Electronic Theses and Dissertations

UC San Diego

Peer Reviewed

Title:
Organic synthesis as an effective approach to chemical, pharmaceutical, and biosynthetic
investigations of natural products
Author:
Suyama, Takashi L.
Acceptance Date:
01-01-2009
Series:
UC San Diego Electronic Theses and Dissertations
Degree:
Ph. D., UC San Diego
Permalink:
http://www.escholarship.org/uc/item/2pn8j589
Local Identifier:
b6296689
Abstract:
The partnership between natural products and synthetic organic chemistry date back to the
origin of organic chemistry itself. While natural products became a major driving force for the
development of novel organic reactions and synthetic strategies, organic synthesis has contributed
in many ways to the elucidation and confirmation of structure, pharmaceutical development,
and biosynthetic studies of natural products. Due to the recent advances in both of these two
disciplines, there are new opportunities and issues surrounding natural products that organic
synthesis can be applied to, and such studies comprise this dissertation. Chapter I introduces the
background information and rationale for the dissertation research, which is based on the history
of organic chemistry and newly emerging research topics in natural products chemistry. Chapter II
describes the isolation, characterization, and toxicological evaluation of polybrominated diphenyl
ethers from a mixed assemblage of a marine red alga and cyanobacteria. Chapter III describes
the isolation, characterization, and biological evaluation of a novel vinylchloride-containing fatty
acid, credneric acid, from a Lyngbya sp. Chapter IV describes the conception of a method for
determining the absolute stereochemistry of natural products lacking proper functional groups for
derivatization. This methodology was applied to a cyclopropane-containing fatty acid and resulted
in a conflict with newly published literature, which is discussed in detail. Chapter V describes
the development of an expedient and efficient total synthesis of a novel alkaloid, epiquinamide,
isolated from the skin of a rainforest frog, Epipedobates tricolor. This work contributed to clarifying
the identity of a potent and selective nicotinic receptor agonistic activity observed in the extract of
E. tricolor. Chapter VI describes the development of a total synthesis of a marine cyanobacterial
metabolite, somocystinamide A, which has been shown to be a potent inhibitor of endothelial
cell proliferation and angiogenesis. The synthesis was later modified for scalability to meet the

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need for further pharmaceutical investigations of this important natural product. Chapter VII
describes the synthetic investigations on scytonemin, a cyanobacterial metabolite possessing UV-
blocking properties. Through this work, many intriguing insights into the biosynthesis of the natural
product were obtained. Finally, Chapter VIII provides the conclusions drawn from this dissertation
research.

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services to the University of California and delivers a dynamic
research platform to scholars worldwide.
 UNIVERSITY OF CALIFORNIA, SAN DIEGO

Organic synthesis as an effective approach to chemical, pharmaceutical, and biosynthetic

investigations of natural products

A dissertation submitted in partial satisfaction of the requirements for the degree of

Doctor of Philosophy

in

Oceanography

by

Takashi L.Suyama

Committee in charge:

Professor William H. Gerwick, Chair

Professor William Fenical
Professor Yoshihisa Kobayashi
Professor Bradley S. Moore
Professor Emmanuel Theodorakis

2009
 Copyright

Takashi L. Suyama, 2009

All rights reserved.

The Dissertation of Takashi L. Suyama is approved, and it is acceptable in quality and

form for publication on microfilm and electronically:

Chair

University of California, San Diego

2009

iii
 DEDICATION

This dissertation is dedicated to my best friend, my counselor, my strength, my

inspiration, my advocate, my guide, my hope, my savior, my hero, and my Lord, Jesus

Christ of Nazareth. For, “we are hard-pressed on every side, yet not crushed; we are

perplexed, but not in despair; persecuted, but not forsaken; struck down, but not

destroyed— always carrying about in the body the dying of the Lord Jesus, that the life of

Jesus also may be manifested in our body.... Therefore we do not lose heart.... For the

things which are seen are temporary, but the things which are not seen are eternal

(2Corinthians 4:8-10, 16, 18, NKJV; emphasis mine).” Indeed, it is because of Him that

even I have persevered through this doctoral program. The credit for all that follows is

due unto His name.

iv
 EPIGRAPH

My Hope is Built.

My hope is built on nothing less

Than Jesus' blood and righteousness.
I dare not trust the sweetest frame,
But wholly lean on Jesus' name.

On Christ the solid rock I stand,

All other ground is sinking sand;
All other ground is sinking sand.

When darkness veils His lovely face,

I rest on His unchanging grace.
In every high and stormy gale,
My anchor holds within the veil.

On Christ the solid rock I stand,

All other ground is sinking sand;
All other ground is sinking sand.

His oath, His covenant, His blood

Supports me in the whelming flood.
When all around my soul gives way,
He then is all my hope and stay.

On Christ the solid rock I stand,

All other ground is sinking sand;
All other ground is sinking sand.

When He shall come with trumpet sound,

O may I then in Him be found!
Dressed in His righteousness alone,
Faultless to stand before the Throne!

On Christ the solid rock I stand,

All other ground is sinking sand;
All other ground is sinking sand.

— Edward Mote, 1797-1874

v
 TABLE OF CONTENTS

Signature Page............................................................................................. iii

Dedication................................................................................................... iv

Epigraph....................................................................................................... v

Table of Contents......................................................................................... vi

List of Abbreviations................................................................................... viii

List of Illustration and Figures.................................................................... x

List of Schemes........................................................................................... xvi

List of Tables............................................................................................... xix

Acknowledgments....................................................................................... xx

Vita and Publications................................................................................... xxiv

Abstract........................................................................................................ xxvi

Chapter I: Introduction................................................................................ 1

Chapter II: Ichthyotoxic brominated diphenyl ethers from a mixed

assemblage of a red alga and cyanobacterium: structure clarification and
biological properties ................................................................................... 60

Chapter III: Discovery of a novel vinylchloride containing fatty acid

from a marine cyanobacterium Lyngbya sp. ............................................... 101

Chapter IV: Absolute stereochemistry of novel fatty acids by synthetic

introduction of a stereochemical reference ................................................. 126

Chapter V: Use of biomimetic starting materials for expedient total

synthesis of epiquinamide enantiomers: true identity of bioactive
metabolite.................................................................................................... 172

Chapter VI: Stereospecific total synthesis of somocystinamide A; an

extremely potent antiangiogenic marine natural product containing
synthetically challenging functional groups................................................ 234

Chapter VII: Biomimetic total synthesis of a cyanobacterial UV-blocking

natural product, scytonemin and insights into its biogenesis...................... 315

vi
Chapter VIII: Conclusions........................................................................... 368

Appendix..................................................................................................... 375

vii
 LIST OF ABBREVIATIONS

CD circular dichroism

CoA coenzyme A

COSY correlation spectroscopy

DCI desorption chemical ionization

DDQ 2,3-dichloro-5,6-dicyano benzoquinone

EC effective concentration

EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide

EtOH ethanol
DMAP 4-dimethylamino pyridine
EI electron impact
ESI electron spray ionization
FAB fast atom bombardment
FLIPR fluorescence laser plate reader
GCMS gas chromatography mass spectrometry
HMBC heteronuclear multiple bond correlation
HMG 3-hydroxy-3-methyl-glutaryl
HMQC heteronuclear multiple quantum coherence
HPLC high performance liquid chromatography
HR high resolution
HSQC heteronuclear single quantum coherence
INADEQUATE incredible natural abundance double quantum transfer
experiment
i-PrOH isopropanol
IR infrared spectroscopy
MeOH methanol
MS mass spectrometry
MsO methanesulfonate
NCS N-chlorosuccinimide
NMR nuclear magnetic resonance

viii
NOE nuclear Overhauser effect

NOESY nuclear Overhauser effect spectroscopy

NRPS non-ribosomal peptide synthase

OR optical rotation

PKS polyketide synthase

ROESY rotating Overhauser effect spectroscopy

rt room temperature

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

TMSOTf trimethylsilyl trifluoromethanesulfonate

TBDMSOTf tert-butyldimethylsilyl trifluoromethanesulfonate

TBSOTf tert-butyldimethylsilyl trifluoromethanesulfonate

TsOH toluenesulfonic acid

UV ultra violet

VLC vacuum liquid chromatography

ix
 LIST OF ILLUSTRATION AND FIGURES

Illustration I.1: Graphical representation of thesis research......................................... 4

Figure I.1: The originally assigned structure (13) of mururin C and its
revised structure (12).................................................................................................... 11
Figure I.2: Types of errors in natural product structure revisions................................. 30
Figure I.3: Structure determination methods used in 2005-2008 for the erroneous
cases.............................................................................................................................. 30
Figure I.4: Structure revision methods used in 2005-2008........................................... 30
Figure I.5: Discrimination of possible structures by HMBC........................................ 31
Figure I.6: HMBC correlations observed and recorded for the labdane diterpenoid... 31
Figure I.7: All new small chemical entities that were approved for clinical use,
01/1981-06/2006, by source......................................................................................... 33
Figure I.8: Drug Development Process......................................................................... 35
Figure II.1: OH-PBDE congeners isolated from the red alga/cyanobacteria assembly
(1 and 2) and an alternative regioisomer of 1 (3)......................................................... 63
Figure II.2: X-ray crystal structure of 1........................................................................ 68
Figure II.3: Ca2+ modulation assay in mouse neocortical neurons for compounds
1-3................................................................................................................................. 72
Figure II.4: Photographs of the red algal voucher sample............................................ 77
Figure II.5: Photograph of the red algal voucher sample............................................. 78
Figure II.6: Isolation scheme for 1 and 3...................................................................... 85
Figure II.7: 1H NMR Spectrum of 1 in CDCl3 ............................................................. 86
Figure II.8: 13C NMR Spectrum of 1 in CDCl3 ............................................................ 87
Figure II.9: 1H-1H COSY Spectrum of 1 in CDCl3 ...................................................... 88
Figure II.10: 1H-13C HSQC Spectrum of 1 in CDCl3 ................................................... 89
Figure II.11: 1H-13C HMBC Spectrum of 1 in CDCl3 .................................................. 90
Figure II.12: 1H NMR Spectrum of 1 in CDCl3 ........................................................... 91
Figure II.13: 13C NMR Spectrum of 3 in CDCl3 .......................................................... 92
Figure II.14: 1H-1H COSY Spectrum of 3 in CDCl3 .................................................... 93
Figure II.15: 1H-13C HSQC Spectrum of 3 in CDCl3 ................................................... 94
Figure II.16: 1H-13C HMBC Spectrum of 3 in CDCl3 .................................................. 95
Figure II.17: 1H NMR Spectrum of 2 in CDCl3 ........................................................... 96

x
Figure II.18: 1H-1H COSY Spectrum of 2 in CDCl3 .................................................... 97
Figure III.1: Jamaicamides A-C.................................................................................... 104
Figure III.2: Malyngamides A-X.................................................................................. 104
Figure III.3: Satellite Image of Lyngbya Collection Site.............................................. 106
Figure III.4: Zoom-up of the Satelite Image of the Lyngbya Collection Site............... 106
Figure III.5: "Green Lyngbya" collected in Papua New Guinea................................... 107
Figure III.6: 1H NMR Spectrum of Credneric Acid (38).............................................. 108
Figure III.7: Credneric Acid and Credneramide........................................................... 112
Figure III.8: N-Acyl Homoserine Lactones (AHLs)..................................................... 112
Figure III.9: Credneric Acid (38) 13C NMR Spectrum in CDCl3.................................. 116
Figure III.10: Credneric Acid (38) 1H-1H COSY Spectrum in CDCl3.......................... 117
Figure III.11: Credneric Acid (38) 1H-13C HSQC Spectrum in CDCl3......................... 118
Figure III.12: Credneric Acid (38) 1H-13C HMBC Spectrum in CDCl3........................ 119
Figure III.13: Credneric Acid (38) 1D ROESY Spectrum: Irradiated at 2.73 ppm in
CDCl3............................................................................................................................ 120
Figure III.14: Credneric Acid (38) 1D ROESY Spectrum: Irradiated at 5.79 ppm in
CDCl3............................................................................................................................ 121
Figure III.15: Credneric Acid (38) 1D ROESY Spectrum: Irradiated at 2.16 ppm in
CDCl3............................................................................................................................ 122
Figure III.16: IR spectrum of 38................................................................................... 123
Figure IV.1: Proposed transition state of alkylation of the enolate of 5 guided by
oxazolidinone
auxiliary........................................................................................................................ 132
Figure IV.2: Dihedral angles predicted from the 1H-1H coupling constants and
Altona equation (A) and select ROESY correlations for 7 and 8 (B)........................... 135
Figure IV.3: History of the absolute stereochemical assignment of cyclopropane
derivative 11.................................................................................................................. 136
Figure IV.4: 1H and 13C NMR assignments and an NOE correlation of 16 in CDCl3 138
Figure IV.5: X-ray crystal structure of 15..................................................................... 139
Figure IV.6: Corrected stereochemistry of 1-m and 1-c............................................... 139
Figure IV.7: Synthetic Introduction of a Stereochemical Reference (SISTER)
method conceptual figure............................................................................................. 141
Figure IV.8: 1H NMR spectrum of 7 in C6D6................................................................ 146
Figure IV.9: 1H NMR spectrum of 7 in CDCl3............................................................. 147
Figure IV.10: 13C NMR spectrum of 7 in CDCl3.......................................................... 148
Figure IV.11: 1H-1H Homonuclear decoupling experiment for 7 in C6D6 – Part 1....... 149
Figure IV.12: 1H-1H Homonuclear decoupling for 7 in C6D6 – Part 2.......................... 150

xi
Figure IV.13: 1H-1H Homonuclear decoupling experiment for 7 in C6D6 – Part 3....... 151
Figure IV.14: 1H-1H COSY spectrum of 7 in C6D6....................................................... 152
Figure IV.15: ROESY spectrum of 7 in C6D6............................................................... 153
Figure IV.16: LR FAB MS spectrum of 7..................................................................... 154
Figure IV.17: IR spectrum of 7..................................................................................... 155
Figure IV.18: 1H NMR spectrum of 8 in C6D6.............................................................. 156
Figure IV.19: 1H NMR spectrum of 8 in CDCl3........................................................... 157
Figure IV.20: 13C NMR spectrum of 8 in CDCl3.......................................................... 158
Figure IV.21: 1H-1H Homonuclear decoupling experiment for 8 in C6D6 – Part 1....... 159
Figure IV.22: 1H-1H Homonuclear decoupling experiment for 8 in C6D6 – Part 2....... 160
Figure IV.23: 1H-1H Homonulcear decoupling experiment for 8 in C6D6 – Part 3 161
Figure IV.24: 1H-1H COSY spectrum of 8 in C6D6....................................................... 162
Figure IV.25: ROESY spectrum of 8 in C6D6............................................................... 163
Figure IV.26: IR spectrum of 8..................................................................................... 164
Figure IV.27: 1H NMR spectrum of 9 in CDCl3........................................................... 165
Figure IV.28: IR spectrum of 9..................................................................................... 166
Figure IV.29: Geometry optimized 3D structures of 7 (above) and 8 (below)............. 167
Figure IV.30: HPLC chromatogram for 4 and 7........................................................... 168
Figure IV.31: HPLC chromatogram for 5 and 8........................................................... 169
Figure IV.32: UV spectra of 7 and 8............................................................................. 170
Figure V.1: Frog skin alkaloids of various core skeletons............................................ 174
Figure V.2: Skin of Epipedobates tricolor contains epibatidine and epiquinamide...... 175
Figure V.3: X-ray crystal structure of epi-epiquinamide (15)....................................... 178
Figure V.4: 1H NMR Spectrum of 11 in CDCl3............................................................ 200
Figure V.5: 13C NMR Spectrum of 11 in CDCl3........................................................... 201
Figure V.6: 1H NMR Spectrum of 12 in CDCl3............................................................ 202
Figure V.7: 13C NMR Spectrum of 12 in CDCl3........................................................... 203
Figure V.8: 1H NMR Spectrum of 13 in CDCl3............................................................ 204
Figure V.9: 13C NMR Spectrum of 13 in CDCl3........................................................... 205
Figure V.10: 1H NMR Spectrum of 14 in CDCl3.......................................................... 206
Figure V.11: 13C NMR Spectrum of 14 in CDCl3......................................................... 207
Figure V.12: 1H NMR Spectrum of 15 in MeOH-d4..................................................... 208
Figure V.13: 13C NMR Spectrum of 15 in MeOH-d4.................................................... 209
Figure V.14: 1H NMR Spctrum of 16 in CDCl3............................................................ 210

xii
Figure V.15: 13C NMR Spectrum of 16 in CDCl3......................................................... 211
Figure V.16: EI-GCMS Chromatogram and Mass Spectrum of 16.............................. 212
Figure V.17: 1H NMR spectrum 29 in CDCl3............................................................... 213
Figure V.18: 13C NMR spectrum of 29 in CDCl3.......................................................... 214
Figure V.19: 1H NMR spectrum of 30 in CDCl3........................................................... 215
Figure V.20: 13C NMR spectrum of 30 in CDCl3.......................................................... 216
Figure V.21: 1H NMR spectrum of 28 in CDCl3........................................................... 217
Figure V.22: 13C NMR spectrum of 28 in CDCl3.......................................................... 218
Figure V.23: 1H NMR spectrum of 31 in CDCl3........................................................... 219
Figure V.24: 13C NMR spectrum of 31 in CDCl3.......................................................... 220
Figure V.25: 1H NMR spectrum of 27 in CDCl3........................................................... 221
Figure V.26: 13C NMR spectrum of 27 in CDCl3........................................................ 222
Figure V.27: 1H NMR spectrum of 32 in CDCl3........................................................... 223
Figure V.28: 1H NMR NMR spectrum of (+)-9 in CDCl3............................................ 224
Figure V.29: 1H NMR spectrum of (+)-9 in CDCl3...................................................... 225
Figure V.30: 13C NMR spectrum of (+)-9 in CDCl3...................................................... 226
Figure V.31: 1H NMR spectrum of (-)-9 in CDCl3....................................................... 227
Figure V. 32: 13C NMR spectrum of (-)-9 in CDCl3..................................................... 228
Figure Vl.1: Antiangiogenic activity of 1 in zebrafish................................................. 236
Figure VI.2: Impact of somocystinamide A (1) on proliferation of paired
neuroblastoma cells lacking caspase 8 expression (triangles) or expressing caspase
8 (squares)..................................................................................................................... 238
Figure VI.3: Extrinsic pathway via activation of caspase-8 leading to apoptosis........ 238
Figure VI.4: Intrinsic pathway via activation of caspase-9 leading to apoptosis......... 239
Figure VI.5: Most stable conformer of somocystinamide A found through molecular
modeling....................................................................................................................... 240
Figure VI.6: Proposed biosynthesis of somocystinamide A (1)................................... 243
Figure VI.7: Enamide containing natural products....................................................... 244
Figure VI.8: Examples of disulfide-containing natural products................................. 247
Figure VI.9: Model compounds used in the study of enamide formation.................... 255
Figure VI.10: Reaction setup for enamide formation using molecular sieves and
glass wool in a Soxhlet extractor.................................................................................. 259
Figure VI.11: Solubility of somocystinamide A (1) in various media.......................... 261
Figure VI.12: 1H NMR spectrum of 22 in CDCl3......................................................... 282
Figure VI.13: 13C NMR spectrum of 22 in CDCl3........................................................ 283

xiii
Figure VI.14: 1H NMR spectrum of 2 in CDCl3........................................................... 284
Figure VI.15: 13C NMR spectrum of 2 in CDCl3.......................................................... 285
Figure VI.16: 1H NMR spectrum of 44 in CDCl3......................................................... 286
Figure VI.17: 13C NMR spectrum of 44 in CDCl3........................................................ 287
Figure VI.18: 1H NMR spectrum of 51 in CDCl3......................................................... 288
Figure VI.19: 13C NMR spectrum of 51 in CDCl3........................................................ 289
Figure VI.20: 1H NMR spectrum of 52 in CDCl3......................................................... 290
Figure VI.21: 13C NMR spectrum of 52 in CDCl3........................................................ 291
Figure VI.22: 1H NMR spectrum of 52-cis in CDCl3................................................... 292
Figure VI.23: 13C NMR spectrum of 52-cis in CDCl3.................................................. 293
Figure VI.24: 1H NMR spectrum of 56 in CDCl3......................................................... 294
Figure VI.25: 13C NMR spectrum of 56 in CDCl3........................................................ 295
Figure VI.26: COSY spectrum of 56 in CDCl3............................................................. 296
Figure VI.27: HMBC spectrum of 56 in CDCl3........................................................... 297
Figure VI.28: 1H NMR spectrum of 57 in CDCl3......................................................... 298
Figure VI.29: 13C NMR spectrum of 57 in CDCl3........................................................ 299
Figure VI.30: 1H NMR spectrum of 3 in 1:1 CDCl3/CD3OD....................................... 300
Figure VI.31: 13C NMR spectrum of 3 in 1:1 CDCl3/CD3OD...................................... 301
Figure VI.32: 1H NMR spectrum of 1 in CDCl3........................................................... 302
Figure VI.33: 13C NMR spectrum of 1 in CDCl3.......................................................... 303
Figure VI.34: 1H NMR spectrum of 70 in CDCl3........................................................ 304
Figure VI.35: 13C NMR spectrum of 70 in CDCl3........................................................ 305
Figure VI.36: 1H NMR spectrum of 71 in CDCl3......................................................... 306
Figure VI.37: 13C NMR spectrum of 71 in CDCl3........................................................ 307
Figure VI.38: 1H NMR spectrum of 73 in CDCl3......................................................... 308
Figure VI.39: 13C NMR spectrum of 73 in CDCl3........................................................ 309
Figure VII.1: Light absorption spectrum of scytonemin (1) (taken from ref. 4).......... 318
Figure VII.2: Select HMBC, COSY, and ROESY correlations of scytonemin
monomer (22) in DMF-d7............................................................................................. 323
Figure VII.3: Scytonemin monomer (22): geometry optimized computer model........ 324
Figure VII.4: 1H NMR spectrum of 22 in DMF-d7....................................................... 325
Figure VII.5: pH-dependent color change of monomer 22.......................................... 328
Figure VII.6: Alternative structures for scytonemin..................................................... 335
Figure VII.7: 1H NMR spectrum of 21 in CDCl3.......................................................... 346
Figure VII.8: 13C NMR spectrum of 21 in CDCl3......................................................... 347

xiv
Figure VII.9: 1H NMR spectrum of 18 in CDCl3......................................................... 348
Figure VII.10: 13C NMR spectrum of 22 in DMF-d7.................................................... 349
Figure VII.11: COSY NMR spectrum of 22 in DMF-d7.............................................. 350
Figure VII.12: HSQC NMR spectrum of 22 in DMF-d7.............................................. 351
Figure VII.13: HMBC NMR spectrum of 22 in DMF-d7............................................. 352
Figure VII.14: NOESY NMR spectrum of 22 in DMF-d7............................................ 353
Figure VII.15: HRMS (EI) spectrum of 22................................................................... 354
Figure VII.16: 1H NMR spectrum of 29 in CDCl3........................................................ 355
Figure VII.17: 13C NMR spectrum of 29 in CDCl3....................................................... 356
Figure VII.18: COSY spectrum of 29 in CDCl3........................................................... 357
Figure VII.19: HSQC spectrum of 29 in CDCl3........................................................... 358
Figure VII.20: HMBC spectrum of 29 in CDCl3.......................................................... 359
Figure VII.21: ROESY spectrum of 29 in CDCl3......................................................... 360
Figure VII.22: HRMS (ESI-FT) spectrum of 29 (showing [M+Na])........................... 361
Figure VII.23: 1H NMR spectrum of 34 in CDCl3........................................................ 362
Figure VII.24: 13C NMR spectrum of 34 in CDCl3....................................................... 363
Figure VII.25: 1H NMR spectrum of 37 in CDCl3........................................................ 364
Figure VII.26: 13C NMR spectrum of 37 in CDCl3....................................................... 365
Figure VIII.1: Natural products isolated during the dissertation research.................... 371
Figure VIII.2: Natural products that were synthetically investigated during the
disseration research....................................................................................................... 372

xv
 LIST OF SCHEMES

Scheme I.1: Echitamine structure elucidation efforts by chemical

degradation and synthetic logic.................................................................................. 8
Scheme I.2: Assigned structures by chemical investigation and X-ray
crystallography............................................................................................................ 8
Scheme I.3: Relationship between 9 and 10 via electrocyclization............................ 9
Scheme I.4: Novartis strategy for the synthesis of discodermolide (17).................... 37
Scheme I.5: Synthetic contributions to the study of brevianamides biosynthesis...... 40
Scheme I.6: Biomimetic synthesis of grossularine-1 (35).......................................... 42
Scheme I.7: Biomimetic synthesis of panepophenanthrin (41) through a Diels-
Alder reaction.............................................................................................................. 42
Scheme I.8: Scytonemin monomer does not dimerize under naturally occurring
conditions.................................................................................................................... 43
Scheme II.1: Synthesis of 3 by Marsh’s route............................................................ 71
Scheme III.1: Divergent biosyntheses of jamaicamide A and curacin A.................... 105
Scheme III.2: Isolation process for credneric acid (38).............................................. 108
Scheme III.3: Substructures of Credneric Acid by 1H NMR, COSY, and HSQC...... 110
Scheme III.4: Stereochemical Assignments of Credneric Acid by 1D ROESY
Correlations................................................................................................................. 111
Scheme III.5: Cyclopropane Fatty Acid and Lyngbyamides...................................... 112
Scheme IV.1: Derivatization of cyclopropane fatty acids 1-m and 1-c...................... 131
Scheme IV.2: Total synthesis of (+)-grenadamide (13) by Baird et al........................ 135
Scheme IV.3: Synthesis of PGME derivatives from 11.............................................. 137
Scheme V.1: Reduction of dehydropiperidine: two stereochemical outcomes........... 176
Scheme V.2: Applying the synthetic logic of quinolizidine biosynthesis to the
synthesis of epiquinamide skeleton............................................................................ 176
Scheme V.3: Biomimetic synthesis of lupinamine by Wanner and Koomen.............. 177
Scheme V.4: Initial Retrosynthetic Analysis of Epiquinamide................................... 177
Scheme V.5: Total synthesis of (1R,9S)-epi-epiquinamide (16)................................. 178
Scheme V.6: Proposed total synthesis of (+)-epiquinamide via boron nucleophilic
addition to acyl imminium ion................................................................................... 180
Scheme V.7: Proposed total synthesis of (+)-epiquinamide via hetero Diels-Alder... 180
Scheme V.8: Retrosynthetic analysis of (+)-epiquinamide......................................... 181
Scheme V.9: Total synthesis of (+)-epiquinamide...................................................... 182

xvi
Scheme V.10: Chelation-controlled hydride reduction of ketone (30)....................... 183
Scheme VI.1: Enamide............................................................................................... 242
Scheme VI.2: Instability of enamide as seen in the facile decomposition of
somocystinamide A..................................................................................................... 243
Scheme VI.3: Enamide synthesis on model systems through acylation of imine....... 246
Scheme VI.4: Different approaches to enamide functional group.............................. 247
Scheme VI.5: Approaches to install disulfide of the epidithiapiperazinedione core.. 248
Scheme VI.6: Total synthesis of glyotoxin involving sensitive installation of
disulfide...................................................................................................................... 248
Scheme VI.7: Synthesis of psammaplin A.................................................................. 249
Scheme VI.8: Retrosynthetic Analysis of Somocystinamide A.................................. 249
Scheme VI.9: Synthesis of vinyl iodide for Suzuki coupling (Scheme VI.10).......... 250
Scheme VI.10: Suzuki coupling with varying coupling partners............................... 250
Scheme VI.11: Ruthenium catalyzed cross metathesis approach............................... 251
Scheme VI.12: Initially planned final steps to Somocystinamide (1)......................... 254
Scheme VI.13: Possible mechanism of enamide formation....................................... 255
Scheme VI.14: Copper-mediated vinylation of a primary amide model compound
(65) and attempted chemoselective methylation......................................................... 257
Scheme VI.15: Final steps to somocystinamide A (1)................................................ 258
Scheme VI.16: Revised synthesis of the bis-methylamide 3...................................... 263
Scheme VII.1: Fragmentation of the reduced form of scytonemin (2) through
ozonolysis in its structure elucidation......................................................................... 319
Scheme VII.2: Biosynthetic gene cluster for scytonemin in Nostoc punctiforme...... 319
Scheme VII.3: Chemical synthesis of a putative biosynthetic precursor of
scytonemin (1), soraphinol A (S-8)............................................................................. 320
Scheme VII.4: Proposed mechanism for the biosynthesis of scytonemin (1) by
Balsksus and Walsh..................................................................................................... 321
Scheme VII.5: Initially conceived synthesis of scytonemin monomer 11.................. 322
Scheme VII.6: Predicted mechanism for regioselectivity of aldol condensation
between ketone 18 and aldehyde 19........................................................................... 322
Scheme VII.7: Synthesis of scytonemin monomer..................................................... 323
Scheme VII.8: Absence of tautomerization and resonance characteristics of
scytonemin monomer (22).......................................................................................... 324
Scheme VII.9: Proposed mcehanism for dimerization of oxidized monomer 25 to
reduced scytonemin 2................................................................................................. 328
Scheme VII.10: Synthesis of methylated monomer 29............................................... 331
Scheme VII.11: Possible role of glycosylation in the biosynthesis of scytonemin.... 332

xvii
Scheme VIl.12: Attempts to synthesize 30 and/or a mimic thereof (e.g. 33)............. 333
Scheme VII.13: Attempts to perform Rubottom oxidation via doubly silylation of
22................................................................................................................................. 334
Scheme VII.14: Newly proposed route for the synthesis of 2.................................... 336
Scheme VII.15: Highly chemo-selective DDQ oxidation of (E)-16-epinormacusine
B (45).......................................................................................................................... 337
Scheme VII.16: Chemo-selective oxidation of benzylic ether by DDQ in a neat
mixture........................................................................................................................ 337
Scheme VII.17: Completion of total synthesis of scytonemin albeit low yield
(yield to be determined).............................................................................................. 338

xviii
 LIST OF TABLES

Table I.1: Use of natural products in the past and present....................................... 6

Table I.2: Structural revisions made in 2005-2008.................................................. 13
Table II.1: Comparison of NMR data for two isolates of 1 and 3 (CDCl3).............. 69
Table II.2: Crystal data and structure refinement for 1............................................ 80
Table II.3: Atomic coordinates ( x 104) and equivalent isotropic displacement
parameters (Å2x103) for 1......................................................................................... 80
Table II.4: Bond lengths [Å] and angles [°] for 1................................................... 82
Table II.5: Anisotropic displacement parameters (Å2x103) for 1............................. 83
Table II.6: Hydrogen coordinates ( x 104) and isotropic displacement parameters
(Å2x103) for 1........................................................................................................... 84
Table III.1: 1H and 13C NMR, COSY, HSQC, and HMBC data for credneric acid
(38)........................................................................................................................... 110
Table IV.1: Dihedral angles calculated or simulated by different methods for 7
and 8......................................................................................................................... 133
Table VI.1: Optimization of ruthenium-catalyzed cross metathesis........................ 253
Table VI.2: Enamide formation via acyl chloride.................................................... 256
Table VII.1: Oxidative dimerization conditions....................................................... 330

xix
 ACKNOWLEDGMENTS

I would like to first thank my PhD advisor, Prof. William Gerwick, who has given

me great opportunities in his laboratory. His enthusiasm and mentorship were invaluable

during my graduate program. Bill has also been a great person to work for, and he has

been greatly patient with me through the hard and slow times that are associated with

scientific research. His approachability undoubtedly contributed to my accomplishments

as well. I am very grateful for the many hours that he has spent editing, reviewing, and

critiquing my manuscripts for this disseration and other publications. I would not like to

ponder what would have happened without his involvement.

It was very helpful to discuss research issues with other members of my PhD

committee. I sincerely thank Professors Emmanual Theodorakis, Yoshihisa Kobashi,

Bradley Moore, and William Fenical for taking their time out to be my advisors. I thank

Professor Fenical for allowing me to work in his laboratory while our new labs at SIO

were undergoing renovation.

I am very thankful for all the postdoctoral researchers, graduate students, and staff

that have worked with me over the years both at OSU and UCSD. I particularly enjoyed

and benefited from bouncing various ideas off Keith Schwartz (Dr. White's lab at OSU)

and my officemate, Karla Malloy. I acknowledge the technical assistance from Dr.

Anthony Mrse, Dr. Yongxuan Su, Dr. Harry Gross, Dr. Arnold Rhinegold and his staff,

Josh Wingerd, and Tara Byrum at UCSD and Roger Kohnert at OSU. I am especially

indebted to Professor Kerry McPhail at OSU, who helped me in the early years of my

scientific career.

xx
 I must not forget to thank Professor David A. Horne at OSU, who first introduced

me to the joy of synthetic organic and bioorganic chemistry and taught me invaluable

skills in his laboratory. Going back further in time, I am thankful for my high school

biology teacher, Mr. Kudara, who inspired me to pursue a career in science back when all

I ever thought about was improving my batting average and hitting a home run some day

(sadly I never did in an official game).

On a more personal note, I am thankful for the ministry by Dr. Kent Hovind and

his associates, who have inspired me to appreciate having a doctoral degree in science for

the Kingdom of our God. I am also very grateful for the church family I have in

Oceanside, CA and in Albany, OR. I cannot replace their fellowship, support, and prayers

with anything else. I am extremely grateful for Pastor Ron Reed, who has been a great

friend and a mentor despite the long distance between us. Knowing that he prays to our

Lord for me everyday is a great treasure to me.

I am deeply thankful for my parents and other family members for their support.

Thank you! I am especially grateful for my mother, who came to the Lord a few years

ago and has given me much joy.

To my dear wife, Janet, I cannot thank God enough for you. Indeed, “whoso

findeth a wife findeth a good thing, and obtaineth favour of the LORD (Proverbs 18:22,

KJV).” You have been a great gift from the Lord. Your support, love, and friendship have

been a great source of strength. Thank you, honey. I look forward to serving our Lord

together with you in the years to come. I also thank you for your help with the tedious

aspects of preparing this thesis manuscript.

xxi
 Lastly, but most definitely not the least, I owe everything to my greatest mentor,

hero, and friend, the Lord Jesus Christ. Lord, You have taken my place and sacrificed

Your life for me, a wretched sinner who was a miserable atheist until six years ago, so

that I may be forgiven and have an eternal life with You. Knowing this fact has been an

infinite source of confidence and strength in all the situations I have faced in the last six

years. Any difficult situation seemed so small compared to the hope You have given me

through Your resurrection. With indescribable gratitude, all I can say is “Now unto Him

[Jesus] that is able to keep you from falling, and to present you faultless before the

presence of His glory with exceeding joy, to the only wise God our Savior, be glory and

majesty, dominion and power, both now and ever. Amen (Jude 24-25).” Hallelujah!

The text of I, in part, will be used for a manuscript to be submitted to an academic

journal. The dissertation author was the primary investigator and author of the research

which forms the basis of this chapter. A coauthor will be William H. Gerwick.

The text of II, in whole, is the manuscript that has been submitted to Toxicon as it

will appear: Takashi L. Suyama, Zhengyu Cao, Thomas F. Murray, and William H.

Gerwick, Ichthyotoxic Brominated Diphenyl Ethers from a Mixed Assemblage of a Red

Alga and Cyanobacterium: Structure Clarification and Biological Properties. The

dissertation author was the primary investigator and author of the research which forms

the basis of this chapter.

The text of III, in part, will be used for a manuscript to be submitted to an

academic journal. The dissertation author was the primary investigator and author of the

xxii
research which forms the basis of this chapter. Coauthors will include Karla L. Malloy

and William H. Gerwick.

The text of IV, in part, will be used for a manuscript to be submitted to an

academic journal. The dissertation author was the primary investigator and author of the

research which forms the basis of this chapter. Coauthors will include Kerry L. Mcphail,

Christopher J. Nannini, and William H. Gerwick.

The text of V, in part, is published material as it appears: Takashi L. Suyama

and William H. Gerwick, Practical Total Syntheses of Epiquinamide

Enantiomers, Organic Letters 2006, 8, 4541-4543. The dissertation author was

the primary investigator and author of the research which forms the basis of this

chapter.

The text of VI, in part, is published material as it appears: Takashi L.

Suyama and William H. Gerwick, Stereospecific Total Synthesis of

Somocystinamide A, Organic Letters 2008, 10, 4449-4452. Another portion of

the text of VI will be submitted to another academic journal. The dissertation

author was the primary investigator and author of the research which forms the

basis of this chapter. A coauthor for the second publication will be William H.

Gerwick.

The text of VII, in part, will be used for two manuscripts to be submitted

to two different academic journals. The dissertation author was the primary

investigator and author of the research which forms the basis of this chapter.

Coauthors will include William H. Gerwick and Carla Sorrels.

xxiii
 VITA

2001-2002 Undergraduate Research Assistant: Biomimetic Synthesis of

Oroidin-derived Alkaloids and Medicinal Chemistry of Psoralens;
Prof. David Horne, Oregon State University

2002-2003 Undergraduate Research Assistant: Novel Methodologies for

Absolute Stereochemistry Determination of Natural Products; Prof.
William Gerwick, Oregon State University

2001-2004 Teaching Assistant; Department of Chemistry, Oregon State

University

2003 Bachelor of Science; Oregon State University

2003-2005 Graduate Research Assistant: Novel Methodologies for Absolute

Stereochemistry Determination of Natural Products and Total
Synthesis of Natural Products; Prof. William Gerwick, University of
California at San Diego

2005-2009 Graduate Research Assistant: Total Synthesis of Natural Products

and Natural Product Chemistry; University of California at San
Diego

2009 Doctor of Philosophy; University of California at San Diego

PUBLICATIONS

•“Practical Total Syntheses of Epiquinamide Enantiomers” Takashi L. Suyama;

William H. Gerwick, Organic Letters 2006, 8, 4541-4543.
•“The marine lipopeptide somocystinamide A triggers apoptosis via caspase 8”
Wolf Wrasidlo; Ainhoa Mieglo; Vicente A. Torres; Simone Barbero; Konstantin
Stoletov; Takashi L. Suyama; Richard L. Klemke; William H. Gerwick; Dennis
A. Carson; Dwayne G. Stupack, Proceedings of National Academy of Science
2008, 105, 2313-2318.
•“Apratoxin D, a Potent Cytotoxic Cyclodepsipeptide from Papua New Guinea
Collections of the Marine Cyanobacteria Lyngbya majuscula and Lyngbya
sordida ” Marcelino Gutierrez; Takashi L. Suyama; Nicolas Engene; Joshua S.

xxiv
Wingerd; Teatulohi Matainaho; William H. Gerwick, Journal of Natural
Products 2008, 71, 1099-1103.
•“Stereospecific Total Synthesis of Somocystinamide A” Takashi L. Suyama;
William H. Gerwick, Organic Letters 2008, 10, 4449-4452.
•“Lipoproteins, lipopetides and analogs, and methods for making and using
them.” William Gerwick; Wolfgang Wrasidlo; Dennis Carson; Dwayne Stupack;
Takashi Suyama. PCT Int. Appl. 2008, 119pp.
•“Ichthyotoxic Brominated Diphenyl Ethers from a Mixed Assemblage of a Red
Alga and Cyanobacterium: Structure Clarification and Biological Properties.”
Takashi L. Suyama; Zhengyu Cao; Thomas F. Murray; William H. Gerwick.
Toxicon, 2009, Manuscript in review.
•“Vinylchloride-Containing Fatty acid and its Neurotoxic Phenethylamide
Derivative from a Papua New Guinea Collection of Lyngbya sp.” Takashi L.
Suyama; Karla L. Malloy; Zhengyu Cao; Thomas F. Murray; William H.
Gerwick. J. Nat. Prod. 2009, Manuscript in preparation.
•“Probing the Enzymatic Potential of Scy1263, a Hypothetical Protein from the
Scytonemin Biosynthetic Gene Cluster in Nostoc punctiforme ATCC 29133”
Carla M. Sorrels; Takashi L. Suyama; William H. Gerwick. Manuscript in
preparation.
•“Expedient Synthesis of α,α-dimethyl-β-hydroxy Carbonyl Scaffolds via Evans'
Aldol Reaction with a Tertiary Enolate” Joshawna K. Nunnery; Takashi L.
Suyama; William H. Gerwick. Manuscript in preparation.
•“Recent Structural Revisions of Natural Products” Takashi L. Suyama; William
H. Gerwick. Manuscript in preparation.
•“Insights into the Biosynthesis of Scytonemin through Synthetic Investigations”
Takashi L. Suyama; William H. Gerwick. Manuscript in preparation.
•“Improved Synthesis of Somocystinamide A” Takashi L. Suyama; William H.
Gerwick. Manuscript in Preparation.

FIELDS OF STUDY

Major field: Organic Chemistry

Studies in Natural Products Chemistry

Professors William H. Gerwick, William H. Fenical, and Bradley S. Moore

Studies in Synthetic Organic Chemistry

Professors Emmanuel Theodorakis and Yoshihisa Kobayashi

xxv
 ABSTRACT OF THE DISSERTATION

Organic synthesis as an effective approach to chemical, pharmaceutical, and biosynthetic

investigations of natural products

by

Takashi L. Suyama

Doctor of Philosophy in Oceanography

University of California, San Diego, 2009

Professor William H. Gerwick, Chair

The partnership between natural products and synthetic organic chemistry date

back to the origin of organic chemistry itself. While natural products became a major

driving force for the development of novel organic reactions and synthetic strategies,

organic synthesis has contributed in many ways to the elucidation and confirmation of

structure, pharmaceutical development, and biosynthetic studies of natural products. Due

to the recent advances in both of these two disciplines, there are new opportunities and

xxvi
issues surrounding natural products that organic synthesis can be applied to, and such

studies comprise this dissertation.

Chapter I introduces the background information and rationale for the dissertation

research, which is based on the history of organic chemistry and newly emerging research

topics in natural products chemistry. Chapter II describes the isolation, characterization,

and toxicological evaluation of polybrominated diphenyl ethers from a mixed assemblage

of a marine red alga and cyanobacteria. Chapter III describes the isolation,

characterization, and biological evaluation of a novel vinylchloride-containing fatty acid,

credneric acid, from a Lyngbya sp. Chapter IV describes the conception of a method for

determining the absolute stereochemistry of natural products lacking proper functional

groups for derivatization. This methodology was applied to a cyclopropane-containing

fatty acid and resulted in a conflict with newly published literature, which is discussed in

detail. Chapter V describes the development of an expedient and efficient total synthesis

of a novel alkaloid, epiquinamide, isolated from the skin of a rainforest frog,

Epipedobates tricolor. This work contributed to clarifying the identity of a potent and

selective nicotinic receptor agonistic activity observed in the extract of E. tricolor.

Chapter VI describes the development of a total synthesis of a marine cyanobacterial

metabolite, somocystinamide A, which has been shown to be a potent inhibitor of

endothelial cell proliferation and angiogenesis. The synthesis was later modified for

scalability to meet the need for further pharmaceutical investigations of this important

natural product. Chapter VII describes the synthetic investigations on scytonemin, a

cyanobacterial metabolite possessing UV-blocking properties. Through this work, many

xxvii
intriguing insights into the biosynthesis of the natural product were obtained. Finally,

Chapter VIII provides the conclusions drawn from this dissertation research.

xxviii
Chapter I

Introduction

1
 2

Abstract

Natural products chemistry and organic synthesis share a rich history of

partnership. While natural products became the major driving force for development of

novel organic reactions and synthetic strategies, organic synthesis has contributed in

many ways to the elucidation and confirmation of structure, pharmaceutical development,

and biosynthetic studies of natural products. Historical and modern examples of these

contributions and the prospect of applying chemical synthesis to current problems in

natural products chemistry will be discussed in detail.

 3

I.1 Origin of Natural Product Chemistry

“I can no longer, as it were, hold back my chemical urine; and I have to let
out that I can make urea without needing a kidney, or even of an animal,
whether of man or dog: the ammonium salt of cyanic acid (cyansäures
Ammoniak) is urea.”
Friedrich Wöhler (1828)1

With the advent of chemical nomenclature and the acceptance of the atomic

theory, chemistry emerged as a scientific discipline during the late eighteenth century. 2

Subsequently, knowledge of inorganic chemistry expanded rapidly. However, scientific

study of organic compounds did not begin until Wöhler synthesized urea from

ammonium cyanate in 1828.3 Prior to this accomplishment, chemists believed that

organic compounds could only be made by “vital forces” in living organisms and that

they obeyed laws other than the physical laws observed for inorganic compounds.2 The

new field of organic chemistry flourished due to the developing confidence that organic

compounds could be studied logically and systematically in the same way inorganic

compounds were being studied. The backbone of this breakthrough was the structural

theory, which explained that the diversity and chemical properties of organic compounds

are due to the way the constituting atoms are connected to one another. The introduction

of this theory is regarded as “of the same importance in the history of science as the

development of the two laws of thermodynamics around 1850, the quantum theory and

the theory of general relativity after 1900, and the explanation of the molecular basis of

genetics after 1950.”4

 4

Illustration I.1: Graphical representation of thesis research

This diagram utilizes the principles of Venn diagram. Where there is an overlap between different
fields, strong interdisciplinary influence is implied. The order of overlaps is meant to roughly depict
the order of emergence of each discipline chronologically.
 5

Due to scientists' constant interest in nature and health, many organic chemists of

the nineteenth century chose to study biological molecules, including medicinal

compounds, isolated from living organisms.5 In time, two sub-disciplines were formed to

study biological compounds. Biochemists studied primary metabolites, which are

essential for normal growth, sustaining of life, and reproduction of the organism.

Examples of primary metabolites include proteins, carbohydrates, fats, and nucleic acids.

In contrast, natural product chemists studied secondary metabolites (Illustration I.1).

Unlike primary metabolites, the absence of secondary metabolites would not result in

immediate death but in long-term disadvantages.5

Secondary metabolites have been unwittingly used throughout the history of

mankind for various purposes. They have been used as medicines for pain relief and

healing of wounds, as poisons for hunting and warfare, as agents for capital punishment,

as narcotics, hallucinogens, or stimulants to relieve tedium, as spice to disguise the bland

flavor of food, as perfume to obscure the odor of dead bodies, and in religious worship

and anointment (Table I-1).5,6 A great many of these natural products are still used today

and often for the same purposes.7 The difference is that the full chemical identities of the

natural products are now mostly known through advances in chemical sciences.

The rise of natural product chemistry not only stimulated the growth of biology,

but it also had a great impact on the art and science of organic synthesis (Illustration I.1).

The complex and elegant structures of many naturally occurring compounds became the

favored targets of total synthesis. In its infancy, the field of organic total synthesis

focused on primary metabolites. Only after a few decades, molecules, such as

 6

Table I.1: Use of natural products in the past and present

Compound Structure Historical usage Modern usage

Ephedrine Treatment for Treatment for asthma,

respiratory aliments hay fever

Salicine Treatment for fever Derivative (aspirin)

used for fever, pain

Strychnine Poison Stimulant, laxative,

pesticide

Coniine Poison (hemlock),

capital punishment

Rotenone Poison, insecticide Insecticide, pesticide

Morphine Narcotic Narcotic

THC Narcotic Narcotic

(tetrahydrocannabinol)

Cocaine Narcotic Narcotic

Caffeine Stimulant Stimulant, dieting

Geraniol Perfume (rose oil) Perfume (rose oil)

Linalol Perfume (lavendar oil) Perfume (lavendar oil)

Cinnamaldehyde Spice (cinnamon) Spice (cinnamon)

Eugenol Spice (cloves) Spice (cloves)

Diallyl sulfide Spice (garlic) Spice (garlic), anti-

cancer agent8
 7

haemin (1)9 and equilenin (2),10 that would be considered complex even by today's

standard were being synthesized.

I.2 Natural Product Structure Elucidation and Organic Synthesis

“Until the mid-1960's, structure determination was an art that could be

likened to solving a complicated detective case, but with the spectacular
advancement in spectroscopy it has become less inspiring, and since the
mid-1980's, in most cases, structure determination has become rather
'routine'.”
Koji Nakanishi (1991)11

In the classical era, many of the natural product chemists had their primary

training in synthetic organic chemistry. Often times, the only possible means of structure

determination of the natural product of interest was to chemically degrade it to known

substances.12 One of the most complex structures to be elucidated by this classical

method is echitamine (8), a tumoricidal alkaloid isolated from the bark of Alstonia

scholaris.13 The presence of an indole nucleus was determined by treatment with

glyoxylic reagent (Scheme I.1). Treatment with aqueous base and acid revealed a methyl

ester and imine-like moiety, respectively. Much structural information around the olefin
 8

Scheme I.1: Echitamine structure elucidation efforts by chemical

degradation and synthetic logic

The conclusion from each chemical reaction is shown in bold letters.

Scheme I.2: Assigned structures by chemical investigation and X-ray crystallography

 9

was obtained by the observed reduction with palladium and the resulting fragments from

ozonolysis. Acetylation added two acetate units, thereby showing that there are two

hydroxyl groups. Further chemical analysis of a similar nature, the molecular formula

derived by mass spectrometry, and the compound's chemical properties finally led to

structure 7 (Scheme I.2).14 Even though 7 was very similar to the correct structure 8, the

true identity of this intriguing alkaloid was not discovered until X-ray crystallography

was applied in 1962. Since the initial isolation of this natural product,15 some 85 years

had passed before its structure determination.

However, with the advent of spectroscopic methodologies, specialization in the

spectroscopic structural analysis of natural products appeared and consequently, the field

of natural product chemistry matured.12,16 Today, in many cases, the processes of natural

product isolation and structural determination are streamlined and expeditious.16

Subsequently, the focus of natural product chemists has shifted from merely describing

the chemical properties of newly found compounds to finding biologically active

Scheme I.3: Relationship between 9 and 10 via electrocyclization

 10

molecules.17 However, the age old partner of natural products chemistry, chemical

synthesis, still plays a major role today in confirmation of the chemical structure of the

natural product, particularly its stereochemistry, and in providing a larger quantity of the

compound as well as analogs for biological testing (vide infra).11

Particularly profound and interesting examples of the contribution from chemical

synthesis to the structural revision of a natural product include tridachiahydropyrone (9)

and mururin C (12). In the first case, the issue concerned stereochemical assignments,

while an incorrect planar structure was assigned to the latter compound. Such revisions

were required for 30 natural products in 2008 alone.18 It is safe to say that the art of

structure elucidation has not achieved perfection and that the partnership between natural

products chemistry and synthetic organic chemistry is still very much in order.

Tridachiahydropyrone (9) was isolated from a marine mollusk Tridachia crispata

by Cimino and coworkers.19 Originally, structure 10 was proposed based on NMR and

NOE spectroscopic analyses. Subsequently, the total synthesis of 10 by Perkins and

coworkers suggested that the correct structure is most likely 9.20 A biogenetic rationale

for structure 9 was inspired by Cimino and Faulkner, who noted that the extensive

conjugation of tridachiahydropyrone and its congeners may serve as defense against

harmful UV rays.21 The putative precursor 11 could be converted to either 9 or 10 through

6π electrocyclization depending on the mode of activation (Scheme I-1). According to the

Woodward-Hoffman rules,22 photochemical 6π electrocyclization would yield the

conrotatory product, 9, whereas the corresponding thermal reaction would yield the

disrotatory product, 10.

 11

Indeed, Moses and coworkers synthesized 11, which was converted to 9 upon

exposure to sunlight.23 Furthermore, this synthetic material, 9, had the same NMR and

UV properties as the authentic sample. All attempts to convert 11 to 10 by thermal means

failed.23 These results not only confirm that 9 is the correct structure for this natural

product, but also suggests that the actual biosynthetic product produced by Tridachia may

be 11 and that it is subsequently converted to the stronger UV absorbent 9 when exposed

to strong sunlight. The biomimetic synthesis of tridachiahydropyrone led to its structural

revision as well as insights into its biosynthesis that could not have been easily obtained

by any other approach.

Figure I.1: The originally assigned structure (13) of mururin C and its
revised structure (12)

Although the majority of structural revisions of natural products have been due to

stereochemical issues involving tetrahedral carbon atoms, perhaps it is more intellectually

interesting to consider the less frequent cases of incorrectly assigned planar structures.

Such was the case with mururin C (12), a natural product isolated from the bark of

Brosimum acutifolium. Originally, structure 13 was incorrectly assigned to this natural

product based on HR-MS and 1D and 2D NMR analyses.24 In the course of the synthesis
 12

of 13, however, it was found that allenes analogous to 13, when formed in situ, were

intercepted readily by nucleophiles in the reaction mixture and the allene species could

not be isolated.25 This observation led to the rationale that if 13 was formed at all in the

course of biosynthesis, the hydroxyl group of the phenol would attack the allenic carbon

and form benzofuran, thereby converting to structure 12. Therefore it is highly unlikely

that 13 was the correct structure. Moreover, comparison of predicted chemical shifts for

12 and 13 revealed that 12 is very likely the correct structure. In this work, synthetic

studies of the natural product eloquently revealed the erroneous feature of the originally

mis-assigned planar structure of mururin C.

In 2005, Nicolaou and Snyder published a fairly comprehensive and inspiring paper

on natural product structural revisions that were made possible by total synthesis in the

period of 1990 to 2004.16 In that paper, the authors articulate the amazement, in contrast

to their expectation, of discovering the large number and profoundness of structural mis-

assignments made in the recent years. In Table I.2, all of the structural revisions 26 of

natural products since 2005 through 2008 are summarized.

 13

Table I.2: Structural revisions made in 2005-2008.

The year in which the initial structure was published is in parentheses. Only the structure
determination methods used for the portion of the structure that is erroneous are mentioned in this
table. Predict.: predictions based on molecular modeling. Mol. Mod.: use of geometry optimized
structure.

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

Total
NOE
Synthesis28

Calafianin27 (2000)

Total
NOESY
Synthesis30
Tribulusterine29 (1999)

Total
Absence of NOE
Synthesis32
Ayapin derivative31 (1999)

1
H & 13C NMR
Total
HMBC
Synthesis34
NOE
Isoaurostatin33 (2001)

1
H & 13C NMR
Total
HMQC
Synthesis36
HMBC
Chloroaurone35 (2001)
 14

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

NOESY Total
HMBC Synthesis38
Feigrisolide A37 (2000)

NOESY Total
HMBC Synthesis38
Feigrisolide B37 (2000)

HMBC Total
NOESY Synthesis40

Feigrisolide C39 (2000)

Derivatization Total
NMR Synthesis42

Agardhilactone41 (1996)

EIMS Total
Fragmentation Synthesis44
Pyrinodemin A43 (2001)

CD
Total
Comparison Synthesis46
NMR

Agelasine C45 (1984)

 15

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

CD
Total
Comparison Synthesis46
NMR

Epi-agelasine C45 (1984)

J-based Total
ROESY Synthesis48

Clavosolide A47 (2002)

X-ray
HMBC
NOE50
Aspernigrin A49 (2004)

1 NMR
H NMR
Comparison52

Suberoretisteroid B51 (2000)

1 NMR
H NMR
Comparison52

Suberoretisteroid B51 (2000)

 16

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

Derivatization
Total
J-based
Synthesis54
Mosher
Amphidinolide W53 (2002)

Comparison
NOE Total
55 Synthesis56
Batzelladine F (1997) DCI-MS
Fragmentation

Total
NOE
Synthesis58

Brevenal57 (2005)

Total
NOE
Synthesis60
Trihydroxycadinane59 (1998)

Total
Comparison
Synthesis62
Aspergione B61 (2003)

Semi-
synthesis
1
H & 13C NMR
NMR
Botcinic acid63 (1993)
Comparison64
(Botcinolide)
 17

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

NMR NMR
Comparison Comparison65
Homobotcinic acid65 (1996)
(Homobotcinolide)

NMR NMR
Comparison Comparison65
Botcinic acid methyl ester66
(1996) (4-O-methylbotcinolide)

EI-MS
HMBC 13
C Prediction
NOE NMR
Peribysin C67 (2004)
comparison68

EI-MS
HMBC 13
C Prediction
NOE NMR
Peribysin C62 (2004)
comparison61

Chemical
ROESY Reactivity
CD predict.70
Pyranonigrin69 (2004)

FAB-MS
HMBC72
Degradation
Shatavarin I71 (1987)
 18

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

EI-MS
Degradation HMBC57
OR predict
Shatavarin IV58 (1987)

HMBC
1D & 2D NMR NMR
Comparison74
Fusapyrone73 (1994)

HMBC
1D & 2D NMR NMR
Comparison74
Deoxyfusapyrone73 (1994)

HMQC
COSY Total
Comparison Synthesis76
Solandelactone A75 (1996)
J-based

Comparison Total
J-based Synthesis76
75
Solandelactone B (1996)

Comparison Total
J-based Synthesis76
75
Solandelactone C (1996)
 19

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

Comparison Total
J-based Synthesis76
75
Solandelactone D (1996)

Comparison Total
J-based Synthesis76
Solandelactone E75 (1996)

Comparison Total
J-based Synthesis76
Solandelactone F75 (1996)

Comparison Total
J-based Synthesis76
75
Solandelactone G (1996)

Comparison Total
J-based Synthesis76
75
Solandelactone H (1996)

NOESY
NOESY78
NMR Comprison

Barlerin77 (2004)
 20

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

NOESY
NOESY78
NMR Comprison

Penstemoside77 (2004)

NOESY
NOESY78
NMR Comprison

Dehydropenstemoside77 (2004)

HR-MS
NMR
NOESY
Comparison78
HMBC

Harpagoside79 (2003)

Degradation
GCMS NOESY78
OR
80
Agnuside (2004)
 21

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

NMR
NOESY78
Comparison

3,4-hydroxybenzoylaucubin80
(2004)

COSY
1
H NMR
HMBC
CD
X-ray82
Camellenodiol81 (1981)

1
H NMR COSY
CD HMBC82

Camelledionol81 (1981)

ROESY
ROESY
Mol. Mod.
J-based
J-based84

Palau'amine83 (1993)

Total
D2O Shift86
Synthesis87
Intricatin85 (1989)
 22

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

Total
Synthesis89
Degradation
First partial
1 D & 2D NMR
synthesis had
an error.90
Kedarcidin88 (1993)

J-based Total
NOESY Synthesis92

Malyngamide U91 (2003)

1D & 2D NMR Total

Comparison Synthesis94
93
Abrotandiol (2007)

1D & 2D NMR Total

Comparison Synthesis94
93
Abrotanone (2007)

Total
NOE
Synthesis96
Biyouyanagin95 (2005)

Total
Synthesis98
Aciphyllene97 (1983)
 23

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

Mosher
Total
J-based
Synthesis100
NOE
Palmerolide A99 (2006)

EI-MS
1 Total
H, 13C NMR
Synthesis102
Benz[g]isochromene-dione101 HETCOR
(1995)

Total
NOE
Synthesis104
Laurentristich-4-ol103 (2005)

1 Total
H, 13C NMR
Synthesis106

Dalmaisione D105 (1972)

Biomimetic
Semi-
synthesis108
Xuxuarine Eα
107
Rzedowskia bistriterpenoid α
(1996)
 24

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

Biomimetic
Semi-
synthesis108
Xuxuarine Eα

Rzedowskia bistriterpenoid α107

(1996)

1D & 2D NMR Total

NOE Synthesis110
Kirkine109 (1995)

1D & 2D NMR Semi-

NOE synthesis112

25-acetoxy-3a-hydroxyolean-12-
en-28-oic acid111 (2004)

ROESY Total
NOE Synthesis114

Callipeltoside C113 (1996)

 25

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

Total
J-based
Synthesis116

Diversifolide115 (1999)

1D & 2D NMR
IR Total
HR EI-MS Synthesis118

NOE
Labdane diterpenoid117 (2006)

Total
NOE
Synthesis120
Netamine E119 (2006)

Total
NOE
Synthesis120

Netamine G119 (2006)

J-based
Mosher Total
Derivatization Synthesis122
Tyroscherin121 (2004)
Optical Rotation
 26

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

Amphidinol 3123 (1999)

Ozonolysis
Combinatorial
Derivatization synthesis of
fragments124
Chiral HPLC
Amphidinol 3 Fragment123

Derivatization Total
Chiral GCMS Synthesis126

Brunsvicamide A125 (2006)

HMBC
ROESY 13
C Predict.
X-ray128
Spiroleucettadine127 (2004)

Mol. Mod.
13
NMR C Predict.130
Comparison
Obtusallene V129 (2000)
 27

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

Mol. Mod.
13
NMR C Predict.130
Comparison
Obtusallene VI129 (2000)

Mol. Mod.
13
NMR C Predict.130
Comparison
Obtusallene VII129 (2000)

INEPT2-
INADEQUATE HMBC
HMBC- X-ray132
Circumdatin A131 (1999) INADEQUATE

INEPT2-
INADEQUATE HMBC
HMBC- X-ray132
INADEQUATE
Circumdatin B131 (1999)
 28

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

HR-MS
X-ray134
1
H NMR

Bromoditerpene133 (1987)

NOE
NMR NOE136
Comparison
Hiyodorilactone B135 (1978)

ESI-MS
X-ray138

Rhizopodin137 (1993)

NMR
Comparison
ESI HR-MS
Semi-
HMBC
synthesis
Bioinformatics
ESI HR-
Microcyclamide 139
(2008) MS140

HMBC
13
Biosynthetic C Predict.
Prediction UV Predict.142
Hassanane141 (1996) Mol. Mod.
 29

Table I.1 continued

Initial Structure Structure

Proposed Structure Determination Revised Structure Revision
Method Method

Comparison
NOESY with synthetic
ESI HR-MS model
compounds144
Zamamistatin 143
(2001) See next entry

Comparison with IR
synthetic model NMR and OR
compounds Comparison145
Zamamistatin144 (2001)
 30

60%

Stereochemical
Constitutional
Constitutional &
Stereochemical

15%

25%

Figure I.2: Types of errors in natural product structure revisions

24% 26%

NOE
Comparison
J-based
Deriv atization
NMR
CD/OR
2% Model
4% Chromatography
MS
18% 4%
6%
12% 6%

Figure I.3: Structure determination methods used in 2005-2008 for the erroneous cases

54%

Total sy nthesis
Other sy nthetic
X-ray
Others

8%
8% 29%

Figure I.4: Structure revision methods used in 2005-2008

 31

Remarkably, a majority of these structures were elucidated in the very recent

years (1990-2008). Of the 83 structure revisions, 40% had constitutional errors (Figure

I.2). This statistic comes as a surprise in this century when NMR tools, such as HMBC, to

connect elucidated substructures are readily available (Figure I.3). However, a close

examination of these structures reveals a subtle problem. Unless all theoretically possible

HMBC correlations are observed, some structures are indistinguishable from their

alternatives on the basis of HMBC spectroscopy only. An example of this type of

Figure I.5: Discrimination of possible structures by HMBC

The HMBC correlations necessary for distinction between the two possible structures are depicted
as blue and orange arrows. If the blue correlation was only present, structure 14 would be correct.
Likewise, if the orange correlation was only present, then structure 15 would be correct.

Figure I.6: HMBC correlations observed and recorded for the labdane diterpenoid108

The HMBC correlations necessary for discrimination of structures 14 and 15, as noted in Figure
I.5, were not recorded (or observed) for this natural product.
 32

problem is shown in Figures I.5 and 6. This and other examples in

Table I.2, particularly the structures with constitutional errors,

illustrate how total synthesis is crucial for confirmation of structure

(Figure I.4).

In addition to the mis-assignment of structures, there has been

one case where total synthesis clarified the true identity of the bioactive metabolite. In

2003, Daly and coworkers isolated a novel quinolizidine alkaloid, epiquinamide (16)

from a rainforest frog, Epipedobates tricolor in a sub-milligram quantity.146 Initially, 16

was believed to be a subtype selective nicotinic receptor agonist and it attracted much

attention from the synthetic community. By 2006, there had been several asymmetric

total syntheses of epiquinamide enantiomers.147 As a result of theses syntheses, it was

found that structure 16 had no activity as a nicotinic receptor agonist.147 Afterwards,

evidence surfaced that the originally observed activity might have been due to a minor

contamination of the sample with epibatidine.148 Awareness of this error was made

possible by the synthetic supply of 16 free of any other bioactive natural product

contaminants. As technologies to elucidate the structures of minute quantities of natural

products advance further, the need for supplying pure samples of natural products by total

synthesis can be expected to increase.

I.3 Natural Products as Pharmaceuticals

Successful attempts have been made to present the case that natural products are a

great resource for drug development.149 Indeed, among all of the drugs that were

approved between 1981 and 2006 for clinical use, 63% had their ultimate origin in
 33

natural products (Figure I.7).93 This recent success of natural products did not emerge

from a vacuum, but it has been nurtured by the partnership with synthetic chemistry since

the conception of organic chemistry. In the earliest era of organic chemistry, it was rarely

taught as an independent discipline, but always in conjunction with medicine, geology,

and other fields.4,5 Even today, it is difficult to find an academic publication in organic

synthesis that does not appeal to medical or ecological interests for a justification of the

research.

A survey of the natural products discovered during the nineteenth century

indicates that many are neurologically relevant compounds, such as caffeine, nicotine,

codeine, and morphine (Table I.1).5 It is disappointing, then, that the pool of clinically

available natural products in neuropharmacology has not expanded significantly since

this earliest era of natural product chemistry. In the last 25 years, only two neurologically

relevant natural products, and an additional two compounds derived from natural

Figure I.7: All new small chemical entities that were approved for clinical use,
01/1981-06/2006, by source.93

N: Natural products. ND: Derived from a natural product and is usually a semisynthetic modification.
S: Totally synthetic drug, often found by random screening/modification of an existing agent. S*:
Made by total synthesis, but the pharmacophore is/was from a natural product. NM: Natural product
mimic.
 34

products, were approved for clinical use.93 In contrast, 21 natural products and 99

compounds derived from natural products were approved for treatments of cancer and

infectious diseases during the same period.93 To respond to this challenge, current efforts

are underway to discover novel neurotoxins from untapped natural sources.150,151

Despite more anti-cancer agents being approved for human treatment than any

other pharmaceutical classes, there is still a great need for new drugs to treat cancer. For

example, there are currently no effective chemotherapeutic treatments available for

pancreatic cancer.152 Even for the types of cancer that are known to be treatable by anti-

cancer agents, severe side-effects and ineffectiveness towards metastasis still plague

patients. With cancer being the second most common cause of death in the USA (23.1%

of all deaths in 2004),153 the search for new and more effective anti-cancer agents is still

warranted indeed. Though outside the scope of this thesis, new anti-microbial agents are

also urgently needed. In recent decades, infectious diseases have emerged as a substantial

threat both in the USA and other countries.

In the past few years, the re-isolation of known compounds has become a

hindrance to the productivity of natural products chemistry efforts.154 Traditionally,

terrestrial plants and microbes have been the major sources of natural products, and

researchers have exhaustively studied those organisms.5 Therefore, the current trend is to

investigate organisms that have not been previously studied in detail. For example,

investigation of poisonous frogs155 and marine organisms91 has enjoyed great productivity

in recent years. Between 1968 and 1998, over 400 alkaloids of over 20 structural classes,

most of which are neurologically relevant, were isolated from amphibians alone.87

Considering the difficulty of obtaining extract samples from amphibian skin, this is a
 35

remarkable achievement. From marine organisms, a relatively new source of secondary

metabolites, a total of 812 new compounds were isolated in 2005 alone (716 for 2004),

and this trend seems to continue.93

I.4 Organic Synthesis to Develop Natural Products as Drugs

With such an outstanding rate of discovery, one would expect to see more natural

products being approved for clinical use. However, this is not the case. One cause for this

unfortunate situation is the lack of follow-up investigation of bioactive natural products

due to an insufficient supply of the compounds.156 Consequently, many compounds with

high potential are never tested in vivo in animals, which often requires more than

hundreds of milligrams of material. Often times, the major barrier in drug development is

this multi-hundred milligram (MHM) quantity, due to the frequent inability to produce

more than several milligrams of a natural product in academic laboratories whether

through synthesis or isolation. Once a compound performs favorably in in vivo assays

and/or pre-clinical trials, pharmaceutical companies with sufficient financial resources

may show interest in carrying out industrial-scale production of the compound for further

testing (Figure I.8). Otherwise, due to financial difficulties, it is virtually impossible for

non-commercial organizations to take a compound to clinical trial.

Identification Clinical Trials

of Compound In vitro testing In vivo testing
Drug in use
Figure I.8: Drug Development Process

Note that “identification of compound” in this figure refers to both lead compound discovery and
medicinal chemistry (analog synthesis and evaluation) processes.
 36

If the natural product is from a bacterium and/or fungus, culturing of the source

organism is often possible at a scale large enough for isolation of the compound to meet

the demands of in vivo assays. For most natural products isolated from animals, such as

amphibians and marine invertebrates, neither large-scale collection or culturing of the

source organism is realistic. Therefore, a scalable chemical synthesis is a necessary

alternative to obtain a sufficient supply of the compound. Because large for-profit

organizations would generally tend to avoid high risk projects, such as a compound at the

in vivo testing stage, academic, non-profit institutions, and benevolent small businesses

must face the challenge of realizing MHM-scale synthesis.

As Paul Wender has articulated, an ideal synthesis is “one in which the target

molecule (natural or designed) is prepared from readily available, inexpensive starting

materials in one simple, safe, environmentally acceptable, and resource-effective

operation that proceeds quickly and in quantitative yield.”157 Employing these principles

for achieving an efficient synthesis while applying creativity and utilizing the vast

knowledge accumulated in the organic synthetic chemistry literature, it is possible to

design and execute a large scale-amenable synthesis of the compound of interest.

Inspiring news hit the synthetic community in 2004 when Novartis disclosed their

total synthesis of 60 g of (+)-discodermolide (17).158 Combining the strengths of the

Discodermolide, 17
 37

Scheme I.4: Novartis strategy for the synthesis of discodermolide (17)

synthetic routes developed by both Smith's and Patterson's laboratories (Scheme I.4),159,160

Novartis pushed the scalability limit of total synthesis. Smith and coworkers devised a

highly convergent and elegant synthesis, in which all three of the units (19, 20, 21)

required for the discodermolide skeleton are derived from a single common precursor

(22) with elaborate stereochemistry.83 However, the preparation for the last coupling in

Smith's synthesis involved a tranformation to a phosphonium salt that required 12.8 kbar

of pressure, which would be too dangerous for an industrial scale synthesis. In the

strategy developed by Patterson and coworkers, the last coupling proceeded with good
 38

diastereoselectivity (21:4) and with excellent yield (87%), and it did not involve any

potentially dangerous reaction conditions.82

"Some 3,000 kg of the sponge--a quantity that probably does not exist--would
have been needed to deliver 60 g of (+)-discodermolide. The large-scale total
synthesis of such a complex natural product in such quantities was a first for
Novartis and probably the entire pharmaceutical industry,“
Stuart J. Mickel, Novartis161

“It’s [discodermolide synthesis by Novartis] probably the best piece of

synthetic work to come out from an industrial company. The ability to make
something at this level of complexity as opposed to extracting it from natural
product sources illustrates the power of modern synthetic chemistry."
Prof. Steven V. Ley, Univ. Cambridge81

Another obstacle to the development of drugs from natural products is the lack of

comprehensive bioactivity profiling due to the limited number of in-house assays

available. Without an extensive network of collaborations for bioassays, a number of

important bioactivities can remain undetected. A marine cyanobacterial natural product,

somocystinamide A's (23)162 remarkable activity against angiogensis (IC50 of 500 fM

against HUVEC and in vivo anti-angiogenic effect in zebrafish at 30 nM) was only

revealed through a new collaborative relationship.163 This promising compound

represents only the tip of an iceberg of pharmacologically important marine natural

Somocystinamide, 23
 39

products. Yet, the discovery and development of these compounds as drugs will depend

on collaborative efforts of chemists and biologists to identify bioactivities that are not yet

known.

After a promising activity is discovered in a structurally defined natural product,

there is usually still work to be done by synthetic chemists. In the example of 23, the

disulfide and enamide are of concerns with regards to human toxicity and compound

stability. These functionalities may need to be replaced in order for the drug to be usable

in clinical situations. Indeed, in Figure I.5, only 6% of the newly approved small

molecule drugs are unmodified natural products, whereas 28% are modified natural

products. This process of modification, medicinal chemistry, is as crucial as finding new

compounds possessing new bioactivities.

I.5 Biosynthesis of Natural Products and Organic Synthesis

As demonstrated above, the organic synthesis of a natural product can have a

multitude of benefits, namely, confirmation or clarification of structure, a solution to the

supply issue, access to analogs, and detailed insights of the chemical properties of the

compound. In addition, mechanistic insight derived from organic synthesis is

indispensable to understanding the details of the biosynthetic processes in nature.# While

working with secondary metabolites, the biosyntheses of such compounds naturally

become of interest. The mechanism of an enzymatic reaction, in particular, is an

intellectually satisfying problem for chemists to solve and can be useful for engineering

of the biosynthetic pathway and in vitro application of the enzyme in chemical synthesis.

Often, this is done by designing and synthesizing an isotope-labelled biosynthetic

 40

intermediate and then analyzing the product of the enzymatic reaction that utilizes the

intermediate.

Brevianamides and their related compounds are a family of natural products that

are biosynthetically derived from tryptophan, proline, and one or two isoprene unit(s).164

Many metabolites of this family share a core structure that appears to be derived from an

intramolecular Diels-Alder reaction, and whether the producers of these metabolites,

fungi Penicilium sp. and Aspergillus sp., possess a “Diels-Alderase,” has been an exciting

topic in the field of bioorganic chemistry. 78,165 Both brevianamide A (33) and

brevianamide B (34) were found to be biosynthetically derived from a common

precursor, deoxybrevianamide E (30) through a feeding experiment of tritium-labelled

30, which was prepared through chemical synthesis.166 When a synthetic precursor similar

to 32 was allowed to undergo a Diels-Alder reaction, an epimer that is not analogous to

any of the isolated natural products was the major product and an isomer that is

analogous to 34 was the minor product (isomeric ratio = 2:1).167 This result gives

Scheme I.5: Synthetic contributions to the study of brevianamides biosynthesis

 41

credence to the hypothesis that the Diels-Alder reaction in Schem I.5 is indeed enzyme-

catalyzed and that 33 and 34 are not artifacts, but natural products.

Occasionally, it is possible for synthetic organic chemistry to more directly

contribute to the biosynthetic study of natural products. By achieving total or partial

synthesis of a natural product in a way that mimics the proposed biosynthetic pathway,

one can gain direct chemical insight into the biochemical reactions of interest, as seen in

the example above with brevianamides. Because a biomimetic synthesis relies upon the

inherent nature of a key intermediate, a complex dimerization reaction of monomers and/

or an elaborate intramolecular reaction are often involved. 168 A recent and elegant

example is the biomimetic total synthesis of grossularine-1 (35), a bisindole alkaloid

isolated from a marine tunicate, Dendrodoa grossularia.169 The dimeric nature of the

molecule is not immediately apparent in the structure (35), but is clearly revealed in the

elaborate sequence of transformations from the monomer 36 to 40.

Another exceptionally elegant example in recent years is panepophenanthrin (41),

whose total synthesis was reported simultaneously by Porco and Baldwin in 2003

(Scheme I.7).170,171 Here the inherent propensity of the monomer (42) for dimerization is

cleverly utilized to effect a stereoselective and hydrogen-bond-assisted Diels-Alder

reaction to produce the natural product (41). Without this biomimetic step, the core

tetracyclic skeleton would require a large number of synthetic steps to construct.

Presumably, the exact same reaction can happen in nature with or without any assistance

from an enzyme, which brings up the question about whether compounds like 41 are truly

biosynthetic metabolites or not. Each case probably requires a separate consideration. In

 42

Scheme I.6: Biomimetic synthesis of grossularine-1 (35)

Scheme I.7: Biomimetic synthesis of panepophenanthrin (41) through a Diels-Alder

reaction

this case, given that 42 requires an extremely high concentration (neat) for smooth

dimerization, it is possible that the transformation to 41 is enzymatically catalyzed.

Finally, a cyanobacterial UV-screening natural product, scytonemin (44)172 is an

example of a dimeric metabolite that is now known to require enzymatic assistance for

dimerization of the corresponding monomer.173 Again, through chemical synthesis, a

putative monomer 45 was prepared and subjected to various oxidizing conditions that

could exist in nature. However, it was found that 45 does not dimerize under such

conditions, thereby providing unique insights into the biosynthesis of scytonemin.

 43

Scheme I.8: Scytonemin monomer does not dimerize under naturally occurring conditions

I.6 Rationale for Thesis Research

Three separate areas in which natural products chemistry has benefited from

organic synthesis were discussed above; namely, structure elucidation and confirmation,

pharmaceutical application, and biosynthetic study of natural products. Simultaneously,

natural products have been the major driving force for the advancement of organic

synthesis. Given this rich history, direct application of the power of organic synthesis to

natural products should improve productivity in all three areas mentioned above. As an

added significant benefit, new chemical transformations and synthetic strategies can be

discovered in the process.

I.7 Overview of Thesis Chapters

Chapter II describes the isolation and characterization of two neurotoxic and

ichthyotoxic polybrominated diphenyl ethers from a mixed assemblage of a red alga and

cyanobacteria. Coauthors contributed the following: Z. Cao conducted the [Ca2+]i assays;

T. F. Murray oversaw the [Ca2+]i assay work and contributed to the corresponding portion
 44

of the article. The text of II, in whole, is the manuscript that has been submitted to

Toxicon as it will appear: Takashi L. Suyama, Zhengyu Cao, Thomas F. Murray, and

William H. Gerwick, Ichthyotoxic Brominated Diphenyl Ethers from a Mixed

Assemblage of a Red Alga and Cyanobacterium: Structure Clarification and Biological

Properties. The dissertation author was the primary investigator and author of the

research which forms the basis of this chapter.

Chapter III describes the discovery of a novel fatty acid containing a vinylchloride

functionality, credneric acid, from a marine cyanobacterium Lyngbya sp. The text of III,

in part, will be used for a manuscript to be submitted to an academic journal. The

dissertation author was the primary investigator and author of the research which forms

the basis of this chapter.

Chapter IV describes the determination of the absolute configurations of a novel

fatty acid containing cyclopropane moiety from a marine cyanobacterium Lyngbya

majuscula through synthetic introduction of a stereochemical reference. A coauthor

contributed the following: K. L. McPhail assisted with the purification of the natural

product and acquisition and interpretation of the NMR data. The text of IV, in part, will

be used for a manuscript to be submitted to an academic journal as it will appear: Takashi

L. Suyama, Kerry L. McPhail, and William H. Gerwick, Absolute Stereochemistry by

Synthetic Introduction of a Stereochemical Reference: Application to an Unusual Marine

Cyanobacterial Lipid. The dissertation author was the primary investigator and author of

the research which forms the basis of this chapter.

Chapter V describes the expedient, efficient and stereoselective total syntheses of

a rainforest frog alkaloid epiquinamide and its enantiomer. The text of V, in part, is
 45

published material as it appears: Takashi L. Suyama and William H. Gerwick,

Practical Total Syntheses of Epiquinamide Enantiomers, Organic Letters 2006, 8,

4541-4543. The dissertation author was the primary investigator and author of

the research which forms the basis of this chapter.

Chapter VI describes the stereoselective and stereospecific total synthesis

of somocystinamide A. The text of VI, in part, is published material as it appears:

Takashi L. Suyama and William H. Gerwick, Stereospecific Total Synthesis of

Somocystinamide A, Organic Letters 2008, 10, 4449-4452. Another portion of

the text of VI will be submitted to another academic journal. The dissertation

author was the primary investigator and author of the research which forms the

basis of this chapter.

Chapter VII describes the biomimetic total synthesis of a cyanobacterial

UV-screening metabolite, scytonemin. The text of VII, in part, will be used for a

manuscript to be submitted to an academic journal. The dissertation author was

the primary investigator and author of the research which forms the basis of this

chapter.

Chapter VIII summarizes my thesis research and provides conclusions

based on the research.

The appendix describes my additional research activities that could not constitute

thesis chapters by themselves.

 46

References and footnotes

1 Kauffman, G. B.; Chooljian, S. H. Friedrich Wöhler (1800-1882), on the Bicentennial of His

Birth. Chem. Educator, 2001, 6, 121-133.

2 Salzberg, H. W. From Caveman to Chemist, 1991, American Chemical Society.

3 Wöhler, F. Ann. Phys. Chem. 1828, 12, 253.

4 Tarbell, D. S.; Tarbell, T. Essays on the history of organic chemistry in the United States,
1875-1955. 1986, K & S Press, Nashville, TN. pp 5.

5 Mann, J. Secondary Metabolism, 2nd Ed., 1987, Oxford Science Publications.

6 Holy Bible, a) The Gospel according to John 19:40 b) Exodus 25:6

7 Indeed, the bioactivities of some of these compounds are still being discovered. For example,
some of the health benefits of garlic are now attributed to the diallyl sulfide compounds found
in it (Table I.1 and ref. 8).

8 Xiao, D.; Li, M.; Herman-Antosiewicz, A.; Antosiewicz, J.; Xiao, H.; Lew, K. L.; Zeng, Y.;
Marynowski, S. W.; Singh, S. V. Diallyl Trisulfide Inhibits Angiogenic Features of Human
Umbilical Vein Endothelial Cells by Causing Akt Inactivation and Down-Regulation of VEGF
and VEGF-R2. Nutr. Canc. 2006, 55, 94-107.

9 Fischer, H.; Zeile, K. Synthese des Hämatoporphyrins, Protoporphyrins und Hämins. Justus
Liebigs Ann. Chem. 1929, 468, 98-116.

10 Bachmann, W. E.; Cole, W.; Wilds, A. L. The total synthesis of sex hormone equilenin. J. Am.
Chem. Soc. 1939, 61, 974.

11 Nicolaou, K. C.; Sorensen, E. J. Classics in Total Synthesis, 1996, VCH publishers.

12 Nakanishi, K. A Wandering Natural Products Chemist, 1991, American Chemical Society, pp

87.

13 Saraswathi, V.; Ramamoorthy, N.; Subramaniam, S.; Mathuram, V.; Gunasekaran, P.;
Govindasamy, S. Inhibition of glycolysis and respiration of sarcoma-180 cells by echitamine
chloride. Chemotherapy 1998, 44, 198-205.

14 Govindachari, T. R.; Rajappa, S. Echitamine. Tetrahedron 1961, 15, 132-143.

15 v. Group-Besanez Ann. Chem. 1875, 176, 88-89.

16 Nicolaou, K. C.; Snyder, S. A. Chasing Molecules That Were Never There: Misassigned
Natural Products and the Role of Chemical Synthesis in Modern Structure Elucidation.
Angew. Chem. Int. Ed. 2005, 44, 1012-1044.
 47

17 To a somewhat lesser extent, compounds of biosynthetic and ecological interests have

attracted natural products chemists' attention as well.

18 SciFinder Scholar search for “structure revision” and other related keywords

19 Gavagnin, M.; Mollo, E.; Cimino, G.; Ortea, J. A new γ-dihydropyrone-propionate from the
caribbean sacoglossan Tridachia crispata. Tetrahedron Lett. 1996, 37, 4259–4262.

20 (a) Jeffery, D. W.; Perkins, M. V.; White, J. M. Synthesis of an Analogue of the Marine
Polypropionate Tridachiahydropyrone. Org. Lett. 2005, 7, 407–409. (b) Jeffery, D. W.;
Perkins, M. V.; White, J. M. Synthesis of the Putative Structure of Tridachiahydropyrone. Org.
Lett. 2005, 7, 1581–1584.

21 Ireland, C.; Faulkner, D. J. The metabolites of the marine molluscs tridachiella diomedea and
tridachia crispata. Tetrahedron 1981, 37, 233–240, Suppl. 1.

22 Fleming, I. Pericyclic Reactions, 1999, Oxford University Press, pp 31-56.

23 Sharma, P.; Griffiths, N.; Moses, J. E. Biomimetic synthesis and structural revision of (±)-
tridachiahydropyrone. Org. Lett. 2008, 10, 4025-4027.

24 Takashima, J.; Asano, S.; Ohsaki, A. Tennen Yuki Kagobutsu Toronkai Koen Yoshishu 2000,
42, 487.

25 Hu, G.; Liu, K.; Williams, L. J. The brosimum allene: a structural revision. Org. Lett. 2008,
10, 5493-5496.

26 Publications that merely claim that the original structures are incorrect, but do not give
revised structures are omitted.

27 Encarnacion, R. D.; Sandoval, E.; Malmstrom, J.; Christophersen, C. Calafianin, a

Bromotyrosine Derivative from the Marine Sponge Aplysina gerardogreeni. J. Nat. Prod.
2000, 63, 874–875.

28 (a) Ogamino, T.; Nishiyama, S. Synthesis and structural revision of calafianin, a member of
the spiroisoxazole family isolated from the marine sponge, Aplysina gerardogreeni.
Tetrahedron Lett. 2005, 46, 1083–1086. (b) Ogamino, T.; Obata, R.; Tomoda, H.; Nishiyama,
S. Total Synthesis, Structural Revision, and Biological Evaluation of Calafianin, a Marine
Spiroisoxazoline from the Sponge, Aplysina gerardogreeni. Bull. Chem. Soc. Jpn. 2006, 79,
134-139.

29 Wu, T.; Shi, L.; Kuo, S. Alkaloids and other constituents from Tribulus terrestris.
Phytochemistry 1999, 50, 1411-1415.

30 Bremner, J.; Sengpracha, W.; Southwell, I.; Bourke, C.; Skelton, B.; White, A. The alkaloids
of Tribulus terrestris: a revised structure for the alkaloid tribulusterine. Acta Horticulturae
2005, 677, 11-17.
 48

31 Baetas, A. C. S.; Arruda, M. S. P.; Mu¨ller, A. H.; Arruda, A. C. J. Braz. Chem. Soc. 1999, 10,
181–183.

32 Maesa, D.; Vervischa, S.; Debenedettib, S.; Davioc, C.; Mangelinckxa, S.; Giubellinaa, N.; De
Kimpe, N. Synthesis and structural revision of naturally occurring ayapin derivatives.
Tetrahedron 2005, 61, 2505-2511.

33 Suzuki, K.; Yahara, S.; Maehata, K.; Uyeda, M. Isoaurostatin, a Novel Topoisomerase
Inhibitor Produced by Thermomonospora alba. J. Nat. Prod. 2001, 64, 204–207.

34 Venkateswarlu, S.; Panchagnula, G. K.; Guraiah, M. B.; Subbaraju, G. V. Isoaurostatin: total

synthesis and structural revision. Tetrahedron 2005, 61, 3013–3017.

35 Atta-Ur-Rahman; Choudhary, M. I.; Hayat, S.; Khan, A. M.; Ahmed, A. Two New Aurones
from Marine Brown Alga Spatoglossum variabile. Chem. Pharm. Bull. 2001, 49, 105–107.

36 Venkateswarlu, S.; Panchagnula, G. K.; Gottumukkala, A. L.; Subbaraju, G. V. Synthesis,

structural revision, and biological activities of 4'-chloroaurone, a metabolite of marine brown
alga Spatoglossum variabile. Tetrahedron 2007, 63, 6909-6914.

37 Tang, Y.-Q.; Sattler, I.; Thiericke, R.; Grabley, S.; Feng, X.-Z. Feigrisolides A, B, C and D,
New Lactones with Antibacterial Activities from Streptomyces griseus. J. Antibiot. 2000, 53,
934-943.

38 Alvarez-Bercedo, P.; Murga, J.; Carda, M.; Marco, J. A. Stereoselective Synthesis of the
Published Structure of Feigrisolide A. Structural Revision of Feigrisolides A and B. Org.
Chem. 2006, 71, 5766–5769.

39 Tang, Y.-Q.; Sattler, I.; Thiericke, R.; Grabley, S.; Feng, X.-Z. Feigrisolides A, B, C and D,
New Lactones with Antibacterial Activities from Streptomyces griseus. J. Antibiot. 2000, 53,
934-943.

40 Kim, W. H.; Jung, J. H.; Lee, E. Feigrisolide C: Structural Revision and Synthesis. J. Org.
Chem. 2005, 70, 8190-8192.

41 Graber, M. A.; Gerwick, W. H. Tetrahedron Lett. 1996, 37, 4635–4638.

42 Miyaoka, H.; Hara, Y.; Shinohara, I.; Kurokawa, T.; Yamada, Y. Synthesis and structural
revision of marine eicosanoid agardhilactone. Tetrahedron Lett. 2005, 46, 7945–7949.

43 Snider, B. B.; Shi, B. Synthesis of pyrinodemins A and B. Assignment of the double bond
position of pyrinodemin A. Tetrahedron Lett. 2001, 42, 1639–1642.

44 Ishiyama, H.; Tsuda, M.; Endo, T.; Kobayashi, J. Asymmetric Synthesis of Double Bond
Isomers of the Structure Proposed for Pyrinodemin A and Indication of Its Structural Revision.
Molecules 2005, 10, 312-316.
 49

45 Nakamura, H.; Wu, H.; Ohizumi, Y.; Hirata, Y. Agelasine-A, -B, -C and -D, novel bicyclic
diterpenoids with a 9-methyladeninium unit possessing inhibitory effects on na,K-atpase from
the okinawa sea sponge Agelas sp. Tetrahedron Lett. 1984, 25, 2989-2992.

46 Marcos, I. S.; Garcia, N.; Sexmero, M. J.; Basabe, P.; Diez, D.; Urones, J. G. Synthesis of (+)-
agelasine C. A structural revision. Tetrahedron 2005, 61, 11672-11678.

47 Rao, M. R.; Faulkner, D. J. Clavosolides A and B, Dimeric Macrolides from the Philippines
Sponge Myriastra clavosa. J. Nat. Prod. 2002, 65, 386-388.

48 Son, J. B.; Kim, S. N.; Kim, N. Y.; Lee, D. H. Total Synthesis, Structural Revision, and
Absolute Configuration of (+)-Clavosolide A. Org. Lett. 2006, 8, 661-664.

49 Hiort, J.; Maksimenka, K.; Reichert, M.; Perovi-Ottstadt, S.; Lin, W. H.; Wray, V.; Steube, K.;
Schaumann, K.; Weber, H.; Proksch, P.; Ebel, R.; Mller, W. E. G.; Bringmann, G. New
Natural Products from the Sponge-Derived Fungus Aspergillus niger. J. Nat. Prod. 2004, 67,
1532-1543.

50 Ye, Y. H.; Zhu, H. L.; Song, Y. C.; Liu, J. Y.; Tan, R. X. Structural Revision of Aspernigrin
A, Reisolated from Cladosporium herbarum IFB-E002. J. Nat. Prod. 2005, 68, 1106-1108.

51 Subrahmanyam, C.; Kumar, S. R. A new polyoxygenated steroid from the gorgonian

Gorgonella. J. Chem. Res. (S) 2000, 182.

52 Zhang, W.; Guo, Y. W.; Gavagnin, M.; Mollo, E.; Cimino, G. Suberoretisteroids A-E, five
new uncommon polyoxygenated steroid 24-ketals from the Hainan gorgonian Suberogorgia
reticulata. Helv. Chim. Acta 2005, 88, 87-94.

53 Shimbo, K.; Tsuda, M.; Izui, N.; Kobayashi, J. Amphidinolide W, a New 12-Membered
Macrolide from Dinoflagellate Amphidinium sp. J. Org. Chem. 2002, 67, 1020.

54 Ghosh, A. K.; Gong, G. Total Synthesis and Revision of C6 Stereochemistry of (+)-

Amphidinolide W. J. Org. Chem. 2006, 71, 1085-1093.

55 Patil, A. D.; Freyer, A. J.; Taylor, P. B.; Carte´, B.; Zuber, G.; Johnson, R. K.; Faulkner, D. J.
Batzelladines F−I, Novel Alkaloids from the Sponge Batzella sp.: Inducers of p56lck-CD4
Dissociation. J. Org. Chem. 1997, 62, 1814-1819.

56 Cohen, F.; Overman, L. E. Evolution of a Strategy for the Synthesis of Structurally Complex
Batzelladine Alkaloids. Enantioselective Total Synthesis of the Proposed Structure of
Batzelladine F and Structural Revision. J. Am. Chem. Soc. 2006, 128, 2594-2603.

57 Bourdelais, A. J.; Jacocks, H. M.; Wright, J. L. C.; Bigwarfe, P. M., Jr.; Baden, D. G. A New
Polyether Ladder Compound Produced by the Dinoflagellate Karenia brevis. J. Nat. Prod.
2005, 68, 2-6.
 50

58 Fuwa, H.; Ebine, M.; Bourdelais, A. J.; Baden, D. G.; Sasaki, M. Total Synthesis, Structure
Revision, and Absolute Configuration of (-)-Brevenal. J. Am. Chem. Soc. 2006, 128,
16989-16999.

59 Ahmed, A. A.; Mahmoud, A. A. Jasonol, a rare tricyclic eudesmane sesquiterpene and six
other new sesquiterpenoids from Jasonia candicans. Tetrahedron 1998, 54, 8141-52.

60 Fang, L.; Bi, F.; Zhang, C.; Zheng, G.; Li, Y. Total synthesis of 4a,5a,10b-trihydroxycadinane
and its C4-isomer: structural revision of a natural sesquiterpenoid. Synlett 2006, 16,
2655-2657.

61 Lin, W.; Brauers, G.; Ebel, R.; Wray, V.; Berg, A.; Sudarsono; Proksch, P. Novel Chromone
Derivatives from the Fungus Aspergillus versicolor Isolated from the Marine Sponge
Xestospongia exigua. J. Nat. Prod. 2003, 66, 57–61.

62 Saito, F.; Kuramochi, K.; Nakazaki, A.; Mizushina, Y.; Sugawara, F.; Kobayashi, S. Synthesis
and Absolute Configuration of (+)-Pseudodeflectusin: Structural Revision of Aspergione B.
Eur. J. Org. Chem. 2006 21, 4796–4799.

63 Cutler, H. G.; Jacyno J. M.; Harwood J. S.; Dulik, D.; Goodrich, P. D.; Roberts, R. G.
Botcinolide: A Biologically Active Natural Product from Botrytis cinerea. Biosci. Biotech.
Biochem. 1993, 57, 1980-1982.

64 Tani, H.; Koshino, H.; Sakuno, E.; Cutler, H. G.; Nakajima, H. Botcinins E and F and
Botcinolide from Botrytis cinerea and structural revision of botcinolides. J. Nat. Prod. 2006,
69, 722–725.

65 Cutler, H. G.; Parker, S. R.; Ross, S. A.; Crumley, F. G.; Schreiner, P. R. A Biologically Active
Natural Homolog of Botcinolide from Botrytis cinerea. Biosci. Biotech. Biochem. 1996, 60,
656-658.

66 Collado, I. G.; Aleu, J.; Herna´ndez-Gala´n, R.; Hanson, J. R. Some metabolites of Botrytis
cinerea related to botcinolide. Phytochemistry 1996, 42, 1621-1624.

67 Yamada, T.; Iritani, M.; Minomura, K.; Kawai, K.; Numata, A. Peribysins A–D, potent cell-
adhesion inhibitors from a sea hare-derived culture of Periconia species. Org. Biomol. Chem.
2004, 2, 2131–2135.

68 Koshino, H.; Satoh, H.; Yamada, T.; Esumi, Y. Structural revision of peribysins C and D.
Tetrahedron Lett. 2006, 47, 4623-26.

69 Hiort, J.; Maksimenka, K.; Reichert, M.; Perovic-Ottstadt, S.; Lin, W. H.; Wray, V.; Steube,
K.; Schaumann, K.; Weber, H.; Proksch, P.; Ebel, R.; Mu¨ller, W. E. G.; Bringmann, G. New
Natural Products from the Sponge-Derived Fungus Aspergillus niger. J. Nat. Prod. 2004, 67,
1532-1543.
 51

70 Schlingmann, G.; Taniguchi, T.; He, H.; Bigelis, R.; Yang, H. Y.; Koehn, F. E.; Carter, G. T.;
Berova, N. Reassessing the structure of pyranonigrin. J. Nat. Prod. 2007, 70, 1180-1187.

71 (a) Ravikumar, P. R.; Soman, R.; Chetty, G. L.; Pandey, R. C.; Sukh, D. Chemistry of
Ayurvedic Crude Drugs: Part VI-(Shatavari-1): Structure of Shatavarin-IV. Ind. J. Chem. 1987,
26B, 1012–1017. (b) Joshi, J.; Sukh, D. Chemistry of Ayurvedic Crude Drugs: Part III-
Shatavari-2: Structure Elucidation of Bioactive Shatavarin-I & Other Glycosides. Ind. J.
Chem. 1988, 27B, 12–16.

72 Hayesa, P. Y.; Jahidina, A. H.; Lehmannb, R.; Penmanb, K.; Kitchinga, W.; De Voss, J. J.
Structural revision of shatavarins I and IV, the major components from the roots of Asparagus
racemosus. Tetrahedron Lett. 2006, 47, 6965-6969.

73 Evidente, A.; Conti, L.; Altomare, C.; Bottalico, A.; Sindona, G.; Segre, A. L.; Logrieco, A.
Fusapyrone and deoxyfusapyrone, two antifungal a -pyrones from Fusarium semitectum. Nat.
Toxins 1994, 2, 4–13

74 Hiramatsu, F.; Miyajima, T.; Murayama, T.; Takahashi, K.; Koseki, T.; Shiono, Y. Isolation
and Structure Elucidation of Neofusapyrone from a Marine-derived Fusarium species, and
Structural Revision of Fusapyrone and Deoxyfusapyrone. The Journal of Antibiotics 2006 59,
704–709.

75 Seo, Y.; Cho, K. W.; Rho, J.-R.; Shin, J.; Kwon, B.-M.; Bok, S.-H.; Song, J.-I.
Solandelactones A-I, Lactonized Cyclopropyl Oxylipins Isolated from the Hydroid Solanderia
secunda. Tetrahedron 1996, 52, 10583-96.

76 Pietruszka, J.; Rieche, A. C. M. Total synthesis of marine oxylipins solandelactones A-H.

Adv. Synth. Catal. 2008, 350, 1407-1412.

77 Delazar, A.; Byres, M.; Gibbons, S.; Kumarasamy, Y.; Modarresi, M.; Nahar, L.; Shoeb, M.;
Sarker, S. D. Iridoid Glycosides from Eremostachys glabra. J. Nat. Prod. 2004, 67,
1584-1587.

78 Jensen, S. R.; Çalış, I.; Gotfredsen, C. H.; Søtofte, I. Structural Revision of Some Recently
Published Iridoid Glucosides. J. Nat. Prod. 2007, 70, 29–32.

79 Ahmed, B.; Al-Rehaily, A. J.; Al-Howiriny, T. A.; El-Sayed, K. A.; Ahmad, M. S.

Scropolioside-D_2 and Harpagoside-B: Two New Iridoid Glycosides from Scrophularia
deserti and Their Antidiabetic and Antiinflammatory Activity. Biol. Pharm. Bull. 2003, 26,
462-467.

80 Ahmad, I.; Afza, N.; Anis, I.; Malik, A.; Fatima, I.; ul-Haq, A.; Tareen, R. B. Iridoid
glycosides and a benzofuran type sesquiterpene from Buddleja crispa. Heterocycles 2004, 63,
1875-1881.

81 Itokawa, H.; Nakajima, H.; Ikuta, A.; Iitaka, Y. Two triterpenes from the flowers of Camellia
japonica. Phytochemistry. 1981, 20, 2539-2542.
 52

82 Yoshikawa, M.; Morikawa, T.; Asao, Y.; Fujiwara, E.; Nakamura, S.; Matsuda, H. Medicinal
flowers. XV. The structures of noroleanane- and oleanane-type triterpene oligoglycosides with
gastroprotective and platelet aggregation activities from flower buds of Camellia japonica.
Chem. Pharmaceut. Bull. 2007, 55, 606-612.

83 Kinnel, R. B.; Gehrken, H.-P.; Scheuer, P. J. Palau'amine: a cytotoxic and immunosuppressive

hexacyclic bisguanidine antibiotic from the sponge Stylotella agminata. J. Am. Chem. Soc.
1993, 115, 3376 – 3377.

84 (a) Koeck, M.; Grube, A.; Seiple, I. B.; Baran, P. S. The pursuit of Palau'amine. Angew.
Chem. Int. Ed. 2007, 46, 6586-6594. (b) Grube, A.; Köck, M. Structural Assignment of
Tetrabromostyloguanidine: Does the Relative Configuration of the Palau'amines Need
Revision? Angew. Chem. Int. Ed. 2007, 46, 2320 – 2324.

85 Wall, M. E.; Wani, M. C.; Manikumar, H. T.; Taylor, H.; McGivney, R. Plant Antimutagens, 6.
Intricatin and Intricatinol, New Antimutagenic Homoisoflavonoids from Hoffmanosseggia
intricata. J. Nat. Prod. 1989, 52, 774-778.

86 Peter, A.; Amenechi, P. I. Isoflavonoid and pterocarpinoid extractives of Lonchocarpus

laxiflorus. J. Chem. Soc. C 1969, 887-896.

87 Siddaiah, V.; Maheswara, M.; Rao, C. V.; Venkateswarlub, S.; Subbarajub, G. V. Synthesis,
structural revision, and antioxidant activities of antimutagenic homoisoflavonoids from
Hoffmanosseggia intricata. Bioorg. Med. Chem. Lett. 2007, 17, 1288–1290.

88 (a) Leet, J. E.; Schroeder, D. R.; Hofstead, S. J.; Golik, J.; Colson, K. L.; Huang, S.; Klohr, S.
E.; Doyle, T. W.; Matson, J. A. Kedarcidin, a new chromoprotein antitumor antibiotic:
structure elucidation of kedarcidin chromophore. J. Am. Chem. Soc. 1992, 114, 7946-7948. (b)
Leet, J. E.; Schroeder, D. R.; Langley, D. R.; Colson, K. L.; Huang, S.; Klohr, S. E.; Lee, M.
S.; Golik, J.; Hofstead, S.J.; Doyle, T. W.; Matson, J. A. Chemistry and structure elucidation of
the kedarcidin chromophore. J. Am. Chem. Soc. 1993, 115, 8432-8443.

89 Ren, F.; Hogan, P. C.; Anderson, A. J.; Myers, A. G. Kedarcidin chromophore: Synthesis of
Its Proposed Structure and Evidence for a Stereochemical Revision. J. Am. Chem. Soc. 2007,
129, 5381–5383.

90 Kawata, S.; Ashizawa, S.; Hirama, M. Synthetic Study of Kedarcidin Chromophore: Revised
Structure. J. Am. Chem. Soc. 1997, 119, 12012–12013.

91 McPhail, K. L.; Gerwick, W. H. Three New Malyngamides from a Papua New Guinea
Collection of the Marine Cyanobacterium Lyngbya majuscula. J. Nat. Prod. 2003, 66, 132.

92 Li, Y.; Feng, J.-P.; Wang, W.-H.; Chen, J.; Cao, X.-P. Total Synthesis and Correct Absolute
Configuration of Malyngamide U. J. Org. Chem. 2007, 72, 2344-2350.

93 Khaliq, S.; Volk, F.-J.; Frahm, A. W. Phytochemical Investigation of Perovskia abrotanoides.

Planta Med. 2007, 73, 77-83.
 53

94 Simmons, E. M.; Yen, J. R.; Sarpong, R. Reconciling Icetexane Biosynthetic Connections

with Their Chemical Synthesis: Total Synthesis of (±)-5,6-Dihydro-6a-hydroxysalviasperanol,
(±)-Brussonol, and (±)-Abrotanone. Org. Lett. 2007, 9, 2705-2708.

95 Tanaka, N.; Okasaka, M.; Ishimaru, Y.; Takaishi, Y.; Sato, M.; Okamoto, M.; Oshikawa, T.;
Ahmed, S. U.; Consentino, L. M.; Lee, K.-H. Biyouyanagin A, an Anti-HIV Agent from
Hypericum chinense L. var. salicifolium. Org. Lett. 2005, 7, 2997-2999.

96 Nicolaou, K. C.; Sarlah, D.; Shaw, D. M. Total synthesis and revised structure of
biyouyanagin A. Angew. Chem. Int. Ed. 2007, 46, 4708-4711.

97 Nii, H.; Furukawa, K.; Iwakiri, M.; Kubota, T. Nippon Nogeikagaku Kaishi 1983, 57, 733–
741; Chem. Abstr. 1983, 99, 200329.

98 Blay, G.; Garcia, B.; Molina, E.; Pedro, J. R. Synthesis of (+)-pechueloic acid and (+)-
aciphyllene. Revision of the structure of (+)-aciphyllene. Tetrahedron 2007, 63, 9621-9626.

99 Diyabalanage, T.; Amsler, C. D.; McClintock, J. B.; Baker, B. J. Palmerolide A, a Cytotoxic

Macrolide from the Antarctic Tunicate Synoicum adareanum. J. Am. Chem. Soc. 2006, 128,
5630 – 5631.

100(a) Nicolaou, K. C.; Guduru, R.; Sun, Y.-P.; Banerji, B.; Chen, D. Y.-K. Total synthesis of the
originally proposed and revised structures of palmerolide A. Angew. Chem. Int. Ed. 2007, 46,
5896-5900. (b) Nicolaou, K. C.; Sun, Y.-P.; Guduru, R.; Banerji, B.; Chen, D. Y.-K. Total
Synthesis of the Originally Proposed and Revised Structures of Palmerolide A and Isomers
Thereof. J. Am. Chem. Soc. 2008, 130, 3633-3644.

101 Solis, P. N.; Lang’at, C.; Gupta, M. P.; Kirby, G. C.; Warhurst, D. C.; Phillipson, J. D. Bio-
active Compounds from Psychotria camponutans. Planta Med. 1995, 61, 62-65.

102 Jacobs, J.; Claessens, S.; De Kimpe, N. First straightforward synthesis of 1-hydroxy-3,4-
dihydro-1H-benz[g]isochromene-5,10-dione and structure revision of a bioactive
benz[g]isochromene-5,10-dione from Psychotria camponutans. Tetrahedron 2008, 64,
412-418.

103 Sun, J.; Shi, D.; Ma, M.; Li, S.; Wang, S.; Han, L.; Yang, Y.; Fan, X.; Shi, J.; He, L.
Sesquiterpenes from the Red Alga Laurencia tristicha. J. Nat. Prod. 2005, 68, 915-919.

104 Chen, P.; Wang, J.; Liu, K.; Li, C. Synthesis and Structural Revision of (±)-Laurentristich-4-
ol. J. Org. Chem. 2008, 73, 339-341.

105 Hastings, J. S.; Heller, H. G. The stereochemistry of aurones [2-substituted

benzylidenebenzofuran-3(2H)-ones] J. Chem. Soc., Perkin Trans. 1972, 1, 2128-2132.

106 Harkat, H.; Blanc, A.; Weibel, J.-M.; Pale, P. Versatile and Expeditious Synthesis of Aurones
via AuI-Catalyzed Cyclization. J. Org. Chem. 2008, 73, 1620-1623.
 54

107 Gunatilaka, A. A. L. In: Herz, W.;Kirby, G. W.; Moore, R. E.; Steglich, W.; Tamm,C.
Progress in the Chemistry of Organic Natural Products vol.67, Springer-Verlag/Wien, New
York 1996, pp. 1–123.

108 Jacobsen, N. E.; Wijeratne, E. M. K.; Corsino, J.; Furlan, M.; Bolzani, V. S.; Gunatilaka, A.
A. L. Biomimetic synthesis of xuxuarines Ea and Eb: Structure revision of Rzedowskia
bistriterpenoids. Bioorg. Med. Chem. 2008, 16, 1884-1889.

109 Bastida, J.; Codina, C.; Peeters, P.; Rubiralta, M.; Orozco, M.; Luque, F. J.; Chharbra, S. C.
Alkaloids from Crinum kirkii. Phytochemistry 1995, 40, 1291-1293.

110 Biechy, A.; Hachisu, S.; Quiclet-Sire, B.; Ricard, L.; Zard, S. Z. The total synthesis of (±)-
fortucine and a revision of the structure of kirkine. Angew. Chem. Int. Ed. 2008, 47,
1436-1438.

111 Sakai, K.; Fukuda, Y.; Matsunaga, S.; Tanaka, R.; Yamort, T. New Cytotoxic Oleanane-Type
Triterpenoids from the Cones of Liquidamber styraciflua. J. Nat. Prod. 2004, 67, 1088-1093.

112 Sun, H.; Fang, W.-S.; Hu, C. An efficient semi-synthesis and structure revision of a cytotoxic
triterpenoid 25-acetoxy-3a-hydroxyolean-12-en-28-oic acid from Liquidamber styraciflua. J.
Asian Nat. Prod. Res. 2008, 10, 271-276.

113 Zampella, A.; D'Auria, M.V.; Minale, L.; Debitus, C.; Roussakis, C. Callipeltoside A: A
Cytotoxic Aminodeoxy Sugar-Containing Macrolide of a New Type from the Marine
Lithistida Sponge Callipelta sp. J. Am. Chem. Soc. 1996, 118, 11085-11088.

114 Carpenter, J.; Northrup, A. B.; Chung, D.; Wiener, J. J. M.; Kim, S.-G.; MacMillan, D. W. C.
Total synthesis and structural revision of callipeltoside C. Angew. Chem. Int. Ed. 2008, 47,
3568-3572.

115 Kuo, Y.-H.; Lin, B.-Y. A New Dinorxanthane and Chromone from the Root of Tithonia
diversifolia. Chem. Pharm. Bull. 1999, 47, 428-429.

116 Matsuo, K.; Yokoe, H.; Shishido, K.; Shindo, M. Synthesis of diversifolide and structure
revision. Tetrahedron Lett. 2008, 49, 4279-4281.

117 Akiyama, K.; Kikuzaki, H.; Aoki, T.; Okuda, A.; Lajis, N. H.; Nakatani, N. Terpenoids and a
Diarylheptanoid from Zingiber ottensii. J. Nat. Prod. 2006, 69, 1637-1640.

118 Boukouvalas, J.; Wang, J.-X. Structure Revision and Synthesis of a Novel Labdane
Diterpenoid from Zingiber ottensii. Org. Lett. 2008, 10, 3397-3399.

119 Sorek, H.; Rudi, A.; Gueta, S.; Reyes, F.; Martin, M. J.; Aknin, M.; Gaydou, E.; Vacelet, J.;
Kashman, Y. Netamines A–G: seven new tricyclic guanidine alkaloids from the marine sponge
Biemna laboutei. Tetrahedron 2006, 62, 8838–8843.
 55

120 Yu, M.; Pochapsky, S. S.; Snider, B. B. Synthesis of 7-Epineoptilocaulin, Mirabilin B, and
Isoptilocaulin. A Unified Biosynthetic Proposal for the Ptilocaulin and Batzelladine Alkaloids.
Synthesis and Structure Revision of Netamines E and G. J. Org. Chem. 2008, 73, 9065-9074.

121 Hayakawa, Y.; Yamashita, T.; Mori, T.; Nagai, K.; Shin-ya, K.; Watanabe, H. J. Structure of
Tyroscherin, an Antitumor Antibiotic against IGF-1-dependent Cells from Pseudallescheria
sp. Antibiot. 2004, 57, 634–638.

122 Katsuta, R.; Shibata, C.; Ishigami, K.; Watanabe, H.; Kitahara, T. Synthesis and structure
revision of tyroscherin, a growth inhibitor of IGF-1-dependent tumor cells. Tetrahedron Lett.
2008, 49, 7042-7045.

123 Murata, M.; Matsuoka, S.; Matsumori, N.; Paul, G. K.; Tachibana, K. Absolute Configuration
of Amphidinol 3, the First Complete Structure Determination from Amphidinol Homologues:
Application of a New Configuration Analysis Based on Carbon−Hydrogen Spin-Coupling
Constants. J. Am. Chem. Soc. 1999, 121, 870–871.

124 Oishi, T.; Kanemoto, M.; Swasono, R.; Matsumori, N.; Murata, M. Synthesis of the 1,5-
Polyol System Based on Cross Metathesis: Structure Revision of Amphidinol 3. Org. Lett.
2008, 10, 5203-5206.

125 Mueller, D.; Krick, A.; Kehraus, S.; Mehner, C.; Hart, M.; Kuepper, F. C.; Saxena, K.; Prinz,
H.; Schwalbe, H.; Janning, P.; Waldmann, H.; Koenig, G. M. Brunsvicamides A-C: sponge-
related cyanobacterial peptides with Mycobacterium tuberculosis protein tyrosine phosphatase
inhibitory activity. J. Med. Chem. 2006, 49, 4871-4878.

126 Walther, T.; Arndt, H.-D.; Waldmann, H. Solid-support based total synthesis and
stereochemical correction of brunsvicamide A. Org. Lett. 2008, 10, 3199-3202.

127 Ralifo, P.; Crews, P. J. A New Structural Theme in the Imidazole-Containing Alkaloids from
a Calcareous Leucetta Sponge. Org. Chem. 2004, 69, 9025–9029.

128 White, K. N.; Amagata, T.; Oliver, A. G.; Tenney, K.; Wenzel, P. J.; Crew, P. Structure
Revision of Spiroleucettadine, a Sponge Alkaloid with a Bicyclic Core Meager in H-Atoms J.
Org. Chem. 2008, 73, 8719–8722.

129 Guella, G.; Mancini, I.; Öztunç, A.; Pietra, F. Conformational Bias in Macrocyclic Ethers and
Observation of High Solvolytic Reactivity at a Masked Furfuryl (=2-Furylmethyl) C-Atom.
Helv. Chim. Acta 2000, 83, 336–348.

130 Braddock, D. C.; Rzepa, H. S. Structural Reassignment of Obtusallenes V, VI, and VII by
GIAO-Based Density Functional Prediction. J. Nat. Prod. 2008, 71, 728-730.

131 Rahbæk, L.; Breinholt, J.; Frisvad, J. C.; Christophersen, C. Circumdatin A, B, and C: Three
New Benzodiazepine Alkaloids Isolated from a Culture of the Fungus Aspergillus ochraceus.
J. Org. Chem. 1999, 64, 1689-1692.
 56

132 Ookura, R.; Kito, K.; Ooi, T.; Namikoshi, M.; Kusumi, T. Structure Revision of Circumdatins
A and B, Benzodiazepine Alkaloids Produced by Marine Fungus Aspergillus ostianus, by X-
ray Crystallography. J. Org. Chem. 2008, 73, 4245-4247.

133 Cafieri, F.; De Napoli, L.; Fattorusso, E.; Santacroce, C. Diterpenes from the red alga
Sphaerococcus coronopifolius. Phytochemistry 1987, 26, 471–473.

134 Smyrniotopoulos, V.; Quesada, A.; Vagias, C.; Moreau, D.; Roussakis, C.; Roussis, V.
Cytotoxic bromoditerpenes from the red alga Sphaerococcus coronopifolius. Tetrahedron
2008, 64, 5184-5190.

135 Takahashi, T.; Eto, H.; Ichimura, T.; Murae, T. Hiyodorilactones A and B, new tumor
inhibitory germacradienolides from Eupatorium sachalinense makino. Chem. Lett. 1978,
1345-1348.

136 Tori, M.; Morishita, N.; Hirota, N.; Saito, Y.; Nakashima, K.; Sono, M.; Tanaka, M.;
Utagawa, A.; Hirota, H. Sesquiterpenoids isolated from Eupatorium glehnii. Isolation of
guaiaglehnin A, structure revision of hiyodorilactone B, and genetic comparison. Chem.
Pharmaceut. Bull. 2008, 56, 677-681.

137 Sasse, F.; Steinmetz, H.; Höfle, G.; Reichenbach, H. Rhizopodin, a new compound from
Myxococcus stipitatus (myxobacteria) causes formation of rhizopodia-like structures in animal
cell cultures. J. Antibiot. 1993, 46, 741–748.

138 Jansen, R.; Steinmetz, H.; Sasse, F.; Schubert, W.-D.; Hagelueken, G.; Albrecht, S. C.;
Mueller, R. Isolation and structure revision of the actin-binding macrolide rhizopodin from
Myxococcus stipitatus (Myxobacteria). Tetrahedron Lett. 2008, 49, 5796-5799.

139 Ziemert, N.; Ishida, K.; Quillardet, P.; Bouchier, C.; Hertweck, C.; Tandeau de Marsac, N.;
Dittmann, E. Microcyclamide Biosynthesis in Two Strains of Microcystis aeruginosa: from
Structure to Genes and Vice Versa. Appl. EnViron. Microbiol. 2008, 74, 1791-1797.

140 Portmann, C.; Blom, J. F.; Kaiser, M.; Brun, R.; Juttner, F.; Gademann, K. Isolation of
aerucyclamides C and D and structure revision of microcyclamide 7806A: Heterocyclic
ribosomal peptides from Microcystis aeruginosa PCC 7806 and their antiparasite evaluation.
J. Nat. Prod. 2008, 71, 1891-1896.

141 Luis, J. G.; Lahlou, E. H.; Andre´s, L. S. Hassananes: C23 terpenoids with a new type of
skeleton from Salvia apiana Jeps. Tetrahedron 1996, 52, 12309-12312.

142 Yang, J.; Huang, S.-X.; Zhao, Q.-S. Structure Revision of Hassanane with Use of Quantum
Mechanical 13C NMR Chemical Shifts and UV-Vis Absorption Spectra. J. Phys. Chem. 2008,
112, 12132-12139.

143 Takada, N.; Watanabe, R.; Suenaga, K.; Yamada, K.; Ueda, K.; Kita, M. Uemura,
Zamamistatin, a significant antibacterial bromotyrosine derivative, from the Okinawan sponge
Pseudoceratina purpurea. Tetrahedron Lett. 2001, 42, 5265-5267.
 57

144 Hayakawa, I.; Teruya, T.; Kigoshi, H. Revised structure of zamamistatin. Tetrahedron Lett.
2006, 47, 155-158.

145 Kita, M.; Tsunematsu, Y.; Hayakawa, I.; Kigoshi, H. Structure of zamamistatin - a correction.
Tetrahedron Lett. 2008, 49, 5383-5384.

146 Fitch, R. W.; Garraffo, H. M.; Spande, T. F.; Yeh, H. J. C.; Daly, J. W. Bioassay-guided
isolation of epiquinamide, a novel quinolizidine alkaloid and nicotinic agonist from an
Ecuadoran poison frog, Epipedobates tricolor. J. Nat. Prod. 2003, 66, 1345-1350.

147 (a) Wijdeven, M. A.; Botman, P. N. M.; Wijtmans, R.; Schoemaker, H. E.; Rutjes, F. P. J. T.;
Blaauw, R. H. Total Synthesis of (+)- Epiquinamide. Org. Lett. 2005, 7, 4005-4007. (b)
Suyama, T. L.; Gerwick, W. H. Practical Total Syntheses of Epiquinamide Enantiomers. Org.
Lett. 2006, 8, 4541-4543. (c) Wijdeven, M. A.; Wijtmans, R.; van den Berg, R. J. F.; Noorduin,
W.; Schoemaker, H. E.; Sonke, T.; van Delft, F. L.; Blaauw, R. H.; Fitch, R. W.; Spande, T. F.;
Daly, J. W.; Rutjes, F. P. J. T. N,N-Acetals as N-Acyliminium Ion Precursors: Synthesis and
Absolute Stereochemistry of Epiquinamide. Org. Lett. 2008, 10, 4001-4003. (d) Ghosh, S.;
Shashidhar, J. Total synthesis of (+)- epiquinamide from -mannitol. Tetrahedron Lett. 2009,
50, 1177-1179. (e) Huang, P.-Q.; Guo, Z.-Q.; Ruan, Y.-P. A Versatile Approach for the
Asymmetric Syntheses of (1R,9aR)- Epiquinamide and (1R,9aR)-Homopumiliotoxin 223G.
Org. Lett. 2006, 8, 1435-1438. (f) Voituriez, A.; Ferreira, F.; Perez-Luna, A.; Chemla, F.
Asymmetric synthesis of (-)-1-hydroxyquinolizidinone, a common intermediate for the
syntheses of (-)-homopumiliotoxin 223G and (-)- epiquinamide. Org. Lett. 2007, 9,
4705-4708. (g) Tong, S.; Barker, D. A concise synthesis of (±) and a total synthesis of (+)-
epiquinamide. Tetrahedron Lett. 2006, 47, 5017-5020. (h) Kanakubo, A.; Gray, D.; Innocent,
N.; Wonnacott, S.; Gallagher, T. The synthesis and nicotinic binding activity of (± )-
epiquinamide and (± )-C(1)-epiepiquinamide. Bioorg. Med. Chem. Lett. 2006, 16, 4648-4651.

148 Fitch, R. W.; Sturgeon, G. D.; Patel, S. R.; Spande, T. F.; Garraffo, H. M.; Daly, J. W.;
Blaauw, R. H. Epiquinamide : A Poison That Wasn't from a Frog That Was. J. Nat. Prod. 2009,
72, 243-247.

149 Newman, D. J.; Cragg, G. M. Natural products as sources of new drugs over the last 25
years. J. Nat. Prod. 2007, 70, 461-477.

150 Füllbeck, M.; Michalsky, E.; Dunkel, M.; Preissner, R. Natural products: sources and
databases. Nat. Prod. Rep. 2006, 23, 347-356.

151 Hill, R. A. Annu. Rep. Prog. Chem. Sect. B, 2003, 99, 183-207.

152 Ghaneh, P.; Costello, E. J P Neoptolemos Biology and management of pancreatic cancer. J.
Postgrad. Med. 2008, 84, 478-497.

153 Heron, M. Deaths: Leading Causes for 2004 National Vital Statistics Reports 2007, 56, 1-96.

154 Sarker, S. D.; Latif, Z.; Gray, A. I. Natural Products Isolation, 2006, Humana Press Inc.
 58

155 Daly, J. W.; Spande, T. F.; Garraffo, H. M. Alkaloids from Amphibian Skin: A Tabulation of
Over Eight-Hundred Compounds. J. Nat. Prod. 2005, 68, 1556-1575.

156 Moores Cancer Center Retreat, 2007, San Diego, California.

157 Wender. P. A. Introduction: Frontiers in Organic Synthesis. Chem. Rev. 1996, 96, 1-2.

158 Mickel, S. J.; Sedelmeier, G. H.; Niederer, D.; Daeffler, R.; Osmani, A.; Schreiner, K.;
Seeger-Weibel, M.; Bérod, B.; Schaer, K.; Chen, R. G.; Chen, W.; Jagoe, C. T.; Kinder, F. R.,
Jr.; Loo, M.; Prasad, K.; Repi, O.; Shieh, W.; Wang, R.; Waykole, L.; Xu, D. D.; Xue, S.
Large-Scale Synthesis of the Anti-Cancer Marine Natural Product (+)-Discodermolide. Part 1:
Synthetic Strategy and Preparation of a Common Precursor. Org. Process Res. Dev. 2004, 8,
92-100.

159 Smith, A.B.; Beauchamp, T. J.; LaMarche, M. J.; Kaufman, M. D.; Qiu, Y.; Arimoto, H.;
Jones, D. R.; Kobayashi, K. Evolution of a Gram-Scale Synthesis of (+)-Discodermolide. J.
Am. Chem. Soc. 2000, 122, 8654-8664.

160 Patterson, I.; Florence, G. J.; Gerlach, K.; Scott, J. P.; Sereinig, N. A Practical Synthesis of
(+)-Discodermolide and Analogues: Fragment Union by Complex Aldol Reactions. J. Am.
Chem. Soc. 2001, 123, 9535-9544.

161 Freemantle, M. Scaled-up synthesis of discodermolide. Science & Technology 2004, 82,
33-35.

162 Nogle, L. M.; Gerwick, W. H. Somocystinamide A, a Novel Cytotoxic Disulfide Dimer from
a Fijian Marine Cyanobacterial Mixed Assemblage. Org. Lett. 2002, 4, 1095-1098.

163 Wrasidlo, W.; Mielgo, A.; Torres, V. A.; Barbero, S.; Stoletov, K.; Suyama, T. L.; Klemke, R.
L.; Gerwick, W. H.; Carson, D. A.; Stupack, D. G. The marine lipopeptide somocystinamide A
triggers apoptosis via caspase 8. Proc. Natl. Acad. Sci. 2008, 105, 2313-2318.

164 Williams, R. M.; Cox, R. J. Paraherquamides, brevianamides, and asperparalines: laboratory

synthesis and biosynthesis. an interim report. Acc. Chem. Res. 2003, 36, 127-139.

165 Williams, R. M. Total synthesis and biosynthesis of the paraherquamides: an intriguing story
of the biological Diels-Alder construction. Chem. Pharm. Bull. 2002, 50, 711-740.

166 Sanz-Cervera, J. F.; Glinka, T.; Williams, R. M. Biosynthesis of brevianamides A and B: in

search of the biosynthetic diels-alder construction. Tetrahedron, 1993, 49, 8471-8482.

167 Williams, R. M.; Kwast, E. Carbanion-mediated Oxidative Deprotection of Non-enolizable

Benzylated Amides. Tetrahedron Lett. 1989, 30, 451-454.

168 For example, see Paul G. Bulger,a Sharan K. Bagalb and Rodolfo Marquez, Recent advances
in biomimetic natural product synthesis, Nat. Prod. Rep., 2008, 25, 254–297 .
 59

169 Miyake, F. Y.; Yakushijin, K.; Horne, D. A. Biomimetic synthesis of grossularines-1. Angew.
Chem. Int. Ed. 2005, 44, 3280 –3282 .

170 Lei, X.; Johnson, R. P.; Porco Jr., J. A. Total Synthesis of the Ubiquitin-Activating Enzyme
Inhibitor (+)-Panepophenanthrin. Angew. Chem. Int. Ed. 2003, 42, 3913-3917.

171 Moses, J. E.; Commeiras, L.; Baldwin, J. E.; Adlington, R. M. Total Synthesis of
Panepophenanthrin. Org. Lett. 2003, 5, 2987–2988.

172 Proteau, P. J.; Gerwick, W. H.; Garcia-Pichel, F.; Castenholz, R. The structure of scytonemin,
an ultraviolet sunscreen pigment from the sheaths of cyanobacteria . Experientia 1993, 49,
825-829.

173 See Chapter VII of this dissertation

 Chapter II

Ichthyotoxic brominated diphenyl ethers from a mixed assemblage of a red alga and

cyanobacterium: structure clarification and biological properties

60
 61

Abstract

Primary fractions from the extract of a tropical red alga mixed with filamentous

cyanobacteria, collected from Papua New Guinea, were active in a neurotoxicity assay.

Bioassay guided isolation led to two natural products (1, 2) with relatively potent calcium

ion influx properties. The more prevalent of the neurotoxic compounds (1) was

characterized by extensive NMR, mass spectrometry, and X-ray crystallography, and

shown to be identical to a polybrominated diphenyl ether metabolite present in the

literature, but reported with different NMR properties. To clarify this anomalous result,

we synthesized a candidate isomeric polybrominated diphenyl ether (3), but this clearly

had different NMR shifts than the reported compound. We conclude that the original

isolate of 3,4,5-tribromo-2-(2,4-dibromophenoxy)phenol was contaminated with a minor

compound, giving rise to the observed anomalous NMR shifts. The second and more

minor natural product (2) isolated in this study was a more highly brominated species.

All three compounds showed a low micromolar ability to increase intracellular calcium

ion concentrations in mouse neocortical neurons as well as toxicity to zebrafish. Because

polybrominated diphenyl ethers have both natural as well as anthropomorphic origins,

and accumulate in marine organisms at higher trophic level (mammals, fish, birds), these

neurotoxic properties are of environmental significance and concern.

 62

II.1 Introduction

Polybrominated diphenyl ethers (PBDEs) have recently attracted considerable

attention due to an increasing awareness that these compounds accumulate in higher

trophic level animals, including sperm whales,1,2 sea gulls,3 seals,2 polar bears,4 as well as

in humans.5,6 Some PBDEs are industrially produced in large quantities for use as flame

retardants, and therefore their presence in animals has generally been assumed to be of

anthropogenic origin.7 Hydroxylated congeners of PBDEs (OH-PBDEs) have also been

found in many of these same higher animals,3,4,8 and recent studies have shown that the

OH-PBDEs found in large marine-associated animals may be of mixed origins with some

deriving from natural sources and the others being derivatives of anthropogenic PBDEs.

Moreover, a variety of anthropogenic PBDEs have been shown to undergo hydroxylation

in rats via oxidative metabolism, thereby producing OH-PBDE congeners.9,10

Nevertheless, a number of OH-PBDEs are known to be natural products of various

marine organisms, such as sponges, tunicates, and cyanobacteria.11,12,13 Environmental

concerns have been raised over the occurrence of PBDEs in higher animals because

PBDEs have been linked to endocrine disruption.2,14 Activities similar to those of

chlorinated aromatic compounds have been observed with PBDEs, such as aryl

hydrocarbon-receptor agonist and antagonist activities, thyroid toxicity, and effects on the

immune system.15 Neonatal exposure to PBDEs has been shown to cause neurotoxicity in

adult animals.14 OH-PBDEs have also been shown to disrupt thyroid hormone

homeostasis presumably due to their structural similarity to the thyroxin-type endogenous

thyroid hormones.4 Recently, BDE-47 and 6-OH-BDE-47 have been shown to trigger an

increase in cytosolic Ca2+ concentration as well as exocytosis of catecholamines in

 63

neuronal cells within a few minutes.16,17 These findings indicate that PBDEs and OH-

PBDEs have the potential to acutely disrupt normal neuronal communication in animals.

Figure II.1: OH-PBDE congeners isolated from the red alga/cyanobacteria

assembly (1 and 2) and an alternative regioisomer of 1 (3).

Our program has been systematically screening marine algae and cyanobacteria

for secondary metabolites with neuropharmacological properties, partially because of the

environmental impacts these substances may impose, and partially to detect agents with

potentially useful biomedical properties.18,19,20,21 Because a variety of cellular events can

trigger an increase in cytosolic Ca2+ levels in neuronal cells, we have employed a FLIPR-

based fluorescence assay which detects Ca2+ modulation in mouse neocortical neurons.

Herein, we describe the assay-guided isolation, structural determination and

neurotoxicological evaluation of two OH-PBDE congeners (1 and 2, Figure II.1) from a

mixed assemblage of marine cyanobacteria and a red alga. In the course of this work, we

have also corrected the assignment of a 13C NMR signal for 1 in the literature which

could be a source of confusion in future efforts with OH-PBDE congeners.

II.2 Materials and Methods

II.2.1 Reagents and algal material

 64

All solvents used were of HPLC grade from EMD and were used without

purification. TLC grade silica gel from Sigma-Aldrich was used for vacuum liquid

chromatography (VLC). Flash chromatography was performed using EMD silica gel

(230-400 mesh). Trypsin, penicillin, streptomycin, heat-inactivated fetal bovine serum,

horse serum and soybean trypsin inhibitor were obtained from Atlanta Biologicals.

Minimum essential medium, deoxyribonuclease (DNase), poly-L-lysine, cytosine

arabinoside, veratridine, aconitine, and deltamethrin were from Sigma-Aldrich. The

fluorescent dye Fluo-3, and pluronic acid F-127 were obtained from Invitrogen

Corporation.

Algal material was collected at Grabo reef in Papua New Guinea at 12-18 m depth

by SCUBA. The algae was identified in the field as Vidalia sp., but subsequent

microscopic examination of the voucher sample identified it to likely be Leptofaucia sp.

Additionally, a small abundance of cyanobacterial filaments similar to Oscillatoria were

observed in the sample by light microscopy.

II.2.2 Bioassay-guided isolation of OH-PBDEs

A total of 2 L of the red algal and cyanobacterial assemblage (dry weight 128 g)

was extracted with CH2Cl2 / MeOH (2:1). Upon removal of the solvents in vacuo, a

portion (4.0 g) of the crude extract (4.6 g) was fractionated into nine fractions using silica

gel vacuum liquid chromatography (hexanes to EtOAc to MeOH), one of which (308 mg,

eluted with 2:3 EtOAc/hexanes) possessed Ca2+ modulating activity in mouse neocortical

neurons. A portion (306 mg) of the active fraction was further fractionated by flash

column chromatography22 on silica gel (hexanes/Et2O 1:9 to 1:8) to obtain seven sub-
 65

fractions, of which two (2 mg and 77 mg) were associated with Ca 2+ modulation activity.

The larger sub-fraction was further purified by HPLC using a Jupiter 10μ C18 300A

column (250 x 10 mm) with a gradient solvent system (2.5 mL/min, 3:2 MeCN/H 2O to

4:1 over 30 min, then to MeCN over 10 min) to obtain 15 mg (0.38% of extract) of

compound 1 (Rt = 52 min) and ~0.5 mg (0.01% of extract) of compound 2 (Rt = 55 min),

both of which were confirmed to have Ca2+ modulating activity.

II.2.3 Neocortical neuron cultures

Primary cultures of neocortical neurons were obtained from embryonic day 16

Swiss-Webster mice. Briefly, pregnant mice were euthanized by CO2 asphyxiation and

their neocortices were collected. Isolated neocortices were then removed of their

meninges, minced by trituration using a Pasteur pipette, and treated with trypsin for 25

minutes at 37ºC. The cells were then dissociated by two successive trituration and

sedimentation steps in soybean trypsin inhibitor and DNase containing isolation buffer,

centrifuged and resuspended in Eagle’s minimal essential medium (MEM) supplemented

with 2 mM L-glutamine, 10% fetal bovine serum, 10% horse serum, 100 IU/mL

penicillin and 0.10 mg/mL streptomycin, pH 7.4. Cells were plated onto poly-L-lysine-

coated 96-well clear-bottomed black-well plate at a density of 1.5 x 105 cells/well. Plates

were incubated at 37ºC with 5% CO2 and 95% humidity. To prevent proliferation of

nonneuronal cells, cytosine arabinoside (10 μM) was added to the culture medium on day

2 after plating. The culture media was changed both on days 5, 8 and 11 using a serum-

free growth medium containing Neurobasal Medium supplemented with B-27, 100 I.U./

mL penicillin, 0.10 mg/mL streptomycin, and 0.2 mM L-glutamine. Neocortical neurons

 66

were used between 11-13 days in vitro (DIV) for Ca2+ influx assay. All animal use

protocols were approved by the Institutional Animal Care and Use Committee (IACUC).

II.2.4 Ca2+ modulation assay

Neocortical neurons cultured in 96-well plate were removed their medium and

replaced with dye loading buffer (100 μL/well) containing 4 μM Fluo-3 and 0.04%

Pluronic F-127 in Lock’s buffer (in mM: 8.6 Hepes, 5.6 KCl, 154 NaCl, 5.6 Glucose, 1.0

MgCl2, 2.3 CaCl2, 0.0001 glycine, pH 7.4). After 1 h incubation in dye loading buffer,

cells were washed five times with Locke’s buffer using automated cell washer (Bioteck

instrument Inc., VT, USA) leaving a final volume of 150 μL in each well. The plate was

then transferred to Fluorescence Laser Plate Reader (FLIPR) (Molecular Devices,

Sunnyvale, CA, USA) chamber. Cells were excited by the 488-nm line of argon laser and

Ca2+ bound fluo-3 emission in the 500–600 nm range was recorded with the charge

coupled device (CCD) camera. Fluorescence readings were taken once every 9 s for 3

min to establish the baseline and then 50 µL of test compound containing solution (4x)

was added to each well from the compound plate, yielding a final volume of 200 μL/well.

The cells were exposed to the testing compounds for another 42 min.

II.2.5 Assay data analysis

Concentration-response relationships were generated by non-linear regression

analysis using GraphPad software (Version 4.0; San Diego, CA).

II.2.6 Structure elucidation

 67

All NMR data were collected in CDCl3 using Varian Inova 300 MHz and 500
MHz instruments. 1H and 13C NMR signals were referenced to the solvent peaks at 7.26
ppm and 77.0 ppm, respectively. High resolution mass spectra were recorded on a
Thermo-Finnigan MAT900XL spectrometer. A crystal was grown from a CH2Cl2 solution
of 1 by diffusional exchange with hexanes for X-ray crystallographic analysis on a
Bruker APEX CCD detector.

II.2.7 Ichthyotoxicity assay

Various quantities of the natural product 1 in 174 μL of EtOH was added to 50 mL

Erlenmeyer flasks containing 40 mL of aquarium water. For controls, 174 μL of EtOH

was added to the water. Both treatments and controls were run in duplicate. Adult male

zebra fish of approximately 4 cm length were randomly selected from aquaria and

individually placed in the Erlenmeyer flasks. Time of death was determined by observing

a lack of gill movement and no visible responses to swirling of the flask for one full

minute.

II.3 Results and Discussion

Fractions from the crude lipid extract of a Papua New Guinea mixed collection of

a red marine alga, tentatively identified as Leptofauchea sp. and a marine cyanobacterium

Oscillatoria sp., showed an ability to increase the intracellular concentration of Ca +2 in

mouse neocortical neurons. Bioassay guided isolation of the active compounds

sequentially used flash column chromatography and HPLC, and yielded one more

prevalent (1) and one minor compound (3).

 68

Figure II.2: X-ray crystal structure of 1.

Analysis by ESI-LCMS (negative mode) revealed the polybrominated nature of

compound 1 and indicated a molecular weight of 1160 (obs. [M -] m/z 1159). Subsequent

analysis by 13C NMR showed 4 protonated and 8 quarternary 13C signals, indicating that

the compound was either a symmetric dimer having a molecular weight of 1160 or that it

was a monomer with a molecular weight of 580 that easily dimerizes in the mass

spectrometer. High resolution EIMS clarified that it was a monomer with an exact mass

of m/z 575.6211, yielding a molecular formula of C12H5O2Br5 (calc. 575.6201). 1H and 13C

NMR, COSY, HSQC, and HMBC data readily identified two benzenoid rings connected

to one another by an ether linkage. By 13C NMR chemical shifts and consideration of the

molecular formula, one of the rings possessed 2 bromines while the other had 3 bromines

and a hydroxyl group. Further analysis of the HMBC spectrum revealed the A ring to be a

2,4-dibromo-phenoxyl group. The regiochemical assignment of the B ring, however,

could not be made confidently based on NMR data alone. Further, the 13C NMR data for
 69

Table II.1: Comparison of NMR data for two isolates of 1 and 3 (CDCl3)

1, ref 25 1, this study 3

13 1 13 1 13 1
Position C H C H C H
1 148.9 148.9 144.5
2 139.9 139.4 143.1
3 113.6 121.1 120.7 7.00
4 119.3 119.2 114.9
5 122.9 122.9 122.4
6 120.8 7.42 120.7 7.44 121.6
1' 151.8 151.6 151.3
2' 112.8 112.7 115.6
3' 136.4 7.79 136.2 7.79 136.5 7.81
4' 116.3 116.2 118.2
5' 131.6 7.29 131.6 7.30 132.1 7.45
6' 115.8 6.41 115.7 6.41 121.5 6.89
 70

1 did not match that reported for any known OH-PBDE. Because 1 deposited well-

formed crystals from CH2Cl2/hexanes, an X-ray crystallographic analysis was conducted

to give an excellent quality crystal structure (Figure II.2). Comparison of this structure

with the literature for naturally occurring OH-PBDEs showed it to be identical to one

previously reported from Dysidea herbacea.25,23

In comparing our 13C NMR data to those reported in the literature for compound

1, a significant discrepancy was noted in the chemical shift for C3 (Table II.1). Only the

Bowden data are included in this table because the only other 13C NMR data available

were recorded in DMSO and not in CDCl3,24 but they were similar to our data. One

hypothesis for this anomalous NMR data was that previous workers had mis-assigned

structure 1 to their material, and that it was in fact the data for another structure. Of the

59 possible regioisomers of 1, 50 could be eliminated based on the fact that the A ring of

Bowden’s isolation of 1 had three protons and the HMBC and COSY correlations were

sufficient to confidently assign this sub-structure. Of the remaining 9 structural

possibilities, two could be disregarded based on HMBC correlations observed by

Bowden. Specifically, the lone proton on the B ring showed HMBC correlations to 4

carbons,25 and thus, the only carbon for which there was not an HMBC correlation should

be para to the carbon bearing this proton. Based on the predicted chemical shifts of the

oxygen bearing carbons, six other isomers could be disregarded, leaving only a single

regioisomer that required consideration (3). Regioisomer 3 is also a known naturally

occurring compound having also been isolated from the marine sponge Dysidea

herbacea.11
 71

Scheme II.1: Synthesis of 3 by Marsh’s route.

13
Because the C NMR data for 3 in CDCl3 has not been published,11 this

compound was synthesized by Marsh's route (Scheme II.1).26 However, comparison of

the 13C NMR shifts of synthetic 3 with those reported by Bowden revealed that they were
13
considerably different (Table II.1). Subsequently, a close inspection of the C NMR

spectrum from the Bowden material revealed a small peak at 121.1 ppm (B. F. Bowden,

personal communication), which corresponds well with our data for C-3 in compound 1.

Because C-3 is para to the only protonated carbon in ring B, this 13C atom is predicted to

relax slowly such that its detection may require longer delay times.27 Moreover, during

our purification of 1-3, it was difficult to remove minor contaminants that had similar

NMR signatures to these OH-PBDEs. In this regard, it is possible that the 113.6 ppm

signal reported for C3 by Bowden et al. was actually the quickly relaxing (e.g.

protonated) resonance of a minor contaminant in the sample.

Having unequivocally established the structure of 1, a minor compound was also

characterized that eluted very closely to 1 by RP-HPLC and showed Ca2+ modulating

activity. A molecular formula of C12H4O2Br6 was determined for compound 2 based on

low resolution negative ion ESI-LCMS (obs. [M-H]- m/z 658.74). The isotopic pattern
 72

observed for the molecular ion was consistent with the presence of 6 bromine atoms.

Although NMR characterization of this compound was challenging due to the small

quantity isolated (~0.5 mg) and the presence of chromatographically similar impurities,
1
H NMR and 1H-1H COSY analysis revealed a three proton spin system consisting of a

vicinally coupled pair (7.29 ppm and 6.39 ppm) and a third (7.79 ppm) which was meta

coupled to δ 7.29. These shifts and coupling pattern matched those reported for synthetic

2 and thus, this material was identified as 3,4,5,6-tetrabromo-2-(2',4'-dibromo-phenoxy)-

phenol (2).

Figure II.3: Ca2+ modulation assay in mouse neocortical neurons for compounds 1-3.

Compounds 1-3 were tested in the Ca2+ modulation assay in mouse neocortical

neurons at various concentrations and their EC50 values were determined to be 16.4 μM
 73

(9.8 μM- 27.2 μM, 95% Confidence Intervals), 27.2 μM (12.3 μM- 59.8 μM, 95%

Confidence Intervals) and 42.4 μM (23.2 μM- 77.5 μM, 95% Confidence Intervals),

respectively (Figure II.3). In light of the finding that 1 may act as an environmental

neurotoxin,16 its toxicity to zebrafish was also evaluated. At 5 μg/mL (8.6 μM) media

concentration, the fish immediately attempted to escape out of the container and its

apparent rate of respiration visibly increased; such behaviors were not observed for the

control group. Within 5 minutes, the treated fish appeared to have difficulty maintaining

their proper orientation, and within 20 minutes, both died. Similar effects were seen at 1

μg/mL (1.7 μM), but it took 50 min for the fish to expire. At 0.5 μg/mL (0.86 μM), the

fish appeared to be unaffected for the duration of the 3 hour assay. Regioisomer 3 had the

same effect but was less potent (8.6 μM, death at 25 min; no effect at 1.7 μM), suggesting

that many congeners of OH-PBDEs may display ichthyotoxicity.

Both the long term and the short term neurotoxic effects of polychlorinated

biphenyls (PCBs) have been studied extensively. Notably, PCBs disrupt the intracellular

Ca2+ homeostasis, which is critical for a variety of cell functions, including release of

neurotransmitters.14 As for the longer term effects, PCBs can modulate muscarinic and

nicotinic receptors and also disrupt neurological development.14 Similarly, PBDEs have

been shown to have detrimental effects to learning and memory functions in mice. These

behavioral effects are likely related to the observation that PBDEs reduced the densities

of nicotinic receptors in the hippocampus of mice. 28 Given the ability of 1, 2, and 3 to

alter cytoplasmic concentrations of Ca+2 ions, the observed ichthyotoxicity of 1 and 2

may involve calcium ion induced neurotoxicity.

 74

These findings are significant in light of recent reports that OH-PBDEs have

wide-spread occurrence in the marine environment.3,4,8 In Disdea herbacea, at least 1~6%

of the dry weight of the organism is composed of OH-PBDEs and visible crystals of these

compounds have been observed in the sponge tissue.13 In addition to sponges, tunicates,

and blue-green algae, we report here that a red alga and/or cyanobacterium living in

association with the red alga as a source of OH-PBDEs.29 Since most of the world's

seaweeds are red algae30 and many invertebrates feed on these algae, the presence of OH-

PBDEs in marine invertebrates may actually reflect the natural occurrence and

bioaccumulation of these compounds into higher trophic levels. Among PBDEs,

pentabrominated congeners are known to be environmentally more mobile due to their

greater water solubility, volatility, Kow (octanol/water partition coefficient), and

lipophilicity.7 OH-PBDEs are expected to be reasonably lipophilic and yet more water

soluble than PBDEs. Therefore, they may be even more prone to bioaccumulation due to

their increased mobility in the environment.

The possibility raised by these findings is that many OH-PBDEs are of natural

occurrence yet possess neurotoxic properties of environmental significance. In this

regard, it is possible that human populations have been exposed to these materials

through seafood consumption for a lengthy period and that this exposure may have had

historical as well as current health implications. PCBs and brominated flame retardants

other than PBDEs are suspected to be responsible for abnormal behavior phenomena such

as the ‘beaching of cetaceans.’31,32 Because of their structurally related nature, it is

possible that OH-PBDEs of natural or anthropogenic origins may contribute to such

events. Thus, it is important to both characterize the origin and occurrence of OH-PBDEs
 75

as well as details of their molecular neurotoxicology.

Acknowledgment

The text of II, in whole, has been submitted for publication to Toxicon at the time

of writing. The dissertation author was the primary author and directed and carried out

the research, which forms the basis for this chapter.

X-ray crystallographic analysis by A. Rhinegold and A. DiPasquale at UCSD is

acknowledged. We thank L. Tan and D. Sherman for help with collection of the algal
13
material, B. F. Bowden for providing copies of their C NMR spectra of 1, and L.

Gerwick for providing zebrafish. We further thank the government of Papua New Guinea

for scientific collection permits and funding from NIH Grant 053398 is acknowledged.
 76

Appendix

Table of Contents of Appendix

1.Photographs of The Red Algal Voucher Sample

2.X-ray Crystallographic Analysis

3.Isolation Scheme for 1 and 2

4.1H NMR Spectrum of 1 in CDCl3

5.13C NMR Spectrum of 1 in CDCl3

6.1H-1H COSY Spectrum of 1 in CDCl3

7.1H-13C HSQC Spectrum of 1 in CDCl3

8.1H-13C HMBC Spectrum of 1 in CDCl3

9.1H NMR Spectrum of 3 in CDCl3

10.13C NMR Spectrum of 3 in CDCl3

11.1H-1H COSY Spectrum of 3 in CDCl3

12.1H-13C HSQC Spectrum of 3

13.1H-13C HMBC Spectrum of 3 in CDCl3

14.1H NMR Spectrum of 2 in CDCl3

15.1H-1H COSY Spectrum of 2 in CDCl3

 77

Figure II.4: Photographs of the red algal voucher sample

 78

Figure II.5: Photograph of the red algal voucher sample

 79

X-ray Crystallographic Analysis

A colorless block 0.12 x 0.08 x 0.08 mm in size was mounted on a Cryoloop with

Paratone oil. Data were collected in a nitrogen gas stream at 100(2) K using phi and

omega scans. Crystal-to-detector distance was 60 mm and exposure time was 5 seconds

per frame using a scan width of 0.5°. Data collection was 98.7% complete to 67.00° in θ.

A total of 8115 reflections were collected covering the indices, -9<=h<=9, -10<=k<=10,

-25<=l<=24. 2597 reflections were found to be symmetry independent, with an R int of

0.0280. Indexing and unit cell refinement indicated a primitive, monoclinic lattice. The

space group was found to be P2(1)/c (No. 14). The data were integrated using the Bruker

SAINT software program and scaled using the SADABS software program. Solution by

direct methods (SIR-2004) produced a complete heavy-atom phasing model consistent

with the proposed structure. All non-hydrogen atoms were refined anisotropically by

full-matrix least-squares (SHELXL-97). All hydrogen atoms were placed using a riding

model. Their positions were constrained relative to their parent atom using the

appropriate HFIX command in SHELXL-97.

 80

Table II.2: Crystal data and structure refinement for 1.

X-ray ID gerw02
Sample/notebook ID 1572D2C
Empirical formula C12 H5 Br5 O2
Formula weight 580.71
Temperature 100(2) K
Wavelength 1.54178 Å
Crystal system Monoclinic
Space group P2(1)/c
Unit cell dimensions a = 7.7959(5) Å = 90°.
b = 8.8184(5) Å = 92.671(2)°.
c = 21.0376(12) Å  = 90°.
Volume 1444.71(15) Å3
Z 4
Density (calculated) 2.670 Mg/m3
Absorption coefficient 16.803 mm-1
F(000) 1072
Crystal size 0.12 x 0.08 x 0.08 mm3
Crystal color/habit colorless block
Theta range for data collection 4.21 to 68.08°.
Index ranges -9<=h<=9, -10<=k<=10, -25<=l<=24
Reflections collected 8115
Independent reflections 2597 [R(int) = 0.0280]
Completeness to theta = 67.00° 98.7 %
Absorption correction Analytical
Max. and min. transmission 0.3467 and 0.2377
Refinement method Full-matrix least-squares on F2
Data / restraints / parameters 2597 / 0 / 174
Goodness-of-fit on F2 1.159
Final R indices [I>2sigma(I)] R1 = 0.0391, wR2 = 0.1018
R indices (all data) R1 = 0.0394, wR2 = 0.1021
Extinction coefficient 0.00126(9)
Largest diff. peak and hole 1.337 and -1.007 e.Å-3
 81

Table II.3: Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x
103) for 1. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.
_____________________________________________________________________________
x y z U(eq)
_____________________________________________________________________________
C(1) 1442(6) 2778(6) 4601(2) 17(1)
C(2) 1873(6) 4251(6) 4435(2) 18(1)
C(3) 2477(6) 5284(6) 4901(2) 17(1)
C(4) 2615(6) 4782(6) 5531(2) 18(1)
C(5) 2167(7) 3329(6) 5701(2) 19(1)
C(6) 1584(6) 2306(6) 5237(2) 18(1)
C(7) 1886(7) 904(6) 3823(2) 19(1)
C(8) 3595(7) 721(6) 3994(2) 19(1)
C(9) 4599(7) -253(6) 3651(2) 19(1)
C(10) 3873(7) -1037(6) 3133(2) 21(1)
C(11) 2171(7) -826(6) 2941(2) 21(1)
C(12) 1183(6) 146(6) 3285(2) 19(1)
O(1) 756(4) 1782(4) 4149(2) 21(1)
O(2) 1203(5) 875(4) 5391(2) 30(1)
Br(1) 1606(1) 4788(1) 3572(1) 23(1)
Br(2) 3061(1) 7263(1) 4676(1) 23(1)
Br(3) 3451(1) 6077(1) 6189(1) 22(1)
Br(4) 5202(1) -2438(1) 2681(1) 24(1)
Br(5) -1156(1) 448(1) 3038(1) 31(1)
_____________________________________________________________________________
 82

Table II.4: Bond lengths [Å] and angles [°] for 1.

_____________________________________________________
C(1)-O(1) 1.383(6) C(7)-O(1) 1.378(6)
C(1)-C(2) 1.389(7) C(7)-C(12) 1.405(7)
C(1)-C(6) 1.401(7) C(8)-C(9) 1.387(8)
C(2)-C(3) 1.403(7) C(8)-H(8) 0.9500
C(2)-Br(1) 1.879(5) C(9)-C(10) 1.388(7)
C(3)-C(4) 1.397(7) C(9)-H(9) 0.9500
C(3)-Br(2) 1.870(5) C(10)-C(11) 1.382(8)
C(4)-C(5) 1.380(7) C(10)-Br(4) 1.896(5)
C(4)-Br(3) 1.888(5) C(11)-C(12) 1.380(8)
C(5)-C(6) 1.390(7) C(11)-H(11) 0.9500
C(5)-H(5) 0.9500 C(12)-Br(5) 1.891(5)
C(6)-O(2) 1.340(6) O(2)-H(2) 0.8400
C(7)-C(8) 1.373(8)
O(1)-C(1)-C(2) 120.8(5) C(8)-C(7)-C(12) 119.5(5)
O(1)-C(1)-C(6) 118.6(4) O(1)-C(7)-C(12) 115.7(5)
C(2)-C(1)-C(6) 120.5(5) C(7)-C(8)-C(9) 120.1(5)
C(1)-C(2)-C(3) 120.6(5) C(7)-C(8)-H(8) 119.9
C(1)-C(2)-Br(1) 117.4(4) C(9)-C(8)-H(8) 119.9
C(3)-C(2)-Br(1) 122.0(4) C(10)-C(9)-C(8) 119.8(5)
C(4)-C(3)-C(2) 117.7(5) C(10)-C(9)-H(9) 120.1
C(4)-C(3)-Br(2) 121.8(4) C(8)-C(9)-H(9) 120.1
C(2)-C(3)-Br(2) 120.5(4) C(11)-C(10)-C(9) 120.9(5)
C(5)-C(4)-C(3) 122.1(5) C(11)-C(10)-Br(4) 118.7(4)
C(5)-C(4)-Br(3) 117.0(4) C(9)-C(10)-Br(4) 120.4(4)
C(3)-C(4)-Br(3) 120.9(4) C(12)-C(11)-C(10) 118.8(5)
C(4)-C(5)-C(6) 119.9(5) C(12)-C(11)-H(11) 120.6
C(4)-C(5)-H(5) 120.0 C(10)-C(11)-H(11) 120.6
C(6)-C(5)-H(5) 120.0 C(11)-C(12)-C(7) 120.8(5)
O(2)-C(6)-C(5) 120.7(5) C(11)-C(12)-Br(5) 119.9(4)
O(2)-C(6)-C(1) 120.2(5) C(7)-C(12)-Br(5) 119.3(4)
C(5)-C(6)-C(1) 119.1(5) C(7)-O(1)-C(1) 117.5(4)
C(8)-C(7)-O(1) 124.9(5) C(6)-O(2)-H(2) 109.5
_____________________________________________________________
 83

Table II.5: Anisotropic displacement parameters (Å2x 103)for 1. The anisotropic displacement
factor exponent takes the form: -22[ h2a*2U11 + ... + 2 h k a* b* U12 ]
_____________________________________________________________________________
U11 U22 U33 U23 U13 U12
_____________________________________________________________________________
C(1) 16(2) 20(2) 14(2) -8(2) 1(2) -3(2)
C(2) 17(2) 26(3) 10(2) 1(2) 2(2) 0(2)
C(3) 16(2) 19(2) 15(2) 0(2) 1(2) 0(2)
C(4) 15(2) 24(2) 15(2) -3(2) -2(2) -1(2)
C(5) 23(2) 22(3) 12(2) 1(2) 0(2) 1(2)
C(6) 16(2) 19(2) 18(3) -1(2) 2(2) 1(2)
C(7) 23(2) 22(2) 12(2) 1(2) 4(2) -5(2)
C(8) 25(3) 18(2) 13(2) -1(2) -2(2) -5(2)
C(9) 23(2) 20(2) 14(2) 2(2) -2(2) 0(2)
C(10) 27(3) 23(3) 13(2) 0(2) 5(2) -3(2)
C(11) 30(3) 21(2) 12(2) -2(2) 0(2) -5(2)
C(12) 17(2) 29(3) 12(2) 1(2) 0(2) -9(2)
O(1) 17(2) 30(2) 16(2) -10(2) 1(1) -4(2)
O(2) 31(2) 22(2) 37(2) 2(2) 7(2) -4(2)
Br(1) 22(1) 34(1) 12(1) 4(1) -1(1) -2(1)
Br(2) 24(1) 20(1) 25(1) 5(1) -1(1) -4(1)
Br(3) 25(1) 24(1) 17(1) -6(1) -2(1) -5(1)
Br(4) 28(1) 26(1) 18(1) -4(1) -1(1) 6(1)
Br(5) 16(1) 53(1) 23(1) -18(1) -1(1) -3(1)
_____________________________________________________________________________
 84

Table II.6: Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 103)
for 1.
_____________________________________________________________________________
x y z U(eq)
_____________________________________________________________________________

H(5) 2255 3028 6135 23

H(8) 4090 1261 4347 23
H(9) 5781 -384 3770 23
H(11) 1689 -1341 2578 25
H(2) 320 595 5179 45
_____________________________________________________________________________
 85

Figure II.6: Isolation scheme for 1 and 3

 86

Figure II.7: 1H NMR Spectrum of 1 in CDCl3

 87

Figure II.8: 13C NMR Spectrum of 1 in CDCl3

 88

Figure II.9: 1H-1H COSY Spectrum of 1 in CDCl3

 89

Figure II.10: 1H-13C HSQC Spectrum of 1 in CDCl3

 90

Figure II.11: 1H-13C HMBC Spectrum of 1 in CDCl3

 91

Figure II.12: 1H NMR Spectrum of 1 in CDCl3

 92

Figure II.13: 13C NMR Spectrum of 3 in CDCl3

 93

Figure II.14: 1H-1H COSY Spectrum of 3 in CDCl3

 94

Figure II.15: 1H-13C HSQC Spectrum of 3 in CDCl3

 95

Figure II.16: 1H-13C HMBC Spectrum of 3 in CDCl3

 96

Figure II.17: 1H NMR Spectrum of 2 in CDCl3

 97

Figure II.18: 1H-1H COSY Spectrum of 2 in CDCl3

 98

References and footnotes

1 Marsh, G.; Athanasiadou, M.; Athanassiadis, I.; Bergman, Å.; Endo, T.; Haraguchi, K.
Identification, quantification, and synthesis of a novel dimethoxylated polybrominated
biphenyl in marine mammals caught off the coast of Japan. Environ. Sci. Technol. 2005, 39,
8684-8690.

2 Vos, J. G.; Becher, G.; van den Berg, M.; de Boer, J.; Leonards, P. E. G. Brominated flame
retardants and endocrine disruption. Pure Appl. Chem. 2003, 75, 2039-2046.

3 Verreault, J.; Gebbink, W. A.; Gauthier, L. T.; Gabrielsen, G. W.; Letcher, R. J. Brominated
flame retardants in glaucous gulls from the Norwegian Arctic: more than just an issue of
polybrominated diphenyl ethers. Environ. Sci. Technol. 2007, 41, 4925-4931.

4 Kelly, B. C.; Ikonomou, M. G.; Blair, J. D.; Gobas, F. A. P. C. Hydroxylated and methoxylated
polybrominated diphenyl ethers in a Canadian arctic marine food web. Environ. Sci. Technol.
2008, 42, 7069-7077.

5 Betts, K. S. Science news: rapidly rising PBDE levels in North America. Environ. Sci.
Technol. 2002, 36, 50A-2A.

6 Athanasiadou, M.; Cuadra, S. N.; Marsh, G.; Bergman, Å.; Jakobsson, K. Polybrominated
diphenyl ethers (PBDEs) and hydroxylated PBDE metabolites in young humans from
Managua, Nicaragua. Environ. Health Perspect. 2008, 116, 400-408.

7 Hale, R. C.; Alaee, M.; Manchester-Neesvig, J. B.; Stapleton, H. M.; Ikonomou, M. G.

Polybrominated diphenyl ether flame retardants in the North American environment. Environ.
Int. 2003, 29, 771-779.

8 McKinney, M. A.; Cesh, L. S.; Elliott, J. E.; Williams, T. D.; Garcelon, D. K.; Letcher, R. J.
Brominated flame retardants and halogenated phenolic compounds in North American West
Coast bald eaglet (Haliaeetus leucocephalus) plasma. Environ. Sci. Technol. 2006, 40,
6275-6281.

9 Malmberg, T.; Athanasiadou, M.; Marsh, G.; Brandt, I.; Bergman, Å. Identification of
hydroxylated polybrominated diphenyl ether metabolites in blood plasma from
polybrominated diphenyl ether exposed rats. Environ. Sci. Technol. 2005, 39, 5342-5348.

10 Marsh, G.; Athanasiadou, M.; Athanassiadis, I.; Sandholm, S. Identification of hydroxylated

metabolites in 2,2',4,4'-tetrabromodiphenyl ether exposed rats. Chemosphere 2006, 63,
690-697.

11 Carte, B.; Faulkner, D. J. Polybrominated diphenyl ethers from Dysidea herbacea, Dysidea
chlorea and Phyllospongia foliascens. Tetrahedron 1981, 37, 2335-2339.

12 Carte, B.; Kernan, M. R.; Barrabee, E. B.; Faulkner, D. J.; Matsumoto, G. K.; Clardy, J.
Metabolites of the nudibranch Chromodoris funerea and the singlet oxygen oxidation products
of furodysin and furodysinin. J. Org. Chem. 1986, 51, 3528-3532.

13 Unson, M. D.; Holland, N. D.; Faulkner, D. J. A brominated secondary metabolite synthesized

 99

by the cyanobacterial symbiont of a marine sponge and accumulation of the crystalline

metabolite in the sponge tissue. Mar. Biol. 1994, 119, 1-11.

14 Mariussen, E.; Fonnum, F. Neurochemical targets and behavioral effects of organohalogen

compounds: an update. Crit. Rev. Toxicol. 2006, 36, 253–289.

15 Hwang, H.; Park, E.; Young, T. M.; Hammock, B. D. Occurrence of endocrine-disrupting

chemicals in indoor dust. Sci. Total Environ. 2008, 404, 26-35.

16 Dingemans, M. M. L.; de Groot, A.; van Kleef, R. G. D. M.; Bergman, Å.; van den Berg, M.;
Henk P.M. Vijverberg, H. P. M.; Westerink , R. H. S. Hydroxylation increases the neurotoxic
potential of BDE-47 to affect exocytosis and calcium homeostasis in PC12 cells. Environ.
Health Perspect. 2008, 116, 637-643.

17 Dingemans, M. M. L.; Ramakers, G. M. J.; Gardoni, F.; van Kleef, R. G. D. M.; Bergman, Å;
Di Luca, M.; van den Berg, M.; Westerink, R. H. S.; Vijverberg, H. P. M. Neonatal exposure to
brominated flame retardant BDE-47 reduces long-term potentiation and postsynaptic protein
levels in mouse hippocampus. Environ. Health Perspect. 2007, 115, 865–870.

18 Berman, F. W.; Murray, T. F. Brevetoxin-induced autocrine excitotoxicity is associated with

manifold routes of Ca2+ influx. J. Neurochem. 2000, 74, 1443-1451.

19 Li, W. I.; Berman, F. W.; Okino, T.; Yokokawa, F.; Shioiri, T.; Gerwick, W. H.; Murray, T. F.,
2001. Antillatoxin is a novel marine cyanobacterial toxin that potently activates voltage-gated
sodium channels. Proc. Natl. Acad. Sci. 2001, 98, 7599-7604.

20 Rogers, K. L.; Fong, W. F.; Redburn, J.; Griffiths, L. R. Fluorescence detection of plant
extracts that affect neuronal voltage-gated Ca2+ channels. Eur J Pharm Sci. 2002, 15, 321-330.

21 Dravid, S. M.; Murray, T. F. Fluorescent detection of Ca2+-permeable AMPA/kainate receptor

activation in murine neocortical neurons. Neurosci. Lett. 2003, 351, 145-148.

22 Still, W. C.; Khan, M.; Mitra, A. Rapid chromatographic technique for preparative separations
with moderate resolution. J. Org. Chem. 1978, 43, 2923-2925.

23 Agrawal, M. S.; Bowden, B. F. Marine sponge Dysidea herbacea revisited: another

brominated diphenyl ether. Mar. Drugs 2005, 3, 9-14.

24 Fu, X.; Schmitz, F. J. New brominated diphenyl ether from an unindentified species of
Dysidea sponge. 13C NMR data for some brominated diphenyl ethers. J. Nat. Prod. 1996, 59,
1102-1103.

25 Bowden, B. F.; Towerzey, L.; Junk, P. C. A new brominated diphenyl ether from the marine
sponge Dysidea herbacea. Aust. J. Chem. 2000, 53, 299-301.

26 Marsh, G.; Stenutz, R.; Bergman, Å. Synthesis of hydroxylated and methoxylated

polybrominated diphenyl ethers – natural products and potential polybrominated diphenyl
ether metabolites. Eur. J. Org. Chem. 2003, 2566-2576.
 100

27 Crews, P.; Rodríguez, J.; Jaspars, M. Organic Structure Analysis, Oxford, 1998, pp. 169-181.

28 Viberg, H.; Fredriksson, A.; Eriksson, P. Neonatal exposure to polybrominated diphenyl ether
(PBDE 153) disrupts spontaneous behaviour, impairs learning and memory, and decreases
hippocampal cholinergic receptors in adult mice. Toxicol. Appl. Pharmacol. 2003, 192,
95-106.

29 Malmvärn, A.; Marsh, G.; Kautsky, L.; Athanasiadou, M.; Bergman, Å.; Asplund, L.
Hydroxylated and methoxylated brominated diphenyl ethers in the red algae Ceramium
tenuicorne and blue mussels from the Baltic Sea. Environ. Sci. Technol. 2005, 39, 2990-2997.

30 Garrison, T., Oceanography 5th Ed. Brooks/Cole publisher, 2005, pp 349-350.

31 Symons, R. K.; Burniston, D.; Jaber, R.; Piro, N.; Trout, M.; Yates, A.; Gales, R.; Terauds, A.;
Pemberton, D.; Robertson, D. Southern hemisphere cetaceans. A study of the POPs
PCDDs/PCDFs and dioxin-like PCBs in stranded animals from the Tasmanian coast.
Organohalogen Compounds 2003, 62, 257-260.

32 Law, R. J.; Allchin, C. R.; deBoer, J.; Covaci, A.; Herzke, D.; Lepom, P.; Morris, S.;
Tronczynski, J.; de Wit, C. A. Levels and trends of brominated flame retardants in the
European environment. Chemosphere 2006, 64, 187–208.
 Chapter III

Discovery of a novel vinylchloride containing fatty acid from a marine cyanobacterium

Lyngbya sp.

101
 102

Abstract

A novel fatty acid containing a vinyl chloride moiety was discovered through

NMR-guided fractionations of an aqueous extract of a marine cyanobacterium, Lyngbya

sp. collected in Papua New Guinea. While no bioactivity has yet been found to be

associated with this natural product, its biogenesis is of significance in light of other

important cyanobacterial compounds containing a similar vinylchloride functional group.

 103

III.1 Introduction

It has been exceptionally productive to search for novel natural products in marine

cyanobacteria, particularly species of Lyngbya.1 For example, jamaicamides A-C and

malyngamides A-X have been found from marine cyanobacteria and nudibranchs (Figure

III.1, III.2),2,3 and nudibranchs probably obtain these compounds from feeding on

cyanobacteria.4 All of these natural products have a common biosynthetic theme in that

they possess a PKS-derived long lipid chain and an NRPS-derived peptidic portion.5

Furthermore, all three jamaicamides and 20 of the 27 malyngamides contain a rather

unusual functionality, a vinylchloride group.2,3 Pyrrolone rings seen in all three

jamaicamides and malyngamides A, Q, R, and X further add to the overall striking

resemblance of these metabolites.

The apparent rule for the construction of malyngamides is that if two acetate units

and a glycine unit are used for the “head group,” then the final product will contain a

linear “head group” that is attached to either a pyrrolone or a pyrrolidinone with the

exception of malyngamide P (19). Alternatively, if three acetate units and a glycine unit

are used, then a cyclohexane with various oxidation states will be the biosynthetic

product for the “head group.” Moreover, the vinylchloride unit resides on the C-1 of the

glycine unit, suggesting that the sp2 carbon bearing the chloride is not derived from S-

adenosyl-methionine (SAM), but from the C-2 carbon of another acetate unit.6 In HMG-

CoA biosynthesis, an intermediate to the 5-carbon unit of terpenes, the same strategy is

employed, where an aldol reaction between an acetoacetyl CoA and an acetyl CoA is

catalyzed by HMG-CoA synthase. Likewise, in jamaicamides,7 the vinlychloride carbon

 104

Figure III.1: Jamaicamides A-C

Figure III.2: Malyngamides A-X

 105

Scheme III.1: Divergent biosyntheses of jamaicamide A and curacin A

The two different biosynthetic pathways leading to 33 and 37 utilize the same chemistry until the
chlorinated intermediate 31, after which the corresponding decarboxylases distinguish the
pathways (32 for jamaicamide A; 34 for curacin A).

has been shown to be derived from the C-2 of acetate.2 Recently, the biosynthesis of

jamaicamides was found to be closely related to that of curacins, where the two different

pathways share a common intermediate (31) as shown in Scheme III.1.8 Herein described

is a novel natural product isolated from Lyngbya that also possesses a vinylchloride

group.

III.2 Collection of the Cyanobacterial Material

Tropical areas are biologically very diverse environments in which many species

compete for space and nutritional resources. Therefore, organisms in such environments
 106

Figure III.3: Satellite Image of Algae Collection Site

The satellite image was obtained through Google Earth™. The site of collection is indicated by
the red circle.

Figure III.4: Zoom-up of the Satelite Image of the Lyngbya Collection Site

The satellite image was obtained through Google Earth™. The site of collection is indicated by
the red circle.
 107

Figure III.5: "Green Lyngbya" collected in Papua New Guinea

are expected to possess a rich collection of secondary metabolites suited for competition.

In order to acquire such metabolites, a sample of Lyngbya sp. was collected in Papua

New Guinea (PNG). The cyanobacterial material was collected at a site between the

northeast coast of New Britain Island and the southwest coast of Credner Island by

snorkeling (S 4º 15.981'; E 152º 19.735'; Figure III.3 and III.4) at the depth of 1 m.9 The

alga had the typical hair-like morphology of Lyngbya, but had a greenish color unlike

typical Lyngbya (Figure III.5).

III-3 Isolation of the Novel Fatty Acid

Since water-soluble organic natural products are very difficult to handle, they are

not as well studied as other organic compounds. Therefore, it is a better likelihood to find

a novel compound in aqueous soluble materials. One of the aims of this study was to

develop a methodology to purify natural products from the aqueous extract. The

cyanobacterial material (WG-1560) was extracted exhaustively with CH2Cl2 and MeOH

(2:1) for the isolation of lipid contents.10 The remaining aqueous layers and the EtOH

used for preservation of the alga were combined and the volatiles were removed under

vacuum. The residues were resuspended in EtOH and the insolubles were filtered off
 108

Scheme III.2: Isolation process for credneric acid (38)

Figure III.6: 1H NMR Spectrum of Credneric Acid (38)

 109

(Scheme III.2). The filtrate was then subjected to two steps of rough normal phase

chromatgraphy to generate a few fractions. The fraction that contained compounds

separable by TLC was subjected to flash column chromatography with silica gel as the

stationary phase and a gradient mixture of MeOH and CH2Cl2 as the mobile phase to

obtain several fractions. One of the fractions contained a fairly pure compound and it was

further purified by semi-prep reverse phase HPLC to obtain 4 mg of a colorless oil. The

compound was named “credneric acid,” after the nearby island where the cyanobacterial

material was collected.

III.4 Structure Elucidation of Credneric Acid

The purified material was first analyzed by 1H NMR (Figure III.6), which

revealed that the compound was a short chain fatty acid. However, one unusual singlet

signal was observed at 5.79 ppm. Further analysis by 13C NMR, COSY, and HSQC (Table

III.1) led to substructures as shown in Scheme III.3. Finally, the two and three bond

heteronuclear coupling information obtained by HMBC analysis allowed connection of

the three substructures (Table III.1). The unknown “R” group was determined to be a

chlorine atom based on the high resolution mass spectroscopy analysis (m/z 217.0990

expected for C11H18O2Cl [M+H]; observed m/z 217.0995) and the NMR chemical shift

(1H 5.79 ppm; 13C 113.1). The stereochemical assignment of the two olefins were made

by the coupling constant between the two vinyl protons (5.42 ppm and 5.49 ppm; J =

15.4 Hz) and 1D ROESY correlations of key protons on and around the olefins (Scheme

III.4).
 110

Table III.1: 1H and 13C NMR, COSY, HSQC, and HMBC data for credneric acid (38)

The HSQC data are reflected in the assignment of the 1H NMR signals to their respective 13C
NMR signals.

13 1
Position C H COSY HMBC

1 178.9 2.44, 2.35

2 33.7 2.44 2.35 2.35

3 27.4 2.35 5.48, 2.44 5.42, 2.44

4 130.5 5.48 2.35 2.73, 2.44, 2.36

5 128.1 5.42 2.73 2.73, 2.36

6 37.9 2.73 5.42 5.79, 5.48, 5.42, 2.16

7 141.4 5.79, 2.73, 2.16, 1.44

8 32.2 2.16 1.44 5.79, 2.73, 1.44, 0.92

9 20.2 1.44 2.16, 2.16, 0.92 2.16, 0.92

10 13.9 0.92 1.44 2.16, 1.44

11 113.1 5.79 2.73, 2.16

Scheme III.3: Substructures of Credneric Acid by 1H NMR, COSY, and HSQC

 111

Scheme III.4: Stereochemical Assignments of Credneric Acid by 1D ROESY

Correlations

III.5 Significance of Crendneric Acid

While the gene cluster responsible for the biosynthesis of jamaicamides has been

identified already,2 the biosynthetic process for this halogenation step is only speculative

and largely based on other related natural products.7 Since there is remarkable

resemblance between credneric acid (38) and the middle portion of the jamaicamides, it

may be advantageous to study the biosynthesis of simpler compounds like 38 to gain

insights into the halogenation process for the jamaicamides and the malyngamides. For

example, biosynthetic precursors necessary for enzymatic reactions with a putative

halogenating enzyme would be easier to synthesize for credneric acid than for the

jamaicamides.

Credneric acid was tested in H-460 cytotoxicity and Na+ channel activation and

blocking assays, but it was found to be inactive (data not shown). It is possible that the

compound is not able to cross cell membranes due to its polar carboxylic acid group.
 112

Figure III.7: Credneric Acid and Credneramide

Scheme III.5: Cyclopropane Fatty Acid and Lyngbyamides

Alternatively, it is possible that this natural product lacks functional groups necessary for

bioactivity. After the discovery of 38, a related natural product, credneramide (39, Figure

III.7), was isolated from the CH2Cl2 / MeOH extract of the same collection of Lyngbya in

our laboratory.11 This amide derivative of 38 was found to be only moderately active in

anti-parasitic assays against malaria, chagas, and leishmania and Na+ channel blocking

assay in Neuro-2a cells. It may be that 38 is merely a biosynthetic precursor for amides,

Figure III.8: N-Acyl Homoserine Lactones (AHLs)

 113

such as 39, and it is not produced as a secondary metabolite with its own biological or

ecological significance.

Cyanobacteria appear to produce various natural products based on some common

foundational biosynthetic units. For example, malyngamides all have lyngbic acid in

common, with minor variations (Figure III.2). Furthermore, grenadamide (lyngbyamide

A), and lyngbyamides B-D have the same cyclopropane fatty acid in common (Scheme

III.5).12 All of these amides have some structural resemblance to bacterial quorum sensing

molecules such as AHLs (N-acyl homoserine lactones; Figure III.8).13 This resemblance

may be related to the biological roles that these amide natural products have in nature.

Likewise, 38 appears to be a foundational biosynthetic unit on which Lyngbya builds

other compounds, such as 39. Given the precedence with lyngbyamides and

malyngamides, discovery of other credneric acid derivatives are expected in the future.

III.6 Conclusion

A novel fatty acid containing a vinylchloride functionality was discovered and

characterized from a PNG collection of Lyngbya. Despite its inactivity in the few

bioassays tested thus far, this natural product may play an important role as a template for

synthesizing bioactive amide derivatives. Credneric acid may also be useful for the study

of the biosynthetic chlorination processes in cyanobacteria.

III.7 Experimental Section

General
 114

Unless noted otherwise, all materials were purchased from commercially available

sources and were used without further purification. Flash chromatorgraphy14 and vacuum

liquid chromatography were performed using EM Science silica gel (230-400 mesh).

TLC was performed using EM Science pre-coated silica gel plates (Merck 60 F254). IR

spectra were recorded on a Nicolet Magna-IR 550. 1H NMR and 13C NMR spectra were

recorded on a Varian Inova spectrometer (500MHz) or on a Varian Inova spectrometer

(300 MHz and 75 MHz, respectively). 1H and 13

C spectra recorded in CDCl3 were

referenced to the residual solvent peaks at 7.26 ppm and 77.0 ppm, respectively. High

resolution mass spectra were recorded on a ThermoFinnigan MAT900XL spectrometer.

Isolation and Characterization of Credneric Acid

The cyanobacterial material (41 g, dry weight) was extracted exhaustively with

CH2Cl2 and MeOH (2:1) for the isolation of lipid contents. The remaining aqueous layers

and the EtOH used for preservation of the alga were combined and the volatiles were

removed under vacuum. The residues were resuspended in EtOH and the insolubles were

filtered off (Scheme III.1). By using vacuum, the filtrate was pulled through a pad of

water-treated silica gel with differing solvents (vacuum liquid chromatography, VLC).

Most of the organic content was eluted with EtOH, but not with other solvents (Scheme

III.1). Upon concentration under vacuum, the residues were subjected to solid phase

extraction (SPE) with silica gel. By eluting with different solvents (1:1 CH 2Cl2/EtOAc;

EtAcO; 1:9 MeOH/CH2Cl2; 3:7 MeOH/CH2Cl2), four fractions (4 mg, 422 mg, 1.08 g,

and 1.09 g, respectively) were obtained (Scheme III.1). Only the third fraction (1:9

MeOH/CH2Cl2 elution) had a TLC profile that appeared suitable for normal phase
 115

separation of compounds. The material in the third SPE fraction was subjected to flash

column chromatography with silica gel as the stationary phase and a gradient mixture of

MeOH and CH2Cl2 as the mobile phase to obtain several fractions. One of the fractions

contained a fairly pure compound (7 mg) and it was further purified by semi-prep reverse

phase HPLC (250 x10.00 mm Synergi 4μ Hydro-RP 80A column, MeCN/H 2O gradient)

to obtain 4 mg of a colorless oil. IR (film on KBr) 3428 (br), 2965, 2930, 2873, 1711,

1632, 1431, 1410, 1284, 1252, 1212, 1158, 968, 795 cm-1. 1H NMR (500 MHz, CDCl3) δ

5.79 (s, 1H), 5.49 (dt, J = 15.4, 6.2 Hz, 1H), 5.42 (dt, J = 15.4, 6.4 Hz, 1H), 2.73 (d, 6.2

Hz, 2H), 2.44 (t, J = 7.4 Hz, 2H), 2.35 (dt, J = 6.4, 7.0 Hz, 2H), 2.16 (t, J = 7.7 Hz, 2H),

1.44 (h, J = 7.5 Hz, 2H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 178.9,

141.4, 130.5, 128.1, 113.1, 37.9, 33.7, 32.2, 27.4, 20.2, 13.9. HRMS (EI): calcd for

C11H18O2Cl (M+H): 217.0990, found 217.0995.

 116

Figure III.9: Credneric Acid (38) 13C NMR Spectrum in CDCl3

 117

Figure III.10: Credneric Acid (38) 1H-1H COSY Spectrum in CDCl3

 118

Figure III.11: Credneric Acid (38) 1H-13C HSQC Spectrum in CDCl3

 119

Figure III.12: Credneric Acid (38) 1H-13C HMBC Spectrum in CDCl3

 120

Figure III.13: Credneric Acid (38) 1D ROESY Spectrum: Irradiated at 2.73 ppm in CDCl3
 121

Figure III.14: Credneric Acid (38) 1D ROESY Spectrum: Irradiated at 5.79 ppm in CDCl3
 122

Figure III.15: Credneric Acid (38) 1D ROESY Spectrum: Irradiated at 2.16 ppm in CDCl3
 123

Figure III.16: IR spectrum of 38

 124

References and Footnotes

1 Gerwick, W. H.; Tan, L. T.; Sitachitta, N. The Alkaloids; Cordell, G. A., Ed.; Academic Press;
San Diego, CA 2001; Vol. 57, pp 75-184.

2 Edwards, D. J.; Marquez, B. L.; Nogle, L. M.; McPhail, K.; Goeger, D. E.; Roberts, M. A.;
Gerwick , W. H. Structure and biosynthesis of the jamaicamides, new mixed polyketide-
peptide neurotoxins from the marine cyanobacterium Lyngbya majuscula. Chemistry &
Biology 2004, 11, 817–833.

3 (a) Appleton, D. R.; Sewell, M. A.; Berridge, M. V.; Copp, B. R. A new biologically active
malyngamide from a New Zealand collection of the sea hare Bursatella leachii. J. Nat. Prod.
2002, 65, 630-631. (b) Kan, Y.; Sakamoto, B.; Fujita, T.; Nagai, H. New malyngamides from
the Hawaiian cyanobacterium Lyngbya majuscula. J. Nat. Prod. 2000, 63, 1599-1602. (c)
Gallimore, W. A.; Scheuer, P. J. Malyngamides O and P from the sea hare Stylocheilus
longicauda. J. Nat. Prod. 2000, 63, 1422-1424. (d) Milligan, K. E.; Marquez, B.; Williamson,
R. T.; Davies-Coleman, M.; Gerwick, W. H. Two New Malyngamides from a Madagascan
Lyngbya majuscula. J. Nat. Prod. 2000, 63, 965-968. (e) Kan, Y.; Fujita, T.; Nagai, H.;
Sakamoto, B.; Hokama, Y. Malyngamides M and N from the Hawaiian red alga Gracilaria
coronopifolia. J. Nat. Prod. 1998, 61, 152-155. (f) Wu, M.; Milligan, K. E.; Gerwick, W. H.
Three new malyngamides from the marine cyanobacterium Lyngbya majuscula. Tetrahedron
1997, 53, 15983-15990. (g) Todd, J. S.; Gerwick, W. H. Malyngamide I from the tropical
marine cyanobacterium Lyngbya majuscula and the probable structure revision of
stylocheilamide. Tetrahedron Lett. 1995, 36, 7837-40. (h) Praud, A.; Valls, R.; Piovetti, L.;
Banaigs, B. Malyngamide G: structure of a new chlorinated amide from a blue-green alga
epiphytic on Cystoseira crinita. Tetrahedron Lett. 1993, 34, 5437-40. (i) Gerwick, W. H.;
Reyes, S.; Alvarado, B. Two malyngamides from the Caribbean cyanobacterium Lyngbya
majuscula. Phytochem. 1987, 26, 1701-4. (j) Ainslie, R. D.; Barchi, J. J., Jr.; Kuniyoshi, M.;
Moore, R. E.; Mynderse, J. S. Structure of malyngamide C. J. Org. Chem. 1985, 50, 2859-62.
(k) Cardellina, J. H., II; Marner, F. J.; Moore, R. E. Malyngamide A, a novel chlorinated
metabolite of the marine cyanophyte Lyngbya majuscula. J. Am. Chem. Soc. 1979, 101,
240-2. (l) Cardellina, J. H., II; Dalietos, D.; Marner, F. J.; Mynderse, J. S.; Moore, R. E. (-)-
trans-7(S)-Methoxytetradec-4-enoic acid and related amides from the marine cyanophyte
Lyngbya majuscula. Phytochem. 1978, 17, 2091-5.

4 Garson M. J. Marine mollusks from Australia and New Zealand: chemical and ecological
studies. Prog. Mol. Subcell. Biol. 2006, 43, 159-74.

5 Fischbach, M. A.; Walsh, C. T. Assembly-line enzymology for polyketide and nonribosomal

peptide antibiotics: Logic, machinery, and mechanisms. Chem. Rev. 2006, 106, 3468-3496.

6 Dewick, P. M. Medicinal natural products; a biosynthetic approach, 2nd Ed. John Wiley &
Sons Ltd. 2001, pp 169-170.

7 Vaillancourt, F. H.; Yeh, E.; Vosburg, D. A.; Garneau-Tsodikova, S.; Walsh, C. T. Nature’s
inventory of halogenation catalysts: oxidative strategies predominate. Chem. Rev. 2006, 106,
3364–3378.
 125

8 Gu, L.; Wang, B.; Kulkarni, A.; Geders, T. W.; Grindberg, R. V.; Gerwick, L.; Håkansson, K.;
Wipf, P.; Smith, J. L.; Gerwick, W. H.; Sherman, D. H. Metamorphic enzyme assembly in
polyketide diversification. Submitted for peer review.

9 This collection was made by Dr. Carla Sorrels.

10 The extracted alga was numbered as “WG-1560” for tracking purposes in the lab.

11 This work was done by Ms. Karla Malloy.

12 Nannini, C. J., “Novel secondary metabolites from a Madagascar collection of Lyngbya

majuscula” (MS thesis, Oregon State University, 2002), Chapter II.

13 Uroz, S.; Dessaux, Y.; Oger, P. Quorum sensing and quorum quenching: the Yin and Yang of
bacterial communication. ChemBioChem 2009, 10, 205-216.

14 Still, W. C.; Khan, M.; Mitra, A. Rapid chromatographic technique for preparative separations
with moderate resolution. J. Org. Chem. 1978, 43, 2923-2925.
 Chapter IV

Absolute stereochemistry of novel fatty acids by synthetic introduction of a

stereochemical reference

126
 127

Abstract

A combined synthetic and spectroscopic method was developed to determine the

absolute stereochemistry of an enatiomeric pair of novel fatty acids isolated from two

collections of a marine cyanobacterium. Use of an oxazolidinone chiral auxiliary to

transport stereochemical information over three bonds is described. However, a

subsequent report of a total synthesis of a related natural product was in conflict with the

results of this approach. There may have been an error in the NMR analysis of the

cyclopropane fatty acid derivative or in the optical rotation measurement of the fatty

acids themselves.
 128

IV.1 Introduction

Cyclopropane containing fatty acid (1)

A Madagascar collection of the marine cyanobacterium Lyngbya majuscula

yielded a series of novel cyclopropyl-ring containing fatty acid derivatives which

possessed antifungal, neurotoxic and cannabinomimetic activities.1 Co-occurring with

these derivatives was significant quantities of the parent fatty acid (1). Subsequently, we

isolated what appeared to be the identical fatty acid, based on 1H, 13C NMR, and FAB-MS

data, from the same genus of cyanobacterium collected in Curaçao. However, the optical

rotation values of these two samples were found to be opposite one another ([α]D25 +19.3˚

for the Madagascar sample 1-m and [α]D25 –9.8° for the Curaçao sample 1-c), a finding

confirmed by converting both to their corresponding 4-bromobenzyl esters (2),

purification, and measurement of the rotation value for these derivatives.2 The

Madagascar-derived 4-bromobenzyl ester gave an [α]D25 +8.8° whereas the Curaçao fatty

acid derivative showed [α]D25 –7.9°.

The finding of two enantiomeric forms of 1 from two different collections of the

same marine cyanobacterium motivated us to determine the absolute stereochemistry of

each in order to gain insight into the biological properties of the two series as well as the
 129

biogenesis of the cyclopropyl ring. However, no enantiospecific synthesis of this class of

fatty acids had been reported at the outset of this project. Initial attempts to crystallize the

4-bromobenzyl-esterified fatty acids for crystallographic analysis failed, and hence, we

were inspired to develop a new strategy for their stereochemical determination. The use

of a Mosher ester was discounted because this technique is limited to the configurational

assignment of a chiral center immediately adjacent to the derivatizable site.3 However, it

was realized that the carboxyl functionality in 1 provided an opportunity for the addition

of a chiral auxiliary which could then be used through a diastereospecific reaction to

introduce a chiral appendage at C-2. We then envisioned that this latter appendage could

be related to chiral centers present in the natural product using a variety of NMR

techniques (nOe and J values). To our knowledge, this strategy which interfaces the

power of chirally-induced chemical synthesis with NMR spectroscopy has never been

applied to a natural product structure elucidation.

IV.2 Synthetic introduction of stereochemical reference (SISTER) method

Both enantiomers of the cyclopropyl fatty acid (1) were isolated from the

Madagascar and Curaçao collections of L. majuscula by a combination of silica gel VLC

and flash column chromatography. The chiral auxiliary, (S)-(+)-4-benzyl-2-oxazolidinone

(3), was condensed with each acid to produce imide derivatives 4 and 5 (Scheme IV.1).

The same reaction was repeated with (R)-(+)-4-benzyl-2-oxazolidinone as the chiral

auxiliary and the Madagascar-derived cyclopropyl fatty acid (1-m) to produce imide

derivative 6. Each of these imides was methylated under standard conditions to produce
 130

4-bromobenzyl ester of cyclopropane fatty acid 1 (2)

α-methyl derivatives 7 (from 4), 8 (from 5) and 9 (from 6). For all three synthetic

sequences, only a single stereoisomer was observed as the final reaction product (7-9).

The 1H NMR of 7 and 8 showed distinctive chemical shifts and splitting patterns

for the cyclopropane ring protons, confirming that they were indeed diasteriomeric to one

other, and thus 1-m and 1-c were indeed enantiomers. However, the 1H NMR spectrum of

8 was identical to that of 9, indicating that these derivatives were enantiomeric. This was

substantiated by measuring similar rotations of opposite sign for 8 (+48°) and 9 (-25°).

Since 8 and 9 have opposite absolute stereo-configurations of both the oxazolidinone (S

and R, respectively) and the cyclopropane ring (+ and -), the stereochemistry of the α-

methyl carbon in these two derivatives must be opposite to each other as well. Because 7

and 9 possess different relative stereochemistries between the cyclopropyl ring and the α-

methyl group, as evidenced by their different NMR spectra, we conclude that the

asymmetric alkylation was exclusively controlled by the oxazolidinone stereochemistry

and not the cyclopropyl ring stereochemistry. The overriding influence of the auxiliary

during the conversion of 6 to 9 was predicted based on the rationale that the long alkyl

chain would be as far away from ionic lithium-chelated six-membered ring as possible

during the alkylation transition state, thus overcoming any mismatching effects during
 131

Scheme IV.1: Derivatization of cyclopropane fatty acids 1-m and 1-c

Note that the depicted absolute stereochemistry is according to the findings obtained through this
SISTER method (vide infra).

this reaction (Figure IV.1). Hence, the stereochemistry of the α-methyl group in 7 and 8

was S whereas it was R in derivative 9.

A variety of NMR techniques (1H-COSY, homonuclear decoupled 1H NMR,

iterative spin simulation, phase-sensitive E.COSY, and 2D ROESY) were applied to

relate the stereochemistry of the α-methyl group to that of the cyclopropyl bridgehead

stereocenters in derivatives 7 and 8. First, all proton chemical shifts were assigned for all

three derivatives (7-9) using 1H-1H COSY data. The values of the key 1H-1H coupling

constants for 7 and 8 were determined by a combination of 1H NMR, homonuclear

 132

Figure IV.1: Proposed transition state of alkylation of the enolate of 5 guided by

oxazolidinone auxiliary

decoupled 1H NMR, and X-WIN NMR spin simulation program. These were in good

agreement with those obtained from the phase-sensitive E.COSY experiment.

Using the Karplus-type equation proposed by Altona,4 the dihedral angles between

the key bonds were estimated (Figure IV.2). Alternatively, the Karplus-type equation

proposed by Barfield and Smith,5 which takes into consideration the bond angles, did not

yield reasonable dihedral angles (Table IV.1). Despite the fact that the key coupling

constants are all similar to each other (around 7 Hz), a qualitative trend was seen in the

calculated dihedral angles. For example, the dihedral angle predicted from the ROESY

analyses for H3α-C3-C1′-H1′ is larger (60°) than that for H3β-C3-C1′-H1′ and the coupling

constant is indeed larger for H3β-H1′ than for H3α-H1′ (Figure IV.2). Detailed analysis of

ROESY experiments led to the identification of 13 relevant dipolar interactions for

derivative 7 and 17 for derivative 8. The relative stereochemistry predicted between the

α-methyl center and cyclopropyl ring centers by these two approaches were in excellent

agreement (Figure IV.2). Finally, the energy minimized structures of both 7 and 8 were

simulated through conformer distribution (molecular mechanics) and geometry

optimization (Hartree-Fock) calculations and compared with those obtained from the

Altona calculations and the ROESY data.6 All three lines of evidence converge to
 133

establish compound 7 as having 1R, 2R, 2´S stereochemistry and compound 8 to have 1S,

2S, 2΄S stereochemistry (Figure IV.2).

Table IV.1: Dihedral angles calculated or simulated by different methods for 7 and 8
Dihedral angles from Spartan were calculated with semi-empirical PM3; those from ROESY
were estimated manually using a molecular model kit; those from Altona & Barfield equations
were calculated from experimental coupling constants.
 134

However, as this work was concluding, Baird and coworkers reported their total

synthesis of (+)-grenadamide, which assigned the absolute configurations of this

cyclopropyl fatty acid to be 1R, 2R (Scheme IV.2).7 This result directly contradicted our

work because (+)-1 was assigned 1S, 2S in our hands whereas (+)-1, one of the

intermediates in Baird's synthesis, was assigned 1R, 2R.8 A number of possible

explanations were considered: 1) Our stereochemical assignement was erroneous, 2) The

optical rotation measured by us or Baid et al. had the wrong sign, 3) The stereochemical

assignment of the chiral starting material that Baird et al. used was erroneous. It should

be noted that the only other asymmetric synthesis of grenadamide was reported by Taylor

and coworkers who simply correlated their absolute stereochemical assignments of their

synthetic products with those of Baird's.9 Therefore, Baird's work is the only source of

contention with the results of our SISTER analysis.

After careful re-examination of our own assignments,10 the third possibility

mentioned above was evaluated through a literature search. The history of absolute

stereochemistry assignment of the chiral cyclopropane fatty acid starting material is

remarkably complex (Figure IV.3). It is interesting that the stereoconfiguration was never

independently determined throughout the chain of citations. Instead, the absolute

stereochemical assignments were correlated to previous literature through chemical

transformations to reference compounds or through comparison of CD spectra. In fact, it

is unclear how the original compound's absolute configuration was assigned in 1927

(Figure IV.3) because the first absolute stereochemical assignments (tartaric acid) were

made in 1951 by Bijvöt et al. through X-ray crystallography.11

 135

Figure IV.2: Dihedral angles predicted from the 1H-1H coupling constants and Altona
equation (A) and select ROESY correlations for 7 and 8 (B).

Scheme IV.2: Total synthesis of (+)-grenadamide (13) by Baird et al.

Here lies the weakness of the modern scientific research; so much of our work

relies heavily on the previous work, but often without any verification due to time

constraints. It is therefore admirable that so many natural product chemists have re-

visited the structures of natural products that were previously characterized and have
 136

Figure IV.3: History of the absolute stereochemical assignment of cyclopropane

derivative 11

found a significant number of them to be incorrect through more or less independent

means.12 Synthetic chemists, by default, synthesize the reported structures and are able to

discover errors in the structure assignment as an additional benefit of the synthetic work.

However, re-visiting a natural product's structure is not an activity that natural product

chemists often perform as it requires much diligence and rigor.

 137

Although it would be difficult to determine at what point in the chain of citations

any erroneous stereochemical assignments may have occurred, it is relatively trivial to

independently verify the absolute configurations of the cyclopropane starting material 11

used by Baird et al. Thus, the synthesis of 11 was performed according to literature.13 X-

ray crystallography was considered the best choice of stereochemical assignment due to

its unequivocal nature. However, various derivatives of 11 could not be crystallized in a

suitable form for crystallographic work.

Subsequently, 11 was converted to 15 for modified Mosher's analysis (Scheme

IV.3). Careful 1D and 2D NMR analysis of 15 and 16 indicated that the C-2 position had

S configuration, which is consistent with Baird's assignment. However, an NOE

correlation was observed between H-1 and H-4, which suggested that the cyclopropane

ring had a trans relative stereochemistry (Figure IV.4). Considering that α-epimerization

of 14 was possible during chromium oxidation to the carboxylic acid (Scheme IV.3), the

C-2 stereocenter was assigned to be S, contradicting Baird's work. To independently

verify or discount this assignment, a suitable crystal of 15 was obtained for X-ray

crystallographic analysis. To our grief, the crystal structure revealed that the

Scheme IV.3: Synthesis of PGME derivatives from 11

Note that the stereochemistry of 14 is drawn as trans. However, it was later discovered to be cis
(vide infra).
 138

Figure IV.4: 1H and 13C NMR assignments and an NOE correlation of 16 in CDCl3
The structure is shown as trans due to the NOE correlation observed between H-1 and H-4.
However, the relative sterechemistry was later shown to be cis based on an X-ray crystal structure
of 15 (Figure IV.5). The 1H and 13C assignments were made through the use of COSY, HSQC, and
HMBC spectra in conjunction with the 1H and 13C NMR spectra. The 1H chemical shifts are
shown in red and the 13C chemical shifts are shown in blue.

cyclopropane ring retained its cis configuration (Figure IV.5). This result unequivocally

assigns the absolute configurations of 1-m to be 1S, 2S (Figure IV.6), thereby re-

affirming Baird's assignment of grenadamide.

The final explanation to be evaluated for reconciling our work and Baird's work

was to ascertain the validity of optical rotation values obtained for 1-m and 1-c. Of the

many structure determination tools utilized by natural product chemists today, the

polarimeter is perhaps the most error-prone instrument. It is extremely sensitive to chiral

contamination, and depending on the condition of the light source, different values can be

obtained for the same sample. Moreover, unlike spectra obtained from NMR or MS,

optical rotation values usually do not leave substantial records of their measurement. The

optical rotation value obtained by C. Nannini ([α]D –7.6° for 1-c)1 was consistent with
 139

Figure IV.5: X-ray crystal structure of 15

This crystal structure unequivocally showed that the stereochemistry of the carboxylic acid 14 is
cis. The NOE correlation between the proton on C24 and C21 (Figure IV.4) could not be
explained.

this work ([α]D –9.8° for 1-c). Therefore, it is unlikely that any human error, such as

mislabeling of the samples, was introduced.

IV.4 Conclusions

A pair of enantiomeric natural products were isolated from the same genus

collected from different locations. We developed a new concept for determining absolute

stereochemistry of natural products (i.e. the SISTER method, Figure IV.7). However, X-

ray crystallographic investigation indicated that either our ROESY and J-based analysis

was incorrect or one of the optical rotation values was wrong. It may require a re-

collection of the algal material to resolve the confusion that has plagued this project.

Figure IV.6: Corrected stereochemistry of 1-m and 1-c

 140

However, as shown in Figure IV.3, the ultimate origin of the absolute stereochemical

assignment of the cyclopropane derivative 11 is unclear. Therefore, it is possible that the

absolute configurations of 11 were never fully determined, but that its arbitrary

assignments happened to be the correct ones. After all, there is always a fifty-fifty chance

in the art of assigning absolute stereochemistry.

IV.5 Experimental Section

General

UV and IR spectra were recorded on a Beckman DU 640B UV spectrophotometer

and a Nicolet 510 spectrophotometer, respectively. NMR spectra were recorded either at a
1
H resonance frequency of 600.04 MHz (Bruker DRX 600) or 400.13 MHz (Bruker DPX

400). The Bruker DRX 600 was equipped with a Bruker Q-Switch TXI probe and the

DPX 400 was equipped with a Bruker BBO probe. All chemical shifts are reported

relative to residual CHCl3 (δH 7.27, δC 77.23) or C6HD5 (δH 7.16) as internal standard.

HRMS were obtained on a Kratos MS 50 TC mass spectrometer. Optical rotations were

measured with a Perkin-Elmer model 243 polarimeter. HPLC was performed using

Waters 515 pumps and a Waters 996 photodiode array spectrophotometer. TLC grade

(10-40 mesh) silica gel was used for vacuum chromatography, and Merck aluminum-

backed TLC sheets (Silica gel 60 F254) were used for TLC. All solvents were purchased

as HPLC grade. THF and triethylamine were freshly distilled before they were used

(from Na-benzophenone and CaH2, respectively). (R)-and-(S)-4-benzyl-2-oxazolidinones,

fatty acids (1-m and 1-c), and the amide derivatives (7, 8, and 9) were dried with a
 141

vacuum pump before use in synthetic reactions. Other reagents were used without any

purification unless specified. All reagents, except for the natural fatty acids 1-m and 1-c

and the solvents were purchased from Sigma-Aldrich. All reactions were carried out

under argon.

Figure IV.7: Synthetic Introduction of a Stereochemical Reference (SISTER) method

conceptual figure
The blue arrows indicate the flow of stereochemical information from a synthetically introduced
chiral group (gray ball).

Isolation of (1'S, 2'S)-3'-(2-heptyl-cyclopropyl)-propionic acid (1-m) and (1R, 2R)-3΄-

(2-heptyl-cyclopropyl)-propionic acid (1-c). Metabolite 1-m was isolated from the

crude extract of a Madagascar collection of L. majuscula by silica gel flash

chromatography. The eluent composition was gradually changed from CH2Cl2 to

acetonitrile/CH2Cl2 (1:9). The same procedure was used to purify 1-c from a Curaçao

collection of this cyanobacterium. A total of 42.7 mg of crude 1-m and 23.3 mg of crude

1-c were isolated. Due to the inherently poor chromatographic properties of these fatty

acids, these crude materials with relatively low purity were used for subsequent reactions

without additional purification.

 142

Synthesis of (1′R, 2′R, 4′′′S)-4′-Benzyl-3′′′-[3-(2′-heptyl-cyclopropyl)-propionyl]-

oxazolidin-2′′′-one (5). In 300 μL of THF were dissolved 23.3 mg crude 1-c which was

cooled to -78°C. To this solution were added pivaloyl chloride (17 μL, 0.14 mmol) and

triethylamine (16 μL, 0.11 mmol). The reaction mixture was stirred and allowed to warm

to RT from -78°C over a period of 1 hour. Meanwhile, (S)-(-)-4-benzyl-2-oxazolidinone

(58.7 mg, 0.331 mmol) was dissolved in 300 μL of THF and cooled to -78°C. To this

solution was added 185 μL of 2.0 M n-butyllithium solution in cyclohexane (0.37 mmol)

and the reaction mixture was stirred for 20 minutes. The lithiated oxazolidinone solution

was transferred to the solution of the mixed anhydride at -78°C. This reaction was stirred

and allowed to warm up to RT over 24 hours, and then quenched with 5 mL of aqueous 1

M NH4Cl solution and the aqueous layer extracted with CH2Cl2 (3 × 5 mL). The organic

layer was subsequently washed with 5 mL of H2O and dried with MgSO4. The

concentrated mixture was analyzed by TLC (MeOH/CH2Cl2, 1:19) which showed that no

starting material (2) was present, thus indicating a yield >95%. The resultant mixture was

purified by silica gel flash chromatography with CH2Cl2 as eluent to yield 36.2 mg of a

pale-yellow oil. This was used for subsequent reactions without further purification. 1H

NMR (CDCl3; 400MHz) δ 0.23 (2H, m, 3′-CH2), 0.49 (2H, m, 1′-CH and 2′-CH), 0.88

(3H, t, J = 6.9 Hz, 7′′-CH3), 1.26 (12H, m, alkyl chain), 1.60 (2H, m, 3-CH 2), 2.75 (1H,

dd, J = 13.2 Hz, 9.7 Hz, Bn-CH2), 3.02 (2H, m, 5′′′-CH2), 3.31 (1H, dd, J = 13.3 Hz, 3.0

Hz, Bn-CH2), 4.18 (2H, m, 2-CH), 4.67 (1H, m, 4′′′-CH), 7.19-7.36 (5H, m, phenyl); MS

(FAB) 372.1 (M+1). Likewise, semi-purified compound 4 (111.4 mg, crude weight) was

synthesized in the same manner starting with 42.7 mg of crude 1-m.

 143

Synthesis of (1′R, 2′R, 2S, 4′′′S)-4′′′Benzyl-3′′′-[3-(2′-heptyl-cyclopropyl)-2-methyl-

propionyl]-oxazolidin-2´΄΄-one (7). The amide 5 (31.2 mg, 84 μmol) was dissolved in

400 μL of THF and the solution was cooled to -78°C. Lithium hexamethyldisilazide (95

μL, 1.0 M solution in THF, 95 μmol) was added dropwise and the mixture was stirred for

20 minutes. Excess methyl iodide (55 μL) was added dropwise at -78°C and the reaction

mixture was allowed to warm to RT over 6 hours. The reaction was then quenched with 4

mL of NH4Cl aqueous solution and extracted with CH2Cl2 (3 ×5 mL). The combined

organic layers were washed with 2 mL of H2O and then dried with MgSO4. The resulting

mixture was concentrated under reduced pressure and passed through a short silica gel

column followed by a C18 column (eluents were CH2Cl2 and MeOH, respectively). After

evaporation of solvents under reduced pressure, 27.7 mg of crude material was recovered,

and this was purified by HPLC using a reverse phase semi-preparative column (YMC,

ODS-AQ, 250 × 10 mm I.D., 5 μm, 120 Å; H2O/MeOH, 0.7:99.3). The major compound

in the reaction mixture was the by-product, pivaloyl oxazolidinone; however, 2.1 mg of 7

were isolated. Based on the HPLC peak integration of 4 and 7 in the reaction mixture, the

yield of 7 was estimated to be 70%. Only one isomer of the methylated product was

observed. 8 and 9 were synthesized in the same manner and only one diastereomer was

observed in each synthesis (50% and 60% yields, resepectively). Colorless oil; UV λ max

255 nm; IR υ 2923cm-1, 1776 cm-1, 1697cm-1; 1H NMR (C6D6; 400MHz) δ 0.27 (2H, m,

3′-CH2), 0.58 (1H, m, 1′-CH), 0.62 (1H, m, 2′-CH), 0.91 (3H, t, J = 6.8 Hz, 7′′-CH3), 1.29

(12H, m, alkyl chain), 1.34 (3H, d, J = 7.0 Hz, 2-CCH3), 1.46 (1H, ddd, J = 13.6 Hz, 6.5

Hz, 6.4 Hz, pro-S-3-CH2), 1.84 (1H, ddd, J = 13.6 Hz, 7.2 Hz, 7.2 Hz, pro-R-3-CH2), 2.29

(1H, dd, J = 13.4 Hz, 9.3 Hz, Bn-CH2), 2.97 (1H, dd, J = 13.4 Hz, 3.0 Hz, Bn-CH 2), 3.20
 144

(1H, dd, 5′′′-CH2), 3.46 (1H, dd, J = 9.1 Hz, 2.6 Hz, 5′′′-CH2), 4.14 (1H, ddt, J = 7.2 Hz,
13
7.0 Hz, 6.4 Hz, 2-CH), 4.25 (1H, m, 4′′′-CH), 6.90-7.10 (5H, m, phenyl); C NMR

(CDCl3; 100 MHz) δ 12.4, 14.3, 16.7, 17.5, 18.9, 22.9, 29.6, 29.7, 29.8, 32.1, 34.4, 38.1,

38.3, 38.4, 55.6, 66.2, 127.6, 129.2, 129.7, 135.6, 153.3, 177.5. [α]D28 = + 48° (c = 1.2,

CHCl3).

(1′R, 2′R, 2S, 4′′′S)-4′′′-Benzyl-3′′′-[3-(2′-heptyl-cyclopropyl)-2΄-methyl-propionyl]-

oxazolidin-2′′′-one (8). Colorless oil; UV λ max 257 nm, IR υ 2918cm-1, 1776 cm-1,

1692cm-1 ; 1H NMR (C6D6; 400MHz) δ 0.23 (1H, m, 3′-CH2), 0.39 (1H, m, 3′-CH2), 0.45

(1H, m, 2′-CH), 0.59 (1H, m, 1′-CH), 0.92 (3H, t, J = 6.9Hz, 7′′-CH3), 1.29 (12H, m,

alkyl chain), 1.33 (3H, d, J = 7.0 Hz, 2-CCH3), 1.43 (1H, ddd, J = 13.7 Hz, 6.9 Hz, 6.0

Hz, pro-S-3-CH2), 1.84 (1H, ddd, J = 13.7 Hz, 7.3 Hz, 7.1 Hz, pro-R-3-CH2), 2.30 (1H,

dd, J = 13.4 Hz, 9.7 Hz, Bn-CH2), 2.98 (1H, dd, J = 13.5 Hz, 2.7 Hz, Bn-CH 2), 3.22 (1H,

dd, 5′′′-CH2), 3.47 (1H, dd, J = 9.0 Hz, 2.5 Hz, 5′′′-CH2), 4.16 (1H, ddq, 7.3 Hz, 7.0 Hz,

6.0 Hz, 2-CH), 4.23 (1H, m, 4′′′-CH), (5H, m, phenyl); 13C NMR (CDCl3, 100MHz) δ

11.9, 14.3, 16.7, 17.4, 19.3, 22.9, 29.6, 29.7, 29.8, 32.1, 34.1, 38.1, 38.2, 55.7, 66.2,

127.5, 129.1, 129.7, 135.6, 153.3, 177.6. [α]D28 = + 42° (c = 2.5, CHCl3).

(1′S, 2′S, 2R, 4′′′R)-4′′′-Benzyl-3′′′-[3-(2′-heptyl-cyclopropyl)-2΄-methyl-propionyl]-

oxazolidin-2′′′-one (9). Colorless oil; IR υ 2924cm-1, 1782 cm-1, 1693cm-1 ; 1H NMR

(C6D6; 400MHz) δ 0.23 (1H, m, 3′-CH2), 0.39 (1H, m, 3′-CH2), 0.45 (1H, m, 2′-CH), 0.59

(1H, m, 1′-CH), 0.92 (3H, t, J = 6.9Hz, 7′′-CH3), 1.29 (12H, m, alkyl chain), 1.33 (3H, d,
 145

J = 7.0 Hz, 2´-CCH3), 1.43 (1H, ddd, J = 13.7 Hz, 6.9 Hz, 6.0 Hz, pro-S-3´-CH2), 1.84

(1H, ddd, J = 13.7 Hz, 7.3 Hz, 7.1 Hz, pro-R-3´-CH2), 2.30 (1H, dd, J = 13.4 Hz, 9.7 Hz,

Bn-CH2), 2.98 (1H, dd, J = 13.5 Hz, 2.7 Hz, Bn-CH2), 3.22 (1H, dd, 5´´-CH2), 3.47 (1H,

dd, J = 9.0 Hz, 2.5 Hz, 5´´-CH2), 4.16 (1H, ddq, 7.3 Hz, 7.0 Hz, 6.0 Hz, 2´-CH), 4.23

(1H, m, 4´´-CH); [α]D28 = – 25° (c = 9.5, CHCl3).

Acknowledgement. We gratefully acknowledge helpful discussions with R. Kohnhert,

the OSU Biochemistry and Biophysics NMR facility for use of the Bruker DRX600

NMR instrument (V. Hsu), the OSU Mass Spectrometry Facility (J. Moore).
 146

Figure IV.8: 1H NMR spectrum of 7 in C6D6

 147

Figure IV.9: 1H NMR spectrum of 7 in CDCl3

 148

Figure IV.10: 13C NMR spectrum of 7 in CDCl3

 149

Figure IV.11: 1H-1H Homonuclear decoupling experiment for 7 in C6D6 – Part 1

 150

Figure IV.12: 1H-1H Homonuclear decoupling for 7 in C6D6 – Part 2

 151

Figure IV.13: 1H-1H Homonuclear decoupling experiment for 7 in C6D6 – Part 3

 152

Figure IV.14: 1H-1H COSY spectrum of 7 in C6D6

 153

Figure IV.15: ROESY spectrum of 7 in C6D6

 154

Figure IV.16: LR FAB MS spectrum of 7

 155

Figure IV.17: IR spectrum of 7

 156

Figure IV.18: 1H NMR spectrum of 8 in C6D6

 157

Figure IV.19: 1H NMR spectrum of 8 in CDCl3

 158

Figure IV.20: 13C NMR spectrum of 8 in CDCl3

 159

Figure IV.21: 1H-1H Homonuclear decoupling experiment for 8 in C6D6 – Part 1

 160

Figure IV.22: 1H-1H Homonuclear decoupling experiment for 8 in C6D6 – Part 2

 161

Figure IV.23: 1H-1H Homonulcear decoupling experiment for 8 in C6D6 – Part 3

 162

Figure IV.24: 1H-1H COSY spectrum of 8 in C6D6

 163

Figure IV.25: ROESY spectrum of 8 in C6D6

 164

Figure IV.26: IR spectrum of 8

 165

Figure IV.27: 1H NMR spectrum of 9 in CDCl3

 166

Figure IV.28: IR spectrum of 9

 167

Figure IV.29: Geometry optimized 3D structures of 7 (above) and 8 (below)

The best conformers were obtained through conformer distribution calculation (molecular
mechianics) and their geometry was optimized using ab initio calculations (HF 3-21G*). The
stereoisomistry depicted here are what was predicted based on the SISTER method. However,
the stereochemistry of the cyclpropanes were later determined to be the opposite of what is
depicted here (see the text above).
 168

Figure IV.30: HPLC chromatogram for 4 and 7

 169

Figure IV.31: HPLC chromatogram for 5 and 8

 170

Figure IV.32: UV spectra of 7 and 8

 171

References and footnotes

1 Nannini, C. J. Novel secondary metabolites from a Madagascar collection of Lyngbya

majuscula. 2002, M.S. Thesis.

2 Complete purification of the fatty acids themselves were not possible by normal means.

3 (a) Teng, Rong-Wei; Shen, Ping; Wang, De-Zu; Yang, Chong-Ren., Bopuxue Zazhi, 2002,
19(2), 203-223. (b) Harada, Nobuyuki; Watanabe, Masataka; Kosaka, Masashi; Kuwahara,
Shunsuke., Yuki Gosei Kagaku Kyokaishi, 2001, 59(10), 985-995. (c) Kusumi, Takenori;
Ohtani, Ikuko I., Biology-Chemistry Interface, 1999, 103-137

4 Haasnoot, C. A.; De Leeuw, F. A. A. M.; Altona, C. The relationship between proton-proton

NMR coupling constants and substituent electronegativities—I : An empirical generalization
of the Karplus equation. Tetrahedron. 1980, 36, 2783-2792.

5 Barfield, M; Smith, W. B. Internal H-C-C angle dependence of vicinal 1H-1H coupling

constants. J. Am. Chem. Soc. 1992, 114, 1574-1581

6 See the experimental section for the geometry optimized 3D structures of 7 and 8.

7 Al Dulayymi, J. R.; Baird, M. S.; Jones, K. The absolute stereochemistry of grenadamide.

Tetrahedron 2004, 60, 341-345.

8 Coxon, G. D.; Al-Dulayymi, J. R.; Baird, M. S.; Knobl, S.; Robertsa, E.; Minnikin , D. E. The
synthesis of (11R,12S)-lactobacillic acid and its enantiomer. Tetrahedron Asymm. 2003, 14,
1211-1222.

9 Avery, T. D.; Culbert, J. A.; Taylor, D. K. The first total synthesis of natural grenadamide.
Org. Biomol. Chem. 2006, 4, 323-330.

10 Alternative interpretations of the ROESY correlations were considered. However, the original
interpretations were more consistent with the molecular modeling results.

11 Bijvöt, J. M.; Peerdeman, A. F.; van Bommel, A. J. Determination of the absolute

configuration of optically active compounds by means of X-rays. Nature 1951, 168, 271-272.

12 See Chapter I of this dissertation

13 Grandjean, D.; Pale, P.; Chuche, J. Enzymatic hydrolysis of cyclopropanes. Total synthesis of
optically pure dictyopterenes A and C. Tetrahedron 1991, 47, 1215-1230.
 Chapter V

Use of biomimetic starting materials for expedient total synthesis of epiquinamide

enantiomers: true identity of bioactive metabolite

172
 173

Abstract

A quinolizidine alkaloid isolated from a rainforest frog, Epipedobates tricolor,

epiquinamide was initially regarded as a neuropharmacologically important compound

due to its apparent selectivity for a nicotinic acetylcholine receptor subtype. In order to

facilitate pharmacological and absolute stereochemical investigation of the molecule,

concise and scalable syntheses of epiquinamide enantiomers were accomplished with

high overall yields and high stereoselectivity from readily commercially available

materials. However, it was found that none of the stereoisomers of epiquinamide was

responsible for the observed bioactivity. Through this error, the importance of total

synthesis of a natural product is clearly highlighted.

 174

V.1 Introduction

Figure V.1: Frog skin alkaloids of various core skeletons

In order to discover novel natural products with biomedically relevant

bioactivities, relatively untapped sources including amphibian skin have been

investigated in the recent decades.1,2,3 In particular, rainforest frogs have proven to be a

rich source of bioactive and structurally complex alkaloids (Figure V.1).1,2 Typically,

these alkaloids have pyrrolidine (1), piperidine (2), pyrrolizidine (3), indole (4),

indolizidine (5), quinolizidine (6), or decahydroquinoline (7) core structures with

relatively low degrees of unsaturation.1,2 Having stereoelectronics similar to endogenous

alkaloidal compounds essential to neurological functions in higher animals, these

secondary metabolites can have a variety of neurological activities both in vitro and in

vivo.3 Many of the frog skin natural products have been traced back to the amphibian's

dietary sources, such as insects and other arthropods.1 However, the ultimate source may
 175

be of microbial origin in some cases.4 This is an ecological theme parallel to that seen

with microbial natural products isolated from marine invertebrates.4,5

Epipedobates tricolor
Photograph from reference 3
Figure V.2: Skin of Epipedobates tricolor contains epibatidine and epiquinamide

Epibatidine (8) was isolated from an Ecuadorian frog Epipedobates tricolor

(Figure V.2) in 1992 by Daly and co-workers and has become one of the cornerstone

compounds in the field of nicotinic acetylcholine receptor (nAChR) studies.2,3,6

Epiquinamide (9) was isolated along with 8 from the same frog species in 20037 and has

attracted much attention due to its potent and selective agonistic activities against β2

nicotinic receptors.2,3,7 Its potential to be a lead compound in the development of

pharmacological agents seemed promising, following the history of epibatidine.6,8

Although a plethora of nicotinic receptor agonists have been discovered, none of

them are known to be sufficiently selective for subtypes of the nicotinic acetylcholine

receptors (nAChR).9 There are varying abundances of nAChR subtypes in different

tissues of the human body, and it is conceivable that by selectively targeting a certain

tissue, the possibility of side effects could be reduced.9 However, the major limiting

factor in testing this compound was its low availability from nature (240 μg from the
 176

Scheme V.1: Reduction of dehydropiperidine: two stereochemical outcomes

Scheme V.2: Applying the synthetic logic of quinolizidine biosynthesis to the synthesis of
epiquinamide skeleton

skins of 183 frogs).7 To meet this need and to explore new chemistry for the construction

of the novel quinolizidine skeleton, a number of research groups have undertaken the

total synthesis of 9.10 To date, there have been four11,12,13,14 enantiospecific total syntheses

of (+)-9 and three12,15,16 enantiospecific total synthesis of (−)-9, respectively. In addition,

there have been two racemic syntheses of 917,18 and two syntheses of the epimer of 9
 177

Scheme V.3: Biomimetic synthesis of lupinamine by Wanner and Koomen

(15).12,19 In the literature on epiquinamide, the optical rotation value of the natural product

was never reported due to its low availability.7 Access to both enantiomers was therefore

of great importance.

V.2 Rationale for epiquinamide synthesis strategy

Scheme V.4: Initial Retrosynthetic Analysis of Epiquinamide

The quinolizidine system could be derived from a piperidine with appropriate

appendages that could be cyclized to give the second ring. The initial synthetic strategy

was formed around the reductive amination reaction to furnish the bicyclic tertiary amine

(Schemes IV.1 through IV.3). This idea was inspired by the observation that there had

been little effort to synthesize cis-2,3-disubstituted piperidines.20,21,22 Some precedence

exists in the literature that suggested that such reductive amination using H2 would give

trans stereochemistry via chelation control22 (Scheme V.2; H2 reaction) while cis

stereochemistry would be achieved if hydride was used20 via a Felkin-Anh type transition

state (Scheme V.2; hydride reaction).23 This latter stereochemical outcome was expected24
 178

Scheme V.5: Total synthesis of (1R,9S)-epi-epiquinamide (16)

Figure V.3: X-ray crystal structure of epi-epiquinamide (15)

for the stereoselective reduction of 2 based on the assumption that the protecting group

would not chelate to the boron atom of NaBH3CN under acidic conditions.

The biosythesis of quinolizidine is generally thought to involve a lysine residue

(Scheme V.3).25 Being inspired by nature's strategy, it was realized that utilization of

ornithine could result in retention of the chiral amine group and stereochemical guidance
 179

by the same group (Scheme V.4). This strategy eliminates the need for introducing

chirality at any point in the synthesis. In order to maximize the flexibility of the

synthesis, a biomimetic approach can be utilized without strictly adhering to a synthetic

route that employs the exact chemistry used in the biosynthesis. For example, Koomen

and Wanner have achieved biomimetic synthesis of quinolizidine alkaloids including

lupinamine (Scheme V.5).26 If the same strictly biosynthetic approach was applied to

epiquinamide, it would be difficult to exert enantioselectivity. By starting out with a

naturally occurring chiral molecule and using it in a biomimetic manner, the synthesis

can be both expedient and cost-effective.

V.3 Attempts to synthesize epiquinamide

The cyclization via reductive amination, however, yielded trans-piperidine in

excellent de (13). The relative stereochemistry of this product was confirmed by an X-ray

crystal structure of the Cbz-protected quinolizidine after the second cyclization (Figure

V.3). This result may be explained by an exchange of ligands on the boron atom; i.e., the

chelation model. If the oxygen atom of the Boc group becomes one of the ligands, the

tethered cyanoborohydride would transfer its hydride from the Si face (when R and R' are

those of 9 in Scheme V.2) rather than the Re face.

Subsequently, two alternative routes that utilize an acyliminium ion as a key

intermediate were devised (Schemes IV.6 and IV.7). One route features addition of vinyl

borylate to the acyliminium ion27 (from 13) and the other route centers around a hetero

Diels-Alder reaction28 with the acyliminum ion (from 22) as the dienophile. Despite some
 180

Scheme V.6: Proposed total synthesis of (+)-epiquinamide via boron nucleophilic

addition to acyl imminium ion

Scheme V.7: Proposed total synthesis of (+)-epiquinamide via hetero Diels-Alder

 181

of the promising precedents in the literature, these approaches proved unsuccessful due to

complications with the unstable acyliminium ion.

V.4 Total synthesis of epiquinamide

According to the chelation model, borohydride reduction of the ketone 29 should

give the anti configuration.29,30 Using a similar synthetic logic as applied to the first

attempted synthesis (Scheme V.1), the first piperidine ring can be synthesized via

cyclization through SN2 displacement as shown in Scheme V.7. The substrate for the SN2

reaction can be easily derived from a corresponding amino alcohol, 　 such as 30, which

could be synthesized from ornithine.

Scheme V.8: Retrosynthetic analysis of (+)-epiquinamide

The synthesis of the intramolecular SN2 substrate began with a commercially

available ornithine derivative 10, which was first converted smoothly to the Weinreb

amide 29 using a common coupling condition (Scheme V.8).31,32 After simple washings,

the product was fairly pure and required no further purification. Upon treatment with an
 182

allyl Grignard reagent,33 we obtained the ketone 30 as white crystals. Chelation-

controlled hydride reduction of 30 yielded a highly crystalline alcohol, 28 (Scheme

V.10).30,34 By 1H and 13
C NMR, none of the other diastereomer was observed in the

crystallized product, whose stereochemistry was ultimately proven by completion of the

total synthesis and comparison to epi-epiquinamide (15).35 Mesylation of the amino

alcohol proceeded smoothly with an excellent yield, and again the product was

crystalline. Therefore, the synthesis of the SN2 substrate 31 was accomplished very

conveniently and without chromatographic purification unless recovery of the residual

amount of the product in the filtrate was desired.

Transformation of the mesylate 31 to the title compound was accomplished as

shown in Scheme V.8. Removal of the Boc group in TFA/CH 2Cl2 followed by

intramolecular SN2 cyclization induced by K2CO3 and subsequent N-alkylation in

acetonitrile yielded the diallyl piperidine 27 in good yield.36,37,38,39,40 The synthesis of the

Scheme V.9: Total synthesis of (+)-epiquinamide

 183

epiquinamide skeleton was concluded by ring-closing metathesis (RCM) reaction on 27

using the Grubbs second-generation catalyst.41,42 It is noteworthy that this was one of the

earlier examples of a basic and relatively unhindered N-allyl group undergoing

ruthenium-catalyzed RCM.43,44 The scope of functional group tolerance by the Grubbs

second-generation catalyst seems to be greater than the consensus at the time.42,43,44

However, the purification of this rather polar RCM product presented difficulties.45 The

problem was overcome when Cho and Kim’s method was employed with a small

modification.46 Since then, a phosphine-free ruthenium catalyst (second-generation

Hoveyda-Grubbs catalyst) became commercially available, thereby making some of the

purification issues obsolete.47,48

Scheme V.10: Chelation-controlled hydride reduction of ketone (30)

After the aluminum hydride source chelates with both the ketone oxygen and the carbamate
nitrogen, it transfers the hydride onto the less hindered face of the ketone. Subsequent hydrolysis
yields the reduced product 28.

Deprotection, alkene reduction, and acetylation were accomplished in one pot by

hydrogenation of 32 in ethanol with acetic anhydride (8 steps overall).49 A better yield

was observed when this process was separated into two steps as shown in Scheme V.8 (9

steps overall).11 By 1H and 13C NMR, a single isomer was observed. This material had the

identical 1H and 13C NMR and IR spectra and HR mass (EI) to those of the authentic

material.7 The reproducible overall yields of (+)-9 beginning from 10 were 38% and 28%
 184

for the longer and shorter sequences, respectively. The specific optical rotation value also

matched that of the material from the previous total synthesis ((+)-9 [α]22D = +24° (c

0.10, CHCl3)).50 Only three chromatographic purification steps were required throughout

the synthesis. The synthesis of the other enantiomer was performed in the same manner

starting with commercially available δN-Boc-αN-Cbz-D-ornithine. This latter synthesis

was accomplished in two weeks, demonstrating the practicality of this synthetic route

((-)-9 [α]22D = –22º, c 0.13, CHCl3).51 In comparison to other enantiospecific syntheses of

9 (12-15 steps),11-17 efficiency of this synthesis is outstanding. This is largely due to the

choice of a biomimetic starting material as mentioned earlier.

V.5 Bioactivity and absolute stereochemistry of epiquinamide

With both of the enantiomers of epiquinamide in hand, the next goal was to

determine the absolute stereochemistry of natural epiquinamide. Neither the optical

rotation value nor the circular

dichroism spectrum of the authentic natural product was ever reported.7 In the absence of

access to the natural sample for comparison in chiral chromatography with the two

synthetic enantiomers, the only plausible method of stereochemistry determination was to

compare the biological activity of the two isomers. If one was as active as the reported

natural product, but not the other stereoisomer, then it can be concluded that the former

isomer possesses the natural stereo-configuration.

However, neither of the compounds were active in the in-house Na+ channel

activation and blocking activity assays using neuroblastoma cells (Neuro 2a),52 the

cytotoxicity assays using human lung cancer cells (H460), or the brine shrimp assay.
 185

Furthermore, Gallagher and coworkers independently reported that racemic epiquinamide

was inactive in their competitive binding assays using [3H]epibatidine.53 With all of these

results combined, it was considered highly likely that the active compound in the original

sample from the frogs was not epiquinamide, but some other exceptionally bioactive

contaminant.

Two years later, Rutjes and coworkers compared their synthetic standards of both

enantiomers of epiquinamide with the authentic sample and determined that the natural

compound is the dextrorotatory isomer ((1S,9S)-(+)-epiquinamide).13 Subsequently, Fitch

and coworkers reported GCMS evidence that the observed bioactivity of epiquinamide

was due to a trace amount of cross-contamination of epibatidine (8).19 This rather

unfortunate finding demonstrates yet another benefit of total synthesis of a natural

product; the total synthesis helps to confirm the identity of the bioactive metabolite.

“While these data are somewhat embarrassing to report, they are not
entirely unusual in the isolation of natural products. Further, the results are
a reminder that the presence of multiple active substances in an extract (in
this case compounded by small amounts of sample available) can
sometimes lead to erroneous results from cross-contamination that are not
always evident a priori. This underscores the importance of obtaining
synthetic material whenever possible to corroborate both structure and
pharmacology. In this case, we were able to produce a correct structure,
but were misled by contamination in the pharmacology. Often with more
complex structures, the converse occurs. Thus,it is important that the
pharmacognosist work in collaboration with the synthetic/medicinal
chemist to produce data that are unequivocal.”
Fitch et al, 200919

V.6 Conclusion

Epiquinamide, a trace natural product isolated from amphibian skin in an effort to

discover a subtype selective nAChR agonists, was enantioselectively synthesized from a

 186

biomimetic starting material in an extremely concise and efficient manner. The

unfortunate realization that the structure 9 does not possess the reported bioactivity

further confirms the contemporary need for total synthesis of novel natural products.

V.7 Experimental Section

General

Unless noted otherwise, all materials were purchased from commercially available

sources and were used without further purification. Anhydrous dichloromethane and

diethyl ether were purchased from VWR. All reactions were carried out under dry argon

atmosphere unless otherwise noted. Flash chromatorgraphy was performed using Fisher

silica gel (230-400 mesh). TLC was performed using EM Science pre-coated silica gel

plates (Merck 60 F254). Melting points were determined on an Electrothermal Mel-Temp

device and are uncorrected. Optical rotations were measured on a Rudolph Research

Autopol III polarimeter and a Jasco P1010 polarimeter. IR spectra were recorded on a

Nicolet Magna-IR 550. For 11-16, 1H NMR and 13C NMR spectra were recorded on a

Bruker spectrometer (300 MHz and 75 MHz, respectively). For 27-32 and 9, 1H NMR

and 13C NMR spectra were recorded on a Varian Inova spectrometer (500MHz) or on a

Varian Inova spectrometer (300 MHz and 75 MHz, respectively). 1H and 13

C spectra

recorded in CDCl3 were referenced to the residual solvent peaks at 7.26 ppm and 77.0

ppm, respectively. High resolution mass spectra were recorded on a ThermoFinnigan

MAT900XL spectrometer.
 187

(S)-methyl 12,12-dimethyl-3,10-dioxo-1-phenyl-2,11-dioxa-4,9-diazatridecane-5-

carboxylate (11). To αN,δN-protected ornithine 10 (718 mg, 1.96 mmol) in CH2Cl2 (10

mL) at 0 ºC were added anhydrous MeOH (300 μL, 7.4 mmol), DMAP (43 mg, 0.35

mmol), EDC·HCl (403 mg, 2.10 mmol), and Et 3N (280 μL, 2.0 mmol) successively. After

the reaction mixture was warmed to rt over night under stirring, the volatiles were

removed under vacuum. The residues were resuspended in Et2O. The solution was

washed with 0.5 M HCl, 1M NaHCO3, and brine successively. Upon drying over

Na2SO4, the solution was passed through a silica gel plug (1:1 EtOAc / Hex) and the

solvents were removed under vacuum. Colorless gummy substance (11; 703 mg, 1.85

mmol, 94%) was obtained, and it was pure enough for the subsequent reactions. TLC Rf

= 0.43 (1:1 EtOAc / Hex). 1H and 13C NMR spectra: see Figures V.4 and 5.

(S)-αN-Cbz, δN-Boc 6,9-diaminonon-1-en-5-one (12). To the suspension of CuCN (330

mg, 3.68 mmol) in THF (7 mL) at −78 ºC was added 1.0 M vinyl magnesium bromide

solution in THF (50 mL) over ~7 min. The solution of 11 (3.14 g, 8.25 mmol) in THF (30

mL) was cannulated in over 5 min. The reaction mixture was gradually warmed to rt over
 188

night under stirring. The mixture turned black. After cooling to 0 ºC, the reaction was

quenched by addition of 1M HCl (200 mL) and the mixture was stirred for 15 min. Then

the mixture was extracted with Et2O thrice. The combined organic layer was washed with

1M NaHCO3, water, and brine successively. Upon drying over Na2SO4, the solution was

concentrated under vacuum and the residues were subjected to flash column

chromatography to obtain off-white solid (12; 3.056 g, 7.56 mmol, 92%). TLC Rf = 0.57

(1:1 EtOAc / Hex). 1H and 13C NMR spectra: see Figures V.6 and 7.

(2R,3S) benzyl-2-(but-3-enyl)piperidin-3-ylcarbamate (13). To the solution of the

ketone 12 (1.72 g, 4.27 mmol) in CH2Cl2 (25 mL) at rt was added TFA (25 mL). The

solution was stirred at rt for 1 hr and the volatiles were removed under vacuum. The oily

residues were dissolved in i-PrOH (40 mL) and the solution was cooled to 0 ºC. NaOH

solution in EtOH was added to adjust the pH to ~3. NaBH3CN (1.38 g, 22.0 mmol) was

added in two portions and the solution was warmed to rt over night under stirring. Then

1M HCl (30 mL) was added to quench the reaction. Evolution of H 2 gas was observed.

The volatiles were removed under vacuum. To the residues were added saturated

NaHCO3 and the aqueous layer was extracted with CH2Cl2 twice and CHCl3 twice. The

combined organic layer was dried over Na2SO4. Upon removal of the solvents under

vacuum, the residues were subjected to flash column chromatography using silica gel
 189

(0.5% Et3N buffer, 1:49 MeOH / CH2Cl2 to 1:4 MeOH / CH2Cl2) to obtain the product

(13; 325 mg, 1.13 mmol, 26% over two steps). 1H and 13C NMR spectra: see Figures V.8

and 9.

(2R,3S) benzyl-2-(4-hydroxybutyl)piperidin-3-ylcarbamate (14). No experimental

details. 1H and 13C NMR spectra: see pages Figures V.10 and 11.

(1S,9aR) benzyl-octahydro-1H-quinolizin-1-ylcarbamate (15). To the solution of the

amino alcohol 14 (56 mg, 0.183 mmol) in CH2Cl2 were added CBr4 (91 mg, 0.27 mmol)

and PPh3 (120 mg, 0.46 mmol) successively at 0 ºC under stirring. The solution was

warmed to rt quickly after 30 min, and it was kept at rt for 2.5 hrs. Then Et3N (64 μL,

0.46 mmol) was added, which made the solution turn yellow. The solution was left

stirring at rt for over night. Upon quenching the reaction with 1M HCl (30 mL), the

organic phase was washed with 1M HCl (20 mL x 3). The combined aqueous phase was

basified with solid NaOH, and it was extracted with CH2Cl2 (30 mL x 4). Upon drying

over K2CO3 and removal of the solvent under vacuum, the residues were subjected to
 190

flash column chromatography using silica gel (1:9 MeOH / CH2Cl2). However, the

product was still contaminated with phosphine compounds and it was further purified by

semi-prep HPLC using a Synergi 4μ Hydro-RP 80A (250 x 10 mm) column (1.2 mL /

min H2O to 1.5 mL / min 5:3 MeCN / H 2O) with acetic acid buffer (0.1%). The product

(15) had a retention time of 6.02 min (31 mg, 0.11 mmol, 59%). 1H and 13C NMR spectra:

see Figures V.12 and 13.

9-epi-epiquinamide (16). No experimental details. 1H and 13C NMR spectra: see Figures

V.14 and 15. GCMS data: see Figure V.16.

(2S)-2-benzyloxycarbonylamino-5-tert-butyloxycarbonylamino-(N-methoxy-N-

methyl)pentanoyl amide (29). To αN,δN-protected ornithine 10 (7.285 g, 19.88 mmol)

in CH2Cl2 (80 mL) stirring vigorously at 0 ºC was added N,O-dimethylhydroxylamine

hydrochloride (2.180 g, 22.35 mmol), Et3N (3.78 mL, 26.90 mmol), (3-dimethylamino-

propyl)-ethyl-carbodiimide hydrochloride (4.216 g, 21.99 mmol), and 4-dimethylamino

pyridine (240 mg, 1.96 mmol) sequentially. The resulting solution was left stirring to

warm up to room temperature over night (18 hours). The volatiles were removed on a
 191

rotary evaporator and the residue was suspended in ethyl acetate (200 mL). The insoluble

solids were filtered off and rinsed with EtAcO. The filtrate was sequentially washed with

0.2 M HCl, water, 1.0 M NaHCO3, water, and brine and was dried over MgSO4. After

filtration using Celite®, the solvents were removed under vacuum and a colorless glass

(29) was obtained and was used without purification for the next step. (7.564 g, 18.47

mmol, 93%). TLC Rf = 0.30 EtAcO/hexane (3:2). [α]D24 –6.3º (c 1.20, CHCl3). IR (film)

3332, 3080, 3024, 2975, 2936, 1709, 1654, 1530, 1450, 1398, 1367, 1250, 1160 cm -1. 1H

NMR (500 MHz, CDCl3) δ 7.26-7.36 (m, 5H), 5.53 (d, J = 9.0 Hz, 1H), 5.08 (m, 2H),

4.73 (m, 1H), 4.57 (m, 1H), 3.77 (s, 3H), 3.20 (s, 3H), 3.12 (m, 2H), 1.70-1.80 (m, 2H),

1.49-1.61 (m, 2H), 1.41 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 172.3, 156.1, 155.8, 136.2,

128.4, 128.0, 127.9, 79.0, 66.8, 61.5, 50.6, 40.0, 30.0, 28.3, 25.8. HRMS (EI): calcd for

C20H32N3O6 (M + H): 410.2286, found: 410.2287.

(5S)-2-benzyloxycarbonylamino-5-tert-butyloxycarbonylamino-4-oxo-1-octene (30).

To the solution of allylmagnesium bromide in Et2O (1.0 M, 100 mL, 100 mmol) at -78 ºC

was added the solution of the Weinreb amide 29 (9.060 g, 22.13 mmol) in Et2O (135 mL)

dropwise over 1 hour while vigorously stirring. The resulting slurry mixture was warmed

to room temperature and stirred for 30 min. Upon cooling to -10 ºC, aqueous HCl

solution (1.0 M, 125 mL) was added carefully and the mixture was stirred for 5 min at

-10 ºC. After partitioning, the aqueous layer was extracted with Et 2O (30 mL × 3) and the
 192

combined organic layer was washed sequentially with cold 0.1 M HCl, cold water, cold

1.0 M NaHCO3, and brine, and then was dried over MgSO4. Upon filtration through

Celite®, the solvents were removed under vacuum. The residue was dissolved in a

mixture of Et2O and CH2Cl2, and hexane was added and the product was re-crystallized

(7.138 g, white needles). The filtrate was concentrated under vacuum and was purified by

flash chromatography (EtAcO / hexane 1:9 to 1:4). The separation of the double bond-

isomerized products from the desired product was difficult. The product (30) was a white

solid (total 7,420 g, 19.00 mmol, 86%). TLC Rf = 0.46 EtAcO / hexane (2:3). Mp 85~86

ºC. [α]D24 +46º (c 0.34, CHCl3). IR (film) 3332, 3080, 3025, 2975, 2936, 1693, 1639,

1525, 1452, 1250, 1168 cm-1. 1H NMR (500MHz, CDCl3) δ 7.29-7.37 (m, 5H), 5.82-5.96

(m, 1H), 5.55 (d, J = 7.2 Hz, 1H), 5.23-5.19 (m, J =11.0 Hz, 1H), 5.13-5.09 (m, J = 11.0

Hz, 1H), 5.09 (s, 2H), 4.57 (bs, 2H), 4.44-4.50 (m, 1H), 3.28 (d, J = 6.6 Hz, 2H),

3.09-3.17 (m, 2H), 1.86-1.97 (m, 2H), 1.46-1.59 (m, 2H), 1.43 (s, 9H). 13C NMR (75

MHz, CDCl3) δ 206.5, 156.0,156.0, 136.2, 129.5, 128.5, 128.2, 128.1, 119.5, 79.3, 67.0,

59.1, 59.0, 44.5, 39.9, 28.6, 28.4, 25.8. HRMS (EI): calcd for C21H31N2O5 (M + H):

391.2227, found: 391.2221.

(4R,5S)-5-benzyloxycarbonylamino-8-tert-butyloxycarbonylamino-4-hydroxy-1-

octene (28). To the suspension of LiAl(Ot-Bu)3H (10.25 g, 40.31 mmol) in EtOH (70

mL) stirring at -78 ºC was added ketone 30 (3.952 g, 10.12 mmol) in EtOH (70 mL)
 193

dropwise over 30 min. After stirring for 5 hours at -70 to -78º C, 10% citric acid (100

mL) was added, and the mixture was stirred for 20 min. This mixture was extracted with

CH2Cl2 four times, and the combined organics were washed with water, saturated

NaHCO3, brine and dried over Na2SO4. Upon concentration under vacuum, the mixture

was dissolved in minimal amount of CH2Cl2 / Et2O (1:2) and small amount of hexane was

added. After slow cooling to -20 ºC, crystals were washed with Et2O / hexane (1:1) and

then dried under vacuum (3.516 g). The filtrate containing the product was purified by

flash chromatography (EtAcO / hexane 1:9 to 1:1) after concentration under vacuum (28;

total 3.703g, 9.435 mmol, 93%). TLC Rf = 0.33 EtAcO / hexane (1:1). Mp 106 ºC. [α]D24

+22º (c 0.37, CHCl3). IR (film) 3348, 3317, 3068, 2944, 1689, 1538, 1452, 1260, 1160.
1
H NMR (300 MHz, CDCl3) δ 7.26-7.38 (m, 5H), 5.74-5.88 (m, 1H), 5.16 (m, 2H),

5.05-5.12 (m, 2H), 5.09 (s, 2H), 4.57 (m, 1H), 3.67 (m, 2H), 3.11 (m, 2H), 2.11-2.32 (m,

2H), 1.51-1.70 (m, 2H), 1.28-1.51 (m, 11H). 13C NMR (300 MHz, CDCl3) δ 156.6, 156.1,

136.4, 134.4, 128.5, 128.1, 128.1, 118.3, 79.2, 73.2, 66.8, 55.0, 40.3, 38.3, 28.4, 26.8,

26.2. HRMS (EI): calcd for C21H33N2O5 (M + H): 393.2384, found 393.2375.

(4R,5S)-5-benzyloxycarbonylamino-8-tert-butyloxycarbonylamino-4-

methanesulfonyloxy-1-octene (31). To the solution of the amino alcohol 28 (4.2807 g,

10.91 mmol) in CH2Cl2 (320 mL) stirring at -15 ºC was added Et3N (3.13 mL, 22.3

mmol). Methanesulfonyl chloride (1.27 mL, 16.4 mmol) was added dropwise, and then 4-
 194

dimethylaminopyridine (702 mg, 5.75 mmol) was added. The solution was left stirring at

temperature ranging from -15 ºC to 0 ºC for 4 hours. Cold water (100 mL) was then

added rapidly at 0 ºC. The mixture was extracted with CH2Cl2 three times and the

combined organics were washed with cold 1.0 M HCl, cold water, cold NaHCO3, and

dried over Na2SO4. Upon removal of the solvents under vacuum, the product was

recrystallized from CH2Cl2 / Et2O / hexane system as white crystals (4.838 g, 10.28

mmol). The residual product in the filtrate was purified by flash chromatography

(EtAcO / hexane 1:9 to 2:3) and white solid (31) was obtained (total 4.978 g, 10.58

mmol, 97 % yield). TLC Rf = 0.70 EtAcO / hexane 1:1. Mp 67ºC. [α] D24 –27º (c 0.50,

CHCl3). IR (film) 3402, 2983, 2944, 1689, 1639, 1525, 1452, 1365, 1339, 1254, 1173,

913 cm-1. 1H NMR (500 MHz, CDCl3) δ 7.26-7.36 (m, 5H), 5.74-5.85 (m, 1H), 5.25 (m,

1H), 5.18 (d, J = 9.5 Hz, 1H), 5.16 (m, 1H), 5.10 (s, 2H), 4.83 (m, 1H), 4.58 (m, 1H),

3.88 (m, 1H), 3.11 (m, 2H), 2.98 (s, 3H), 2.46-2.54 (m, 1H), 2.37-45 (m, 1H), 1.54-1.66

(m, 2H), 1.27-1.49 (m, 11). 13C NMR (75 MHz, CDCl3) δ 156.1, 156.0, 136.4, 132.3,

128.4, 128.0, 127.9, 119.2, 83.5, 79.2, 66.8, 53.0, 39.9, 38.5, 36.1, 28.3, 26.7, 25.4.

HRMS (EI): calcd for C22H34N2O7S: 470.2081, found 470.2083.

(2S, 3S)-2-allyl-3-benzyloxycarbonylaminopiperidine (27). To the solution solution of

the mesylate 31 (2.734 g, 5.809 mmol) in CH2Cl2 (43 mL) at 0ºC was added
 195

trifluoroacetic acid (11 mL) dropwise over 5 min while stirring. Immediately the reaction

mixture was warmed to room temperature and was stirred for 45 min. Upon cooling the

reaction mixture to 0ºC again and diluting the mixture with CH 2Cl2 (100 mL), 2M K2CO3

solution (100 mL) was added carefully. This mixture was partitioned and the aqueous

phase was extracted with CH2Cl2 (50 mL × 4). The combined organic phase was dried

over anhydrous K2CO3 and was filtered through celite. The solvents were removed on a

rotary evaporator. The residues were then dissolved in CH3CN (450 mL), and K2CO3

(4.20 g, 30.4 mmol) was added in two portions over 3 hours. After stirring the mixture for

24 hours, the mixture was gradually heated up to 70ºC over 1.5 hours. The consumption

of the primary amine was confirmed on TLC, and the mixture was filtered through celite

and was concentrated in vacuum to ca. 90 mL. After the addition of K 2CO3 (1.78g, 12.9

mmol), the mixture was stirred for 30 min at room temperature. Then allylbromide

(800μL, 9.19 mmol) and additional K2CO3 (0.98 g, 7.1 mmol) were added and the

mixture was left stirring at room temperature for 30 hours. Additional allylbromide

(200μL, 2.30 mmol) and K2CO3 (0.54 g, 3.9 mmol) was added and the mixture was

stirred at room temperature for 4 hours. Upon filtration of the mixture, the solvents were

removed on a rotary evaporator. To the residues were added Et 2O (200 mL) and saturated

NaHCO3 (150 mL). After partitioning the phases, the aqueous phase was extracted with

Et2O (50 mL × 4). The combined organic phases were dried over Na2SO4, and filtered

through Celite®. After the removal of the solvents under vacuum, the residue was purified

by flash chromatography (EtAcO / hexane, 1:19 to 1:4), yielding a colorless oil (27;

1.382 g, 4.395 mmol, 76 % over 2 steps). TLC Rf = 0.45 EtAcO / hexane 1:1. [α] D24 =

+16º (c 0.23, CHCl3). IR (film) 3427, 3078, 3029, 2942, 2864, 2811,1720, 1640, 1500,
 196

1456, 1341, 1214, 919 cm-1. 1H NMR (300 MHz, CDCl3) δ 7.28-7.38 (m, 5H), 5.86-5.71

(m, 2H), 5.48 (m, 1H), 5.16-4.96 (m, 7H), 3.84 (m, 1H), 3.29 (m, 1H), 2.92 (m, 1H), 2.80

(m, 1H), 2.35-2.46 (m, 2H), 2.15-2.03 (m, 2H), 1.84 (m, 1H), 1.64 (m, 1H), 1.47 (m, 1H),

1.39 (m, 1H). 13C NMR (75 MHz, CDCl3) δ 155.5, 136.7, 134.9, 134.4, 128.3, 127.9,

127.9, 117.6, 117.2, 66.3, 63.0, 56.3, 52.6, 48.1, 33.9, 29.5, 20.8. Both 1H and 13C NMR

spectra contain rotamer signals.55 HRMS (EI): calcd for C19H26N2O2 (M): 314.1989,

found 314.1994.

(1S, 10S)-1-benzyloxycarbonylamine-7,8-dehydroquinolizidine (31). To the solution

of the 1,2-diallyl piperazine 27 (1.131 g, 3.597 mmol) in CH2Cl2 (360 mL) was added

Grubb’s 2nd generation catalyst (269 mg, 0.360 mmol) in two portions over 2 hours. The

mixture was brought to reflux slowly and the temperature was reduced to room

temperature after 2 hours for the second addition of the catalyst. Then the mixture was

refluxed again for 5 hours (while monitoring by TLC) and cooled again to room

temperature. Silica gel (3.0 g) was added and the mixture was stirred for 10 min. The

solids were filtered through Celite® and rinsed with CH2Cl2 thoroughly. To the filtrate was

added activated charcoal (ca. 20 g) and the mixture was left stirring at room temperature

over night. Upon filtration through Celite®, the filtrate was concentrated on a rotary

evaporator. The residue was purified by flash chromatography on silica gel twice to yield
 197

transparent brown oil still contaminated with a miniscule amount of the ruthenium

catalyst (32; 855 mg, 2.986 mmol, ). TLC Rf = 0.24 CH2Cl2 / MeOH 49:1. [α]D24 –40º (c

0.33 CHCl3). IR (film) 3431, 3055, 2960, 2807, 2758, 1718, 1644, 1500, 1337, 1217,

1095 cm-1. 1H NMR (500 MHz, CDCl3) δ 7.28-7.35 (m, 5H), 5.68-5.71 (m, 1H), 5.57 (m,

1H), 5.10 (s, 2H), 3.77 (dd, J = 9.2, 2.5 Hz, 1H), 3.19 (m, J = 16.4 Hz, 1H), 2.89 (dd, J =

11.2, 2.2 Hz, 1H), 2.68 (m, J = 16.4 Hz, 1H), 2.35 (m, J = 8.3 Hz, 1H), 2.19-2.26 (m,

1H), 2.03 (td, J = 12.2, 3.1 Hz, 1H), 1.93 (m, 1H), 1.90 (m, 1H), 1.75 (m, J = 12.9, 3.8

Hz, 1H), 1.54 (m, J = 14.6 Hz, 1H), 1.49 (dt, J = 13.3, 3.7 Hz, 1H). 13C NMR (75 MHz,

CDCl3) δ 156.2, 136.7, 128.4, 127.9, 127.8, 124.2, 123.4, 66.4, 58.9, 56.1, 54.3, 49.1,

29.8, 27.8, 20.3. Both 1H and 13C NMR spectra contain rotamer signals.54 HRMS (EI):

calcd for C17H22N2O2 (M): 286.1676, found 286.1677.

(+)-epiquinamide (9). Method A. To the solution of the dehydroquinolizidine 32 (200

mg, 0.698 mmol) in EtOH (3.7 mL) was added Pd/C (10% wt, 180 mg) carefully. Argon

was evacuated and H2 was bubbled into the mixture for 5 min. Then the reaction mixture

was left stirring under H2 atmosphere over night using balloon pressure. Acetic anhydride

(170 μL, 1.80 mmol) was added in three portions over 1 hour. The reaction mixture was

left stirring again over night and then was filtered through Celite® and the filter case was

rinsed with ethanol. Upon concentration, the residue was purified by flash

chromatography on silica gel (CH2Cl2 / MeOH / 28% NH4OH(aq) 99:0.9:0.1 to 90:9:1) to

 198

yield pale yellow solid (9; 87 mg, 0.44 mmol, 63 % yield). Rf = 0.39 CH2Cl2 / MeOH /

28% NH4OH(aq) 90:9:1. Mp 130ºC. [α]D24 +24º (c 0.10, CDCl3). IR (film) 3427, 2949,

2862, 2814, 2766, 1637, 1540, 1376, 1295, 1211 cm-1. 1H NMR (300 MHz, CDCl3) δ

6.24 (bs, 1H), 3.92 (m, J = 8.8, 2.0 Hz, 1H), 2.78 (m, 2H), 2.01 (s, 3H), 1.94 (m, 3H),

1.85 (m, J = 13.4 Hz, 1H), 1.72 (m, J = 13.2 Hz, 2H), 1.19-1.63 (m, 7H). 13C NMR (75

MHz, CDCl3) δ 169.6, 64.4, 56.7, 56.6, 48.0, 29.5, 28.9, 25.4, 23.9, 23.4, 20.4. HRMS

(EI): calcd for C11H21N2O1 (M + H): 197.1648, found 197.1650.

(+)-epiquinamide. Method B. To the solution of the dehydroquinolizidine 32 (290 mg,

1.01 mmol) in EtOH (3.0 mL) was added Pd/C (10% wt, 100 mg) carefully. Argon was

evacuated and H2 was bubbled into the mixture for 5 min. Then the reaction mixture was

left stirring under H2 atmosphere over night using balloon pressure. The reaction mixture

was left stirring again over night and then was filtered through Celite ® and the filter cake

was rinsed with ethanol. The solvent was removed under vacuum, and the residues were

dissolved in dioxane (10 mL). While stirring, aqueous solution of NaOH (1.0 M, 10 mL)

was added slowly at room temperature. After 2 hours, the reaction mixture was extracted

with CH2Cl2 (20 mL × 4). The organic layer was dried over Na2SO4 and was concentrated

under vacuum. The residue was purified by flash chromatography on silica gel (CH2Cl2 /

MeOH / 28% NH4OH(aq) 99:0.9:0.1 to 90:9:1) to yield pale yellow solid (9; 173 mg,

0.881 mmol, 87 % yield).

 199

(-)-epiquinamide. TLC Rf = 0.39 CH2Cl2 / MeOH / 28% NH4OH(aq) 90:9:1. [α]D24 –22º

(c 0.13, CHCl3). . 1H and 13C NMR: identical to (+)-9. HRMS (EI): calcd for C11H21N2O1

(M + H): 197.1648, found 197.1648.

 200

NMR Spectra

Figure V.4: 1H NMR Spectrum of 11 in CDCl3

 201

Figure V.5: 13C NMR Spectrum of 11 in CDCl3

 202

Figure V.6: 1H NMR Spectrum of 12 in CDCl3

 203

Figure V.7: 13C NMR Spectrum of 12 in CDCl3

 204

Figure V.8: 1H NMR Spectrum of 13 in CDCl3

 205

Figure V.9: 13C NMR Spectrum of 13 in CDCl3

 206

Figure V.10: 1H NMR Spectrum of 14 in CDCl3

 207

Figure V.11: 13C NMR Spectrum of 14 in CDCl3

 208

Figure V.12: 1H NMR Spectrum of 15 in MeOH-d4

 209

Figure V.13: 13C NMR Spectrum of 15 in MeOH-d4

 210

Figure V.14: 1H NMR Spctrum of 16 in CDCl3

 211

Figure V.15: 13C NMR Spectrum of 16 in CDCl3

 212

Figure V.16: EI-GCMS Chromatogram and Mass Spectrum of 16

 213

Figure V.17: 1H NMR spectrum 29 in CDCl3

 214

Figure V.18: 13C NMR spectrum of 29 in CDCl3

 215

Figure V.19: 1H NMR spectrum of 30 in CDCl3

 216

Figure V.20: 13C NMR spectrum of 30 in CDCl3

 217

Figure V.21: 1H NMR spectrum of 28 in CDCl3

 218

Figure V.22: 13C NMR spectrum of 28 in CDCl3

 219

Figure V.23: 1H NMR spectrum of 31 in CDCl3

 220

Figure V.24: 13C NMR spectrum of 31 in CDCl3

 221

Figure V.25: 1H NMR spectrum of 27 in CDCl3

 222

Figure V.26: 13C NMR spectrum of 27 in CDCl3

 223

Figure V.27: 1H NMR spectrum of 32 in CDCl3

 224

Figure V.28: 13C NMR spectrum of 32 in CDCl3

 225

Figure V.29: 1H NMR spectrum of (+)-9 in CDCl3

 226

Figure V.30: 13C NMR spectrum of (+)-9 in CDCl3

 227

Figure V.31: 1H NMR spectrum of (-)-9 in CDCl3

 228

13
Figure V.32:34:
Figure
Figure 33:C13C
NMR
1H spectrum
NMR
NMR of (-)-9
spectrum
spectrum of in CDCl3
of (-)-9
(+)-9
 229

References and footnotes

1 Daly, J. W. Ernest Guenther award in chemistry of natural products. amphibian skin: a

remarkable source of biologically active arthropod alkaloids. J. Med. Chem. 2003, 46,
445-452.

2 Daly, J. W.; Spande, T. F.; Garraffo, H. M. Alkaloids from amphibian skin: a tabulation of over
eight-hundred compounds. J. Nat. Prod. 2005, 68, 1556-1575.

3 Daly, J. W. Nicotinic agonists, antagonists, and modulators from natural sources. Cell. Mol.
Neurobiol. 2005, 25, 513-552.

4 Daly, J. W. Marine toxins and nonmarine toxins: convergence or symbiotic organisms? J. Nat.
Prod. 2004, 67, 1211-1215.

5 Simmons, T. L.; Coates, R. C.; Clark, B. R.; Engene, N.; Gonzalez, D.; Esquenazi, E.;
Dorrestein, P. C.; Gerwick, W. H. Biosynthetic origin of natural products isolated from marine
microorganism–invertebrate assemblages. Proc. Natl. Acad. Sci. 2008, 105, 4587–4594.

6 Spande, T. F.; Garraffo, H. M.; Edwards, M. W.; Yeh, H. J. C.; Pannell, L.; Daly, J. W.
Epibatidine: a novel (chloropyridyl)azabicycloheptane with potent analgesic activity from an
Ecuadoran poison frog. J. Am. Chem. Soc. 1992, 114, 3475-3478.

7 Fitch, R. W.; Garraffo, H. M.; Spande, T. F.; Yeh, H. J. C.; Daly, J. W. Bioassay-guided
isolation of epiquinamide, a novel quinolizidine alkaloid and nicotinic agonist from an
Ecuadoran poison frog, Epipedobates tricolor. J. Nat. Prod. 2003, 66, 1345-1350.

8 Baker, D. D.; Alvi, K. A. Small-molecule natural products: new structures, new activities.
Curr. Opin. Biotechnol. 2004, 15, 576-583.

9 Gotti, C.; Riganti, L.; Vailati, S.; Clementi, F. Brain Neuronal Nicotinic Receptors as New
Targets for Drug Discovery. Curr. Pharm.Design 2006, 12, 407-428.

10 12 groups in total, 11 of which have published their work so far.

11 Wijdeven, M. A.; Botman, P. N. M.; Wijtmans, R.; Schoemaker, H. E.; Rutjes, F. P. J. T.;
Blaauw, R. H. Total Synthesis of (+)- Epiquinamide. Org. Lett. 2005, 7, 4005-4007.

12 Suyama, T. L.; Gerwick, W. H. Practical Total Syntheses of Epiquinamide Enantiomers. Org.

Lett. 2006, 8, 4541-4543.

13 Wijdeven, M. A.; Wijtmans, R.; van den Berg, R. J. F.; Noorduin, W.; Schoemaker, H. E.;
Sonke, T.; van Delft, F. L.; Blaauw, R. H.; Fitch, R. W.; Spande, T. F.; Daly, J. W.; Rutjes, F. P.
J. T. N,N-Acetals as N-Acyliminium Ion Precursors: Synthesis and Absolute Stereochemistry
of Epiquinamide. Org. Lett. 2008, 10, 4001-4003.

14 Ghosh, S.; Shashidhar, J. Total synthesis of (+)- epiquinamide from -mannitol. Tetrahedron
Lett. 2009, 50, 1177-1179.
 230

15 Huang, P.-Q.; Guo, Z.-Q.; Ruan, Y.-P. A Versatile Approach for the Asymmetric Syntheses of
(1R,9aR)- Epiquinamide and (1R,9aR)-Homopumiliotoxin 223G. Org. Lett. 2006, 8,
1435-1438.

16 Voituriez, A.; Ferreira, F.; Perez-Luna, A.; Chemla, F. Asymmetric synthesis of (-)-1-
hydroxyquinolizidinone, a common intermediate for the syntheses of (-)-homopumiliotoxin
223G and (-)- epiquinamide. Org. Lett. 2007, 9, 4705-4708.

17 Tong, S.; Barker, D. A concise synthesis of (±) and a total synthesis of (+)- epiquinamide.
Tetrahedron Lett. 2006, 47, 5017-5020.

18 Kanakubo, A.; Gray, D.; Innocent, N.; Wonnacott, S.; Gallagher, T. The synthesis and nicotinic
binding activity of (± )- epiquinamide and (± )-C(1)-epiepiquinamide. Bioorg. Med. Chem.
Lett. 2006, 16, 4648-4651.

19 Fitch, R. W.; Sturgeon, G. D.; Patel, S. R.; Spande, T. F.; Garraffo, H. M.; Daly, J. W.; Blaauw,
R. H. Epiquinamide : A Poison That Wasn't from a Frog That Was. J. Nat. Prod. 2009, 72,
243-247.

20 Pal, K.; Behnke, M. L.; Tong, L. A general stereocontrolled synthesis of cis-2,3 disubstituted
pyrrolidines and piperidines. Tetrahedron Lett. 1993, 34, 6205-6208.

21 Singh, R.; Ghosh, S. K. Synthesis of enantiomerically pure all cis-2,3,6-trisubstituted

piperidine: a silicon mediated total synthesis of (+)-carpamic acid. Tetrahedron Lett. 2002, 43,
7711-7715.

22 (a) Gómez-Monterrey, I.; González-Muniz, R.; Herranz, R.; García-López, M. T.

Stereospecific synthesis of (2R,3S)-3-amino-2-piperidineacetic acid derivatives for use as
conformational constraint in peptides. Tetrahedron Lett. 1993, 34, 3593-3594. (b) Jefford, C.
W.; Wang, J. B. An enantiospecific synthesis of solenopsin A. Tetrahedron Lett. 1993, 34,
2911-2914.

23 (a) Anh, N. T.; Eisenstein, O. Nouv. J. Chim. 1977, 1, 61-70. (b) Lodge, E. P.; Heathcock, C.
H. Acyclic stereoselection. 40. Steric effects, as well as .sigma.*-orbital energies, are
important in diastereoface differentiation in additions to chiral aldehydes. J. Am. Chem. Soc.
1987, 109, 3353-3361.

24 The corresponding transition states were modeled at semi-empirical (PM3) level and the
relative energy difference between the two transition states leading to the two stereoisomers
was calculated. Based on this result, Si-face addition of hydride was predicted (data not
shown).

25 Rana, Jatinder; Robins, David J. Quinolizidine alkaloid biosynthesis : incorporation of

cadaverine-1-amino-15N,1-13C into lupinine. J. Chem. Soc. Chem. Comm. 1984, 2, 81-2.

26 Wanner, M. J.; Koomen, G. Biomimetic synthesis of quinolizidine alkaloids. Tetrahedron

1991, 47, 8431-8442.
 231

27 Batey, R. A.; MacKay, D. B.; Santhakumar, V. Alkenyl and aryl boronates – mild
nucleophiles for the stereoselective formation of functionalized N-heterocycles. J. Am. Chem.
Soc. 1999, 121, 5075-5076.

28 Speckamp, W. N.; Moolenaar, M. J. New developments in the chemistry of N-acyliminium

ions and related intermediates. Tetrahedron 2000, 56, 3817-3856.

29 Guarna, A.; Occhiato, E. G.; Machetti, F.; Scarpi, D. A Concise route to 19-nor-10-azasteroids,
a new class of steroid 5α-reductase Inhibitors. Synthesis of (+)-19-nor-10-azatestosterone and
(+)-17β-(acetyloxy)-(5β)-10-azaestr-1-en-3-one. J. Org. Chem. 1998, 4111-4115.

30 So, R. C.; Ndonye, R.; Izmirian, D. P.; Richardson, S. K.; Guerrera, R. L.; Howell, A. R.
Straightforward synthesis of sphinganines via a serine-derived Weinreb amide. J. Org. Chem.
2004, 69, 3233-3235.

31 Nahm, S.; Weinreb, S. M. N-methoxy-N-methylamides as effective acylating agents.

Tetrahedron Lett. 1981, 22, 3815-3818.

32 Mentzel, M.; Hoffmann, H. M. R. N-Methoxy-N-methylamides (Weinreb amides) in modern

organic synthesis. J. Prakt. Chem. 1997, 339, 517-524.

33 Grignard, V. Sur quelques nouvelles combinaisons organométaliques du magnésium et leur

application à des synthèses d'alcools et d'hydrocabures. Compt. Rend. 1900, 130, 1322–1325.

34 Haug, B. E.; Rich, D. H. Synthesis of a Gln-Phe hydroxy-ethylene dipeptide isostere. Org.

Lett. 2004, 6, 4783-4786.

35 The bridgehead proton of 9 has a chemical shift of δ 3.91 ppm, and that of 15 has a shift of δ
3.95 ppm in MeOH-d4. Otherwise, the two compounds have exactly the same carbon
framework and molecular weight by NMR and LRMS.

36 Abe, H.; Aoyagi, S.; Kibayashi, C. Total synthesis of the tricyclic marine alkaloids (-)-
lepadiformine, (+)-cylindricine C, and (-)-fasicularin via a common intermediate formed by
formic acid-induced intramolecular conjugate azaspirocyclization. J. Am. Chem. Soc. 2005,
127, 1473-1480.

37 Wang, Q.; Sasaki, N. A. Versatile approach to enantiopure 2,6-disubstituted piperidin-3-ol

framework: application to the total synthesis of (+)-deoxoprosopinine. J. Org. Chem. 2004, 69,
4767-4773.

38 Cyclization was extremely slow in the absence of base.

39 Sundberg, R. J.; Amat, M.; Fernando, A. M. Analogs of the iboga alkaloids. Synthesis and
reactions of (.+-.)-15-oxo-20-deethylcoronaridine derivatives. J. Org. Chem. 1987, 52,
3151-3159.

40 Kinderman, S. S.; Wekking, M. M. T.; van Maarseveen, J. H.; Schoemaker, H. E.; Hiemstra,
H.; Rutjes, F. P. J. T. A stereodivergent approach to substituted 4-hydroxypiperidines. J. Org.
 232

Chem. 2005, 70, 5519-5527.

41 Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Synthesis and activity of a new generation of
ruthenium-based olefin metathesis catalysts coordinated with 1,3-dimesityl-4,5-
dihydroimidazol-2-ylidene ligands. Org. Lett. 1999, 1, 953-956.

42 Nicolaou, K. C.; Bulger, P. G.; Sarlah, D. Metathesis reactions in organic synthesis. Angew.
Chem. Int. Ed. 2005, 44, 4490-4527.

43 Hong, S. H.; Grubbs, R. H. Highly active water-soluble olefin metathesis catalyst. J. Am.
Chem. Soc. 2006, 128, 3508-3509.

44 Vernall, A. J.; Abell, A. D. Cross metathesis of nitrogen-containing systems. Aldrichimica

Acta 2003, 36, 93-105.

45 Compain, P. Olefin metathesis of amine-containing systems: beyond the current consensus.

Adv. Synth. Catal. 2007, 349, 1829 – 1846.

46 Published methods for the removal of the ruthenium catalyst are better suited for nonpolar
compounds. For examples, see: (a) Maynard, H. D.; Grubbs, R. H. Purification technique for
the removal of ruthenium from olefin metathesis reaction products. Tetrahedron Lett. 1999,
40, 4137-4140. (b) Haack, K. L.; Ahn, Y. M.; Georg, G. I. A convenient method to remove
ruthenium byproducts from olefin metathesis reactions using polymer-bound
triphenylphosphine oxide (TPPO). Mol. Diversity 2005, 9, 301-303.

47 Amir H. Hoveyda, Dennis G. Gillingham, Joshua J. Van Veldhuizen, Osamu Kataoka, Steven
B. Garber, Jason S. Kingsbury and Joseph P. A. Harrity Ru complexes bearing bidentate
carbenes: from innocent curiosity to uniquely effective catalysts for olefin metathesis. Org.
Biolmol. Chem. 2004, 2, 8-23.

48 Michrowska, A; Grela, K. Quest for the ideal olefin metathesis catalyst. Pure Appl. Chem.
2008, 80, 31–43.

49 Shinada, T.; Hayashi, K.; Yoshida, Y.; Ohfune, Y. Squaric acid derivatives prevent the removal
of N-Cbz and N-Fmoc groups under catalytic hydrogenation reaction. Synlett 2000, 10,
1506-1508.

50 The literature value of (+)-9 was [α]20D = +28° (c 0.23, CHCl3) (see ref 11) and [α]22D = +26.2°
(c 0.23, CHCl3) (see ref 17).

51 The literature value of (-)-9 was [α]20D = - 25° (c 0.26, CHCl3) (see ref 15).

52 LePage, K. T.; Dickey, R. W.; Gerwick, W. H.; Jester, E. L.; Murray, T. F. On the use of
neuro-2a neuroblastoma cells versus intact neurons in primary culture for neurotoxicity
studies. Critical Reviews in Neurobiology 2005, 17, 27-50.

53 Kanakubo, A.; Gray, D.; Innocent, N.; Wonnacott, S.; Gallagher, T. The synthesis and nicotinic
binding activity of (±)-epiquinamide and (±)-C(1)-epiepiquinamide. Bioorg. Med. Chem. Lett.
 233

2006, 16, 4648-4651.

54 Moraczewski, A. L.; Banazsynski, L. A.; From, A. M.; White, C. E.; Smith, B. D. Using
hydrogen bonding to control carbamate C−N rotamer equilibria. J. Org. Chem. 1998, 63,
7258-7262.
 Chapter VI

Stereospecific total synthesis of somocystinamide A; an extremely potent antiangiogenic

marine natural product containing synthetically challenging functional groups.

234
 235

Abstract

A marine cyanobacterial metabolite, somocystinamide A, has been shown to have

exceptional anti-angiogenic activities through caspase-8-mediated apoptosis of

endothelial cells. Due to its promising bioactivity profile, total synthesis of this natural

product was accomplished despite synthetically challenging features including an

enamide and disulfide. After an initial successful route was developed, the total synthesis

was revised to be suitable for larger-scale synthesis to provide for in vivo evaluations and

analog production.
 236

VI.1 Introduction

Over the last few decades, natural products

have been directly or indirectly responsible for

over 60% of the new small molecule drugs tested

in clinical trials.1 Of various sources of natural

products, marine organisms represent a relatively

new frontier.1,2 Marine cyanobacteria of the genus

Somocystinamide A
Lyngbya have been an exceptionally rich source of

biologically significant natural products.2 Somocystinamide A (1) was isolated from a

mixed assemblage of Schizothrix and Lyngbya majuscula collected in Somo Somo, Fiji.3

The initial report by Nogle and Gerwick cited its activity against Neuro-2a neuroblastoma

cells with an IC50 value of 1.8 µM.3

Subsequently, 1 was found to have extremely potent cytotoxic activity against

human umbilical vein endothelial cells (HUVEC) with an IC50 of 500 fM.4 HUVEC cells

Control 1.6µM 0.16µM

3µm

3.0 uM 0.30 uM 0.08µM

Figure VI.1: Antiangiogenic activity of 1 in zebrafish

Transgenic Tg(fli1:EGFP) zebrafish embryos in which GFP is expressed in endothelial cells were
incubated without (control) or with increasing concentrations of 1: 3 μM, 1.6 μM, 300 nM, 160
nM, or 80 nM. The GFP gene was coupled to the VEGF gene so that fluorescence would be
observed at sites where angiognesis (promoted by VEGF) was occurring. Blood vessel
morphology was recorded by fluorescence microscopy.
 237

are primary endothelial cells derived from human tissues and are a good model for

studying angiogenesis.5 In cancer therapy, it is crucial to have control over angiogenesis

in the tissues surrounding a tumor. Growing tumors need an even greater supply of

nutrients from the blood than normal tissue. Therefore, a compound that inhibits the

growth of HUVEC cells would be expected to be advantageous against cancer through

anti-angiogenic effects. Furthermore, 1 was shown to be cytotoxic against A549 (human

lung carcinoma) cells with an IC50 value of 46 nM.4

Vascular endothelial growth factor (VEGF) is a key growth factor that is involved

in angiogenesis.5 In rapidly developing organisms, the presence of this protein in a

particular area indicates active formation of blood vessels. The use of transgenic

zebrafish embryos, which are transparent, allows direct observation of a drug's effects on

angiogensis through the visualization of green fluorescent protein (GFP) that is placed

under the control of a VEGF promotor.6 In such a study by our collaborators at the

Moores Cancer Center, somocystinamide A (1) was found to significantly reduce the

level of GFP, and thus indicate a reduced level of VEGF activity present in zebrafish

embryos even at low drug concentrations (<0.1 μM, the media concentration).4 Equally

significant, the zebrafish were not adversely affected by the compound even at 1.6 μM,4

providing a large potential therapeutic window (Figure VI.1).

Of the various mechanisms by which a cell can be killed by a chemical agent,

apoptosis is the most effective mechanism. This is because certain forms of apoptotic

processes can be initiated, in principle, by as little as a single molecule coming into

contact with the corresponding receptor on the cell surface.7 Therefore, when extremely

potent cytotoxicity is observed, it is reasonable to hypothesize that the cytotoxicity is

 238

XTT Cell Proliferation Assay on neuroblastoma NB-7

110
IC50
90 WG-144 (NB-7 cell) 8.369e-007

% Cell proliferation
WG-144(NB-7-c-8 cell) 1.589e-008
70

50

30

10

-10
-12.5 -10.0 -7.5 -5.0 -2.5 0.0
Log Drug Concentration

Figure VI.2: Impact of somocystinamide A (1) on proliferation of paired neuroblastoma

cells lacking caspase 8 expression (triangles) or expressing caspase 8 (squares)4
Caspase-8 potentiates the activity of 1 by 50 fold.

Cell Membrane
Cell membrane

Nucleus

Figure VI.3: Extrinsic pathway via activation of caspase-8 leading to apoptosis

FAS: Fas death receptor, TNFR: tumor necrosis factor receptor, FADD: Fas-associated protein
with death domain, CAD: caspase activated DNase, ICAD: inhibitor of CAD. Scissors indicate
proteolytic activities.
 239

Figure VI.4: Intrinsic pathway via activation of caspase-9 leading to apoptosis

Upon binding of cytochrome c and dATP, Apaf-1 (apoptotic peptidase activating factor 1) recruits
procaspase-9 through its caspase recruitment domain (CARD) and releases the active form of
caspase-9. The rest of the apoptotic events are essentially identical to those of the extrinsic
pathway (Figure VI.3).

caused via an apoptotic mechanism. A study conducted using two neuroblastoma-7 cell

lines, one with caspase 8 expression and the other without it, lends further evidence to the

hypothesis that somocystinamide A is an apoptosis inducer.8 The former cell line was

found to be 50-fold more susceptible to the effects of 1 than the latter (Figure VI.2).4

Caspase 8 is normally an inactive protease, but once activated through recruitment by

FADD proteins at the death receptor and subsequent self-proteolysis of the pro-domain, it

can activate executioner caspases (caspases-3,6,7), which will exercise their destructive

proteolytic ability and cause apoptosis (Figure VI.3).7 Because the initial recruitment of
 240

Figure VI.5: Most stable conformer of somocystinamide A found through molecular

modeling
Molecular mechanics-level calculation was used for conformer search. The distance between the
amide NH proton and the amide oxygen in the center of the figure is only 2.2 Å.

the pro-caspase 8 is induced through an extracellular signal, this process is called an

extrinsic pathway.7 Interestingly, 1 was also found to intrinsically trigger apoptosis

through the caspase-9 pathway (Figure VI.4), but to a lesser extent.4 Moreover, when 1

was reduced to the corresponding monomeric thiol, its bioactivity was lost, suggesting

that either the dimeric nature or the disulfide functionality of 1 is necessary for the

activity.4 Conformer distribution calculation of 1 revealed evidence for intramolecular

hydrogen bonding between the acetamide groups, which would hold the molecule in a

pair-of-forceps fashion (Figure VI.5). Given that the enamide functionality appears to be

necessary for the bioactivity, it is possible that each enamide functionality in 1 is

responsible for recruitment of a pro-caspase-8 protein and that a pair of pro-caspase-8

proteins are brought together through the virtue of the aforementioned conformation.

Furthermore, examination of A549 cells stained with DAPI, anti-ceramide, and

anti-caspase-8 suggested that the mechanism of somocystinamide A-mediated apoptosis

 241

involved structural alterations to the plasma membrane.4 To determine whether the

observed activity was simply due to the lipophilic nature of the natural product, an analog

of 1 that lacks any functionalization in the middle of the chain was tested (2).9 This

analog (2) was found to be completely inactive. Likewise, the hydrolysis product of 1,

bis-methylamide 3 was also inactive.10 Therefore, the enamide as well as some or all of

the other functional groups present in 1 seem to be necessary for induction of apoptosis.

Docosanedioic bis-enamide analog (2) and hydrolyzed analog of 1 (3).

Somocystinamide A (1) completely and readily intercalates within liposomes and

it retains potent anti-proliferative activity when administered as such.4 Therefore, if the

solubility of 1 or its analogs prove to be an issue in the course of development, use of

liposomes would be a potentially viable mechanism for delivery of the drug. The

combination of the anti-proliferative activities and the ability to induce apoptosis in

endothelial cells is an excellent biological profile for potential treatment of cancer. 11 An

endogenous protein, angiostatin,12 has a similar bioactivity profile, and it has advanced to

phase II clinical trial as of 2008 for treatment of lung cancer. 13 Given its bioactivity

profile, somocystinamide A (1) presents itself as a promising cancer therapeutic.

 242

VI.2 Synthetically challenging features of somocystinamide A

Scheme VI.1: Enamide

When an olefin is introduced immediately adjacent to an amide, the characteristics

of the compound changes more drastically compared to the alkylamide analog. One of the

most noticeable changes is the shift in UV absorption spectrum. Enamide, by which is

meant a vinyl amide herein, possesses conjugation of the carbonyl double bond and the

alkenyl group. This conjugated system absorbs UV at 230-240 nm,14 which can be a

useful physical property when the presence of an enamide compound is sought by HPLC

techniques. Easily accessible enamides will almost always have a proton at R3 position

(Scheme VI.1). The proton is also very diagnostic for an enamide moiety in that it gives

rise to a unique doublet 1H NMR signal at 7.0-7.2 ppm.15

Chemically, enamides can be significantly more unstable than amides due to the

possible tautomerism to become an acyl imine or an acyl iminium ion that is further

activated by the adjacent carbonyl group. For example, pronounced instability of

somocystinamide A (1) was noted when it underwent complete hydrolysis in CDCl3 in an

NMR tube due to the residual moisture and acid (HCl) present in CDCl3 (Scheme VI.2).3

Given this precedence, it is possible that some natural products, as we know them, are

actually artifacts of hydrolytic decomposition of enamides.

 243

Scheme VI.2: Instability of enamide as seen in the facile decomposition of

somocystinamide A

Figure VI.6: Proposed biosynthesis of somocystinamide A (1)

The depicted mechanism of the terminal olefin formation is reminiscent of the predicted
biosynthesis of curacin A, which also contains a terminal olefin.18
 244

Figure VI.7: Enamide containing natural products

The enamide functional group comes in different forms: a secondary enamide,

where R1 is a proton, or a tertiary enamide, where R1 is an alkyl group (Scheme VI.1). As

will be discussed later, this distinction becomes significant for devising a strategy to

introduce the enamide functionality. In fact, most of the accomplishments in enamide

synthesis have been for secondary enamides, and the same approaches have been found

ineffective for tertiary enamides.16

 245

As discussed earlier, the enamide is a labile functional group and therefore care

must be taken to avoid inadvertent decomposition during handling of the compound.

Growing sophistication in natural product isolation techniques have no doubt contributed

to the increasing number of enamide-containing secondary metabolites that have been

isolated in recent years (Figure VI.7). All of these natural products appear to have been

assembled through hybrid PKS-NRPS systems (except for 10, a peptide derivative),17

where the bulk of the carbon skeletons are constructed through PKS catalytic activities

and amino acid residues are embedded via NRPS catalytic activities to provide the

nitrogen of the enamide moieties. In particular, the biosynthesis of somocystinamide A

(1) was proposed as shown in Figure VI.6.18 Most of these natural products have been

shown to possess strong bioactivity. Enamide-containing natural products can be

categorized into secondary enamides (6-10) and tertiary enamides (11, 12, 1). The latter

group generally has a methyl group on the enamide nitrogen that is most likely derived

from S-methy-adenosylmethionine.19,20

Phomoenamide (6), for example, was shown to have a minimum inhibitory

concentration (MIC) value of 6.25 μg/mL against Mycobacterium tuberculosis H37Ra.21

Salicylihalamide A (7) is strongly cytotoxic against NCI melanoma cell line (GI50 = 7

nM)22 and is a very potent inhibitor of vacuolar H+-ATPases (IC50 < 1 nM),23 whose

involvement is implicated in diseases such as osteoporosis and cancer (Figure VI.7).24

Likewise, lobatamides, which are structurally related to the salicylihalamides, also

possesses cytotoxicity against human cancer cell lines and inhibitory activity against

vacuolar H+-ATP ases.25 The peptidic compounds terpeptin (8) and chondriamide A (10)

have cell-cycle inhibition activity and cytotoxicity against colon cancer cells (LOVO
 246

cells), respectively.26,27 Ulapualides, isolated from sponges and nudibranchs, have been

shown to have antifungal and ichthyotoxic properties, as well as cytotoxicity against

leukemia cells.28 Interestingly, almost all of the recently isolated enamide natural products

are of marine origin. These newly discovered and bioactive enamide natural products

should drive innovation in the synthesis of enamides.

There have been extensive studies on enamide chemistry in the context of

heterocycle and asymmetric synthesis, where enamides are used as synthons.29,30 There is

one review on the preparation of this labile, yet crucial functional group.15 In Scheme

VI.3, four major types of approaches for synthesizing enamides are summarized.

In devising a strategy for the synthesis of 1, the stabilities of both the disulfide

and the enamide became of concern. Since the installment of the enamide was envisioned

to be performed at a very late stage, if not at the last step, it was desirable to assess the

difficulty of synthesizing a tertiary enamide as part of a model system. The most

straightforward and expeditious approach to the desired enamide is to acylate the

corresponding carboxylic acid with the fully furnished imine (Scheme VI.3).31 This

approach was successful with simple substrates bearing carboxylic acids and no other

functional groups (Scheme VI.4).

Scheme VI.3: Enamide synthesis on model systems through acylation of imine

 247

Furthermore, the presence of the disulfide group in 1 requires great care and

consideration during the course of synthesis.32 For example, synthetic investigation of

epidithiapiperazine-dione natural products (such as 24 in Figure VI.8) has met with much

difficulty in the installation of the disulfide (Scheme VI.6).32,33 To date, only one complete

total synthesis of a compound of this class has been reported.34 Another case in point is

Scheme VI.4: Different approaches to enamide functional group

A: direct acylation of imine, B: vinylation of amide, C: amide addition to alkyne, D:
condensation of aldehyde to amide. Curtius rearrangement of α,β-unsaturated acyl azides was
excluded due to its rarity. Typically, other approaches also utilize one of these reactions to
prepare the starting enamide material.

Figure VI.8: Examples of disulfide-containing natural products

 248

Scheme VI.5: Approaches to install disulfide of the epidithiapiperazinedione core

In the first and the third case, a multi-step maneuver of functional groups is required to install
disulfide. In the second case, a very harsh condition (S2Cl2 and NaH) is required. None of these
approaches would be ideal for a total synthesis of a natural product with labile functional
groups.34

Scheme VI.6: Total synthesis of glyotoxin involving sensitive installation of disulfide

After a lengthy investigation, a strategy was devised in which the disulfide is only formed in the
last step. Any attempts to install it in earlier stages were not successful.34
 249

Scheme VI.7: Synthesis of psammaplin A

The installation of the labile disulfide had to be reserved until the last step.35

psammaplin A (25); in all three of the published syntheses of 25, the sulfur atoms were

introduced as a disulfide in the final step so as to avoid side reactions (Scheme VI.7).35

VI.3 Attempted synthesis of somocystinamide A through Suzuki coupling

With a method to construct a simple enamide in hand, retrosynthetic analysis of

the natural product led to the proposed synthetic approach shown in red in Scheme VI.8.

Suzuki coupling is well known as a robust reaction to connect a vinyl or aryl halide to a

vinyl or aryl borylate.36 The reaction is somewhat less reliable if the boron is on an sp 3

Scheme VI.8: Retrosynthetic analysis of somocystinamide A

The initial strategy is shown in red while the successful approach is shown in blue.
 250

Scheme VI.9: Synthesis of vinyl iodide for Suzuki coupling (Scheme VI.10)

Scheme VI.10: Suzuki coupling with varying coupling partners

For convenience, a commercially available olefin 48 was used despite the wrong length of the
alkyl chain.

carbon.36,37 Thus, besides the enamide synthesis, other steps of concern in the proposed

synthesis were the Takai vinyl iodide synthesis38 and the Suzuki coupling. The Takai

reaction was tested on a commercially available aldehyde derived from proline, which is

structurally very similar to the actual substrate except for the absence of sulfur. This
 251

Scheme VI.11: Ruthenium catalyzed cross metathesis approach

reaction proceeded with good yield (70%). The necessary vinyl halide for the Suzuki

coupling could be synthesized via the Takai protocol from the corresponding aldehyde. 38

Finally late-stage dimerization at the sulfur atom via air oxidation and installation of the

enamide moiety were planned to complete the synthesis of 1.

The synthesis of 1 began with the putative biosynthetic precursor, L-cysteine,

whose stereochemistry translates into the final product. The cysteine was then protected

as a Boc-thiazolidine 41 with the use of benzaldehyde and Boc anhydride.39 Following

reduction of the carboxylic acid 41 to an alcohol 42 and oxidization back up to the

aldehyde 43 via a Swern reaction,39,40 the aldehyde was treated with chromium (II)

chloride and iodoform (Takai protocol).38 The Takai reaction seemed to give a good yield

by direct TLC analysis of the reaction mixture. However, after work-up and

chromatography, only a minute amount of the desired product was recovered, possibly

due to the presence of excess iodoform (elutes closely with the product) and

decomposition of the vinyl iodide. Brief efforts at optimizing this reaction were not

successful.
 252

The vinyl iodide 44 was tested for the Suzuki coupling reaction and only a trace

amount of the desired product was formed. The difficulty of this reaction was attributed

to the combination of functional group incompatibility and, possibly, the excess 9-BBN

that could have reduced the palladium-vinyl complex to the terminal alkene.41 To

examine the latter possibility, the same Suzuki coupling reaction was carried out with a

trifluoroboronate substrate, which is a solid that can be isolated.42 However, this approach

also proved unfruitful.

VI.4 Cross metathesis approach to somocystinamide A skeleton

To circumvent the Suzuki coupling problem, a cross metathesis approach was

employed.43 Conveniently, commercially available methyl 10-undecenoate (48) would

give the correct chain length for the natural product because the terminal sp 2 carbon

would be cleaved off in the process. The metathesis reaction was carried out using 48 and

the vinyl thiazolidine 51, which was synthesized from the aldehyde 43 via a simple

Wittig reaction.44 Different Ruthenium catalysts and various amounts of each substrate

and the catalyst were screened to find an optimum condition for this coupling reaction

(Table VI.1).

The production of the dimeric ester 53 seemed much faster than the desired

product by TLC analysis. This observation seemingly implied that the dimerization of

the ester happens first and then the dimeric ester is used in the actual metathesis.

Therefore it was hypothesized that the yield could be improved by utilizing pre-prepared

dimeric ester for the metathesis, which would presumably reduce the work load for the

catalyst. However, this hypothesis was discounted because the use of 12 as one of the
 253

coupling partners did not improve the yield and apparently opened another reaction

pathway for the starting material (entry 5, Table VI.1). In any case, the second generation

Hoveyda catalyst was found to be the best catalyst for this purpose.45

Table VI.1: Optimization of ruthenium-catalyzed cross metathesis.

catalyst conc.a esterb yield of 52 c

1 Grubbs I, 20 mol % 0.04 48, 10 equiv 0%
2 Grubbs II, 20 mol % 0.04 48, 10 equiv 26% (na)
3 Grubbs II, 4 mol % 0.04 48, 3 equiv 23% (57%)
4 Hoveyda II, 5 mol % 0.03 48, 3 equiv 81% (94%)
5 Hoveyda II, 5 mol % 0.03 53, 1.5 equiv 53% (55%)
6 Hoveyda II, 2.5 mol % 0.03 48, 3 equiv 44% (na)
d
7 Hoveyda II, 5 mol % 0.04 48, 3 equiv 73% (83%)
8e Hoveyda II, 5 mol % 0.2 48, 2.2 equiv 82% (82%)

(a) Concentration of 9 (M); (b) Equivalents of 48 or 53 with respect to 51; (c) Isolated yields.
Yields in parentheses are based on recovered starting material; (d) 3.2 g scale (ca. 10 times more
than entries 1~6); (e) 5.5 g scale. The E/Z ratio was 18:1.
 254

VI.5 Total synthesis of somocystinamide A

With a reliable coupling protocol in hand, a new synthetic route was devised, as

shown in Scheme VI.12. The deprotection of the cysteine residue was effected by

reduction of the benzylic carbon with sodium in liquid ammonia in excellent yield (54).13

Upon methylation with diazomethane, the resulting thiol (55) was treated with TFA and

subsequently with acetic anhydride and triethylamine to replace the Boc protecting group

with acetate, which results in acylation of the thiol group as well (56). Both the removal

of the unwanted acetate on the thiol and hydrolysis of the methyl ester were achieved in

one step by treatment with lithium hydroxide in aqueous THF, which also causes the

oxidative dimerization of the thiol if performed in air (somocystinoic acid, 57).46 This air

oxidation only occurred with small scale reactions (<100 mg). Attempts to effect the

Scheme VI.12: Initially planned final steps to Somocystinamide (1)

 255

dimerization with conventional means, such as treatment with I2, did not give good

yields.

Scheme VI.13: Possible mechanism of enamide formation

The method developed from the model study was used to effect the acylation with

the corresponding imine. Various conditions were investigated to couple the in situ

generated imine 58 to the corresponding di-acyl chloride 60, whose formation was

verified by the reaction with methylamine to produce 3 (Scheme VI.13, Table VI.2).

Model compounds (63-65) were also tested in this investigation for enamide formation

(Figure VI.9, Table VI.2). In most cases, the starting material decomposed while in some

cases a trace amount of 1 was observed (Table VI.2). This result was curious because, in

Figure VI.9: Model compounds used in the study of enamide formation

 256

Table VI.2: Enamide formation via acyl chloride

Substrate Conditions Result

1 63 1. SOCl2 (excess), CH2Cl2 Decomp.
2. MeNH2, 4-pentenal, MS3Å
2 63 1. (COCl)2, DMF, CH2Cl2 Decomp.
2. MeNH2, 4-pentenal, MS3Å
3 64 1. SOCl2 (excess), CH2Cl2 Decomp.
2. MeNH2, 4-pentenal, MS3Å
4 64 1. (COCl)2, DMF, CH2Cl2 Decomp.
2. MeNH2, 4-pentenal, MS3Å
5 64 1. (COCl)2, DMF, CH2Cl2 Methyl Ester, low
2. MeOH, Et3N yield
6 65 1. SOCl2 (excess), CH2Cl2 Decomp.
2. MeNH2, 4-pentenal, MS3Å
7 65 1. SOCl2 (1.5 eq), CH2Cl2 Methyl ester,
2. MeOH, Et3N moderate yield
8 57 1. (COCl)2 (excess), CH2Cl2 Trace of 1
2. MeNH2, 4-pentenal, MS3Å
9 57 1. SOCl2 (excess), CH2Cl2 Decomp.
2. MeNH2, 4-pentenal, MS3Å
10 57 1. (COCl)2, DMF, CH2Cl2 Methyl Ester, low
2. MeOH, Et3N yield
11 57 1. (COCl)2, DMF, CH2Cl2 Trace of 1
2. MeNH2, 4-pentenal, MS3Å
12 23 1. EtCO2Cl, Et3N, CH2Cl2 No Acylation
2. MeNH2, 4-pentenal, MS3Å
13 64 1. EtCO2Cl, Et3N, CH2Cl2 No Acylation
2. MeNH2, 4-pentenal, MS3Å
14 65 1. EtCO2Cl, Et3N, CH2Cl2 No Acylation
2. MeNH2, 4-pentenal, MS3Å
15 21 1. EDCA·HCl, Et3N, CH2Cl2 No Acylation
2. MeNH2, 4-pentenal, MS3Å
16 21 MeNH2, 4-pentenal, MS3Å ~70% yield
17 23 1. (COCl)2 (excess), CH2Cl2 ~70% yield
2. MeNH2, 4-pentenal, MS3Å
 257

addition to the model study, there are reports of synthesis of simple enamides via

acylation of the corresponding acid chloride with imine.47 It is possible that the putative

acyl iminium ion intermediate 61 is intercepted intramolecularly due to the dimeric

nature of the substrate (Scheme VI.13).48 In support of this hypothesis, the tautomer 59

was not observed at all by 1H or 13C NMR in CD2Cl2.49

Scheme VI.14: Copper-mediated vinylation of a primary amide model compound (65)

and attempted chemoselective methylation
For the methylation step, the investigated conditions are the following: K2CO3 in EtOH, MeCN,
and dioxane; NaHMDS in THF, NaH in THF.

Conventionally, protocols for enamide formation include, in addition to acylation

of imines, direct addition of amides to alkynes, condensation of aldehyde with amide, and

the olefination of amides (Scheme VI.3). Most of these protocols suffer from low yields

and/or lack of stereocontrol on the double bond geometry. The most recent collective

effort in enamide synthesis has been on the development of amide vinylation

methodology. In 1991, Suzuki and coworkers first reported successful vinylation of

amides with vinyl halide in the presence of copper (I) iodide.50 At the time when there

were only a few methods to regiospecifically and stereospecifically produce enamides,

 258

this noble method guaranteed both aspects of reaction specificity. Since the

regiospecificity and stereospecificity are solely dictated by the position of the halide on

the olefin, this methodology could be universally applied to any enamide systems as long

as all the functional groups are tolerated. In the past decade, Buchwald and coworkers

have improved this reaction through identification of a more suitable ligand for the

copper catalyst.16a As such, a Cu-mediated vinylation approach was explored. However,

as expected from the literature,16a direct vinylation of acyclic secondary amide was not

possible. While it was possible to vinylate a primary amide model compound, the

required chemoselective methylation of the enamide moiety was not successful (Scheme

VI.14).

The observation that the hydrolytic decomposition of 1 to 3 seems to have

relatively low activiation energy3 inspired us to carry out the opposite reaction, namely

condensation of the aldehyde 551 with 3 (Scheme VI.15). Because of the need to remove

water during the course of reaction, the use of a Soxhlet extraction apparatus was more

Scheme VI.15: Final steps to somocystinamide A (1)

 259

Figure VI.10: Reaction setup for enamide formation using molecular sieves and glass
wool in a Soxhlet extractor

convenient for small scale reactions and it also allowed for use of solvents heavier than

water (Figure VI.10).52 Observing that the putative intermediate 21 did not yield 1, we

hypothesized that the E1 path way was not viable (Scheme VI.13). In support of this

hypothesis, use of a more polar solvent, THF, had an adverse effect in comparison to 1,2-

dichloroethane, a more non-polar solvent.53 The best result was obtained when TsOH was

used as the catalyst, which gave a 47% yield (Scheme VI.15).54 Thus, a total synthesis of

1 was completed. This synthesis is fairly robust for all steps but the last two such that >1

g of 57 was prepared. However, scale-up for the production of 3 proved difficult due to

complications in the purification procedure as a consequence of the extremely poor

 260

solubility of 3. Therefore, a modified synthetic route or improved purification protocols

are necessary for larger-scale production of 1 and its analogs.

VI.6 Bioassays of the synthetic sample of somocystinamide A

Upon the completion of a total synthesis of somocystinamide A (1), its in vivo

activity was investigated in brine shrimp. While 1 was not lethal to the brine shrimp even

at 130 μM (media concentration) after 24 hours of exposure, their motility was

significantly impaired even at 1.3 μM. A much decreased quantity of intestinal contents

were found in the treated group. Thus, 1 seems to possess biological properties not yet

understood.

In an A549 (human lung carcinoma) cytotoxicity assay, the synthetic sample of

somocystinamide A (1) possessed anti-proliferative activity, but to a lesser extent than the

natural sample. Initially there was concern about the possibility that 1 was not the

extremely bioactive metabolite in the earlier investigation.55 To investigate the cause of

this decreased activity, a hypothesis was formed and examined; synthetic 1 is purer than

natural 1 and the natural sample contained contaminants that assisted in solubilizing 1,

whereas the synthetic sample had poor solubility. While much better than 3, the solubility

of 1 is not satisfactory for many purposes. Indeed, in the brine shrimp assays, the samples

at 100 μM were cloudy with incompletely dissolved 1. In 96-well plate cytotoxicity

assays, serial dilutions are made from the highest concentration solution. Therefore, if

any portion of 1 precipitates in the highest concentration solution, all of the wells

containing solutions of 1 also have decreased concentrations of 1.

 261

Figure VI.11: Solubility of somocystinamide A (1) in various media

The solubility was measured by the UV absorbance (Y-axis) of RP-HPLC PDA dectector. For
each run, 10 μL of a 100 μM solution was injected. The X-axis denotes the presence or absence
of additives. The 4th and 5th runs were duplicates.

Because all the in vitro bioassays are done in aqueous environments, the solubility

of somocytinamide A (1) in various types of aqueous solutions were examined (Figure

VI.11). The solubility was determined by injecting a sample of 1 onto a RP-HPLC

column and measuring the PDA-detected UV-absorbance of 1. As expected, more of the

compound was detected when only organic solvents were used (the first two entries in

Figure VI.9). However, when a solution of 1 was diluted with H2O, the measured

solubility decreased significantly. Phase-transfer catalyst-like additives (CTAB,

dodecamide, DMEM) eliminated the solubility of 1. Given these results, any future
 262

bioassay of 1 must be carried out with care. It is recommended that the maximum amount

of organic solvents tolerated by the cells be used in the sample solutions.

VI.7 Scalable synthesis of somocystinamide A

While the above synthetic route is sufficient for small-scale synthesis in a

laboratory, it has three main bottlenecks in terms of scaling-up.

1.The conversion of 52 to 56 requires 5 steps for what is essentially a deprotection-

reprotection maneuver.

2.The conversion of 57 to 3 cannot be readily scaled-up due to the insolubility of 3. If 3

readily dissolved in solvents such as EtOAc, the workup and purification would be

simply a matter of aqueous acid/base washings for this amine coupling step.

3.The final step of introducing the enamide moiety has not been optimized beyond 41%

yield.

In order to remedy all three of these issues, an efficient alternative method must

be first developed for the production of 3, which would enable optimization studies on

the finals step from 3 to 1. When investigating the enamide formation reaction with

simpler substrates, such as undecanoic acid, it was observed that the resulting tertiary

enamide had excellent solubility in common solvents such as CH2Cl2 and MeOH.

However, the dimeric equivalent of this compound, 2, had extremely poor solubility in

these same solvents. Suspecting that the insolubility of bis-amide 3 was at least partially

due to its dimeric nature and the methyl amide functionality, a synthetic route for 3 was

devised in which the intermediates remain as monomers until the enamide formation step

as shown in Scheme VI.16.

 263

Scheme VI.16: Revised synthesis of the bis-methylamide 3

This scheme shortened the synthesis by two steps. However, the cross metathesis

step suffered low yields (~40%) under the same condition as the previous synthesis. After

examination of the reaction by-products, a large amount of dimer 74 was found as an

insoluble solid. In the previous reaction (51 to 52), the dimeric ester by-product was able

to be recycled in the reaction (Table IV.1, entry 5), which was probably a large

contributing factor to the excellent yield (82%) despite the faster formation of 53 than 52.

Therefore, it was hypothesized that if a solvent system that solubilize 74 was utilized, the

yield would increase due to recycling of 74. When 9:1 CH2Cl2 / MeOH was used, the

yield decreased and most of the starting material (51) was left intact. When CHCl3 was

used, the cross metathesis proceeded smoothly with excellent yield (81-84%). It was
 264

noted, however, that the purification of the product (71) was more troublesome than 52

due to the increased polarity of 71.

Afterwards, the conversion of 71 to 73 was straightforward. As a result of this

revised synthesis, the use of a potentially dangerous and toxic reagent, diazomethane was

also eliminated.56 The hydrolysis/oxidation step from 73 to 3 only required washing of the

precipitated product, 3, thereby taking advantage of its insolubility. However, the yield

was somewhat less than ideal (65%). Purification methods such as washing of a solid is

well-suited for future adaptation to industrial scale production should the compound

become a pharmaceutical product.57 All of these steps were done at fairly large scales and

>1 g of 73 and >500 mg of 3 were obtained, the latter of which would not have been

reasonable without this revision of the synthesis. Finally, the penultimate natural product

3 (301 mg) could be converted to somocystinamide A (1, 119 mg, 33% yield) through the

previously established method (Scheme VI.15), thereby completing a sufficiently large-

scale synthesis58 required for future in vivo investigations. The overall yield of this

revised synthetic route was 11% over 7 steps from the known aldehyde 43, whereas the

overall yield of the earlier synthesis was 9% over 10 steps from 43. Despite a small

difference in the overall yield, the smaller number of steps required for the revised route

makes it significant improvement.

VI.8 Conclusions

A stereoselective and stereospecific total synthesis of a promising anti-angiogenic

marine natural product, somocystinamide A (1) was accomplished through ruthenium-

catalyzed olefin cross metathesis, molecular oxygen and base-promoted dimerization of

 265

the thiol, and enamide formation through condensation of aldehyde with amide. This

synthesis provided material for further bioassays and helped to identify the solubility

problem of this natural product. Furthermore, the synthesis was successfully revised to

suit the future needs for synthesis of analogs and a large supply of the compound for in

vivo evaluations.
 266

VI.9 Experimental section

General Experimental Procedures and Materials

Unless noted otherwise, all materials were purchased from commercial sources

and were used without further purification. Anhydrous benzene was purchased from

EMD. Tetrahydrofuran (THF) was distilled from sodium / benzophenone. Et3N and

CH2Cl2 were distilled from CaH. Ac2O was distilled from quinone. Dimethyl sulfoxide

(DMSO), oxalyl chloride, and trifluoroacetic acid (TFA) were distilled without desiccant.

All reactions were carried out under dry argon atmosphere unless otherwise noted.

Reaction temperatures herein recorded are external temperatures unless otherwise noted.

Flash chromatorgraphy was performed using EMD silica gel (230-400 mesh) in all

cases.59 Thin layer chromatographic (TLC) analysis was performed using EM Science

pre-coated silica gel plates (Merck 60 F254). Melting points were determined on an

Electrothermal Mel-Temp device and are uncorrected. Optical rotations were measured

on a Jasco P-2000 polarimeter. IR spectra were recorded on a Nicolet Magna-IR 550. 1H

NMR and 13C NMR spectra were recorded on a Varian Inova spectrometer (500 MHz and

125 MHz, respectively) or on a Varian Inova spectrometer (300 MHz and 75 MHz,

respectively). 1H and 13
C spectra recorded in CDCl3 (or in CD3OD/CDCl3) were

referenced to the residual solvent peaks at 7.26 ppm (CHCl3) and 77.0 ppm (CDCl3),

respectively. High resolution mass spectra were recorded on a ThermoFinnigan

MAT900XL spectrometer.
 267

Preparation of Known Aldehyde 43

43

The first step was done according to reference 59.60 The rest of the steps to the aldehyde

(43) were carried out according to reference 38.39

 268

Synthesis of enamides 22 and 2. To a solution of the aldehyde 5 (97 μL, 0.98 mmol) in

CH2Cl2 (5 mL) was added a 2M solution of MeNH2 in THF (0.49 mL, 0.98 mmol) and

MgSO4 (710 mg) at 0˚C. Immediately, the mixture was warmed to rt and left stirring for

1.5 hrs. Then neat acetylchloride (70 μL, 0.98 mmol) was added. After 10 min, pyridine

(95 μL, 1.18 mmol) was added, and the mixture was left stirring for 3 hrs at rt. The

reaction mixture was filtered and was poured into a cold saturated solution of NaHCO3.

Upon phase separation, the organic layer was washed with saturated NaHCO 3 and was

dried over Na2SO4. The volatiles were removed in vacuo and a brown oil was obtained,

which was mostly pure by TLC and 1H NMR (96 mg, ~70% yield). In order to obtain an

analytically pure sample, the brown oil was subjected to flash column chromatography

using silica gel (1:6 EtOAc / Hexanes to 1:5 EtOAc / Hexanes). A colorless oil (51 mg,

0.37 mmol, 37%) was obtained. TLC Rf = 0.41 (3:7 EtOAc/Hexanes). The bis-enamide 2

was synthesized in the same manner except that two equivalents of 5 and MeNH2 were

used. 1H and 13C NMR spectra: see Figures VI.10-13.

 269

(2R,4R)-tert-butyl 4-((E)-2-iodovinyl)-2-phenylthiazolidine-3-carboxylate (44). CrCl2

(5.189 g, 42.22 mmol) was flame-dried in a flask under vacuum. Upon cooling to rt, THF

(31 mL) was added and the suspension was stirred at rt for 25 min. Then the mixture was

cooled to 0°C. A solution of the aldehyde 43 (1.972 g, 6.693 mmol) and CHI3 (5.270 g,

13.38 mmol) in THF (30 mL) was cannulated dropwise into the CrCl 2 suspension over 20

min. An additional amount of THF (10 mL) was used to rinse the flask containing 43 and

CHI3, which was cannulated again into the CrCl2 suspension. At this point, TLC analysis

showed that there was little starting material left, and that a significant amount of the

desired product had formed. This mixture was taken out of the ice bath immediately and

was stirred at rt for 4 hrs. The mixture was diluted with Et2O (30 mL), and it was poured

into brine, which was extracted with Et2O (20 mL x 2). The organic layer was washed

with brine, and was dried over Na2SO4. Upon removal of the solvents, the residues were

subjected to flash column chromatography (hexanes to 1:4 EtOAc/hexanes) to obtain 124

mg of the product (44) as a pale yellow solid (4.4% yield). TLC Rf = 0.30 (1:4 EtOAc /

hexanes). 1H and 13C NMR spectra: see Figures VI.15 and 16.

(2R,4R)-2-phenyl-4-vinylthiazolidine-3-tert-butyl carboxylate (51). To the suspension

of MePPh3·Br (13.37 g, 37.43 mmol) in THF (150 mL) at –78°C (dry ice bath) was added

1.0 M NaHMDS (43 mL, 43 mmol) dropwise under vigorous stirring, which resulted in a

yellow solution. The dry ice bath was removed and the solution stirred for 45 min at rt.
 270

The solution was cooled again to –78°C and a solution of the aldehyde (8.302 g, 28.30

mmol, synthesized according to literature2) in THF (100 mL) was added dropwise. Then

the solution was warmed to rt over night under stirring. Consumption of the aldehyde was

confirmed by TLC analysis. Then 1.0 M aqueous NH4Cl (100 mL) and Et2O (100 mL)

were added successively. The aqueous phase was extracted with Et2O (100 mL x 2). The

combined organic phase was washed with brine, dried over Na2SO4, and was filtered

through a plug of silica gel. Upon evaporation of solvents, the residue was purified by

flash column chromatography (3:97 to 1:9 Et2O / hexane) and a white solid (6.04 g, 20.7

mmol) was obtained (74% yield). Mp 44-45 ºC. TLC Rf 0.39 Et2O / hexane (1:4). [α]D23

+136.2º (c 6.50, CHCl3). IR (film, KBr) νmax 2977, 1698, 1367, 1164 cm-1. 1H NMR (300

MHz, CDCl3) δ 7.22-7.39 (m, 5H), 6.10 (s, 1H), 6.03 (m, 1H), 5.37, (d, 17.1 Hz, 1H),

5.25 (dt, J=10.2, 0.9 Hz, 1H), 4.83 (m, 1H), 3.27 (dd, J=11.7, 6.6 Hz, 1H), 2.85 (dd,

J=11.7, 5.1 Hz, 1H), 1.33 (s, 9H). 13C NMR (75 MHz, CDCl3) δ 153.7, 141.6, 137.8,

128.2, 127.5, 126.3, 117.2, 80.7, 66.3, 64.5, 36.1, 28.2. HRMS (EI): calcd for

C16H21O2N1S1: 291.1288, found 291.1284.

(2R,4R)-3-tert-butoxycarbonyl-4-((E)-11-methoxy-11-oxoundec-1-enyl)-2-

phenylthiazolidine (52). To the solution of the thiazolidine olefin 51 (5.500 g, 18.87

mmol) in CH2Cl2 (95 mL) was added methyl undecenoate (9.2 mL, 41 mmol) at rt. The
 271

solution was then subjected to vacuum-sonication-Ar introduction cycle three times for

degassing. Then second generation Hoveyda-Grubbs catalyst (592 mg, 0.944 mmol) was

addedin one portion. After 20 min of stirring at rt, the solution was refluxed for 16 hrs.

Then the solvents were removed under vacuum and the residue was subjected to flash

column chromatography (hexane to 1:9 Et2O / hexane) to obtain colorless oil (7.174 g,

15.54 mmol, 82% yield). Along with the desired product was isolated the cis isomer

(TLC Rf=0.52 Et2O / hexane 3:7, 539 mg, 1.17 mmol, 6% yield cis). TLC Rf 0.47 Et2O /

hexane (3:7). [α]D23 –81.7º (c 1.85, CHCl3). IR (film, KBr) νmax 2978, 2928, 2853, 1740,

1696, 1367, 1166 cm-1. 1H NMR (300 MHz, CDCl3) δ 7.21-7.38 (m, 5H), 6.08 (s, 1H),

5.78 (dt, J = 15.3 Hz, 6.6 Hz, 1H), 5.63 (dd, J = 15.3 Hz, 7.6 Hz, 1H), 4.81 (ddd, J = 7.6,

6.5, 4.6 Hz, 1H), 3.65 (s, 3H), 3.24 (dd, J=11.7, 6.5 Hz, 1H), 2.79 (dd, J = 11.7, 4.6 Hz,

1H), 2.29 (t, J = 7.6 Hz, 2H), 2.06 (q, J = 6.8 Hz, 2H), 1.61 (m, 2H), 1.28-1.41 (m, 20H).
13
C NMR (75 MHz, CDCl3) δ 174.2, 153.5, 141.8, 133.9, 129.4, 128.1, 127.3, 126.2,

80.4, 66.0, 63.8, 51.3, 36.5, 34.0, 32.1, 29.2, 29.1, 29.1, 29.0, 29.0, 28.2, 24.9. HRMS

(EI): calcd for C26H39O4N1S1: 461.2594, found 461.2592.

52-cis. 1H NMR (500 MHz, CDCl3) δ 7.40 – 7.16 (m, 5H), 6.13 (s, 1H), 5.63 (t, J = 9.7

Hz, 1H), 5.58 – 5.45 (m, 1H), 5.13 (d, J = 6.1 Hz, 1H), 3.65 (d, J = 1.7 Hz, 3H), 3.20 (dd,

J = 11.6, 6.4 Hz, 1H), 2.72 (dd, J = 11.6, 5.5 Hz, 1H), 2.29 (t, J = 7.5 Hz, 2H), 2.25 –

2.10 (m, 2H), 1.60 (dd, J = 13.6, 6.8 Hz, 2H), 1.51 – 1.14 (m, 20H). 13C NMR (125 MHz,

CDCl3) δ 174.15, 153.59, 141.92, 132.08, 129.72, 128.11, 127.30, 126.01, 80.46, 66.05,

59.14, 51.33, 36.75, 33.99, 29.58, 29.24, 29.19, 29.11, 29.02, 28.17, 27.60, 24.84.
 272

(E)-11-((2R,4R)-3-(tert-butoxycarbonyl)-2-phenylthiazolidin-4-yl)undec-10-enoic

acid (52-OH). The solution of the methyl ester 52 (11.40 g, 24.70 mmol) in THF (240

mL) was cooled to 0°C. A solution of LiOH·H2O (10.07 g, 240.2 mmol) in H2O (120 mL)

was added in one portion. Then the reaction mixture was stirred and warmed gradually

over night to rt. Upon seeing some intact starting material on TLC, MeOH (25 mL) was

added at rt. 6 hrs later, the solution was cooled to 0°C and Et2O (200 mL) and 1M

NaHSO4 solution (300 mL) were added, after which the pH was ca 2~3. Upon phase

separation, the aqueous layer was extracted with Et2O (150 mL x 3). The combined

organic layer was washed with brine (250 mL), dried over MgSO4, and was filtered.

Upon removal of the solvents, a gummy oil (11.27 g) was obtained, which was used in

the next step without further purification. TLC Rf 0.70 EtAcO / hexane (3:2). [α]D23

+69.5º (c 4.4, CHCl3). IR (film, KBr) νmax 2975, 2928, 2855, 1701, 1369, 1164 cm-1. 1H

NMR (300MHz, CDCl3) δ 7.21-7.38 (m, 5H), 6.08 (s, 1H), 5.78 (dt, J = 15.3, 6.6 Hz,

1H), 5.63 (dd, J = 15.3, 7.6 Hz, 1H), 4.82 (ddd, J = 7.6, 6.4, 4.7 Hz, 1H), 3.24 (dd, J =

12.2, 6.4 Hz, 1H), 2.80 (dd, J = 12.2, 4.7 Hz, 1H), 2.34 (t, J = 7.5 Hz, 2H), 2.07 (q, J =

6.7 Hz, 2H), 1.63 (m, 2H), 1.30-1.43 (m, 19H). 13C NMR δ 179.5, 153.6, 141.8, 134.0,

129.4, 128.1, 127.4, 126.3, 80.5, 66.1, 63.9, 36.6, 34.2, 32.2, 29.21, 29.15, 29.04, 28.99

(3 C's), 28.2, 24.6. HRMS (EI): calcd for C25H37O4N1S1: 447.2438, found 447.2441.
 273

(R,E)-methyl 12-acetamido-13-(acetylthio)tridec-10-enoate (56). To the solution of the

thiazolidine 52-OH (11.145 g from the previous step) in THF (10 mL) was added NH3(l)

(350 mL) that was distilled over Na. To this mixture was added cubic pieces (5~7 mm) of

Na under vigorous stirring until dark blue color persisted. The mixture was kept refluxing

at rt and Na pieces were added as necessary to keep the color for a total of 2 hrs. Then

NH4Cl was carefully added until the color disappeared. NH3 was removed under gentle

stream of N2 to obtain a colorless oil (8.260 g).

The carboxylic acid (8.030 g, from the previous step) was dissolved in Et 2O (50

mL) and was cooled to 0°C. Ethereal solution of CH2N2 (0.14 M, 170 mL, 24 mmol) was

added dropwise over 30 min. Then N2 was bubbled in for 45 min to remove excess

CH2N2. Upon removal of the solvent in vacuo, white crystals were obtained (8.74 g).

These crystals were dissolved in CH2Cl2 (200 mL) and was cooled to 0°C. Then

TFA (50 mL) was added dropwise over 30 min. Upon further stirring at 0°C for 30 min,

the volatiles were removed in vacuo to yield orange oil. Residual TFA was removed

under hi-vacuum for 1 hr. This TFA salt was dissolved in CH 2Cl2 (220 mL) and the

solution was cooled to 0°C. Then Et3N (30.6 mL, 220 mmol) and Ac2O (10.4 mL, 110

mmol) were added slowly under stirring. The solution was let warm to rt over night under

stirring. Then the volatiles were removed in vacuo. The residues were dissolved in Et2O

(200 mL) and 0.5 M HCl solution (200 mL) was added. Upon phase separation, the

aqueous phase was extracted with Et2O (200 mL x 2). The combined organic phase was
 274

washed with H2O (150 mL), saturated NaHCO3 solution (100 mL), H2O (100 mL), and

brine (100 mL) successively. After flash column chromatography (15:85 EtAcO / hexane

to 1:1 EtAcO / hexane), the desired product was obtained as a white solid (~4.2 g, 12

mmol, 50% yield over 5 steps). The disulfide methyl ester was also isolated as a colorless

oil (173 mg, 0.275 mmol, 1% yield). Mp 70-71 ºC. TLC Rf 0.38 EtAcO / hexane (3:2).

[α]D23 –13.2º (c 6.10, CHCl3). IR (film, KBr) νmax 3278, 3081, 2985, 2926, 2852, 1737,

1691, 1647, 1547, 1434, 1373, 1186, 1139, 968, 630 cm-1. 1H NMR (500MHz, CDCl3) δ

5.97 (d, J = 8.1 Hz, 1H), 5.57 (dtd, J = 15.5, 6.6, 1.2 Hz, 1H), 5.29 (ddt, J = 15.5, 6.4, 1.2

Hz, 1H), 4.50 (quin, J = 6.8 Hz, 1H), 3.60 (s, 3H), 3.01 (d, J = 6.6 Hz, 2H), 2.28 (s, 3H),

2.24 (t, J = 7.7 Hz, 2H), 1.94 (q, J = 7.8 Hz, 2H), 1.90 (s, 3H), 1.55 (quin, J = 7.4 Hz,
13
2H), 1.21-1.30 (m, 12H). C NMR (125MHz, CDCl3) δ 195.9, 174.1, 169.3, 133.1,

127.1, 51.3, 50.9, 33.9, 33.4, 32.0, 30.4, 29.0, 29.0, 28.9, 28.8, 28.8, 24.7, 23.1. HRMS

(EI): calcd for C18H32O4N1S1 (M+H): 358.2047, found 358.2045.

Somocystinoic acid (57). To the solution of the methyl ester (487 mg, 1.36 mmol) in

THF (40 mL) was added 1M NaOH solution (20 mL) at rt under stirring. O2 was

continuously bubbled into the mixture while stirring at rt until the consumption of the
 275

thiol was observed by TLC (20 hrs). The reaction mixture was diluted with Et2O (30 mL)

and 1M KHSO4 solution (40 mL) was added at 0°C. Upon phase separation, the aqueous

layer was extracted with Et2O (30 mL) and EtAcO (30 mL x 2). The combined organic

layer was dried over MgSO4 and was filtered. Removal of the solvents in vacuo yielded

pale yellow oil (458 mg), which was used without further purification for the next step.

TLC Rf 0.41 AcOH / MeCN / EtAcO (1:9:90). [α]D23 +41.6º (c 0.66, CHCl3). IR (film,

KBr) νmax 3272(br), 3079(br), 2927, 2855, 1712, 1648, 1550, 1374, 969 cm -1. 1H NMR

(300 MHz, CDCl3) δ 6.23 (d, J = 8.1 Hz, 2H), 5.66 (dtd, J = 15.5, 6.8, 0.7 Hz, 2H), 5.43

(dd, J = 15.5, 6.2 Hz, 2H), 4.70 (quin, J = 6.5 Hz, 2H), 3.06 (dd, J = 13.5, 6.0 Hz, 2H),

2.85 (dd, J = 13.5, 6.2 Hz, 2H), 2.35 (t, J = 7.1 Hz, 4H), 2.04 (m, 4H), 2.03, (s, 6H), 1.64

(quin, J = 7.4 Hz, 4H), 1.26-1.43 (m, 20H). 13C NMR (75 MHz, CDCl3) δ 178.7, 170.1,

133.7, 127.6, 50.8, 44.5, 33.9, 32.0, 28.7, 28.7, 28.6, 28.5, 28.3, 24.6, 23.3. HRMS

(FAB): calcd for C30H52O6N2S2 (M+H): 601.3340, found 601.3337.

Somocystinoic acid di-N,N-methyl amide (17). To the suspension of somocystinoic acid

(72 mg, 0.1198 mmol) in CH2Cl2 (8 mL) was added MeNH3Cl (24 mg, 0.36 mmol), Et3N

(84 µL, 0.60 mmol), and EDC·HCl (50 mg, 0.26 mmol), and DMAP (5 mg) successively
 276

at 0°C under stirring. The reaction was immediately warmed to rt and was stirred at rt for

5 hrs. After 3 hrs into the reaction, white precipitates appeared. The solvents were

removed under vacuum. The residues were taken up in CH2Cl2 / MeOH (2:1) and the

mixture was washed with 0.5 M HCl solution and saturated NaHCO3 solution and was

dried over Na2SO4. Upon removal of the solvents, sufficiently pure material for the next

step was obtained as a white solid (52 mg, 0.083 mmol, 69% yield over 2 steps). Mp

154-155 ºC. TLC Rf 0.36 MeOH / CH2Cl2 (1:9). [α]D23 –17.8º (c 1.4, CHCl3/MeOH 1:1).

IR (film, KBr) νmax 3306, 3276, 2918, 2849, 1642, 1540 cm-1. 1H NMR (300 MHz,

CD3OD/CDCl3 1:1, rotamers) δ 7.55 (d, J =8.3 Hz, 2H), 7.25 (bs, 2H), 5.31 (dt, J = 15.5,

6.7 Hz, 2H), 5.06 (dd, J = 15.5, 6.6 Hz, 2H), 4,24 (m, 2H), 2.53 (d, J = 7.1 Hz, 4H), 2.38

& 2.39 (s, 6H), 1.83 (t, J = 7.8 Hz, 4H), 1.68 (q, J = 6.5 Hz, 4H), 1.64 (s, 6H), 1.25 (quin,

J = 7.4 Hz, 4H), 0.95-1.05 (m, 20H). 13C NMR (75 MHz, CD3OD/CDCl3 1:1, rotamers)

δ 175.2, 175.1, 170.7, 170.6, 132.7, 127.2, 50.1, 43.3, 35.6, 35.5, 31.6, 28.6, 28.5, 28.5,

28.4, 28.3, 25.2, 25.1, 21.7,. 21.6. HRMS (EI): calcd for C32H59O4N4S2 (M+H): 627.3972,

found 627.3973.
 277

Somocystinamide A (1). The suspension of the methyl amide (42 mg, 0.0670 mmol) in

1,2-dichloroethane (20 mL) was degassed three times by vacuum-sonication-Ar

introduction cycle. Then 4-pentenal (100 µL, 1.01 mmol, synthesized according to

literature)51 and TsOH·H2O (3 mg, 0.015 mmol) were added successively at rt. The

reaction mixture was refluxed for 14 hours under stirring in a Soxhlet apparatus that is

equipped with glass wool and molecular sieves 4Å so as to remove residual H 2O. Then

another batch of 4-pentenal (100 μL, 1.01 mmo) and TsOH·H 2O (3 mg, 0.015 mmol)

were added. After 1.5 hours, 4-pentenal (100 μL, 1.01 mmol) was added again. Upon

further refluxing and stirring for 1.5 hours, the reaction mixture was cooled to 0°C, and

Et3N (100 µL) was added. Most of the solvent was removed in vacuo, but ca. 1 mL of

solvent was retained, which was directly subjected to flash column chromatography

(EtAcO to 1:1:8 MeOH / CH2Cl2 / EtAcO) to obtain somocystinamide A (21 mg, 0.0277

mmol, 41% yield) as an off-white amorphous solid. Mp 93-95 ºC. TLC Rf 0.56 MeOH /

CH2Cl2 / EtAcO (1:1:8). [α]D22 +19.1º (c 0.59, CHCl3). IR (film, KBr) νmax 3286(br), 2926,

2854, 1645, 1541, 1395, 1289, 1087, 968 cm-1. 1H NMR (300 MHz, CDCl3, rotamers) δ

7.35 (d, J = 14.4 Hz 0.6H), 6.67 (dt, J = 13.7, 1.3 Hz, 1.4H), 6.28 (d, J = 7.8 Hz, 2H),

5.84 (m, 2H), 5.65 (dtd, J = 15.5, 6.7, 1.1 Hz, 2H), 5.43 (dd, J = 15.5, 6.2 Hz, 2H),

4.91-5.11 (m, 4H), 4.67 (quin, J = 6.5 Hz, 2H), 3.07 and 3.08 (s, 6H), 3.06 (m, 2H), 2.82

(m, 6H), 2.42 (m, 4H), 2.03 (m, 4H), 2.01 (s, 6H), 1.64 (quin, J = 7.5 Hz, 4H), 1.25-1.38

(m, 20H). 13C NMR (75 MHz, CDCl3, rotamers) δ 171.7, 169.1, 137.4, 137.0, 133.7,

129.2, 128.1, 127.6, 115.4, 115.0, 109.0, 108.4, 50.8, 44.7, 34.5, 34.5, 34.4, 33.8, 32.3,

32.2, 29.7, 29.7, 29.3, 29.2, 29.0, 28.9, 25.0, 23.4. UV (MeOH) λ max 234 nm (ε 3.2×105).

HRMS (EI): calcd for C42H70O4N4S2 (M+): 758.4833, found 758.4823.

 278

N-methylundec-10-enamide (70). To a solution of 10-undecenoic acid (25.136 g, 136.40

mmol) in CH2Cl2 (350 mL) was added MeNH2·HCl (18.45 g, 273.3 mmol) at rt. This

mixture was cooled to –15ºC and Et3N (57.0 mL, 408 mmol) was added. Then EDC·HCl

(27.29 g, 142.4 mmol) and DMAP (238 mg, 1.95 mmol) were added successively under

vigorous stirring. The mixture was very cloud at this point. The mixture was gradually

warmed to rt, and was stirred over night. The mixture appeared milky. Upon removal of

the volatiles in vacuo, the solid residues were triturated with EtOAc (500 mL). This

mixture was filtered, and the solids were rinsed with EtOAc. Then the EtOAc solution

was poured into 1M HCl (250 mL), and the phases were separated. The organic layer was

washed with H2O (150 mL), saturated NaHCO3 (150 mL), H2O (100 mL), and brine (100

mL) successively. After the solution was dried over Na2SO4 and the solvent was removed,

a white solid (70) resulted, which was sufficiently pure for the subsequent step. 1H NMR

(300 MHz, CDCl3) δ 5.80 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.50 (s, 1H), 5.05 – 4.86 (m,

2H), 2.80 (d, J = 4.9 Hz, 3H), 2.15 (t, J = 7.6 Hz, 2H), 2.02 (dd, J = 14.1, 6.9 Hz, 2H),

1.70 – 1.53 (m, 3H), 1.45 – 1.18 (m, 15H). 13C NMR (75 MHz, CDCl3) δ 173.80, 139.15,

114.10, 58.15, 36.71, 33.75, 29.28, 29.03, 28.86, 26.24, 25.75.

 279

(2R,4R)-tert-butyl 4-((E)-11-(methylamino)-11-oxoundec-1-enyl)-2-phenyl

thiazolidine -3-carboxylate (71). To the thiazolidine (51) (2.063 g, 7.079 mmol) and the

methylamide (70) (2.270 g, 11.50 mmol) was added CHCl3 (45 mL) that was passed

through basic alumina (activity I). Then the reaction mixture was degassed in vacuum

with ultrasonic assistance three times. Grubbs-Hoveyda II catalyst (95 mg, 0.152 mmol)

was added. After stirring at rt for 30 min, the reaction mixture was refluxed (bath

temperature = 80ºC). Over the course of 18 hrs, additional amounts of 70 (1.932 g, 9.791

mmol) and Grubbs-Hoveyda II catalyst (354 mg, 0.564 mmol, 5.8 mol%) were added in

four portions. Then the mixture was cooled to rt, concentrated under vacuum, and

subjected to silica gel flash column chromatography (1:9 EtOAc / Hex to 2:9:9 acetone /

EtOAc / Hex). Some of the colored impurities were not completely removed. A dark-

brown thick oil (2.752 g, 5.974 mmol, 84%) was obtained. TLC Rf = 0.47 (2:9:9 MeOH/

EtOAc/hexanes). [α]22D +58.6º (c 3.6, CHCl3). IR (film, KBr) νmax 3298(br), 2927, 2854,

1696, 1650, 1554, 1368, 1163, 1109, 697 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.37 – 7.19

(m, 5H), 6.05 (s, 1H), 5.83 – 5.70 (m, 1H), 5.61 (dd, J = 15.3, 7.4 Hz, 1H), 4.79 (s, 1H),

3.22 (dd, J = 11.7, 6.5 Hz, 1H), 2.81 – 2.75 (m, 1H), 2.75 (d, J = 4.9 Hz, 3H), 2.17 – 2.08

(m, 1H), 2.08 – 1.99 (m, 2H), 1.64 – 1.53 (m, 2H), 1.41 – 1.20 (m, 21H). 13C NMR (100

MHz, CDCl3) δ 173.8, 153.5, 141.7, 134.0, 129.3, 128.1, 127.4, 126.2, 80.4, 66.0, 63.8,

36.6, 36.5, 32.1, 29.2, 29.2, 29.1, 29.0, 28.9, 28.1, 26.1, 25.7. HRMS (ESI): calcd for

C26H40N2O3SNa [M+Na+]: 483.2652, found 483.2654.

 280

(R,E)-S-2-acetamido-13-(methylamino)-13-oxotridec-3-enyl ethanethioate (73).

Liquid NH3 (150 mL) was distilled from Na onto a solution of 71 (3.098 g, 6.725 mmol)

in THF (10 mL). Then Na pieces (~ 5 mm cubes) were added to keep the color of the

solution dark blue for 2 hrs under reflux (at rt) and vigorous stirring. Solid NH 4Cl was

added carefully until the blue color disappeared. NH3 was evaporated under a stream of

N2. Then 1M NaHSO4 (300 mL) was added, which was extracted with EtOAc (200 mL x

3). The organic layer was washed with brine and dried over Na2SO4. Upon removal of the

solvent in vacuo, amorphous pale brown solid (2.531 g) resulted, which was used in the

next step without purification. This solid (2.531 g) was dissolved in CH2Cl2 (75 mL) and

freshly distilled TFA (18 mL) was added at 0ºC under stirring. After the solution was kept

at 0ºC under stirring for 30 min, the volatiles were removed in vacuo. The residues were

placed under high vacuum for 2 hours, which resulted in a thick orange oil. This oil was

dissolved in CH2Cl2 (100 mL) and the solution was cooled to 0ºC. Et3N (15.8 mL, 113.2

mmol) and acetic anhydride (5.3 mL, 56.6 mmol) were added successively at 0ºC under

stirring. The solution was gradually allowed to warm to rt. After 16 hrs of stirring at rt,

the volatiles were removed in vacuo. The residues were treated with 1M NaHSO4 (250

mL), which was extracted with EtOAc (200 mL x 3). The combined organic layer was

washed with brine (150 mL) and dried over Na2SO4. Most of the solvent was removed

under vacuum, and the crude solution was let stand in a -20ºC freezer over night. Pale

brown solids were precipitated and filtered, which was rinsed with hexanes. Upon drying
 281

under high vacuum, 73 was obtained as a pale brown solid (1.112 g). The filtrate

contained some of the product, and it was purified by flash column chromatography (1:1

EtOAc/hexanes to 2:9:9 MeOH/EtOAc/hexanes). A total of 1.406 g of 73 was obtained

(59% yield). Mp 95-97ºC. TLC Rf = 0.22 (2:9:9 MeOH/EtOAc/hexanes). [α]22D –8.0º (c

0.90, CHCl3). IR (film, KBr) νmax 3314, 3286, 3089(br), 2925, 2853, 1693, 1646, 1553,

1412, 1373, 1133, 968 cm-1. 1H NMR (300 MHz, CDCl3) δ 6.03 (d, J = 8.3 Hz, 1H), 5.90

(s, 1H), 5.59 (ddd, J = 7.9, 7.3, 4.0 Hz, 1H), 5.43 – 5.21 (m, 1H), 4.52 (p, J = 6.3 Hz,

1H), 3.04 (d, J = 6.6 Hz, 2H), 2.76 (d, J = 4.8 Hz, 3H), 2.32 (s, 3H), 2.13 (t, J = 7.6 Hz,

2H), 2.00 – 1.95 (m, 2H), 1.93 (s, 3H), 1.72 – 1.42 (m, 2H), 1.26 (m, 10H). 13C NMR (75

MHz, CDCl3) δ 196.2, 173.9, 169.5, 133.1, 127.8, 58.1, 51.0, 36.5, 33.5, 32.0, 30.5, 29.1,

29.0, 29.0, 28.7, 26.2, 25.6, 23.2. HRMS (ESI): calcd for C18H32N2O3SNa [M+Na+]:

379.2026, found 379.2029.

 282

Figure VI.12: 1H NMR spectrum of 22 in CDCl3

 283

Figure VI.13: 13C NMR spectrum of 22 in CDCl3

 284

Figure VI.14: 1H NMR spectrum of 2 in CDCl3

 285

Figure VI.15: 13C NMR spectrum of 2 in CDCl3

 286

Figure VI.16: 1H NMR spectrum of 44 in CDCl3

 287

Figure VI.17: 13C NMR spectrum of 44 in CDCl3

 288

Figure VI.18: 1H NMR spectrum of 51 in CDCl3

 289

Figure VI.19: 13C NMR spectrum of 51 in CDCl3

 290

Figure VI.20: 1H NMR spectrum of 52 in CDCl3

 291

Figure VI.21: 13C NMR spectrum of 52 in CDCl3

 292

Figure VI.22: 1H NMR spectrum of 52-cis in CDCl3

 293

Figure VI.23: 13C NMR spectrum of 52-cis in CDCl3

 294

Figure VI.24: 1H NMR spectrum of 56 in CDCl3

 295

Figure VI.25: 13C NMR spectrum of 56 in CDCl3

 296

Figure VI.26: COSY spectrum of 56 in CDCl3

 297

Figure VI.27: HMBC spectrum of 56 in CDCl3

 298

Figure VI.28: 1H NMR spectrum of 57 in CDCl3

 299

Figure VI.29: 13C NMR spectrum of 57 in CDCl3

 300

Figure VI.30: 1H NMR spectrum of 3 in 1:1 CDCl3/CD3OD

 301

Figure VI.31: 13C NMR spectrum of 3 in 1:1 CDCl3/CD3OD

 302

Figure VI.32: 1H NMR spectrum of 1 in CDCl3

 303

Figure VI.33: 13C NMR spectrum of 1 in CDCl3

 304

70

Figure VI.34: 1H NMR spectrum of 70 in CDCl3

 305

70

Figure VI.35: 13C NMR spectrum of 70 in CDCl3

 306

Figure VI.36: 1H NMR spectrum of 71 in CDCl3

 307

Figure VI.37: 13C NMR spectrum of 71 in CDCl3

 308

Figure VI.38: 1H NMR spectrum of 73 in CDCl3

 309

Figure VI.39: 13C NMR spectrum of 73 in CDCl3

 310

References and footnotes

1 Newman, D. J.; Cragg, G. M. Natural products as sources of new drugs over the last 25 years.
J. Nat. Prod. 2007, 70, 461-477.

2 Gerwick, W. H.; Tan, L. T.; Sitachitta, N. The Alkaloids; Cordell, G. A., Ed.; Academic Press;
San Diego, CA 2001; Vol. 57, pp 75-184.

3 Nogle, L. M.; Gerwick, W. H. Somocystinamide a, a novel cytotoxic disulfide dimer from a

fijian marine cyanobacterial mixed assemblage. Org. Lett. 2002, 4(7), 1095-8.

4 Wrasidlo, W.; Mielgo, A.; Torres, V. A.; Barbero, S.; Stoletov, K.; Suyama, T. L.; Klemke, R.
L.; Gerwick, W. H.; Carson, D. A.; Stupack, D. G. The marine lipopeptide somocystinamide A
triggers apoptosis via caspase 8. Proc. Natl. Acad. Sci. 2008, 105, 2313-2318.

5 Park, H.-J.; Zhang, Y.; Georgescu, S. P.; Johnson, K. L.; Kong, D.; Galper, J. B. Human
umbilical vein endothelial cells and human dermal microvascular endothelial cells offer new
insights into the relationship between lipid metabolism and angiogenesis. Stem Cell Reviews
and Reports 2006, 2, 93-101.

6 Goishi, K.; Klagsbrun, M. Vascular endothelial growth factor and its receptors in embryonic
zebrafish blood vessel development. Current Topics in Developmental Biology 2004, 62,
127-152.

7 EarnShaw, W. C.; Martins, L. M.; Kaufmann, S. H. mammalian caspases: structure, activation,

substrates, and functions during apoptosis. Annu. Rev. Biochem. 1999, 68, 383-424.

8 There is some evidence that 1 also induces intrinsic apoptosis, which involves caspase-9, in
addition to the extrinsic apoptotic pathway involving caspase-8 (see ref. 4).

9 This compound was synthesized as shown in Scheme VI.4.

10 This compound was obtained through unintentional hydrolysis of 1 (see Scheme VI.2).

11 Bukowski, R. M. AE-941, a multifunctional antiangiogenic compound: trials in renal cell

carcinoma. Expert Opin. Investig. Drugs 2003, 12, 1403-1411.

12 Wahl, M. L.; Kenan, D. J.; Gonzalez-Gronow, M.; PizzoMore, S. V. Angiostatin’s Molecular

Mechanism: Aspects of Specificity and Regulation Elucidated . J. Cell. Biochem. 2005, 96,
242-261.

13 National Instituted of Health. Safety and Efficacy Study of rhAngiostatin Administered in

Combination With Paclitaxel and Carboplatin to Patients With Non-Small-Cell Lung Cancer.
http://www.clinicaltrials.gov/ct/gui/show/NCT00049790;jsessionid=91EE48B7C2F7D935B96
F12E31D5609E3?order=1 (accessed on 05/19/2009).

14 Crews, P.; Rodrǐguez, J.; Jaspars, M. Optical and chiroptical techniques: Ultraviolet
spectroscopy. In Organic Structure Analysis; Oxford University Press: Oxford, 1998, pp
349-373.
 311

15 Tracey, M. R.; Hsung, R. P.; Antoline, J.; Kurtz, K. C. M.; Shen, L.; Slafer, B. W.; Zhang, Y.
Product class 4: N-Arylalkanamides, ynamides, enamides, dienamides, and allenamides.
Science of Synthesis 2005, 21, 387-475.

16 (a) Jiang, L.; Job, G. E.; Klapars, A.; Buchwald, S. L. Copper-Catalyzed Coupling of Amides
and Carbamates with Vinyl Halides . Org. Lett. 2003, 5, 3667–3669. (b) Dehli, J. R.; Legros,
J.; Bolm, C. Synthesis of enamines, enol ethers and related compounds by cross-coupling
reactions . Chem. Commun. 2005, 973–986. (c) Han, C.; Shen, R.; Su, S.; Porco, J. A., Jr.
Copper-Mediated Synthesis of N-Acyl Vinylogous Carbamic Acids and Derivatives: Synthesis
of the Antibiotic CJ-15,801. Org. Lett. 2004, 6, 27–30.

17 (a) Ramaswamy, A. V.; Flatt, P. M.; Edwards, D. J.; Simmons, T. L.; Han, B.; Gerwick, W. H.
The secondary metabolites and biosynthetic gene clusters of marine cyanobacteria.
Applications in biotechnology. Frontiers Mar. Biotech. 2006, 175-224. (b) Moore, Bradley
S. Biosynthesis of marine natural products: macroorganisms (Part B). Nat. Prod. Rep, 2006,
23, 615-629.

18 Chang, Z.; Sitachitta, N.; Rossi, J. V.; Roberts, M. A.; Flatt, P. M.; Jia, J.; Sherman, D, H.;
Gerwick, W. H. Biosynthetic Pathway and Gene Cluster Analysis of Curacin A, an Antitubulin
Natural Product from the Tropical Marine Cyanobacterium Lyngbya majuscula. J. Nat. Prod.
2004, 67, 1356-1367.

19 Cantoni, G. L. The Nature of the Active Methyl Donor Formed Enzymatically from L-
Methionine and Adenosinetriphosphate. J. Am. Chem. Soc. 1952, 74, 2942-2943.

20 Dewick, P. M. Secondary metabolism: The building blocks and construction mechanisms. In

Medicinal natural products: A biosynthetic approach; 2nd Ed. John Wiley & Sons, LTD:
London, 2001, pp 7-34.

21 Rukachaisirikul, V.; Sommart, U.; Phongpaichit, S.; Sakayaroj, J.; Kirtikara, K. Metabolites
from the endophytic fungus Phomopsis sp. PSU-D15. Phytochem. 2008, 69, 783-787.

22 (a) Erickson, K. L.; Beutler, J. A.; Cardellina, J. H., II; Boyd, M. R. Salicylihalamide A and B,
novel cytotoxic macrolides from the marine sponge Haliclona sp. J. Org. Chem. 1997, 62,
8188–8192. (b) Erickson, K. L.; Beutler, J. A.; Cardellina, J. H., II; Boyd, M. R.
Salicylihalamides A and B, Novel Cytotoxic Macrolides from the Marine Sponge Haliclona
sp. [Erratum]. J. Org. Chem. 2001, 66, 1532.

23 Boyd, M. R.; Farina, C.; Belfiore, P.; Gagliardi, S.; Kim, J. W.; Hayakawa, Y.; Beutler, J. A.;
McKee, T. C.; Bowman, B. J.; Bowman, E. J. Discovery of a novel antitumor benzolactone
enamide class that selectively inhibits mammalian vacuolar-type (H+)-ATPases. J.
Pharmacol. Exp. Ther. 2001, 297, 114–120.

24 Sun-Wada, G.-H.; Wada, Y.; Futai, M. Diverse and essential roles of mammalian vacuolar-type
proton pump ATPase: toward the physiological understanding of inside acidic compartments.
Biochim. Biophys. Acta 2004, 1658, 106–114.
 312

25 Huss, M.; Wieczorek, H.; Inhibitors of V-ATPases: old and new players. J. Exp. Biol. 2009,
212, 341-346.

26 Palermoa, J. A.; Flowera, P. B.; Seldes, A. M. Chondriamides A and B, new indolic

metabolites from the red alga Chondria sp. Tetrahedron Lett. 1992, 33, 3097-3100.

27 Jorge A. Palermo, Patricia Blanch Flower and Alicia M. Seldes. Chondriamides A and B, New
Indolic Metabolites Red Alga Chondria sp. Tetrahedron Lett. 1992, 33, 3097-3100.

28 Pattenden, G.; Ashweek, N. J.; Baker-Glenn, C. A. G.; Kempson, J.; Walker, G. M.; Yee , J. G.
K. Total synthesis of (−)-ulapualide A, a novel tris-oxazole macrolide from marine
nudibranchs, based on some biosynthesis speculation . Org. Biolmol. Chem. 2008, 6,
1478-1497.

29 Su, S.; Kakeya, H.; Osada, H.; Porco, J. A. Jr. Synthesis and cell cycle inhibition of the peptide
enamide natural products terpeptin and the aspergillamides . Tetrahedron 2003, 59,
8931-8946.

30 (a) Ninomiya, I. Applications of enamide chemistry to the synthesis of heterocyclic

compounds. Heterocycles 1980, 14, 1567–1579. (b) Davies, D. T.; Kapur, N.; Parsons, A. F.
Preparation of N-Heterocycles by Radical Cyclisation of Enamides Mediated by
Manganese(III) or Copper(I). A Comparison of Cyclisation Methods. Tetrahedron 2000, 56,
3941–3949. (c) Baker, S. R.; Burton, K. I.; Parsons, A. F.; Pons, J.; Wilson, M. Tandem radical
cyclisation of enamides mediated by tin hydride; pyrrolizidinone or indolizidinone ring
formation. J. Chem. Soc., Perkin Trans. 1 1999, 4, 427–436. (d) Brodney, M. A.; Padwa, A.
Generation and Trapping of N-Acyliminium Ions Derived from Isomünchnone Cycloadducts.
A Versatile Route to Functionalized Heterocycles. J. Org. Chem. 1999, 64, 556–565. (e)
Okano, T.; Sakaida, T.; Eguchi, S. Generation of 6-(Trifluoromethyl)-4,5-dihydro-2(3H)-
pyridone and the Application to Synthesis of Some Fused Nitrogen Heterocycles Carrying a
Trifluoromethyl Group on the Bridgehead Position via Radical Cyclization of
Dihydropyridones. J. Org. Chem. 1996, 61, 8826–8830. (f) Schultz, A. G.; Guzzo, P. R.;
Nowak, D. M. Asymmetric Organic Synthesis. Radical Cyclizations of Chiral Enamides. J.
Org. Chem. 1995, 60, 8044–8050.

31 a) Cook, G. R.; Stille, J. R. Stereochemical consequences of the lewis acid-promoted 3-aza-

cope rearrangement of N-alkyl-N-allyl enamines. Tetrahedron 1994, 50, 4105-4124. b) Padwa,
A.; Heidelbaugh, T. M.; Kuethe, J. T.; McClure, M. S.; Want, Q. Tandem pummerer/mannich
cyclization cascade of α-sulfinylamides as a method to prepare aza-heterocycles. J. Org.
Chem. 2002, 67, 5928-5937.

32 Overman, L. E.; Sato, T. Construction of Epidithiodioxopiperazines by Directed Oxidation of

Hydroxyproline-Derived Dioxopiperazines. Org. Lett. 2007, 9, 5267-5270.

33 Middleton, M. D.; Peppers, B. P.; Diver, S. T. Studies directed toward the synthesis of the
scabrosins: validation of a tandem enyne metathesis approach . Tetrahedron 2006, 62,
10528-10540.
 313

34 Fukuyama, T.; Nakatsuka, S.; Kishi, Y. Total synthesis of gliotoxin, dehydrogliotoxin, and
hyalodendrin. Tetrahedron 1981, 37, 2045-2078.

35 (a) Nicolaou, K. C.; Hughes, R.; Pfefferkorn, J. A.; Barluenga, S.; Roecker, A. J.
Combinatorial Synthesis through Disulfide Exchange: Discovery of Potent Psammaplin A
Type Antibacterial Agents Active against Methicillin-Resistant Staphylococcus aureus
(MRSA) . Chem. Eur. J. 2001, 7, 4280-4295. (b) Hoshino, O.; Murakata, M.; Yamada, K. An
improved synthesis of psammaplin A. Bioorg. Med. Chem. Lett. 1992, 2, 1561-1562. (c)
Hoshino, O.; Murakata, M.; Yamada, K. A convenient synthesis of a bromotyrosine derived
metabolite, psammaplin A, from Psammaplysilla sp. Bioorg. Med. Chem. Lett. 1992, 12,
1561-1562.

36 Chemler, S. R.; Trauner, D.; Danishefsky, S. J. The B-alkyl Suzuki-Miyaura cross-coupling

reaction: development, mechanistic study, and applications in natural product synthesis.
Angew. Chem. Int. Ed. 2001, 40, 4544-4568.

37 Nicolau, K. C.; Bulger, P. G.; Sarlah, D. Palladium-catalyzed cross-coupling reactions in total

synthesis. ibid. 2005, 44, 4442-4489.

38 a) Takai, K.; Nitta, K.; Utimoto, K. Simple and selective method for aldehydes (RCHO)
.fwdarw. (E)-haloalkenes (RCH:CHX) conversion by means of a haloform-chromous chloride
system. J. Am. Chem. Soc. 1986, 108, 7408-7410. b) Concellon, J. M.; Bernad, P. L.; Mejica,
C. Synthesis of enantiopure (S)-(E)-1-haloalk-1-ene-3-amines with total or very high
diastereoselectivity by halomethylenation of α-amino aldehydes promoted by CrCl2.
Tetrahedron Lett. 2005, 46, 569-571.

39 Deroose, F. D.; De Clercq, P. J. Novel enantioselective syntheses of (+)-biotin. J. Org. Chem.

1995, 60, 321-330.

40 Mancuso, A. J.; Huang, S. L.; Swern, D. Oxidation of long-chain and related alcohols to
carbonyls by dimethyl sulfoxide "activated" by oxalyl chloride. J. Org. Chem. 1978, 43,
2480-2482.

41 There is evidence that 1 also induces intrinsic apoptosis in addition to the extrinsic apoptotic
pathway involving caspase-8. However, initiation of the extrinsic pathway, which is more
sensitive, appears to be the predominant mechanism of action.

42 Molander, G. A.; Ito, T. Cross-Coupling Reactions of Potassium Alkyltrifluoroborates with

Aryl and 1-Alkenyl Trifluoromethanesulfonates. Org. Lett. 2001, 3, 393-396.

43 Nicolaou, K. C.; Bulger, P. G.; Sarlah, D. Metathesis reactions in total synthesis. Angew.
Chem. Int. Ed. 2005, 44, 4490-4527.

44 Wittig, G.; Schöllkopf, U. Über Triphenyl-phosphin-methylene als olefinbildende Reagenzien.

Ber. 1954, 87, 1318.

45 Garber, S. B.; Kingsbury, J. S.; Gray, B. L.; Hoveyda, A. H. Efficient and Recyclable
Monomeric and Dendritic Ru-Based Metathesis Catalysts. J. Am. Chem. Soc. 2000, 122,
 314

8168-8179.

46 I took the liberty to name this compound (57) somocystinoic acid because it is a new
compound.

47 (a) Cook, G. R.; Stille, J. R. Stereochemical consequences of the lewis acid-promoted 3-aza-
cope rearrangement of N-alkyl-N-allyl enamines. Tetrahedron 1994, 50, 4105–4124. (b)
Gangadasu, B.; Narender, P.; Kumar, S. B.; Ravinder, M.; Rao, B. A.; Ramesh, C.; Raju, B. C.;
Rao, V. J. Facile and selective synthesis of chloronicotinaldehydes by the Vilsmeier reaction.
Tetrahedron 2006, 62, 8398–8403. (c) Cook, G. R.; Barta, N. S.; Stille, J. R. Lewis acid-
promoted 3-aza-Cope rearrangement of N-alkyl-N-allyl enamines. J. Org. Chem. 1992, 57,
461–467. (d) Couture, A.; Dubiez, R.; Lablache-Combier, A. Secondary enamide and
thioenamide photochemistry. A new spiroannulation method. J. Org. Chem. 1984, 49, 714–
717. (e) Pattenden, G.; Ashweek, N. J.; Baker-Glenn, C. A. G.; Kempson, J.; Walker, G. M.;
Yee, J. G. K. Total synthesis of (-)-ulapualide A, a novel tris-oxazole macrolide from marine
nudibranchs, based on some biosynthesis speculation. Org. Biomol. Chem. 2008, 6, 1478–
1497. (f) Morales, O.; Seide, W.; Watson, S. E. Synthesis of 7,6-fused bicyclic lactams for use
as beta-turn mimics. Synth. Commun. 2006, 36, 965–973.

48 The acetamide, disulfide, or already formed enamide of the other half of the dimeric
intermediate may act as a nucleophile to attack the iminium ion. For examples of nucleohilic
additions to iminium ions, see: Moonen, K.; Stevens, C. V. Synthesis 2005, 20, 3603–3612.

49 In separate studies, reaction of 17 with simple unfunctionalized acid chlorides formed

enamides, thus indicating the presence of an acyliminium ion intermediate like 21. Because 19
was not observed by NMR, an acylenaminium intermediate is not likely

50 Ogawa, T.; Kiji, T.; Hayami, K.; Suzuki, H. Stereospecific one-pot synthesis of enamides and
enimides by the copper iodide promoted vinylic substitution. Chem. Lett. 1991, 1443-1446.

51 4-pentenal (5) was synthesized according to the following paper: Griffith, G. A.; Percy, J. M.;
Pintat, S.; Smith, C. A.; Spencer, N.; Uneyama, E. Towards novel difluorinated sugar
mimetics; syntheses and conformational analyses of highly-functionalised difluorinated
cyclooctenones. Org. Biomol. Chem. 2005, 3, 2701–2712.

52 Instead of a thimble, glass wool and molecular sieves were used to capture any moisture
distilled as an azeotrope.

53 (a) Saunders, W. H. Jr. Acc. Chem. Res. 1976, 9, 19-25. (b) Carey, F. A.; Sundberg, R. J.
Advanced Organic Chemistry Part A 4th Ed, Kluwer Academic / Pleunum Publishers; New
York, 2000, 378-383.

54 Since the publication of this synthesis, the condition for enamide formation was slightly
modified (higher concentration and more equivalents of the aldehyd) to increase the yield
from 41 to 47%.

55 If this is the case, it would not be the first time. See Chapter V for the bioactivity of the
synthetic sample of epiquinamide.
 315

56 de Boer, T. J.; Backer, H. J. Diazomethane. Org. Synth. 1963, 4, 250-253.

57 Lee, S. Process development : fine chemicals from grams to kilograms. Oxford ; New York :
Oxford University Press, 1995.

58 Unfortunately, the 200 mg mark was not met for technically being a multi-hundred-milligram
(MHM) scale synthesis. See Chapter I of this dissertation for the discussion on MHM
synthesis.

59 Still, W. C.; Khan, M.; Mitra, A. Rapid chromatographic technique for preparative separations
with moderate resolution. J. Org. Chem. 1978, 43, 2923-2925.

60 Chavan, S. P.; Chittiboyina, A. G.; Ramakrishna, G.; Tejwani, R. B.; Ravindranathan, T.;
Kamat, S. K.; Rai, B.; Sivadasan, L.; Balakrishnan, K.; Rmalingam, S.; Deshpande, V. H. An
unusual stereochemical outcome of radical cyclization: synthesis of (C)-biotin . Tetrahedron,
2005, 61, 9273-9280.
 Chapter VII

Biomimetic total synthesis of a cyanobacterial UV-blocking natural product, scytonemin

and insights into its biogenesis

316
 317

Abstract

Scytonemin is a cyanobacterial alkaloid that has UV-screening properties. In order

to gain insights into the biogenesis of the unique structure and to produce a chelation-free

sample, synthetic studies were conducted on scytonemin. Through a successful synthesis

of the proposed monomeric precursor of scytonemin, its spontaneous dimerization was

shown to be impossible under ambient conditions. Various attempts to oxidatively

dimerize this monomer and explanations for their failure are described herein. As a result,

an alternative structure for the monomeric precursor of scytonemin was proposed.

 318

VII.1 Introduction

Cyanobacteria can be commonly found in

environments that are exposed to harmful levels of

UV radation as well as sun light necessary for

photosynthesis.1 In order to avoid the deleterious

effects of UVA (320-400 nm), UVB (280-320 nm),

and UVC (190-280 nm) radiations, pigmented metabolites, including scytonemin (1),2 are

stored in the extracellular sheaths of many cyanobacteria.1,3 Indeed, this dark-greenish

brown indole alkaloid is particularly well suited for absorbing UV light in the blue

wavelengths (Figure VIII.1).4

Figure VII.1: Light absorption spectrum of scytonemin (1) (taken from ref. 4)

This natural product is a dimer of a unit that appears to be derived from L-

tryptophan and chorismate.5 Under anaerobic conditions, scytonemin can exist in a

reduced form (2) as shown in Scheme VII.1.2 Almost 150 years after the initial report of

the compound,6 the structure of 1 was finally elucidated in 1993 by Gerwick and
 319

coworkers through NMR and MS analysis of 1, as well as those of the ozonolysis product

of 2 (Scheme VII.1).2 Incidentally, 3 is also a natural product (nostodione) isolated from a

terrestrial cyanobacteria, Nostoc commune, and it is known to disrupt mitotic spindle

formation in sea-urchin eggs.7

Due to the unique carbon skeleton and ecological significance of this metabolite,

there is a considerable interest in its biosynthesis.8 The first study in this regard by

Scheme VII.1: Fragmentation of the reduced form of scytonemin (2) through ozonolysis in its
structure elucidation (Gerwick et al. 1993)

Scheme VII.2: Biosynthetic gene cluster for scytonemin in Nostoc punctiforme5,6

 320

Scheme VII.3: Proposed mechanism for the biosynthesis of scytonemin (1) by Balsksus and
Walsh5

Biosynthetic products that were analytically characterized (6 and 7) are highlighted by

rectangles. Note the proposed structure of the monomer (11). In reality, the putative monomeric
precursor is exclusively in the keto form (vide infra).

Garcia-Pitchel and coworkers reported the identification of the scytonemin biosynthetic

gene cluster in 2007 (Scheme VII.2).5,8 The involvement of tryptophan and chorismate in

the biosynthesis were implicated by the presence of genes NpR1261-1266 and by the

presence of genes NpR1260, 1267, and 1269, respectively. Subsequently, Balskus and

Walsh provided biochemical evidence for the biosynthetic intermediacy of α-hydroxy

ketone 7 and p-hydroxyphenylpyruvic acid 4.5 The proposed mechanism shown in

 321

Scheme VII.3 is based on the observed activity of the ThDP (thiamine diphosphate)-

dependent acetolactate synthase (NpR1276 in Scheme VII.2 and 3).

Phosphorylated thiamine is essential for the normal function of the nervous

system in many species.9 Having a ylide functionality, thiamine possesses both

nucleophilicity and electrophilicity. Consequently, 5 is able to undergo nucleophilic

addition to the ketone of α-ketoacid 4, facilitate aldol condensation with another α-

ketoacid 6, and eliminate as an ylide to regenerate itself (Scheme VII.3). Decarboxylation

of 7 is expected to be necessary due to the steric hindrance by the carboxylic acid in the

cyclization step. Although structure 8 appears to be suitable for a rapid intramolecular

cyclization reminiscent of the Pictet-Spengler reaction, catalysis is apparently necessary. 10

Scheme VII.4: Chemical synthesis of a putative biosynthetic precursor of scytonemin (1),

soraphinol A (S-8)

Balskus and Walsh were able to synthesize α-hydroxy ketone S-8 without spontaneous

cyclization through an independent route, and this material was used as a synthetic

standard for comparison with the acid-catalyzed decarboxylation product of 7 (Scheme

VII.4).5

As can be seen in Scheme VII.3, there are many steps remain to be determined in

the biosynthesis of scytonemin. For example, despite genetic evidence, the intermediacy
 322

Scheme VII.5: Initially conceived synthesis of scytonemin monomer 11

Dissection “a” represents the initially conceived approach. Dissection “b” represents the revised
approach. While the biomimetic approach of “a” is attractive, the starting material 16 would
require many steps to prepare.

Scheme VII.6: Predicted mechanism for regioselectivity of aldol condensation between ketone
18 and aldehyde 19

of the monomer 11 has not been established. Furthermore, it is unclear from the gene

cluster analysis whether the oxidative dimerization step is enzyme-catalyzed or

spontaneous under ambient conditions. The most straightforward approach to answering

this question is to synthesize the putative monomer precursor. If the monomer does not

dimerize spontaneously, synthetic attempts to dimerize it could provide insight into the

chemistry that must occur for this important step in the biosynthesis of scytonemin (1).
 323

Scheme VII.7: Synthesis of scytonemin monomer

The product of the condensation of 18 and 19 was exclusively in the keto form (22) and not in
the enol form (11) as predicted by others. Therefore, this product is given a new compound
number (22).

Figure VII.2: Select HMBC, COSY, and ROESY correlations of scytonemin monomer
(22) in DMF-d7
1
H NMR shifts are shown in red and 13C NMR shifts are shown in blue.
 324

Scheme VII.8: Absence of tautomerization and resonance characteristics of scytonemin

monomer (22)

Figure VII.3: Scytonemin monomer (22): geometry optimized computer model

A dihedral angle constraint was placed around the single bond between the phenyl ring and the
exocyclic double bond so as to keep the phenyl ring perpendicular to the tricyclic system
(according to the NMR data). Ab initio (Hartree-Fock 3-21G*) geometry optimization was
performed using Spartan®. A conformer distribution calculation at a molecular mechanics level
also yielded the depicted geometry as one of the best conformers.

It has been reported that 1 possesses anti-proliferative and anti-inflammatory

activities.11 However, due to its strong metal-binding properties (especially for zinc), a

pure sample of 1 could not be obtained for further biological assays. Therefore, a metal-

free synthetic sample of 1 would be valuable for pharmaceutical investigation.

 325

Figure VII.4: 1H NMR spectrum of 22 in DMF-d7

VII.2 Synthesis of scytonemin monomer

At the outset, a synthetic route that involves a putative biosynthetic precursor was

considered for the synthesis of monomer 11 (Scheme VII.5), where diketone 16 is

predicted to undergo a cyclization reaction similar to the cyclization of 8 in Scheme

VII.3. However, due to the fairly large number of steps 16 would require to synthesize,

another approach was conceived through the use of an alternative intermediate 17 instead

of 16 (Scheme VII.5). In this strategy (dissection b), aldol condensation of ketone 18 with

aldehyde 19 was envisioned. The required regioselectivity was expected based on the
 326

predicted transition state involving a six-membered ring chelation between 18 and 19

(Scheme VII.6).

Fortunately, ketone 18 is a known compound that has been synthesized in the

course of investigating rhodium-catalyzed decomposition of a diazo group.12 Thus, the

synthesis of scytonemin monomer began with indole-2-acetic acid (20), which was

activated for acylation by ethylchloroformate and N-methylmorpholine to yield diazo

ketone 21. After the solvent was changed from diethyl ether (literature12) to THF, the

yield increased to 81% from 50~60%, presumably due to the improved solubility of the

starting material. The rhodium-catalyzed decomposition of the diazo group was sluggish

despite the literature precedence,12 and the product (18) was somewhat unstable during

purification. This material (18) slowly converts to a pink-purple compound that further

converts to an insoluble blue material upon standing in solution. The identity of these

colored compounds could not be determined due to their lack of solubility.

The aldol condensation of 18 and 19 was performed by refluxing in a Soxhlet

extractor equipped with a plug of glass wool and 3 Å molecular sieves for removal of

water.13 Catalysis by toluenesulfonic acid appeared to have proceeded as predicted

(Scheme VII.6) and the other possible regioisomer (22) and over-condensed product (23)

could not be found in the reaction mixture. The major product's color was bright yellow

in a dilute solution and bright orange when dried. This solid had good solubility in DMF

and DMSO, moderate solubility in pyridine and MeOH, and somewhat poor solubility in

CH2Cl2 and EtOAc, and was insoluble in diethyl ether, hexane, and water. This material's

structure was fully elucidated by NMR and HR-MS analyses (Figure VII.2). It should be

noted that it was difficult to remove all of the unreacted 4-hydroxybenzaldehyde (19),
 327

and complete purification was only possible by RP-HPLC or by another round of flash

column chromatography.

The regiochemistry of the site of aldol condensation was determined based on

HMBC and ROESY correlations. The ROESY correlation between the indole NH proton

and meta-proton on the phenol indicated that the geometry of the exocyclic double bond

is E (Figure VII.2). The 1H NMR spectra in both MeOH-d4 and DMF-d7 revealed that the

ketone was exclusively in the keto form (22) and not at all in the enol form (11), as has

been suggested by others.5 This finding is supported by the calculated energy difference

between the keto and enol forms (24 kcal/mol).14 This is possibly due to both the anti-

aromaticity of the cyclopentadiene in the enol form (11) and the electron-donating ability

of the phenol and electron-withdrawing ability of the ketone (Scheme VII.8). Due to the

NMR equivalence of the two pairs of protons on the phenol moiety (H-14, H-14' and

H-15, H-15'; see Scheme VII.9 for numbering), the phenyl ring is expected to be

perpendicular to the tricyclic system, and not in direct conjugation with the rest of the

molecule (Figure VII.3). Finally, the color of a solution of 22 could be changed to bright

red when treated with a base (Figure VII.4). Upon neutralization, the color of the solution

changed back to bright yellow and a TLC analysis showed that 22 was intact.

VII.3 Oxidative dimerization of scytonemin monomer

Now with the monomer 22 available, oxidative dimerization was all that was

necessary for the total synthesis of scytonemin (1). Once the methylene carbon in 22

becomes oxidized to a methine (25), tautomerization should be able to render that carbon
 328

both electrophilic and nucleophilic (Scheme VII.9). This dual property of 25 may

promote spontaneous dimerization.

Scheme VII.9: Proposed mcehanism for dimerization of oxidized monomer 25 to reduced

scytonemin 2

Note that the conversion of 2 to 1 is known to occur spontaneously under ambient conditions.2

Figure VII.5: pH-dependent color change of monomer 22

Left: a solution of 22 in MeOH. Right: a solution of 22 in MeOH that was treated with NaOH.
 329

Inspired by the above hypothesis, 22 was initially subjected to naturally-occurring

oxidative conditions, such as O2, UV light, and the combination thereof to oxidize the

methylene carbon selectively. However, even after prolonged exposure to aerial O2 and

UV light (1 week), the monomer 22 remained intact (entry 1 in Table VII.1). Therefore, it

was concluded that either this oxidative dimerization step is a non-spontaneous

enzymatic reaction or the oxidation of the methylene carbon occurs earlier in the

biosynthesis of 1.

In order to evaluate the two possible hypotheses, an isotope-labeled monomer

(22) could be synthesized and fed into scytonemin-producing organisms, such as Nostoc

punctiforme, to look for incorporation of the labeled monomer. The synthesis of 22 was

repeated with [1-13C] 4-hydroxybenzaldehyde15 for the feeding study (see Scheme

VII.7).16 If incorporation occurs, it could be concluded that 22 is a true biosynthetic

intermediate and that oxidation of the methylene carbon does not happen earlier in the

biosynthesis. If incorporation does not occur, nothing can be firmly concluded due to the

possibility that 22 cannot cross the cell membrane of the cyanobacterium.

 330

Table VII.1: Oxidative dimerization conditions

Entry Reagents Result

1 UV light, air, MeOH No Rxn
2 O2, MeOH No Rxn
3 Fe(III) citrate, pyridine, H2O2 No Rxn
4 MeOH, NaOCl Decomp.
5 m-CPBA, MeOH No Rxn
6 Ce(SO4)2, H2SO4 No Rxn
7 NCS, MeOH No Rxn
8 Phosphomolybdic acid, MeOH Green soln, but no desired products
(1 or 2) by LCMS
9 CrO3, AcOH, THF, H2O Decomp.
10 Base, Cu(RCO2)2, THF17 Green products, but no desired
products (1 or 2) by LCMS
11 NaOH, DMSO, AgNO3 Same mass as nostodione (3), but 1H
NMR did not match
12 DDQ, THF Trace amount of 2 by LCMS
13 Pd(OAc)2, O2, DMSO Mostly decomposition, but a trace of
1 was observed by LCMS
14 Pd(OAc)2, O2, pyridine Most of 22 remained intact, the
remainder decomposed
15 Fremy's salt Decomp.
16 1. formylation Unknown products
2. diazonization
3. Rh2(OAc)4
17 SeO2, DMSO Decomp.
18 (PhCO)2Se, DMSO Decomp.
19 ZrO2, air, MeOH/DMF
20 Rose bengal, UV, air, DMF
 331

Given the knowledge that the monomer 22 does not spontaneously oxidize under

ambient conditions, various oxidizing reagents were tested for selective oxidation of 22

as shown in Table VII.1. These reaction conditions included two-electron oxidants, such

as hypochlorite, N-chlorosuccinimide, m-chloroperbenzoic acid and one-electron

oxidants, such as iron (III) and dichloro-dicyanobenzoquinone. However, none of these

conditions yielded scytonemin (1) or its reduced form (2) in amounts detectable by

LCMS or 1H NMR except for entry 13, but because the detected amount was so little, the

presence of 1 in the reaction mixture could not be unequivocally established. Moreover,

even after most of 22 decomposed in this Pd(OAc)2 catalyzed reaction, a significant

amount of 22 was still intact, which presumably meant that oxidative decomposition of 1

(if it was formed at all) was much faster than oxidative dimerization of 22.

In light of these failed reactions, it was hypothesized that at least some of the

failed oxidation reactions were due to chemoselective oxidation of the phenol and not the

cyclopentenone of 22. This hypothesis seemed reasonable due to the electron-rich nature

of the phenolic ring upon deprotonation. To overcome this potential problem, a

methylated version of 22 was synthesized according to Scheme VII.10. However, even

Scheme VII.10: Synthesis of methylated monomer 29

Note that the yield could be increased to ~70% if a significant excess of 28 was used.
However, the chromatographic purification became increasingly difficult due to the
unreacted 28, which almost completely coeluted with 29.
 332

this methylated monomer 28 could not be oxidatively dimerized under various

conditions. A significantly different synthetic approach was clearly necessary.

In the gene cluster for the biosynthesis of 1 (Scheme VII.2), there are a few genes

whose roles are not clearly understood in light of the proposed biosynthesis (Scheme

VII.3). For example, NpR1270 (a putative glycosyltransferase) may or may not be

involved in the dimerization step. One hypothesis has been brought forth in P. Proteau's

thesis as follows. The enzyme from NpR1270 is hypothesized to glycosylate the ketone

oxygen, converting the keto form to the enol form, and the glycosylated enol is

transported across the cell membrane to be transported into the extracellular sheaths,

Scheme VII.11: Possible role of glycosylation in the biosynthesis of scytonemin

In 30, resonance forms with a negative charge on the cyclopentadiene are preferred due to
creation of aromaticity, which renders the ring susceptible to oxidation due to its rich electron
density. Once oxidized to 31, dimerization could occur in the same manner as in Scheme VII.9.
 333

where the electron-rich enol α-carbon is oxidized and coupled to another monomer

molecule (Scheme VII.11).18

Scheme VIl.12: Attempts to synthesize 30 and/or a mimic thereof (e.g. 33)

In order to construct a mimic of the glycosylated monomer 30, per-acetylation

was attempted to form 33 (Scheme VII.12). However, the only product isolated from the

reaction was a mono-acetylated monomer 34. By taking advantage of the protection of

the reactive phenolic hydroxy group in 34, glycosylation of the corresponding enolate

was attempted. It is noteworthy that there have not been rigorous synthetic studies on the

glycosylation of enolate-systems even though this process appears to be widely utilized in

nature.19 Treatment of 34 with per-acetylated glucose derivative 35 and potassium tert-

butoxide resulted in the loss of the acetyl group and reversion to monomer 22.

Similarly, double silylation of 22 to form 36 was attempted, but it only resulted in

either decomposition or mono-silylation to produce 37 (Scheme VII.14). In order to form

a silyl enol ether of the ketone in 36, it was treated with LiHMDS and TBDMSOTf. A

new product was formed in this reaction, but due to its unstable nature, it could not be
 334

isolated for full characterization. Therefore, this crude material was treated with m-CPBA

to oxidize the α carbon. However, the resulting product showed intact methylene protons

in its 1H NMR spectrum.

Scheme VII.13: Attempts to perform Rubottom oxidation via doubly silylation of 22

VII.4 Chemical insights on the biosynthesis of scytonemin

After repeated failure to dimerize 22, the legitimacy of the structure of

scytonemin 1 was questioned. While the HMBC correlations noted by Gerwick et al. are

fairly conclusive, they leave some room for three additional possibilities (Figure VII.6).20

Particularly, the structure 38 is interesting because reduction and ozonolysis of 38 in the

same manner as the conversion of 1 to 2 would also result in the formation of nostodione

(3). However, there is no other way to evaluate these structures unless they are actually

synthesized and their spectral data are compared with the authentic compound, which

was not feasible due to time constraints.

 335

Subsequently, the intermediacy of 22 was also questioned. Given that 22 cannot

assume the enol form to enrich the electron density on C-5, presumably due to the

consequential anti-aromaticity (Scheme VII.8), selective oxidation of C-5 is not readily

realizable. To eliminate the anti-aromaticity problem and to be able to convert C-5 to sp2

hybridization, a carbon other than C-5 in the cyclopentenone ring could be converted to

an sp3 carbon, thereby breaking the conjugation of the system. For example, the reason

for the ketone 18 being able to undergo an aldol condensation via acid-catalyzed

enolilzation is likely owing to the non-conjugated nature of the enol form of the

cyclopentenone ring. The most feasible site of oxidation level change is the carbonyl

carbon in the cyclopentenone ring. Thus, the synthesis of 22 was modified for the

preparation of cyclopentene-ol 43 (Scheme VII.14). The execution of this synthesis is

ongoing at the time of this writing.

Figure VII.6: Alternative structures for scytonemin

 336

Scheme VII.14: Newly proposed route for the synthesis of 2

Neither 44a or 44b suffers from anti-aromaticity. However, an important resonance form of 25
suffers from anti-aromaticity (Scheme VII.9), and it may not be possible to synthesize 25 at all. If
this route is successful, credence to the intermediacy of desilylated version of 44 will be
provided.

Incidentally, the desylilated version of 43 is identical to a biosynthetic

intermediate (10) proposed by Balskus and Walsh (Scheme VII.4).5 It is perfectly

reasonable to propose that 10 is the true monomeric precursor, and not 22, provided that

C-5 of 10 is readily oxidizable (Scheme VII.14). Therefore, chemical investigations of 43

and/or 10 are expected to provide evidence for or against the alternative intermediacy of

10. Moreover, while enolate-glycosylation of 34 was not possible, the same

transformation may be possible for 42, which would allow access to glycosylated
 337

Scheme VII.15: Chemo-selective oxidation of benzylic ether by DDQ in a neat mixture

Scheme VII.16: Completion of total synthesis of scytonemin albeit very low yield

monomer 30. Testing for incorporation of 13C-labeled 30 as well as the desilylated version

of 43 would be of interest with regard to the evaluation of the putative glycosylase

(NpR1270) in the biosynthesis of scytonemin.

VII.4 Completion of the total synthesis of scytonemin

As seen in entry 12 of Table VII.1, treatment of 22 with DDQ produced trace

amounts of 1 and 2. The presence of these two compounds was detected by RP-HPLC

coupled with ESI-MS, in which the retention time and mass spectra of 1 and 2 were

matched with the crude reaction products. However, the integration of the peaks

corresponding to 1 and 2 were too small to be certain that they were not due to some

background noise. DDQ oxidation of a methylene carbon attached to C-3 of indole is

 338

well known in the literature (Scheme VII.15).21 Therefore, this reaction was revisited to

improve the yield so as to determine the identity of the reaction product.

Scheme VII.17: Highly chemo-selective DDQ oxidation of (E)-16-epinormacusine B (45)

As mentioned earlier, complete purification of 22 was difficult due to 19 eluting in

close proximity to 22 during flash chromatography. Therefore, many of the batches of 22

used for the oxidative dimerization studies were contaminated with significant amounts

of 19. In order to avoid any possible side reactions involving 19, such as reduction of the

oxidant by 19, fine-tuned flash column chromatography conditions (1:19 to 1:4 THF /

hexanes) were developed to completely purify 22. Moreover, being inspired by Li and

coworkers' work on so-called dehydrogenative coupling involving DDQ (Scheme

VII.16),22 a concentrated solution (0.44 M) of very pure 22 in 1,4-dioxane was used in

this DDQ oxidation (Scheme VII.17). In this attempt, distinct chromatographic peaks

corresponding to 1 and 2 were observed after flash column purification. Semi-prep HPLC

purification of the reaction products is under way at the time of this writing. Due to the

low yield, however, it is still thought that the alternative intermediacy of 43 is possible. It

is possible that an unnatural reaction pathway with a high activation energy is operating

in the DDQ reaction with the compound 22.

 339

VII.5 Conclusions

A cyanobacterial alkaloid scytonemin possesses a unique dimeric structure that

raises many interesting biosynthetic questions. Through chemical synthetic investigations

of this alkaloid, a few important insights into its biosynthesis were obtained. The

biosynthetic precursor proposed by Walsh (11) was found to be a structure that does not

exist because the equilibrium between 11 (enol form) and 22 (keto form) lies exclusively

towards 22. Through various failed attempts to oxidatively dimerize 22 (although a trace

amount of 1 and 2 appeared to have been produced under certain conditions), this newly

proposed monomeric precursor was found to have physico-chemical properties that

appear to be incompatible with the oxidation required for conversion to the natural

product 1. Therefore, if 22 is a biosynthetic precursor, its dimerization must be catalyzed

by an enzyme with unusual chemical properties. An alternative conclusion from this work

is that a compound other than 22 is the biosynthetic precursor. In order to further evaluate

the intermediacy of 22, a stable isotope-labeled version of it is being prepared for a

feeding study. The intermediacy of an alternative precursor, such as 44, was proposed and

it is being evaluated through chemical synthesis. Finally a small yield of 1 and 2 were

obtained after revisiting the DDQ oxidation of 22. Further studies are required to improve

the yield of this reaction to a meaningful level.

VII.6 Experimental Section

General
 340

Unless noted otherwise, all materials were purchased from commercial sources

and were used without further purification. Anhydrous benzene, 1,4-dioxane, and 1,2-

dichloroethane were purchased from EMD. Anhydrous N,N-dimethylformamide was

purchased from Acros. Tetrahydrofuran (THF) was distilled from sodium /

benzophenone. Pyridine, Et3N, and CH2Cl2 were distilled from CaH. Ac2O was distilled

from quinone. CHCl3 was passed through a column of basic alumina activity I. All

reactions were carried out under dry argon atmosphere unless otherwise noted. Reaction

temperatures herein recorded are external temperatures unless otherwise noted. Flash

chromatorgraphy was performed using EMD silica gel (230-400 mesh) in all cases.23

Thin layer chromatographic (TLC) analysis was performed using EM Science pre-coated

silica gel plates (Merck 60 F254). Melting points were determined on an Electrothermal

Mel-Temp device and are uncorrected. Optical rotations were measured on a Jasco

P-2000 polarimeter. IR spectra were recorded on a Nicolet Magna-IR 550. NMR spectra

were recorded on a Varian Inova spectrometer (500 MHz and 125 MHz, respectively for
1
H and 13C), Varian Mercury spectrometer (400 MHz and 100 MHz, respectively for 1H

and 13C), Varian Mercury spectrometer (300 MHz and 75 MHz, rspectively for 1H and
13
C), or Varian Inova spectrometer (300 MHz and 75 MHz, respectively for 1H and 13C).
1
H and 13C spectra recorded in CDCl3 were referenced to the residual solvent peaks at

7.26 ppm (CHCl3) and 77.0 ppm (CDCl3), respectively. 1H and 13C spectra recorded in

DMF-d7 were referenced to the residual solvent peaks at 8.03 ppm (CHNO(CH3)2) and

163.15 ppm (CDCl3), respectively. High resolution mass spectra were recorded on a

ThermoFinnigan MAT900XL spectrometer.

 341

1-diazo-3-(1H-indol-3-yl)propan-2-one (21). This synthesis was done mostly according

to the literature procedure12 with minor modifications, such as the solvent and

concentration. A CH2N2 solution in Et2O was prepared according to a literature

procedure.24 The concentration of CH2N2 was determined to be 0.24 M by treatment with

an excess amount of benzoic acid and back titration with NaOH in the presence of phenol

red. To a solution of indole-3-acetic acid (2.500 g, 14.27 mmol) in THF (100 mL) at 0ºC

were added ethyl chloroformate (1.42 mL, 14.9 mmol) and N-methyl morpholine (1.64

mL, 14.9 mmol) successively under stirring. After 15 min of stirring at 0ºC, the solution

of CH2N2 was added in one portion (225 mL, 54.0 mmol). This solution was allowed to

gradually warm to rt over night. A pale brown-orange solution with black tar-like residues

resulted. Most of the volatiles were evaporated under a stream of N2. The residues that

dissolved in EtOAc were subjected to flash column chromatography using deactivated

basic alumina (activity III) as the stationary phase (1:9 EtOAc / hexanes to 1:1) to obtain

an orange oil (2.31 g, 11.6 mmol, 81% yield). TLC Rf = 0.39 (1:1 EtOAc / hexanes). 1H

NMR (400 MHz, CDCl3) δ 8.64 (bs, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 8.1 Hz,

1H), 7.28 – 7.19 (m, 1H), 7.19 – 7.12 (m, 1H), 7.04 (d, J = 2.4 Hz, 1H), 5.19 (s, 1H), 3.77

(s, 2H). 13C NMR (101 MHz, CDCl3) δ 194.5, 136.2, 126.9, 123.6, 122.2, 119.7, 118.5,

111.4, 54.3, 37.9.

 342

3,4-dihydrocyclopenta[b]indol-2(1H)-one (18). This synthesis was done exactly

according to according to the literature procedure.12 However, the yield was consistently

lower than reported (20~40%). TLC Rf = 0.37 (3:7 THF / hexanes). 1H NMR (300 MHz,

CDCl3) δ 8.16 (s, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.39 (dd, J = 4.7, 3.4 Hz, 1H), 7.25 –

7.12 (m, 2H), 3.54 (s, 2H), 3.53 (d, J = 1.0 Hz, 2H).

(E)-3-(4-hydroxybenzylidene)-3,4-dihydrocyclopenta[b]indol-2(1H)-one (22). The

best overall yield was obtained when a crude mixture or 18 was used in this reaction. To a

solution of the diazoketone 21 (277 mg, 1.39 mmol) in CH2Cl2 at rt was added Rh2(OAc)4

(6 mg, 0.014 mmol). Vigorous evolution of gas was observed. After 2 hrs, no starting

material was detected by TLC analysis. A reddish orange solution resulted. The reaction

mixture was filtered through a plug of silica gel, which was rinsed with 1:1

EtOAc/hexanes (100 mL). Upon removal of the solvents in vacuo, 155 mg of crude

material was obtained (a mixture of white and orange solids). To a solution of this
 343

material in 4:1 CH2Cl2/1,2-dichloroethane (10 mL) were added 4-hydroxybenzaldehyde

19 (144 mg, 1.18 mmol), toluenesulfonic acid monohydrate (16 mg, 0.080 mmol), and

molecular sieves 3 Å (155 mg) at rt. This mixture was brought to reflux in a flask

equipped with a condenser. After 14 hrs of refluxing, additional amounts of 19 (27 mg,

0.22 mmol) and molecular sieves 3 Å (60 mg) were added. After 4 hrs, the reaction

mixture was filtered through a plug of silica gel, which was rinsed with EtOAc (200 mL).

Upon concentration under vacuum, this solution was subjected to flash column

chromatography (1:19 THF/hexanes to 1:4) to obtain a bright orange solid (58 mg, 0.211

mmol, 15% yield from 21 over 2 steps). Mp >210ºC. IR (film, KBr) νmax 3375(br), 2963,

2921, 1712, 1599, 1483, 1441, 1274, 1231, 1165, 1123, 745 cm -1. 1H NMR (300 MHz,

DMF-d7) δ 11.11 (s, 1H), 10.22 (s, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.60 (d, J = 8.1 Hz, 1H),

7.57 (d, J = 8.0 Hz, 1H), 7.23 (ddd, J = 8.2, 7.2, 1.3 Hz, 1H), 7.12 (ddd, J = 7.9, 4.5, 0.6

Hz, 1H), 7.06 (s, 1H), 6.97 (dd, J = 6.1, 2.4 Hz, 2H), 3.56 (s, 2H). 13C NMR (75 MHz,

DMF-d7) δ 205.3, 160.2, 141.6, 141.4, 131.7, 127.6, 127.3, 125.4, 125.2, 124.5, 121.2,

120.9, 120.4, 117.3, 113.9, 36.9. HRMS (EI): calcd for C18H13NO2[M+]: 275.0941, found

275.0945 (Δ 1.6 ppm).

(E)-3-(4-methoxybenzylidene)-3,4-dihydrocyclopenta[b]indol-2(1H)-one (29). This

compound was synthesized in the same manner as 22. Yellow-green solid. 1H NMR (300

MHz, CDCl3) δ 8.21 (s, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 8.0 Hz, 1H), 7.35 (d, J

= 8.1 Hz, 1H), 7.25 (t, J = 7.2 Hz, 1H), 7.17 (s, 1H), 7.16 (dd, J = 7.4 Hz, 1H), 7.04 (d, J

= 8.7 Hz, 2H), 3.90 (s, 3H), 3.55 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 204.6, 160.3,

140.2, 138.8, 129.6, 128.7, 127.8, 124.3, 124.3, 120.9, 120.3, 119.8, 114.8, 111.8, 55.5,
 344

36.5. HRMS (EI): calcd for C19H15NO2Na [M+Na]: 312.0995, found 312.0998 (Δ 1.0

ppm).

(E)-4-((2-oxo-1,2-dihydrocyclopenta[b]indol-3(4H)-ylidene)methyl)phenyl acetate

(34). To a solution of 22 (21 mg, 0.076 mmol) in pyridine (200 μL) was added Ac2O (200

μL) at rt. This solution was left at rt over night. Most of the volatiles were removed in

vacuo, and the residues (dark orange oil) was subjected to flash column chromatography

(hexanes to 1:9 acetone/hexanes) to obtain 16 mg of an yellow solid (0.050 mmol, 66%

yield). TLC Rf = 0.64 (1:2 acetone/hexanes). 1H NMR (400 MHz, CDCl3) δ 8.29 (s, 1H),

7.64 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.27 (m, 1H),

7.23 (d, J = 8.6 Hz, 2H), 7.16 (m, 1H), 7.14 (s, 1H), 3.56 (s, 2H), 2.36 (s, 3H). 13C NMR

(101 MHz, CDCl3) δ 204.3, 169.5, 150.8, 139.6, 139.1, 134.0, 131.9, 129.1, 124.7, 124.0,

122.9, 122.5, 121.5, 121.0, 119.9, 112.0, 36.4, 21.2. LRMS (ESI, negative): 316 [M-H].

(E)-3-(4-(trimethylsilyloxy)benzylidene)-3,4-dihydrocyclopenta[b]indol-2(1H)-one

(37). To a solution of 22 (40 mg, 0.145 mmol) in THF (1.1 mL) at -78ºC were added 2,6-

lutidine (42 μL, 0.426 mmol) and TMSOTf (52 μL, 0.29 mmol) dropwise under stirring.

After stirring for 5 min at -78ºC, the solution (orange-yellow color) was warmed

immediately to rt. After stirring at rt for 3 hrs, saturated NaHCO3 solution (10 mL) was

added to quench, which was extracted with EtOAc (20 mL x 3). The combined organic

layer was washed with H2O (15 mL) and brine (25 mL) successively, and was dried over

Na2SO4. This product readily decomposed to 22 on a silica gel TLC plate. Upon removal

of the solvents under vacuum, 34 mg of amorphous yellow-orange solid was obtained

 345

(0.098 mmol, 67% yield). 1H NMR (300 MHz, CDCl3) δ 8.26 (s, 1H), 7.55 (d, J = 8.3 Hz,

2H), 7.35 (d, J = 8.0 Hz, 1H), 7.26 (t, J = 7.2 Hz, 1H), 7.17 (t, J = 7.2 Hz, 1H), 7.16 (s,

1H), 6.99 (d, J = 8.6 Hz, 2H), 3.53 (s, 2H), 0.36 (s, 9H). 13C NMR (75 MHz, CDCl3) δ

204.6, 156.2, 140.1, 138.9, 129.6, 127.9, 125.5, 124.4, 124.3, 124.3, 120.9, 120.8, 119.8,

116.3, 111.8, 36.5, 30.3, 0.32.

 346

Figure VII.7: 1H NMR spectrum of 21 in CDCl3

 347

Figure VII.8: 13C NMR spectrum of 21 in CDCl3

 348

Figure VII.9: 1H NMR spectrum of 18 in CDCl3

 349

Figure VII.10: 13C NMR spectrum of 22 in DMF-d7

Note that this sample contains a significant amount of 4-hydroxybenzaldehyde (19), and its 13C
shifts include 191.8, 164.9, 133.1, 130.2, 117.0, as were established by HMBC.
 350

Figure VII.11: COSY NMR spectrum of 22 in DMF-d7

 351

Figure VII.12: HSQC NMR spectrum of 22 in DMF-d7

Note that this sample contains a significant amount of 4-hydroxybenzaldehyde (19), and its 13C
shifts include 191.8, 164.9, 133.1, 130.2, 117.0 ppm, as were established by HMBC. 1H shifts of
19 include 9.88, 7.83, 7.04 ppm.
 352

Figure VII.13: HMBC NMR spectrum of 22 in DMF-d7

There are some impurity peaks such as the one at 1.5 ppm (1H).
 353

Figure VII.14: 1D NOESY NMR spectrum of 22 in DMF-d7

 354

D:\Xcalibur\data\09_16_2008_scyor_b-c1 09/17/08 10:47:29 AM

09_16_2008_scyor_b-c1 #8 RT: 1.45 AV: 1 NL: 4.47E6

T: + c EI Full m s [ 263.88-282.84]
275.0945
100

95

90

85

80

75

70

65

60
Relative Abundance

55

50

45

40

35

30

25
276.0987

20
280.9824
15

10
268.9824
274.0876 277.1047
5
272.0715 273.0780
267.9909 269.9863 271.0820
274.9935
276.0002 277.0111 278.1091 278.9859 279.9927 281.9864

268 269 270 271 272 273 274 275 276 277 278 279 280 281 282
m /z

Figure VII.15: HRMS (EI) spectrum of 22

Reference: Perfluorokerosene. Two pink lines are reference peaks.
 355

Figure VII.16: 1H NMR spectrum of 29 in CDCl3

 356

Figure VII.17: 13C NMR spectrum of 29 in CDCl3

 357

Figure VII.18: COSY spectrum of 29 in CDCl3

 358

Figure VII.19: HSQC spectrum of 29 in CDCl3

 359

Figure VII.20: HMBC spectrum of 29 in CDCl3

 360

Figure VII.21: ROESY spectrum of 29 in CDCl3

 361

MScMn #28-30 RT: 0.60-0.65 AV: 3 NL: 8.08E4

T: FTMS + p ESI Full ms [200.00-400.00]
312.0998
100

95

90

85

80

75

70

65

60
Relative Abundance

55

50

45

40

35

30

25

20 313.1032

15

10

5
310.5966 311.2948 313.2053 314.1069 314.5144 315.1631 315.5026
311.5897 312.3627 316.7042
0
310.5 311.0 311.5 312.0 312.5 313.0 313.5 314.0 314.5 315.0 315.5 316.0 316.5
m/z

Figure VII.22: HRMS (ESI-FT) spectrum of 29 (showing [M+Na])

 362

Figure VII.23: 1H NMR spectrum of 34 in CDCl3

 363

Figure VII.24: 13C NMR spectrum of 34 in CDCl3

 364

Figure VII.25: 1H NMR spectrum of 37 in CDCl3

 365

Figure VII.26: 13C NMR spectrum of 37 in CDCl3

 366

References and Footnotes

1 Garcia-Pitchel, F.; Sherry, N. D.; Castenholz, R. W. Evidence for an ultraviolet sunscreen role
of the extracellular pigment scytonemin in the terrestrial cyanobacterium Chlorogloeopsis sp.
Photochem. Photobiol. 1992, 56, 17-23.

2 Proteau, P. J.; Gerwick, W. H.; Garcia-Pitchel, F.; Castenholz, R. The structure of scytonemin,
an ultraviolet sunscreen pigment from the sheaths of cyanobacteria. Experientia 1993, 49,
825-829.

3 Dillon, J. G.; Castenholz, R. W. Scytonemin, a cyanobacterial sheath pigment, protects against

UVC radiation: implications for early photosynthetic life. J. Phycol. 1999, 35, 673-681.

4 Sinha, R. P.; Häder, D. UV-protectants in cyanobacteria. Plant Sci. 2008, 174, 278-289.

5 Balskus, E. P.; Walsh, C. T. Investigating the initial steps in the biosynthesis of cyanobacterial
sunscreen scytonemin. J. Am. Chem. Soc. 2008, 130, 15260-15261.

6 Nägeli, C. Gattungen einzelliger Algen, physiologisch und systematisch bearbeitet. Neue

Denkschrift Allg. Schweiz. Natur Ges. 1849, 10, 1-138.

7 Kobayashi, A.; Kajiyama, S.; Inawaka, K.; Kanzaki, H.; Kawazu, K. Nostodione A, a novel
mitotic spindle poison from a blue-green alga Nostoc commune. Z. Naturforsch. 1994, 49c,
464–470.

8 Tanya Soule, V. Stout, W. D. Swingley, J. C. Meeks, and F. Garcia-Pichel, Molecular Genetics

and Genomic Analysis of Scytonemin Biosynthesis in Nostoc punctiforme ATCC 29133 . J.
Bacteriol. 2007, 189, 4465-4472.

9 Matthews, C. K.; van Holde, K. E.; Ahern, K. G. Biochemistry, 3rd Ed. 2000, Addison Wesley
Longman, Inc.

10 Normally cyclization of indole at C-2 to form a five-membered ring is a rapid process.

11 (a) Stevenson, C. S.; Capper, E. A.; Roshak, A. K.; Marquez, B.; Grace, K.; Gerwick, W. H.;
Marshall, L. A. Inflamm. Res. 2002, 51, 112–114. (b) Stevenson, C. S.; Capper, E. A.; Roshak,
A. K.; Marquez, B.; Eichman, C.; Jackson, J. R.; Mattern, M.; Gerwick, W. H.; Jacobs, R. S.;
Marshall, L. A. J. Pharmacol. Exp. Ther. 2002, 303, 858–866.

12 (a) Erick Cuevas-Yanez,a,* Joseph M. Muchowskib and Raymundo Cruz-Almanza ,

Rhodium(II) catalyzed intramolecular insertion of carbenoids derived from 2-pyrrolyl and 3-
indolyl α-diazo-β-ketoesters and α-diazoketones. Tetrahedron 60 (2004) 1505–1511. (b)
Mohamed Salim and Alfredo Capretta, Intramolecular Carbenoid Insertions: the Reactions
of α-Diazoketones Derived from Pyrrolyl and Indolyl Carboxylic Acids with
Rhodium(II) Acetate . Tetrahedron 56 (2000) 8063–8069.

13 This system worked well in the synthesis of somocystinamide A (see Chapter VI).

14 HF 3-21G* single point energy calc of dihedral angle constraint.

 367

15 Such is available from Aldrich.

16 The feeding study is not completed at the time of writing.

17 DeMartino, M. P.; Chen, K.; Baran, P. S. Intermolecular Enolate Heterocoupling: Scope,

Mechanism, and Application . J. Am. Chem. Soc. 2008, 130, 11546-11560.

18 Proteau, P. J. Oxylipins from temperate marine algae and a photoprotective sheath pigment
from blue-green algae. 1993, M.S. Thesis.

19 Sugimoto, T.; Ueda, M. Biomimetic synthesis of a leaf-opening factor, potassium

isolespedezate, by direct formation of enol-glycoside. Chem. Lett. 2004, 33, 976-977.

20 All of these structures (38-40) require at least one four-bond HMBC correlation, which is not
unheard of in a polycyclic aromatic system like scytonemin.

21 Yu, J.; Wang, T.; Wearing, X. Z.; Ma, J.; Cook, J. M. Enantiospecific Total Synthesis of (-)-
(E)16-Epiaffinisine, (+)-(E)16-Epinormacusine B, and (+)-Dehydro-16-epiaffinisine as well as
the Stereocontrolled Total Synthesis of Alkaloid G . J. Org. Chem. 2003, 68, 5852-5859.

22 Li, C. Cross-Dehydrogenative Coupling (CDC): Exploring C-C Bond Formations beyond

Functional Group Transformations . Accounts Chem. Res. 2009, 42, 335-344.

23 Still, W. C.; Khan, M.; Mitra, A. Rapid chromatographic technique for preparative separations
with moderate resolution. J. Org. Chem. 1978, 43, 2923-2925.

24 Leonard, J.; Lygo, B.; Procter, G. Advanced Practical Organic Chemistry, 2nd Ed. 1998,
Stanley Thomas Ltd.
Chapter VIII

Conclusions

368
 369

Abstract

Conclusions drawn from the research activities described in Chapters I through

VII are discussed herein.

 370

VIII.1 Thesis

Natural products chemistry has benefited from organic synthesis in the areas of

structure elucidation and confirmation, pharmaceutical application, and biosynthetic

study of natural products. Simultaneously, natural products have been a major driving

force for the advancement of organic synthesis. Given this rich history, direct application

of organic synthesis to issues and opportunities surrounding natural products should

improve productivity in all three areas mentioned above. As a significant added benefit,

new chemical transformations and synthetic strategies may be discovered in the process.

VIII.2 Natural products isolated through the dissertation research

In order to evaluate the thesis that contributions from organic synthesis enhance

the productivity of natural products chemistry, various studies were conducted, which

included applying the techniques learned through the mastery of organic synthesis to

natural product isolation and structure elucidation. The structures of the natural products

that were isolated in this endeavor are summarized below in Figure VIII.1. A chemist

well-trained in synthetic organic chemistry usually has mastered the art of

chromatographic purification of unstable compounds. This skill most certainly increases

the chance of isolating natural products of interest with a good yield and purity.

Moreover, a synthetic chemist has seen a large number of NMR spectra of many different

classes of compounds, which is an experience that can aid in the structure elucidation

process.
 371

Figure VIII.1: Natural products isolated during the dissertation research

Cyclopropyl fatty acid 4-m was isolated from a Madagascar collection of Lyngbya majuscula.
Likewise, 4-c was isolated from a Curaçao collection of the same organism. The stereochemistry
of these two natural products are updated as per Chapter IV.

Water-soluble organic compounds are difficult to purify and to handle. Therefore,

many natural products remain undiscovered due to their polarity and solubility in water.

Skills nurtured through organic synthesis were utilized to isolate credneric acid (1), a

metabolite found in the aqueous extract of a Lyngbya sp. from Papua New Guinea. In the

pursuit to identify neurologically important marine natural products, two polybrominated

diphenyl ethers (2 and 3) were obtained through a bioassay-guided isolation approach.

The literature 13C NMR values for 2 contradicted our findings, and this discrepancy was

unequivocally resolved through synthesizing alternative structures to evaluate the

contradicting 13C NMR data.

Although unresolved, a novel concept (synthetic introduction of a stereochemical

reference, SISTER method) was developed to elucidate the absolute stereochemistry of

natural products that do not have a functional group appropriate for traditional

derivatizations, such as synthesis of a Mosher's ester. In the course of this investigation,

 372

an interesting concept regarding the 'phylogenetic nature' of absolute stereochemical

assignments was noted.

VIII.3 Synthetic investigations of biologically important natural products

Through the total synthesis of both enantiomers of epiquinamide (5), a

contribution was made to resolve the confusion about the identity of the potent nicotinic

receptor agonist found in the skin of a rainforest frog, Epipedobates tricolor.

Unfortunately, 5 was found to be inactive in many bioassays tested thus far. However,

through the concept of using a biomimetic starting material (an ornithine derivative), a

very expedient and practical synthetic route to 5 was developed. Had 5 been a bioactive

molecule, this synthesis should have been able to provide sufficient quantities of the

natural product for pharmaceutical investigations.

A total synthesis of a marine cyanobacterial metabolite, somocystinamide A (7),

an exceptionally potent inhibitor of endothelial cell proliferation and angiogenesis, was

also accomplished. The challenges presented by the enamide functionality were

overcome after rigorous investigations. Surprisingly, the disulfide group, historically

known to be a difficult functional group to synthesize, was relatively easily introduced

after resorting to a somewhat biomimetic condition (molecular oxygen atmosphere in the

presence of an aqueous base). Finally, a revision of the synthesis was successfully made

to improve the scalability of the first synthesis, thereby providing a sufficient quantity of

7 for in vivo studies. This latter synthesis should enable future investigations of

somocystinamide A analogs for development in the treatment of cancer.

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Figure VIII.2: Natural products that were synthetically investigated during the disseration
research

Synthetic studies were conducted on a structurally unique alkaloid, scytonemin

(8), a UV-blocking metabolite produced by cyanobacteria. The first synthesis of the

putative monomeric precursor (9) of this natural product was accomplished in one step

from a known indole derivative. Access to the monomer 9 allowed experiments to show

that dimerization of 9 cannot occur spontaneously under ambient conditions and that such

a reaction is probably enzymatically catalyzed (if it occurs at all). Moreover, many

chemical and biosynthetic insights were gained through attempts to dimerize 9, which

was successful at a small yield albeit a very low yield. The synthesis of 9 and its

derivatives contributed to a concurrent biosynthetic investigation of scytonemin as well.

 374

Interestingly, pure 9 was tested in a mechanism-based anti-cancer assay and found to

have unique and potentially valuable properties.

VIII.4 Final conclusions.

Through the chemical investigations of natural products mentioned above, the

benefits of applying organic synthesis to natural products chemistry were clearly

demonstrated. The approaches taken in this dissertation should continue to be productive

in the foreseeable future.

 Appendix

UV spectrum for scytonemin monomer

3.5

2.5

1.5

0.5

-0.5
210 260 310 360 410 460 510 560

λmax : 255 nm (ε = 3.8 m2/mol); 289 nm (ε = 2.9 m2/mol); 401 nm (ε = 6.1 m2/mol)
Taken in 99:1 MeCN/THF (0.1 mg/ mL)

375
 376

Feeding study with 13C-labeled scytonemin monomer

The experimental results for the feeding study are summarized in Scheme 1.
Labeled scytonemin's identity was confirmed through its retention time, which coincided
with that of an authentic sample of scytonemin.
It is apparent that monomer 22 can be converted to 1 by a scytonemin-producing
cyanobacterium. Moreover, two different hypotheses can be postulated based on the
absence of singly labeled scytonemin (Figure 1). It is possible that presence of 22
suppressed any production of scytonemin as well as its monomer in the organism through
a feedback mechanism. In this hypothesis, the observation of the unlabeled scytonemin is
explained by small basal production of 1 prior to the UV radiation treatment and the

Scheme .1: Incorporation of 13C-labeled monomer 22

Surprisingly, singly labeled scytonemin was not observed at all. However, almost equal amounts
of unlabeled and doubly labeled scytonemin were observed. Tolypothrix distorta is a
cyanobacterum that is known to be able to sustain moderate UV radiation through producing
scytonemin.

Figure 1: ESI mass spectra of control and 13C-labeled monomer treatment scytonemin

The peak 543.3 corresponds to scytonemin ([M-H]) while the 545.3 peak corresponds to doubly
labeled scytonemin ([M-H]).

introduction of 22. Alternatively, it is possible that 22 blocked the UV radiation, which

could result in suppression of the production of scytonemin in the cell. Given that most
scytonemin molecules are found in the sheath and not in the cyanobacterial cell, it is
 377

possible that the dimerization process only occurs in the sheath. If so, the labeled
monomer that penetrated into the sheath would be converted only to doubly labeled
scytonemin due to the suppressed production of scytonemin monomer in the cell.
 378

Progress Report on N2024

The above scheme summarizes the experiments done on N2024 so far. The 13C and 1H
NMR spectra of N-Bz-O-Ac-N2024 were consistent with the expected structure. The
decision was made to protect the free amines as amides because of the expected side
reaction of the amine nitrogens attacking the ester(s) in the hydrolysis step (thereby
forming lactams).

The attempt to perform ethenolysis by olefin metathesis was not successful even after
prolonged reaction time. The 1H NMR spectrum of the resulting material revealed olefin
peaks, indicating that there was no reaction. However, oxidative ozonolysis seemed to
have proceeded well based on the crude 1H NMR taken on the product, in which the
olefin peaks had disappeared. Subsequently, barium hydroxide mediated hydrolysis was
carried out. After work-up, there were a few different compounds in the mixture and upon
HPLC purification, only one of them was isolated in sufficient quantity for NMR
analysis. Despite the 1.7 mm probe, it was not possible to obtain 1H NMR spectrum of
good quality for this material. This disappointing result may be due to two factors; 1)
insufficient starting material, 2) the final product's poor solubility. The latter issue may
have been worsened by the presence of an additional carboxylic acid moiety created by
the oxidative ozonolysis step. For the next time, doubly reductive ozonolysis may be for
performed, where the resulting ketone and aldehyde are reduced to alcohol. One
complication would be the introduction of a new stereo center, the secondary alcohol.

Another approach that could be taken either independently from the above chemistry or
in conjunction is to utilize the newly installed carbon-sensitive probe for 13C NMR
experiments. With this probe, experiments like INADEQUATE and 13C-13C COSY can be
performed without the need for 13C enrichment, thereby clarifying 13C peaks through
decoupling.
 379

Experimental Details for Determination of Racemerization Rate of (S)-2-

methylbutanal

Pier Lucio Anelli, Fernando Montanari, and Silvio Quici Organic Syntheses, 1993, 8,
367-372.

(S)-2-methylbutanal was synthesized according to the published procedure (above).

The optical rotation was measured on a JASCO P-2000 polarimeter in the time-course
measurement mode. Measurements were taken at 589 nm and at 22 ºC.

For the DMF/water run, 20.2 mg of (S)-2-methylbutanal was dissolved in 300 μL of

DMF. Then immediately after the addition of 1.2 mL H2O, the mixture was swirled and
its optical rotation was measured at 30 minute intervals.

For the MeOH run, 46.4 mg of (S)-2-methylbutanal was dissolved in 1.5 mL of MeOH.
Immediately the mixture's optical rotation was measured at 1 minute intervals.

Below is a typical experimental result in which racemerization of (S)-2-methylbutanal is

clearly seen through gradual decrease in the optical rotation value.

40

35

30

25

20

15

10

0
0 20 40 60 80 100 120 140 160 180 200