Chembiomolecular science at the frontier of chemistry and biology

Takashi Fujita Laboratory of Molecular Genetics, Institute for Virus Research , Kyoto University , Kyoto , Japan Laboratory of Molecular Cell Biology , Graduate School of Biostudies, K

Chembiomolecular Science

Masakatsu Shibasaki  Masamitsu Iino

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

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© Springer Japan 2013

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To understand biological functions at the molecular level and create new ceuticals 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 fi eld 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 fi eld will promote world-class life science research in Japan to the international scienti fi c 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 fi eld 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 mentals of life science based on chemical and biological approaches, and the devel-opment of novel strategies for discovering new drug leads

funda-We are very pleased to be able to publish the proceedings of this exciting symposium

Preface

Part I Chembiomolecular Chemistry

Chemistry of Mycolactones, the Causative Toxins

of Buruli Ulcer 3Yoshito Kishi

Practical Synthesis of Tamiflu and Beyond 15Motomu Kanai

An Approach Toward Identification of Target Proteins

of Maitotoxin Based on Organic Synthesis 23Tohru Oishi, Keiichi Konoki, Rie Tamate, Kohei Torikai,

Futoshi Hasegawa, Takeharu Nakashima, Nobuaki Matsumori,

and Michio Murata

Inhibitors of Fatty Acid Amide Hydrolase 37Dale L Boger

Small Molecule Tools for Cell Biology and Cell Therapy 51Motonari Uesugi

Toward the Discovery of Small Molecules Affecting

RNA Function 59Shiori 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 69

Go Hirai, Ayako Tsuchiya, and Mikiko Sodeoka

The Deep Oceans as a Source for New Treatments for Cancer 83William Fenical, James J La Clair, Chambers C Hughes,

Paul R Jensen, Susana P Gaudêncio, and John B MacMillan

Contents

Search for New Medicinal Seeds from Marine Organisms 93Motomasa Kobayashi, Naoyuki Kotoku, and Masayoshi Arai

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 183

Kaya Shigaki, Kazuaki Tokunaga, Yuki Mihara, Yota Matsuo,

Yamato Kojimoto, Hiroaki Yagi, Masayuki Igarashi, and Tokio Tani

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

ix Contents

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

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

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 ,

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 Scienti fi c 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 Scienti fi c 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 Scienti fi c 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, Grif fi th 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

xiii Contributors

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

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 Scienti fi c 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 Scienti fi c 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 Scienti fi c 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

xv Contributors

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 ,

Institute for Chemical Research, Kyoto University, Kyoto, Japan

Shiori Umemoto Department of Regulatory Bioorganic Chemistry , The Institute

of Scienti fi c and Industrial Research, Osaka University , Osaka , Japan

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 Scienti fi c and Industrial Research, Osaka University , Osaka , Japan

Slava Ziegler Chemical Biology Department , Max Planck Institute of Molecular Physiology , Dortmund , Germany

Part I

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

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 ease 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: kishi@chemistry.harvard.edu

Toxins of Buruli Ulcer

Yoshito Kishi

Stereochemistry

For the proposed gross structure of mycolactone A/B, 1,024 stereoisomers are sible Considering the limited availability, as well as the noncrystallinity, of mycolac-tone A/B, we recognized the dif fi culties that might be encountered in the assignment

pos-of its stereochemistry Coincidentally, we were then engaged in the development pos-of the universal NMR database approach to assign the relative and absolute con fi guration

of unknown compounds without degradation or derivatization, and we noticed that the universal NMR database approach was uniquely suited to assign the stereo-chemistry 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 mycolac-tone 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 - D 4 ¢ ,5 ¢ - and E - D 4 ¢ ,5 ¢ isomers, respectively, in the unsaturated fatty acid side chain

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

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 li fl andii and the fi sh 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 fl exible, modular synthesis (vide infra), we undertook a new approach to elucidate the struc-ture of the mycolactone congeners For an illustration of this approach, we use the

case of mycolactone F isolated from the fi sh 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 differ-ent physicochemical properties in a chiral environment Following the synthesis

outlined later, we uneventfully synthesized both 2 and 3 Under photochemical

con-ditions, 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 mophore 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

1: Complete Structure of Mycolactone A/B

5

Me

OH Me

15'

Me Me O

Fig 2 Complete structure of mycolactone A/B Wavy line indicates that this bond exists as a

mixture of E - and Z -geometric isomers

6 Y Kishi

mycolactone F was available, we needed an analytical method with a high ity and opted to use chiral HPLC For this search, we purposely used the photo-

sensitiv-chemically 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,

O

Me

Me Me

Fig 3 Upper panel: structure of mycolactones F and dia -F isolated from Mycobacterium

mari-num in freshwater and saltwater fi sh, 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 mers Lower panel: HPLC comparison of synthetic, photochemically isomerized mycolactones F

iso-and dia -F ( a ) 1 , synthetic mycolactone F; 2 , synthetic mycolactone dia -F; 3 , their 1:1 mixture ( b ) 1 , mycolactone isolated from freshwater fi sh pathogen ( M marinum BB170200); 2 , mixed with synthetic mycolactone F; 3 , mixed with synthetic mycolactone dia- F ( c ) 1 , mycolactone

isolated from saltwater fi sh pathogen ( M marinum DL240490); 2 , mixed with synthetic

mycolactone dia- F; 3 , mixed with synthetic mycolactone F

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 fi sh 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 fi nding, it is interesting to quote the Stinear claim that

mycolactone-producing mycobacteria have all evolved from a common M

mari-num progenitor [ 18 ] This fi nding may suggest that, at some stage of evolution, the absolute con fi guration in question was switched between the mycolactone F and mycolactone A/B series Before the isolation of mycolactone F from marine fi sh 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 con fi rm the assigned structure For this reason, we carried out a total synthesis of mycolactone A/B and con fi rmed 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

chal-lenging 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-de fi ned and homogeneous materials in suf fi cient quantities for further study and continued synthetic work, yielding a con-vergent, fl exible, and ef fi cient 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 saponi fi cation The

cou-pling of the unsaturated fatty acid with the core, followed by tetrabutylammonium

fl uoride (TBAF)-promoted t -butyldimethylsilyl (TBS)-deprotection, furnished mycolactone A/B It is worthwhile noting that (1) this synthesis is scalable to pre-pare mycolactone A/B and its congeners with high optical purity and (2) this

O Me

10

5 1 15

O

OH

Me OH

1'

16' 20

15'

O

Me

Me Me

Fig 4 Structurally well-de fi ned mycolactones Wavy line indicates that this bond exists as a

mixture of E - and Z -geometric isomers

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 munity, not only for their biological activity, but also for being the fi rst 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 tone and a highly unsaturated fatty acid side chain (Fig 4 ) The macrolactone core

macrolac-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 de fi ned 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 fi sh pathogens bear shorter unsaturated

fatty acids Two mycolactones from the frog pathogen M li fl andii 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 fi sh pathogen M marinum share the same

pentadecanoic acid However, they occur as a mirror image

MeO

I

OPMB

Me O

Me I

10 Y Kishi

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 fi eld 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 fl uorogenic 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

develop-ing 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 fi sh 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

mycolac-tone plays a central role in the pathogenesis of M ulcerans disease; injection of 100 m g

of the toxin was suf fi cient to cause characteristic ulcers in guinea pig skin

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 )

Signi fi cant progress has been made in the characterization of the biological ity of mycolactones, including cytotoxic and immunosuppressive effects [ 1– 3, 8, 9 ] However, bioactivity studies have been limited to only mycolactone A/B and its immediate derivatives thus far [ 31 ]

Despite efforts from many research groups, the molecular target of mycolactones remains unknown In this connection, we should note that Dr Jackson of our labora-

tory has recently synthesized analogue 4a and demonstrated that (1) 4a is useful to prepare a mycolactone conjugate and (2) the amide 4b , derived from 4a , exhibits

cytotoxicity (30 nm) against L929 fi broblasts at one third of the potency (10 nm) of mycolactone A/B (Fig 7 )

Prospects

The chemistry of mycolactones, including structure determination/analysis, total synthesis, and highly sensitive detection methods, has been well developed Because

of the slow growth of M ulcerans , it has been a challenging task to secure

mycolac-tone A/B in quantities by cultivation The convergent, scalable, and fl exible sis developed can now provide not only chemically well-de fi ned and homogeneous materials, but also mycolactone analogues for study In our view, this is an exciting time to witness a new phase in mycolactone science

Acknowledgments We are grateful to the National Institutes of Health (CA 22215) and Eisai

USA Foundation for generous fi nancial support

Me O

Fig 7 A possible precursor for the preparation of mycolactone conjugates A wavy line indicates

that this bond exists as a mixture of E - and Z -geometric isomers

12 Y Kishi

References

1 Asiedu K, Scherpbier R, Raviglione M (eds) (2000) Buruli ulcer: Mycobacterium ulcerans

infection World Health Organization, Geneva

2 Johnson PDR, Stinear T, Small PLC, Plushke G, Merritt RW, Portaels F, Huygen K, Hayman

JA, Asiedu K (2005) Buruli ulcer ( M ulcerans infection): new insights, new hope for disease

control PLoS Med 2:282–286

3 Demangel C, Stinear TP, Cole ST (2009) Buruli ulcer: reductive evolution enhances

pathoge-nicity of Mycobacterium ulcerans Nat Rev 7:50–60

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C, Carbonnelle B (2002) Aquatic insects as a vector for Mycobacterium ulcerans Appl

Environ Microbiol 68:4623–4628

5 Johnson PDR, Azuolas J, Lavender CJ, Wishart E, Stinear TP, Hayman JA, Brown L, Jenkin

GA, Fyfe JAM (2007) Mycobacterium ulcerans in mosquitoes captured during outbreak of

Buruli Ulcer, Southeastern Australia Emerg Infect Dis 13:1653–1660

6 George KM, Chatterjee D, Gunawardana G, Welty D, Hayman J, Lee R, Small PLC (1999)

Mycolactone: a polyketide toxin from Mycobacterium ulcerans required for virulence Science

283:854–857

7 George KM, Pascopella L, Welty DM, Small PLC (2000) A Mycobacterium ulcerans toxin,

mycolactone, causes apoptosis in guinea pig ulcers and tissue culture cells Infect Immun 68:877–883

8 Hong H, Demangel C, Pidot SJ, Leadlay PF, Stinear T (2008) Mycolactones: sive and cytotoxic polyketides produced by aquatic mycobacteria Nat Prod Rep 25:447–454

9 Kishi Y (2011) Chemistry of mycolactones, the causative toxins of Buruli ulcer Proc Natl Acad Sci USA 108:6703–6708

10 Gunawardana G, Chatterjee D, George KM, Brennan P, Whittern D, Small PLC (1999) Characterization of novel macrolide toxins, mycolactones A and B, from a human pathogen,

Mycobacterium ulcerans J Am Chem Soc 121:6092–6093

11 Kobayashi Y, Lee J, Tezuka K, Kishi Y (1999) Toward creation of a universal NMR database for the stereochemical assignment of acyclic compounds: the case of two contiguous propi- onate units Org Lett 1:2177–2180

12 Seike F, Ghosh I, Kishi Y (2006) Attempts to assemble universal NMR database without thesis of NMR database compounds Org Lett 8:3861–3864

13 Benewoitz AB, Fidanze S, Small PLC, Kishi Y (2001) Stereochemistry of the core structure of the mycolactones J Am Chem Soc 123:5128–5129

14 Fidanze S, Song F, Szlosek-Pinaud M, Small PLC, Kishi Y (2001) Complete structure of the mycolactones J Am Chem Soc 123:10117–10118

15 Hong H, Stinear T, Porter J, Demangel C, Leadlay P (2007) A novel mycolactone toxin obtained by biosynthetic engineering Chembiochem 8:2043–2047

16 Kim H-J, Kishi Y (2008) Total synthesis and stereochemistry of mycolactone F J Am Chem Soc 130:1842–1844

17 Kim H-J, Jackson KL, Kishi Y, Williamson HR, Mosi L, Small PLC (2009) Heterogeneity in

the stereochemistry of mycolactones isolated from M marinum : toxins produced by fresh vs

saltwater fi sh pathogens Chem Commun:7402–7404

18 Yip MJ, Porter JL, Fyfe JAM, Lavender CJ, Portaels F, Rhodes M, Kator H, Colorni A, Jenkin

GA, Stinear T (2007) Evolution of Mycobacterium ulcerans and other mycolactone-producing

mycobacteria from a common Mycobacterium marinum progenitor J Bacteriol 189:2021–2029

19 Judd TC, Bischoff A, Kishi Y, Adusumilli S, Small PLC (2004) Structure determination of mycolactone C via total synthesis Org Lett 6:4901–4904

20 Aubry S, Lee RE, Mahrous EA, Small PLC, Beachboard D, Kishi Y (2008) Synthesis and structure of mycolactone E isolated from frog mycobacterium Org Lett 10:5385–5388

21 Spangenberg T, Aubry S, Kishi Y (2010) Synthesis and structure assignment of the minor metabolite arising from the frog pathogen Mycobacterium li fl andii Tetrahedron Lett 51: 1782–1785

22 Song F, Fidanze S, Benowitz AB, Kishi Y (2002) Total synthesis of the mycolactones Org Lett 4:647–650

23 Song F, Fidanze S, Benowitz AB, Kishi Y (2007) Total synthesis of mycolactones A and B Tetrahedron 63:5739–5753

24 Jackson KL, Li W, Chen C-L, Kishi Y (2010) Scalable and ef fi cient synthesis of the tone core Tetrahedron 66:2263–2272

25 Alexander MD, Fontaine SD, La Clair JJ, DiPasquale AG, Rheingold AL, Burkart MD (2006)

Synthesis of the mycolactone core by ring-closing metathesis Chem Commun : 4602–4604

26 Feyen F, Jantsch A, Altmann K-H (2007) Synthetic studies on mycolactones: synthesis of the mycolactone core structure through ring-closing ole fi n metathesis Synlett:415–418

27 van Summeren RP, Feringa BL, Minnaard AJ (2005) New approaches towards the synthesis of the side-chain of mycolactones A and B Org Biomol Chem 3:2524–2533

28 Yin N, Wang G, Qian M, Negishi E (2006) Stereoselective synthesis of the side chains of mycolactones A and B featuring stepwise double substitutions of 1,1-dibromo-1-alkenes Angew Chem Int Ed 45:2916–2920

29 Wang G, Yin N, Negishi E (2011) Highly stereoselective total synthesis of fully protected mycolactones A and B and their stereoisomerization upon deprotection Chem Eur J 17:4118–4130

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M Shibasaki et al (eds.), Chembiomolecular Science: At the Frontier

of Chemistry and Biology, DOI 10.1007/978-4-431-54038-0_2, © Springer Japan 2013

Introduction

In fl uenza viruses pose a serious threat to world public health In particular, the rently spreading avian H5N1 virus strain is a great menace because of its high lethality rate, and strains of this virus have already spread to many countries in Asia, Europe, and Africa There are now increasing concerns that this virus might acquire infectious ability among humans, leading to a worldwide pandemic Two of the drugs currently used to treat in fl uenza patients are Tami fl u [(−)-oseltamivir phos-phate; Fig 1 , 1 ] [ 1 ] and Relenza (zanamivir) [ 2] , both of which inhibit viral neuraminidase Tami fl u is an orally active prodrug, whereas Relenza has low bio-availability and is administered by inhalation Because neuraminidase is a funda-mental enzyme for the life cycle of general in fl uenza viruses, the neuraminidase inhibitors are effective against all in fl uenza virus types including H5N1

There are three current major concerns related to Tami fl u First, Tami fl u is duced and supplied by Roche using a natural product, (−)-shikimic acid, as the starting material [ 3, 4 ] Production of (−)-shikimic acid with consistent purity, how-ever, requires a long time and high cost In addition, dependence on a single syn-thetic route for the supply of such an important drug is unwise Therefore, alternative practical syntheses of Tami fl u starting from easily available starting materials are important for a stable supply of Tami fl u [ 5, 6 ] Second, in quite rare cases, abnormal behaviors (such as hallucinations and impulsive behavior) have been reported in Japanese patients (especially under the age of 20) after taking Tami fl u Molecular-level studies using appropriate biological tools are required to conclude whether there is any correlation between Tami fl u medication and the abnormal behaviors

M Kanai ( * )

Graduate School of Pharmaceutical Sciences, The University of Tokyo ,

7-3-1 Hongo, Bunkyo-ku , Tokyo 113-0033 , Japan

e-mail: kanai@mol.f.u-tokyo.ac.jp

Practical Synthesis of Tami fl u and Beyond

Motomu Kanai

Third, Tami fl u-resistant in fl uenza viruses are emerging and spreading widely In Japan, for example, nearly 100% of the 2008–2009 seasonal in fl uenza (H1N1) acquired resistance to the drug In addition, some of the highly virulent avian

in fl uenza H5N1 are now Tami fl u resistant [ 7 ] New drugs that are effective against Tami fl u-resistant in fl uenza viruses are in high demand

In this chapter, I review our endeavor to tackle those three concerns related to Tami fl u, based on the development of the original catalytic asymmetric synthesis

Catalytic Asymmetric Synthesis of Tami fl u

After establishing the fi rst- and second-generation synthesis of Tami fl u relying on

the catalytic desymmetrization of meso -aziridines with TMSN 3 [ 8, 9 ] , we developed

a signi fi cantly improved third-generation synthesis relying on a novel catalytic asymmetric Diels–Alder reaction [ 10 ] The synthetic scheme of the third-generation route is shown in Scheme 1 The catalytic asymmetric Diels–Alder reaction between

siloxy diene ( 2 ) and dimethyl fumarate ( 3 ) proceeded in the presence of a barium

complex of F 2 -FujiCAPO ( 5 ) (2.5 mol%) and cesium fl uoride cocatalyst, affording product 4 in 91% yield with a 5:1 diastereomeric ratio and 95% ee for the desired

a -hydroxy isomer The reaction was scalable, and up to 58 g-scale reaction was conducted without dif fi culty The reaction could be performed in the presence of

1 mol% catalyst with slightly decreased enantioselectivity (91% ee) Lewis

acid-catalyzed reactions between 2 and 3 , however, produced complex mixtures because

2 is labile under acidic conditions

The proposed catalytic cycle of the novel barium-catalyzed asymmetric Diels–Alder reaction is shown in Scheme 2 The active catalyst is a trinuclear barium

complex, 14 , and the reaction proceeds through catalytic activation of siloxy diene

2 via transmetalation to a chiral barium dienolate complex 15 The cocatalyst, cesium fl uoride, should generate pentavalent silicate 16 , which is active for the key

17 Practical Synthesis of Tami fl u and Beyond

Scheme 1 Catalytic asymmetric synthesis of Tami fl u

Scheme 2 Proposed catalytic cycle of asymmetric Diels–Alder reaction

yield, as summarized in Scheme 1 A mixture of diastereomers 4 was hydrolyzed to

afford corresponding carboxylic acids, which were treated with Shioiri reagent (diphenylphosphoryl azide, DPPA) to produce diastereomerically pure hydroxy dia-

cyl azide 6 Products derived from a minor b -isomer decomposed during these

transformations Curtius rearrangement and subsequent trap of the resulting C-4

and C-5 isocyanate groups by the C-2 hydroxy group and t -BuOH, respectively,

proceeded in one pot, and cyclic carbamate 7 was obtained after selective tion One recrystallization of 7 in 95% ee afforded enantiomerically pure 7 in high

acetyla-ef fi ciency

Regio- and stereoselective allylic substitution proceeded when cyclic carbamate

7 was heated with acyl anion equivalent 8 [ 11] in the presence of 2 mol%

Pd 2 (dba) 3 CHCl 3 and 4 mol% dppf, and product 9 was obtained in 95% yield Epoxidation of 9 with in situ-generated tri fl uoroperacetic acid afforded a -epoxide

10 as a sole product, possibly the result of the directing effect of the neighboring acetamide moiety at C-4 Treatment of 10 with K 2 CO 3 in EtOH revealed an ethoxy-

carbonyl group, and subsequent E 2 epoxide opening proceeded concomitantly to

produce a -allyl alcohol 11 in one pot Mitsunobu esteri fi cation of a -alcohol 11

with p -nitrobenzoic acid, and one-pot ethanolysis of the resulting ester, produced

C-3 b -alcohol 12 Mitsunobu aziridine synthesis from 12 was successfully

per-formed using Me 2 PPh and DIAD in the presence of 21 mol% of Et 3 N, producing

the key aziridine intermediate 13 in 76% yield The ring-opening reaction of 13

with 3-pentanol was performed using BF 3 ·OEt 2 , affording Boc-protected tamivir in 75% yield Cleavage of the Boc group with TFA and salt formation with

(−)-osel-phosphoric acid produced Tami fl u ( 1 ) in 73% yield We also succeeded in

develop-ing further improved route by usdevelop-ing only one Mitsunobu reaction at the late stage

biomole-appropriate linker (see 19 and 20 in Scheme 3 ) For synthesis of the designed logical tools, our synthetic route described in Scheme 1 is useful because we can introduce the linker and resin at a late stage of the synthesis using an aziridine

intermediate (related to 13 )

19 Practical Synthesis of Tami fl u and Beyond

Considering the functional group compatibility, we used aziridine 21 containing

an allyl ester as the key intermediate (Scheme 3 ) The reaction between 21 and linear azido alcohol 22 in the presence of BF 3 ·Et 2 O produced ether 24 in 78% yield;

25 with a branched linker was also synthesized using 23 following the same

proce-dure After protecting group shuf fl ing from N -Boc to N -F moc (from 24 and 25 to 26

and 27 , respectively), the allyl ester moiety was cleaved under palladium catalysis

to afford 28 and 29 in high yields Reduction of the azide in 28 and 29 in the

pres-ence of zinc powder preactivated with dibromoethane and TFA/EtOH solvent

afforded 30 and 31 in excellent yields Because 30 and 31 are water soluble and

highly polar, they were only partially puri fi ed by fi ltration through celite to nate the excess zinc and resulting zinc salts

The fi nal step of the synthesis was linking 30 and 31 to the chromatographic resin, Af fi -Gel 10 ( 32 ) The coupling reaction was performed under slightly basic

conditions (pH 8) in the presence of Et 3 N in MeOH at room temperature for 1 h After the coupling reaction, the resin was separated by fi ltration and washed with MeOH Finally, removal of the F moc group and blocking of the unreacted activated

ester on 32 were conducted simultaneously using excess piperidine in DMF

We con fi rmed that 19 and 20 indeed bind to in fl uenza neuraminidase We are

currently studying identi fi cation of human-derived proteins that can interact with those resins

Design and Synthesis of New Tami fl u Analogues

That May Be Effective for Resistant Viruses

According to the crystal structure of Tami fl u-resistant virus neuraminidase (H274Y) reported by Gamblin et al [ 13 ] , the mechanism for gaining resistance is as follows: substitution of histidine-274 by the bulkier tyrosine residue pushes the hydrophilic

Scheme 3 Synthesis of immobilized oseltamivir acid on resin

side chain of proximate glutamic acid-276 into the binding site of Tami fl u The charged carboxylic acid group disrupts the otherwise hydrophobic pocket that nor-mally accommodates the pentyloxy substituent of Tami fl u On the other hand, the structure of the H274Y–Relenza complex shows that the hydrophilic side chain of Relenza can interact with the pushed glutamic acid side chain Therefore, there is no erosion in activity of Relenza against Tami fl u-resistant viruses

Based on this structural information, we planned to generate Tami fl u–Relenza hybrid molecules to overcome the resistant viruses (Fig 2 ) We envisioned that Tami fl u derivatives having hydrophilic functionalities at the C-3 ether side chain to interact with pushed Glu 276 would remain active even against Tami fl u-resistant viruses By optimizing the physical properties of analogues, it might be also possi-ble to maintain the oral availability Our synthetic route shown in Scheme 1 is again useful for the synthesis of the Tami fl u–Relenza hybrid because various C-3 chains

can be introduced to key aziridine intermediate 13 at a later stage of the synthesis

We are currently working on such a research direction

Conclusion

Based on the development of the original catalytic asymmetric synthesis of the

anti-in fl uenza drug Tami fl u, we addressed three maanti-in problems related to Tami fl u: scale supply, origin of possible adverse effects, and resistance Synthesis-based biological studies will be more and more important and powerful in the medical science fi eld in future

Acknowledgments I thank Professor Masakatsu Shibasaki for his kind support and guidance of

this project I also thank Drs Kenzo Yamatsugu, Liang Yin, Shin Kamijo, Mr Yasuaki Kimura, and Kenta Saito of The University of Tokyo for their contribution Professor Takashi Kuzuhara and

Dr Noriko Echigo are acknowledged for collaboration This work was partly supported by the Uehara Memorial Foundation

Fig 2 Tami fl u–Relenza hybrid to overcome resistance while maintaining oral availability

21 Practical Synthesis of Tami fl u and Beyond

References

1 Kim CU, Lew W, Williams MA, Liu H, Zhang L, Swaminathan S, Bischofberger N, Chen MS, Mendel DB, Tai CY, Laver G, Stevens RC (1997) In fl uenza neuraminidase inhibitors possess- ing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-in fl uenza activity J Am Chem Soc 119:681–690

2 Itzstein MV, Wu WY, Kok GB, Pegg MS, Dyason JC, Jin B, Phan TV, Smythe ML, White HF, Oliver SW, Colman PM, Varghese JN, Ryan DM, Woods JM, Bethell RC, Hotham VJ, Cameron JM, Penn CR (1993) Rational design of potent sialidase-based inhibitors of in fl uenza virus replication Nature (Lond) 363:418–423

3 Abrecht S, Harrington P, Iding H, Karpf M, Trussardi R, Wirz B, Zutter U (2004) The synthetic development of the anti-in fl uenza neuraminidase inhibitor oseltamivir phosphate (Tami fl u ® ): a challenge for synthesis & process research Chimia 58:621–629

4 Abrecht S, Federspiel MC, Estermann H, Fisher R, Karpf M, Mair HJ, Oberhauser T, Rimmler

G, Trussardi R, Zutter U (2007) The synthetic-technical development of oseltamivir phosphate Tami fl u™: a race against time Chimia 61:93–99

5 Shibasaki M, Kanai M (2008) Synthetic strategies for oseltamivir phosphate Eur J Org Chem 2008:1839–1850

6 Shibasaki M, Kanai M, Yamatsugu K (2011) Recent development in synthetic strategies for oseltamivir phosphate Isr J Chem 51:316–328

7 Le QM, Kiso M, Someya K, Sakai YT, Nguyen TH, Nguyen KH, Pham ND, Ngyen HH, Yamada S, Muramoto Y, Horimoto T, Takada A, Goto H, Suzuki T, Suzuki Y, Kawaoka Y (2005) Avian fl u: isolation of drug-resistant H5N1 virus Nature (Lond) 437:1108

8 Fukuta Y, Mita T, Fukuda N, Kanai M, Shibasaki M (2006) De novo synthesis of Tami fl u via

a catalytic asymmetric ring-opening of meso -aziridines with TMSN 3 J Am Chem Soc 128:6312–6313

9 Mita T, Fukuda N, Roca FX, Kanai M, Shibasaki M (2007) Second generation catalytic metric synthesis of Tami fl u: allylic substitution route Org Lett 9:259–262

10 Yamatsugu Y, Yin L, Kamijo S, Kimura Y, Kanai M, Shibasaki M (2009) A synthesis of Tami fl u by using a barium-catalyzed asymmetric Diels–Alder-type reaction Angew Chem Int

13 Collins PJ, Haire LF, Lin YP, Liu J, Russell RJ, Walker PA, Skehel JJ, Martin SR, Hay AJ, Gamblin SJ (2008) Crystal structures of oseltamivir-resistant in fl uenza virus neuraminidase mutants Nature (Lond) 453:1258–1262

M Shibasaki et al (eds.), Chembiomolecular Science: At the Frontier

of Chemistry and Biology, DOI 10.1007/978-4-431-54038-0_3, © Springer Japan 2013

Introduction

Maitotoxin (MTX) is the principal toxin of ciguatera, a common form of seafood poisoning caused by consumption of fi sh from subtropical and tropical regions that

carry the epiphytic dino fl agellate Gambierdiscus toxicus Ciguatera is characterized

by gastrointestinal, cardiovascular, and neurological disorders, and affects more

than 50,000 people annually MTX is produced by G toxicus living on macroalgae,

and the toxin accumulates in fi sh as a result of transfer through the food chain [ 1 ] Although limited supplies of MTX from natural sources have hampered efforts to determine its molecular structure, the Yasumoto and Murata group ultimately eluci-dated the structure through extensive nuclear magnetic resonance (NMR) analysis [ 2, 3 ] , and the complete stereochemistry of MTX was determined by the Kishi and Tachibana groups (Fig 1 ) [ 4– 7 ]

MTX is one of the largest secondary metabolites described to date (MW 3422) and is related to the so-called ladder-shaped polyethers (LSPs), which include ciguatoxin (CTX) and brevetoxin B (BTXB) The large MTX molecule is composed

of 32 cyclic ethers containing 98 stereogenic centers (Fig 1 ) and can be divided into either hydrophobic (upper part) or hydrophilic (lower part) regions, depending upon

T Oishi ( * ) • K Torikai

Department of Chemistry, Faculty and Graduate School of Sciences, Kyushu University ,

6-10-1 Hakozaki, Higashi-ku , Fukuoka 812-8581 , Japan

e-mail: oishi@chem.kyushu-univ.jp

K Konoki

Graduate School of Agricultural Science, Tohoku University , 1-1 Tsutsumidori Amamiyamachi, Aoba-ku , Sendai 981-8555 , Japan

R Tamate • F Hasegawa • T Nakashima • N Matsumori • M Murata

Department of Chemistry , Graduate School of Science, Osaka University ,

1-1 Machikaneyama , Toyonaka , Osaka 560-0043 , Japan

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

the distribution of hydroxy groups and sulfate esters MTX is one of the most toxic compounds known in mammals, with an LD 50 in mice of 50 ng/kg (i.p.) [ 2, 3 ] (for a review, see [ ] ) In addition, MTX elicits remarkable biological activities at extremely low concentrations; for instance, MTX causes hemolysis of red blood cells at 15 nM [ 9 ] The most striking biological activity associated with MTX is its ability to cause a profound in fl ux of Ca 2+ into cells at concentrations as low as 0.3 nM, a phenomenon that has been demonstrated in all cell types examined to date, including rat glioma C6 cells [ 10 ] Because of its capacity to induce Ca 2+

in fl ux, MTX has been used as a reagent for physiological studies

Despite the large number of pharmacological and biophysical investigations that have focused on MTX, its precise mode of action at the molecular level has not been elucidated In the 1980s, MTX was thought to be a speci fi c activator of voltage-gated

Ca 2+ channels [ 11 ] , but it was subsequently suggested to activate Ca 2+ -permeable nonselective cation channels [ 12– 14 ] Recently, it was reported that a plasmalemmal

Ca 2+ -ATPase is one of the target proteins of MTX, and that the Ca 2+ pump is verted to a Ca 2+ -permeable cation channel by the action of MTX [ 15 ] However, attempts to identify the target proteins using molecular probes derived from the natu-ral product, such as tritium- or photoaf fi nity-labeled probes, have been hampered, primarily by nonspeci fi c binding brought on by the molecule’s large structure, as well as the limited availability of MTX from natural sources [ 16 ] Among the family

con-of LSPs, BTXB and CTX (Fig 1 ) are unusual in that their molecular targets have been identi fi ed These toxins share a common binding site, the so-called site 5 on an

a -subunit of voltage-sensitive Na + channels composed of a number of

transmem-brane a -helices, to which they bind with very high af fi nity ( K D = 1.6 nM) [ 17 ] On the other hand, it has been reported that MTX-induced Ca 2+ in fl ux in rat glioma C6 cells

is inhibited by BTXB, with an EC 50 estimated at 13 m M [ 10 ]

Our strategy for exploring the target proteins of MTX is shown in Fig 2 Hypothetically, when MTX elicits a biological response, the hydrophobic portion

of the molecule would be inserted in the lipid membrane and bind to the target protein, because of its structural similarity with BTXB [ 10, 18 ] In the presence of other LSPs such as BTXB, biological responses induced by MTX diminish because

of competitive binding to the target Therefore, the partial structure corresponding

to the hydrophobic region would competitively bind to the target of MTX and result in inhibition of the biological activities elicited by MTX more potently than BTXB A photoaf fi nity probe with a low capacity for nonspeci fi c binding derived from the partial structure of MTX could thus be used as a tool for identifying target proteins

Design and Synthesis

We initially selected the W–C ¢ ring system of MTX because it is hydrophobic and the structure resembles the C–I ring system of BTXB (Fig 2 ) From the aspect of synthesis, constructing the WXYZ ring system in a convergent manner is a daunting

26 T Oishi et al.

challenge because of the presence of contiguous angular methyl groups on the Y and Z rings, although a linear synthesis of the WXYZA ¢ ring system has been reported [ 19 ] Our plan to synthesize the WXYZA ¢ B ¢ C ¢ ring system ( 1 ) of MTX is

shown in Scheme 1 During the course of our synthetic studies of LSPs, we oped a convergent method via a -cyano ethers [ 20 ] , which was effectively utilized for synthesizing not only naturally occurring LSPs [ 21 ] but also arti fi cial LSPs (vide infra) [ 22 ] We envisaged extensive utilization of the convergent method via a -cyano

devel-ethers to construct the heptacyclic ether ( 1 ), which was to be derived from the W ( 5 ), Z ( 4 ), and C ¢ ( 2 ) ring units through construction of the XY and the A ¢ B ¢ ring

systems However, it remained uncertain whether our method was applicable to the WXYZ ring system with its contiguous angular methyl groups

As shown in Scheme 2 , synthesis of the WXYZ ring system ( 3 ) started with coupling of the Z ring diol ( 4 ) and the W ring aldehyde ( 5 ) through acetal formation, followed by regioselective opening of the seven-membered ring acetal ( 6 ), and elimination of the resulting primary alcohol to form a -cyano ether ( 7 ) as an insepa- rable mixture of epimers Reduction of the nitrile ( 7 ) producing aldehyde, followed

by treatment with vinyllithium, yielded allylic alcohol ( 8 ) Ring-closing metathesis

of diene ( 8 ) by the action of the second-generation Grubbs catalyst proceeded smoothly, and subsequent oxidation of the alcohol gave the enone ( 9 ) Hydrogenation

Fig 2 Hypothetical scheme for the inhibition of MTX-induced biological activities by

ladder-shaped polyethers ( LSP )