. The supernatant was discarded and the pellets were resuspended in EtOH, moved to preweighed glass vials and dried in a 37 C incubator overnight. The biomass weight was then determined for each sample and the biomass concentration (g/L) was then plotted against the turbidity. The standard curve was generated using Gr aphPad Prism 5 by nonlinear curve fitting. Fo r the m icrotiter plate based bioassay 100 L of a diluted overnight liquid shake culture was added to each well of a 96 well plate. 1 L of the test compound in EtOH was then added and the initial OD was measured at 600 nm using a plate reader. The plate was incubated in the plate reader at the optimum temperature for each organism, and the turbidity was measured every hour for 24 h. The OD values were converted to biomass using the standard curve. The retardat ion of biomass growth was determined using the following equation:

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4 9 (2 1) Where R is the retardation of growth (%), ( C24 C0) is the biomass growth in the control wells, and ( T24 T0) is the biomas s growth in the sample wells. The calculated values were used to determine the IC50 value using GraphPad Prism 5 by nonlinear curve fitting.

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50 Table 21. NMR spectroscopic d ata for p itiprolamide ( 2 1 ) in b enzened6 ( in ppm, J in Hz) at 600 MHz (1H) and 125 MHz (13C) at 20 C Unit C/H # C H ( J ) HMBC a Pro 1 1 173.9 2, 3, 44 2 59.4 5.62, dd (8.3, 3.4) 3, 5a, 5b 3 29.4 1.64, m 2, 5a, 5b 4a 4b 24.6 1.38, m 1.86, m 2, 3, 5b 5a 5b 47.6 3.82, ddd (13. 9, 9.2, 4.2) 4.10, ddd (9.2, 7.7, 7.5) 2, 3, 4b Dpv 6 170.5 7 7 55.7 5.28, dd (11.5, 9.8) 8, 9a, 9b, 16, NH (2) 8 39.2 2.74, m 7, 9a, 9b, 16 9a 9b 39.9 2.25, m 3.30, dd (12.9, 1.5) 7, 16, 11/15 10 141.1 16, 11/15, 12/14 11/15 129.8 7.34, d (7.5) 16, 12/14, 13 12/14 128.7 7.23, m 11/15, 13 13 126.3 7.11, m 11/15, 12/14 16 15.5 0.90, d (6.7) 7, 9a, 9b NH (2) 9.17, d (9.7) Pro 2 17 171.6 7, 18, 19a 18 61.9 4.33 b 19a, 20a 19a 19b 32.3 1.46, m 2.42, m 18, 20b, 21a 20a 20b 22.0 1 .27, m 1.60, m 18, 21a, 21b 21a 21b 46.5 3.51, m 3.64, m 18, 19b Pro 3 22 170.8 23, 24a. 24b 23 59.4 4.33 b 24a, 24b, 25a, 25b, 26a, 26b, 28

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51 Table 2 1. Continued Unit C/H # C H ( J ) HMBC a 24a 24b 28.1 1.11, m 1.28, m 23 25a 25b 24.8 1 .05, m 1.40, m 23 26a 26b 47.2 3.17, m 3.57, m 24a, 24b, 25a, 25b Hiva 27 167.3 23, 28, 29 28 77.7 4.58, d (8.6) 29, 30, 31 29 30.7 2.27, m 28, 30, 31 30 18.3 1.12, d (6.7) 28, 29, 31 31 18.8 0.85, d (6.5) 28, 29, 30 Pro 4 32 172.6 28, 33, 34 33 59.4 4.46, dd (8.6, 6.2) 28, 34, 35a, 35b, 36a, 36b 34 29.6 1.50, m 33, 35a, 35b, 36a, 36b 35a 35b 25.2 1.21, m 0.92, m 33, 34, 36a, 36b 36a 36b 46.8 2.89, m 3.00, m 33, 34, 35a, 35b Val 37 170.1 38, 39, NH (6) 38 55.3 5.21, dd (8.8, 1.9) 40, 41 39 32.5 2.25, m 38, 40, 41 40 17.4 1.54, d (6.8) 38, 39, 41 41 21.5 1.31, d (6.7) 38, 39, 40 NH (6) 7.73, d (8.8) Dmhha 42 173.9 44, 48, 49, NH (6) 43 47.2 44, 48, 49 44 79.3 5.01, dd (11.4, 1.6) 48, 49 45a 45b 32.2 1.61, m 1.77, m 44, 46a, 46b, 47

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52 Table 21. Continued. Unit C/H # C H ( J ) HMBC a 46a 46b 19.5 1.05, m 1.20, m 44, 45a, 45b, 47 47 13.5 0.61, t (7.4) 45b, 46a 48 23.3 1.28, s 44, 49 49 26.1 1.73, s 44, 48 a Protons showing longrange correlation to indicated carbon. b Multiplicity could not be unambiguously deduced because t he peaks for those two protons as well as minor peaks from minor conformer are overlapping

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53 Table 22. Crystal d ata and structure r efinement for p itiprolamide ( 2 1 ). Empirical formula C49H79N6O13.50 Formula weight 968.18 Temperature 100(2) K Wavelength 0.71073 Crystal system Orthorhombic Space group P 212121 Unit cell dimensions a = 10.534(2) = 90. b = 14.952(3) = 90. c = 33.511(7) = 90. Volume 5278.0(19) 3 Z 4 Density (calculated) 1.218 Mg/m3 Absorption coefficient 0.089 mm1 F(000) 2092 Crystal size 0.13 x 0.09 x 0.04 mm3 Theta range for data collection 1.22 to 25.00. Index ranges Reflections collected 39870 Independent reflections 9317 [R(int) = 0.1990] Completeness to theta = 25.00 100.0 % Absorption correction Numerical Max. and min. transmission 0.9968 and 0.9883 Refinement method Fullmatrix l east squares on F2 Data / restraints / parameters 9317 / 0 / 622 Goodness of fit on F2 0.727 Final R indices [I>2sigma(I)] R1 = 0.0499, wR2 = 0.0626 [3819] R indices (all data) R1 = 0.1639, wR2 = 0.0812 Absolute structure parameter 0.4(13) Largest diff. peak and hole 0.336 and 0.443 e.3 R1 = (||F o | |F c ||) / |F o | wR2 = [ w(F o 2 F c 2 ) 2 ] / w F o 2 2 ]] 1/2 S = [ w(F o 2 F c 2 ) 2 ] / (n p)] 1/2 w= 1/[ 2 (F o 2 )+(m*p)2+n*p], p = [max(F o 2 ,0)+ 2* F c 2 ]/3, m & n are constants.

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54 Table 23. Atomic coordinates ( 104) and e quivalent i sotropic d ispl acement p arameters ( 2 103) for p itiprolamide ( 2 1 ). x y z U(eq) a O1 1119(3) 2625(2) 3170(1) 31(1) O2 1880(3) 4265(2) 3968(1) 21(1) O3 1540(3) 6532(2) 4094(1) 28(1) O4 5591(3) 8418(2) 3294(1) 29(1) O5 5986(3) 6446(2) 3614(1) 22(1) O6 8194(3) 5470( 2) 3775(1) 19(1) O7 7624(3) 5907(2) 4391(1) 26(1) O8 6020(3) 3980(2) 3782(1) 20(1) O9 6487(3) 1162(2) 3205(1) 32(1) O10 3167(3) 2852(2) 3359(1) 19(1) O11 538(3) 7403(2) 4762(1) 40(1) O12 7851(3) 9570(2) 3272(1) 42(1) O13 398(5) 373(4) 3201(2) 35(2) O14 8247(4) 6738(2) 5125(1) 47(1) N1 2803(3) 4662(2) 3397(1) 15(1) N2 3521(4) 6246(2) 3864(1) 21(1) N3 3725(4) 7991(2) 3562(1) 24(1) N4 6892(4) 7616(2) 3927(1) 21(1) N5 8056(3) 3712(2) 3958(1) 14(1) N6 5924(3) 2607(2) 3266(1) 20(1) C1 1975(5) 3114( 3) 3236(1) 22(1) C2 1915(5) 4118(3) 3169(1) 19(1) C3 2195(4) 4357(3) 2728(1) 26(1) C4 3589(4) 4633(3) 2739(1) 28(1) C5 3739(4) 5120(3) 3136(1) 25(1) C6 2683(4) 4700(3) 3794(1) 17(1) C7 3631(4) 5321(3) 4010(1) 18(1) C8 3507(4) 5260(3) 4469(1) 21(1) C9 4018(4) 4339(3) 4612(1) 22(1) C10 3681(5) 4174(3) 5041(1) 21(1) C11 2456(5) 3917(3) 5139(2) 27(1) C12 2076(5) 3823(3) 5536(2) 41(2) C13 2924(7) 4014(3) 5842(2) 49(2)

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55 Table 23. Continued. x y z U(eq) a C14 4152(6) 4264(3) 5752(2) 43(2) C15 4530( 5) 4326(3) 5352(2) 30(1) C16 4274(4) 6021(3) 4674(1) 27(1) C17 2550(5) 6792(3) 3953(2) 26(1) C18 2830(5) 7807(3) 3887(2) 25(1) C19 1628(5) 8312(3) 3746(1) 39(2) C20 1716(5) 8211(5) 3295(2) 87(3) C21 3068(5) 8191(3) 3186(1) 43(2) C22 4988(5) 8162(3) 3588(2) 25(1) C23 5636(5) 8031(3) 3987(1) 22(1) C24 5937(5) 8930(3) 4190(1) 27(1) C25 7158(4) 8713(3) 4420(1) 27(1) C26 7922(4) 8123(3) 4131(1) 28(1) C27 6946(5) 6825(3) 3739(1) 24(1) C28 8246(4) 6417(3) 3658(1) 20(1) C29 8499(4) 6417(3) 3214(1) 24( 1) C30 9753(4) 5940(3) 3118(1) 41(2) C31 8502(5) 7385(3) 3054(1) 29(1) C32 7894(4) 5335(3) 4158(2) 20(1) C33 7997(4) 4359(3) 4283(1) 18(1) C34 9272(4) 4225(3) 4504(1) 20(1) C35 9618(4) 3252(3) 4412(1) 21(1) C36 9255(4) 3177(3) 3975(1) 18(1) C37 701 7(5) 3565(3) 3733(1) 18(1) C38 7168(4) 2891(3) 3396(1) 17(1) C39 7971(4) 3290(3) 3049(1) 25(1) C40 7293(4) 4079(3) 2847(1) 33(2) C41 8332(4) 2555(3) 2751(1) 36(2) C42 5650(5) 1728(3) 3207(1) 21(1) C43 4258(4) 1489(3) 3117(1) 22(1) C 44 3322(5) 1876(3 ) 3426(1) 21(1)

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56 Table 23. Continued. x y z U(eq) a C45 3692(5) 1770(3) 3860(1) 25(1) C46 2599(4) 1972(3) 4146(1) 28(1) C47 2945(4) 1763(3) 4578(1) 33(1) C48 3917(4) 1812(3) 2693(1) 28(1) C49 4078(4) 465(3) 3134(1) 34(2) a U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.

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57 Table 24. Hydrogen b onds for p itiprolamide ( 2 1 ) [ and ] D H...A d(D H) d(H...A) d(D...A) <(DHA) O11 H11...O3 0.98 1.93 2.794(4) 146.0 O12 H12"...O4 0.97 1.97 2.939(5) 177.0 O13 H 13'...O1 0.85 2.60 3.454(7) 179.4 O14 H14'...O7 0.96 1.91 2.834(4) 161.4 N2 H2...O5 0.85 1.96 2.745(5) 153.4 N6 H6...O10 0.87 2.25 2.943(5) 136.6 O11 H11"...O14#1 1.07 2.10 3.153(5) 169.4 O12 H12'...O9#2 1.06 1.75 2.790(4) 166.4 O13 H13"...O12# 3 1.06 1.95 2.949(7) 154.9 O14 H14"...O11#4 1.12 1.87 2.880(5) 146.8

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58 Table 25. Results for a ntimycobacterium d isc d iffusion a ssay; Mycobacterium tuberculosis [ATCC 25177]. Compound Diameter of Zone of Inhibition (mm) Pitiprolamide 100 g 50 g 10 g 23 13 0 Streptomycin 10 g 5 g 1 g 40 30 0

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59 Figure 21 S tructure s of pitiprolamide ( 2 1 ) and its closely related analogues dolastatin 16 and homodolastatin 16.

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60 Figure 22 Important HMBC correlations f or sequence i dentification for p itiprolamide ( 2 1 )

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61 Figure 23. MS/MS f ragmentation p attern for p itiprolamide ( 2 1 ).

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62 Figure 24. 1H NMR spectrum for p itiprolamide ( 2 1 ) in benzened6 at 600 MHz at 25 C shows conformational changes over time. Initial spectrum (top), spectrum after 60 h (middle), spectrum after 84 h (bottom). Peak changes were more significant for the residues involved in prolyl amide linkages, as in Dpv and Val protons. This was not the case with acetone, a polar solvent with higher dielectric constant, where only one major isomer was prominent.

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63 A B C Figure 2 5. Crystal structure of pitiprolamide ( 2 1 ) shows intraand intermolecular hydrog en bonding. A) ORTEP diagram for p itiprolamide ( 2 1 ) showing the molecule and water solvent molecules. Hydrogen bonding is shown as dashed lines B) Packing d iagram showing the h ydrogen b onding b etween pitiprolamide ( 2 1 ) and solvent w ater p rotons C) Ster eo ORTEP d rawing of p itiprolamide ( 2 1 ) with h ydrogen bonds s hown as d ashed l ines.

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64 Figure 26 Residue sequences for dolastatin 16, homodolastatin 16, kulokekahilide1 and pitiprolamide ( 2 1 ) ( from top to bottom ). Conserved units are shown in red. Substituted hexanoic and isohexanoic acid residues are shown in blue. Green lines indicate ester linkages. Dotted lines indicate connected residues. The configuration of the fourth unit in all the analogues is noted in bold indicating configurational change at that position.

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65 CHAPTER 3 PITIPEPTOLIDES C F, ANTIMYCOBACTERIAL CYCLODEPSIPEPTIDES FROM THE MARINE CYANOBACTERIUM LYNGBYA MAJUSCULA FROM GUAMa Introduction Pitipeptolides A ( 3 1 ) and B ( 3 2 ) are cyclic depsipeptides isolated from a Guamanian sample of the marine cyanobacterium Lyngbya majuscula.70 Both compounds contain standard proteinogenic as well as N hydroxy acid, and are characterized by the presence of the unique hydroxy fatty acid units 2,2 dimethyl 3 hydroxy 7 octynoic (Dhoya) or 2,2dimethyl 3 hydroxy 7 octenoic (Dhoea) acids (Figure 3 1 ). Both compounds were reported to possess weak cytotoxic and moderate antimycobacterial activities. Moreover, the total synthesis of pitipeptolide A w as achieved, which confirmed its structure and the assigned configuration.83 Revisiting the cyanobacterium on a larger scale led to the identification of the new prolinerich cyclic dep sipeptide pitiprolamide84 and four more pitipeptolide analogues named pitipeptolides C F ( 3 3 3 6 ). Here we report the isolation, structure determination as well as the biological characterization of pitipeptolides C F Isolation and Structure Determination The cyanobacterial sample was extracted three times with EtO Ac MeOH mixtures. This organic extract was subjected to solvent partitioning steps, yielding 7.2 g of a semi polar n BuOH fraction. The n BuOH fraction was fractionated by silica gel chromatography. The presence of pitipeptolides in one fraction was identi fied by 1H a Reproduced in part with permission from Montaser, R., Paul, V. J., Luesch, H Phytochemistry 2011, 72 2068 2074. Copyright (2011) Elsevier B.V.

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66 NMR, and subsequent purification of this fraction by reversedphase HPLC yielded pitipeptolides C F The 1H NMR spectrum of pitipeptolide C ( 3 3 ) indicated close structural similarity to pitipeptolide A ( 3 1 ). HRESIMS suggested a molecular formula of C44H70N5O9 ( m/z 812.5178 for [M + H] +), which is four mass units higher than that of pitipeptolide A Furthermore, the acetylenic carbons were absent in the 13C NMR for this analogue, and two carbon resonances for an additional methylene as well as a methyl carbon were present instead. All this data indicated that compound 3 3 is a tetrahydroanalogue of pitipeptolide A, containing the completely saturated fatty acid derived unit 2,2dimethyl 3 hydroxy octanoic acid (Dhoaa) (Figure 3 1) Pitipeptolid e D ( 3 4 ) had the molecular formula C43H63N5O9 as suggested by the HRESIMS data ( m/z 816.4524 for [M + Na]+). This molecular formula suggested a pitipeptolide A analogue with a lower degree of methylation. Further analysis of 1H and 13C NMR spectra showed that the Phe residue in compound 3 4 lacks the N Me modification (Figure 3 1) The additional amide proton resonates as a doublet at H 6.21 ppm (Table 3 1 protons in the Val ( H 4.17 ppm) and Phe ( H 4 .25 ppm) units, while a ROESY correlation was evident proton in Val ( H 4.17 ppm) and the amide proton in Phe ( H 6.21 ppm), indicating a trans peptide bond. The absence of the N Me group did not influence the amide linkages conformation; th protons in the Val and Phe units was also missing in compound pitipeptolide C and a strong ROESY proton in Val ( H 4.71 ppm) and the N methyl

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67 group ( H 2.78 ppm) in N Me Phe, whic h points to a trans conformation for the amide linkage between both units. The two constitutional isomers pitipeptolides E ( 3 5 ) and F ( 3 6 ) co eluted during the first round of reversedphase HPLC purification of the silica gel chromatography fraction containing pitipeptolides. Both compounds showed 1H NMR spectra very similar to each other and to pitipeptolide A (Table 32) Moreover, HRESIMS (m/z 816.4521 for [M + Na]+ for 3 5 ; m/z 816.4518 for [M + Na]+ for 3 6 ) showed that both compounds have the same molecular formula as pitipeptolide D and are additional pitipeptolide A analogues with one less methylene group. Further analysis of the NMR spectra showed that compound 3 5 compound 3 6 displacement compared t o pitipeptolide A ( Figure 31, Table 32) The structures of compounds 3 3 3 6 were further confirmed by analyzing their MS/MS fragmentation patterns (Figure 3 2 ). hydroxy acids in pitipeptolides C F were determined using chiral HPLC after acid hydrolysis. The configurations of the fatty acid derived units were determined by comparing NMR and optical rotation data with those of pitipeptolide A The closely matching NMR chemical shifts and optical rotations of all the analogues indicated the same relative and absolute configuration. All chiral centers in pitipeptolides C F had S configuration. Related Analog ue s Thacker and Paul have shown that despite the isolation of a plethora of novel secondary metabolites fr om different globally distributed collections of L. majuscula, these chemical compounds could not be strictly associated to specific geographical regions.85 In accordance, we noticed a close chemical relationship between this

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68 population of L. majuscula from Guam and another collection from Madagascar that yielded the cyclodepsipeptides antanapeptins.86 The collection from Madagascar has yielded dolastatin 16 as well as antanapeptins A D. Similarly, this collection from Guam has yielded the dolastatin 16 analogue pitiprolamide and pitipept olides A F; the latter show structural homology to antanapeptins. Moreover, the structural differences between antanapept in A D are the same as between pitipeptolides A C and E displacement. The structural homology between dolastatin 16 and pitiprolamide has been noted before (Chapter 2) .84 For simplicity, we compared the structural differences between anta napeptin A and pitipeptolide A (Figure 33 ), which could be summarized as follows: (1) while both compounds have substituted octynoic acid units at the same position, anantapeptin A has a monomethyl fatty acid derivative (Hmoya) instead of a dimethyl fatty acid unit (Dhoya) in 3 1 ; (2) anantapeptin A incor porates a Hiva unit instead of Hmpa in 3 1 which is a common displacement as mentioned before, and therefore this particular difference probably does not require a genetic variation, (3) the biosyntheti c gene cluster responsible for anantapeptin A is expec ted to have an additional N methyltransferase domain associated with the Ile module, (4) anantapeptin A lacks the Gly unit, giving a hexacyclodepsipeptide instead of the heptacyclodepsipeptide as in 3 1 Because of these differences between both groups o f depsipeptides, antanapeptins may have failed to show any toxicity against brine shrimp or any antibacterial activities.86 The recently reported marine cyanobacterial depsipeptides cocosam ides A and B represent another example of cyanobacterial metabolites that share some structural

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69 features with pitipeptolides.87 Cocosamides were isolated from the marine cyanobacterium Lyngbya majuscula from Cocos Lagoon, Guam, and showed weak cytotoxic activities against HT 29 and MCF7 cancer cell lines. Biological Activity Evaluation Pitipeptolides A ( 3 1 ) and B ( 3 2 ) have been previously shown to possess weak cytotoxicity towards cancer cells as well as moderate antimycobacterial activities.70 It is unclear if the relevant targets in euk aryotic and prokaryotic cells are related or the same. However, pitipeptolides C F appeared to be less cytotoxic against cancer cells than pitipeptolides A and B in HT 29 colon adenocarcinoma and MCF7 breast cancer cell lines (Table 33). The same structur al features required for cytotoxic activity against cancer cells do not appear to be critical for the antimycobacterial effect, since compounds 3 3 and 3 5 showed similar antimycobacterial activities compared to 3 1 and 3 2 and strikingly, compound 3 6 sh owed the highest potency in the disc diffusion assay against Mycobacterium tuberculosis Notably, 3 4 lacked significant activities against both mammalian and bacterial cells. The above findings lead to the following structure activity relationship conclus ions: (1) N methylation in the Phe unit is important for both cytotoxic and antibacterial activities; (2) the system in the fatty acid unit is one of the essential features for the cytotoxic activity in mammalian cells, but it is not essential for the antibacterial activity; (3) decreasing the steric effects at certain units anticancer activity ( 3 5 and 3 6 ), while on the other hand, particularly 3 6 possessed increased antibacterial potency. This indicates that the cytotoxic activity in cancer cells could be separated from the antimycobacterial activity, and the selectivity of the antibacterial compounds could be increased through structural modifications. Pitipeptolides did not show any significant antibacterial

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70 activities against either the Gram negative bacterium Pseudomonas aeruginosa or the Gram positive bacteria Bacillu s cereus and Staphylococcus aureus The structures of pitipeptolides suggest they are biosynthesized by a mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) pathway. N methylation is one of the modifications characterizing nonribosomally synthesized peptides. This modification plays an important role in protecting the peptide bond against proteolytic cleavage, and thus influences the biological activity of the peptide.88, 89 Thus, cellular stabilit y might be one explanation for the much reduced activities in all our assays of pitipeptolide D ( 3 4 ) which lacks this modification. Nonribosomal peptide synthetases are also characterized by moderate substrate specificity, which results in structural div ersity. This specificity can even be lost when it comes to discrimination between two similar substrates,89, 90 which may be the reason for the simultaneous production of the two analogues 3 5 and 3 6 that differ fr om 3 1 by only one methylene group. The abundance of pitipeptolides in this sample suggests that this class of cyclic depsipeptides may play an important ecological role. Pitipeptolide A is the major depsipeptide in this series (378 mg, 0.14% of total dry weight) followed by pitipeptolide B (70.5 mg, 0.024% of total dry weight). The production of pitipeptolides C, E and F (23 times lower than that of pitipeptolide A and only of pitipeptolide D was isolated, possibly indicating that pitipeptolide A is the most benefic ial analogue for this organism. In accordance with this conclusion, pitipeptolide A

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71 was previously reported as a defensive s econdary metabolite that acts as a feeding deterrent at natural concentrations against a range of marine grazers.91 Towards Target Identification of Pitipeptolide A Once a hit compound has been characterized in a phenotypic screen, the identification of its cellular target and mechanism of action remains a bottleneck for its development into a marketed drug. T his mechanistic knowledge is crucial to anticipate the potential side effects and, consequently, to avoid costly clinical failures. It also allows for lead optimization by medicinal chemists for better drug target interaction and possibly lower off target effects .60, 61 Affinity based biochemical isolation of molecular targets remains o ne of the common yet powerful methods for target identification.92 The bioactive molecule can be tagged with a biotin entity and used in a pull down assay utilizing strept ( avidin ) resin ,9397 based on the st rong binding affinity between biotin and strept(avidin) .98, 99 For example, this method was effective in the identification of the molecular target of the cytotoxic marine natural product pateamine A.100 A pull down assay utilizing the biotinylated derivative showed that this m arine metabolite binds to eIF4A a key factor in eukaryotic translation initiation, and causes a subsequent inhibition of translation initiation and protein synthes is .94 Another efficient modification for target identification purpose is linking the bioactive molecule to a fluorescent moiety, which facilitates the localization of the drug in cellular components. For exam ple, the subcellular localization of the marine antifungal compound theonellamide F101 in yeast cells was determined using a fluorescent derivative which showed that this compound is distributed in the same manner as some lipid molecules in Schizosaccharomyces pombe, and further binding assays proved that this compound is a sterol binding molecule that causes

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72 membrane damage.102 However, i n order to run the above classical methods, modif ication of the bioactive molecule should be done in a way that r etains its biological activity and enables for further use of this derivative to probe the drug action. Therefore, the identification of the major structure activity relationship features is essential. Design of Synthetic Probes to Investigate Mechanism of Action Pitipeptolide A appears to be a good candidate for target identificati on studies for the following reasons: 1. Phenotypic cytotoxicity and antimycobacterial screens suggested that the terminal alkyne in pitipeptolide A Dhoya moiety (Figure 3 1) is essential for the cytotoxic activity in mammalian cells but not for the antimycobacterial activity in M. tuberculosis. Accordingly, this terminal alkyne appears to be a suitable site for modification to probe the mechanism of action of pitipeptolide A in mycobacterial cells. 2. The terminal alkyne offers a feasible site for chemical mod ification since it could be easily linked to any molecule with a terminal azide group through Cu(I)catalyzed Huisgen cycloaddition reaction ( click chemistry ) .103106 3. Pitipeptolide A was isolated in relatively abundant amounts, which offers enough material for further target identification studies. Accordingly, two molecular probes were designed and semi synthesized for pitipeptolide A ( 3 1 ) ; a biotinylated derivative ( p itiA b iotin ( 3 7 )) for affinity chromatography experiments, and a fluorescent derivative ( p itiA f l u o r (3 8 )) (Figure 3 4) for monitoring the subcellular localization of the drug molecule. A bi functional probe was designed and synthesized to include the affinity tag biotin at one end to be used as the affinity bait, and an azide terminal at the other end to be linked to the bioactive compound pitipeptolide A ( 3 1 ) (Figure 3 4 3 5 ). The two ends are linked by a spacer arm containing a poly ethylene glycol (PEG) part and an aliphatic linear chain. Thi s linker part was designed to offer the following advantages: (1) it offers an extra space between biotin and pitipeptolide A, which decreases steric

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73 hindrance and extend the biotin tag far enough to fit into the strept ( avidin ) binding pocket during affini ty purification99, 107, 108; (2) t he PEG linker offers the advantage of increasing the water solubility of the molecule.109 The synthesis scheme started with a 3 n PEG, where the two hydroxy terminals were converted into good leaving groups through tosylation110112 to give compound 3 9 in good yield (74.5%) Using 1 equivalent of sodium azide, the tosyl group at one end in 3 9 was converted to the corr esponding azide113, 114 ( 3 10, 55% ). The other terminal was converted to the bromoderivative 3 11 (84%) followed by the reduction of the azide group to the corresponding amine which was Boc protected ( 3 12, 60% ) to ensure its stability until the next step is performed. After deprotection of 3 12, t he intermediate was coupled to the commercially available Boc protected valeric acid to give 3 1 3 in 48% yield followed by another azidation reaction to introduce the azi de terminal ( 3 1 4, 91% yield) that will be used for click chemistry. Compound 3 14 was then coupled to the activated biotin succinimide ester to yield the bifunctional probe 3 15 (69%) which was linked to the alkyne terminal in pitipeptolide A ( 3 1 ) through Cu(I) catalyzed Huisgen cycloaddition reaction (click chemistry)104, 106 to yield p itiA b iotin ( 3 7 80% ) (Figure 3 5 ). The fluorescent probe ( 3 8 ) was synthesized by linking pitipeptolide A ( 3 1 ) to the commerc ially available rhodaminetype fluor escent molecule azidef luor 488 (Click Chemistry Tools, Scottsdale, AZ)b through click chemistry (67% yield for the major isomer ) (Figure 3 6) Bioactivity Evaluation of the Synthetic P robes In order to confir m that the prepared derivatives 3 7 and 3 8 retained the antimycobacterium activity, dis c diffusion assay was repeated for the three compounds, b Azide fluor compound was purchased as a mixture of two isomers. Isomers differ in the a ttachment of the PEG linker to the phenyl ring at the 5or 6position.

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74 pitipeptolide A ( 3 1 ) piti A biotin ( 3 7 ) a nd piti A fl uor ( 3 8 ) Both derivatives were still active against M. tuberculos is when tested at 50 g (Table 3 4). Therefore, both probes could be used for further studies to iden tify and validate drug targets. Conclusion A population of the cyanobacterium Lyngbya majuscula from Piti Bomb Holes in Guam produces a class of bioactive cyclodepsipeptides named pitipeptolides. In addition to the previously reported major analogues pitipeptolides A ( 3 1 ) and B ( 3 2 ), the cyanobacterium contained more analogues, four of which are reported here ( 3 3 3 6 ). All pitipeptolide analogues have minor structural differences. However, the structural variations gave a better insight into the contribution of some structural features to cancer cytotoxic and antimycobacterial activities. Pitipeptolides might prove to be privileged structures that could interact with different biological targets. Consequently, those compounds showed several biological activities besides their ecological role. Despite different environmental and geographical regions, chemical similarity was detected between this Guamanian sample and another L. majuscula sample from Madagascar. The structural similarity between the isolated secondary metabolites from both samples suggests genetic similarity including the putative gene cluster encoding biosynthetic enzymes, and pitipeptolides could be considered analogues of antanapeptins. Notably, those structural modifications uniquely granted the pitipeptolides cytotoxic and antibacterial activities that were not found in the antanapeptins. Therefore, we could consider pitipeptolides as lead compounds and we expect further structural optimization to selectively enhance their cytotoxic or antibacterial activities. The availability of a biotinylated and a fluorescent probe for

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75 pitipeptolide A through chemical synthesis allows for further mechanistic investigations to identify the biological targets of this secondary metabolite in M. tuberculosis Experimental Section General Experimental Procedures Optical rotation was measured on a PerkinElmer 341 polarimeter. UV and optical density were measured on a SpectraMax M5 (Molecular Devices), and IR data were obtained on a PerkinElmer Spectrum One FT IR Spectrometer. 13C NMR spectrum was recorded on a Bruker 500 MHz spectrometer operating at 125 MHz. 1H and 2D NMR spectra were recorded on a Bruker Av ance II 600 MHz spectrometer. All spectra were obtained in CDCl3 using residual solvent signals ( H 7.26, C 77.16 ppm) as internal standards. HSQC and HMBC experiments were optimized for 1JCH = 145 and 1JCH = 7 Hz, respectively. HRMS data was recorded on an Agilent LC TOF mass spectrometer equipped with an APCI/ESI multimode ion source detector in positive ion mode. LC MS data were obtained using an API 3200 triple quadrupole MS (Applied Biosystems) equipped with a Shimadzu LC system. ESIMS fragmentation data were recorded on an API 3200 by direct injection with a syringe driver. 2Hydroxy isovaleric acid (Hiva) standards were purchased from SigmaAldrich. 2 Hydroxy 3 methyl pentanoic acid (Hmpa) standards were synthesized from isoleucine.115 Azide Fluor 488 was purchased from Click Chemistry Tools (Click Chemistry Tools, Scottsdale, AZ) Marine Cyanobacterial Sample The sample of the marine cyanobacterium Ly ngbya majuscula (recollection of UOG strain VP627) was collected at Piti Bomb Holes, Guam, in February 2000 by V. Paul A voucher sample has been preserved at the Smithsonian Marine Station at Fort Pierce, FL.

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76 Extraction and Isolation The freeze dried organism was extracted three times with EtOAc MeOH (1:1) to afford the crude organic extract (35.5 g). The resulting extract was partitioned between hexanes and 80% aqueous MeOH; the methanolic phase was concentrated to dryness, and the residue was further par titioned between n BuOH and H2O. After concentrating the n BuOH extract in vacuo, the resulting residue (7.2 g) was subjected to flash chromatography over silica gel, eluting with CH2Cl2 followed by increasing gradients of i PrOH in CH2Cl2, and finally wit h MeOH. The fraction eluting with 4% i PrOH in CH2Cl2 was fractionated on a semi preparative reversedphase HPLC column (YMC Pack ODS AQ, 250 10 mm, 5 m, 2 mL/min; UV detection at 220/254 nm) using a MeOH/H2O linear gradient (75% to 100% over 30 min, and then 100% MeOH for 10 min) to yield 10 collected fractions. Pitipeptolides A ( 3 1 ) (378 mg) and B ( 3 2 ) (70.5 mg) eluted at tR 21.3 min (fraction 4) and tR 23.8 m in (fraction 7), respectively. Pitipeptolide C ( 3 3 ) (23 mg) eluted at tR 25 min as a single peak (fraction 9). Further purification of fractions 2 and 3 gave compounds 3 4 3 5 and 3 6 (see below). Pitipeptolide D ( 3 4 ) was further purified from fraction 2 (eluting between tR 19.2 to 20.2 min using the conditions mentioned above) using semipreparative reversedphase HPLC (Luna C18, 250 10 mm, 5 m, 2.0 mL/min; UV detection at 200/220 nm) with a MeOH/H2O linear gradient (75% to 100% aqueous MeOH over 20 minutes then 100% MeOH for 10 min). The peak eluting at tR 19.9 min was subjected to further purification on another semipreparative reversedphase HPLC column (Phenomenex Phenylhexyl, 250 10 mm, 5 m, 2.0 mL/min; UV detection at 220/200 nm) using a

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77 MeOH/H2O linear gradient (85% to 100% aqueous MeOH over 35 min, and then 100% MeOH for 10 min) t o afford 1.1 mg of the pure compound at tR 20.1 min. Pitipeptolides E ( 3 5 ) and F ( 3 6 ) co eluted at tR 20.6 min (fraction 3 from the first HPLC run mentioned above on the YMC Pack ODS AQ column) to give a total of 68 mg of an impure fraction. 5 mg of thi s mixture was further purified several times on an analytical reversedphase HPLC column (Allure Restec C18, 250 4.6 mm, 5 m, 1.0 mL/min; UV detection at 220/200 nm) using a MeOH/H2O linear gradient (75% to 100% aqueous MeOH over 35 min, and then 100% M eOH for 10 min) to yield compound 3 5 at tR 12.2 min (2.2 mg), and compound 3 6 at tR 12.7 min (1.9 mg). Extrapolating those values suggests a total of about 30 mg of 3 5 and 26 mg of 3 6 in this sample. Pitipeptolide C ( 3 3 ) Colorless, amorphous solid; [ 20 D 121 ( c 0.11, MeOH); UV (MeOH) max (log ) 202 (4.57) nm; IR (film) max 3403, 2961, 2932, 2874, 1727, 1653, 1511, 1464, 1415, 1371, 1188, 1030, 736, 702 cm 1 ; 1H NMR, 13C NMR and HMBC data, see Table 3 1; HRESI/APCIMS m/z 812.5178 for [M + H]+ ( calcd for C44H70N5O9 812.5168). Pitipeptolide D ( 3 4 ) 20 D 112 ( c 0.12, MeOH); UV (MeOH) max (log ) 202 (4.29) nm; IR (film) max 3410, 2966, 2935, 2876, 1735, 1654, 1513, 1455, 1370, 1180, 1039, 735, 701 cm 1 ; 1H NMR, 1 3C NMR and HMBC data, see Table 3 1; HRESI/APCIMS m/z 816.4524 for [M + Na]+ (calcd for C43H63N5O9Na 816.4518). Pitipeptolide E ( 3 5 ) 20 D 105 ( c 0.13, MeOH); UV (MeOH) max (log ) 202 (4.34) nm; IR (film) max 2967, 2372, 2343, 2297, 1986,

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78 1655, 1512, 1175, 804, 702 cm 1 ; 1H NMR, 13C NMR and HMBC data, see Table 3 2; HRESI/APCIMS m/z 816.4521 for [M + Na]+ (calcd for C43H63N5O9Na 816.4518). Pitipeptolide F ( 3 6 ) 20 D 101 ( c 0.10, MeOH); U V (MeOH) max (log ) 202 (4.34) nm; IR (film) max 3404, 2964, 1726, 1646, 1506, 1413, 1370, 1275, 1177, 1094, 1032, 967, 805, 733, 701 cm 1; 1H NMR, 13C NMR and HMBC data, see Table 3 2; HRESI/APCIMS m/z 816.4518 for [M + Na]+ (calcd for C43H63N5O9Na 816.4518). Acid Hydrolysis and Enantioselective Analysis A sample of compounds 3 3 3 6 (0.1 mg each) was hydrolyzed with 6N HCl (0.5 mL) at 110 C for 24 h. The hydrolysate was concentrated to dryness, resuspended in 100 L H2O, and then analyzed by chiral HPLC. Amino acid units were analyzed by HPLC/MS chiral analysis [column: Chirobiotic TAG (250 4.6 mm), Supelco; solvent: MeOH/10 mM NH4OAc (40:60, pH 5.6); flow rate 0.5 mL/min; detection by ESIMS in positive ion mode (MRM scan)]. The retention times ( tR product) of the authentic amino acids were as follows: LDPro (32), LDVal (17.0), LDIle (20.5), LPhe (15.1; DPhe (22.5), N Me -L2), N Me -DPhe (37.0). The hydrolysates showed peaks corresponding to Lamino acids at tR 12.3, 7.7, 8.2, 15.1 and 21.2 min. The MS parameters used were as follows: DP 32 EP 4 CE 21.8 CXP 2.8 CUR 30, CAD medium, IS 4500, TEM 700, GS1 65, and GS2 65. The absolute hydroxy acid units were analyzed by chiral HPLC analysis under different conditions; [column: CHIRALPAK MA (+) (50 4.6 mm); solvent: Me CN/2 mM CuSO4 (10:90); flow rate 1 mL/min; detection by UV (254 nm)]. The retent ion times ( tR

PAGE 79

79 min) of the authentic standards were as follows: (2S ,3 S ) Hmpa (30.5), (2 R ,3 R ) Hmpa (17.0), (2 S ,3 R ) Hmpa (24.1), (2R ,3 S ) Hmpa (14.0), (2S ) Hiva (8.9), (2 R ) Hiva (5.0). The retention times of the samples corresponded to (2S ,3 S ) Hmpa (30.5) in c ompounds 3 3 3 4 and 3 6 and (2S ) Hiva (8.9) in 3 5 Cell V iability A ssay Cells were propagated and maintained in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) at 37 C humidified air and 5% CO2. Cells were seeded in 96well plates (MCF7 10,500 cells/well; HT 29 13,000 cells/well). After 24 h, cells were treated with various concentrations of the test compound, or solvent control (1% EtOH). After 48 hours of incubati on, cell viability was measured using MTT according to the manufacturers instructions (Promega, Madison, WI). Disc D iffusion A ssay An inoculum of Mycobacterium tuberculosis [ATCC # 25177] was grown in a liquid shake culture (Middlebrook 7H9 broth with gly cerol and Middlebrook ADC enrichment) for 12 days at 37 C and adjusted to OD600 inoculate the agar plates. The test compounds were dissolved in EtOH and three different amounts were loaded on sterile 6mm filter paper discs The discs were kept at room temperature for 10 minutes to dry, and then loaded on the inoculated agar plates. The plates were incubated in a humidified environment at 37 C for 12 days, after which the diameter of zone of inhibition (mm) was measured. St reptomycin was used as positive control and the solvent (10 L) was used as negative control. The experiment was done in duplicate.

PAGE 80

80 Synthetic Procedures (Ethane 1,2 di ylbis(oxy))bis(ethane 2,1 diyl) bis(4 methylbenzenesulfonate) ( 3 9 ) : Triethylamine (28 mL) was added to a solution of triethylene glycol (9 g, 8 mL 5.99 mmol, 1 eq ) in DCM (150 mL) and cooled to 0C in an ice bath Toluenesulfonyl chloride (24 g 12.5 mmol, 2.1 eq ) was added in several portions, and the solution was stirred at 0C for an hour until a white precipitate was formed The solution was warmed up to room temperature and stirred overnight. The salt was filtered, washed with methylene chloride and the solvent was removed. The residue was redissolved in methylene chloride (10 0 m L ) and washed with water (3 50 mL). The organic layer was dried over MgSO4, filtered, and the solvent was evaporated in vacuo to give the pure product (20.5 g, 74.5%) as a white solid. 1H NMR (400 MHz, CDCl3) : = 7.79 (d, J = 8.2 Hz, 4 H), 7.34 (d, J = 8.0 Hz, 4 H), 4. 13 ( t, J = 4.7 Hz 4 H), 3. 65 ( t, J = 4.7 Hz 4 H), 3.53 (s, 4 H), 2.44 (s, 6 H). 13C NMR (10 0 MHz, CDCl3) : = 145.13, 133.09, 130.09, 128.16, 70.88, 69.47, 68.93, 21.87 LRMS: m/z 459.4 [M + H]+, 481.3 [M + Na]+. 2 (2 (2 azidoethoxy)ethoxy)ethyl 4 me thylbenzenesulfonate ( 3 10) : Sod ium azide (425.7 mg, 6.5 mmol, 1.5 eq) was added to a solution of the ditosylate 3 9 (2 .0 g, 4.3 mmol, 1 eq) in DMF (20 mL). The solution was stirred at 50 C for 24 hours and then quenched by adding water (10 mL) and EtOAc (50 mL). The organic layer was separated and washed with water (3 20 mL), dried over MgSO4 and the solvent was removed in vacuo to give 1.3 g of crude product. The crude product was purified using silica gel column chromatography (hexanes/EtOAc, 3:1) to give the pure azide (580 mg, 55%). 1H NMR (400 MHz, CDCl3) : = 7.79 ( br d, J = 8. 0 Hz, 2 H), 7.34 ( br d, J = 8.0 Hz,

PAGE 81

81 2 H), 4. 16 ( t, J = 4.7 Hz 2 H), 3. 70 ( t, J = 4.9 Hz 2 H), 3. 64 ( t, J = 4.9 Hz, 2 H ), 3.6 (s, 4H), 3.36 ( t, J = 5.1 Hz, 2 H ) 2.44 (s, 3 H). LRM S: m/z 284.2 [M + H]+. 1 azido2 (2 (2 bromoethoxy)ethoxy)ethane ( 3 11) : Sodium bromide (6.0 g, 58.66 mmol, 5 eq) was added to a solution of the azide 3 10 (3.3 g, 11.7 mmol, 1 eq) in DMSO (100 mL). The solution was refluxed overnight in an oil bath at 60C. The reaction was quenched by adding water (30 mL) and the product was extracted with EtOAc (3 100 mL). The organic layer was washed with water (3 30 mL), dried over MgSO4 and the solvent was removed. The crude product (3.2 g) was purified using silica gel column chromatography (hexanes/EtOAc, 3:1) to give the pure compound (2.3 g, 84 %). 1H NMR (400 MHz, CDCl3) : = 3.82 ( t, J = 6.3 Hz 2 H), 3. 71 3.68 ( m, 6H ), 3. 48 ( t, J = 6.3 Hz, 2 H ), 3.39 ( t, J = 5.2 Hz, 2 H ). LRMS: m/z 260.0, 262.0 (1:1) [M + Na ]+. Tert butyl (2 (2 (2 bromoethoxy)ethoxy)ethyl)carbamate ( 3 12 ) : Triphenyl phosphine (4.0 g) was added to a solution of the azide 3 11 (2.3 g) in THF (60 m L ). The mixture was stirre d at r.t for 2 h, then 60 mL of distilled water was added and th e mixture was stirred for 16 h The solvent was then removed in vacuo CH3CN (88 mL ) was added to the crude product, followed by (Boc)2O (3.4 g) and 1N NaOH (11 m L ). The reaction mixture w as stirred again for 4 h at r.t, and then the mixture was filtered and the solvent removed. The filter cake was washed with EtOAc and the solution was dried over MgSO4 and then filtered again. The solvent was removed to give the crude product as a white solid (8.76 g) which was further purified by column chromatography ( hexanes/Et OAc, 1:1) to yield the pure product (1.8 g, 60 %). 1H NMR (400 MHz, CDCl3) : = 3.79 (t, J = 6.2 Hz, 2H), 3.68 3.59 (m, 4H), 3.53 (t, J = 4.9 Hz, 2H), 3.47 (t,

PAGE 82

82 J = 6.2 Hz, 2H), 3.30 (d, J = 4.7 Hz, 2H), 1.42 (s, 9H). 13C NMR (10 0 MHz, CDCl3) : = 156.12, 79.59, 71.37, 70.61, 70.49, 70.34, 40.53, 30.47, 28.63. Tert butyl (5 ((2 ( 2 (2 bromoethoxy)ethoxy)ethyl)amino 5 oxopentyl)carbamate ( 3 13): Deprotection of 3 12 was performed using 4M HCl. Compound 3 12 (1.7 g, 5.7 mmol, 1 eq) was stirred in a solution of HCl (4M solution in EtOAc, 1.7 mL, 57.2 mmol, 10 eq) for 2 h at r.t. After TLC showed complete reaction, the solvent was removed and the residue was washed with Et2O and dried again. The free amine (1.4 g, 5.7 mmol, 1 eq) was dissolved in DCM (60 mL). N methyl morpholine (0.7 mL, 6.2 mmol, 1.1 eq) was added to the solution at 0 C and stirred for 10 min. The commercially available Boc protected 5aminovaleric acid ( 1.2 g, 5.7 mmol, 1 eq) was then added to the solution followed by DCC (1.3 g, 6.3 mmol, 1.1 eq) and HOBt (0.9 g, 6.2 mmol, 1.1 eq). The solution mixture was st irred at r.t for 24 h and then the solution was filtered, washed with 5% Na2CO3 followed by brine and the solvent was removed. The crude product (4 g) was purified by column chromatography (hexanes/ acetone, 1:3) to give the pure product (1.12 g, 48 %). 1H NMR (400 MHz, CDCl3) : = 6.15 (s, 1 H), 4.71 (s, 1 H), 3.78 (t, J = 6.1 Hz, 2H), 3.67 3.57 (m, 4 H), 3. 52 (t, J = 5 .1 Hz, 2H), 3.49 3.37 (m, 4H) 2.17 (t, J = 7. 6 Hz, 2H), 1.62 (dt, J = 12.5, 7.6 Hz, 2 H), 1.53 1.42 (m, 4 H), 1.39 (s, 9 H). LRMS: m/z 433. 2, 435.2 (1:1) [M + Na]+. Tert butyl (5 ((2 (2 (2 azidoethoxy)ethoxy)ethyl)amino) 5 oxopentyl)carbamate ( 3 14): Compound 3 13 (1.12 g, 2.7 mmol, 1 eq) was dissolved in DMSO (10 mL). Sodium azide (888 mg, 13.6 mmol, 5 eq) was then added and the solution was stirred at 50 C in an oil bath for 24 h. The reaction was quenched by adding water (5 mL) and the product was extracted with EtOAc (2 30 m L ). The

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83 organic layer was washed with water (4 10 m L ), dried over MgSO4 and the solvent was evaporated in vacuo to give the pure product (920 mg, 91 %) as a yellow liquid. 1H NMR (400 MHz, CDCl3) : = 6.10 (s, 1 H), 4.70 ( s, 1 H), 3.69 3.59 (m, 6 H), 3.53 (t, J = 5.1 Hz, 2 H), 3.45 3.36 (m, 4 H), 2.20 2.15 (m, 2 H), 1.68 1.59 (m, 2 H), 1.53 1.44 (m, 2 H), 1.41 (s, 9H). LR MS: m/z 396.2 [M + Na]+. N (2 (2 (2 azidoethoxy)ethoxy)ethyl) 5 (5 ((3a S ,4 S ,6a R ) 2 oxohexahydro1 H theio[3,4 d ]imidazol 4 yl)pentanamido)pentamide ( 3 15) : D biotin (500 mg, 2.0 mmol, 1 eq) was dissolved in DMF (45 m L ). N hydroxysuccinimide (282.7 mg, 2.4 m mol, 1.2 eq) was then added to the solution, followed by EDC (471.2 mg, 2.4 mmol, 1.2 eq). The reaction mixture was stirred at r.t for 24 h and then the solvent was removed in vacuo using toluene (4 50 m L ). The remaining residue was dissolved in DCM (50 m L washed with 5% Na2CO3 (3 30 m L ) and brine (2 20 m L ). The organic layer was dried over MgSO4 and the solvent was removed in vacuo to give the activated biotin product (340 mg, 71.5%) as a white solid. The BOC protected azide 3 14 (500 mg, 1.3 mmol, 1 eq) was first deprotected by stirring in HCl (4M solution in EtOAc; 3.5 mL, 10 eq) at r.t. for 2 h. The solvent was evaporated, the product was washed with Et2O (3 1 mL) and the solvent was removed again to yield the free amine (415 mg). The free amin e (415 mg, 1.3 mmol, 1 eq) was dissolved in DMF (20 m L ). The activated biotin (480 mg, 1.3 mmol, 1.05 eq) was then added to the solution followed by a slow addition of triethylamine (0.6 mL, 4.0 mmol, 3 eq), and the solution was stirred at r .t for 24 h Th e reaction was then quenched by azeotropic distillation using toluene to give the crude product as a yellow solid (1.2 g). The product was purified using silica gel column chromatography (CHCl3/ MeOH, 8:1) to

PAGE 84

84 give the pure product as white crystals (462.5 mg, 69 %). 1H NMR (400 MHz, DMSO ) : = 7. 84 ( t J = 5.2 Hz, 1 H), 7. 78 ( t J = 5.2 Hz, 1H) 6. 41 ( s, 1 H), 6.36 (s, 1H), 4.32 4.25 (m, 1H), 4.14 4.09 (m, 1H), 3. 58 ( t, J = 4.7 Hz 2H), 3.51 (dq, J = 5.8, 3.2 Hz, 4H), 3.37 (m, 4 H), 3.16 (dd, J = 11.5, 5.6 Hz, 2H), 3.11 2.94 (m, 4 H), 2.80 (dd, J = 12.4, 5.1 Hz, 1H), 2.03 (q, J = 7.0 Hz, 4H), 1.64 1.54 (m, 1H), 1.53 1.37 (m, 5H), 1.36 1.22 (m, 4H). 13C NMR (10 0 MHz, DMSO ) : = 172.74, 172.46, 163.38, 70.28, 70.25, 69.94, 69.84, 61.70, 59.86, 56.10, 5 0.65, 45.95, 39.08, 38.81, 35.88, 35.61, 29.48, 28.90, 28.71, 26.01, 23.45. HRESI m/z 500.2653 [M + H ]+ (calcd C21H37N7O5S 500.2650) and m/z 522.2476 [M + Na ]+ (calcd C21H37N7O5S Na 522.2469) Piti A b iotin ( 3 7 ) : Pitipeptolide A ( 3 1 ) (3.65 mg, 0.0045 mmol 1 eq) and the biotinylated probe 3 15 (4.51 mg, 0.009 mmol, 2 eq) were suspended in a 1:1 mixture of water and tert butyl alcohol (0.3 mL). Sodium ascorbate (22.6 L of freshly prepared 0.1 M solution in water, 0.5 eq) was added, followed by CuSO4 (15.1 L of 0.03 M solution in water, 0.1 eq) and the mixture was stirred overnight at r.t. The reaction was quenched by removing the solvent in vacuo The crude product was then purified using semi preparative reversedphase HPLC (Column: YMC Pack ODS AQ, 250 x 10 mm, 5 m, 2 mL/min; UV detection at 200/220 nm) using a MeOH/H2O linear gradient (75% to 100 % over 30 min, and then 100% MeOH for 10 min) to afford the pure p itiA b iotin ( 3 7 ) product (4.8 mg, 80 %) at tR 19.2 min as a colorless oil. 1H NMR (500 MHz, CDCl3) : = 7.84 (d, J = 8.0 Hz, 1H), 7.60 (s, 1H), 7.32 7.22 (m, 8H), 7.12 (d, J = 7.1 Hz, 2H), 6.85 (t, J = 5.3 Hz, 1H), 6.64 (t, J = 5.4 Hz, 1H), 6.45 (d, J = 9.5 Hz, 1H), 6.09 (d, J = 9.1 Hz, 1H), 5.85 (s, 1H), 5.09 (s, 1H), 5.05 5.00 (m, 1H), 4.94 (d, J = 6.3 Hz, 1H), 4.70 (dd, J = 9.1, 2.0 Hz, 1H), 4.62 (dd, J = 9.6, 7.2 Hz, 3H), 4.51 (dd, J = 13.0, 7.1 Hz, 2H), 4.35

PAGE 85

85 4.30 (m, 1H), 4.11 4.06 (m, 1H), 4.03 3.97 (m, 3H), 3.86 (dd, J = 11.2, 3.8 Hz, 1H), 3.73 3.53 (m, 8H), 3.50 (dd, J = 10.9, 5.8 Hz, 3H), 3.44 3.38 (m, 2H), 3.30 3.19 (m, 4H), 3.18 3.08 (m, 2H), 2.91 (dd, J = 12.8, 4.9 Hz, 1H), 2.79 (s, 4H), 2.78 2.69 (m, 2H), 2.67 2.58 (m, 1H), 2.56 2.50 (m, 1H), 2.25 (t, J = 7.2 Hz, 2H), 2.20 (t, J = 7.2 Hz, 2H), 2.04 1.91 (m, 4H), 1.75 (d, J = 23.4 Hz, 14H), 1.72 1.50 (m, 16H), 1.46 (dd, J = 14.6, 7.1 Hz, 4H), 1.30 (s, 3H), 1.28 1.16 (m, 3H), 1.14 (s, 4H), 1.00 (d, J = 6.7 Hz, 3H), 0.95 0.86 (m, 18H). 13C NMR ( 125 MHz, CDCl3) : = 129.08, 128.83, 127.17, 122.70, 78.19, 65. 97, 61.85, 61.61, 61.13, 60.24, 55.53, 53.53, 37.13, 35.30, 29.62, 23.09, 20.42, 19.42, 16.16, 15.82, 14.84, 11.84, 10.75. HRESI m/z 1329.7260 for [M + Na]+ (calcd for C65H102N12O14SNa 1329.7251). Piti A f luor ( 3 8 ) : Pitipeptolide A ( 3 1 ) (3 mg, 0.0037 mmol, 1 eq) and the commercially available azidefluor compoundc ( 4.3 mg, 0.0074 mmol, 2 eq) were suspended in a 1:1 mixture of water and tert butyl alcohol (0.3 mL). Sodium ascorbate (19 L of freshly prepared 0.1 M solution in water, 0.5 eq) was added, fol l owed by CuSO4 ( 15 L of 0.03 M solution in water, 0.1 eq). The mixture was stirred overnight. The reaction was quenched by removing the solvent in vacuo The crude product was then purified using semipreparative reversedphase HPLC (Column: Luna Phenyl He xyl column, 250 x 10 mm, 5 m, 2 mL/min; UV detection at 220/254 nm) using a MeCN/H2O linear gradient (50% over 10 min, then 50% to 75% over 10 min, followed by 70% to 100 % over 10 min, and then 100% MeCN for 10 min) to afford the major p itiA f luor isome r ( 3 8 ) (3.5 mg) at tR 17.2 min. HRESIMS: m/z 1404.6840 for [M + Na]+ (calcd for C73H95N11O16Na 1404.6850), m/z 1382.7012 for [M + H ]+ (calcd for c Azide fluor compound was purchased as a mixture of two isomers. Isomers differ in the attachment of the PEG linker to the phenyl ring at the 5or 6position.

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86 C73H9 6N11O16 1382.7031), m/z 691.8556 for [M + 2H ]2+ (calcd for C73H9 7N11O16 691.8552), m/z 702.8469 for [M + H + Na ]2+ (calcd for C73H9 6N11O16Na 702.8462).

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87 Table 31. NMR spectroscopic data for pitipeptolides C ( 3 3 ) and D ( 3 4 ) in CDCl3 ( in ppm, J in Hz) at 600 MHz Unit C/H # Pitipeptolide C ( 3 3 ) Pitipeptolide D ( 3 4 ) C H ( J ) HMBC a C H ( J ) HMBC a Dhoaa b /Dhoya c 1 175.7 qC 3, 2 (Val), 9, 10, NH (Val) 175.9 qC 3, 2 (Val), 9, 10, NH (Val) 2 45.7 qC 3, 9, 10 46.0 qC 3, 9, 10 3 77.6 CH 4.92 dd (9.3, 3.2) 9, 10 77.3 CH 5.11 dd (10.2, 2.3) 4, 5, 9, 10 4 30.3 CH 2 1.56 m 1.45 m 28.4 CH 2 1.79 m 1.53 m 2, 5, 6 5 25.7 CH 2 1.23 m 3, 4 24.4 CH 2 1.43 m 3, 4, 6 6 31.9 CH 2 1.24 m 4, 7, 8 18.0 CH 2 2.27 m 2.19 m 3, 4, 5 7 22.5 CH 2 1.29 m 6, 8 83.6 qC 5, 6, 8 8 14.4 CH 3 0.88 t (7.0) 7 69.3 CH 1.96 t (2.6) 6 9 19.8 CH 3 1.28 s 3, 10 18.5 CH 3 1.25 s 3, 10 10 23.1 CH 3 1.13 s 9 23.8 CH 3 1.16 s 3, 9 Val 1 172.1 qC 2, 3 (Dhoaa), 10 ( N Me Phe) 171.5 qC 2, 3, NH, NH (Phe) 2 53.4 CH 4.71 dd (9.4 1.8) 5, NH 57.6 CH 4.17 dd (8.6, 4.2) 3, 4, 5, NH 3 29.8 CH 1.75 m 2, 5 30.6 CH 1.89 m 2, 4, 5 4 16.1 CH 3 1.00 d (6.8) 2, 3, 5 17.0 CH 3 0.79 d (6.7) 2, 3, 5 5 20.5 CH 3 0.89 d (6.7) 2, 3, 4 19.7 CH 3 0.78 d (6.7) 2, 3, 4 NH 6.11 d (9.1) 5.90 d (8.7) N Me Phe b /Phe c 1 172.7 qC 2 172.6 qC 2, 3, 2 (Hmpa), NH 2 65.9 CH 3.84 dd (11.3, 3.9) 3, 10 55.0 CH 4.25 ddd (8.2, 6.6, 4.1) 3, NH 3 34.1 CH 2 3.20 dd (14.3, 3.7) 3.12 dd (14.3, 11.3) 2, 5/9 35.3 CH 2 3.27 dd (14.5, 4.1) 3.11 dd (14.5, 10.8) 2, 5/6 4 137.8 qC 2, 3, 6/8 137.3 qC 2, 3, 6/8 5/9 129.2 CH 7.11 d (7.1) 3, 6/8, 7 129.4 CH 7.14 d (7.2) 3, 6/8 6/8 128.9 CH 7.28 m 5/9, 7 128.8 CH 7.29 m 5/6, 7 7 127.2 CH 7.24 m 5/9 127.1 CH 7.24 m 5/9 6/8 10 39.4 CH 3 2.78 s 2 NH 6.21 d (6.6) Hmpa 1 169.8 qC 2 169.3 qC 2 2 78.4 CH 4.94 d (7.3) 6 78.3 CH 4.91 d (6.5) 3, 4, 6

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88 a Protons showing longrange correlation to indicated carbon. b Refers to compound 3 3 c Refers to compound 3 4 Table 3 1. Continued Unit C/H # Pitipeptolide C ( 3 3 ) Pitipeptolide D ( 3 4 ) C H ( J ) HMBC a C H ( J ) HMBC a 4 25.2 CH 2 1.58 m 1.15 m 2, 5, 6 25.5 CH 2 1.21 m 1.66 m 2, 5, 6 5 11.9 CH 3 0.89 t (6.7) 4 11.0 CH 3 0.90 t (7.4) 3, 4 6 14.6 CH 3 0.93 d (6.9) 2, 4 14.7 CH 3 0.97 d (7.2) 3, 4 Pro 1 170.4 qC 2 170.4 qC 2, NH (Ile) 2 61.4 CH 4.63 d (6.9) 3, 4 61.2 CH 4.59 d (7.5) 3, 4 3 31.4 CH 2 2.67 m 1.93 m 2, 4, 5 31.5 CH 2 2.60 m 1.99 m 2, 5 4 21.9 CH 2 1.97 m 1.76 m 2, 3, 5 21.8 CH 2 2.00 m 1.77 m 2, 5, 6 5 46.5 CH 2 3.70 m 3.54 dd (10.3, 9.1) 2, 3, 4 46.6 CH 2 3.70 m 3 .58 dd (11.5, 9.8) 2, 3 Ile 1 171.9 qC 2, 2 (Gly), NH (Gly) 171.4 qC 2, 2 (Gly), NH (Gly) 2 61.1 CH 4.22 dd (8.7, 8.6) 3, 6, NH 60.5 CH 4.25 dd (8.6, 8.1) 6 3 35.3 CH 2.05 m 2 35.6 CH 2.05 m 2, 4, 5, 6 4 25.9 CH 2 1.58 m 1.23 m 2, 6 25.5 CH 2 1.5 8 m 1.21 m 2, 5, 6 5 11.1 CH 3 0.87 t (7.0) 4 11.0 CH 3 0.89 t (7.5) 4 6 16.1 CH 3 1.00 d (6.8) 2, 4 14.8 CH 3 0.98 d (7.2) 4 NH 7.97 d (8.4) 7.64 d (8.1) Gly 1 170.2 qC 2, 3 (Dhoaa), NH (Ile) 169.9 qC 2, 3 (Dhoya) 2 41.3 CH 2 4.60 dd (17. 8, 9.3) 3.97 d (17.8) 41.4 CH 2 4.34 dd (18.0, 8.6) 3.95 dd (18.0, 2.0) NH 6.38 d (8.9) 6.50 d (7.4)

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89 Table 32 NMR spectro scopic data for pitipeptolides E ( 3 5 ) and F ( 3 6 ) in CDCl3 ( in ppm, J in Hz) at 600 MHz Unit C/H No. Pitipeptolide E ( 3 5 ) Pitipeptolide F ( 3 6 ) C H ( J ) HMBC a C H ( J ) HMBC a Dhoya 1 175.2 qC 2 (Val), 3, 9, 10, NH (Val) 175.3 qC 2 (Val), 3, 9, 19, NH (Val) 2 45.2 qC 3, 4, 9, 10 45.3 qC 9, 10 3 77.1 CH 4.97 dd (9.3, 2.9) 5, 9 77.2 CH 4.95 dd (9.6, 2.0) 1 (Gly), 1, 2, 4, 9, 10 4 28.8 CH 2 1.82 m 1.58 m 3, 5 28.9 CH 2 1.83 m 1.48 m 3, 5, 6 5 24.4 CH 2 1.43 m 3, 4, 6 24.4 CH 2 1.43 m 3, 4, 6 6 18.1 CH 2 2.22 m 3, 5, 7 18.1 CH 2 2.22 m 5 7 83.6 qC 5, 6, 8 83.4 qC 5, 6, 8 8 69.3 CH 1.97 t (2.6) 6 69.4 CH 1.96 t (2.6) 6, 7 9 19.4 CH 3 1.30 s 10 19.4 CH 3 1.25 s 3, 10 10 23.0 CH 3 1.16 s 3, 5, 9 23.0 CH 3 1.16 s 3, 9 Val 1 171 .6 qC 2, 3, NH, 10 ( N Me Phe) 171.7 qC 2, 3, NH, 10 ( N Me Phe) 2 53.4 CH 4.69 dd (9.6 1.8) 3, 4, 5, NH 53.5 CH 4.70 dd (9.0, 1.4) 4, 5, NH 3 29.5 CH 1.76 m 2, 4, 5 29.5 CH 1.76 m 2, 4, 5 4 16.0 CH 3 0.90 d 2, 3, 5 16.0 CH 3 0.90 d (7.1) 2, 3, 5 5 20.3 CH 3 0.89 d 2, 3, 4 20.5 CH 3 0.89 d (6.9) 2, 3, 4 NH 6.06 d (8.9) 6.09 d (9.0) N Me Phe 1 172.3 qC 2, 3 171.8 qC 2 2 65.9 CH 3.86 dd (11.6, 3.2) 3, 10 65.9 CH 3.85 dd (11.2, 3.4) 3, 10 3 33.9 CH 2 3.21 dd (14.2, 3.5) 3.12 dd (14.2, 11.2) 2, 5/9 34.0 CH 2 3.21 dd (14.4, 3.3) 3.11 dd (14.4, 11.4) 1, 2, 5/9 4 137.3 qC 2, 3, 6/8 137.3 qC 2, 3, 6/8 5/9 129.2 CH 7.12 d (7.5) 6/8, 7 129.4 CH 7.10 d (7.2) 6/8 6/8 128.9 CH 7.29 m 5/9, 7 129.0 CH 7.28 m 5/6, 7 7 127.2 CH 7.24 m 5/9, 6/8 127.3 CH 7.24 m 5/9, 6/8 10 39.3 CH 3 2.80 s 2 39.3 CH 3 2.78 s 2 Hiva b /Hmpa c 1 169.2 qC 2 172.3 qC 2 2 78.5 CH 4.90 d (6.7) 3, 4, 5 78.4 CH 4.92 d (6.8) 4, 6 3 30.7 CH 2.05 m 2, 4, 5 37.3 CH 1.81 m 2, 5, 6

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90 Table 3 2. Continued Unit C/H No. Pitipeptolide E ( 3 5 ) Pitipeptolide F ( 3 6 ) C H ( J ) HMBC a C H ( J ) HMBC a 5 18.2 CH 2 0.98 d 2, 3, 4 11.7 CH 3 0.89 t (8.1) 4 6 14.6 CH 3 0.93 d (7.2) 2, 3 Pro 1 170.0 qC 2, 3, NH (Ile) 170.4 qC 2, NH (Ile) 2 61.2 CH 4.6 2 d (7.8) 3 61.3 CH 4.62 d (6.5) 3, 4, 5 3 31.4 CH 2 2.65 m 1.96 m 2, 4, 5 31.4 CH 2 2.64 m 1.97 m 2, 4, 5 4 21.8 CH 2 1.97 m 1.76 m 2, 3, 5 21.8 CH 2 1.98 m 1.77 m 2, 3, 5 5 46.5 CH 2 3.70 m 3.55 dd (10.4, 10.2) 2, 3, 4 46.5 CH 2 3.70 m 3.56 dd (12. 4, 9.7) 2, 3, 4 Ile b /Val c 1 171.4 qC 2, 2 (Gly), NH (Gly) 171.4 qC 2, 2 (Gly), NH (Gly) 2 61.1 CH 4.20 dd (9.3, 8.5) 3, 6, NH 62.4 CH 4.13 dd (9.8, 8.5) 3, 4, 5, NH 3 35.2 CH 2.04 m 2, 4, 5, 6, NH 29.2 CH 2.24 m 2, 4, 5 4 25.9 CH 2 1.58 m 1.24 m 2, 5, 6 19.5 CH 3 1.01 d (6.9) 3, 5 5 10.8 CH 3 0.87 t (7.5) 4 19.7 CH 3 1.03 d (7.0) 3, 4 6 15.8 CH 3 1.00 d (6.8) 2, 3, 4 NH 7.91 d (8.2) 7.88 d (8.3) Gly 1 169.9 qC 2, 3 (Dhoya), NH 169.9 qC 2, 3 (Dhoya), NH 2 41.0 CH 2 4.62 dd (1 7.9, 8.7) 3.99 d (17.9) 41.2 CH 2 4.62 dd (18.0, 9.4) 4.01 d (18.0) NH 6.39 d (9.3) 6.40 d (9.0) a Protons showing longrange correlation to indicated carbon. b Refers to compound 3 5 c Refers to compound 3 6 *Could not deduce coupling cons tants due to overlapping peaks

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91 Table 33 Cytotoxicity in cancer cells and antimycobacterial activities of pitipeptolides A F ( 3 1 3 6 ) a Experiment done in duplicate. Antimycobacterial Activity Cytotoxicity IC 50 ( M) a Diameter of Zone of Inhibition (mm ) a HT 29 MCF7 100 g 50 g 10 g Pitipeptolide A ( 3 1 ) 28 23 9 13 13 Pitipeptolide B ( 3 2 ) 30 24 14 13 11 Pitipeptolide C ( 3 3 ) 26 21 18 67 73 Pitipeptolide D ( 3 4 ) 10 0 0 > 100 > 100 Pitipeptolide E ( 3 5 ) 21 15 0 75 > 100 Pitipeptolide F ( 3 6 ) 40 30 10 87 83 10 g 5 g 1 g Streptomycin 40 30 0 Paclitaxel 0.007 0.006

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92 Table 3 4 Antimycobacterial activities of the semi synthetic probes piti A biotin ( 3 7 ) and piti A fluor ( 3 8 ) Compo und Diameter of Zone of Inhibition (mm ) a Pitipeptolide A ( 3 1 ) 23 b Piti A b iotin ( 3 7 ) 1 0 b Piti A f luor ( 3 8 ) 1 4 b Streptomycin 30 c a Experiment run in duplicate. b Compounds tested at 50 g (corresponding to 6.2 mM of pitipeptolide A, 3.8 mM of pitiA biotin and 3.6 mM of piti A fluor) c Positive control tested at 5 g.

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93 Figure 31. Structures of pitipeptolides A F ( 3 1 3 6 ).

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94 Figure 32. ESI MS/MS fragmentation patterns of pitipeptolides C F ( 3 3 3 6 ).

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95 Figure 33. Structural comparison between pitipeptolide A ( 3 1 ) (left) and antanapeptin A (right). Subunit sequences are shown below the structures; dotted lines indicate connected residues. The absolute configuration of the Hmoya unit in ana ntapeptin A is still unknown.

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96 Figure 3 4 Structures of the probes p itiA b iotin ( 3 7 ) and p itiA f lu o r ( 3 8 )

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97 Figure 3 5 Synthetic scheme to obtain p itiA b iotin ( 3 7 )

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98 Figure 36. Synthetic reaction to obtain piti A fluor ( 3 8 ).

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99 CHAPTER 4 MARINE CYANOBACTERIAL FATTY ACID AMIDES ACTING ON CANNABINOID RECEPTORSa Introduction The fact that lipids are utilized by diverse organisms suggests an evolutionarily conserved role of this class of compounds.116 Inde ed, lipidomics is emerging as a crucial field of research because of the key role of lipid biomolecules in a wide array of physiological functions.117 Research has also uncovered some vital lipid protein interactions in which lipid molecules bind to specific protein domains to mediate physiological effects. The endocannabinoid system is a representative example; here two characterized G proteincoupled cannabinoid receptors CB1 and CB2 are modulated by endogenous lipids known as endocannabinoids. Importantly, this system has been implicated in various pathophysiologies, includi ng neurodegenerative diseases, eating disorders, pain, inflammation, and cancer.118129 Therefore, a better understanding of this system has become of significant interest, and the cannabinoid receptors are viewed as possible targets for different diseases.116, 127 The classical concept that all agonists at a given GPCR induce a similar repertoire of downstream events is now uncertain, and the latest experimental evi dence supports the existence of ligand specific functional selectivity at the cannabinoid receptors .118 Consequently, the identification of new structural scaffolds that can bi nd to the cannabinoid receptors remains an essential tool for digging further into this complex system. a Reproduced with per mi ssion from Montaser, R., Paul, V. J., Luesch, H. ChemBioChem. 2012, 13, 2676 2681 Copyright (2012) John Wiley & Sons, Inc.

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100 Anandamide ( N arachidon oylethanolamine) (Figure 4 1) was the first endogenous ligand to be identified among the endocannabinoid family.130 Influenced by its structure, the discovery of this fatty acid amide suggested that other natural and synthetic fatty acid amides might also function as cannabinoid receptor ligands. M arine cyanobacteria of the genus Lyngbya have a characteristic metabolic profile that is rich in fatty acid amides (in addition to peptides),36 and therefore represent a potential source of new model compounds acting on the cannabinoid receptors. Support for this assumption comes from r eports of metabolites isolated from Lyngbya samples that can interact with the cannabinoid receptors. To our knowledge, only five marine cyanobacterial fatty acid amides with binding affinities to the cannabinoid receptors have been identified; grenadamide,131 semiplenamides A, B, and G,132 and the recently rep orted metabolite serinolamide A133 ( Figure 4 1 ). However, none of them has been tested in functional assays befo re, so i t remains unknown whether those metabolites act as receptor agonists or antagonists. Exploring a Lyngbya sample from Piti Bomb Holes from Guam led us to isolate and characterize the new fatty acid amide serinolamide B ( 4 1 ), a closely related analogue to the recentl y reported lipid serinolamide A (Figure 4 1 ). Based on structural features, we evaluated the ability of compound 4 1 to bind to both human cannabinoid receptors CB1 and CB2 with a functional outcome. The marine cyanobacteria Lyngbya spp. are also well known for the production of a large class of fatty acid amides known as malyngamides.38 More than 30 malyngamide analogues with a broad spectrum of bioactivities are known. Although malyngamides usually contain different amine portions and sometimes vary in the length of the fatty acyl chain, they generally have a unique

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101 and characteristic structural scaffold of an N substituted amide of a long chain 7methoxy fatty acid with monounsaturation at C4 (Figure 4 2 ). As malyngamides are the most abundant fatty acid amides found in Lyngbya spp., we examined whether the malyngamidetype structural features could also bind to the cannabinoid receptors, although they usually possess more complicated amine portions and somewhat different fatty acid side chains from the known endogenous cannabinoids. For this, we tested malyngamide B ( 4 2 ) (Figure 4 2) for its potential as a cannabimimetic compound. Interestingly, malyngamide B ( 4 2 ) can bind to both CB1 and CB2 receptors with moderate potencies. Here, we report the results of our studies on two marine cyanobacterial metabolites; the newly identified analogue serinolamide B ( 4 1 ) and a member of a large group of cyanobacterial fatty acid amides, malyngamide B ( 4 2 ). Isolation and Structure Determination A cyanobacterial sample from Guam was extracted three times with EtOAc MeOH mixtures. Solvent partitioning of the organic extract yielded 7.2 g of a semi polar n BuOH fraction, which was then fractionated by si lica gel chromatography. Compound 4 1 was purified by reversedphase HPLC from a silica column fraction that also contained pitipeptolides.134 The m olecular formula C22H43NO3 was established by HRESIMS ( m / z 370.3316 for [M + H]+). NMR profiles of this compound were characteristic of a fatty acid derivative, where typical chemical shifts of a monounsaturated fatty acid chain were prominent (Table 4 1 ): 1H and 13C NMR spectra showed a number of overlapping methylene groups ( H C carbonyl ( C 172.9 ppm), an methylene group ( H 2.27 ppm and C 36.4 ppm), a terminal methyl group ( H 0.88 ppm and C 13.9 ppm) and characteristic olefinic methines ( H 5.39 ppm, C 127.8 ppm and H 5.49, C 132.1 ppm). Additionally, the

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102 compound appeared to be a fatty acid amide; where the amide proton at H 6.16 ppm showed HMBC correlation to the carbonyl carbon as well as a COSY correlation to a m ethine proton at 4.07 ppm ( C 50.3 ppm). Three additional oxygenated groups were also identified based on their chemical shifts; two methylene groups ( H 3.58, 3.53 ppm, C 73.6 ppm; and H 3.66, 3.82 ppm, C 64.2 ppm) and a methoxy group ( H 3.36 ppm and C 59.2 ppm), where the latter showed HMBC correlation to the first oxygenated methylene group ( C 73.6 ppm) (Table 4 1 ). Further analysis of COSY, TO CS Y and HMBC data allowed for the construction of the amine part of this molecule as a monomethyl serinol and located the olefinic system in the fatty acid chain between C4C5 ( Figure 4 1 Table 4 1 ). Finally, to complete the molecular formula suggested by MS data, the number of methylene groups forming the fatty acid chain was assigned to construct an 18car bon monounsaturated fatty acid. The absolute configuration of the chiral center in the serinol moiety was determined through Jones oxidation of the primary alcohol to its corresponding carboxylic acid, followed by acid hydrolysis to liberate O Me serine. E nantioselective analysis revealed S configuration of the amino acid and consequently R configuration in the parent compound. Additionally, the doublebond geometry was assigned as trans based on the chemical shifts of the adjacent methylene groups C3 and C 6 133, 135 and the absence of NOESY correlations between the two olefinic protons. While we were investigating the biological activity of this metabolite, the Gerwick group reported the closely related analogue seri nolamide A (Figure 4 1 ).133 Notably, 1H and 13C chemical shifts and the stereochemical assignments for 4 1 match those reported for serinolamide A

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103 Biological Activity Evaluation Given the structural similarity of serinolamide A to the endocannabinoids anandamide ( Figures 4 1 ) and 2arachidono yl glycer ol, it was tested for binding to the human cannabinoid receptors CB1 and CB2; it appeared to possess more than 5fold selectively for the CB1 receptor with a moderate binding affinity ( Ki = 1.3 M).133 As the only structural difference between the two serinolamide analogues is a secondary amide in 4 1 instead of the tertiary amide N Me group in serinolamide A we evaluated the cannabimimetic activity of serinolamide B ( 4 1 ). This compound can also bind to both CB1 and CB2 receptors with moderate to weak binding affinities (Figure 4 3 A Table 4 2 ). Howe ver, 4 1 showed an opposite trend in binding affinities compared to serinolamide A ; as it exhibited a moderate affinity and higher selectivity for CB2 ( Ki = 5.2 M) over CB1 receptor ( Ki = 16.4 M). Notably, t he endocannabinoid anandamide shows higher selectivity for CB1 receptor ( Ki for CB1 = 32 nM; Ki for CB2 = 1.9 M),136 thus suggesting that the presence of a secondary rather than a tertiary amide is not the main determinant for receptor selectivity. Malyngamides have been reported with different biological activities, including cytotoxic, anti inflammatory and quorum sensing actions.51, 53, 55 Yet, t o the best of our knowledge, the malyngamide structural features were not probed before for cannabinoid receptor interactions Accordingly, we were interested in testing a representative analogue so as to determine whether this molecular archit ecture is capable of possessing cannabimimetic effects. We obtained malyngamide B ( 4 2 ) from our marine natural products library and tested it for CB1 and CB2 binding Interestingly, 4 2 appeared to possess moderate binding affinities to both receptors wit h Ki values of 3.6 M for CB1 and 2.6 M for CB2 ( Figure 4 3 A Table 4 2 ). Those results are noteworthy,

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104 since 4 2 shows a more complex amine portion than the endocannabinoids and other cyanobacterial fatty acid amides which possess the same biological act ivity ( Figures 4 1 and 4 2 ) It has not yet been determined whether the marine cyanobacterial fatty acid amides that can bind to the cannabinoid receptors act as agonists or antagonists. In order to clarify this we tested the functional response induced by the binding of 4 1 and 4 2 to the cannabinoid receptors Cannabinoid receptors are G protein coupled receptors that are functionally coupled to the inhibition of adenylyl cyclase and subsequent inhibition of cAMP accumulation (Figure 4 3B ).128 C ompounds 4 1 and 4 2 were able to inhibit forskolinstimulated cAMP accumulation through both CB1 and CB2 receptors with moderate potencies ( Figure 4 3C Table 4 2 ), which proves that those metabolites act as cannabinoid rec eptor agonists. Intriguingly, serinolamide B ( 4 1 ) appeared to be more CB2receptor selective in the binding as well as the functional assays, but malyngamide B ( 4 2 ) appeared to bind to both receptors similarly with comparable functional outcomes ( Figure 4 3C Table 4 2 ) It is known that anandamide signaling is terminated by the enzyme fatty acid amide hydrolase (FAAH), which catalyzes anandamide hydrolysis. Therefore, one emerging pharmacological approach to augment the endocannabinoid activity is direct ed towards FAAH inhibition.137 Thus, we tested the ability of metabolites 4 1 and 4 2 to inhibit this enzyme. However, no considerable inhibitory effects were detected for either compound when tested at 10 and 100 M It has been repeatedly shown that cannabimimeti c compounds could also mediate anti inflammatory responses.116, 129, 138 From that perspective, we tested the

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105 ability of compounds 4 1 and 4 2 to exert anti inflammatory effec ts in lipopolysaccharide (LPS) induced murine macrophages RAW 264.7. Serinolamide B ( 4 1 ) showed a weak effect with an IC50 > 25 M ; h owever, malyngamide B ( 4 2 ) was more potent, inhibiting NO production with an IC50 of 6.2 M without affecting cellular vi ability up to 25 M. Although evidence suggests that the anti inflammatory effects of some cannabimimetic compounds are mediated through cannabinoid receptors, particularly CB2, it is questionable if this is also the case with malyngamide B ( 4 2 ). Notably, Mukhopadhyay et al. showed that no detectable CB2 receptors were apparent in RAW 264.7 cells unless they are stimulated by LPS.139 Therefore, the effect of 4 2 on LPS induced inflammation is probably not totally me diated through the cannabinoid receptors, since it was able to prevent the early stimulation by LPS. Further experiments using CB2 -receptor selective antagonists or CB2receptor deficient cells will help ascertain the presence or absence of a cannabinoidr eceptor mediated anti inflammatory effect for 4 2 Some malyngamides can also reduce NO accumulation under similar anti inflammatory assay conditions; malyngamide F acetate and malyngamide 2 ( Figure 4 2 ) have IC50 values of 7.1 M55 and 8 M ,53 respectively M alyngamide F acetate was shown to possess a distinctive cytokine profile and appeared to be selectively inhibiting the MyD88 dependent pathway.55 Notably, malyngamide F acetate and malyngamide 2 share common structural features such as oxidized cyclohexyl rings whereas malyngami de B ( 4 2 ) has a signif icantly different amine entity ( Figure 4 2 ) To our knowledge, this is the first report of such activity for an anti inflammatory malyngamide

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106 with the pyrrolidone ring in the amine portion rather than the common six membered cyclic ketone or lactone. We also tested the cytotoxic effects of compounds 4 1 and 4 2 against cancer cells. Serinolamide B ( 4 1 ) failed to show significant cytotoxicity against HT 29 colon adenocarcinoma and MCF7 breast cancer cell lines up to 100 M. It is imp ortant to point out here that serinolamide A showed some cytotoxic properties in a different cell line.133 In contrast, malyngamide B ( 4 2 ) is known as a feeding deterrent140 and in our hands, 4 2 was cytotoxic to HT 29 cells with an IC50 value of 26 M, but it remains unclear if its cannabimimetic activity contributes to this cytotoxic effect. Conclusion We identified the new cannabimimetic marine cyanobacterial fatty acid amide serinol amide B ( 4 1 ). S erinolamide B with a secondary amide had higher CB2 receptor selectivity and lower cytotoxicity than its analogue with a tertiary amide. In agreement, other reports showed that several structural features can increase the affinity for CB2 receptor, including an E double bond at position 4 an amide proton, and additional substituents in the amine part.116, 138 Testing analogues serinolamide A and B side by side under the same experimental conditions will unequivocally clarify this comparison. We show that malyngamide B ( 4 2 ) also possesses cannabimimetic properties ; this provides new insight into the biological activities of malyngamides the most abundant marine fatty acid amide class in Lyngbya spp This finding introduces a new structural lead to the cannabimimetic field from the marine environment and should foster the cannabimimetic evaluation of further analogues Additionally, our finding that both metabolites act as receptor agonists proposes that they can mediate certain physiological effects through this pathway, and therefore opens more research

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107 avenues. S everal malyngamides have been subjected to total chemical syntheses and some well established synthetic routes are already available,141146 these can assist structural optimization efforts towards more potent analogues Experimental Procedures General Experimental Procedures The optical rotation was measured on a PerkinElmer 341 polarimeter. IR data were obtained on a PerkinElmer Spectrum One FT IR Spectrometer Chemi luminescence and fluorescence measurements were performed on a Sp ectraMax M5 (Molecular Devices) The 1H and 2D NMR spectra were recorded on a Bruker Avance II 600 MHz spectrometer. Al l spectra were obtained in CDCl3 using residual solvent signals ( H 7.26, C 77.16 ppm) as internal standards. HSQC and HMBC experiments were optimized for 1JCH= 145 and 1JCH =7 Hz, respectively. HRMS data was recorded on an Agilent LC TOF mass spectrometer equipped with an APCI/ESI multimode ion source detector in posi tive ion mode. LC MS data were obtained using an API 3200 triple quadrupole MS (Applied Biosystems) equipped with a Shimadzu LC system. Extraction and Isolation The sample of the marine cyanobacterium Lyngbya majuscula (recollection of UOG strain VP627) w as collected at Piti Bomb Holes, Guam, in February 2000 by V. Paul A voucher sample (voucher specimen number EC025) has been preserved at the Smithsonian Marine Station at Fort Pierce, FL. The freezedried organism was extracted with EtOAc MeOH (1:1 3 ) to give a crude organic extract (35.5 g), which was partitioned between hexanes and 80% aqueous MeOH. After the methanolic phase had been dried the residue was partitioned between n BuOH and H2O. The concentrated n BuOH residue (7.2 g) was subjected to f lash chromatography over silica gel, eluting with

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108 increasing gradients of i PrOH in CH2Cl2, and finally with MeOH. The fraction eluting with 4% i PrOH/ CH2Cl2 was fractionated on a semi preparative reversedphase HPLC column (YMC Pack ODS AQ, 250 10 mm, 5 m, 2 mL/min; UV detection at 220/254 nm) using a MeOH/H2O linear gradient (75 100 % aqueous MeOH over 30 min, and then 100% MeOH for 10 min) to afford 10 fractions. Repurification of five fractions yielded pitiprolamide84 and pitipeptolides.134 Compound 4 1 eluted as a single peak (fraction 10) at tR 28.8 min. Serinolamide B ( 4 1 ) 20 D 7.9 ( c 0.075, CHCl3); IR (film) max 3290, 3077, 2955, 2920, 2851, 1641, 1542, 1465, 1377 c m 1 ; 1H NMR, 13C NMR and HMBC data, see Table 4 1 ; HRESI/APCIMS m/z 370.3324 [ M + H]+ (calcd C22H44NO3 370.3316). Jones Oxidation and Enantioselective Amino Acid Analysis by HPLC/MS Compound 4 1 (1 mg) was dissolved in acetone (1 mL) followed by the addit ion of freshly prepared Jones reagent (CrO3 in diluted H2SO4, 50 L). The reaction mixture was stirred at room temperature for 1 h The reaction was then quenched by the addition of a few drops of i PrOH and the mixture was fi ltered through a pad of celit e. The reaction mixture was dried down under nitrogen and the residue was redissolved in water and partitioned between water and EtOAc three times. The organic layer was dried under nitrogen and the crude product was then purified using HPLC (YMC Pack ODS AQ, 250 10 mm, 5 m, 2 mL/min; UV detection at 220/200 nm) using a MeOH/0.05% aqueous TFA linear gradient (75 100% aqueous MeOH over 20 min, then 100% MeOH for 10 min) to give the oxidized compound (0.7 mg) at tR 27.7 min (68% yield). Then, 6N HCl ( 400 L) w as added to the product ( 100 L ) and stirred at

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109 110C overnight. The reaction mixture was dried and reconstituted in water ( 100 L ) and subjected to HPLC /MS enantioselective analysis. For the standards, ( S ) O Me Ser standard (0.3 mg) (Waterstone Technology Carmel, IN, USA ) was subjected to partial epimerization to obtain the ( R ) O Me Ser standard. The compound was dissolved in water ( 80 L) followed by the addition of triethylamine ( 32 L ) and acetic anhydride ( 32 L ) The reaction mixture was stirred at 60C for 1 h and then dried down. The residue was re dissolved in 6N HCl ( 100 L) and stirred at 110C overnight and then dried again. The ratio of S : R enantiomers obtained from the partial epimerization reaction was 9:1. S tandards as well as the test c ompound were s ubjected to HPLC/MS chiral analysis (MRM monitoring ) under the following conditions : CUR 10, CAD medium, IS 5500, TEMP 600, GS1 55, GS2 55, positive ion mode, MRM pair [120 74], tR: ( S ) O Me Ser (10.6 min), ( R ) O Me Ser (15.4 min), oxidized moiety from compound 4 1 (10.6 min). Cannabinoid CB1/CB2 Receptor Binding Assays Assays were done by Caliper Life Sciences (Hanover, MD, USA) Human recombinant CB1 (Bmax = 1.5 pmol/mg protein) or CB2 (Bmax = 8 pmol/mg protein) receptors were expressed i n HEK 293 cells. [3H]CP 55940 was used as the radioligand with a final concentration of 0.5 nM ( Kd for CB1 = 0.6 nM and for CB2 = 4.2 nM). HU210 (1 M) was used as nonspecific binding determinant ( Ki values of 1.1 and 3.0 nM for CB1 and CB2 receptors, re spectively ) Reactions were carried out in TRIS HCl buffer ( 50 mM, pH 7.4) containing EDTA ( 2.5 mM ) MgCl2 ( 5 mM ) and BSA ( 0.1% ) at 30 C for 90 min The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined using liquid scintillation

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110 spectrometry and compared to control values in order to ascertain any interactions of test compound with the CB1 or CB2 binding sites. cAMP Functional Assay CHO K1 cells expressing CB1 or CB2 recepto rs were used, and cAMP levels were determined after forskolin stimulation using cAMP Hunter express GPCR assay kits ( DiscoverX Fremont, CA, USA ) according to the manufacturers procedures Briefly, cells were seeded in 96well plates (3 104 cells pre we ll) and incubated at 37 C humidified air with 5% CO2. After 24 h, the medium was aspirated, and cell assay buffer with cAMP antibody reagent w as added to the wells. Test compound and forskolin were dissolved in DMSO. The cells were then stimulated with di fferent concentrations of the test compound in the presence of forskolin (20 M) for 30 min at 37 C Cell lysis and chemiluminescent signal detection were performed with the detection reagents according to the recommended protocol. FAAH I nhibitor E nzyme A ssay FAAH inhibitor screening assay kit was purchased from Cayman Chemical (Ann Arbor, MI, USA) and used as recommended. In a black 96well plate, FAAH enzyme (10 L) was added to the assay buffer (170 L), followed by the addition of the test compound or the solvent control (10 L). The reactions were initiated by adding the substrate AMC arachidonoyl amide (10 L, 20 M), and the plate was incubated for 30 min at 37 C After incubation the signal was detected at an ex = 350 nm and em = 455 nm using a microplate reader. NO Assay RAW 264.7 mouse macrophage cells were cultured and maintained in Dulbeccos modified Eagle medium (Invitrogen, Carlsbad, CA USA ) supplemented with

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111 10% fetal bovine serum (HyClone Laboratories, Logan, UT USA ) in a humidified environment with 5% CO2. Cells were seeded in 96well plates (2 104 cells/well), and after 24 h the cells were treated with different concentrations of the test compound or solvent control ( 1% EtOH), followed by LPS (0.5 g/mL ) to stimulate an inflamm at ory response. The production of NO was assessed by measuring the nitrite conc entration in the culture medium after 24 h using Gr ie ss reagent. Briefly, sulfanilamide (1% w / v) in phosphoric acid (5% v/ v) (50 L ) was added to the cell culture supernatant ( 50 L) and incubated for 5 min at room temp erature in the dark followed by the addition of naphthylethylenediamideHCl (0.1% w/v, 50 L ) After 5 min incubation at room temp erature in the dark, the absorbance of the reaction mixture was measured at 540 nm us ing a microplate reader. Assays were run in duplicates Nitrite quantification was determined relative to a nitrite standard curve (0100 M). Cell Viability Assays Cells were propagated and maintained in Dulbeccos modified Eagle medium (Invitrogen, Carl sbad, CA USA ) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT USA ) at 37 C humidified air and 5% CO2. Cells were seeded in 96well plates ( HT 29 ; 1 1,000 cells per well ). After 24 h, cells were treated with various concentrations of the test compound, or solvent control (1% EtOH). After 48 h of incubation, cell viability was measured using MTT (Promega, Madison, WI USA ) according to the manufacturers instructions.

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112 Table 4 1 1H and 13C NMR spectroscopic d ata for s erinolamid e B ( 4 1 ) in CDCl3 ( in ppm, J in Hz) at 600 MHz Unit C/H # C H ( J ) HMBC a Fatty acid 1 2 3 a b 4 5 6 7 8 15b 16 17 18 172.9 qC 36.4 CH2 28.4 CH2 127.8 CH 132.1 CH 32.3 CH2 29.3 CH2 29.4 CH2 31.8 CH2 22.4 CH2 13.9 CH 3 2.27 dd (6.6, 2.1) 2.28 d (6.9) 2.23 m 5.39 m 5.49 m 1.96 ddd (7.7,7.5, 6.8) 1.32 m 1.25 m 1.25 m 1.29 m 0.88 t (6.8) 2, 3, 19, NH 3b, 4 2, 4, 5 3a, 3b, 5, 6 3b, 4, 6, 7 4, 5, 7, 8 6, 8 6, 7 17, 18 16, 18 16, 17 Serinol ether 19 20 a b 21 a b 22 NH 50.3 CH 64.2 CH2 73.6 CH2 59.2 CH3 4.07 m 3.82 dd (11.2, 4.1) 3.66 d br (11.2) 3.58 dd (9.3, 4.2) 3.53 dd (9.3, 4.2) 3.36 s 6.16 d (6.8) 20a, 21a, 21b, NH 19, 21a, 21b 19, 21a, 20b, 22 21a, 21b a Protons showing longrange correlation to indicated car bon. b Overlapping peaks

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113 Table 4 2 Cannabinoid receptors affinities ( Ki) and consequent functional effects on cAMP accumulation (EC50) by compounds 4 1 and 4 2 CB 1 CB 2 Compound K i ( M) EC 50 ( M) [ a ] K i ( M) EC 50 ( M) a HU 210 b 0.00069 0. 0011 CP 55940 c 0.00024 0.00036 4 1 16.4 11.8 5. 2 1.8 4 2 3.6 5.3 2. 6 8.8 a Results from cAMP functional assays. EC50 is the agonist concentration to cause half maximal inhibition of forskolininduced cAMP accumulation. b Positive control for binding assays. c Positive control for functional assays.

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114 Figure 4 1. Structures of the endocannabinoid anandamide and fatty acid amides from marine cyanobacteria with binding affinities to the cannabinoid receptors.

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115 Figure 4 2. General structural scaffold of malyngamides and the structures of three malyngamides.

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116 Figure 4 3 Cannabimimetic effects of serinolamide B ( 4 1 ) and malyngamide B ( 4 2 ). A) Binding of compounds 4 1 and 4 2 to CB1 (l eft) and CB2 (right) receptors, represented as percent inhibition of the binding of a radioactive ligand; B) Simplified diagrammatic representation of the cannabinoid receptors and the consequences of agonist binding; C) Effect of compounds 4 1 (upper) and 4 2 (lower) on forskolininduced cAMP accumulation. A higher level of cAMP produces a higher luminescence reading (RLU).

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117 CHAPTER 5 CHARACTERIZATION AND SYNTHESIS OF BIOACTIVE LIPID S FROM A GUAMANIAN MARINE CYANOBACTERIUMa Introduction Lipids are essential for all living organisms. Microorganisms which live in the unique marine environment produce specialized lipid molecules which are involved in biological functions, integrity of the cellular membrane, signal transduction and defensive roles such as anti bacterial lipids.147 Fatty acid metabolites could themselves possess biological activities or be used as building blocks or precursors for the biosynthesis of other bioactive molecules. For example, the marine cyanobacter ium Pseudoalteromonas haloplanktis produces the simple short chain acids isovaleric acid and 2methyl butyric acids which act as antibacterial compounds.148 In addition, 2methyl butyric acid is a building block of the cyanobacterial neurotoxin kalkitoxin.149 Another example is the production of lyngbic acid by marine cyanobacteria of the genus Lyngbya, which is a C14 monounsaturated fatty acid with O Me group at C7.56 This fatty acid derivative itself is a bioactive metabolite with anti quorum sensing activity,150 and al so acts as a building block for a large number of cyanobacterial fatty acid amides known as malyngamides with wide range of biological activities.50, 51, 5355, 87, 151155 Furthermore, polyunsaturated long chain fatty acids from marine sources are also well known bioactive molecules which offer several benefits to human health. The FDA approved marine drug Lovaza is a mix ture of ethyl esters of omega3 fatty acids (mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) sourced from a Reproduced in part with permission from Organic Letters submitted for publication. Unpublished work copyright (2013) American Chemical Society.

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118 fish oils. It is approved as an anti lipidemic drug to lower very high triglyceride levels.18, 20 Thus, marine fatty acids and their derivatives represent an interesting structural class of oftentimes bioactive molecules. During our efforts to discover novel bioactive compounds from marine cyanobacteria, we explored a population of the marine cyanobacterium Lyngbya majuscula collected from a channel at the north end of Piti Bay at Guam, through NMR guided fractionation. This collection contained a major fatty acid that dominated all of the generated fractions, beside some minor fatty acid analogues. Further purification of some fractions yielded the new fatty acid 5methylene decanoic ac id ( 5 1 ) as the major compound and another minor chlorinated ester derivative ( 5 2 ) (Figure 5 1). As with the examples mentioned earlier, the simple major fatty acid ( 5 1 ) showed anti quorum sensing activity in Pseudomonas aeruginosa and it acts as a buil ding block to yield another biologically active chlorinated ester ( 5 2 ) which prevents the induction of proinflammatory cytokine expression in THP 1 macrophages. Isolat ion and Structure Determination The EtOAc MeOH extract was subjected to solvent solvent partitioning to yield 5.47 g of a semi polar n BuOH fraction, which was fractionated by silica gel chromatography. 1H NMR profiles of the generated silica fractions showed a major simple fatty acid ( 5 1 ) dominating most of the fractions, which was then purified using HPLC from one of the fractions that eluted with 10% i PrOH in DCM as colorless oil. The 1H NMR spectrum of 5 1 showed typical peaks for fatty acids: two methylene groups at H H 0.89 ppm, an methylene group at H 2.35 ppm, and the fatty acid carbonyl carbon appeared in the 13C NMR spectrum at C 180.2 ppm. Additionally, a methylene group at H 4.73 ppm and C 109.5 ppm

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119 showed an HMBC c orrelation to a quaternary carbon at C 148.7 ppm indicating the presence of an exodouble bond along the fatty acid chain. The information obtained from 1D, 2D NMR spectra and MS data (HRESIMS: m/z 183.1398 for [M H]corresponding to C11H19O2) constructe d the simple fatty acid 5 methylene decanoic acid ( 5 1 ) as the major fatty acid in this sample (Figure 5 1, Table 5 1). HPLC purification of another silica fraction that eluted with 40% MeOH in DCM yielded another fatty acid compound, where the 1H and 13C NMR spectra included all the peaks corresponding to the major fatty acid 5methylene decanoic acid ( 5 1 ) beside peaks for five additional carbons and attached protons: one carbonyl at C 173.5 ppm, an oxygenated methine at H 5.09 and C 70.7 ppm, a methylene at H 2.64 and C 32.5 ppm and olefinic methines at H 5.9; C 127.3 ppm and H 6.12 and C 121.4 ppm (Table 5 1). Investigating COSY, TOCSY and HMBC data led to the assignment of the additional portion as a 2hydroxy pent 4 enoic acid, where an attachment to the terminal olefinic methine was still missing. Finally, a chloride attachment at this site was suggested by the remaining molecular mass and the isotopic cluster detected in the HRESIMS analysis ( m/z 315.1384, 317.1348 (3:1) for [M H]corresponding to C16H24 35ClO4 and C16H24 37ClO4, respectively). Notably, the chemical shift of the carbonyl carbon in the decanoic acid part was shifted upfield compared to the free fatty acid (T able 5 1), and therefore the compound appeared to be a fatty acid ester 5 2 (Figure 5 1). The proposed structure was also confirmed by the detected mass fragments in the LRMS (m/z 315.2/317.1 (3:1) [M H]-, 183.1 [M C5H7ClO2](acid fragment), 148.9/150.9 ( 3:1) [M C11H19O](alcohol fragment), 131.1/132.9 (3:1) [M C11H21O](alcohol fragment, McLafferty rearrangement or loss of water)) (Figure 52) The geometry of the alkene in

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120 the 2hydroxy 5 chloro pent 4 enoic acid moiety was determined to be ( E ) based on the large vicinal coupling constant (3JH,H 13.4 Hz) between the olefinic protons. In order to assign the stereochemistry of the chiral center C2 (Figure 5 1), the compound was subjected to ozonolysis followed by oxidative workup to yield the corresponding malic acid, which was then analyzed by chiral HPLC compared to malic acid standards. The analysis revealed the S configuration of this chiral center. The abundance of 5methylene decanoic acid ( 5 1 dry weight) suggested that it has an important ecological role for this organism, and was sufficient for biological activity evaluation in several assays. However, the chlorinated ester ( 5 2 ) was only isolated in minute amo 900 g) which was sufficient for structural characterization but did not allow for further biological characterization. Since 5 2 has an intriguing chemical structure (Figure 5 1 ), we decided to synthesize this compound to obtain more material for f urther biological evaluation. Synthesis of the Chlorinated Ester Our initial retrosynthetic strategy was to synthesize the chlorinated alcohol part and couple it to the abundant 5methylene decanoic acid ( 5 1 ) isolated from this sample to obtain the semi synthesized ester 5 2 (Figure 5 3 strategy 1). The selective introduction of the E vinyl chloride in the alcohol moiety could be achieved through Takai olefination reaction156 from an hydroxy 4 oxobutanoic acid. The latter could be obtained by selective reduction of malic acid at C4 (Figure 5 3 strategy 1a). The hydroxy group in the commercially available Lmalic acid was protected as TBDPS ether ( 5 3 ) with 87% yield. A bulky protecting group was chosen to introduce steric hindrance adjacent to the C1 carboxylic acid terminus and allow for selective reduction at the C4 terminus only (Figure 5 4 A ). However, the selective reduction to 5 4

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121 was not totally achieved as multiple aldehyde peak s w ere present in the 1H NMR spectrum, but the maj or aldehyde was the desired product. Therefore, we continued with the next steps in this scheme with the intention to purify the desired product at a later stage. Takai olefination reaction for 5 4 proceeded with a low yield (17%) to give compound 5 5 Not ably, the purification of 5 5 using silica gel column chromatography was tedious and we had to repurify the product several times using TLC plates. The TBDPS protected hydroxy group in 5 5 was then deprotected using TBAF in THF to obtain the free alcohol 5 6 (32%). However, after purifying the product from the deprotection step, 1H NMR spectrum clearly showed that the product is a mixture of and hydroxy acids with the ratio of 2:1. There are two explanations for obtaining this isomer mixture: first the non selective DIBAL reduction and the subsequent difficulty in separating the desired aldehyde is one reason, where two compounds have possibly reacted in Takai reaction; the compound with an aldehyde at C4 which will yield the hydroxy moiety, and the compound with the aldehyde at C1 which will yield the hydroxy moiety. The second explanation is that the alcohol part in compound 5 2 is unstable and can undergo dehydration/rehydration, which could lead to nonselective introduction of the hydroxy g roup giving a mixture of and hydroxy acids, specially that the ratio of those two isomers after the deprotection step is relatively close (2:1, : ). Additionally, m/z corresponding to the dehydrated alcohol fragment is always present upon LR MS analy sis of compound 5 2 ( m/z 131.1/133.2 (3:1)). Accordingly, we had to seek alternatives for this synthetic strategy. In order to solve this problem we faced in reduction selectivity starting from Lmalic acid, we followed the method by Padron et al.,157 where the di Boc protected aspartic

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122 acid ( 5 8 ) could be selectively reduced to the semialdehyde 5 9 Accordingly, the retrosynthetic strategy was modified to start from Laspartic acid instead of Lmalic acid (Figure 5 3 retrosynthetic scheme 1b). Therefore, strategy 1b includes an additional diazotization step to convert the amino to hydroxy group with retention of configuration158 before the final coupling step to the acid 5 1 (Figure 5 4 B ). For this, the primary amine in the commercially available Ldimethyl aspartate was protected with two Boc groups over two successive steps157 with good yields (84% for 5 7 and 95% for 5 8 ). Indeed, the selective reduction to the corresponding semialdehyde ( 5 9 ) at C4 proceeded smoothly with a 67% yield. Although this strategy appeared successful at those early steps, Takai olefination156 for the aspartate semialdehyde 5 9 to the chlorinated compound 5 10 proceeded with a low yield (32%) and relatively low selectivity ( E : Z determined by analyzing the 1H NMR spectrum, which indicates that the Boc group is not sufficiently compatible with this reaction. Since Boc protected amino groups are acid labile, we repeated the same steps using the acidstable amine protecting group Fmoc. However, under the same reaction conditions the Fmoc protected dimethyl aspartate ( 5 11 ) could not be selectively reduced at one terminus to give compound 5 12 as with the di Boc prot ection. Therefore, we had to change the strategy again to a more feasible one. In our next retrosynthetic analysis (Figure 5 3 strategy 2), we tried to avoid obtaining the free alcohol part to prevent any potential stability problems. Therefore, this stra tegy aimed to primarily introduce the vinyl chloride terminus by Takai reaction to a C4 moiety with 1,2diol. The secondary alcohol could be coupled first to the acid 5 1

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123 before the primary alcohol at C1 could be converted to the carboxylic acid terminal ( Figure 5 3 strategy 2). This way, dehydration of the secondary alcohol could be avoided by protecting it as an ester, which may also be the natural function of its presence as an ester in the producing organism. This attempt started from the commerciall y available acetonide ( S ) 2 (2,2 dimethyl 1,3 dioxolan4 yl)ethanol (Figure 5 5 ), where the terminal alcohol group was oxidized to the corresponding aldehyde 5 13 in 76% yield using PCC in DCM.159 Ta kai olefination156 for compound 5 13 appeared to proceed with higher selectivity ( E : Z previous trials from the first strategy. Acetonide opening for 5 14 was done using DOWEX in MeOH160 to give the 1,2diol ( 5 15 rimary alcohol in 5 15 was then protected as the TBDMS ether (47% yield) and the free secondary alcohol was coupled to the fatty acid 5 1 to furnish compound 5 17 (76% yield). The next step was to deprotect the primary alcohol and oxidize it to the corresp onding carboxylic acid to obtain the final ester 5 2 First attempts to deprotect the primary alcohol in 5 17 using TBAF161 were faced with the challenge of acyl mi gration from the secondary to the primary alcohol. After several modifications, minimal acyl migration was obtained by using a neutral mixture of TBAF and AcOH in THF and by limiting the reaction time to two hours giving the free primary alcohol 5 18 in go od yield (72%; 85% BRSM). Unreacted starting material was recovered and deprotected again. For the final oxidation step, we first pursued several mild oxidation attempts, since we were concerned about the stability of the ester linkage and the vinyl chlor ide functionalities by any harsh reaction conditions. The traditional PDC mediated oxidation

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124 in DMF was ineffective.162 Additionally, two step oxidation protocols to the aldehyde and then to the acid were also inefficient to produce the desired carboxylic acid, including Dess Martin oxidation followed by NaClO2 or PDC treatment, and TEMPO catalyzed oxidations with NaOCl/NaClO2 system.163 Notably, the aldehyde could be obtained in some trials, but not the desired acid. Also, the exomethylene group in the fatty acid part appeared to be affect ed by the NaOCl/NaClO2 oxidizing system, as the corresponding peaks for those olefinic protons disappeared in the 1H NMR spectrum. Another strategy for onepot deprotection of the silyl ether catalyzed by Bi(OTf)3 followed by TEMPO catalyzed oxidation164 did not give desirable results either. Finally, we decided to try Jones oxidation165 although it employs the usage of the strong acid H2SO4. Off all conditions tested, Jones oxidation was the only method that gave the final d esired product, HPLC purification was essential, which appeared to significantly lower the yield of the pure product (yield drops to 20% after HPLC purification). NMR spectr al data and optical rotation for the synthetic compound matched those for the natural product 5 2 (Figure 5 6 ). The problems faced with the stability and the purification of this chlorinated ester could provide one explanation for the low amount of the nat ural product that we were able to isolate from the cyanobacterium. However, sufficient amount was successfully prepared to allow for further biological characterization. Biological Activity Evaluation Since 5methylene decanoic acid ( 5 1 ) was produced in r elatively large amounts in this sample, this suggested that 5 1 might have an ecological role for this organism. One of the common biological roles of fatty acid compounds produced by bacteria in large quantities is the interference with quorum sensing (communication between

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125 bacteria in response to high population densities). This was shown in several previous reports, including by our group where we reported the anti quorum sensing activity of lyngbyoic acid.150 Accordingly, we tested the fatty acid 5meth ylene decanoic acid ( 5 1 ) for its ability to interfere with quorum sensing in P. aeruginosa by monitoring its effect on the transcription and the production of two virulence factors, the elastase LasB enzyme and the pigment pyocyanin. Compound 5 1 signific antly reduced the transcript levels of the enzyme lasB and the pyocyanin biosynthetic member phzG1 after 6 hours at 1 mM and 100 M as assessed by RT qPCR (Figure 57) Additionally after 6 hours, the levels of LasB and pyocyanin in the culture supernatants were also significantly reduced by 5 1 at 1 mM, as evaluated by an enzymatic assay for LasB and quantitat ive evaluation for p yocyanin (Figure 5 7 ). Measuring the bacterial cell density (OD600) showed that this fatty acid did not affect cellular viabilit y at th e tested concentrations. Compound 5 2 has an intriguing chemical structure which combines several functional groups. It is a fatty acid derivative which comprises an ester linkage, double bonds and a chlori d e termin us Similar structural features w ere also present in the recently reported anti inflammatory marine cyanobacterial fatty acid esters honaucins which also enclose a terminal chl oride.166 Accordingly, we initiated the biological characterization of 5 2 by testing its effect on LPS induced inflammatory responses in human acute monocytic leukemia cell line THP 1 after differentiation to macrophages. In preliminary assays, this chlorinated ester was able to decrease the transcript levels of the proinfl ammatory cytokines TNF and IL 6 after 4 h of LPS stimulation (Figure 5 8 ). Additionally, the anti inflammatory effect was also sustained after 24 h, where the

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126 mRNA levels of IL 6 IL and IL 8 were significantly reduced (Figure 5 8 ). Notably, IL 6 was the most affected cytokine at all time points. Ongoing structureactivity relationship studies for the chlorinated ester 5 2 will enable us to determine whether the whole ester or the alcohol part alone is responsible for the anti inflammatory effect. It r emains interesting to explore whether ester hydrolysis takes place in vivo to release the anti quorum sensing acid part ( 5 1 ) of the molecule while keeping the anti inflammatory activity. This could highlight this compound as a hybrid molecule with dual bi ological activity. Experimental Section General Experimental Procedures Optical rotations were measured on a Perkin Elmer 341 polarimeter, whereas UV was measured on a SpectraMax M5 (Molecular Devices). 13C NMR spectra were recorded on a Varian 400 MHz spectrometer operating at 100 MHz, whereas 1H and 2D NMR spectra were acquired on a Bruker Avance II 600 MHz spectrometer or Varian 400 MHz spectrometer. Spectra obtained in CDCl3 using residual solvent signals ( H 7.26, C 77.16 ppm) as internal standards. HSQC and HMBC experiments were optimized for 1JCH = 145 and 1JCH = 7 Hz, respectively. HRMS data was recorded on an Agilent LC TOF mass spectrometer equipped with an APCI/ESI multimode ion source detector in positive or negative ion mode, whereas LRMS data were obtained using an API 3200 triple quadrupole MS (Applied Biosystems). ESIMS fragmentation data were recorded on an API 3200 by direct injection with a syringe driver. Malic acid standards were purchased f rom Sigma.

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127 Extraction and Isolation Lyngbya majuscula sample was collected from a channel at the north end of Piti Bay, Guam in July 2000 by V. Paul The freeze dried sample was extracted three times with EtOAc MeOH (1:1) to afford an organic extract (24. 3 g). The resulting extract was partitioned between hexanes and 80% aqueous MeOH; the methanolic phase was evaporated to dryness and the residue was further partitioned between n BuOH and H2O. After concentrating the n BuOH extract in vacuo, the resulting residue (5.47 g) was subjected to flash chromatography over silica gel, eluting with DCM followed by increasing gradients of i PrOH in DCM then MeOH in DCM, and finally with MeOH. The silica fraction eluting with 10% i PrOH/DCM was fractionated on a semi preparative reversedphase HPLC column (Synergi HydroRP, 250 10 mm, 5 m, 2 mL/min; UV detection at 220/254 nm) using a MeOH/0.05% aqueous TFA linear gradient (80% to 100% over 20 min and then 100% MeOH for 10 min) to give the pure fatty acid ( 5 1 ) as t he major peak at tR 16.3 min as a colorless oil. The silica fraction eluting with 40% MeOH/DCM was fractionated on a semi preparative reversedphase HPLC column (YMCPack ODS AQ, 250 10 mm, 5 m, 2 mL/min; UV detection at 200/220 nm) using a MeOH/0.05% aqueous TFA linear gradient (60% to 100% over 30 min and then 100% MeOH for 10 min) to yield 13 fractions. The fraction eluting at tR 32.8 min was repurified on a semi preparative reversedphase HPLC column (Luna C18, 250 10 mm, 5 m, 2.0 mL/min; UV dete ction at 200/220 nm) using a MeOH/0.05% aqueous TFA linear gradient (75% to 100% over 20 min followed by 100% MeOH for 10 min) to yield 0.9 mg of the pure chlorinated ester ( 5 2 ) at tR 16.2 min.

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128 5 methylene decanoic acid ( 5 1) : colorless oil; 1H NMR, 13C NMR and HMBC data, see Table 5 1; HRESI/APCIMS m/z 183.1398 [M H](calcd C11H19O2 183.1391). Chlorinated ester ( 5 2) : 20 D 12 ( c 0.05, MeCN); 1H NMR, 13C NMR and HMBC data, see Table 5 1; HRESI/APCIMS m/z 315.1384/ 317.1348 (3:1) [M H](calcd C16H24ClO4 315.1369/ 317.1369). Enantioselective Analysis A sample of compound 5 2 (100 g) was dissolved in 3 mL DCM and subjected to ozonolysis at 78C for 10 min. The solvent was evaporated and the residue was dissolved in HCOOH/H2O2 (2:1) and heated at 75C for 30 min. After removing the solvent, the residue was subjected to hydrolysis with 6 N HCl at 110C f or 12 h. The hydrolyzed product was dried and subjected to chiral HPLC (column: Phenomenex Chirex phase 3126 N S dioctyl (D) penicillamine, 4.6 250 mm, 5 m; solvent: 0.5 mM Cu(OAc)2, 0.1 M NH4OAc in 85:15 H2O/MeCN, pH 4.6; flow rate: 1.0 mL/min; detecti on: 254 nm). Retention times were as follows: Lmalic acid eluted at tR 7.0 min, Dmalic acid eluted at tR 13.8 min. Malic acid in the hydrolysate eluted after 7.0 min, indicating the presence of ( S ) hydroxy acid in compound 5 2 Synthetic P rocedures ( S ) d imethyl 2 ((tertbutyldiphenylsilyl)oxy)succinate ( 5 3 ) : In an ice bath, tert butyl diphenylsilyl chloride (3.38 g, 0.012 mol, 1 eq) was added, over 5 min, to a mixture of dimethyl Lmalate (2 g, 0.012 mol, 1 eq), pyridine (5 mL) and methyl imidazole (1.1 mL). The mixture was stirred for 1 h on icebath and for another 3 h at room temperature. Once TLC showed a complete reaction, the mixture was concentrated at 60 C under reduced pressure. The residue was cooled to room temperature; acidified water (20 mL, pH 2.5) and diethyl ether (20 mL) were added. The

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129 layers were separated and the aqueous layer was extracted twice with diethyl ether. The organic phases were combined, washed once with 5% citric acid (15 mL) and twice with water (2 15 mL), dried over Mg SO4, filtered and the solvent was removed to give 5.4 g of the crude product. The crude product was purified using silica gel column chromatography (Hex: EtOAc 1:9) to give 4.3 g (87%) of the pure product as a colorless liquid. 1H NMR (400 MHz, CDCl3): 7.68 (dd, J = 7.9, 1.4 Hz, 2H), 7.64 (dd, J = 7.9, 1.4 Hz, 2H), 7.44 7.33 (m, 6H), 4.56 (t, J = 5.9 Hz, 1H), 3.60 (s, 3H), 3.50 (s, 3H), 2.75 (t, J = 5.9 Hz, 2H), 1.06 (s, 9H). 13C NMR (100 MHz, CDCl3): 172.60 170.55, 136.13, 136.03, 133.10, 132.96 1 30.1 1, 130.03, 127.85, 12 7.73, 69.83, 51.98, 40.10, 26.97. LRMS: m/z 423.2 [M + Na]+. ( S ) dimethyl 2 ((tertbutyldiphenylsilyl)oxy) 4 oxobutanoate ( 5 4): Compound 5 3 (0.2 g, 0.5 mmol, 1 eq) was stirred in anhydrous ether (4 mL) and cooled down to 78C. D IBAL H (0.5 mL, 1.1 eq, from 1 M solution in Toluene) was added drop wise. The reaction mixture was stirred at 78 C for 15 min and then the reaction was quenched by warming up to r.t and adding water (0.05 mL, 7 eq). After water addition, the reaction was stirred at r.t. for 30 min till a gelatinous precipitate started to form. MgSO4 was then added to the reaction flask, and after mixing, the reaction product was filtered through a pad of celite in a fritted funnel. The solvent was removed to give 186 mg of the crude product. LRMS showed product peaks: m/z 425.4 [M + Na]+. However, 1H NMR showed nonselective reduction (more than one aldehyde peak). ( S E ) methyl 5 chloro2 hydroxypent 4 enoate ( 5 6 ): To a dry flask, anhydrous CrCl2 (1.65 g, 13.5 mmol, 10 eq) was added and then heated using a heat gun under vacuum. After cooling, the vacuum was released under argon, and dry THF

PAGE 130

130 (5 mL) was added. The greenish suspension was stirred in an oil bath and heated to 65 C. The aldehyde 5 4 (500 mg, 1.35 mmol, 1 eq) was dissolved in 2 mL dry THF in another vial, and mixed with dry CHCl3 (436 L, 5.4 mmol, 4 eq). This solution was then added drop wise to the heated flask containing chromous chloride, which turned violet upon stirring. The reaction mixture was stirred for 3 h at 65 C, and then cooled down to r.t. The mixture diluted with brine, filtered and extracted three times with Et2O. The combined organic layers were dried over MgSO4, filtered and the solvent was removed. The crude green residue was then purified using silica gel column chromatography (Hex/ Acetone 5:1) to yield the yellow oily product (94.2 mg). This product was repurified on a TLC plate two times, but the 1H NMR spectrum after several rounds of purification did not show the pure product 5 5 Th e semi pure product 5 5 (15 mg, 0.0373 mmol, 1 eq) was dissolved in THF (0.1 mL), and TBAF (70 L of 1 M solution in THF, 2 eq) was added drop wise. The solution was stirred at 0 C for one hour and then quenched by adding 0.2 mL of brine. The product was extracted 2 with EtOAc, dried over MgSO4, filtered and the solvent was removed to give a crude product as yellow oil (40 mg). Purification using silica gel column chromatography (Hex/EtOAc 5:1) yielded 3.8 mg of the deprotected product as a mixture of i somers (mixture of cis and trans hydroxy and hydroxy isomers). (2 S ) dimethyl 2 tertbutoxycarbonylaminobutanodioate ( 5 7 ): Laspartic acid dimethyl (1g, 5.06 mmol) was dissolved in dry MeOH (20 mL). Et3N (4.5 mL, 32.89 mmol, 6.5 eq) was then added fol lowed by (Boc)2O (1.2 g, 5.56 mmol, 1.1 eq) and the reaction mixture was stirred overnight at r.t. The solvent was then removed; the residue was suspended in Et2O and washed through a pad of celite in a fritted funnel. The

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131 solvent was removed and the crude product (1.93 g) was purified using silica gel column chromatography (Hex/EtOAc 3:1) to yield 1.26 g (84%) of the pure product as a white solid. 1H NMR (400 MHz, CDCl3): 5.48 (d, J = 6.9 Hz, 1H ), 4.56 (m,1H), 3.74 (s, 3H), 3.68 (s, 3H ), 2.98 (dd, J = 17.0 Hz 3.1 Hz, 1H ), 2.81 (dd, J = 17.0 Hz 4.6 Hz, 1H), 1.43 (s, 9H ). 13CNMR (100 MHz, CDCl3): 171.67, 171.5 1, 155.52, 80.20, 52.7, 52.11, 50.05, 36.76, 28.40 LRMS: m/z 284.3 [M + Na]+, 262.4 [M + H]+, 228.1 [M C(CH3)3]+, 162.2 [M Boc]+. (2 S ) dimethyl 2 ( ( tert butoxy) N [(tertbutyl)oxy carbonyl]carbonylamino) butanedioate (5 8): Compound 5 7 (1g, 3.6 mmol, 1 eq) was dissolved in dry MeCN (12 mL). DMAP (88 mg, 0 .72 mmol, 0.2 eq) followed by (Boc)2O (1.2 g, 5.4 mmol, 1.5 eq) were added to the reaction mixture and stirred overnight at r.t. The solvent was removed, and the crude product (1.8 g) was purified using silica gel column chromatography (Hex/EtOAc 4:1) to yield 1.3 g (95%) of the pure product as yellow oil. 1H NMR (400 MHz, CDCl3): 5.44 (dd, J = 6.7, 6.7 Hz, 1H), 3.72 (s, 3H), 3.69 (s, 3H ), 3.24 (dd, J = 16.4 Hz 7.1 Hz, 1H), 2.72 (dd, J = 16.4 Hz 6.4 Hz, 1H ), 1.49 (s, 18H). 13C NMR (100 MHz, CDCl3): 171.31, 170.31, 151.74, 8 3.74, 55.09, 52.75, 52.19, 35.91, 28.42. LRMS: m/z 384.3 [M+Na]+, 362.4 [M+H]+, 284.2 [M Boc]+,162.2 [M 2 Boc]+. (2 S ) methyl 2 4 oxobutanoate ( 5 9): Compound 5 8 (1 g, 2.77 mmol, 1 eq) was stirred in anhydrous ether (20 mL) and cooled down to 78 C. DIBAL H (3 mL, 1.1 eq, from 1 M solution in Toluene) was added drop wise. The reaction mixture was stirred at 78C for 15 min and then the reaction was quenched by warming up to r.t and adding water (0.4 mL, 7 eq). After water addition, the reaction was stirred at r.t. for 30 min till a

PAGE 132

132 gelatinous precipitate started to form. MgSO4 was then added to the reaction flask, and after mixing, the reaction product was filtered through a pad of celite in a fritted funnel. The solvent was removed, and the crude product was purified using silica gel column chromatography (Hex/EtOAc 4:1) to yield 500 mg (55%) of the pure product as colorless oil. 1H NMR (400 MHz, CDCl3): 9.78 (s, 1H ), 5.52 (t, J = 6.4 Hz, 1H ), 3.72 (s, 3H ), 3.41 (dd, J = 17.9 Hz 6.8 Hz, 1H), 2.82 (dd, J = 17.9 Hz 5.9 Hz, 1H ), 1.49 (s, 18H ). 13C NMR (100 MHz, CDCl3): 198. 61, 170.25, 151.80, 83.89, 53.09, 52.80, 45.16, 28.14. LRMS: m/z 354.4 [M + Na]+. ( S E ) methyl 2 (( tertbutoxy) N [( tertbutyl)oxycarbonyl]carbonylamino) 5 chloropent 4 enoate ( 5 10): To a dry flask, anhydrous CrCl2 (184.6 mg, 1.51 mmol, 10 eq) was added and then heated using a heat gun under vacuum. After cooling, the vacuum was rel eased under argon, and dry THF (2 mL) was added. The greenish suspension was stirred in an oil bath and heated to 65 C. The aldehyde 5 9 (50 mg, 0.151 mmol, 1 eq) was dissolved in 1 mL dry THF in another vial, and mixed with dry CHCl3 (40.6 L, 0.5 mmol, 3.3 eq). This solution was then added drop wise to the heated flask containing chromous chloride, which turned violet upon stirring. The reaction mixture was stirred for 3 h at 65 C, and then cooled down to r.t. The mixture diluted with brine, filtered and extracted three times with EtOAc. The combined organic layers were dried over MgSO4, filtered and the solvent was removed. The crude green residue was then purified using silica gel column chromatography (Hex/ EtOAc 4:1) to yield the pure yellow oily product as a mixture of isomers (12.6 mg, 32%, E : Z Notably, one Boc group was lost during this reaction; the amide proton appears in the 1H NMR and the integration of the t butyl methyl protons at 1.4 ppm indicates the

PAGE 133

133 presence of only one t butyl g roup. 1H NMR (400 MHz, CDCl3) : 6.01 (d, J = 13.2 Hz, 1H), 5.09 (d, J = 6.7 Hz, 1H), 4.39 (d, J = 6.5 Hz, 1H), 3.72 (s, 3H), 2.49 (d br, 2H), 1.41 (s, 9H). 13C NMR (100 MHz, CDCl3) : 172.29, 155.33, 127.89, 120.89, 80.16, 67.10, 52.66, 34.16, 28.46. LRMS : m/z 264.3/266.3 (3:1) [M + H]+. ( S ) dimethyl 2 ((((9H fluoren9 yl)methoxy)carbonyl)amino)succinate ( 5 11): Laspartic acid dimethyl (150 mg, 0.76 mmol, 1 eq) was dissolved in water (0.8 mL) and cooled down to 0 C. NaHCO3 (150 mg, 1.9 mmol, 2 eq) was added to the solution, followed by a solution of Fmoc Cl (196 mg, 0.76 mmol, 1 eq) in dioxane (0.8 mL). The reaction was warmed up to room temperature and stirred for 2 h. Once TLC showed a complete reaction, it was diluted with water (1 mL) and dioxane was removed under reduced pressure. The aqueous layer was extracted three times with EtOAc. The organic layer was then washed twice with brine, dried over MgSO4 and the solvent was removed. The crude product ( 360 mg) was then purified using silica gel column chromatography (Hex/EtOAc 2:1) to give 290 mg (99%) of pure product. 1H NMR (400 MHz, CDCl3) : 7.70 (d, J = 7.4 Hz, 2H), 7.56 (m, 2H), 7.34 (t, J = 7.3 Hz, 2H), 7.25 (t, J = 7.3 Hz, 2H), 6.05 (d, J = 8.2 Hz, 1H), 4.69 4.62 (m, 1H), 4.36 (m, 2H), 4.18 (t, J = 6.8 Hz, 1H), 4.07 (q, J = 7.1 Hz, 1H), 3.70 (s, 3H), 3.63 (s, 3H), 2.97 (dd, J = 16.9, 3.8 Hz, 1H), 2.85 (dd, J = 16.9, 3.8 Hz, 1H). 13C NMR (100 MHz, CDCl3) : 171.42, 171.24, 156.22, 144.08, 143.96, 141.47, 127.91, 127.26, 125.31, 120.18, 67.36, 52.93, 52.18, 50.66, 47.30, 36.56. LRMS: m/z 406.3 [M + Na]+, 384.2 [M + H]+ ( S ) 2 ((((9H flu oren9 yl)methoxy)carbonyl)amino) 4 oxobutanoic acid ( 5 12): Compound 5 11 (100 mg, 0.26 mmol, 1 eq) was stirred in anhydrous ether (2 mL) and cooled down to 78C. DIBAL H (0.26 mL from 1 M solution in toluene, 0.26 mmol,

PAGE 134

134 1 eq) was added drop wise. The re action mixture was stirred at 78 C for 15 minutes and then the reaction was quenched by warming up to r.t and adding water (0.03 mL, 7 eq). After water addition, the reaction was stirred at r.t. for 30 min. till a gelatinous precipitate started to form. MgSO4 was then added to the reaction flask, and after mixing, the reaction product was filtered through a pad of celite in a fritted funnel. The solvent was removed, and the crude product was purified using silica gel column chromatography (Hex/EtOAc 1:1) to yield 14 mg (17%) of a product mixture (major : minor 7:1). 1H NMR (400 MHz, CDCl3) : 9.73 (s, 1H), 9.64 (s, minor product), 7.76 (d, J = 7.4 Hz, 4H), 7.59 (d, J = 6.5 Hz, 4H), 7.40 (t, J = 7.3 Hz, 4H), 7.32 (t, J = 7.3 Hz, 4H), 5.71 (d, J = 7.4 Hz, 1H), 4.66 (m, 1H), 4.39 (m, 4H), 4.23 (d, J = 6.5 Hz, 2H), 3.76 (s, 3H), 3.71 (s, minor pr oduct), 3.17 (dd, J = 18.5, 4.7 Hz, 1H), 3.06 (dd, J = 18.5, 4.7 Hz, 1H). LRMS: m/z 376.3 [M + Na]+, 354.3 [M + H]+. ( S ) 2 (2,2 dimethyl [1,3]dioxolan 4 yl) acetaldehyde ( 5 13): PCC (10.6 g, 49 mmol, 3.6 eq) was added slowly to a suspension of freshly acti vated molecular sieves (3A, 10.6 g) in dry DCM (50 mL). The alcohol (2 g, 13.6 mmol, 1 eq) was dissolved in 5 mL DCM and added to the above mixture which was stirred at r.t for 2 h. The mixture was then diluted using Et2O, filtered through silica gel and concentrated under reduced pressure, to give 1.8 g of the pure aldehyde as a colorless oil (76% yield). 1H NMR (400 MHz, CDCl3): 9.80 (s, 1H ), 4.53 (quint, J = 6.6 Hz, 1H ), 4.18 (dd, J = 8.2, 6.3 Hz, 1H ), 3.58 (dd, J = 8.2, 7.0 Hz, 1H ), 2.84 (ddd, J = 17.1, 6.7, 1.6 Hz, 1H ), 2.64 (ddd, J = 17.1, 6.2, 1.6 Hz, 1H), 1.41 (s, 3H), 1.36 (s, 3H ). 13C NMR (100 MHz, CDCl3): 200.22 109.46, 70.84, 69.33, 48.05, 26.99, 25.84. LRMS: m/z 167.2 [M + Na]+, 145.4 [M + H]+.

PAGE 135

135 ( S E ) 4 (3 chloroallyl) 2,2 dimethyl 1,3 d ioxolane ( 5 14): To a dry flask, anhydrous CrCl2 (4 g, 33.3 mmol, 10 eq) was added and then heated using a heat gun under vaccum. After cooling, the vacuum was released under argon, and dry THF (30 mL) was added. The greenish suspension was stirred in an oil bath and heated to 65C. The aldehyde ( 5 13 ) (500 mg, 3.4 mmol, 1 eq) was dissolved in 5 mL dry THF in another vial, and mixed with dry CHCl3 (1.3 mL, 16.7 mmol, 4 eq). This solution was then added drop wise to the heated flask containing chromous chlor ide, which turned violet upon stirring. The reaction mixture was stirred for 3 h at 65 C, and then cooled down to r.t. The mixture diluted with brine, filtered and extracted three times with Et2O. The combined organic layers were dried over MgSO4, filtere d and the solvent was removed. The crude green residue was then purified using silica gel column chromatography (Hex/ Et2O 14:1) to yield the pure yellow oily product as a mixture of isomers (226 mg, 37 %, E : Z 1H NMR (400 MHz, CDCl3): 6.05 (d, J = 13.3 Hz, 1H ), 5.89 (m, 1H), 4.12 (quint, J = 6.2 Hz, 1H ), 4.01 (dd, J = 8.0, 6.1 Hz, 1 H ), 3.55 (dd, J = 7.9, 6.9 Hz, 1H), 2.32 (m, 2H), 1.40 (s, 3H), 1.33 (s, 3H ). 13C NMR (100 MHz, CDCl3): 129.13 119.81 109.48, 74.87, 68.83, 35.26, 27.06, 25.75. The compound could not be detected by LRESIMS in both positive and negative modes. ( S E ) 5 chloropent 4 ene 1,2 diol ( 5 15): Compound 5 14 (122 mg) was dissolved in MeOH (1 mL). 200 mg of MeOH washed DOWEX 50WX4 were added to the solution and stirred at 50 C for 3 h. The mixture was filtered through celite using Et2O, and residual MeOH and H2O were removed using toluene via azeotropic distillation to give 90.3 mg (95%) of the diol product, which was used for the next step without further purification. 1H NMR (400 MHz, CDCl3): 6.05 (d, J = 13.3 Hz, 1H), 5.91

PAGE 136

136 (m, 1H ), 3.70 (m, 1H), 3.61 (dd, J = 11.2, 2.3 Hz, 1H ), 3.42 (dd, J = 11.2, 7.5 Hz, 1H), 2.19 (m, 2H ). 13C NMR (100 MHz, CDCl3): 129.57, 119.83, 71.41, 66.17, 34.84. LRMS: m/z 159.2/161.3 (3:1) [M + Na]+. ( S E ) 1 ((tertbutyldimethylsilyl)oxy) 5 chloropent 4 en 2 ol ( 5 16): Imidazole (32.5 mg, 0.478 mmol, 1.3 eq) and TBS Cl (66.5 mg, 0.441 mmol, 1.2 eq) were added sequentially to a solution of diol 5 15 ( 50 mg, 0.367 mmol, 1 eq) in DMF (0.5 mL). The solution was allowed to warm up to r.t and stirred for 4 h. The reaction mixture was then diluted with EtOAc, washed three times with saturated NaHCO3, washed twice with brine, dried over MgSO4 and the solvent was removed under reduced pressure. The crude product (74 mg) was then purified by column chromatography (Hex /Et2O, 12:1) to yield 43.1 mg (47%) of pure product. 1H NMR (400 MHz, CDCl3): 6.03 (d, J = 13.3 Hz, 1H), 5.9 (m, 1H), 3.67 (m, 1H ), 3.61 (dd, J = 9.9, 3.7 Hz, 1H ), 3.44 (dd, J = 9.9, 6.6 Hz, 1H), 2.22 (dd br, 6.7 Hz, 2H), 0.89 (s, 9H), 0.06 (s, 6H ). 13C NMR (100 MHz, CDCl3): 129.89, 119.29, 71.00, 66.49, 34.76, 26.07, 5.20, 5.17 HRESIMS: m/z 273.1051/ 275.1034 (3:1) [M + Na]+. ( S E ) 1 ((tertbutyldimethylsilyl)oxy) 5 chloropent 4 en 2 yl 5 methylenedecanoate ( 5 17): Et3N (13.6 L, 0.098 mmol, 1 eq) was added to a solution of 5 1 (18 mg, 0.098 mmol, 1 eq) in dry DCM (0.2 mL). EDC.HCl (19 mg, 0.098 mmol, 1 eq) and DMAP (4 mg, 0.033 mmol, 0.3 eq) were then added and the mixture was stirred at r.t. for 10 min. A solution of HOAT (14.6 mg, 0.12 mmol, 1.1 eq) in DMF (0.12 mL) was then added to the above mixture and cooled to 0 C. Compound 5 16 (15 mg, 0.059 mmol, 0.6 eq) was then dissolved in 0.2 m L DCM and added to the cooled mixture, which was then stirred at r.t. overnight. The reaction was then quenched by adding 0.7

PAGE 137

137 mL of Sorensen buffer (0.4 M, pH 7). The mixture was extracted three times with DCM, dried over MgSO4 and the solvent was removed under reduced pressure to give 33.4 mg of crude product. Purification using preparative TLC (Hex/Et2O 2:1) yielded 18.3 mg of the pure ester 5 17 as a colorless oil (73%). 1H NMR (400 MHz, CDCl3): 6.01 (d, J = 13.2 Hz, 1H), 5.85 (m, 1H), 4.88 (m, 1H), 4.75 (s, 1H), 4.72 (s, 1H), 3.63 (m, 2H ), 2.29 (t, J = 7.4 Hz, 2H ), 2.04 (t, J = 7.3 Hz, 2H ), 1.98 (t, J = 7.7 Hz, 2H ), 1.75 (m 2H), 1.41 (m, 2H), 1.32 (m, 2H), 1.28 (m, 2H), 0.88 (t, 3H), 0.88 (s, 9H), 0.04 (s, 6H ) 13C NMR (100 MHz, CDCl3): 173.29, 149.08, 128.94, 119.86, 109.67, 72.95, 63.54, 36.02, 35.51, 34.13, 32.24, 31.84, 29.94, 27.64, 26.01, 23.18, 22.79, 14.31, 5.21, 5.19. HRESIMS: m/z 439.2410/ 441.2388 (3:1) [M + Na]+. ( S E ) 5 chlor o 1 hydroxypent 4 en 2 yl 5 methylenedecanoate ( 5 18): AcOH (3.4 L, 2.5 eq) was added to TBAF (33 L of a 1 M solution in THF, 2 eq) to give a neutral reagent, which was added to a solution of compound 5 17 (6.8 mg, 0.017 mmol, 1 eq) in THF (0.2 mL) at r. t. The reaction mixture was stirred at r.t. for 3 h, and then quenched by adding 0.7 mL of saturated NaHCO3 solution. The aqueous layer was extracted 3 with EtOAc, dried over MgSO4 and concentrated under reduced pressure. Purifica tion using preparative T LC (Hex /Et2O 2:1) yielded 3.5 mg of the pure alcohol 5 18 as a colorless oil (72%, 85% BRMS). 1H NMR (400 MHz, CDCl3): 6.06 (d, J = 13.3 Hz, 1H), 5.87 (m, 1H), 4.93 (m, 1H), 4.75 (s, 1H), 4.72 (s, 1H), 3.69 (m, 2H), 2.40 (m, 2H ), 2.35 (t, J = 7.4 Hz, 2H ), 2.05 (t, J = 7.4 Hz, 2H ), 2.00 (t, J = 7.4 Hz, 2H), 1.78 (m, 2H), 1.46 1.26 (m, 6H ), 0.89 (t, J = 6.65 Hz, 3H ). 13C NMR (100 MHz, CDCl3): 173.83, 148.98, 128.47, 120.36, 109.76, 73.69, 63.96, 35.97, 35.44, 34.03, 32.26, 31.81, 27.62, 23.11, 22.78, 14.29. HRESIMS: m/z 325.1541/327.1517 (3:1) [M + Na]+.

PAGE 138

138 ( S E ) 5 chloro2 ((5 methylenedecanoyl)oxy)pent 4 enoic acid ( 5 2): Freshly prepared Jones reagent (1.1 mL) was added over 5 min to a solution of alcohol 5 18 (30.6 mg, 0.1 mmol) in acetone (7 mL) at 0 C. The mixture was warmed up to r.t. and stirred for 1 h. Excess reagent was quenched by the addition of i PrOH (1.5 mL) followed by filtration through a pad of celite. The solvent was removed and then the residue was resuspended in water and extracted t wice with EtOAc. The combined EtOAc layers were dried over MgSO4, filtered and the solvent was removed. The crude product was purified using silica gel column chromatography to give 18 mg of a semi pure fraction, which was repurified using reversedphase HPLC (YMC Pack ODS AQ, 250 10 mm, 5 m, 2 mL/min; UV detection at 220/200 nm) using a MeOH/0.05% aqueous TFA linear gradient (60% to 100% over 30 min and then 100% MeOH for 10 min) to give 8 mg of the pure product as a colorless oil (mixture of Z and E isomers; Z : E 20 D 3.4 ( c 0. 32, DCM). 1H NMR (400 MHz, CDCl3) : 6.12 (d, J = 13.3 Hz, 1H ), 5.95 5.86 (m, 1H), 5.11( br dt, J = 5.1 Hz, 1H), 4.75 (s, 1H), 4.72 (s, 1H), 2.64 (m, 2H), 2.41 (td, J = 7.4, 3.2 Hz, 2H), 2.07 (t, J = 7.4 Hz, 2H), 1.99 (t, J = 7.6 Hz, 2H), 1.79 (m, 2H), 1. 46 1.36 (m, 2H), 1.36 1.22 (m, 4H ), 0.89 (t, J = 7.0 Hz, 3H ). 13C NMR (100 MHz, CDCl3): 173.22, 148.91, 127.14, 121.51, 109.85, 35.98, 35.35, 33.62, 32.62, 31.83, 27.63, 22.93, 22.79, 14.3 LRMS: m/z 315.2/317.0 [M H]-, 182.9 [M C5H7ClO2](acid fr agment), 149.0/150.8 (3:1) [M C11H19O](alcohol fragment), 130.8/132.8 (3:1) [M C11H21O](alcohol fragment, McLafferty rearrangement or loss of water).HRESIMS: m/z 315.1375/317.1357 (3:1) [M H]-, 631.2816/ 633.2802 [2M H]-. Pyocyanin and Elastase Qu antitation in Pseudomonas aeruginosa150 An overnight culture of Pseudomonas aeruginosa (strain PAO1) was diluted 100fold and incubated at 37 C for 2 h with shaking. This was followed by another 100fold

PAGE 139

139 dilution and incubation for 1 h. Then, 100 L of this culture were transferred to another culture tube containing 890 L LB broth and 10 L compound (1 mM final concentration) or EtOH control, and incubated at 37 C with shaking for 6 h. The culture was then spun down for 10 min at maximum speed, and the s upernatant was filtered using 0.2 M Eppendorf filters. 100 L of this sterile supernatant was added to 900 L Elastin Congo Red (ECR) suspension (prepared in 1 mM CaCl2, 100 mM Tris buffer, pH 7.2) and incubated at 37 C with shaking for 18 h. After 18 h, the solid ECR was removed by centrifugation and the UV absorbance was measured at 495 nm for the quantitation of the soluble Congo red liberated by the enzyme LasB, reflecting the enzyme activity. For Pyocyanin quantification, 500 L of the sterile supern atant (stored overnight at 80 C and used next day) were added to 500 L CHCl3 in an Eppendorf tube. Tube shaking allowed for the extraction of Pyocyanin in the CHCl3 layer. This layer was then added to 150 L of 0.2 N HCl in another Eppendorf tube. After shaking, the aqueous layer containing Pyocyanin turned red. 100 L of this layer were transferred to a clear bottomed 384well plate and the absorbance was measured at 385 nm to quantify the amount of Pyocyanin. Data was analyzed using GraphPad Prism 5 so ftware. RT qPCR in Pseudomonas aeruginosa150 A culture of Pseudomonas aeruginosa (strain PAO1) was grown overnight and diluted as mentioned above. Then, 100 L of this culture were transferred to another culture tube containing 890 L LB broth and 10 L co mpound or EtOH control, and incubated at 37 C with shaking for 6 h. The culture was then spun down for 10 min at maximum speed; the supernatant was removed (used for quantitation mentioned above) and the cell pellet was resuspended in 500 L LB broth. 1 m L of RNAprotect

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140 b acteria r eagent (Qiagen) was added to the resuspended pellet, mixed and incubated at r.t for 5 min to stabilize RNA After centrifugation at maximum speed for 10 min, the supernatant was decanted and RNA was extracted using the RNeasy Kit (Qiagen) according to the manufacturers instructions (using enzymatic lysis and proteinase K digestion protocol). On column DNase digestion was done using the RNaseFree DNase set (Qiagen). DNA contamination was quantified by qPCR of the RNA samples usi ng a primer / probe set for rpsL.150 To further reduce DNA contamination, the TURBO DNA free kit (Ambion) was used according the manufacturers protocol (rigorous DNase treatment). RNA samples were then requantified by UV absorbance (Nanodrop 8000, Thermo) and RNA integrity was assessed using Agilent 2100 Bioanalyzer. Samples used for RT qPCR showed RIN values > 9.2. Total RNA (2 g) was reversetranscribed using Superscript II reverse transcriptase (Invitrogen) and Oligo (dT)1218 Primers (Invitrogen). For qPCR, 0 .5 L of the synthesized cDNA was added to 12.5 L of 2 TaqMan gene expression master mix, 1.25 L of 20 TaqMan gene expression assay mix and 10 .75 L sterile water, in a total of 25 L reaction volume. TaqMan primers/probes used were custom designed by Applied Biosystems150 for the target genes lasB and phzG1 and the endogenous control rpoD Real time PCR was performed on an ABI 7300 sequence detection system with the following thermocycler program: 2 min at 50 C, 10 min at 95 C, 40 cycles of 15 sec at 95 C and 1 min at 60 C. Experiments were done in triplicate. Data was analyzed using GraphPad Prism 5 software.

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141 THP 1 C ell C ulture and RTqPCR THP 1 human acute monocytic leukemia cells were purchased from American Type Culture Collection (ATC C, TIB 202). The cells were maintained and propagated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 0.05 mM mercaptoethanol at 37 C humidified air and 5% CO2. For the anti inflammatory assays, cells were seeded (5 106 cells/ w ell) in 6 well plate in RPMI 1640 medium supplemented with 10% FBS. The monocytes were differentiated to macrophages by incubating the cells in the presence of 100 nM PMA (Sigma) for 48 h. The medium was then aspirated and the adherent macrophages were was hed twice with PBS and fresh medium was added. Cells were treated with the compound (100 M in DMSO) for 1 h before they were stimulated with LPS (5 g/mL) for 4, 12 or 24 h. At the end of each time point, the medium was aspirated and the RNeasy mini kit ( QIAGEN) was used for RNA extraction and purification according to the manufacturers protocol T otal RNA was quantified using UV absorbance. Total RNA (2 g) was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and Oligo (dT)121 8 Primers (Invitrogen). For qPCR, 0 .5 L of the synthesized cDNA was added to 12.5 L of 2 TaqMan gene expression master mix, 1.25 L of 20 TaqMan gene expression assay mix and 10 .75 L sterile water, in a total of 25 L reaction volume. TaqMan primers /probes (Applied Biosystems) used for this experiment were for the target genes TNF IL 6 IL and IL 8 and the endogenous control GAPDH. Real time PCR was performed on an ABI 7300 sequence detection system with the following thermocycler program: 2 mi n at 50 C, 10 min at 95 C, 40 cycles of 15 sec at 95 C and 1 min at 60 C. Experiments were done in triplicate. Data was analyzed using GraphPad Prism 5 software.

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142 Table 5 1. NMR spectroscopic data for 5 methylene decanoic acid ( 5 1 ) and its chlorinated ester ( 5 2 ) in CDCl3 ( in ppm, J in Hz) at 600 MHz C/H No. 5 methylene decanoic acid ( 5 1 ) Chlorinated ester ( 5 2 ) C H ( J ) HMBC a C H ( J ) HMBC a 1 180.1 qC 2, 3 173.3 qC 2, 3 2 33.6 CH 2 2.35 t (7.4) 3, 4 33.4 CH 2 2.40 dt (7.8, 6.7) 3, 4 3 22.5 CH 2 1.77 m 2 4 22.7 CH 2 1.79 m 2, 4 4 35.0 CH 2 2.06 t (7.5) 3, 6, 11 35.2 CH 2 2.06 t (7.3) 2, 3, 6, 11 5 148.7 qC 3, 4, 6, 7, 11 148.9 qC 3, 4, 6, 7, 11 6 35.6 CH 2 1.99 t (7.5) 4, 7, 11 35.8 CH 2 1.99 t (7.6) 4, 7, 11 7 27.3 CH 2 1.42 m 6, 8 27.4 CH 2 1.42 m 6, 8 8 31.6 CH 2 1.26 m 7, 9, 10 31.7 CH 2 1.26 m 6, 7, 9, 10 9 22.4 CH 2 1.31 m 7, 8, 10 22.6 CH 2 1.32 m 8, 10 10 13.9 CH 3 0.89 t (7.1) 8, 9 14.1 CH 3 0.89 t (7.1) 8, 9 11 109.5 CH 2 4.72 s, 4.75 s 4, 6 109.8 CH 2 4.72 s, 4.75 s 4, 6 1 173.5 qC 2 2 70.7 CH 5.09 t (5.6) 3, 4, 5 3 32.5 CH 2 2.64 m 2, 4, 5 4 127.3 CH 5.9 m 2, 3, 5 5 121.4 CH 6.12 d (13.4) 3, 4 a Protons showing long range correlation to indicated carbon.

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143 F igure 5 1. Chemical structure of 5methylene decanoic acid ( 5 1 ) and it s chlorinated ester ( 5 2 ).

PAGE 144

144 Figure 5 2. LRMS data for compound 5 2 ( ve mode). McLafferty rearrangement (shown in the figure) or loss of water from the alcohol fragment could produce the fragment detected at m/z 131.1/132.9.

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145 Figure 5 3 Retrosynthetic strategies for the semi synthesis of the chlorinated ester ( 5 2 ). Natural products are shown in blue; starting points for each strategy are shown in red.

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146 Figure 5 4 Schemes showing the initial trials to obtain the alcohol fragment for coupling with the acid 5 1 to obtain the chlorinated ester 5 2 (from retrosynthetic strategies 1a and 1b).

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147 Figure 5 5 Scheme for semi synthesis of the chlorinated ester 5 2 (from retrosynthetic strategy 2).

PAGE 148

148 Figure 56 Comparison of 1H NMR spectra of the natural product ( 5 2 ) and the synthetic compound.

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149 A B Figure 57 Quorum sensing inhibition by the fatty acid 5 methylene decanoic acid ( 5 1 ) in P. aeruginosa (PAO1). A) Effect on the production of the virulence factors p yocyan in pigment and the elastase Las B. B) Effect on virulence factor gene ex pression assessed by RT qPCR P < 0.05, P < 0.01. Lyngbyoic acid1 50 was used as the positive control.

PAGE 150

150 Figure 5 8 Effect of the chlorinated ester ( 5 2 ) on the transcript levels of proinflammatory cytokines IL 6 IL IL 8 and TNF in differentiated THP 1 cells. Effect monitored after 4 h, 12 h and 24 h of LPS stimulation (5 g/mL). Data shown are for compound 5 2 tested at 100 M, relative to DMSO control. P value < 0.1, ** P value < 0.05.

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151 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS Drug discovery efforts have a massive impact on life expectancy and quality of life. In addition, drug development has indirectly aided the basic research community by generating small molecule probes for pharmacological studies.167 Our continuous efforts to discover new drug leads and pharmacological probes from marine cyanobacteria led to the identification of a group of compounds which belong to two main structural classes; peptides and fatty acids. Pitipeptolides are a group of cyclodepsipeptides with antimycobacterial properties. The work presented here shows the identification of their biological activity as well as the preparation of two probes for pitipeptolide A which could be used for target identification purposes. The prepared biotinylated derivative of pitipeptolide A could be employed in a pull down assay to purify and identify the directly interacting molecular target of this compound after incubation with the cell lysate. Additionally, the availability of a fluorescent derivative could add more information regarding the cellular localization of this compound, which could guide further experimental approaches. The ide ntification of the mechanism of action of pitipeptolides in Mycobacterium tuberculosis will offer a great advantage which could allow for further compound optimization to obtain a more potent antibiotic, and could possibly uncover a nov el drug target for t uberculosis treatment. Fatty acids are important structural components which also play important roles in several biological processes. Serinolamide B, a new fatty acid amide analogue, was identified as a cannabinoid receptor agonist. More interestingly, t he known cyanobacterial fatty acid amide malyngamide B was also proved to act as a cannabinoid

PAGE 152

152 receptor agonist for the first time. This latter finding is interesting because it opens further research avenues for the cannabimimetic evaluation of other maly ngamide analogues. Since there are more than 30 known malyngamide analogues identified to date, cannabimimetic evaluation of other analogues could lead to the identification of a more potent cannabinoid receptor agonist. 5 methylene decanoic acid is a sim ple but novel lipid which was produced in large amounts by the source organism, which indicates its ecological importance for this organism. One hypothesis was that this fatty acid is helping the organism to survive by acting as a defensive chemical to inhibit quorum sensing in other bacteria. Indeed, in the experimental model we used, this fatty acid appeared to inhibit quorum sensing (QS) in Pseudomonas aeruginosa. Since there are other model organisms available for investigating the effect on QS activity this fatty acid might show higher potency against other organism s. Additionally, a minor chlorinated ester analogue was synthesized after its chemical characterization which allowed for the identification of its anti inflammatory biological activity. Fur ther structureactivity relationship studies could provide insight into the essential structural features for this biological activity and allow for t he development of a more potent analogue. The continuous discovery of novel molecules from marine organis ms designates the marine environment as a wealthy reservoir that still encloses a large number of unidentified bioactive entities awaiting discovery. In addition, t he work presented here highlights the importance of the integration of multiple disciplines, including analytical chemistry, biology and chemical synthesis for the successful characterization of a

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153 bioactive molecule from natural sources, which could be subsequently developed into a useful drug.

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154 APPENDIX A NMR DATA OF ISOLATED MARINE CYANOBACTERIAL SECONDARY METABOLITES The following pages contain 1D and 2D NMR spectral data for the natural products isolated from Lyngbya samples mentioned earlier. NMR data include 1H, 13C, COSY, TOCSY, ROESY, HSQC and HMBC spectra.

PAGE 155

155 Figure A 1 1H NMR Spectrum of Pitiprolamide ( 2 1 ) in Benzened6 (600 MHz) at 20 C.

PAGE 156

156 Figure A 2 13C NMR Spectrum of Pitiprolamide ( 2 1 ) in Benzened6 ( 125 MHz) at 20 C.

PAGE 157

157 Figure A 3 COSY Spectrum of Pitiprolamide ( 2 1 ) in Benzened6 (600 MHz) at 20 C

PAGE 158

158 Figure A 4 TOCSY Spectrum of Pitiprolamide ( 2 1 ) in Benzened6 (600 MHz) at 20 C

PAGE 159

159 Figure A 5 ROESY Spectrum of Pitiprolamide ( 2 1 ) in Benzened6 (600 MHz) at 20 C

PAGE 160

160 Figure A 6 HSQC Spectrum of Pitiprolamide ( 2 1 ) in Benzened6 (600 MHz) at 20 C

PAGE 161

161 Figur e A 7 HMBC Spectrum of Pitiprolamide ( 2 1 ) in Benzened6 (600 MHz) at 20 C

PAGE 162

162 Figure A 8 1H NMR Spectrum Pitiprolamide ( 2 1 ) in Acetoned6 (400 MHz) at 25 C

PAGE 163

163 Figure A 9. 1H NMR Spectrum of Pitipeptolide C ( 3 3 ) in CDCl3 (600 MHz).

PAGE 164

164 Figure A 10. 13C NMR Spectrum of Pitip eptolide C ( 3 3 ) in CDCl3 ( 125 MHz)

PAGE 165

165 Figure A 11. COSY Spectrum of Pitip eptolide C ( 3 3 ) in CDCl3 (600 MHz)

PAGE 166

166 Figure A 12. TOCSY Spectrum of Pitip eptolide C ( 3 3 ) in CDCl3 (600 MHz).

PAGE 167

167 Figure A 1 3. ROESY Spectrum of Pitip eptolide C ( 3 3 ) in CDCl3 (600 MHz).

PAGE 168

168 Figure A 14. HSQC Spectrum of Pitip eptolide C ( 3 3 ) in CDCl3 (600 MHz)

PAGE 169

169 Figure A 15. HMBC Spectrum of Pitip eptolide C ( 3 3 ) in CDCl3 (600 MHz)

PAGE 170

170 Figure A 16. 1H NMR Spectrum of Pitip ept olide D ( 3 4 ) in CDCl3 (600 MHz).

PAGE 171

171 Figure A 17. COSY Spectrum of Pitip eptolide D ( 3 4 ) in CDCl3 (600 MHz)

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172 Figure A 18. TOCSY Spectrum of Pitip eptolide D ( 3 4 ) in CDCl3 (600 MHz).

PAGE 173

173 Figure A 19. ROESY Spectrum of Pitip eptolide D ( 3 4 ) in CDCl3 (600 M Hz)

PAGE 174

174 Figure A 20. HSQC Spectrum of Pitip eptolide D ( 3 4 ) in CDCl3 (600 MHz)

PAGE 175

175 Figure A 21. HMBC Spectrum of Pitip eptolide D ( 3 4 ) in CDCl3 (600 MHz)

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176 Figure A 22. 1H NMR Spectrum of Pitip eptolide E ( 3 5 ) in CDCl3 (600 MHz).

PAGE 177

177 Figure A 23. COSY Spe ctrum of Pitip eptolide E ( 3 5 ) in CDCl3 (600 MHz)

PAGE 178

178 Figure A 24. TOCSY Spectrum of Pitip eptolide E ( 3 5 ) in CDCl3 (600 MHz)

PAGE 179

179 Figure A 25. ROESY Spectrum of Pitip eptolide E ( 3 5 ) in CDCl3 (600 MHz)

PAGE 180

180 Figure A 26. HSQC Spectrum of Pitip eptolide E ( 3 5 ) in CDCl3 (600 MHz)

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181 Figure A 27. HMBC Spectrum of Pitip eptolide E ( 3 5 ) in CDCl3 (600 MHz)

PAGE 182

182 Figure A 28. 1H NMR Spectrum of Pitipeptolide F ( 3 6 ) in CDCl3 (600 MHz).

PAGE 183

183 Figure A 2 9. COSY Spectrum of Pitip eptolide F ( 3 6 ) in CDCl3 (600 MHz)

PAGE 184

184 Figur e A 30. TOCSY Spectrum of Pitip eptolide F ( 3 6 ) in CDCl3 (600 MHz).

PAGE 185

185 Figure A 31. ROESY Spectrum of Pitip eptolide F ( 3 6 ) in CDCl3 (600 MHz).

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186 Figure A 32. HSQC Spectrum of Pitip eptolide F ( 3 6 ) in CDCl3 (600 MHz)

PAGE 187

187 Figure A 33. HMBC Spectrum of Piti p eptolide F ( 3 6 ) in CDCl3 (600 MHz)

PAGE 188

188 Figure A 34. 1H NMR Spectrum of Serinolamide B ( 4 1 ) in CDCl3 (600 MHz)

PAGE 189

189 Figure A 35. COSY Spectrum of Serinolamide B ( 4 1 ) in CDCl3 (600 MHz).

PAGE 190

190 Figure A 36. TOCSY Spectrum of Serinolamide B ( 4 1 ) in CDCl3 (60 0 MHz)

PAGE 191

191 Figure A 37. NOESY Spectrum of Serinolamide B ( 4 1 ) in CDCl3 (600 MHz).

PAGE 192

192 Figure A 38. HSQC Spectrum of Serinolamide B ( 4 1 ) in CDCl3 (600 MHz).

PAGE 193

193 Figure A 39. HMBC Spectrum of Serinolamide B ( 4 1 ) in CDCl3 (600 MHz).

PAGE 194

194 Figure A 40. 1H NMR Sp ectrum of 5 M ethylene D ecanoic A cid ( 5 1 ) in CDCl3 (600 MHz).

PAGE 195

195 Figure A 41. COSY Spectrum of 5M ethylene D ecanoic A cid ( 5 1 ) in CDCl3 (600 MHz).

PAGE 196

196 Figure A 42. TOCSY Spectrum of 5 M ethylene D ecanoic A cid ( 5 1 ) in CDCl3 (600 MHz).

PAGE 197

197 Figure A 43. NOESY Spectrum of 5 Methylene Decanoic Acid ( 5 1 ) in CDCl3 (600 MHz).

PAGE 198

198 Figure A 44. HSQC Spectrum of 5 M ethylene D ecanoic A cid ( 5 1 ) in CDCl3 (600 MHz).

PAGE 199

199 Figure A 45. HMBC Spectrum of 5 Methylene Decanoic Acid ( 5 1 ) in CDCl3 (600 MHz).

PAGE 200

200 Figure A 4 6 1H NMR Spectrum of Chlorinated Ester ( 5 2 ) in CDCl3 (600 MHz).

PAGE 201

201 Figure A 4 7 COSY Spectrum of Chlorinated Ester ( 5 2 ) in CDCl3 (600 MHz).

PAGE 202

202 Figure A 48. TOCSY Spectrum of Chlorinated Ester ( 5 2 ) i n CDCl3 (600 MHz).

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203 Figure A 49. NOESY Spectrum of Chlor inated Ester ( 5 2 ) in CDCl3 (600 MHz).

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204 Figure A 50. HSQC Spectrum of Chlorinated Ester ( 5 2 ) in CDCl3 (600 MHz).

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205 Figure A 51. HMBC Spectrum of Chlorinated Ester ( 5 2 ) in CDCl3 (600 MHz).

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206 APPENDIX B NMR DATA OF SYNTHETIC COM P O UNDS The following pages contain 1H, 13C NMR spectra for synthetic compounds and intermediates mentioned earlier. NMR data are for synthetic schemes mentioned in Chapter 3 and Chapter 5

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207 Figure B 1 1H NMR Spectrum of I ntermediate 3 9 in CDCl3 ( 4 00 MHz).

PAGE 208

208 Figure B 2 13C NMR Spectrum of I ntermediate 3 9 in CDCl3 ( 1 00 MHz).

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209 8 Figure B 3 1H NMR Spectrum of I ntermediate 3 10 in CDCl3 ( 4 00 MHz). Residual solvent (EtOAc) peaks are denoted with asterisks.

PAGE 210

210 Figure B 4. 1H NMR Spectrum of I ntermediate 3 11 in CDCl3 (400 MHz ). Residual solvent (EtOAc) peaks are denoted with asterisks.

PAGE 211

211 Figure B 5 1H NMR Spectrum of I ntermediate 3 12 in CDCl3 ( 4 00 MHz).

PAGE 212

212 Figure B 6 13C NMR Spectrum of intermediate 3 12 in CDCl3 ( 1 00 MHz).

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213 Figure B 7 1H NMR Spectrum of I ntermediate 3 13 in CDCl3 ( 4 00 MHz).

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214 Figure B 8 1H NMR Spectrum of I ntermediate 3 14 in CDCl3 ( 4 00 MHz).

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215 Figure B 9 1H NMR Spectrum of I ntermediate 3 15 in CDCl3 ( 4 00 MHz).

PAGE 216

216 Figure B 10. 13C NMR Spectrum of I ntermediate 3 15 in CDCl3 ( 1 00 MHz).

PAGE 217

217 Figure B 11. 1H NMR Spectrum of PitiA Biotin ( 3 7 ) in CDCl3 (400 MHz ).

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218 Figure B 12. APT Spectrum of PitiA Biotin 3 7 in CDCl3 ( 1 00 MHz).

PAGE 219

219 Figure B 13. 1H NMR Spectrum of I ntermediate 5 3 in CDCl3 ( 4 00 MHz). Residual solvent (EtOAc) peaks are denoted with asteris ks.

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220 Figure B 14. 13C NMR Spectrum of I ntermediate 5 3 in CDCl3 ( 1 00 MHz). Residual solvent (EtOAc) peaks are denoted with asterisks.

PAGE 221

221 Figure B 15. 1H NMR Spectrum of I ntermediate 5 4 in CDCl3 (400 MHz). Spectrum shown is for the crude product. Multiple aldehyde peaks indicate nonselective reduction.

PAGE 222

222 Figure B 16. 13C NMR Spectrum of I ntermediate 5 4 in CDCl3 ( 1 00 MHz ). Spectrum shown is for the crude product

PAGE 223

223 Figure B 17. 1H NMR Spectrum of Intermediate 5 6 in CDCl3 (400 MHz) showing mixture of is omers ( and hyroxy acids with E vinyl chloride, and Z and E isomers of hydroxy acid).

PAGE 224

224 Figure B 1 8 1H NMR Spectrum of I ntermediate 5 7 in CDCl3 (400 MHz). Residual solvent (EtOAc) peaks are denoted with asterisks.

PAGE 225

225 Figure B 1 9 13C NMR Spectrum of I ntermediate 5 7 in CDCl3 ( 1 00 MHz).

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226 Figure B 20. 1H NMR Spectrum of I ntermediate 5 8 in CDCl3 ( 4 00 MHz). Residual solvent (EtOAc) peaks are denoted with asterisks.

PAGE 227

227 Figure B 2 1 13C NMR Spectrum of I ntermediate 5 8 in CDCl3 ( 1 00 MHz).

PAGE 228

228 Figure B 2 2 1H NMR Spectrum of I ntermediate 5 9 in CDCl3 ( 4 00 MHz). Residual solvent (EtOAc) peaks are denoted with asterisks.

PAGE 229

229 Figure B 2 3 13C NMR Spectrum of I ntermediate 5 9 in CDCl3 ( 1 00 MHz).

PAGE 230

230 Figure B 2 4 1H NMR Spectrum of I ntermediate 5 10 in CDCl3 ( 4 00 MHz).

PAGE 231

231 Figure B 2 5 13C NMR Spectrum of I ntermediate 5 10 in CDCl3 ( 1 00 MHz).

PAGE 232

232 Figure B 2 6 1H NMR Spectrum of I ntermediate 5 11 in CDCl3 ( 4 00 MHz). Residual solvent (EtOAc) peaks are denoted with asterisks.

PAGE 233

233 Figure B 2 7 13C NMR Spectrum of I nt ermediate 5 11 in CDCl3 ( 1 00 MHz). Residual solvent (EtOAc) peaks are denoted with asterisks.

PAGE 234

234 Figure B 2 8 1H NMR Spectrum of I ntermediate 5 12 in CDCl3 (400 MHz ). Spectrum shown is for the crude product. Two aldehyde peaks indicate nonselective reduct ion. Residual solvent (EtOAc) peaks are denoted with asterisks.

PAGE 235

235 Figure B 2 9 1H NMR Spectrum of I ntermediate 5 13 in CDCl3 ( 4 00 MHz). Residual solvent ( DCM ) peak is denoted with asterisks.

PAGE 236

236 Figure B 30. 13C NMR Spectrum of I ntermediate 5 13 in CDCl3 ( 1 00 MHz).

PAGE 237

237 Figure B 3 1 1H NMR Spectrum of I ntermediate 5 14 in CDCl3 ( 4 00 MHz).

PAGE 238

238 Figure B 3 2 13C NMR Spectrum of I ntermediate 5 14 in CDCl3 ( 1 00 MHz).

PAGE 239

239 Figure B 3 3 1H NMR Spectrum of I ntermediate 5 1 5 in CDCl3 ( 4 00 MHz).

PAGE 240

240 Figure B 3 4 13C NMR Sp ectrum of I ntermediate 5 15 in CDCl3 ( 1 00 MHz).

PAGE 241

241 Figure B 35. 1H NMR Spectrum of I ntermediate 5 16 in CDCl3 ( 4 00 MHz).

PAGE 242

242 Figure B 36. 13C NMR Spectrum of I ntermediate 5 16 in CDCl3 ( 1 00 MHz).

PAGE 243

243 Figure B 3 7 1H NMR Spectrum of I ntermediate 5 17 in CDCl3 ( 4 00 MHz). Residual solvent (EtOAc) peaks are denoted with asterisks.

PAGE 244

244 Figure B 38. 13C NMR Spectrum of I ntermediate 5 17 in CDCl3 ( 1 00 MHz).

PAGE 245

245 Figure B 3 9 1H NMR Spectrum of I ntermediate 5 18 in CDCl3 ( 4 00 MHz). Residual solvent (Et2O ) peaks are denoted with asterisks.

PAGE 246

246 Figure B 40. 13C NMR Spectrum of I ntermediate 5 18 in CDCl3 ( 1 00 MHz).

PAGE 247

247 Figure B 4 1 1H NMR Spectrum of the Synthesized Chlorinated Ester 5 2 in CDCl3 ( 4 00 MHz).

PAGE 248

248 Figure B 4 2 13C NMR Spectrum of the Synthesized Chlorinated Ester 5 2 in CDCl3 ( 1 00 MHz).

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249 LIST OF REFERENCES 1. Houghten, R. A. Curr. Biol 1994, 4, 564 567. 2. Ortholand, J. Y.; Ganesan, A. Curr. O pin. C hem Biol. 2004, 8, 271280. 3. Class, S. Chem Eng News 2002, 80, 3949. 4. Henkel, T.; Brun ne, R. M.; Mller, H.; Reichel, F Angew Chem Int. Ed 1999 38, 643647. 5. Newman, D. J.; Cragg, G. M. J. Nat Prod 2012, 75, 311335. 6. Harvey, A. Drug D iscov. T oday 2000, 5, 294300. 7. Census of Marine Life. www .coml.org 8. Fenical, W. Trends Biotechnol 1997 15 339 41. 9. Blunt, J. W.; Copp, B. R.; Munro, M. H.; Northcote, P. T.; Prinsep, M. R. Nat Prod Rep 2011, 28, 196 268. 10. Blunt, J. W.; Copp, B. R.; Munro, M. H.; N orthcote, P. T.; Prinsep, M. R. N at Prod Rep 2010, 27, 165 237. 11. Galeano, E.; Rojas, J. J.; Martnez, A. Nat Prod Commun 2011, 6, 287300. 12. Mayer, A. M.; Glaser, K. B.; Cuevas, C.; Jacobs, R. S.; Kem, W.; Little, R. D.; McIntosh, J. M.; Newman, D. J.; Potts, B. C.; Shuster, D. E. Trends Pharmacol Sci 2010, 31, 25565. 13. Molinski, T. F.; Dalisay, D. S.; Lievens, S. L.; Saludes, J. P. Nat Rev Drug Discov 2009, 8, 69 85. 14. Montaser, R.; Luesch, H. Future Med. Chem 2011, 3, 1475 89. 15. Munro, M. H.; Blunt, J. W.; Dumdei, E. J.; Hickford, S. J.; Lill, R. E.; Li, S.; Battershill, C. N.; Duckworth, A. R. J. Biotechnol 1999 70, 15 25. 16. Kong, D. X.; Jiang, Y. Y.; Zhang, H. Y. Drug Discov Today 2010, 15, 884 6. 17. FDA approves Adcetris to treat two types of lymphoma. www.fda.gov 18. Gerwick, W. H.; Moore, B. S. Chem Biol. 2012, 19, 85 98. 19. Huyck, T. K.; Gradishar, W.; Manuguid, F.; Kirkpatrick, P. Nat Rev Drug Discov 2011, 10 1734. 20. Koski, R. R. P. T. 2008 33, 271.

PAGE 250

250 21. Ol ivera, B. M In : Drugs from the Sea. Fusetani N (Ed.). Karger Publishers, Basel, Switzerland 2000 75 85. 22. Rinehart, K. L. Med Res Rev 2000, 20, 127. 23. Bai, R. L.; Paull, K. D.; Herald, C. L.; Malspeis L.; Pettit, G. R.; Hamel, E. J Biol. Chem 1991, 266, 158829. 24. Hirata, Y.; Uemura, D. Pure Appl Chem 1986, 58, 701 710. 25. Luesch, H.; Moore, R. E.; Paul, V. J.; Mooberry, S. L.; Corbett, T. H. J. Nat Prod 2001, 64, 90710. 26. Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Pr insep, M. R. Nat Prod Rep 2012, 29, 144 222. 27. Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Nat Prod Rep 2013, 30, 237 323. 28. de Silva, E. D.; Scheuer, P. J. Tetrahedron Lett 1980, 21, 16111614. 29. Glaser, K. B.; Jacobs, R. S. Biochem. Pharmacol. 1986 35, 449. 30. Lombardo, D.; Dennis, E. A. J. Biol Chem 1985, 260, 72347240. 31. Folmer, F.; Jaspars, M.; Schumacher, M.; Dicato, M.; Diederich, M. Biochem Pharmacol 2010, 80, 1793800. 32. McGeer, E. G .; Olney, J W.; McGeer, P. L. In: Kainic acid as a tool in neurobiology Raven Press New York 1978. 33. Victor Nadler, J. Life S ci 1981, 29, 20312042. 34. Oln ey, J. W.; Rhee, V.; Ho, O. L. Brain R es 1974, 77, 507. 35. Moloney, M. G. Nat P rod R ep 1998, 15, 205 219. 36. Burja, A. M.; Banaigs, B.; AbouMansour, E.; Grant Burgess, J.; Wright, P. C. Tetrahedron 2001, 57, 93479377. 37. Nunnery, J. K.; Mevers, E.; Gerwick, W. H. Curr Opin Biotechnol 2010, 21 78793. 38. Tan, L. T. Phytochemistry 2007, 68, 95479. 39. Davidson, S. K.; Allen, S. W.; Lim, G. E.; Anderson, C. M.; Haygood, M. G. App Environ. Microbiol 2001 67, 4531 4537.

PAGE 251

251 40. TrindadeSilva, A. E.; Lim Fong, G. E.; Sharp, K. H.; Haygood, M. G. Curr. O pin B iotech nol. 2010, 21, 834842. 41. Pettit, G. R.; Kamano, Y.; Herald, C. L.; Fujii, Y.; Kizu, H.; Boyd, M. R.; Boettner, F. E.; Doubek, D. L.; Schmidt, J. M.; Chapuis, J.C.; Michel, C. Tetrahedron 1993, 49, 91519170. 42. Liu, L.; Rein, K. S. Mar Drugs 2010 8, 181737. 43. Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.; Corbett, T. H. J. Am Chem Soc 2001, 123, 541823. 44. Tripathi, A.; Puddick, J.; Prinsep, M. R.; Rottmann, M.; Tan, L. T. J. Nat Prod 2010, 73, 18104. 45. Tripathi, A.; Puddick, J.; Prinsep, M. R.; Rottmann, M.; Chan, K P.; Chen, D. Y.; Tan, L. T. Phytochemistry 2011, 72, 236975. 46. Dai, L.; Chen, B.; Lei, H.; Wang, Z.; Liu, Y.; Xu, Z.; Ye, T. Chem Commun 2012, 48, 86978699. 47. Nogle, L. M.; Gerwick, W. H. Org Lett 2002, 4, 10958. 48. Gerwick, W. H.; Proteau, P J.; Nagle, D. G.; Hamel E.; Blokhin, A.; Slate, D. L. J. Org. Chem. 1994 59, 12435. 49. Chang, Z.; Sitachitta, N.; Rossi, J. V.; Roberts, M. A.; Flatt, P. M.; Jia, J.; Sherman, D. H.; Gerwick, W. H. J. Nat Prod 2004, 67, 135667. 50. Gross, H.; McPh ail, K. L.; Goeger, D. E.; Valeriote, F. A.; Gerwick, W. H. Phytochemistry 2010, 71, 172935. 51. Kwan, J. C.; Teplitski, M.; Gunasekera, S. P.; Paul, V. J.; Luesch, H. J. Nat Prod 2010, 73, 4636. 52. Gekwick, W. H.; Reyes, S.; Alvarado, B. Phytochemist ry 1987, 26, 17014. 53. Malloy, K. L.; Villa, F. A.; Engene, N.; Matainaho, T.; Gerwick, L.; Gerwick, W. H. J. Nat Prod 2011, 74, 958. 54. Appleton, D. R.; Sewell, M. A.; Berridge, M. V.; Copp, B. R. J. Nat Prod 2002 65, 6301. 55. Villa, F. A.; Lie ske, K.; Gerwick, L. Eur J. Pharmacol 2010, 629, 140 6. 56. Cardellina ll, J. H.; Dalietos, D.; Marner, F. J.; Mynderse, J. S.; Moore, R. E. Phytochemistry 1978, 17, 20915.

PAGE 252

252 57. Engene, N.; Choi, H.; Esquenazi, E.; Rottacker, E. C.; Ellisman, M. H.; Dorrestein, P. C.; Gerwick, W. H. Environ. Microbiol 2011, 13, 1601 10. 58. Engene, N.; Rottacker, E. C.; Katovsk, J.; Byrum, T.; Choi, H.; Ellisman, M. H.; Komrek, J.; Gerwick, W. H. Int J Syst Evol Microbiol 2012, 62, 11718. 59. Engene, N.; Gunasek era, S. P.; Gerwick, W. H.; Paul, V. J. Appl Environ Microbiol 2013, 79, 18828. 60. Chan, J. N.; Nislow, C.; Emili, A. Trends Pharmacol Sci 2010, 31, 82 8. 61. Hart, C. P. Drug Discov Today 2005, 10, 5139. 62. Molinski, T. F. Curr. O pin B iotechnol 2010, 21, 819 826. 63. Fellenberg, M.; oksezen, A.; Meyer, B. Angew Chem Int. Ed 2010 49, 2630 3. 64. Dorman, D. E.; Bovey, F. A. J. Org Chem 1973, 38, 23792383. 65. Eberhardt, E. S.; Loh, S. N.; Hinck, A. P.; Raines, R. T. J. Am Chem Soc 1992, 114, 54379. 66. Tonelli, A. E. J. Am Chem Soc 1973 95, 59468. 67. Jhon, J. S.; Kang, Y. K. J. Phys Chem A 1999, 103, 54369. 68. Wedemeyer, W. J.; Welker, E.; Scheraga, H. A.. Biochemistry 2002 41, 1463744. 69. Lu, K. P.; Finn, G.; Lee, T. H.; N icholson, L. K. Nat Chem Biol. 2007 3, 61929. 70. Luesch, H.; Pangilinan, R.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J. J. Nat Prod 2001, 64, 3047. 71. Pettit, G. R.; Xu, J. P.; Hogan, F.; Williams, M. D.; Doubek, D. L.; Schmidt, J. M.; Cerny, R. L. ; Boyd, M. R. J. Nat Prod 1997, 60, 7524. 72. Pettit, G. R.; Smith, T. H.; Xu, J. P.; Herald, D. L.; Flahive, E. J.; Anderson, C. R.; Belcher, P. E.; Knight, J. C. J. Nat Prod 2011 74, 1003 8. 73. Herald, D. L.; Cascarano, G. L.; Pettit, G. R.; Srira ngam, J. K. J. Am Chem Soc 1997, 119, 69626973. 74. Davies Coleman, M. T.; Dzeha, T. M.; Gray, C. A.; Hess, S.; Pannell, L. K.; Hendricks, D. T.; Arendse, C. E. J. Nat Prod 2003, 66, 7125. 75. Kimura, J.; Takada, Y.; Inayoshi, T.; Nakao, Y.; Goetz, G.; Yoshida, W. Y.; Scheuer, P. J. J. Org Chem 2002, 67, 17607.

PAGE 253

253 76. Tan, L. T.; Sitachitta, N.; Gerwick, W. H. J. Nat Prod 2003 66, 764 71. 77. Taniguchi, M.; Nunnery, J. K.; Engene, N.; Esquenazi, E.; Byrum, T.; Dorrestein, P. C.; Gerwick, W. H. J Nat Prod 2010, 73, 3938. 78. Pettit, G. R.; Xu, J.p.; Dorsaz, A. C.; Williams, M. D.; Boyd, M. R.; Cerny, R. L. Bioorg. Med Chem Lett. 1995, 5, 1339 1344. 79. Mohammed, R.; Peng, J.; Kelly, M.; Hamann, M. T. J. Nat Prod 2006 69, 173944. 80. Schmi dt, G.; Grube, A.; Koeck, M. European J Org Chem 2007 38, 410310 81. Paterson, I.; Anderson, E. A. Science 2005 310, 4513. 82. Casey, J. T.; O'Cleirigh, C.; Walsh, P. K.; O'Shea, D. G. J. Microbiol Methods 2004, 58, 32734. 83. Peng, Y.; Pang, H.; Xu, Z.; Ye, T. Lett Org Chem 2005 2, 703706. 84. Montaser, R.; Abboud, K. A.; Paul, V. J.; Luesch, H. J. Nat Prod 2011, 74, 109 12. 85. Thacker, R. W.; Paul, V. J. Appl Environ Microbiol 2004 70, 3305 12. 86. Nogle, L. M.; Gerwick, W. H. J. Nat Prod 2002, 65, 214. 87. Gunasekera, S. P.; Owle, C. S.; Montas er, R.; Luesch, H.; Paul, V. J. J. Nat Prod 2011, 74, 8716. 88. Schaller, A. Phytochemistry 1998, 47, 605 12. 89. Marahiel, M. A.; Stachelhaus, T.; Mootz, H. D. Chem Rev 1997, 97, 265174. 90. Magarvey, N. A.; Beck, Z. Q.; Golakoti, T.; Ding, Y.; Huber, U.; Hemscheidt, T. K.; Abelson, D.; Moore, R. E.; Sherman, D. H. ACS Chem Biol. 2006, 1, 76679. 9 1. Cruz Rivera, E.; Paul, V. J. J. Chem Ecol 2007 33, 2137. 92. Sato, S.; Murata, A. ; Shirakawa, T.; Uesugi, M. Chem Biol. 2010, 17, 616 23. 93. Kaida, D.; Motoyoshi, H.; Tashiro, E.; Nojima, T.; Hagiwara, M.; Ishigami, K.; Watanabe, H.; Kitahara, T.; Yoshida, T.; Nakajima, H.; Tani, T.; Horinouchi, S.; Yoshida, M. Nat Chem Biol. 2007 3, 57683. 94. Low, W. K.; Dang, Y.; Schneider Poetsch, T.; Shi, Z.; Choi, N. S.; Merrick, W. C.; Romo, D.; Liu, J. O. Mol Cell. 2005, 20, 709 22.

PAGE 254

254 95. Sato, S.; Kwon, Y.; Kamisuki, S.; Srivastava, N.; Mao, Q.; Kawazoe, Y.; Uesugi, M. J. Am Chem Soc 2007 129, 87380. 96. Shimogawa, H.; Kwon, Y.; Mao, Q.; Kawazoe, Y.; Choi, Y.; Asada, S.; Kigoshi, H.; Uesugi, M. J. Am Chem Soc 2004, 126, 346171. 97. Sin, N.; Meng, L.; Wang, M. Q.; Wen, J. J.; Bornmann, W. G.; Crews, C. M. Proc Natl. Acad Sci U S A 1997, 94, 6099103. 98. Pugliese, L.; Coda, A.; Malcovati, M.; Bolognesi, M. J. Mol Biol. 1993 231, 698710. 99. Livnah, O.; Bayer, E. A.; Wilchek, M.; Sussman, J. L. Proc Natl. Acad Sci U S A 1993, 90, 507680. 100. Northcote, P. T.; Blunt, J. W. ; Munro, M. H. G. Tetrahedron Lett 1991, 32, 6411 14. 101. Matsunaga, S.; Fusetani, N.; Hashimoto, K.; Walchli, M. J. Am Chem Soc 1989, 111, 25828. 102. Nishimura, S.; Arita, Y.; Honda, M.; Iwamoto, K.; Matsuyama, A.; Shirai, A.; Kawasaki, H.; Kakeya, H.; Kobayashi, T.; Matsunaga, S.; Yoshida, M. Nat Chem Biol. 2010, 6, 51926. 103. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew Chem Int. Ed Engl 2001, 40, 200421. 104. Bock, V. D.; Hiemstra, H.; van Maarseveen, J. H. European J Org Chem 2006 2006, 5168. 105. Kolb, H. C.; Sharpless, K. B. Drug Discov Today 2003 8, 1128 37. 106. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew Chem Int. Ed Engl 2002, 41, 25969. 107. Diamandis, E. P.; Christopoulos, T. K. Clin Che m 1991 37, 625 36. 108. Wilchek, M.; Bayer, E. A. Methods Enzymol 1990, 184, 513. 109. Roberts, M. J.; Bentley, M. D.; Harris, J. M. Adv Drug Deliv Rev 2002, 54, 459 76. 110. Bonger, K. M.; van den Berg, R. J.; Heitman, L. H.; IJzerman, A. P.; Oosterom, J.; Timmers, C. M.; Overkleeft, H. S.; van der Marel, G. A. Bioorg Med Chem 2007, 15, 484156. 111. Kazemi, F.; Massah, A. R.; Javaherian, M. Tetrahedron 2007 63, 50837.

PAGE 255

255 112. Mohler, D. L.; Shen, G. Org Biomol Chem 2006, 4, 20827. 113. Patel K.; Angelos, S.; Dichtel, W. R.; Coskun, A.; Yang, Y. W.; Zink, J. I.; Stoddart, J. F. J. Am Chem Soc 2008, 130, 23823. 114. Meunier, S. J.; Wu, Q.; Wang, S.N.; Roy, R. Can J. C hem 1997, 75, 147282. 115. Van Draanen, N. A., Arseniyadis, S., Crimm ins, M.T., Heathcock, C.H. J. Org. Chem. 1991 ; 56, 2499 2506. 116. Gertsch, J. Planta Med 2008 74, 638 50. 117. Wenk, M. R. Nat Rev Drug Discov 2005 4, 594610. 118. Bosier, B.; Muccioli, G. G.; Hermans, E.; Lambert, D. M. Biochem Pharmacol 2010, 80, 1 12. 119. De Petrocellis, L.; Melck, D.; Bisogno, T.; Di Marzo, V. Chem Phys Lipids 2000, 108, 191209. 120. Di Marzo, V.; Petrocellis, L. D. Annu Rev Med 2006 57, 553 74. 121. Guindon, J.; Hohmann, A. G. CNS Neurol. Disord Drug Targets 2009, 8, 40321. 122. Guindon, J.; Beaulieu, P. Curr. Mol Pharmacol 2009, 2, 1349. 123. Izzo, A. A.; Camilleri, M. Pharmacol Res 2009 60, 11725. 124. Lambert, D. M.; Fowler, C. J. J. Med Chem 2005, 48, 505987. 125. Marchalant, Y.; Cerbai, F.; Brothers, H. M.; Wenk, G. L. Neurobiol Aging 2008, 29, 1894901. 126. Matias, I.; Bisogno, T.; Di Marzo, V. Int. J Obes (Lond) 2006 30, S7 S12. 127. Di Marzo, V. Nat Rev Drug Discov 2008 7, 43855. 128. Felder, C. C.; Glass, M. Annu Rev Pharmacol Toxicol 1998, 38, 179200. 129. Klein, T. W. Nat Rev Immunol 2005, 5, 40011. 130. Devane, W. A.; Hanus, L.; Breuer, A.; Pertwee, R. G.; Stevenson, L. A.; Griffin, G.; Gibson, D.; Mandelbaum, A.; Etinger, A.; Mechoulam, R. Science 1992, 2 58 19469. 131. Sita chitta, N.; Gerwick, W. H. J Nat Prod 1998 61, 6814. 132. Han, B.; McPhail, K. L.; Ligresti, A.; Di Marzo, V.; Gerwick, W. H. J. Nat Prod 2003, 66, 13648.

PAGE 256

256 133. Gutirrez, M.; Pereira, A. R.; Debonsi, H. M.; Ligresti, A.; Di Marzo, V.; Gerwick, W. H J. Nat Prod 2011, 74, 231317. 134. Montaser, R.; Paul, V. J.; Luesch, H. Phytochemistry 2011, 72, 206874. 135. Gunstone, F. D.; Polard, M. R.; Scrimgeour, C. M.; Vedanayagam, H. S. Chem Phys. Lipids 1977 18, 11529. 136. Vemuri, V. K.; Makriyannis, A. In : Reggio, P. H., Ed. Humana Press: 2009; 21 48. 137. Cravatt, B. F.; Lichtman, A. H. Curr. Opin Chem Biol. 2003, 7, 46975. 138. Raduner, S.; Majewska, A.; Chen, J. Z.; Xie, X. Q.; Hamon, J.; Faller, B.; Altmann, K. H.; Gertsch, J. J. Biol. Chem 2 006 281, 14192 206. 139. Mukhopadhyay, S.; Das, S.; Williams, E. A.; Moore, D.; Jones, J. D.; Zahm, D. S.; Ndengele, M. M.; Lechner, A. J.; Howlett, A. C. J. Neuroimmunol 2006, 181, 8292. 140. RW, T.; DG, N.; VJ, P. Mar. Ecol. Prog. Ser. 1997, 147, 212 9. 141. Chen, J.; Li, Y.; Cao, X.P. Tetrahedron: Asymmetry 2006, 17, 933 941. 142. Chen, J.; Fu, X. G.; Zhou, L.; Zhang, J.T.; Qi, X.L.; Cao, X. P. J. Org Chem 2009, 74, 414957. 143. Chen, J.; Shi, Z.F.; Zhou, L.; Xie, A.L.; Cao, X.P. Tetrahedron 2010, 66, 3499 3507. 144. Feng, J. P.; Shi, Z.F.; Li, Y.; Zhang, J.T.; Qi, X. L.; Chen, J.; Cao, X.P. J. Org Chem 2008, 73, 687376. 145. Li, Y.; Feng, J.P.; Wang, W. H.; Chen, J.; Cao, X.P. J. Org Chem 2007, 72, 234450. 146. Zhang, J. T.; Qi, X. L.; Chen, J.; Li, B. S.; Zhou, Y. B.; Cao, X. P. J. Org Chem 2011, 76, 394659. 147. de Carvalho, C. C.; Caramujo, M. J. Mar. Drugs 2012, 10, 26982714. 148. HayashidaSoiza, G.; Uchida, A.; Mori, N.; Kuwahara, Y.; Ishida, Y. J. Appl Microbiol 2008, 1 05, 16727. 149. Berman, F. W.; Gerwick, W. H.; Murray, T. F. Toxicon 1999, 37, 16458. 150. Kwan, J. C.; Meickle, T.; Ladwa, D.; Teplitski, M.; Paul, V.; Luesch, H. Mol Biosyst 2011 7, 1205 16.

PAGE 257

257 151. Tan, L. T.; Okino, T.; Gerwick, W. H. J Nat Prod 2000 63, 952 5. 152. Praud, A.; Valls, R.; Piovetti, L.; Banaigs, B. Tetrahedron Letters 1993 34, 543740. 153. Mynderse, J. S.; Moore, R. E. J. Org Chem 1978, 43, 435963. 154. Wan, F.; Erickson, K. L. J. Nat Prod 1999 62, 16989. 155. McPhail, K. L.; Gerwick, W. H. J Nat Pr od 2003, 66, 132 5. 156. Takai, K.; Nitta, K.; Utimoto, K. J. Am Chem Soc 1986, 108, 740810. 157. S. Tetrahedron: Asymmetry 1998, 9, 338194. 158. Ileby, N.; Kuzma, M.; Heggvik, L. R.; Srbye, K.; Fiksdahl, A. Tetrahedron: Asymmetry 1997, 8, 21938. 159. Ermolenk o, L.; Sasaki, N. A.; Potier, P. Synlett 2001, 2001, 15656. 160. Marshall, J. A.; Piettre, A.; Paige, M. A.; Valeriote, F. J. Org Chem 2003, 68, 17719. 161. David Crouch, R. Tetrahedron 2004 60, 5833 71. 162. Corey, E. J.; Schmidt, G. Tetrahedron Let ters 1979, 20, 399402. 163. Huang, L.; Teumelsan, N.; Huang, X. Chemistry 2006 12, 5246 52. 164. Barnych, B.; Vatle, J. M. Synlett 2011 2011, 204852. 165. Bowden, K.; Heilbron, I. M.; Jo nes, E. R. H.; Weedon, B. C. L. J. Chem Soc 1946, 39 45. 166. Choi, H.; Mascuch, S. J.; Villa, F. A.; Byrum, T.; Teasdale, M. E.; Smith, J. E.; Preskitt, L. B.; Rowley, D. C.; Gerwick, L.; Gerwick, W. H. Chem Biol 2012, 19, 58998. 167. Crews, C. M. Chem Biol 2010, 17, 551 5.

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258 BIOGRAPHICAL SKETCH Rana Montaser received her b achelors degree in Pharmaceutical Sciences from the Faculty of Pharmacy, Ain Shams University in Cairo, Egypt in 2006. She was appointed as a teaching assistant in the department of Pharmacognosy and Phytotherapy in the same university in 2007. She joined the College of Pharmacy at the University of Florida in August 2008, where she started as a graduate student in Dr. Hendrik Lueschs lab in the M edicinal C hemistry D epartment. There, she worked on chemical and biological characterization of bioactive secondary metabolites from marine cyanobacteria. She received her Ph D degree from the University of Florida in the spring of 2013.