Cutting-Edge Organic Synthesis and Chemical Biology of Bioactive Molecules: The Shape of Organic Synthesis to Come [1st ed.] 978-981-13-6243-9;978-981-13-6244-6

Table of contents :
Front Matter ....Pages i-xxv
Microbial Fraction Library: A Screening Source for Drug Discovery (Toshihiko Nogawa, Julius Adam V. Lopez, Hiroyuki Osada)....Pages 1-20
Efficient Total Synthesis of Ōmura Natural Products (Toshiaki Sunazuka, Tomoyasu Hirose, Satoshi Ōmura)....Pages 21-47
Enantioselective Total Synthesis of the Antitumor Polycyclic Natural Products FR182877 and Taxol (Masahisa Nakada)....Pages 49-73
Synthetic Approaches on the Pluramycin-Class Antibiotics (Yoshio Ando, Kei Kitamura, Takashi Matsumoto, Keisuke Suzuki)....Pages 75-100
Recent Progress on the Total Synthesis of Duocarmycins A and SA, Yatakemycin, and PDE-I and PDE-II (Juri Sakata, Hidetoshi Tokuyama)....Pages 101-124
Structure-Activity Relationship Studies of Maitotoxin Based on Chemical Synthesis (Tohru Oishi)....Pages 125-143
Substitution of Allylic Picolinates with Various Copper Reagents and Synthetic Applications (Yuichi Kobayashi, Miwa Shimoda)....Pages 145-169
Total Synthesis of Ingenol (Tyler F. Higgins, Jeffrey D. Winkler)....Pages 171-192
Strategies for the Synthesis of Anti-inflammatory Metabolites of Unsaturated Fatty Acids (Yuichi Kobayashi, Masao Morita)....Pages 193-231
Biosynthesis, Biological Functions, and Receptors of Leukotriene B4 and 12(S)-Hydroxyheptadecatrienoic Acid (Toshiaki Okuno, Takehiko Yokomizo)....Pages 233-246
Synthesis of Classical/Nonclassical Hybrid Cannabinoids and Related Compounds (Thanh C. Ho, Marcus A. Tius)....Pages 247-289
Exploring Bioactive Marine Natural Products and Identification of Their Molecular Targets (Masayoshi Arai)....Pages 291-303
Target Protein Chemical Modification (Hiroyuki Nakamura)....Pages 305-333
Target Identification of Bioactive Compounds by Photoaffinity Labeling Using Diazido Probes (Suguru Yoshida, Takamitsu Hosoya)....Pages 335-355

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Yuichi Kobayashi Editor

Cutting-Edge Organic Synthesis and Chemical Biology of Bioactive Molecules The Shape of Organic Synthesis to Come

Cutting-Edge Organic Synthesis and Chemical Biology of Bioactive Molecules

Yuichi Kobayashi Editor

Cutting-Edge Organic Synthesis and Chemical Biology of Bioactive Molecules The Shape of Organic Synthesis to Come

Editor Yuichi Kobayashi Department of Biotechnology Tokyo Institute of Technology Yokohama, Japan

ISBN 978-981-13-6243-9    ISBN 978-981-13-6244-6 (eBook) https://doi.org/10.1007/978-981-13-6244-6 © Springer Nature Singapore Pte Ltd. 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

The purpose of organic synthesis is to provide target compounds by selective methods with high optical purity. Many transition metal-catalyzed asymmetric reactions and reactions catalyzed by organocatalysts have been developed to efficiently reach the goals. Reducing and oxidizing reagents that are highly chemoselective and/or environmentally friendly are also indispensable tools toward the purpose. On the other hand, analytical techniques have advanced to allow swift determination of the efficiency of reactions and the structures. For example, LC-MS analysis equipped with a chiral column could determine the chirality of the structure. Such new reactions and the advances in analytical techniques produced new power for organic synthesis to foray into fields, which used to be considered out of organic synthesis. As a consequence, many interdisciplinary areas have emerged, attracted attention of chemists, and rapidly grown. This book was planned to grasp the latest and excellent achievements in organic synthesis, which are expected to be grown more in the future. On the other hand, the background of each chapter is explained. I hope that the chapters will be understood easily by researchers, especially young, and stimulate research in the future. Chapters “Microbial Fraction Library: A Screening Source for Drug Discovery”, “Efficient Total Synthesis of Ōmura Natural Products”, “Enantioselective Total Synthesis of the Antitumor Polycyclic Natural Products FR182877 and Taxol”, “Synthetic Approaches on the Pluramycin-Class Antibiotics”, “Recent Progress Toward the Total Synthesis of Duocarmycins A and SA, Yatakemycin, and PDE-I and –II”, “Structure-Activity Relationship Studies of Maitotoxin Based on Chemical Synthesis”, “Substitution of Allylic Picolinates with Various Copper Reagents and Synthetic Applications”, “Total Synthesis of Ingenol” present isolation, elucidation, and organic syntheses of several compounds that are naturally occurring. Organic synthesis and biosynthesis of metabolites of polyunsaturated fatty acids are described in chapters “Strategies for the Synthesis of Anti-inflammatory Metabolites of Unsaturated Fatty Acids” and “Biosynthesis, Biological Functions, and Receptors of Leukotriene B4 and 12(S)-Hydroxyheptadecatrienoic Acid”, respectively. Chapter “Synthesis of Classical/Nonclassical Hybrid Cannabinoids and Related Compounds” deals with natural and unnatural cannabinoids and would be helpful to design new v

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Preface

medical cannabinoids. Chapters “Exploring Bioactive Marine Natural Products and Identification of Their Molecular Targets”, “Target Protein Chemical Modification”, “Target Identification of Bioactive Compounds by Photoaffinity Labeling Using Diazido Probes” explain chemical biology focusing on tools that are designed based on organic reaction and/or interaction with target proteins. The project of publishing this book began with an invitation of Mr. S. Koizumi, Springer Japan. I sincerely acknowledge Ms. A.  Komada of Springer and Ms. M. Shimoda in our laboratory for checking the chapters carefully. Yokohama, Japan 

Yuichi Kobayashi

Contents

1 Microbial Fraction Library: A Screening Source for Drug Discovery��������������������������������������������������������������������������������������������������    1 Toshihiko Nogawa, Julius Adam V. Lopez, and Hiroyuki Osada 2 Efficient Total Synthesis of Ōmura Natural Products��������������������������   21 Toshiaki Sunazuka, Tomoyasu Hirose, and Satoshi Ōmura 3 Enantioselective Total Synthesis of the Antitumor Polycyclic Natural Products FR182877 and Taxol��������������������������������������������������   49 Masahisa Nakada 4 Synthetic Approaches on the Pluramycin-­Class Antibiotics����������������   75 Yoshio Ando, Kei Kitamura, Takashi Matsumoto, and Keisuke Suzuki 5 Recent Progress on the Total Synthesis of Duocarmycins A and SA, Yatakemycin, and PDE-I and PDE-II������������������������������������������������������������������������������������������������  101 Juri Sakata and Hidetoshi Tokuyama 6 Structure-Activity Relationship Studies of Maitotoxin Based on Chemical Synthesis ����������������������������������������������������������������������������  125 Tohru Oishi 7 Substitution of Allylic Picolinates with Various Copper Reagents and Synthetic Applications ����������������������������������������������������  145 Yuichi Kobayashi and Miwa Shimoda 8 Total Synthesis of Ingenol������������������������������������������������������������������������  171 Tyler F. Higgins and Jeffrey D. Winkler 9 Strategies for the Synthesis of Anti-­inflammatory Metabolites of Unsaturated Fatty Acids���������������������������������������������������������������������  193 Yuichi Kobayashi and Masao Morita

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Contents

10 Biosynthesis, Biological Functions, and Receptors of Leukotriene B4 and 12(S)-Hydroxyheptadecatrienoic Acid������������  233 Toshiaki Okuno and Takehiko Yokomizo 11 Synthesis of Classical/Nonclassical Hybrid Cannabinoids and Related Compounds ������������������������������������������������������������������������  247 Thanh C. Ho and Marcus A. Tius 12 Exploring Bioactive Marine Natural Products and Identification of Their Molecular Targets��������������������������������������  291 Masayoshi Arai 13 Target Protein Chemical Modification��������������������������������������������������  305 Hiroyuki Nakamura 14 Target Identification of Bioactive Compounds by Photoaffinity Labeling Using Diazido Probes����������������������������������  335 Suguru Yoshida and Takamitsu Hosoya

List of Figures

Fig. 1.1 Screening strategy of interesting secondary metabolites................. 2 Fig. 1.2 Morphological profiles of HeLa and srcts-NRK cancer cells treated with pyrrolizilactone.................................................... 5 Fig. 1.3 Concept of the fraction library......................................................... 7 Fig. 1.4 Culture condition and preparation of crude extracts and fractions....................................................................... 8 Fig. 1.5 A basic concept of 2D separation for the preparation of fractions....................................................................................... 9 Fig. 1.6 Mass distribution of fractions.......................................................... 9 Fig. 1.7 General activity profile of the fraction library evaluated at 100 μg/mL.................................................................................... 10 Fig. 1.8 NPPlots and unusual alignments...................................................... 11 Fig. 1.9 NPPlots and specific metabolite group of Streptomyces sp. RK88–1355...................................................................................... 11 Fig. 1.10 Structure and putative biosynthetic pathway of verticilactam......... 12 Fig. 1.11 Structures and putative biosynthetic pathway of spirotoamides...... 13 Fig. 1.12 Structures of octaminomycins A and B........................................... 14 Fig. 1.13 NPPlot of Streptomyces sp. RK88–1355 and new antimycin-related metabolites.......................................................... 15 Fig. 1.14 Structural diversity of the antimycin class of metabolites............... 16 Fig. 1.15 Variety of the functional group at C-3 position............................... 17 Fig. 2.1 Fig. 2.2 Fig. 2.3 Fig. 2.4 Fig. 2.5 Fig. 2.6 Fig. 2.7 Fig. 2.8 Fig. 2.9 Fig. 2.10

Our strategy...................................................................................... 22 Structures of novel Ōmura natural products.................................... 23 Structures of pyripyropenes............................................................. 24 Biosynthesis of pyripyropene A....................................................... 24 Structures of pyripyropene analogues.............................................. 26 Structure-activity relationships of pyripyropenes............................ 27 Structure of PP8201 (Afidopyropen)............................................... 27 Structures of arisugacins.................................................................. 28 Computer simulation of arisugacin A 8 docking with AChE........... 30 Structures of lactacystin and salinosporamide A............................. 31

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List of Figures

Fig. 2.11 Regulatory functions of the eukaryotic proteasome and target for lactacystin......................................................................... 31 Fig. 2.12 Structures of lactacystin analogues.................................................. 32 Fig. 2.13 Inhibitory mode of macrosphelide A on cell adhesion.................... 33 Fig. 2.14 Structures of macrosphelides A and B............................................. 34 Fig. 2.15 Structures of madindolines A 41 and B 42...................................... 36 Fig. 2.16 The mode of action of madindoline A............................................. 39 Fig. 2.17 Structures of neoxaline 59 and oxaline 60....................................... 40 Fig. 2.18 Collaboration between the natural products and the organic synthesis for drug discovery................................................ 44 Fig. 3.1 Structures of (−)-FR182877 (1) and (−)-FR182876........................ 50 Fig. 3.2 X-ray crystal structure of 29............................................................ 58 Fig. 3.3 Immunostaining images obtained using control (I), paclitaxel (10 nM) (II), and 31 (200 μM) (III) (left) and the mitotic indexes (right)................................................................ 59 Fig. 3.4 Structure of (−)-taxol....................................................................... 60 Fig. 4.1 Fig. 4.2 Fig. 4.3 Fig. 4.4

Pluramycins...................................................................................... 77 Hedamycin and conformation of α-C-glycosyl vancosamine.......... 78 Synthetic challenges and lability of the pluramycins...................... 78 Strategies for the skeletal construction of the pluramycin aglycon.......................................................................... 79 Fig. 4.5 The rare deoxyamino sugars on pluramycins.................................. 84 Fig. 4.6 Platforms for installing bis-C-glycoside.......................................... 90 Fig. 4.7 Further utilities of bis-C-glycosyl monocycles................................ 92 Fig. 5.1 Fig. 5.2 Fig. 5.3 Fig. 5.4

Structures of duocarmycins and the related compounds.................. 103 Proposed mechanism of DNA alkylation......................................... 103 Structures of PDE-I (5) and PDE-II (6)........................................... 104 Proposed structure of yatakemycin (40) by Igarashi and coworkers.................................................................... 109

Fig. 6.1 Fig. 6.2 Fig. 6.3 Fig. 6.4 Fig. 6.5

Structure of maitotoxin (MTX)........................................................ 126 Structure of brevetoxin B (BTXB)................................................... 126 Hypothesis of mode of action.......................................................... 127 Partial structures of MTX................................................................ 127 Structure of artificial ladder-shaped polyether ALP7B.................... 139

Fig. 7.1 Synthetic targets using the allylic substitution................................. 152 Fig. 7.2 Synthetic targets of the quaternary carbon-forming allylic substitution............................................................................ 159 Fig. 8.1 (a) Ingenol 1 alongside a 3-D model that highlights the highly contorted trans-­intrabridgehead stereochemistry, (b) trans- and cis-­intrabridgehead stereochemistry in bicyclo[4.4.1]undecanes, and (c) the Paquette ingenane analog with cis-intrabridgehead stereochemistry............................. 172

List of Figures

Fig. 8.2 Fig. 8.3 Fig. 8.4 Fig. 8.5 Fig. 8.6 Fig. 9.1 Fig. 9.2 Fig. 9.3 Fig. 9.4 Fig. 9.5 Fig. 9.6 Fig. 9.7 Fig. 9.8 Fig. 9.9 Fig. 9.10 Fig. 9.11

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(a) Taxol and (b) retrosynthetic approach to AB system of taxol........................................................................ 173 Taxol AB ring system retrosynthetic analysis via intramolecular dioxenone photocycloaddition-fragmentation...... 174 (a) Ingenol in which the tricyclic core is highlighted in blue and (b) retrosynthetic analysis for the synthesis of the tricyclic core 19............................................................................. 175 (a) Oxygen functionality and unsaturation previously installed. (b) Oxygen functionality and unsaturation yet to be installed................................................................................ 177 Models depicting rationale for stereochemistry of (a) 1,2-addition and (b) cyclization............................................... 185 ω-3 Fatty acids and their metabolites............................................ 194 Metabolites of arachidonic acid.................................................... 195 The substructure of the metabolites in this section....................... 198 RvE1 analogues synthesized by Kobayashi/Ogawa...................... 203 Structural difference of the aspirin-triggered (AT) and endogenous metabolites.......................................................... 203 The substructure of the metabolites in this section....................... 211 Naturally occurring RvE3............................................................. 213 The substructure of the metabolites in this section....................... 216 The substructure of the metabolites in this section....................... 217 Structural similarity between 5,18-DiHETE and resolvin E2....... 226 Purification of compounds by using a recycle HPLC: YMC, LC-forte/R, normal phase (silica gel) column with eluents specified.................................................................... 228

Fig. 10.1 Fig. 10.2 Fig. 10.3 Fig. 10.4 Fig. 10.5

Biosynthesis pathways of leukotriene B4 (LTB4) and LTC4 ......... 235 LTB4 inactivation pathways........................................................... 236 Structure of LTB4 and related fatty acids...................................... 238 PGH2 biosynthesis pathway.......................................................... 240 Biosynthesis and metabolism pathway of 12-HHT and TxA2 .................................................................... 242

Fig. 11.1

Structural classification of cannabinoids with representative compounds............................................................. 249 Three pharmacophores and retrosynthetic analysis of classical tricyclic cannabinoids................................................. 250 By-products detected in the condensation of olivetol with (+)-p-mentha-2,8-dien-1-ol........................................................... 252 Structures of optically active monoterpenes commonly  used in cannabinoid synthesis....................................................... 253

Fig. 11.2 Fig. 11.3 Fig. 11.4

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Fig. 11.5 Fig. 11.6 Fig. 11.7 Fig. 12.1 Fig. 12.2 Fig. 12.3 Fig. 12.4 Fig. 12.5 Fig. 12.6 Fig. 12.7 Fig. 13.1

List of Figures

Examples of a classical cannabinoid, a nonclassical cannabinoid, and a hybrid cannabinoid......................................... 263 Structure of some fused multicyclic cannabinoids........................ 272 Structure of some heterocyclic cannabinoids and a phenanthrene-derived cannabinoid............................................. 277 Chemical structures of previously discovered bioactive marine natural products................................................. 294 Inhibition of protein complex formation by furospinosulin-1 (A) and structure of nucleotide probe (B)..................................... 296 Chemical structures of furospinosulin-1 probe and dummy probe.......................................................................... 297 Expected mechanism of action of furospinosulin-1...................... 298 Summary of the phage display method......................................... 299 Chemical structure (A) and activity (B) of the dictyoceratin probes...................................................................... 300 Strategy of target identification using M. smegmatis transformed with genomic DNA library....................................... 302

(a) Pre-translational protein modifications that require genetic manipulation and (b) posttranslational protein modification with small chemicals................................................ 306 Fig. 13.2 The photoaffinity functional groups and their reactive intermediates.................................................................... 308 Fig. 13.3 A series of α-D-mannoside photoaffinity probes conjugated with an azide (1), a diazirine (2), and a benzophenone (3)........... 309 Fig. 13.4 Typical chemical modification reactions of proteins at lysine residues........................................................................... 310 Fig. 13.5 Typical chemical modification reactions of proteins at cysteine residues........................................................................ 311 Fig. 13.6 Typical chemical modification reactions of proteins at tyrosine residues........................................................................ 312 Fig. 13.7 Other amino acid residue-specific chemical modifications........... 317 Fig. 13.8 Typical trifunctional chemical probes for target identification........................................................................ 318 Fig. 13.9 The photoaffinity labeling/photo-cross-linking process from the non-covalent binding to the detection and identification of the tagged proteins.................................................................... 318 Fig. 13.10 Post-photoaffinity labeling modification of a saccharide-binding protein, concanavalin A (ConA).................... 319 Fig. 13.11 The affinity labeling modification followed by the hydrazone/oxime exchange reaction............................................. 320 Fig. 13.12 Schematic illustration of LDT chemistry for labeling endogenous proteins in living native cells..................................... 321

List of Figures

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Fig. 13.13 Schematic illustration of the strategy for the quenched ligand-directed tosylate (Q-LDT)-mediated construction of turn-on fluorescent biosensors....................................................... 321 Fig. 13.14 A fluorescent dye transfer strategy for identifying the target protein of marinopyrrole A.................................................. 322 Fig. 13.15 Schematic illustration of ligand-directed acyl imidazole (LDAI) chemistry for selective protein modification. Lg ligand, Nu nucleophilic amino acid.......................................... 322 Fig. 13.16 Schematic of “turn-on” fluorescent labeling of a target protein by the O-NBD method. A small bifunctional O-NBD unit is contained in the ligand. Lg ligand............................................. 323 Fig. 13.17 Ligand-tethered-DMAP-catalyzed acyl transfer reaction for lectin.......................................................................... 324 Fig. 13.18 Acyl transfer reaction for lectin catalyzed by DMAP................... 324 Fig. 13.19 Catalytic covalent modification of a specific side chain of a substrate peptide using coiled-coil assembly to drive localization of a dirhodium metallopeptide. Label subscripts represent the position, on a helical-wheel model, of key residues that deviate from the parent E3 or K3 sequences................................. 325 Fig. 13.20 Covalent modifications of various E3gX peptides with the styryl-diazo reagent 8 by dirhodium metallopeptide catalysts (K3a,eRh2)...................................................................... 326 Fig. 13.21 LDRP system for target-selective (A) modification and (B) oxidative inactivation.............................................................. 327 Fig. 13.22 EGFR knockdown using Ru-gefitinib in A431 cells..................... 327 Fig. 13.23 Structures of [Ru(bpy)3]2+-conjugated with the GU40C peptoid (RuGU40C) and RIP1 (RuRIP1) for CALI targeting to VEGF and the 26S proteasome, respectively............................ 328 Fig. 13.24 Selective isolation and modification of target proteins................. 330 Fig. 14.1 Fig. 14.2 Fig. 14.3 Fig. 14.4 Fig. 14.5 Fig. 14.6 Fig. 14.7 Fig. 14.8 Fig. 14.9 Fig. 14.10

Chemical methods for target identification................................... 337 Typical photoreactive groups used for PAL probes....................... 338 Typical detectable groups used for PAL probes............................ 339 Diazido probe method................................................................... 340 Diazido PAL probes developed by other groups........................... 341 Photoreaction of an equimolar mixture of azides 1a and 1b......... 342 Cerivastatin (4) and diazido probe photovastatin CAA1 (5)......... 343 1BnTIQ (10) and diazido probe 11............................................... 344 Dantrolene (18) and PAL probes 19 and 20.................................. 346 Synthesis of biaryl-type diazido compounds using borylated building block 21........................................................... 347 Fig. 14.11 Synthesis of aryl azides 24 by the formal C–H azidation............. 348 Fig. 14.12 Diazido building blocks prepared from diazido compounds 14, 27, and 28............................................................. 350

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List of Figures

Fig. 14.13 Other bifunctional PAL probe candidates of dantrolene............... 351 Fig. 14.14 Bifunctional PAL probe and unit bearing (ethynyldifluoromethyl)diazirinyl group....................................... 352 Fig. 14.15 Bifunctional PAL probe library..................................................... 353 Fig. 14.16 Bifunctional PAL probe 53 of palmitic acid (54).......................... 353 Fig. 14.17 Minimalist photo-crosslinkers....................................................... 354 Fig. 14.18 Minimal all-in-one PAL unit......................................................... 354 Fig. 14.19 Commercially available building blocks for bifunctional PAL probe synthesis...................................................................... 354

List of Schemes

Scheme 2.1 Retrosynthetic analysis of pyripyropene A�������������������������������   25 Scheme 2.2 Total synthesis of pyripyropene A��������������������������������������������   26 Scheme 2.3 Total synthesis of arisugacin A (part 1)������������������������������������   28 Scheme 2.4 Total synthesis of arisugacin A (part 2)������������������������������������   29 Scheme 2.5 Total synthesis of lactacystin����������������������������������������������������   32 Scheme 2.6 Total synthesis of macrosphelide A������������������������������������������   35 Scheme 2.7 Strategy for a combinatorial synthesis of macrosphelide analogues����������������������������������������������������������������������������������   35 Scheme 2.8 Asymmetric oxidative ring closure of tryptophol���������������������   36 Scheme 2.9 First-generation strategy for total synthesis of madindolines�����������������������������������������������������������������������������   37 Scheme 2.10 Second-generation strategy for total synthesis of madindolines�����������������������������������������������������������������������������   38 Scheme 2.11 The synthesis of the indoline spiroaminal framework of neoxaline (part 1)������������������������������������������������������������������   41 Scheme 2.12 The synthesis of the indoline spiroaminal framework of neoxaline (part 2)������������������������������������������������������������������   41 Scheme 2.13 Synthesis of cyclization precursor��������������������������������������������   42 Scheme 2.14 Construction of indoline spiroaminal���������������������������������������   43 Scheme 2.15 Total synthesis of neoxaline������������������������������������������������������   44 Scheme 3.1 Scheme 3.2 Scheme 3.3 Scheme 3.4 Scheme 3.5 Scheme 3.6 Scheme 3.7

Sorensen’s and Evans’ total syntheses of (−)FR182877 (1)����������������������������������������������������������������������������   51 Construction of the AB-ring moiety by the stereoselective IMDA reaction��������������������������������������������������   51 Construction of the CD-ring moiety by the stereoselective IMHDA reaction�����������������������������������������������   52 Transformation of 2 to 7 via the stereoselective IMDA-IMHDA reaction cascade����������������������������������������������   52 Stereoselective reduction of 7 (Table 3.1)��������������������������������   53 Preparation of 10�����������������������������������������������������������������������   53 The palladium-catalyzed 7-exo-trig cyclization of 10a and 10b (Table 3.2)��������������������������������������������������������   54 xv

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Scheme 3.8 Scheme 3.9 Scheme 3.10 Scheme 3.11 Scheme 3.12 Scheme 3.13 Scheme 3.14 Scheme 3.15 Scheme 3.16 Scheme 3.17 Scheme 3.18 Scheme 3.19 Scheme 3.20 Scheme 3.21 Scheme 3.22 Scheme 3.23 Scheme 3.24 Scheme 3.25 Scheme 3.26 Scheme 3.27 Scheme 3.28 Scheme 3.29 Scheme 4.1 Scheme 4.2 Scheme 4.3 Scheme 4.4 Scheme 4.5 Scheme 4.6 Scheme 4.7

List of Schemes

Possible pathway from 11 to 13a and energetic difference between 13a and 13b calculated by the PM3 method��������������   55 Isomerization of allylic alcohol 11 to α-methyl ketone 13a (Table 3.3)���������������������������������������������������������������������������������   55 Enantioselective total synthesis of (−)-FR182877��������������������   56 Structures of (−)-FR182877 (1) and 17, and retrosynthetic analysis of 17����������������������������������������������������������������������������   57 Preparation of 19�����������������������������������������������������������������������   57 Preparation of 18�����������������������������������������������������������������������   58 Preparation of 17 and 29–32�����������������������������������������������������   58 Formation of the eight-membered ring of taxol in the convergent synthesis�����������������������������������������������������������������   61 Intramolecular B-alkyl Suzuki-Miyaura coupling reactions of 33 and 34���������������������������������������������������������������   62 Retrosynthetic analysis of (−)-taxol�����������������������������������������   63 Preparation of the A-ring fragment 41-I from 43���������������������   63 Enantioselective preparation of 47-I from 46-I via organocatalysis��������������������������������������������������������������������������   63 The SAD of 49-I, 50-I, and 50-Br�������������������������������������������   64 Transformation of 51-Br to 41-Br via 52���������������������������������   65 Attempted aldol reaction and Nozaki-Hiyama-type reaction and envisioned reliable stereoselective coupling of 41 and 54 to afford 55����������������������������������������������������������   65 Preparation of 54 via the baker’s yeast reduction of 56������������   65 Assembly of the A- and C-ring fragments and the intramolecular B-alkyl Suzuki-­Miyaura coupling reaction of 60����������������������������������������������������������������������������   66 Formation of 63 by palladium-catalyzed alkenylation of methyl ketone 62������������������������������������������������������������������   67 Preparation of methyl ketone 68 via 1,5-hydride shift�������������   68 Formal total synthesis of (−)-taxol via the palladium-catalyzed alkenylation of 68������������������������   68 Kuwajima successful epimerization of 75 and our failed example���������������������������������������������������������������������������   69 Transformation of 73 to Nicolaou’s advanced synthetic intermediate 79�������������������������������������������������������������������������   70 Synthesis of O-methyl kidamycinone���������������������������������������   80 Total synthesis of γ-indomycinone�������������������������������������������   80 Total synthesis of (S)-espicufolin���������������������������������������������   80 Friedel–Crafts cyclization in the synthetic study of kidamycin���������������������������������������������������������������������������������   81 Total synthesis of γ-indomycinone�������������������������������������������   82 Total synthesis of AH-1763 IIa�������������������������������������������������   82 Total synthesis of BE-26554A and heraclemycin B�����������������   82

List of Schemes

xvii

Scheme 4.8 Scheme 4.9 Scheme 4.10 Scheme 4.11 Scheme 4.12 Scheme 4.13 Scheme 4.14 Scheme 4.15 Scheme 4.16 Scheme 4.17 Scheme 4.18 Scheme 4.19 Scheme 4.20 Scheme 4.21 Scheme 4.22 Scheme 4.23 Scheme 4.24

Total synthesis of (R)-espicufolin���������������������������������������������   83 Synthesis of pluraflavin A aglycon�������������������������������������������   84 Total synthesis of γ-indomycinone�������������������������������������������   84 Baer and Georges synthesis������������������������������������������������������   85 Brimble synthesis����������������������������������������������������������������������   85 McDonald synthesis������������������������������������������������������������������   86 Suzuki synthesis������������������������������������������������������������������������   86 Kahne degradation��������������������������������������������������������������������   87 Giuliano synthesis���������������������������������������������������������������������   87 Nicolaou synthesis��������������������������������������������������������������������   88 McDonald synthesis������������������������������������������������������������������   89 Parker synthesis������������������������������������������������������������������������   89 Doi and Takahashi synthesis�����������������������������������������������������   89 Suzuki synthesis������������������������������������������������������������������������   90 O→C-Glycoside rearrangement�����������������������������������������������   91 Bis-C-glycosidation of monocyclic platforms��������������������������   92 Reverse polarity strategy for construction of bis-C-glycoside���������������������������������������������������������������������   93 Synthetic study of kidamycin���������������������������������������������������   94 Synthetic study of pluraflavin A�����������������������������������������������   94 Total synthesis of isokidamycin������������������������������������������������   95 Total synthesis of isokidamycin������������������������������������������������   96 Total synthesis of saptomycin B�����������������������������������������������   98

Scheme 4.25 Scheme 4.26 Scheme 4.27 Scheme 4.28 Scheme 4.29 Scheme 5.1 Scheme 5.2 Scheme 5.3 Scheme 5.4 Scheme 5.5 Scheme 5.6 Scheme 5.7 Scheme 5.8 Scheme 5.9 Scheme 5.10 Scheme 5.11 Scheme 5.12 Scheme 5.13 Scheme 5.14 Scheme 5.15 Scheme 5.16 Scheme 5.17 Scheme 5.18 Scheme 5.19

Copper-­mediated intramolecular aryl amination����������������������  105 Synthesis of indoline 13������������������������������������������������������������  105 Preparation of left-hand segment 18�����������������������������������������  106 Terashima’s synthesis of right-hand segment 22����������������������  106 Preparation of right-hand segment 28���������������������������������������  107 Fukuyama’s total synthesis of (+)-duocarmycin A (1)�������������  107 Fukuyama’s total synthesis of (+)-duocarmycin SA (2)�����������  108 Synthesis of the left-hand segment of the putative yatakemycin (40)����������������������������������������������������������������������  110 Boger’s synthesis of proposed yatakemycin (40)���������������������  111 Initial structural revision of yatakemycin���������������������������������  111 Boger’s synthesis of revised left-hand segment 60�������������������  112 The second structural revision of yatakemycin������������������������  112 Boger’s total synthesis of (+)-yatakemycin (4)������������������������  113 Synthesis of the middle segment 73�����������������������������������������  114 Synthesis of left-hand segment 60��������������������������������������������  114 Boger’s second-generation total synthesis of (+)-yatakemycin (4)������������������������������������������������������������������  115 Synthesis of middle segment 87�����������������������������������������������  116 Synthesis of amino alcohol 93��������������������������������������������������  116 Synthesis of left-hand segment 100������������������������������������������  117

xviii

List of Schemes

Scheme 5.20 Synthesis of right-hand segment 105����������������������������������������  118 Scheme 5.21 Fukuyama’s total synthesis of (+)-yatakemycin (4)�����������������  118 Scheme 5.22 Tokuyama’s synthetic strategy based on their copper-mediated double amination�������������������������������������������  119 Scheme 5.23 Preparation of the substrate for the double amination reaction��������������������������������������������������������������������  120 Scheme 5.24 Result of the initial trial of the double arylamination����������������������������������������������������������������������������  120 Scheme 5.25 Proposed reaction pathway�������������������������������������������������������  121 Scheme 5.26 Tokuyama’s first-generation synthesis of PDE-II (6)���������������������������������������������������������������������������������  121 Scheme 5.27 Tokuyama’s synthesis of PDE-I (5)������������������������������������������  122 Scheme 5.28 Tokuyama’s second-generation synthesis of PDE-II (6)���������������������������������������������������������������������������������  122 Scheme 6.1

Convergent method via two-rings construction (1): α-cyano ether method���������������������������������������������������������������  128 Scheme 6.2 Convergent method via two-rings construction (2)������������������  129 Scheme 6.3 Synthetic methods of 6/6/6-tricyclic ether systems based on Achmatowicz reaction�����������������������������������������������  130 Scheme 6.4 Convergent synthesis of the WXYZ ring (1)����������������������������  130 Scheme 6.5 Convergent synthesis of the WXYZ ring (2)����������������������������  131 Scheme 6.6 Convergent synthesis of the WXYZA′B′C′ ring�����������������������  133 Scheme 6.7 Synthesis of the QRS ring���������������������������������������������������������  134 Scheme 6.8 Synthesis of the C′D′E′F′ ring���������������������������������������������������  135 Scheme 6.9 Synthesis of the LMNO and ent-LMNO rings�������������������������  136 Scheme 6.10 Synthesis of the NOPQR(S) ring����������������������������������������������  138 Scheme 7.1 Scheme 7.2 Scheme 7.3 Scheme 7.4 Scheme 7.5 Scheme 7.6 Scheme 7.7 Scheme 7.8 Scheme 7.9 Scheme 7.10 Scheme 7.11 Scheme 7.12 Scheme 7.13

Allylic substitutions using prim- and sec-allylic esters������������  146 Allylic substitution of secondary allylic esters published by 2007���������������������������������������������������������������������  147 Picolinoxy (Pic) leaving group for allylic substitution�������������  148 A double activation of the Pic group����������������������������������������  149 Preliminary results using various secondary allylic esters with phenylmetal reagents: Table 7.1�����������������������������  149 Allylic substitution of enantioenriched picolinates with Grignard-based copper reagents���������������������������������������  150 Substitution with PhLi-based copper reagents: Table 7.2������������������������������������������������������������������������������������  150 Allylic substitution with RLi-based copper reagents����������������  151 1,4-Addition reactions of classical copper reagents to enone�������������������������������������������������������������������������������������  152 Synthesis of sesquichamaenol��������������������������������������������������  152 Synthesis of equol���������������������������������������������������������������������  153 Synthesis of the inhibitor of ACAT�������������������������������������������  154 Synthesis of the bioactive form of loxoprofen��������������������������  155

List of Schemes

xix

Scheme 7.14 Scheme 7.15 Scheme 7.16 Scheme 7.17

Synthesis of trans-2,6-disubstituted cyclohexanones���������������  156 Allylic substitution with alkynyl reagents��������������������������������  157 Synthesis of the Stork’s prostaglandin intermediate 61������������  158 Quaternary carbon-forming allylic substitution with Ph and n-Bu copper reagents: Table 7.3�����������������������������������  158 Formation of enantioenriched quaternary carbons with Ph copper reagents derived from PhMgBr or PhLi����������  159 Synthesis of mesembrine����������������������������������������������������������  160 Synthesis of the verapamil intermediate�����������������������������������  161 Synthesis of LY426965�������������������������������������������������������������  162 Construction of a quaternary carbon on a cyclohexane and possible targets����������������������������������������������  162 Construction of a quaternary carbon: Table 7.4������������������������  163 Conformation-controlled allylic substitution����������������������������  164 Synthesis of cyclobakuchiols A and B��������������������������������������  165 Synthesis of anastrephin�����������������������������������������������������������  166 Synthesis of axenol�������������������������������������������������������������������  167

Scheme 7.18 Scheme 7.19 Scheme 7.20 Scheme 7.21 Scheme 7.22 Scheme 7.23 Scheme 7.24 Scheme 7.25 Scheme 7.26 Scheme 7.27 Scheme 8.1 Scheme 8.2 Scheme 8.3 Scheme 8.4 Scheme 8.5 Scheme 8.6 Scheme 8.7 Scheme 8.8 Scheme 8.9 Scheme 8.10 Scheme 8.11 Scheme 8.12 Scheme 8.13 Scheme 8.14 Scheme 8.15 Scheme 8.16 Scheme 8.17 Scheme 8.18

Baldwin’s dioxenone intermolecular photocycloaddition-fragmentation��������������������������������������������  173 Synthesis of trans-bicyclo[5.3.1]undecane�������������������������������  174 Synthesis of the tricyclic core of the ingenane ring system��������������������������������������������������������������������������������  175 Synthesis of 3-oxygenated ingenane diterpenes�����������������������  176 Synthesis of the first biologically active ingenane analog������������������������������������������������������������������������  177 Elaboration of the A and B rings of the ingenane skeleton������  178 Retrosynthetic analysis for D ring incorporation����������������������  179 Attempted elimination of the mesylate of the C-14 hydroxyl produced an unexpected transannular aldol reaction����������������������������������������������������������������������������  180 Incorporation of the D ring cyclopropane of ingenol���������������  181 First total synthesis of ingenol��������������������������������������������������  181 Completion of the first total synthesis of ingenol���������������������  182 Tanino and Kuwajima’s original approach to ingenane synthesis����������������������������������������������������������������  184 Synthesis of ingenane skeleton 79 via key Me3Al-mediated rearrangement������������������������������������������������  184 Functionalization of A and B rings of ingenol��������������������������  185 Completion of Tanino and Kuwajima’s ingenol synthesis�������  186 Ring-closing metathesis approach to the synthesis of ingenol����������������������������������������������������������������������������������  187 Improved ring-closing metathesis toward the synthesis of ingenol������������������������������������������������������������  187 Functionalization of the A ring of ingenol��������������������������������  188

xx

List of Schemes

Scheme 8.19 Scheme 8.20 Scheme 8.21 Scheme 8.22

Completion of Wood’s ingenol synthesis���������������������������������  189 Baran’s retrosynthetic analysis of ingenol��������������������������������  190 Cyclase phase for the Baran synthesis of ingenol��������������������  190 Oxidase phase and completion of Baran’s ingenol synthesis�����������������������������������������������������������������������  191

Scheme 9.1 Scheme 9.2 Scheme 9.3 Scheme 9.4 Scheme 9.5 Scheme 9.6 Scheme 9.7 Scheme 9.8 Scheme 9.9

Construction of conjugated olefins�������������������������������������������  196 Synthesis of E-iodo olefins�������������������������������������������������������  197 Synthesis of RvE1 methyl ester by Petasis/Serhan�������������������  199 Improved synthesis of RvE1 by Schwartz��������������������������������  199 Synthesis of key intermediates�������������������������������������������������  200 Synthesis of the three intermediates 36, 40, and 45�����������������  201 Synthesis of RvE1 by Kobayashi/Ogawa���������������������������������  202 Improved synthesis of RvD3 by Petasis/Serhan�����������������������  204 Synthesis of PD1 and (18R)-PD1 (AT-PD1) by Petasis/Serhan����������������������������������������������������������������������  205 Preparation of intermediates 60 and 61������������������������������������  205 Synthesis of PD1 by Kobayashi/Ogawa�����������������������������������  206 Preparation of intermediates 70 and 72������������������������������������  206 Synthesis of the PD1 intermediates by Spur�����������������������������  207 Synthesis of PD1 by Hansen�����������������������������������������������������  207 Synthesis of 10-epi-PD1 methyl ester by Balas�����������������������  208 Synthesis of MaR1 and (7S)-isomer by Inoue/Arita using Julia-Kocienski coupling�������������������������������������������������  209 Synthesis of MaR1 by Kobayashi/Ogawa via Suzuki-Miyaura coupling���������������������������������������������������  210 Synthesis of MaR1 by Spur via Sonogashira coupling������������  210 Synthesis of MaR1 by Hansen via Sonogashira coupling��������  211 Three approaches to RvD4�������������������������������������������������������  212 Synthesis of RvD4 intermediates 127 and 129�������������������������  212 Synthesis of RvD4 by Kobayashi/Morita���������������������������������  213 Kinetic stability of a dihydroxy acid����������������������������������������  213 Synthesis of RvE3 by Inoue/Arita��������������������������������������������  214 Synthesis of RvE3 by Kobayashi����������������������������������������������  215 Synthesis of MaR2 by Spur������������������������������������������������������  215 Synthesis of RvD1 and RvD2 by Spur�������������������������������������  216 Synthesis of RvD2 by Rizzacasa����������������������������������������������  216 Synthesis of RvD1 by Kobayashi/Morita���������������������������������  217 Approaches to RvE2�����������������������������������������������������������������  218 Synthesis of the intermediates for synthesis of RvE2��������������  219 Synthesis of RvE2 by Inoue/Arita��������������������������������������������  219 Synthesis of RvE2 by Kobayashi/Ogawa���������������������������������  220 Ozonolysis of E- and Z-allylic alcohol derivatives (R = Et, i-Pr, C5H11)������������������������������������������������������������������  220 Synthesis of (14R,15S)-diHETE by Kobayashi������������������������  221

Scheme 9.10 Scheme 9.11 Scheme 9.12 Scheme 9.13 Scheme 9.14 Scheme 9.15 Scheme 9.16 Scheme 9.17 Scheme 9.18 Scheme 9.19 Scheme 9.20 Scheme 9.21 Scheme 9.22 Scheme 9.23 Scheme 9.24 Scheme 9.25 Scheme 9.26 Scheme 9.27 Scheme 9.28 Scheme 9.29 Scheme 9.30 Scheme 9.31 Scheme 9.32 Scheme 9.33 Scheme 9.34 Scheme 9.35

List of Schemes

xxi

Scheme 9.36 Scheme 9.37 Scheme 9.38 Scheme 9.39 Scheme 9.40 Scheme 9.41 Scheme 9.42 Scheme 9.43

Synthesis of RvE2 by Spur�������������������������������������������������������  221 Synthesis of RvE2 by Shuto�����������������������������������������������������  221 Synthesis of RvD6 by Spur�������������������������������������������������������  222 Approaches to RvD5�����������������������������������������������������������������  222 Synthesis of RvD5 by Spur�������������������������������������������������������  223 Synthesis of RvD5 by Kobayashi/Ogawa/Morita���������������������  223 Synthesis of (14S,20R)-dihydroxy-DHA by Inoue/Arita���������  224 Synthesis of (17R,18S)-epoxy-(12S)-hydroxy-EPA by Inoue/Arita���������������������������������������������������������������������������  224 Synthesis of (14S,21R)-DiHDHA by Kobayashi/ Morita/Hong�����������������������������������������������������������������������������  225 Synthesis of Maresin-like 1 by Kobayashi et al.����������������������  226 Retrosynthesis of (5S,18R)-DiHETE����������������������������������������  226 Synthesis of HHTE and 12S-HHT��������������������������������������������  227

Scheme 9.44 Scheme 9.45 Scheme 9.46 Scheme 9.47

Scheme 11.1 Synthesis of (−)-Δ8- and (−)-Δ9-THCs from (−)-verbenol���������������������������������������������������������������������  251 Scheme 11.2 Synthesis of (−)-Δ8- and (−)-Δ9-THCs from (+)-p-mentha-2,8-dien-1-ol����������������������������������������������  251 Scheme 11.3 Synthesis of AM-411����������������������������������������������������������������  253 Scheme 11.4 Synthesis of AMG-3�����������������������������������������������������������������  253 Scheme 11.5 Synthesis of HU-210�����������������������������������������������������������������  254 Scheme 11.6 Synthesis of AM-708����������������������������������������������������������������  254 Scheme 11.7 Synthesis of ajulemic acid��������������������������������������������������������  255 Scheme 11.8 Synthesis of nabilone����������������������������������������������������������������  255 Scheme 11.9 Condensation of resorcinols with diacetates 40 and 41������������  256 Scheme 11.10 Synthesis of AM-993 and AM-994�������������������������������������������  257 Scheme 11.11 Synthesis of AM-2389 and canbisol�����������������������������������������  257 Scheme 11.12 Synthesis of 9β-hydroxymethyl-HHC from HU-210���������������  257 Scheme 11.13 Synthesis of AM-7499��������������������������������������������������������������  258 Scheme 11.14 Synthesis of a 9-ketocannabinoid bearing a non-bulky C3 side chain�����������������������������������������������������������������������������  259 Scheme 11.15 Synthesis of a series of hexahydrocannabinols via a common intermediate�������������������������������������������������������  259 Scheme 11.16 Structures of (−)-CP-47,497 and (−)-CP-55,244 and the synthesis of (−)-CP-55,940������������������������������������������  260 Scheme 11.17 Synthesis of KLS-13019�����������������������������������������������������������  261 Scheme 11.18 Synthesis of PRS-211,375��������������������������������������������������������  261 Scheme 11.19 Synthesis of HU-308�����������������������������������������������������������������  261 Scheme 11.20 Synthesis of HU-910�����������������������������������������������������������������  262 Scheme 11.21 Synthesis of (−)-6β-hydroxymethyl-Δ9-THC���������������������������  263 Scheme 11.22 C6-diastereoselective synthesis of hybrid cannabinoids�����������������������������������������������������������������������������  264 Scheme 11.23 Total synthesis of hybrid cannabinoid AM-919 and derivatives��������������������������������������������������������������������������  265

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List of Schemes

Scheme 11.24 Synthesis of hybrid cannabinoid 130 via heteroDiels−Alder reaction�����������������������������������������������������������������  267 Scheme 11.25 Preparation of the aliphatic aldehyde 125��������������������������������  267 Scheme 11.26 Preparation of resorcinol 31������������������������������������������������������  268 Scheme 11.27 Synthesis of AM-4030��������������������������������������������������������������  269 Scheme 11.28 Synthesis of aliphatic aldehyde 140�����������������������������������������  269 Scheme 11.29 Synthesis of acetophenone 141�������������������������������������������������  270 Scheme 11.30 Synthesis of AM-960����������������������������������������������������������������  271 Scheme 11.31 Synthesis of C3-adamantyl hybrid cannabinoids via oxymercuration-demercuration�������������������������������������������������  271 Scheme 11.32 Synthesis of C3-adamantyl hybrid cannabinoids via hetero-Diels−Alder cyclization�������������������������������������������������  271 Scheme 11.33 Synthesis of tetracyclic analogs of Δ8-THC������������������������������  272 Scheme 11.34 Synthesis of rotationally restricted tetrahydrocannabinol ethers������������������������������������������������������  273 Scheme 11.35 Synthesis of some cannabinoid quinones���������������������������������  274 Scheme 11.36 Synthesis of cannabinoid quinones via photooxygenation�������  274 Scheme 11.37 Synthesis of cannabinol via von Pechmann condensation��������  275 Scheme 11.38 Synthesis of cannabilactones via Suzuki coupling reaction����������������������������������������������������������������������  275 Scheme 11.39 Synthesis of C-ring cannabinoid lactones���������������������������������  276 Scheme 11.40 Asymmetric synthesis of levonantradol������������������������������������  277 Scheme 11.41 Synthesis of a pentacyclic hybrid cannabinoid�������������������������  278 Scheme 11.42 Structure of rimonabant and the synthesis of chromenopyrazoles���������������������������������������������������������������  279 Scheme 11.43 Synthesis of chromenopyrazolediones�������������������������������������  280 Scheme 11.44 Synthesis of chromenoisoxazoles���������������������������������������������  281 Scheme 11.45 Structures of resorcinol−AEA hybrids and the synthesis of representative compound CB-25��������������������������  281 Scheme 11.46 Synthesis of O-2220�����������������������������������������������������������������  282 Scheme 11.47 Structures of 2-AG and the synthesis of a resorcinol−2AG hybrid���������������������������������������������������������������������������������  282 Scheme 13.1 The photoredox mechanism of the tyrosine residue-specific modification reaction catalyzed by ruthenium complex ([Ru(bpy)3]2+)����������������������������������������������������������������������������  313 Scheme 13.2 PTAD-based chemical modification�����������������������������������������  314 Scheme 13.3 Luminol-based tyrosine residue-specific chemical modification������������������������������������������������������������������������������  315 Scheme 13.4 MAUra-based tyrosine residue-specific chemical modification������������������������������������������������������������������������������  315 Scheme 13.5 Strain-promoted cycloaddition of 1,2-quinones converted from tyrosine residues specifically oxidized by mushroom tyrosinase�����������������������������������������������������������  316 Scheme 14.1 Photoreaction of triazido compound 2��������������������������������������  342

List of Schemes

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Scheme 14.2 Synthesis of photovastatin CAA1 (5)���������������������������������������  343 Scheme 14.3 PAL experiment using photovastatin CAA1 (5) and recombinant HMGR�����������������������������������������������������������������  344 Scheme 14.4 Synthesis of diazido PAL probe 11�������������������������������������������  345 Scheme 14.5 PAL experiment using diazido probe 11 in subcellular fractions of rat whole brain homogenates���������������������������������  345 Scheme 14.6 Facile synthesis of various diazido building blocks bearing connecting groups (CGs)���������������������������������������������  348 Scheme 14.7 Synthesis of diazido compounds 14 and 25������������������������������  349 Scheme 14.8 Synthesis of diazido compounds 27 and 28������������������������������  349 Scheme 14.9 Photoreaction of diazirine 40a��������������������������������������������������  351 Scheme 14.10 Photoreaction of diazirine 40b��������������������������������������������������  351 Scheme 14.11 Synthesis of 3-aryl-3-(azidodifluoromethyl)diazirine unit 48���������������������������������������������������������������������������������������  352 Scheme 14.12 Photoreactions of aryl(azidodifluoromethyl)diazirine 48���������  352

List of Tables

Table 1.1 Pros/cons of biological and chemical screenings���������������������������    2 Table 1.2 Pros/cons of natural products and synthetic compound�����������������    3 Table 3.1 Stereoselective reduction of 7 (Scheme 3.5)����������������������������������   53 Table 3.2 The palladium-catalyzed 7-exo-trig cyclization of 10a and 10b (Scheme 3.7)����������������������������������������������������������������������������   54 Table 3.3 Isomerization of allylic alcohol 11 to α-methyl ketone 13a (Scheme 3.9)�����������������������������������������������������������������������������������   55 Table 6.1 Inhibitory activity of the synthetic specimens against Ca2+ influx induced by MTX����������������������������������������������������������  138 Table 7.1 Results of Scheme 7.5: Preliminary results using various secondary allylic esters with phenylmetal reagents������������������������  149 Table 7.2 Results of Scheme 7.7: Substitution of rac-12a with PhLi-based copper reagents������������������������������������������������������������  150 Table 7.3 Results of Scheme 7.17: Quaternary carbon-forming allylic substitution���������������������������������������������������������������������������  158 Table 7.4 Results of Scheme 7.23: Feasibility study of the quaternary carbon construction�������������������������������������������������������������������������  163

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Chapter 1

Microbial Fraction Library: A Screening Source for Drug Discovery Toshihiko Nogawa, Julius Adam V. Lopez, and Hiroyuki Osada

Abstract  Natural products are an important source for drug screening because of their diverse chemical properties and biological activities. However, there are several drawbacks in this field such as the duplicate isolation of previously reported metabolites and the inherent difficulty of obtaining a pure compound with significant bioactivity, which requires considerable amount of work, time, and resources. Researchers are continuously devising ways to efficiently discover and isolate novel compounds by developing new screening and sample preparation methods. In this chapter, the current situation of microbial product research is described by introducing screening and sample preparation methods with focus on fraction libraries including our own strategy and discoveries. Keywords  Natural product · Microbial metabolite · Fraction library · Screening · Antimycin · Biological activity

1  Introduction Natural products have been used as a source for the screening of drugs and drug leads [1]. In particular, microbial secondary metabolites not only have extensive structural diversity but also various biological activities, many of which have been developed as drugs, pesticides, and agrochemicals [2–4]. They also have an important role in chemical biology studies as bioprobes, which are chemical tools for the investigation of biological functions [5, 6]. Although the versatility of natural products holds promise for drug discovery research, in reality, it accompanies several challenges. A major difficulty lies in the sample screening and the isolation and purification of compounds from natural sources such as microbial culture broths and plant extracts.

T. Nogawa · J. A. V. Lopez · H. Osada (*) RIKEN Center for Sustainable Resource Science, Chemical Biology Research Group, Wako, Saitama, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2019 Y. Kobayashi (ed.), Cutting-Edge Organic Synthesis and Chemical Biology of Bioactive Molecules, https://doi.org/10.1007/978-981-13-6244-6_1

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The screening methods of natural products are divided into two approaches: biological and chemical screenings (Fig. 1.1, Table 1.1). Biological screening is based on a biological activity test, for example, antimicrobial activity or cytotoxicity against tumor cells, and focuses on searching biologically active compounds. Using a biological screening throughout the separation process of natural sources, such as a microbial broth, to isolate metabolites with specific activity is called activity-­ guided isolation. The emergence of high-throughput screening (HTS) technology Natural sources (broths/extracts)

Biological screening

Chemical screening

For interesting activity

For novel structure

Phenotypic screening

Target-based screening

LC/MS

NMR

Fig. 1.1  Screening strategy of interesting secondary metabolites Table 1.1  Pros/cons of biological and chemical screenings Screening methods Biological Phenotypic screening screening

Target-based screening

Pros Discovery of activity with unknown mode of action Confirmation of activity against whole organism Easy to apply to HTS Focus on specific activity

Chemical screening

LC/MS

NMR

Applicable for most of hydrophobic compounds Applicable for complex mixtures by LC separation High sensitivity High throughput Applicable for most compounds Obtainable precise structural information such as specific functional groups

Cons Necessity to identify the molecular target Low throughput Uncertainty on the specific activity Unexpected side effects/off-target effects Difficult to apply to polar compounds

Not applicable to non-ionized compounds Low sensitivity No separation system like LC Low throughput

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has greatly improved the speed of biological screening in terms of the large number of samples that can be tested in a single run. Nevertheless, such large number of samples does not guarantee the discovery of first-in-class drug leads. For example, a synthetic compound library created via combinatorial chemistry is commonly applied to HTS to secure a large set of compounds; however, this does not assure structural diversity compared to natural products and may contain unsuitable compounds as drug candidates. In addition, not all samples can be easily applied to the HTS format, especially natural products, because it is difficult to maintain a large number of purified and high-quality sample set (Table 1.2) [7, 8]. On the other hand, chemical screening is a structure-oriented approach that focuses on properties that give information about compound structure such as UV and IR absorption spectra, molecular weight, and other spectroscopic data. The corresponding spectroscopic techniques are spectrophotometry, liquid chromatography/mass spectrometry (LC/ MS), and nuclear magnetic resonance (NMR). The major drawback of this approach is the discovery of new compounds without significant bioactivity. Moreover, whether implementing biological or chemical screening, the isolation process entails several steps such as extraction, partition, and possibly many types of chromatography including high-performance liquid chromatography (HPLC) on normal- and reversed-phase modes, to obtain a pure compound. Also, the limited amount of isolated compounds oftentimes hinders their full structural and bioactivity characterization. Clearly, the whole process from raw sample to pure compound takes a lot of time, work, and resources. These issues have caused drug discovery research to shift from natural products to synthetic compound libraries prepared by combinatorial chemistry. However, these synthetic compound libraries fail to represent the entire chemical space, which is defined as “all possible small organic molecules, including those present in biological systems” [9]. Using statistics to compare chemical properties, natural products were significantly similar to developed drugs, while the distribution of synthetic compound libraries was limited, suggesting that natural products are more suitable for drug screening. Accordingly, many of the drugs developed in the past 30 years are natural products or synthetic compounds based on or inspired by natural ­products Table 1.2  Pros/cons of natural products and synthetic compound Sources Natural product

Synthetic compound

Pros Structural diversity Variety of biological activity Discovery of unexpected structures and biological activity Simple structures and low molecular weight Easy to secure a large set of compound library for HTS Focused library can be prepared for specific structures and biological activity

Cons Time-consuming work for isolation and purification of compounds Re-isolation of known compounds Difficulty to secure a large set of compound library for HTS Relatively low structural diversity

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[1]. Therefore, natural products continue to be a vital source and may be more productive sources for drug screening [10] once we can find a way to overcome the challenges mentioned above. In this chapter, screening methods based on bioactivity and chemical structure are described in brief followed by a discussion on natural product fraction libraries and our own strategy and findings.

2  Biological Screening Biological activity screening has been a major way of discovering bioactive natural products, and many of medicinally important compounds have been discovered in this manner [11]. It is mainly categorized into phenotypic and target-based screenings. Phenotypic screening focuses on the identification of compounds or molecules that cause changes to the phenotypes of cells, tissues, or whole organisms without prior knowledge of any specific targets. Another form of phenotypic screening is high-content screening (HCS), wherein multiple information such as spatial distribution and morphology can be recorded in different timescales using automated imaging [12]. Meanwhile, target-based screening operates on a putative or known target such as a protein and searches for compounds that will induce an effect on it [13–15]. Between 1999 and 2008, 23% and 37% of first-in-class drugs approved by the US Food and Drug Administration (FDA) were products of target-based and phenotypic screenings, respectively [13]. Later on, Eder et al. reported the contrary that between 1999 and 2013, 69% and 7% of FDA-approved first-in-class drugs were from target-based and phenotypic approaches, respectively [16]. However, the authors pointed out that phenotypic screening is not inferior to but rather a complement to target-based screening and further suggested it to be a new discipline [15, 16]. In this point of view and in our search for new anticancer compounds, we have developed a phenotypic screening system based on cell morphology changes and created a database called MorphoBase [17, 18]. MorphoBase contains the data of morphological changes of various cancer cell lines treated with over 200 well-­ characterized drugs. It is anticipated that a new molecular mechanism of action will be revealed upon detection of a unique cell morphology induced by a new agent in comparison with existing profiles in the database. In the course of our phenotypic screening, pyrrolizilactone – a novel fungal metabolite characterized by a unique ketone-linked pyrrolizidinone and decalin structure  – was found. By using MorphoBase, pyrrolizilactone was classified as a proteasome inhibitor, and this was later confirmed by a proteome-based profiling analysis (Fig. 1.2) [19, 20]. Based on these facts, biological screening is an integral part of natural product research.

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Fig. 1.2 Morphological profiles of HeLa and srcts-NRK cancer cells treated with pyrrolizilactone

3  Chemical Screening Nowadays, LC/MS and NMR spectrometers are routine analytical instruments. Upgrades are continually being developed to acquire higher sensitivity and resolution. In minutes, one can obtain precise structural information of a trace amount of compound by a single analysis of a complex mixture with no separation and purification, which is suitable for the fast screening of structurally interesting compounds. Each technique has its advantages and disadvantages (Table 1.1) [21]. LC/MS is a method of choice due to its high sensitivity and resolution and the separation that LC provides. With the development of ultrahigh-performance liquid chromatography (UHPLC), higher throughput was achieved by utilizing specialized columns that can withstand high pressures resulting to higher resolution and shorter run times. For example, a typical 30-min HPLC run can be done in 5 min or less with UHPLC. Various mass spectrometers are available for LC/MS such as quadrupole (Q), ion trap (IT), time-of-flight (Tof), Fourier-transform (FT), as well as tandem or hybrid systems which include triple quadrupole (TQ or QqQ), quadrupole + time of flight (QTof), and quadrupole + ion trap (QTrap). In addition, different ion sources can be used such as electron impact (EI), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption/ionization (MALDI), in which ESI is the preferred option for sample screening because it can ionize most compounds in the liquid form leading to high compatibility with typical LC systems [22, 23]. In our laboratory, we use a UHPLC-ESI-QqQ to generate mass data for compound libraries and an LC/ESI-QTof for high-­ resolution measurements in both MS and MS/MS modes, which afford accurate estimation of the chemical formula of molecular and fragment ions, respectively. The determination of the exact molecular formula is the initial and a critical step in the structure elucidation of a compound. Some issues associated with LC/MS are non-ionizable and highly polar compounds, although ambient mass spectrometry (AMS) with dual ionization source and hydrophilic interaction chromatography

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(HILIC) have been reported as possible alternatives [24, 25]. Another drawback is that structural isomers cannot be distinguished by mass data alone, though there has been tremendous effort in utilizing fragmentation data (MS/MS) to address this matter. Fragmentation patterns of compounds can be considered as unique signatures; thus, structural similarity or difference may be distinguished by comparing MS/MS data. For this purpose and to streamline dereplication, which is a way to avoid re-isolation of known compounds, a molecular networking platform was developed, and the Global Natural Product Social Molecular Networking (GNPS) database was established [26–28]. NMR spectroscopy provides more specific structural information such as functional groups and their corresponding connections, which are indispensable for structure elucidation. NMR-based screening, NMR-directed isolation, LC/NMR, and NMR fingerprinting method are some strategies that have led to the discovery of new compounds [29–32]. However, NMR remains less popular than LC/MS due to its low sensitivity and throughput and the high cost of deuterated solvents [31]. Both LC/MS and NMR measurements afford huge sets of data, which includes retention time, m/z value, and intensity for LC/MS and chemical shift and intensity for NMR. To manage and conveniently analyze these data sets, statistical tools such as the principle component analysis (PCA) have been applied to prioritize samples in large extract libraries [33].

4  Natural Product Fraction Libraries After deciding on and developing the screening method, it is time to apply it to sample extracts. Traditionally, the preparation of natural sources for screening was accomplished by simple extraction using an organic solvent such as methanol and subsequent concentration. However, these crude extracts are complex and consist of many unknown metabolites including undetectable minor constituents. The active component may be at a very low concentration to have any significant effect or masked by interference or nuisance compounds. In addition, both inhibitors and promoters may be present, and synergistic effects may be in place. These concerns suggest that biological activity may not be accurately estimated using extracts rendering them unsuitable for HTS. Moreover, viscosity and solubility problems are also encountered [34]. To solve this problem, several groups including ourselves worked on fractions instead of crude extracts for screening programs and the construction of natural products libraries that range from 103 to over 106 fractions [34]. Different pre-­ fractionation techniques were reported such as solid-phase extraction (SPE), C18 HPLC, SPE-C18 HPLC, polyaromatic adsorbent-C18 HPLC, and so on [32, 34–39]. By using SPE-C18 HPLC fractionation with MS monitoring and subsequent NMR analysis with a cryoprobe, the cytotoxic plakinidines were identified [35]. Without fractionation, these minor compounds could have been easily overlooked because of more predominant cytotoxic metabolites. Meanwhile, the Quinn group tested

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various adsorbents to find which one would best retain compounds with “drug-like” properties [36]. This created a unique fraction library, in which the NMR fingerprinting method was applied leading to the isolation of iotrochotazine A – a chemical probe for Parkinson’s disease [32]. Recently, the US National Cancer Institute (NCI) has initiated the establishment of the NPNPD (NCI Program for Natural Product Discovery) fraction library, which is envisioned to become the largest in the world [37]. The program targets to create >1,000,000 fractions prepared from >125,000 extracts from marine, microbial, and plant sources collected worldwide. The library will be free and publicly accessible to support screening platforms and stimulate drug discovery research. These reports show that fraction libraries are promising sources for the discovery of new lead compounds.

5  The Fraction Library at RIKEN We have constructed a fraction library using microbial culture broths by a combination of liquid partition and chromatographic techniques for the efficient discovery and identification of structurally interesting secondary metabolites with interesting biological activity (Fig. 1.3) [38, 39]. This led to the isolation of several new compounds: verticilactam, spirotoamides, and antimycin-related metabolites. The construction and advantages of the fraction library as well as the details of the isolation and chemical biology are described in this section.

Culture broth

Rich source of various secondary metabolites Fractionation by basic chromatographic techniques

Fraction library

LC/MS analysis

Structure inf ormation

Bioassay

Activity inf ormation

Combine the results

Novel structure with interesting activity Fig. 1.3  Concept of the fraction library

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5.1  Construction of a Microbial Metabolite Fraction Library In reality, it is not practical to repeatedly collect samples from natural sources, such as microbes, plants, and marine invertebrates, to cover a suite of samples for HTS and, eventually, for isolation purposes. For this reason, we focus on soil bacteria and fungi which can be cultured in the laboratory, thus eliminating the need for resampling. To create the fraction library (Fig. 1.4), microbial culture broths were prepared from selected strains in our in-house repository. The strains were selected based on their production of various secondary metabolites such as polyketides, terpenoids, and peptides, which were identified by previous works and LC/MS analysis. The optimum culture conditions were established by pre-investigation using several conditions accompanied by LC/MS chemical profiling. For complete biological and chemical screening, 20–30 L cultures were prepared to obtain several dried fractions that were approximately 2  mg each. Culture broths were directly mixed with the same volume of acetone to extract metabolites and filtered. The filtrate was concentrated under reduced pressure, and the extract was partitioned with ethyl acetate and water to afford hydrophobic and hydrophilic materials. The hydrophobic part was fractionated by a 2D separation method, which combined both normal- and reversed-phase chromatographies to obtain well-separated fractions (Fig. 1.5). The first separation was done on silica gel by medium-pressure liquid chromatography (MPLC) with a chloroform/methanol solvent system to afford six to eight MPLC fractions. Then, for the second separation, each MPLC fraction was subjected to reversed-phase HPLC with an acetonitrile/0.05% aqueous formic acid solvent system to afford 48 fractions. Similarly, the water-soluble material was separated by reversed-phase MPLC in the same solvent system to afford 48 fractions. Hence, the fraction library core consists of 48 fractions each from ethyl acetate and water layers per sample. A portion of each fraction was used for LC/MS analysis and several bioassays. The remaining fraction was dried and stocked for the identification and isolation of metabolites. In total, over 10,000 fractions were prepared

Fig. 1.4  Culture condition and preparation of crude extracts and fractions

start elution

1D: Normal phase chromatography

1  Microbial Fraction Library: A Screening Source for Drug Discovery

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metabolite(s) separated by normal phase

metabolite(s) separated by reversed phase start

2D: Reverse phase chromatography

elution

Fig. 1.5  A basic concept of 2D separation for the preparation of fractions Fig. 1.6  Mass distribution of fractions

10%

65%

25%

> 2.0 mg 0.5 ~ 2.0 mg < 0.5 mg

from 30 microbial strains comprising of actinomycetes and fungi. Approximately 65% of the fractions were over 2.0 mg, and 25% were within 0.5–2.0 mg, which were sufficient to check for various biological activities (Fig. 1.6). The remaining 10% was under 0.5 mg but were still enough to test on selected bioassays, such as basic cytotoxicity and antimicrobial activities.

5.2  Evaluation of Biological Activities As typical examples of bioassay, the fraction library was evaluated against the human promyelocytic leukemia cell line (HL-60) for cytotoxicity and Escherichia coli HO141 and Pyricularia oryzae kita-1 for antimicrobial activities (Fig.  1.7). Approximately 25% of fractions showed single or multiple activities at a final concentration of 100  μg/mL.  It is noteworthy that compared to earlier results using crude extracts, more active samples were observed using the fractions, and some fractions prepared from previous non-active extracts showed significant activity. By using a fraction library as a screening source, bioactive compounds in low

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Fig. 1.7  General activity profile of the fraction library evaluated at 100 μg/mL

concentrations or in coexistence with some inhibitors or interfering compounds in the crude extract can be unraveled.

5.3  NPPlot as a Spectral Database A spectral database is useful and necessary for easy and preliminary identification of metabolites, specifically for dereplication, to save time and resources. However, typical spectral databases put priority on the identification of known compounds rather than targeting novel compounds. To create an interface that will aid in the location of potential new compounds, we constructed an original spectral database named NPPlot (Natural Products Plot) by combining UV, mass, and retention time data in a single plot. Data was acquired from an LC/MS instrument equipped with a diode-array detector (LC-DAD/MS). NPPlot shows a simple distribution map of metabolites (Figs. 1.8 and 1.9), which appear as spots in 2D (retention time vs m/z). Usually 100–200 metabolites are detected from the fraction library of one strain, and it is time-consuming to check each data and look for distinct characteristics manually. With the NPPlot, the distribution patterns for each strain can be visualized and compared. For example, unusual alignments, such as spots having the same m/z value or a specific difference of m/z value with slightly different retention times, were detected in our samples (Fig. 1.8). Moreover, the UV spectrum can be readily accessed for each spot adding an extra layer of information to easily gauge structure similarity. These data were crucial and led to the isolation of verticilactam, spirotoamides, octaminomycins, RK88–1355A and RK88–1355B, and antimycin-­ related compounds. The NPPlot provided a convenient means of analyzing a combination of spectral data, which helped us focus on characteristic patterns of metabolites or metabolite groups resulting to the determination of several new compounds.

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Fig. 1.8  NPPlots and unusual alignments Each spot is a metabolite observed in fractions. The plot is an overlay of three different Streptomyces strains represented by colors. Expanded areas show the unusual alignments and their typical UV spectra 1500

Specific metabolite group observed in Streptomyces sp. RK88-1355

3-hydroxyquinaldic acid 210

wave length (nm)

500

Identical UV spectrum with m/z values of around 1150 m/z

Isolation from the fraction Structure elucidation

0.00 0.00

retention time (min)

45.00

R = Me: RK-1355A R = Et: RK-1355B

Fig. 1.9  NPPlots and specific metabolite group of Streptomyces sp. RK88–1355 Each spot is a metabolite observed in the fractions. The plot is an overlay of five different Streptomyces strains represented by colors. The highlighted area is the specific region of Streptomyces sp. RK88–1355, wherein each spot showed identical UV spectra characteristic of 3-hydroxyquianldic acid. Two of them were isolated, and the structures were elucidated as new quinomycin derivatives, RK-1355A and RK-1355B

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5.4  S  tructurally Interesting Metabolites Discovered from the Fraction Library by NPPlot Screening The first step to find potentially new metabolites from numerous compounds detected by LC/MS is to narrow down the search by looking for a specific metabolite group. For instance, a metabolite may show some relationship such as similar UV spectra, close m/z values, or adjacent retention times. In the NPPlot, such metabolite groups basically appeared as characteristic alignments. For example, the NPPlots of Streptomyces spiroverticillatus and Streptomyces griseochromogenes included unusual alignments, in which three and four spots were aligned with the same m/z values and identical UV spectra, respectively (Fig. 1.8). This pattern suggested that they were possibly structural isomers. These were isolated from their respective fractions, and the structures were elucidated as the novel compounds verticilactam [40] and spirotoamides [41]. Verticilactam contained a decalin skeleton and a rare 16-membered lactam with a β-keto amide moiety, which was the first example as a natural product. Recently, we have identified a new Diels-Alderase that controls decalin formation in the fungal metabolite, phomasetin, which is biosynthesized by polyketide synthase (PKS)/ nonribosomal peptide synthase (NRPS) hybrid enzymes [42]. The decalin skeleton of verticilactam might be formed by a potential Diels-Alder cycloaddition after the production of a putative 24-membered lactam by polyketide condensations of type I PKS followed by lactamization (Fig. 1.10). On the other hand, the spirotoamides had a 6,6-spiroacetal skeleton and a terminal carboxamide moiety. Reveromycin A, which belongs to the polyketide family and was isolated from Streptomyces reveromyceticus, had the same 6,6-spiroacetal skeleton [43, 44]. We recently reported the

lactamization

polyketide chain

24-membered lactam

tetrahydrofuran formation

Diels-Alder reaction 16-membered lactam

Fig. 1.10  Structure and putative biosynthetic pathway of verticilactam

verticilactam

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identification of a key enzyme for the stereospecific formation of the spiroacetal core structure of reveromycin A [45]. This prompted us to search for an enzyme homolog in S. griseochromogenes but found none suggesting that the spiroacetal skeleton in spirotoamides might be synthesized nonenzymatically from a putative polyketide chain (Fig. 1.11). The other type of unusual alignment was observed in the NPPlot of Streptomyces sp. RK85–270, in which four metabolites with a sequential mass difference of 14 Da were found, implying the addition of a methylene group (Fig. 1.8). Two new compounds were isolated from the corresponding fraction, and the structures were elucidated as new octadepsipeptides, octaminomycins A and B (Fig.  1.12) [46]. Remarkably, the compounds had both D- and L-leucines in their structures and exhibited moderate antimalarial activity with no antimicrobial activity nor cancer cell cytotoxicity. The mass difference of 14 Da was derived from acylation at the threonine nitrogen by a propionyl or acetyl group. Other than structural analogs, metabolites that are specific to one strain can also be recognized. These groups are more clearly identified by the comparison and overlay of NPPlots generated from several strains (Fig. 1.9). The NPPlot generated from Streptomyces sp. RK88–1355 showed a distinctive group of spots with m/z values of around 1100. They had characteristic and identical UV spectra suggesting the presence of 3-hydroxyquinaldic acid. Some of them were identified to be known quinomycins [47], but there were still unidentified analogs. These were isolated from the fraction, and the structures were identified as new quinomycin derivatives RK-1355A and RK-1355B [48]. They showed potent cytotoxicity against cancer cells and moderate antibacterial activity and were the first sulfoxide-containing quinolone-type natural products. In addition, they showed antimalarial activity against P. falciparum through DNA intercalation [49].

polyketide chain Non-enzymatic spiroacetal formation

6,6-spiroacetal

spirotoamide A

spirotoamide B

Fig. 1.11  Structures and putative biosynthetic pathway of spirotoamides

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L-Pro-1

D-Leu-2

L-Leu-1

N-Me-L-Tyr L-Pro-2

L-Phe

N-Acyl-L-Thr

R = Et: octaminomycin A R = Me: octaminomycin B

Fig. 1.12  Structures of octaminomycins A and B

5.5  A  ntimycin-Related Novel Metabolites from the Fraction Library Since the first isolation of antimycin A in 1949 from a species of Streptomyces, the antimycin class of metabolites has constantly drawn the attention of many researchers due to their structural diversity and various biological activities such as cytotoxicity, antimicrobial, and antiviral activities [50, 51]. The number of possible structures continues to increase as new types of analogs are isolated even 70 years later [52]. In this regard, UV and mass spectra obtained through an NPPlot search implied that Streptomyces sp. RK88–1355 may also produce antimycin-related metabolites other than the quinomycins as described above. They had similar UV spectra comparable to antimycins and were grouped by m/z values in the range of 500–700 (Fig. 1.13). One of them was identified as a known neoantimycin analog, SW-163A [53], and the others were potentially new analogs with distinct molecular weights. They were isolated from the corresponding fractions, and their structures were elucidated as new antimycin-related metabolites, unantimycin A [54] and opantimycin A [55]. The antimycins are depsipeptides containing a macrolide core and a 2-hydroxy-­ 3-formylaminobenzoic acid moiety connected by an amide linkage at the C-3 position. They have been isolated from various actinomycetes, and structural variety is derived from the size of macrolide core, 9-, 12-, 15-, and 18-membered, with several types of substitutions (Fig.  1.14). The 15-membered antimycins are specifically named as neoantimycins since the first isolation of neoantimycin A in 1967 from Streptomyces orinoci [56]. Variations of the 2-hydroxy-3-formylaminobenzoic acid moiety, an essential functional group for the bioactivity [57], has been reported such

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NPPlot for Streptomyces sp. RK88-1355 Quinomycins

tR: 27.65 min m/z: 626 [M+H]+

m/z

Antimycin related metabolites

Interesting spots by similar UV spectra

0.00

Unantimycin A

retention time (min)

Unantimycin A

Opantimycin analog

tR: 21.32 min m/z: 557 [M+H]+

Opantimycin A tR: 22.77 min m/z: 539 [M+H]+

SW-163A tR: 27.27 min m/z: 671 [M+H]+

Unantimycin analog

tR: 24.28 min m/z: 685 [M+H]+

45.00

Opantimycin A

Fig. 1.13  NPPlot of Streptomyces sp. RK88–1355 and new antimycin-related metabolites

as 2-hydroxy-aminobenzoic acid, 2,3-hydroxybenzoic acid, and benzoic acid (Fig. 1.15). Isoneoantimycin was the first ring-opened neoantimycin analog to be reported [58], and, more recently, ring-opened analogs of 9-membered antimycins, antimycins B1 and B2, were isolated from the marine-derived actinomycete, Streptomyces lusitanus (Fig.  1.14) [52]. A comprehensive review on antimycins covering up to 2016 was published by Liu and colleagues [50]. Unantimycin A had a 3-hydroxybenzoic acid instead of 2-hydroxy-3-­ formylaminobenzoic acid, and this type of neoantimycin was the first of its kind (Fig. 1.13). It is speculated that 3-hydroxybenzoic acid is mainly incorporated as the starter unit in the biosynthesis of unantimycin A with enzyme flexibility for substrate recognition [59, 60]. On the other hand, opantimycin A had a 2-hydroxy-­3formylaminobenzoic acid; however, it lacked a macrolide core and contained a γ-butyrolactone instead. So far, only three ring-opened antimycins have been reported—antimycins B1 and B2 and isoneoantimycin—and only isoneoantimycin had a γ-butyrolactone (Fig.  1.14). In addition, isoneoantimycin is composed of identical units of neoantimycin in contrast to opantimycin A, which had a different unit in the form of a dehydrated threonine. Thus, unantimycin A and opantimycin A are unique antimycins and important additions to its growing structural diversity.

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i) Representative 9-membered antimycin analogue

ii) Representative 12-membered antimycin analogue

3

3

antimycin A (antimycinA1a) JBIR-06 iii) Representative 15-membered antimycin analogue

iv) Representative 18-membered antimycin analogue

3

3

neoantimycin (neoantimycin A) respirantin v) Ring-opened antimycin analogues

antimycin B1

isoneoantimycin antimycin B2

Fig. 1.14  Structural diversity of the antimycin class of metabolites

It was reported that antimycins and neoantimycins showed potent antimicrobial activities [50], but unantimycin A did not show any remarkable effect on microbes, which is probably due to the lack of a 2-hydroxy-3-formylaminobenzoic acid unit. This was supported by the strong antimicrobial activity of SW-163A, which had a 2-hydroxy-3-formylaminobenzoic acid unit on the same macrolide core. Opantimycin A also did not show any effect on microbes, even though it had a 2-hydroxy-3-formylaminobenzoic acid unit. These results suggest that a ­2-hydroxy-­3-formylaminobenzoic acid unit and a macrolide core are necessary for antimicrobial activity. Both unantimycin A and opantimycin A showed moderate cytotoxicity against various cancer cell lines similar to SW-163A [54, 55].

1  Microbial Fraction Library: A Screening Source for Drug Discovery

2-hydroxy3-formylaminobenzoic acid (representative group)

2-hydroxy3-amino-benzoic acid

2,3-hydroxybenzoic acid

17

2-aminobenzoic acid

2-formylaminobenzoic acid

benzoic acid Fig. 1.15  Variety of the functional group at C-3 position

5.6  Mechanism of Action of Unantimycin A We recently constructed a phenotypic screening system that focused on bioenergetic profiles and characteristic proteome changes for inhibitors of cancer metabolisms [61]. This screening system uses a Seahorse XFe96 analyzer (Seahorse Bioscience, Inc.) to monitor metabolic properties including oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), which are simultaneously measured, and our original ChemProteoBase [62], which is a target identification platform based on proteomic analysis using 2D difference gel electrophoresis (2D-DIGE). Both systems used human cervical epidermoid carcinoma cell lines (HeLa) for evaluation. Based on the screening system, we can screen a compound that inhibits glycolysis/mitochondrial respiration and unexpected targets by time course monitoring of OCR and ECAR by Seahorse XFe96 and proteomic profiling on ChemProteoBase. We applied this screening system to the RIKEN Natural Products Depository (NPDepo), a public chemical bank in Japan which contains commercially available natural compounds and their derivatives as well as the natural products that we have isolated [63]. Through the screening on NPDepo, unantimycin A was found to show remarkable decrease of OCR and increase of ECAR, indicating the inhibitory effect on oxidative phosphorylation [61]. In addition, unantimycin A was identified as an upregulator of a glycolytic enzyme by proteome

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analysis. These results suggested that unantimycin A was an inhibitor of the mitochondrial electron transport chain and the molecular target was determined to be complex III similar to antimycin A [64].

6  Conclusion In this chapter, screening strategies of biologically and structurally interesting compounds from natural sources were discussed. There are advantages in using either biological or chemical screening methods, and the disadvantages can be overcome by combining both methods. The creation of fraction libraries is a powerful way to screen natural products more efficiently and is a key aspect for high-content and high-throughput screening programs. Compounds with unprecedented skeleton and interesting activity have been discovered by using fraction libraries. Natural products continue to play an important role in drug discovery research. Acknowledgment  We acknowledge Dr. Y. Futamura, Ms. A. Okano, and all the members of our laboratory for their assistance during the work described here. This work was supported in part by JSPS KAKENHI Grant Numbers JP17H06412, JP18H03945, and JP17K07784, AMED under Grant Number JP18cm0106112 (Project for Cancer Research and Therapeutic Evolution/P-CREATE), and the Project of the NARO Bio-oriented Technology Research Advancement Institution (Research program on development of innovative technology).

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Chapter 2

Efficient Total Synthesis of Ōmura Natural Products Toshiaki Sunazuka, Tomoyasu Hirose, and Satoshi Ōmura

Abstract  Naturally occurring novel bioactive microbial metabolites have been discovered by a variety of customized screening methods in the Kitasato Institute, which have been promising to subsequently develop into novel and extremely useful pharmaceutical products. This chapter provides an overview of this research, encompassing the isolation, the total synthesis, and the analogue synthesis as well as the determination of the absolute stereochemistry of bioactive microbial metabolites, such as pyripyropenes (cholesterol lowering agents), arisugacins (acetylcholinesterase inhibitors), lactacystin (proteasome inhibitor), macrosphelides (cell-cell adhesion inhibitors), madindolines (IL-6 modulators), and neoxaline (cell proliferation inhibitor). Keywords  Ōmura natural products · Microbial metabolites · Novel structures · Biological activities · Pharmaceutical products

1  Introduction Bioactive natural products produced by microbes have limitless potential in pharmaceutical applications, and the organic synthesis of such products as lead compounds will result in the creation of new, highly practical, and widely used pharmaceutical products. With a focus on the drug discovery process, the Kitasato Institute (KI) is using cutting-edge, unique screening techniques to discover useful bioactive natural products from microbial metabolites. These novel natural products have distinctive structures and attractive bioactivities. Through a comprehensive and dynamic research program, the KI has discovered more than 500 novel bioactive microbial metabolites over the past four decades. Among them, 26 compounds have been developed into commercially important biological reagents, and 7 compounds are first-line, globally used medicines [1, 2]. T. Sunazuka (*) · T. Hirose · S. Ōmura Kitasato Institute for Life Sciences, Kitasato University and The Kitasato Institute, Tokyo, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2019 Y. Kobayashi (ed.), Cutting-Edge Organic Synthesis and Chemical Biology of Bioactive Molecules, https://doi.org/10.1007/978-981-13-6244-6_2

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The key challenge in synthetic organic chemistry is how to more efficiently and convergently synthesize target compounds with unique, novel molecular skeletons using short process pathways. The construction of novel molecular skeletons necessitates the development of new synthetic strategies and key reactions, which leads to further progress in synthetic organic chemistry (Fig. 2.1). If efficient synthetic methods can be established, it will be possible to quantitatively supply natural products that are presently only naturally available in trace amounts, and this will contribute to a more thorough elucidation of their bioactivities. In addition, it will become possible to determine, using synthetic techniques, the relative and absolute configuration and structures of compounds for which only trace amounts can be extracted from natural sources. Furthermore, use of newly developed molecular skeleton construction methods will allow the creation of a wide range of analogues, thereby leading to the production of compounds with properties that surpass those found in nature and heralding the promise of bioactivity. Hence, the discovery of bioactive natural products with novel molecular skeletons will lead to advances in synthetic organic chemistry that are dynamically related to the elucidation and development of bioactive materials (Fig. 2.1). Target compounds of new natural products may only be available from natural sources in trace amounts, so that the KI has built on the above concepts to develop efficient, rational, and highly flexible construction methods for the production of compounds with novel molecular skeletons and useful bioactivities. To date, 50 types of bioactive natural products have been successfully synthesized. The research program also calls for the application of established methods to synthesize related compounds, elucidating their structure-activity relationships and contributing to the creation of improved bioactive compounds.

Natural Products

New Skeletons

Bioactivity 1) Structure-activity Relationships 2) The Mode of Action 3) Development of Medicine

Fig. 2.1  Our strategy

Organic Synthesis 1) New Synthetic Strategy 2) New Synthetic Method 3) Supply of Natural Products 4) Determination of Stereochemistry 5) Synthesis of New Analogues

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Fig. 2.2  Structures of novel Ōmura natural products

This chapter provides an overview of this research, encompassing the isolation, mode of action, total synthesis, and analogue synthesis as well as the determination of the absolute stereochemistry of Ōmura natural, bioactive microbial metabolites, such as pyripyropenes (cholesterol-lowering agents), arisugacins (acetylcholinesterase inhibitors), lactacystin (proteasome inhibitor), macrosphelides (cell-cell adhesion inhibitors), madindolines (IL-6 modulators), and neoxaline (cell proliferation inhibitor) (Fig. 2.2).

2  Total Synthesis of Pyripyropenes A promising, fundamentally new approach to the prevention and treatment of atherosclerosis is based upon inhibition of acyl-CoA cholesterol acyltransferase (ACAT), the enzyme that catalyzes intracellular esterification of cholesterol. This strategy may permit suppression of three distinct, ACAT-dependent steps in the pathology of atherosclerosis: absorption of dietary cholesterol in the gut, hepatic synthesis of lipoproteins, and deposition of oily cholesteryl esters within the developing arterial lesions. Therefore, inhibitors of ACAT may be promising new types of antiatherosclerotic agents [3]. In the course of our screening of microbial metabolites that inhibit the activity of ACAT, we isolated potent and selective inhibitors of ACAT, pyripyropenes A–D 1–4, from Aspergillus fumigatus FO-1289 [4–6]. These novel, polyoxygenated

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Fig. 2.3  Structures of pyripyropenes

Fig. 2.4  Biosynthesis of pyripyropene A

polyketide-terpenoid hybrid metabolites (meroterpenoid) contain a fused pyridyl α-pyrone moiety and eight contiguous stereocenters; subsequently, we determined the relative and absolute stereochemistries of 1 as shown in Fig.  2.3, employing NOE difference and Mosher ester NMR studies in conjunction with X-ray crystallography [7]. The pyripyropenes A–D 1–4 not only rank as the most effective naturally occurring ACAT inhibitors in  vitro, with IC50 values of 58, 117, 53, and 268 nM, respectively, but also display oral bioavailability in hamsters. Biosynthetic pathway of pyripyropene A 1 was investigated using feeding experiments with 13C-labeled precursors and degradation experiments. 1 denoted that a nicotinic acid primer condenses with two acetates in a head-to-tail fashion, forming the pyridino-α-pyrone moiety, which is linked with a sesquiterpene to create the core skeleton. Then, three acetyl residues are introduced into core (Fig. 2.4). This was the first demonstration that an intact nicotinic acid works as an acyl primer unit for oligoketide formation in fungal secondary metabolites [8]. We have succeeded the first total synthesis of the most active member of this family, (+)-pyripyropene A, via a flexible, concise, and highly efficient route [9].

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Scheme 2.1  Retrosynthetic analysis of pyripyropene A

From the retrosynthetic perspective (Scheme 2.1), we envisioned construction of advanced ketone 5 via acylation of the known hydroxy α-pyrone 6 with acid chloride 7 in the presence of an acid catalyst; isomerization to the C-acyl pyrone and ring closure would then deliver 5 with the requisite anti geometry at the BC ring fusion (Scheme 2.1). The sesquiterpene subunit 7 was derived from (+)-Wieland-Miescher ketone (WMK) [10] in 11 steps. The crucial sequence joining hydroxy pyrone 6 with AB subunit 7 proceeded readily in trifluoroacetic acid (80 °C, 4 h), and O-acylation followed by in situ 1,3-acyl migration and 1,4-cyclization formed the pentacyclic ketone (+)-5 in 47% yield for the three steps; the requisite anti BC ring junction in 5 derived from conjugate addition and selective protonation of enolate due to the C(12) angular methyl group. Stereoselective reduction of 5 then furnished synthetic (+)-pyripyropene A 1 (Scheme 2.2). Importantly, the successful approach is designed to provide flexibility in construction of congeners B–D 2–4 as well as a range of potentially bioactive analogues. Modification and structure-activity relationships of ACAT inhibitor pyripyropenes were examined. Over 300 derivatives of pyripyropenes have been synthesized [11–15]. The pyridine ring of 1 was replaced by the benzene ring (PR-264), which proved to be 100-fold less active than that of 1. This suggests that the pyridine moiety plays a significant role in binding to the enzyme. Some of them, such as PR-86, PR-45, and PR-109, have shown inhibitions on the order of nanomolar (Fig. 2.5). PR-109 showed the most potent (IC50 6 nM) in vitro inhibitory activity. PR-86 also displayed strong ACAT inhibition (IC50 19 nM). From in vivo experiments using hamsters, PR-86 (ED50 = 10 mg/kg) was found to be approximately ten times more effective than pyripyropene A (ED50 = ca. 100 mg/kg) in the inhibition of cholesterol absorption from intestines [16]. Based on the derived structure-activity relationships, binding model of ACTA was proposed for the most potent derivative, PR-109 (Fig. 2.6). The phenyl group of the PR-109 11-benzylidene acetal moiety is located in an equatorial position, suitably fitting a hydrophobic site of ACAT. Another hydrophobic pocket might exist for the 7-O-valeryl group. 7-O-Valeryl, including the carboxyl group, 13-hydroxy, and pyridine moieties, appears to be very important for binding to ACAT [16].

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Scheme 2.2  Total synthesis of pyripyropene A

Fig. 2.5  Structures of pyripyropene analogues

Meanwhile, we found that pyripyropene A 1 exhibited high aphicidal activity through insecticidal screening tests of natural products. Over 150 additional derivatives of pyripyropenes have been synthesized. Among them, PP8201 (Afidopyropen) (Fig. 2.7), having two cyclopropanecarbonyl groups at the C-1 and C-11 positions and a hydroxyl group at the C-7 position, showed the most excellent insecticidal activity against aphid and whitefly and proved to be very safe and highly effective. Very recently, we clarify that Afidopyropen is a modulator of insect transient receptor potential channels. Consequently, Afidopyropen was put on the agricultural market as a pesticide in 2018 [17].

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Fig. 2.6  Structure-activity relationships of pyripyropenes Fig. 2.7  Structure of PP8201 (Afidopyropen)

O HO Me O

1 O

O

N

Me O

Me 7

OH

O 11 O

These results show that Ōmura natural products have a lot of possibility to create a variety of novel medicines.

3  Total Synthesis of Arisugacins Synthetic inhibitors of acetylcholinesterase (AChE) recently have attracted particular attention, since 1-benzyl-4-[(5,6-dimethoxy-1-oxaindan-2-yl)methyl]piperidine (E2020) was approved by the US Food and Drug Administration (FDA) for the treatment of Alzheimer’s disease (AD) [18]. In the course of our screening of microbial metabolites that inhibit the activity of AChE in order to create novel medicines for AD, we have isolated potent and selective inhibitors of AChE, arisugacins A 8 and B 9, from a culture broth of Penicillium sp. FO-4259 (Fig. 2.8) [19–21]. We determined the relative stereochemistry of arisugacin A, employing NOE difference NMR studies, and the absolute stereochemistry of 8, employing Mosher ester NMR studies [22]. The arisugacins A and B not only rank as the most potent naturally occurring AChE inhibitors in vitro, with IC50 values of 1 and 26 nM, respectively, but also protect against amnesia induced by treatment with scopolamine in mice

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Fig. 2.8  Structures of arisugacins

Scheme 2.3  Total synthesis of arisugacin A (part 1)

[23]. Unfortunately, the original source has produced a very small quantity of arisugacins. We have achieved the first total synthesis of arisugacins A and B, via a flexible, concise, and highly effective route [24–30]. We envisioned the construction of advanced olefin 10 via a Knoevenagel-type reaction of the known 4-hydroxy 2-pyrone 11 with α,β-unsaturated aldehyde 12, which was derived from α-ionone, in the presence of L-proline; amine elimination of 13 and six-electron electrocyclic ring closure of 14 then delivered 10 with the requisite geometry at the BC ring fusion as a single compound in 61% yield (Scheme 2.3). All attempts of epoxidation of 2H-pyran 10 failed to produce the desired epoxide owing to the steric hindrance of the angular methyl groups (β-face) and C-1 axial hydroxy group (α-face). We reasoned that inversion of C-1 α-OH to β-OH might lead to formation of the desired epoxide. Oxidation of 10 followed by stereoselective reduction afforded β-OH 15. Epoxidation of 2H-pyran 15 using AcOOH led to the C-1 OAc-diol 16 in 41% yield and β-epoxide 17 in 38% yield.

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Scheme 2.4  Total synthesis of arisugacin A (part 2)

The removal of the activated allylic hydroxy group was carried out by Et3SiH and TFA, followed by solvolysis to afforded 18. After oxidation to ketone 19, phenylselenylation of 19 and oxidative elimination furnished (+)-arisugacin A (8) (Scheme 2.4). To demonstrate the applicability of our strategy, we prepared the analogue (+)-arisugacin B 9 using the 4-methoxy-α-pyrone instead of 3,4-dimethoxy-α-pyrone. Because we were interested in the structure-activity relationships of arisugacins, we tested the activities of the synthetic intermediates against AChE. We found that only 19 showed activity, and this was 80-fold lower than that of arisugacin A. We determined that 1-keto of 10, lacking both an enone moiety on the A ring and a 12aα-hydroxy group, and 18, lacking the enone moiety, no longer inhibited AChE. Furthermore, we found that 8 was 25-fold potent than 9. Consequently, we suggested that the enone moiety in ring A, the hydroxy group at position 12a, and the E ring substitution play important roles in the inhibition of AChE by arisugacins [31]. In collaboration with Dr. Itai of the Institute of Medicinal Molecular Design, a three-dimensional view of arisugacin A 8 docking with AChE could be simulated using an automated computer docking program, ADAM, in which a basket-like structure in blue represents the putative cavity in the AChE protein, to which the substrate of inhibitory molecule binds. As is evident in Fig. 2.9, the long-stretched arisugacin A 8 molecule is well buried along the long and narrow cavity of the enzyme, and hydrogen bondings are also apparent.

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Fig. 2.9 Computer simulation of arisugacin A 8 docking with AChE

4  Total Synthesis of Lactacystin Neurotrophic factors (NTFs) [32] are proteins essential for the survival and function of nerve cells. Decreased availability of NTFs is thought to cause various nerve disorders including Alzheimer’s disease, leading to speculation that NTF-like substances might be therapeutically useful [33, 34]. In the course of our screening of microbial metabolites that promote the differentiation of the mouse neuroblastoma cell line Neuro 2a in order to create novel medicines for AD, lactacystin 20 was isolated from a culture broth of Streptomyces sp. OM-6519 and the novel γ-lactam thioester structure elucidated via 1H and 13C NMR; single-crystal X-ray analysis subsequently revealed the absolute stereochemistry (Fig. 2.10) [35, 36]. Lactacystin induces neuritogenesis with a characteristic parallel array of microtubules and neurofilaments and also causes transient increases in intracellular cAMP levels as well as acetylcholine (ACh) esterase activity in the Neuro 2a neuroblastoma cells [37]. Schreiber described its mode of action appears to be inhibition of the 20S proteasome peptidase activity via acylation of the amino-terminal threonine (Fig. 2.11) [38]. Recently, Fenical reported that salinosporamide A 21, the similar structure of lactacystin, was isolated from a marine bacterium and showed a high cytotoxicity and a stronger proteasome inhibition (Fig. 2.10) [39]. The intriguing structures and significant pharmacological potential of these substances have stimulated considerable interest. We have developed a concise approach to lactacystin 20, designed to afford easy access to the natural product and a variety of analogues [40–49].

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Fig. 2.10  Structures of lactacystin and salinosporamide A

Fig. 2.11  Regulatory functions of the eukaryotic proteasome and target for lactacystin

As our point of departure, we required 22, which was derived from the (2R,3S)3-hydroxyleucine [50]. Aldol condensation with formaldehyde via the Seebach protocol [51] then gave 23 exclusively (85% yield, >98% de) (Scheme 2.5). Oxidation of primary alcohol 23 proved troublesome under a variety of conditions. Fortunately, Moffatt oxidation did provide the requisite aldehyde 24. Deformylation to oxazoline 22 (syn/anti-mixture) occurred quite readily during extraction and silica gel chromatography, so the aldehyde was isolated via nonaqueous workup and subjected without purification to Brown asymmetric allylboration with (E)crotyl(diisopinocampheyl)borane [52, 53]. This sequence afforded a 4:1 diastereo mixture of the desired homoallylic alcohol 25 in 70% overall yield from alcohol 23. Cleavage of the vinyl group in 25 with ozonolysis, subsequent chlorite oxidation, and catalytic transfer hydrogenation followed by saponification gave γ-lactam acid 26. To complete the synthesis, we employed the two-step sequence devised by Corey [42]. Following thioesterification of 26 with bis(2-oxo-3-oxazolidinyl)phos-

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Scheme 2.5  Total synthesis of lactacystin

Fig. 2.12  Structures of lactacystin analogues

phinic chloride (BOP-Cl) and N-acetyl-L-cysteine allyl ester followed by deallylation provided (+)-lactacystin 20 (Scheme 2.5). Because the mechanism of action of lactacystin apparently involves amine acylation [38], we envisioned that related active esters could also induce neuritogenesis. In addition, we have sought to develop analogues with lower cytotoxicity indices in relation to lactacystin 20 itself. As expected, synthetic precursor 26, which are not reactive acylating agents, showed no activity (Fig. 2.12). Interestingly, β-lactone 27, so-called Ōmuralide [38], proved to be as active as lactacystin, and analogues 28 and 29 proved to be significantly more potent than 20 in the neurite outgrowth bioassay. Moreover, we discovered that the descarboxy

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analogue 29 displayed a low toxicity. As such, 29 represents a significantly more potent nonprotein neurotrophic agent than lactacystin [54].

5  Total Synthesis of Macrosphelides Critical early events in inflammation, the allergic response, and tumor metastasis involve interactions between leukocytes and endothelial cells. A variety of cytokinins and related chemical mediators control both leukocyte adhesion and subsequent intercellular invasion by regulating the expression of cellular adhesion molecules. Inhibition of cell-cell adhesion thus holds promise for the treatment of diverse pathologies [55]. In the course of our screening of microbial metabolites that inhibit the adhesion of human leukemia HL-60 cells to human umbilical vein endothelial cells (HUVEC), we have discovered macrosphelides A 30 and B 31 [56, 57]. These novel macrolides, produced by Microsphaeropsis sp. FO-5050, are the first 16-membered ring antibiotics embodying three lactone linkages (i.e., macrotriolides). Macrosphelides strongly inhibit their adhesions in dose-dependent fashion (IC50 3.5 and 36  μM, respectively). Preliminary studies suggest that 30 and 31 prevent cell-cell adhesion by inhibiting the binding of sialyl Lewis x to E-selectin (Fig. 2.13). Macrosphelide A 30 also proved to be orally active against lung metastasis of B16/BL6 melanoma in mice (50 mg/kg). No acute toxicity was observed upon intraperitoneal injection into BDF1 mice (200 mg/kg for 5 days). In conjunction with our continuing program

Fig. 2.13  Inhibitory mode of macrosphelide A on cell adhesion

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Fig. 2.14  Structures of macrosphelides A and B

directed toward the structure elucidation and synthesis of important bioregulatory natural products, we have determined the complete relative and absolute stereochemistries of macrosphelides A 30 and B 31 and have succeeded the first total synthesis of these materials [58]. Initially, we deduced the connectivity of 30 and 31 via a series of NMR studies and chemical characterization of the derived di- and monoacetates, respectively. Single-crystal X-ray diffraction has now been employed to elucidate the relative stereochemistry of 1 and verify the planar structure (Fig. 2.14) [59, 60]. We next have determined the absolute configuration of 30 via the Kakisawa-­ Kashman modification of the Mosher NMR method [60, 61]. To secure the relative and absolute stereochemistries of 31, we subjected (+)-macrosphelide A to PDC oxidation. Synthetic 31 proved to be indistinguishable from the natural product. Accordingly, the configurations of (+)-macrosphelides A 30 and B 31 are (3S,8R,9S,14R,15S) and (3S,8R,9S,15S), respectively. These assignments were confirmed by total synthesis [58]. Our approach to the construction of 30 and 31 entailed the enantioselective preparation of two differentially protected derivatives of trans-(4R,5S)-4,5-dihydroxy-­2hexenoic acid. As our point of departure, we selected the asymmetric dihydroxylation [61] of (E,E)-hexa-2,4-dienoic acid tert-butyl ester 32, which afforded the (4S,5S)diol 33 (Scheme 2.6). Selective monosilylation of 33 followed by Mitsunobu inversion at C(4) furnished 34. After protection of 34 as the MEM ether 35, saponification gave 36, whereas desilylation generated the second building block 37. Condensation of carboxylic acid 36 and alcohol 37 via the Keck protocol [62] and desilylation of the resulting ester produced 38. The third fragment, TBS ether 39, was prepared from (3S)-3-hydroxybutyric acid and coupled with 38 followed by removal of the silyl and tert-butyl moieties providing seco acid 40, which smoothly underwent Yamaguchi macrolactonization [63]. Finally, deprotection of 40 gave synthetic 30. In summary, a highly convergent, stereocontrolled first total synthesis of (+)-macrosphelide A 30 has been achieved in 11 steps from sorbic acid ester, with a 20% overall yield (corresponding to an 88% average yield per step). Moreover, we have demonstrated the combinatorial synthesis of natural product-­ like library based on macrosphelide. The combinatorial synthesis of a 122-member macrosphelide library including macrosphelides A, C, E, and F has been achieved based on a unique strategy for a three-component coupling utilizing a palladium-­ catalyzed chemoselective carbonylation and an unprecedented macrolactonization on a polymer support (Scheme 2.7) [64].

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Scheme 2.6  Total synthesis of macrosphelide A

C

3) carbonlylative esterification 1) attachment

CO

A

W

HO

I polymer-support

HO Y

OR1

4) carbonlylative macrolactonization O

Br O Y

B

D

O

2) esterification

HO

O O

Z

OR2

O

W O

Z

Br OH CO

5) cleaveage

HO Y

W O

O O

Z

O

O

E 4 X 4 X 8 = 128

Scheme 2.7  Strategy for a combinatorial synthesis of macrosphelide analogues

6  Total Synthesis of Madindolines Interleukin 6 (IL-6) [65, 66] is a multifunctional cytokine involved in the regulation of differentiation and antibody production. In addition, uncontrolled IL-6 activity plays a central role in a variety of serious diseases, including cancer cachexia, Castleman’s disease, rheumatoid arthritis, hypercalcemia, and multiple myeloma. Because no effective therapeutic agents for these diseases have been developed, a low molecular weight compound that modulates the function of IL-6 has been sought [67].

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Fig. 2.15  Structures of madindolines A 41 and B 42

Scheme 2.8  Asymmetric oxidative ring closure of tryptophol

In our program to discover new IL-6 modulators, we have isolated madindolines A 41 and B 42, comprised of a 3a-hydroxyfuroindoline ring connected at nitrogen via a methylene bridge to a cyclopentene-1,3-dione ring from Streptomyces nitrosporeus K93–0711 (Fig. 2.15) [68, 69]. Structural assignments were based on NMR studies; the relative and absolute configurations, however, remained undefined. They were stereoisomers at the C-2′ position. Bioassays revealed potent, selective inhibition of IL-6 activity in the IL-6-dependent cell line MH60; importantly the response was dose-dependent. In addition, madindoline A 41, the more potent congener, inhibited the differentiation of osteoblast cells. Unfortunately, the original source no longer produces these antibiotics. Intrigued by the novel architecture, the significant IL-6 inhibitory activity, and the scarcity of these natural products, we have developed the first total synthesis and assignment of the relative and absolute configurations of madindolines A 41 and B 42 [70–72]. As a prelude to total synthesis, we devised an efficient, asymmetric synthesis of the 3a-hydroxyfuroindoline ring system 44 from tryptophol 43 with Sharpless asymmetric epoxidation protocol (Scheme 2.8). Having secured a viable asymmetric protocol to access the 3a-­hydroxyfuroindoline ring, we envisioned the total synthesis of 41 and 42. Next, the stereoselective aldol reaction of α-hydroxyester 45 with methacrolein furnished a mixture of diol 46. The mixture of diol 46 was subjected directly to metathesis to obtain 47. Protection and oxidation of 47, conjugate addition, followed by phenylselenylation of the derived enolate, and oxidative elimination furnished a mixture comprised of 48 and the exomethylene isomer (1:1). Treatment of the mixture with RhCl3 converted the exo congener to 48. Stereoselective reduction

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Scheme 2.9  First-generation strategy for total synthesis of madindolines

of 48, silylation, and reduction followed by Dess-Martin oxidation furnished 49. However, all attempts to achieve reductive coupling of the derived aldehyde 49 with tryptophol 43 or 3a-hydroxyfuroindoline 44 proved unsuccessful. Formation of the intermediate imine appeared to be the problem, presumably due to the poor nucleophilicity of the indole or furoindoline nitrogen. With the more nucleophilic indoline 50, reductive alkylation furnished 51 in high yield as a diastereomeric mixture. Having achieved the union of 51 with indoline, all that remained to arrive at the madindolines was generation of the enedione and indole moieties and elaboration of the 3a-hydroxyfuroindoline ring. To this end, removal of the silyl groups in 51, followed in turn by selective silylation of the primary hydroxyl, oxidation, and acid hydrolysis, afforded indole 52. Oxidative ring closure of 52 then yielded ­(+)-madindoline A 41 and (−)-madindoline B 42 (2.2:1) (Scheme 2.9). Our (−)-madindoline B 42 is the enantiomer of natural (+)-42. Confirmation of the relative configurations in 41 and 42 was achieved by X-ray analysis of synthetic (+)-41. Therefore, the absolute configuration of natural (+)-madindoline A is 3aR,8aS,2’R and (+)-madindoline B is 3aR,8aS,2’S. Next, we developed a more efficient and practical total synthesis of (+)-madindolines A (+)-41 and B (+)-42 [73, 74]. Reductive amination of optically active 3a-hydroxyfuroindoline (−)-44 with aldehyde 53 using acetic acid, followed by iminium reduction, silylation, hydrolysis, and oxidation followed by esterification furnished the methyl ester 54. The final stages of the synthesis involved diastereoselective acylation of ester 54 carrying a

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Scheme 2.10  Second-generation strategy for total synthesis of madindolines

3a-hydroxyfuroindoline moiety with the acid chloride 57. The ester 54 was treated with LDA followed by treatment with acid chloride 57 to afford the desired compound 55 as a single isomer in quantitative yield. We predicted that the lithium enolate of compound 54 would coordinate with oxygen of the furan ring on the chiral 3a-hydroxyfuroindoline to make a rigid conformation and that diastereoselective acylation would occur, producing 55 (Scheme 2.10). Finally, an intramolecular endo cyclization of allylsilane 55 using tris(dimethylamino)sulfur(trimethylsilyl) difluoride (TASF) directly led to (+)-madindoline B (+)-42. In the total synthesis of (+)-madindoline A (+)-41, the stereoselective acylation of 54 with acid chloride 58 predominantly afforded the desired compound 56 in high yield. The intramolecular endo cyclization of allylsilane 56 with tetrabutylammonium triphenyldifluorosilicate (TBAT) directly led to (+)-madindoline A (+)-41. Since madindolines are non-peptidal low molecular specific inhibitors of IL-6, studies on their action mechanism to IL-6 functions are expected to provide ­significant information about IL-6-dependent diseases as well as possible therapeutic uses of the compounds. After completion of the total syntheses of madindolines, we have prepared the radioactively labeled madindoline A to facilitate the studies on its mode of action. We revealed the binding site of (+)-41 in the complex of gp130, IL-6, and IL-6 receptor (IL-6R) by the use of [3H]-(+)-41. In a dose-dependent manner, [3H]-(+)-41 binds to gp130, which is a signal-­ transducing 130-kDa glycoprotein, but formation of the trimeric complex IL-6/IL-6 receptor/gp130 was not inhibited, suggesting that madindoline A suppresses dimerization of trimeric complexes. Not only did madindoline A markedly inhibit IL-6and IL-11-induced osteoclastogenesis in vitro, but it also inhibited IL-6-stimulated serum amyloid A production and bone resorption in an experimental model of post-

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Fig. 2.16  The mode of action of madindoline A

menopausal osteoporosis in vivo by a different mechanism from that of 17β-estradiol. Madindoline A has a highly selective inhibitory effect on IL-6 and IL-11 activities by inhibiting a gp130 activity while suppressing bone loss in ovariectomized mice. Madindoline A is anticipated as a lead compound for treatment of hormone-­ dependent postmenopausal osteoporosis, which has no serious side effects, and as a new mechanism of action, gp130 blocking. IL-6 has three topological binding sites (sites I, II, and III), whereas gp130 has two binding sites (sites 1 and 2). IL-6 binds to the IL-6R via its site I and then to gp130 via site II, forming a trimeric IL-6/ IL-6R/gp130 complex. The trimeric complex then induces homodimerization of gp130 and forms a hexameric complex. We found that (+)-madindoline A binds to gp130 and inhibits the actions of IL-6 activity without inhibiting the formation of the IL-6/IL-6R/gp130 complex (Fig. 2.16) [75]. In summary, in the second-generation synthesis, the syntheses of 41 and 42 are highly efficient, proceeding in 16% and 19% overall yield for nine linear steps, respectively, stereocontrolled, and amenable to gram-scale production. Actually, we have synthesized 3 g of madindoline A. We also confirmed that synthetic madindoline A 41 markedly inhibited osteoclastogenesis in vitro and inhibited bone resorption in ovariectomized (OVX) mice in vivo, and the use of tritiated [3H]-(+)-madindoline A revealed the mode of action for manifestation of inhibitory activity. We believe that madindolines can serve as lead compounds for development of new drugs to treat refractory diseases known to involve IL-6, such as cancer cachexia, Castleman’s disease, rheumatoid arthritis, and hypercalcemia [75].

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7  Total Synthesis of Neoxaline During the course of our chemical screening of microbial metabolites, neoxaline 59 [76] was isolated from the culture broth of Aspergillus japonicus Fg-551, together with the structurally related known compound oxaline 60 [77]. Neoxaline 59 and oxaline 60 (Fig. 2.17) are members of a novel class of biologically active indole alkaloids characterized by a unique indoline spiroaminal framework and substitution of a 1,1-dimethylallyl (“reverse-prenyl”) group at the benzylic ring junction. The relative stereochemistry of 60 has been previously established by X-ray analysis. Hence, the structure of 59 was estimated by comparison with 60; however, the relative and absolute configurations of 59 remain undefined. 59 and 60 were found to inhibit cell proliferation and arrest the cell cycle during M phase in Jurkat cells. Compounds 59 and 60 bind to tublin at, or near, the colchicine binding site, which results in inhibition of tubulin polymerization [78]. The highly complex indoline spiroaminal framework of the neoxalines was recognized as an attractive target for total synthesis. So, we have first developed the concise stereoselective synthesis of the indoline spiroaminal framework 61 of 59 and 60 [79]. Regioselective alkylation of excess indole with chiral epoxide 62 with Yb(OTf)3 gave the excellent yield. Silylation of the secondary hydroxy group, followed by Boc protection of the amino group and desilylation, afforded the alcohol 63. Next, selenylation-induced ring closure with N-phenylselenophthalimide (N-­ PSP) [80] provided the separable diastereo mixture (1:1) of 3a-selenylated furoindolines 64 and 65. Treatment of 64 with methyl triflate and prenyltri(n-butyl) stannane introduced the reverse prenyl group to the desired position to give 66 with either stereochemistry (Scheme 2.11). BOC deprotection of 66, reprotection with Alloc group, methyl ester hydrolysis, and condensation with glycine amide 67 afforded 68. Subsequent deprotection of the Alloc group gave 69 in high yield (Scheme 2.11). Treatment of aminal 69 with AlMe3 facilitated transcyclization to afford diaminal 72, through the iminium intermediate 71, in good yield. Subsequent tungstate-­ catalyzed oxidation of 72 gave nitrone 73, which was then treated with silica gel to afford spiroaminal compound, followed by methylation affording the desired

Fig. 2.17  Structures of neoxaline 59 and oxaline 60

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Scheme 2.11  The synthesis of the indoline spiroaminal framework of neoxaline (part 1)

Scheme 2.12  The synthesis of the indoline spiroaminal framework of neoxaline (part 2)

indoline spiroaminal framework 61 (Scheme 2.12). Compound 61 is a versatile intermediate for the synthesis of the neoxaline family. Moreover, first total synthesis of neoxaline 59 was accomplished [81, 82]. Optically active 44, which was prepared from tryptophol 43 under modified Katsuki-Sharpless asymmetric epoxidation conditions, was protected by Alloc and trichloroacetimidate. Next, the allylation took place with prenyl tributylstannane in the presence of BF3· OEt2 to afford 75 as a single isomer. After removal of the Alloc

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Scheme 2.13  Synthesis of cyclization precursor

group, reductive ring opening of the tetrahydrofuran, reprotection with an Alloc group, and the hydroxyl group was oxidized to give the corresponding aldehyde 76. Next, boric acid-mediated addition of isocyanoacetate 77 afforded α-hydroxyamide 78 as an inseparable diastereomeric mixture (9R:9S = 1:2). The diastereomeric ratio of 78 could be changed to 9R:9S = 9:1 by oxidation of the hydroxyl group to the ketone followed by a reduction process, and TBS protection subsequent removal of the Alloc group and treatment of 2 M NH3 afforded amide (9R)-79 (Scheme 2.13). Oxidation of (9R)-79 with H2O2·urea in the presence of NaWO4 provided nitrone (9R)-80 and diaminal (9R)-81, followed by treatment with Et3N affording (9R)-81 in excellent yield. The cyclic diaminal (9R)-81 was oxidized with Pb(OAc)4 to afford the N-oxoamidine (9R)-82. Next, treatment of tetrabutylammonium hydroxide (TBAOH) was the most efficient reagent for generation of indoline spiroaminal (9R)-83 in excellent yield as a single diastereomer (Scheme 2.14). With the core framework assembled, the final issue was the introduction of the conjugated imidazole at C-12. The hydroxyl group and amide of 83 needed to be protected by TBS and 2-(trimethylsilyl)ethoxymethyl (SEM) groups, respectively. The aldol reaction with 84 and imidazolyl aldehyde 85 proceeded smoothly to give 86 together with a minor diastereomer (a/b = 5.7:1). The elimination reaction of 86 provided (Z)-dehydrohistidine 87 as a single isomer in good yield. Next, selective deprotection of the N-hydroxide by TBAF followed by methylation provided methoxide 88. Removal of all of the protecting groups generated 90, the geometrical isome of neoxaline, in two steps. Fortunately, in the treatment with Me3Al to remove the SEM and Boc groups, the C9 stereogenic center was completely epimerized to the S configuration. Finally, the photoisomerization of unnatural (Z)neoxaline 90 upon mercury lamp irradiation (λ  >  254  nm) provided natural (E)-neoxaline 59 in unsatisfactory yield (26% with 58% recovery of the Z isomer 90). In the course of studying the isomerization, we found that the geometric isomers of neoxaline were at equilibrium under the photoirradiation conditions. Thus, to improve the efficiency of the isomerization, we attempted selective excitation of the unnatural Z isomer 90. Since the maximum absorption wavelengths of the Z and

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Scheme 2.14  Construction of indoline spiroaminal

E isomers were 314 and 330 nm, respectively, a short-pass filter that blocked light with λ > 325 nm was applied. As a result, the desired isomerization was efficiently induced, affording natural (E)-59 in good yield (55% with 35% recovery of Z isomer 90; 77% in three cycles) (Scheme 2.15). In conclusion, a first asymmetric total synthesis and determination of the absolute configuration of neoxaline 59 has been accomplished through the highly ­stereoselective introduction of a reverse prenyl group to create a quaternary carbon stereocenter using (−)-3a-hydroxyfuroindoline 44 as a building block, construction of the indoline spiroaminal via cautious stepwise oxidations with cyclizations from the indoline, assembly of (Z)-dehydrohistidine, and photoisomerization of unnatural (Z)-neoxaline to the natural (E)-neoxaline as the key steps.

8  Concluding Remarks Natural products isolated or derived from microorganisms frequently embody “privileged structures,” which bind to various protein-receptor surfaces [83]. Naturally occurring bioactive microbial metabolites were discovered by different screening methods in order to create novel medicines in the Kitasato Institute. We also have achieved efficient and concise total synthesis, as well as determination of the absolute stereochemistry, of these bioactive microbial metabolites such as pyripyropenes (cholesterol-lowering agents), arisugacins (acetylcholinesterase inhibitors), lactacystin (proteasome inhibitor), macrosphelides (Cell-cell adhesion inhibitors), madindolines (IL- 6 modulators), and neoxaline (cell proliferation inhibitor). It is very important to collaborate between the natural products and the organic synthesis in order to create the novel medicines (Fig. 2.18).

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Scheme 2.15  Total synthesis of neoxaline

Fig. 2.18 Collaboration between the natural products and the organic synthesis for drug discovery

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Acknowledgments  We thank all of our co-workers who carried out the research described here; their names are listed in the references. This work was supported in part by a Grant of the twenty-­ first Century COE Program, Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Japan Science and Technology Corporation (JST), a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan, and the Japan Keirin Association.

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6 5. T. Hirano, Int. Rev. Immunol. 16, 249 (1998) 66. T. Kishimoto, S. Akira, T. Taga, Science 258, 593 (1992) 67. B. Stein, S. Kung, S. May, Drug Discovery Today 3, 202 (1998) 68. M. Hayashi, Y.-P. Kim, S. Takamatsu, A. Enomoto, M. Shinose, Y. Takahashi, H. Tanaka, K. Komiyama, S. Ōmura, J. Antibiot. 49, 1091 (1996) 69. S. Takamatsu, Y-P. Kim, A. Enomoto, M. Hayashi, H. Tanaka, K. Komiyama, S. Ōmura, J. Antibiot. 50, 1069 (1997) 70. T. Sunazuka, T. Hirose, T. Shirahata, Y. Harigaya, M. Hayashi, K. Komiyama, S. Ōmura, A.B. Smith, III, J. Am. Chem. Soc. 122, 2122 (2000) 71. S. Hosokawa, K. Sekiguchi, K. Hayase, Y. Hirukawa, S. Kobayashi, Tetrahedron Lett. 41, 6435 (2000) 72. C.C. McComas, J.B. Perales, D.L. Van Vranken, Org. Lett. 4, 2337 (2002) 73. T. Hirose, T. Sunazuka, D. Yamamoto, K. Kojima, T. Shirahata, Y. Harigaya, I. Kuwajima, S. Ōmura, Tetrahedron 61, 6015 (2005) 74. T. Sunazuka, T. Hirose, S. Ōmura, J. Synth. Org. Chem. Jpn. 63, 1090 (2005) 75. M. Hayashi, A. Fukami, M-C. Rho, Y. Sekiguchi, T. Sunazuka, T. Hirose, K. Komiyama, S. Ōmura, Proc. Natl. Acad. Sci. USA 99, 14728 (2002) 76. A. Hirano, Y. Iwai, R. Masuma, K. Tei, S. Ōmura, J. Antibiot. 32, 781 (1979) 77. P.S. Steyn, Tetrahedron 26, 51 (1970) 78. Y. Koizumi, M. Arai, H. Tomoda, S. Ōmura, Biochem. Biophys. Acta 1693, 47 (2004) 79. T. Sunazuka, T. Shirahata, S. Tsuchiya, T. Hirose, R. Mori, Y. Harigaya, I. Kuwajima, S. Ōmura, Org. Lett. 50, 453 (2005) 80. K.M. Depew, S.P. Marsden, D. Zatorska, A. Zatorski, W.G. Bornmann, S.J. Danishefsky, J. Am. Chem. Soc. 121, 11953 (1999) 81. T. Ideguchi, T. Yamada, T. Shirahata, T. Hirose, A. Sugawara, Y. Kobayashi, S. Ōmura, T. Sunazuka, J. Am. Chem. Soc. 135, 12568 (2013) 82. T. Yamada, T. Ideguchi, T. Hirose, T. Shirahata, R. Hokari, A. Ishiyama, M. Iwatsuki, A. Sugawara, Y. Kobayashi, K. Otoguro, S. Ōmura, T. Sunazuka, Chem. Eur. J. 21, 11855 (2015) 83. R. Breinbauer, I.R. Vetter, H. Waldmann, Angew. Chem. Int. Ed. 41, 2878 (2002)

Chapter 3

Enantioselective Total Synthesis of the Antitumor Polycyclic Natural Products FR182877 and Taxol Masahisa Nakada

Abstract  A total synthesis of (−)-FR182877 is described that features a one-pot stereoselective intramolecular Diels-Alder and hetero-Diels-Alder reaction cascade to form the ABCD ring system of (−)-FR182877, the palladium-mediated 7-exo-­ trig reaction to construct the strained seven-membered F-ring, and the iridium-­ mediated isomerization of an allylic alcohol to an α-methyl ketone followed by stereoselective transformations. The enantioselective synthesis of the DEF-ring moiety of (−)-FR182877 was also accomplished. Derivatives of the resulting product could induce mitotic arrest by interfering with the microtubule dynamics, similar to the cellular effects induced by paclitaxel. The eight-membered ring of taxol was synthesized by a palladium-catalyzed intramolecular alkenylation of a methyl ketone in 97% yield, allowing for an efficient formal total synthesis of (−)-taxol. To the best of our knowledge, this is the first example of a palladium-catalyzed intramolecular alkenylation to form an eight-membered carbocyclic ring in natural product synthesis. During the preparation of a substrate for such palladium-catalyzed reaction, a rearrangement of an epoxy benzyl ether including a 1,5-hydride shift unexpectedly took place generating the C3 stereogenic center and C1–C2 benzylidene, and this was successfully used to attain the formal total synthesis of (−)-taxol. Keywords  Cascade reaction · Cycloaddition · Enantioselective synthesis · Natural product · Palladium-catalyzed reaction · 1,5-Hydride shift

M. Nakada (*) Department of Chemistry and Biochemistry, Faculty of Science and Engineering, Waseda University, Tokyo, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2019 Y. Kobayashi (ed.), Cutting-Edge Organic Synthesis and Chemical Biology of Bioactive Molecules, https://doi.org/10.1007/978-981-13-6244-6_3

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1  Introduction We decided to pursue the enantioselective total synthesis of polycyclic natural products endowed with important biological activities due to the fact that such bioactive compounds pose a synthetic challenge that arises from their complex and unique structures requiring the formation of many bonds and stereogenic centers. Herein, the enantioselective total syntheses of antitumor polycyclic antibiotics such as (−)FR182877 and taxol achieved by us are reviewed highlighting the key reactions in each synthesis. Moreover, the design, synthesis, and biological evaluation of new derivatives of (−)-FR182877 that induce mitotic arrest are also described.

2  (−)-FR182877 2.1  Introduction A research group at Fujisawa Pharmaceutical Co. (now Astellas Pharma) isolated WS9885B and its congener from Streptomyces sp. No.9885. These compounds, which were later named as (−)-FR182877 (1) [1–4] and (−)-FR182876 [5] (Fig. 3.1), respectively, bind to tubulin and stabilize microtubules and exhibit potent cytotoxic activity against human cancer cell lines. The cytotoxic potency of 1 was found to be comparable to that of taxol [1–5], and further in  vivo assays using mouse models recognized 1 as a promising antitumor lead compound for development of chemotherapeutic agents [3]. (−)-FR182877 (1) possesses a unique hexacyclic structure that includes 12 contiguous stereogenic centers and a vinylogous carbonate, which is a reactive push-pull alkene [3] distorted by an ethylene bridge. The intriguing biological activity and mode of action of this molecule together with its unprecedented structural features make 1 an attractive compound for synthetic [6–17] and chemical biology studies [18–22]. The research groups of Sorensen [6, 7] and Evans [8, 9] have independently reported the elegant enantioselective total synthesis of 1 employing similar synthetic strategies, namely, stereoselective transannular cycloadditions (Scheme 3.1). We were also interested in the complex structure of 1 as well as its potent bioactivity and unique mode of action [11–14] and successfully accomplished its e­ nantioselective

Fig. 3.1  Structures of (−)-FR182877 (1) and (−)-FR182876

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Scheme 3.1  Sorensen’s and Evans’ total syntheses of (−)-FR182877 (1)

Scheme 3.2  Construction of the AB-ring moiety by the stereoselective IMDA reaction

total synthesis based on an alternative approach [10] that led to the discovery of new derivatives of 1 displaying mitotic arrest activity [15].

2.2  Enantioselective Total Synthesis of (−)-FR182877 We have reported the stereoselective syntheses of the AB- and the CD-ring moieties of 1 via an intramolecular Diels-Alder (IMDA) [11, 12] and intramolecular hetero-­ Diels-­Alder (IMHDA) reaction [13, 14], respectively, as shown in Scheme 3.2 and Scheme 3.3. It was envisioned that these cycloadditions could proceed sequentially, i.e., the IMDA-IMHDA reaction cascade of the acyclic substrate 4 could afford the tetracyclic compound 6 via 5 (Scheme 3.4). Aiming to investigate the above IMDA-IMHDA reaction cascade, compound 3 was prepared from the previously reported intermediate 2 (Scheme 3.4) [14]. The C6 hydroxy group of 2 was protected as a TES ether and the subsequent removal of the acetyl group afforded 3, which was then reacted with MnO2. When this reaction was carried out in methylene chloride, the formation of a complex mixture of products including aldehyde 4 was observed. However, when the MnO2 oxidation of 3 was conducted in toluene at 80 °C, the desired product 6 was obtained in 28% yield. Extensive analysis of the reaction products did not reveal the presence of any other tetracyclic isomers, though undesired side-products with a five-six fused ring and an aldehyde residue were detected in the mixture.

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Scheme 3.3  Construction of the CD-ring moiety by the stereoselective IMHDA reaction

Scheme 3.4  Transformation of 2 to 7 via the stereoselective IMDA-IMHDA reaction cascade

Although the reason for the observed stereoselectivity has not been completely clarified, it can be assumed that the stereoelectronic effect induced by the C6 allylic substituent could influence the diastereoselectivity [12, 23]. It should be noted that the overall yield of 6 from 2 was slightly improved when compared with that of our stepwise synthesis of 6 [14]. However, the desired compound 6 could be more easily separated from other products in the tandem ­IMDA-­IMHDA reaction when compared with the stepwise protocol. To the best of our knowledge, this cascade cycloaddition reaction has not been described before for acyclic compounds [24]. Before addressing the construction of the strained EF-ring moiety of 1, the configuration of the C6 stereogenic center of 6, which is necessary to achieve a stereoselective IMDA reaction, had to be inverted [11–14]. However, after removal of the TES protecting group of compound 6, Mitsunobu inversion of the resulting C6 hydroxy group afforded no products. Hence, the generated alcohol was first converted to 7 by Dess-Martin oxidation and then subjected to stereoselective reduction. The reduction of 7 (Scheme 3.5) was investigated in the presence of a variety of reducing reagents (Table 3.1). The use of LiEt3BH and Li(s-Bu)3BH afforded C6-epi 8 as the sole product (Table 3.1, entries 1 and 2). Reactions performed with less-­ hindered reagents, such as NaBH4 or LiBH4, resulted in low stereoselectivity (Table 3.1, entries 3 and 4). Finally, the reduction of 7 with BH3 afforded the desired

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Table 3.1  Stereoselective reduction of 7 (Scheme 3.5) Entry 1 2 3 4 5 6 a

Reagents LiEt3BH Li(s-Bu)3BH NaBH4 LiBH4 BH3·THF BH3·THF

Solvent THF THF MeOH THF THF THF

Temp (°C) −78 −78 0 −78 0 −20

Time (min) 60 30 30 60 15 30

Yield of 8 (%)a 0 0 23 45 57 72

Yield of C6-epi-8 (%)a 63 75 77 45 9 3

Ratio of 8:C6-epi-8 0:1 0:1 1:3.3 1:1 6.4:1 24:1

Isolated yield

Scheme 3.5  Stereoselective reduction of 7 (Table 3.1)

Scheme 3.6  Preparation of 10

β-alcohol 8 as the major product (57%, dr = 6.4/1) (Table 3.1, entry 5). Moreover, the yield and selectivity were considerably improved by conducting the reaction at −20 °C (75%, dr = 24/1) (Table 3.1, entry 6). After having achieved the synthesis of the A-D ring unit of 1, we next focused on the preparation of the F-ring moiety. After several attempts, the palladium-catalyzed 7-exo-trig cyclization [25–35] of 10 was found to allow for the construction of the F-ring moiety. The preparation of 10 is shown in Scheme 3.6. Treatment of the TBS ether of 8 with LDA and iodine afforded 9, which then underwent removal of the TBS group, Dess-Martin oxidation, and treatment with vinylzinc bromide to provide a separable mixture of 10a and 10b. The use of a Grignard reagent in place of vinylzinc bromide afforded a complex mixture of products most likely due to its reaction with the methyl ester functionality. The reaction of 10a with Pd(PPh3)4 (Scheme 3.7) did not afford the desired product (Table 3.2, entry 1), while the use of Pd2(dba)3 and DPPF in refluxing acetonitrile successfully afforded 11b in 59% yield (Table 3.2, entry 2). The yield of 11a decreased when the reaction was carried out in DMF (Table 3.2, entry 3), while an improved yield of 88% was obtained in toluene at 100 °C (Table 3.2, entry 4). When compound 10b, the diastereomer of 10a, was subjected to the same reaction

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Table 3.2  The palladium-catalyzed 7-exo-trig cyclization of 10a and 10b (Scheme 3.7) Entry 1b 2 3 4 5 6 7f

Substrate 10a 10a 10a 10a 10b 10a + 10bd 10a + 10bd

Solvent MeCN MeCN DMF Toluene Toluene Toluene Toluene

Temp (°C) Reflux Reflux 100 100 100 100 100

Time (h) 24 4 1 1 1 1 7.5

Yield (%)a –c 11a (59) 11a (23) 11a (88) 11b (73) 11a + 11be (71) 11a + 11be (60)

Isolated yields Pd(PPh3)4 was used c No products except the deiodonated compound were obtained d 10a:10b = 1:1 e 11a:11b = 1.3:1 f Pd2(dba)3 (15 mol %) and DPPF (30 mol %) were used a

b

Scheme 3.7  The palladium-catalyzed 7-exo-trig cyclization of 10a and 10b (Table 3.2)

­conditions, 11b was isolated in 73% yield (Table 3.2, entry 5). The reaction of a mixture of 10a and 10b also afforded the corresponding products in 71% yield (Table 3.2, entry 6), while a reduced amount of catalyst (15 mol %) afforded products 11a and 11b in 60% yield (Table 3.2, entry 7). Next, the C18 methylene group needed to be converted into a methyl group. Theoretical calculations (PM3) indicated that the desired compound 13a might be energetically favored when compared with its diastereomer 13b (Scheme 3.8). These results suggested that the epimerization of 13b to 13a might be favored. Hence, the 1,4-reduction of 12 was first attempted, but a mixture of products including 14 (Table 3.3) was formed owing to the conjugating α,β-unsaturated ester. Another approach was therefore pursued based on the isomerization of 11 to 13a, not only such route is advantageous over the oxidation-reduction sequence in terms of synthetic steps, but also, even when a mixture of 13a and 13b is formed, the epimerization of the mixture is expected to afford thermodynamically stable 13a as mentioned above. Hence, we examined the isomerization of allylic alcohol 11 to α-methyl ketone 13a (Scheme 3.9) under different conditions (Table 3.3). Allylic alcohols and ethers are known to easily isomerize to the corresponding vinyl alcohols and ethers, respectively, under transition metal catalysis.

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Scheme 3.8  Possible pathway from 11 to 13a and energetic difference between 13a and 13b calculated by the PM3 method

Table 3.3  Isomerization of allylic alcohol 11 to α-methyl ketone 13a (Scheme 3.9) Entry 1b 2b 3 4b 5

Reagents (equiv) [RuCl2(p-cymene)]2 (1.0), K2CO3 (3.0) RuCl2(PPh3)3 (1.0), K2CO3 (3.0) RhH(PPh3)3 (1.0) RhH(PPh3)3 (0.3) [IrCl(COD)]2 (10.0), K2CO3 (20.0)

Solvent THF Toluene THF THF THF

Time (h) 4 12 0.5 5.5 24

Yield (%)a 13a 14 – –c – –d 53 28 50 27 62 –

Isolated yields Without DBU treatment c 92% of 12 was isolated d 67% of 12 was isolated a

b

Scheme 3.9  Isomerization of allylic alcohol 11 to α-methyl ketone 13a (Table 3.3)

Among various reagents [36], a ruthenium-mediated isomerization [37, 38] was first attempted; however, the treatment of a mixture of compounds 11a and 11b with Ru(II) catalysts resulted in the oxidation to dienone 12 (Table 3.3, entries 1 and 2).

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Scheme 3.10  Enantioselective total synthesis of (−)-FR182877

The reaction with RhH(PPh3)3, which was prepared in situ by reacting the Wilkinson complex with n-BuLi [39], smoothly afforded a mixture of 13a, 13b, and 14. Treatment of this mixture with DBU in benzene at room temperature induced epimerization affording a mixture of 13a (53%) and 14 (28%) (Table 3.3, entry 3). Longer reaction time (5.5 h) and a catalytic amount of RhH(PPh3)3 (30 mol %) promoted the epimerization affording a mixture of 13a (50%) and 14 (27%) (Table 3.3, entry 4), which was difficult to separate by silica gel column chromatography. Finally, the reaction with [IrCl(COD)]2 [40] successfully provided a mixture of 13a and 13b without formation of 14; treatment of this mixture with DBU led to 13a in 62% yield as a single isomer (Table 3.3, entry 5). Large amounts of reagents were used when the reaction was carried out in a small scale; however, catalytic amounts could be used for large-scale reactions. The stereoselective reduction of ketone 13a with NaBH4 afforded the desired alcohol 15 as the sole product (Scheme 3.10). Complete removal of the silyl protecting groups of 15 with HF·Py to afford 16, cleavage of the methyl ester with TMSOK, and lactonization in the presence of Mukaiyama reagent [41] finalized the total synthesis of (−)-FR182877. The synthesized compound was proved to be identical to the natural product in all respects, as confirmed by 1H-NMR, IR, MS, [α]D, and 13C-NMR. In summary, an enantioselective total synthesis of (−)-FR182877 has been achieved that features a one-pot stereoselective IMDA-IMHDA reaction cascade to form the ABCD ring system of (−)-FR182877, palladium-mediated 7-exo-trig reaction to construct the strained seven-membered F-ring, and iridium-mediated isomerization of an allylic alcohol to an α-methyl ketone followed by epimerization and stereoselective reduction.

2.3  E  nantioselective Synthesis and Bioactivity of the Pharmacophore of (−)-FR182877 Since biological studies [21] of (−)-FR182877 (1) revealed that this compound irreversibly binds to a previously unidentified site on the outer wall of microtubules, we became interested in evaluating the biological activity of the proposed

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Scheme 3.11  Structures of (−)-FR182877 (1) and 17, and retrosynthetic analysis of 17

Scheme 3.12  Preparation of 19

pharmacophore of 1, i.e., the highly strained DEF-ring moiety. Hence, we started the synthesis of compound 17 (Scheme 3.11). The DEF-ring moiety of 1 is highly strained owing to the ethylene bridge between C15 and C19, and thereby, the C2–C17 alkene of 1 is highly distorted and reactive and easily oxidized in air to afford the corresponding epoxide [3]. In view of this, it is reasonable to synthesize lactone 17 from 18 (Scheme 3.11). Compound 18 could be obtained in turn from 19, which could be prepared via the IMHDA reaction of 20. This type of cycloaddition is challenging and has never been reported before. It can be expected that the reaction would proceed due to the fact that the electron-­ withdrawing ester group and the electron-donating methyl group are attached at suitable positions of the oxa-butadiene and the terminal alkene, respectively. Compound 20 could be prepared by oxidation of 21, which could be derived from 22, prepared in turn from 23. Thus, aldehyde 24, prepared in three steps from a commercially available material [42], was subjected to Evans aldol reaction with 25 to afford 23 (Scheme 3.12). Compound 23 was converted to Weinreb amide, followed by protection of the hydroxy group as a TES ether. The DIBAL-H reduction of 26, and subsequent reaction with an aluminum reagent, which was generated in situ by the reaction of methyl propiolate and DIBAL-H [43], afforded 21. Next, Dess-Martin oxidation of 21 furnished 20. The IMHDA reaction of 20 was carried out in toluene at 100 °C in the presence of BHT. The reaction required 4 days to afford 19 in 63% yield as a single isomer.

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Scheme 3.13  Preparation of 18 Scheme 3.14 Preparation of 17 and 29–32

Fig. 3.2  X-ray crystal structure of 29

We next attempted the conversion of 19 to 18 under the same conditions employed in the total synthesis of (−)-FR182877, but interestingly, the ester was not cleaved due to the stability of the vinylogous carbonate system. Since all attempts to prepare 18 from 19 failed, we decided to replace the methyl ester with a PMB ester, which could be removed by treatment with DDQ or acid. While the conversion of 19 to the corresponding PMB ester failed, 21 was successfully converted to PMB ester 27 using Otera’s catalyst [44] (Scheme 3.13). Dess-Martin oxidation of 27 and subsequent IMHDA reaction afforded 28 as a single isomer, which was treated with trifluoroacetic acid to successfully provide 18. The reaction of 18 with the Mukaiyama reagent (2-chloro-1-methylpyridinium iodide) [41] successfully afforded 17 (Scheme 3.14). However, this product was not isolated as such but in the crystalline form 29. The absolute structure of 29 was elucidated by X-ray crystallographic analysis (Fig.  3.2). It can be assumed that

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Fig. 3.3  Immunostaining images obtained using control (I), paclitaxel (10  nM) (II), and 31 (200 μM) (III) (left) and the mitotic indexes (right)

compound 29 was formed upon reaction of 17 with water during the workup phase. Thus, 17 was highly reactive, and in situ treatment with methanol after its formation easily afforded 30. Compounds 29 and 30 were stable, and 29 was successfully converted to acetate 31 and benzoate 32. Since 17 was difficult to isolate due to its high reactivity, the stable compounds 29–32 were subjected to the tubulin polymerization assay. Interestingly, although 29–32 showed no activity toward tubulin, 31 induced cell cycle arrest during the M phase in HeLa cells within 12 h of treatment (Fig. 3.3). This is consistent with the effects of paclitaxel, which causes disruption of the microtubule dynamics preventing normal mitotic progression and leading to mitotic arrest. As shown in the immunostaining images in Fig. 3.3, bipolar spindle formation was severely inhibited in HeLa cells after treatment with both paclitaxel (10 nM) and 31 (200 μM). These data strongly indicated that 31 has the ability to induce mitotic arrest by interfering with the microtubule dynamics, and its cellular effects of 31 are similar to those of paclitaxel. In summary, the enantioselective and highly stereoselective synthesis of 17, which corresponds to the DEF-ring moiety of (−)-FR182877 (1), was accomplished via the IMHDA reaction. Compound 17 was found to be highly reactive and easily afforded 29 upon reaction with water. Compound 31, which is the acetate of 29, showed no activity in the tubulin polymerization assay. However, interestingly, 31 induced cell cycle arrest during the M phase in HeLa cells. The activity of 31 was not as high as that of paclitaxel, but such compound was nonetheless shown to induce mitotic arrest by interfering with the microtubule dynamics, exhibiting cellular effects similar to those of paclitaxel.

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3  Formal Total Synthesis of Taxol 3.1  Introduction Taxol (Fig. 3.4), which was isolated from the bark of the Pacific yew tree (Taxus brevifolia) [45, 46], has been widely used as an anticancer drug in the twenty-first century [47]. From a structural standpoint, taxol features a highly distorted 6–8-6 tricyclic bridged scaffold with nine stereogenic centers including two all-carbon quaternary centers, one of which is stereogenic, a variety of functional groups (such as oxetane, trans-1,2-diol, acyloin, secondary and tertiary alcohols, and a bridgehead double bond), and an amino alcohol side chain linked by an ester. The potent bioactivity and unprecedented complex structure has made taxol an attractive synthetic target. Indeed, more than 200 papers that describe synthetic studies [48–56] and 9 total syntheses of taxol (including formal total syntheses) have been published thus far [57–75]. Since medium rings (8–11-membered rings) are difficult to form due to steric strain, transannular interactions, and entropical effects, taxol is considered as a difficult synthetic target. Herein, our formal total synthesis of (−)-taxol via a convergent approach is reviewed.

3.2  C  onstruction of the Eight-Membered Ring of Taxol by Intramolecular B-Alkyl Suzuki-Miyaura Coupling Reaction A convergent total synthesis approach allows for the preparation of building blocks in parallel, which not only increases the synthetic efficiency but also enables independent transformations of functional groups that are otherwise incompatible with each other. Furthermore, a convergent synthesis is useful for preparing new derivatives with diverse structures. Thus, the convergent synthesis of taxol, i.e., the preparation and coupling of the A- and C-ring fragments and subsequent formation of the eight-membered B-ring followed by further transformations, has been investigated by many research groups; six convergent total syntheses of taxol [59–65, 70–74], including formal syntheses [72–75], have been reported to date. However, the formation of the eight-membered B-ring poses a challenge in organic synthesis as Fig. 3.4  Structure of (−)-taxol

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Scheme 3.15  Formation of the eight-membered ring of taxol in the convergent synthesis

described above and demonstrated by the reported convergent total syntheses (Scheme 3.15). The yield for the pinacol coupling described by Nicolaou et  al. was 23–25% [59–63], while the Heck reaction reported by Danishefsky et al. required a stoichiometric amount of the palladium reagent to afford the product in 46% yield (Scheme 3.15) [64, 65]. The Kuwajima group succeeded in the cyclization of a relatively simple substrate by reacting a silyl dienol ether with an acetal; however, the yield was 59% over two steps [70, 71]. Takahashi et al. used microwave irradiation to achieve the alkylation of a cyanohydrin ethoxy ethyl ether, though the yield was 49% [72]. Recently, the Chida’s group reported the formation of the eight-­membered ring using SmI2 in 66% yield, but their synthesis required some additional steps to introduce the double bond into the bridgehead position since the SmI2-mediated reaction was accompanied by a double bond migration [74, 75]. The reported strategies summarized in Scheme 3.15 indicate the difficulties associated with this step. The development of efficient methods for forming eight-­ membered rings could be valuable for natural product synthesis since a number of terpenoids including eight-membered rings have been reported. Furthermore, the developed methods could be applied for constructing other medium-sized rings. Hence, we decided to start an investigation into efficient methods for constructing eight-membered rings that could be applied to the total synthesis of taxol and disclosed a highly efficient formation of the B-ring of taxol by palladium-catalyzed cyclization (Scheme 3.15). We focused on the construction of eight-membered rings by palladium-catalyzed reactions in view of their high efficiency and mild reaction conditions [76–78]. The B-alkyl Suzuki-Miyaura coupling reaction was reported in 1986 [79] and was followed by an intramolecular version [80–86]; this reaction has also been utilized for natural product synthesis. However, to the best of our knowledge, the B-alkyl

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Scheme 3.16 Intramolecular B-alkyl Suzuki-Miyaura coupling reactions of 33 and 34

Suzuki-Miyaura coupling reaction has only been utilized for generating ten-­ membered rings. It was expected that a palladium-mediated reaction could be effective for forming eight-membered rings since it proceeds via reductive elimination of a nine-membered palladacycle intermediate, which is presumed to be more unstable than the eight-membered carbocyclic product to be formed. Thus, we investigated the formation of eight-membered rings by the B-alkyl Suzuki-Miyaura coupling reaction. It was found that the reaction of 33 with Pd(PPh3)4 (50 mol %) and NaOH (4.0 equiv) in MeCN/H2O (10:1) under reflux afforded 35 in 70% yield (Scheme 3.16). The same reaction conditions applied to 34, which bears diverse protecting groups, furnished compound 36 in 82% yield indicating that the selection of the C1 and C2 protecting groups is important to attain high yields, and a cyclic protecting group probably increased the strain in the transition states, resulting in a lower yield [87].

3.3  Preparation of the A-Ring Fragment We then examined to apply an intramolecular B-alkyl Suzuki-Miyaura coupling reaction to the convergent total synthesis of (−)-taxol. It was envisioned that the total synthesis of taxol could be accomplished via allylic oxidation of 38 at the C10 position (Scheme 3.17). Thus, 38 was selected as a key synthetic intermediate, which could be obtained via the B-alkyl Suzuki-Miyaura coupling reaction of 39. This compound could be prepared in turn from 40 by introducing the C3 stereogenic center, while 40 could derive from the assembly of A- and C-ring fragments. Since no commercially available compounds suitable for use as starting materials were found, we decided to prepare both fragments via asymmetric catalysis. 3.3.1  P  reparation of the A-Ring Fragment of Taxol Via Silicon-Tethered Intramolecular Alkylation At the preliminary stage of our total synthesis, the A-ring fragment 41-I was prepared from known 43 (Scheme 3.18) [88]. In this synthesis, we developed a silicon-­ tethered intramolecular alkylation of a ketone to generate the chiral tertiary alcohol [89]. Treatment of 44, which was prepared from 43, with tert-BuLi underwent a

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Scheme 3.17  Retrosynthetic analysis of (−)-taxol

Scheme 3.18  Preparation of the A-ring fragment 41-I from 43

Scheme 3.19  Enantioselective preparation of 47-I from 46-I via organocatalysis

rapid halogen-lithium exchange reaction and intramolecular alkylation of the ketone. Subsequent Tamao oxidation [90–92], acetonide formation, and Dess-­ Martin oxidation afforded 45, which was then subjected to eight conventional steps to successfully afford 41-I. 3.3.2  P  reparation of the A-Ring Fragment of Taxol Via an Enantioselective Organocatalytic Reaction In a further study, we explored an alternative short approach to the preparation of the A-ring fragment [93]. In particular, the enantioselective formation of an α-hydroxy aldehyde from 46-I (Scheme 3.19) using an organocatalytic method attracted our attention. The α-aminoxylation of an aldehyde with nitrosobenzene catalyzed by proline or its derivatives has been reported to afford the corresponding chiral α-hydroxyaldehyde [94–96]. However, the organocatalytic enantioselective α-oxygenation of α-branched aldehydes is rare [97–99], and the enantiomeric excess (ee) values of the products generally do not exceed 45%. List et al. reported

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the organocatalytic enantioselective α-benzoyloxylation of α-branched aldehydes and enals [100]. However, this protocol could not be utilized for 46-I, since the α-hydroxy aldehyde derived from 46-I was assumed to decompose via fragmentation under the basic reaction conditions required for removing the benzoate group. It was found that proline 48a and its derivatives 48b and 48c were effective to promote the enantioselective transformation of 46-I. However, owing to the unsatisfactory yield, an alternative short route for the preparation of the A-ring fragment was explored. 3.3.3  P  reparation of the A-Ring Fragment of Taxol Via Sharpless Asymmetric Dihydroxylation (SAD) Next, we investigated the preparation of the A-ring fragment via a Sharpless asymmetric dihydroxylation (SAD) [93]. The SAD of 49-I in the presence of a catalytic amount of (DHQ)2PHAL and K2OsO2·2H2O afforded 47-I in 83% yield (Scheme 3.20); however, the ee was low (45% ee). The use of DHQ-PHN led to an improvement of the ee to 76%, which nonetheless was still disappointing. We then examined the SAD of the silyl enol ether of 50-I, which was expected to give better results in view of the highly enantioselective SAD of a similar compound reported by Kuwajima and co-workers [101]. After several attempts, the SAD of 50-I using a catalytic amount of DHQ-PHN (15.0 mol %) and K2OsO2·2H2O (5.0 mol %) was found to afford 51-I in 64% yield and 91% ee, while the reaction of 50-Br [102] with DHQ-PHN (10.0 mol %) and K2OsO2·2H2O (5.0 mol %) furnished 51-Br in 92% yield and 91% ee. These results were reproducible in a gram-scale reaction. Compound 51-Br was successfully converted to 41-Br via formation of benzylidene intermediate 52, regioselective cleavage of the benzylidene acetal by DIBAL-H, and Dess-Martin oxidation (Scheme 3.21). Enantiomerically pure 41-Br was obtained following recrystallization. Compound 41-Br was prepared from a commercially available compound in seven steps. Hence, the preparation of 41-Br via

Scheme 3.20  The SAD of 49-I, 50-I, and 50-Br

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Scheme 3.21  Transformation of 51-Br to 41-Br via 52

Scheme 3.22  Attempted aldol reaction and Nozaki-Hiyama-type reaction and envisioned reliable stereoselective coupling of 41 and 54 to afford 55

Scheme 3.23  Preparation of 54 via the baker’s yeast reduction of 56

SAD represents the shortest route for the preparation of the A-ring fragment. Analogue 41-I was also prepared from 51-I according to the same procedure illustrated in Scheme 3.21.

3.4  E  nantioselective Preparation of the C-Ring Fragment of Taxol We attempted the stereoselective coupling reaction between the A- and C-ring fragments using model compounds. However, the coupling of 53 with either enolates or allylic metal compounds did not take place, probably owing to the steric hindrance (Scheme 3.22). Hence, we selected compound 54 as C-ring fragment to be used for the addition reaction with compound 41 to afford 55 and planned to form the C3 stereogenic center after the coupling. We have previously reported the preparation of 57 by the baker’s yeast reduction of 56 (Scheme 3.23) [103, 104]. Compound 57 possesses two stereogenic centers which have the same configurations as those of C-ring fragment 54. The transformation of 57 into 54 is shown in Scheme 3.23. Removal of the benzyl group of 57 by hydrogenolysis, benzylidene formation, and subsequent transformation by Barton’s protocol afforded iodide 58. The reductive cleavage of the benzylidene moiety with

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DIBAL-H and subsequent Dess-Martin oxidation provided an aldehyde, which successfully underwent the Takai reaction to give 54 in high yield.

3.5  C  oupling of the A- and C-Ring Fragments and Construction of the Taxane Scaffold by an Intramolecular B-Alkyl Suzuki-Miyaura Coupling Reaction The coupling of the A- and C-ring fragments met with success, as shown in Scheme 3.24. The reaction of 54 with n-butyllithium generated the corresponding alkenyllithium species, which subsequently reacted with 41-I to furnish 59 as a single isomer. The configuration of 59 shown in Scheme 3.24 was determined by X-ray crystallographic analysis, indicating that the reaction took place at the Si-face of the aldehyde. This stereoselectivity can be well explained according to the chelation model. Compound 59 was successfully converted to 60, which underwent an intramolecular B-alkyl Suzuki-Miyaura coupling reaction to afford 61 possessing a taxane scaffold in a gratifying 71% yield. However, the introduction of the oxygen atom at the C10 position on the B-ring was found to be difficult and could not be achieved despite our extensive efforts [105, 106]. Nevertheless, we found that the palladium-catalyzed cyclization was effective for the formation of the eight-­ membered ring. This fact encouraged us to explore another palladium-catalyzed reaction to construct the taxane scaffold.

3.6  C  onstruction of an Eight-Membered Ring by Palladium-­ Catalyzed Alkenylation of a Methyl Ketone It was assumed that a palladium-catalyzed coupling reaction could be a unique method for the formation of the taxane scaffold since the iodoalkene of the A-ring could undergo an oxidative addition with palladium. Hence, our attention was next turned to the palladium-catalyzed alkenylation of a methyl ketone [107]. In 1997, the groups of Miura [108], Buchwald [109], and Hartwig [110] independently reported the palladium-catalyzed intermolecular arylation of ketones, which led to

Scheme 3.24  Assembly of the A- and C-ring fragments and the intramolecular B-alkyl Suzuki-­ Miyaura coupling reaction of 60

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Scheme 3.25  Formation of 63 by palladium-catalyzed alkenylation of methyl ketone 62

the development of the alkenylation of ketones as well as its intramolecular variant [111–114], and their application to natural product synthesis [115–117]. However, the yield of such reaction was not high, and no palladium-catalyzed eight-­membered ring formation by alkenylation has been reported to date. We first examined the intramolecular alkenylation of methyl ketone 62 to evaluate its feasibility for the formation of the eight-membered ring (Scheme 3.25). The reaction was carried out under the reaction conditions described by the Solé group’s, i.e., the reaction using 30 mol % of Pd(PPh3)4 and 3.0 equivalents of t-BuOK in THF. Compound 63 was thus obtained in 58% yield, and a certain amount of the de-iodinated derivative of 62 was also formed. However, when the reaction was conducted in toluene, the yield increased to 82%. Finally, the use of PhOK [118, 119] afforded 63 in 96% yield. To the best of our knowledge, this is the first example of the construction of an eight-membered ring by palladium-catalyzed alkenylation of a methyl ketone.

3.7  F  ormal Total Synthesis of (−)-Taxol Via Palladium-­ Catalyzed Alkenylation of a Methyl Ketone The palladium-catalyzed alkenylation of a methyl ketone was applied to the total synthesis of taxol. The A-ring fragment 41-I and the C-ring fragment 64 were assembled to afford 65-I as a single isomer via a halogen-lithium exchange reaction (Scheme 3.26). Subsequent epoxidation with TBHP and VO(OEt)3 exclusively afforded 66-I. Treatment of 66-I with BF3·OEt2 induced a stereoselective 1,5-hydride shift and benzylidene formation cascade, which proceeded smoothly even if the substrate and products contained an acid-sensitive ethoxyethyl group. The ethoxyethyl group was partly removed under acidic conditions; therefore, the products were treated with hydrochloric acid to obtain 67-I (66%, 2 steps). The primary hydroxy group of 67-I was selectively oxidized; this was followed by TES ether formation at the secondary hydroxy group, reaction of the aldehyde with MeMgI, and Dess-Martin oxidation of the resultant alcohol to afford methyl ketone 68-I. The palladium-catalyzed alkenylation of 68-I successfully afforded 69 in 97% yield (Scheme 3.27). We confirmed that the palladium-catalyzed alkenylation of 68-Br also afforded 69 in 89% yield. When compared with the yields of other methods

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Scheme 3.26  Preparation of methyl ketone 68 via 1,5-hydride shift

Scheme 3.27  Formal total synthesis of (−)-taxol via the palladium-catalyzed alkenylation of 68

used for constructing the B-ring of taxol (Scheme 3.15), 97% yield is an exceptionally high yield. Considering that nearly 20 steps are required to achieve the total synthesis of taxol after the construction of the taxane scaffold, an efficient ring-­ closing method is desirable to supply the key synthetic intermediate.

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Scheme 3.28  Kuwajima successful epimerization of 75 and our failed example

Acetylation of the C4 hydroxy group did not proceed in the case of many synthetic intermediates; however, after extensive studies, it was found that the acetylation was possible when a hydroxy group was introduced at the C10 position of 72. Compound 72 was prepared from compound 69 through the removal of the TES group, Dess-Martin oxidation, and Takai methylenation to afford 70, followed by conversion via intermediate 71 based on reported reaction conditions [59–62]. The C10 hydroxy group was only introduced into a limited number of compounds, too, owing to the specific structure of the taxane scaffold. We also found that only few compounds underwent epimerization at the C10 position. Kuwajima and co-workers reported the successful epimerization of 75 to 76 (Scheme 3.28). However, our structurally related compound 77 did not undergo epimerization under the same reaction conditions, and 78 was not formed despite our extensive efforts. The reaction of 77 carried out in the presence of D2O did not result in any incorporation of deuterium into 77, which indicates that the enol or enolate of 77 was not generated. Thus, although the structural differences between 75 and 77 are apparently small, the properties of such compounds are different. Pleasingly, we finally disclosed that the reaction of 72 with KHMDS and Davis’ reagent successfully allowed to introduce the C10 hydroxy group, and subsequent acetylation and treatment with DBN led to a complete epimerization to afford 73 (Scheme 3.27). Hydrogenolysis of the benzyl or benzylidene groups of intermediates or derivatives of taxol bearing an oxetane group is known to be troublesome. In fact, it has been reported that the C2 hydroxy group liberated upon hydrogenolysis may attack the proximal oxetane moiety to form a tetrahydrofuran ring. We surmised that impurities included in the palladium catalyst could cause this side reaction. Specifically, acidic impurities included in the palladium catalyst could activate the oxetane ring during the hydrogenolysis since most palladium catalysts are prepared from PdCl2. Hence, we optimized the conditions for this step and found that use of alumina as additive was effective for suppressing the side reaction. Thus, this issue could be perfectly solved by conducting the hydrogenolysis of 73 in the presence of Pd(OH)2/C and alumina in CPME at −5  °C, affording the desired product without formation of the tetrahydrofuran ring. Finally, the f­ ormation of a cyclic carbonate at the C1 and C2 diol as well as the C7 TES ether led to Nicolaou’s synthetic intermediate 74, thereby accomplishing the formal total synthesis of (−)-taxol. We also achieved the synthesis of another synthetic intermediate developed by the Nicolaou’s group, namely, compound 79, starting from 73 in three steps

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Scheme 3.29 Transformation of 73 to Nicolaou’s advanced synthetic intermediate 79

(Scheme 3.29). Thus, the C1–C2 benzylidene of 73 was converted into a C2 benzoate by the Ru-catalyzed oxidation [120]. Only the C2 benzoate was formed upon oxidation since the formation of the C1 benzoate was hindered by steric strain. Oxidation at the C13 allylic position and benzyl ether was not observed during the ruthenium-­catalyzed reaction. Subsequent hydrogenolysis of the benzyl group and a TES ether formation afforded 79. In summary, the eight-membered ring of taxol could be efficiently formed by the palladium-catalyzed intramolecular alkenylation of a methyl ketone. This palladium-­ catalyzed reaction afforded the desired product in an excellent yield (97%), allowing for an efficient formal total synthesis of (−)-taxol by a convergent approach. To the best of our knowledge, this is the first example of a palladium-catalyzed intramolecular alkenylation that leads to the formation of an eight-membered carbocyclic ring in natural product synthesis. During the preparation of a substrate for the palladium-catalyzed reaction, a rearrangement of an epoxy benzyl ether including a 1,5-hydride shift took place, generating the C3 stereogenic center and subsequently forming the C1–C2 benzylidene, and this was further employed to achieve the concise synthesis of (−)-taxol. The number of linear steps via 79 from a commercially available compound to taxol was 37 when 41-Br was used. Although this is one of the shortest syntheses of (−)-taxol reported, the overall yield (0.22%) still needs improvement. In our synthesis, the number of linear steps from a commercially available compound to (−)-taxol via compound 74 was 42 with a 0.75% overall yield when 41-I was used and 38 with a 0.31% overall yield when 41-Br was employed.

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Chapter 4

Synthetic Approaches on the Pluramycin-­ Class Antibiotics Yoshio Ando, Kei Kitamura, Takashi Matsumoto, and Keisuke Suzuki

Abstract  The pluramycins constitute a group of antibiotics sharing C-glycosylated anthrapyranone skeleton. Their significant bioactivities and intriguing chemical structures attracted synthetic chemists, and many challenges have been performed toward the total synthesis. This chapter focuses on the synthetic approaches on the pluramycins. The Introduction describes the historical background, structural features, and synthetic problems. In the second section, the synthetic approaches to the aglycons are summarized. The methods for the preparation of the rare deoxyamino sugars are overviewed in the third section. Finally, the synthetic studies toward the C-glycosyl tetracyclic skeletons and the completed total syntheses are described. Keywords  Pluramycins · Bis-C-glycoside · Anthrapyranone · Angolosamine · Vancosamine · Antitumor antibiotic

1  Introduction The pluramycins constitute a family of antibiotics, sharing a unique structural feature consisting of two amino C-glycosides attached to the polyketide-derived 4H-anthra[1,2-b]pyran-4,7,12-trione chromophore (Fig. 4.1) [1]. Pluramycin A was discovered by Umezawa and Kondo in 1956 as the first member of this family from a terrestrial Streptomyces pluricolorescens [2, 3]. Since then, a large number of congeners have appeared [1]. In 1974, Furukawa and Iitaka determined the chemical structure of kidamycin for the first time [4–9], where the X-ray diffraction analysis played a key role, identifying the unusual structure features: two C-glycosyl Y. Ando · K. Suzuki (*) Department of Chemistry, Tokyo Institute of Technology, Tokyo, Japan e-mail: [email protected] K. Kitamura Department of Applied Chemistry for Environment, School of Science and Technology, Kwansei Gakuin University, Sanda, Hyogo, Japan T. Matsumoto School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan © Springer Nature Singapore Pte Ltd. 2019 Y. Kobayashi (ed.), Cutting-Edge Organic Synthesis and Chemical Biology of Bioactive Molecules, https://doi.org/10.1007/978-981-13-6244-6_4

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bonds with rare deoxyamino sugars, as d-angolosamine [2,3,6-trideoxy-­3(dimethylamino)-d-arabinohexose, E-ring] at C8 and N,N-dimethyl-l-­vancosamine [2,3,6-trideoxy-3-(dimethylamino)-3-C-methyl-l-lyxohexose, F-ring] at C10. This report paved the way for all subsequent structure determinations of the pluramycins, including pluramycin A [3, 10], hedamycin [11–13], and the saptomycins [14, 15]. In the naturally occurring pluramycins, the hydroxy group of the E-ring is never acetylated. In contrast, the F-ring hydroxy group is acetylated depending on the congeners including pluramycin A. Anthrapyranones bearing only the C10 sugar, such as saptomycin H [15], belong to also the pluramycin family. Pluramycinones, corresponding to the aglycons, such as γ-indomycinone [16], are the family of pluramycins, which sometimes can be found in the fermentation broths besides the glycosylated compounds. Recently, a new class of the congeners is reported. Altromycin A lacks the C8 sugar and has a disaccharide at C10 and an intriguing C-glycoside bond through a quaternary center at C5 [17, 18]. Pluraflavin A also has a C-linked disaccharide at C10 and a hydroxymethyl group at C5 bearing a β-3-epivancosamine residue [19]. As can be seen in Fig. 4.1, the absolute stereochemistry of the side chains at C2 on most of the pluramycins is still not determined. The conformation of the α-C-glycosyl vancosamine is flexible and takes a chair form, a boat-like form, or a flipped chair form. As for hedamycin, found by Séquin in 1978 (Fig.  4.2), the structure was determined by a spectral comparison with kidamycin, and the X-ray diffraction analysis elucidated the full stereochemical structure, absolute and relative configuration including the side chain moiety [11– 13]. Interestingly, the vancosamine moiety adopts a chair conformation in the crystalline state, disposing the aryl group axial, while the prevalence of a twisted conformation in solution was inferred by NMR analysis. On the other hand, kidamycin manifests a different structural feature in which the vancosamine adopts a boat-­ like conformation, as suggested by the X-ray as well as the NMR analyses [9]. Furthermore, such conformational preference seems delicate as inferred by the deacetylation of saptomycin D to deacetylsaptomycin D, inducing a conformational change from chair to boat, in the C-vancosaminyl moiety [20]. The pluramycins show a broad spectrum of antimicrobial activities. Also they show potent antitumor activities that are attributed to the intercalation into DNA, where two amino sugars play key roles for the sequence selectivity [21]. Interestingly, when the β-vancosamine moiety adopts a chair conformer, the bioactivities decrease in comparison with the α-isomer. The diminished antitumor and antibacterial activities indicate that the boat-form F-ring of the pluramycins has also biological relevance. In addition to such biological importance, the challenging structural features have attracted sizable interests of the synthetic communities. Three key synthetic challenges include (1) selective construction of the highly oxygenated tetracyclic core; (2) preparation of sufficient amounts of the deoxyamino sugars, commercially unavailable; and (3) regio- and stereoselective installation of two C-glycosides, d-angolosamine at C8 and N,N-dimethyl-l-vancosamine at C10 (Fig.  4.3).

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Fig. 4.1 Pluramycins

Furthermore, the pluramycins readily undergo isomerization at the anomeric position of vancosamine under acidic conditions [8, 9], and the angolosamine at C8 position is transformed into the glycal upon irradiation with UV- or daylight under air [22, 23]. This chemical lability adds a further complication to the synthesis but also the manipulation. Especially, stereoselective construction of the α-C-glycoside at C10 is extremely difficult. Therefore, only two total syntheses of the pluramycins have been accomplished that possess β-C-glycoside at C10. For more detailed historical background and chemical properties of the pluramycins, see the account by Séquin in 1986 [1]. The current review will focus on the synthetic studies toward the pluramycins and is divided to three parts: (Sect. 2) synthesis of aglycons, anthrapyranone chromophores with differing side chain structures; (Sect. 3) preparation of the rare

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Fig. 4.2  Hedamycin and conformation of α-C-glycosyl vancosamine

Fig. 4.3  Synthetic challenges and lability of the pluramycins

deoxyamino sugars, l-vancosamine and d-angolosamine; and (Sect. 4) total syntheses covering approaches toward the C-glycosylated tetracyclic framework.

2  Approaches to the Aglycons This section outlines various strategies for the construction of the anthrapyranone framework of the pluramycins (pluramycinones) [24] which are divided into three types (type 1–3) differing in the ring finally constructed (Fig. 4.4). Type 1 describes methods for constructing the A-ring and is further divided into five approaches I–V.  Type 2, which deals with construction of the D-ring, is subdivided into approaches VI and VII, and type 3 comprises of approach VIII to build up the B-ring. In the following, representative examples are shown for each approach, focusing attention to the construction of the pyranone ring (the A-ring). The two total syntheses of the pluramycin-class antibiotics, isokidamycin and saptomycin B, relied on approach IV. Approach I Hauser exploited an oxidative cyclization of dienone 2 to construct the pyranone ring, achieving the synthesis of the kidamycinone methyl ether (Scheme 4.1) [25,

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Fig. 4.4  Strategies for the skeletal construction of the pluramycin aglycon

26]. By contrast, the attempted acid-promoted cyclization of β-diketone 4 gave an undesired product 5. Approach II In 2004, Krohn reported an effective construction of the anthrapyranone skeleton by assembling the β-diketone side chain via the Baker–Venkataraman rearrangement followed by an acid-catalyzed cyclization [27, 28]. Scheme 4.2 shows its application to the synthesis of γ-indomycinone in 2007 [29]. In the same year, Tietze reported the synthesis of (S)-espicufolin by using a 1,3-diketone cyclization to construct the pyranone moiety (Scheme 4.3) [30]. Approach III In 2005, McDonald reported the syntheses of kidamycinone and altromycinone by utilizing intramolecular Friedel–Crafts reactions of 14 and 15 for the construction of the pyranone moiety (Scheme 4.4) [31]. Later, the same group synthesized diphenol 20 by double Friedel–Crafts reaction of dicarboxylic acid 18 [32]. Unfortunately,

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Scheme 4.1  Synthesis of O-methyl kidamycinone [25]

Scheme 4.2  Total synthesis of γ-indomycinone [29]

Scheme 4.3  Total synthesis of (S)-espicufolin [30]

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Scheme 4.4  Friedel–Crafts cyclization in the synthetic study of kidamycin [31]

however, the attempted installation of two amino sugar moieties on the kidamycin skeleton was not fruitful as described in Scheme 4.25. Approach IV Approach IV relies on the pyranone formation by cyclization of the anthrol derivative having an alkynoyl side chain. Depending on the substrate structure, the desired 6-endo cyclization is often interfered by the competing 5-exo cyclization. Based on the report of Mzhelskaya [33], in 2006, Suzuki and Matsumoto achieved the desired 6-endo cyclization by using amine adduct 23 en route to γ-indomycinone (Scheme 4.5) [34]. In the same year, Tietze synthesized the putative stereoisomers of AH-1763 IIa (Scheme 4.6) [35]. The pyranone cyclization proceeded smoothly as well by treatment of alkynone 27 with Cs2CO3 in acetone to give pyranone 28. Approach V In 2013, Stewart reported the synthesis of BE-26554A by employing β-ketosulfoxide for constructing the A-ring (Scheme 4.7) [36, 37]. Anthracene ester 29 was treated with lithium methylsulfinylmethide to give β-ketosulfoxide 30, which was combined with propionaldehyde in the presence of a catalytic amount of piperidine to furnish anthrapyranone 31. Oxidation of 31 and elongation of the side chain gave the anthraquinone 32. Detachment of the isopropyl group and epoxidation afforded the natural product. This method was also applied for the synthesis of heraclemycin B [38].

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Scheme 4.5  Total synthesis of γ-indomycinone [34]

Scheme 4.6  Total synthesis of AH-1763 IIa [35]

Scheme 4.7  Total synthesis of BE-26554A and heraclemycin B [36, 37]

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Scheme 4.8  Total synthesis of (R)-espicufolin [39, 41]

Approach VI Approaches VI and VII are characterized by construction of the D-ring at the final stage. Uno reported an approach by using anionic Fries rearrangement of 33 to install an alkynyl side chain to the naphthalene skeleton followed by cyclization to form the pyranone ring in 36 (Scheme 4.8). After conversion into quinone 37, the Diels–Alder reaction with methoxycyclohexadiene and a thermal retro-Diels–Alder reaction allowed construction of the D-ring, giving tetracycle 38. Several steps converted 38 to the natural enantiomer of the target, (R)-espicufolin [39–41]. Approach VII Danishefsky reported an approach employing a 1,3-dipolar cycloaddition of nitrile oxide to install the 1,3-diketone side chain followed by the pyranone cyclization under acidic conditions to form the A-ring. The Diels–Alder reaction of chloroquinone 43 and siloxydiene 44 and further oxidative manipulations of the D-ring gave the pluraflavin A aglycon (Scheme 4.9) [42]. In a recent report, bromo-substituted siloxydiene 46 was used for the Diels–Alder reaction to give bromo-anthrapyranone 47, which was employed to the installation of the C-glycoside moiety, targeting the total synthesis of pluraflavin A [43]. Approach VIII In 2015, Collet reported an approach based on the B-ring annulation by the Diels– Alder reaction of juglone (48) and diene 49 bearing the A-ring moiety to give cycloadduct 50, which was converted to γ-indomycinone (Scheme 4.10) [44, 45].

3  Glycosyl Donors: Monosaccharide This section covers the preparation of the rare deoxyamino sugars, d-angolosamine, and l-vancosamine (Fig. 4.5). They are not commercially available, so that the efficient syntheses are required for the formation of the aryl C-glycoside [46]. However,

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Scheme 4.9  Synthesis of pluraflavin A aglycon [42]

Scheme 4.10  Total synthesis of γ-indomycinone [44]

Fig. 4.5  The rare deoxyamino sugars on pluramycins

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the scalable protocols enabling the total synthesis of the pluramycins are still limited.

3.1  Synthesis of Angolosamine d-Angolosamine is the N,N-dimethyl analog of d-acosamine, which is a component of the antibiotic, angolamycin, and also found in kidamycin and the related C-glycoside antibiotics. The first reported preparation of d-angolosamine, as the hydrochloride, was performed by the Eschweiler–Clarke methylation of d-acosamine derivative 54 in 1977 by Baer and Georges [47]. By that time, two syntheses of d-acosamine had been reported, one from methyl α-d-mannopyranoside via keto sugar intermediate 56 [48] and the other from methyl α-d-glucopyranoside via aldol reaction of dialdehyde 57 with nitromethane (Scheme 4.11) [47, 49]. In 2002, Brimble reported a short step synthesis of azido acetate 62 as an angolosamine donor (Scheme 4.12) [50, 51]. Starting from diacetyl d-rhamnal (59), azido acetate 62 was obtained in three steps, but as an inseparable mixture of four diastereomers. This glycosyl donor 62 was employed in the Martin’s total synthesis of isokidamycin (Scheme 4.27) [52, 53]. McDonald reported a stereoselective synthesis of 69 from a noncarbohydrate, acyclic starting material (Scheme 4.13) [32, 54]. Enone 63 was subjected to CBS reduction, giving the corresponding alcohol in 97% ee, and subsequent Katsuki– Sharpless epoxidation gave epoxyalcohol 64 in a stereo-defined form. Titanium-­ promoted regioselective anti-addition of benzoic acid followed by desilylation gave diol 65. After several steps, alkynone 66 was cycloisomerized with catalytic W(CO)6

Scheme 4.11  Baer and Georges synthesis [47]

Scheme 4.12  Brimble synthesis [50]

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in the presence of DABCO under photo-irradiation conditions to provide glycal 67 in high yield. After removal of the silyl group in 67, introduction of an azide group with stereoinversion gave azide 68. Treatment of glycal 68 with hot aqueous acid followed by acetylation gave acetate 69 as an angolosamine glycosyl donor. Suzuki employed a more steady route to angolosamine donor 75 starting from the mannose-derived keto sugar 56 (Scheme 4.14) [55] in their total synthesis of saptomycin B (Scheme 4.29). Based on the earlier report by Mallams and Lukacs [56], keto sugar 56 was converted into 6-deoxy glycoside 72 via the stereoselective reduction with LiAlH4, mesylation, NBS-promoted fragmentation of the benzylidene acetal moiety, and hydrogenolysis of the C6–Br bond over Pd/C. Introduction of an azide group was performed with NaN3 in the presence of TBAI.  Basic ­hydrolysis of the benzoate group and protection with a benzyl group afforded azide 74, which was derived to acetate 75 as a glycosyl donor.

3.2  Synthesis of N,N-Dimethylvancosamine l-Vancosamine and its N,N-dimethyl derivative are found as the sugar constituents of several antibiotics. For example, l-vancosamine is an essential component of vancomycin, a glycopeptide antibiotic, while the N,N-dimethyl analog occurs in the pluramycin-class C-glycoside antibiotics. Although it is possible to obtain

Scheme 4.13  McDonald synthesis [32]

Scheme 4.14  Suzuki synthesis [55]

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l-vancosamine by acid degradation of commercially available vancomycin, many methods have been reported for the chemical synthesis of the vancosamine derivatives starting from either carbohydrates or noncarbohydrates. Kahne degradation (Scheme 4.15) [57]: Treatment of vancomycin hydrochloride with alloc-succinimide followed by acidic methanolysis afforded methyl glycoside 76, which was acetylated to give 77. Martin modified the protecting groups for the amino group (Alloc→Cbz) in this method and applied to their total synthesis of isokidamycin (Scheme 4.28) [52, 53]. Giuliano synthesis (Scheme 4.16) [58]: Glycal 80 was prepared from either enone 78 or benzylidene acetal 79 by treatment with methyllithium. Acylation and the Ferrier rearrangement of 80 afforded allylic ester 81, which is followed by deacylation with ion-exchange resin to give allylic alcohol. Inversion of the C4 alcohol was realized by Mitsunobu reaction. After cleavage of the labile nitrobenzoyl group, allylic alcohol 82 was treated with NaH in neat Me2NCN to give isourea 83. An electrophilic cyclization of 83 was performed by treatment with Hg(OCOCF3)2 followed by NaBH4 to afford oxazoline 84. After hydrolysis of the oxazoline in 84 and O-benzoylation, the l-vancosamine derivative 85 was obtained. Nicolaou synthesis (Scheme 4.17) [59]: Intermolecular Kishi–Nozaki coupling of vinyl iodide 86 with enantioenriched aldehyde 87 gave the corresponding alcohol as a mixture of diastereomers, which was oxidized with Dess–Martin periodinane to give ketone 88. Stereoselective Luche reduction of 88 followed by the reaction with aryl isocyanate in the presence of catalytic DBU afforded carbamate 89. Removal of the silyl protecting groups in 89 with HF·(py)n, selective oxidation of the primary

Scheme 4.15  Kahne degradation [57]

Scheme 4.16  Giuliano synthesis [58]

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Scheme 4.17  Nicolaou synthesis [59]

alcohol by IBX, and protection of the anomeric hydroxy group with PMB gave PMB-glycoside 90. Formation of the key strategic bond between a nitrogen and a quaternary carbon at C3 was achieved by IBX-mediated cyclization of 90 to give cyclic carbamate 91. Subsequent deprotection by CAN and basic hydrolysis completed the synthesis of l-vancosamine. McDonald synthesis (Scheme 4.18) [60]: The synthesis is featured by the endo cyclization of acyclic alkynol to form the amino sugar 98. Staudinger cycloaddition of imine 92 with the ketene, derived from acyl chloride 93, gave the β-lactam 94 as a single diastereomer. Upon addition of methyllithium, nucleophilic opening of the β-lactam in 94 occurred to give ketone 95 without forming any of the corresponding tertiary alcohol as byproduct. Reduction of ketone 95 under Luche conditions proceeded to give alcohol 96. The stereochemical course of the reduction was explained by a Felkin–Anh model. After switching the amino protection by a Cbz group to reduce the Lewis basicity, the tungsten-catalyzed cycloisomerization of alkynol 97 gave glycal 98 in excellent yield. Subsequent hydrolysis of the enol ether afforded vancosamine derivative 99. Later, the asymmetric route using an optical resolution was also reported by this group [32]. Parker synthesis (Scheme 4.19) [61]: Diastereoselective addition of (P)allenylstannane 100 [62, 63] to (S)-lactic aldehyde 101 gave alkynol 102. Treatment of alcohol 102 with trichloroacetyl isocyanate followed by methanolysis and ­debenzylation gave alkynol 103, which was subjected to tungsten-catalyzed cycloisomerization to afford glycal 104 followed by rhodium-catalyzed C–H insertion of the carbamate nitrogen via a nitrene to give cyclic carbamate 105. After N-methylation, reduction with LiAlH4 and silylation gave N,N-dimethylvancosamine glycal 106. Doi and Takahashi synthesis (Scheme 4.20) [64]: Starting from (S)-furfuryl alcohol 107, Achmatowicz reaction with NBS followed by the Boc protection gave enone as a mixture of anomers, which was separated by silica gel column chromatography to afford enone 108. After replacing the Boc group by a PMB group under Pd-catalyzed conditions, introduction of the C3-methyl group was performed via the C3 iodination followed by Migita–Stille coupling with Me4Sn to afford enone 110. Stereoselective reduction of ketone 110, Mitsunobu reaction, and methanolysis

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Scheme 4.18  McDonald synthesis [60]

Scheme 4.19  Parker synthesis [61]

Scheme 4.20  Doi and Takahashi synthesis [64]

gave allylic alcohol 111, which was converted to isocyanate 90 for IBX-mediated cyclization as described above. Suzuki synthesis (Scheme 4.21) [65, 66]: Ketone 56, derived from d-mannose, was combined with O-methylhydroxylamine to give oxime ether 112. Stereoselective

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nucleophilic addition of methylcerium reagent to oxime 112 and treatment with TFAA and pyridine gave amide 113. Reductive cleavage of the N–O bond in 113 with SmI2 gave amide 114, which was subjected to a regioselective cleavage of the benzylidene acetal by NBS to give benzoate 115. Elimination of HBr in 115 by DBU gave exocyclic enol ether 116, which underwent stereoselective hydrogenation by using Wilkinson catalyst to give benzoate 117.

4  Synthetic Studies and Total Synthesis Construction of the bis-C-glycoside is the major problem in the total synthesis of the pluramycins [46]. Three key issues are (1) availability of the rare sugars as stated above (Sect. 3), (2) the regioselective installation of two different sugars at C8 and C10 on the tetracyclic chromophore, and (3) the stereoselective formation of the thermodynamically unfavored α-C-glycoside at C10. Particularly, the third issue still remains to be solved. Several studies for installing the bis-C-glycosides have been reported. They are roughly classified into two categories, early or late, by the timing of the bis-C-­ glycosylation (Fig.  4.6). The early-stage approach uses mono- or bicyclic compound as C-glycosyl acceptors. For example, the authors reported resorcinol derivative I as a monocyclic platform, enabling the installation of two sugars by repeated O→C-­ glycoside rearrangement [55]. Parker reported an interesting approach, using naphthoquinone II as a bicyclic platform to accept two glycal

Scheme 4.21  Suzuki synthesis [65, 66]

Fig. 4.6  Platforms for installing bis-C-glycoside

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anions followed by dienone–phenol-type rearrangement [67]. Unfortunately, such approaches inevitably need long subsequent steps, and to date, no total syntheses have been completed. By contrast, the late-stage approach, if viable, is ideal in terms of the convergency and simplicity. However, the critical difficulty is to ensure the regiochemical control and the sufficient reactivity for the C-glycosylation as exemplified by tetracyclic derivative IV, which demanding an extra phenol to introduce the two sugars [32]. The effective intermediates toward the total synthesis are tricyclic anthrone III and the tetracyclic derivative V with C-glycoside at C8 already formed, which was constructed by intramolecular benzyne cycloaddition of a C-glycosyl furan in the early stage of the synthesis. This section will discuss the studies of the construction of C-glycosyl tetracyclic skeletons and the successful total syntheses.

4.1  Suzuki Approach Suzuki reported an early-stage approach by utilizing the “O→C-glycoside rearrangement” [68, 69], which allows a selective and high-yield C-glycosylation of phenol derivatives (Scheme 4.22). The process is composed of three steps. Step 1 is the activation of glycosyl donor A by Lewis acid (LA) to generate oxonium species B, which is trapped with the phenolic oxygen to give O-glycoside D. Step 2 is the rearrangement of O-glycoside D to C-glycoside F, which is responsible for the oxonium–phenolate ion pair E being generated by Lewis acid activation of D. Recombination by C–C bond formation gives C-glycoside F. Step 3 is the anomerization of aryl C-glycoside F via the intermediary quinone methide species G, generated by the Lewis acid activation of the endocyclic oxygen in F coupled with an electron donation by the ortho-hydroxy lone pair, which gives the thermodynamically favored β-C-glycoside H. 1,3-Dihydroxybenzene derivatives with various functional groups at the C2 position worked nicely as monocyclic platforms for the bis-C-glycosidation with amino sugar donors (Scheme 4.23) [55]. The reaction of resorcylic ester 118 with vancos-

Scheme 4.22  O→C-Glycoside rearrangement

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amine donor 119 smoothly proceeded by using Sc(OTf)3 in the presence of Drierite® to give C-glycoside 120. After deprotection, the second C-glycosylation with angolosamine donor 122 was again effected by Sc(OTf)3 and Drierite® to furnish bisC-glycoside 123. In a similar manner, bis-C-glycoside 125 possessing an iodine at C2 was obtained by using 2-iodoresorcinol derivative 124 as the starting material. Bis-C-glycosides 123 and 125, thus obtained, were successfully converted to o-toluic acid derivative 126 and triflate 128, respectively, which could serve as the precursors to zwitterion 127 and benzyne species 129, respectively, with potential for annulation reactions toward the tetracyclic structure of natural products (Fig. 4.7).

4.2  Parker Approach Parker applied their “reverse polarity strategy” for the early-stage construction of bis-C-glycoside structure (Scheme 4.24) [67]. Glycosyl anion 131, generated from the corresponding glycal by deprotonation with t-BuLi (0 °C, THF), was allowed to react with benzoquinone 130 at −78 °C to give 1,2-adduct 132, which was subjected to the same conditions to afford cyclohexadienediol 133. Treatment of 133 with

Scheme 4.23 Bis-C-glycosidation of monocyclic platforms

Fig. 4.7  Further utilities of bis-C-glycosyl monocycles

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Scheme 4.24  Reverse polarity strategy for construction of bis-C-glycoside

ZnCl2 induced a dienone–phenol-type rearrangement to give phenyl bis-C-glycal 134. Use of naphthoquinone as a bicyclic platform made bis-C-glycosyl naphthalene derivative 135 accessible. This approach is quite unique, while further research toward pluramycins has not been reported.

4.3  McDonald Approach McDonald reported a synthetic study of kidamycin, featuring an attempt at the late-­ stage introduction of two amino sugars onto the anthrapyran aglycon 20 (for the preparation, see Scheme 4.4) having an additional free hydroxy group at C9 in the hope of facilitating the dual C-glycosylation (Scheme 4.25) [32]. The first C-glycosylation of tetracycle 20 with d-angolosamine donor 69 was cleanly achieved by using SnCl4, forming the C-glycosidic bond at the C8 position to give C-glycoside 136 in 70% yield. However, the second C-glycosylation of 136 was hampered by the severe steric hindrance. The reaction of 136 with l-vancosamine donor 137 under similar conditions gave bis-C-glycoside 138 only in low yield, unfortunately.

4.4  Danishefsky Approach Danishefsky reported a synthetic study of pluraflavin A based on a late-stage installation of carbohydrates (Scheme 4.26) [43]. Enantioenriched bromoanthrapyran 47, which was prepared by the same group as described in Scheme 4.9 [42], was combined with stannyl glycal 139 by Stille coupling, giving C-glycosyl glycal 140 in 80% yield. The next challenge was the stereoselective hydrogenation of the glycal,

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Scheme 4.25  Synthetic study of kidamycin [32]

Scheme 4.26  Synthetic study of pluraflavin A [43]

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for which heterogeneous conditions in a less polar solvent were effective. C-Glycosyl glycal 140 was converted to quinone monoacetal 141, which was hydrogenated to give C-glycoside 142, favoring the desired α-isomer. The dimethyl acetal in 142 conferred the stability during reduction of the glycal double bond. Further steps including the O-glycosylation gave the advanced intermediate 143.

4.5  Martin Synthesis The total synthesis of isokidamycin was reported by Martin, exploiting a silicon tether for the regiocontrol in the intramolecular Diels–Alder reaction of a benzyne and a furan, and a Sc(OTf)3-promoted O→C-glycoside rearrangement for achieving C-glycosylation of vancosamine [55, 70] (Schemes 4.27 and 4.28) [52, 53]. The first C-glycosylation was realized by the Friedel–Crafts reaction of furan with azido sugar 62, corresponding to the angolosamine subunit. Glycosyl furan 144 was converted to furyl C-glycoside 145, to which a β-hydroxyethylsilyl tether was introduced and combined with naphthol 147 via the Mitsunobu reaction to give the intermediate 148 in 92% yield, thereby setting the stage for intramolecular Diels– Alder reaction. Treatment of 148 with n-BuLi at −25 °C delivered via A oxabicycle 149 as a diastereomer mixture in 92% yield [71]. Removal of the silicon tether and opening of the oxabicyclic ring in 149 gave the corresponding C-glycosyl anthracene 150.

Scheme 4.27  Total synthesis of isokidamycin [52, 53]

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Scheme 4.28  Total synthesis of isokidamycin [52, 53]

After regioselective bromination, the resulting anthracene 151 was combined with alkynal 152 via halogen–lithium exchange to give ynone 153. Pyranone formation was realized by Lewis acid-promoted cyclization of the intermediary ­vinylogous diethylamide to provide tetracycle 154. For introducing the vancosamine unit to anthrol 154, acetate 155 was used for the O→C-glycoside rearrangement. Extensive screening of Lewis acids revealed that Sc(OTf)3 and Drierite® allowed the highyield formation of β-C-glycoside 156. Further manipulation including removal of the protecting groups on the amino sugars and oxidative demethylation of the central ring of the anthracene core gave the final product.

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4.6  Suzuki Synthesis Saptomycin B was isolated from Streptomyces sp. HP530, which shows antitumor activity against human and murine tumor cell lines. Structurally, it shares a common anthrapyranone chromophore containing two amino C-glycosides and a six-carbon side chain. In 2014, Suzuki and Matsumoto reported a convergent total synthesis and the structure assignment of saptomycin B (Scheme 4.29) [72, 73]. The synthesis was achieved by assembly of four building blocks,  i.e. a tricyclic platform, two amino sugars, and an alkynal through ten synthetic steps. Use of tricycle 159 allowed the stepwise and regiocontrolled installation of two amino sugars. The reaction of l-vancosaminyl acetate 160 with 159 in the presence of Sc(OTf)3 and Drierite® gave the mono-C-glycoside 161 in 82% yield. d-Angolosaminyl acetate 75 was combined with C-glycoside 161 under the similar conditions to give bis-C-glycoside 162 in 96% yield. The anomeric configurations of the C-glycoside moieties were both β. For elaboration of the tetracyclic aglycon structure, tricycle 163 was enolized by LDA and allowed to react with enantiopure aldehyde 164, giving aldol 165 in 89% yield as a diastereomeric mixture. Alkynal 164 was prepared in both enantiomeric forms via asymmetric alkylation by using Seebach’s auxiliary [74]. Alcohol 165 was then oxidized with IBX to give 1,3-­diketone 166. The A-ring closure was realized by treating 166 with K2CO3 in MeOH to provide pyranone 167 via a 6-endo cyclization [24]. Further synthetic steps to the final product were performed under light shielding for avoiding the potential photoinduced degradation of the pluramycins [22, 23]. In the further investigation, this photoinduced side reaction served as new transformation of quinones [75–79]. Tetracycle 167 was carefully oxidized by PhI(OCOCF3)2 in aqueous MeCN to the corresponding naphthoquinone, which was directly treated with DBU and then exposed to air to give anthraquinone 168 in 67% overall yield. Finally, conversion of two azide groups into N,N-dimethylamino groups [80] and removal of all protecting groups gave the final product, saptomycin B. Following this synthetic route, the enantiomer of alkynal 164 was also incorporated in the final product in similar overall yield. Careful comparison of the two synthetic samples revealed that the configuration of the C14 stereogenic center of the natural product is R.

5  Conclusion This chapter has outlined strategies toward the pluramycin-class antibiotics. Several methods for construction of the tetracyclic structure and preparation of the rare deoxyamino sugars have been described. Because of the synthetic challenges by extreme complexity and delicate functionalities, only two completed total syntheses have been reported, and thus, the absolute stereochemistry of the side chains on most of the pluramycins have not been determined yet. The remained problems are

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Scheme 4.29  Total synthesis of saptomycin B [72, 73]

(1) stereoselective construction of α-C-glycoside at C10, (2) stereoselective synthesis of the highly oxidized side chain, and (3) easier access to the deoxyamino sugars. Further investigations on new synthetic methods and routes to enable their total synthesis are required. Acknowledgments  The authors are very grateful to Prof. Takenori Kusumi (Tokyo Institute of Technology) for invaluable discussions on the chemistry of the pluramycins and related natural products.

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References 1. U. Séquin, in Progress in the Chemistry of Organic Natural Products, ed. by W. Herz, H. Grisebach, G.W. Kirby, C. Tamm (Springer, New York, 1986), p. 57 2. K. Maeda, T. Takeuchi, K. Nitta, K. Yagishita, R. Utahara, T. Osato, M. Ueda, S. Kondo, Y. Okami, H. Umezawa, J. Antibiot. 9, 75 (1956) 3. S. Kondo, M. Miyamoto, H. Naganawa, T. Takeuchi, H. Umezawa, J. Antibiot. 30, 1143 (1977) 4. N. Kanda, J. Antibiot. 24, 599 (1971) 5. N. Kanda, M. Kono, K. Asano, J. Antibiot. 25, 553 (1972) 6. N. Kanda, J. Antibiot. 25, 557 (1972) 7. M. Furukawa, A. Itai, Y. Iitaka, Tetrahedron Lett. 14, 1065 (1973) 8. M. Furukawa, Y. Iitaka, Tetrahedron Lett. 15, 3287 (1974) 9. M. Furukawa, I. Hayakawa, G. Ohta, Y. Iitaka, Tetrahedron 31, 2989 (1975) 10. M. Ceroni, U. Séquin, Helv. Chim. Acta 65, 302 (1982) 11. U. Séquin, C. T. Bedford, S. K. Chung, A. I. Scott, Helv. Chim. Acta 60, 896 (1977) 12. U. Séquin, Tetrahedron 34, 761 (1978) 13. M. Zehnder, U. Séquin, H. Nadig, Helv. Chim. Acta. 62, 2525 (1979) 14. N. Abe, Y. Nakakita, T. Nakamura, N. Enoki, H. Uchida, M. Munekata, J. Antibiot. 46, 1530 (1993) 15. N. Abe, N. Enoki, Y. Nakakita, H. Uchida, T. Nakamura, M. Munekata, J. Antibiot. 46, 1536 (1993) 16. R.W. Schumacher, B.S. Davidson, D.A. Montenegro, V.S. Bernan, J. Nat. Prod. 58, 613 (1995) 17. M. Jackson, J.P. Karwowski, R.J. Theriault, D.J. Hardy, S.J. Swanson, G.J. Barlow, P.M. Tillis, J.B. McAlpine, J. Antibiot. 43, 223 (1990) 18. G.M. Brill, J.B. McAlpine, D.N. Whittern, A.M. Buko, J. Antibiot. 43, 229 (1990) 19. L. Vértesy, F.P. Barbone, E. Cashmen, H. Decker, K. Ehrlich, B. Jordan, M. Knauf, D. Schummer, M.P. Segeth, J. Wink, G. Seibert, J. Antibiot. 54, 718 (2001) 20. N. Abe, N. Enoki, Y. Nakakita, H. Uchida, T. Nakamura, M. Munekata, J. Antibiot. 46, 692 (1993) 21. M.R. Hansen, L.H. Hurley, Acc. Chem. Res. 29, 249 (1996) 22. A. Fredenhagen, U. Séquin, J. Antibiot. 38, 236 (1985) 23. Fredenhagen, U. Séquin, Helv. Chim. Acta 68, 391 (1985) 24. K. Kitamura, Y. Ando, Y. Maezawa, T. Matsumoto, K. Suzuki, Heterocycles 90, 1240 (2015) 25. F.M. Hauser, R.P. Rhee, J. Am. Chem. Soc. 101, 1928 (1979) 26. F.M. Hauser, R.P. Rhee, J. Org. Chem. 45, 3061 (1980) 27. K. Krohn, J. Vitz, Eur. J. Org. Chem. 209 (2004) 28. K. Krohn, A. Vidal, J. Vitz, B. Wastermann, M. Abbas, I. Green, Tetrahedron: Asymmetry 17, 3051 (2006) 29. K. Krohn, H.T. Tran-Thien, J. Vitz, A. Vidal, Eur. J. Org. Chem. 1905 (2007) 30. L.F. Tietze, K.M. Gericke, R.R. Singidi, I. Schuberth, Org. Biomol. Chem. 5, 1191 (2007) 31. Z. Fei, F.E. McDonald, Org. Lett. 7, 3617 (2005) 32. Z. Fei, F.E. McDonald, Org. Lett. 9, 3547 (2007) 33. M.A. Mzhelskaya, I.D. Ivanchikova, N.E. Polyakov, A.A. Moroz, M.S. Shvartsberg, Russ. Chem. Bull. 53, 2798 (2004) 34. D.-S. Hsu, T. Matsumoto, K. Suzuki, Chem. Lett. 35, 1016 (2006) 35. L.F. Tietze, K.M. Gericke, R.R. Singidi, Angew. Chem. Int. Ed. 45, 6990 (2006) 36. J.E. Rixson, B.W. Skelton, G.A. Koutsantonis, K.M. Gericke, S. G. Stewart, Org. Lett. 15, 4834 (2013) 37. J.E. Rixson, J.R. Abraham, Y. Egoshi, B.W. Skelton, K. Young, J. Gilbert, J.A. Sakoff, K.M. Gericke, A. McCluskey, S.G. Stewart, Bioorg. Med. Chem. 23, 3552 (2015) 38. K.D. Jones, J.E. Rixson, B.W. Skelton, K.M. Gericke, S.G. Stewart, Asian J. Org. Chem. 4, 936 (2015) 39. H. Uno, K. Sakamoto, E. Honda, N. Ono, Chem. Commun. 1005 (1999)

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4 0. K. Sakamoto, E. Honda, N. Ono, H. Uno, Tetrahedron Lett. 41, 1819 (2000) 41. H. Uno, K. Sakamoto, E. Honda, K. Fukuhara, N. Ono, J. Tanaka, M. Sakanaka, J. Chem. Soc., Perkin Trans. 1, 229 (2001) 42. B.J.D. Wright, J. Hartung, F. Peng, R. Van de Water, H. Liu, Q.-H. Tan, T.-C. Chou, S.J. Danishefsky, J. Am. Chem. Soc. 130, 16786 (2008) 43. J. Hartung, B.J.D. Wright, S.J. Danishefsky, Chem. Eur. J. 20, 8731 (2014) 44. M. Pantin, D. Zon, R. Vomiandry, L. Foulgoc, D. Sissouma, A. Guingant, S. Collet, Tetrahedron Lett. 56, 2110 (2015) 45. T. Mabit, A. Siard, M. Pantin, D. Zon, L. Foulgoc, D. Sissouma, A. Guingant, M. MathéAllainmat, J. Lebreton, F. Carreaux, G. Dujardin, S. Collet, J. Org. Chem. 82, 5710 (2017) 46. K. Kitamura, Y. Ando, T. Matsumoto, K. Suzuki, Chem. Rev. 118, 1495 (2018) 47. H.H. Baer, F.F.Z. Georges, Can. J. Chem. 55, 1100 (1977) 48. D. Horton, W. Weckerle, Carbohydr. Res. 44, 227 (1975) 49. F.M. Hauser, S.R. Ellenberger, Chem. Rev. 86, 35 (1986) 50. M.A. Brimble, R.M. Davey, M.D. McLeod, Synlett 1318 (2002) 51. M.A. Brimble, R.M. Davey, M.D. McLeod, M. Murphy, Aust. J. Chem. 56, 787 (2003) 52. B.M. O’Keefe, D.M. Mans, D.E. Kaelin Jr., S.F. Martin, J. Am. Chem. Soc. 132, 15528 (2010) 53. B.M. O’Keefe, D.M. Mans, D.E. Kaelin Jr., S.F. Martin, Tetrahedron 67, 6524 (2011) 54. F.E. McDonald, K.S. Reddy, Y. Díaz, J. Am. Chem. Soc. 122, 4304 (2000) 55. M. Shigeta, T. Hakamata, Y. Watanabe, K. Kitamura, Y. Ando, K. Suzuki, T. Matsumoto, Synlett 2654 (2010) 56. P. Bartner, D.L. Boxler, R. Brambilla, A.K. Mallams, J.B. Morton, P. Reichert, F.D. Sancilio, H. Surprenant, G. Tomalesky, G. Lukacs, A. Olesker, T.T. Thang, L. Valente, S. Omura, J. Chem. Soc., Perkin Trans. 1 1600 (1979) 57. C. Thompson, M. Ge, D. Kahne, J. Am. Chem. Soc. 121, 1237 (1999) 58. G.R. Smith, R.M. Giuliano, Carbohydr. Res. 323, 208 (2000) 59. K.C. Nicolaou, P.S. Baran, Y.-L. Zhong, J.A. Vega, Angew. Chem. Int. Ed. 39, 2525 (2000) 60. W.W. Cutchins, F.E. McDonald, Org. Lett. 4, 749 (2002) 61. K.A. Parker, W. Chang, Org. Lett. 5, 3891 (2003) 62. J.A. Marshall, Z.-H. Lu, B.A. Johns, J. Org. Chem. 63, 817 (1998) 63. J.A. Marshall, C.M. Grant, J. Org. Chem. 64, 8214 (1999) 64. T. Doi, K. Shibata, A. Kinbara, T. Takahashi, Chem. Lett. 36, 1372 (2007) 65. D.-S. Hsu, T. Matsumoto, K. Suzuki, Synlett 469 (2006) 66. K. Kitamura, M. Shigeta, Y. Maezawa, Y. Watanabe, D.-S. Hsu, Y. Ando, T. Matsumoto, K. Suzuki, J. Antibiot. 66, 131 (2013) 67. K.A. Parker, Y.-H. Koh, J. Am. Chem. Soc. 116, 11149 (1994) 68. T. Kometani, H. Kondo, Y. Fujimori, Synthesis (1988) 69. T. Matsumoto, M. Katsuki, K. Suzuki, Tetrahedron Lett. 29, 6935 (1988) 70. A. Ben, T. Yamaguchi, T. Matsumoto, K. Suzuki, Synlett 225 (2004) 71. D.E. Kaelin Jr., S.M. Sparks, H.R. Plake, S.F. Martin, J. Am. Chem. Soc. 125, 12994 (2003) 72. K. Kitamura, Y. Ando, T. Matsumoto, K. Suzuki, Angew. Chem. Int. Ed. 53, 1258 (2014) 73. K. Kitamura, Y. Maezawa, Y. Ando, T. Kusumi, T. Matsumoto, K. Suzuki, Angew. Chem. Int. Ed. 53, 1262 (2014). 74. T. Hintermann, D. Seebach, Helv. Chim. Acta 81, 2093 (1998) 75. Y. Ando, T. Matsumoto, K. Suzuki, Synlett 28, 1040 (2017) 76. Y. Ando, A. Hanaki, R. Sasaki, K. Ohmori, K. Suzuki, Angew. Chem. Int. Ed. 56, 11460 (2017) 77. Y. Ando, F. Wakita, K. Ohmori, K. Suzuki, Bioorg. Med. Chem. Lett. 28, 2663 (2018) 78. F. Wakita, Y. Ando, K. Ohmori, K. Suzuki, Org. Lett. 20, 3928 (2018) 79. Y. Ando, K. Suzuki, Chem. Eur. J. in press, https://doi.org/10.1002/chem.201801064 80. H. Kato, K. Ohmori, K. Suzuki, Synlett 1003 (2001)

Chapter 5

Recent Progress on the Total Synthesis of Duocarmycins A and SA, Yatakemycin, and PDE-I and PDE-II Juri Sakata and Hidetoshi Tokuyama

Abstract  Duocarmycin A, duocarmycin SA, (+)-CC-1065, and (+)-yatakemycin are site-selective DNA alkylating agents isolated from Streptomyces sp. In addition to their significant biological activity, their characteristic structural features, which arise from the presence of both the cyclopropapyrroloindole motif, responsible for the DNA alkylation, and a highly functionalized heterocyclic segment that acts as a DNA-binding site, have attracted a great deal of attention from the synthetic community. To date, a number of total syntheses as well as numerous synthetic studies on the partial structures of these compounds have been reported. In particular, over the last 15  years, a series of synthetic strategies and tactics have emerged, and remarkable progresses have been made in this area. Since several reviews on the biological function of these compounds have been already reported, this chapter mainly deals with selected examples on their representative total syntheses. Keywords  Total synthesis · Duocarmycins · Yatakemycin · CC-1065 · Antitumor agent · DNA alkylating agent

Abbreviations Ac Acetyl Bn Benzyl Boc tert-Butoxycarbonyl Bu Butyl Cbz Benzyloxycarbonyl dba Dibenzylideneacetone DMAP 4-(N,N-Dimethylamino)pyridine J. Sakata · H. Tokuyama (*) Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2019 Y. Kobayashi (ed.), Cutting-Edge Organic Synthesis and Chemical Biology of Bioactive Molecules, https://doi.org/10.1007/978-981-13-6244-6_5

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DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide Fmoc 9-Fluorenylmethyloxycarbonyl HOBt 1-Hydroxybenzotriazole Ms. Methanesulfonyl Ns 2-Nitrobenzenesulfonyl Ph Phenyl PPTS Pyridinium p-toluenesulfonate quant Quantitative TBAF Tetra-n-butylammonium fluoride TBS tert-Butyldimethylsilyl Tf Trifluoromethanesulfonyl TFA Trifluoroacetic acid THF Tetrahydrofuran TMG 1,1,3,3-Tetramethylguanidine TMP 2,2,6,6-Tetramethylpiperidine TMS Trimethylsilyl TPAP Tetra-n-propylammonium perruthenate Troc 2,2,2-Trichloroethoxycarbonyl WSCD Water-soluble carbodiimide

1  Introduction Duocarmycin A (1), duocarmycin SA (2), (+)-CC-1065 (3), and (+)-yatakemycin (4) are extremely potent antitumor antibiotics that share the cyclopropapyrroloindole (CPI) segment (colored in red in Fig. 5.1). Among these compounds, (+)-CC-­ 1065 (3) was first isolated from the culture of Streptomyces zelensis by a research group at Upjohn in 1978 [1, 2]. Then, (+)-duocarmycin A (1) was isolated from Streptomyces DO-88 at Kyowa Hakko Kogyo in 1988 [3–6], and 2  years later, (+)-duocarmycin SA (2) was isolated from Streptomyces DO-113 by the same group [7, 8]. More recently, in 2003, (+)-yatakemycin (4), which exhibits the most potent cytotoxicity (IC50 = 3 pM against L1210 cells) among this class of compounds, was isolated from Streptomyces sp. TP-A0356 by Igarashi and coworkers [9]. According to the detailed studies on the relationships between structure, chemical reactivity, and biological properties of these compounds [10–13], their biological effects are achieved through DNA alkylation via addition of adenine-N3 to the least substituted carbon of the dienone cyclopropane moiety after binding on AT-rich sites of the DNA minor groove. The alkylation by 1–3 proceeds in  1000 nM).

4.4  Nitrogen-Containing Heterocyclic Cannabinoids Since the first synthesis of (−)-Δ9-THC was published, there have been many reports of the incorporation of heteroatoms such as nitrogen or sulfur into the structure of cannabinoids or replacement of the oxygen atom in the B-ring of tricyclic cannabinoids with a methylene group [170–181]. Some examples of these cannabinoids are illustrated in Fig. 11.7. The discussion that follows focuses on the synthesis of some typical nitrogen-containing cannabinoids that have been developed in the last two decades.

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Fig. 11.7  Structure of some heterocyclic cannabinoids and a phenanthrene-derived cannabinoid

Scheme 11.40  Asymmetric synthesis of levonantradol

Johnson and Milne at Pfizer reported the non-stereoselective synthesis of levonantradol (224; Scheme 11.40) [178], a compound that is more potent than (−)-Δ9-THC (1) in activating the CB1 and CB2 receptors and possesses analgesic and antiemetic properties [133]. It is currently not in clinical use because of its toxicity. An asymmetric synthesis of levonantradol (224) in 11 linear steps with 16.2% overall yield was recently described (Scheme 11.40) [182]. Key steps in the enantioselective synthesis involve the aza-Michael addition, the Williamson ether synthesis, and the Robinson annulation to form the A ring. Treatment of the triflate salt of 3,5-dimethoxyaniline (214) with the chelating Michael acceptor 213 in the presence

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of (R)-BINAP−Pd (II) catalyst led to the (3S)-isomer of adduct 215 in good yield (93%) and high enantioselectivity (97.5% ee). Optical purity is further improved after a single recrystallization from toluene. Hydrolysis of 215 with potassium hydroxide in methanol to 216, followed by acid-mediated intramolecular cyclization and cleavage of the phenolic methyl ethers in a single step, led to quinolone 217 in good yield. Coupling of 217 (100% ee) with 218 (97% ee) afforded 219 in 88% yield as a single diastereomer (97% ee). Enantiomer 218 was prepared via the kinetic resolution of the hemiphthalate of racemic 5-phenylpentan-2-ol using (S)-αmethylbenzylamine as the resolving agent. The temporary introduction of a formyl α to the keto group in 219 to improve reactivity for the next step was accompanied by N-formylation. Following conjugate addition to methyl vinyl ketone, the α-formyl group was removed under mild basic conditions to give 221 as a 3:2 mixture of epimers. The aldol condensation step of the Robinson annulation of 221 with sodium methoxide in methanol at reflux gave tricyclic compound 222 in 70% yield, with removal of the N-formyl group in the same step. Enone reduction with Li/NH3 afforded a 2:1 mixture of trans- and cis-(6a,10a) isomers, and the desired transisomer 223 was isolated in 55% yield after acetylation of the phenolic hydroxyl group. Finally stereoselective reduction of the 9-keto group with sodium borohydride at −60 °C led to levonantradol (224) as a single diastereomer (99% ee). It is notable that the stereogenic center created in the aza-Michael addition step (at the C3 position in 215) directs the stereoselectivity at C6a, C9, and C10a in a chiral relay process. In the class of aminoalkylindole cannabinoids that is typified by WIN-55,212-2 (5), some 1-alkyl-3-(1-naphthoyl)indoles which lack the aminoalkyl group (e.g., 225; Scheme 11.41) are very potent cannabinoids, both in vitro and in vivo [183, 184]. Petrzilka’s approach has been used to prepare pentacyclic hybrid cannabinoid 228 (Scheme 11.41), which combines the benzopyran ABC-tricyclic nucleus of the classical cannabinoids with the 1-pentylindole moiety of JWH-007 (225, R1 = n-­ C5H11, R2  =  CH3) [185]. Acid-catalyzed condensation of (+)-trans-p-mentha-2,8-­ dien-­1-ol (11) with N-benzoyl-2,4-dihydroxycarbazole (226) afforded pentacyclic compound 227 in modest yield along with the regioisomer and the abnormal com-

Scheme 11.41  Synthesis of a pentacyclic hybrid cannabinoid

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pound. Hydrolysis of the amide function in 227 with base and in situ treatment of the reaction mixture with n-pentyl bromide led to the product of both N- and O-alkylation. Treatment with sodium thiopropoxide cleaved the n-pentyl phenolic ether leading to hybrid cannabinoid 228 in 60% yield. Hybrid cannabinoid 228 has affinity for the CB1 receptor approximately equal to that of Δ8-THC (9) (Ki = 19.3 ± 3 nM) and shows comparable potency in vivo using the mouse model of cannabimimetic activity which measures spontaneous activity, antinociception (as tail flick), and rectal temperature. Jagerovic and coworkers have reported the synthesis of chromenopyrazole cannabinoids in which the pyrazole ring, featured in SR-141716A (rimonabant, 4) and other diarylpyrazole cannabinoids, was introduced to the structure of classical cannabinoids (Scheme 11.42) [186, 187]. The synthesis involves the construction of the pyrazole ring through the condensation of β-ketoaldehyde 230 with an appropriate hydrazine as the key step. Thus, exposure of 5-(1′,1′-dimethylheptyl)resorcinol (29) with 3,3-dimethylacrylic acid followed by α-formylation gave chromanone 230 in good yield. Condensation of β-ketoaldehyde 230 with alkylhydrazines, cyclohexyl hydrazine, or arylhydrazines gave pairs of isomeric chromenopyrazoles 231 and 232. Most chromenopyrazoles 231 and 232 showed significant to high affinity and selectivity for CB1, for example, an analog of 231 (R1 = Et) has binding affinity Ki = 4.5 ± 0.6 nM for hCB1 and Ki > 40,000 nM for hCB2. The full CB1 selectivity over CB2 can be explained by the presence of the pyrazole ring in the structure, as observed in rimonabant (4), which is a selective CB1 antagonist [43]. Like the classical cannabinoids, chromenopyrazoles can be modified easily to increase the CB2

Scheme 11.42  Structure of rimonabant and the synthesis of chromenopyrazoles

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selectivity by removing or masking the phenolic hydroxyl group [187]. O-Alkyl chromenopyrazoles 233 were prepared in moderate yields by treatment of the corresponding 231 or 232 with an appropriate alkyl halide in base. The alkylation also led to N-alkylated and N-O-dialkylated by-products that decreased the yield of the desired O-alkyl compounds. Cyclodehydration of chromenopyrazoles 233 (R2 = CH2CH2CH2OH) with phosphorus pentoxide provided the condensed cyclic ether 234. Analog 233 has high to moderate CB2 affinity and selectivity, while the tetracyclic analog 234 showed moderate CB2 affinity but elicited excellent CB2 selectivity, for example, an analog of 234 (R1  =  N2-Et) has binding affinity Ki > 40,000 nM for hCB1 and Ki = 121.6 ± 43.5 nM for hCB2. Jagerovic and coworkers have carried out the synthesis of o- and p-chromenopyrazolediones via regiocontrolled oxidation of the phenol moiety in chromenopyrazoles with hypervalent iodine reagents (Scheme 11.43) [188, 189]. Oxidation of chromenopyrazoles 231 and 232 with [bis(trifluoroacetoxy)iodo]benzene in aqueous acetonitrile at room temperature led to regioselective formation of p-chromenopyrazolediones 235 [188], while o-quinone derivatives 236 were conveniently prepared using 2-iodoxybenzoic acid in DMF [189]. p-Quinone 235 (PM-49, R = H) has weak binding affinity for both receptors (Ki  =  134  ±  21  nM for hCB2 and Ki = 324 ± 235 nM for hCB1). Interestingly o-quinone 236 (R = N1-Et) is completely selective for CB2 (Ki = 529 ± 26 nM for hCB2 and Ki > 40,000 nM for hCB1). Further exploration of the chromenopyrazole scaffold prompted the Jagerovic group to synthesize chromenoisoxazoles in which the pyrazole was replaced by the isoxazole (Scheme 11.44) [187]. Isoxazole 237 was prepared in excellent yield via acid-catalyzed condensation of β-diketone 230 with hydroxylamine hydrochloride, and this ligand exhibited high affinity for both CB1 and CB2 receptors with Ki values in the low nanomolar range. Phenolic alkylation of 237 yielded methoxy 238 (PM-226) and hydroxypropoxy 239 chromenoisoxazole derivatives. Chromenoisoxazole 238 showed high affinity for hCB2 with a Ki value of 12.8 ± 2.4 nM and was over 3000-fold selective for CB2 over CB1 (Ki > 40,000 nM for hCB1).

Scheme 11.43  Synthesis of chromenopyrazolediones

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Scheme 11.44  Synthesis of chromenoisoxazoles

Scheme 11.45  Structures of resorcinol−AEA hybrids and the synthesis of representative compound CB-25

4.5  Resorcinol−Endocannabinoid Hybrids Anandamide (6; Fig.  11.1) and other endogenous arachidonic acid-based lipids (endocannabinoids) are very unstable and are rapidly hydrolyzed by FAAH [33] or MAGL [34, 35] and/or oxidized. In order to improve metabolic stability, a series of resorcinol−anandamide hybrid ligands 240 (Scheme 11.45) that consist of both a rigid aromatic portion, as in the classical cannabinoids, and a flexible chain, as in anandamide, were designed and prepared [190, 191]. These compounds were obtained through a brief sequence of reactions via O-alkylation of a phenol or resorcinol with an ω-bromoester followed by conversion of the ester to an amide. Some dialkylation took place, and in the case of 4-hexylresorcinol, regioisomers were formed; therefore, the desired monoalkylated products were isolated in moderate yield. Compound 242 (CB-25) exhibits high binding affinity for the cannabinoid receptors (Ki = 5.2 nM for rCB1 and Ki = 13 nM for mCB2). Other analogs of 242 (n = 7, R = OH, R1 = 1′,1′-dimethylheptyl, R2 = cyclopropyl) also show high binding affinity for the cannabinoid receptors (Ki = 5.6 nM for rCB1 and Ki = 7.9 nM for mCB2) and potent antinociceptive activity in vivo. The synthesis of related compounds, such as O-2220 (248) and analogs containing an unsaturated aliphatic moiety, has also been reported (Scheme 11.46) [192]. Electrophile 247 was prepared from homopropargyl alcohol 243 in seven steps in which the Sonogashira coupling of bromide 244 with methyl hex-5-ynoate was followed by the partial reduction of the diyne over nickel boride catalyst. O-2220 (248)

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Scheme 11.46  Synthesis of O-2220

Scheme 11.47  Structures of 2-AG and the synthesis of a resorcinol−2-AG hybrid

has high binding affinity for the cannabinoid receptors (Ki = 8.55 ± 1.83 nM for rCB1 and Ki = 21.48 ± 0.40 nM for mCB2). A series of resorcinol−2-AG and resorcinol−2-AGE ether hybrids have been prepared via O-alkylation of a resorcinol with a bromoalkyl-glycerol ester or with a bromoalkyl-glycerol ether [193]. The synthesis of resorcinol−2-AG hybrid 252 is summarized in Scheme 11.47. O-Alkylation of 5-(1′,1′-dimethylheptyl)resorcinol (29) with bromoalkyl-glycerol ester 250, which was obtained via esterification of the bromoacid with commercially available cis-1,3-benzylideneglycerol, furnished the protected compound 251 in moderate to good yield. No dialkylated compound was isolated. Hydrolytic cleavage of acetal 251 under acidic conditions gave 252 in good yield. Hybrid cannabinoid 252 exhibits high binding affinity and selectivity for CB1 (Ki = 0.01 ± 0.002 μM for rCB1 and Ki = 0.79 ± 0.07 μM for mCB2).

5  Summary and Outlook During the last three decades along with the synthesis of analogs of classical tricyclic cannabinoids and nonclassical bicyclic cannabinoids, various hybrid cannabinoids and related compounds with structures derived from the combination of classical cannabinoids with other classes of cannabinoids or with other groups of

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chemical compounds have been designed and synthesized. Some ligands exhibit very high binding affinity and selectivity for the receptors (e.g., hybrid cannabinoids with a chiral center at C6 such as AM-4030 and chromenoisoxazole PM-226). Like the classical cannabinoids, some hybrid cannabinoids and related compounds contain a benzopyran ABC-tricyclic framework, and as such the classical approaches developed by Petrzilka or by the team at Lilly have been used to prepare them. Hybrid cannabinoids with a chiral center at C6 have been synthesized through an intramolecular hetero-Diels−Alder reaction of an o-quinone methide. Other compounds have been obtained from classical cannabinoids, for example by oxidation of (−)-Δ8-THC to o- and p-quinones. Simple and brief syntheses of resorcinol/ endocannabinoid hybrids have also been described. Acknowledgments  We would like to express our thanks to Professor Alexandros Makriyannis and his research group for a wonderful and rewarding collaboration with our group that has spanned nearly a quarter century and to NIDA for providing funding.

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Chapter 12

Exploring Bioactive Marine Natural Products and Identification of Their Molecular Targets Masayoshi Arai

Abstract  Phenotypic screening is a method that allows detection of compounds that can induce desired phenotypes in cells and microbes. The method shows great potential for discovering compounds with novel mechanisms of action. Discoveries of first-in-class drugs indicate high efficacy of phenotypic screening. Target identification of compounds discovered by phenotypic screening is expected to uncover new metabolic pathways and functions that will lead to the development of novel drug target. Marine organisms like sponges and marine microbes exist in unique habitats and, therefore, produce distinctive secondary metabolites varying from those of terrestrial organisms. They are recently attracting attention as a “seedbed” for novel drug discoveries. We have previously focused on unique phenotypic alterations of cells and pathogenic microbes at the pathologic site of the human body and established phenotypic screening systems searching for bioactive marine natural products, which can regulate such unique phenotypic alterations. Further, implementing the methods of molecular biology and chemical biology, we identified the molecular targets of bioactive marine natural products that we discovered. In this chapter, we introduce our phenotypic screening to explore “medicinal seeds” from marine medicinal resources for novel drug discoveries and also provide identification of molecular targets (binding proteins) of isolated compounds. Keywords  Marine natural products · Molecular target · Furospinosulin-1 · Dictyoceratin-C · Agelasine D

M. Arai (*) Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2019 Y. Kobayashi (ed.), Cutting-Edge Organic Synthesis and Chemical Biology of Bioactive Molecules, https://doi.org/10.1007/978-981-13-6244-6_12

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1  Introduction Pathways for exploring bioactive natural products in the quest for novel drug discovery “seeds” may be broadly classified into two categories: target-specific screening and phenotypic screening. Target-specific screening identifies compounds effective for an existing drug targets, while phenotypic screening identifies compounds that can elicit desired phenotypes. Target-specific screening is the main choice of many pharmaceutical manufacturers as it is a high-throughput screening method, which does not require target identification (molecular target identification). In contrast, low-throughput phenotypic screening method requires elucidation of mechanisms of action, as well as target identification in order to develop the compounds as a lead. However, phenotypic screening has led to discovery of many first-in-class drugs with novel mechanisms of action proving its remarkable efficiency. Indeed, first-in-class drugs discovered by phenotypic screening make up a significant amount of all pharmaceuticals approved by FDA (USA) within a 10-year span from 1999 to 2008 [1]. Further, the target identification of compounds discovered by phenotypic screening unravels hitherto unknown metabolic pathways and functions in cells and microbes, increasing the possibility for discovery of novel target drugs. The example of such discovery can be lactacystin, a bioactive natural product isolated from the actinomycetes by Omura et al., which induces neurite outgrowth and elongation in mouse neuroblastoma Neuro 2a cells [2, 3]. β subunits of 20S proteasome were identified as molecular targets of lactacystin by analyzing the binding proteins using 3H-labeled compound [4]. Moreover, lactacystin was used to analyze intracellular proteasome functions. Proteasomes were identified as potential targets for cancer treatment leading to development of bortezomib, a therapeutic drug for multiple myeloma. In another study performed by Schreiber et al., target identification of the immunosuppressant FK506 (tacrolimus) using FK506 solid-­ phase carriers (Affligel-10) led to the discovery of FK506-binding proteins and novel immunity-regulating mechanism of FK506-binding proteins; this research laid the foundations for chemical biology [5, 6]. Due to their unique habitats, marine organisms, e.g. sponges and microbes, produce secondary metabolites varying from those of terrestrial organisms. Recently, bioactive natural products derived from marine organisms were used to develop drugs such as Halaven and Yondelis. Currently, over 20 clinical trials of compounds and derivatives originating from marine medicinal resources are in progress. In light of these developments, marine medicinal resources have attracted much attention as a “seedbed” for novel drug discoveries. This chapter provides an introduction to our phenotypic screening to explore “medicinal seeds” from marine medicinal resources for novel drug discoveries and also introduces identification of molecular targets (binding proteins) of isolated compounds.

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2  B  ioactive Marine Natural Products Isolated from Phenotypic Screening Systems Due to recent advances in cellular biology and bacteriology, culturing of various cells and pathogenic microbes has become relatively easy. Consequently, the quest for new bioactive natural products, utilizing cells and pathogenic microbes, is being vigorously pursued. However, it has been recognized that the cells and pathogenic microbes in pathologic site of the human body display unique phenotypes different from those in the general culture conditions. For instance, vascular endothelial cells located in tumor area experience phenotypic alterations stimulated by angiogenesis promoting factors produced by cancer cells. This results in the formation of new blood vessels to the tumor. Further, a hypoxic and low-nutrient environment is created within some parts of the tumor because of fragile and disordered vascular network. Prolific production of cytokines from cancer cells adapted to this environment is associated with tumor growth and metastasis. Therefore, the ability of cancer cells to adapt to such environments contributes to cancer progression. Similar phenotypic alternations in pathogenic microbes have also been reported. Numerous pathogenic microbes that form biofilms at the locus of infection were known to act as barriers against hosts immunological mechanisms and antibacterial agents. One more example is tuberculosis-causing bacteria Mycobacterium tuberculosis. It is known that, after the infection, environmental stimuli within the granuloma formed at the locus of infection cause phenotypic alterations resulting in resistance to conventional antituberculosis drugs and subsequent long-term dormancy of M. tuberculosis. Given the current situation of phenotypic screening research, we have previously focused on unique phenotypic alterations of cells and pathogenic microbes at the pathologic site of the human body and established phenotypic screening systems searching for bioactive natural products, which can regulate such unique phenotypic alterations. Some of the compounds that we discovered are the following: cortistatins [7, 8], novel steroid alkaloids isolated from the marine sponge, which inhibit angiogenesis in cancer; furospinosulin-1 [9, 10], a furanosesterterpene also isolated from marine sponge, which selectively inhibits growth in cancer cells adapted to hypoxic environment; and dictyoceratin-C (hypoxia-selective growth inhibitor) [11–13], a sesquiterpene phenol and biakamides (selective growth inhibitors of cancer cells adapted to lower-nutrient environment) [14], which are novel polyketides. Other compounds exploiting specific phenotypic alterations in pathogenic microbes that were assessed are ophiobolin-type sesterterpenes produced by marine fungi of the genus Emericella [15] and desferrioxamine E [16], a siderophore produced by the marine actinomycetes. Both ophiobolin-type sesterterpenes and desferrioxamine E inhibit biofilm formation by bacteria in the genus Mycobacterium, even when used below concentrations indicating antibacterial activity. Compounds, such as halicyclamines [17–19], agelasines [20], and melophlins from marine sponges [21], as well as nybomycin [22] and trichoderins [23] produced by marine microorganisms, show antibacterial activity against dormant tubercle bacilli (Fig. 12.1).

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Fig. 12.1  Chemical structures of previously discovered bioactive marine natural products

Thus, while chemical structures and biological activities of bioactive natural products discovered through specific phenotype screening are extremely fascinating, the identification of their detailed mechanisms of action and molecular targets is indispensable for their development into “drug leads” for novel drug discoveries. In addition, the target identification of bioactive natural products is also tied to the development of novel target drugs. The following section will introduce specific examples of the target identification of bioactive natural products that we discovered.

3  T  arget Identification of Furospinosulin-1 Based on the Methods of Biology and Molecular Biology Furospinosulin-1, a furanosesterterpene isolated from the Dactylospongia elegans of marine sponge, selectively inhibits growth in DU145 human prostate cancer cells adapted to hypoxic environments. When orally administered, it also demonstrated

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efficacy against S180 mouse sarcoma cells transplanted to mice. This compound selectively inhibits growth under hypoxic culture conditions. However, it is known that it has no effect on the expression of hypoxia-inducible factor 1α (HIF-1α), which is an important transcription factor for adaptation to hypoxic environments in cancer cells. Therefore, this compound was believed to have novel mechanisms of action. When our research began, no data was available on the biological activity and binding proteins of furospinosulin-1. To identify molecular targets for furospinosulin-­1, we performed a comparative study of genetic variations between untreated cells and those treated with furospinosulin-1, utilizing pathway-specific Oligo GEArray (SABiosciences), which can analyze 116 types of genetic variations related to cancer cell adaptations to hypoxia. The results suggested that furospinosulin-­1 inhibits the expression of insulin-like growth factor-2 (IGF-2) at the transcriptional level. Expression of IGF-2 is induced by hypoxic environment; therefore, furospinosulin-1 selectively inhibits growth in cancer cells adapted to hypoxic environments. These results coincided with those of IGF-2 signal transduction analysis by the western blotting method. Next, the IGF-2 gene promoter region affected by furospinosulin-1 was analyzed by a gel shift assay. Interestingly, furospinosulin-1 inhibited protein complex formation, uniquely observed in hypoxic environments along the Sp-1-like consensus sequence of the IGF-2 P3 promoter (Fig. 12.2) [9]. After showing that furospinosulin-1 can act as a hypoxia-selective growth inhibitor in cancer cells, we attempted to identify furospinosulin-1-binding proteins based on the earlier findings. In this experiment, molecular probes consisting of specific oligonucleotides, including the Sp-1-like consensus sequence, were used. The structures of the nucleotide probes used are shown in Fig. 12.2. Protein complexes specifically formed in hypoxic environments and observed on the oligonucleotides were comprehensively analyzed by LC-MS/MS.  Then, we attempted to identify furospinosulin-1-binding proteins by formation of protein complexes when furospinosulin-­1 or inactive furospinosulin-1 analog was added to the system. Consequently, we have found two transcriptional regulators, p54nrb and LEDGF/p75, which could potentially bind to furospinosulin-1. Neither of these proteins has been reported to have correlation with cancer cell adaptation to hypoxia. At this point, utilizing a synthesized furospinosulin-1 probe and a dummy probe, we further investigated whether selected transcriptional regulators bind directly to furospinosulin-1 (Fig. 12.3). The results showed furospinosulin-1 binding directly to both proteins. Interestingly, binding to p54nrb occurs only when it was cultured in a hypoxic environment, but not to p54nrb cultured under normal conditions. This phenomenon signifies that p54nrb undergoes some posttranslational modifications in a hypoxic environment and that furospinosulin-1 is capable of selectively distinguishing modified p54nrb. To determine the efficacy of p54nrb and LEDGF/p75 as drug targets in cancer cells adapted to hypoxic environments, we investigated the phenotypes of DU145 cells where p54nrb and LEDGF/p75 expression had been silenced by siRNA. The results showed that all DU145 cells, where either p54nrb or LEDGF/p75 expression had been silenced, demonstrated hypoxia-selective growth inhibition. Further, an

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Fig. 12.2  Inhibition of protein complex formation by furospinosulin-1 (A) and structure of nucleotide probe (B). (Reprinted with permission from [10] Copyright 2018 John Wiley and Sons)

examination of HIF-1α and IGF-2 expression levels in DU145 cells, where p54nrb or LEDGF/p75 expression had been silenced, revealed no observable effect on HIF-1α expression in both cell groups. This finding supports our previous observation that furospinosulin-1 is not an inhibitor of HIF-1α. However, in DU145 cells, where p54nrb expression had been silenced, hypoxia-selective silencing of IGF-2 ­expression was observed. Meanwhile, in DU145 cells, where LEDGF/p75 expression had been silenced, no silencing of IGF-2 expression was observed. These findings suggest that furospinosulin-1 suppresses IGF-2 production by binding to p54nrb that has undergone some posttranslational modifications under hypoxic conditions (Fig. 12.4A). Further, when it binds to LEDGF/p75, the gene expression characteristic to cancer cell adaptation to hypoxia is silenced, signaling hypoxia-selective growth inhibitory activity (Fig.  12.4B) [10]. Although in  vivo growth studies of

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Fig. 12.3  Chemical structures of furospinosulin-1 probe and dummy probe. (Reprinted with permission from [10] Copyright 2018 John Wiley and Sons)

DU145 cells, where p54nrb and LEDGF/p75 expression had been silenced, are required, our findings suggest that p54nrb and LEDGF/p75 have a strong potential to become novel targets for drug discovery, replacing HIF-1α. Thus, based on the alterations in gene expression, we elucidated the mechanism of action of furospinosulin-1, which led to the identification of its molecular targets.

4  T  arget Identification of Dictyoceratin-C from Phage Library Using Probe Molecule Isolated from the same sponge as furospinosulin-1, dictyoceratin-C is a sesquiterpene that selectively inhibits the growth of cancer cells adapted to hypoxic environments. We previously proved that parahydroxybenzyl esters in the chemical structures of dictyoceratin-C are essential for its bioactivity based on the study of structure-activity relationships using structurally related natural products. Dictyoceratin-A, which contains the same structural components, exhibited the same bioactivity as dictyoceratin-C [11]. It was shown that orally administered dictyoceratin-­C also demonstrated efficacy against S180 mouse sarcoma cells transplanted to mice [13]. Additionally, analysis of its mechanism of action showed that it reduced HIF-1α expression levels in DU145 cells in hypoxic environments [11]. We then started identifying the binding proteins of dictyoceratin-C. At the beginning of target identification of dictyoceratins, our research group succeeded in both total synthesis of dictyoceratins and synthesis of their analogs [12, 13]. Since the synthesis of dictyoceratin probes based on the result of structure-­ activity relationships was possible, analysis of the binding proteins was performed

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Fig. 12.4  Expected mechanism of action of furospinosulin-1. (Reprinted with permission from [10] Copyright 2018 John Wiley and Sons)

using the phage display method. In this method, a library of bacteriophages, which displayed proteins or peptides on the head, was used as a source for searching the binding protein of bioactive compounds. Phages that bind to the probe molecules were selected therefrom. Finally, based on a genetic analysis of the selected phages, the molecular targets of the compounds were determined by identifying the binding proteins or peptides on the head of phages (Fig. 12.5). Then, a phage library, which displayed various peptides derived from proteins of DU145 cells, was created based on the mRNA derived from the DU145 cells by reverse transcription and amplification using random primers. Three types of probes were used in this investigation: Probe A, which retained hypoxic selectivity and bioactivity; Probe B, which retained growth inhibitory activity, but had no hypoxic selectivity; and Probe C, which had no bioactivity (Fig. 12.6).

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Utilizing Probe A, we implemented the phage display method. Consequently, 10 kinds of phages were selected. Next, we investigated whether these 10 kinds of phages with displayed peptides could also bind to Probe B or Probe C. The results showed selective binding between Probe A and the phages displaying peptide components from any of the following proteins: RBM28 (RNA-binding motif protein 28), RPAP3 (RNA polymerase II-associated protein 3), MIA3 (melanoma inhibitory activity protein 3), EIF5AL1 (eukaryotic translation initiation factor 5A-1-­ like), and TRMT6 (tRNA methyltransferase 6). We then tested whether these peptides could also bind Probe A when used in full-length protein form. The results showed that RBM28, RPAP3, and MIA3 proteins did bind to the Probe A, while EIF5AL1 and TRMT6 did not. These results strongly suggest that RBM28, RPAP3, and MIA3 are molecular targets of dictyoceratins. Next, we investigated whether DU145 cells, where the expression of each of these three protein types had been silenced by siRNA, would experience hypoxia-selective growth inhibition, just as

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the DU145 cells treated with dictyoceratin-C did. The results revealed that only DU145 cells, where RPAP3 expression had been silenced, showed hypoxia-­selective growth inhibition, as well as lower HIF-1α expression levels. Based on these results, we concluded that RPAP3 is a binding protein for dictyoceratin-C. We are currently investigating the efficacy of RPAP3 as a drug target.

5  T  arget Identification of Anti-mycobacterial Substance Using Transformant Library with Genomic DNA Although it is relatively easy to identify the binding proteins by utilizing probe molecules, as in the example of the target identification of dictyoceratins, the design and synthesis of probe molecules that retain bioactivity are essential. In cases like target identification of furospinosulin-1, the binding proteins are unraveled step by step based on their effects on signal transduction pathways; therefore, the method requires an extensive period before the binding proteins can be identified. Therefore, the development of a method capable of identifying the molecular targets of

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bioactive natural products effectively and easily has become a priority. Jacobs et al. demonstrated that enoyl-ACP reductase (inhA), a part of type-2 fatty acid synthetase, is a molecular target of isoniazid, an antituberculosis drug. They suggested that resistance to isoniazid exhibited by M. smegmatis transformants overexpressing inhA could be utilized for the identification of molecular drug targets [24]. At the same time, if the gene contributing to resistance against antibacterial substance with undetermined molecular targets can be found, those molecular targets can also be identified. Exploiting this concept, we created a method for identifying molecular targets without modifying the antibacterial substances discovered, by utilizing transformants with a genomic DNA library. Specifically, we created a genomic DNA library from the M. bovis BCG genome. Based on this library, through the transformation of M. smegmatis, we constructed a library consisting of approximately 4000 transformant strains randomly overexpressing the M. bovis BCG genome. After finding the strains resistant to the target compound, we analyzed the M. bovis BCG genes inserted into these strains. Next, fragmentation of the inserted M. bovis BCG genes was performed, and fragmented genes were cloned into expression vectors. M. smegmatis transformants overexpressing the fragmented genes were created, and strains resistant to the target compound were selected. By repeatedly performing this procedure, we attempted to identify the genes contributing to resistance against the target compound (Fig. 12.7). The following section introduces an actual example where the method described above was applied to agelasine D, a compound derived from marine sponges from the genus Agelas. Agelasine D is known as an antibacterial agent effective against latent tubercle bacilli. We screened our library of approximately 4000 M. smegmatis transformants for resistance to agelasine D and succeeded in isolating 6 strains. An analysis of the M. bovis BCG genome inserted into each agelasine D resistant strain revealed that M. bovis BCG genomic sequence 3475.051–502.901 kb (27.85 kb) has been inserted in all agelasine D resistant strains, signifying that the gene (molecular target) contributing to agelasine D resistance was located within this shared sequence. We then divided this 27.85 kb of genomic fragment into 10 regions (S1–S10). After cloning, M. smegmatis transformants overexpressing each region were created. An assessment of sensitivity to agelasine D was then performed to each strain. Only those with high expression levels of region S4, which contained four open reading frames (ORF), were resistant to agelasine D. Next, transformant strains with high expression levels for each of the four ORFs subsumed in region S4 were created, and the same assessment was performed. The results revealed that resistance to agelasine D was only detected in the strain overexpressing BCG3185c gene, which encodes a protein presumed to be a dioxygenase. To determine whether agelasine D and BCG3185c protein bind directly to each other, recombinant BCG3185c protein with His-tag was prepared, and their binding affinity was analyzed using the Biacore system. According to the results, the KD value between agelasine D and recombinant BCG3185c protein was 2.42 μM. Further, when the same assessment was performed on streptomycin targeting 23S rRNA, the KD value was 1.47 mM. These results conclusively attest to the fact that BCG3185c protein is a molecular target of agelasine D [20].

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Fig. 12.7  Strategy of target identification using M. smegmatis transformed with genomic DNA library

When the discovered bioactive natural product is an antibacterial substance and transformants resistant to the compound can be successfully acquired, the method described above is highly efficient for identifying molecular targets. We have also applied this method to trichoderin A and halicyclamine A, both of which are antibacterial agents effective against dormant tubercle bacilli. We have successfully demonstrated that their molecular targets are ATP synthetase and DedA family membrane proteins with indeterminate functions, respectively [19, 25]. DedA membrane protein family is nonexistent in humans; therefore the protein has a potential to become a novel drug target against M. tuberculosis.

6  Concluding Remarks As described above, we conducted the search for bioactive marine natural products using established phenotypic screening systems and analyzed molecular targets of isolated compounds. Based on our experience, many of the molecular targets of bioactive marine natural products, isolated by phenotypic screening systems, were molecules that were not previously considered candidates for drug targets. From the perspective of novel drug discovery, this approach, pivoting on the target identification of bioactive marine natural products, is extremely productive. In recent years, the lack of novel drug targets has emerged as a significant problem. It is our hope that future research employing this approach will lead to breakthroughs that will alleviate this scarcity.

12  Exploring Bioactive Marine Natural Products and Identification of Their Molecular… 303 Acknowledgments  I would like to express my gratitude to professor emeritus Motomasa Kobayashi and Dr. Naoyuki Kotoku (present affiliation: Colleges of Pharmaceutical Sciences, Ritsumeikan University) for providing their invaluable guidance, comments, suggestions, and cooperation throughout the projects. I am also grateful for the assistance given by all my students and collaborators. These studies were financially supported by Naito Foundation, Hoansha Foundation, Takeda Science Foundation, JST A-STEP (AS242Z00800Q), Osaka University Project MEET, KAKENHI from JSPS or MEXT, and Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP18am0101084.

References 1. D.C. Swinney, J. Anthony, Nat. Rev. Drug Discov. 10, 507 (2011) 2. S. Omura, T. Fujimoto, K. Otoguro, K. Matsuzaki, R. Moriguchi, H. Tanaka, Y. Sasaki, J. Antibiot. 44, 113 (1991) 3. S. Omura, K. Matsuzaki, T. Fujmoto, K. Kosuge, T. Furuya, S. Fujita, A. Nakagawa, J. Antibiot. 44, 117 (1991) 4. G. Fenteany, R.F. Standaert, W.S. Lane, S. Choi, E.J. Corey, S.L. Schreiber, Science 268, 726 (1995) 5. M.W. Harding, A. Galat, D.E. Uehling, S.L. Schreiber, Nature 341, 758 (1989) 6. J. Liu, J.D. Farmer Jr., W.S. Lane, J. Friedman, I. Weissman, S.L. Schreiber, Cell 66, 807 (1991) 7. S. Aoki, Y. Watanabe, M. Sanagawa, A. Setiawan, N. Kotoku, M. Kobayashi, J. Am. Chem. Soc. 128, 3148 (2006) 8. S. Aoki, Y. Watanabe, D. Tanabe, M. Arai, H. Suna, K. Miyamoto, H. Tsujibo, K. Tsujikawa, H. Yamamoto, M. Kobayashi, Bioorg. Med. Chem. 15, 6758 (2007) 9. M. Arai, T. Kawachi, A. Setiawan, M. Kobayashi, ChemMedChem 5, 1919 (2010) 10. M. Arai, T. Kawachi, N. Kotoku, C. Nakata, H. Kamada, S. Tsunoda, Y. Tsutsumi, H. Endo, M. Inoue, H. Sato, M. Kobayashi, ChemBioChem 17, 181 (2016) 11. M. Arai, T. Kawachi, H. Sato, A. Setiawan, M. Kobayashi, Bioorg. Med. Chem. Lett. 24, 3155 (2014) 12. Y. Sumii, N. Kotoku, A. Fukuda, T. Kawachi, Y. Sumii, M. Arai, M. Kobayashi, Bioorg. Med. Chem. 23, 966 (2015) 13. Y. Sumii, N. Kotoku, A. Fukuda, T. Kawachi, M. Arai, M. Kobayashi, Mar. Drugs 13, 7419 (2015) 14. N. Kotoku, R. Ishida, H. Matsumoto, M. Arai, K. Toda, A. Setiawan, O. Muraoka, M. Kobayashi, J. Org. Chem. 82, 1705 (2017) 15. M. Arai, H. Niikawa, M. Kobayashi, J. Nat. Med. 67, 271 (2013) 16. S. Ishida, M. Arai, H. Nikawa, M. Kobayashi, Biol. Pharm. Bull. 34, 917 (2011) 17. M. Arai, M. Sobou, C. Vilchèze, A. Baughn, H. Hashizume, P. Pruksakorn, S. Ishida, M. Matsumoto, W.R. Jacobs Jr., M. Kobayashi, Bioorg. Med. Chem. 16, 6732 (2008) 18. M. Arai, S. Ishida, A. Setiawan, M. Kobayashi, Chem. Pharm. Bull. 57, 1136 (2009) 19. M. Arai, L. Liu, T. Fujimoto, A. Setiawan, M. Kobayashi, Mar. Drugs 9, 984 (2011) 20. M. Arai, Y. Yamano, A. Setiawan, M. Kobayashi, ChemBioChem 15, 117 (2014) 21. M. Arai, Y. Yamano, K. Kamiya, A. Setiawan, M. Kobayashi, J. Nat. Med. 70, 467 (2016) 22. M. Arai, K. Kamiya, P. Pruksakorn, Y. Sumii, N. Kotoku, J.P. Joubert, P. Moodley, C. Han, S. Dayoung, M. Kobayashi, Bioorg. Med. Chem. 23, 3534 (2015) 23. P. Pruksakorn, M. Arai, N. Kotoku, C. Vilchèze, A.D. Baughn, P. Moodley, W.R. Jacobs Jr., M. Kobayashi, Bioorg. Med. Chem. Lett. 20, 3658 (2010) 24. M.H. Larsen, C. Vilchèze, L. Kremer, G.S. Besra, L. Parsons, M. Salfinger, L. Heifets, M.H. Hazbon, D. Alland, J.C. Sacchettini, W.R. Jacobs Jr., Mol. Microbiol. 46, 453 (2002) 25. P. Pruksakorn, M. Arai, L. Liu, P. Moodley, W.R. Jacobs Jr., M. Kobayashi, Biol. Pharm. Bull. 34, 1287 (2011)

Chapter 13

Target Protein Chemical Modification Hiroyuki Nakamura

Abstract  Protein modification is an important technology for investigating the protein functions and their signaling networks in cells, tissues, and organs and for understanding complex mechanisms in living systems. Although various tag protein fusion technologies have been developed for these purposes, genetic manipulation is necessary, and fusion of the relatively large tag proteins with target proteins may inhibit biophysical functions. Therefore, chemical modification with small molecules has attracted much attention as an alternative protein modification technology. In this chapter, several key bioconjugation reactions leading to non-specific chemical modifications and the recent amino acid residue-specific chemical modifications of proteins are first summarized in Sect. 2. Based on these bioconjugation reactions, the recent development of target protein-selective modifications is described in Sect. 3. Furthermore, selective isolation and modification of target proteins is described in Sect. 4. Keywords  Chemical modification · Protein engineering · Posttranslational modification · Amino acid residue-specific · Single electron transfer (SET) · Chromophore-assisted light inactivation (CALI)

1  Introduction A living system comprising prokaryotes and eukaryotes involves the assembly of well-organized biomolecules. One of the key players in living systems is a protein that is translated from DNA through mRNA according to the central dogma. It is reported that the total number of proteins in a HeLa cell, a human cervical cancer cell, is estimated to be 3.0 × 109 [1]. Therefore, determining protein functions and their signaling networks in cells, tissues, and organs is critical for understanding complex mechanisms in living systems. “Protein modification” is an important H. Nakamura (*) Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2019 Y. Kobayashi (ed.), Cutting-Edge Organic Synthesis and Chemical Biology of Bioactive Molecules, https://doi.org/10.1007/978-981-13-6244-6_13

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Fig. 13.1 (a) Pre-translational protein modifications that require genetic manipulation and (b) posttranslational protein modification with small chemicals

technology for investigating the protein profiles. Various tag protein fusion technologies have been developed (Fig.  13.1a). For example, autofluorescent protein (AFP) tags are widely used to visualize target proteins not only in cells but also in tissues and organs in living systems [2]. SNAP-tag [3] and CLIP-tag [4] are self-­ labeling proteins that are mutated forms of the human suicide protein O6-­ alkylguanine-­DNA alkyltransferase (hAGT) and its variants that react with small chemicals, O6-benzylguanine derivatives and O2-benzylcytosine derivatives, respectively. HaloTag is another protein that reacts with chloroalkyl derivatives [5]. Affinity peptide tag systems, such as polyhistidine tag (His-tag), polyarginine tag (Arg-tag), and FLAG tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys peptide tag), are useful for purifying and detecting recombinant proteins while minimally affecting the tertiary structure and biological activity [6]. Although these technologies are useful for real-time monitoring of target proteins in living systems, genetic manipulation is necessary, and the relatively large

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tag proteins fused with target proteins sometimes inhibit biophysical functions due to undesirable interactions with other molecules. Therefore, modifying target proteins with small chemicals has attracted much attention as an alternative protein modification technology (Fig. 13.1b). Gene-coded protein modification is referred to as a pre-translational modification, whereas the chemical modification is referred to as a posttranslational modification because it occurs after protein biosynthesis. In this chapter, several key bioconjugation reactions used for chemical modification are first summarized based on their amino acid residues, and then recent development of target protein-selective chemical modifications is described.

2  Protein Modification Reactions 2.1  Non-specific Modifications Protein chemical modification reactions are organic reactions that proceed in aqueous media under physiological conditions. Therefore, the modification reagents must be inert to water but react with proteins in aqueous media. To satisfy these requirements, light (UV) irradiation strategy has been widely used to generate reactive species that react with amino acid residues of proteins, usually non-specifically, in a proximity-dependent manner to form covalent bonds between the modification reagents and proteins. Westheimer and co-workers were the first to report the generation of carbene from a diazoacetyl group which was introduced into an enzyme, using photolysis to induce an internal attack on the enzyme itself under mild conditions of temperature and pH conditions; they proposed the concept of photoaffinity labeling of proteins in 1962 [7]. Since their discovery, tremendous efforts have been devoted to the development of photoaffinity labeling systems in life science. During photoaffinity labeling, the photoreactive groups are conjugated to the ligand and converted into the corresponding reactive species by light irradiation to bind the ligand to its target macromolecules. Consequently, this modification can be used to investigate ligand-­ protein interactions, identify the location of an enzyme inhibitor, isolate and identify unknown proteins, or identify amino acid residues at protein-protein or protein-lipid interfaces. So far, three photoaffinity functional groups, aryl azide, diazirine, and benzophenone, are known to be widely used for the conventional non-­ specific photoaffinity labeling reactions of proteins. Aryl azide was first introduced as a photoaffinity functional group for antibody modification by Knowles and co-workers [8]. Aryl azide undergoes photolysis to generate aryl nitrene species that undergo insertion reactions with amino acid residues of proteins to form a carbon-nitrogen bond. Alternatively, aryl nitrene species are converted to ketimines that react with nucleophilic side chains of amino acid residues to form the corresponding azepine adducts (Fig. 13.2a) [9, 10]. Aryl azides are easily prepared, and their photoaffinity functional group is relatively small,

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Fig. 13.2  The photoaffinity functional groups and their reactive intermediates

resulting in minimal structural changes in ligands. However, aryl azides require the short wavelength excitation lights (320 nm and can be used for photoaffinity labeling without degradation of biomacromolecules. Polyfluorinated aryl azides such as pentafluorophenyl azides generate stabilized nitrene species that readily undergo the insertion reaction preventing the ring expansion pathway to afford the stable covalent adducts [17]. Insertion of photoaffinity probes into six amino acids, threonine, serine, tyrosine, isoleucine, lysine, and arginine, was examined, and their unique selectivity was investigated. Azide 1 selectively undergoes insertion with isoleucine, whereas diazirine 2 undergoes insertion with threonine, lysine, and arginine, and benzophenone 3 reacts with isoleucine and lysine, presumably via the insertion. These photoaffinity probes also showed selective insertion with several peptides.

2.2  Lysine Residue-Specific Modification Reactions The most widely used biorthogonal modification reactions rely on electrophilic reagents that target nucleophilic amino acids, such as lysine and cysteines. Lysines have a primary amine moiety in their side chain that reacts as a nucleophile, although it is protonated under physiological pH. Lysines undergo the nucleophilic substitution with various electrophilic reagents, such as N-hydroxysuccinimide (NHS) esters (Fig.  13.4A) and sulfonyl chlorides to form the corresponding amides and sulfonamides, respectively. The reactions proceed without exogenous reagents such

Fig. 13.3  A series of α-D-mannoside photoaffinity probes conjugated with an azide (1), a diazirine (2), and a benzophenone (3)

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Fig. 13.4  Typical chemical modification reactions of proteins at lysine residues

as bases which are usually required for peptide bond formation. Lysines also undergo the nucleophilic addition with isocyanates and isothiocyanates (Fig. 13.4B) to afford the corresponding ureas and thioureas, respectively, and 1H-benzo[d][1,3] oxazine-2,4-dione reacts with lysines to form the corresponding ring opening ortho-­ aminobenzamide. The resulting modified proteins can be further reacted with NaIO4 and a dialkyl acyl phenylenediamine to install functional groups into the modified lysine residues (Fig.  13.4C) [18]. Reductive amination is also used for lysine residue-­specific modification. The reaction of lysines with aldehydes rapidly and reversibly generates imines or Schiff bases which are irreversibly reduced by water-­ compatible sodium cyanoborohydride to afford N-alkylating lysine proteins [19]. Tanaka, Katsumura, and co-workers developed a 6π-aza-electrocyclization-based pyridinium ring formation (Fig. 13.4D). In this modification reaction, generation of imines between lysines and α,β,γ,δ-unsaturated aldehydes is essential for achieving the 6π-aza-electrocyclization [20]. Using this modification reaction, positron emission tomography (PET) probes were prepared to realize a first visualization sialic acid-dependent circulatory residence of glycoproteins [21]. It should be noted that lysine-modifying reagents also react with the N-termini of proteins. Lysines undergo reductive amination with aldehydes in the presence of reductive agents such as sodium cyanoborohydride to afford the corresponding N-alkylated proteins (Fig. 13.4E). The formation of imines from lysine residues and aldehydes is essential, and a relatively higher temperature (56 °C) is needed to achieve the reductive amination. Francis and McFarland developed a lysine-residue-specific N-alkylation by iridium-catalyzed hydrogen transfer [22]. This reductive amination approach

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provides higher modification efficacy than with the sodium cyanoborohydride-­ based reductive amination approach under physiological conditions.

2.3  Cysteine Residue-Specific Modification Reactions Cysteine is an alternative nucleophilic amino acid widely used for residue-specific modification reactions with electrophilic reagents, because the thiol group of the cysteine side chain is more nucleophilic than a primary amine, especially below pH below 9, at which the primary amine moiety of lysines is protonated. Therefore, cysteine undergoes modification reactions much faster than lysine, resulting in cysteine rather than lysine residue-specific modification. Cysteine thiols are often observed as disulfide dimer forms in proteins. Consequently, free thiols occur at a relatively low level of occurrence on the surfaces of many proteins (Fig. 13.5A). The thiol group of cysteine undergoes conjugate addition with Michael acceptors such as maleimides under physiological conditions. Maleimides are relatively stable under biological conditions and are widely used for selective cysteine residue-­ specific modification (Fig.  13.5B) [23]; α-haloketones are also often used for alkylating the thiol group of cysteine (Fig. 13.5C). Davis and co-workers developed a two-step modification of cysteines. The first step involves oxidative elimination of the thiol group of cysteine by treating with O-mesitylenesulfonylhydroxyamine (MSH) to afford dehydroalanine. The resulting dehydroalanine residue can be used for further modification reactions such as conjugate addition of thiols to form thioethers [24].

Fig. 13.5  Typical chemical modification reactions of proteins at cysteine residues

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2.4  Tyrosine Residue-Specific Modification Reactions The phenolic side chain of tyrosine also reacts as a nucleophile with various electrophilic reagents, and the modification reactions usually take place at the α position of the phenolic hydroxyl group of tyrosine residues. The development of tyrosine residue-­specific modifications was initiated by Francis and co-workers. They found that tyrosine reacts with aromatic diazonium salts with electron-withdrawing groups such as nitro and carbonyl groups at the para position [25]. The resulting azo derivatives are further reduced to the corresponding anilines, which react with functionalized acrylamides to form stable benzoxazines with a nitro group (Fig.  13.6A) or undergo condensation to afford the corresponding stable oxime ethers with a carbonyl group (Fig. 13.6B) [26]. Francis and co-workers also reported a Mannich-­ type three-component coupling reaction for the tyrosine residue-specific modification (Fig. 13.6C) [27]. Chymotrypsinogen A was exposed to a 100–1000-­ fold excess of formaldehyde and aniline derivatives at room temperature and subjected to trypsin digestion to identify the modification sites by matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS) analysis. Three potentially reactive tyrosine residues on the surface of chymotrypsinogen A are modified, and the modified chymotrypsinogen A retains its enzymatic activity. Tanaka and co-workers reported that cyclic imines covalently react with phenols, including tyrosine residues, in aqueous solution (Fig. 13.6D). They synthesized a fluorescent dansyl group-conjugated cyclic imine and demonstrated covalent

Fig. 13.6  Typical chemical modification reactions of proteins at tyrosine residues

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l­abeling of proteins. However, the modification rate was slow, and it took 4 days to modify proteins at 37 °C [28]. The phenol group of tyrosine residues reacts with phenylenediamine and anisidine derivatives under oxidative conditions (Fig.  13.6E). Francis and co-workers reported the oxidative coupling reaction of a TRT with electron-rich amino acid residues such as tyrosine and tryptophan residues in the presence of cerium(IV) ammonium nitrate (CAN) as an oxidation reagent [29]. Sato and Nakamura reported a tyrosine residue-specific modification with N′-acyl-N,N-dimethyl-1,4-­ phenylenediamines (tyrosyl radical trapping agent, TRT) using the ruthenium photocatalyst ([Ru(bpy)3]2+). The modification reaction is induced by visible light irradiation under mild conditions (pH  6.0–7.4) at an ambient temperature. This modification reaction is based on the oxidative dimer formation from two tyrosine residues catalyzed by the ruthenium photocatalyst under visible light irradiation, reported by Kodadek and co-workers [30]. The photoredox mechanism of the tyrosine residue-specific modification reaction is shown in Scheme 13.1. The ruthenium(II) complex is excited by visible light irradiation, and the excited ruthenium(II) complex is oxidized by molecular oxygen to generate the ruthenium(III) complex and a superoxide anion radical. Single electron transfer (SET) from the tyrosine residue to the ruthenium(III) complex affords a tyrosyl radical and an initial ground-state ruthenium(II) complex. The resulting tyrosyl radical reacts with the TRT in the presence of a superoxide anion radical to afford the corresponding TRT-coupled protein. The SET step from the tyrosine residue to the ruthenium(III) complex is accelerated in the presence of oxidants, such as ammonium persulfate. This modification system can be applied to target protein-­ selective protein modification by tethering a ligand of the protein of interest into the ruthenium photocatalyst. The detailed approach is described in Sect. 3.3.

Scheme 13.1  The photoredox mechanism of the tyrosine residue-specific modification reaction catalyzed by ruthenium complex ([Ru(bpy)3]2+)

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Barbas and co-workers developed 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD) as a tyrosine residue-specific modification agent. PTAD is readily prepared oxidatively from 4-phenyl-1,2,4-triazolidine-3,5-dione (4) with N-bromosuccinimide (NBS) and pyridine in DMF and reacts with a tyrosine residue at the α position of the phenolic hydroxyl group to form a carbon-nitrogen bond (Scheme 13.2). The reaction of PTAD was conducted in an equimolar mixture of six nucleophilic amino acids, tyrosine, histidine, tryptophan, serine, cysteine, and lysine, and only tyrosine modification was detected by 1H NMR analysis, indicating a high specificity of PTAD toward tyrosine [31]. Although PTAD is an efficient reagent for tyrosine-­specific modification, it is unstable under physiological conditions. PTAD also undergoes hydroxylation in aqueous media to afford the corresponding isocyanate which reacts with lysine to afford lysine residue-modified side products during protein modification [32]. Therefore, PTAD must be prepared before use and used immediately without purification. Sato and Nakamura focused on the luminol reaction, which is a chemiluminescence reaction and is useful for detecting trace amounts of blood that is used to detect trace in criminal investigations (Scheme 13.3). In the luminol reaction, iron in hemoglobin acts as a catalyst for oxidizing luminol with hydrogen peroxide (H2O2) to generate an oxidized luminol intermediate (6), which has a cyclic diazodicarboxyamide structure common to PTAD. Although the luminol-dependent chemiluminescence is caused by emission of the excited 3-aminophthalate generated from 6, the oxidized luminol intermediates specifically reacted with tyrosines. Especially, N-methylated luminol derivatives (5: R1 = CH3) are highly reactive with tyrosine residues in peptides and proteins under oxidative conditions in the presence of hemin and H2O2. The modification efficiency of N-methylated luminol is more than twofold higher than that of PTAD in the modification of bovine serum albumin (BSA) [33]. Furthermore, 1-methyl-4-arylurazole (MAUra), an N-methylated

Scheme 13.2  PTAD-based chemical modification

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Scheme 13.3  Luminol-based tyrosine residue-specific chemical modification

Scheme 13.4  MAUra-based tyrosine residue-specific chemical modification

d­ erivative of the PTAD precursor, is also synthesized as a novel tyrosyl radical trapping agent for effective modification of tyrosine residues with the ruthenium photocatalyst (Scheme 13.4) [34]. In general, radical protein labeling reagents such as tyramide have an effective labeling distance of about 20  nm with the peroxidase catalyst; however, MAUra reacts with tyrosine residues within a few nanometers of the ruthenium photocatalyst. Such a modification reagent should be effective for selective modification and identification of proteins of interest. Micklefield and co-workers reported enzyme-based tyrosine residue-specific modification. This approach relies on the tandem enzymatic reaction of a fungal tyrosinase and the mammalian catechol-O-methyltransferase (COMT). The tyrosinase induces the sequential hydroxylation of the phenolic group to afford an intermediate catechol moiety that is subsequently O-alkylated by COMT.  The tyrosinase-COMT combination enables the highly selective alkoxylation of tyrosine residues in peptides and proteins and provides highly versatile and regioselective modification of a diverse range of substrates including peptide antitumor agents, hormones, cyclic peptide antibiotics, and model proteins [35]. Van Delft and co-­ workers reported alternative enzyme-based tyrosine residue-specific modification. In this system, mushroom tyrosinase is used to convert a phenol moiety of tyrosine into 1,2-quinone which reacts with various bicyclo[6.1.0]nonyne derivatives (7) to afford the modified protein (Scheme 13.5). Fluorophore labeling of laminarinase A and site-specific preparation of an antibody-drug conjugate were demonstrated using this method [36].

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mushroom tyrosinase O

O HO

O

O

OH antibody

1,2-quinone form strain-promoted cycloaddition

O 7

O HN

O N H

O O

O O O

O

N H

O antibody

Scheme 13.5  Strain-promoted cycloaddition of 1,2-quinones converted from tyrosine residues specifically oxidized by mushroom tyrosinase

2.5  Other Amino Acid Residue-Specific Modifications The selective modification of carboxylic acid groups in glutamic acid, aspartic acid, or C-termini of proteins proceeds with exogenous amines in phosphate or HEPES buffer (pH 7.4) at room temperature to form an amide bond (Fig. 13.7a). The choice of coupling reagents is important: the use of EDC generates the O-acylisouronium intermediate which undergoes rearrangement, resulting in the formation of N-acylurea as a side product; however, the use of an excess of hydroxybenzotriazole (HOBt) decreases the formation of this side product and affords the desired amide bond-forming product [26]. Epoxides react with various nucleophilic amino acid residues and are thus used for random modification of protein surfaces. However, selective modification of histidine residues is observed in some cases (Fig.  13.7b). Sames and co-workers reported that oxirane-2-methanol reacts with human carbonic anhydrase II (HCA II) in >90% yield for 20 h at room temperature. MS analysis of the modified protein reveals that 1 equiv. of oxirane-2-methanol is attached and that modification occurs at a single residue (His-64) outside the active site [37]. Methylglyoxal is an endogenous metabolite that increases in diabetes and is a common intermediate in the Maillard reaction (glycation). Uchida and co-workers developed the modification of arginine residues with methylglyoxal. In this modification, two molecules of methylglyoxal react with two of the nitrogens in the guanine to form a tetrahydropyrimidine-type adduct (Fig.  13.7c) [38]. Although the reaction of methylglyoxal with Nα-acetyl-L-arginine is slow and the cyclized adduct is observed in 16% yield after 14 days at 37 °C, about 78% of the arginine and 27%

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Fig. 13.7  Other amino acid residue-specific chemical modifications

of lysine residues of BSA react with methylglyoxal 24 h after incubation at 37 °C, suggesting that the arginine residues of proteins represented primary targets for reaction with methylglyoxal. Francis and co-workers reported a unique modification of tryptophan with a diazo compound using a rhodium catalyst (Fig. 13.7d). In this modification reaction, a rhodium carbenoid generated in situ from rhodium acetate and a diazo compound is a reactive intermediate that reacts with tryptophan to afford the corresponding alkylated proteins. The modification of 3-methylindole with a diazo compound catalyzed by rhodium acetate in aqueous media gave a 1.4:1 mixture of 2-alkylated and N-alkylated products in 51% combined yield [39].

3  Target Protein-Selective Modification In the last decade, various amino acid residue-specific chemical modifications were developed as described in Sect. 2. Using these modification methods, we can introduce various functions into protein(s) in vitro. However, in order to define protein functions and their signaling networks in cells, tissues, or organs, the chemical modification of a specific target protein must be performed in a protein mixture, such as in living cells or in cell lysates, where a variety of other nontarget proteins and reactive biomolecules are present. Therefore, it is necessary to develop technologies that allow chemical modification reactions to proceed within close proximity of a target protein.

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3.1  Small-Molecule Target Identification Trifunctional chemical probes have been developed for the target protein identification and characterization from endogenous proteins in the living system. In general, the chemical probes used for the target protein identification consist of three functions: recognition function (ligand) for the target protein, a detectable function for labeling, and a reactive function for covalent binding (Fig. 13.8). Maximal target protein binding with minimal non-specific interactions is essential for designing the chemical probes. Photoaffinity groups have been widely used as reactive functional groups for the affinity-based chemical probes to create covalent cross-links between the target protein and probe molecule, because the photoaffinity labeling reactions can be controlled in the proximity of the ligand-binding site. Typical photoaffinity groups are aryl azide, diazirine, and benzophenone, which are already described in Sect. 2.1. There are three stages in the affinity-based protein labeling process (Fig. 13.9). The first stage is a macro-level target identification. During this stage, proteins that Fig. 13.8 Typical trifunctional chemical probes for target identification. (Modified with permission from Ref. [14]. Copyright 2016 American Chemical Society)

Fig. 13.9  The photoaffinity labeling/photo-cross-linking process from the non-covalent binding to the detection and identification of the tagged proteins. (Reprinted with the permission from Ref. [14]. Copyright 2016 American Chemical Society)

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were covalently cross-linked with the probe molecules are identified by SDS-PAGE, immunoblotting, radioautography, and/or fluorography. Especially, fluorescent or biotin labels are particularly useful in complex proteome mining. Biotinylated proteins and peptides can be purified using streptavidin beads; thus, isolated proteins and peptides can be directly subjected to proteome analysis. The second stage is a semimacro-level, where the specifically labeled proteins are isolated and subjected to enzymatic digestion or chemical cleavage at specific amino acid residues to identify the binding domains or interaction subdomains of the chemical probes. The last stage is a micro-level, where key amino acid residues of the receptor that is involved in binding with the ligand or participates in the protein-­protein interaction are identified by MS/MS analysis. Structural information regarding the major structural determinants of the contact interface can be obtained during this stage [14].

3.2  Ligand-Directed Target Protein Modification Hamachi and co-workers have developed several ligand-directed protein labeling methods. They first reported the “post-photoaffinity labeling modification” (Fig.  13.10) for constructing a fluorescent saccharide-biosensor based on a saccharide-­binding protein, concanavalin A (ConA). The photoaffinity labeling reagent, which consists of dithiomannoside as an affinity ligand-conjugated with 3-(trifluoromethyl)-3-phenyldiazirine as a labeling moiety through a disulfide bond,

Fig. 13.10  Post-photoaffinity labeling modification of a saccharide-binding protein, concanavalin A (ConA)

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Fig. 13.11 The affinity labeling modification followed by the hydrazone/oxime exchange reaction

is incorporated into the sugar-binding site, and the UV irradiation generates a reactive carbene species from the phenyldiazirine moiety which reacts with the target protein to form a covalent bond in the proximity of the ligand-binding site. The resulting modified ConA is treated with dithiothreitol (DTT) to produce a mercaptobenzyl site that reacts with an iodoacetylated dansyl group as a labeling reagent. These two-step chemical modifications proceed almost quantitatively [40]. A maleimide-conjugated fluorescent labeling reagent also reacts with the mercaptobenzyl site of the modified ConA [41]. However, this method is unstable for many proteins having cysteine residues with a free SH. Hamachi and co-workers also reported the affinity labeling modification followed by a hydrazone/oxime exchange reaction (Fig. 13.11). Benzenesulfonamaide, as a ligand of human carbonic anhydrase II (hCAII), is conjugated with an epoxide group as a reactive group through a hydrazone linkage. The affinity labeling is directed to the modification site close to the active site, and the hydrazone linkage between the ligand and the reaction site is subsequently replaced by an oxime bond. These two reactions proceed in a sequential manner under mild conditions to achieve post-labeling modification and reopen the active site cavity of the target enzyme to restore the enzymatic activity of the modified hCAII [42]. As a similar strategy, ligand-directed “tosyl” (LDT) chemistry was reported by the same research group (Fig. 13.12). In this system, an electrophilic phenylsulfonate (tosyl) group is conjugated with ligand molecules as a linker, and this ligand-­ conjugated phenylsulfonate ester specifically reacts with the nucleophilic amino acid side chains on the surfaces of target proteins through an SN2-type reaction, resulting in concomitant release of the ligand moiety and retention of the protein activity [43]. Furthermore, a quenched ligand-directed tosylate (Q-LDT) reagent was designed by linking an organic dye to a conjugate of a protein ligand and a fluorescence quencher through a tosyl linker (Fig. 13.13). When the Q-LDT reagent reacts with the target protein, its fluorescence is initially quenched by the organic dye linked to the molecule, and the fluorescence is enhanced (turned on) only in the presence of specific analytes due to the expulsion of the ligand-quencher fragment [44]. Acylphenol also shows a similar reaction to phenylsulfonate. Fenical and co-­ workers reported a fluorescent dye transfer strategy for identifying a target protein of marinopyrrole A, a natural product (Fig. 13.14). In this system, a carboxylic acid terminal tag containing a fluorescent dye is conjugated to a phenol in the natural

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Fig. 13.12  Schematic illustration of LDT chemistry for labeling endogenous proteins in living native cells

Q-LDT Reagent Fluorophore Quencher Ligand

Fig. 13.13  Schematic illustration of the strategy for the quenched ligand-directed tosylate (Q-LDT)-mediated construction of turn-on fluorescent biosensors. (Modified with permission from Ref. [44]. Copyright 2009 American Chemical Society)

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Fig. 13.14  A fluorescent dye transfer strategy for identifying the target protein of marinopyrrole A

Fig. 13.15  Schematic illustration of ligand-directed acyl imidazole (LDAI) chemistry for selective protein modification. Lg ligand, Nu nucleophilic amino acid. (Reprinted with permission from Ref. [46]. Copyright 2017 Authors licensed under CC BY 4.0)

product through an acyl linker, and the acyl carbonyl moiety of the acylphenol group undergoes an SN2-type reaction with nucleophilic amino acid residues to transfer the dye to the target protein [45]. Using this strategy, actin was identified as a target protein of marinopyrrole A, and the dye in the marinopyrrole probe was selectively transferred to the side chain amine of Lys115 in actin. Acyl imidazole can be used for ligand-directed selective modification of proteins. Hamachi and co-workers demonstrated the visualization of native α-amino-3-­ hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs), which are essential for cognitive functions without genetic manipulation using AMPA ligand-conjugated acyl imidazoles (Fig. 13.15) [46]. The ­diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices are characterized by using this method [47].

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Fig. 13.16  Schematic of “turn-on” fluorescent labeling of a target protein by the O-NBD method. A small bifunctional O-NBD unit is contained in the ligand. Lg ligand

Sodeoka and co-workers reported a unique modification system. They focused on O-nitrobenzoxadiazole (O-NBD), which is non-fluorescent, although amino NBD (N-NBD) exhibits a strong fluorescence at a long wavelength (λem 520– 550 nm), and thus designed the ligand-conjugated O-NBD probes (Fig. 13.16) [48]. In this system, the ligand-conjugated O-NBD probes bind to the target protein, and the O-NBD unit undergoes an SN2-type reaction with nucleophilic amino acid residues near the ligand-binding site of the target protein to afford an N-NBD-modified protein with “turn-on” fluorescence. They designed O-NBD-conjugated N,N-­ dialkyl-­2-phenylindol-3-ylglyoxylamides (PIGAs) and translocator protein (TSPO) ligands and used these to visualize mitochondria expressing TSPO in living cells by means of turn-on fluorescence. The O-NBD-conjugated PIGA probes selectively modified a partner protein of TSPO, a voltage-dependent anion channel (VDAC) in living cells.

3.3  Catalytic Ligand-Directed Target Protein Modification Catalytic target protein-selective modification was first reported by Hamachi and co-workers. They focused on 4-dimethylaminopyridine (DMAP), which is widely used as a catalyst for acylation in organic synthesis, and designed ligand-conjugated DMAP catalysts for target protein-selective chemical modification (Fig. 13.17). In this strategy, the catalysts first selectively bind to a target protein, and then an acyl donor reacts with a DMAP catalyst to generate an activated acyl group that undergoes an SN2-type reaction with nucleophilic amino acid residues to afford the sitespecifically acylated proteins, and the ligand-conjugated DMAP catalyst is regenerated. This modification technology has been demonstrated for labeling

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Sugar -tethered-DMAP

Acyl Donor

(a)

(b)

Lectin

(c)

Complexation

N

N

S Nu

O

Labeled lectin

Acyl Transfer Reaction +

N

N

O Nu

N

N

Nu

O

Fig. 13.17  Ligand-tethered-DMAP-catalyzed acyl transfer reaction for lectin. (Reprinted with permission from Ref. [49]. Copyright 2008 American Chemical Society)

Fig. 13.18  Acyl transfer reaction for lectin catalyzed by DMAP. (Reprinted with permission from Ref. [51]. Copyright 2015 American Chemical Society)

various proteins such as lectins; sugar-binding proteins, such as congerin II, concanavalin A, and wheat germ agglutinin [49]; and the ligand-binding proteins, such as the SH-domain and FKBP12 [50]. The acyl transfer reaction is accelerated by increasing the number of DMAP groups: multivalent DMAP catalysts modify the target protein more efficiently than a monovalent DMAP catalyst [50]. Antibodies can be used as ligands for the DMAP catalyst-based chemical modification. Hamachi and co-workers developed DMAP catalyst-antibody conjugates that enabled selective introduction of small chemical probes into receptor proteins such as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) on the surface of living cells (Fig. 13.18). The DMAP catalyst

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moiety is conjugated with HER2 scFv or anti-EGFR affibody molecules through proline-based linkers via a conventional cysteine-maleimide coupling reaction. An optimum length of the proline linkers exists for efficient modification of each antibody due to different accessibilities of the DMAP moiety to the nucleophilic amino acids on the antibodies [51]. Kunishima and co-workers reported alternative catalytic target protein-selective modification using a combination of 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N,N-dimethylglycine esters as a catalyst. A biotin-conjugated N,N-­ dimethylglycine ester was designed as a ligand-conjugated catalyst for selective chemical modification of avidin as a target protein. The N,N-dimethyl amino group of the catalyst bound to avidin reacts with CDMT to generate an activated ammonium intermediate which readily reacts with the carboxyl groups of aspartate or glutamate residues at the protein surface in the vicinity of the biotin-binding pocket to afford the modified avidin [52]. Popp and Ball reported the site-specific protein modification of aromatic side chains with the styryl-diazo reagent 8 by dirhodium metallopeptide catalysts (Fig.  13.19) [53]. They focused on stable dirhodium metallopeptide complexes formed via two glutamate side chain carboxylates on peptides in water and conducted the structure-selective peptide modification induced by peptide-peptide molecular recognition between dirhodium metallopeptide catalysts and polypeptides. Coiled-coil assembly of substrate peptides with dirhodium metallopeptide

Fig. 13.19  Catalytic covalent modification of a specific side chain of a substrate peptide using coiled-coil assembly to drive localization of a dirhodium metallopeptide. Label subscripts represent the position, on a helical-wheel model, of key residues that deviate from the parent E3 or K3 sequences. (Reprinted with permission from Ref. [53]. Copyright 2010 American Chemical Society)

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Fig. 13.20  Covalent modifications of various E3gX peptides with the styryl-diazo reagent 8 by dirhodium metallopeptide catalysts (K3a,eRh2)

catalysts enables modification of a wide range of amino acid residue side chains, such as tryptophan, tyrosine, phenylalanine, cysteine, glutamine, asparagine, glutamic acid, aspartic acid, arginine, serine, histidine, and lysine. Covalent modifications of various E3gX peptides with 8 using dirhodium metallopeptide catalysts (K3a,eRh2) are summarized in Fig. 13.20 [54]. Sato and Nakamura developed ligand-directed ruthenium photocatalysts (LDRPs) that activate tyrosines in the proximity of ligand-binding sites, and the generated tyrosyl radicals react with TRTs to afford the modified proteins (path A in Fig. 13.21). The modification reaction is based on a single electron transfer (SET) from the [Ru(bpy)3]2+ photocatalyst (Scheme 13.1) described in Sect. 2.4. Benzenesulfonamide, a ligand of carbonic anhydrase (CA), was conjugated with ruthenium photocatalysts and examined for the target protein-selective chemical modification. Using the photocatalytic probes and biotin-conjugated TRT, bovine carbonic anhydrase II (CA) was confirmed to be selectively modified in mouse erythrocyte lysates [55]. This system can be used for EGFR tyrosine kinase-­selective modification using gefitinib, an EGFR tyrosine kinase inhibitor, conjugated with ruthenium photocatalysts in cells. Interestingly, the LDRPs induce ­chromophore-­assisted light inactivation (CALI) of target proteins under visible light irradiation in  vitro and within cells in the absence of a TRT (path B in Fig. 13.21). Figure 13.22 shows EGFR knockdown in A431 cells, which overexpress EGFR on the surface of the cell membrane, using the gefitinib-conjugated ruthenium photocatalyst. Gefitinib, an EGFR tyrosine kinase inhibitor, binds to the ATP-binding pocket of EGFR tyrosine kinase. The gefitinib-conjugated ruthenium

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(A) Target-selective modification

Target protein

SET

Protein mixture

(B) Target-selective oxidative inactivation

Fig. 13.21  LDRP system for target-selective (A) modification and (B) oxidative inactivation

Fig. 13.22  EGFR knockdown using Ru-gefitinib in A431 cells

photocatalyst inhibits the phosphorylation of EGFR at 30 μM without affecting the downstream phosphorylation cascade, such as Akt and PKCα, or home proteins, such as actin and tubulin, whereas the [Ru(bpy)3]2+ photocatalyst does not inhibit the phosphorylation of EGFR under visible light irradiation, revealing that the CALI takes place on the ligand binding protein, EGFR. A mechanistic study suggests that a [Ru(bpy)3]2+ photocatalyst generates a single oxygen 1O2, which ­oxidizes proteins, making them inactive. The oxidation of histidine, methionine, and tryptophan residues is identified by MS analysis under light irradiation of various peptides in the presence of the [Ru(bpy)3]2+ photocatalyst, and the TRT scavenges 1O2 con-

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Fig. 13.23  Structures of [Ru(bpy)3]2+-conjugated with the GU40C peptoid (RuGU40C) and RIP1 (RuRIP1) for CALI targeting to VEGF and the 26S proteasome, respectively

comitantly with the coupling reaction to the tyrosyl radical generated by [Ru(bpy)3]2+. Consequently, the LDRPs can be used not only for target-selective protein modification but also for protein knockdown by CALI. CALI and selective protein modification can be regulated by the [Ru(bpy)3]2+ photocatalysts in the absence or presence of a TRT [56]. Kodadek and co-workers also reported two types of the peptoid-conjugated [Ru(bpy)3]2+ photocatalysts, RuGU40C4 and RuRIP1 (Fig. 13.23), for CALI targeting to vascular endothelial growth factor (VEGF) and the 26S proteasome, respectively [57]. The RuRIP1 peptoid is more labile than RuGU40C, because the indole side chain and the adenine ring involving in RuRIP1 probably undergo the self-­ oxidation with the [Ru(bpy)3]2+ photocatalysts. It is clear that the rates of CALI induced by both peptoid-conjugated photocatalysts are substantially faster than the self-oxidation rate.

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4  Selective Isolation and Modification of Target Proteins Various target protein-selective chemical modification strategies are now available as described in the previous section. However, the isolation of target proteins from a protein mixture is usually performed using affinity chromatography. Several matrices (beads or resins) are now available for affinity chromatography. Toshima and co-workers were first to demonstrate the selective isolation and chemical modification of target proteins from a protein mixture using a solid-phase affinity labeling method. They designed solid-supported chemical tools containing three functionalities in the molecular structure: (i) a targeting ligand moiety that selectively binds to a target protein, (ii) an alkylating group to covalently bind to the target protein, and (iii) a ligand exchange group to remove the labeled target protein from the solid support. Poly[acryloyl-bis(aminopropyl)polyethylene glycol] (PEGA) resin, which is porous, and hydrophilic, suitable for both chemical and enzymatic reactions, was chosen as the solid support, and a hydrazone group was used as a ligand exchange group to undergo a hydrazine oxime ligand exchange reaction. Figure 13.24a shows the strategy involving selective isolation and chemical modification of target proteins. Recognition of the target protein by the ligand moiety in the solid-supported chemical tool triggers alkylation of the nucleophilic amino acid residues on the target protein with 2-bromoacetamide, an alkylating reagent. The resulting alkylated proteins react with the hydrazone oxime and are removed from the solid support. Selective isolation and chemical modification of peanut agglutinin (PNA) was achieved with β-D-galactoside as a PNA ligand. Interactions between monosaccharides and lectins are known to be generally weak, and the association constant for binding of methyl β-D-galactoside to PNA is 1.18 × 103 M−1. Nevertheless, PNA was selectively modified in a mixture of four proteins, BSA, fetuin, PNA, and ribonuclease A (RNase A). Furthermore, when benzenesulfonamide was used as a ligand, the corresponding target protein, hCAII, was selectively isolated and modified in human red blood cell lysate [58]. Sakurai and co-worker reported multivalent carbohydrate photoaffinity probes using gold nanoparticles (AuNPs) to identify carbohydrate-binding proteins (Fig. 13.24b). In this system, a carbohydrate ligand and a benzophenone photoreactive group are assembled on the surface of AuNPs through bivalent S-Au bonds. UV light irradiation generates photo-cross-links between the benzophenone moiety and carbohydrate-binding proteins, and the resulting probe-cross-linked proteins are separated from the unreacted proteins in solution by centrifugation. The modified proteins are removed from the AuNPs by treating with a harsh protein denaturing buffer. The target proteins are identified by SDS-PAGE and MS analysis. Although this method is useful for target protein identification, the final step involving the harsh protein denaturing buffer resulted in the loss of the original protein functions [59]. Sato and Nakamura reported selective purification and chemical labeling of a target protein in a protein mixture simultaneously achieved on the surface of affinity beads functionalized with ligands and ruthenium photocatalysts (Fig. 13.24c).

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(A) Target protein

Target protein

Protein Lg mixture PEGA

Active OH

O

(B)

Target protein

X

X = C, N, O, S

OH X

Lg S S

S

Protein mixture

S

S S

HS S

OH

S SH SH

AuNP (C)

Inactive

O

OH O

Ru cat.

Lg FG

Protein mixture

HN

Ru cat.

Ru cat.

HN OH N

N

Blue light (455 nm)

Reusable

Active

Fig. 13.24  Selective isolation and modification of target proteins. (a) Alkylation of target proteins on the surface of PEGA resin followed by removal from solid supports with hydrazone oxime for modification. (b) Photo-cross-links on the surface of AuNPs followed by removal from AuNPs by treating with a harsh protein denaturing buffer. (c) Tyrosine modification of target proteins with a TRT through SET on the surface of FG beads. The functionalized FG beads are easily recovered from the reaction mixture with a magnet and can be reused several times without diminishing their capture and catalytic abilities

They applied target protein-selective chemical modification using LDRPs with a TRT to selective purification and chemical labeling to avoid the loss of the protein functions. The target protein ligands and ruthenium photocatalysts are introduced into magnetic FG beads functionalized with an NHS ester. FG beads are multiple ferrite particles with approximately 200  nm in diameter coated with a glycidyl methacrylate polymer; these have several characteristics, including low non-specific protein binding, easy recovery from mixtures with a magnet, and high dispersibility in aqueous solution. The conventional ruthenium photocatalyst functionalized on the surface of FG beads causes non-specific protein interactions probably due to its lipophilic properties, whereas the ruthenium photocatalyst functionalized with more hydrophilic carboxylate groups decreases the non-specific protein interactions, resulting in efficient selective purification and modification of target proteins efficiently. The FG beads functionalized with target protein ligands and ruthenium photocatalysts can be easily recovered from the reaction mixture with a magnet

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and reused several times without diminishing their capture and catalytic abilities. The purified and modified protein maintains sufficient enzymatic activity [60]. Importantly, this system can be used for the high-sensitivity detection of low-­ abundance target proteins or minor off-targets by chemical labeling with biotin-­ TRT on the affinity beads, unlike the conventional silver staining method, which is the most widely used tool for visualizing proteins with a subpicomolar detection limit. For example, an amine-conjugated methotrexate (MTX) derivative was introduced into the ruthenium photocatalyst-functionalized FG beads, and endogenous dihydrofolate reductase (DHFR), a target protein of MTX, was isolated and labeled with biotin-TRT for chemiluminescence detection from the diluted cell lysate sample even at a 0.1  mg/mL protein concentration, whereas the isolated DHFR was not detected by conventional silver staining at the same protein concentration.

5  Conclusion Protein chemical modifications were initially developed for investigating ligand-­ protein interactions, locating enzyme inhibitors, isolating and identifying unknown proteins, and identifying amino acid residues at protein-protein or protein-lipid interfaces. However, in the last decade, chemical modification of target proteins with small chemicals has been developed without losing the original functions of target proteins after modification, because real-time monitoring and control of target proteins in living systems are essential for understanding molecular mechanisms and dynamics and for protein engineering. In addition, chemical modification of target proteins has the potential to control both the structures and functions of proteins. Enzymatic posttranslational chemical modifications such as phosphorylation, farnesylation, glycosylation, and acetylation are natural events in regulating cell signals in living systems. Therefore, further development of technologies that enable artificial chemical modification of target proteins is urgently required for protein and cell engineering as well as in medicine. In this regard, understanding of enzymatic chemical modifications based on organic chemistry must be essential for designing chemical probes for artificial protein modification.

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Chapter 14

Target Identification of Bioactive Compounds by Photoaffinity Labeling Using Diazido Probes Suguru Yoshida and Takamitsu Hosoya

Abstract This chapter reviews a photoaffinity labeling (PAL) method which employs “diazido” probes bearing aromatic and aliphatic azido groups. Since our first report in 2004, the target molecules of various bioactive compounds have been identified by this method. It is a two-step target identification method involving the conjugation of the bioactive diazido probe with the target molecules through the selective photoreaction of the aromatic azido group and subsequent introduction of a detectable tag through a click reaction to the target molecules at the remaining aliphatic azido group. An overview of the history and recent progresses of this method, including facile methods for preparing diverse diazido building blocks, is presented, focusing mainly on the chemical aspects. The relevant methods using a bifunctional probe bearing photoreactive and bioorthogonal groups are also briefly summarized. Keywords  Photoaffinity labeling · Target identification · Azide · Diazirine · Photoreaction · Click reaction

Abbreviations 9-BBN 9-Borabicyclo[3.3.1]nonane AIBN 2,2′-Azobis(isobutyronitrile) Boc tert-Butyloxycarbonyl CG Connecting group CuAAC Copper-catalyzed azide–alkyne cycloaddition DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DIBAL–H Diisobutylaluminum hydride

S. Yoshida · T. Hosoya (*) Laboratory of Chemical Bioscience, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), Tokyo, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2019 Y. Kobayashi (ed.), Cutting-Edge Organic Synthesis and Chemical Biology of Bioactive Molecules, https://doi.org/10.1007/978-981-13-6244-6_14

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DPPA Diphenylphosphoryl azide dtbpy 4,4′-Di-tert-butyl-2,2′-bipyridyl FG Functional group HDAC Histone deacetylase HMGR Hydroxymethylglutaryl-coenzyme A reductase HTS High-throughput screening MALDI Matrix-assisted laser desorption/ionization MASCOT Modular approach to software construction operation and test MS Mass spectrum NBS N-Bromosuccinimide PAL Photoaffinity labeling pin Pinacolato RI Radioisotope SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis TBS tert-Butyldimethylsilyl TBTA Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine THP 2-Tetrahydropyranyl TMS Trimethylsilyl TOF Time of flight Ts p-Toluenesulfonyl

1  Introduction Target identification of bioactive compounds is an important matter in life science researches, including in the fields of pharmaceutical science and molecular biology. Recent advances in high-throughput screening (HTS) assays using a chemical library, particularly those based on biological phenotypes, have led to the identification of a number of promising bioactive compounds with the mechanism of action unknown. Target identification of these compounds paves the way to develop innovative drugs that work based on novel mechanisms. Moreover, the identification of new target molecules of existing drugs may help the elucidation of the mechanism of their adverse effects and may also offer the possibility of applying them to other cases. This chapter summarizes a target identification method focusing on a radioisotope (RI)-free photoaffinity labeling technique using a “diazido probe” that we previously developed.

2  Chemical Methods for Target Identification Affinity purification and photoaffinity labeling (PAL) are chemical methods that are widely used for the target identification of bioactive compounds (Fig. 14.1). These methods are based on the strong interaction between the bioactive compound (ligand) and its target molecules.

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Fig. 14.1  Chemical methods for target identification

Affinity purification is performed by treating a biological sample that contains target molecules with a ligand-immobilized polymer such as sepharose and agarose (Fig. 14.1, panel A) [1, 2]. This method can easily identify the target biomolecules to which the ligand binds with high affinity. The method developed by Handa and coworkers using magnetic beads immobilized with a bioactive ligand is an efficient method as exemplified by the target identification of bioactive small compounds such as thalidomide [2]. The key to success in target identification by affinity purification is to find an appropriate position in the ligand when immobilizing the polymer so that the interaction between the ligand and the target molecules is not affected. PAL is another efficient chemical method for identifying the target molecules of bioactive compounds by capturing their target molecules by means of a photoactivatable probe (Fig.  14.1, panel B) [3–5]. In contrast to the affinity purification method, the PAL method identifies the targets through covalent bond formations with the PAL probe. Generally, PAL probes are equipped with a photoreactive group and a detectable group, which are introduced to the original bioactive compounds of interest. The key to success in this method is to design probes that show sufficient bioactivity compared with the original compounds. Photoirradiation of a mixture between a biological sample that contains target

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molecules and the PAL probe results in the formation of a covalent bond between the probe and the target molecules. The analysis of the photolabeled molecules is guided by the detectable group installed in the probe; this analysis allows for the identification of specific target molecules from among the numerous molecules in the biological sample. In principle, the binding site of the probe compound on the target molecules can be identified by analyzing the fragments obtained by further enzymatic digestion of the photolabeled molecules. For example, Colca and coworkers demonstrated that linezolid binds specifically to 23S ribosomal RNA through a PAL study using a PAL probe bearing a photoreactive aromatic azido group and a detectable radioisotope iodine-125 group [6]. Several photoreactive groups have been employed in PAL studies (Fig.  14.2). These groups have both advantages and disadvantages, and the most suitable is selected for PAL probe synthesis depending on the research subjects. The aromatic azido group is one of the most widely used photoreactive groups because of its ease for preparing PAL probes (Fig. 14.2, panel A) [7]. Generally, irradiation of aromatic azides with 254 nm ultraviolet (UV) light causes the transient generation of a nitrene intermediate with the liberation of nitrogen from the azido group. Then, ring expansion of the nitrene species proceeds rapidly to form dihydroazepine, which is attacked by a nucleophilic amino acid residue close to the PAL probe to form a covalent bond that results in the capture of the target molecules.

Fig. 14.2  Typical photoreactive groups used for PAL probes

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In addition, since azido group is prone to be reduced, the use of aromatic azido-­ installed PAL probes is not recommended under reductive conditions. The diazirinyl group, in particular, 3-trifluoromethyldiazirin-3-yl group has also been widely used as a photoreactive group for PAL probes (Fig. 14.2, panel B) [8, 9]. Although the synthesis of aryl trifluoromethyldiazirines involves multiple steps, including difficult transformations, they are widely used owing to their preferable photoreactivity. Highly reactive carbene intermediates that can react with diverse amino acid residues are generated by irradiating diazirines with 365 nm UV light accompanied by the removal of nitrogen. Photoirradiation of benzophenones generates biradical intermediates reversibly. These intermediates react with various amino acid residues. However, since the benzophenone skeleton is polarized and large, the bioactivity of this type of probes is often decreased compared with that of the original compounds (Fig. 14.2, panel C) [10, 11]. Recently, Hirai, Sodeoka, and coworkers found that thienyl-substituted α-ketoamides serve as a less hydrophobic photoreactive group and are suitable for PAL studies [12]. Various detectable groups that allow the detection of photolabeled molecules have been employed in PAL studies (Fig. 14.3). Conventionally, radioisotopes (RIs), biotin, and fluorescent groups have been used (Fig. 14.3, panels A–C). However, probes labeled with RI cannot be used in the direct analysis of the photolabeled

Fig. 14.3  Typical detectable groups used for PAL probes

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molecules, and RI-free methods using probes installed with a biotin or a fluorescent group often result in a significant decrease of the bioactivity [3–5, 13]. To address these problems, we developed a two-step photoaffinity labeling method using a bioorthogonal functional group as the latent detectable tag (Fig. 14.3, panel D) [14].

3  Diazido Probe Method 3.1  Overview In 2004, we reported the diazido probe method as a new RI-free photoaffinity labeling method (Fig.  14.4) [14]. This is a two-step target identification method involving a photoreaction and subsequent click reaction [15, 16] using diazido PAL

Fig. 14.4  Diazido probe method

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probes bearing two types of azido groups—a photoreactive aromatic azido group and a relatively photostable aliphatic azido group. As the first step, a mixture of a bioactive diazido probe and a biological sample is irradiated with 254 nm UV light to generate nitrene species for capturing the target molecules through the formation of covalent bonds. The second step is the introduction of a detectable tag, such as a fluorescent group, to the remaining aliphatic azido group [14]. Then, the photolabeled molecules are identified after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis or after further digestion and following sequence analysis. This method enables preparation of bioactivity-retained diazido probe candidates because only small structural and polarity changes from the original bioactive compound are needed. Indeed, several groups have succeeded in the target identification of bioactive compounds using this method (Fig. 14.5). In the following section, the development history of the diazido probe method and studies on its applications conducted with our collaborators are described.

Fig. 14.5  Diazido PAL probes developed by other groups

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3.2  D  iscovery of the Photoreactivity Difference Between Aromatic and Aliphatic Azido Groups The diazido PAL probe method was developed based on the discovery of a large difference in the photoreactivity between aromatic and aliphatic azido groups. We found that irradiation of an equimolar mixture of phenyl azide (1a) and benzyl azide (1b) in acetonitrile with 254 nm UV light resulted in the selective consumption of 1a within 5 min and most (>90%) of 1b remained intact (Fig. 14.6). Aromatic azido group-selective photoreaction was also observed for C2-symmetrical triazido compound 2 (Scheme 14.1). Thus, the photoirradiation of 2 in the presence of excess diethylamine afforded 3H-azepine 3 via the generation of nitrene I, ring expansion to form II, and nucleophilic attack to II by the amine. These results clearly show that the aromatic azido group is remarkably more photoreactive than the aliphatic azido group.

N3 1a

N3

+ Ph 1b

h (254 nm) CH3CN

decomposed + 1a + 1b products

Fig. 14.6  Photoreaction of an equimolar mixture of azides 1a and 1b

Scheme 14.1  Photoreaction of triazido compound 2

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3.3  Photovastatin: The First Diazido PAL Probe To demonstrate the effectiveness of the diazido probe method, we prepared photovastatin CAA1 (5) as a diazido probe of cerivastatin (4) and examined whether it could photolabel the major target protein of 4, that is, human 3-hydroxy-3-­ methylglutaryl coenzyme A reductase (HMGR) (Fig. 14.7) [17–19]. Probe 5 was designed by replacing the methoxy group of 4 with 3-azido-5-(azidomethyl) benzyloxy group. Starting from known 3,5-bis(hydroxymethyl)aniline (6), 3-azido-­ 5-(azidomethyl)benzyl alcohol (7) was prepared by azidation via diazotization, monobromination of the hydroxy group, and SN2-type azidation (Scheme 14.2). Then, SN2-type etherification of deprotonated alcohol 7 with aryl bromide 8, which is the protected main structure of the probe, was conducted, which was followed by the deprotection of tert-butyl ester and 1,3-diol moiety to afford the desired 5, leaving the azido groups unreacted. Importantly, the synthesized probe 5 showed a potent inhibitory effect on the recombinant human HMGR similar to that exhibited by original compound 4.

Fig. 14.7  Cerivastatin (4) and diazido probe photovastatin CAA1 (5)

Scheme 14.2  Synthesis of photovastatin CAA1 (5)

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Scheme 14.3  PAL experiment using photovastatin CAA1 (5) and recombinant HMGR

Fig. 14.8  1BnTIQ (10) and diazido probe 11

The PAL experiment using the probe 5 and recombinant human HMGR was conducted by photoirradiating their mixture with 254 nm UV light (Scheme 14.3). The resulting mixture was treated with fluorescein-conjugated phosphine 9 to introduce the fluorescent group to the photolabeled protein at the remaining azido group by the Staudinger–Bertozzi ligation [20]. The SDS-PAGE analysis showed a dosedependent increase in the intensity of the fluorescent signal. In addition, a decrease in the intensity of the fluorescent signals was observed when the photolabeling with the probe 5 was conducted in the presence of original compound 4. These results indicate that the enzyme was photolabeled specifically with the probe 5. Moreover, the sequence analysis of the digested photolabeled protein provided the binding site information, which was in good agreement with the X-ray co-crystallographic structures of HMGR and cerivastatin [18].

3.4  Diazido PAL Probe of 1BnTIQ In a collaborative study with Ohta and coworkers, we successfully developed diazido probe 11 of 1BnTIQ (10) [21], which is considered to act as an endogenous neurotoxin (Fig. 14.8) [22]. Since 1BnTlQ has a benzyl moiety at C2 position in the

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Scheme 14.4  Synthesis of diazido PAL probe 11

Scheme 14.5  PAL experiment using diazido probe 11 in subcellular fractions of rat whole brain homogenates

tetrahydroisoquinoline skeleton, we designed diazido probe 11, which was assumed to be prepared by the palladium-catalyzed Suzuki–Miyaura cross-coupling reaction between 1-azido-3-(azidomethyl)-5-iodobenzene (14) and organoboron compound 17 (Scheme 14.4) [23]. Actually, diazido building block 14 was synthesized from 3,5-dinitrobenzyl alcohol (12) in six steps: monoreduction of one of the nitro groups, iodination of the resulting amino group via diazotization, reduction of the remaining nitro group, azidation via diazotization, mesylation of the hydroxyl group, and SN2-type azidation. Organoboron compound 17 was separately prepared by deprotonation and N-Boc-protection of dihydroquinolinium salt 15 and subsequent hydroboration using 9-BBN. Cross-coupling between 14 and 17 and deprotection of the Boc group afforded diazido probe 11, which had neurotoxicity comparable to that of the original 1BnTIQ (10). The target protein of 1BnTIQ (10) was determined to be tubulin β based on a PAL study using diazido probe 11 for subcellular fractions of rat whole brain homogenates (Scheme 14.5). Irradiation of the fractionated rat whole brain homogenates containing 11 with 254  nm UV light, the Staudinger–Bertozzi ligation by treatment with fluorescein–phosphine 9, SDS-PAGE analysis or two-dimensional (2D)-electrophoresis, sequence analysis by MALDI-TOF/TOF-MS of the photolabeled proteins, and the MASCOT analysis revealed that tubulin α and tubulin β as the target candidates. Further, immunoprecipitation method indicated that the direct target of 1BnTIQ (10) is tubulin β.

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3.5  Diazido PAL Probe of Dantrolene In collaboration with Ikemoto, Endo, and coworkers, we developed PAL probes of dantrolene, which is a muscle relaxant used as a clinical drug for the treatment of malignant hyperthermia (Fig. 14.9). Based on previous studies using iodine-125-­ labeled PAL probe 19 [24], we prepared the corresponding diazido probe 20, where the iodine-125 was replaced with an azidomethyl group [25]. By using these PAL probes, skeletal-type neuroendocrine-specific protein-like 1 (sk-NSPl1) was identified as one of the target molecules of dantrolene; this target is involved with the membrane translocation of GLUT4 induced by contraction/exercise [26].

4  S  ynthesis of Various Diazido Building Blocks for Expeditious Photoaffinity Probe Development 4.1  Background HTS of the chemical library has become one of the conventional approaches for the initial stage of drug development study. In particular, with the technical advances in phenotypic assays, target-unknown hit compounds are expected to increase. To contribute to their target identification by expediting the development of diazido PAL probes, we prepared various types of 3-azido-5-(azidomethyl)benzene derivatives bearing connecting groups.

Fig. 14.9  Dantrolene (18) and PAL probes 19 and 20

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4.2  D  iazido Arylboronic Acid Pinacol Ester for the Facile Synthesis of Biaryl-Type Diazido Probe Candidates To make a wide range of biaryl-type diazido compounds easily synthesizable, 3-azido-5-(azidomethyl)phenylboronic acid pinacol ester (21) was prepared by the Miyaura borylation [27] of 1-azido-3-(azidomethyl)-5-iodobenzene (14) with bis(pinacolato)diboron leaving the azido groups untouched (Fig. 14.10) [28]. The Suzuki–Miyaura cross-coupling [29] of 21 with a wide range of (hetero)aryl bromides proceeded smoothly using tetrakis(triphenylphosphine)palladium(0) as the catalyst in the presence of potassium phosphate in DMF at 80 °C to afford the corresponding biaryl 22 in high yields. These results indicate that 21 is a useful diazido building block for preparing biaryl-type diazido compounds without damaging the azido groups. By using this method, another type of diazido dantrolene analog 22e, which exhibited bioactivity similar to that of the previous probe 20a, was prepared [28].

Fig. 14.10  Synthesis of biaryl-type diazido compounds using borylated building block 21

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4.3  F  acile Synthesis of Various Diazido Building Blocks via the Formal C–H Azidation A facile method for preparing various diazido building blocks from inexpensive 1,3-disubstituted benzenes via formal C–H azidation was also developed (Scheme 14.6) [30, 31]. The formal C–H azidation was achieved by iridium-catalyzed regioselective C–H borylation [32] of m-halobenzoic acid esters and m-alkoxycarbonylor m-cyano-substituted toluenes followed by copper-catalyzed deborylazidation [33]. Further transformations of one-carbon unit (C1, alkoxycarbonyl and methyl groups) to an azidomethyl group afforded several diazido building blocks bearing functional groups, which were further transformed to various connecting groups (CGs) (vide infra). Four types of aryl azides 24a–24d bearing a one-carbon unit (C1) and a functional group (FG) were efficiently prepared from commercial 1,3-disubstitued benzenes 23 by a one-pot procedure involving the iridium-catalyzed C–H borylation, removal of the solvent under reduced pressure, and subsequent copper-catalyzed azidation using trimethylsilyl azide under air (Fig. 14.11) [30]. A previous attempt to borylate

Scheme 14.6  Facile synthesis of various diazido building blocks bearing connecting groups (CGs)

Fig. 14.11  Synthesis of aryl azides 24 by the formal C–H azidation

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Scheme 14.7 Synthesis of diazido compounds 14 and 25

Scheme 14.8  Synthesis of diazido compounds 27 and 28

3-azido-5-(azidomethyl)benzene by the iridium-catalyzed C–H borylation was unsuccessful, and a small amount of the corresponding aniline was obtained via the reduction of the azido group. Nevertheless, the corresponding anilines were not obtained in the formal azidation of 23, which was performed in the presence of iridium catalyst and bis(pinacolato)diboron that could reduce the azido group. This result suggests that the deactivation of the iridium catalyst by air oxidation is the key to avoid the undesired reduction of the azido group. Reduction of the ester moiety of 24a and 24b using DIBAL–H at −78 °C and the subsequent azidation of the resulting alcohols by the Mitsunobu–Merck method [34] using diphenylphosphoryl azide (DPPA) and 1,8-diazabicyclo[5.4.0]undec-7-­ ene (DBU) afforded diazido compounds 14 and 25 in good yields (Scheme 14.7). This method greatly improved the availability of 14 from the previous method shown in Scheme 14.4. Diazido benzoic acid ester 27 and diazido benzonitrile 28 were prepared from azides 24c and 24d by radical bromination using N-bromosuccinimide (NBS) and a catalytic amount of azobisisobutyronitrile (AIBN), followed by SN2-type azidation of the resulting benzyl bromides 26a and 26b (Scheme 14.8). Diazido compounds 14, 27, and 28 bearing an iodo, methoxycarbonyl, and cyano groups, respectively, were transformed to various diazido building blocks bearing various types of connecting groups, leaving the two azido groups untouched (Fig.  14.12). These building blocks include arylboronic acid ester 21, phenol 29, benzoic acid 30, succinimidyl ester 31, benzoyl chloride 32, benzyl alcohol 7, benzyl bromide 33, benzaldehyde 34, benzamide 35, aniline 36, and amidoxime 37. These diazido building blocks can be conjugated with various molecules through various types of reactions including condensation, addition, substitution, and transition metal-catalyzed coupling reactions to afford diazido probe candidates very easily.

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Fig. 14.12  Diazido building blocks prepared from diazido compounds 14, 27, and 28

5  O  ther Bifunctional Units Bearing Photoreactive and Bioorthogonal Groups 5.1  A  ryl Trifluoromethyldiazirine Derivatives Bearing a Clickable Group The diazido probe concept was expanded to other bifunctional probes bearing photoreactive and clickable groups in combinations other than aromatic and aliphatic azido groups. For example, in the study of dantrolene-based PAL probe synthesis, we prepared three other probe candidates 38 bearing aromatic azido and ethynyl groups and aryl trifluoromethyldiazirine derivatives 39a and 39b bearing azidomethyl and ethynyl group, respectively (Fig.  14.13) [25]. Using model substrates 40a and 40b, the photoreactivity of trifluoromethyldiazirinyl moiety was examined (Schemes 14.9 and 14.10). Sequential irradiation of these compounds with 365 and 302 nm UV light in methanol-d4 afforded denitrogenative methanol-d4 adducts, 41a and 41b, respectively, indicating that the generation of carbenes such as III and their insertion in methanol-d4 proceeded smoothly without damaging the clickable azido and ethynyl groups. The remaining ethynyl group was shown to be clickable with an azide as demonstrated in the CuAAC reaction between 41b and benzyl azide.

5.2  B  ifunctional PAL Units Bearing an “All-in-One” Substituent Because the minimal modification of the original compounds is preferable to retain the bioactivity when preparing PAL probes, in 2007, we developed a small building block 48 bearing an (azidodifluoromethyl)diazirinyl group, which includes

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Fig. 14.13  Other bifunctional PAL probe candidates of dantrolene

Scheme 14.9  Photoreaction of diazirine 40a

Scheme 14.10  Photoreaction of diazirine 40b

photoreactive diazirinyl and clickable azido groups (Scheme 14.11) [35]. This bifunctional PAL unit with an “all-in-one” substituent was prepared with aryl bromide 43 as the starting compound. Thus, α-chloro-α,α-difluoroacetophenone 44 was prepared from 43 via lithiation of the bromo group. Azidation of 44 and the following transformation of the carbonyl group of resulting 45 to the diazirinyl group by oxime formation, O-tosylation, treatment with liquid ammonia, and oxidation afforded (3-(azidodifluoromethyl)diazirin-3-yl)benzene derivative 48 in high yield. Irradiation of 48 in methanol-d4 with 365 nm UV light afforded an approximately 2:1 mixture of insertion product 49 and linear diazo compound 50 leaving the azido group untouched (Scheme 14.12). The diazo 50 was transformed to 49 by subsequent irradiation with 302 nm UV light. The remaining azido group of photoproduct 49

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Scheme 14.11  Synthesis of 3-aryl-3-(azidodifluoromethyl)diazirine unit 48

Scheme 14.12  Photoreactions of aryl(azidodifluoromethyl)diazirine 48 Fig. 14.14 Bifunctional PAL probe and unit bearing (ethynyldifluoromethyl) diazirinyl group

was shown to react with phenylacetylene under the CuAAC reaction conditions to afford triazole 51. These results indicate that 3-aryl-3-(azidodifluoromethyl) diazirine derivatives can be applied for two-step PAL studies. In 2009, Young and Kumar reported a similar bifunctional PAL probe 52 bearing (ethynyldifluoromethyl)diazirinyl group as an all-in-one unit, though it was synthesized via a linear route (Fig. 14.14) [36]. The photoreaction of 52 in methanol and subsequent click conjugation with a biotin-conjugated azide were also demonstrated.

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5.3  P  AL Probes Bearing a Clickable Functional Group Developed by Other Groups A wide variety of bifunctional PAL units bearing photoreactive and clickable groups has been developed [37]. In 2012, Cravatt and Cisar proposed an excellent approach using a “fully functionalized” small molecule library comprising terminal alkyne-­ substituted 5-benzoyl indole (BzIndole) and 7-benzoylbenzo-1,4-diazepin-2,5-­ dione (BzBD) scaffolds, which was used for phenotypic screening and target identification (Fig. 14.15) [38]. The same group recently extended the method to target discovery by fragment-based screening in human cells [39]. In 2013, Haberkant and coworkers developed a diazirinyl- and terminal alkyne-containing fatty acid 53 as a PAL probe of palmitic acid (54) to study protein–lipid interactions in living cells (Fig. 14.16) [40]. Yao and coworkers developed various “minimalist linkers” bearing the alkyl diazirine moiety, a clickable group, and a connecting group (CG) that are small building blocks preferred for preparing PAL probes (Fig.  14.17) [41, 42]. Recently, Woo and coworkers reported 3-(ethynyldifluoromethyl)diazirin-3-ylmethanol (55) as a minimal all-in-one PAL unit (Fig.  14.18) [43]. Several bifunctional building blocks bearing connecting groups such as 56 and 57 are currently commercially available, indicating the increasing demand for these types of PAL probes (Fig. 14.19).

6  Summary This chapter described the history and recent advances in photoaffinity labeling methods using diazido and other relevant probes. The easy availability of various types of bifunctional building blocks bearing a connecting group has expedited the development of photoaffinity probes. Through the target identification of bioactive compounds, these methods are expected to contribute to the advancement of basic molecular biology and innovative drug development studies.

Fig. 14.15  Bifunctional PAL probe library

Fig. 14.16  Bifunctional PAL probe 53 of palmitic acid (54)

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Fig. 14.17 Minimalist photo-crosslinkers

Fig. 14.18  Minimal all-in-one PAL unit

Fig. 14.19  Commercially available building blocks for bifunctional PAL probe synthesis Acknowledgments  This work was supported by Japan Agency for Medical Research and Development (AMED) under Grant Numbers JP18am0101098 (Platform Project for Supporting Drug Discovery and Life Science Research, BINDS) and JP18am0301024 (the Basic Science and Platform Technology Program for Innovative Biological Medicine); JSPS KAKENHI Grant Numbers JP15H03118 and JP18H02104 (B; T.  H.), JP16H01133 and JP18H04386 (Middle Molecular Strategy; T. H.), JP17H06414 (Organelle Zone; T. H.), and JP26350971 (C; S. Y.); the Cooperative Research Project of Research Center for Biomedical Engineering; and the Naito Foundation (S. Y.).

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