Chembiomolecular science at the frontier of chemistry and biology

Chembiomolecular Science


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Masakatsu Shibasaki Masamitsu Iino
Hiroyuki Osada
Editors

Chembiomolecular Science
At the Frontier of Chemistry and Biology


Editors
Masakatsu Shibasaki
Director
Institute of Microbial Chemistry
3-14-23 Kamiosaki, Shinagawa-ku
Tokyo 141-0021, Japan
Hiroyuki Osada
Director
Antibiotics Laboratory
Chemical Biology Core Faculty,
RIKEN Advanced Science Institute
2-1 Hirosawa, Wako
Saitama 351-0198, Japan

Masamitsu Iino
Professor
Department of Pharmacology

Graduate School of Medicine
The University of Tokyo
7-3-1 Hongo, Bunkyo-ku
Tokyo 113-0033, Japan

ISBN 978-4-431-54037-3
ISBN 978-4-431-54038-0 (eBook)
DOI 10.1007/978-4-431-54038-0
Springer Tokyo Heidelberg New York Dordrecht London
Library of Congress Control Number: 2012948879
© Springer Japan 2013
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Preface

To understand biological functions at the molecular level and create new pharmaceuticals that can contribute to improving human health, the integration of both
chemical and biological approaches is indispensable. Chemical biology, taking
advantage of the creativity of chemistry to explore biology, is currently a very
important stream in life science. Here we propose “chembiomolecular science” as a
further advancement in the field of life science through the integration of chemical
biology with molecular-level biological studies. Chembiomolecular science will
facilitate the elucidation of new biological mechanisms as potential drug targets and
will enhance the creation of new drug leads. This new field will promote worldclass life science research in Japan to the international scientific community.
In 2009, the Uehara Memorial Foundation announced a 3-year research program
focused on chembiomolecular science. To date, 20 research groups in Japan have
been funded under this program. The aim of the symposium was to bring together
leading scientists in the field of chembiomolecular science to discuss their latest
research. The main topics to be addressed in the symposium were:
1. Chembiomolecular chemistry
2. Chembiomolecular biology
3. Chembiomolecular medicinal chemistry
The explicit aims of this symposium were to contribute to understanding the fundamentals of life science based on chemical and biological approaches, and the development of novel strategies for discovering new drug leads.
We are very pleased to be able to publish the proceedings of this exciting
symposium.
Tokyo, Japan

Masakatsu Shibasaki

v

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Contents

Part I

Chembiomolecular Chemistry

Chemistry of Mycolactones, the Causative Toxins
of Buruli Ulcer .................................................................................................
Yoshito Kishi
Practical Synthesis of Tamiflu and Beyond ..................................................
Motomu Kanai
An Approach Toward Identification of Target Proteins
of Maitotoxin Based on Organic Synthesis ...................................................
Tohru Oishi, Keiichi Konoki, Rie Tamate, Kohei Torikai,
Futoshi Hasegawa, Takeharu Nakashima, Nobuaki Matsumori,
and Michio Murata

3
15

23

Inhibitors of Fatty Acid Amide Hydrolase ...................................................
Dale L. Boger

37

Small Molecule Tools for Cell Biology and Cell Therapy ...........................
Motonari Uesugi

51

Toward the Discovery of Small Molecules Affecting
RNA Function..................................................................................................
Shiori Umemoto, Changfeng Hong, Jinhua Zhang, Takeo Fukuzumi,
Asako Murata, Masaki Hagihara, and Kazuhiko Nakatani
New Insights from a Focused Library Approach
Aiming at Development of Inhibitors of Dual-Specificity
Protein Phosphatases ......................................................................................
Go Hirai, Ayako Tsuchiya, and Mikiko Sodeoka
The Deep Oceans as a Source for New Treatments for Cancer ..................
William Fenical, James J. La Clair, Chambers C. Hughes,
Paul R. Jensen, Susana P. Gaudêncio, and John B. MacMillan

59

69
83

vii


viii

Contents

Search for New Medicinal Seeds from Marine Organisms .........................
Motomasa Kobayashi, Naoyuki Kotoku, and Masayoshi Arai

93

Identification of Protein–Small Molecule Interactions
by Chemical Array.......................................................................................... 103
Hiroyuki Osada and Siro Simizu
Part II

Chembiomolecular Biology

Small Molecule-Induced Proximity ............................................................... 115
Fu-Sen Liang and Gerald R. Crabtree
High-Throughput Screening for Small Molecule
Modulators of FGFR2-IIIb Pre-mRNA Splicing ......................................... 127
Erik S. Anderson, Peter Stoilov, Robert Damoiseaux,
and Douglas L. Black
Identification of Signaling Pathways That Mediate Dietary
Restriction-Induced Longevity in Caenorhabditis elegans .......................... 139
Masaharu Uno, Sakiko Honjoh, and Eisuke Nishida
Roles for the Stress-Responsive Kinases ASK1
and ASK2 in Tumorigenesis ........................................................................... 145
Miki Kamiyama, Takehiro Sato, Kohsuke Takeda, and Hidenori Ichijo
Tailored Synthetic Surfaces to Control Human Pluripotent
Stem Cell Self-Renewal................................................................................... 155
Laura L. Kiessling
Cell-Surface Glycoconjugates Controlling Human
T-Lymphocyte Homing: Implications for Bronchial
Asthma and Atopic Dermatitis ...................................................................... 167

Reiji Kannagi, Keiichiro Sakuma, and Katsuyuki Ohmori
Establishment of a Novel System for Studying
the Syk Function in B Cells ............................................................................ 177
Tomohiro Kurosaki and Clifford A. Lowell
Visual Screening for the Natural Compounds
That Affect the Formation of Nuclear Structures .........................................
Kaya Shigaki, Kazuaki Tokunaga, Yuki Mihara, Yota Matsuo,
Yamato Kojimoto, Hiroaki Yagi, Masayuki Igarashi, and Tokio Tani

183

Versatile Orphan Nuclear Receptor NR4A2 as a Promising
Molecular Target for Multiple Sclerosis and Other
Autoimmune Diseases ..................................................................................... 193
Shinji Oki, Benjamin J.E. Raveney, Yoshimitsu Doi,
and Takashi Yamamura


Contents

ix

Antiviral MicroRNA ....................................................................................... 201
Ryota Ouda and Takashi Fujita
Synaptic Function Monitored Using
Chemobiomolecular Indicators ..................................................................... 207
Masamitsu Iino
Part III

Chembiomolecular Medicinal Chemistry

Practical Catalytic Asymmetric Synthesis of a Promising
Drug Candidate ............................................................................................... 219
Masakatsu Shibasaki
Hunting the Targets of Natural Product-Inspired Compounds.................. 229
Slava Ziegler and Herbert Waldmann
Chemical Approaches for Understanding and Controlling
Infectious Diseases .......................................................................................... 239
Hirokazu Arimoto
Nongenomic Mechanism-Mediated Renal Fibrosis-Decreasing
Activity of a Series of PPAR-g Agonists ........................................................ 249
Hiroyuki Miyachi
Novel Carbohydrate-Based Inhibitors That Target
Influenza A Virus Sialidase ............................................................................ 261
Mark von Itzstein
Multidrug Efflux Pumps and Development of Therapeutic
Strategies to Control Infectious Diseases ...................................................... 269
Kunihiko Nishino
Enzymes as Chemotherapeutic Agents ......................................................... 281
Ronald T. Raines
Mechanism of Action of New Antiinfectious Agents
from Microorganisms ..................................................................................... 293
Nobuhiro Koyama and Hiroshi Tomoda
Correction of RNA Splicing with Antisense Oligonucleotides
as a Therapeutic Strategy for a Neurodegenerative Disease ....................... 301
Yimin Hua, Kentaro Sahashi, Frank Rigo, Gene Hung, C. Frank Bennett,
and Adrian R. Krainer
Modulation of Pre-mRNA Splicing Patterns with Synthetic
Chemicals and Their Clinical Applications .................................................. 315
Masatoshi Hagiwara

Index ................................................................................................................. 321


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Contributors

Erik S. Anderson Molecular Biology Institute, University of California, Los Angeles,
CA, USA
The David Geffen School of Medicine, University of California, Los Angeles,
CA, USA
Masayoshi Arai Graduate School of Pharmaceutical Sciences, Osaka University,
Osaka, Japan
Hirokazu Arimoto Graduate School of Life Sciences, Tohoku University,
Sendai, Japan
C. Frank Bennett Isis Pharmaceuticals, Carlsbad, CA, USA
Douglas L. Black Howard Hughes Medical Institute, University of California,
Los Angeles, CA, USA
Department of Microbiology, Immunology and Molecular Genetics, University
of California, Los Angeles, CA, USA
Dale L. Boger Department of Chemistry, The Scripps Research Institute,
La Jolla, CA, USA
Gerald R. Crabtree Howard Hughes Medical Institute, Stanford University
School of Medicine, Stanford, CA, USA
Robert Damoiseaux Molecular Screening Shared Resource, University of
California, Los Angeles, CA, USA
Yoshimitsu Doi Department of Immunology, National Institute of Neuroscience,
National Center of Neurology and Psychiatry, Tokyo, Japan
William Fenical Center for Marine Biotechnology and Biomedicine, Scripps

Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA

xi


xii

Contributors

Takashi Fujita Laboratory of Molecular Genetics, Institute for Virus Research,
Kyoto University, Kyoto, Japan
Laboratory of Molecular Cell Biology, Graduate School of Biostudies, Kyoto
University, Kyoto, Japan
Takeo Fukuzumi Department of Regulatory Bioorganic Chemistry, The Institute
of Scientific and Industrial Research, Osaka University, Osaka, Japan
Susana P. Gaudêncio Center for Marine Biotechnology and Biomedicine,
Scripps Institution of Oceanography, University of California, San Diego, La Jolla,
CA, USA
Masaki Hagihara Department of Regulatory Bioorganic Chemistry, The Institute
of Scientific and Industrial Research, Osaka University, Osaka, Japan
Masatoshi Hagiwara Department of Anatomy and Developmental Biology,
Graduate School of Medicine, Kyoto University, Kyoto, Japan
Futoshi Hasegawa Department of Chemistry, Graduate School of Science, Osaka
University, Osaka, Japan
Go Hirai Synthetic Organic Chemistry Laboratory, RIKEN Advanced Science
Institute, Saitama, Japan
Changfeng Hong Department of Regulatory Bioorganic Chemistry, The Institute
of Scientific and Industrial Research, Osaka University, Osaka, Japan
Sakiko Honjoh Department of Cell and Developmental Biology, Graduate School
of Biostudies, Kyoto University, Kyoto, Japan

Yimin Hua Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
Chambers C. Hughes Center for Marine Biotechnology and Biomedicine,
Scripps Institution of Oceanography, University of California, San Diego, La Jolla,
CA, USA
Gene Hung Isis Pharmaceuticals, Carlsbad, CA, USA
Hidenori Ichijo Laboratory of Cell Signaling, Graduate School of Pharmaceutical
Sciences, The University of Tokyo, Tokyo, Japan
Masayuki Igarashi Laboratory of Disease Biology, Institute of Microbial
Chemistry, Tokyo, Japan
Masamitsu Iino Department of Pharmacology, Graduate School of Medicine,
The University of Tokyo, Tokyo, Japan
Mark von Itzstein Institute for Glycomics, Griffith University, Gold Coast
Campus, Southport, QLD, Australia
Paul R. Jensen Center for Marine Biotechnology and Biomedicine, Scripps
Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA


Contributors

xiii

Miki Kamiyama Laboratory of Cell Signaling, Graduate School of Pharmaceutical
Sciences, The University of Tokyo, Tokyo, Japan
Motomu Kanai Graduate School of Pharmaceutical Sciences, The University of
Tokyo, Tokyo, Japan
Reiji Kannagi Research Complex for Medical Frontiers, Aichi Medical University,
Aichi, Japan
Department of Molecular Pathology, Aichi Cancer Center, Nagoya, Japan
Laura L. Kiessling Departments of Chemistry and Biochemistry, University of
Wisconsin-Madison, Madison, WI, USA

Yoshito Kishi Department of Chemistry and Chemical Biology, Harvard University,
Cambridge, MA, USA
Motomasa Kobayashi Graduate School of Pharmaceutical Sciences, Osaka
University, Osaka, Japan
Yamato Kojimoto Department of Biological Sciences, Graduate School of Science
and Technology, Kumamoto University, Kumamoto, Japan
Keiichi Konoki Graduate School of Agricultural Science, Tohoku University,
Sendai, Japan
Naoyuki Kotoku Graduate School of Pharmaceutical Sciences, Osaka University,
Osaka, Japan
Nobuhiro Koyama Graduate School of Pharmaceutical Sciences, Kitasato
University, Tokyo, Japan
Adrian R. Krainer Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
Tomohiro Kurosaki Laboratory for Lymphocyte Differentiation, WPI Immunology
Frontier Research Center, Osaka University, Osaka, Japan
RIKEN Research Center for Allergy and Immunology, Kanagawa, Japan
James J. La Clair Xenobe Research Institute, San Diego, CA, USA
Fu-Sen Liang Howard Hughes Medical Institute, Stanford University School of
Medicine, Stanford, CA, USA
Clifford A. Lowell Department of Laboratory Medicine, University of California,
San Francisco, CA, USA
John B. MacMillan Center for Marine Biotechnology and Biomedicine, Scripps
Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA
Nobuaki Matsumori Department of Chemistry, Graduate School of Science,
Osaka University, Osaka, Japan
Yota Matsuo Department of Biological Sciences, Graduate School of Science
and Technology, Kumamoto University, Kumamoto, Japan


xiv

Contributors

Yuki Mihara Department of Biological Sciences, Graduate School of Science
and Technology, Kumamoto University, Kumamoto, Japan
Hiroyuki Miyachi Graduate School of Medicine, Dentistry and Pharmaceutical
Sciences, Okayama University, Okayama, Japan
Asako Murata Department of Regulatory Bioorganic Chemistry, The Institute of
Scientific and Industrial Research, Osaka University, Osaka, Japan
Michio Murata Department of Chemistry, Graduate School of Science, Osaka
University, Osaka, Japan
Takeharu Nakashima Department of Chemistry, Graduate School of Science,
Osaka University, Osaka, Japan
Kazuhiko Nakatani Department of Regulatory Bioorganic Chemistry, The Institute
of Scientific and Industrial Research, Osaka University, Osaka, Japan
Eisuke Nishida Department of Cell and Developmental Biology, Graduate School
of Biostudies, Kyoto University, Kyoto, Japan
Kunihiko Nishino Laboratory of Microbiology & Infectious Diseases, Institute
of Scientific and Industrial Research, Osaka University, Osaka, Japan
Katsuyuki Ohmori Department of Clinical Pathology, Kyoto University School
of Medicine, Kyoto, Japan
Tohru Oishi Department of Chemistry, Faculty and Graduate School of Sciences,
Kyushu University, Fukuoka, Japan
Shinji Oki Department of Immunology, National Institute of Neuroscience,
National Center of Neurology and Psychiatry, Tokyo, Japan
Hiroyuki Osada Chemical Biology Department, RIKEN Advanced Science
Institute, Saitama, Japan
Ryota Ouda Laboratory of Molecular Genetics, Institute for Virus Research,
Kyoto University, Kyoto, Japan
Laboratory of Molecular Cell Biology, Graduate School of Biostudies, Kyoto

University, Kyoto, Japan
Ronald T. Raines Department of Biochemistry, University of Wisconsin-Madison,
Madison, WI, USA
Benjamin J.E. Raveney Department of Immunology, National Institute of
Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
Frank Rigo Isis Pharmaceuticals, Carlsbad, CA, USA
Kentaro Sahashi Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA


Contributors

xv

Keiichiro Sakuma Research Complex for Medical Frontiers, Aichi Medical
University, Aichi, Japan
Department of Molecular Pathology, Aichi Cancer Center, Nagoya, Japan
Takehiro Sato Laboratory of Cell Signaling, Graduate School of Pharmaceutical
Sciences, The University of Tokyo, Tokyo, Japan
Masakatsu Shibasaki Institute of Microbial Chemistry, Tokyo, Japan
Kaya Shigaki Department of Biological Sciences, Graduate School of Science
and Technology, Kumamoto University, Kumamoto, Japan
Siro Simizu Chemical Biology Department, RIKEN Advanced Science Institute,
Saitama, Japan
Department of Applied Chemistry, Faculty of Science and Technology, Keio
University, Yokohama, Japan
Mikiko Sodeoka Synthetic Organic Chemistry Laboratory, RIKEN Advanced
Science Institute, Saitama, Japan
Peter Stoilov Department of Biochemistry, West Virginia University, Morgantown,
WV, USA
Kohsuke Takeda Division of Cell Regulation, Graduate School of Biomedical

Sciences, Nagasaki University, Nagasaki, Japan
Rie Tamate Department of Chemistry, Graduate School of Science, Osaka
University, Osaka, Japan
Tokio Tani Department of Biological Sciences, Graduate School of Science and
Technology, Kumamoto University, Kumamoto, Japan
Kazuaki Tokunaga Department of Biological Sciences, Graduate School of
Science and Technology, Kumamoto University, Kumamoto, Japan
Hiroshi Tomoda Graduate School of Pharmaceutical Sciences, Kitasato University,
Tokyo, Japan
Kohei Torikai Department of Chemistry, Faculty and Graduate School of Sciences,
Kyushu University, Fukuoka, Japan
Ayako Tsuchiya Synthetic Organic Chemistry Laboratory, RIKEN Advanced
Science Institute, Saitama, Japan
Motonari Uesugi Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto
University, Kyoto, Japan
Institute for Chemical Research, Kyoto University, Kyoto, Japan
Shiori Umemoto Department of Regulatory Bioorganic Chemistry, The Institute
of Scientific and Industrial Research, Osaka University, Osaka, Japan


xvi

Contributors

Masaharu Uno Department of Cell and Developmental Biology, Graduate School
of Biostudies, Kyoto University, Kyoto, Japan
Herbert Waldmann Chemical Biology Department, Max Planck Institute of
Molecular Physiology, Dortmund, Germany
Hiroaki Yagi Department of Biological Sciences, Graduate School of Science
and Technology, Kumamoto University, Kumamoto, Japan

Takashi Yamamura Department of Immunology, National Institute of
Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
Jinhua Zhang Department of Regulatory Bioorganic Chemistry, The Institute of
Scientific and Industrial Research, Osaka University, Osaka, Japan
Slava Ziegler Chemical Biology Department, Max Planck Institute of Molecular
Physiology, Dortmund, Germany


Part I

Chembiomolecular Chemistry


Chemistry of Mycolactones, the Causative
Toxins of Buruli Ulcer
Yoshito Kishi

Introduction
Buruli ulcer is a severe and devastating skin disease caused by Mycobacterium
ulcerans infection, yet it is one of the most neglected diseases (Fig. 1) (for recent
reviews on Buruli ulcer, see [1–3]). Among the diseases caused by mycobacterial
infection, Buruli ulcer occurs less frequently than tuberculosis (Mycobacterium
tuberculosis) and leprosy (Mycobacterium leprae). However, it is noted that the
occurrence of Buruli ulcer is increasing and spreading in tropical countries, and that
the incidence of the disease may exceed that of leprosy and tuberculosis in highly
affected areas. Infection with M. ulcerans, probably carried by aquatic insects and
mosquitoes [4, 5], results in painless necrotic lesions that, if untreated, can extend
to 15% of a patient’s skin surface. Surgical intervention has been the only practical
curative therapy for Buruli ulcer.
Most pathogenic bacteria produce toxins that play an important role(s) in disease. However, there has been no evidence thus far to suggest toxin production by

M. tuberculosis and M. leprae. Interestingly, the presence of a toxin in M. ulcerans
had been noticed for many years, but the toxin was not isolated until 1999 when
Small and co-workers succeeded in isolation and characterization of mycolactone
A/B from this bacteria [6]. Intradermal inoculation of mycolactone A/B into guinea
pigs produces lesions similar to that of Buruli ulcer in humans, demonstrating their
direct correlation with Buruli ulcer ([7]: for recent reviews on mycolactones, see
[8, 9]).

Y. Kishi (*)
Department of Chemistry and Chemical Biology, Harvard University,
12 Oxford Street, Cambridge, MA 02138, USA
e-mail:
M. Shibasaki et al. (eds.), Chembiomolecular Science: At the Frontier
of Chemistry and Biology, DOI 10.1007/978-4-431-54038-0_1, © Springer Japan 2013

3


4

Y. Kishi

Fig. 1 Buruli ulcer lesion (taken from [1])

Structure
Gross Structure
The gross structure of mycolactone A/B was elucidated by Small and co-workers
via a variety of spectroscopic methods; coupled with mass spectroscopy (MS),
ultraviolet (UV), and infrared (IR) studies, extensive two-dimensional nuclear magnetic resonance (2D NMR) experiments led them to suggest the gross structure of
mycolactone A/B [10].

Stereochemistry
For the proposed gross structure of mycolactone A/B, 1,024 stereoisomers are possible. Considering the limited availability, as well as the noncrystallinity, of mycolactone A/B, we recognized the difficulties that might be encountered in the assignment
of its stereochemistry. Coincidentally, we were then engaged in the development of
the universal NMR database approach to assign the relative and absolute configuration
of unknown compounds without degradation or derivatization, and we noticed that
the universal NMR database approach was uniquely suited to assign the stereochemistry of the mycolactone A/B ([11, 12] and references cited therein). Indeed,
with use of this approach, we could establish the complete structure of the mycolactone A/B (Fig. 2) [13, 14]. Mycolactone A/B exists as a 3:2 equilibrating mixture,
with the major and minor components corresponding to the Z-D4¢,5¢- and E-D4¢,5¢isomers, respectively, in the unsaturated fatty acid side chain.


Chemistry of Mycolactones, the Causative Toxins of Buruli Ulcer
Me

Me

5

20

Me
15

OH

Me

OH

10

O
1

O
Me
5

O

Me

Me

Me

Me

OH
15'

1'

Me

10'
5'

O

OH

OH

1: Complete Structure of Mycolactone A/B

Fig. 2 Complete structure of mycolactone A/B. Wavy line indicates that this bond exists as a
mixture of E- and Z-geometric isomers

Structure Determinations of Mycolactone Congeners
Following the isolation of mycolactone A/B, several mycolactone congeners were
reported from clinical isolates of M. ulcerans from Africa, Malaysia, Asia, Australia,
and Mexico. In addition, mycolactone-like metabolites were isolated from the frog
pathogen Mycobacterium liflandii and the fish pathogen Mycobacterium marinum.
As these metabolites were available only in very minute quantities, their structure
determination posed a major challenge. The structure information available on these
metabolites was often limited to the molecular formula by mass spectroscopy.
Having established the complete structure of mycolactone A/B as well as a flexible,
modular synthesis (vide infra), we undertook a new approach to elucidate the structure of the mycolactone congeners. For an illustration of this approach, we use the
case of mycolactone F isolated from the fish pathogen M. marinum.
Based on the mass spectroscopic data, Leadlay and co-workers suggested the
gross structure of mycolactone F [15]. Considering its structural similarity to
mycolactone A/B, we speculated 2 to be the likely structure (Fig. 3). However, we
thought that 3 should be included for our structure analysis. In our terminology, 3 is
a remote diastereomer of 2, a diastereomer as a result of the stereocenter(s) present
outside a self-contained box(es) [11, 12]. Importantly, remote diastereomers exhibit
virtually identical physicochemical properties in an achiral environment but different physicochemical properties in a chiral environment. Following the synthesis
outlined later, we uneventfully synthesized both 2 and 3. Under photochemical conditions, they exhibited a facile geometric isomerization, furnishing a 5:2:2 mixture
of three predominant isomers: note the 1,3,5-trimethyl groups present in the chromophore of mycolactone F versus the 1,3-dimethyl groups in the chromophore of
mycolactone A/B.

With both diastereomers 2 and 3 in hand, we began to search for an analytical
method to distinguish them. Given the fact that only a very minute amount of natural


6

Y. Kishi
Me
Me

Me

10'

Me
Me

10

O

O
Me

1

5'

O

15

OH

OH

Me
OH

OH

2: Mycolactone dia-F
Me

5

Me

Me

1'

20

Me

Me
10'

1'

Me

O

5'

O

Me
OH

OH

3: Mycolactone F

a

b

c

1

1

1
*

2

2

2

3

3

3
*

10.0

15.0

10.0

15.0

10.0

15.0

Retention time (min)

Fig. 3 Upper panel: structure of mycolactones F and dia-F isolated from Mycobacterium marinum in freshwater and saltwater fish, respectively. Under photochemical conditions (300 nm,
acetone), both mycolactones smoothly isomerize, to furnish a 5:2:2 mixture of three predominant
regioisomers. Wavy line indicates that this bond exists as a mixture of E- and Z-geometric isomers. Lower panel: HPLC comparison of synthetic, photochemically isomerized mycolactones F
and dia-F. (a) 1, synthetic mycolactone F; 2, synthetic mycolactone dia-F; 3, their 1:1 mixture.

(b) 1, mycolactone isolated from freshwater fish pathogen (M. marinum BB170200); 2, mixed
with synthetic mycolactone F; 3, mixed with synthetic mycolactone dia-F. (c) 1, mycolactone
isolated from saltwater fish pathogen (M. marinum DL240490); 2, mixed with synthetic
mycolactone dia-F; 3, mixed with synthetic mycolactone F

mycolactone F was available, we needed an analytical method with a high sensitivity and opted to use chiral HPLC. For this search, we purposely used the photochemically equilibrated 2 and 3 with the hope that each of their geometric isomers
might give a distinct retention time. Thus, HPLC comparison could be performed
on the basis of six, instead of two, distinct retention times. After numerous attempts,


Chemistry of Mycolactones, the Causative Toxins of Buruli Ulcer

7

we eventually found that a Chiralpak IA chiral column employing a mobile phase of
toluene–isopropanol can distinguish all six remote diastereomers (Fig. 3). Finally,
we subjected the natural product to this HPLC analysis, thereby demonstrating that
mycolactone from the fish pathogen M. marinum is, surprisingly, 3 [16].
The 1,3-diol present in the unsaturated fatty acid side chain of 3 occurs curiously
in the mirror image of the 1,3-diol present in other mycolactones. The mycolactone
F used for this study was isolated from M. marinum from cultured European sea
bass. Intriguingly, we later found that the mycolactone isolated from M. marinum
from freshwater silver perch in Israel corresponds to 2, referred to as mycolactone
dia-F [17]. Related to this finding, it is interesting to quote the Stinear claim that
mycolactone-producing mycobacteria have all evolved from a common M. marinum progenitor [18]. This finding may suggest that, at some stage of evolution, the
absolute configuration in question was switched between the mycolactone F and
mycolactone A/B series. Before the isolation of mycolactone F from marine fish
populations, all the other mycolactones had been isolated from species located in or
around freshwater habitats.
The approach described for the structure elucidation of mycolactone F was used

to establish the structure of mycolactones C–E, and E ketone (Fig. 4) [19–21].

Total Synthesis
As the structure of mycolactone A/B was elucidated by application of the newly
developed logic and method, we believed it was necessary to confirm the assigned
structure. For this reason, we carried out a total synthesis of mycolactone A/B and
confirmed that the assigned structure was indeed correct [22]. During this work, we
realized that organic synthesis could play an additional critical role to advance
mycolactone science. Because of the slow growth of M. ulcerans, it has been a challenging task to secure mycolactone A/B in quantities by cultivation. In addition,
mycolactone A/B from the natural source is often contaminated with various
unknown compounds, including mycolactone congeners. We believed that organic
synthesis could supply chemically well-defined and homogeneous materials in
sufficient quantities for further study and continued synthetic work, yielding a convergent, flexible, and efficient synthesis of the mycolactone class of natural
products.
The core is assembled from the three building blocks A, B, and C, each of which
is synthesized using asymmetric reactions as the key steps (Fig. 5). The building
blocks A, B, and C are then assembled with cross-coupling reactions to furnish the
mycolactone core. The unsaturated fatty acid is prepared from the building blocks
D and E via the Horner–Emmons reaction, followed by saponification. The coupling of the unsaturated fatty acid with the core, followed by tetrabutylammonium
fluoride (TBAF)-promoted t-butyldimethylsilyl (TBS)-deprotection, furnished
mycolactone A/B. It is worthwhile noting that (1) this synthesis is scalable to prepare mycolactone A/B and its congeners with high optical purity and (2) this


Me

Me

20

Me

15

OH

Me

OH

10

O
1

O
Me
5

Me

O
Mycolactone Core
Mycolactone Unsaturated Fatty Acyl Side Chains
Isolated from human
pathogen M. ulcerans

Me

Me

Me

OH

16'

Me

1'

mycolactone A/B
O

OH
Me

mycolactone C

Me

16'

Me

1'

O

OH
Me

mycolactone D

OH

Me

Me

Me

Me

OH

OH

16'

Me

1'

O

OH

Isolated from frog
pathogen M. liflandii

Me

Me

OH

Me
15'

1'

Me

mycolactone E
O

OH
Me

mycolactone E
ketone

Me

15'

1'

Me

O

OH

Isolated from fish
pathogen M. marinum
mycolactone F
(saltwater fish)

Me

Me

O

Me

14'

Me

1'

O

OH
Me

mycolactone dia-F
(freshwater fish)

OH

Me

Me

14'

Me

1'

O

OH

Me

OH

OH

Fig. 4 Structurally well-defined mycolactones. Wavy line indicates that this bond exists as a
mixture of E- and Z-geometric isomers


Chemistry of Mycolactones, the Causative Toxins of Buruli Ulcer

a

b

9

c
13

I
MeO 1

Me

7

I

Me

O

Me

O

8

d

20

Me

I 14

O

TBSO

Me

OPMB

Me

OTBS

e
Me
1'

MeO2C

Me

P(O)(OEt)2
8'

Me

OTBS

9'

16'

Me

OHC
TBSO

OTBS

Fig. 5 Five building blocks used in a convergent and flexible synthesis of the mycolactone class
natural products

synthesis is modular in nature and can be adjusted to prepare various mycolactone
stereoisomers or analogues [23, 24].
The mycolactones have attracted considerable attention from the synthetic community, not only for their biological activity, but also for being the first examples of
polyketide macrolides isolated from a human pathogen. Indeed, several other groups
have reported the syntheses of the mycolactone core and/or the unsaturated fatty
acid side chain [25–29].

Structural Diversity in the Mycolactone Class of Natural Products
All the mycolactones reported to date are composed of a 12-membered macrolactone and a highly unsaturated fatty acid side chain (Fig. 4). The macrolactone core
is conserved in all the members in the mycolactone class of natural products. On the
other hand, a remarkable structural diversity is observed in the unsaturated fatty
acid portion, including the length of the fatty acid backbone, degree of unsaturation,
degree of hydroxylation, stereochemistry of hydroxylation, oxidation state of
alcohols, and the number of methyl groups.
The three mycolactones A/B, C, and D from clinical isolates of M. ulcerans are

structurally well defined. All are composed of a hexadecanoic acid backbone with a
pentaenoate chromophore but differ in the number of hydroxyl and methyl groups.
Mycolactones isolated from frog and fish pathogens bear shorter unsaturated
fatty acids. Two mycolactones from the frog pathogen M. liflandii are composed of
a pentadecanoic acid backbone with the tetraenoate chromophore but differ in the
oxidation level; that is, 1,3-diol versus 1,3-hydroxyketone at C11¢ and C13¢.
Mycolactones F and dia-F from the fish pathogen M. marinum share the same
pentadecanoic acid. However, they occur as a mirror image.


10

Y. Kishi

Fig. 6 Thin-layer chromatography (TLC) detection of mycolactones A/B and C. (a) Synthetic
mycolactones A/B (left), C (right), and their mixture (middle). (b) Synthetic mycolactone A/B
(left), a lipid extract of an African strain of M. ulcerans (right), and their mixture (middle).
(c) Synthetic mycolactone C (left), a lipid extract of an Australian strain of M. ulcerans (right), and
their mixture (middle)

Detection and Structure Analysis
Combination treatments with rifampicin and either streptomycin or amikacin have
recently been reported to prevent the growth of the bacteria in early lesions [1–3],
pointing out the importance of diagnosing the disease at its preulcerative stage.
Polymerase chain reaction of M. ulcerans DNA is commonly used to detect
M. ulcerans infection. Undoubtedly, there is an urgent need for development of a
cost- and time-effective method, ideally simple enough for field use in remote areas,
to detect M. ulcerans infection. Knowing that mycolactones are the causative toxins
of Buruli ulcer, we noticed the possibility of using mycolactones as a marker to
detect M. ulcerans infection or to diagnose Buruli ulcer.

With this background, we have recently developed a boronate-assisted fluorogenic
chemosensor that can detect as small as 2 ng of mycolactone A/B in a semiquantitative
manner [30]. We recognize two possible areas to apply this analytical method. First, it
appears to be suited for the mycolactone-based chemotaxonomy of M. ulcerans. To
illustrate its feasibility, we analyzed the crude lipid extracts of African and Australian
strains of M. ulcerans (Fig. 6). Second, we began this study with the hope of developing a cost- and time-effective method to detect M. ulcerans infection. To this end, we
have shown that this method can detect mycolactone A/B in pig and fish skin and
muscle tissues doped with mycolactone A/B. There are a few issues still to address,
but we are cautiously optimistic in achieving the ultimate goal.

Biological Activity
Various in vitro and in vivo studies in mice and guinea pigs demonstrated that mycolactone plays a central role in the pathogenesis of M. ulcerans disease; injection of 100 mg
of the toxin was sufficient to cause characteristic ulcers in guinea pig skin.