New Tide of Natural Product Chemistry 9819917131, 9789819917136

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Hayato Ishikawa Hiromitsu Takayama Editors

New Tide of Natural Product Chemistry

New Tide of Natural Product Chemistry

Hayato Ishikawa · Hiromitsu Takayama Editors

New Tide of Natural Product Chemistry

Editors Hayato Ishikawa Chiba University Chiba, Japan

Hiromitsu Takayama Chiba University Chiba, Japan

ISBN 978-981-99-1713-6 ISBN 978-981-99-1714-3 (eBook) https://doi.org/10.1007/978-981-99-1714-3 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 This work is subject to copyright. All rights are solely and exclusively licensed 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, expressed 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

Introduction

A vast number and extremely diverse structures of organic compounds have been discovered in living organisms. The remarkable and useful biological activity exhibited by many of these natural products has driven the development of various fields of natural science, such as chemistry, biology, medical physiology, plant science, and analytical technologies. Indeed, natural products have historically been one of the most medicinally important resources and the study of them has contributed greatly to the development of therapeutic drugs. In fact, more than 50% of smallmolecule medicines approved by the Food and Drug Administration between 1981 and 2019 are natural product-related compounds (e.g., unaltered natural products, botanical drugs, natural product derivatives, and mimics of natural products). In addition, natural products are important tools for research that attempt to understand various life phenomena in nature at the molecular level. For example, it has become clear that natural small molecules are used for association and communication between different classes of organisms; several findings that have attracted increasing attention. Recently, with the advancement of biotechnology, genetic engineering, synthetic methodology, and analytical and computational sciences, natural product chemistry related to classical as well as many interdisciplinary areas has made significant progress. Under such circumstances, this book has been edited to introduce research at the forefront of this field conducted by up-and-coming researchers. In addition to the important achievements accumulated so far, the author’s own cutting-edge results are included. I am confident that new entrants as well as senior researchers in related fields will benefit from this overview of the latest research in natural product chemistry. The book consists of four parts, which cover a wide range of research areas related to natural product chemistry. The first part contains five chapters that describe novel approaches to the search for new bioactive natural products and chemical biology studies using natural products. Chapter 1 introduces a novel synthetic biology approach that has enabled the fungal production of meroterpenoids never before found in nature. Chapter 2 describes the discovery, structure determination, and mechanism of action of novel v

vi

Introduction

natural products that act as SERCA inhibitors from marine cyanobacteria. In Chapter 3, the discovery of marine natural products that act specifically in a tumor microenvironment is presented and the chemical approach used to identify their target molecules is described. In Chapter 4, possible mechanisms for the inhibitory effect of aplyronine A (ApA), a marine antitumor macrolide, on microtubule dynamics are proposed based on molecular dynamics simulation studies on the actin–ApA– tubulin heterotrimeric complex. The development of simple side-chain analogues of ApA that potently bind to and depolymerize actin is also introduced. In Chapter 5, immunological functions and chemical syntheses of bacterial lipid A derivatives are summarized. Representative natural products appearing in Part I are as follows.

MeO

O

OH OMe

O

OH O

O HO P O HO O O

O O

HN

O

HO

O

O

O

HO O

O

O HN O P OH O OH

HO

O

O

O

HO

H

FDDP K (C14) (C10) (C14) (C14)

(C14)

N

(C12)

S

Alcaligenes faecalis lipid A

NH

iezoside

N

O

O

O O

O OH

O

N

biakamide A

S

N O

O O OH

O

OMe NMe2

Cl

O

O O

OMe

OH

N

OH NMe2

MeO

aplyronine A

O

OAc

Me N CHO

Introduction

vii

The second part consists of three chapters and summarizes the state-of-the-art in studies of the biosynthesis of secondary metabolites. Chapter 6 summarizes studies on the biosynthesis of three kinds of natural products from filamentous fungi having distinctive structural characteristics and useful biological activities. Chapter 7 covers new trends in biosynthetic studies of fungal polyketides based on the phylogenetic analysis of polyketide synthases and describes the chemical space of related polyketides. In Chapter 8, structural and functional analysis of key biosynthetic enzymes of some β-amino-acid-containing macrolactam polyketides and biosynthetic engineering for the production of non-natural macrolactam polyketides are described. Representative natural products appearing in Part II are as follows. OH

OH

OH

OH

O OH

phialotide A aglycone MeO HO

O

O N

O

H3C N H OH

Cl S

O

H N

O O

N H

aspirochlorine

S vicenistatin

The third part, containing six chapters, introduces the total synthesis of natural products with diverse structures. Efficient synthetic routes are required for the synthesis of structurally complex natural products. To this end, each researcher plans their own retrosynthetic route and devises methods to achieve the critical and difficult steps in the synthesis. In Chapter 9, a great deal of synthetic effort is described for the structure determination of a marine natural product, symbiodinolide, which is a 62-membered polyol macrolide with a molecular weight of 2860 and 61 chiral centers. In Chapter 10, the efficacy of a bioinspired strategy is demonstrated by realizing the collective total synthesis of eighteen monoterpene indole alkaloids by following a biosynthetic tree diagram. Chapter 11 presents efficient total syntheses of four kinds of structurally different oxacyclic natural products by incorporating cascade reactions. In Chapter 12, total syntheses of three kinds of highly functionalized natural products utilizing newly developed radical coupling reactions are introduced. Chapter 13 summarizes the achievement of efficient total syntheses of polycyclic alkaloids by applying the devised nucleophilic addition reaction toward the usually inactive amide function. Chapter 14 introduces a concise total synthesis of a complex indole alkaloid, which was accomplished in an elegant manner with equilibrium-controlled stereoselective tandem cyclizations. Representative natural products appearing in Part III are as follows.

viii

Introduction OH HO

OH

O

Me

OH

OH

OH

O

OH

Me

OH

OH

HO

OSO3Na OH

HO

Me

Me

OH OH

O

O

OH OH HO

OH

OH OH

OH

OH O

HO

OH Me

O O

OH OH OH OH

OH

OH HO

OH

H O

HO OH

O

OH

OH

NH

OH

O

NH

N H H

Me

OH OH

O

OH

OH HO H

OH

HO

H

OH

O O

O

HO

OH

O

O

O O

HO

O

MeO

Me OH

OH HO

symbiodinolide

OH

O

HO

OH

strictosidine

lysidicin A OMe

OH

OH

O

H2N

H 2N

HO O

N N

OH

OH

O

OH OH NH2

HO

H H

O H

O O

H O

N

H

protostemonine O

OH

NH

OH

hikizimycin

O

O

N H

tronocarpine

OH

H

O

Introduction

ix

In fourth part, consisting of three chapters, new approaches for drug discovery using natural products are introduced. Chapter 15 reviews the development of high-throughput searches using natural product libraries to discover inhibitors of aggregation of amyloidogenic proteins. Chapter 16 introduces an innovative highthroughput strategy; namely, one-bead-one-compound-based functional enhancement and modulation of two kinds of peptidic natural products, which led to the successful discovery of natural product analogues that exhibit enhanced and altered functions. The development of novel innate-like T-cell modulators through the modification of natural ligands is summarized in Chapter 17. Representative natural products appearing in Part IV are as follows. OH

HO H

O

CO2H

HO

OH

HO

OH

OH

O

O

OH

H

OH

O

O

OH

myricitrin

uncarinic acid C

O NH

N

OCH3

O

H N

HN

O OH O

HO

OH OH

5-OP-RU

NH

H 2N O HO

NH HN

O H 2N

O

O HN NH

H N

O O HN

O OH

NH O

NH

O O

NH

NH

O

O N

HN O O

HO NH

O

HO

HN O HN NH

lysocin E

H2N

As described above, each chapter begins with a brief and easy-to-understand introduction and then leads the reader toward the research frontlines of the individual specialty. Thus, this book will not only be a practical and essential reference resource for natural product chemists, medicinal chemists, synthetic organic

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Introduction

chemists, biochemists, and pharmacologists, as well as the pharmaceutical and biotechnological industries, but will also be a useful guide to understanding new and emerging trends in this field. I hope that readers will enjoy the diversity, development, dynamics, and potential of natural product chemistry through this book. Finally, I would like to express my gratitude to the authors who contributed to this book and to the editorial team at Springer Nature. Chiba, Japan

Hiromitsu Takayama

Contents

Part I

Exploring Novel Bioactive Natural Products and Uncovering Life Phenomena with Natural Products

1

Synthetic Biology-Based Natural Product Discovery . . . . . . . . . . . . . . Teigo Asai

2

Sarco/Endoplasmic Reticulum Ca2+ -ATPase (SERCA) Inhibitors Isolated from Subtropical Marine Cyanobacteria in Japan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arihiro Iwasaki

3

Marine Natural Products Targeting Tumor Microenvironment . . . . Naoyuki Kotoku

4

Chemical Biology Studies on Aplyronine A, A PPI-Inducing Antitumor Macrolide from Sea Hare . . . . . . . . . . . . . . . . . . . . . . . . . . . . Masaki Kita

5

Chemical Synthesis and Immunological Functions of Bacterial Lipid A for Vaccine Adjuvant Development and Bacterial-Host Chemical Ecology Research . . . . . . . . . . . . . . . . . . Atsushi Shimoyama

Part II

3

17 35

59

77

Uncovering Biosynthesis of Natural Products

6

Dissecting Biosynthesis of Natural Products Toward Drug Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Yuta Tsunematsu

7

A New Trend in Biosynthetic Studies of Natural Products: The Bridge Between the Amino Acid Sequence Data and the Chemical Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Atsushi Minami

xi

xii

8

Contents

Biosynthesis of β-Amino Acid-Containing Macrolactam Polyketides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Akimasa Miyanaga

Part III Total Synthesis of Complex Natural Products by Innovative Strategies 9

Synthetic Approach Toward Structural Elucidation of Marine Natural Product Symbiodinolide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Hiroyoshi Takamura

10 Collective Synthesis of Monoterpenoid Indole Alkaloids Using Bioinspired Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Hayato Ishikawa 11 Total Syntheses of Bioactive Oxacyclic Natural Products . . . . . . . . . . 235 Yusuke Ogura 12 Total Syntheses of Densely Oxygenated Natural Products by Radical-Based Decarbonylative Convergent Assembly . . . . . . . . . 259 Masanori Nagatomo 13 Nucleophilic Addition to Amides Toward Efficient Total Synthesis of Complex Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 Takaaki Sato 14 Equilibrium-Controlled Stereoselective Sequential Cyclizations Enabled Concise Total Synthesis of Complex Indole Alkaloid, Tronocarpine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 Atsushi Nakayama Part IV New Approach for Drug Discovery Using Natural Products 15 High-Throughput Searches for Natural Products as Aggregation Modulators of Amyloidogenic Proteins . . . . . . . . . . . . 313 Kazuma Murakami 16 Discovery of Natural Product Analogues with Altered Activities by a High-Throughput Strategy . . . . . . . . . . . . . . . . . . . . . . . 333 Hiroaki Itoh 17 Development of Novel Ligands That Modulate Innate-Like T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 Shinsuke Inuki

Part I

Exploring Novel Bioactive Natural Products and Uncovering Life Phenomena with Natural Products

Chapter 1

Synthetic Biology-Based Natural Product Discovery Teigo Asai

Abstract Natural products are historically one of the most important pharmaceutical sources for drug development. In the post-genomic era, next generation sequencing analyses of microorganisms show the presence of a vast number of biosynthetic gene resources that have been recognized as attractive sources for novel natural products and novel biosynthetic machineries. A synthetic biology approach based on genome mining and heterologous expression enables us to translate those gene resources into natural products. In addition, we can make artificial biosynthetic pathways in the heterologous host by combinatorial biosynthesis to afford natural products that have not been programmed in nature. This review summarizes the results on the structure development of fungal diterpenoid pyrones through a synthetic biology approach based on reconstructing and redesigning biosynthetic pathways in the heterologous host by using fungal biosynthetic gene resources. Keywords Natural product · Fungi · Genome mining · Heterologous expression · Synthetic biology · Combinatorial biosynthesis

1.1 Introduction Despite significant advances in modern medicine, there remain many diseases that are difficult or impossible to treat, such as cancer and dementia. The development of therapeutic drugs to treat these diseases is thus desirable. In addition, the emergence and re-emergence of infectious diseases caused by drug-resistant bacteria and new viruses, such as the one that caused the COVID-19 pandemic, has become a substantial problem for global public safety. The development of new drugs for treating these diseases is an urgent and continuous challenge for the international community. Natural products have historically been one of the most medicinally important resources and have contributed greatly to the development of therapeutic drugs for T. Asai (B) Graduate School of Pharmaceutical Sciences, Tohoku University, 3-8-1 Aza-aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_1

3

4

T. Asai

various conditions, especially cancer and infectious diseases [1–3]. The discovery of natural products with novel structures and biological activities, and research into the production thereof, is thus an important aspect of the field of natural product chemistry. The traditional style of exploratory research on natural products involves obtaining the products from extracts of naturally occurring species (or cultures thereof). Typically, the product that is obtained is chosen at the time the species is selected (Fig. 1.1). In other words, the success of exploratory research on natural products relies on researchers having a good instinct for selecting appropriate bio-resources. Exploratory research on natural products has thus come to be seen as something of a treasure hunt. With the development of analytical instruments such as nuclear magnetic resonance (NMR) equipment and separation devices that permit us to determine the structures of natural products using minute quantities, it is still possible to discover natural products with new chemical structures. However, in the long history of natural product discovery research, this has become increasingly difficult to find natural products with new chemical structures. Although the chemical structures and biological activities of many existing natural products are certainly useful for drug development, the facts that the research methods used to identify these products rely on serendipity and that a stable supply of such products may be difficult to obtain means that the inconvenience of using natural products hinders their industrial use, with the result that natural products have retreated from the frontline of industrial drug developments. Recent advances in genome-sequencing technology have revealed the existence of vast quantities of biosynthetic gene resources for the production and development of natural products, and most of these gene resources have thus far been silent [4–6]. Thus, these silent biosynthetic gene resources have been recognized as an attractive resources for the development of novel natural products. A number of approaches to awakening these silent biosynthetic genes to induce the production of the natural products they encode have been developed [7–10]. For example, a chemical epigenetic approach by using inhibitors of histone deacetylase and DNA methyl transferase Fig. 1.1 Conventional and post-genomic schemes for natural-product exploration

Bio resources Plant, Bacteria, Fungi etc

Isolation

Natural Products Isolation

Genome sequence

Bioinformatics analyses

Genomic information

Genome mining Heterologous expression

Heterologous host

Conventional natural product discovery Natural product discovery in post-genomic era

1 Synthetic Biology-Based Natural Product Discovery

5

is one of the most successful methods to access fungal natural products produced by silent biosynthetic genes [11–23]. Furthermore, the rapid accumulation of research on the biosynthesis of natural products in recent years has made it possible to find the desired biosynthetic gene clusters by genome mining. In addition, excellent heterologous expression systems, which allow the genes from one organism to be inserted into another host organism, causing the latter to produce a substance that is produced naturally by the former, have been developed [24–26]. Against this backdrop, there have been dramatic developments in research on the ways in which organisms produce natural products—a field known as synthetic biology. In this field, the biosynthetic pathways by which organisms produce the natural products are mined and reconstructed, and the natural products we want are biosynthesized by heterologous hosts. In other words, the production of natural products can now be achieved using genomic information (Fig. 1.1). Therefore, a synthetic biology approach based on genome mining and heterologous expression systems allows easier access to natural products, some of which may be new or rare even in nature [27–30]. In addition, it is possible to design heterologous production systems such that they overexpress the product of interest. In other words, the synthetic biology approach can produce high yields. Further, once constructed, a production system can be used repeatedly to cultivate more of the heterologous host organism, ensuring a stable supply of the product. It is therefore likely that the weaknesses inherent in the conventional strategy used for research into natural products can be overcome. As a result, the applicability of these products for drug discovery research is likely to increase. The synthetic biology of natural products therefore has the potential to cause a paradigm shift in the exploration of natural products and to revitalize drug discovery research. Using the synthetic biology approach, reconstructing a biosynthetic gene cluster from a fungal strain in a heterologous host affords the production of a corresponding natural product as well as its intermediates (Fig. 1.2a). Although these products are biosynthesized in a heterologous host, they are recognizable natural products that are programmed in nature. By discovering an unknown biosynthetic gene cluster and reconstructing it in a heterologous host, it is possible to explore a novel natural product along with its new intermediates. We thus have successfully discovered a wide range of novel natural products from untapped gene resources in fungi via genome mining using a heterologous expression involving Aspergillus oryzae, a model host for fungal heterologous expression studies (Fig. 1.2b) [31–37]. Further, we are able to introduce any gene in heterologous expression system. In other words, we can express not only genes from one organism, but also a combination of genes from multiple organisms. Using the advantages of heterologous expression, we can construct artificial natural product biosynthetic pathways that do not exist in nature, and thereby creating natural products that are not programmed in nature. This combinatorial biosynthetic approach thus allows us to investigate the previously unexplored chemical spaces of natural products (Fig. 1.2). Here, we describe a synthetic biology-based natural product discovery through genome mining, reconstruction, and redesign of fungal

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T. Asai

Fig. 1.2 Synthetic biology of natural products based on genome mining and heterologous expression, (a) a scheme of reconstruction of biosynthetic gene cluster to discover novel natural products and examples of novel natural products we isolated by using the synthetic biology approach, (b) combinatorial biosynthesis based on redesigning biosynthetic gene clusters to create natural products that are not programmed in nature

1 Synthetic Biology-Based Natural Product Discovery

7

natural product biosynthetic pathways, and we propose this as a new and potent structural development method for natural products [38].

1.2 Diterpenoid Pyrones (DDPs) with a Decalin Skeleton Produced by Filamentous Fungi Decalin-type diterpenoid pyrones (DDPs) produced by filamentous fungi are a member of meroterpenoids that are hybrids of polyketides and diterpenes, and many biologically active DDPs have been discovered (Fig. 1.3) [39–44]. DDPs are pharmaceutically important natural products and composed of two privileged structures, pyrone and decalin, which are associated with pharmacological activity. In this group, since slight structural differences between molecules result in their exhibiting different activities, it is likely that creating a variety of analogs will facilitate the construction of a useful library of substances containing a wide range of biological active compounds. Thus far, DDPs have attracted attention as targets for total synthesis because of their structures and activities, and the complete synthesis of several DDPs has been achieved [45–48]. O

Fig. 1.3 Typical fungal DDPs

O

O

OH

AcO

OH

H

O

Sesquicillin A Acremonium sp.

O

O

H

Subglutinol A Fusarium subglutinans

MeO

O

O OMe O

OH

O

HO H

O

H

HO

Higginsianin A Colletotrichum higginsianum

Colletotrichin Colletotrichum nicotianae

8

T. Asai

However, because of their complicated structures, supplying DDPs in volume and synthesizing analogs thereof has been difficult, so the biological activities of DDPs have not yet been fully explored. We have therefore attempted to construct a DDP library containing various analogs by using synthetic biology techniques based on genome mining and heterologous expression. We performed genome mining using SubA (Fig. 1.4) [49], which is a polyketide synthase (PKS), as an indicator, using our laboratory’s own draft genome library and the genome database of the National Center for Biotechnology Information (NCBI) to search for DDP biosynthetic gene clusters. We identified candidate gene clusters in the genomes of five filamentous fungi: Fusarium graminearum (Fg), Macrophomina phaseolina (Mp), Colletotrichum higginsianum (Ch), Metarhizium anisopliae (Ma), and the fungus isolated from a spider, Arthrinium sacchari (As) (Fig. 1.5a). A gene group encoding enzymes thought to be involved in the construction of the skeleton of DDPs (PKS, geranylgeranyl pyrophosphate synthase [GGPPs], prenyltransferase [PT], flavin-dependent epoxidase [FMOep], and terpene cyclase [TC]) was highly conserved in each of the clusters, which suggests that the biosynthetic pathways are encoded via common intermediate 1 (Fig. 1.4). In addition, each strain had a different gene composition related to structural modifications, and we envisage that the DDP biosynthetic pathways branch out from common intermediate 1 and diversify. Next, using bioinformatic analysis, we deduced the conversion pathways after common intermediate 1 through a detailed comparative analysis of the modified enzymes contained in each cluster. First, we classified the enzymes as short-chain dehydrogenase/reductases (SDR), methyltransferases (MT), cytochrome P450s (P450), flavin-dependent monooxygenase (FMO), and berberine bridge enzymelike oxidases (BBE), based on the amino acid sequences we had deduced. Next, we

Fig. 1.4 A hypothetical biosynthetic pathway for a DDP common intermediate 1

1 Synthetic Biology-Based Natural Product Discovery

9

Fig. 1.5 (a) Putative biosynthetic clusters for DDPs, (b) Comparative analysis of DDP biosynthetic enzyme genes, (c) Hypothetical biosynthetic pathways programmed in nature for natural DDPs, (d) Pathways not programmed in nature and shunt pathways for extra-modified DDPs

defined an amino acid sequence identity of more than 45% as the criterion for homologous enzymes having the same function, and we arranged these sequences as shown in Fig. 1.5b. Subsequently, since enzymes with the same function convert the same substrate to the same product, and based on the basic idea that enzymes common to

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T. Asai

more pathways tend to operate further upstream, we drew the transformation pathways encoded in the five clusters as shown in Fig. 1.5c. Based on the isolated DDPs that have been reported thus far and the fungal strains used for the production of these DDPs, we predicted that the dpch and dpma pathways would produce higginsianin A and subglutinol A, respectively. We predicted that the dpfg, dpmp, and dpas pathways, which were found in the genera Fusarium, Macrophomina, and Arthrinium and for which no DDPs have yet been reported, would produce novel DDPs.

1.3 Reconstruction of Natural DDP Biosynthetic Pathways and Discovery of Naturally Programmed DDPs Based on the putative biosynthetic pathways shown in Fig. 1.5c, we reconstructed the biosynthetic pathways of all five natural DDPs in the Aspergillus oryzae heterologous expression system. First, by heterologously expressing the five skeleton-forming genes of each cluster in Aspergillus oryzae, we confirmed that 1 was a common intermediate. We used the Aspergillus strain into which the skeleton-forming gene of dpas pathway was introduced (which produced approximately 90 mg/L of common intermediate 1) as the platform for our subsequent heterologous expression experiments. By sequentially introducing genes for modifying enzymes into this strain, we reconstructed the natural DPP synthesis pathways observed in all five fungal genera in a stepwise manner. We were thus able to heterologously produce all intermediates and final products, yielding 11 DDPs including five novel analogs (Fig. 1.6). As we had anticipated based on the predicted biosynthesis pathways, dpma and dpch encoded the biosynthesis of subglutinols (2, 3) and higginsianin A (7), respectively, and the biosynthesis of the novel DDPs was encoded by the previously unexplored dpas, dpmp, and dpfg pathways. Our results also suggested that in the dpas pathway, FMO synthesizes not only the subglutinols (2, 3) but also a novel DDP (4), which has an enone structure in the C5 unit from common intermediate 1. This therefore suggests that this FMO is a multifunctional oxidase. In addition, the dpfg and dpmp pathways both synthesized novel DDPs (9, 10, 11) with highly modified pyrone moieties. Based on the correspondence between the transgenes and the structures of the biosynthesized compounds, we were able to elucidate the entire picture regarding the biosynthesis pathways of DDPs, including the functions of all the modifying enzymes in each pathway.

1.4 Discovery of DDPs not Programmed in Nature by Redesign of the DDP Biosynthetic Pathways Figure 1.6 summarizes the reactions of each enzyme in all the pathways. Enzymes involved in modifying the side chain C5 unit were specifically distributed in the

1 Synthetic Biology-Based Natural Product Discovery

11

Fig. 1.6 DDPs discovered in this study

dpma, dpas, and dpch pathways, while those involved in modifying the pyrone moiety were localized in the dpmp and dpfg pathways. However, there were no pathways containing enzymes for modifying both the C5 unit and the pyrone moiety. With the aim of creating DDPs that are more highly modified than the final products of each of these DDP pathways, therefore, we designed extra-modified biosynthetic pathways that combined these two modification pathways (Fig. 1.5d). We succeeded in producing DDPs 14–22, which were more highly modified than the naturally programmed DDPs (Fig. 1.6). In addition, we also constructed one shunt pathway,

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T. Asai

which created 12 and 13. Including the reconstructed biosynthetic pathways, therefore, we constructed a total of 10 biosynthesis pathways (five natural, four artificial, and one shunt) using genome mining and the Aspergillus oryzae heterologous expression system, and we discovered a total of 22 DPP analogs with various modifications. The new compounds, 4, 8, 11, 9, 10, 13, 18–22, 17, and 14–16 were named as FDDP A–O, respectively. The yield of all the DDPs biosynthesized in this study was much higher (10–100 mg/L) than that obtained via the ordinary exploitation of natural products, and these DPPs can be obtained easily by repeatedly culturing the corresponding transformants.

1.5 Evaluation of Biological Activities of the DDPs We evaluated various activities of the DDPs we biosynthesized in this study. We discovered previously unreported biological activities using the information on simple structure–activity relationships, such as strong anti-HIV activity, inhibition of cancer-stem-cell proliferation, insect-paralyzing activity, suppression of innate immunity in insect, and inhibition of amyloid β aggregation. During our investigation of the inhibitory activity of DDPs against amyloid β aggregation, we demonstrated that DDPs with a highly modified pyrone structure, 9 and 22, significantly suppressed amyloid β aggregation. We demonstrated that 9 and 22 formed a covalent bond with lysine 16 in amyloid β 42 via the highly modified pyrone structure by LC–MS analysis and a model chemical reaction (Fig. 1.7).

Fig. 1.7 Reaction between a lysine derivative and 9

1 Synthetic Biology-Based Natural Product Discovery

13

1.6 Conclusion In this study, we have demonstrated that a synthetic biology-based approach based on genome mining and heterologous expression is a promising and practical avenue for expanding the chemical space of biologically active natural products. Focusing on filamentous fungal DDPs, we used candidate biosynthetic gene clusters discovered via genome mining and data from bioinformatics analyses and experiments to design five natural pathways, four artificial pathways, and one shunt pathway. We constructed these pathways in a heterologous host Aspergillus oryzae and achieved the simultaneous heterologous production of 22 kinds of DDP, including 15 novel analogs. The compounds derived from the artificial pathways are certainly not encoded in nature, but the process of in vivo biosynthesis is the same as that of natural products, and just like with natural products, we expect these compounds to retain properties that are desirable in pharmaceutical seeds. The fact that unique, biologically active compounds were discovered based on the artificial pathways indicates that this method for discovering natural products is effective for drug discovery research. All the DDP-producing strains showed high production yields, and heterologous production in heterologous hosts results in few contaminants, easy purification, and a stable supply. This implies that producing a sufficient quantity of products for performing everything from structure determination to testing for pharmacological activity is feasible. As a result, biological activities that have not previously been identified for DDPs could be discovered. In this way, synthetic biology-based research on natural products may lead to substantial improvements in supply-side challenges, which have long been a problem in conventional natural-product exploration. In the post-genome era, synthetic biology, which can turn ever-increasing quantities of genetic information into natural products, will undoubtedly bring about dramatic innovations that will support drug discovery research in the future.

References 1. Newman, D.J., Cragg, G.M.: Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. J. Nat. Prod. 83, 770–803 (2020) 2. Rossiter, S.E., Fletcher, M.H., Wuest, W.M.: Natural products as platforms to overcome antibiotic resistance. Chem. Rev. 117, 12415–12474 (2017) 3. Wright, G.D.: Opportunities for natural products in 21st century antibiotic discovery. Nat. Prod. Rep. 34, 694–701 (2017) 4. Sanchez, J.F., Somoza, A.D., Keller, N.P., Wang, C.C.C.: Advances in Aspergillus secondary metabolite research in the post-genomic era. Nat. Prod. Rep. 29, 351–371 (2012) 5. Khaldi, N., Seifuddin, F.T., Turner, G., Haft, D., Nierman, W.C., Wolfe, K.H., Fedorova, N.D.: Genomic mapping of fungal secondary metabolite clusters. Fungal Genet. Biol. 47, 736–741 (2010) 6. Scherlach, K., Hertweck, C.: Triggering cryptic natural product biosynthesis in microorganisms. Org. Biomol. Chem. 7, 1753–1760 (2009)

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Chapter 2

Sarco/Endoplasmic Reticulum Ca2+ -ATPase (SERCA) Inhibitors Isolated from Subtropical Marine Cyanobacteria in Japan Arihiro Iwasaki Abstract Marine cyanobacteria are prolific producers of natural products with novel structures and potent biological activities. We have investigated the secondary metabolites of marine cyanobacteria that inhabit subtropical regions in Japan. Recently, we found that marine cyanobacteria tend to produce inhibitors of sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA). SERCA is a Ca2+ pump on the endoplasmic reticulum that has been recognized as a promising drug target. This chapter describes the isolation, structure determination, total synthesis, biological activities, and clarification of the mode of action of SERCA inhibitors from marine cyanobacteria collected in Japan. In addition, we suggest a plausible ecological role for these SERCA inhibitors in the coral reef ecosystem. Keywords Cyanobacteria · Sarco/endoplasmic reticulum calcium Ca2+ -ATPase (SERCA) · Iezoside · Kurahyne

2.1 Introduction Cyanobacteria were the first group of bacteria to perform oxygen-evolving photosynthesis on Earth. They have existed on this planet for at least 2.7 billion years and are known to have contributed to the formation of the current oxygen-rich atmosphere. Today, cyanobacteria are found in a variety of habitats, including freshwater, seas, deserts, and polar regions [1], and their secondary metabolites have been investigated for years, revealing several significant bioactive compounds [2]. Among these bacteria, marine cyanobacteria have been recognized as prolific producers of novel natural products. Recent advances in genome analysis technology have shown that certain marine cyanobacteria devote 20% of their genome to the production of

A. Iwasaki (B) Faculty of Science and Technology, Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Kanagawa, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_2

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natural products and possess an average of thirty-eight kinds of secondary metabolite biosynthetic gene clusters [3]. This high content rate is comparable to that of the actinomycete genus Streptomyces, which is a prominent producer of antibiotics. Based on this information, natural products from marine cyanobacteria have been investigated, mainly by chemists in the United States who focused on samples collected from the Caribbean Sea and South Pacific Ocean and discovered a number of novel bioactive compounds (Fig. 2.1). For example, curacin A [4] gatorbulin1 [5], and dolastatin 10 (originally isolated from invertebrates [6], but reisolated from the marine cyanobacterium Symploca sp. [7]) destabilize microtubules by binding them and show potent cell growth-inhibitory activity. Lyngnyastatin 1 [8] and lyngbyabellin A [9] disrupt the normal regulation of actin filaments. Largazole, a peptide-polyketide hybrid possessing a thioester group, potently inhibits the activity of histone deacetylase and exhibits the anticancer activities against colon cancer [10–12]. Gallinamide A (symplostatin 4) shows strong antiparasitic activity against Plasmodium falciparum (the causative organism of malaria) and Trypanosoma cruzi (the causative organism of Chagas disease) by inhibiting their cysteine proteases [13–16].

Fig. 2.1 Structures of representative natural products isolated from marine cyanobacteria

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The secondary metabolites of marine cyanobacteria inhabiting eastern Asia have been investigated mainly by Japanese chemists, and several intriguing natural products have been discovered. Bisebromoamide, an acyclic peptide possessing unusual moieties, such as methyl proline and pivalic acid, exhibits potent cytotoxicity by stabilizing actin filaments [17–19]. Heptavalinamide A is an extensively N-methylated linear nonapeptide possessing a rare N,N-dimethylvaline residue at the N-terminal [20]. Caldorazole is an acyclic thiazole-containing polyketide that selectively inhibits complex I in the mitochondrial respiratory chain [21]. In this context, over the past decade we have focused on marine cyanobacteria inhabiting subtropical regions in Japan to investigate their natural products. We discovered greater than sixty new natural products and reported on their structures and biological activities. In the following sections, we describe three sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) inhibitors isolated from marine cyanobacteria in Japan. SERCA is a membrane protein on the endoplasmic reticulum (ER) that transports Ca2+ from the cytosol into the ER. As calcium signaling is associated with various biological phenomena such as muscle contraction and neural function, SERCA has attracted attention as a promising target for drug development. Here, we introduce the isolation, structure determination, total synthesis, and biological activities of these SERCA inhibitors along with the characteristic techniques that we used in the studies. In addition, the possible ecological role of cyanobacterial SERCA inhibitors in coral reef ecosystems is discussed.

2.2 Iezoside In July 2020, we visited the east coast of Ie Island, a small subtropical island famous as a fierce battlefield in the Pacific War. On the reef, we discovered small cyanobacterial colonies that we had not observed on other shores, and collected 250 g of these organisms (Fig. 2.2). The crude EtOH extract of this sample showed potent growth inhibition activity against HeLa cells, with the IC50 value of 79 ng/mL. Further chromatographic separations directed by the cell growth-inhibitory activity allowed us to isolate 5.4 mg of iezoside (1) as the compound responsible for the strong cytotoxicity (IC50 4.6 ng/mL) [22]. The structural determination of iezoside (1) commenced with a prediction using artificial intelligence. Small molecule accurate recognition technology (SMART) is an artificial intelligence-based structure prediction tool recently developed by Gerwick, Cottrell, et al. [23, 24]. The tool uses proton and proton-attached carbon chemical shifts of a query compound to pick up structurally related known compounds from a large database, and the degree of similarity of each compound is indicated by a cosine score. The SMART results suggested that several polyene natural products such as 7-desmethylcitreoviridin (cosine score: 0.7842) [25], asteltoxin F (cosine score: 0.7633) [26], and archazolid D (cosine score: 0.7579) [27] were similar to iezoside (1), but not to a high degree. Therefore, we predicted that iezoside (1) would be a novel natural product with a polyene skeleton.

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Fig. 2.2 Structure of iezoside and a photograph of its producer, Leptochromothrix valpauliae

Detailed analyses of the 2D NMR data revealed that iezoside (1) is a hybrid compound of three partial structures: peptide, pyranoside, and polyketide moieties, whose biosynthetic pathways are completely different from each other. The peptide is composed of N-Me-alanine, leucine, and thiazole which is derived from a cysteine residue. The absolute configurations of leucine and N-Me-alanine were determined to be l and d, respectively, based on acid hydrolysis followed by analyses using chiral-phase HPLC and Marfey’s method [28]. The pyranoside was determined to be 2,3-O-dimethyl-α-l-rhamnoside based on proton coupling constants and a modified Moser’s method [29]. The polyketide contained two polyene parts, as predicted by the SMART analysis. The geometries of the four olefines were established based on the NOESY correlations, coupling constants, and carbon chemical shifts of the vinyl methyl groups. As described above, we clarified the structure of iezoside (1), except for the stereochemistry of two chiral centers in the polyketide chain (C-18 and C-19). The last remaining issue for the structural determination of iezoside (1) was the clarification of the absolute configurations at C-18 and C-19. Despite several attempts to degrade and derivatize iezoside (1), we were unable to obtain any useful products for structure determination. Therefore, we used a computational approach to resolve this issue. Iezoside (1) is a linear compound that has many rotatable bonds, and to conduct a conformational search for the entire structure of iezoside (1), we had to consider more than 4,800,000,000 conformations. Hence, we designed simplified model compounds which lack the peptide moiety and the terminal methyl group in the polyketide chain. These partial structures are considered not to affect the conformation around C18 and C-19, thereby successfully reducing the number of conformers to 104,976 (Fig. 2.3a). First, we roughly estimated stable conformers using the relatively lowlevel theories, MMFF [30] and HF/3-21G [31], to reduce the number of candidates. Second, we performed more sophisticated calculations at the B3LYP/6-31G* level [32] against the remaining conformers. Finally, we regarded the conformers within 10 kJ/mol of the global minimum as stable conformers that should be considered in the evaluations. In the calculations, the Boltzmann distribution based on the electronic energy of each conformer significantly differed from that based on the free energy of each conformer. Compared to the former value, the latter included the effect

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a

b

Fig. 2.3 Structures of iezoside and its simplified model compounds (a) and evaluations of each isomer based on computational chemistry (b)

of the entropy term calculated by vibrational analyses. Iezoside (1) has a linear flexible skeleton; therefore, it was expected that the change in conformation would significantly affect the entropy term. Hence, we used the free energy to calculate the Boltzmann distribution of the stable conformers. As plausible stable conformers were obtained for each possible diastereomer, we calculated the theoretical chemical shifts of each conformer using the gaugeindependent atomic orbital (GIAO) method [33] at the B3LYP/6-31G* level and averaged them based on the Boltzmann distribution. The results were statistically

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analyzed with experimental chemical shifts according to the previous report by Goodman et al. [34] and are summarized in Fig. 2.3b. Each DP4 score indicated the expected value of the corresponding diastereomer. Based on the carbon DP4 data, the 18R,19S and 18S,19S isomers were clearly ruled out. Comparing 18R,19R and 18S,19R, the former was more plausible based on the DP4 value. However, both RMSD values were within the Goodman’s standard deviation (13 C:2.306 ppm) and did not show significant differences. For the proton data, the RMSD values of all isomers were in the range of Goodman’s standard deviation (1 H:0.185 ppm); therefore, we could not discuss the configuration based on these data. Finally, we compared the theoretical proton coupling constant between H18 and H19 of each isomer with that of iezoside (1). As shown in Fig. 2.3(B), only the 18R,19R isomer perfectly reproduced the experimental value, 3 J H18-H19 (6.5 Hz). Therefore, 18R,19R was the most plausible candidate, based on theoretical NMR data analyses. Using another approach, we focused on the ECD spectrum of iezoside (1). Iezoside (1) has two polyene parts in its linear polyketide chain, which are divided by the two chiral centers, C-18 and C-19. Hence, the torsional angle of these two polyene parts and the resultant Cotton effect are expected to be affected by the C-18 and C-19 configurations. Based on these considerations, we calculated the theoretical ECD spectra of each model compound at the B3LYP/6-31G* level and compared them with those of iezoside (1). Both 18R,19R and 18S,19R isomers reproduced the ECD data well. In contrast, 18R,19S and 18S,19S showed the opposite Cotton effect and were explicitly excluded as candidates. In summary, our computational study of the configuration of C-18 and C-19 of iezoside (1) revealed that the 18R,19R isomer was the most plausible structure. However, we could not clearly rule out 18S,19R. Therefore, we synthesized (18R,19R)- and (18S,19R)-iezoside and compared their spectral properties with those of the natural iezoside (1) to verify our structure prediction. The synthetic scheme for (18R,19R)-iezoside (1) is shown in Scheme 2.1. First, we synthesized a polyketide chain of iezoside. The elongation of the carbon chain was mainly achieved by Wittig and Horner-Wadsworth-Emmons reactions, and the two chiral centers in the polyketide chain were constructed by a syn Evans aldol reaction [35]. The resulting polyketide chain was condensed with the rhamnose and peptide moieties, respectively, to produce the (18R,19R)-iezoside (1) (4.4% overall yield). Similarly, (18S,19R)-iezoside was prepared (1.5% overall yield). Finally, we compared the spectral data of both synthetic compounds with those of natural iezoside. The (18R,19R)-iezoside (1) showed identical spectral data to those of the natural compound, and thus we determined the complete structure of iezoside (1). We then focused on the mode of action of the potent cell growth-inhibitory activity of iezoside (1) (IC50 6.7 nM). We evaluated the cytotoxicity of iezoside (1) using the Japanese Foundation for Cancer Research 39 (JFCR39) anticancer drug screening system, which is similar to the NCI-60 Human Tumor Cell Lines Screen developed by the National Cancer Institute [36]. The results suggested that two known compounds, thapsigargin [37–39] and A23187 [40, 41], possess a mode of action analogous to that of iezoside (1). Both induced the elevation of cytosolic Ca2+ concentration, but the origin of Ca2+ was completely different. As A23187 acts as an ionophore, it

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Scheme 2.1 Total synthesis of (18R,19R)-iezoside

transports extracellular Ca2+ into the cytosol. Thapsigargin inhibits the activity of SERCA, a Ca2+ ion pump on the ER, and releases Ca2+ stored in ER. Therefore, we evaluated the cytosolic Ca2+ concentration in iezoside-treated cells in a Ca2+ -free medium using Fura-2 [42] and detected an increased Ca2+ concentration, indicating that iezoside (1) possesses a thapsigargin-like mode of action. Finally, we prepared a microsome fraction containing SERCA from rabbit white skeletal muscle and evaluated the SERCA-inhibitory activity of iezoside (1) using a coupled enzyme assay [43]. The results indicated that iezoside (1) significantly inhibited the activity

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of SERCA with a K i value of 7.1 nM, which is the same concentration range as that of the growth-inhibitory activity (IC50 6.7 nM). The potency was comparable to that of thapsigargin, which is the strongest SERCA inhibitor currently identified (K i 0.2–1.3 nM), and we concluded that iezoside (1) is the second strongest SERCA inhibitor known at present.

2.3 Kurahyne In March 2013, we collected small granular cyanobacterial assemblages, mainly consisting of Lyngbya sp. at Kuraha on the main island of Okinawa (Fig. 2.4). The sample contained a variety of secondary metabolites, such as kuarahamide [44], maedamide [45, 46], and yoshinone A [47], and careful purification of the extracts afforded 29.9 mg of kurahyne (3) as a moderate cytotoxic compound (IC50 3.9 μM against HeLa cells) [48]. The planar structure of kurahyne (3) was clarified based on the detailed analyses of NMR data, and the absolute configuration was established by conventional methods, acid hydrolysis followed by HPLC analyses. Interestingly, kurahyne (3) possesses two different functional groups, a terminal alkyne and ketone, at each terminal. Both functional groups are expected to be useful for the construction of chemical probes. To verify the structure, we achieved the first total synthesis of kurahyne (3) and obtained a sufficient amount of compound to evaluate biological activities (Scheme 2.2) [49]. We then attempted to clarify the mode of action of the cell growth-inhibitory activity of kurahyne (3) [50]. The JFCR39 anticancer drug screening assay [36] of kurahyne (3) suggested that thapsigargin [37–39] had a similar mode of action. As described in the previous section, thapsigargin inhibits SERCA activity by binding it, inducing ER stress in cells. Therefore, we evaluated the ER stress-inducing activity of kurahyne (3) by using the reverse transcription-polymerase chain reaction (RTPCR) [51] and detected the expression of the ER stress markers, BiP and CHOP [52, 53], in kurahyne-treated cells.

Fig. 2.4 Structure of kurahyne and a photograph of its producers, cyanobacterial assemblages

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Scheme 2.2 Total synthesis of kurahyne

To evaluate the subcellular localization of kurahyne (3), we designed and prepared two fluorescein probes using the terminal alkyne and ketone groups as scaffolds (Scheme 2.3). The N-terminal-modified probe 4 was synthesized by Huisgen cycloaddition between the terminal alkyne and the azide group. The C-terminalmodified probe 5 was prepared by oxime formation between the ketone group and the hydroxylamine. We evaluated the ER stress-inducing activities of these probes by RT-PCR51 and found that only the N-terminal-modified probe 4 induced the expression of BiP, indicating that 4 retained the original biological activity of kurahyne (3). Therefore, we used 4 for the evaluation of subcellular localization and revealed that the localizing area overlapped the ER where the SERCA existed. Next, we designed and synthesized a biotin-conjugated probe 6 to verify the direct binding between kurahyne (3) and SERCA. Based on the structure-activity relationship of fluorescein probes 4 and 5, we attached a biotin linker at the N-terminal of kurahyne (3). Affinity purification of HeLa cell lysate using 6 followed by western blotting analysis revealed

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Scheme 2.3 Synthesis of chemical probes of kurahyne

that kurahyne (3) directly bound to SERCA [54, 55]. Finally, we verified the SERCAinhibitory activity of kurahyne (3) using the coupled enzyme assay as described in the previous section [43]. We did not calculate the exact inhibitory constant of kurahyne (3), but its potency was considerably weaker than that of iezoside (1) (K i circa 10 μM).

2.4 Biselyngbyasides and Their Aglycons, Biselyngbyolides In 2009, Teruya, Suenaga, et al. isolated an 18-membered macrolide glycoside, which they called biselyngbyaside (7), from the Lyngbya sp. marine cyanobacterium collected at Bise on the main island of Okinawa (Fig. 2.5) [56]. The structure of this compound was established by a combination of spectral analyses, degradation reactions, and chemical synthesis of degradation products. In 2012 and 2014, Morita, Ohno, Suenaga, et al. discovered new analogs of biselyngbyasides (8–12) [57, 58] and their aglycons, biselyngbyolides (13–15) [59, 60], from Lyngbya sp. marine cyanobacteria collected at Ishigaki island and Tokunoshima island, respectively. Their structures were clarified mainly by spectral analyses and were verified by total synthesis achieved by several groups [61–66]. Although biselyngbyaside (7) showed moderate growth-inhibitory activity against HeLa cells (IC50 0.30 μM), the aglycons, biselyngbyolides (13–15) and showed approximately ten times greater growthinhibitory activities (IC50 0.04–0.05 μM). In 2015, Morita, Suenaga, Toyoshima,

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Fig. 2.5 Structures of biselyngbyasides and biselyngbyolides

et al. clarified that SERCA is the target molecule of biselyngbyasides and biselyngbyolides by cocrystal X-ray structure analysis [67]. Interestingly, despite the significant difference in cytotoxicity, the inhibitory constants of biselyngbyaside (7) and biselyngbyolide (13) were comparable (K i circa 17-19 nM), suggesting that the sugar moiety reduced the cell membrane permeability of the molecule.

2.5 Possible Ecological Role of SERCA Inhibitors Produced by Marine Cyanobacteria Based on the studies described above, we noticed an interesting relationship between marine cyanobacteria and SERCA inhibitors. SERCA is a relatively rare target for natural products, and several compounds that inhibit it have been reported. Figure 2.6 shows representative compounds that have been shown to inhibit the activity of SERCA [68–71]. The most potent inhibitor is thapsigargin, which is a sesquiterpene lactone isolated from a terrestrial plant, while the second and third strongest compounds, iezoside (1) and biselyngbyaside (7), are derived from marine cyanobacteria. In addition, the structures of the three cyanobacterial SERCA inhibitors in Fig. 2.6, are completely different from each other. Therefore, we are convinced that there is a reason that marine cyanobacteria produce a wider variety of SERCA inhibitors than other organisms. From a chemical ecological point of view, we propose that SERCA inhibitors produced by marine cyanobacteria are chemical weapons used to combat corals. Corals live symbiotically with photosynthetic zooxanthellae to acquire nutrients; therefore, they need scaffolds to receive enough sunlight. However,

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cyanobacteria also require scaffolds to perform photosynthesis, indicating that both cyanobacteria and corals are potential competitors in the field. In fact, cyanobacteria producing SERCA inhibitors are benthic, and we often observe their growth on dead corals on a reef. We then consider the reason for cyanobacteria choosing the SERCA-inhibitory activity to attack corals. In 2017, Ruiz-Jones and Palumbi reported interesting observations as follows [72]. Reef corals inhabiting an intertidal zone are routinely exposed to strong sunlight, especially at low tide, which increases their body temperature. As a result, ER stress is induced, which activates the unfolded protein response in corals to cope with this environmental stress. Based on that report, corals living in an intertidal zone are considered vulnerable to ER stress because they are exposed to ER stress induced by environmental factors. Marine cyanobacteria use this as an opportunity to attack corals using SERCA inhibitors, which induce the accumulation of unfolded proteins in the ER, leading to ER stress. Therefore, SERCA inhibitors produced by cyanobacteria enhance ER stress in corals to intolerable levels that eventually kill them. In fact, the amino acid sequence homology of SERCA-like

Fig. 2.6 Structures of representative SERCA inhibitors

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proteins between corals and humans is relatively high (approximately 70%), indicating the effectiveness of the SERCA inhibitors against corals. In contrast, the low sequence homology of SERCA-like proteins between humans and cyanobacteria (approximately 36%) indicates the existence of a self-resistance mechanism in marine cyanobacteria. Future studies aimed at experimentally verifying this hypothesis will provide new insights into cryptic chemical communication in coral reefs.

2.6 Conclusion In this chapter, novel and potent SERCA inhibitors isolated from marine cyanobacteria that inhabit subtropical regions of Japan are described. Recently, SERCA has attracted attention as a target for cancer treatment [73]. In fact, mipsagargin, a thapsigargin-based prodrug, has been developed as an anti-prostate cancer drug [74, 75]. Compared with thapsigargin, the SERCA-inhibitory activity of iezoside (1) is slightly weaker, but the structure is much simpler. The synthesis of artificial analogs and clarification of the structure-activity relationship can allow for the development of attractive drug lead compounds that are comparable to mipsagargin. In addition, our hypothesis that marine cyanobacteria use SERCA inhibitors to attack corals indicates that marine cyanobacteria are a promising resource for the discovery of new SERCA inhibitors. As the preliminary evaluation of SERCAinhibitory activity is readily conducted using Fura-2 [42], further exploration of cyanobacterial extracts using this assay system could reveal the unknown structural diversity of natural SERCA inhibitors. The discovery of new compounds from nature is one of the most fundamental studies in natural product chemistry. These recent technological developments have simplified the incorporation of a variety of techniques into natural product chemistry from other fields, such as computational chemistry, organic synthesis, chemical biology, biosynthesis, and informatics. Therefore, modern natural product chemists are expected to not only isolate and determine the structure of new natural products, but also to reveal hidden values of the compounds by integrating interdisciplinary approaches.

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Chapter 3

Marine Natural Products Targeting Tumor Microenvironment Naoyuki Kotoku

Abstract Discovery of marine natural products targeting tumor microenvironment, as well as the chemical approach toward the compounds is highlighted in this chapter. Screening and separation of the extracts of marine sponge guided by bioassay focusing on the typical phenotypes of the tumor microenvironment enabled us to find the active constituents: hypoxia-selective growth inhibitors (furospinosulin-1 and dictyoceratin-C), and selective growth inhibitors under glucose-deficient condition (N-methylniphatyne A, polybrominated diphenyl ethers, fasciospyrinadinone, fasciospyrinadinol, and biakamides A-D). Chemical approach such as total synthesis, structure–activity relationship (SAR) study, and development of affinity probe opened the way to structure elucidation, in vivo evaluation, and target identification. Keywords Marine natural products · Tumor microenvironment · Hypoxia · Nutrient starvation · Target identification

3.1 Introduction Marine natural products have been regarded as a rich and promising source of drug candidates, especially in the search for the drug leads with novel mode-ofaction against intractable diseases such as cancer or infection by multi-drug resistant pathogen [1, 2]. Historically and analytically, challenging but promising way for the discovery of new chemical entities leading to first-in-class drug is the screening focusing on the typical phenotype of the diseases and the following target identification [3–5]. In this chapter, our recent works about the search for marine natural products through phenotype screening targeting tumor microenvironment, and the chemical approaches toward the active compounds leading to the target identification are described. N. Kotoku (B) College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu 525-8577, Shiga, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_3

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3.2 Screening of Hypoxia-Selective Growth Inhibitors It is now widely recognized that the hypoxic nature of tumor environments is an important factor for tumor growth, angiogenesis, and metastasis. Tumor cells in hypoxic environments also exhibit resistance to chemotherapy and irradiation, and promote angiogenesis [6]. As the hypoxic environment of a tumor is unlike that in normal tissues, selective growth inhibitor against tumor cells in hypoxic environments might be promising anticancer drug leads [7]. Cancer cells are known to adapt to hypoxia through some regulation systems, in which hypoxia-inducible factor 1 (HIF-1) signaling is one of the most wellknown systems [8, 9]. HIF-1 is a heterodimeric transcription factor composed of oxygen-regulated α-subunit and a constitutively expressed β-subunit, and the cellular responses to hypoxia mediated by HIF-1 has been studied extensively. As the activity of HIF-1α is closely related to tumor phenotype such as growing angiogenesis, most drug discovery projects targeting hypoxia have focused on HIF-1 or HIFrelated proteins/signaling pathway. A number of natural products have been found to inhibit HIF-1α or its signaling pathway. Some examples are depicted in Fig. 3.1: rapamycin [10] and verucopeptin [11, 12] as inhibitors of HIF-1α protein synthesis, geldanamycin [13–16] and manassantin B [17, 18] as HIF-1α protein degraders, and echinomycin [19, 20] and chetomin [21] as inhibitors of the interaction between HIF-1 and DNA. On the other hand, it has been reported that the expression levels of over 1000 genes are altered under hypoxic conditions, so the regulation systems for the adaptation of cancer cells toward hypoxia other than HIF-1 might exist [22, 23]. Under the above background, a new screening system to identify hypoxia-selective growth inhibitors as potential anticancer drug leads with novel mode of action other than HIF-1 or related regulation system were established. Thus, human prostate cancer DU145 cells are incubated in the presence of the testing sample under normoxic or hypoxic condition (1% O2 , 94% N2 , and 5% CO2 in which condition the elevated expression level of HIF-1α are observed), and the samples exhibiting selective growth inhibition under hypoxic condition were selected. Screening of extracts of marine sponge and marine-derived microorganisms provided an active sample, extract of the Indonesian marine sponge Dactylospongia elegans. Bioassay-guided separation resulted in the isolation of two active constituents: furospinosulin-1 (1) and dictyoceratin-C (2) (Fig. 3.2). As there have been no reports that those compounds exhibited hypoxia-selective growth inhibitory activity, we engaged in the detailed studies about bioactivity, mechanism of action, and synthetic approach toward them.

3.2.1 Bioactivity and Action Mechanism of Furospinosulin-1 The furanosesterterpene furospinosulin-1 (1) [24] has been known to exhibit several biological activities, including weak cytotoxicity against HCT-116 cells

3 Marine Natural Products Targeting Tumor Microenvironment

OMe

O

O H OH OMe O

HO NH N HN

N H

HO N

O

O

OMe

O O O O O O

OH

O

37

OH

O OMe

N

O OH O

O N

verucopeptin

HN

rapamycin

O MeO

O N H

O

O

HO

O

MeO OH

MeO

O OMe

OH

OMe

O

OMe O

OCONH2

geldanamycin N N

O

O

H N O

O

N

O S

O O

NS SN

O N

N H

OMe

manassantin B

N

O S

O O

N O

O

H N O

N

OH O

N H

N

N N S S H H N O

HO

N

chetomin

echinomycin

Fig. 3.1 Structure of natural products targeting HIF-1

HO

H

COOMe

O

furospinosulin-1 (1)

dictyoceratin-C (2)

Fig. 3.2 Hypoxia-selective growth inhibitors isolated from the extract of Dactylospongia elegans

(IC50 = 155 μM) [25], toxicity toward brine shrimp (IC50 = 10 μg/mL) [26], and inhibitory activity toward Cdc25A (IC50 = 2.5 μM) [27]. 1 was found to exhibit selective growth inhibition against hypoxic DU145 cells in a dose-dependent manner ranging from 1.0 to 100 μM, and maximal growth inhibition of 60% was achieved

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Fig. 3.3 (a) Hypoxia-selective growth inhibition of furospinosulin-1 (1) against DU145 cells (5 × 104 cells/mL), (b) Inhibition of IGF-2 expression and autophosphorylation of the IGF-1R and IR by 1

with 300 μM treatment under hypoxic conditions (Fig. 3.3a). The conventional antitumor drug such as cisplatin did not exhibit selective growth inhibition under hypoxic condition. Furthermore, in vivo antitumor effect of 1 was evaluated. 25 mg kg−1 oral administrations of 1 suppressed tumor growth with 74% decreases in tumor weight relative to the control. No significant side effects such as weight loss and/or diarrhea were observed during the evaluation period. To elucidate the action mechanism of 1, the gene expression profile of DU145 cells cultured under normoxic/hypoxic conditions in the presence/absence of 1 (30 μM) were analyzed using a pathway-specific OligoGE array. Among 113 genes involved in hypoxia-related signaling pathway, 41 genes were up-regulated under hypoxic conditions, and 4 out of the 41 genes: interleukin-8 (IL-8), insulin-like growth factor-2 (IGF-2), the 60 kDa HIV-1 Tat-interacting protein (HTATIP), and Bcl2-associated X protein (BAX), were then decreased after 12 h treatment with 1 under hypoxic conditions. The following evaluation of the protein expression level of the genes revealed that the alteration of the protein level of IGF-2 was well correlated with IGF-2 gene expression profile. In addition, hypoxia-induced autophosphorylation of IGF-1R and IR proteins, the response against the binding of IGF-2 [28], were suppressed by the treatment with 1 (Fig. 3.3b). Furthermore, proliferation of DU145 cells was selectively inhibited under hypoxic conditions with the treatment of anti-IGF-2 antibody (0.3–100 μg/mL) for 24 h in a dose-dependent manner. Those findings indicated that 1 suppressed the expression of IGF-2 induced under hypoxic conditions, probably through inhibition of IGF-2 gene expression [29].

3 Marine Natural Products Targeting Tumor Microenvironment

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3.2.2 Synthesis and Evaluation of Affinity Probe of Furospinosulin-1 It is known that human hepatocarcinoma HepG2 cells in hypoxia showed increased expression of IGF-2 mRNA, and the P3 region of the IGF-2 promoter is important for the transcription of IGF-2 under hypoxic conditions [30]. Considering the above experimental data, furospinosulin-1 (1) suppresses IGF-2 transcription by inhibiting the complex formation of nuclear proteins with the P3 region of the promoter through binding to the protein(s) of them. The unique bioactivity of 1 attracted us to identify the binding protein using affinity probe molecule derived from 1, which could lead to the identification of unknown molecules/signal pathway responsible for adaptation to hypoxia. The structure of 1 is quite simple having a 3-substituted furan ring with a long side chain consisting of four iterative isoprene units. As there are no functional groups such as hydroxyl or amino groups in the molecule that can be used for derivatization to affinity probes, de novo synthesis of an analogue appended an appropriate anchoring group for derivatization without losing bioactivity of the parent compound is needed. In order to prepare various analogues of 1 efficiently, convergent synthetic method using sulfone-mediated coupling was developed [31]. PCC oxidation of the known alcohol 3 [32] and subsequent Wittig olefination gave an α,β-unsaturated ester 4. Reduction of the ester moiety in 4 using DIBAL and subsequent bromination of 5 provided an allylic bromide 6. Then coupling reaction between 6 and the known phenylsulfone 8 [33] using t-BuOK [34], and reductive desulfonylation of 7 with LiBHEt3 in the presence of Pd(dppp)Cl2 [35] afforded 1 by 60% overall yield (Scheme 3.1) [36]. We then analyzed the detailed SAR of 1 through syntheses and evaluation of analogues. Using the combination of the two fragments: allylic bromide and prenyl phenylsulfone, various analogues changing aromatic ring, elongation/truncation of the methyl group near the furan ring and side chain, and functionalization of the side

OH O

3

1) PCC

CO2Et 1) DIBAL

2) Ph3P=CHCO2Et O

2) CBr4, Ph3P

4 R 1) 8, KOtBu

O 5: R = OH 6: R = Br

O 2) LiBHEt3 Pd(dppf)Cl2 PhO2S 8

Scheme 3.1 Total synthesis of furospinosulin-1 (1)

R 7: R = SO2Ph 1: R = H

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N. Kotoku

O

Y

X

analog ue 9

Z

100

Y = O, CH2 Z = A c, propa rg yl, e tc.

O furosp ino su lin-1 (1)

R R = H, Et

n n = 0~2

growth inhibition (%)

hypoxia

X = O, NH, S

80

normoxia

60 40 20 0 1

3

10

30

100

300

concentration (µM)

Fig. 3.4 SAR of furospinosulin-1 (1) and bioactivity of an analogue 9 against DU145 cells

chain tail were prepared according to the synthetic route in Scheme 3.1 with ease. Biological evaluation of analogues revealed that most of the structural modifications resulted in significant loss of hypoxia-selectivity, clearly indicating that the whole chemical structure was important for the binding to the target molecule. On the other hand, some analogues with modified side chain tail were found to retain hypoxiaselective growth inhibitory activity, and the C-propargylated analogue 9 (X = O, Y = CH2 , Z = C≡CH in Fig. 3.4) showed potent and hypoxia-selective growth inhibitory activity in a dose-dependent manner. Surprisingly, the analogue 9 exhibited more potent in vivo antitumor effects than 1: 25 mg/kg oral administration of the analogue 9 suppressed tumor weight by 11% of the control group. Then, synthesis of an affinity probe molecule derived from the analogue 9 to find the target molecule of 1 was examined. We planned to use a photo-cross-linking strategy to covalently capture the target molecule, because the binding affinity of 1 to its target molecule might be predicted as not so strong, interacting only through hydrophobic interactions. To maximize the cross-linking efficiency of the photoreactive group, the distance between the bioactive ligand and photoreactive group should be optimized [37–39]. Therefore, we designed the affinity probe as shown in Fig. 3.5. Thus, the alkyne-tailed furospinosulin-1 analogue 9 and the common functional unit consisted of biotin tag for detection/purification [40] and the benzophenone for photocrosslink with target protein [41] were assembled through a spacer unit (10), so that the position of the photoreactive group could be adjusted by using the spacer of an appropriate length for the efficient covalent bond formation. And the three components were coupled through selective ligation reaction, i.e., thiol-iodoacetamide ligation and copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction [42]. According to the assembling strategy, four photoaffinity probes equipped with the spacer of various lengths (11a–11d) were systematically obtained. Next, electrophoretic mobility shift assay (EMSA) analysis was executed to evaluate the probe molecules (Fig. 3.6). As described above, furospinosulin-1 (1) might inhibit the formation of the nuclear protein complex onto the P3 region of the

3 Marine Natural Products Targeting Tumor Microenvironment benzophenone for photo-crosslink

O

analogue 9

O O

4

11a: X = –(CH2OCH2)3– 11b: X = –(CH2OCH2)2– 11c: X = –(CH2)3– 11d: X = –CH2–

NH O

O

S

O

41

4

N H

X

N N N

HN

N N N

O 3

O

N3

O

HN

HN

NH H

H S

I spacer 10

N H

X

O

biotin tag for detection/purification

O

Target protein

O NH O

O

S

O

N N N

4

N H

X

HN

Optimization of spacer length

efficient photo-crosslink

Fig. 3.5 Design and synthetic strategy of photoaffinity probe of furospinosulin-1 (1)

lane :

1

2

3

4

5

6

7

8

9 O S N N N

X

N H

11a: X = –(CH2OCH2)3– 11b: X = –(CH2OCH2)2– 11a: X = –(CH2)3– 11a: X = –CH2–

nuclear protein : compound : (100 µM)

-

+ -

+ 1

+

+

+

+

+

11a 11a* 11b 11b* 11c

+ 11d

Fig. 3.6 Result of the EMSA analysis of affinity probes 11a–11d. An arrow indicates the supershifted band of the complex with Sp1 oligonucleotide and nuclear protein of DU145 cells cultured under hypoxic condition *: 300 μM treatment

promoter. Indeed, the nuclear proteins of DU145 cells cultured under hypoxic condition formed a complex with an oligonucleotide corresponding to the Sp1 consensus sequence responsible for the IGF-2 transcription, which was abrogated by 1 (lane 2 and 3). It suggested that the probe molecule with an appropriate spacer length is expected to bind to nuclear proteins and to inhibit the complex formation. As expected, the super-shifted band of the complex with the nuclear proteins were significantly diminished by the treatment (100 μM) of the probes 11a and 11b having the

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N. Kotoku

longer spacer (lanes 4 and 6). The complex formation was completely inhibited upon 300 μM treatment (lanes 5 and 7). In contrast, no inhibition occurred with the shortest probes 11c and 11d (lanes 8 and 9). Therefore, the spacer length is found to be critical for the efficient binding with the target protein as expected, and the probes 11a and 11b can capture the target protein contained in the transcription factor complex [43].

3.2.3 Target Identification of Furospinosulin-1 Furthermore, target identification of 1 was examined as follows. The result of EMSA shown above clearly indicates that the target molecule of 1 is one (or more) of the nuclear proteins of DU145 cells cultured under hypoxic condition, forming complex with Sp1 consensus sequence. So, the oligonucleotide corresponding to the Sp1 consensus sequence was used as a useful tool to narrow down the candidates. Through detailed MS/MS analyses of the binding protein profile to the oligonucleotide in various conditions (nuclear proteins from DU145 cells in normoxic/hypoxic condition, in the presence/absence of 1, etc.) and the following western blotting analysis, two candidate proteins: non-POU domain-containing octamer-binding protein (p54nrb ; also known as NONO), and PC4 and SFRS1-interacting protein (LEDGF/p75, also known as PSIP1) were identified. Then, binding experiment with the synthesized photoaffinity probe was executed (Fig. 3.7). Western blotting analysis showed that probe 12, having a spacer of the optimized length, bound to p54nrb in cell lysate of DU145 cells cultured in hypoxic condition (Fig. 3.7a, lane 1), which was inhibited by the addition of 1 (100 μM) (lane 2) and was retained by the addition of inactive analogue 14 (100 μM) instead of 1 (lane 3). p54nrb also weakly bound to probe 13 containing farnesyl group as a dummy ligand (lane 4). As probe 12 did not bind to p54nrb of DU145 cells cultured under normoxic conditions (lane 5), post-translational modification of p54nrb under hypoxic conditions might be occurred. On the other hand, LEDGF/p75 bound to probe 12 irrespective to the presence of competitor and the cell culture condition (Fig. 3.7b, lanes 1, 2, and 5), and did not bind to probe 13 (lane 4). Finally, knockdown experiment using siRNA indicated that knockdown of the two proteins resulted in the selective growth inhibition of DU145 cells under hypoxic condition, similar with the phenotype of furospinosulin-1 (1) treatment. On the other hand, the expression of IGF-2 in p54nrb knockdown cells was suppressed but no change was observed in the LEDGF/p75 knockdown cells. This result indicates that the role of p54nrb for adaptation to hypoxia is well correlated to the phenomena that occurred by 1, whereas further investigations are needed to elucidate the mechanism of how LEDGF/p75 contributes to hypoxia adaptation in cancer cells. At any rate, the target molecules of 1 are elucidated in these studies as p54nrb post-translationally modified under hypoxic conditions and LEDGF/p75, and the precisely designed affinity probe is the powerful tool for target identification [44].

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Fig. 3.7 Binding experiment of photoaffinity probe 12 and 13 with (a) p54nrb and (b) LEDGF/p75

3.2.4 Structure Confirmation of Dictyoceratins-A and -C by Total Synthesis From the extract of Dactylospongia elegans, the same sponge as that contained furospinosulin-1 (1), sesquiterpene phenol dictyoceratin-C (2) [45] was identified as another hypoxia-selective growth inhibitor (Fig. 3.2). From the SAR study with some previously isolated sponge-derived sesquiterpenoids, a closely related compound dictyoceratin-A (smenospondiol) (15) [46, 47] possessing a p-hydroxybenzoyl ester moiety was found to exhibit similar hypoxia-selective growth inhibitory activity. On the other hand, all the tested sesquiterpene quinones like ilimaquinone (16) did not show hypoxia-selective growth inhibition [48] (Fig. 3.8). In the literature, the absolute stereostructure of 2 and 15 remained unclear. Only the absolute configuration of 15 was determined through comparison of the degradation product with that of (–)-16 [46] which was ascertained by total synthesis [49, 50]. And, absolute configuration of 2 should be the same as 15 because of the structural similarity and the same sign of their specific rotation. On the other hand, Kondracki and co-workers isolated a sesquiterpenoid named smenospondiol, the absolute configuration of which was proposed as an antipode of 2 [47]. To confirm the absolute stereochemistry, and to supply a sufficient amount of the compounds for in vivo evaluation, total synthesis of both enantiomers of 2 and 15 was examined at first.

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N. Kotoku

Among the known synthetic strategies of sesquiterpene quinones/phenols [51], we decided to apply the method reported by Samadi and co-workers for the scalable synthetic method of 16 using the enone 17 [52] because the optically pure 17 and its enantiomer (ent-17) could be easily prepared using d- or l-phenylalanine as chiral catalyst, respectively [53]. Starting from 17, the needed transformations, i.e., basemediated coupling under heating condition; stereoselective introduction of the C-8 methyl group; removal of all protecting groups and transesterification; and Wittig olefination, proceeded smoothly, and total synthesis of 2 was accomplished. All the spectroscopic properties of the synthetic 2 were identical to those of natural product, including optical rotation [synthetic: [α]D + 17.3 (c 0.12, CHCl3 ), natural: [α]D + 16.7 (c 0.03, CHCl3 )] [54]. As the optical rotation of ent-2, prepared from ent-17 [[α]D –17.7 (c 0.28, CHCl3 )] was found to be opposite, the absolute stereochemistry of (+)-dictyoceratin-C was determined as 5R,8R,9S,10R. Using the same manner, the absolute stereochemistry of dictyoceratin-A (15) was determined as depicted in Fig. 3.8. Considering these results, smenospondiol is the same compound including absolute stereochemistry as 15 [55] (Scheme 3.2). MeO

O H

OH

R HO

O

17

15 1

OH

COOMe

H 9

8

4

ref. 46 11

HO

18

H

?

12

(+)-dictyoceratin-C (2, R = H) (+)-dictyoceratin-A (15, R = OH)

16) confirmed (total synthesis)

COOMe

(+)-smenospondiol (proposed structure)

Fig. 3.8 Structure of dictyoceratins-C (2) and -A (15) and related compounds

R R

RO

R

RO Br

RO

COOtBu H

H O

COOtBu O

H

COOtBu

O

O O

17 both enantiomers available

O

O

Scheme 3.2 Outline of the total synthesis of dictyoceratins-C (2) and -A (15)

2 or 15

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3.2.5 SAR and Target Identification of Dictyoceratin-C Then, SAR of 2 was analyzed focusing mainly on the four moieties: hydroxyl group/methyl ester moiety at aromatic part, and C-4 methylene/C-8 methyl group at decalin part. Various analogues depicted in Fig. 3.9 were obtained using the synthetic intermediate for the total synthesis of 2. Biological evaluation revealed that the 17hydroxyl group might be necessary for hypoxia-selective growth inhibition, whereas the 18-hydroxyl group was partially contributed to growth inhibitory activity. In addition, all the structural modification at decalin part led to the diminished activity and selectivity. These results indicate that not only the p-hydroxybenzoyl moiety but also the decalin part play an essential role for 2 and 15 to exhibit hypoxia-selective growth inhibitory activity. Among the prepared sample, only the propargyl amide analogue 18 showed hypoxia-selective growth inhibitory activity with the potency exceeding that of 2 at a high dose (30 μM). On the other hand, the potency of 18 at lower dose was weaker than the parent compound 2, implying that affinity of 2 with the target molecule might be higher than 18 [56]. Following the above SAR, we utilized three types of affinity probe molecules (19–21) to identify the target molecule (binding protein) of dictyoceratins-C (2) and -A (15) (Fig. 3.10). Biological activity against DU145 cells revealed that the probe A (19) exhibited the selective growth inhibitory activity under hypoxic conditions. On the other hands, the probe B (20) with 18-hydroxyl group showed the nonselective growth inhibition between normoxic and hypoxic conditions. In addition, the probe C (21) derived from 4-keto analogue exhibited no growth inhibitory activity irrespective to the oxygen concentration. Considering the biological properties of these compounds, the target molecule can be captured by probe 19 and the nonspecific binding proteins can be excluded using probes 20 and 21. Then the target molecule of 2 and 15 was investigated by phage display method using bacteriophage T7. The peptide-displayed phage library was constructed from mRNA of DU145 cells cultured under hypoxic conditions, and seven rounds of biopanning gave eight displayed peptides of target molecule candidate which bound to probe 19. Then, three out of eight candidates could be excluded through the binding selectivity test against the above three types of affinity probes 19–21, and the binding

R1, R2 = H, C9H19, R1O propargyl

OR2

H N R 18

HO

propargyl amide analogue 18: potent hypoxia-selective growth inhibition at 30 µM

O

17

COOCH3

H 8

CH3 H CH3

OH O

4

Fig. 3.9 SAR of dictyoceratin-C (2)

H H

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N. Kotoku OR3

Fig. 3.10 Structures of affinity probe for target identification of dictyoceratin-C (2) and -A (15)

probe A (19): R1 = CH2, R2 = H, R3 = S

R2O

probe B (20): R1 = CH2, R2 = S, R3 = H H

COOCH3

probe C (21): R1 = O, R2 = H, R3 = S O

S= R1

N N N

O

HN

H N

NH

10

4

O

experiment of the candidate proteins in DU145 cell lysate against probe 19 further narrowed down the candidates by three proteins, RNA-binding protein 28 (RBM28), RNA polymerase II-associated protein 3 (RPAP3), and melanoma inhibitory activity protein 3 (MIA3). Finally, knockdown experiment using siRNA of three proteins clearly indicated that knockdown of RPAP3 correlated with the selective growth inhibition of DU145 cells under hypoxic condition whereas no relationships were observed on RBM28 and MIA3 knockdown cells. This result strongly suggested that RPAP3 is the binding protein for 2 and 15, which was complemented by the protein expression profile of the RPAP3 knockdown cells. Thus, knockdown of RPAP3 reduced the expression levels of HIF-1α and related genes such as HK2 and VEGF in DU145 cells. This expression profile is quite similar with that of 2- or 15-treated DU145 cells. As the binding region of 2 and 15 in RPAP3 is in the vicinity of TRP1 domain which is essential for the complex formation with HSP90 [57, 58], the plausible action mechanism of 2 and 15 might be the dysfunction of mTOR through R2TP/PEDL/HSP90 complex formation [59, 60] and the following reduction of amounts of HIF-1α in DU145 cells, leading to the growth inhibition under hypoxic conditions [61].

3.3 Search for Selective Growth Inhibitors Under Glucose-Starved Condition Another characteristic of tumor microenvironment is nutrient starvation. Due to the structurally and functionally disordered formation of vasculature, some regions in solid tumor are exposed to oxygen- and nutrient-deficient condition [62]. From the recent studies the activation of phosphoinositide 3-kinase (PI3k) and/or v-akt murine thymoma viral oncogene homolog 1 (Akt) together with mammalian target of rapamycin (mTOR) signaling pathway were found to be important for the adaptation of cancer cells to nutrient starvation. In addition, activation of the unfolded protein response (UPR) such as induction of glucose-related protein 78 (GRP78) is also known as another adaptation system against nutrient starvation [63, 64]. Because the cancer cells that have adapted to these environments in tumors are generally thought to stimulate the pathological progression of cancer by promoting tumor growth, angiogenesis, metastasis, and drug resistance [65, 66], compounds that selectively

3 Marine Natural Products Targeting Tumor Microenvironment

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inhibit the growth of cancer cells under these conditions should have potential for anticancer drugs. Based on the background, there have been some reports regarding the search for selective growth inhibitors against tumor cells adapted to nutrient-starved conditions, so-called “anti-austerity compound” [67, 68]. Especially, Esumi et al. have found several anti-austerity agents from microbial secondary metabolites or traditional herbal medicines. For example, a polycyclic xanthone derivative kigamicin D, isolated from the culture broth of Amycolatopsis sp., showed selective cytotoxic activity against pancreatic cancer cells under the nutrient-starved condition compared with general culture conditions through inhibition of Akt signaling [69]. In addition, arctigenin, a lignan from the fruit of Arctium lappa used in the Kampo medicine, also showed the selective cytotoxicity under nutrient-starved condition [70]. In particular, phase I clinical trial of GBS-01, an extract of A. lappa as an orally administered drug rich in arctigenin, has been executed for the patients of advanced pancreatic cancer refractory to gemcitabine and further development was expected [71]. Moreover, the inhibitors of mitochondrial functions such as antimycin A (respiratory chain complex III inhibitor) or rotenone (complex I inhibitor) are known to exhibit the selective growth inhibitory activity against the cancer cells adapted to the nutrient-starved conditions [72] (Fig. 3.11). Following this background, we also established a screening system using human pancreatic cancer PANC-1 cells utilizing the glucose-deficient culture medium, to search selective growth inhibitors against the cancer cells adapted to the glucosestarved conditions. Screening and separation guided by the above bioassay provided

O OH O

N

O

HO OH

MeO O MeO

H

O

O

O O

OH

O

O OMe

HO MeO

O

OH

O

O

kigamicin D

O

arctigenin

OMe O 2-5

H O

O O

O

O H HO NH

H O

NH

H

O O

MeO

O

O

O

OMe

antimycin A

rotenone

Fig. 3.11 Reported natural products exhibiting selective growth inhibition under nutrient-starved condition

48

N. Kotoku Br

OH N

O OMe Br

Br

Br

R

N-methylniphatyne A (22)

N

polybrominated diphenyl ether 23: R = H, 24: R = Br 19

R1

N

S

R2

13

25

N

20

N

14

15

4

6

17

O 12

9

1

O

N 16

Cl

21

R

26

OH

biakamide A (27): 9E-isomer R = fasciospyrinadinone (25): R1 = R2 = O biakamide B (28): 9Z-isomer fasciospyrinadinol (26): R1 = H, R2 = OH biakamide C (29): 9E-isomer biakamide D (30): 9Z-isomer R =

23

27

OMe

Fig. 3.12 Structure of anti-austerity compounds identified by our research group

Table 3.1 Growth inhibition of compounds 22–30 against PANC-1 cells IC50 against PANC-1 (μM) 22

23

24

25

26

27

28

29

30

glucose (–)

16

3.8

2.1

13

18

1.0

4.0

0.5

0.5

glucose (+)

>100

>100

>100

>100

>100

>100

>100

50

35

some active compounds from the marine medicinal resources: N-methylniphatyne A (22), a new 3-alkylpyridine alkaloid isolated from an Indonesian marine sponge of Xestospongia sp. [73]; polybrominated diphenyl ethers 23 and 24 from an Indonesian marine sponge of Dysidea sp. [74]; 3-alkylpyridine sesquiterpenoids fasciospyrinadinone (25)/fasciospyrinadinol (26) [75] and unique polyketides biakamides AD (27–30) from an Indonesian marine sponge of Petrosaspogia sp. In particular, structure elucidation, mechanistic analysis, and SAR study toward biakamides were highlighted above (Fig. 3.12) and (Table 3.1).

3.3.1 Structure Elucidation of Biakamides A-D by NMR and Total Synthesis Biakamides A–D (27–30) were isolated from the MeOH extract of a marine sponge Petrosaspongia sp., collected in Biak, Indonesia in 2005 through bioassay-guided partition and further successive fractionation by SiO2 column chromatography and reversed-phase HPLC. High-resolution MS analyses suggested that chlorine and sulfur atoms might be contained in the molecule. In addition, many exceptionally broadened and doubled signals were observed in the 1 H and 13 C NMR spectra of 1, anticipating that some difficulties might arise in the structure determination. Brief analyses of some 2D-NMR spectra revealed that two N-methylamide moieties were

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contained in the molecule, which might be the principal cause of the broadening of the NMR signals. Through detailed analyses of some 2D-NMR spectra (correlation spectroscopy [COSY], heteronuclear single quantum coherence [HSQC], and heteronuclear multiple bond correlation [HMBC]) of 27, the presence of seven partial structures, including characteristic moieties in some marine natural products such as 2-substituted thiazole A [76, 77] and chloromethylene moiety E [78, 79]. Although most of the connectivity of the partial structures were clarified by careful analyses of the correlation signals in HMBC and/or the nuclear Overhauser effect spectroscopy (NOESY) spectra, no correlation signals around the partial structure D were observed in the HMBC and H–H COSY spectra of 27. It might be caused by the restricted bond rotation around the N-methylamide group of partial structure B, because NMR signals corresponding to the left half of the molecule were exceptionally broadened. Then, NMR experiment at elevated temperature may provide the sharpened NMR signals through accelerated bond rotation around the region. As expected, NMR measurement at 50 °C in DMSO-d 6 resulted in the observation of key HMBCs and magnetization relay in the total correlation spectroscopy [TOCSY] around the partial structure D, and all partial structures could be connected definitely (Fig. 3.13). On the other hand, the relative/absolute stereostructure of the two stereocenters at C-4 and C-6 for biakamides A–D (27–30) could not be determined because the 1 H-NMR signals of the compounds around the positions were too broadened to apply empirical methods [80] to analyze the differences in 1 H-NMR chemical shifts. Then, we decided to determine the absolute stereostructures of these moieties of 27–30 by total synthesis. F

B

O

D

A C

N N

G

N

E

COSY H

O

O

C

H

HMBC

S Cl O N

N N

TOCSY OH

O

S

H

Cl

O N 4

O

OH

N

6

HMBC d 6)

N S

C

Cl

Fig. 3.13 Partial structures and 2D-NMR correlations of biakamide A (27)

50

N. Kotoku

An outline of the total synthesis of biakamides is depicted in Scheme 3.3. To prepare all stereoisomers for structure determination, the monoprotected 2,4dimethyl-1,5-pentanediol 31 was used as a starting material. The possible four chiral isomers (31a-d) could be obtained through enzymatic desymmetrization/kinetic resolution of the syn- and anti-isomer, respectively [81]. Then, Corey-Seebach coupling reaction [82] between the 1,3-dithiane 32 and the nitrogen-contained alkyl halide, and additional reaction steps provided the central polyketide skeleton 33. The following introduction of the terminal thiazole amide, oxidative hydrolysis of 1,3-dithiane, and Wittig reaction to install the chloromethylene moiety proceeded smoothly to give 34, the common precursor of all biakamides. Finally, condensation with the corresponding carboxylic acid and the separation of the geometric isomer at C-9 by reversed-phase HPLC afford all the possible stereoisomers of biakamides A–D (27–30) [83]. Then, a comparison of the spectroscopic data of the synthetic compounds with those of the naturally occurring ones to ascertain the absolute stereochemistry. It revealed that the 1 H-NMR spectra of the (4S, 6R)- and (4R, 6S)-isomers of biakamide A (27) were found to be quite similar to those of the natural 27 throughout the molecule. However, only the multiplicity of the signal corresponding to H-12 of the (4S, 6R)-isomer was different from that of the (4R, 6S)-isomer and the natural product (Fig. 3.14A). In addition, the obvious differences between the chemical shifts of the signals corresponding to H-6 and H-7 in the 1 H-NMR (4S, 6S)- and (4R, 6R)-isomers and those of natural 27 helped us to rule out the possibility of those configurations (Fig. 3.14B). Finally, the comparison of the specific rotation and the CD spectra concluded that the absolute stereochemistry of biakamides A (27) is (4R, 6S), and the following analyses clearly showed that those of biakamides B-D (28–30) were found to be the same as 27. As the bioactivities of synthetic (4R, 6S)-isomers of

RO

OH

RO

OH

31a RO

RO

OH

*

RO

31b

S

32a-d (R = TBDPS)

OH

31c

*

31d

S

available by enzymatic desymmetrization / resolution N

*

*

EtO

S

O

N

S

Boc

O

33a-d N S

*

N

S

34a-d

*

NH Cl

O *

*

N 4

6

N

Comparison of NMR spectra (4R, 6S)

O Cl all isomers of biakamides A-D (27-30)

Scheme 3.3 Outline of the total synthesis of biakamides A-D (27–30) for the structure determination

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(b)

(a)

(4R,6R)-27

R

R

(4S,6R)-27

1.30, 1.30 1.30, 1.20 1.10

(4R,6S)-27

(4S,6S)-27

S

S

1.32, 1.32 1.32, 1.12 1.21

O natural 27

12

OH

26

natural 27

5

7 1.28, 1.41 1.41, 1.16 1.16

H-26 (cis)

H-12 (trans) H-25

15

14 H-26 (trans)

N

H-6, H-7A

H-12 (cis)

H-14 H-5B, H-7B

H-15

H-5A

Fig. 3.14 Comparison of 1 H-NMR spectra of natural biakamide A (27) and the synthetic isomers

27–30 were almost identical, the chemical structure of biakamides was certified as depicted [83].

3.3.2 Bioactivity and SAR of Biakamides As mentioned above, cancer cells can adapt to nutrient starvation through activation of PI3k/Akt/mTOR signaling pathway and UPR. In addition, inhibition of mitochondrial function or GRP78 leads to selective growth inhibition of cancer cells cultured under glucose-deprived conditions [72, 84]. Indeed, induction of Akt phosphorylation and GRP78 expression occurred in PANC-1 cells cultured under glucose-starved conditions, that were inhibited by the treatment with compounds 23 and 24 [74]. So, the effect of biakamides on these signaling pathways or mitochondrial function were analyzed. Western blot analysis revealed that induction of Akt phosphorylation and GRP78 expression in PANC-1 cells under glucose-starved conditions was inhibited by biakamide C (29). As the similar inhibition was observed by antimycin A, a known anti-austerity agent that inhibits the mitochondrial respiratory chain complex III. Mito Check Complex Activity Assays (Cayman Chemical) showed that 29 selectively inhibited complex I in the mitochondrial respiratory chain with an IC50 of 0.45 μM, comparable to the IC50 against PANC-1 cells under glucose-starved conditions (Table 3.1).

52

N. Kotoku O O

N

N

Cl

OMe

S

N

HO

36: IC50 10 µM

N

N

N

O

37: IC50 3.0 µM

O

analogue 35: IC50 0.6 µM

38: IC50 1.0 µM S

O N

Cl

N OMe

N

O

39: IC50 >100 µM

40: IC50 >100 µM S N

O MeO

O N H

N

caldorazole

S

Fig. 3.15 Bioactivity of synthetic analogues of biakamides. IC50 values against PANC-1 cells under glucose-starved conditions are depicted

The SAR study of biakamides was then executed through synthesis and evaluation of various analogues. It revealed that stereoisomers at C-4/C-6 of all biakamides, prepared as shown in Scheme 3.3, exhibited almost the same activities as those of natural products. This result clearly indicates the unnecessariness of two methyl groups here, and the 14,15-dinor analogue (35) actually showed comparable antiausterity activity (IC50 0.6 μM) without losing selectivity. Based on the compound 35, easily accessible because of the removal of stereocenters, some more analogues were prepared to analyze the participation of the substructures to the growth inhibitory activity, in other words, affinity toward target protein. As shown in Fig. 3.15, conversion of terminal acyl group (analogue 36) resulted in the dramatically dropped activity, whereas little differences were observed in changing the chloromethylene or thiazole amide moieties (analogues 37 and 38). It implies that the terminal acyl chain is important for binding with the target molecule, and transformation of amide part at the opposite terminal might be tolerated without losing activity. On the other hand, compounds 39 and 40, corresponding to the fragment of each terminal, completely lost the growth inhibitory activity. As the reported inhibitors of mitochondrial respiratory chain complex I such as caldorazole [85] have similar linear skeleton, relatively long aliphatic chain in their structure is needed for inhibiting the complex I [86]. It is known that mammalian mitochondrial complex I is an assembly of 45 different subunits [87], and the binding site of the inhibitors is dependent on their structure

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[88]. Further analysis of the binding region of biakamides using the affinity probe, with rational design considering the result of SAR and the tactics described here, will be executed in due course.

3.4 Conclusion In this chapter, search for marine natural products targeting tumor microenvironment, especially focused on the hypoxic and glucose-deficient condition, and the following chemical approaches were described. Application of in vivo cellbased phenotypic screening instead of in vitro target-based screening resulted in the discovery of active compounds with a novel mode of action or unprecedented chemical structure, and the detailed consideration on the compounds from the viewpoint of chemistry and biology was essential for finding the needle in the haystack. Indeed, the precise design of the photoaffinity probe enabled us to identify the target molecules of furospinosulin-1 (1), responsible for adaptation to hypoxia in cancer cells other than HIF-1, and the structures of biakamides A-D (27–30) were unambiguously determined only after the total synthesis of all the possible isomers were accomplished. In addition, careful analysis may provide some intriguing gifts: We found that the enantiomer of dictyoceratins-C and -A (ent-2 and ent-15) showed almost the same hypoxia-selective growth inhibitory activity as those of naturally occurring 2 and 15, respectively [55]. As the SAR study showed that the whole chemical structure is needed for binding to RPAP3, an endogenous protein composed of chiral α-amino acids, ent-2 and ent-15 should not be able to bind to RPAP3 and another target(s) should exist. The “unnatural” product could uncover the mystery, which is only accessible by organic synthesis, and continuing analysis for the other compounds might lead to some more attractive findings.

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69. Lu, J., Kunimoto, S., Yamazaki, Y., Kaminishi, M., Esumi, H.: Kigamicin D, a novel anticancer agent based on a new anti-austerity strategy targeting cancer cells’ tolerance to nutrient starvation. Cancer Sci. 95, 547–552 (2004) 70. Awale, S., Lu, J., Kalauni, S.K., Kurashima, Y., Tezuka, Y., Kadota, S., Esumi, H.: Identification of arctigenin as an antitumor agent having the ability to eliminate the tolerance of cancer cells to nutrient starvation. Cancer Res. 66, 1751–1757 (2006) 71. Ikeda, M., Sato, A., Mochizuki, N., Toyosaki, K., Miyoshi, C., Fujioka, R., Mitsunaga, S., Ohno, I., Hashimoto, Y., Takahashi, H., Hasegawa, H., Nomura, S., Takahashi, R., Yomoda, S., Tsuchihara, K., Kishino, S., Esumi, H.: Phase I trial of GBS-01 for advanced pancreatic cancer refractory to gemcitabine. Cancer Sci. 107, 1818–1824 (2016) 72. Momose, I., Ohba, S., Tatsuda, D., Kawada, M., Masuda, T., Tsujiuchi, G., Yamori, T., Esumi, H., Ikeda, D.: Mitochondrial inhibitors show preferential cytotoxicity to human pancreatic cancer PANC-1 cells under glucose-deprived conditions. Biochem. Biophys. Res. Commun. 392, 460–466 (2010) 73. Arai, M., Kamiya, K., Shin, D., Matsumoto, H., Hisa, T., Setiawan, A., Kotoku, N., Kobayashi, M.: N-Methylniphatyne A, a New 3-alkylpyridine alkaloid as an inhibitor of the cancer cells adapted to nutrient starvation, from an Indonesian marine sponge of Xestospongia sp. Chem. Pharm. Bull. 64, 766–771 (2016) 74. Arai, M., Shin, D., Kamiya, K., Ishida, R., Setiawan, A., Kotoku, N., Kobayashi, M.: Marine spongean polybrominated diphenyl ethers, selective growth inhibitors against the cancer cells adapted to glucose starvation, inhibits mitochondrial complex II. J. Nat. Med. 71, 44–49 (2017) 75. Matsumoto, H., Hisa, T., Toda, K., Ishida, R., Setiawan, A., Arai, M., Kotoku, N.: Fasciospyrinadinone and fasciospyrinadinol, novel 3-alkylpyridine sesquiterpenoids from an indonesian marine sponge, as selective growth inhibitors of the cancer cells under nutrient starvation. Heterocycles 103, 827–838 (2021) 76. Orjala, J., Gerwick, W.H.: Barbamide, a chlorinated metabolite with molluscicidal activity from the caribbean cyanobacterium Lyngbya majuscula. J. Nat. Prod. 59, 427–430 (1996) 77. Unson, M.D., Rose, C.B., Faulkner, D.J., Brinen, L.S., Steiner, J.R., Clardy, J.: New polychlorinated amino acid derivatives from the marine sponge Dysidea herbacea. J. Org. Chem. 58, 6336–6343 (1993) 78. Shaala, L.A., Youssef, D.T.A., McPhail, K.L., Elbandy, M.: Malyngamide 4, a new lipopeptide from the red sea marine cyanobacterium Moorea producens (formerly Lyngbya majuscula). Phytochem. Lett. 6, 183–188 (2013) 79. Edwards, D.J., Marquez, B.L., Nogle, L.M., McPhail, K., Goeger, D.E., Ann Roberts, M., Gerwick, W.H.: Structure and biosynthesis of the jamaicamides, new mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya majuscula. Chem. Biol. 11, 817–833 (2004) 80. Schmidt, Y., Lehr, K., Colas, L., Breit, B.: Assignment of relative configuration of desoxypropionates by 1 H NMR spectroscopy: method development, proof of principle by asymmetric total synthesis of xylarinic Acid A and applications. Chem. Eur. J. 18, 7071–7081 (2012) 81. Fujita, K., Mori, K.: Synthesis of (2R,4R)-Supellapyrone, the sex pheromone of the crownbanded cockroach, Supella longipalpa, and its three stereoisomers. Eur. J. Org. Chem. 493–502 (2001). 82. Seebach, D., Corey, E.J.: Generation and synthetic applications of 2-Lithio-1,3-dithianes. J. Org. Chem. 40, 231–237 (1975) 83. Kotoku, N., Ishida, R., Matsumoto, H., Arai, M., Toda, K., Setiawan, A., Muraoka, O., Kobayashi, M.: Biakamides A-D, Unique polyketides from a marine sponge, act as selective growth inhibitors of tumor cells adapted to nutrient starvation. J. Org. Chem. 82, 1705–1718 (2017) 84. Cha, M.-R., Yoon, M.-Y., Son, E.-S., Park, H.-R.: Selective cytotoxicity of Ponciri Fructus against glucose-deprived PANC-1 human pancreatic cancer cells via blocking activation of GRP78. Biosci. Biotechnol. Biochem. 73, 2167–2171 (2009) 85. Ohno, O., Iwasaki, A., Same, K., Kudo, C., Aida, E., Sugiura, K., Sumimoto, S., Teruya, T., Tashiro, E., Simizu, S., Matsuno, K., Imoto, M., Suenaga, K.: Isolation of caldorazole, a

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thiazole-containing polyketide with selective cytotoxicity under glucose-restricted conditions. Org. Lett. 24, 4547–4551 (2022) 86. Ishida, R., Matsumoto, H., Ichii, S., Kobayashi, M., Arai, M., Kotoku, N.: Structure-activity relationship of biakamide, selective growth inhibitors under nutrient-starved condition from marine sponge. Chem. Pharm. Bull. 67, 210–223 (2019) 87. Carroll, J., Fearnley, I.M., Skehel, J.M., Shannon, R.J., Hirst, J., Walker, J.E.: Bovine complex I is a complex of 45 different subunits. J. Biol. Chem. 281, 32724–32727 (2006) 88. Esposti, M.D., Ghelli, A., Ratta, M., Cortes, D., Estornell, E.: Natural substances (Acetogenins) from the family Annonaceae are powerful inhibitors of mitochondrial NADH dehydrogenase (Complex I). Biochem J. 301, 161–167 (1994)

Chapter 4

Chemical Biology Studies on Aplyronine A, A PPI-Inducing Antitumor Macrolide from Sea Hare Masaki Kita

Abstract The antitumor macrolide aplyronine A (ApA) disturbs microtubule (MT) dynamics by inducing the protein–protein interaction (PPI) between actin and tubulin. However, the detailed binding modes of actin–ApA–tubulin heterotrimeric complex (HTC) and the inhibitory mechanism of ApA on MT dynamics have been unclear. Blind protein–protein docking and molecular dynamics simulations afforded two plausible actin–ApA–tubulin HTC models. Based on the superposed models with the MT lattice, internal and capping HTC models to potently destabilize MT structure have been proposed. Next, to develop new antitumor PPI inducers, several aplyronine analogs with the C1–C9 macrolactone moiety and the C24–C34 side-chain were synthesized. While these analogs showed potent actin-depolymerizing activity, they did not interact with MTs. Docking simulation studies revealed that the C1–C9 part was not enough to fix the conformation of TMSer ester moiety, which protrudes from the actin surface on the actin–ApA complex. Meanwhile, structurally simplified C29– C34 side-chain ApA analogs were developed, which potently depolymerize actin. Binding kinetics analysis, docking simulations, and pull-down experiments revealed that these analogs were useful actin-affinity tags to study cytoskeletal dynamics and develop new drug leads. Keywords Actin · Antitumor macrolide · Chemical probe · Microtubule · Molecular dynamics simulation · Protein–protein interaction

4.1 Introduction The higher-order structure of actin (called as microfilament) forms a fiber network by the association of monomer actin (G-actin), along with a variety of endogenous binding proteins. Regulation of the protein–protein interactions (PPIs) of actin can modulate cytoskeletal dynamics and a variety of cellular signaling pathways [1, 2]. M. Kita (B) Graduate School of Bioagricultural Sciences, Nagoya University, Furo-Cho, Chikusa 464-8601, Nagoya, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_4

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A number of actin-targeting macrolides were discovered from marine invertebrates, such as aplyronines A–C (1–3) [3–10], mycalolides A and B [11–17], and reidispongiolide A [18–20] (Fig. 4.1). They have structurally related 11-carbon side-chains terminating in an N-methyl enamide group, with similar functionality and stereochemistry. Among them, aplyronine A (1) was recently shown to be a unique PPI inducer between actin and tubulin, and to inhibit microtubule (MT) dynamics [21, 22]. PPIs have received considerable attention and show great potential as intervention targets in various diseases such as cancer, neurodegenerative diseases, and infectious diseases [23]. MT is a common target for various anticancer drugs, and its mechanism of action has been well established [24, 25]. On the other hand, binding modes of the actin–ApA complex on tubulin and their inhibitory mechanism of MT dynamics remain unclear. This chapter describes the recent developments in chemical biology studies on the antitumor macrolide aplyronine A. To establish the binding position of the actin– ApA complex on the tubulin α,β-heterodimer and MT, molecular dynamics (MD) simulation studies were performed. We have aimed to clarify how the PPI-inducing effect of 1 is involved in the expression of antitumor activity. Furthermore, through the structure–activity relationship (SAR) studies, we have also aimed to develop simplified side-chain analogs of 1 as versatile actin-affinity tags. Our studies on 1 and the actin-affinity tags conjugated with bioactive ligands would highly contribute to the development for PPI-based new anticancer drug leads and research tools.

4.2 Biochemical Evaluations of ApA Marine organisms have a variety of bioactive secondary metabolites which are potential as anticancer properties [26, 27]. Among them, ApA (1) is a 24-membered antitumor macrolide isolated from the sea hare Aplysia kurodai. Its structural properties include the C1–C23 macrolactone part, the C24–C34 side-chain moiety with an Nmethyl enamide group and two kinds of amino acid esters [N,N,O-trimethylserine (TMSer, S/R = 54:48) and N,N-dimethylalanine (DMAla, S/R = 72:28)]. To date, eight analogs of aplyronines A–H have been isolated from the same animal. Among them, aplyronine B (ApB, 2) is a congener of ApA, in which the TMSer ester was migrated from C7 to C9. Another congener aplyronine C (ApC, 3) lacks the TMSer ester [5]. Despite the potent actin-depolymerizing activities of 2 and 3, it has been a mysterious question why they are much less cytotoxic than 1. The function of 1 was initially characterized as an actin-targeting compound that interferes with actin cytoskeleton dynamics by forming a 1:1 complex with a monomeric globular actin (G-actin), and depolymerizes fibrous actin (F-actin) [28–31]. X-ray crystallographic analysis of the actin–ApA complex showed that it intercalates into the hydrophobic cleft between actin subdomains (SD) 1 and 3 with its side-chain moiety [32, 33]. X-ray structures of complexes of actins with several macrolides with similar side-chain of 1 have also been established, such as swinholide A [19], sphinxolide B [34], reidispongiolides A and C [34], and kabiramide

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Fig. 4.1 Several structurally related actin-depolymerizing marine macrolides

C [35], all of which similarly bind to actin. It was also revealed that 1 exhibits potent antitumor activities in vivo against several cancer cells, such as Lewis lung carcinoma and P388 leukemia [5]. Another question regarding 1 was that why it exhibits potent

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cytotoxicity against cancer cells such as HeLa S3 (IC50 10 pM), while the concentration needed for actin cytoskeleton disassembly was much higher (~100 nM) than above. In 2013, the second target of 1 was established by using photoaffinity probes and related biochemical and cell biology experiments. In fact, 1 formed a 1:1:1 heterotrimeric complex (HTC) with actin and tubulin, and inhibited MT formation [21]. The synergistic binding effect of actin and 1 on tubulin was supported by the surface plasmon resonance (SPR) and gel-permeation HPLC analysis. Interestingly, ApC (3) was unable to bind to tubulin or interfere with MT dynamics. Thus, this minor analog was quite useful to demonstrate the highly potent activity of 1, this meant “diversified natural products-oriented structure–activity relationship (SAR) study”. The microtubule (MT) cytoskeleton is involved in a variety of biological processes, i.e., cell motility, cell shape maintenance, intracellular transport, and mitosis [36]. There are several microtubule-targeting agents which have potential anticancer efficacies [37, 38]. For example, colchicine is the most commonly prescribed gout drug, vinca alkaloids such as vinblastine and vincristine are the first MT targeting agents approved for the treatment of lymphomas, and paclitaxel is an ordinary medicine for the treatment of ovarian, breast, bladder, prostate, and lung malignancies [39]. To our knowledge, however, 1 is the first PPI inducer between actin and tubulin to prominently inhibit MT dynamics [2]. Since little is known about how 1 inhibits tubulin polymerization and induces MT disassembly in association with actin, we have examined MD simulations of the actin–ApA–tubulin HTCs to clarify its binding mode [40]. For the structure–activity relationship studies, two analogs of 1 were recently synthesized, which have the ApA macrolactone moiety and the side-chain moieties of mycalolide B [41] or swinholide A [42]. In fact, the latter compound showed induced potent PPI between actin and tubulin. On the other hand, little is known about the simplification of the macrolactone moiety of 1. To develop new analogs that modulate actin-related PPIs as new drug leads and research tools, we have examined the simplification of the macrolactone and side-chain parts of aplyronines [43, 44].

4.3 Binding Mode of Actin–ApA–Tubulin HTC Revealed by MD Simulation Since neither crystallographic nor solution structures of the actin–ApA–tubulin HTC have been established, scoring and posing were important to propose a reliable complex by molecular docking. Thus, by using the Molecular Operating Environment (MOE) program, protein–protein docking simulations were examined, in which the binding site of 1 on the SD1 and SD3 of actin (PDB: 1WUA) could interact with the

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whole surface of α/β-tubulin heterodimer (PDB: 1TUB). This “blind molecular docking” method [45, 46] gave 100 binding HTC poses with a variety of binding energies and positions. For the 10 best models within 17.76 kcal/mol in energy, the actin–ApA complex bound to the α/β-tubulin at various positions including C-terminal domain (CTD), intermolecular domain (IMD), and N-terminal domain (NTD). Next, MD simulations using YASARA software [47] were examined for further screening of the most reasonable HTCs. Among the 10 best models evaluated under conditions that mimic the cellular environment (pH 7.4 at 37 °C), the two HTC models (Models 1 and 2) were found to maintain the PPI between actin and tubulin, as indicated by small fluctuations and ligand movement dynamics during the simulations (Fig. 4.2). Notably, only the Models 1 and 2 maintained interactions between the C7 TMSer ester of 1 and tubulin through the 20 ns simulations. On the Model 1, the TMSer ester interacted with Gln336 and Asn337 at CTD of β-tubulin, and was close to CTD of αβ-tubulin. On the Model 2, the TMSer ester interacted with Arg123 and Glu127 at NTD of β-tubulin. The ligand–protein contact area of Models 1 and 2 after 20 ns MD simulations (1122.9 and 1417.2 Å2 , respectively) were much larger than that of actin–ApA complex (881.8 Å2 ). Furthermore, the ApA photoaffinity probes [21, 48] formed covalent bonds with both actin and tubulin, which was clearly explained by these models. In both Models 1 and 2, it was revealed that the SD1 and SD3 of actin faced α- and β-tubulin, respectively. Multiple ligand–protein interactions were observed at the C7 TMSer ester with β-tubulin, and the side-chain part with actin, including hydrophobic interactions and electrostatic attractions by opposite charge residues. These specific interactions might enable 1 to induce and stabilize PPIs between actin and α/β-tubulin in both models. To understand the binding modes of the actin–ApA complex on MTs, superposed models with the MT lattice (PDB: 5SYF) were examined (Fig. 4.3). On the Model 1, the actin–ApA complex located on the outer surface of MT, since the actin directly interacted with CTD of α-tubulin located MT outside. Viewing the MT lattice from the plus end, the actin of Model 1 protruded to the counter-clockwise side, and partially overlap the adjacent α/β-tubulin. This interaction might inhibit the tight binding between the lateral tubulin heterodimers and destabilize the MT structure. Meanwhile, Model 2 was supposed to bind to the MT polymerization terminus, in which the actin–ApA complex might overlap with another α/β-tubulin. Thus, Model 2 might inhibit the elongation and polymerization of MT at the plus end by the capping effect. Based on these models, inhibitory mechanisms for MT dynamics were proposed. First, the actin–ApA–tubulin HTC might be formed to reduce the concentration of liberated α/β-tubulin, which may delay its elongation into MT. In addition, the actin– ApA complex would bind to the middle part of MT to form the internal HTC (i.e. Model 1) to cause the MT disassembly. Furthermore, the formed HTC might bind to the plus end of MT as a capping structure (i.e. Model 2) to inhibit tubulin assembly. These superposed models could explain that 1 prominently depolymerizes MT and inhibits tubulin polymerization in association with actin [22].

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Fig. 4.2 MD simulation of the actin–ApA–tubulin HTC. (a) Conformational stability and (b) ApA movement dynamics of two HTC models simulated at 37 °C, pH 7.4 for 20 ns, (c) Proposed structures of the actin–ApA–tubulin HTC after 20 ns simulations. ApA (1) is shown in green sphere models

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Fig. 4.3 Possible mechanisms for the inhibitory effect of ApA on MT dynamics. (a) Models 1 and 2 superposed on the MT lattice (PDB: 5SYF, and α/β-tubulin are shown in grey and pale blue), Actin, α- and β-tubulin of two HTCs are highlighted in magenta, yellow, and cyan, (b) Two possible mechanisms to inhibit MT dynamics

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4.4 Synthesis and Biological Activities of ApA Analogs Previous structure–activity relationship studies showed that the side-chain parts in ApA and structurally related macrolides are important to interact with and depolymerize actin [28–31]. For example, the C21–C34 side-chain analog 4 substantially depolymerize F-actin, but has little cytotoxicity in cancer cells (Fig. 4.4). In fact, the C7 TMSer ester, the C9 OH group, and the C3–C6 conjugated diene moiety were important for potent cytotoxicity of 1. To develop new molecules that induce PPI between actin and tubulin, two analogs 5 and 6 were designed, which have the C1–C9 lactone part with or without the C7 TMSer ester and the C24–C34 side-chain part. The conjugated diene in 5 was expected to contribute to conformational fixation of the C1–C9 part, which directs the TMSer ester to the desired position on the actin. Furthermore, two side-chain analogs 7 and 8 were designed by the modification of C23 (OAc) and C29 (DMAla or OAc) functional groups, while the methyl and OH groups at C24–C26 were eliminated to simplify the structures. Analogs 5 and 6 were synthesized by the condensation of the C1–C9 carboxylic acids and the C23–C34 side-chain, followed by the selective TMSer esterification, according to the previous synthetic studies on aplyronines and mycalolides [49]. Meanwhile, to concisely synthesize analogs 7 and 8, we examined another route (Scheme 4.1). Mukaiyama–Paterson aldol condensation with Sn(OTf)2 [50] between enal 9 and ethyl ketone 10 provided syn-β-hydroxyketone 11. anti-Selective reduction of 11 with Me4 NBH(OAc)3 under acidic conditions and acetonide

Fig. 4.4 Structures of ApA derivatives 4–8

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protection of the 1,3-diol provided 12. Catalytic hydrogenation of the olefin and removal of the Bn group using Pd/C, and oxidation of the primary alcohol with 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) and PhI(OAc)2 [51] gave aldehyde 13. Subsequent Takai olefination [52] using CrCl2 and CHI3 yielded iodoolefin 14. Buchwald amidation with N-methylformamide [30, 53, 54] gave the (E)enamide, and removal of TBS group afforded a common intermediate 15. tertbutyldiphenylsilyl (TBDPS) protection in 15, removal of acetonide group, and regioselective esterification with l-N,N-dimethylalanine (DMAla) at the less-hindered C29 secondary alcohol with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and DMAP followed by the acetylation gave diester 16. Finally, the removal of the TBDPS group and subsequent acetylation gave analog 7. Similarly, the removal of the acetonide group in 15 and acetylation provided analog 8. We compared the activities of analogs 5–8 to those of aplyronines. By using pyrenyl actin and ultracentrifugation methods, the potent actin-depolymerizing activity of ApA (1) were established (EC50 1.3 ~ 1.4 μM for 3 μM monomer actin) (Table 4.1) [43, 55, 56]. Both analogs 5 and 6 strongly depolymerized actin at less than 5 μM, being more potent than 4 (EC50 7.9 μM against 3.7 μM actin) [49]. As for cytotoxicity, 5 and 6 moderately inhibited the proliferation of HeLa S3, a human cervical carcinoma cell line (IC50 1.4 and 1.1 μM, respectively). ApA analog 5 was >

Scheme 4.1 Synthesis of analogs 7 and 8

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Table 4.1 Biological activity of aplyronines and their side-chain derivatives Compound

Cytotoxicity, IC50 (nM)

Actin-depolymerizing activity, EC50 (μM) Ultracentrifugation method

Pyrenyl actin method

ApA (1)

0.010 (0.45)

1.4

1.3 [1.6]

ApB (2)

(2.9)

–

–

ApC (3)

17 (22)

–

1.6

4

(>19.000)

–

[7.9]

5

1,400

10,000

31

–

The data calculated as free salts are shown in parentheses. The data used 3.7 μM for monomeric actin are shown in square brackets

13 times more cytotoxic than 4, but 140,000- and 82-fold less cytotoxic than natural 1 and 3. The cytotoxicity of 5 and 6 was similar, and this did not reflect significant differences in the cytotoxicity between 1 and 3. Consequently, while the C1–C9 parts in 5 and 6 might partially potentiate the activities of C24–C34 side-chain part, ApA analog 5 did not induce PPIs between actin and tubulin in both cellular and in vitro experiments. For the side-chain simplified analogs, triacetate 8 showed weak actindepolymerizing activity (EC50 31 μM). Meanwhile, activity of 7 was more potent (EC50 8.8 μM, 16% residual activity of 1), being comparable to that of full sidechain analog 4 [29]. Thus, both the DMAla ester at C29 and the acetyl group at C23 in 7 were important to interact with actin, the latter of which mimicked the C1 lactone moiety of 1. Meanwhile, both 7 and 8 did not show cytotoxicity or affect actin cytoskeleton effects even at 10 μM in cancer cells, while ApA and related macrolides rapidly disassemble actin filaments at sub-μM concentrations [21].

4.5 Molecular Modeling Studies of ApA and Its Side-Chain Analogs To get more information to design structurally simplified ApA derivatives, molecular modeling studies between the ligands and actin were examined. First, we examined conformational search for ApA (1) bound to actin, in which the C24–C34 moiety was fixed on the rigid actin model. In the most stable conformer of 1, the entire macrolactone moiety as well as the C7 TMSer ester overlapped significantly with those of the actin–ApA complex [root mean square deviation (RMSD) 0.48 Å] (Fig. 4.5). This data suggested that modeling study is suitable for the prediction of ApA analog conformers on actin. To consider why the ApA analog 5 was ineffective on the

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PPI between actin and tubulin, similar docking simulations were performed. In fact, the C1–C9 part of the dominant conformer of 5 (90.3% occupation among the 17 conformers within 30 kJ/mol) had a highly different orientation from that of ApA (RMSD 4.98 Å), by the hydrogen bonding between the OMe group of the C7 TMSer ester and the Asp25 carboxylate. In contrast, the fourth stable conformer of 5 (ΔE + 11.9 kJ/mol) had a similar structure with 1 (RMSD 0.87 Å), in which the C7 TMSer ester protruded from the actin surface, and the C9 OH group interacted with the Glu334 carboxylate. However, due to the low proportion of the desired conformer, 5 might be unable to cause PPI between actin and tubulin. Next, interactions between the analogs 7 and 8 and actin were investigated by the docking simulations by using induced-fit models (Fig. 4.6 and Table 4.2). As expected by the potent actin-depolymerizing activity, 7 had a higher negative energy score (ΔG –8.60 kcal/mol) than 8 (–8.13 kcal/mol). The binding mode of 7 on actin was close to that of ApA (1) (RMSD 1.23 Å). Further LIGPLOT analysis showed that the hydrophobic contacts of 7 on actin were also similar to those of 1. This correct binding pose between the SD1 and SD3 hydrophobic cleft of actin would appropriately contribute to its actin-depolymerizing activity. Meanwhile, the binding pose of analog 8 was highly different from that of 1 (RMSD 2.90 Å). Among the marine macrolides that depolymerize actin, the C29 functional group has structural diversity (i.e., ketones for mycalolide A and reidispongiolide A; 2,3-dimethoxypropionate for mycalolide B) (Fig. 4.1). Still, our findings suggested that the positive-charged

Fig. 4.5 Molecular modeling studies of 1 and 5. (a) The most stable conformer of 1 (green) on actin. ApA (yellow) in the actin—ApA complex is superimposed. (b, c) The most stable (green) and (d, e) the fourth stable (cyan) conformers of analog 5 on actin. Reproduced from Ref. 43 (https:// pubs.acs.org/doi/10.1021/acsomega.9b01099), with permission from American Chemical Society, Copyright 2019

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Fig. 4.6 Binding position of the side-chain analogs 7 (blue) and 8 (yellow) on actin. Docking simulations were conducted with the induced-fit refinements. ApA (1) on the actin–ApA complex (red, PDB: 1WUA) is superimposed

Table 4.2 Actin-depolymerizing activity and binding free energy of side-chain analogs Compound Relative actin-depolymerizing RMSD for 1 (Å) Free binding energy (kcal/mol) activity for 1 (%) 1

100

–

−12.58

4

20

1.98

−8.95

7

16

1.23

−8.60

8

4.5

2.90

−8.13

C29 DMAla ester, found in five aplyronines (1, 2, 3, aplyronines G and H),9 might contribute to the specific conformations and strong interactions with actin.

4.6 Development of Versatile Actin-Affinity Tags Designed from the ApA Side-Chain Part Previously we examined the affinity purification of ApA target proteins by using biotin probes [57]. To demonstrate the specific binding of side-chain analog 7 with actin, interactions, and binding kinetics were analyzed. Because analog 7 had the moderate actin-depolymerizing activity and the good binding pose on actin among the synthetic side-chain analogs, a biotin derivative 7-bio was prepared from the common intermediate 15 in three steps. With the use of 7-bio (Fig. 4.7), potent bindings were observed at 1.25 μM actin. Bio-Layer Interferometry (BLI) analysis established that the dissociation constant (K D ) of 7-bio was 10.1 μM. While this value was slightly larger than those of ApA biotin derivative (1-bio) (5.16 μM with the BLI method and 4.02 μM with SPR analysis) [58], it was successfully showed that 7-bio was a moderate actin binder in this set. Finally, affinity pull-down experiments using cell lysate were examined. The HCT116 cell lysate was incubated with 7-bio bound to the Neutravidin agarose. Competitive elution with 7 detected the actin as a nearly single band, as with the

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Fig. 4.7 Pull-down experiments using cancer cell lysate. (a) Schematic representation of the affinity purification of target proteins. (b) Structures of biotin probes. (c) HCT116 cell lysate was loaded on Neutravidin agarose pretreated with 7-bio and 1-bio. The bound actin (arrowhead, 43 kDa) was competitively eluted with analog 7, and detected with silver stain

case of 1-bio prepared from natural 1 [57]. Thus, the side-chain analog 7 had potent affinity to disrupt the interactions of these two biotin probes with actin. We also found that analog 7 bound to actin in a highly specific but non-covalent manner, and that both 7 and 7-bio possessed desired properties as chemical probes with high actin-affinity.

4.7 Conclusion In summary, this chapter focused on the mode of action studies on aplyronine A, a potent antitumor macrolide from sea hare. This molecule has a unique PPI-inducing effect between actin and tubulin, two major cytoskeleton proteins in eukaryotic cells. Most natural actin-depolymerizing macrolides have been considered to be merely toxin, since they cause toxic effects against normal cells. However, our study revealed

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that 1 inhibits MT dynamics and causes MT disassembly at a much lower concentration in tumor cells which little affects the microfilament dynamics. Even today, it is difficult to predict the correct structures of molecular complexes without crystal structure data, homology models, or templates. We focused on the interactions of the TMSer ester, a characteristic important structure for the potent activity of 1. We successfully simulated the interaction between the actin–ApA complex and the α/βtubulin, and obtained two reliable HTC models that can explain the mechanism of 1’s inhibitory effect on the MT dynamics in association with actin. This identification of the binding modes of the actin–ApA–tubulin HTCs and the proposed mechanism for the effect of 1 on MT disassembly might greatly contribute to the design and development of future PPI-based drug leads. We also developed simple side-chain analogs of 1 that potently binds to and depolymerizes actin. The actin-depolymerization, binding kinetics, and affinity pulldown experiments revealed that these analogs well served as actin-affinity tags. Further conjugation of these tags with several bioactive ligands instead of biotin has the potential to provide new actin-associated PPI tools that can modulate cellular functions. It may be possible to mimic inhibition of MT assembly similar to 1 at the concentrations much lower than those required for actin filament disassembly in future. The use of such chemical probes as versatile actin-affinity tags should accelerate the study of mechanism of action associated with cytoskeletal dynamics and the development of PPI-based drug leads. The author hopes to elucidate the functions of fascinating natural products that are rich in structural and functional diversity beyond human knowledge and computational science. Acknowledgements I would like to express my sincere gratitude to Professor Hideo Kigoshi (University of Tsukuba) for his kind guidance and encouragement. I also thank all collaborators, including Dr. Didik H. Utomo, Ms. Akari Fujieda, Mr. Kentaro Futaki, and Ms. Momoko Takahashi, for their fruitful discussion and efforts in contributing to this research. This work is supported in part by JSPS grants (19H02839) and the Naito Foundation and the Uehara Memorial Foundation.

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Chapter 5

Chemical Synthesis and Immunological Functions of Bacterial Lipid A for Vaccine Adjuvant Development and Bacterial-Host Chemical Ecology Research Atsushi Shimoyama Abstract Lipopolysaccharide (LPS), an outer membrane component of gramnegative bacteria, is a representative immune activator, and its active principle is the glycolipid lipid A; thus, LPS is a potential adjuvant candidate. However, canonical Escherichia coli LPS is known to be an endotoxin, as it can induce lethal sepsis due to a hyperinflammatory immune response. Therefore, to apply them as adjuvants, it is necessary to structurally modify LPS and lipid A to minimize their toxic effects while maintaining their adjuvant effects Chemical ecology research considers the various life phenomena that occur between organisms as molecular interactions and has mainly focused on plants. Recently, as a new trend, the author hypothesized that LPS and lipid A mediate the bacterial-host chemical ecology and regulate various biological phenomena in the host, especially immunity. The author also predicted that parasitic and symbiotic bacteria that inhabit their hosts would have a low-toxicity immunomodulator owing to chemical structural changes in LPS as a result of coevolution with the host. To confirm these hypotheses and apply lipid A to low-toxicity and safe adjuvants, the author researched the chemical synthesis and functional evaluation of lipid As. In this chapter, chemical synthesis of lipid A, its structure–activity relationship, and its potential as a vaccine adjuvant are discussed. Keywords Glycolipid · Lipopolysaccharide · Lipid A · Adjuvant · Bacterial-host chemical ecology

5.1 Introduction Bacteria-derived components are known to modulate the host immune system [1]. Cancer shrinkage due to bacterial infections has been reported for more than 300 years A. Shimoyama (B) Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka 560-0043, Osaka, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_5

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[2]. The first cancer immunotherapy was an anti-cancer therapy conducted by Coley et al. (1893) using Streptococcus pyogenes and Serratia marcescens. In the 1900s, immunostimulatory effects of killed bacteria were found (killed Salmonella typhimurium and Mycobacterium tuberculosis in 1916 and 1924, respectively). These immunostimulatory actions are now widely recognized in the innate immune system. It is now understood that innate immunity is triggered by the recognition of characteristic molecular patterns of pathogens and microorganisms by various innate immune receptors in multicellular organisms. In recent years, innate immune stimulators have attracted attention as adjuvants for optimizing vaccine efficacy. Adjuvants are categorized as those that promote antigen uptake and elicit immune activation. Since innate immune stimulators also activate acquired immune responses, such as antigen–antibody interactions and cell-mediated immunity, they are developed mainly as adjuvants for the latter. However, most innate immune stimulators have undesired secondary reactions, such as inflammatory effects leading to toxicity; therefore, optimizing their function for use in vaccines is necessary. Lipid A is the first bacteria-derived molecule with innate immunostimulatory functions that can be put to practical use as an adjuvant. Lipopolysaccharide (LPS), a major glycoconjugate in the outer membrane of gram-negative bacteria, is a representative innate immune stimulator. Lipid A, a glycolipid bound to the terminus of the polysaccharide moiety of LPS via the specific acidic sugar Kdo (2-keto-3-deoxy-d-mannoctanoic acid), is the active principle of LPS [3, 4] The chemical structure of canonical Escherichia coli lipid A (1) is shown in Fig. 5.1. The recognition of LPS/lipid A by the Toll-like receptor (TLR) 4 and myeloid differentiation protein (MD-2) on host innate immune cells triggers various immune responses, such as cytokine production, nitric oxide production, reactive oxygen species production, leukocyte migration, and lymphocyte activation, thus triggering the host defense mechanism against pathogens. In contrast, LPS and lipid A cause severe inflammation. As endotoxins, they are the main cause of lethal sepsis, triggering severe systemic illnesses that cause multiple organ failure, hypotension, and septic shock. Canonical E. coli LPS is thus highly toxic, and its application as an adjuvant requires improvement to reduce its inflammatory effects and toxicity. Furthermore, lipid A can be attenuated by modifying its chemical structure. The monophosphoryl lipid A (MPL) derivative 3D-MPL (2) (Fig. 5.2), has already been developed by GlaxoSmithKline (GSK) and approved as a vaccine adjuvant [5]. Here, we introduce the chemical synthesis and structure–activity relationship of lipid A and a strategy to develop lipid A as an adjuvant, specifically, to develop symbiotic bacterial lipid A as an attenuated adjuvant, based on the concept of bacterial-host chemical ecology.

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Fig. 5.1 E. coli LPS and Kdo-lipid A Fig. 5.2 Chemical structure of 3D-MPL

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5.2 Bacterial Glycolipid Lipid A, an Innate Immune Stimulator In 1892, Pfeiffer showed that Vibrio cholerae produced two types of toxins: a heatsensitive exotoxin and a heat-stable endotoxin. In 1945, Westphal reported that LPS is the active ingredient of endotoxins. In 1957, glycolipid lipid A, an acylated disaccharide at the LPS terminus, was reported as the active principle of LPS [6]. Shiba and Kusumoto, in collaboration with a German research group, submitted the correct E. coli lipid A (1) structure (Fig. 5.1) and achieved its first total synthesis, establishing that lipid A is the active principle of LPS [7–9]. The lipid A structure from a heptose-less mutant of Salmonella typhimurium was identified by Qureshi and Takayama almost simultaneously [10]. Shiba and Kusumoto also synthesized lipid IVa (3) (Fig. 5.4), a biosynthetic precursor of E. coli lipid A (1). Lipid IVa (3) was found to exhibit immunostimulatory effects in mice but worked as an antagonist in humans. The discovery of antagonists suggests the existence of receptors and has led to exploratory studies of LPS receptors. In 1996, Hoffman showed that the Toll gene, which regulates dorsoventral axis formation in Drosophila, is essential for defense mechanisms against fungi. This led to a breakthrough in the discovery of various innate immune receptors [11]. In 1997, TLRs were identified as human homologs of Drosophila Toll protein [12], and in 1998, TLR4 was identified by Beutler as the LPS receptor [13]. Hoffman and Beutler were jointly awarded the 2011 Nobel Prize in Physiology or Medicine for their discoveries concerning the activation of innate immunity. Ten types of TLRs (TLRs 1–10) in humans and twelve types (TLRs 1–9 and TLRs 11–13) in mice have been identified so far, and Akira has contributed to many of these innate immune receptor discoveries [14]. The membrane glycoprotein TLRs are composed of an ectodomain with a leucinerich repeat motif and a cytoplasmic signaling domain homologous to the interleukin 1 receptor (IL-1R) and Toll/IL-1R (TIR) domains. Downstream signaling of TLR4 is transduced through various adaptor molecules (such as MyD88 and TRIF) that contain TIR domains (Fig. 5.3). MyD88-dependent signaling triggers the activation of NF-κB, a transcription factor that plays a central role in immune responses, and induces the production of inflammatory cytokines, such as IL-6. Inflammatory cytokines are produced as part of the defense response against infections. In contrast, TRIF-dependent signaling leads to the activation of interferon (IFN) regulator 3 (IRF3), which induces the production of antiviral type I IFN. Canonical E. coli LPS triggers the robust activation of both signals simultaneously, leading to an intense inflammatory response and, in severe cases, causing lethal septic shock. The recognition mechanism of LPS and lipid A by the TLR4 has been analyzed at the molecular level. MD-2, an accessory protein of TLR4, is essential for TLR4 activation triggered by LPS and was discovered by Miyake [15]. A tritium-labeled E. coli lipid A analog 4 (Fig. 5.5) was synthesized by Fukase, and Miyake used it to elucidate the interaction of the TLR4/MD-2 complex with lipid A [16]. The species

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Fig. 5.3 Innate immune system activation via TLR4/MD-2

specificity of TLR4/MD-2 is owing to the differences in lipid A recognition by MD2, as revealed by Miyake [17]. The binding mode of agonists and antagonists for TLR4/MD-2 was revealed by X-ray crystallography. In 2007, the crystal structure of human MD-2 in complex with lipid IVa (3) was revealed by Ohto and Satow [18] and the crystal structure of mouse TLR4/MD-2 in complex with the TLR4 antagonist eritoran (5) (Fig. 5.5) developed by Eisai was revealed shortly thereafter by Lee [19]. In 2009, X-ray crystallography of human TLR4/MD-2 in complex with E. coli LPS was reported by Lee [20]. Five of the six fatty acid chains of E. coli lipid A (1) were found to be accommodated within the hydrophobic pocket of MD-2, and the remaining fatty acid chain interacted with the hydrophobic surface consisting of the leucine-rich repeat of the adjacent TLR4. This interaction causes dimerization of the TLR4/MD-2 complex, which activates the innate immune system (Fig. 5.4). In contrast, an agonist with six fatty acid chains (E. coli lipid A [1]) and the antagonist lipid IVa (3), which has four fatty acid chains, cannot cause TLR4/MD-2 dimerization because all fatty acid chains are located inside the MD-2 pocket, and no overflowing fatty acid chains are present (Fig. 5.4). Incidentally, the antagonist bound to MD-2 with the lipid A moiety rotated 180° was comparable to the agonist. In addition, as mentioned above, lipid IVa (3) works as an antagonist against human TLR4/MD-2 and as an agonist in mice. Ohto revealed this mechanism by identifying the crystal structure of mouse TLR4/MD-2 with lipid IVa (3) [21]. In mice, three of the four fatty acid chains of lipid IVa (3) fit within the MD-2 pocket, and the remaining chain interacts with the hydrophobic surface of the adjacent TLR4 to form a dimer.

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Fig. 5.4 Molecular mechanism of TLR4/MD-2 dimerization

Fig. 5.5 Chemical structure of lipid As

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Previous structure–activity relationship studies [3, 22, 23] have revealed that, in addition to the number of fatty acid chains, TLR4/MD-2 functions can also be modulated by their chain length and number of phosphate groups. For example, MPL504 (6) (Fig. 5.6), an E. coli lipid A (1) without 1-phosphate, showed weaker IL-6 induction activity than E. coli lipid A (1) [24–26]. This is because TLR4/MD-2 dimerization in response to MPL is much lower than that in response to E. coli lipid A (1). In addition, MPL504 (6) is less dependent on CD14, a glycosylphosphatidylinositolanchored receptor known to be a co-receptor for TLR4. MPL504 (6) showed CD14independent but MyD88-dependent TNFα-producing ability and TRIF-dependent CD86 upregulation and IFN-β inducing ability [25]. MPL505 (7), which is an E. coli lipid A (1) without 4' -phosphate, exhibited weaker immunostimulatory effects than E. coli lipid A (1), similar to that of MPL504 (6); however, MPL504 (6) and MPL505 (7) showed a slightly different trend in IL-18 induction. That is, MPL504 (6) indicated lower IL-18-inducing activity than E. coli lipid A (1), but MPL505 (7) showed the same IL-18-inducing activity as E. coli lipid A (1) [26]. The polysaccharide portion of LPS (Fig. 5.1) is divided into two parts: an Oantigen polysaccharide portion, which is characteristic of each bacterial species, and a core oligosaccharide part, which has a highly common chemical structure among bacterial species. The effect of the unique acidic sugar Kdo in the core oligosaccharide moiety on the function of lipid A was investigated. There are R-mutant bacteria consisting of LPS lacking the O-antigen polysaccharide moiety and the E. coli Remutant containing a Re-LPS (8) consisting of lipid A linked to the Kdo disaccharide. Kusumoto synthesized Re-LPS (8) and Kdo-506 (9) (Fig. 5.6) and showed, for the first time, that lipid A activity is enhanced by the addition of Kdo [27].

5.3 Practical Synthetic and Semi-Synthetic Lipid A Adjuvants The advances in elucidating the structure–activity relationship of lipid A have promoted the development of attenuated lipid A, enabling the use of lipid A as a vaccine adjuvant. GSK attenuated lipid A by optimizing the lipid A structure, particularly the phosphate and acyl groups, and developed 3D-MPL (2) (Fig. 5.2) with a 4' -monophosphate structure similar to MPL504 and 3-deacyl structure. Furthermore, 3D-MPL (2) selectively activated the TRIF-dependent pathway (Fig. 5.3) and exhibited antiviral activity without excessive inflammatory response [5]; it is currently derivatized and manufactured from Salmonella minnesota R595 LPS. GSK adjuvants, including 3D-MPL, have already been applied and are being considered for practical use in vaccines. The adjuvant system (AS) 01, a mixture of 3D-MPL (2), cholesterol, and QS21 (a saponin derived from the tree Quillaya saponaria, native to South America), was developed in liposomal form. Shingrix®, the herpes zoster vaccine consisting of recombinant glycoprotein E of varicella-zoster virus and AS01, has already been commercialized. In addition, the

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Fig. 5.6 Partial structures of LPS

malaria vaccine candidate RTS, S/AS01, targeting Plasmodium falciparum sporozoite surface proteins, has been developed and is currently in Phase III clinical trials. AS02, consisting of 3D-MPL (2), squalene, oil emulsion, and QS21, has also been developed, and the malaria vaccine RTS,S/AS02 is also currently in Phase III clinical trials. GSK has also developed the adjuvant AS04, a mixture of 3D-MPL (2) and aluminum salts. AS04 has been commercialized as an adjuvant for the human papillomavirus (HPV) vaccine Cervarix® and HBV vaccine Fendrix®. RC-529 (Fig. 5.12), an MPL mimic, was approved in Argentina as an adjuvant for the hepatitis B virus vaccine.

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5.4 Lipid A Adjuvant Development Based on Bacterial-Host Chemical Ecology 5.4.1 Lipid A Mediates Bacterial-Host Chemical Ecology The field of chemical ecology, which considers various biological phenomena among organisms as molecular interactions, has developed mainly focusing on plants. As a new trend of chemical ecology research, the author focused on bacterial-host chemical ecology, especially that mediated by lipid A (Fig. 5.7). The author hypothesized that the co-evolved parasitic and symbiotic bacterial components would moderately modulate host immunity with low toxicity. Most studies on immunomodulation by parasitic and symbiotic bacterial components have focused on low-molecular-weight metabolites. In this host immunomodulation, it is obvious that LPS/lipid A, which are representative immunomodulatory molecules, are the primary key factors; however, because they are macromolecules or middle-size molecules with heterogeneous and complex structures, their molecular-level studies are still under development. Therefore, the author established a systematic synthetic strategy for these parasitic [26, 28] and symbiotic [29] bacterial lipid As and evaluated their immune functions. The author demonstrated that bacteria regulate host immune responses through differences in lipid A structure, elucidated the molecular basis of immune modulation and symbiosis, and applied these findings to developing safe and useful adjuvants.

Fig. 5.7 Bacterial-host chemical ecology mediated by lipid A

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5.4.2 Parasitic Bacterial Lipid A 5.4.2.1

Characteristic Structures of the Parasitic Bacterial Lipid A

Helicobacter pylori resides in the stomach and causes gastric ulcers, and Porphyromonas gingivalis resides in dental plaque and causes periodontal disease. LPS extracted from these parasitic bacteria has a weak immunostimulatory activity and has been suggested to be related to chronic inflammation and atherosclerosis [30– 33]. It has been suggested that the ability of these parasitic bacterial LPS to modulate TLR receptors contributes to the development of the aforementioned diseases (chronic inflammation and atherosclerosis) related to parasitic bacteria [34]. H. pylori and P. gingivalis have several lipid A species with structures that differ from that of a typical E. coli lipid A (1), particularly with their fatty acid chains and phosphate groups [35–38]. That is, H. pylori lipid A 10 and 11 (Fig. 5.8) have fewer (three– four) and longer (C16–C18) fatty acid chains, respectively, in contrast to canonical E. coli lipid A (1) (Fig. 1.1), which consists of six fatty acid chains (C12–C14). Regarding the phosphate group, H. pylori lipid A 10 and 11 are each found to have an MPL structure, whereas E. coli lipid A (1) has two phosphate groups at the 1 and 4' positions. Specifically, 10a and 11a each have a normal phosphate group only at 1-position, and 10b and 11b each have an ethanolamine phosphate group only at 1-position. P. gingivalis lipid As 13–16 have three to five fatty chains (C15–C17), including a terminal branched chain, and only 1-position is phosphorylated. As such, the parasitic bacterial lipids As 10–16 have common structural features, including diverse and heterogeneous structures compared to those of canonical E. coli lipid A (1), especially longer fatty acids and a 1-monophosphate group. The author hypothesized that these parasitic bacterial lipid A functions derived from their characteristic structures would be involved in establishing parasitic relationships (based on the low toxicity of lipid A) and chronic inflammation. To demonstrate this, the author synthesized these lipids as with diverse structures.

5.4.2.2

Chemical Synthesis of H. Pylori and P. Gingivalis Lipid AS

The author designed key disaccharide intermediate 19 with orthogonal protecting group patterns applicable to various lipid A syntheses with different acyl and phosphate group patterns and established a diversity-oriented lipid A synthetic strategy (Fig. 5.9) [28]. Each protecting group in 19 (i.e., 1-O-allyl, 2-Nallyloxycarbony (Alloc), 2' -N-2,2,2-trichloroethyloxycarbonyl (Troc), 3' -O-paramethoxybenzyl (MPM), and 4' ,6' -benzylidene) could be selectively removed to enable the sequential introduction of the Kdo moiety, acyl groups, or phosphate groups at the appropriate position. H. pylori lipid A 10 [28] and 11 [26] and P. gingivalis lipid As 13–16 [26] were synthesized by condensing long-chain fatty acids using the Shiina method and introducing a phosphate group by the phosphoramidite

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Fig. 5.8 Partial structures of parasitic bacterial LPS

Fig. 5.9 Diversity-oriented lipid A synthetic strategy

or acid anhydride method to the intermediate 19, which was prepared through βselective glycosylation between glucosamine derivatives 17 and 18 by neighboring group participation of the Troc group. The detailed synthetic scheme of H. pylori lipid A 10 from intermediate 19 was described in Fig. 5.10. 2' -N-Troc group of 19 was removed by Zn-Cu couple and the given amino group was acylated with the fatty acid 22 using 2-methyl-6nitrobenzoic anhydride (MNBA) [39] to yield 23. Other coupling reagents (O-(7azabenzotriazol-1-yl)-N,N,N' ,N'-tetramethyluronium hexafluorophosphate (HATU) or (1H-benzo[d][1,2,3]triazol-1-yl)oxy)tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyBOP)) gave considerable amounts of symmetric acid anhydrides from fatty acids, which showed low reactivity in this acylation. Subsequently, the

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2-N-Alloc group of 23 was cleaved by Pd(PPh3 )4 and dimethylaminotrimethylsilane (TMSDMA), [40] and given amino group was acylated with fatty acid 24 by using HATU to give 25. 1-O-Allyl groups in 25 were isomerized to the propenyl group with Ir complex [41], and the resulting 1-propenyl group was then removed by iodine and water to yield 26. For the synthesis of H. pylori lipid A 10a, 1-O-α-phosphorylation in 26 was carried out with lithium hexamethyldisilazide (LHMDS) and tetrabenzyl pyrophosphate in THF at –78 °C to give the desired 27 [42]. All protecting groups in 27 were then removed by hydrogenolysis with Pd-black under H2 gas in THF to yield H. pylori lipid A 10a, quantitatively. For the synthesis of H. pylori lipid A 10b, the phosphitylation of 26 was performed using the phosphoramidite [43, 44] to give the 1-O-phosphite, which was then oxidized to the desired 1-O-α-phosphorylated 28 using dimethyldioxirane (DMDO) [45]. All protecting groups in 28 were removed by hydrogenolysis with Pd(OH)2 /C under H2 gas in THF/H2 O/AcOH to give H. pylori lipid A 10b. To investigate the effect of Kdo addition on H. pylori lipid A activity, Kdo lipid A 12 was synthesized. Kdo has a 2-keto-3-deoxy structure, and its glycosylation has the following problems:(1) because of the 3-deoxy structure, neighboring group participation cannot be used for the stereoselectivity of glycosylation; and (2) βhydrogen elimination between the 2 and 3-positions is easily induced, producing serious amounts of by-product glycal. Kusumoto and Shiba found that by using Kdo fluorides protected with isopropylidene at 4 and 5 position, α-selective glycosylation proceeds due to the steric hindrance of the isopropylidene group [46, 47]. Kdo donors protected with isopropylidene groups at 4 and 5 position have a boat-shaped conformation and are highly reactive but tend to afford glycal formation. In contrast, the Kdo donor protected with TBS groups at 4 and 5 position has a chair conformation, which reduces reactivity but suppresses glycal formation [27]. However, the activation of conventionally used Kdo fluorides usually requires stoichiometric amounts of Lewis acids, and acid-sensitive protecting groups such as isopropylidene group are undesirably cleaved. Therefore, the author developed an acid-catalyzed Kdo glycosylation by using N-phenyl trifluoroacetimidate as a leaving group, which was developed by Yu [48–51]. In the glycosylation between the disaccharide intermediate and the Kdo donor protected with TBS groups, the reaction did not proceed because of its low reactivity of TBS protected Kdo donor, however, the desired trisaccharide 21 was successfully produced using Kdo donor 20 protected with an isopropylidene group (Fig. 5.9) [28]. Specifically, the glycosylation reaction between Kdo donor 20 with N-phenyl trifluoroacetimidate as the leaving group and the disaccharide acceptor 29 obtained by selective benzylidene opening of the disaccharide intermediate 19 was investigated (Fig. 5.11). After optimization of the acid catalyst and the solvent, the desired trisaccharide 21 was obtained in good yield (70%) and high selectivity (α: β = 95: 5) when TBSOTf was used as the acid catalyst in cyclopentyl methyl ether (CPME) using 5 equivalents of Kdo donor 20 for 29 (entry 1). The α-promoting solvent effect of cyclopentyl methyl ether (CPME) was employed here [52, 53]. When TfOH was used in CH3 CN, the glycosylation reaction proceeded quantitatively, even though the α-selectivity (α: β = 75:25) was reduced (entry 3). While, considerable amounts of

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Fig. 5.10 Synthesis of the H. pylori lipid A 10

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Fig. 5.11 Kdo glycosylation under microfluidic conditions

glycal 30 derived from donor 20 were by-product in batch reaction system. Generally, in the synthesis of ketosides with 2-keto-3-deoxy structure such as Kdo and sialic acid, 3-position hydrogen is relatively acidic and liable to cause β-hydrogen elimination. Thus, an excess amount of Kdo donor 20 was required due to the byproduction of glycals 30. Fukase previously reported α-selective sialylation with the N-phenyltrifluoroacetimidate donors by using a microflow reactor, which enables efficient mixing and rapid heat transfer, to improve the stereoselectivity and suppress the glycals formation [54]. Therefore, the author investigated the use of microflow reactors for Kdo glycosylation. A CPME solution of Kdo donor 20 and acceptor 29 was mixed with a CPME solution of TBSOTf using IMM micromixer and the reaction mixture allowed to react in a tube reactor for 42 s before quenching (Fig. 5.11). The microflow conditions allowed to reduce the amount of Kdo donor 20 to 1.5 equivalents and still give trisaccharide 21 in almost the same yield as in the batch reaction (entry 1, 2). Trisaccharide 21 was efficiently produced because of the suppression of glycal by-product formation through the efficient removal of reaction and mixing heat by the large surface area of the microflow reactor, as well as the promotion of intermolecular reactions by high-efficiency mixing [28, 55]. H. pylori Kdo-Lipid A 12 was synthesized by introducing acyl and phosphate groups to trisaccharide 21 as with lipid A synthesis [28].

5.4.2.3

Immunomodulatory Functions of H. Pylori and P. Gingivalis Lipid A

The immunomodulatory functions (cytokine-inducing activity) of the chemically synthesized parasitic LPS substructure 10–16 in human peripheral whole blood were

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evaluated. Lipids A 10a, 11a, 13, and 14, with three to four fatty acid chains and monophosphate group, exhibited antagonistic activity in the induction of inflammatory cytokines, such as IL-6 and TNF-α. In contrast, lipid A 10b and 11b, with three or four fatty acid chains and mono-ethanolamine phosphate group, and lipid A 15 and 16 with four to five fatty acid chains and monophosphate group, showed much weaker but definite IL-6 and TNF-α inductions than that of E. coli LPS. As mentioned above, the addition of Kdo enhanced the activity of E. coli lipid A (1) [27]. In contrast, in the case of H. pylori, 12a with Kdo added to antagonist 10a showed stronger antagonistic effects on the induction of IL-6 than 10a. Furthermore, 12b with Kdo added to the weak agonist 10b switched to an antagonist.28 Thus, it was observed that the function of lipid A could be switched depending on the pattern of acyl and phosphate groups and the presence or absence of Kdo. In addition, for H. pylori LPS, in contrast to other LPS, Kdo lipid A, not lipid A itself, was found to be the active principle. On the other hand, the parasitic bacterial lipid As was shown to induce IL-12 and -18, which are involved in chronic inflammatory diseases caused by parasitic bacteria, with a different tendency from the induction of IL-6 and TNF-α. The parasitic bacterial lipid As 10–16 induced IL-12 and -18. Moreover, 10a, 11a, 13, and 14 were found to mediate a significant selective induction of IL-12 and IL-18. These results suggested that parasitic lipid As can regulate TLR4 downstream signaling. As the induction of IL-6 and TNF-α is MyD88-dependent, it is certain that 10a, 11a, 13, and 14 inhibit the MyD88-dependent pathway (Fig. 5.3). On the other hand, TRIF-dependent [56] and TRIF-independent pathways [57] have been reported for LPS-mediated IL-18 induction, and it remains unclear whether 10a, 11a, 13, and 14 can selectively activate the TRIF pathway. Incidentally, since the IL-12 and -18 combination induces IFN-γ, which is involved in anti-tumor and anti-allergic reactions, parasitic bacterial lipid A, which selectively induces IL-12 and IL-18 while suppressing pro-inflammatory cytokines, has the potential as an adjuvant.

5.4.3 Symbiotic Bacterial Lipid A Parasitic bacterial lipid A research suggested that parasitic bacteria evolve to escape the host immune responses, as their LPS/lipid A showed antagonistic that favors infection to the host or weak agonistic effects. Some of the parasitic bacteria lipid As selectively induced cytokines involved in chronic inflammation while suppressing the induction of inflammatory cytokines. These results indicate that parasitic bacteria may induce chronic inflammatory diseases while evading the antibacterial effects derived from acute inflammation (host immune response), suggesting that lipid A activity strongly reflects bacterial characteristics. Inspired by these results, the author hypothesized that bacterial lipid A regulates host immunity, that is, that there is a lipid A-mediated bacterial-host chemical ecology (Fig. 5.7). Therefore, the author considered that symbiotic bacteria have immunomodulators with extremely low toxicity

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and they are involved in the maintenance of homeostasis. Thus, the author expected symbiotic bacterial lipid A to be a potential pool of safer immunomodulators. Gram-negative bacterium Alcaligenes faecalis, known as an opportunistic bacterium, was found to inhabit Peyer’s patch, a gut-associated lymphoid tissue (GALT), and plays an important role in maintaining homeostasis [58–60]. The author considered that A. faecalis lipid A might have a homeostatic function and could be applied as a safe, low-toxicity immunomodulator. Therefore, we conducted a collaborative study with Kiyono and Kunisawa, extracted the LPS fraction from dried A. faecalis, determined the structure of A. faecalis LPS in collaboration with Molinaro[29], and performed functional analysis of extracted A. faecalis LPS [61, 62]. Canonical E. coli produces LPS consisting of tens to hundreds of sugar residues, while some species produce lipooligosaccharides (LOSs) with short sugar chains. A. faecalis produces LOS composed of a nonasaccharide [29]. The extracted A. faecalis LOS fraction was found to remarkably enhance IgA antibody production comparable to that of toxic E. coli LPS, while showing no harmful effects. As these effects were TLR4-dependent, A. faecalis lipid A was expected to be a promising safe adjuvant candidate [61]. Since A. faecalis lipid A was found to be a mixture of 31–33 with four to six fatty acid chains (Fig. 5.12), the author established a strategy to synthesize A. faecalis lipid A systematically. The author developed a diversity-oriented synthetic strategy for parasitic bacterial MPL based on the key intermediate 19 [28] (Fig. 5.9). On the other hand, A. faecalis lipid A 31–33 were bis-phosphorylated. Therefore, in this study, the author applied intermediate 19 to the synthesis of bis-phosphorylated lipid A. The detailed synthetic scheme of A. faecalis lipid A 33 from intermediate 19 was described in Fig. 5.13. Fatty acid 34 was introduced to the 3-position of 19 in the presence of MNBA to give 35. Then, 2' -N-Troc group of 35 was removed by a Zn-Cu couple and given amino group was acylated with fatty acid 36 using MNBA. 2-N-Alloc group of 37 was then removed by Pd(PPh3 )4 and TMSDMA [40], and fatty acid 38 was

Fig. 5.12 Synthesized A. faecalis lipid As

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Fig. 5.13 Synthesis of the hexa-acylated A. faecalis lipid A 33

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introduced to the free 2-amino group using HATU to give 39. After the cleavage of the MPM group at 3-position by 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) oxidation, fatty acid 34 was introduced using MNBA to yield 40. Subsequently, 4' ,6' O-benzylidene group of 40 was removed by trifluoroacetic acid (TFA), and then the trityl (Tr) group was selectively introduced to 6' -position. 1-O-Allyl groups in 41 was then removed [41] to yield 1,4' -dihydroxyl 42. The simultaneous phosphitylation of 1 and 4' -position using phosphoramidite gave the desired 1, 4' -O-diphosphite, which was then oxidized to 1,4' -O-diphosphorylated 43 by DMDO [45]. All protecting groups of 43 were removed by hydrogenolysis with Pd(OH)2 /C under H2 gas in THF/H2 O/AcOH to give hexaacylated A. faecalis lipid A 33. Nuclear factor (NF)-κB activation was evaluated by a secretory alkaline phosphatase (SEAP) reporter assay in HEK-Blue™ hTLR4 cells to evaluate the effective immuno-activation of A. faecalis lipid A 31–33 through TLR4. Only hexaacylated A. faecalis lipid A 33 showed immunostimulatory activity (Fig. 5.14), which was almost comparable to that of the extracted A. faecalis LOS, thus confirming that A. faecalis lipid A 33 is the active principle of A. faecalis LOS [49]. Furthermore, in vivo studies using mice showed that A. faecalis lipid A 33 exhibited useful adjuvant effects (enhancement of antigen-specific IgA and IgG production Th17 mediated protective immunity) without toxicity [63–65] In particular, enhancement of antigenspecific IgA and IgG production was observed in mice intranasally administered with antigen and A. faecalis lipid A 33 adjuvant [63, 66]. Thus, both mucosal and systemic immunity can be activated by intranasal administration of A. faecalis lipid A 33, indicating that 33 is a promising intranasal adjuvant candidate. The efficacy of 33 as a safe intranasal vaccine adjuvant was demonstrated in a Streptococcus pneumoniae infection model [63]. Thus, it was found that lipid A 33 derived from A. faecalis, a resident in GALT, which is responsible for the regulation of intestinal mucosal immunity, can regulate the induction of IgA, which is responsible for the homeostasis of mucosal immunity. Lipid A 33 was suggested to function as a regulator of intestinal mucosal immunity.

Fig. 5.14 Synthesized A. faecalis lipid A and extracted A. faecalis LOS mediated NF-κB activation in HEK-Blue™ hTLR4 cells was evaluated by SEAP reporter assay. E. coli LPS O111 was used as positive control

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Therefore, by focusing on symbiotic bacteria living in mucosal immune regulatory tissues, the author identified the key compound for intestinal mucosal immunity and discovered the promising adjuvant candidate that can safely control mucosal immunity.

5.4.4 Lipid A in Fermented Foods as Adjuvants Lipid A in fermented foods has attracted attention as safe adjuvant candidates. Kurozu (black vinegar), an Asian fermented food, contains LPS derived from the gram-negative bacteria Acetobacter spp. that oxidizes sugars or ethanol and produces acetic acid during fermentation. The chemical structure of A. pasteurianus lipid A (44) is shown in Fig. 5.15. The phosphate group at the anomeric position, which is relatively unstable under acidic conditions, is replaced by glucuronic acid[67]. Although extracted A. pasteurianus LPS showed weaker immunostimulatory activity than E. coli LPS [67, 68], A. pasteurianus LPS and its lipid A would be safe owing to food experience and are expected to be safe adjuvant candidates with high chemical stability. Pantoea agglomerans, a gram-negative bacterium widely present in soils and food plants such as wheat, rice, sweet potatoes, apples, and pears, was detected during the fermentation of rye bread [69]. Since P. agglomerans LPS could show immunostimulatory effects upon oral administration, it has the potential as an oral adjuvant. P. agglomerans lipid A is a mixture of E. coli lipid A (1) and S. minnesota lipid A (45), both of which are agonists.

Fig. 5.15 Chemical structures of various lipid As

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5.5 Conclusion In this chapter, the structure–activity relationship of TLR4 ligands, especially lipid A, and their potential as vaccine adjuvants were discussed. Chemical structural modifications of lipid A can regulate TLR4/MD-2 function, and specific lipid A derivatives can be used to regulate cell-mediated, humoral, or mucosal immune responses, respectively. Lipid A-based adjuvants, such as the AS0X series (GSK), are already being used in the clinic as components of various vaccines, including anti-cancer and antimalarial vaccines, and their importance is expected to increase in the future. These studies also suggest the potential of a strategy for developing useful functional molecules based on bacterial-host chemical ecology, which focuses on the characteristics of each bacterium and then focuses on bacteria that can be used as a pool of desired functional molecules. This strategy is applicable to various subjects, and the author expects that it will lead to the discovery of numerous useful natural products in the future.

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30. Hynes, S.O., Ferris, J.A., Szponar, B., Wadstrom, T., Fox, J.G., O’Rourke, J., Larsson, L., Yaquian, E., Ljungh, A., Clyne, M., Andersen, L.P., Moran, A.P.: Comparative chemical and biological characterization of the lipopolysaccharides of gastric and enterohepatic helicobacters. Helicobacter 9, 313–323 (2004) 31. Nielsen, H., Birkholz, S., Andersen, L.P., Moran, A.P.: Neutrophil activation by Helicobacter pylori lipopolysaccharides. J. Infect. Dis. 170, 135–139 (1994) 32. Perez-Perez, G.I., Shepherd, V.L., Morrow, J.D., Blaser, M.J.: Activation of human THP-1 cells and rat bone marrow-derived macrophages by Helicobacter pylori lipopolysaccharide. Infect. Immun. 63, 1183–1187 (1995) 33. Danesh, J., Wong, Y., Ward, M., Muir, J.: Chronic infection with Helicobacter pylori, Chlamydia pneumoniae, or cytomegalovirus: population based study of coronary heart disease. Heart 81, 245–247 (1999) 34. Triantafilou, M., Gamper, F.G., Lepper, P.M., Mouratis, M.A., Schumann, C., Harokopakis, E., Schifferle, R.E., Hajishengallis, G., Triantafilou, K.: Lipopolysaccharides from atherosclerosisassociated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced inflammatory responses in human vascular endothelial cells. Cell Microbiol. 9, 2030–2039 (2007) 35. Suda, Y., Ogawa, T., Kashihara, W., Oikawa, M., Shimoyama, T., Hayashi, T., Tamura, T., Kusumoto, S.: Chemical structure of lipid A from Helicobacter pylori strain 206–1 lipopolysaccharide. J. Biochem. 121, 1129–1133 (1997) 36. Moran, A.P., Lindner, B., Walsh, E.J.: Structural characterization of the lipid A component of Helicobacter pylori rough- and smooth-form lipopolysaccharides. J. Bacteriol. 179, 6453–6463 (1997) 37. Ogawa, T.: Chemical structure of lipid A from Porphyromonas (Bacteroides) gingivalis lipopolysaccharide. FEBS Lett. 332, 197–201 (1993) 38. Kumada, H., Haishima, Y., Umemoto, T., Tanamoto, K.: Structural study on the free lipid A isolated from lipopolysaccharide of Porphyromonas gingivalis. J. Bacteriol. 177, 2098–2106 (1995) 39. Shiina, I., Ushiyama, H., Yamada, Y.-K., Kawakita, Y.-I., Nakata, K.: 4(Dimethylamino)pyridine N-oxide (DMAPO): an effective nucleophilic catalyst in the peptide coupling reaction with 2-methyl-6-nitrobenzoic anhydride. Chem. Asian J. 3, 454–461 (2008) 40. Merzouk, A., Guibe, F., Loffet, A.: On the use of silylated nucleophiles in the palladium catalyzed deprotection of allylic carboxylates and carbamates. Tetrahedron Lett. 33, 477–480 (1992) 41. Baudry, D., Ephritikhine, M., Felkin, H.: Isomerization of allyl ethers catalyzed by the cationic iridium complex [Ir(cyclo-octa-1,5-diene)(PMePh2)2]PF6. A highly stereoselective route to trans-propenyl ethers. J. Chem. Soc., Chem. Commun. 694–695 (1978) 42. Inage, M., Chaki, H., Kusumoto, S., Shiba, T.: A convenient preparative method of carbohydrate phosphates with butyllithium and phosphorochloridate. Chem. Lett. 1281–1284 (1982) 43. Campbell, A., Fraser-Reid, B.: Support studies for installing the phosphodiester residues of the Thy-1 glycoprotein membrane anchor. Bioorg. Med. Chem. 2, 1209–1219 (1994) 44. Prestwich, G. D., Marecek, J. F., Mourey, R. J., Theibert, A. B., Ferris, C. D., Danoff, S. K., Snyder, S. H.: Tethered IP3. Synthesis and biochemical applications of the 1-O-(3-aminopropyl) ester of inositol (1,4,5)-trisphosphate. J. Am. Chem. Soc. 113, 1822–1825 (1991) 45. Murray, R.W., Jeyaraman, R.: Dioxiranes: synthesis and reactions of methyldioxiranes. J. Org. Chem. 50, 2847–2853 (1985) 46. Imoto, M., Kusunose, N., Matsuura, Y., Kusumoto, S., Shiba, T.: Preparation of novel pyranosyl fluorides of 3-deoxy-d-manno-2-octulosonic acid (KDO) feasible for synthesis of KDO alpha -glycosides. Tetrahedron Lett. 28, 6277–6280 (1987) 47. Imoto, M., Kusunose, N., Kusumoto, S., Shiba, T.: Synthetic approach to bacterial lipopolysaccharide. Preparation of trisaccharide partial structures containing KDO and 1-dephospho lipid A. Tetrahedron Lett. 29, 2227–2230 (1988)

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Part II

Uncovering Biosynthesis of Natural Products

Chapter 6

Dissecting Biosynthesis of Natural Products Toward Drug Discovery Yuta Tsunematsu

Abstract Microorganisms have developed diverse metabolic systems for enduring in a competitive society of nature. While some microbial species threaten human beings as the cause of infectious disease, there are many examples how people have utilized microbial potential to produce fermented foods, beverages, and pharmaceuticals, all of which are indispensable for maintaining the quality of human life. A significant factor that triggers biological phenotypes in the recipient is microbeproducing small organic molecules, also known as natural products. In the current post-genomic era, many of the specialized biosynthetic pathways of natural products have been identified and linked to their biosynthesis genes, enabling us to design, harness, and create the pathway to obtain more valuable substances. In this chapter, we initially introduce the biosynthesis of aspirochlorine, a potent antifungal agent produced by a beneficial fungus, Aspergillus oryzae, which has been utilized in East Asia for brewing fermented products. Then, we focus on potential drug candidates, fumagillin and pseurotins, produced by a human-pathogenic fungus Aspergillus fumigatus. As toxin and medicine are two sides of the same coin, deciphering and manipulating the biosynthetic pathways of bioactive natural products will facilitate the discovery of untapped therapeutics. Keywords Biosynthesis · Fungal natural products · Enzyme · Metabolism · Drug discovery

6.1 Introduction Filamentous fungi are highly skilled synthetic chemists that exist in nature. These organisms can generate an expansive array of secondary metabolites [1]. Penicillin, the most iconic example of an antibiotic, was discovered in Penicillium chrysogenum [2]. Furthermore, lovastatin, a cholesterol-reducing agent from Aspergillus terreus Y. Tsunematsu (B) Graduate School of Bioagricultural Sciences, Nagoya University, Furo-Cho, Chikusa, Nagoya 464-8601, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_6

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[3], and cyclosporine A [4], an immunosuppressant drug from entomopathogenic Tolypocladium inflatum, have been utilized in chemotherapy. Consequently, filamentous fungi have been recognized as a valuable resource for drug discovery [5]. Conversely, certain fungi are utilized to produce fermented foods and beverages globally [6]. The green-colored edible mold Penicillium roqueforti is supplemented to create blue cheese, while Rhizopus fungi are employed in the fermentation of Tempeh, a traditional Indonesian food made from soybeans. Additionally, the red Monuscus fungi, which also produce lovastatin (monacolin K), are used in the production of Tohuyo, a traditional food in the Okinawa region of Japan. Furthermore, various Aspergillus fungi are essential for brewing alcoholic beverages such as Shochu and Awamori in Japan, which are made using potato, rice, barley, and other cereal crops as fermentation ingredients [7].

6.2 Aspirochlorine Biosynthesis One of the most significant fungi in Japanese food culture and industry is Aspergillus oryzae, also known as koji mold. A. oryzae has been used to create various fermented seasonings and beverages, such as soy sauce, miso, and sake (rice wine) [8]. Thus, biological studies of A. oryzae, including molecular genetics and cell biology, have primarily been conducted in Japan [9]. It is worth noting that A. oryzae auxotrophic mutants (e.g., strain NSAR1, adeA– , niaD– , /\argB, sC – ) developed by Japanese research groups have been utilized as versatile hosts for the heterologous expression of exogenous fungal genes [10]. This heterologous expression system can investigate the biosynthetic machinery of complex secondary metabolites and has been widely utilized worldwide [11–13]. An advantage of using A. oryzae is that it produces a limited number of secondary metabolites in small quantities under laboratory culture conditions, making it straightforward to identify specific metabolites derived from exogenous genes. Similar to other fungi, A. oryzae also possesses more than 50 biosynthetic gene clusters (BGCs) of secondary metabolite within its genome [14]. Some compounds, such as kojic acid, 13-deoxypaxilline, 2-oxocyclopiazonic acid, and diaporthin, have been identified as the metabolites produced by this fungus (Fig. 6.1) [15]. Aspirochlorine (1), a chlorinated peptidic compound, is a metabolite produced by A. oryzae and found in various species of Aspergillus fungi [16, 17]. 1 exhibits a variety of biological activities, including antifungal [18, 19], antibacterial, antiviral [20], anticancer [21], and SARS-CoV2 Mpro inhibitory properties [22]. As a molecular mechanism of action, it has been proposed that 1 inhibits protein synthesis in eukaryotic cells such as Saccharomyces cerevisiae [23]. The distinctive structural characteristic of 1 featuring a spiroaminal ring, an N-methoxyamide, and an intramolecular transannular disulfide bond is expected to be vital for its biological actions. Disulfide-containing cyclic dipeptidyl compounds are found in nature and encompass a large and structurally diverse group, epidithiodiketopiperazines (ETPs) [24]. A representative example of

6 Dissecting Biosynthesis of Natural Products Toward Drug Discovery

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Fig. 6.1 Natural products identified from koji mold, Aspergillus oryzae

ETPs is gliotoxin (2) [25], a pathogenic factor produced by Aspergillus fumigatus [26]. Over the past few decades, numerous studies have been conducted on the multifaceted biological activities of 2, all of which are likely associated with the disulfide bridge “warhead” [27]. The biogenesis of the disulfide bridge within the peptide backbone has been of interest, and it was disclosed in gliotoxin biosynthesis (Fig. 6.2). The disulfur atom derives from glutathione, which is incorporated into a diketopiperazine through sequential catalysis by P450 monooxygenase (GliC) and glutathione S-transferase (GST, GliG), resulting in the formation of 3 [28]. Subsequently, the enzymes γ-glutamyl cyclotransferase (GGCT, GliK) and dipeptidase (GliJ) catalyze the hydrolysis of two peptide bonds in the glutathione moiety of 3, leading to the formation of 4 [29, 30]. The next step involves a remarkable transformation as the carbon-sulfur bond is cleaved by C–S lyase (GliI), a pyridoxal 5’-phosphate (PLP) dependent enzyme [31]. Finally, thioredoxin-type oxidase (GliT) can oxidize the dithiol substrate (5) with flavin-adenine dinucleotide (FAD) as a cofactor to give the disulfide compound 6 [32, 33]. Due to the widespread conservation of the homologous genes GliC/G/K/J/I/T among other ETP biosynthesis gene clusters, most ETPs are biosynthesized through a comparable pathway (Fig. 6.4). While many ETPs feature a 6-membered disulfide bridge connecting the α-positions of each amino acid, 1 is unique in that it harbors a rearranged skeleton with a seven-membered disulfide and a spiroaminal ring. Its disulfide is linked from the α-position of glycine to the β-position of phenylalanine, which motivated our group to investigate how its unusual structure could be constructed in A. oryzae. The aspirochlorine biosynthesis gene cluster was discovered by Chankhamjon et al. in 2014 (Fig. 6.3) [21]. They disrupted the halogenase-encoding aclH gene in A. oryzae and identified dechloroaspirochlorine 7 as the compound that specifically

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gliotoxin (2)

Fig. 6.2 Biosynthetic pathway of gliotoxin (2)

accumulated in /\aclH (/\aclH: aclH gene deletion mutant, Fig. 6.4). The conversion of 7 to 1 was demonstrated in the presence of recombinant AclH enzyme, thereby corroborating the involvement of AclH in the biosynthesis of 1. The chemical structure of 1 initially indicated that 1 is biosynthesized from Phe and Gly. However, isotope labeling experiments verified that Gly was not incorporated into the peptide backbone, and instead, an epidithiodiketopiperazine (11) comprising two molecules of Phe emerged as an unexpected biosynthetic intermediate. This hypothesis was further substantiated by isolating 1.2 mg of 11 from 100 L of A. oryzae culture and performing a chemical complementation assay; the administration of 11 could restore the production of 1 in the /\aclP mutant, which was a null mutant of non-ribosomal peptide synthetase (NRPS) gene, aclP. These results raised a question as to how the monophenyl-type product could be generated from the biphenyl intermediate. An obstacle for determining the biosynthesis of 1 was that production titers of aspirochlorines in cultured A. oryzae were trace level (~0.01 mg/L), preventing the structure elucidation of biosynthetic intermediates in each acl-gene deletion mutant. Therefore, the author focused on the transcription factor gene aclZ within the BGC of 1. The transcription factors present within the BGC are referred to as pathwayspecific activators that enhance transcription of entire genes within the BGC [34–36]. The results of genetic engineering experiments showed that the production of 1 was completely abolished in the aclZ gene disruption strain but was 100-fold increased in the overexpression of aclZ with a starch-inducible glaA promoter [37]. Based on the strain with enhanced production of 1 (strain AOKW8), the post-spiro-formation pathway of 1 was successfully elucidated. The enzyme responsible for producing 11, the previously isolated intermediate, was identified as AclL, a cytochrome P450 monooxygenase, which catalyzes aromatic oxidation at C3 of 10 (Fig. 6.4). The subsequent transformation is conducted by another P450 AclO enzyme, which introduces oxygen at the N13 position of the amide of 11 to yield 12 bearing

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Fig. 6.3 Biosynthetic gene cluster of aspirochlorine (1). Abbreviations, ABC: ATP-binding cassette, A: adenylation domain, T: thiolation domain, C: condensation domain

the N-hydroxylamide. AclU, a methyltransferase, then catalyzes the methylation of the N-hydroxylamide structure to form 13, which is labile and quickly converts to the monophenyl-type compound 7, the precursor of 1. This conversion, including the removal of the phenyl moiety, is considered retro aldol-type fragmentation, whereby the carbon–carbon bond is cleaved to generate a benzaldehyde. Although monophenyl compounds (14–16) were isolated as shunt products from the aclL, aclO, and aclU disruptants, the biphenyl-type compounds were predominantly produced in the early stage of cultivation. Conversely, we observed that 7 was the major product, and only a trace amount of 13 was detected in /\aclH. While an enzyme might facilitate the conversion of 13 to 7, we showed that the cleavage of the C–C bond is sufficiently accelerated by the formation of N-methoxyamide in 13 (Fig. 6.5a). This model was supported by recent synthetic studies demonstrating that the N-methoxyamide can be attacked by nucleophiles [38], indicating the enhanced electrophilicity of the carbonyl carbon compared to typical amides. Upon functional analysis, it was found that enzymes AclL, O, and U could only accept biphenyl-structured substrates, while AclH catalyzed reactions only in monophenyl substrates. The distinct substrate recognition modes in these biosynthetic enzymes and nature’s convergent strategy for synthesizing 1 will explain the physiological role of secondary metabolites. Our group next identified the enzyme capable of synthesizing spiroaminal and 7membered disulfide structures in the aspirochlorine pathway. The author conducted a search for biosynthetic intermediates without a spirocycle in the structure for each acl-gene disruptant. Non-target comparative metabolomics analysis of LC-MS data (control vs. /\aclR) and subsequent rapid estimation of its structure through isotope labeling experiment, allowed us to identify a candidate compound 9 with a massto-charge ratio of 445 [M-H]− , specifically accumulated in the /\aclR mutant [39].

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13

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HO

MeO O N

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N H S

1

Fig. 6.4 Biosynthetic pathway of aspirochlorine (1)

Unfortunately, the aclR disruptant generated from the aclZ-expressed strain was unable to produce 9 for an unexplained reason. In order to elucidate the structure, 0.9 mg of 9 was purified from a 5.0 L of culture of the /\aclR strain expressing aclP, which encodes the NRPS for the production of cyclo-(Phe-Phe). The chemical structure of 9 was unexpectedly found to contain an acetoxy group at C7, a feature that has not been previously observed in any other 1-related molecules (Fig. 6.4). Bioinformatics analysis revealed that AclR belongs to the flavin-dependent thioredoxin oxidoreductase family [40] and that the representative enzyme GliT functions as a disulfide-forming enzyme in gliotoxin (2) biosynthesis and has a conserved CysX-X-Cys (CXXC) domain as a catalytic site. In this family, two cysteine residues in

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H147

FAD

Fig. 6.5 a. Proposed reaction mechanism for the production of 7 from 13, b. Proposed mechanism of the spiroaminal forming reaction, c. Crystal structure of AclR. The rounded rectangle represents the expanded view of the catalytic CXXH motif in AclR

the CXXC motif organize an intramolecular disulfide bond, facilitating the oxidative disulfide bond formation to substrates [33, 41]. However, one cysteine was replaced by a histidine residue in the AclR protein, suggesting a specialized role in the biosynthesis. We confirmed that recombinant AclR enzyme obtained as a homogeneity from E. coli could transform 9 into 10 with the sprioaminal formation. The X-ray crystal structure of AclR (PDB: 7BR0, Fig. 6.5c) was found to be similar in conformation to that of GliT, despite their disparate catalytic functions. Further mutagenesis studies revealed that Cys144 is essential for AclR catalysis, and the substitution of His147 with Ala/Cys significantly decreased enzymatic activity.

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These findings demonstrated that AclR, possessing the unique Cys-X-X-His (CXXH) motif, catalyzes an unconventional reaction involving carbon-sulfur migration and spiroaminal formation. We propose the reaction mechanism of AclR depicted in Fig. 6.5b. Initially, Cys144 residue of AclR captures the intramolecular disulfide bond of the substrate 9 to generate an intermolecular disulfide bond between enzyme and substrate, yielding a thiolate anion, which forms an intermediary thiirane upon removal of the acetoxy group (path b). The subsequent 1,2-sulfamyl migration, facilitated by electron donation from the nitrogen atom, possibly affords a seven-membered disulfide bridge, the hallmark of 1. It is also suggested that a His147ND1-initiated formation of the ortho-quinone methide may prompt 1,2-sulfamyl migration (path a). Our research has demonstrated that AclR, structurally similar to the disulfide-forming enzyme, is a specialized enzyme for creating spirocyclic structures with the deviated active site of CXXH. Similar to AclR, some enzymes with a CXXH motif can be found in genome databases, indicating that there is potential for discovering specialized new ETP compounds through genome mining approaches.

6.3 Fumagillin Biosynthesis Contrary to safety fungi such as A. oryzae, there are many fungi that cause pathogenesis in plants, insects, and animals, including humans [42]. These fungi have evolved to produce specialized metabolites and utilized them for their effective infection. Therefore, extracting biologically active substances from pathogenic fungi has been a popular approach to identify potential drug candidates, with many natural products exhibiting unique modes of action being discovered. A well-known example of this is fumagillin (17), a terpene-polyketide hybrid molecule that was initially identified in 1951 as an amoebicidal substance from A. fumigatus [43]. Due to its various attractive properties of 17 such as anti-angiogenesis [44], anti-obese [45] effects on humans, anti-malaria [46], and anti-giardia [47], several pharmaceutical companies have attempted to develop it as a novel medication over three decades [48]. The primary strategy for developing 17 was semisynthesis, a type of chemical derivatization of secondary metabolites obtained from natural sources such as masscultured microbes and plants. As the polyene dicarboxylic acid portion (polyketide portion) of 17 was readily hydrolyzed in a basic condition, the resulting secondary alcohol at C5 in the terpene portion could be chemically converted to improve the activity and pharmacokinetics/pharmacodynamics properties more suitable for the drug (Fig. 6.6). Fumagillin analogues TNP-470 [49], PPI-2458 [50], and CKD-732 [51], were investigated in a clinical trial for cancer treatment over 20 years ago. More recently, beloranib and ZGN-1061 were developed to treat obesity, diabetes, and related genetic diseases, Prader-Willi syndrome [52, 53]. Despite some clinical trials reaching phase III, fumagillin-based semisynthetic compounds have not been approved for clinical use due to safety concerns such as neurotoxic effects and vein thrombosis [52]. Currently, natural 17 (Flisint) is only approved as an orphan drug in

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France for treating intestinal microsporidiosis that is caused in HIV-infected patients and transplant recipients, despite occasional observations of hematological toxicity [54]. In addition to its therapeutic use in humans, 17 has also been utilized to control nosema infection in apiculture [44, 55, 56]. The primary target of 17 was identified as type 2 methionine aminopeptidase (MetAP2) in 1997 by two independent research groups using a similar “molecular fishing” approach [57, 58]. They synthesized biotinylated fumagillin derivatives that were capable of capturing its binding protein, MetAP2, from the cell lysates of mammalian endothelial cells. X-ray crystallography further confirmed the covalent binding of MetAP2 with 17, revealing that the electrophilic C1,2 epoxide of fumagillin was the warhead of the activity and received a nucleophilic attack from the His231 residue of MetAP2 [59]. The biosynthetic gene cluster of 17 in A. fumigatus was first reported in 2013 by Tang et al. (Fig. 6.7) [60]. They first demonstrated that polyene dicarboxylate of 17 was biosynthesized by a highly reducing polyketide synthase (HR-PKS), designated Fma-PKS (Afu8g00370), through a gene deletion experiment. They further identified a terpene cyclase Fma-TC (Afu8g00520), which was initially annotated as an integral membrane protein in the genome database. This protein catalyzes the transformation from farnesyl pyrophosphate (FPP) to β-trans-bergamotene (18), the sesquiterpene scaffold of 17 (Fig. 6.8). Fma-TC was found to be a new class of terpene cyclase with a UbiA prenyltransferase domain (PFAM01040). During this period, our research group had also independently identified the biosynthetic genes of 17. As a result, the authors initiated a collaborative effort with Tang et al. to clarify the biosynthetic pathway of 17. One particularly fascinating biocatalyst in the fumagillin pathway was Fma-P450 (Afu8g00510), a multifunctional cytochrome P450 oxygenase that transformed 18 into 19 [61]. Fma-P450 is likely to catalyze quadruple oxidation in this transformation, as depicted in Fig. 6.9a. We proposed that the initial step in the catalytic process involves the abstraction of a hydrogen atom at C5 in 18, resulting in the production of tertiary alcohol (25). Subsequent C-H activation at C9 leads to the formation of an unstable radical species, which initiates cleavage of the C-C bond between C5 and C8 due to the strain inherent in the bicyclo[3.1.1]heptane scaffold. After the hydroxyl group is attached to the C5 radical, two of the three olefins are sequentially oxidized by the same P450 enzyme to form the bisepoxide (19). To our

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Fig. 6.7 Biosynthetic genes of fumagillin-pseurotin supercluster. Abbreviations. KS: ketosynthase, AT: acyltransferase, KR: ketoreductase, DH: dehydratase, ACP: acyl carrier protein, FMO: flavindependent monooxygenase, ABM: antibiotic biosynthesis monooxygenase

knowledge, this quadruple oxidation catalyzed by a single enzyme is unprecedented, despite the recent report of an enzyme installing triple oxygen into the substrate in the biosynthesis of novofumigatonin [62]. We then focused on unraveling the later biosynthetic pathway. We generated recombinant proteins of Fma-C6H (Afu8g00480), Fma-MT (Afu8g00390), FmaKR (Afu8g00490), Fma-AT (Afu8g00380), and Fma-PKS (Afu8g00370) as homogeneity from E. coli. In vitro test using the enzymes with the substrates revealed that Fma-C6H, an α-ketoglutarate (KG)-dependent oxygenase, introduced a hydroxyl group at C6 in 19 to generate 20, which was subsequently transformed into a methylated product 21 through the catalysis of the methyltransferase Fma-MT. We next confirmed that the reduction to afford fumagillol (22) from 21 was carried out by Fma-KR, which encoded a partial PKS containing a dehydratase (DH) and a ketoreductase (KR) domains. Interestingly, Fma-KR exhibits a high degree of similarity to a polyketide synthase–non-ribosomal peptide synthetase (PKS–NRPS) hybrid

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megasynthase, PsoA (Afu8g00540) [63], which is responsible for producing pseurotin A (26), a distinct natural product. In Sect. 6.4, we will delve into the specifics of the biosynthesis genes for 17 and 26, which are located in a contiguous region on chromosome VIII of A. fumigatus, forming an intertwined supercluster [64]. It was hypothesized that Fma-KR originated as a duplicate of psoA and evolved to function in the fumagillin pathway. In addition to the terpene pathway mentioned above, Fma-PKS formed a polyene hexaketide–ACP (23) through iterative catalysis by domains of KS–AT–DH–ER–KR–ACP, which did not possess a terminal domain for releasing the products from PKS. This acyl thioester intermediate bound to ACP was subsequently transesterified at the C5 position of 22 by Fma-AT, an acyltransferase, yielding prefumagillin (24), which was identical to the compound isolated from the /\fma-ABM (Afu8g00470) mutant. It was challenging to obtain recombinant protein of Fma-ABM from E. coli, possibly due to the presence of a transmembrane domain in the N-terminus. The biotransformation from 24 to 17 was observed when Fma-ABM was expressed in S. cerevisiae, confirming that Fma-ABM was responsible for producing 17 at the final stage of biosynthesis. The reaction employed by Fma-ABM was also unprecedented in its ability to cleave an olefin with regioselectivity, yielding the carboxylic acid in 17. We proposed that Fma-ABM catalyzed multistep oxidations in Fig. 6.9b. despite further validation of the enzymatic reaction was required.

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Fig. 6.9 Unique enzymatic transformations in the fumagillin pathway. a. Proposed mechanism of the unique transformation catalyzed by Fma-P450, b. Proposed mechanism for the production of 17 by the membrane-bound oxidase Fma-ABM

6.4 Pseurotin/Synerazol Biosynthesis The discovery of fumagillin biosynthesis genes was surprising due to the close proximity of the terpene cyclase gene (Fma-TC, Afu8g00520) to the biosynthetic gene cluster of pseurotin A (26), a structurally different metabolite. Pseurotins were originally obtained from the filamentous fungus Pseuroeurotium ovalis, and the gene encoding the PKS–NRPS hybrid megasynthase responsible for their biosynthesis, PsoA (Afu8g00540), was identified in A. fumigatus through a knockout study (Fig. 6.7) [63]. The biosynthetic genes required for the production of 17 and 26 were found to be physically intertwined within a single supercluster located at a subtelomeric region of chromosome VIII in A. fumigatus, with a single transcription factor fapR acting as the positive regulator [64]. In contrast to 17, which is composed of a terpene-polyketide hybrid, the chemical scaffold of 26 was determined to be derived from malonate, propionate, and l-phenylalanine, through the feeding study of isotope-labeled precursors [65]. The structural complexity of pseurotins containing a spirocycle scaffold, prompted us to determine how 26 is biosynthesized in A. fumigatus. The PK–NRP scaffold of 26 is synthesized by two dedicated enzymes, PsoA and PsoF, using propionate, malonates, l-phenylalanine, and S-adenosylmethionine

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(SAM) as substrates (Fig. 6.11c) [66]. One of the unique characteristics of the PsoA enzyme is that it can utilize propionyl-CoA as a starter unit, as demonstrated by in vitro study of recombinant PsoA enzyme [67]. PsoA is composed of KS–AT– DH–MT0 –KR–ACP domains for its PKS module and C–A–T–R domains for its NRPS module (Fig. 6.11a). Bioinformatic analysis suggested that function of the MT0 domain in PsoA could potentially be inactivated as the SAM binding motif was not conserved in its primary sequence. Alternatively, a different protein encoded in the pso gene cluster, PsoF, possesses a C-terminus methyltransferase (MT) domain with an active catalytic motif [68]. PsoF is uniquely equipped with a catalytic domain of flavin-dependent monooxygenase (FMO) at its N-terminus, rendering it a bifunctional enzyme (as depicted in Fig. 6.11b). The PsoF-MT domain exhibits similarity to the C-methyltransferase domain of iterative type HR-PKS in fungi, rather than other N- or O-methyltransferases involved in natural products biosynthesis. We uncovered that PsoF-MT was capable of transferring a methyl group to an elongating propionyltriketide intermediate (Fig. 6.11c). This C-methylation acted as a gatekeeping step in the condensation of the polyketide and l-phenylalanine, as the absence of PsoF resulted in only compounds with polyketide structure. In vitro studies confirmed that PsoB, an α,β-hydrolase, was not essential, but it accelerated the production of the compound with a molecular weight of 353 [67], which was proposed as a plausible precursor of azaspirene (28) [69]. Although the precise function has remained enigmatic, PsoG, a membrane-bound oxidase, has been shown to be responsible for the production of 28 as de novo production of 28 was observed in yeast expressing psoA, psoF, psoB, and psoG [67]. Further investigation revealed the later stages of pseurotin biosynthesis from 28 through in vitro enzymatic analysis and in vivo biotransformation assays using a yeast expression system (Fig. 6.10) [66, 70]. A cytochrome P450, designated PsoD, constructed a benzoyl moiety through consecutive C-H activation reactions at C17 of 28. Subsequently, PsoC, a methyltransferase, was able to selectively methylate C8-OH of 29 to generate presynerazol (30), despite the presence of a secondary hydroxyl group at C9 in the substrate. 30 was only obtainable from a mutant strain of A. fumigatus lacking the gene encoding psoE, a glutathione S-transferase, indicating that 30 was a substrate of PsoE. Therefore, in order to clarify the function of PsoE, we incubated 30 and recombinant PsoE with glutathione. However, we did not observe any reaction products under the condition. In contrast, when we added recombinant PsoF and its cofactor, NADPH, to the aforementioned reaction condition, we observed a rapid transformation from 30 to synerazol (31). This tandem reaction, which included trans-to-cis isomerization and epoxidation, required an equimolar amount of GSH and NADPH, respectively. To identify a reaction intermediate of PsoE and PsoF, we successfully obtained a crystal structure of a ternary complex of PsoE-presynerazol-GSH (PDB: 5F8B, 5FHI, Fig. 6.11e) [58]. To our surprise, the sulfur atom of GSH was covalently bound to C13 of 30, forming a possible precursor (32) for the subsequent epoxidation by PsoF in the crystal structure of the PsoE protein. A proposed reaction mechanism is depicted in Fig. 6.11d. The GSH was first transferred to C13 of 30 in the catalytic pocket of PsoE to generate the intermediate (32), which was then oxidized to form sulfoxide (33) by the FAD-binding

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Fig. 6.10 Biosynthetic pathway of pseurotin/synerazol

monooxygenase, PsoF. This sulfoxide intermediate was likely unstable, leading to the production of a cis olefin (34) via syn-elimination. Finally, the resulting 10,11trans olefin of 34 was epoxidized by the same oxidase PsoF to yield 31. Initially, we proposed that the PsoF protein carried out two different reactions, including Cmethylation during polyketide elongation and epoxidation in post-PKS modification. However, our detailed analysis revealed a third role for PsoF’s catalysis as the unusual S-oxidation, resulting in cis-olefin formation. The final step in pseurotin biosynthesis involves hydrolysis of the epoxide. We confirmed that 31 was gradually converted to 26 and 27 in an aqueous solution, suggesting that pseurotins may be generated spontaneously from 31 under culture conditions. Through the investigation of pseurotin biosynthesis, we were able to obtain over 20 new congeners of pseurotins from genetic mutants with altered biosynthesis genes. As pseurotin/synerazol has exhibited various biological activities, it is possible that these related compounds may also show attractive biological activities. 31 was originally reported to act as an antifungal agent, inhibiting the growth of Candida albicans and Cryptococcus neoformans at a minimum inhibitory concentration (MIC) of 12.5 μg/mL, although it did not demonstrate efficacy in curing C. albicansinfected mice at a dose of 200 mg/kg [71]. Igarashi et al. reported that 31 possesses cytocidal activity against mammalian cells, and its fluorinated congeners, 19- or 20-fluorosynerazoles, which were produced through directed biosynthesis with the administration of fluorinated phenylalanine in the growth medium, are capable of inhibiting angiogenesis [72]. Conversely, in the epoxide-opening form of 31, 26 was reported to act as an inhibitor of immunoglobulin E production, which plays a role in the progression of chronic inflammatory disorders such as asthma [73]. However, the molecular target(s) and detailed mode-of-action of synerazol/pseurotins remain elusive. Asami et al. reported that 28 inhibited the Raf1-activation of the MAPK

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Fig. 6.11 Domain components of PsoA (a.) and PsoF (b.). c. Proposed biosynthetic pathway to form the oxaspirocycle in pseurotin/synerazol. d. Proposed mechanism to produce 31 from 30. e. Crystal structure of PsoE–32 complex. The rounded rectangle represents the expanded view of the ligand (32) in PsoE. Abbreviations, MT0 : inactive methyltransferase, R: reductase

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pathway in VEGF-treated HUVEC cells [74]. As synerazol-class molecules show efficacy against a wide range of eukaryotic cells, understanding their mechanism of action, including their direct binding partners, is a compelling focus of chemical biology research.

6.5 Closing Remarks This chapter summarized our recent investigations on the biosynthesis of natural products from filamentous fungi. The biosynthesis of natural products is not sorely composed of simple transformation reactions such as methylation, but also involves a wealth of drastic and unpredictable transformations. Identifying such intriguing enzymatic conversion through bioinformatic analysis remains challenging even in the post-genomic era. In other words, it is necessary to carry out detailed studies using wet experiments such as gene disruption and in vitro assay using recombinant proteins based on the decoded genome information. There are still numerous exciting enzymes concealed in nature. Recently, A. oryzae has been developed as a host for gene expression in filamentous fungi, which has greatly accelerated research on natural product biosynthesis. This technology has significant potential as a platform for the creation of novel materials, as it is capable of biosynthesizing a vast amount of rare and valuable natural products and their derivatives. The author hopes that this technology will facilitate the development of compounds that can contribute to industrialization and enhance human health, rather than merely determining the chemical pathway of natural product biosynthesis. Acknowledgements I am deeply grateful to Professor Kenji Watanabe of the University of Shizuoka for his invaluable support in facilitating the research endeavors in his laboratory. I would also like to express my appreciation to Professor Christian Hertweck of the Leibniz Institute for Natural Product Research and Infection Biology-Hans Knöll Institute (HKI) for their guidance and insightful suggestions. Dr. Tsuyoshi Yamamoto, Ms. Manami Fukutomi, and Mr. Naoya Maeda of Watanabe’s laboratory alumni significantly contributed to the above research and were greatly acknowledged for their efforts.

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45. Lijnen, H.R., Frederix, L., Van Hoef, B.: Fumagillin reduces adipose tissue formation in murine models of nutritionally induced obesity. Obesity (Silver Spring) 18, 2241–2246 (2010) 46. Chen, X., Xie, S., Bhat, S., Kumar, N., Shapiro, T.A., Liu, J.O.: Fumagillin and fumarranol interact with P. falciparum methionine aminopeptidase 2 and inhibit malaria parasite growth in vitro and in vivo. Chem. Biol. 16, 193–202 (2009) 47. Padia, J., Kulakova, L., Galkin, A., Herzberg, O.: Discovery and preclinical development of antigiardiasis fumagillol derivatives. Antimicrob. Agents Chemother. 64, e00582-e620 (2020) 48. Kornienko, A., Evidente, A., Vurro, M., Mathieu, V., Cimmino, A., Evidente, M., van Otterlo, W.A., Dasari, R., Lefranc, F., Kiss, R.: Toward a cancer drug of fungal origin. Med. Res. Rev. 35, 937–967 (2015) 49. Bhargava, P., Marshall, J.L., Rizvi, N., Dahut, W., Yoe, J., Figuera, M., Phipps, K., Ong, V.S., Kato, A., Hawkins, M.J.: A phase I and pharmacokinetic study of TNP-470 administered weekly to patients with advanced cancer. Clin. Cancer Res. 5, 1989–1995 (1999) 50. Arico-Muendel, C.C., Benjamin, D.R., Caiazzo, T.M., Centrella, P.A., Contonio, B.D., Cook, C.M., Doyle, E.G., Hannig, G., Labenski, M.T., Searle, L.L., et al.: Carbamate analogues of fumagillin as potent, targeted inhibitors of methionine aminopeptidase-2. J. Med. Chem. 52, 8047–8056 (2009) 51. Shin, S.J., Jeung, H.C., Ahn, J.B., Rha, S.Y., Roh, J.K., Park, K.S., Kim, D.H., Kim, C., Chung, H.C.A: Phase I pharmacokinetic and pharmacodynamic study of CKD-732, an antiangiogenic agent, in patients with refractory solid cancer. Invest. New Drugs 28, 650–658 (2010) 52. McCandless, S.E., Yanovski, J.A., Miller, J., Fu, C., Bird, L.M., Salehi, P., Chan, C.L., Stafford, D., Abuzzahab, M.J., Viskochil, D., et al.: Effects of MetAP2 inhibition on hyperphagia and body weight in prader-willi syndrome: a randomized, double-blind, placebo-controlled trial. Diabetes Obes. Metab. 19, 1751–1761 (2017) 53. Wentworth, J.M., Colman, P.G., Zafgen Study, G.: The methionine aminopeptidase 2 inhibitor ZGN-1061 improves glucose control and weight in overweight and obese individuals with type 2 diabetes: a randomized, placebo-controlled trial. Diabetes Obes. Metab. 22, 1215–1219 (2020) 54. Maillard, A., Scemla, A., Laffy, B., Mahloul, N., Molina, J.M.: Safety and efficacy of fumagillin for the treatment of intestinal microsporidiosis. A French prospective cohort study. J. Antimicrob. Chemother. 76, 487–494 (2021) 55. van den Heever, J.P., Thompson, T.S., Curtis, J.M., Pernal, S.F.: Stability of dicyclohexylamine and fumagillin in honey. Food Chem. 179, 152–158 (2015) 56. Higes, M., Nozal, M.J., Alvaro, A., Barrios, L., Meana, A., Martin-Hernandez, R., Bernal, J.L., Bernal, J.: The stability and effectiveness of fumagillin in controlling Nosema ceranae (microsporidia) infection in honey bees (Apis mellifera) under laboratory and field conditions. Apidologie 42, 364–377 (2011) 57. Sin, N., Meng, L., Wang, M.Q., Wen, J.J., Bornmann, W.G., Crews, C.M.: The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, MetAP-2. Proc. Natl. Acad. Sci. U.S.A. 94, 6099–6103 (1997) 58. Griffith, E.C., Su, Z., Turk, B.E., Chen, S., Chang, Y.H., Wu, Z., Biemann, K., Liu, J.O.: Methionine aminopeptidase (type 2) is the common target for angiogenesis inhibitors AGM1470 and ovalicin. Chem Biol. 4, 461–471 (1997) 59. Liu, S., Widom, J., Kemp, C.W., Crews, C.M., Clardy, J.: Structure of human methionine aminopeptidase-2 complexed with fumagillin. Science 282, 1324–1327 (1998) 60. Lin, H.C., Chooi, Y.H., Dhingra, S., Xu, W., Calvo, A.M., Tang, Y.: The fumagillin biosynthetic gene cluster in Aspergillus fumigatus encodes a cryptic terpene cyclase involved in the formation of β-trans-Bergamotene. J. Am. Chem. Soc. 135, 4616–4619 (2013) 61. Lin, H.C., Tsunematsu, Y., Dhingra, S., Xu, W., Fukutomi, M., Chooi, Y.H., Cane, D.E., Calvo, A.M., Watanabe, K., Tang, Y.: Generation of complexity in fungal terpene biosynthesis: discovery of a multifunctional cytochrome P450 in the fumagillin pathway. J. Am. Chem. Soc. 136, 4426–4436 (2014) 62. Mori, T., Zhai, R., Ushimaru, R., Matsuda, Y., Abe, I.: Molecular insights into the endoperoxide formation by Fe(II)/α-kg-dependent oxygenase NvfI. Nat. Commun. 12, 4417 (2021)

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63. Maiya, S., Grundmann, A., Li, X., Li, S.M., Turner, G.: Identification of a hybrid PKS/NRPS required for pseurotin a biosynthesis in the human pathogen Aspergillus fumigatus. ChemBioChem 8, 1736–1743 (2007) 64. Wiemann, P., Guo, C.J., Palmer, J.M., Sekonyela, R., Wang, C.C., Keller, N.P.: Prototype of an intertwined secondary-metabolite supercluster. Proc. Natl. Acad. Sci. U.S.A. 110, 17065– 17070 (2013) 65. Mohr, P., Tamm, C.: Biosynthesis of pseurotin-A. Tetrahedron 37, 201–212 (1981) 66. Tsunematsu, Y., Fukutomi, M., Saruwatari, T., Noguchi, H., Hotta, K., Tang, Y., Watanabe, K.: Elucidation of pseurotin biosynthetic pathway points to trans-acting C-methyltransferase: generation of chemical diversity. Angew. Chem. Int. Ed. 53, 8475–8479 (2014) 67. Zou, Y., Xu, W., Tsunematsu, Y., Tang, M., Watanabe, K., Tang, Y.: Methylation-dependent Acyl transfer between polyketide synthase and nonribosomal peptide synthetase modules in fungal natural product biosynthesis. Org. Lett. 16, 6390–6393 (2014) 68. Kishimoto, S., Tsunematsu, Y., Matsushita, T., Hara, K., Hashimoto, H., Tang, Y., Watanabe, K.: Functional and structural analyses of trans C-methyltransferase in fungal polyketide biosynthesis. Biochem. 58, 3933–3937 (2019) 69. Asami, Y., Kakeya, H., Onose, R., Yoshida, A., Matsuzaki, H., Osada, H.: Azaspirene: a novel angiogenesis inhibitor containing a 1-Oxa-7-azaspiro[4.4]non-2-ene-4,6-dione skeleton produced by the fungus Neosartorya sp. Org. Lett. 4, 2845–2848 (2002) 70. Yamamoto, T., Tsunematsu, Y., Hara, K., Suzuki, T., Kishimoto, S., Kawagishi, H., Noguchi, H., Hashimoto, H., Tang, Y., Hotta, K., Watanabe, K.: Oxidative trans to cis isomerization of olefins in polyketide biosynthesis. Angew. Chem. Int. Ed. 55, 6207–6210 (2016) 71. Ando, O., Satake, H., Nakajima, M., Sato, A., Nakamura, T., Kinoshita, T., Furuya, K., Haneishi, T.: Synerazol, a new antifungal antibiotic. J. Antibiot. (Tokyo) 44, 382–389 (1991) 72. Igarashi, Y., Yabuta, Y., Sekine, A., Fujii, K., Harada, K., Oikawa, T., Sato, M., Furumai, T., Oki, T.: Directed biosynthesis of fluorinated pseurotin a, synerazol and gliotoxin. J. Antibiot. (Tokyo) 57, 748–754 (2004) 73. Ishikawa, M., Ninomiya, T., Akabane, H., Kushida, N., Tsujiuchi, G., Ohyama, M., Gomi, S., Shito, K., Murata, T.: Pseurotin A and its analogues as inhibitors of immunoglobulin e production. Bioorg Med. Chem. Lett. 19, 1457–1460 (2009) 74. Asami, Y., Kakeya, H., Komi, Y., Kojima, S., Nishikawa, K., Beebe, K., Neckers, L., Osada, H.: Azaspirene, a fungal product, inhibits angiogenesis by blocking Raf-1 activation. Cancer Sci. 99, 1853–1858 (2008)

Chapter 7

A New Trend in Biosynthetic Studies of Natural Products: The Bridge Between the Amino Acid Sequence Data and the Chemical Structure Atsushi Minami Abstract Significant advances in analyzing the genomic information of natural product (NP)-producing microorganisms have triggered a paradigm shift in the field of NP biosynthesis. Specifically, the accumulation of sequence data of NP biosynthetic genes might provide opportunities for understanding long-standing and intrinsic questions about the chemical space of NPs, such as whether known NPs adequately cover the potential NPs that microorganisms can synthesize and whether predicting the chemical structure of NPs from the genomic information is possible. This chapter covers new trends in biosynthetic studies of fungal polyketides (PKs) based on the phylogenetic analysis of iterative polyketide synthases (iPKSs) and describes the chemical space of fungal PKs. A unified stereochemical rule contributing to the structural prediction and structure determination of fungal reduced PKs is also discussed. Keywords Polyketide · Polyketide synthase · Chemical space · Stereochemical rule · Phylogenetic analysis

7.1 Introduction Natural products (NPs) are promising molecules for developing lead compounds for medicinal use because they are compounds structurally “optimized” during the evolution of organisms for selective interaction with specific targets within the cell. In fact, 62% of the small molecules approved by the Food and Drug Administration (FDA) between 1981 and 2019 are NP-related compounds (e.g., unaltered natural products, botanical drugs, natural product derivatives, and mimics of natural products) [1]. The target-specific interaction mainly depends on their characteristic chemical structures. Specifically, NPs typically have multiple stereogenic centers and hydrogen bonding donor/acceptor sites on their diverse ring scaffold [2, 3]. Those chemical A. Minami (B) Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_7

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features have been classified using several computational tools [4–6]. As a result, it was shown that the chemical space of NPs is wider than that of combinatorial compounds and covers almost the same area as that of drugs [2]. With these results in mind, attempts to synthesize pseudo-NPs have been actively examined in the field of synthetic chemistry [7, 8]. These are good examples of how understanding and exploring the chemical space of NPs and pseudo-NPs are central topics in the field of natural product chemistry. From a biosynthetic point of view, we have now entered an era in which we engage with big genomic data. Linking biosynthetic genes, which are blueprints for synthesizing NPs, to chemical structures is a hot-topic in the field of biosynthesis because it provides direct evidence for understanding the chemical space and provides opportunities for isolating novel NPs [9–13]. This trend is accelerated by the development of gene prediction tools such as anti-SMASH [14] and SMURF [15] and the establishment of a reliable heterologous expression system [16–20]. Most recently, machine learning has also been applied to genome mining approaches [21]. This chapter describes our approaches using phylogenetic analysis to elucidate the chemical space of fungal polyketides (PKs). A unified stereochemical rule contributing to structural prediction and structure determination of fungal reduced PKs is also discussed.

7.2 Fungal Polyketide Synthases 7.2.1 Classification of Fungal Polyketide Synthases Fungi produce a large repertoire of structurally diverse polyketides (PKs), including lovastatin (1) (characteristic structural motif: the decalin moiety), tenellin (2) (the pyridone moiety), radicicol (3) (the macrolactone ring with an aromatic moiety), brefeldin A (4) (the macrolactone ring), griseofulvin (5) (the spirocyclic moiety), and T-toxin (6) (the linear structure) (Fig. 7.1). Their polyketide backbones are synthesized by single modular iterative polyketide synthases (iPKSs), which can be divided into two groups based on the domain organization: highly-reducing PKS (HR-PKS) and non-reducing PKS (NR-PKS) [22–24]. HR-PKS is basically composed of βketoacyl synthase (KS), malonyl-CoA:ACP transacylase (AT), dehydratase (DH), methyltransferase (MT), active/inactive enoylreductase (ER/ER0 ), ketoreductase (KR), and acyl carrier protein (ACP) domains. In some cases, HR-PKS also makes a fusion protein with a non-ribosomal peptide synthetase (NRPS) to give a PKS-NRPS hybrid (hybrid-PKS). On the other hand, NR-PKS lacks β-keto processing domains (KR, DH, and ER) but has unique domains, including the starter unit: ACP transacylase (SAT), product template (PT), and thioesterase (TE) domains. The function of the NR-PKSs that synthesize aromatic PKs has been extensively reviewed elsewhere [24]. This chapter focuses on HR-PKSs and hybrid-PKSs.

7 A New Trend in Biosynthetic Studies of Natural Products: The Bridge … O

HO O

O

HO

OH O

OH O

O O

H

O MeO

Cl

O

radicicol (3)

tenellin (2) Cl

O

O

HO

N O OH

lovastatin (1) HO H

125

OH OH O

MeO O

O

OH O

O H

O

O

OH O

O

OH O

OMe O

HO

brefeldin A (4)

griseofulvin (5)

T-toxin (6)

Fig. 7.1 Chemical structures of fungal PKs. The characteristic structural motif is highlighted by grey color

7.2.2 General Polyketide Chain Elongation Mechanism Scheme 7.1 summarizes the chain elongation processes catalyzed by HR-PKS [22, 23, 25]. KS catalyzes a decarboxylative Claisen condensation of two units of malonyl thioesters to afford β-ketothioester IntA, which undergoes methylation at the αposition by the action of MT to afford IntB. Subsequently, IntB is reduced by KR to give IntC. Syn-elimination by DH results in the formation of IntD, which is then subjected to an enoylreduction by ER to furnish IntE. It should be noted that all intermediates make a covalent bond with an ACP domain of HR-PKS, thereby requiring a chain-releasing reaction to afford the corresponding PK. This reaction is, in most cases, catalyzed by an additional enzyme. The well-established chain-releasing reactions are as follows: (1) an acyltransferase-catalyzing PK transfer (biosynthesis: 1) [26] (2) a thioesterase-catalyzing macrolactonization (4) [27], and hydrolysis (1) [28], (3) a SAT-catalyzing PK transfer (monocillin II) [29], (4) a condensation (C) domain-catalyzing PK transfer (thermolide A) [30]. Recently, our group reported a fifth mechanism that a C domain of the truncated NRPS-like enzyme composed of C and adenylation (A) domains catalyzes a hydrolysis reaction to afford the corresponding carboxylic acid during the biosynthetic studies of phialotide A and phomenoic acid (discussed in Sect. 7.4) [25]. We also reported that HR-PKS in the biosynthesis of betaenone has a built-in reduction (R) domain that catalyzes a reduction of the PKS-tethered thioester intermediate to afford the corresponding aldehyde [31]. This built-in system resembles that in hybrid-PKSs, which affords a hybrid-PK possessing a PK chain and an amino acid moiety. A non-enzymatic cleavage accompanying pyrone formation has also been reported in HR-PKSs for the biosyntheses of solanapyrone [32] and alternapyrone [33]. In 2021, the first structural analysis of HR-PKS, LovB, was reported [34]. The overall architecture is basically the same as that of vertebrate fatty acid synthases [35]. The inactive ER0 domain of LovB lacks the space for accommodating a PK

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Scheme 7.1 Polyketide chain elongation process catalyzed by HR-PKS. Dotted lines show the optional pathways in the biosynthesis of fungal reduced PKs. In the O R/O S model, a carboxy terminal chain (RC ) has higher priority than a methyl terminal chain (RM ). The configuration is described by a capital letter and the domain used to install the substituent is described by a subscript. Carbons with a substituent installed by MT, KR, and ER domains are labeled with blue, red, and green circles, respectively

chain and NADP+ . The function of the ER0 domain is complemented by a trans-acting ER (trans-ER), LovC [36, 37], which interacts with the AT domain of LovB in the LovBC complex to catalyze the enoylreduction. Mutational experiments indicated the importance of the interaction in maintaining the integrity of the catalytic chamber. Although the complex lacks structural information about C-terminal domains, ACP, and C domains, this pioneering work would provide opportunities for understanding the molecular dynamics closely related to the reaction mechanism.

7.3 Phylogenetic Analysis of Hybrid-PKSs to Predict the Chemical Space of Hybrid-PKs A phylogenetic analysis using amino acid sequences provides opportunities for understanding evolutionary implications of the proteins. When focusing on polyketide synthases (PKSs), it has been used to predict sequential diversity [38, 39], interkingdom gene transfer [40], and evolutionary processes of secondary metabolites [41]. Because biosynthetic enzymes with different amino acid sequences catalyze different chemical reactions, however, the amino acid sequence data should inherently include information about the function. In other words, phylogenetic analysis can help understand the functional diversity of the biosynthetic enzymes, which leads to understanding the chemical diversity of natural products (NPs). This section will discuss our phylogenetic analysis of fungal PKS-NRPS hybrids (hybrid-PKSs) to predict the chemical space of the resultant hybrid-polyketides (hybrid-PKs) [42]. The phylogenetic tree of 884 numbers of hybrid-PKSs using amino acid sequences of the PKS region shows the phylogenetic signal with respect to the structural diversity of the hybrid-PKs (Fig. 7.2) [42]. Of particular importance is that the phylogenetic

7 A New Trend in Biosynthetic Studies of Natural Products: The Bridge … Phylogenetic tree of hybrid-PKSs

Major clade

FAS, NR-PKS

Intermediate

!

Ia (313)

24

II (128)

25

Ib (189)

24

Ib

III (254) III

!

Representative hybrid PK

Predicted NP clade

!

Ia-A

pyranonigrin A (79)

Ia-B, -C

cyclopiazonic acid etc. (31)

D-1: tenellin (52) Ia-D-1, -2 D-2: leporin A (26) Ia-D-3 D-3: illicicolin H (13)

Ia

II

NP clade

127

II-A

fusarin C (36)

II-B, -C II-D

pseurotin A etc. (45)

Ib-A

burnettramic acid A (24)

Ib-B, -C Ib-D

B: Sch210972 (20) C: equisetin (50) D: fusaridione (5)

25

III-A

chaetoglobosin A (19)

tetronic acid

III-B

acyltetronic acid (13)

III-C

oxaleimide A (40)

III-D

cytochalasin K (36)

! IaIaIaIaIa-

(16) (17) (14) (10) (20)

II- (11) II- (18) II- (18)

Ib- (25) Ib- (6) Ib- (16)

Not examined

25

Fig. 7.2 Phylogenetic analysis of hybrid-PKSs. This phylogeny revealed four major clades, Ia, Ib, II, and III, in which hybrid-PKSs synthesize either a tetramic acid (24) (highlighted by yellow color), a pyrrolinone (25) (highlighted by blue color), or a tetronic acid (highlighted by grey color) as biosynthetic intermediate. Each major clade includes the NP clades, in which hybrid-PKSs synthesize known hybrid-PKs. This figure also showed predicted NP clades, which might synthesize hybrid-PKs with a novel structural motif. The number of each major/NP clades is described in the parenthesis

cladogram has a high correlation with the characteristic structural motif of the resultant hybrid-PKs. In other words, hybrid-PKSs classified into the same group give a PK backbone possessing the essential substituents, which undergo further modification reactions to synthesize the characteristic structural motif commonly found in the product hybrid-PKs. This is further supported by the fact that the hybrid-PKS genes classified into the same group accompany the same modification enzyme genes. We named the set of hybrid-PKSs to synthesize the same structural motif as NP clade. At present, our group identified 18 NP clades as follows (Figs. 7.3 and 7.4): [42] Ia-A (the number of hybrid-PKSs in this group; 79)::pyranonigrin A (7) [43], Ia-B (9)::xyrrolin (8) [44], Ia-C (22)::cyclopiazonic acid (9) [45], Ia-D-1 (52)::tenellin (2) [46, 47], Ia-D-2 (26)::leporin A (10) [48], Ia-D-3 (13)::illicicolin H (11) [49], Ib-A (24)::burnettramic acid A (12) [50], Ib-B (20)::Sch210972 (13) [51], Ib-C (50)::equisetin (14) [52], Ib-D (5)::fusaridione (15) [52], II-A (36)::fusarin C (16) [53], II-B (10)::himeic acid A (17) [54], II-C (12)::flavipucine (18) [55], II-D (23)::pseurotin A (19) [56], III-A (19)::chaetoglobosin A (20) [57], III-B (13)::acyltetronic acid (21) [58], III-C (40)::oxaleimide A (22) [59], and III-D (36)::cytochalasin K (23) [60, 61]. Key modification enzymes to synthesize the characteristic structural motif of each hybrid-PK are summarized in Table 7.1. These enzymes are used for the Level II classification of hybrid-PKSs discussed later. We also found that the borders of each NP clade were found to be almost identical to the apparent borders of the phylogenetic tree. This finding allowed us to identify 11

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Fig. 7.3 Chemical structures of representative PKs in clades Ia and Ib derived from a tetramic acid intermediate (highlighted by yellow color). The 2-pyridone moiety found in 2, 10, and 11 is constructed by oxidative ring expansion of the tetramic acid intermediate. Therefore, it is also highlighted by yellow color. The BGC name of each hybrid-PK is shown in the parenthesis. Asterisk indicates tentative BGC name for xyrrolin (xyr)

predicted NP clades; 171 hybrid-PKSs are classified into these groups. The hybridPKS gene in these predicted NP clades accompany uncharacterized modification enzyme genes, thus preventing us to identify the corresponding PKs at this stage. Of particular note is that there are small clades in the phylogeny that were not classified at this stage. One interpretation of these small groups is that they are currently growing PKSs. Overall, the phylogenetic analysis-based approach revealed that we covered less than 62% (18 out of 29) of the chemical space of hybrid-PKs. In other words, we can obtain novel hybrid-PKs if we conduct genome mining focusing on the biosynthetic genes classified into 11 predicted NP clades. This phylogeny-based analysis also revealed an early-stage structural diversification mechanism in nature (Scheme 7.2). The key biosynthetic intermediates are a tetramic acid (24) and a pyrrolinone (25), both of which are synthesized from a non-ribosomal peptide synthetase (NRPS)-tethered β-ketoamide intermediate (26)

7 A New Trend in Biosynthetic Studies of Natural Products: The Bridge …

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Fig. 7.4 Chemical structures of PKs in clades II and III derived from a pyrrolinone intermediate (highlighted by blue color). Himeic acid and flavipucine undergo oxidative ring cleavage of the pyrrolinone moiety, thereby preventing us to highlight it. Exceptional is acyltetronic acid, which is synthesized from a tetronic acid (highlighted by grey color). The BGC name of each hybridPK is shown in the parenthesis. Asterisk indicates tentative BGC names for flavipucine (fla) and chaetoglobosin (chgg)

composed of a PK chain and an amino acid. The former is synthesized by the action of a Dieckmann cyclization domain [62], while the latter is synthesized by a coupling reaction of a reductase domain of the hybrid-PKS and a stand-alone α, β-hydrolase via a hypothetical aldehyde (27) [53, 61, 63]. The α, β-hydrolase was named putative “Knoevenagelase (pKN)” [42]. These two pathways are easily distinguished by a modification enzyme search focusing on the pKN gene. The products 25 and 26 undergo further modifications leading to hybrid-PKs. Taken together, our group proposed hierarchical classification of hybrid-PKSs. Level I classification focusing on the pKN gene allowed us to identify three major clades: I, II, and III (Fig. 7.2 and Table 7.1). Clade I is composed of hybrid-PKSs in which biosynthetic gene clusters (BGCs) lack a pKN gene. Therefore, the hybridPKSs in this clade synthesize 24 as a common biosynthetic intermediate. This clade apparently separated into two distinct subclades, Ia and Ib, in Fig. 7.2. In contrast,

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Table 7.1 Key modification enzymes for the Levels I and II classifications. The function of each modification enzyme is summarized in ref [42] clade

Ia-A

Knoevenagelase (Level I)

−

Key modification enzymes (Level lI) Representative hybrid NP clade

Ib-A

Knoevenagelase (Level I)

−

Key modification enzymes (Level lI) Representative hybrid NP

Ia-C

Ia-D

FMO

reductase

PT

expandase (P450)

P450 (Ia)

−

−

7

8

9

2

Ib-B

Ib-C

Ib-D

III-B

P450 (Ib)

aldolase

DAase (type II)

−

P450 (III)

−

transaminase

−

−

α-KG

12

13

14

15

21

clade

II-A

Knoevenagelase (Level I)

pKN

II-B

II-C

II-D

III-A

III-C

III-D

P450 (IIB)

monooxygenase

P450 (IID)

DAase (type IIβ)

DAase (type IIβ)

DAase (type IIβ)

P450 (IIA)

−

−

−

−

−

−

16

17

18

19

20

22

23

Key modification MT enzymes (Level lI)

Representative hybrid NP

Ia-B

hybrid-PKSs in clade II accompany a pKN gene for synthesizing 25. Hybrid-PKSs in clade III are unique in that relatively small BGC groups possessing or lacking the pKN gene are mixed, making it difficult to identify uncharacterized NP clades. These major clades include the NP clades described above, which can be identified by the modification enzyme search focusing on the key modification enzymes summarized in Table 7.1 (Level II classification). This hierarchical classification allows us to predict the structural motif of the hybrid-PKs synthesized by a target hybrid-PKS (Scheme 7.3). Functional analysis of the key modification enzymes would be essential to identify unexplored NP clades and to improve the accuracy of prediction.

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Scheme 7.2 Two alternative pathways leading to key biosynthetic intermediates, 24 and 25, of hybrid-PKs. The tetramic acid and pyrrolinone core is highlighted by yellow and blue color, respectively

7.4 Focused Functional Analysis of HR-PKSs Using the Aspergillus Oryzae Expression System As in the case of PKS-NRPS hybrids (hybrid-PKSs), a phylogenetic analysis of highly-reducing PKSs (HR-PKSs) also revealed the relationship between the phylogeny and the characteristic structure [25]. Specifically, HR-PKSs involved in the biosynthesis of maleidride anhydrides (Fig. 7.5), such as zopfiellin (28) [64, 65], phomoidride B (29) [66, 67], byssochlamic acid (30) [68], were classified into a single clade. During the analysis, our group also found a clade including an HR-PKS, PhomA [25, 69], for the biosynthesis of phomenoic acid (31). We named this clade as PMA clade (phomenoic acid). 31 is a unique PK possessing multiple hydroxyand methyl groups on a single polyketide chain. Interestingly, this clade includes the uncharacterized HR-PKS of PhiaA from Pseudophialophora sp. BF-0158, which is a known producer of polyhydroxy PK, phialotide A (32) [70]. PKSs from the producers of known polyhydroxy PKs such as cubensic acid (33) (strain; Xylaria cubensis) [71] and TMC-171C (34) (Clonostachys rosea) [72] are also included in this PMA clade. This circumstantial evidence suggests that PKSs in this clade participate in synthesizing the backbone structure of polyhydroxy PKs. A focused analysis of PKSs in the PMA clade was conducted through a heterologous expression study using Aspergillus oryzae (AO) [17, 73, 74]. A bioinformatics analysis of modification enzyme genes located adjacent to the HR-PKS gene in the PMA clade revealed that a trans-enoylreductase (ER) (PhiaB or PhomB) gene and a truncated-nonribosomal peptide synthetase (NRPS) (PhiaC or PhomC) gene were highly conserved in their biosynthetic gene clusters (BGCs), strongly suggesting

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Scheme 7.3 Schematic view of the proposed hierarchical classification. The key intermediates, 24 and 25, in early-stage biosynthesis are identified from the modification enzyme search focusing on pKN (Level I classification). Subsequent Level II classification focusing on other modification enzyme genes (Table 7.1) provides information about the structural motif (highlighted by grey color) of the resultant hybrid-PK. In this scheme, examples for identifying clades Ib-C and III-D are summarized

that they are essential genes for the biosynthesis. Therefore, these conserved biosynthetic genes were co-expressed with the corresponding HR-PKS gene. A transformant AO-phiaABC produced the PK backbone 35 of 32, while a transformant AOphiaAB did not (Scheme 7.4), suggesting that all three genes are responsible for the production of 35. In contrast, AO-phiaAC afforded a non-reduced product 36 of 35. The production of 36 showed that the trans-ER PhiaB catalyzes a reduction of the double bond on the PKS-tethered intermediate. The function of PhiaC was examined by a mutational analysis of the highly conserved HHxxxDG motif [75] of the condensation (C) domain. Given that the transformants possessing a PhiaC mutant, AHxxxDG or AAxxxDG, lost the production of 35, as in the case of AO-phiaAB, it was proposed that the C domain mediates a hydrolysis reaction instead of a canonical amide-bond formation. Similarly, the production of phomenoic acid lactone 37 was demonstrated by the heterologous expression of PhomA, PhomB (trans-ER),

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Fig. 7.5 Chemical structures of (A). fungal maleidride anhydrides and (B). polyhydroxy polyketides. Carbons with a substituent installed by MT, KR, and ER domains are labeled with blue, red, and green circles, respectively. This labeling allows us to understand the apparent absolute configuration of the stereogenic center installed by the each domain. The same labeling is also applied in Figs. 7.6–7.9 and Scheme 7.4

and PhomC (truncated-NRPS) (Fig. 7.6). Interestingly, the PMA clade also includes an HR-PKS, ACRTS2 [76], involved in the biosynthesis of ACR-toxin (38). Unlike other PKSs in the PMA clade, the BGC for synthesizing 38 was not deposited in the public database, preventing us from searching for the modification enzyme genes. However, the successful production of 39, a decarboxylation product of 38 [77, 78], in AO-ACRTS2, showed that a truncated-NRPS is not essential in the biosynthesis of 38, possibly due to an intramolecular non-enzymatic cyclization (Fig. 7.5), as in the case of shimalactone biosynthesis [79]. The above results demonstrated that heterologous expression in A. oryzae is a powerful method for characterizing the function of multifunctional enzymes, and suggested that PKSs in the PMA clade participate in the biosynthesis of polyhydroxy PKs. Interestingly, this PMA clade is located adjacent to a clade, including HR-PKSs for the synthesis of macrolactam PKs such as thermolides. This clade is named TML clade (thermolide). These PKs are composed of a polyhydroxy PK moiety and amino acids, synthesized by the collaborative action of two multifunctional enzymes, HR-PKS and NRPS. Unlike PhiaC and PhomC, these NRPSs are composed of four domains: condensation (C), adenylation (A), thiolation (T), and terminal

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acetyl-CoA malonyl-CoA S-adenosylmethionine

PK construction

PhiaA MT KR DH + PhiaB

ACP S O

ACP

MT KR

S O

ACP

MT KR DH

ACP

S

S

O

O

HO

HO

OH2

HO

HO

PhiaA: HR-PKS

OH

OH

OH

O

PhiaC OH

PhiaB: t-ER PhiaC: truncatedNRPS

OH

hydrolysis

35 OH

OH

OH

OH

O OH

No enoylreduction

HO

HO

36

Scheme 7.4 Proposed biosynthetic pathway leading to 35 and 36

Fig. 7.6 a. Chemical structures of 37 and 39. b. Proposed degradation pathway from 38 to 39

condensation-like (CT ) domains. A detailed functional analysis of the NRPSs, especially focusing on the functional differences of the C domain, will provide opportunities for elucidating an evolutionary relationship between truncated-NRPSs and complete-NRPSs.

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7.5 Unified Stereochemical Rule of Fungal PKSs 7.5.1 Stereochemical Course to Synthesize the Backbone Structure of Polyhydroxy PKs Classified into PMA Clades Polyketide synthases (PKSs) in the PMA clade produce polyhydroxy polyketides (PKs) with multiple stereogenic centers installed by the action of a PKS and a trans-enoylreductase (ER), providing opportunities for discussing the stereochemical course during PK elongation. For this purpose, the absolute configurations of 35 and 37 were determined by chemical degradation followed by comparison of the degradation products with synthetic standards [25]. The absolute configuration of 38 was reported in the previous paper [80, 81]. Based on the results, the stereochemical course leading to 35, 37, and 38 was firmly established. To simplify discussions about the stereochemical course, our group proposed the O R/O S model (Scheme 7.1) [25]. In this model, the absolute configuration installed by the action of a highly-reducing PKSs (HR-PKSs) and a trans-ER was discussed, focusing on the direction of the PK chain elongation without considering the absolute configurations of other substituents located nearby. Briefly, following the formation of IntA, a methyltransferase (MT) domain installs an O RMT -methyl group of IntB. Subsequently, a ketoreductase (KR) domain reduces the β-keto group to afford an O RKR -hydroxy group. The resultant IntC undergoes syn-elimination to afford IntD with an E-olefin through the action of a dehydratase (DH) domain. It should be pointed out that the stereochemical course leading to E-olefin via O RKR -hydroxythioester indicates close stereochemical and biosynthetic relationships between these three domains [25]. Finally, a transER gives IntE possessing an O S ER -methyl group. This stereochemical course is basically the same as that of fatty acid synthesis [82] and can explain the reported stereochemistry of 34 (Fig. 7.6) [72, 83], a polyhydroxy PK possibly synthesized by the action of HR-PKS in the PMA clade.

7.5.2 Proposal of a Unified Stereochemical Rule The above example clearly shows that there were two prerequisites to understanding the inherent stereoselectivity of HR-PKSs. First, the function of HR-PKSs and the backbone structure of the PKS products were experimentally determined. The other is that the absolute configuration of the PKS product was determined by reliable methods, such as X-ray structural analysis and asymmetric total synthesis. Considering these prerequisites, a chemoinformatics analysis of fungal reduced PKs and a literature search of functionally characterized PKSs were conducted [25]. As a result, it was proposed that most fungal reduced PKs follow the proposed stereochemical rule. Examples are AKML A (40) [84], CIML A (41) [84], squalestatin (42) [85, 86],

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Fig. 7.7 Selected examples of fungal reduced PKs, which follow the proposed stereochemical rule. Other examples are summarized in detail in ref [25]

chaetoviridin H (43) [87, 88], lovastatin (1) [36, 89, 90] and emericellamide A (44) [91, 92] (Fig. 7.7). Although enoylreduction catalyzed by ER basically installs the O S ER -methyl group, exceptional ERs installing the O R ER -methyl group were also summarized [25]. These ERs participate in the biosyntheses of fumonisin B1 (45) [93, 94], betaenone B (46) [31, 95], and scyphostatin (47) [96, 97] (Fig. 7.8). Considering the fact that this optional selectivity contributes to constructing the structural diversity of fungal reduced PKs, it was hypothesized that the optional selectivity has been retained during the evolution of the HR-PKS. This agrees with the fact that the function of the ER domain is independent of that of other domains. The generality of the stereochemical rule was also examined through a bioinformatics analysis focusing on the stereoselectivity of the KR domain, since it was well-established in bacterial PKSs. A-type KR installs the O S KR -hydroxy group while B-type does the O RKR -hydroxy group [98, 99]. The multiple amino acid sequence alignment of KR domains revealed that KR domains of HR-PKSs have a typical conserved LDD motif of bacterial B-type KR, as in the case of mammalian fatty acid synthase. This result agrees with the fact that most fungal reduced PKs have O RKR hydroxy groups on the PK backbone and synthesize E-olefins by a syn-elimination of the O RKR -hydroxy groups. Site-specific violations of the stereochemical rule should also be pointed out. The violation discussed here is that one domain catalyzes the reaction in an opposite stereochemical manner only at a specific stage during the chain elongation process. For example, in the case of phaeospelide A (48) [100] (Fig. 7.9), the methyl-terminal stereogenic center (O S) with a hydroxy group goes against the proposed rule while the remaining follow it. A detailed chemoinformatics analysis of fungal reduced PKs revealed that similar violations occur specifically at the methyl-terminal position. Selected examples are as follows: hypothemycin (49) (violation; ketoreduction) [101–103], brefeldin A (4) (ketoreduction) [27, 104], dehydrocurvularin (50) (ketoreduction) [105, 106], monocerin (51) (ketoreduction) [107, 108], atpenin B

7 A New Trend in Biosynthetic Studies of Natural Products: The Bridge … O

O R

S ACP oS

O

O

ER

137

proposed stereochemistry

R

S ACP oR

ER

alternative stereochemistry

OH O OH

OH

O

O

OH

S-Enz OH

O O

NH2

OH

O OH

O

fumonisin B1 (45) HO O

O H

O

H

S-Enz HO

O

H

OH

betaenone B (46) O

O O

MeO

S-Enz OH NH

scyphostatin (47)

O

Fig. 7.8 Chemical structures of fungal PKs, which have the o RER -Me installed by alternative enoylreduction. The putative PKS-tethered intermediate is described in the brackets. The methyl groups with the o RER stereochemistry are enclosed by dotted line. Substituents installed by the action of modification enzymes are highlighted by yellow color

(52) (enoylreduction) [109, 110], and aspyridone A (53) (enoylreduction) [111, 112] (Fig. 7.9). A functional analysis of each domain would clarify the mechanistic reasons for how the violation occurs. Determining the absolute configuration of NPs with multiple stereogenic centers is still challenging, especially in the case of NPs with a flexible structure, although powerful methods such as X-ray single-crystal diffraction, NMR-based methods, and Circular Dichroism-based methods, and crystalline sponge methods have been established [113, 114]. The proposed stereochemical rule might highlight an error in the absolute configuration of the reported structure of fungal PKs. Once we found PKs with questionable absolute configurations, the synthetic approach of the model compound would allow us to conclude the structure. In fact, our group proposed the revised structure of thermolides by this approach [25]. In addition, we also pointed out that the proposed stereochemical rule is also useful for predicting the absolute configurations of fungal reduced PKs, for which only planar structures have been reported [25].

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Fig. 7.9 Examples of the site-specific violations with (A). a (O S KR )-hydroxy group and (B). a (O RER )-methyl group. The ACP-tethered intermediate leading to the corresponding PK is described in the brackets. A mismatched stereochemistry is labeled by an asterisk. Substituents installed by the action of modification enzymes are highlighted by yellow color

7.6 Conclusion and Perspectives This chapter describes recent advances in biosynthetic studies of fungal PKSs based on phylogenetic analysis. A phylogenetic analysis of fungal HR-PKSs and hybridPKSs provides opportunities for understanding the molecular diversity in nature. Most recently, a similar phylogenetic analysis focusing on a chain length factor, which participates in the PK processing in bacterial type II PKSs, revealed the molecular landscape of aromatic PKs [115]. These results suggest that phylogenetic analysis accelerates the bioinformatics-guided discovery of novel bacterial and fungal PKs. The proposed stereochemical rule would support the structural determination of novel PKs. This showcases that functional analysis of biosynthetic enzymes leads to elucidating the missing link between biosynthetic genes and natural products. This strategy will hopefully introduce a new era in natural product chemistry by exploring the chemical space of NPs.

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Acknowledgements This work was financially supported by Japan Society for the Promotion of Science Grants (JP22H02204 and JP23H04533), The Uehara Memorial Foundation, and Institute for Fermentation, Osaka (IFO) (G-2022-3-011).

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92. Chiang, Y.–M., Szewczyk, E., Nayak, T., Davidson, A.D., Snachez, J.F., Lo, H.–C., Wen-Yueh, H., Simityan, H., Kuo, E., Praseuth, A., Watanabe, K., Oakley, B.R., Wang, C.C.C.: Molecular genetic mining of the Aspergillus secondary metabolome: discovery of the emericellamide biosynthetic pathway. Chem. Biol. 15, 527–532 (2008) 93. Pereira, C.L., Chen, Y.-H., McDonald, F.E.: Total synthesis of the sphingolipid biosynthesis inhibitor fumonisin B1. J. Am. Chem. Soc. 131, 6066–6067 (2009) 94. Yu, F., Zhu, X., Du, L.: Developing a genetic system for functional manipulations of FUM1, a polyketide synthase gene for the biosynthesis of fumonisins in Fusarium verticillioides. FEMS Microbiol. Lett. 248, 257–264 (2005) 95. Miki, S., Sato, Y., Tabuchi, H., Oikawa, H., Ichihara, A., Sakamura, S.: Synthesis of (–)probetaenone I: structural confirmation of biosynthetic precursor of betaenone B. J. Chem. Soc. Perkin Trans. 1, 1228–1229 (1990) 96. Pitsinos, E., Athinaios, N., Xu, Z., Wang, G., Negishi, E.: Total synthesis of (+)-scyphostatin featuring an enantioselective and highly efficient route to the side-chain via Zr-catalyzed asymmetric carboalumination of alkenes (ZACA). Chem. Commun. 46, 2200–2202 (2010) 97. HR-PKS involved in the biosynthesis of scyphostatin has not been characterized. However, origin of the branched methyl groups are obvious because, to our knowledge, no modification enzymes are reported to install a methyl branch at the aliphatic position in the post-PKS modification process of fungal reduced PKs. 98. Baerga-Ortiz, A., Popovic, B., Siskos, A.P., O’Hare, H.M., Spiteller, D., Williams, M.G., Campillo, N., Spencer, J.B., Leadlay, P.F.: Directed mutagenesis alters the stereochemistry of catalysis by isolated ketoreductase domains from the erythromycin polyketide synthase. Chem. Biol. 13, 277–285 (2006) 99. Keatinge-Clay, A.T.: Stereocontrol within polyketide assembly lines. Nat. Prod. Rep. 33, 141–149 (2016) 100. Morishita, Y., Zhang, H., Taniguchi, T., Mori, K., Asai, T.: The discovery of fungal polyene macrolides via a postgenomic approach reveals a polyketide macrocyclization by trans-acting thioesterase in Fungi. Org. Lett. 21, 4788–4792 (2019) 101. Agatsuma, T., Takahashi, A., Kabuto, C., Nozoe, S.: Revised structure and stereochemistry of hypothemycin. Chem. Pharm. Bull. 41, 373–375 (1993) 102. Reeves, C.D., Hu, Z., Reid, R., Kealey, J.T.: Genes for the biosynthesis of the fungal polyketides hypothemycin from Hypomyces subiculosus and radicicol from Pochonia chlamydosporia. Appl. Environ. Microbiol. 74, 5121–5129 (2008) 103. Zhou, H., Gao, Z., Qiao, K., Wang, J., Vederas, J.C., Tang, Y.: A fungal ketoreductase domain that displays substrate-dependent stereospecificity. Nat. Chem. Biol. 8, 331–333 (2012) 104. Wu, Y., Gao, J.: Total synthesis of (+)-brefeldin A. Org. Lett. 10, 1533–1536 (2008) 105. Allu, S.R., Banne, S., Jiang, J., Qi, N., Guo, J., He, Y.: A unified synthetic approach to optically pure curvularin-type metabolites. J. Org. Chem. 84, 7227–7237 (2019) 106. Cochrane, R.V.K., Gao, Z., Lambkin, G.R., Xu, W., Winter, J.M., Marcus, S.L., Tang, Y., Vederas, J.C.: Comparison of 10,11-dehydrocurvularin polyketide synthases from Alternaria cinerariae and Aspergillus terreus highlights key structural motifs. ChemBioChem 16, 2479– 2483 (2015) 107. Ghosh, A.K., Lee, D.S.: Enantioselective total synthesis of (+)-monocerin, a dihydroisocoumarin derivative with potent antimalarial properties. J. Org. Chem. 84, 6191–6198 (2019) 108. HR-PKS involved in the biosynthesis of monocerin has not been characterized. However, the biosynthetic pathway was proposed based on the feeding experiments with putative intermediates. Therefore, it was included in Figure 7.9. 109. Ohtawa, M., Ogihara, S., Sugiyama, K., Shiomi, K., Harigaya, Y., Nagamitsu, T., Omura, S.: Enantioselective total synthesis of atpenin A5. J. Antibiot. 62, 289–294 (2009) 110. Bat-Erdene, U., Kanayama, D., Tan, D., Turner, W.C., Houk, K.N., Ohashi, M., Tang, Y.: Iterative catalysis in the biosynthesis of mitochondrial complex II inhibitors harzianopyridone and atpenin B. J. Am. Chem. Soc. 142, 8550–8554 (2020)

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111. Wasil, Z., Pahirulzaman, K.A.K., Butts, C., Simpson, T.J., Lazarus, C.M., Cox, R.J.: One pathway, many compounds: heterologous expression of a fungal biosynthetic pathway reveals its intrinsic potential for diversity. Chem. Sci. 4, 3845–3856 (2013) 112. Xu, W., Cai, X., Jung, M.E., Tang, Y.: Analysis of intact and dissected fungal polyketide synthase-nonribosomal peptide synthetase in vitro and in Saccharomyces cerevisiae. J. Am. Chem. Soc. 132, 13604–13607 (2010) 113. Huo, Z.-Q., Zhu, F., Zhang, X.-W., Zhang, X., Liang, H.-B., Yao, J.-C., Liu, Z., Zhang, G.-M., Yao, Q.-Q., Qin, G.-F.: Approaches to configuration determinations of flexible marine natural products: Advances and prospects. Mar. Drugs 20, 333 (2022) 114. Zigon, N., Duplan, V., Wada, N., Fujita, M.: Crystalline sponge method: X-ray structure analysis of small molecules by post-orientation within porous cystals—Principle and proofof-concept studies. Angew. Chem. Int. Ed. 60, 25204–25222 (2021) 115. Chen, S., Zhang, C., Zhang, L.: Investigation of the molecular landscape of bacterial aromatic polyketides by global analysis of type II polyketide synthases. Angew. Chem. Int. Ed. 61, e202202286 (2022)

Chapter 8

Biosynthesis of β-Amino Acid-Containing Macrolactam Polyketides Akimasa Miyanaga

Abstract β-Amino acid-containing macrolactam antibiotics are an important class of macrocyclic polyketides in Actinobacteria. These macrolactam antibiotics are biosynthesized from various β-amino acid starter units, contributing to their structural diversity. In this chapter, the biosynthetic mechanisms of these β-amino acid-containing macrolactam polyketides are summarized. Conserved biosynthetic machinery is used to incorporate a β-amino acid starter unit into the polyketide skeleton. The VinN-type adenylation enzyme loads a specific β-amino acid unit onto an acyl carrier protein, and the VinM-type adenylation enzyme aminoacylates the β-amino acid moiety to form a dipeptidyl unit, which is subsequently loaded onto polyketide synthases for polyketide chain elongation. A terminal aminoacyl moiety on biosynthetic intermediates is a characteristic feature in the biosynthesis of β-amino acid-containing macrolactam polyketides. Structural analysis of key biosynthetic enzymes has revealed the basis of selective recognition of β-amino acids and dipeptidyl moieties of polyketide intermediates. Biosynthetic engineering strategies have enabled the production of macrolactam polyketide derivatives in which a β-amino acid substrate analog is incorporated. Keywords Polyketide · β-Amino acid · Macrolactam · Biosynthesis · Mutasynthesis

8.1 Introduction Polyketides form a large family of natural products with various biological activities. Polyketides show a high degree of structural diversity, although they are biosynthesized from simple acyl starter and extender units [1]. The use of unique acyl starter units, such as short-chain (branched) fatty acids, aromatic acids, and amino acids, contributes to the production of structurally diverse polyketides. The property of the A. Miyanaga (B) Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-8657, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_8

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acyl starter unit provides important structural and biological features to the polyketide natural product in many cases. β-Amino acid-containing macrolactam polyketide antibiotics produced by Actinobacteria are a relatively small but growing class of natural products [2–4]. Approximately 100 β-amino acid-containing macrolactam compounds have been isolated thus far [3]. Many of these macrolactam compounds exhibit potent biological activities. These compounds are biosynthesized from a β-amino acid as a polyketide starter unit. The β-amino group of the β-amino acid is essential for the formation of their characteristic macrocyclic structures. These macrolactam compounds contain various β-amino acid starter unit moieties, including 3-aminoisobutyric acid, β-alanine, 3-aminobutyric acid, β-phenylalanine, and 3-aminofatty acid, in their polyketide skeletons, contributing to their structural diversity. Because it is anticipated that swapping the β-amino acid unit by biosynthetic engineering could lead to the production of unnatural bioactive compounds, accumulation of knowledge about their biosynthetic machinery is important. The biosynthetic gene clusters for several of these macrolactam polyketides have been identified. Functional and structural analyses of key biosynthetic enzymes have elucidated the conserved biosynthetic machinery that incorporates a β-amino acid unit into the polyketide backbone. Here, these β-amino acid-containing macrolactam polyketide natural products are first classified into five families based on the type of β-amino acid starter unit. The biosynthetic pathways for the representative β-amino acid-containing macrolactam polyketides, vicenistatin, fluvirucin B2 , incednine, hitachimycin, and cremimycin, are then summarized, with a focus on function and structure of key biosynthetic enzymes. Biosynthetic engineering for the production of macrolactam polyketide derivatives is also described.

8.2 3-Aminoisobutyric Acid Unit-Containing Macrolactams Such as Vicenistatin 8.2.1 Chemical Structures and Biological Activities of 3-Aminoisobutyric Acid Unit-Containing Macrolactams Vicenistatin, verticilactam, piceamycin, ciromicins, and niizalactams are classified into the family of 3-aminoisobutyric acid-containing macrolactam polyketides (Fig. 8.1). Vicenistatin (1), isolated from Streptomyces halstedii HC34 and Streptomyces parvus SCSIO Mla-L010, has a 20-membered macrolactam aglycon with an aminosugar vicenisamine [5, 6]. 1 shows antitumor activity against human colon carcinoma Co-3 in a xenograft model. Verticilactam (2), which is produced by Streptomyces spiroverticillatus JC-8444, has a 16-membered macrolactam aglycon conjugated with an octalin skeleton [7]. Although the production of 2 was unstable in the original producing strain, heterologous expression of its biosynthetic gene cluster in

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Fig. 8.1 Vicenistatin and other 3-aminoisobutyric acid-containing macrolactam polyketides. The 3-aminoisobutyric acid-derived moieties are shown in red

Streptomyces avermitilis SUKA17 strain resulted in sufficient production of 2 [8]. Piceamycin (3), which was isolated from Streptomyces sp. GB4-2 and Streptomyces sp. SD-53, is a 23-membered macrolactam antibiotic that contains a bicyclic structure with an internal five-membered carbocycle [9, 10]. 3 shows inhibitory activities against Gram-positive bacteria and various human tumor cell lines. Streptomyces sp. SD-53 also produces related macrolactam compounds, bombyxamycin A, B, and C [11]. Ciromicins A–B and niizalactams A–C were isolated by a combinedculture technique [12]. Ciromicin A (4) and its rearrangement product ciromicin B (5) were isolated from a Nocardiopsis sp. FU40 ΔapoS strain after coculturing it with Rhodococcus wratislaviensis [13]. Similarly, niizalactams A–C (6–8) were isolated from Streptomyces sp. NZ-6 by coculturing it with the mycolic acid-containing bacterium Tsukamurella pulmonis TP-B0596 [14]. Ciromicin A (4), ciromicin B (5), niizalactam A (6), and niizalactam B (7) contain a pyrrolidinol substructure.

8.2.2 Vicenistatin Biosynthesis To elucidate the biosynthetic mechanisms of 3-aminoisobutyric acid unit-containing macrolactams, the author’s research group has identified the biosynthetic gene cluster for vicenistatin (1) in 2004 [15]. The author’s group also conducted biochemical analysis of its biosynthetic enzymes and elucidated the incorporation mechanism of

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the β-amino acid unit into the vicenistatin (1) polyketide skeleton (Fig. 8.2) [16]. In the biosynthetic pathway of 1, l-glutamic acid is first converted to (2S,3S)3-methylaspartic acid (3-MeAsp) by a cobalamin-dependent glutamate mutase consisting of VinH and VinI [17]. The adenylation enzyme VinN selectively recognizes (2S,3S)-3-MeAsp as a β-amino acid, and transfers it onto the standalone acyl carrier protein (ACP) VinL to produce (2S,3S)-3-MeAsp–VinL [16]. The pyridoxal 5’-phosphate (PLP)-dependent enzyme VinO then decarboxylates the (2S,3S)3-MeAsp moiety to generate 3-aminoisobutyryl–VinL. Thus, the decarboxylation occurs after the VinN-catalyzed reaction. A set of the cobalamin-dependent glutamate mutase and the PLP-dependent decarboxylase is encoded in the biosynthetic gene clusters for verticilactam (2) and ciromicin A (4), suggesting that the same strategy is used to provide a 3-aminoisobutyrate unit in these polyketide biosynthetic pathways [8, 13]. Next, the 3-aminoisobutyrate unit is aminoacylated with l-alanine by the adenylation enzyme VinM to give a dipeptidyl–VinL. The trans-acting acyltransferase (AT) VinK subsequently transfers the dipeptidyl group to the ACP domain of the loading module of polyketide synthase (PKS) VinP1. The terminal alanyl group remains attached during the polyketide elongation reaction catalyzed by modular PKSs VinP1, P2, P3, and P4, and is then removed from the elongated polyketide chain by the amidohydrolase VinJ to allow the formation of vicenilactam by the thioesterase (TE) domain of VinP4. Finally, the glycosyltransferase VinC glycosylates vicenilactam using dTDP–vicenisamine to produce vicenistatin (1) [18]. The terminal alanyl moiety of the biosynthetic intermediates is a characteristic feature in the vicenistatin (1) biosynthetic pathway. The alanyl group likely serves as a “protecting group” to avoid undesired spontaneous formation of a six-membered lactam during polyketide chain elongation (Fig. 8.3). Homologous genes for VinN, VinL, VinM, VinK, and VinJ are fully conserved in biosynthetic clusters of other β-amino acid-containing macrolactams, suggesting that each β-amino acid unit is loaded onto the modular PKSs with the same protecting group strategy in their biosynthetic pathways. It has been proposed that the methyl group derived from (2S,3S)-3-MeAsp is epimerized during the vicenistatin (1) biosynthetic pathway, although the timing of the epimerization has not been elucidated. In the biosynthesis of verticilactam (2) and ciromicin A (4), the polyketide aglycons are proposed to be modified by post-PKS reactions [8, 13].

8.2.3 Structural Analysis of Vicenistatin Biosynthetic Enzymes The author’s group has conducted structural and biochemical analyses of VinN, VinK, and VinJ, providing detailed insights into their substrate recognition mechanisms, which are key for the biosynthesis of vicenistatin (1).

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Fig. 8.2 Biosynthesis of vicenistatin

8.2.3.1

Structural Analysis of VinN

The adenylation enzyme VinN plays an important role in the selective incorporation of the β-amino acid unit, (2S,3S)-3-MeAsp, in vicenistatin (1) biosynthesis. Most adenylation enzymes use an α-amino acid substrate, whereas VinN accepts a βamino acid as the substrate. To elucidate the β-amino acid recognition mechanism of VinN, the author’s group determined the crystal structure of VinN in complex with (2S,3S)-3-MeAsp (Fig. 8.4) [19]. Residue Asp230 of VinN recognizes the βamino group of the substrate, analogous to the conserved Asp residue in α-amino acid adenylation enzymes. However, the architecture of the substrate-binding pocket of VinN is different from that of α-amino acid adenylation enzymes. The structure of VinN revealed two key structural motifs for β-amino acid recognition. VinN has a bulky residue (Phe231) adjacent to the Asp residue (Asp230), and the length of the substrate-binding loop (Gly325–Ile332) of VinN is one-residue shorter than that of α-amino acid-selective adenylation enzymes. VinN has two basic residues in the substrate-binding pocket—Lys330 and Arg331—to recognize the C1 carboxy group of the substrate (2S,3S)-3-MeAsp (Fig. 8.4) [19]. VinN prefers (2S,3S)-3-MeAsp as a substrate and shows low activity toward l-aspartic acid. The substrate preference of VinN might be attributed to the interaction of Phe231 with the methyl group of (2S,3S)-3-MeAsp.

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Fig. 8.3 Vicenistatin biosynthetic pathway focused on the alanyl protecting group. The alanyl group is shown in blue

Asp230

Lys330

Phe231

Arg331

Fig. 8.4 The structure of VinN in complex with (2S,3S)-3-MeAsp (PDB code: 3WN5). The (2S,3S)3-MeAsp molecule is shown using cyan sticks. Salt bridge interactions are shown as broken lines

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153

Structural Analysis of VinK

In vicenistatin (1) biosynthesis, the trans-acting AT VinK catalyzes the transfer of the dipeptidyl group from VinL to the ACP domain of the loading module of the PKS VinP1 [16]. Biochemical analysis of VinK suggested that VinK distinguishes dipeptidyl–VinL from 3-aminoisobutyryl–VinL by recognizing the terminal alanyl group of dipeptidyl–VinL (Fig. 8.3) [16]. To clarify the substrate recognition mechanism of VinK, the author’s group determined the crystal structure of VinK in the ligand-free form [20]. VinK has a larger substrate-binding tunnel than that of ATs that use malonyl-CoA thioester as the substrate. The malonyl-CoA-specific ATs have a conserved Arg residue that interacts with the carboxy group of the malonyl substrate in the substrate-binding tunnel, but VinK has Leu131 at the corresponding position, which provides sufficient space for the dipeptidyl group. Docking analysis suggested that the terminal alanyl group is recognized by Glu109 and Leu131 (Fig. 8.5a). Thus, VinK seems to have a unique substrate-binding cavity that is specific for the dipeptidyl group. To visualize specific interactions between VinK and VinL, the author’s group conducted structural analysis of VinK complexed with VinL [20]. Because the interactions between VinK and VinL are weak and transient, structural characterization of VinK–VinL complex was difficult. Therefore, the author’s group developed a covalent cross-linking strategy using a 1,2-bismaleimidoethane cross-linking reagent to trap the transient VinK–VinL complex. The cross-linking reaction between the VinK S266C mutant and VinL in the presence of 1,2-bismaleimidoethane gave a covalent VinK–VinL complex (Fig. 8.5b), enabling structural determination of the VinK–VinL complex. In the VinK–VinL complex structure, Arg153 and Arg299 of VinK form salt bridges with Glu47 and Asp35 of VinL, respectively (Fig. 8.5c). Met206 of VinK forms hydrophobic contacts with VinL. VinK R153A, M206A, and R299A mutants showed significantly decreased affinity for VinL, confirming the importance of these residues of VinK. Recently, the author’s group also determined the crystal structure of a VinK–VinL covalent complex formed with a synthetic pantetheine-type probe [21]. The binding interface between VinK and VinL was essentially the same in the two VinK–VinL complex structures.

8.2.3.3

Structural Analysis of VinJ

In vicenistatin (1) biosynthesis, the amidohydrolase VinJ catalyzes the hydrolytic cleavage of the terminal alanyl group from the elongated polyketide chain [16]. To clarify the substrate recognition mechanism of VinJ, the author’s group determined the crystal structure of VinJ in the ligand-free form [22]. VinJ has a long hydrophobic substrate-binding tunnel that extends from the surface to the active site (Fig. 8.6), suggesting that VinJ recognizes the hydrophobic polyketide chain of fully or mostly elongated N-alanyl-polyketide intermediates. Thus, VinJ seems to regulate the timing of hydrolytic removal of the alanyl protecting group to avoid undesired spontaneous formation of a six-membered lactam during polyketide elongation (Fig. 8.3).

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(a)

(b) Ser106

VinL

SH

O O

N

N

O

HS

VinK

O VinL

Ser266

O

S

O

N

N

O

S

VinK

O Leu131

Glu109

(c) Leu43

Glu47

VinL Asp35

Arg153 Met206

Arg299 VinK

Fig. 8.5 Crystal structure of VinK. (a) The structure of VinK (PDB code: 5CZC) with the docked dipeptidyl substrate molecule. The docked dipeptidyl substrate molecule is shown using cyan sticks. (b) Cross-linking reaction of VinK S266C mutant with holo-VinL. (c) The binding interface between VinK and VinL in the structure of the VinK–VinL cross-linked complex (PDB code: 5CZD). Salt bridge interactions are shown as broken lines

8.2.4 Production of Vicenistatin Derivative (2S,3S)-3-MeAsp is used as a starter unit in vicenistatin (1) biosynthesis [19]. The vinI gene, encoding the cobalamin-dependent glutamate mutase that converts l-glutamic acid to (2S,3S)-3-MeAsp, was disrupted for functional confirmation of the vinI gene in S. halstedii HC34 [17]. As a result, the ΔvinI strain did not produce vicenistatin (1). Production of 1 was restored when 3-MeAsp was fed into culture of the ΔvinI strain. Interestingly, the production of a vicenistatin derivative, desmethylvicenistatin (9), was observed in the ΔvinI strain (Fig. 8.7). Instead of (2S,3S)-3-MeAsp, l-aspartic acid, which is a proteinogenic α-amino acid, was incorporated into the vicenistatin (1) biosynthetic pathway in the ΔvinI strain. The incorporation of l-aspartic acid could be attributed to the relatively broad substrate specificity of the vicenistatin (1)

8 Biosynthesis of β-Amino Acid-Containing Macrolactam Polyketides

Phe197

155

Tyr46

Phe201 Ser110

Tyr211

Fig. 8.6 Crystal structure of VinJ (PDB code: 3WMR) with docked N-alanyl secovicenilactam thioester substrate molecule. The docked substrate molecule is shown as cyan sticks

biosynthetic enzymes. Importantly, the adenylation enzyme VinN, which functions as a gatekeeper for selective incorporation of the β-amino acid starter unit, has activity toward l-aspartic acid, although the activity is relatively low [19]. The observed outcomes could clearly inspire attempts at mutasynthesis to produce “unnatural” βamino acid-containing macrolactam compounds as described later (Sects. 8.4.4 and 8.5.4).

Fig. 8.7 Production of desmethylvicenistatin in the ΔvinI strain of S. halstedii HC34

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Fig. 8.8 Fluvirucins. The β-alanine-derived moieties are shown in red

8.3 β-Alanine Unit-Containing Macrolactams Such as Fluvirucin B2 8.3.1 Chemical Structures and Biological Activities of Fluvirucins Fluvirucins are classified into the family of β-alanine-containing macrolactam polyketides (Fig. 8.8). Fluvirucins are 14-membered macrolactam polyketides that show antifungal and antiviral activities [23–27]. Various fluvirucin derivatives with several combinations of alkyl side-chains, and deoxyaminosugar units have been isolated from several actinomycetes. For example, fluvirucin B1 (10), which is produced by Actinomadura vulgaris, has a 14-membered macrolactam polyketide aglycon with ethyl side-chains at the C2 and C10 positions and a methyl side-chain at the C6 position, and contains 3-amino-3,6-dideoxy-l-talopyranose. Fluvirucin B2 (11) (also known as Sch38518), which is produced by Actinomadura fulva subsp. indica ATCC 53,714, has a 14-membered macrolactam polyketide aglycon with ethyl side-chains at the C2, C6, and C10 positions, and contains l-mycosamine.

8.3.2 Fluvirucin Biosynthesis The Schnarr’s research group identified the PKS genes that are responsible for fluvirucin B1 (10) biosynthesis in 2013 [28]. The author’s group identified the biosynthetic gene cluster for fluvirucin B2 (11) in 2016 [29]. The author’s group also conducted biochemical analysis of the fluvirucin B2 (11) biosynthetic enzymes, and elucidated its biosynthetic mechanism. In the biosynthetic pathway of 11, the VinN-type adenylation enzyme FlvN selectively recognizes l-aspartic acid (l-Asp) as a β-amino acid and transfers it onto the standalone ACP FlvL to produce lAsp–FlvL (Fig. 8.9) [29, 30]. Next, the PLP-dependent enzyme FlvO decarboxylates the l-Asp moiety to form the β-alanine unit. Biochemical analysis showed that FlvN has a marked preference for l-aspartic acid over β-alanine, suggesting that

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157

the decarboxylation of the α-carboxy group occurs after the FlvN-catalyzed reaction. The resulting β-alanyl–FlvL is aminoacylated with l-alanine by the VinM-type adenylation enzyme FlvM to give a dipeptidyl–FlvL. The resulting dipeptidyl unit is transferred onto PKS FlvP1 by the trans-acting AT FlvK. After polyketide chain elongation by PKSs FlvP1, FlvP2, and FlvP3, the amidohydrolase FlvJ catalyzes the removal of the terminal alanyl group from the elongated polyketide chain. The TE domain of PKS FlvP3 then catalyzes macrocyclization to form the aglycon of fluvirucin B2 (11). Finally, the glycosyltransferase FlvS5 glycosylates the aglycon using dTDP–l-mycosamine to produce fluvirucin B2 (11).

Fig. 8.9 Biosynthesis of fluvirucin B2

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8.4 3-Aminobutyric Acid Unit-Containing Macrolactams Such as Incednine 8.4.1 Chemical Structures and Biological Activities of 3-Aminobutyric Acid Unit-Containing Macrolactams Incednine, sipanmycins, salinilactam, micromonolactam, lobosamides, mirilactams, silvalactam, pretilactam, and dracolactams are classified into the family of 3aminobutyric acid-containing macrolactam polyketides (Fig. 8.10). Incednine (12), which was isolated from Streptomyces sp. ML694-90F3, has a 24-membered polyene macrolactam aglycon with a disaccharide unit [31]. 12 has suppressive activity of the antiapoptotic function of Bcl-2/Bcl-xL oncoproteins. Sipanmycin (13), which is produced by Streptomyces sp. CS149, is a glycosylated 24-membered macrolactam polyketide [32]. Auroramycin, whose structure is identical to that of 13, was also isolated from Streptomyces roseosporus by using a CRISPR–Cas9 gene cluster activation strategy [33]. Auroramycin was reported to show potent antibacterial activity against methicillin-resistant Staphylococcus aureus. Salinilactam (14), which is produced by marine actinobacterium Salinispora tropica CNB-440, is a 26-membered polyene macrolactam polyketide [34]. Micromonolactam (15), which is produced by Micromonospora sp. CMS I2-32, is a 26-membered polyene macrolactam polyketide [35]. Lobosamides A–C, which are produced by the marine actinobacterium Micromonospora sp. RL09-050-HVF-A, are 26-membered polyene macrolactam polyketides. Lobosamide A (16) shows growth inhibition activity toward the protozoan parasite Trypanosoma brucei [36]. Actinosynnema mirum NBRC 14,064 produces mirilactams A (17) and B, whose structures are similar to those of lobosamides A–C [36]. Silvalactam (18), which is produced by Streptomyces sp. Tü 6392, is a glycosylated 24-membered macrolactam polyketide [37]. 18 shows potent cytotoxic activity toward various cancer and noncancer cell lines. Pretilactam (19), which is produced by Actinosynnema pretiosum ATCC 31,565, is a 26-membered polyene macrolactam polyketide that contains a dihydroxy tetrahydropyran moiety [38]. Dracolactams A–B and mirilactams C–E were isolated using a combined-culture technique: [12, 39] Dracolactam A (20) and B were isolated from Micromonospora wenchangensis HEK797 by coculturing it with T. pulmonis TP-B0596, and mirilactams C, D, and E were isolated from A. mirum NBRC 14,064 by coculturing it with T. pulmonis TP-B0596.

8.4.2 Incednine Biosynthesis To elucidate the biosynthetic mechanisms of 3-aminobutyric acid unit-containing macrolactams, the author’s group has identified the biosynthetic gene cluster for

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Fig. 8.10 Incednine and other 3-aminobutyric acid-containing macrolactam polyketides. The 3aminobutyric acid-derived moieties are shown in red

incednine (12) in 2013 [40]. The author’s group also conducted biochemical analysis of the biosynthetic enzymes of 12, and elucidated its biosynthetic mechanism. In the biosynthetic pathway of 12, l-glutamic acid is first converted to β-glutamic acid by the glutamate 2,3-aminomutase IdnL4 (Fig. 8.11). The PLP-dependent enzyme IdnL3 decarboxylates the β-glutamic acid to (S)-3-aminobutyric acid. A set of glutamate 2,3-aminomutase and β-glutamate decarboxylase genes is encoded in the biosynthetic gene clusters for sipanmycin (13), salinilactam (14), micromonolactam (15), and lobosamide A (16), suggesting that the same strategy is used to provide the 3-aminobutyric acid starter unit in these polyketide biosynthetic pathways [34–36, 41]. Next, the VinN-type adenylation enzyme IdnL1 selectively transfers (S)-3-aminobutyric acid onto the standalone ACP IdnL6 [42]. The resulting 3-aminobutyryl–IdnL6 is aminoacylated with l-alanine by the VinM-type adenylation enzyme IdnL7 to give a dipeptidyl–IdnL6 [43]. The resulting dipeptidyl unit is transferred onto PKS IdnP1 by the trans-acting AT IdnL2. After polyketide chain elongation by PKSs IdnP1, IdnP2, IdnP3, IdnP4, and IdnP5, the amidohydrolase IdnL5 catalyzes the removal of the terminal alanyl group from the elongated polyketide chain. The TE domain of PKS IdnP5 then catalyzes macrocyclization to form the aglycon of incednine (12). In the post-PKS modification reaction, IdnO1 catalyzes hydroxylation at the C10 position, and the glycosyltransferases IdnS4 and IdnS14 glycosylate the aglycon to produce incednine (12).

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Fig. 8.11 Biosynthesis of incednine

8.4.3 Structural Analysis of Incednine Biosynthetic Enzymes The author’s group has conducted structural and biochemical analyses of IdnL1 and IdnL7, both of which are an adenylation enzyme involved in incednine (12) biosynthesis.

8.4.3.1

Structural Analysis of IdnL1

The VinN-type adenylation enzyme IdnL1 functions as a gatekeeper for incorporation of the (S)-3-aminobutyric acid unit in incednine (12) biosynthesis [42]. IdnL1 adenylates (S)-3-aminobutyric acid, and then uses the resulting (S)-3-aminobutyryl– adenylate intermediate to produce (S)-3-aminobutyryl–IdnL6 (Fig. 8.12a). Biochemical analysis showed that IdnL1 exhibits good activity toward 3-aminopentanoic acid at similar level to its natural substrate 3-aminobutyric acid, although IdnL1 exhibits only low activity toward β-alanine [42]. To elucidate the substrate recognition mechanism of IdnL1, the author’s group determined the crystal structure IdnL1 in complex with (S)-3-aminobutyryl–adenylate intermediate (Fig. 8.12b) [42]. IdnL1 has the βamino acid-selective motifs as observed in the structure of VinN, suggesting that IdnL1 employs the same mechanism to distinguish a β-amino acid from an α-amino acid. IdnL1 has a narrow hydrophobic substrate-binding tunnel for the recognition

8 Biosynthesis of β-Amino Acid-Containing Macrolactam Polyketides

(a)

(b)

O H2N

IdnL1

OH HS

ATP

IdnL6

O

Asp216

NH2 N

O O P O

H2N

O

N

N

N

O HO

O S

H2N

Phe217

OH

IdnL6

Leu220

O

(c)

161

IdnL7

OH

ATP

NH2

H2N

Lys500

(d)

O S

IdnL6

NH2 O

N

O O P O

NH2 O N NH2 H

O

N

N N

Asp216

O HO O S

OH IdnL6

Thr318

Fig. 8.12 Crystal structures of adenylation enzymes involved in incednine biosynthesis. Salt bridge interactions are shown as broken lines. (a) Reaction catalyzed by IdnL1. (b) The structure of IdnL1 in complex with 3-aminobutyryl–adenylate (PDB code: 5JJQ). The 3-aminobutyryl–adenylate molecule is shown using cyan sticks. (c) Reaction catalyzed by IdnL7. (d) The structure of IdnL7 in complex with an l-alanyl–adenylate analog (PDB code: 6AKD). The l-alanyl–adenylate analog molecule is shown using cyan sticks

of aliphatic β-amino acid substrates [42]. IdnL1 has residue Leu220 at the bottom of the shallow substrate-binding tunnel to accommodate short-chain β-amino acids such as 3-aminobutyric acid and 3-aminopentanoic acid (Fig. 8.12b).

8.4.3.2

Structural Analysis of IdnL7

The VinM-type adenylation enzyme IdnL7 adenylates l-alanine, and then uses the resulting l-alanyl–adenylate intermediate for amide bond formation with a β-aminoacyl–IdnL6 to produce dipeptidyl–IdnL6 (Fig. 8.12c). IdnL7 has broad substrate specificity for small α-l-amino acids, whereas IdnL7 shows no activity

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toward d-alanine, suggesting that IdnL7 is an l-amino acid adenylation enzyme [43]. To elucidate the substrate recognition mechanism of IdnL7, the author’s group determined the crystal structure of IdnL7 in complex with an l-alanyl–adenylate intermediate analog [43]. The amino and carboxy groups of the alanyl moiety interact with Asp216 and Lys500 of IdnL7, respectively (Fig. 8.12d). The relatively small side-chain of residue Thr318 provides space in front of the methyl group of the alanyl moiety in the substrate-binding pocket. This structural observation explains why IdnL7 shows relaxed substrate specificity toward a variety of small α-l-amino acids. VinM-type adenylation enzyme catalyzes the transfer of an l-alanyl group onto a β-aminoacyl–ACP, while it does not catalyze the transfer of an l-alanyl group onto holo-ACP, suggesting that the VinM-type adenylation enzyme has a substrate-binding tunnel that is specific for the β-aminoacyl–phosphopantetheine group of VinL-type ACP. The IdnL7 structure does not provide any clue to provide the basis of the specificity of the β-aminoacyl–phosphopantetheine group. Structural determination of a VinM-type enzyme complexed with a VinL-type ACP would be necessary to understand the recognition mechanism of β-aminoacyl–ACP during the amide bond formation.

8.4.4 Production of Incednine Derivatives Mutasynthesis is an attractive strategy to produce “unnatural” natural products. Mutasynthesis essentially uses a non-producing mutant strain in which a gene in the target biosynthetic pathway is disrupted to block the normal pathway at a crucial step [44]. The disrupted pathway can be (re)activated by feeding analogs of the missing biosynthetic intermediate. The fed analog compound is incorporated into the target biosynthetic pathway, resulting in the production of a compound with a modified structure. Because the VinN-type adenylation enzyme IdnL1 shows relatively relaxed substrate specificity, the author’s group attempted to produce incednine derivatives via mutasynthesis strategy. To explore the possibility of production of an incednine derivative by substituting the 3-aminobutyric acid unit with 3-aminopentanoic acid, the glutamate 2,3aminomutase idnL4 gene, which is responsible for supplying (S)-3-aminobutyric acid, was disrupted in Streptomyces sp. ML694-90F3 [45]. As a result, the ΔidnL4 strain did not produce incednine (12). Production of 12 was restored when racemic 3aminobutyric acid was fed into culture of the ΔidnL4 strain. Moreover, when racemic 3-aminopentanoic acid was fed into culture of the ΔidnL4 strain, production of an incednine derivative, 28-methylincednine (21), was observed (Fig. 8.13) [45]. This implies that all of the downstream biosynthetic enzymes accommodate the modified β-amino acid moiety. 21 inhibits the antiapoptotic function of oncoprotein Bcl-xL (as does 12) [45]. When β-alanine was fed into culture of the ΔidnL4 strain, the incednine derivative was also produced. However, the production level of this incednine derivative was very low. This mutasynthesis strategy likely depends significantly on the substrate specificity of IdnL1.

8 Biosynthesis of β-Amino Acid-Containing Macrolactam Polyketides

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Fig. 8.13 Production of 28-methylincednine in the ΔidnL4 strain of Streptomyces sp. ML694-90F3

Similarly, the Olano’s research group obtained a sipanmycin derivative by a mutasynthesis strategy in which 3-aminopentanoic acid was fed into culture of a ΔsipL4 strain of Streptomyces sp. CS149, in which the glutamate 2,3-aminomutase gene sipL4 was disrupted [46]. However, production of sipanmycin derivatives was not observed following feeding of β-Ala, β-Leu, or β-Phe. The failure to obtain sipanmycin derivatives with β-Ala, β-Leu, or β-Phe could be explained by the substrate specificity of SipL1, which shares high sequence identity with IdnL1.

8.5 β-Phenylalanine Unit-Containing Macrolactams Such as Hitachimycin 8.5.1 Chemical Structures and Biological Activities of β-Phenylalanine Unit-Containing Macrolactams Hitachimycin and viridenomycin are classified into the family of β-phenylalaninecontaining macrolactam polyketides (Fig. 8.14). Hitachimycin (22) (also known as stubomycin), which was isolated from Streptomyces scabrisporus, is a 19-membered macrolactam antibiotic that contains a bicyclic structure with an internal fivemembered carbocycle [47]. 22 shows antimicrobial, antiprotozoal, and antitumor activities [48]. 22 also exhibits inhibitory activity toward yeast plasma membrane ATPase 1 [49]. Viridenomycin (23), which was isolated from Streptomyces viridochromogenes and Streptomyces gannmycicus, is a 24-membered macrolactam antibiotic that contains a bicyclic structure with an internal five-membered carbocycle and one ester linkage [50, 51]. 23 shows antitumor activity.

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Fig. 8.14 Hitachimycin and viridenomycin. The β-phenylalanine-derived moieties are shown in red

8.5.2 Hitachimycin Biosynthesis To elucidate the biosynthetic mechanisms of β-phenylalanine unit-containing macrolactams, the author’s group has identified the biosynthetic gene cluster for hitachimycin (22) in 2015 [52]. The author’s group also conducted biochemical analysis of the biosynthetic enzymes of 22, and elucidated its biosynthetic mechanism. In the biosynthetic pathway of 22, l-phenylalanine is first converted to (S)-βphenylalanine [(S)-β-Phe] by the phenylalanine 2,3-aminomutase HitA (Fig. 8.15) [52]. Next, the VinN-type adenylation enzyme HitB selectively transfers (S)-β-Phe onto the standalone ACP HitD [53]. After aminoacylation catalyzed by the VinMtype adenylation enzyme HitE, the resulting dipeptidyl unit is transferred onto PKS HitP1 by the trans-acting AT HitC. After polyketide chain elongation by PKSs HitP1, HitP2, HitP3, HitP4, and HitP5, the amidohydrolase HitF catalyzes the removal of the terminal alanyl group from the elongated polyketide chain. The TE domain of PKS HitP3 then catalyzes the formation of the macrolactam ring. The bicyclic structure with an internal five-membered carbocycle is proposed to be constructed by postPKS enzymes HitM1, HitM2, HitM3, HitM4, and HitM5, although the formation mechanism of internal five-membered carbocycle has not been elucidated. Finally, the methyltransferase HitM6 catalyzes O-methylation to produce hitachimycin (22).

8.5.3 Structural Analysis of HitB The adenylation enzyme HitB functions as a gatekeeper for incorporation of the (S)β-Phe unit in hitachimycin (22) biosynthesis [54]. Biochemical analysis showed that HitB exhibits good activity toward (S)-β-Phe analogs [(S)-β-o-F-Phe, (S)-β-m-F-Phe, and (S)-β-p-F-Phe] that have a fluorine atom (which is small) at the ortho, meta, or para position [54]. In addition, HitB tolerates (S)-β-Phe analogs that have a relatively small hydrophobic functional group (Cl, Br, or CH3 ) at the meta position, but it is not tolerant of ortho- or para-substituted (S)-β-Phe analogs. To obtain the structural basis for the substrate specificity of HitB, the author’s group determined the crystal structure of HitB in complex with an (S)-β-Phe–adenylate analog (Fig. 8.16a) [54].

8 Biosynthesis of β-Amino Acid-Containing Macrolactam Polyketides

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Fig. 8.15 Biosynthesis of hitachimycin

HitB has three aromatic residues, Phe222, Phe225, and Phe328, forming a wide hydrophobic substrate-binding pocket for the recognition of the phenyl side-chain of the substrate (S)-β-Phe (Fig. 8.16b). Conformational flexibility around Phe328 could explain why HitB also accommodates meta-substituted (S)-β-Phe analogs. HitB is necessary to distinguish HitD from other ACPs in the producer strain for functional substrate transfer. To visualize the protein–protein interactions between HitB and HitD, the author’s group conducted structural analysis of the HitB–HitD complex [53]. Because the transient HitB–HitD complex is difficult to crystallize, the author’s group developed a covalent cross-linking strategy using a synthetic pantetheine-type probe to trap the transient HitB–HitD complex. In this strategy, a synthetic pantetheine-type probe that has an electrophilic bromoacetamide group was chemoenzymatically loaded onto apo-HitD to generate a crypto-HitD. The bromoacetamide group of the resulting crypto-HitD efficiently reacted with the HitB D221C mutant to generate a covalently cross-linked HitB–HitD complex (Fig. 8.16b), enabling the structural determination of the HitB–HitD complex. In the HitB–HitD complex structure, Glu41 and Glu47 of HitD form salt bridges with

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(b)

(a)

O

HitB

SH

Br

D221C mutant

Asp221

O O P O

O

HitD

O

O

crypto-HitD

O S

HitB

OH

H N

H N

N H

N H

OH

H N

H N O

O O P O

O

HitD

O

cross-linked HitB–HitD complex Phe222 Phe225

Phe328

(c)

Phe16

HitD Glu41

Glu47 Trp247

Val40

Arg249 Arg275 HitB

Fig. 8.16 Crystal structures of HitB and HitB–HitD cross-linked complex. Salt bridge interactions are shown as broken lines. (a) The structure of HitB in complex with an (S)-β-Phe–adenylate analog (PDB code: 7DQ5). The (S)-β-Phe–adenylate analog molecule is shown using cyan sticks. (b) Cross-linking reaction of the HitB D221C mutant with crypto-HitD. (c) The binding interface between HitB and HitD in the structure of the HitB–HitD cross-linked complex (PDB code: 6M01)

Arg275 and Arg249 of HitB, respectively (Fig. 8.16c). Phe16 and Val40 of HitD form hydrophobic contacts with HitB. The importance of these four HitD residues was confirmed by mutational analysis, in which HitB showed significantly decreased (S)-β-Phe transfer activity toward the HitD mutants F16A, V40A, E41A, and E47A. Phe16, Val40, Glu41, and Glu47 of HitD are highly conserved among VinL-type ACPs, suggesting similar binding interactions between other VinN-type enzymes and VinL-type ACPs.

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8.5.4 Production of Hitachimycin Derivatives Because the VinN-type adenylation enzyme HitB shows relatively relaxed substrate specificity, the author’s group attempted to produce several hitachimycin derivatives via mutasynthesis strategy. To explore the possibility of producing hitachimycin derivatives by substituting the (S)-β-Phe unit with (S)-β-Phe analogs, various (S)-βPhe analogs were fed into culture of a ΔhitA strain of S. scabrisporus JCM11712, in which the phenylalanine 2,3-aminomutase gene hitA was disrupted. Supplementation with (S)-β-o-F-Phe, (S)-β-m-F-Phe, or (S)-β-p-F-Phe led to production of the corresponding hitachimycin derivatives (24–26) (Fig. 8.17) [54]. Supplementation with (S)-β-m-Br-Phe also led to production of the hitachimycin derivative (27), whereas supplementation with (S)-β-o-Br-Phe or (S)-β-p-Br-Phe did not lead to production of a hitachimycin derivative. Similarly, supplementation with (S)-β-m-CH3 -Phe or (S)-β-m-Cl-Phe led to production of hitachimycin derivatives (28 and 29). Thus, the productivity of hitachimycin derivatives in the ΔhitA strain was consistent with the substrate specificity of HitB. These findings also imply that all of the downstream biosynthetic enzymes accommodate the modified β-amino acid moieties, as in the cases of the incednine (12) and sipanmycin (13) biosynthetic systems. However, supplementation of the ΔhitA strain with (S)-β-m-CF3 -Phe or racemic β-m-I-Phe did not lead to production of hitachimycin derivatives, even though HitB shows good activities toward these (S)-β-Phe analogs. In these cases, the downstream biosynthetic enzymes might be unable to accept the modified β-Phe moieties that have relatively bulky substituent groups, such as –CF3 at the meta position. Among the other tested β-amino acids, supplementation with (S)-3-amino-3-(3-thienyl)propanoic acid and (S)-3-amino-3-(2-thienyl)propanoic acid also led to production of hitachimycin derivatives (30 and 31). Thus, 3-thienyl and 2-thienyl groups at the β-position of the β-amino acid are also accepted by the hitachimycin biosynthetic machinery. The obtained hitachimycin derivatives (24–31) exhibit different levels of cytotoxic activity in HeLa cells and budding yeast [54]. Hitachimycin derivatives (25–27 and 29) in which the phenyl moiety is halogenated at the meta position or para position show increased cytotoxicity toward HeLa cells compared with the original compound hitachimycin (22) (Fig. 8.17). However, hitachimycin derivatives (25, 27, and 29) that are halogenated at the meta position of the phenyl group show decreased cytotoxicity toward yeast cells. The compounds might target different molecules in HeLa cells and yeast.

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Fig. 8.17 Hitachimycin derivatives produced by the ΔhitA strain of S. scabrisporus JCM11712. IC50 values of these derivatives against HeLa cells are shown in parentheses

8.6 3-Aminofatty Acid Unit-Containing Macrolactams Such as Cremimycin 8.6.1 Chemical Structures and Biological Activities of 3-Aminofatty Acid Unit-Containing Macrolactams Cremimycin, BE-14106, aureoverticillactam, and heronamides are classified into the family of 3-amino fatty acid-containing macrolactam polyketides (Fig. 8.18). Cremimycin (32) has a fully saturated side-chain [55], whereas BE-14106 (33), aureoverticillactam (34), and heronamides (35–37) have unsaturated side-chains [56–59]. Cremimycin (32), which was isolated from Streptomyces sp. MJ635-86F5, is a glycosylated 19-membered macrolactam antibiotic that contains a bicyclic structure with an internal five-membered carbocycle [55]. 32 shows broad antimicrobial activity against Gram-positive bacteria. BE-14106 (33), which was isolated from Streptomyces spheroids A14106, is a 20-membered macrolactam antibiotic that shows antibacterial, antifungal, and anticancer activities [56]. Aureoverticillactam (34), which is produced by Streptomyces aureoverticillatus, is a 22-membered macrolactam antibiotic that shows cytotoxicity toward various tumor cell lines [57]. Heronamides A–C were isolated from Streptomyces sp. CMB-M0406 [58]. Heronamide C

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Fig. 8.18 Cremimycin and other 3-aminofatty acid-containing macrolactam polyketides. The 3aminofatty acid-derived moieties are shown in red

(35) (also known as ML-449) is a 20-membered polyene macrolactam compound. Heronamide A (36) and B (37) are derived from heronamide C (35) by transannular intramolecular cycloadditions [60–62]. Although 36 and 37 have no reported biological activity, 35 shows inhibitory activity toward fission yeast cells, the basis of which is unique targeting of the lipid membrane [59]. Heronamides D–F, whose side-chains are two carbons shorter than those of heronamides A–C (35–37), were isolated from Streptomyces sp. CCSIO 03,032 [63].

8.6.2 Cremimycin Biosynthesis To elucidate the biosynthetic mechanisms of 3-aminofatty acid unit-containing macrolactams, the author’s group has identified the biosynthetic gene cluster for cremimycin (32) in 2013 [64]. The author’s group also conducted biochemical analysis of the biosynthetic enzymes of 32, and elucidated its biosynthetic mechanism. In the biosynthetic pathway of 32, the 3-aminononanoic acid starter unit is constructed through a polyketide pathway. PKSs CmiP2, CmiP3, and CmiP4 construct (E)non-2-enoyl-ACP thioester from a methylmalonyl-CoA and three malonyl-CoAs (Fig. 8.19). The standalone TE CmiS1 catalyzes the Michael addition of glycine to the (E)-non-2-enoyl-ACP thioester, and the subsequent hydrolysis of the thioester to produce (R)-N-carboxymethyl-3-aminononanoic acid. Structural analysis of the CmiS1 homolog SAV606 from S. avermitilis provides mechanistic insights into

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the multistep reaction of CmiS1 [65]. Next, the flavin adenine dinucleotide (FAD)dependent oxidase CmiS2 catalyzes the oxidation of the N-carboxymethyl moiety of (R)-N-carboxymethyl-3-aminononanoic acid, followed by spontaneous hydrolysis to produce (R)-3-aminononanoic acid [66]. A set of CmiS1-type TE and CmiS2type FAD-dependent oxidase genes is encoded in the biosynthetic gene clusters for BE-14106 (33) and heronamides (35–37), suggesting that the same strategy is used to provide the 3-aminofatty acid starter unit in these polyketide biosynthetic pathways [67–69]. Next, the VinN-type adenylation enzyme CmiS6 selectively transfers (R)-3-aminononanoic acid onto the standalone ACP CmiS4. After aminoacylation catalyzed by the VinM-type adenylation enzyme CmiS3, the resulting dipeptidyl unit is transferred onto PKS CmiP1 by the trans-acting AT CmiS5. After polyketide chain elongation by PKSs CmiP1, CmiP5, CmiP6, CmiP7, and CmiP8, the amidohydrolase CmiM6 catalyzes the removal of the terminal alanyl group from the elongated polyketide chain. The TE domain of PKS CmiP6 then catalyzes the formation of the macrolactam ring. The bicyclic structure with an internal five-membered carbocycle is proposed to be constructed by post-PKS enzymes CmiM1, CmiM2, CmiM3, CmiM4, and CmiM7 through the same mechanism as in the hitachimycin (22) biosynthetic pathway. Finally, the glycosyltransferase CmiM5 glycosylates the bicyclic aglycone using dTDP–digitoxose with the methylation catalyzed by the methyltransferase CmiC1 to produce cremimycin (32).

8.6.3 Structural Analysis of CmiS6 The VinN-type adenylation enzyme CmiS6 functions as a gatekeeper for incorporation of the (R)-3-aminononanoic acid unit in cremimycin (32) biosynthesis [42]. Biochemical analysis showed that CmiS6 exhibits the highest activity toward a cognate substrate 3-aminononanoic acid [42]. CmiS6 also exhibits a relatively good activity toward other medium-chain β-amino acids such as 3-aminoheptanoic acid and 3-aminoundecanoic acid. To elucidate the substrate recognition mechanism of CmiS6, the author’s group determined the crystal structure CmiS6 [42]. CmiS6 has a narrow and long hydrophobic substrate-binding tunnel to accommodate mediumchain β-amino acids such as 3-aminononanoic acid (Fig. 8.20). CmiS6 has Gly220 at the corresponding position of Leu220 in IdnL1, allowing the tunnel to extend over the position of Gly220 and accept the aliphatic side-chain of (R)-3-aminononanoic acid. The CmiS6 G220L mutant completely lost activity against 3-aminononanoic acid, while it retained activity against 3-aminobutyric acid, suggesting that the size of amino acid residue at the 220 position is crucial for the selection of an aliphatic β-amino acid substrate in CmiS6 and IdnL1.

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Fig. 8.19 Biosynthesis of cremimycin

8.7 Conclusions and Future Perspectives In this chapter, the biosynthetic mechanisms of β-amino acid-containing macrolactam natural products are described. Various β-amino acid-containing macrolactam natural products have been isolated, and their biosynthetic gene clusters have been identified. Homologous genes for five starter-related enzymes are fully conserved in these biosynthetic gene clusters, suggesting that the biosynthetic machinery of β-amino acid-containing macrolactam compounds employs the same mechanism for incorporating a β-amino acid unit into the polyketide backbone. Biochemical and structural analyses have revealed the substrate recognition mechanisms of biosynthetic enzymes such as VinN-type adenylation enzymes. Using the relaxed β-amino acid substrate specificity of VinN-type enzymes, β-amino acid-containing macrolactam derivatives were successfully obtained via mutasynthesis strategies. Some derivatives show increased biological activity compared with the original compound. However, because the mutasynthesis strategy depends on the substrate specificities of VinN-type enzymes, protein engineering and swapping of VinN-type adenylation

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Asp216

Gly220

Fig. 8.20 The structure of CmiS6 (PDB code: 5JJP) with docked 3-aminononanoyl–adenylate. The docked 3-aminononanoyl–adenylate molecule is shown using cyan sticks. Salt bridge interactions are shown as broken lines

enzymes would be necessary to further expand the repertoire of β-amino acid starter units in macrolactam polyketide skeletons. In addition, modular PKSs are an attractive target for engineering to produce natural product derivatives [70]. The replacement and swapping of PKS modules and domains could lead to new derivatives. Recently accumulated structural information on polyketide biosynthetic enzymes, such as modular PKSs, could support the rational redesign of biosynthetic machinery. The author believes that the production of natural product derivatives by redesigning of biosynthetic pathway would be a new trend in the field of natural product chemistry and natural product biosynthesis.

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Part III

Total Synthesis of Complex Natural Products by Innovative Strategies

Chapter 9

Synthetic Approach Toward Structural Elucidation of Marine Natural Product Symbiodinolide Hiroyoshi Takamura

Abstract Structural determination of natural products is a significant research topic in natural product chemistry. The combination of spectroscopic analysis and chemical synthesis is well recognized as a reliable method for the structural elucidation of natural products. Symbiodinolide is a 62-membered polyol macrolide marine natural product that has a molecular weight of 2860 and possesses 61 chiral centers. The gross structure of symbiodinolide was revealed by extensive 2D NMR analysis. However, the stereostructure of symbiodinolide remains an open question because of its huge and complicated chemical structure. Therefore, the degradation of natural symbiodinolide and chemical synthesis of each fragment, including the stereoisomers, have been examined in efforts to establish stereochemical details of this natural product. This chapter describes the degradation of natural symbiodinolide and stereodivergent synthesis of each fragment, which has led to the stereostructural elucidation of portions corresponding to 70% of the whole structure of symbiodinolide. Keywords Structural elucidation · Stereodivergent synthesis · Stereoisomer · Common synthetic intermediate · Symbiodinolide

9.1 Introduction Structural elucidation of biologically active natural products is a fundamental and significant research topic in natural product chemistry. Study on structural determination of natural products leads to structure–activity relationship study, elucidation of the pharmacophores, design and synthesis of molecular probes, identification of target molecules in vivo, clarification of the mode of action, and the creation of novel biologically active molecules. The combination of spectroscopic analysis and chemical synthesis is well recognized as a reliable method for the structural elucidation of natural products [1]. In the structural determination of natural products, H. Takamura (B) Department of Chemistry, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushimanaka, Kita-Ku, Okayama 700-8530, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_9

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if the natural product possesses a very large molecular size, acyclic systems, or a number of functional groups, then organic synthesis is often a useful tool to employ for unambiguous structural establishment [2–6]. A diversity of organic compounds have been isolated from marine organisms [7, 8]. Among these secondary metabolites, polyether and polyol marine natural products, whose structural feature is a polyoxygenated long carbon backbone, have attracted attention due to their remarkable chemical structures and potent biological activities [9–11]. Symbiodinolide (1) is a 62-membered polyol macrolide marine natural product. In 2007, it was isolated from an 80% aqueous ethanol extract of the cultured dinoflagellate Symbiodinium sp. (Fig. 9.1) [12, 13]. The natural product 1 is a structural congener of zooxanthellatoxins, which are polyol macrolides isolated from the same dinoflagellate [14–21]. Biological evaluation of 1 has revealed that this natural product exhibits voltage-dependent N-type Ca2+ channel-opening activity at 7 nM and COX-1 inhibition (65%) at 2 μM. Moreover, 1 ruptures the tissue surface of the acoel flatworm Amphiscolops sp. at 2.5 μM. The gross structure of natural product 1 was assigned by extensive 2D NMR spectroscopic techniques. However, the stereostructure of this natural product remains an open question because of its huge and complicated chemical structure—it has a molecular weight of 2860 and 61 chiral centers. Therefore, the degradation of natural 1 and the chemical synthesis of each fragment of 1, including the stereoisomers, have been investigated toward the full stereochemical assignment of this natural product. This chapter addresses the degradation of the natural product 1 and the stereodivergent synthesis [22–24] of each fragment, which has resulted in the elucidation of stereostructures of the portions corresponding to 70% of the whole structure of 1.

9.2 Synthesis and Absolute Configuration of the C1' –C25' Fragment Prior to examining the synthetic approach toward 1, degradation of the natural product 1 was carried out. When natural 1 was treated with KOH in MeOH/H2 O, hydrolysis at the C1 and C1' positions proceeded, to give the C1' –C25' carboxylic acid fragment 2 as a degraded product (Scheme 9.1) [12]. Detailed NMR analysis and comparison of the NMR data of the degraded product 2 with data reported in the research on zooxanthellatoxins [14–21] established the relative configuration of the C1' –C25' fragment 2. Next, the stereoselective synthesis of the C1' –C25' fragment 2 was investigated toward elucidation of the absolute configuration of this fragment. In the retrosynthetic analysis, the C1' –C25' fragment 2 could be synthesized by constructing the trisubstituted alkene moiety from the left-hand fragment 3 and the right-hand fragment 4 (Scheme 9.2). The left side chain domain of 3 could be introduced by the coupling between 1-nonyne and triflate 5. By contrast, the right-hand fragment 4 could be transformed from the starting material d-xylose.

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Fig. 9.1 Structure of symbiodinolide (1)

Scheme 9.1 Alkaline hydrolysis of symbiodinolide (1)

Stereoselective synthesis of the left-hand fragment 15 is shown in Scheme 9.3. Eschenmoser–Claisen rearrangement of allylic alcohol 6, which was prepared from tri-O-acetyl-d-glucal in two steps [25], with N,N-dimethylacetamide dimethyl acetal proceeded in xylene under reflux to give amide 7 in 93% yield. Treatment of 7 with I2 in THF/H2 O afforded iodolactone 8. Methanolysis of the lactone 8 and subsequent epoxidation afforded methyl ester 9. The regio- and stereoselective epoxide opening of 9 with TFA in H2 O took place to yield trihydroxy lactone 10 [26, 27]. The resulting three hydroxy groups of 10 were protected as the TBDPS ether and the acetonide to

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Scheme 9.2 Retrosynthetic analysis of the C1' –C25' fragment 2

Scheme 9.3 Synthesis of PT-sulfone 15

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give 11. The stereochemistries of the tetrahydropyran 11 were confirmed by coupling constants and NOE observations. Thus, the value of 3 J 14' ,15' (9.7 Hz) indicated that both H-14' and H-15' were oriented in axial positions. The equatorial configuration of H-13' was determined by the value of 3 J 13' ,14' (5.5 Hz). In addition, the NOE correlations of H-11' /H-12' and H-11' /H-15' revealed them to be in syn relationships with each other. The lactone 11 was converted to triflate 12 in six steps. The triflate 12 reacted with n-C7 H15 CCLi, generated from 1-nonyne and n-BuLi, to give alkyne 13 in 76% yield [28]. The latter was exposed to Birch reduction conditions, to yield the corresponding (E)-alkene, which was transformed to syn-diol 14 by Sharpless asymmetric dihydroxylation [29]. The transformation by a six-step sequence from 14 afforded the PT-sulfone 15, which is the coupling precursor of the left-hand fragment. Stereoselective synthesis of the right-hand fragment 19, which is the coupling partner of the PT-sulfone 15, was next carried out (Scheme 9.4). The chiral oxazolidinone was introduced to carboxylic acid 16, which was synthesized from D-xylose in 12 steps, to yield imide 17. Asymmetric methylation of 17 according to the Evans protocol [30] afforded methylated product 18 as a single diastereomer. Reductive removal of the chiral auxiliary of 18 with LiAlH4 followed by Parikh–Doering oxidation [29] of the resulting alcohol provided the aldehyde 19. With the two coupling precursors 15 and 19 in hand, coupling and completion of the synthesis of the C1' –C25' fragment 2 was examined (Scheme 9.5). Julia– Kocienski olefination [32–34] between the PT-sulfone 15 and the aldehyde 19 with LDA afforded the desired (E)-alkene 20 in 37% yield together with the (Z)-alkene in 24% yield. The geometry of the trisubstituted alkene part of 20 was determined by ROE observations between Ha and Hb. Deprotection of the TBS ether 20 and subsequent stepwise oxidation afforded carboxylic acid 21. Finally, the benzyl, methoxymethyl, and acetonide protecting groups were fully removed to give the C1' –C25' fragment 2. The NMR data and specific rotation of the synthetic product 2 were identical to those of the degraded C1' –C25' fragment obtained from natural 1. Therefore, the absolute configuration of this fragment was elucidated to be that of 2 [35].

9.3 Synthesis and Relative Configuration of the C1–C13 Fragment As degradation of natural 1, methanolysis with Et3 N/MeOH followed by oxidative cleavage with Grubbs II catalyst/NaClO was carried out to give the C1–C13 fragment 22 (Scheme 9.6) [36, 37]. The stereochemistry of 22 was next analyzed to reduce the number of possible diastereomers of this fragment. 1 H NMR data analysis of the degraded product 22 revealed that the chemical shifts of H-5 and H-7 were the same (3.97 ppm), and the two vicinal coupling constants 3 J 5,6 and 3 J 6,7 were also the same (4.5 Hz), as shown in Fig. 9.2a. These findings suggest that the relative

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Scheme 9.4 Synthesis of aldehyde 19

Scheme 9.5 Synthesis of the C1' –C25' fragment 2

configurational relationships at the C5 and C7 positions to the C6 position in the 1,2,3triol system are the same [38]. Therefore, among the eight possible diastereomers of this fragment, four candidate compounds 22a–22d were selected (Fig. 9.2b), wherein 22a and 22c are anti/anti (C5,C6/C6,C7) compounds, and 22b and 22d possess syn/syn (C5,C6/C6,C7) relationships. Stereoselective synthesis of the four candidate compounds 22a–22d was next examined. A stereodivergent synthetic route [22–24] toward the four candidate compounds 22a–22d was planned (Scheme 9.7). β-Hydroxy ketone 23 was designed as the common synthetic intermediate for delivering 22a–22d, that is, the first branching

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Scheme 9.6 Methanolysis and subsequent oxidative cleavage of symbiodinolide (1)

Fig. 9.2 (a) 1 H NMR data of the degraded product 22 and (b) four candidate compounds 22a–22d

point in the synthetic scheme. Utilizing the C5 stereocenter, diastereoselective reductions of the β-hydroxy ketone 23 with different reductants could produce syn-diol 24a and anti-diol 24b. The diols 24a and 24b are the second branching points. Deprotection of 24a and oxidation of the resulting allylic alcohol would give the first candidate compound 22a. Inversion of the C6 stereochemistry of 24a would afford the second candidate compound 22b. Moreover, the transformation from 24b, which is similar to that planned from 24a, could provide the third and fourth candidate compounds 22c and 22d. An aldol reaction of methyl acetoacetate (25) and aldehyde 26 was performed with NaH/n-BuLi as bases to give β-hydroxy ketone 27 bearing the desired C5 stereochemistry in 94% yield at the 6:1 diastereomeric ratio (Scheme 9.8) [39]. Treatment of the β-hydroxy ketone 27 with Et2 BOMe/NaBH4 [40] afforded syn-diol 28a in 98% yield as a single diastereomer. A protection and deprotection sequence from 28a resulted in 29, which was converted to unsaturated aldehyde 30. Finally, removal of the acetonide moiety of 30 and subsequent deprotection of the TES ether

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Scheme 9.7 Stereodivergent synthetic plan of four candidate compounds 22a–22d

were carried out with TiCl4 [41] in one-pot, affording the first candidate compound 22a. For the synthesis of the second candidate compound 22b, which is the C6epimer of 22a, the oxymethyne portion at the C6 position of 28a was oxidized to give ketone 31. Diastereoselective reduction of 31 by a Felkin–Anh model with NaBH4 proceeded smoothly to give the desired alcohol 32 in 98% yield as the sole product. A conversion similar to that used for the synthesis of 22a was carried out, affording 22b. Stereoselective synthesis of the third and fourth candidate compounds 22c and 22d was next carried out (Scheme 9.9). The β-hydroxy ketone 27 reacted with NaBH(OAc)3 [42] to give the desired anti-diol 28b, which is the C3-epimer of 28a, in 95% yield. The synthetic route from 28b to 22c and 22d was analogous to that used in the synthesis of 22a and 22b. Thus, 22c and 22d were obtained from 28b through the synthetic intermediates 33 and 34, respectively. With the four candidate compounds 22a–22d in hand, the 1 H NMR data of these synthetic products were next compared with the data of the degraded product 22. The 1 H NMR data of the synthetic 22b were identical to the data of the degraded 22. By contrast, the 1 H NMR data of the synthetic products 22a, 22c, and 22d differed from the data of the degraded 22. These findings revealed the relative configuration of the C1–C13 fragment of 1 to be that as shown in 22b [43].

9.4 Synthesis and Absolute Configuration of the C14–C24 Fragment Cross-metathesis of 1 with ethylene, using Hoveyda–Grubbs II catalyst, was performed as a degradation reaction to yield the C14–C24 fragment 35, the C23–C34 fragment 36, and the C33–C42 fragment 37 (Scheme 9.10) [36]. The C14–C24 fragment 35 possesses three chiral centers, wherein the two stereocenters at the C17 and

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Scheme 9.8 Stereodivergent and stereoselective synthesis of candidate compounds 22a and 22b

C21 positions are the oxymethyne domains. Before commencing the synthetic study of this fragment, the modified Mosher method [44] was applied to the degraded C14–C24 fragment (Fig. 9.3). Thus, (S)- and (R)-MTPA esters 38 were prepared by reaction of the degraded C14–C24 fragment 35 with MTPACl. The signs of the Δδ S–R values of the (S)- and (R)-38 at the left side of the C17 position were negative and those at the right side of the C21 position were positive. Therefore, the absolute configurations at the C17 and C21 positions were determined to be 17R and 21R, respectively, as shown in Fig. 9.3 [45]. Next, stereodivergent and stereoselective synthesis of the two stereoisomers at the C18 positions was investigated.

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Scheme 9.9 Stereodivergent and stereoselective synthesis of candidate compounds 22c and 22d

Fig. 9.3 Chemical shift differences (Δδ S–R ) of (S)- and (R)-38

Scheme 9.10 Cross-metathesis of symbiodinolide (1) with ethylene

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Scheme 9.11 Stereoselective synthesis of (18R)-diol 45a

To obtain the two stereoisomers at the C18 positions in a concise synthesis, it was planned that these two stereoisomers would be synthesized from the same synthetic intermediate by changing the reaction conditions. Thus, the asymmetric aldol reaction under Crimmins conditions [46, 47] was selected as a branching step from the common synthetic intermediate toward the synthesis of the two C18-stereoisomers. The stereoselective synthesis of (18R)-diol 45a is summarized in Scheme 9.11. α,βUnsaturated aldehyde 39 reacted with propionyl oxazolidinethione 40 (1.5 equiv) in the presence of TiCl4 (1.6 equiv)/(–)-sparteine (3.8 equiv) to give the Evans-type syn-aldol product 41 bearing the desired C18 stereochemistry [46, 47]. The stereochemical inversion at the C17-oxymethyne part was next carried out. Thus, changing the oxazolidinethione 41 to the Weinreb amide and oxidation of the allylic alcohol afforded α,β-unsaturated ketone 42. Diastereoselective reduction of the ketone 42 with LiBHs-Bu3 proceeded smoothly to give the desired alcohol 43. α,β-Unsaturated aldehyde 44, which was derived from 43 by a two-carbon elongation, was subjected to Keck asymmetric allylation conditions [48] to provide the corresponding alcohol. Deprotection of the TBS ether and methyl acetalization at the C14 position took place, affording the (18R)-diol 45a. The other target molecule (18S)-diol 45b was synthesized by changing the conditions in the Crimmins asymmetric aldol reaction (Scheme 9.12). Thus, treatment of the common synthetic intermediate 39 with propionyl oxazolidinethione 40 (2.0 equiv)/TiCl4 (4.0 equiv)/(–)-sparteine (2.2 equiv) yielded the non-Evans-type syn-aldol adduct 46 with the desired stereochemistries at the C17 and C18 positions [46, 47]. The (18S)-diol 45b was synthesized from 46 by transformation analogous to that used in the synthesis of 45a. Next, the NMR data of the two synthetic products 45a and 45b were compared with the data of the degraded C14–C24 fragment. 1 H NMR data of the synthetic product 45a were found to be in excellent agreement with the data of the degraded product. 1 H NMR data of the synthetic 45b differed from the data of the degraded product. Significant differences were observed in the chemical shifts at the C17 and

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Scheme 9.12 Stereoselective synthesis of (18S)-diol 45b

Fig. 9.4 Chemical shift (in ppm) of the H-17 and H-18 in the degraded product and the synthetic products 45a and 45b

C18 positions (Fig. 9.4). Furthermore, the 1 H NMR data of (S)- and (R)-MTPA esters, prepared from the synthetic product 45a, matched the data of the (S)- and (R)-38 derived from the degraded C14–C24 fragment, respectively. Therefore, the absolute stereostructure of the C14–C24 fragment was determined to be 17R, 18R, and 21R, as depicted in 45a [49, 50].

9.5 Synthesis and Absolute Configuration of the C23–C34 Fragment As in the case of the C14–C24 fragment, the stereochemistries at the C26 and C32 positions in the C23–C34 fragment were analyzed by utilizing the modified Mosher method [44] in the degraded product of this fragment (Fig. 9.5). Thus, the degraded product diol 36 was transformed to (S)-and (R)-47, the corresponding bis-MTPA esters, and the chemical shift deviations between (S)-47 and (R)-47 were calculated. The signs of the Δδ S–R values at the left side of the C26 position were negative while those at the C33 and C34 positions were positive. From these results, the absolute configurations at the C26 and C32 positions were determined to be 26S and 32S, respectively, as shown in Fig. 9.5 [45]. Therefore, the number of possible stereoisomers of the C23–C34 fragment could be decreased from 16 to 4, and four candidate compounds 48a–48d were selected as the synthetic targets in this fragment (Fig. 9.6). A stereodivergent synthetic plan [22–24] of the selected four candidate compounds 48a–48d is shown in Scheme 9.13. Allylic alcohol 49 was set as the common synthetic intermediate for affording 48a–48d, and the stepwise introduction

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Fig. 9.5 Chemical shift differences (Δδ S–R ) of (S)and (R)-47

Fig. 9.6 Four candidate compounds 48a–48d

Scheme 9.13 Stereodivergent synthetic plan of four candidate compounds 48a–48d

of the two contiguous epoxide moieties was planned. Thus, reagent-controlled asymmetric epoxidation of the allylic alcohol 49 could introduce the C29,C30-epoxide domains, stereoselectively, to give epoxides 50a and 50b. The second epoxidation of the allylic alcohols 50a and 50b could supply the four target diepoxides 48a and 48b, and 48c and 48d, respectively. Sharpless asymmetric epoxidation [51, 52] of allylic alcohol 51 with TBHP/(–)DET/Ti(Oi-Pr)4 proceeded smoothly to give epoxy alcohol 52a in 89% yield at the 12:1 diastereomeric ratio (Scheme 9.14). By contrast, the use of (+)-DET in Sharpless asymmetric epoxidation [51, 52] afforded epoxy alcohol 52b in 84% yield with 20:1 diastereoselectivity. The two-carbon elongation of 52a gave allylic alcohol 53a. Treatment of 53a with mCPBA afforded syn-diepoxide 54a and anti-diepoxide 54b

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in 45% yields. The syn-diepoxide 48a and the anti-diepoxide 48b were, respectively, synthesized from 54a and 54b in a parallel manner by introducing the homoallylic alcohol portions via Roush asymmetric allylation [53] and the right side terminal alkene moieties by Grieco protocol [54]. The conversion from 52b, which is analogous to that from 52a to 48a and 48b, was carried out to give the syn-diepoxide 48c and the anti-diepoxide 48d. After completion of the synthesis of all four candidate compounds 48a–48d, the 1 H NMR data of these four synthetic products were compared with the data of the degraded product in this fragment. Only the 1 H NMR spectrum of 48c was identical to that of the degraded product. Furthermore, the specific rotation of the synthesized 48c, [α]D 24 –14.2 (c 0.20, MeOH), matched that of the degraded C23–C34 fragment, [α]D 21 –11.2 (c 0.05, MeOH). Therefore, the absolute stereostructure of the C23–C34 fragment was determined to be as in 48c [55].

Scheme 9.14 Stereodivergent synthesis of four candidate compounds 48a–48d

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9.6 Synthesis and Absolute Configuration of the C33–C42 Fragment The degraded product 37 corresponding to the C33–C42 fragment was also submitted to stereochemical analysis by the modified Mosher method (Fig. 9.7) [44]. Thus, bisMTPA esters (S)- and (R)-55 were prepared from the degraded C33–C42 fragment 37. The signs of the chemical shift differences (Δδ S–R ) in the (S)- and (R)-55 were negative at the left side of the C36 position and positive at the C41 and C42 positions. Therefore, the absolute stereochemistry of this fragment was determined to be (36S, 40S), as depicted in Fig. 9.7 [45]. Stereoselective synthesis of this fragment was next examined for confirmation of the stereochemical assignment (Scheme 9.15). The asymmetric allylation of α,βunsaturated aldehyde 56 was conducted under Keck conditions [48] to give allylic alcohol 57 in 41% yield at the 10:1 diastereomeric ratio. Protection of 57 with TBDPSCl and selective removal of the primary TBS group were carried out to give alcohol 58. Introduction of the alkene portion under Grieco conditions [54] and deprotection of the resulting bis-silyl ether afforded diol 59. The 1 H NMR spectrum of the synthesized 59 was in full agreement with that of the degraded product. Moreover, the synthetic diol 59 was transformed to the corresponding bis-(S)- and (R)-MTPA esters. These bis-MTPA esters were compared with the bis-MTPA esters (S)- and (R)-55, derived from the degraded product, in terms of the NMR data. The 1 H NMR spectra of the synthetic bis-(S)- and (R)-MTPA esters were identical to those of the bis-MTPA esters (S)- and (R)-55, respectively. Therefore, the absolute configuration of this fragment was confirmed to be (36S, 40S), as shown in 59 [56, 57]. Fig. 9.7 Chemical shift differences (Δδ S–R ) of (S)and (R)-55

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Scheme 9.15 Stereoselective synthesis of the C33–C42 fragment 59

Fig. 9.8 Stereochemical analysis of the C83–C104 fragment

9.7 Synthesis and Relative Configuration of the C79–C104 Fragment The relative stereochemistry of the C79–C104 fragment was determined by NMR analysis of natural 1. Thus, the relative configuration of this fragment, including the spiroacetal and hemiacetal moieties and the C91–C99 carbon chain portion, was elucidated by the 3 J H,H coupling constants and observed NOE correlations (Fig. 9.8) [12]. First, the stereoselective synthesis of the C79–C104 fragment bearing the proposed stereostructure was investigated.

9.7.1 Synthesis of the Proposed C79–C104 Fragment Asymmetric synthesis of the C79–C93 fragment PT-sulfone 65 is summarized in Scheme 9.16. The chiral epoxide 60, which was synthesized from L-aspartic acid, reacted with 3-butenylmagnesium bromide/CuI, affording epoxide 61. The epoxide 61 was coupled with alkyne 62 to provide ketone 63. Removal of the three TBS groups of 63 and subsequent spiroacetalization were conducted by CSA in MeOH

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Scheme 9.16 Stereoselective synthesis of the C79–C93 fragment 65

Scheme 9.17 Stereoselective synthesis of the C94–C104 fragment 72

in one-pot to give alcohol 64 in 95% yield. The absolute stereochemistry of 64 was determined by the NOE observations of H-83/H-91. The obtained alcohol 64 was transformed into the PT-sulfone 65. Enantioselective synthesis of the C94–C104 fragment aldehyde 72, which is the coupling partner of 65, was next examined (Scheme 9.17). Achmawictz rearrangement [60] of the chiral furyl alcohol 66 was performed with NBS in THF/H2 O gave the corresponding hemiacetal, which was treated with (MeO)3 CH/BF3 ‧OEt2 , to afford acetal 67. The α,β-unsaturated ketone 67 was transformed to aldehyde 68 through dihydroxylation of the alkene moiety and deoxygenation of the carbonyl group. Coupling reaction between 68 and dithiane 69 took place to yield alcohol 70 as a single diastereomer. The resulting stereochemistry at the C98 position of 70 was undesired; hence, the C98 stereochemistry was inverted by the combination of Dess–Martin oxidation and diastereoselective reduction with DIBAL-H, affording

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Scheme 9.18 Stereoselective synthesis of the proposed C79–C104 fragment 74a

alcohol 71 in 84% yield in two steps. The alcohol 71 was converted to the coupling precursor 72. Completion of synthesis of the proposed C79–C104 fragment 74a is summarized in Scheme 9.18. The C79–C93 fragment PT-sulfone 65 and the C94–C104 fragment aldehyde 72 were coupled by Julia–Kocienski olefination [32–34] under optimized conditions to give the expected (E)-alkene in 70% yield. The vicinal diol part at the C93 and C94 positions was introduced via Sharpless asymmetric dihydroxylation [29] to give syn-diol 73 in 91% yield, stereoselectively. Finally, deprotection of the benzyl ether 73 by hydrogenation followed by removal of the acetonide and TBS protecting groups in one-pot with HCl in MeOH afforded the proposed C79–C104 fragment 74a in 79% yield in two steps. With the C79–C104 fragment 74a possessing the proposed relative configuration in hand, the 13 C NMR data of the synthesized 74a were then compared with the data of the C79–C104 portion of natural symbiodinolide (1). Unexpectedly, the chemical shifts in the C91–C99 carbon chain domain of the synthetic 74a did not match those of the natural product (Fig. 9.9). The chemical shift difference at the C95-Me group was particularly critical (Δδ 1–74a = + 17). These results indicated that the stereochemistry at the C91–C99 carbon chain portion of 1 needs to be reexamined [58].

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Fig. 9.9 13 C NMR chemical shift differences between natural symbiodinolide (1) and the synthetic product 74a (Δδ = δ 1 – δ 74a in ppm)

9.7.2 Strategy for the Stereostructural Elucidation There are seven chiral centers in the C91–C99 carbon chain portion. Therefore, the number of possible diastereomers of this moiety is 26 = 64. If we could synthesize these 64 diastereomers, we could elucidate the stereochemistry of the C79– C104 domain of 1 by comparing the NMR data between the natural product and the synthetic products. However, chemical synthesis of 64 possible diastereomers in the C91–C99 carbon chain portion is impractical. Therefore, as a more efficient synthetic approach toward the structural elucidation of the C79–C104 moiety of 1, the C79–C104 fragment 74 was divided into two fragments: the C79–C97 fragment 75 and the C94–C104 fragment 76 (Scheme 9.19). The stereogenic center at the C95 position is contained in both the C79–C97 fragment 75 and the C94–C104 fragment 76. Therefore, if the stereochemistries of 75 and 76 could be clarified, then the stereostructure of the C79–C104 fragment 74 could finally be revealed by relating each stereochemistry of the two fragments 75 and 76 through the C95 stereogenic center.

Scheme 9.19 Strategy for the stereostructural elucidation of the C79–C104 fragment 74

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9.7.3 Synthesis and Relative Configuration of the C79–C97 Fragment The C79–C97 fragment bears three unidentified chiral centers at the C93, C94, and C95 positions in the carbon chain part; therefore, there are eight possible diastereomers of this fragment, as described in Fig. 9.10. Chemical synthesis of all eight possible diastereomers 75a–75h in a stereodivergent manner was pursued. An addition reaction of the anion, which was prepared from dithiane 78 and n-BuLi, to aldehyde 77 was performed. Alcohols 79a and 79b were obtained, in 33% yields; they are the C93-stereoisomers to each other (Scheme 9.20). This reaction is the first stereodiversification step. The obtained dithiane 79a was hydrolyzed to give α-hydroxy ketone 80, which is the key common synthetic intermediate for the target molecules 75a and 75b. Transformation from the α-hydroxy ketone 80 is the second stereodiversification step. After protection of the alcohol 80 with TBSOTf/2,6-lutidine, a Felkin–Anh-type reduction of the resulting α-siloxy ketone, using the C93 stereochemistry, was investigated. As a result, treatment of the α-siloxy ketone with DIBAL-H in CH2 Cl2 at –100 °C afforded the desired alcohol 81 in 88% yield, in two steps, as a single diastereomer. Finally, the TBS and benzyl protecting groups were removed to give the tetraol 75a. Next, stereoselective synthesis of the tetraol 75b, which is the C94-epimer of 75a, was carried out. After a detailed survey of the reaction conditions in the chelation-controlled reduction of the α-hydroxy ketone 80, it was proven that reaction of 80 with L-Selectride in the presence of ZnCl2 [59] as a chelating reagent gave the expected anti-diol 82 in 86% yield as the sole product. Global deprotection of 82 yielded the tetraol 75b. In a similar way, the tetraols 75c and 75d, which are the C93-epimers of 75b and 75a, respectively, were synthesized from the alcohol 79b. Furthermore, the tetraols 75e–75h, which are the

Fig. 9.10 Eight possible diastereomers of the C79–C97 fragment

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Scheme 9.20 Stereodivergent synthesis of possible diastereomers 75a–75d

C95-epimers of 75a–75d, respectively, were also obtained using the enantiomer of the dithiane 78. Next, comparison of the 13 C NMR data between the synthetic products 75a– 75h, which are the eight possible diastereomers for the C79–C97 fragment, and the natural product was carried out. Figure 9.11 displays chemical shift deviations in the 13 C NMR data at the C91, C93, C94, and C95 positions between natural 1 and the synthesized 75a–75h. The chemical shift differences of the two diastereomers 75a and 75f were smaller than those of the other six diastereomers. These findings suggest the relative configuration of the C79–C97 fragment to be that shown in either 75a or 75f.

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Fig. 9.11 13 C NMR chemical shift differences between natural symbiodinolide (1) and the synthetic products 75a–75h (Δδ = δ 1 – δ 75 in ppm)

9.7.4 Synthesis and Relative Configuration of the C94–C104 Fragment There are three undefined stereocenters at the C95, C97, and C98 positions in the acyclic portion of the C94–C104 fragment. Therefore, as in the case of the C79–C97 fragment, the number of possible diastereomers of this fragment is 23 = 8 (Fig. 9.12).

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Fig. 9.12 Eight possible diastereomers of the C94–C104 fragment

Stereodivergent synthesis of all these eight possible diastereomers 76a–76h was examined. Hydrolysis of the dithiane 71 gave α-hydroxy ketone 83 (Scheme 9.21). The αhydroxy ketone 83 was subjected to the chelation-controlled reduction conditions with Zn(BH4 )2 to give anti-diol 84 in 90% yield as a single diastereomer. The PMB, acetonide, and TBS protecting groups in 84 were removed, affording the hexaol 76a. By contrast, the Felkin–Anh-type reduction of the α-siloxy ketone, derived from the alcohol 83, was conducted with DIBAL-H to give the desired alcohol 85 in 84% yield, in two steps, as a single product. Deprotection of 85 afforded the hexaol 76b, which is the C97-epimer of 76a. The other six diastereomers 76c–76h were obtained using similar transformation, stereodivergently. With all eight possible diastereomers 76a–76h in hand, the 13 C NMR data of the synthetic products 76a–76h were next compared with those of the natural product. Figure 9.13 shows the 13 C NMR chemical shift differences at the C95, C97, C98, and C99 positions between natural 1 and the synthetic products 76a–76h. The chemical shifts of the two diastereomers 76a and 76e were proven to be similar to those of the natural product compared with those of the other six diastereomers. These results indicate the relative stereochemistry of the C94–C104 fragment to be that represented in either 76a or 76e.

9.7.5 Stereochemical Revision of the C79–C104 Fragment As shown in Scheme 9.22, four candidate compounds of the C79–C104 fragment could be suggested by relating the relative configurations of the C79–C97 fragment and the C94–C104 fragment through the C95 stereogenic center. The combinations of the two fragments are 75a + 76a in 74a, ent-75a + 76e in 74b, ent-75f + 76a in 74c, and 75f + 76e in 74d. Toward the stereochemical clarification of the C79–C104 fragment, other three candidates 74b–74d were prepared in a manner similar to the synthesis of compound 74a shown in Scheme 9.18. The C93,C94-syn-diol 74b was synthesized using Julia–Kocienski olefination [32–34] and Sharpless asymmetric

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Scheme 9.21 Stereodivergent synthesis of possible diastereomers 76a and 76b

dihydroxylation [29] of the resulting (E)-alkene as key reactions. The C93,C94anti-diols 74c and 74d were obtained by the combination of Wittig olefination and Sharpless asymmetric dihydroxylation [29] of the resulting (Z)-alkenes. After the unified synthesis of all four candidate compounds 74a–74d, the synthetic products 74a–74d were submitted to detailed 2D NMR analysis, and their NMR data were compared with the data of the natural product. As is evident in Fig. 9.14, only the candidate compound 74b showed similar 13 C NMR chemical shifts to those of the natural product. The chemical shift deviations of the candidate compounds 74a, 74c, and 74d were significant, especially in the C91–C99 carbon chain portion. Therefore, it was concluded that natural 1 possesses the relative configuration in the C79–C104 domain as shown in 74b [61].

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Fig. 9.13 13 C NMR chemical shift differences between natural symbiodinolide (1) and the synthetic products 76a–76h (Δδ = δ 1 – δ 76 in ppm)

9.8 Conclusion In this chapter, as an example of synthetic efforts toward the structural clarification of natural products, the stereodivergent synthesis of each fragment in symbiodinolide (1) is described. The very large molecular size of the natural product 1 has hampered the stereochemical assignment of this natural product. Therefore, prior to commencing with the synthetic approach toward 1, degradation of the natural

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Scheme 9.22 Four candidate compounds of the C79–C104 fragment

product was examined. Stereostructural analysis of the degraded products, which were obtained from the natural product 1, reduced the number of targeted stereoisomers. The selected target stereoisomers were synthesized in a stereodivergent route, wherein the target molecules were supplied by branching from the common synthetic intermediates in a unified manner. Spectroscopic data of the synthetic products were compared with those of the degraded products, which then led to the configurational determination of the C1' –C25' , C1–C13, C14–C24, C23–C34, and C33–C42 fragments (Fig. 9.15). In the C79–C104 fragment, stereodivergent synthesis of the partial structure corresponding to this domain of 1 was attempted. The relative configuration of the C79–C104 fragment was elucidated by synthesizing 20 diastereomers

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Fig. 9.14 13 C NMR chemical shift differences between natural symbiodinolide (1) and the synthetic products 74a–74d (Δδ = δ 1 – δ 74 in ppm)

among 64 possible diastereomers in the C91–C99 carbon chain moiety of this fragment and comparing the NMR data between the synthetic products and the natural product. Further synthetic approach toward the complete structural elucidation of 1 is currently underway in the author’s research group. Over the past several decades, remarkable advancements have been made in the development of instruments and techniques of NMR spectroscopic analysis to clarify the gross structure and the configuration of natural products. For instance, J-based configuration analysis (JBCA), which was developed by Murata and coworkers [62], is a powerful tool for the relative stereochemical determination of acyclic organic compounds upon combination with NOE. Kishi and coworkers developed a universal NMR database approach [38, 63–67], wherein the relative stereochemistry of an authentic sample is assigned by comparing its 3 J H,H coupling constants and 13 C and 1 H NMR chemical shifts with those of the reference structural motifs. Kishi’s universal NMR database approach can be also utilized for the absolute stereostructural determination of secondary alcohols using chiral NMR solvents [68–71]. Nonetheless, the configurational elucidation of natural products, especially those bearing acyclic motifs or remote stereogenic centers, needs careful spectroscopic analysis, and the synergy of spectroscopic analysis and chemical synthesis is effective for the unambiguous structural determination [1–6]. In addition, chemical synthesis can supply structural analogues as well as parent natural products, which can lead to the structural confirmation/determination of natural products and structure–activity relationship studies. Biosynthetic consideration [72] and computational chemistry [73–76] are also useful in elucidating/predicting configurations of natural products. The synergistic combination of these methodologies and synthetic approaches will

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Fig. 9.15 Portions in which synthesis and structural determination have been completed

enhance our ability to determine the configurations of structurally complex natural products. Acknowledgements This work was supported by JSPS KAKENHI Grant Number JP21H01938. The author dedicates this chapter to the memory of Professor Daisuke Uemura, who sadly passed away on April 13, 2021.

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Chapter 10

Collective Synthesis of Monoterpenoid Indole Alkaloids Using Bioinspired Strategies Hayato Ishikawa

Abstract In recent years, collective total synthesis, in which natural products are synthesized exhaustively from common intermediates, has attracted attention in academia and industry. In this chapter, collective total syntheses of monoterpenoid indole alkaloids found in higher plants are highlighted. Secologanin and strictosidine, which are key branching intermediates in biosynthesis, were synthesized in gram-scale quantities. Then, 18 indole alkaloid syntheses from secologanin tetraacetate or its aglycones were accomplished. These total syntheses, using bioinspired strategies reminiscent of a tree diagram, can provide several different skeletal natural products in one scheme. Fewer than 13 steps are required for the total synthesis of the compounds, and the total yield typically exceeded 10%. Keywords Collective synthesis · Monoterpenoid indole alkaloid · Bioinspired reaction · Strictosidine · Secologanin

10.1 Introduction Monoterpenoid indole alkaloids (MTIAs) are widely known as naturally occurring secondary metabolites found in higher plants, and more than 3000 compounds have been isolated to date [1–11]. Many of these compounds exhibit potent biological activity. Two examples are vinblastine, which is used as an anticancer drug, and strychnine, a well-known natural poison (Fig. 10.1). The structures are also diverse. For example, quinine and camptothecin, which have quinoline structures, are derived from indole in biosynthesis and thus belong to the monoterpenoid indole alkaloids. Synthetic chemists have been attracted by the interesting biological activities and complex structures of such compounds, which has led to numerous total syntheses [12–19]. In addition, a large number of studies have been reported in which the attractive biosynthesis of these alkaloids is reproduced in flasks [20, 21]. On the other H. Ishikawa (B) Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8675, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_10

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N

OH Et N H H

N H MeO2C

N H

N O

MeO

H HO

OH vinblastine

N Me H

OAc CO2Me

strychnine

O N HO H

MeO

N

N

quinine

N O OH O camptothecin

Fig. 10.1 Typical monoterpenoid indole alkaloids

hand, the biomimetic syntheses reported to date have mostly mimicked biosynthesis only in key reactions. If biosynthesis could be reproduced continuously (like a tree diagram) in a flask, resulting in the exhaustive synthesis of multiple natural products by the same synthetic scheme, it would represent a new bioinspired strategy. Total synthesis can enable libraries of natural products to be prepared and can lead to the discovery of new drug leads through subsequent biological screening. Therefore, in pursuit of new drug candidates, we were challenged to achieve the collective total synthesis of monoterpene indole alkaloids by a bioinspired strategy following a biosynthetic tree diagram approach.

10.2 Biosynthesis of Monoterpenoid Indole Alkaloids In the biosynthesis of MTIAs, several important molecules serve as starting points for branching in the biosynthetic tree diagram. The most important molecule is strictosidine (1), the starting material for over 3000 MTIAs (Scheme 10.1). Strictosidine (1) is biosynthesized from tryptamine, which has an indole moiety, and secologanin (2), which belongs to the monoterpenes [22–30]. The diastereoselective enzymatic Pictet–Spengler reaction mediated by strictosidine synthase directs the C3 position of 1 to the S configuration. Strictosidine (1) has a dense array of highly reactive functional groups (indole ring, β-acrylate moiety, acetal, terminal olefin, secondary amine) and is converted into unique alkaloids with various skeletons. For example, if

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the sugar moiety of 1 is not cleaved and the ring-closing, condensation, and transannular reactions proceed, it is converted into strictosamide (3), neonaucleoside A (4), or cymoside (5). On the other hand, when the sugar chain is cleaved by β-glycosidase, the secondary amine or indole, and β-acrylate or yielded hemiacetal moiety cyclize in various patterns in the early stages of biosynthesis and are transformed into polycyclic indole alkaloids through subsequent cascade reactions (e.g., tetrahydroalstonine (6), dihydrocycloakagerin (7), naucleamide E (8)). Further biosynthesis, starting with β-glycosidase, flows downstream, eventually branching off into a vast number of alkaloids. Strictosidine-related alkaloid glycosides O N H H

N

3S

O 3S

N H H O

-D-Glc

O

H

O

9

4

MeO 11

7

strictosidine synthase

1

NH H

15

OH

MeO

22

18

20

OH

O 21

O

16

OH

O

O

OH

HO

3

O

HO

19

17

O

O

3S

14

O

cymoside (5)

5

N H H

HO 1

H CO2Me

9 11

10

7

-D-Glc

-D-Glc

O

NH2

8

O NH

MeO

neonaucleoside A (4)

N H tryptamine +

5

O O

MeO

strictosamide (3)

6

H

N

O

O

H

H H H N 3S

H

O

-D-Glc O

strictosidine (1)

HO secologanin (2)

-glucosidase

Early stage generated monoterpenoid indole alkaloids

N H H

4

N H

21

H 19

Me

1 H N 17

MeO

O

NMe H

N H O

3

4

N

O 22

OH

O 21

H

H

17

O tetrahydroalstonine (6)

dihydrocycloakagerine (7)

naucleamide E (8)

Scheme 10.1 Biosynthetic pathway of monoterpenoid indole alkaloids

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10.3 Asymmetric Total Synthesis of Secologanin A practical and concise asymmetric total synthesis of secologanin (2) is the first task for a unified chemical transformation along with MTIA biosynthesis. Our developed synthetic route that enabled the quantitative supply of 2 is shown in Scheme 10.2 [31]. The key sequence to construct the chiral dihydropyran ring was the organocatalytic Michael reaction followed by Fukuyama reduction [32]/spontaneous cyclization reaction using 9, which was prepared from commercially available 3-(trimethylsilyl)2-propynal by the Knoevenagel condensation reaction. Thus, when compound 9, which has high electrophilicity, and thiophenyl-containing aldehyde 10 were treated with 3 mol% diphenylprolinol silyl ether catalyst, the desired Michael adduct was obtained with fully controlled stereocenters and with excellent conversion. Subsequent selective conversion of thioester into an aldehyde using triethylsilane and Pd-C followed by spontaneous enolization and cyclization reactions yielded dihydropyran 11 in superb yield. The enantiomeric excess of 11 was >99% ee. The sterically unregulated hemiacetal moiety then became a single isomer when the Schmidt glycosylation reaction introduced the sugar chain [33]. The hydroboration of the alkyne moiety to give an aldehyde followed by the construction of the terminal double bond by sulfoxide elimination resulted in the preparation of secologanin tetraacetate (13) on a decagram scale. Finally, the total synthesis of secologinin (2) was achieved by removing the acetyl group of the sugar chain. 3 mol% N H

TMS

Ph Ph OTMS

SPh TMS Pd/C Et3SiH

PhS(CH2)3CHO (10) SEt

MeO

THF 76% (2 steps from 9)

Et2O

O

O 9 E:Z=1:1

1) AcO Cl3C O

H

O

O MeO

-D-Glc(OR)4

O O

hydrolysis 94%

R = Ac secologanin tetraacetate (13) R = H secologanin (2)

OH

OAc O

2O,

OAc OAc

MS 3 , 87%

2) TBAT, quant. 3) cat. Cp2ZrHCl, 9-BBN; H2O2, 70% 3) PO(OMe)3, 68%

Scheme 10.2 Asymmetric total synthesis of secologanin

O

O 11 (dr = 1:1) > 99% ee

12

NH BF3

MeO

10 Collective Synthesis of Monoterpenoid Indole Alkaloids Using …

215

10.4 Development of Diastereoselective Pictet–Spengler Reaction, and Total Synthesis of Strictosidine Having a sufficient amount of secologanin tetraacetate (13) available, the Pictet– Spengler reaction was investigated to reach key intermediate strictosidine (1) for the bioinspired collective total synthesis of MTIAs [34]. The stereochemistry at the C3 position of the cyclized product, which is completely regulated by strictosidine synthases in biosynthesis, could not be regulated by simple mixing of 13 and tryptamine in the presence of trifluoroacetic acid (TFA). Therefore, we developed a diastereoselective Pictet–Spengler reaction using optically active α-cyanotryptamine (14) derived from tryptophan (Scheme 10.3). Cyano groups attached to adjacent carbons of nitrogen incorporated into the product can be removed under reductive conditions. The first attempt employed (S)-14 as a substrate derived from ltryptophan. As a result, the desired C3S form was obtained with a slight preference (3S/3R = 1.5:1). However, this ratio was not satisfactory for efficient total synthesis. On the other hand, (R)-14 was employed under similar conditions, and, to our surprise, the selectivity was improved dramatically. Thus, the desired C3S product was obtained as almost a single isomer. Next, we investigated whether tryptophan methyl ester had the same tendency. Thus, a Pictet–Spengler reaction was performed using l- and d-tryptophan methyl ester. When l-tryptophan methyl ester was employed, the C3S product predominated at a ratio of 2.6:1, but the stereoselectivity in the case of d-tryptophan was completely reversed compared to (R)-14. Therefore, it was clarified that when α-cyanotryptamine and tryptophan methyl ester were used in the Pictet–Spengler reaction, they proceeded via different transition states. R 5

NH2 N H tryptophan derivative

TFA MS 4Å

N H

5

-D-Glc(OAc)4 O

CH2Cl2

+

MeO

secologanin tetraacetate (13) Tryptophan derivative

R

N H H O

Time

Yield

Product ratio

quant.

Tryptophan derivative

3S : 3R = 1.5 : 1

N H (S)-14

O

Time

Yield

Product ratio

3h

quant.

3S : 3R = 2.6 : 1

3h

quant.

3S : 3R = 1 : 1.8

CO2Me NH2

N H L-tryptophan methyl ester R

CN NH2

N H (R)-14

-D-Glc(OAc)4 O

O

S

3 min

R

NH H

MeO

O

S CN

NH2

N H H

3S

R

3 min

quant.

3S : 3R = >10 : 1

CO2Me NH2

N H D-tryptophan methyl ester

Scheme 10.3 Diastereoselective Pictet–Spengler reaction

216

H. Ishikawa

To clarify the reaction mechanisms, various secologanin derivatives were synthesized and the stereoselectivity of the resulting Pictet–Spengler reaction was monitored (see reference 34 for details). Furthermore, the thermodynamically stable transition state of each tryptophan derivative was determined by DFT calculations (Fig. 10.2). In the transition state, when (R)-α-cyanotryptamine ((R)-14) is employed, a clear hydrogen bond is observed between the carbonyl group of the β-acrylate moiety of secologanin and the proton of the secondary amine, indicating the formation of a unique eight-membered ring. Furthermore, the steric effect of the cyano group at the C5 position and of the chiral center at C15 induced the high diastereoselectivity. On the other hand, in the transition state of l-tryptophan methyl ester, it was found that a hydrogen bond is present in a five-membered ring between the methoxycarbonyl group of the tryptophan and the amine proton; hydrogen bonding with the β-acrylate moiety is not possible as in α-cyanotryptamine (14). In conclusion, the Pictet–Spengler reaction proceeds through completely different transition states for α-cyanotryptamine (14) and tryptophan methyl ester. With the desired diastereoselective Pictet–Spengler reaction in hand, the total synthesis of strictosidine (1) was promoted (Scheme 10.4). Thus, after the Pictet– Spengler reaction with secologanin tetraacetate (13) and (R)-α-cyanotryptamine ((R)14), reductive decyanation using NaBH3 CN in the presence of acid was performed. Strictosidine tetraacetate (16) was then obtained with perfect control of the C3 stereocenter, in excellent yield (85% over two steps). This two-step protocol proceeded OMe 5R

N H H

N H

C

5S

N N H H

N

H O

Me O

15S

MeO

H

O

MeO

O

O O Me

Fig. 10.2 The transition state of diastereoselective Pictet–Spengler reaction

O H

10 Collective Synthesis of Monoterpenoid Indole Alkaloids Using …

H

O

-D-Glc(OAc)4 O

(R)-14 TFA MS 4Å

217 CN

N H H

3S

NH H

-D-Glc(OAc)4 O

O

MeO O

O

reductive decyanation NaBH3CN AcOH

K2CO3 MeOH N H H MeO

O

MeO

13

NH H

HO O

O

O

OH 91% OH

O HO strictosidine (1) Total 20% over 10 steps

N H H MeO

15 85% (2 steps)

NH H

-D-Glc(OAc)4 O O

O strictosidine tetraacetate (16)

Scheme 10.4 Total synthesis of strictosidine

on a gram scale, and the performance of strictosidine synthase in biosynthesis was completely reproduced in the flask. After the removal of the acetyl groups of 16, the first total synthesis of strictosidine (1) was achieved. The product was obtained in just 10 steps and the overall yield was 20%, resulting in a very efficient synthesis [34].

10.5 Bioinspired Total Syntheses of Strictosamide, Neonaucleoside A, and Cymoside Strictosamide (3) has been isolated from several species, such as Nauclea, Palicourea, Ophiorrhiza, Psychotria, Triosteum, Strychnos, Vinca, and Sarcocephalus [35–46]. In addition, a range of biological activities have been reported, including antiplasmodial, antiviral, antiproliferative, anticarcinogenic, acetyl-/butyrylcholinesterase inhibiting, anti-inflammatory, antidiabetic, and antioxidative activities [47–51]. Strictosamide (3) has a lactam ring (D ring) that is constructed by the ring closure of the secondary amine at N4 and the ester moiety at C22 of strictosidine (1) (Scheme 10.5). Thus, lactamization proceeded by simple heating of strictosidine tetraacetate (16) under neutral conditions, and subsequent removal of the acetyl group led to strictosamide (3) in good yield (69%, 2 steps) [34].

218

H. Ishikawa

N D

N H H

2) K2CO3, MeOH 69% (2 steps)

O

H

O

OH O

N H H

4

strictosamide (3)

NH H

O

HO

OH

-D-Glc(OAc)4

OH

O

MeO 22

OH OH

HO

O

O stricosidine tetraacetate (16) 1) 13, NaBH3CN AcOH, MS 4Å MeOH, 79%

O

O OH

O N H H

2) K2CO3, MeOH 74%

N O

H

MeO

MeO

O

O

OH

O O

neonaucleoside A (4) HO

OH OH

Scheme 10.5 Bioinspired total synthesis of strictosamide and neonaucleoside A

In 2003, neonucleoside A (4), consisting of two secologanin units and a tryptamine moiety, was isolated from Neonauclea sessilifolia [52] and Psychotria bahiensis [53], respectively. To synthesize neonaucleoside A (4), strictosidine tetraacetate (16) was treated with 1 equivalent of secologanin tetraacetate (13) in the presence of NaBH3 CN. The condensed product obtained an excellent yield (79%). After the hydrolysis reaction, the desired neonaucleoside A (4) was obtained in 74% yield. As a result, the stereochemistry of the C3 position of 4 was determined by this first total synthesis [34]. Cymoside (5) was isolated from Chimarrhis cymosa [54]. This unusual monoterpenoid indole alkaloid 5 has a hexacyclic skeleton with a propellane-type structure. Eight densely packed asymmetric centers, including three continuous quaternary chiral centers other than a sugar moiety, are present. In addition, a unique biosynthetic pathway involving sequential oxidation and cyclization reactions has been proposed. The first total synthesis of this natural product was achieved by Vincent et al. in 2020 [55]. Our biogenetically inspired total synthesis is shown in Scheme 10.6. Compound 15, with an α-cyano group at the C5 position, was chosen as the substrate for the key bioinspired transformations. Thus, stereoselective oxidation of the indole C7 position was carried out by reaction with mCPBA in the presence of TFA. The oxidation

10 Collective Synthesis of Monoterpenoid Indole Alkaloids Using …

219

proceeded on the opposite side of the C5 cyano group to give the desired β-hydroxy compound. The resulting C7 β-hydroxyl group spontaneously attacked the C17 position of the β-acrylate moiety nucleophilically (Michael reaction), and the generated anion at the C16 position attacked the imine at the C2 position (Mannich reaction). This formal [3 + 2] cycloaddition inspired by biosynthesis led to the construction of the complex ring system of cymoside (5). Another feature is that secondary amine, which are protected in ordinary synthesis, are used as salts to prevent side reactions. The resulting compound 18 was subjected to decyanation and methanolysis of the acetyl group to cymoside (5). The total synthesis of one of the most complex monoterpenoid indole alkaloids was accomplished in only 11 steps by following the biosynthetic pathway [34].

OH mCPBA TFA

CN NH H

N H H

N

CF3CO2 NH2 H

H

42% O

CN

7 2

-D-Glc(OAc)4

MeO

16 17

O O

17

15

H

NC

-D-Glc(OAc)4

O

O

MeO

O

H H N

H

O

-D-Glc(OAc)4

CF3CO2 H H H N NC

O O NH

7

CO2Me

1) NaBH3CN, AcOH, 73% 2) K2CO3, MeOH, 84% HO

H H

OH

O O

O O NH

O NH

H CO2Me

OH

HO

cymoside (5)

Scheme 10.6 Bioinspired total synthesis of cymoside

O

17

CO2Me

17

18

H H N

O

16

2

H

H H

-D-Glc(OAc)4

220

H. Ishikawa

10.6 Synthetic Strategies for Monoterpenoid Indole Alkaloids Produced in the Early Stages of Biosynthesis, and Synthesis of Secologanin Aglycone Silyl Ether The main structural diversity of the monoterpenoid indole alkaloids begins with the removal of glucose from strictosidine (1) by β-glycosidases in biosynthesis (Scheme 10.7) [56–61]. The resulting strictosidine aglycone has several electrophilic and nucleophilic sites and undergoes a variety of skeletal transformations upstream of biosynthesis. For example, ring-opening of the hemiacetal of aglycon 19, then ring closure of the resulting C21 aldehyde with a secondary amine (N4), and subsequent addition of the resulting C17 hydroxyl group to the C19 position, generates a heteroyohimbine-type indole alkaloid (e.g., tetrahydroalstonine (6)). On the other hand, when the ring closure from the C17 hydroxyl group in the biosynthetic pathway of heteroyohimbine-type alkaloids does not proceed, corynantheine-type indole alkaloids are generated (e.g., dihydrocorynantheine (20) and corynantheidine (21)). When N4 is methylated before the hemiacetal opens, aglycone 19 is converted into another skeleton. Thus, after methylation, the hemiacetal is opened, and the resulting C21 aldehyde is generated by nucleophilic attack from the indole nitrogen to provide akagerine-related alkaloids (e.g., akagerine (22)). Furthermore, when the lactam is formed by condensing the N4 and C22 positions before the hemiacetal opens, naucleaoral-related indole alkaloids (e.g., naucleaoral B (23)) are obtained. These unique bioinspired transformations were achieved in the flask by introducing small modifications to the substrate as described below [62]. The supply of strictosidine aglycone (19) was essential to achieving these bioinspired transformations. For the protecting group of the aglycon, a silyl ether was selected, which can be removed under both basic and acidic conditions (Scheme 10.8). Thus, when the hemiacetal of optically active dihydropyran 11 was treated with TBSCl in silver nitrate, only the less sterically hindered α-hydroxy isomer under the equilibrium of hemiacetal reacted to give silyl ether 24 as a single diastereomer. The hydroboration/oxidation reaction of 24 followed by oxidation of the thioether moiety gave 25 in 79% yield. Subsequent sulfoxide elimination provided secologanin aglycon silyl ether 26 on a gram scale. Subsequent sequential diastereoselective Pictet–Spengler cyclization and reductive decyanation gave sufficient strictosidine aglycone silyl ether 28 for further use.

10 Collective Synthesis of Monoterpenoid Indole Alkaloids Using …

4

N H H

N H

21

H 19

MeO

Me

N4 C21 C17-O C19

N4

221

N H H

C21

tetrahydroalstonine (6) heteroyohimbine type

strictosidine (1)

21

H

MeO bioispired transformation

O

N H

20

O 17

4

-glycosidases in biosynthesis

1

N H H

4

NH H

OMe

O 20R; dihydrocorynantheine (20) 20S; corynantheidine (21) corynantheine type

19

OH 21

MeO

O

22 17

O strictosidine aglycone (19)

1

HO

NMe H

N 17

bioispired transformation

H O akagerine (22) akagerine-related alkaloids

4

N H H

C17

N4

C22

O 22

H N1

N

H O

O

naucleaoral B (23) naucleaoral-related alkaloids

Scheme 10.7 Bioinspired transformations from strictosidine aglycone

10.7 Bioinspired Transformations to Nacycline, Cathenamine, and Tetrahydroalstonine from Strictosidine Aglycone Heteroyohimbine-type alkaloids (e.g., cathenamine (34), and tetrahydroalstonine (6)) are found in Rauwolfia, Alstonia, Vinca, and Uncaria species and are characterized by a continuous A to E ring structure [63–76]. The biosynthesis of these compounds involves complex and sequential stereoselective transformations of ringopening and ring-closing reactions starting from a strictosidine aglycon. To reproduce this fascinating biosynthetic reaction in a flask, the silyl group of 28 was removed (Scheme 10.9). First, TBS was removed under acidic conditions. As a result, the basicity of the amine at N4 was inactivated under strongly acidic conditions due to salt formation, and the nucleophilic attack to the oxonium ion generated at C21 proceeded from the indole nitrogen. The complex pentacyclic compound obtained in high yield was an alkaloid known as nacycline (30) [77, 78]. Next, a desilylation reaction was carried out under conditions that maintained the nucleophilicity

222

H. Ishikawa SPh SPh H

TBSCl AgNO3

TMS OH

OTBS O

MeO

86% O

MeO

9-BBN ; H2O2 79%

O

24 single isomer

O 11

O

H

O

PO(OMe)3 o-C6H4Cl2 O OTBS reflux

SPh

H OTBS

MeO

O

74% MeO

O (R)-14, TFA CH2Cl2 quant.

diastereoselective

26

O 25

O

cyclization

CN N H H

3S

MeO

NH H

NaBH3CN AcOH OTBS O

O

27 single isomer

N H H

NH H

OTBS

89%

reductive decyanation

MeO

O

O strictosidine aglycone silyl ether 28

Scheme 10.8 Total synthesis of strictosidine aglycone silyl ether

of amine at the N4 position. Thus, when compound 28 is treated with tetrabutylammonium fluoride (TBAF) in the presence of acetic acid, the generated hemiacetal was smoothly converted into 31 through a ring-opening reaction. The resulting C21 aldehyde underwent rapid ring closure to form a D ring with the amine at N4 to form intermediate 32. The latter was further converted into cathenamine (34) through a stereoselective oxy-Michael reaction from the C17 oxygen to C19 of α, β-iminium ion intermediate 33 via the electronically stable Z-olefin. The isolated yield at this stage was 67% (cathenamine (34) was an unstable molecule that was difficult to handle). The enamine moiety of cathenamine (34) can be stereoselectively reduced with NaBH(OAc)3 . The transformations could be applied from silyl ether 28 in a one-pot operation to deliver tetrahydroalstonine (6) a single isomer in 77% yield.

10 Collective Synthesis of Monoterpenoid Indole Alkaloids Using …

223

Cl NH H

N H H

1

excess HCl OTBS

MeO

dioxane /CH2Cl2

O

1

NH2 H

N H H

87%

21

O

MeO

O

OMe O nacycline (30)

O 29

28

H

N1 C21

17

NH H

H N H 21 O

Total 24% yield over 9 steps

TBAF AcOH

4

H

4

NH H

H

O 21

OH

MeO

N H

21

19 20

N4 C21

MeO

MeO

OH

17

O

33 (Z-olefin)

32

H 19 Me O A

N

B

NH

H

20

67%

E

D 15

H H CO2Me

single isomer

OH

17

O

O 31

C

N H

H

NaBH(OAc)3 77% in one pot from 28

N H H

N H

17-O C19 19

Me

O

MeO 17

tetrahydroalstonine (6)

O cathenamine (34)

Total 21% yield over 10 steps

Total 18% yield over 9 steps

Scheme 10.9 Bioinspired transformations to nacycline, cathenamine, and tetrahydroalstonine

To summarize, total syntheses of nacycline (30), heteroyohimbine alkaloids, cathenamine (34), and tetrahydroalstonine (6) were performed in fewer than 10 steps in a total yield of over 18% (30; total 9 steps, 24% overall yield, 34; total 9 steps, 18% overall yield, 6; total 10 steps, 21% overall yield) [62].

10.8 Bioinspired Transformations to Corynantheine-Type Indole Alkaloids Corynantheine-type alkaloids are characterized by a tetracyclic skeleton consisting of an A–D ring system. Dihydrocorynantheine (20) and corynantheidine (21), which are representative compounds of this class of alkaloids, were isolated from Uncaria, Cephalanthus, Corynanthe, Pausinystalia, Mitragyna, and Pseudcinchona species [79–87], and several biological activities have been reported (the difference in the structure of 20 and 21 is the stereochemistry at the C20 position). For the development of bioinspired transformations toward corynantheine-type indole alkaloids in a flask, the double bond at C18–C19 of 28 was reduced to avoid the E ring closure that occurs in the biosynthesis of heteroyohimbine-type alkaloids describe above

224

H. Ishikawa

(Scheme 10.10). Thus, key substrate 35 was obtained by hydrogenation of 28 in quantitative yield. Then, the silyl group of 35 was removed by TBAF treatment. As a result, after the hemiacetal was opened, N4-C21 cyclization was promoted and 37 was obtained as an E and Z enol mixture. Next, the enamine was reduced by following two different protocols. Thus, when the enamine was reduced with NaBH(OAc)3 to the mixture of 37, a thermodynamically stable α-ethyl group was constructed to provide 38. The C17 enol of 38 was converted into the methyl ether via dimethoxy acetal formation followed by E1cB elimination of methanol to construct the trans methyl acrylate moiety (51%, four steps from 35). In conclusion, dihydrocorynantheine (20), a corynantheine-type indole alkaloid, was synthesized with a total yield of 14% over 13 steps. On the other hand, in the hydrogenation reaction of intermediate 37 with platinum oxide, the β-ethyl group was formed selectively, and methyl etherification on the β-acrylate residue was carried out in the same manner as in 20 to give corynantheidine (21) (44%, 4 steps; total 12% yield over 13 steps) [62].

10.9 Bioinspired Transformations to Akagerine-Related Indole Alkaloids Akagerine (22), dihydrocycloakagerine (44), and other akagerine-related alkaloids are monoterpenoid indole alkaloids found in Strychnos species (Scheme 10.11) [88– 96]. Structural features of this class include methylation at the N4 position and linkage of the indole N1 and C17 positions by a hemiacetal (or acetal) motif. In addition, these alkaloids lack the β-acrylate moiety that most MTIAs possess. According to Brandt’s enzymatic semisynthesis, 22 was formed from the N4-Me stryctosidine derivative via decarboxylation and cyclization of the β-acrylate residue [97]. Therefore, N4-Me strictosidinic acid aglycone 41 was synthesized as a substrate for the bioinspired reaction to induce decarboxylation and seven-membered ring construction reactions in the flask. After several trials to prepare 41, hydrolysis of the β-acrylate residue was found to be required before the Pictet–Spengler reaction. Thus, when secologanin aglycon 26 was treated with triethylamine as a weak organic base in an aqueous acetonitrile solution, the aldehyde was hydrated, and then an intramolecular lactonization proceeded to provide hemiacetal 40 in excellent yield. Hemiacetal 40, which is equivalent to the aldehyde, was directly subjected to a diastereoselective Pictet–Spengler reaction with (R)-cyanotryptamine 14 to construct the C3 chiral center, followed by decyanation and methylation of the N4 to provide the desired 41 in superb yield. The TBS group of strictosidinic acid aglycone 41 was removed with TBAF/AcOH. As a result, the C19–C20 bond was cleaved to yield bisaldehyde 42. Intermediate 42 undergoes further decarboxylation due to the C17 aldehyde and isomerization of the double bond due to the C21 aldehyde (yielding a stable (E)-α,β-unsaturated aldehyde), leading to 43. A seven-membered ring was then formed between N1 and the C17 aldehyde, and akagerine (22) was generated in 88% yield in this domino reaction (11 steps and a total 23% yield from commercially available materials). The natural

10 Collective Synthesis of Monoterpenoid Indole Alkaloids Using …

Pd/C H2

28

NH H

N H H

225

TBAF AcOH

18 19

OTBS

quant. MeO

O 35

O 4

H

N H

H OH

MeO O

MeO

; PtO2, H2

N H

N H H

OH

OH

N H H

17

O 39 1) CH(OMe)3 p-TsOH, MeOH 2) t-BuOK

51% (4 steps)

N H

20

MeO

17

38

36

site selective reduction

20

MeO

OH O

; NaBH(OAc)3

N H

O

N4 C21

37

thermodynamic protonation /reduction

N H H

NH H

21

20

OMe

MeO 17

O 44% (4 steps)

N H H

N H

20

OMe

MeO 17

O dihydrocorynantheine (20)

O corynantheidine (21)

Total 14% yield over 13 steps

Total 12% yield over 13 steps

Scheme 10.10 Bioinspired transformations to dihydrocorynantheine and corynantheidine

226

H. Ishikawa

HO Et3N

26

O

O MeCN:H2O 96%

OTBS 1) (R)-14, TFA 2) NaBH3CN, AcOH

N H H

3) HCHO, NaBH3CN 87% (3 steps)

O 40 dr = 1.2:1

3S

NMe H

HO

OTBS O

O 41 single isomer TBAF AcOH

1

HO

NMe

N H

88%

17

H O

N H H

1

NMe O H 21

NMe H H

N H H 2

N1 C17

21

O

17

O

43

akagerine (22) Total 23% yield over 11 steps

NaBH4 CeCl3 7H2O MeOH/THF 95%

O

21

HO

17

HCl

H

H N

H

O

95% H

HO

42

NMe

N

17

O

17 21

21-O C17

O

NMe H H

21

21-dihydroakagerine (44)

dihydrocycloakagerine (7) Total 21% yield over 13 steps

Scheme 10.11 Bioinspired transformations to akagerine and dihydrocycloakagerine

product akagerine (22) was subsequently transformed into 21-dihydroakagerine (44) by Luche reduction and further converted into the naturally occurring pentacyclic compound, dihydrocycloakagerine (7), by acid treatment (13 steps and a total 21% yield). In these total syntheses, interesting biomimetic domino reactions proceed in the flask to yield the target natural products with high efficacy [62].

10.10 Bioinspired Transformations to Naucleaoral-Related Indole Alkaloids Nauclea species that belong to Rubiaceae plants are a rich source of MTIAs derived from strictosamide (3); these alkaloids have as a common structural motif a lactam

10 Collective Synthesis of Monoterpenoid Indole Alkaloids Using …

227

on the D ring from N4–C22 coupling of strictosidine (1) [98–111]. To achieve a divergent total synthesis of naucleaoral-related alkaloids using bioinspired transformations, lactamization of the D ring was performed using strictosidine aglycone silyl ether 28 as a substrate (Scheme 10.12). Thus, when 28 was heated in toluene without any reagents, strictosamide aglycone silyl ether 45, which is a key molecule of these transformations was obtained in 95% yield. Instead of the deglycosylation of strictosamide (3) in biosynthesis, removal of the silyl group of 45 led to cleavage of the E ring and isomerization of the double bond C18–19, to produce the desired naucleaoral B (23) in excellent yield (90%). The Z geometry of the double bond at C16–C17 of 23 is adopted because of the intramolecular hydrogen bonding. The total synthesis of 23 was performed in only 10 steps and a total yield of 24%. The MTIAs found in Nauclea species were branched further from naucleaoral B (25) into various skeletons. For example, naucleidinal (48), in which the 17-OH and C19 are ring-closed through an oxy-Michael reaction, was synthesized in high yield by heating 23 under aqueous conditions in the presence of pyridine [112]. On the other hand, conversion of α,β-unsaturated aldehydes in 23 into allyl alcohols by Luche reduction resulted in a spontaneous Michael reaction from 21-OH to C17, yielding naucleofficines 50 and 51 with hemiacetal structures. The synthesized naucleofficines were dehydrated at low temperatures and converted into nauclefiline (52). The synthesis was accomplished in 12 steps with an overall yield of 18%. Further over-reduction of 23 with NaBH4 reduced the C17 aldehyde (enol form was depicted) and the C21 aldehyde to afford naucleamide A (53) as the major diastereomer. Finally, bioinspired oxidation and ring-closing reactions at the 21-OH and C3 positions developed by Jia et al. [113] led from natural product 53 to naucleamide E (8). To summarize bioinspired total synthesis of naucleaoral-related alkaloids, six bioinspired transformations from a key lactam natural product 23 to prepare seven MTIAs were discovered. This is a good example of reduction and oxidation reactions occurring in nature that produce a variety of alkaloids in a series of processes like a tree diagram.

10.11 Conclusion In this chapter, the collective total synthesis of natural products following the biosynthetic pathway was described. Biosynthesis is not always the most efficient of the possible synthetic pathways for natural products, but some of them provide high efficiency that is difficult to realize using only our knowledge of chemical reactions (biosynthesis guidance is necessary). In the case of the MTIA presented here, many attractive natural products have been derived from key intermediates such as strictosidine, as shown in the tree diagram. All of the total syntheses described here can usually be synthesized in a short sequence of steps and can be generated in large quantities. In fact, from secologanin tetraacetate (13) or its aglycones, supplied by total synthesis, we have succeeded in the total syntheses of 18 varieties of alkaloids. Fewer than 13 steps are required for all total syntheses, and the total yield of most of

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toluene 4

N H H

NH OTBS H

MeO

4

N H H

95% N4 C22

TBAF AcOH

O 22

90%

17

H

O

O

21

19

22

OTBS

30

O

N D

strictosamide aglycone silyl ether (45) N

N H H NaBH4, 65% (2 steps)

CeCl3 7H2O NaBH4

O

H O

17

H

O

19

naucleaoral B (23) Total 24% yield over 10 steps

N H H

N

3

O 17

H 21

OH

N

N H H

OH

21

OH

49

Total 18% yield over 11 steps

N

N H H

76% (2 steps) 50:51 = 2.1:1

DDQ, 75%

OH

17

H

naucleamide A (53)

pyridine/H2O

O

21-O C17

O

17

H

OH 19

46 N

N H

O

3 17

HO

OH

N

3

N H H

16

H

H

21

21

O H

OH

17

21-O C3

94% (2 steps) dr = 1:1

Total 20% yield over 11 steps

N

N H

TMSOTf O N H O 21

3

N

17-O C19

O

17R; naucleofficine D (50) 17S; naucleofficine III (51)

54

O

O 17

H 20

19

O

Me

O OH H

N

N H H

O 17

H

20R; 47 20S; naucleidinal (48) Total 12% yield over 11 steps

O

naucleamide E (8) Total 13% yield over 12 steps

nauclefiline (52) Total 18% yield over 12 steps

Scheme 10.12 Bioinspired transformations to naucleaoral-related alkaloids

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them exceeded 10%. In addition, because they mimic reactions that occur in nature, they do not require special reactions such as transition-metal-mediated reactions; that is, simple reactions such as oxidation, reduction, and acid or base treatment are often used. We believe that total synthesis using bioinspired strategies will be a new trend in the field of synthetic organic chemistry and natural product chemistry in the future.

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83. Goh, S.H., Junan, S.A.A.: Alkaloids of Uncaria callophylla. Phytochemistry 24, 880–881 (1985) 84. Kanatani, H., Kohda, H., Yamasaki, K., Hotta, I., Nakata, Y., Segawa, T., Yamanaka, E., Aimi, N., Sakai, S.: The active principles of the branchlet and hook of Uncaria sinensis Oliv. Examined with a 5-hydroxytryptamine receptor binding assay. J. Pharm. Pharmacol. 37, 401–404 (1985) 85. Kitajima, M., Misawa, K., Kogure, N., Said, I.M., Horie, S., Hatori, Y., Murayama, T., Takayama, H.: A new indole alkaloid, 7-hydroxyspeciociliatine, from the fruits of Malaysian Mitragyna speciosa and its opioid agonistic activity. J. Nat. Med. 60, 28–35 (2006) 86. Wang, K., Zhou, X.-Y., Wang, Y.-Y., Li, M.-M., Li, Y.-S., Peng, L.-Y., Cheng, X., Li, Y., Wang, Y.-P., Zhao, Q.-S.: Macrophyllionium and macrophyllines A and B, oxindole alkaloids from Uncaria macrophylla. J. Nat. Prod. 74, 12–15 (2011) 87. Liu, Y., Yu, H.-Y., Xu, H.-Z., Liu, J.-J., Meng, X.-G., Zhou, M., Ruan, H.-L.: Alkaloids with immunosuppressive activity from the bark of Pausinystalia yohimbe. J. Nat. Prod. 81, 1841–1849 (2018) 88. Angenot, L., Dideberg, O., Dupont, L.: Isoluloh and structure of akagerine: a new type of indole alkaloid. Tetrahedron Lett. 16, 1357–1358 (1975) 89. Rolfsen, W., Bohlin, L., Yeboah, S.K., Geevaratne, M., Verpoorte, R.: New indole alkaloids of Strychnos dale and Strychnos elaeocarpa. Planta Med. 34, 264–273 (1978) 90. Rolfsen, W.N.A., Olaniyi, A.A., Hylands, P.J.: New tertiary alkaloids of Strychnos decussata. J. Nat. Prod. 43, 97–102 (1980) 91. Marini-Bettolo, G.B., Messana, I., Nicoletti, M., Patamia, M., Galeffi, C.: On the alkaloids of Strychnos. XXXV. The occurrence of akagerine in South American Strychnos. J. Nat. Prod. 43, 717–720 (1980) 92. Verpoorte, R., Joosse, F.T., Groenink, H., Svendsen, A.B.: Alkaloids from Strychnos floribunda. Planta Med. 42, 32–36 (1981) 93. Leclercq, J., De Pauw-Gillet, M.C., Bassleer, R., Angenot, L.: Screening of cytotoxic activities of Strychnos alkaloids (methods and results). J. Ethnopharmacol. 15, 305–316 (1986) 94. Massiot, G., Thepenier, P., Jacquier, M.J., Men-Olivier, L.L., Verpoorte, R., Delaude, C.: Alkaloids of Strychnos johnsonii. Phytochemistry 26, 2839–2846 (1987) 95. Wright, C.W., Bray, D.H., O’Neill, M.J., Warhurst, D.C., Phillipson, J.D., Quetin-Leclercq, J., Angenot, L.: Antiamoebic and antiplasmodial activities of alkaloids isolated from Strychnos usambarensis. Planta Med. 57, 337–340 (1991) 96. Thongphasuk, P., Suttisri, R., Bavovada, R., Verpoorte, R.: Alkaloids and a pimarane diterpenoid from Strychnos vanprukii. Phytochemistry 64, 897–901 (2003) 97. Brandt, V., Tits, M., Penelle, J., Frederich, M., Angenot, L.: Main glucosidase conversion products of the gluco-alkaloids dolichantoside and palicoside. Phytochemistry 57, 653–659 (2001) 98. Hotellier, F., Delaveau, P., Pousset, J.-L.: Naucleidinal and epinaucleidinal, alkaloids of Nauclea latifolia. Phytochemistry 19, 1884–1885 (1980) 99. Mao, L., Xin, L., Dequan, Y.: Alkaloids of Nauclea officinalis. Planta Med. 50, 459–461 (1984) 100. Shigemori, H., Kagata, T., Ishiyama, H., Morah, F., Ohsaki, A., Kobayashi, J.: Naucleamides A-E, new monoterpene indole alkaloids from Nauclea latifolia. Chem. Pharm. Bull. 51, 58–61 (2003) 101. Sun, J., Lou, H., Dai, S., Xu, H., Zhao, F., Liu, K.: Indole alkoloids from Nauclea officinalis with weak antimalarial activity. Phytochemistry 69, 1405–1410 (2008) 102. Sichaem, J., Surapinit, S., Siripong, P.-P., Khumkratok, S., Jong-aramruang, J., Tip-pyang, S.: Two new cytotoxic isomeric indole alkaloids from the roots of Nauclea orientalis. Fitoterapia 81, 830–833 (2010) 103. Li, Q., Zhang, Y., Wu, B., Qu, H.: Identification of indole alkaloids in Nauclea officinalis using high-performance liquid chromatography coupled with ion trap and time-of-flight mass spectrometry. Eur. J. Mass Spectrom. 17, 277–286 (2011)

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Chapter 11

Total Syntheses of Bioactive Oxacyclic Natural Products Yusuke Ogura

Abstract Natural products with cyclic ether structural motifs have attracted considerable attention from natural products and medicinal chemists because of their potentially useful biological activities. This chapter describes the total syntheses of four bioactive oxacyclic natural products, lysidicin A, amphirionin-4, anthecularin, and celafolin B-3. In these synthetic studies, the application of cascade or one-pot reactions helped reduce the number of reaction steps and improve the overall yield. Appropriate and efficient synthetic methods were established to construct the required stereocenters of each natural product based on their three-dimensional characteristics. Keywords Oxacyclic natural product · Total synthesis · One-pot reaction · Lysidicin · Amphirionin · Anthecularin · Diydro-β-agarofuran · Isocleorbicol

11.1 Introduction Oxacycle moieties are characteristic substructures found in many natural products, and ether ring structures have been reported to exhibit various biological activities. For example, paclitaxel (anticancer drug), artemisinin (antimalarial drug), and avermectin (antiparasitic drug) are famous examples of drugs that have been developed through natural product drug discovery programs. Therefore, oxacyclic natural products have attracted considerable attention from medicinal and natural product chemists. As with these great examples, many oxacyclic natural products listed as lead compounds have synthetically challenging structures, which have actively engaged synthetic organic chemists in synthetic studies. In order to supply sufficient amounts of complex natural products, it is important to establish efficient synthetic routes with the minimum number of steps. In addition, to achieve efficient total syntheses, stereocontrolled construction of stereocenters is necessary, which remains challenging even with modern chemical technology. The author focused on syntheses Y. Ogura (B) Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-Ku, Tokyo 113-8657, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_11

235

236

Y. Ogura

O

O

O

HO H

OH

OH

O

O

O

O

O O

HO

O

O

OH

HO O O (+)-celafolin B-3

OH lysidicin A OH

O O O

O OH

(+)-anthecularin

Fig. 11.1 Chemical structures of representative oxacyclic natural products

of lysidicin A, amphirionin-4, anthecularin, and celafolin B derivatives (Fig. 11.1), which are ether ring-containing natural products that exhibit remarkable biological activity. Synthetic studies concentrated on the stereoselective construction of the pivotal chiral centers. In this chapter, details of the synthetic studies undertaken to access complex oxacyclic natural products are described.

11.2 Lysidicin A Lysidicins A (1), B (2), and C (3) are natural products that were isolated in 2006 from Lysidice rhodostegia Hance (Fabaceae), which is traditionally used as a Chinese medicinal plant (Fig. 11.2) [1]. Although the biological activities of these phloroglucinol derivatives have not been clarified, other compounds showing vasodilator activity have been isolated from the plant. After a report on the isolation of 1–3, another series of structurally related compounds, lysidicins D–J, V, and W, were isolated [2–5]. Among the 12 lysidicins isolated, lysidicin A (1) has a unique and distinctive spiro[furan-furofuran] ring system. No analogues of 1 have been reported. In addition, constructing the novel and complex core framework of 1 is a synthetically challenging task; in particular, 1 is known to be converted into 2 and 3 under acidic conditions, making the chemical synthesis of lysidicin A (1) even more interesting. We attempted to establish synthetic protocols to efficiently construct the unusual skeleton of lysidicin A (1). Scheme 11.1 shows the retrosynthetic analysis of lysidicin A (1). The spiro[furanfurofuran] unit, which forms the core framework of this natural product, would be constructed by acetalization of the diketone produced by the oxidative cleavage of

11 Total Syntheses of Bioactive Oxacyclic Natural Products

O

237

HO H

OH

O

O

O O

HO

OH

HO

OH

O OH lysidicin A (1) O

HO H

OH

O O

HO

O

HO

OH

OH

O OH

O

O

OH O

O

HO

O

OH

HO O

OH lysidicin B (2)

OH lysidicin C (3)

Fig. 11.2 Structures of lysidicins A–C (1–3)

the two exo-olefins of diene 4. Triple acylations of the pivotal core framework would introduce three isovaleryl groups at appropriate positions, leading to lysidicin A. The key step would be the triple (single and cascade) Claisen rearrangements of 5, bearing three allyl aryl ethers, to give 4; this step would both deliver three aromatic rings to the correct positions and install the two exo-methylenes. Triether 5 would be obtained from triol 6 and the phloroglucinol derivative 7. The synthesis of 1 commenced with the preparation of precursor 5a for the Claisen rearrangement (Scheme 11.2). First, the allylic alcohol moiety of the diol obtained by reduction of the readily available dimethyl itaconate (8) was protected with a tertbutyldimethylsilyl (TBS) group to give 9. Subsequent iodination of the remaining hydroxy group of 9, followed by reaction with triphenylphosphine, afforded phosphonium salt 10. Wittig reaction between phosphonium ylide, generated by treatment of 10 with n-butyllithium (n-BuLi), and separately prepared dioxinone 11 [6], furnished the protected triol 12. The chemical yield of 12 was 77% from alcohol 9 in the three-step reaction sequence. Exposure of 12 to hydrous acetic acid led to the removal of the TBS and acetalic protecting groups of 12, providing the desired triol 6 in a yield of 84%. The latter was then subjected to a Mitsunobu reaction with phloroglucinol dibenzyl ether 7a [7], to afford the pivotal precursor of the key triple Claisen rearrangement, triallyl aryl ether 5a, in moderate yield. Using the key precursor 5a, conditions for conducting the continuous triple Claisen rearrangement (such as heating in various solvents and addition of a wide variety of Lewis acids) were screened (Scheme 11.3). It was found that the desired

238

Y. Ogura HO H

OH R O O

HO

HO

PO

OH

R OH

lysidicin A (1) (R = isovaleryl)

OP

[O]

R

O

PO

OP

OH

OP

HO

acetalization acylation

P = protecting group

R = isovaleryl equivalent

OP

4

OP

PO

OP

OH

OH

OH

HO

3 X Claisen rearrangement PO

O

OH

6

O

O

+ OP

OP HO

OP

5

7

OP

Scheme 11.1 Retrosynthetic analysis of lysidicin A (1) 1) I2, Imid., PPh3 CH3CN-Et2O, rt

1) DIBAL, THF CO2Me MeO2C

2) TBSCl, NaH 8

OH

TBSO

2) PPh3, CH3CN reflux

9

41% in 2 steps O O O 11 n-BuLi, DME PPh3I

TBSO

HO DEAD, PPh3

O

rt, 84%

O

12 OBn 7a OBn

OH

HO

TBSO

77% in 3 steps

10

THF/H2O/AcOH (1 : 1 : 1)

BnO

OBn

BnO

OBn

O

O

OBn

O OH 6

5a OBn

Scheme 11.2 Preparation of triaryl allyl ether 5a

11 Total Syntheses of Bioactive Oxacyclic Natural Products

239

single and cascade Claisen rearrangement proceeded smoothly upon treatment of 5a with trimethylaluminum, affording diene 4a in high yield. It was noted that the rate of this rearrangement reaction increased over time. This feature was explained by considering that, over time, moisture in the atmosphere would contaminate the reaction vessel, forming a complex of aluminum and water that would act as a catalyst and accelerate the reaction [8]. Subsequently, the three hydroxy groups produced in the rearrangement reaction were protected with acetyl groups, and the two exo-olefins underwent oxidative cleavage with ozone to form diketones. The acyl protection of the hydroxy groups was essential because ozonolysis of non-O-acetylated substrates induced degradation of the substrate. Removal of the three acetyl groups of the diketone afforded the corresponding triphenol, which was then subjected to an acetalization reaction under acidic conditions to furnish the desired spiro[furan-furofuran] skeleton as the major component of a 7:1 separable mixture of diastereomers at the spirocenter. The remaining phenolic alcohol was protected as the benzyl ether to give 13. Although the simultaneous introduction of three isovaleryl groups into spiro[furan-furofuran] 13 was highly challenging because of the fragility of the spiro[furan-furofuran] skeleton against some Lewis acids, after screening a range of reaction conditions, exposure of 13 to a mixture of isovaleryl chloride and AgOTf in methylene chloride at low temperatures promoted the triple Friedel–Crafts acylation to introduce isovaleryl groups to each of the three aryl groups [9]. Global deprotection of the benzyl groups of the corresponding triacylated compound through hydrogenolysis afforded a separable mixture of positional isomers of the isovaleryl groups of lysidicin A (14a and 14b). The desired lysidicin A (1) was not observed in this reaction. Finally, treatment of either 14a or 14b with p-TSA allowed the desired isomerization of the acetal group to be achieved in good yield, which enabled the total synthesis of lysidicin A (1). An efficient synthetic route for lysidicin A (1) was established in this study. The key features of the total synthesis of 1 included the triple Claisen rearrangements of 5a, bearing three allyl aryl ethers, to introduce the three aromatic rings and afford diene 4a, and the triple isovalerylation of the core spiro[furan-furofuran] skeleton utilizing a mixture of isovaleryl chloride and AgOTf. The overall yield was 3.5% from 8 over 15 steps [10, 11]

11.3 Amphirionin-4 Amphirionin-4 (15), a polyketide isolated from the cultured algal cells of the benthic dinoflagellate Amphidinium sp. strain KCA09051 by Tsuda et al. in 2014, promotes the proliferation of mouse bone marrow stromal cells ST2, which can be differentiated into osteoblasts, at very low concentrations (Fig. 11.3) [12]. Currently, the main therapeutic agents used in the medical treatment of osteoporosis improve symptoms by suppressing osteoclast activity and activating osteoblasts. On the other hand, 15 is expected to resolve the imbalance in bone metabolism by stimulating the proliferation

240

Y. Ogura BnO

OBn

4a

7:1 OBn

OBn 1) Ac2 2) O3, CH2Cl2,

OBn

HO

84%

5a

OH

OH

BnO

OBn CH2Cl2

O

OBn

BnO

OBn

Me3Al

O

O

BnO

OBn

OBn

BnO H

OBn

3

72% in 2 steps

O O

BnO

3) K2CO3, MeOH 4) pTsOH, CH2Cl2 in 2 steps

O OBn

BnO 13

O

OBn O 1)

HO H

OH Cl

Ob

OH

AgOTf O O

HO

2) H2, Pd(OH)2 EtOH-EtOAc. rt 62% in 2 steps

O

O OH HO H

OH

O

O O

HO

HO 63% from a 73% from b

OH

HO at a/b = 5 : 4 14a/b

pTsOH DCM/Et2O

O

a

OH O

OH

O OH lysidicin A (1)

Scheme 11.3 Total synthesis of lysidicin A (1)

of osteoblasts. This natural product 15 is a natural seed compound with the potential to lead to the development of therapeutic drugs for osteoporosis-related disorders based on new ideas that differ from conventional methods. In addition, two related compounds, amphirionin-2 (16) [13] and amphirionin-5 (17) [14], were isolated from

11 Total Syntheses of Bioactive Oxacyclic Natural Products

241

OH OH

O 15)

OH H O

O H HO

O H H O (+)-amphirionin-2 (16)

O

H

H

O

O

H

H

OH

O

O 17)

Fig. 11.3 Structure of amphirionin-2, -4, and -5 (15–17)

Amphidinium sp. strain KCA09051 and strain KCA09053, respectively. In particular, amphirionin-5 (17) is as interesting as amphirionin-4 (15) because it strongly promotes the proliferation of osteoblastic MC3T3-E1 cells. In terms of the chemical structure of amphirionin-4 (15), it bears characteristic features such as an all-cistrisubstituted hydroxytetrahydrofuran structure and a polyene side chain containing a skipped diene. Thus, this polyketide is also of great interest from the viewpoint of synthetic organic chemistry. The remarkable biological profile of 15, its attractive structural architecture, and limited availability, prompted synthetic studies that culminated in two total syntheses [15–17]. This author describes a new enantioselective total synthesis of 15 using an eight-pot sequence that incorporates four one-pot transformations. Scheme 11.4 outlines the retrosynthetic analysis of amphirionin-4 (15). The compound would be synthesized by Stille coupling between two segments: vinyl iodide 18 and vinylstannane 19. The exomethylene-containing dihydroxy tetrahydrofuran segment 18 would be obtained from tetrahydrofuranol 20 via acylation with the dioxinone derivative 21 followed by intramolecular alkylation of the resulting keto ester and some additional steps. Compound 21 could be prepared by the alkylation of a dianion derived from 24 with bromide 25. The stannane segment 19, however, could be synthesized via Horner–Wadsworth–Emmons (HWE) olefination between 22 and 23, followed by two-carbon elongation with TMS-acetylene and regioselective hydrostannylation. The synthesis of 15 commenced with the preparation of hydroxytetrahydrofuran derivative 20 and dioxinone derivative 21 (Scheme 11.5). Starting from known allyl alcohol (±)-26 [18], kinetic optical resolution via Sharpless asymmetric epoxidation afforded 27. Treatment of epoxyalcohol 27 with Red-Al® caused regioselective epoxide opening to provide 28. The latter was subjected to iodoetherification, furnishing hydroxytetrahydrofuran 20 with complete all-cis-stereoselectivity. All by-products from the kinetic optical resolution of (±)-26 and the reduction of the epoxide were separated chromatographically in this step. The stereochemistry of the constructed secondary hydroxy group was confirmed by using the modified Mosher’s method after converting 20 into the corresponding MTPA ester 29, with

242

Y. Ogura OH O amphirionin-4 (15)

OH

Stille Coupling

OH

+

I

O

n-Bu3Sn 19

OH

TMS

18 O OH +

OHC

22 O

O 20

+

EtO2C

I

O

P(OEt)2

23

O

+

t-BuO2C

I

21

24

I

Br

O

25

Scheme 11.4 Retrosynthetic analysis of amphirionin-4 (15)

a good enantiomeric excess of 96%. The dioxinone 21 was also synthesized in two straightforward steps. The dianion generated from t-butyl acetoacetate (24) was alkylated with bromide 25 [19], and the product 30 was reacted with sulfuric acid in the presence of acetone and acetic anhydride to furnish the desired dioxinone 21 [20]. For the synthesis of the polyene side chain moiety containing a skipped triene system (Scheme 11.6), a known phosphonate 22 [21] and aldehyde 23 [22] were subjected to HWE olefination to form triene 31 (E/Z = 11:1). Interestingly, changing

OH

OH

OH

Ti(Oi-Pr)4 MS 4Å, CH2Cl2

Red-Al

O

OH 28

27

(±)-26 OR

42% from (R)-25

I

O 20: R = H 29: R = MTPA I

t-BuO2C

t-BuO2C O 24

I2, NaHCO3

I

Br

H2SO4, Ac2O

25

30

I

O

acetone

O

NaH, n-BuLI, THF

68% from 25

Scheme 11.5 Preparation of iodofuranol 20 and dioxinone 21

O

O 21

11 Total Syntheses of Bioactive Oxacyclic Natural Products O

243

23

OHC

40:1

n-BuLi

P(OEt)2

hexane

EtO2C

EtO2C

31

22 DIBAL 22 one pot

TMS-acetylene n-BuLi, CuCN

X

THF

32: X= OH 33: X= Br

NBS, Me2S MS 4Å, CH2Cl2

(n-Bu3Sn)2 n-BuLi CuCN

X

THF 34: X= TMS 35: X= H

60% from 32

KF, aq DMF rt, 88%

45%

n-Bu3Sn 19

Scheme 11.6 Synthesis of vinylstannane 19

the reaction solvent from THF to hexane induced significant improvement in the geometrical selectivity (E/Z = 40:1). Furthermore, the subsequent direct addition of DIBAL to the reaction mixture containing 31 furnished the corresponding alcohol 32 in one pot, in satisfactory yield. The alcohol 32 was then derivatized to the unstable bromide 33, which, without purification, was treated with lithium TMS acetylide in the presence of copper cyanide to afford 34. After removing the TMS group of 34, the corresponding terminal alkyne 35 was treated with lithium bis(tributylstannyl)cuprate, and endo-selective hydrostannylation was performed to afford the desired vinylstannane 19 [23–25]. Coupling of the three fragments 19, 20, and 21 was performed, as shown in Scheme 11.7. First, heating a mixture of 20 and 21 in toluene under reflux gave the corresponding acylated product 36, to which sodium hydride was added to induce intramolecular alkylation, providing 37. Exposure of the reaction mixture containing 37 to a solution of TBSOTf in the presence of triethylamine resulted in the formation of silyl enol ether-protected intermediate 38 as a single geometrical isomer in one pot from 20 and 21. Subsequent reduction of the lactone moiety of 38 proceeded uneventfully to give 39, and benzoylation of the latter followed by removal of the TBS group upon treatment with TBAF afforded enone 40 in one pot. Corey–Bakshi– Shibata (CBS) reduction of the ketone group of 40 followed by reductive removal of the benzoyl group with DIBAL afforded a 25:1 mixture of diol 18 and its C8 epimer, in one pot, which, upon chromatographic purification, furnished 18 in 88% yield. Finally, the Stille coupling of 18 with the right-hand stannane segment 19 completed the asymmetric total synthesis of amphirionin-4 (15). The enantioselective total synthesis of amphirionin-4 (15) was accomplished from (±)-26 in 9.4% overall yield in eight steps [26]. Utilization of four one-pot transformations enabled a short and efficient synthetic route to be established for the potent proliferation promotive polyketide 15 against bone marrow stromal ST-2 cells.

244

Y. Ogura

O

+

20

O

toluene

21

NaH I

reflux

O I

O

36

TBSOTf Et3

O

O

I O

I

61% from 20 one pot

O 38

OH

OH I

O 39

71% from 38 one pot

OTBS (R)-CBS BH3

OBz I

then DIBAL, THF

O 40

OTBS

BzCl, DMAP

DIBAL

O

O

O

O 37

toluene

reflux

OH O

one pot

I

8

OH 18 19, Pd2(dba)3 Ph3As, CuTC

OH

59% O OH

amphirionin-4 (15)

Scheme 11.7 Enantioselective total synthesis of amphirionin-4 (15)

11.4 Anthecularin Malaria is one of the three major infectious diseases in the world. Although the natural product artemisinin has contributed to the development of therapeutic agents for malaria, malaria parasites (Plasmodium falciparum) resistant to the agent have emerged. Therefore, the development of new malaria therapeutics is desirable [27, 28]. Anthecularin (41), isolated from a lipophilic extract of the aerial parts of the Greek–Roman chamomile Anthemis auriculata, is a sesquiterpene lactone that exhibits antimalarial activity against the drug-resistant Plasmodium falciparum K1 strain (Fig. 11.4) [29]. This intriguing biological property of 41 has been attributed to its specific inhibition of PfFabI and PfFabG, key enzymes of the plasmodial fatty acid synthase, which is a type II multiple enzyme complex and differs radically from human type I fatty acid synthase. In addition, biosynthetically related sesquiterpene derivatives 42–44 have been isolated from the same plant [30]. The structural features of anthecularin (41) include a novel tetracyclic fused ring system composed

11 Total Syntheses of Bioactive Oxacyclic Natural Products

245

of oxabicyclo[3.2.1]octene, cyclohexene, and γ-butyrolactone rings, as well as four contiguous asymmetric centers. Thus, the attractive pharmacological profiles and the structural features of anthecularin (41) make it an intriguing synthetic target. Scheme 11.8 outlines a retrosynthetic analysis of anthecularin (41). A crucial point for the total synthesis of this compound would be the stereoselective construction of the doubly allylic tetrasubstituted C8 chiral center. In this synthetic plan, the vinyl addition/ring-closing metathesis (RCM) sequence would be conducted twice in a stepwise manner (48→ 47→ 46→ 45→ 41), although the diastereoselective addition of the second vinyl group to 46 to form 45 could be a challenging task for the stereoselective construction of the C8 chiral center. The optically active bicyclic compound 48 would be obtainable by a sequence of reactions involving the Evans asymmetric aldol reaction between 51 and 49, installation of the C2 side chain using 50, and an intramolecular Claisen-type cyclization to construct the protected cyclic hemiacetal moiety. R

O

O

O

H

O O (+)-anthecularin (41)

O

R = H : (+)-anthecotulide (42) R = OH : (+)-4-hydroxyanthecotulide (43) R = OAc : (+)-4-acetoxyanthecotulide (44)

Fig. 11.4 Structure of (+)-anthecularin (41) and related natural products 42–44

8

etherification RCM

O

8

O O (+)-anthecularin (41)

HO 45

O

OH

OP

OH

Li vinylation

HO

46

stereoselective vinylation

OP

O

RCM HO

Li

HO

47

OH

P = protecting group

O CHO 49

+

I

H

OP

O

+

O

CO2Me

N

50

Scheme 11.8 Retrosynthetic analysis of (+)-anthecularin (41)

O 51

O

2

O

OP 48

246

Y. Ogura

The synthesis of anthecularin (41) commenced with the preparation of the protected bicyclic lactol compounds 56a/b, as key intermediates (Scheme 11.9). First, the Evans asymmetric aldol reaction of aldehyde 49 was attempted; however, attempts to generate the starting aldehyde 49 through oxidation of 3-methyl-3-buten1-ol under various conditions gave only 3-methyl-2-butenal, with an isomerized double bond. Therefore, the starting material was changed to the dibromo alcohol 52 [31]. Oxidation of 52 using 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) afforded the desired dibromo aldehyde, which, without purification, was subjected to Evans asymmetric aldol reaction with the oxazolidinone derivative 51 [32]. Treatment of the resulting aldol with acid led to lactone cyclization, which was followed by the reductive elimination of the two bromines to afford the desired intermediate 53 as a separable mixture of C3 diastereomers (11:1) in 51% yield in four steps from the dibromo alcohol 52. The undesired 3-epi-53 was readily isomerized to the desired isomer 53 by heating in benzene in the presence of DBU (53/3-epi-53 = 10:1). Subsequently, removal of the chiral auxiliary by hydrolysis of 53, followed by reduction of the mixed anhydride derived from the newly generated acid, led to the corresponding primary alcohol, which was protected with a TBS group as lactone 54. Alkylation with iodide 50 of the enolate generated from 54 upon deprotonation with LDA in the presence of HMPA, gave the alkylated derivative; although the stereochemistry at the C2 position was not determined, the alkylated product was obtained as a single isomer. The TBS group on the primary alcohol was removed by treatment with TBAF, and the corresponding alcohol was condensed with formic acid to afford formate 55 in excellent yield. Exposure of 55 to a solution of KHMDS induced an intramolecular Claisen-type reaction, and, upon reaction of the resulting hemiacetalic alkoxide with MeOTf, the desired key intermediate bicyclic lactone 56a was obtained. The configuration of the stereochemical centers was confirmed by observation of NOE correlations, as depicted in Scheme 11.9. Compound 56b, bearing a 2-methoxyethyl protecting group, was also prepared by using a similar procedure. With the key intermediates 56a/b in hand, construction of the C8 chiral center was attempted (Scheme 11.10). First, the nucleophilic addition of one equivalent of vinyl lithium to 56a proceeded smoothly to give 57a. The cyclic hemiacetal 57a was then subjected to RCM conditions, followed by treatment with silica gel to afford an inseparable mixture of 58a and its ring-opened form 59a in a 5:1 ratio. Exposure of the mixture of 58a and 59a to an excess of vinyl lithium allowed the installation of the chiral center at C8 (dr = 17:1). However, the total yield of the three steps from 56 to 60a (17%) was unsatisfactory. On the other hand, exposure of the 3:1 mixture of 58b and 59b to an excess of vinyl lithium in THF successfully furnished 60b from 56b with excellent stereoselectivity (dr 27:1) in a moderate overall yield of 54%; the relative configuration was determined by NOE experiments, as depicted in Scheme 11.10. The 2-methoxyethoxy moiety of 59b might serve as a handle to direct the addition of vinyl lithium, significantly enhancing diastereoselectivity. Treatment of 60b with MsCl in pyridine containing a catalytic amount of DMAP enabled the construction of the tetrahydrofuran ring to afford 61 in excellent yield. Subsequent removal of the 2-methoxyethyl protecting group using

11 Total Syntheses of Bioactive Oxacyclic Natural Products

247

1) TEMPO, aq. NaClO, KBr CH2Cl2 2) 51, n-Bu2BOTf, Et3

Br Br OH

O 3

O

N O

4) Zn, THF, AcOH, rt 51% in 4 steps

52

TBSO

2

55

O 3) HCO2H, EDCI, DMAP CH2Cl2

54

KHMDS, THF, O

then a) TfOMe

OCHO

50 , LDA, HMPA

I O

64% in 2 steps

O

53 1)

2O H2 2) PivCl, Et3 then NaBH4

O

O

O

80% b) TfO(CH2)2OMe

H OR O 56a: R = Me OMe 56b: R =

80%

NOEs

O

O

H H H

O

2

H H

O

H OMe

56a

Scheme 11.9 Preparation of bicyclic lactone 56a/b

zinc bromide was followed by oxidation of the corresponding lactol with tetrapropylammonium perruthenate (TPAP)-N-methylmorpholine N-oxide (NMO) to furnish the desired lactone 62. Finally, RCM of 62 completed the enantioselective total synthesis of anthecularin (41). The specific rotation of the synthetic 41 [[α]D 26 +24.0 (c 0.28, CHCl3 )] was consistent with that of the natural product [[α]D 26 +23.9 (c 0.15, CHCl3 )]. Therefore, the absolute configurations of anthecularin (41) were determined to be 2R,3R,4S,8R, which was consistent with the suggestions previously reported for the synthetic studies of the biosynthetically related natural sesquiterpenoids 42 and 43 [33, 34]. The enantioselective total synthesis of (+)-anthecularin (41) was achieved in 3.9% overall yield in 18 steps from dibromo alcohol 52. The highly stereocontrolled addition of vinyllithium to the lactone 59b, bearing a 2-methoxyethoxy group, enabled the efficient construction of the tetrasubstituted chiral center at C8. In addition, the absolute configuration of anthecularin was determined by this first asymmetric total synthesis to be 2R,3R,4S,8R [35].

248

Y. Ogura

O

Li (1 equiv.)

O

H

O

OR

O 57a: R = Me 57b: R =

OMe

OH

+

OH 58a: R = Me 58b: R =

8

MsCl, DMAP

HO HO 60b

OH

O

2) TPAP, NMO, MS 4Å CH2Cl2 93% in 2 steps

NOEs

H

8

HO H OR O

HO

60a: R = Me, 17% (from 56a, dr 17:1) OMe, 54% 60b: R = (from 56b, dr 27:1) O

94%

OMe

1) ZnBr2, MeCN, rt

OMe

OR

OH 59a: R = Me OMe 59b: R =

OMe

then SiO2, CH2Cl2, rt

THF

HO

OR

OR

Li (5 equiv.)

O

O

Grubbs' II benzene, reflux

H

Et2O,

O 56a: R = Me 56b: R =

OH

61

OH

O

OMe

8

Grubbs 2n cat.

O O

toluene, reflux 72%

O 62

O 4

3

2

O O (+)-anthecularin (41)

Scheme 11.10 Asymmetric total synthesis of (+)-anthecularin (41)

11.5 Celafolins B-1, B-2, B-3 Widely found in tropical and temperate zones around the world, plants of the Celastraceae family are rich sources of bioactive natural products. In China, East Asia, South America, and North Africa, its extracts have long been used as drugs in traditional medicine and agriculture. Among them, many sesquiterpenoids with a dihydroβ-agarofuran (DHβAF; 63) skeleton have been isolated from plants of this family [36–38]. This group of natural products is composed of a characteristic sesquiterpenoid skeleton consisting of a trans-decalin ring fused with a THF ring, and they possess an extremely diverse structure based on the number, position, and stereochemistry of the hydroxy group attached to the common tricyclic skeleton. Moreover, the hydroxy groups are condensed with various carboxylic acids to form a wide variety of its polyesters. To date, more than 500 compounds belonging to the family of DHβAF natural products have been identified. Their various biological activities

11 Total Syntheses of Bioactive Oxacyclic Natural Products 9

OH OH

1 2

7

5

4

249

9

1

3

O

O

dihydro- -agarofuran (63)

OH HO

OH 2

=

HO O 64)

O OR1

O

OR2

O

65: R1 = Ac, R2 = H ((+)-celafolin B-1) 66: R1 = H, R2 = Ac ((+)-celafolin B-2) 67: R1 = Ac, R2 = Bz ((+)-celafolin B-3)

Fig. 11.5 Structure of dihydro-β-agarofuran sesquiterpenoids; (–)-isocelorbicol (64) and celafolins B-1, B-2, and B-3 (65–67)

have attracted many synthetic chemists, leading to many synthetic studies. However, few total syntheses of DHβAF natural products have been reported [39–43]. (–)-Isocelorbicol (64) is a DHβAF-triol isolated from an alkaline hydrolysis product of the seed oil of Celastrus orbiculatus [44], which forms the parent nucleus of DHβAF-triesters, celafolins B-1 (65), B-2 (66), and B-3 (67) (Fig. 11.5) [45]. Although the biological activity of 64 is unknown, the antitumor activity and cytotoxicity of 66, and the neuroprotective activity of 67, have been reported [46, 47]. Isocelorbicol (64), featuring seven densely packed stereocenters and six axially oriented substituents on the decalin ring, has attracted considerable attention from the synthetic community. As a result, four total syntheses of isocelorbicol (64) have been reported [48–51]. However, there is still room for improvement in their syntheses regarding the number of steps, yield, and stereoselectivity. Furthermore, none of the total syntheses of celafolin B derivatives have been reported. The author’s group embarked on the synthesis of isocelorbicol (64) to establish an efficient synthetic route for the construction of the DHβAF skeleton by controlling the stereochemistry, and to develop the first total syntheses of celafolins B-1, B-2, and B-3, the bioactive ester derivative. Scheme 11.11 outlines the retrosynthetic analysis of celafolins B-1, B-2, and B-3 (65–67). The series of derivatives would be derived from isocelorbicol (64) by regioselective acylation of the three hydroxy groups at C1, C2, and C9. Isocelorbicol (64) could be synthesized by RCM of 68, followed by etherification and dihydroxylation. Compound 68 would be obtainable by the reduction of appropriate spirolactone 69 followed by olefin formation via the Wittig reaction. The construction of stereochemistry at the C4-position, which had been a common issue in previous syntheses, would be achieved by the stereoselective 1,4-reduction of the butenolide moiety of 69. Spirolactone 69 would be prepared via the nucleophilic addition of an alkyne to the ketone 70 and subsequent lactone ring formation. A quaternary chiral carbon

250

Y. Ogura O OH OH OR1

O

OR2

regioselective acylation

OH

RCM etherification

O O 65 67) PO

dihydroxylation

(-)-isocelorbicol (64) PO

stereoselective 1,4-reduction 4

HO

decarboxylation olefination P = protecting group

68 PO

Li

R

4

R

O 69

lactone formation

O

semipinacol rearrangement 10

O

O 70

(R )-carvone (71)

Scheme 11.11 Retrosynthetic analysis of celafolins B-1, B-2, and B-3 (65–67)

at C10 of 70 can be installed in a few steps involving a semipinacol rearrangement from (R)-carvone (71). The author set out to synthesize (–)-isocelorbicol (64) (Scheme 11.12). Initially, (R)-carvone (71) was epoxidized stereoselectively to the known compound 72 [52]. Conformational analysis of 72 by computational chemistry suggested it adopted a half-chair conformation due to the effect of the epoxide and isopropenyl group. Upon treatment with vinyllithium, the nucleophilic addition proceeded selectively from the sterically less hindered bottom face, affording 73 as a single isomer. The stereochemistry of 73 was confirmed by NOE correlations, as depicted in the scheme. Upon subsequent treatment of 73 with BF3 ·OEt, the desired semipinacol rearrangement proceeded as expected, furnishing the desired 74 in excellent yield as a single isomer. The stereochemistry of the hydroxy group at C9 was inverted by the Mitsunobu reaction to p-nitrobenzoyl ester 70a, the undesired C9-epimer of which was separated by silica gel column chromatography. Subsequently, the nucleophilic addition of lithium propiolate to 70a gave tertiary alkoxide 75 with complete stereoselection, followed by acylation of the resulting alcohol with methyl malonyl chloride in one pot to afford 76. Upon exposure of 76 to Cs2 CO3 in DMSO in one pot, conversion of the acetylenic malonate 76 into 77 via 5-exo-dig cyclization was conducted to allow efficient butenolide ring formation, providing 77 in 93% yield from 70a in a one-pot operation. The axially oriented vinyl group at C10 would have promoted an axial attack of the acetylide to the carbonyl group of 70a, providing the desired stereochemistry of the tertiary alkoxide 75.

11 Total Syntheses of Bioactive Oxacyclic Natural Products

O

95%

10

H

THF H

77%

72

PNBO

OEt2

DIAD, PPh3, PNBOH

CH2Cl2

O

9

O

51% (C9-epimer 4%)

74

semipinacol rearrangement

70a then O

PNBO Li

HO 73

OH BF3

O

Li

MeOH

(R)-carvone (71)

NOE

H

O

H2O2 NaOH O

251

CO2Me

O

Cl

OMe

CO2Me O

75

PNBO

PNBO CO2Me

Cs2CO3 CO2Me OMe

O 76

O

O

one pot

CO2Me

O

93% from 70a 77

O

Scheme 11.12 Preparation of spirocyclic lactone 77

With the spirobutenolide 77 in hand, the stereoselective construction of the chiral center at C4, a crucial task of this synthesis, was attempted (Scheme 11.13). Treatment of 77 with one equivalent of LiCl in DMSO caused Krapcho decarboxylation to regioselectively remove methoxycarbonyl group at the γ-position, yielding 78 in high yield. Upon exposure of spirolactone 78 to a suspension of a mixture of NaBH4 and triethylamine in THF, 1,4-reduction proceeded in a completely diastereoselective manner to furnish the desired 79 as a single isomer. The bulky hydride complex (NaBH4 /Et3 N) [53] would have approached from the less hindered β-face, avoiding steric hindrance due to the equatorially oriented C10 methyl group. The stereochemistries at C5 and C4 were confirmed unambiguously by NOE experiments with 79, as depicted in the scheme. The subsequent decarboxylation of 79, followed by reduction with DIBAL, afforded lactol 81 in excellent yield. Notably, diethyl ether was required for the chemoselective reduction of 80. To prepare the precursor for the RCM, lactol 81 was subjected to a Wittig reaction utilizing (methylene)triphenylphosphorane (Scheme 11.14). Unfortunately, undesired Grob fragmentation (through the mechanism depicted via 82) product 83 was obtained as the major product, although the desired product 68a was afforded in an unsatisfactory yield of 6%. Fortunately, this unexpected problem was circumvented

252

Y. Ogura PNBO

PNBO CO2Me CO2Me

O 77

LiCl (1 eq.) DMSO, H2O

O

78 PNBO

O PNBO

NaBH4, Et3N

LiCl

4

CO2Me

O

87% stereoselective 1,4-reduction

CO2Me

O

DMSO, H2O

O

79

O O

80

=

DIBAL, Et2O

NOE H CO2Me H PNBO

PNBO

O

H O

O 79

81

OH

Scheme 11.13 Construction of the chiral methyl group at C4

by using the stabilized ylide [(methoxycarbonyl)methylene]triphenylphosphorane, providing 84 in satisfactory yield (E/Z = 6.3:1). RCM of 84 in the presence of a catalytic amount of Grubbs catalyst proceeded slowly at 50 °C, but the starting material 84 was not completely consumed even under reflux in toluene and with the additional catalyst loading. But, conducting RCM under reduced pressure (2.3 kPa) [54, 55] led to complete consumption of 84, furnishing the bicyclic compound 85. Subsequent reaction of 85 with triflic acid led to THF ring formation, which allowed the construction of the DHβAF skeleton. Dihydroxylation of the corresponding double bond followed by deprotection of the p-nitrobenzoyl group completed the total synthesis of (–)-isocelorbicol (64), the structure of which was confirmed by X-ray crystallography. Having established the high-yielding and highly stereocontrolled synthetic route to (–)-isocelorbicol (64), the first total synthesis of celafolins B-1, B-2, and B-3 (65–67) was attempted (Scheme 11.15). According to the X-ray crystallographic and 1 H NMR spectroscopic analyses of isocelorbicol (64), the presence of hydrogen bonding between oxygen in the THF ring and the C9-hydroxy group was suggested, which meant lower reactivity of the hydroxy group at C9 than those at C1 and C2. Indeed, treatment of (–)-isocelorbicol (64) with Ac2 O in the presence of triethylamine and DMAP and with BzCl in pyridine caused the regioselective acylation of the hydroxy group at C1 and C2, respectively, affording the diester 87 in satisfactory yield. Subsequent cinnamoylation of the hydroxy group at C9, however, did not afford celafolin B-3 but gave rise to the migration of the acetyl group. Faced

11 Total Syntheses of Bioactive Oxacyclic Natural Products PNBO

253

PNBO

Ph3PMeBr KHMDS toluene, 0 C to rt

O

HO

OH

81

68a (6% : desired)

PNBO

O PNBO Ph3P

PNBO

PNBO

85

CO2Me

mesitylene, 50 C 2.3 kPa, 76%

HO

OH

84 (E/Z= 6.3:1)

1) TfOH, THF

PNBO

2) OsO4, Py. then NaHSO3 aq. rt, 73%

OH OH

OH OH

89% HO

83 (21%)

Grubbs 2nd cat.

PhMe, reflux 66%

O 81

CO2Me

O

OPNB

82

O 86

OH

K2CO3 MeOH, rt 88%

O ( )-isocelorbicol (64)

Scheme 11.14 Stereocontrolled total synthesis of (–)-isocelorbicol (64)

with this problem, cinnamoylation of the least reactive hydroxy group at C9 was first attempted. Isocelorbicol (64) was converted into acetonide 88, followed by cinnamoylation and subsequent deprotection in one pot to afford cinnamate 89. Exposure of 89 to Ac2 O in pyridine under heating conditions caused mono acetylation, yielding a separable mixture of celafolin B-1 (65: 39%) and B-2 (66: 37%). The conversion of 66 into 65 in 48% yield was possible by heating 66 to reflux with DMAP in pyridine. Finally, benzoylation of 65 completed the total synthesis of celafolin B-3 (67). The highly stereocontrolled total synthesis of (–)-isocelorbicol (64) was achieved in 14 steps from (R)-carvone (71) in 7.4% overall yield, which enabled the first total synthesis of natural dihydro-β-agarofuran triesters, celafolins B1–B3 (65–67) [56]. A semipinacol rearrangement of epoxy alcohol 73 to install a quaternary carbon at C10, and diastereoselective conjugate reduction of the spirocyclic butenolide 78 for the construction of chiral methyl group at C4, were exploited for the high-yielding synthesis of (–)-isocelorbicol (64).

254

Y. Ogura

OH OH 9

1

2

1) Ac2O, Et3N DMAP, THF OH rt, quant.

O OH O O

celafolin B-3 (67)

O

2) BzCl, Py.

O

O 87

(-)-isocelorbicol (64) 2,2-dimethoxypropane CSA CH2Cl2, rt, 81% OH

O

O

OH

O

O

OH then aq. HCl, rt, 91%

O

(Cin = cinnamoyl)

O

88

O 89

OH

O

O 2

O O

DMAP Py., reflux 65: 48% 66: 51%

a) Ac2 65: 39%, 66: 37% b) triethyl orthoacetate CSA, THF, rt

celafolin B-2 (66)

65: 24%, 66: 62%

+ O

O

BzCl, DMAP

O

O

1

O celafolin B-1 (65)

O

O O

OH

O O

79%

O

O celafolin B-3 (67)

Scheme 11.15 Elaboration to dihydro-β-agarofuran triesters, celafolins B1–B3 (65–67)

11.6 Conclusion In this chapter, the total syntheses of lysidicin A, amphirionin-4, anthecularin, isocelorbicol, and celafolin B-3 have been described as examples of synthetic studies of oxacyclic natural products with intriguing biological activities. In these studies, the use of cascade and one-pot reactions helped reduce the number of reaction steps and improve the overall yield. In so doing, appropriate and efficient synthetic methods have been established to construct stereocenters according to the threedimensional characteristics of each natural product. There are many natural organic

11 Total Syntheses of Bioactive Oxacyclic Natural Products

255

compounds with cyclic ether structures in nature, and their chemical structures and biological activities are quite diverse. Such compounds offer a source of endless interest to natural product chemists and synthetic chemists. The author intends to continue synthetic studies on other oxacyclic natural products with interesting chemical structures and biological activities.

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17. Ghosh, A.K., Nyalapatla, P.R.: Total syntheses of both enantiomers of amphirionin 4: a chemoenzymatic based strategy for functionalized tetrahydrofurans. Tetrahedron 73, 1820– 1830 (2017) 18. Kim, Y.G., Cha, J.K.: Stereoselective synthesis of hydroxy-substituted tetrahydrofurans. Tetrahedron Lett. 29, 2011–2013 (1988) 19. Kleinbeck, F., Fettes, G.J., Fader, L.D., Carreira, E.M.: Total synthesis of bafilomycin A1 . Chem. Eur. J. 18, 3598–3610 (2012) 20. Shojaei, H., Li-Böhmer, Z., von Zezschwitz, P.: Iromycins: a new family of pyridone metabolites from Streptomyces sp. II. Convergent total synthesis. J. Org. Chem. 72, 5091–5097 (2007) 21. Hashim, A.-B., Elie, A.-J., Noel, C.: New and efficient synthesis of (E)-diethoxyphosphonyl2-methyl-2-butenal and of Ethyl (E)-4-diethoxyphosphonyl-2-methyl-2-butenoate, important building blocks in retinoid chemistry. Tetrahedron Lett. 36, 393–396 (1995) 22. Vora, K.P., Lochow, C.F., Miller, R.G.: Rhudium-catalyzed hydroacylation of ethylene with 4-pentenal. Reactioins of 4-hexenal-1-d. J. Organomet. Chem. 192, 257–264 (1980) 23. Singer, R.D., Hutzinger, M.W., Oehlschlager, A.C.: Additions of copper cyanide (CuCN)derived stannylcuprates to terminal alkynes: a comparative spectroscopic and chemical study. J. Org. Chem. 56, 4933–4938 (1991) 24. Barbero, A., Pulido, F.J.: The regiochemistry of the stannylcupration of allenes: synthesis of allylstannanes using the lower order cuprate (Bu3 Sn)CuCNLi. Tetrahedron Lett. 45, 3765–3767 (2004) 25. Fuwa, H., Nakajima, M., Shi, J., Takeda, Y., Saito, T., Sasaki, M.: A Convergent synthesis of the C1–C16 segment of goniodomine A via palladium-catalyzed organostannane-thioester coupling. Org. Lett. 13, 1106–1109 (2011) 26. Ogura, Y., Sato, H., Kuwahara, S.: Total synthesis of amphirionin-4. Org. Lett. 18, 2399–2402 (2016) 27. Miotto, O., Amato, R., Ashley, E.A., MacInnis, B., Almagro-Garcia, J., Amaratunga, C., Lim, P., Mead, D., Oyola, A.O., Dhorda, M., et al.: Genetic architecture of artemisinin-resistant Plasmodium falciparum. Nature Genet. 47, 226–234 (2015) 28. Blasco, B., Leroy, D., Fidock, D.A.: Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic. Nat. Med. 23, 917–928 (2017) 29. Karioti, A., Skaltsa, H., Linden, A., Perozzo, R., Brun, R., Tasdemir, D.: Anthecularin: a novel sesquiterpene lactone from Anthemis auriculata with antiprotozoal activity. J. Org. Chem. 72, 8103–8106 (2007) 30. Theodori, R., Karioti, A., Ranˇci´c, A., Skaltsa, H.: Linier sesquiterpene lactone from Anthemis auriculata and their antibacterial activity. J. Nat. Prod. 69, 662–664 (2006) (For correction, 72, 804 (2009)) 31. Cane, D.E., Weiner, S.W.: Cyclization of farnesyl diphosphate to pentalenene. Orthogonal stereochemistry in an enzyme-catalyzed SE’ reaction. Can. J. Chem. 72, 118–127 (1994) 32. Hajra, S., Giri, A.K., Karmakar, A., Khatua, S.: Asymmetric aldol reactions under normal and inverse addition modes of the reagents. Chem. Commun. 43, 2408–2410 (2007) 33. Hodgson, D.M., Talbot, E.P.A., Clark, B.P.: Catalytic asymmetric synthesis of (+)-anthecotulide using enyne and meyer-schuster rearrangements. Org. Lett. 13, 5751–5753 (2011) 34. Hodgson, D.M., Talbot, E.P.A., Clark, B.P.: Synthesis of the anti-tripanosomal agent (±)hydroxyanthecotulide by Cr(II)-catalysed allylation an meyer-schuster rearrangement. Chem. Commun. 48, 6349–6350 (2012) 35. Ogura, Y., Okada, S., Mori, N., Ishigami, K., Watanabe, H.: Enantioselective total synthesis of (+)-anthecularin. Org. Lett. 20, 3888–3891 (2018) 36. Spivey, A.C., Weston, M., Woodhead, S.: Celastraceae sesquiterpenoids: biological activity and synthesis. Chem. Soc. Rev. 31, 43–59 (2002) 37. Gao, J.-M., Wu, W.-J., Zhang, J.-W.: Konishi Y. Dihydro-β-agarofuran sesquiterpenoids. Nat. Prod. Rep. 24, 1153–1189 (2007) 38. Brinker, A.M., Ma, J., Lipsky, P.E., Raskin, I.: Medicinal chemistry and pharmacology of genus Tripterygium (Celastraceae). Phytochemistry 68, 732 (2007)

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Chapter 12

Total Syntheses of Densely Oxygenated Natural Products by Radical-Based Decarbonylative Convergent Assembly Masanori Nagatomo

Abstract Convergent synthetic strategies have been developed that include designing acyl tellurides capable of generating α-hetero carbon radicals and performing intermolecular decarbonylative radical addition reactions to three kinds of unsaturated bonds (C=C, C=N, C=O) via radical exchange under redox neutral conditions. The feasibility of this synthetic strategy was verified by the expeditious and diastereoselective total syntheses of highly functionalized bioactive compounds: manzacidin A, polyoxins, and hikizimycin. The synthetic strategies described here should have broad applications for the synthesis of densely functionalized natural products and pharmaceuticals. Moreover, as exemplified by the synthesis of polyoxins, these new synthetic strategies will accelerate the divergent total syntheses and detailed biological studies of natural products and related compounds to enhance their therapeutic utility. Keywords Convergent synthesis · Natural products · Organic synthesis · Pharmaceuticals · Radical · Total synthesis

12.1 Introduction Bioactive natural products are a significant source of lead structures for developing novel therapeutic agents [1]. A characteristic feature of these natural products is the presence of numerous sp3 -hybridized carbon stereogenic centers. Indeed, the oxygensubstituted functional groups of natural products are three-dimensionally defined by the stereocenters within the carbon skeleton. These functional groups often act as hydrogen bond acceptors/donors during interaction with their target biomolecules. Consequently, natural products that are densely packed with oxygen-substituted stereocenters tend to display more excellent bioactivity than corresponding molecular frameworks containing fewer oxygen atoms. As such, compounds rich in M. Nagatomo (B) Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_12

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oxygen-substituted stereocenters are regarded as promising lead structures for new pharmaceutical products [2]. However, the chemical synthesis of compounds with multiple oxygen-substituted stereocenters is problematic. Current synthetic technologies are unable to generate sufficient quantities of these natural products at an affordable cost yet. As a result, we set out to develop efficient and robust synthetic strategies for natural products with high levels of structural complexity and diversity. Such a strategy requires mild reaction conditions, high selectivity, and excellent functional group tolerance. To achieve this goal, we chose to examine a radical-based chemistry approach. Here, radical-based convergent strategies were developed for the assembly of three highly oxygenated natural products that display potent biological activities (Fig. 12.1). As representative examples, total syntheses of manzacidin A (1, neuroprotective agent), polyoxins (2, antifungal), and hikizimycin (3, anthelmintic) are described. Interested readers can also consult the cited original papers [3–7]. The focus of this chapter is to demonstrate the current state of synthetic chemistry for the total synthesis of complex natural products. I hope this review will encourage the further development of novel synthetic schemes in natural product chemistry.

Fig. 12.1 Retrosynthetic production of α-hetero radicals as synthons from three highly oxygenated natural products

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12.2 Design of Radical-Based Convergent Strategies for the Synthesis of Highly Oxygenated Natural Products Increased complexity of target natural products often necessitates the development of laborious synthetic approaches, which inevitably reduces product yield. Addressing this issue requires the adoption of novel strategies to enhance synthetic efficiency. Currently, the total synthesis of highly oxygenated natural products employs either linear or convergent synthetic strategies. A convergent strategy entails coupling synthetic fragments with preinstalled polar functional groups to generate the target molecule. In general, a convergent approach is favored over a linear approach because the number of synthetic steps is reduced, and complexity in the target molecule can be enhanced during coupling, which diminishes the requirement for functional group manipulation after the coupling reaction [8–10]. The convergent synthesis should entail the stereoselective introduction of polar functional groups into the building blocks to generate C(sp3 )-centers. However, this approach is technically challenging because chemo- and stereoselective C(sp3 )−C(sp3 ) coupling strategies are limited compared to their C(sp2 )−C(sp2 ) counterparts. Carbon-centered radicals are useful reactive chemical species that are widely utilized in complex molecular synthesis [11–13]. These radical species are precious in forming C(sp3 )−C(sp3 ) bonds because the reactions can be performed efficiently under mild conditions with high selectivity and good functional group tolerance. Thus, it was reasoned that convergent assembly of highly oxygenated natural products might be achieved via radical-mediated C(sp3 )−C(sp3 ) coupling reactions. This radical-based convergent strategy was assessed by devising novel synthetic routes to three natural products (Fig. 12.1): manzacidin A (1), polyoxins (2), and hikizimycin (3). These natural products were selected because they comprise highly oxygenated carbon skeletons that elicit potent biological activities. A retrosynthetic approach was adopted to facilitate the assembly of 1–3 by homolytic cleavage to generate the highly functionalized radicals Aa−Ad with α-hetero (alkoxy/amino) functionalities. A key step in the total syntheses of 1−3 involved the formation and coupling of α-hetero radicals Aa−Ad under mild conditions without interfering with the pre-existing functional groups. There are numerous methods for generating α-hetero radical A (X = O/NR1 ) [14]. For example, single-electron reduction of the sulfone B yields radical A (Fig. 12.2). Otherwise, with an X, Y-acetal compound C, where the Y is iodine, bromine, selenide, or telluride, the weak C−Y bond can be cleaved homolytically to form radical A. Alternatively, in a more modern strategy, carboxyl radical intermediate E may be produced via a redox process using photocatalysis from carboxylic acid or carboxylic acid derivative D, which can then undergo decarboxylation to produce radical A. However, the formation of radicals from compounds with complex chemical structures raises concerns as to whether the desired facilities can tolerate the reaction conditions. For example, side reactions may occur before the expected reaction

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proceeds in making a sterically congested bond. Specifically, one-electron reduction and oxidation from A under the reaction conditions may lead to α-hetero anion F and cation H, respectively, negating the radical coupling. Thus, under singleelectron reduction conditions, further single-electron reduction of radical A gives rise to carbanion F, which will readily undergo β-elimination of the adjacent alkoxide to yield vinyl ether G. Alternatively, the X, Y-acetal is inherently unstable, leading to homolytic cleavage of Y to give heterocarbenium ion H. Finally, radical A generated under redox conditions may be further oxidized to give H again. To overcome these constraints, we used Et3 B/O2 as a radical initiator and αheteroacyl telluride 4 as a radical precursor, which can be easily prepared from the corresponding carboxylic acid (Fig. 12.3A) [3, 15–17]. Under mild conditions, A is formed from 4 via radical exchange without reduction/oxidation in this reaction system. Et3 B/O2 generates an Et radical [18], and a subsequent radical exchange reaction via C–Te homolysis transforms 4 to acyl radical I. Because of the favorable orbital interaction between the σ* bond and the adjacent nitrogen/oxygen lone pair (nO ), I undergoes rapid C–CO scission to generate the desired radicals [19, 20]. A also functions as a stable nucleophilic radical due to the orbital interactions with the lone pair [21–23]. Next, coupling of the fragments of the target structures 1−3 must be performed. In this regard, three ways to trap the radicals Aa−Ad were employed (Fig. 12.3B). The nucleophilic radical A adds to electron-deficient C=C, C=N, and C=O bonds before Et3 B, acting as a radical terminator, generates the coupling adducts 6, 8, and 10, respectively. First, radical 1,4-addition of A to the electron-deficient olefin 5

Fig. 12.2 Generation of α-hetero radicals

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Fig. 12.3 Three key reactions of α-hetero radicals generated from α-heteroacyl telluride

and subsequent capture of the resultant oxyl radical J with Et3 B furnishes the boron enolate K, which yields 6 via protonation. Using this strategy, we completed the total synthesis of manzacidin A (1). Second, for the total synthesis of polyoxins (2) and hikizimycin (3), radical 1,2-addition of A to oxime 7 and aldehyde 11 was performed. Although the unstable amidyl radical L and oxyl radical N formed by the addition of A may be easily reversed via β-scission [24], Et3 B converts L and N to more stable polar intermediates M and O, respectively. The hydrolysis of coupling products M and O delivers hydroxylamine 8 and alcohol 10, respectively. The following section summarizes the convergent total syntheses of 1−3, focusing specifically on the C(sp3 )−C(sp3 ) radical coupling reactions of the fragments, their stereoselective outcomes, and advanced intermediate post-transformations. Here, the aim is to illustrate the broad applicability and efficiency of this radical-based convergent strategy for the total synthesis of highly oxygenated natural products.

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12.3 Total Synthesis of Manzacidin A Manzacidin A (1), a novel bromopyrrole alkaloid, was isolated from a marine sponge Hymeniacidon sp. collected at the Manza beach of Okinawa Island, Japan [25]. The structural features of 1 include a γ-aminobutyric acid structure, a 4-bromopyrrole ester, and a unique tetrahydropyrimidine skeleton with two (C4 and C6) tetra- and tri-substituted stereocenters. The unusual structural features of 1, together with its scarcity from natural sources and its recently reported neuroprotective activity, make it an attractive target for total synthesis [26]. The most challenging aspect of the synthetic scheme of the targeted 1 is the assembly of the C6-tetrasubstituted stereogenic carbon center bearing a nitrogen atom. Several well-designed approaches have been used to address this problem [27, 28]. The total synthesis of manzacidin A (1), by utilizing decarbonylative addition of an α-amino radical to acrylate [29] as a key C(sp3 )−C(sp3 ) bond formation reaction, is shown in Fig. 12.4. For this purpose, we designed the enantiopure oxazolidine-acyl telluride 15 as the coupling partner of acrylate 16. The key α-aminoacyl telluride 15 was prepared in one step from the known N-Boc-protected-α-amino acid 13, which is readily obtained in five steps from d-serine [30, 31]. Specifically, N-Boc-protected 2methyl-d-serine can be converted into a reactive ester by condensation with isobutyl chloroformate. Subsequent in situ treatment with PhTeNa, prepared from (PhTe)2 and NaBH4 , gave α-aminoacyl telluride 15 in good yield. Telluride 15 was not degraded in the presence of air, light, or silica gel. Treatment of 15 with 16 at 50 °C under radical coupling conditions, that is, the reagent combination of Et3 B and (Me3 Si)3 SiH under air, gave adduct 18 in 83% yield. In this reaction, hydrogen transfer from (Me3 Si)3 SiH was found to be effective in forming products from α-carbonyl radical intermediates in coupling with acrylates as the radical acceptor. Despite the temporary loss of stereochemistry at the C6 position of radical Aa, which was generated from 15, the β-oriented t-Bu group on the remaining aminal stereocenter leads to an exclusive α-attack of 16, resulting in the complete C6 stereoselectivity of 18 (dr 1:1 at C4) [32]. With the crucial coupling reaction completed, the N-phthaloyl group of 18 was subsequently detached using hydrazine to yield amine 19. The Boc group was then removed to generate cyclic formadine by the addition of CF3 CO2 H and CH(OMe)3 . Next, aqueous HCl was added to the crude mixture to induce hydrolysis of the methyl ester and achieve removal of the pivalaldehyde N,O-acetal, producing tetrahydropyrimidine 20. Finally, using NaH, the 4-bromopyrrole ester was formed from 20 and 21, yielding pure manzacidin A (1) and its C4 epimer upon HPLC purification. This decarbonylative radical coupling offers the advantage of simplicity of operation using mild reaction conditions that are suitable for a wide range of polar functional groups to facilitate the efficient intermolecular formation of hindered bonds. Indeed, this radical coupling strategy enables the convergent syntheses of complex target compounds such as alkaloids, natural or artificial amino acids/peptides, and pharmaceuticals.

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Fig. 12.4 Total synthesis of manzacidin A

12.4 Unified Total Synthesis of Polyoxins Polyoxins J (2a) and L (2b) [33] are nucleoside antibiotics that belong to a class of natural products with a wide range of biological activities (Fig. 12.5) [34]. For example, these compounds are powerful antifungal agents that are non-toxic to mammals. Consequently, polyoxins are attractive agents for treating systemic fungal infections. Polyoxin structure-activity relationship studies have revealed that the C5substituents of nucleobases significantly affect their biological activity. Accordingly, we were interested in preparing artificial analogs with C5-CF3 (2c) and C5-F (2d) functionalities as the target molecules to establish whether these fluorine substitutions modulate the biological activity of 2a and 2b. The densely functionalized structures

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Fig. 12.5 Unified total synthesis of four polyoxins

M. Nagatomo

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2a–d have a common ribofuranosyl α-amino acid and a carbamoylated trihydroxy αamino acid but different pyrimidine nucleobases [35]. A unified synthetic scheme was formulated by introducing four different non-protected pyrimidines. The right- and left-hand α-amino acids were generated via two radical coupling reactions between the α-alkoxyacyl tellurides and a chiral glyoxylic oxime ether [3, 4]. Subsequent condensation and elaboration yielded the target structures. Initially, the α-alkoxyacyl tellurides 23a–d and 28 were, respectively, generated in three steps from commercially available ribonucleosides 22a–d and acetonideprotected l-threitol derivative 27. The radical coupling reactions utilized Oppolzer’s (−)-camphorsultam derivative of glyoxylic oxime ether 23 as the chiral acceptor [36]. When 22a–d were each subjected to 24 and Et3 B in CH2 Cl2 (22a, 22b) or in benzene (22c, 22d) under air at room temperature, a facile radical addition to the C=N bond took place. Adducts 25a–d in 64%–79% yield was generated with the stereoselective introduction of the two C(sp3 )-centers highlighted by the pink and cyan circles. These experiments demonstrated the efficient production of the desired diastereomers 25a–d without affecting the reactive nucleobase. The general applicability of the Et3 B/O2 -mediated reaction was further demonstrated by coupling 28 and 24, which produced the protected α-amino acid 29 with complete control of the two newly formed stereocenters. Stereoselective control of the key radical addition of 23a–d and 28 can be rationalized in terms of the structure of the radical intermediates Ab-a–d and Ac. The methyl group of the bicyclic dioxolane Ab-a–d and the siloxymethyl group of the monocyclic dioxolane Ac prevent the approach of oxime 24 from the opposite face, resulting in the formation of the desired stereocenters shown in pink. In turn, the sulfonamide oxygen atom of 24 is positioned perpendicular to the C=O bond in the preferred conformation to obstruct radical addition from the upper side. Consequently, radical species Ab-a–d and Ac add to the C=N bond specifically from the bottom face of 24 to generate the stereocenters highlighted in cyan. Next, the two α-amino acids were incorporated into the polyoxins 2a–d. Initially, 29 and 25a–d were converted into carboxylic acid 31 and amines 26a–d, respectively. The benzyloxy group of 29 was exchanged with the Boc group during hydrogenolysis in the presence of Boc2 O. The resulting sultam auxiliary was then cleaved by the addition of aqueous LiOH and H2 O2 to yield carboxylic acid 31. Under hydrogenolysis conditions, the benzyloxyamines 25a–d were converted to the primary amines 26a–d, respectively. Condensation of amines 26a–d and acid 31 was then accomplished with 1H-benzotriazol-1-yloxy-tri(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) and 1-hydroxy-7-azabenzotriazole (HOAt) to give 32a–d. The TBS group was then removed from 32a–d using n-Bu4 NF, and the carbamoyl group was attached to 33a–d with p-nitrophenyl chloroformate to furnish the corresponding carbonates. Each of these carbonates was then treated with aqueous NH3 to generate carbamates 34a–d. Next, the hindered camphor sultam of 34a–d was hydrolytically removed by the addition of aqueous n-Bu4 NOH and H2 O2 in dimethoxyethane. The addition of aqueous CF3 CO2 H to the reaction mixture simultaneously removed the acidlabile Boc and two acetonide groups. Consequently, the four compounds 34a–d were

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Table. 12.1 Antimicrobial activity (MIC [μg/mL]) of the four polyoxinsa Strains

Polyoxins 2a (Z=CH3 )

2b (Z=H)

2c (Z=CF3 )

2d (Z=F)

Pyricularia oryzae NBRC5279

8

8

16

32

Trichophyton mentagrophytes TIMM2789

8

8

128

8

Staphylococcus aureus Smith ATCC13709

>128

>128

>128

32

Methicillin-resistant S. aureus MRSA4

>128

>128

>128

32

Vancomycin-resistant S. aureus HIP14300

NDb

NDb

NDb

16

True fungi

Gram-positive bacteria

a Antimicrobial

activities against true fungi and Gram-positive bacteria were determined by the microdilution method. b ND: not determined.

successfully transformed into natural polyoxins 2a and 2b, as well as fluorinated polyoxins 2c and 2d. Notably, the reactive C1' -aminal, C5'' -carbamoyl, and C1'' amide functionalities remained unaffected during these transformations. Preliminary evaluation of the newly synthesized 2a–d showed that the structural alteration of C5–CH3 (2a) and C5–H (2b) to CF3 (2c) and F (2d) markedly modulated the antimicrobial activity of the corresponding parent molecule (Table 12.1). While 2a and 2b showed potent activities against plant and human pathogenic true fungi, 2d was the only compound displaying activity against Gram-positive bacteria, including methicillin-resistant and vancomycin-resistant Staphylococcus aureus. These results demonstrate the applicability of the present radical-based strategy for synthesizing highly oxygenated natural products and their analogs. Moreover, the potential benefits of a unified total synthetic approach for the efficient identification of novel biologically active molecules are evident.

12.5 Total Synthesis of Hikizimycin Hikizimycin (3, Fig. 12.6) is a nucleoside antibiotic composed of a cytosine base, a 3-amino-3-deoxyglucose sugar (kanosamine), and a complex 4-amino-4deoxyundecose sugar containing 1 amino and 10 hydroxy groups (hikosamine) [37, 38]. These rare structural characteristics make 3 the most synthetically challenging compound among the known nucleoside antibiotics [39, 40]. Compound 3 blocks peptide-forming reactions during protein synthesis and is a potent anthelmintic

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Fig. 12.6 Total synthesis of hikizimycin

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agent. The synthetic strategy involved convergently building the hikosamine structure, which includes 10 contiguous stereocenters, by performing a radical coupling reaction between an α-alkoxy radical and an aldehyde. Hikizimycin (3) was retrosynthetically divided into four parts, 36, 38, 41, and 45. While α-alkoxyacyl telluride 36 and aldehyde 38 correspond to the left and righthalf of hikosamine, respectively, bis-TMS-cytosine 41 and 3-azide-3-deoxyglucose derivative 45 are known compounds that could be used to append the cytosine base and kanosamine moieties. First, radical precursor 36 and radical acceptor 38 were generated from dgalactose derivative (35) and d-mannose (37) in nine and six steps, respectively. Next, an unprecedented radical coupling of the highly substituted fragments was performed. A mixture of α-alkoxyacyl telluride 36, aldehyde 38, and Et3 B in CH2 Cl2 was exposed to air at −30 °C to furnish 39-α along with the minor C6-epimer 39-β in 65% combined yield (39-α/39-β = 2.2: 1). Hence, the desired 39-α with its 10 contiguous stereocenters was constructed by stereoselectively generating the hindered C(sp3 )–C(sp3 ) bond under mild reaction conditions. The result also highlights the facile radical initiating and terminating characteristics of Et3 B. Stereoselectivity at the carbon atoms (highlighted in pink and cyan) can be rationalized from the spatial arrangement of the carefully selected protective groups of Ad and 38. Pyran Ad adopts a boat conformation, in which the C1-radical stabilizes secondary orbital interactions with the oxygen lone pair and the co-planar σ*-orbital of the C4– N bond [41, 42]. The bulky β-oriented C4-NPhth of Ad (highlighted in gray) only facilitates α-approach by 38 to install the desired C5α-stereochemistry. By contrast, the steric hindrance with the methyl group of the acetonide of the 6/6-cis-fused bicycle (highlighted in gray) constrains Ad to approach 38, resulting in the desired C6α-stereochemistry. A total synthesis of 4 was concluded by stepwise attachment of the cytosine and kanosamine moieties to 39 and global deprotection. The protective groups were transformed in three reactions prior to conducting the two glycosylation steps under acidic conditions. The secondary alcohol of 39 was protected as the acid-resistant benzyl ether of 40 using N-phenyl-2,2,2-trifluoroacetimidate and TfOH. Chemoselective removal of the acid-labile acetonides of 39 using BF3 ·OEt2 and 1,3-propane dithiol generated the tetraol, which was peracetylated to yield 40. The C1-acetoxy group of polyacylated 40 was chemo- and stereoselectively transformed to α-oriented benzoylcytosine 42 by the addition of bis-TMS-cytosine 41 and TMSOTf, followed by treatment with BzCl and pyridine. After releasing the C6-alcohol from benzyl ether 43 with DDQ, TMSOTf-promoted glycosylation of 44 with trichloroacetimidate 45 gave rise to C12β-kanosamine 47 as the major diastereomer [43]. For the two glycosylation reactions, the C2O- and C13O-benzoyl groups facilitated the transaddition of the incoming nucleophiles to ensure the requisite stereoselectivities. Lastly, all 12 acyl groups of 47 at the 10 hydroxy and 2 amino groups were simultaneously removed by treatment with n-BuNH2 in MeOH. Hydrogenolysis using Lindlar catalyst chemoselectively reduced the C14-azide substituent over the unsaturated cytosine ring to afford hikizimycin (3).

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Overall, the convergent total synthesis of hikizimycin (3) was achieved in 17 steps from 35 without additional carbon extension or oxygen atom introduction. This short synthetic route was accomplished by adopting a novel radical coupling approach involving α-alkoxyacyl telluride 36 and aldehyde 38. Notably, the C5αand suitably introduced C4-NPhth and bis-acetonide structures directed the desired C6α-stereochemical outcomes. Thus, due to its broad functional group compatibility, intermolecular radical addition to aldehydes is advantageous for shortening the synthetic route to target compounds rich in oxygen and nitrogen functionalities [44, 45].

12.6 Summary and Perspective In conclusion, we have developed several novel radical-based strategies for the efficient assembly of highly oxygenated carbon skeletons. Specifically, we successfully realized the efficient convergent total syntheses of three structurally distinct natural products: manzacidin A (1), polyoxins (2), and hikizimycin (3). The crucial α-hetero carbon radical intermediates were generated by utilizing α-heteroacyl tellurides as precursors and Et3 B/O2 as initiators. This common reagent system was utilized for the three different types of coupling reactions. The radical terminating role of Et3 B was exploited, which facilitated intermolecular addition of the α- hetero carbon radicals to electron-deficient C=C, C=N, and C=O bonds to afford 1, 2, and 3, respectively. The rational design of the fragments, including the protective groups, enabled the stereoselectivity of the coupling reactions to be controlled. As exemplified by the three total syntheses, the developed radical-based reactions are neutral (neither oxidative nor reductive) and efficient in generating hindered C(sp3 )–C(sp3 ) bonds in the presence of polar functional groups. These characteristics are key for the synthesis of complex target molecules. Moreover, this strategy facilitates the production of structurally related compounds (e.g., 2a–d) in a unified fashion by simply altering the structures of the corresponding fragments. Taken together, this approach represents a new convergent methodology for the efficient total synthesis of highly oxygenated natural products or their analogs. Hopefully, the radical-based strategy described here will provide new insights for retrosynthetic analysis. Further applications for the synthesis of a wide range of sp3 -rich natural products and related compounds will facilitate a comprehensive analysis of structure-activity relationships [46, 47]. In this respect, additional improvements to make the convergent strategies even more efficient will benefit future studies in the chemical and biological sciences field. Acknowledgements I am profoundly grateful to Professor Masayuki Inoue at the University of Tokyo for his direction, unremitting support, and encouragement. I gratefully acknowledge my highly talented co-workers whose names and contributions are referenced. This research was financially supported by Grants-in-Aid for Scientific Research (C) (JP22K06521) and for Transformative Research Areas (A) (JP22H05341) from JSPS, and a research grant from The University of Tokyo Excellent Young Researcher program and Kowa Life Science Foundation.

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Chapter 13

Nucleophilic Addition to Amides Toward Efficient Total Synthesis of Complex Alkaloids Takaaki Sato

Abstract Nucleophilic addition to amides has been recognized as a promising transformation for the total synthesis of polycyclic alkaloids. The reaction produces substituted amines from easily available amides while avoiding the unwanted high reactivities of amines and use of extra-protecting group manipulations. In this chapter, we introduce the development of two types of amide-selective nucleophilic addition reactions and their applications to total synthesis. The first topic is the total synthesis of gephyrotoxin based on the reductive allylation of an N-methoxylactam. The reaction takes place in the presence of a more electrophilic methyl ester by taking advantage of the N-methoxy group and the Schwartz reagent [Cp2 ZrHCl]. The second topic is the unified total synthesis of stemoamide-type alkaloids by chemoselective assembly of five-membered rings. The most challenging step is an iridium-catalyzed reductive nucleophilic addition to a γ-lactam. A γ-lactone was installed to a γ-lactam carbonyl without affecting the γ-lactone found in tetracyclic protostemonamide, resulting in the total synthesis of pentacyclic protostemonines. Keywords Amide · Chemoselectivity · Gephyrotoxin · Nucleophilic addition · Stemona alkaloid

13.1 Nucleophilic Addition to Amides Recent total syntheses of biologically active natural products require compounds of ever-increasing complexity, especially when applications to drug discovery are considered. Polycyclic alkaloids are among the most attractive synthetic targets due to their promising biological activities [1]. However, their high structural complexity containing basic nitrogen atoms often requires a number of steps and inhibits sufficient supplies of the target molecules. To achieve an efficient total synthesis of polycyclic alkaloids, the proper choice of nitrogen-containing functional groups is crucial T. Sato (B) Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Kohoku-Ku, Yoko-Hama 223-8522, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_13

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Fig. 13.1 Representative functional groups used in the synthesis of polycyclic alkaloids

(Fig. 13.1). For example, amine 1 is the representative functional group used in the synthesis of alkaloids. However, this group often encounters high reactivity involving nucleophilicity, basicity and instability under oxidative conditions. To circumvent the high reactivity of amine 1, a protected derivative, such as a Boc-amine 2, has been widely utilized. However, the use of protecting group manipulations decreases the efficiency of the synthetic route. Another synthetic issue when employing either amine 1 or protected amine 2 is the limited C–C bond formation available at the carbon atom connected to the nitrogen atom, often requiring extra transformations to construct carbon frameworks. Our research group is engaged in a program devoted to the total syntheses of polycyclic alkaloids by taking advantage of amide 3 as a central functional group [2]. Synthesis of amide 3 has been extensively studied, which makes it a promising starting material. Amide 3 remains intact under various reaction conditions and shows higher stability than amine 1, allowing for total syntheses without unnecessary protecting groups. In addition, the amide carbonyl group enables direct C–C and C–N bond formations such as an aldol reaction at the α-position, resulting in the quick construction of carbon frameworks of natural products. The crucial step using amide 3 is reductive nucleophilic addition to give substituted amine 4 embedded in polycyclic alkaloids [3–5]. Reductive nucleophilic addition to amides starts with semi-reduction of amide carbonyl 3 to generate hemiaminal 5, which is converted to iminium ion 6 (Scheme 13.1). The resulting iminium ion 6 undergoes nucleophilic addition with organometallic reagent R1 M to provide substituted amine 4. However, reductive nucleophilic addition to amide 3 is not trivial for the following reasons. In general, the reduction of amide carbonyls requires harsh reaction conditions due to the high stability derived from the resonance effect of the nitrogen atom. The second issue is the instability of the resulting hemiaminal 5, which is often converted to aldehyde 7 and amine 8 instead of iminium ion 6. The over-reduction is a significant issue because iminium ion 6 is more electrophilic than amide 3 itself. These three issues must be overcome at a later stage of the total synthesis. The synthetic intermediates of polycyclic alkaloids have a variety of functional groups. Therefore, reductive nucleophilic addition to amides requires high chemoselectivity. If these issues are successfully addressed, the synthetic route to polycyclic alkaloids can become highly straightforward.

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Scheme 13.1 Nucleophilic addition to amides

13.2 Total Synthesis of Gephyrotoxin 13.2.1 Gephyrotoxin Gephyrotoxin (10) was isolated from the skin extracts of the Colombian tropical poison dart frog, Dendrobates histrionicus, in 1977 (Fig. 13.2) [6]. Structurally, it possesses a tricyclic framework with two distinct side chains. The most conspicuous structural feature of gephyrotoxin (10) is an array of five stereogenic carbon centers, three of which are connected to the nitrogen atom. Its intriguing structure, as well as an array of neurological activities including mild muscarinic activities, inspired a number of synthetic organic chemists in the 1980s, culminating in the landmark first total synthesis by Kishi (1980) [7, 8] based on a set of stereoselective hydrogenations to establish all five stereocenters. Soon after Kishi’s report, the groups of Hart (1983) [9] and Overman (1983) [10] documented their total syntheses, whose key steps were cyclization via the N-acyliminium ion and the Diels-Alder reaction of the amino diene, respectively. In the 2010s, gephyrotoxin (10) was revisited as a synthetic target to demonstrate the utility of new methodologies. The groups of Sato/Chida (2014) [11], Smith (2014) [12], Nemoto/Hamada (2014) [13], and Amat (2015) [14] accomplished the total synthesis of 10 with their own unique approaches. In this section, we introduce the development of amide-selective reductive nucleophilic addition and its application to the total synthesis of gephyrotoxin (10). Fig. 13.2 Structure of gephyrotoxin

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13.2.2 Chemoselective Reductive Nucleophilic Addition to N-Methoxyamides To overcome the three inherent issues shown in Scheme 1.1, we took advantage of the unique properties of N-methoxyamide 11 and the highly oxophilic Schwartz reagent [Cp2 ZrHCl] [15] (Scheme 13.2) [11]. The first step of our reaction is the reduction of N-methoxyamide 11 with the Schwartz reagent to form chelated five-membered intermediate 12. In the well-known Weinreb ketone synthesis, intermediate 12 (R' = Me) is hydrolyzed to give the corresponding aldehyde 15. However, when the appropriate acid is added, activation of intermediate 12 generates N-oxyiminium ion 13, which undergoes nucleophilic addition to form substituted amine 14. In general, N-methoxyamide 11 shows higher electrophilicity than ordinary amides, allowing for a mild reduction. Formation of five-membered intermediate 12 chelated through the methoxy group prevents both unfavorable hydrolysis and extra addition of hydride. In addition, the selection of the reducing reagents is essential. Compared with conventional reducing reagents such as DIBAL-H, the Schwartz reagent is known to exhibit much higher oxophilicity, enabling differentiation of the amide carbonyl group from other carbonyl groups such as esters. Overall, reductive nucleophilic addition becomes highly amide selective and is then applied to the total synthesis of polycyclic alkaloids. The reductive allylation of N-methoxyamides 11 was realized when using allyltributylstannane and a catalytic amount of Sc(OTf)3 (Scheme 13.3). Treatment of a solution of N-methoxyamide 11a in (CH2 Cl)2 with Cp2 ZrHCl (1.6 equiv) at room temperature initiated the semi-reduction of the amide carbonyl group. Subsequent addition of allyltributylstannane and Sc(OTf)3 (20 mol%) gave substituted amine 17a in 86% yield. It is noteworthy that the reaction proceeded without affecting the more electrophilic methyl ester. Although the aliphatic methyl ester is more electrophilic than the aromatic ester, the reaction took place in high yield (17b: 84%). The developed conditions were compatible with a variety of functional groups. The nitro group is known to be sensitive to reductive conditions, but it remained intact (17c: 90%). In contrast, the reduction of the nitrile group with the Schwartz reagent slightly competed with allylation (17d: 68%).

Scheme 13.2 Nucleophilic addition to N-methoxyamides

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Scheme 13.3 Chemoselective reductive allylation of N-methoxyamides. Reaction conditions: 11 (1 equiv), Cp2 ZrHCl (1.6 equiv), (CH2 Cl)2 (0.1 M), rt, 10 min; CH2 =CHCH2 SnBu3 (1.2 equiv), Sc(OTf)3 (20 mol %), rt, 1 h. Yields of the isolated product after purification by column chromatography. a) Sc(OTf)3 (30 mol %) was used

An aryl bromide did not disturb the reaction, which highlighted its utility upon further transformation with transition-metal-catalyzed reactions (17e: 92%). Conventional protecting groups of amines such as carbamate and sulfonamide were well tolerated (17f:87%; 17g: 90%). Concerning the hydrozirconation of double bonds, the nucleophilic addition took place in the presence of a terminal olefin in good yields (17h: 87%). Use of a Lewis acid was essential to activate the five-membered chelated intermediate, but the acetal group was not affected under these acidic conditions (17l: 91%). The N-oxyiminium ions derived from N-methoxyamides were applicable to C–C bond formation (Table 13.1). Reductive propargylation with allenylstannane introduced the terminal acetylene, which could undergo further transformations such as a click reaction (Entry 1). The reductive Strecker reaction took place with TMSCN in 81% yield (Entry 2). A Friedel-Crafts-type reaction was possible when using an electron-rich N-methylindole (Entry 3). Mukaiyama-type Mannich reactions with a silyl ketene acetal and a silyl enol ether proceeded in good yields (Entries 4–6). The vinylogous Mannich reaction with a siloxyfuran installed a butenolide group in 78% yield (Entry 7).

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Table 13.1 Variation of the nucleophile in the reductive nucleophilic addition to Nmethoxyamidesa

Entry

Nucleophile

Product

Yield [%]b

1

71

2 3c

81 75

4d

88

5d

83

6

74

7

78 (dr = 1.6:1)

a 11a

(1 equiv), Cp2 ZrHCl (1.6 equiv), (CH2 Cl)2 (0.1 M), rt, 10 min; nucleophile (1.2 – 2.0 equiv), Sc(OTf)3 (10 mol %), rt, 1 h. b Yield of isolated product after purification by column chromatography. c The reaction was performed with Sc(OTf) (20 mol %) at –40 °C. d BF ·Et O (1.6 equiv) was 3 3 2 used instead of Sc(OTf)3

13.2.3 Total Synthesis of Gephyrotoxin With the promising reductive nucleophilic addition to N-methoxyamides in hand, we turned our attention to the total synthesis of gephyrotoxin (10) (Scheme 13.4). Our central strategy was the utilization of a methoxy group as a reactivity control element for amides [16]. The carbonyl oxygen atom is the most nucleophilic site in ordinary amide 25. However, N-methoxyamide 11 possesses increased nucleophilicity at the nitrogen atom by incorporation of the methoxy group in amide 25 (Effect A). As described in the above section, the N-methoxy group also increases the electrophilicity of the amide carbonyl group (Effect B) and exhibits a chelation effect (Effect C). Our basic vision toward the total synthesis was the application of these unique reactivities to develop a new transformation of amides, which was not realized without the assistance of the methoxy group. Ultimately, the use of these reactivities enabled us to achieve the concise total synthesis of gephyrotoxin (10). The first key reaction was the direct coupling reaction of N-methoxyamide 26 with aldehyde 27 to give N-methoxylactam 29. In general, intermolecular condensation

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Scheme 13.4 Synthetic plan for the total synthesis of gephyrotoxin

between an ordinary amide and an aldehyde is highly challenging due to the poor nucleophilicity of the amide nitrogen atom. However, the increased nucleophilicity of the amide nitrogen in 26 enabled direct coupling with aldehyde 27. The resulting N-acyliminium ion 28 underwent intramolecular allylation to give N-methoxylactam 29. The second key reaction was reductive allylation of N-methoxylactam 30. If the lactam selectivity is realized in the presence of the methyl ester, the method reduces the number of steps in the total synthesis. Our synthesis commenced with the condensation of 4-pentenoic acid 34 with N-methoxyamine to give N-methoxyamide 35 (Scheme 13.5). The allylsilane was introduced by a cross-metathesis reaction with the Grubbs II catalyst to give 26 in 61% yield as an E/Z diastereomeric mixture, which ultimately proved to have no effect on the stereoselectivity in the subsequent cyclization. Addition of BF3 ·Et2 O to a solution of N-methoxyamide 26 and aldehyde 27 in CH2 Cl2 at –20 °C provided N-methoxylactam 29 in 79% yield as a single diastereomer. Interestingly, a control experiment using N-methyl-substituted amide instead of N-methoxyamide 26 did not provide the corresponding lactam, which confirmed the critical role of the Nmethoxy group as a reactivity control element. Ozonolysis of the terminal olefin in 29 and subsequent Ando Z-selective olefination [17] of aldehyde 36 gave a mixture of 37 and 38 in 84% yield over two steps. Stereocontrol of the Z-configuration was crucial in the subsequent radical cyclization. Bicyclic lactam 30 was obtained in 97% yield as a single diastereomer under conventional radical conditions. The Zconfiguration likely induced steric repulsion between the N-methoxyamide and the carbomethoxy group in conformer 39', and the Z-enoate would point outside the lactam ring, resulting in the desired stereoselective cyclization to give 30. With bicyclic lactam 30 in hand, we turned our attention to the crucial reductive allylation (Scheme 13.6). DIBAL-H reduction and subsequent allylation using allyltributylstannane and Sc(OTf)3 (1.3 equiv) [18] provided the desired product 33

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Scheme 13.5 Synthesis of bicyclic lactam 30 through amide-aldehyde coupling

Scheme 13.6 Chemoselective reductive allylation of N-methoxylactam 30

and its diastereomer 40 in 14% combined yield (dr = 2.5:1). Initially, we expected that DIBAL-H would differentiate the N-methoxylactam from the methyl ester due to the enhanced electrophilicity caused by the N-methoxy group. Although the electrophilicity of the N-methoxylactam was slightly higher, the reduction of the methyl ester also competed to give aldehydes 41 and 42 in 36% combined yield. In contrast, the use of the Schwartz reagent achieved complete lactam selectivity, giving allylated products 33 and 40 in 82% yield (33:40 = 4.9:1) without producing aldehydes 41 and 42. In addition, after reduction with DIBAL-H, the subsequent allylation required 1.3 equivalents of Sc(OTf)3 . In contrast, allylation after reduction with the Schwartz reagent allowed for use of a catalytic amount of Sc(OTf)3 (30 mol %). Thus, we achieved the amide-selective reductive allylation by taking advantage of the N-methoxy group and the oxophilic Schwartz reagent.

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The remaining issues regarding the total synthesis were construction of the pyrrolidine ring and installation of the two side chains (Scheme 13.7). Hydroborationoxidation of the terminal olefin in 33 with thexylborane provided primary alcohol 43, which underwent Parikh-Doering oxidation and a one-pot Wittig reaction [19] to give unsaturated ester 44. The N-methoxy group used as a reactivity control element was smoothly cleaved with activated zinc in AcOH/H2 O at 60 °C. A subsequent azaMichael addition gave tricyclic compound 45 in 98% yield with 1.8:1 diastereoselectivity. The next transformation required solving two challenging tasks: (i) differentiation of the methyl ester from the t-butyl ester and (ii) semi-reduction of the methyl ester. After extensive studies, the use of NaAlH(Ot-Bu)i-Bu2 (SDBBA) [20] overcame these issues in a single operation to provide aldehyde 46 in 87% yield. The enyne side chain was installed by the Z-selective Wittig reaction of 46, followed by Sonogashira coupling. Finally, reduction of the t-butyl ester in 48 was realized by sequential reduction with DIBAL-H and NaBH4 . Thus, the total synthesis of gephyrotoxin (10) was achieved in 14 steps with an overall yield of 9.4% from commercially available 4-pentenoic acid.

Scheme 13.7 Total synthesis of gephyrotoxin

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13.3 Unified Total Synthesis of Stemoamide-Type Alkaloids 13.3.1 Stemoamide-Type Alkaloids and Their Synthetic Plan Approximately, 200 Stemona alkaloids have been isolated from Stemonaceae plants. These can be structurally classified into eight groups [21]. The stemoamide-type alkaloids are one of the largest groups in the Stemona alkaloids and comprise tricyclic, tetracyclic and pentacyclic frameworks (Scheme 13.8a). Tricyclic stemoamide (49) [22] including a γ-lactam and a γ-lactone is the representative natural product in this group. Tetracyclic derivatives of stemoamide (49) have an additional γ-lactone. For example, protostemonamide (50) [23] possesses an additional γ-lactone on the γ-lactone side of stemoamide (49). Pentacyclic natural products as represented by protostemonine (51) [24] have two γ-lactones on stemoamide (49). As seen in bisdehydroprotostemonine (52), [25] another structural feature in this class is the pyrrole group, which could be generated by oxidation of the pyrrolidine rings in the biosynthesis. Extracts of Stemonaceae plants have been traditionally used in Chinese and Japanese folk medicines as antitussive and insecticidal agents. Recent biological studies revealed their potential for drug discovery. In particlular, protostemonine (51) has been investigated as an anti-inflammatory agent without detectable toxic effects on vital organs [26]. However, the scarcity of the natural stemoamide-type alkaloids has precluded systematic structural-activity relationship studies which would be solved by collective total synthesis. To achieve unified total synthesis of stemoamide-type alkaloids, we envisioned the chemoselective assembly of five-membered building blocks (Scheme 13.8b). The quickest way to pentacyclic natural products would be a building-block strategy through three coupling reactions of four five-membered heterocycles such as γlactone 57 and γ-lactam 58. Although the building-block strategy has been recognized as an efficient method, in order to apply it to the synthesis of stemoamide-type alkaloids, the chemoselectivity issue between γ-lactone 57 and γ-lactam 58 must be solved. Another challenge to unified total synthesis is the construction of the totally substituted butenolide 59, which is widely observed in this class of natural products. This motif is sterically hindered and highly oxygenated with two tetrasubstituted olefins. Specifically, stereocontrol of the 1,2-enediol structure is challenging even with the tools available in modern organic synthesis. The third issue is the direct oxidation of the pyrrolidine ring in 60 to pyrrole derivative 61 at the last stage of the synthesis. In this section, we introduce the development of tertiary amide-selective nucleophilic addition [5, 27–29] and its application to the total synthesis of tricyclic, tetracyclic and pentacyclic stemoamide-type alkaloids [30, 31].

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Scheme 13.8 a Representative stemoamide-type alkaloids and their synthetic plan, b Synthetic challenges for total synthesis

13.3.2 Iridium-Catalyzed Reductive Nucleophilic Addition to Tertiary Amides Although we have achieved reductive nucleophilic addition to N-methoxyamides, unified total synthesis of stemoamide-type alkaloids requires reaction conditions to be applicable to more general tertiary amides (Scheme 13.9). In 2009, the Nagashima group reported hydrosilylation of tertiary amide 62 by using a catalytic amount of the Vaska complex [IrCl(CO)(PPh3 )2 ] and (Me2 HSi)2 O to give enamine 64 via N,Oacetal 63 [32]. We considered that addition of an appropriate acid would convert nucleophilic enamine 64 to electrophilic iminium ion 65, which could undergo nucleophilic addition to provide substituted amine 66. To test our hypothesis, the reductive Mannich reaction of tertiary amides was surveyed by using tertiary amide 67a (Scheme 13.10). Treatment of a solution of 67a with 1 mol% of IrCl(CO)(PPh3 )2 and (Me2 HSi)2 O in toluene initiated the

Scheme 13.9 Design of reductive nucleophilic addition to tertiary amides

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Scheme 13.10 Chemoselective reductive nucleophilic addition to tertiary amides. Reaction conditions: 67 (1 equiv), IrCl(CO)(PPh3 )2 (1 mol %), (Me2 HSi)2 O (1.5 equiv), toluene (0.2 M), rt, 30 min, then CH2 =C(OTBS)(OEt) (1.2 equiv), PPTS (1.1 equiv), rt, 1 h. Yields of isolated product after purification by column chromatography

hydrosilylation at room temperature. The subsequent Mannich reaction proceeded with silylketene acetal in the presence of PPTS. This one-pot reaction took place in 85% yield without affecting the methyl ester due to the high oxophilicity of the iridium-catalyzed hydrosilylation (68a). The reductive Mannich reaction was applicable in the presence of an aliphatic methyl ester (68b: 94%). Other electrophilic functional groups including nitro, nitrile and aryl bromide were reasonably tolerated (68c: 64%; 68d: 74%; 68e: 79%). Carbamates such as a Boc group were successfully differentiated from the tertiary amide despite having similar electrophilicity (68f: 92%). While a sulfone amide had an acidic proton, it did not inhibit the reductive Mannich reaction (68g: 91%). Hydrosilylation of double bonds and hydrolysis of acetal groups were not observed under these conditions (68h: 93%; 68i: 93%). Although iminium ions generated from tertiary amides are less electrophilic than N-oxyiminium ions derived from N-methoxyamides, the developed conditions allowed for the use of various nucleophiles if the appropriate acids were selected (Table 13.2). The Mannich reaction using the sterically hindered dimethyl silyl ketene acetal provided product 69 in 75% yield without disturbing the amide selectivity. The vinylogous Mannich reaction installed the five-membered ring, which was applicable to the synthesis of stemoamide-type alkaloids (70: 83%). Allylation and cyanation

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Table 13.2 Variation of nucleophiles in reductive nucleophilic addition to tertiary amidesa

entry 1 2 3 4

RM

acid additive BF3·Et2O MeCN

products

PPTS none TFA MeCN Sc(OTf)3d none

Yield (%)b 75 83c 77 87

a 67a

(1 equiv), IrCl(CO)(PPh3 )2 (1 mol %), (Me2 HSi)2 O (1.5 equiv), toluene (0.2 M), rt, 30 min, then RM (1.2–3.0 equiv), acid (1.1 equiv), additive (none or 0.2 M), rt, 1 h. b Yields of isolated product after purification by column chromatography. c The diastereomeric ratio was 1.6:1. d 10 mol % of Sc(OTf)3 was used

were possible when utilizing allyltributylstannane and TMS cyanide, respectively (71: 77%; 72: 87%).

13.3.3 Unified Total Synthesis of Stemoamide-Type Alkaloids 13.3.3.1

Gram-Scale Total Synthesis of Tricyclic Stemoamide

To achieve total synthesis of pentacyclic alkaloids, a sufficient supply of tricyclic stemoamide (49) was essential. Although a number of total syntheses of stemoamide (49) have been reported due to its relatively simple structure [33], a gram-scale total synthesis had not been achieved before our report (Scheme 13.11). Our synthesis commenced with DIBAL-H reduction of ethyl ester 73 to provide aldehyde 27, which was subjected to Trost’s enantioselective alkynylation [34], giving propargylic alcohol 74 in 78% yield with 98%ee. The semi-hydrogenation of 74 with Sajiki’s catalyst, Pd/PEI [35], followed by acid-promoted cyclization gave chiral γ-lactone 53 in 75% yield. We then investigated the first coupling reaction of γ-lactone 53 with siloxypyrrole 75 derived from the five-membered lactam. Treatment of γ-lactone 53 and siloxypyrrole 75 with SnCl4 promoted the Friedel-Crafts-type Michael reaction to form bicyclic intermediate 76. In this carbon–carbon bond formation, use of 0.5

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Scheme 13.11 Gram-scale total synthesis of stemoamide by vinylogous Michael/reduction sequence

equivalent of SnCl4 was essential. When using more than 0.5 equivalent, unfavorable retro-Michael addition was observed. Addition of TiCl4 , Et3 SiH and MeOH in a one-pot process initiated protonation at the α-position of the amide carbonyl, giving enamide 77, which underwent reduction to give bicyclic compound 78 in 90% yield with 4.4:1 diastereoselectivity. Although the factors for controlling the stereoselectivity have yet to be elucidated, choice of the DMPM group [36] was crucial in the successful coupling regarding both yield and diastereoselectivity at the C9a carbon center. The cleavage of the DMPM group took place with TFA in the presence of anisole as a cation-trapping agent. The seven-membered ring of 80 was constructed with NaH, which was premixed with TMSOTf (20 mol %) and TBAI (10 mol %) to remove the residual sodium hydroxide. Finally, the regioselective and stereoselective methylation of 80 developed by Narasaka provided stemoamide (49) [37]. Thus, the coupling reaction of chiral γ-lactone 53 with γ-lactam derivative 75 enabled the total synthesis of tricyclic stemoamide (49) in 7 steps from commercially available ethyl ester 73 in 26% yield on a gram scale (49: 1.07 g).

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13.3.3.2

289

Total Synthesis of Tetracyclic Protostemonamide

The next stage was the total synthesis of tetracyclic protostemonamide (50) from stemoamide (49) by lactone-selective coupling (Scheme 13.12). Although the lactone selectivity issue was easily solved by taking advantage of the inherent electrophilicity of the γ-lactone, the building-block synthesis approach was not trivial due to the totally substituted butenolide. Ultimately, we developed a stereodivergent three-step synthesis through silver-mediated elimination. First, the reductive vinylogous aldoltype reaction installed a five-membered ring on the lactone side of stemoamide (49). Treatment of stemomide (49) with DIBAL-H initiated a lactone-selective partial reduction to generate hemiacetal derivative 81. After the remaining DIBAL-H was quenched with benzaldehyde, addition of siloxypyrrole 82 and BF3 ·Et2 O gave tetracyclic intermediate 83 in 70% yield in a one-pot process. Although the product was obtained as a mixture of four diastereomers, they could potentially be converted to the same tetrasubstituted olefin. The diastereomeric mixture was deprotonated with NaHMDS at –78 °C, followed by regioselective bromination, to afford bromides 84 in 96% combined yield. We found that the selective synthesis of either protostemonamide (50) or isoprotostemonamide (85) was feasible by changing the kinetic or thermodynamic conditions. The kinetic elimination of bromides 84 with AgOTf at 40 °C gave protostemonamide (50) containing a Z-tetrasubstituted olefin as the major product (85%, 50:85 = 3.0:1). In contrast, after the formation of protostemonamide (50), subsequent addition of TfOH at 40 °C promoted isomerization of the tetrasubstituted olefin to give isoprotostemonamide (85) in 80% yield with 4.0:1

Scheme 13.12 First total synthesis of tetracyclic protostemonamides including totally substituted butenolide

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diastereoselectivity as a thermodynamic product. Thus, we achieved stereodivergent total synthesis of protostemonamide (50) and isoprotostemoamide (85) bearing totally substituted butenolide in 10 steps from a commercially available compound.

13.3.3.3

Total Synthesis of Pentacyclic Stemoamide-Type Alkaloids

With tetracyclic protostamonamide (50) in hand, the remaining tasks were the lactam-selective coupling reaction and the direct oxidation of the pyrrolidine ring (Scheme 13.13). Treatment of protostemonamide (50) with 1 mol% of the Vaska complex [IrCl(CO)(PPh3 )2 ] and (Me2 HSi)2 O initiated the hydrosilylation of the lactam carbonyl group. Subsequent addition of siloxyfuran 86 in the presence of 2-nitrobenzoic acid provided pentacyclic products 87 and 88 in 56% combined yield with 1.2:1 diastereoselectivity. The reaction took place with complete stereocontrol at the C3 carbon center, but slight diastereoselectivity was observed at the C18 carbon center. However, DBU-promoted isomerization was found to be possible at C18. It is noteworthy that the iridium-catalyzed coupling reaction proceeded in a highly lactam-selective fashion in the presence of a more electrophilic butenolide. The stereoselective and regioselective hydrogenation of 87 with Rh/Al2 O3 (20 wt%) gave protostemonine (51) in 93% yield as a single diastereomer. The direct oxidation of the pyrrolidine ring in 51 proved to be highly challenging due to the presence of the two γ-lactones. The epimerization at C18 in 51 was problematic since elimination and subsequent recyclization pathway could occur. Finally, we found the use of an

Scheme 13.13 First total synthesis of pentacyclic protostemonines through lactam-selective reductive nucleophilic addition and direct oxidation of pyrrolidine

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excessive amount of MnO2 at –20 °C suppressed the epimerization, resulting in the isolation of bisdehydroprotostemonine (52) in 53% yield. Thus, we achieved chemoselective assembly of five-membered building blocks. The three chemoselective coupling events enabled quick access to tricyclic, tetracyclic and pentacyclic stemoamide-type alkaloids within 13 steps from a commercially available ethyl ester.

13.4 Conclusions In this chapter, we discussed our total syntheses of polycyclic alkaloids based on nucleophilic addition to amides. Use of stable amides as main functional groups would solve the inherent issues seen in alkaloids syntheses such as the high reactivities of amines and limited C–C bond formation around the nitrogen atom. In the late stage of the synthesis, chemoselective nucleophilic addition to amides provided highly substituted amines embedded in complex alkaloids. In the first section, the N-methoxy group was utilized as a reactivity control element, which enabled the direct coupling of an N-methoxyamide with an aldehyde, as well as the reductive allylation of an N-methoxylactam with the Schwartz reagent. High amide selectivity in the reductive nucleophilic addition was useful, resulting in the concise total synthesis of gephyrotoxin in 14 steps. In the second section, the chemoselective assembly of five-membered heterocyclic rings was developed in the total synthesis of stemoamide-type alkaloids. The key to success was an iridium-catalyzed reductive nucleophilic addition to a γ-lactam without affecting a more electrophilic γ-lactone. The developed method enabled quick access to the synthesis of tricyclic, tetracyclic and pentacyclic stemoamide-type alkaloids. Until recently, nucleophilic addition to amides required harsh reaction conditions, which prevented its application to the total synthesis of functionalized complex alkaloids. However, we demonstrated that the development of amide-selective nucleophilic addition enabled quick access to polycyclic alkaloids. Achievement of a higher level of chemoselective control especially for carbon–carbon bond formations will become more crucial and cause dramatic changes in the future total synthesis.

References 1. For a recent and selected review on total synthesis of complex alkaloids, see: Crossley, S.W.M., Shenvi, R.A.A.: Longitudinal study of alkaloid synthesis reveals functional group interconversions as bad actors. Chem. Rev. 115, 9465–9531 (2015) 2. For a review on total synthesis of complex alkaloids through nucleophilic addition to amides, see: Sato, T., Yoritate, M., Tajima, H., Chida, N.: Total synthesis of complex alkaloids by nucleophilic addition to amides. Org. Biomol. Chem. 16, 3864–3875 (2018) 3. Pace, V., Holzer, W., Olofsson, B.: Increasing the reactivity of amides towards organometallic reagents: an overview. Adv. Synth. Catal. 356, 3697–3736 (2014)

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4. Huang, P.-Q.: Direct transformations of amides: tactics and recent progress. Acta Chim. Sinica 76, 357–365 (2018) 5. Matheau-Raven, D., Gabriel, P., Leitch, J.A., Almehmadi, Y.A., Yamazaki, K., Dixon, D.J.: Catalytic reductive functionalization of tertiary amides using Vaska’s complex: synthesis of complex tertiary amine building blocks and natural products. ACS Catal. 10, 8880–8897 (2020) 6. Daly, J.W., Witkop, B., Tokuyama, T., Nishikawa, T., Karle, I.L.: Gephyrotoxins, histrionicotoxins and pumiliotoxins from the neotropical frog Dendrobates histrionicus. Helv. Chim. Acta 60, 1128–1140 (1977) 7. Fujimoto, R., Kishi, Y., Blount, J.F.: Total synthesis of (±)-gephyrotoxin. J. Am. Chem. Soc. 102, 7154–7156 (1980) 8. Fujimoto, R., Kishi, Y.: On the absolute configuration of gephyrotoxin. Tetrahedron Lett. 22, 4197–4198 (1981). The absolute structure of gephyrotoxin remains inconclusive due to the limited supply of the natural sample. 9. Hart, D.J.: Synthesis of (±)-gephyrotoxin. J. Org. Chem. 46, 3576–3578 (1981) 10. Overman, L.E., Lesuisse, D., Hashimoto, M.: Importance of allylic interactions and stereoelectronic effects in dictating the steric course of the reaction of iminium ions with nucleophiles. An efficient total synthesis of (±)-gephyrotoxin. J. Am. Chem. Soc. 105, 5373–5379 (1983). 11. Shirokane, K., Wada, T., Yoritate, M., Minamikawa, R., Takayama, N., Sato, T., Chida, N.: Total synthesis of (±)-gephyrotoxin by amide-selective reductive nucleophilic addition. Angew. Chem. Int. Ed. 53, 512–516 (2014) 12. Chu, S., Wallace, S., Smith, M.D.: A cascade strategy enables a total synthesis of (–)gephyrotoxin. Angew. Chem. Int. Ed. 53, 13826–13829 (2014) 13. Nemoto, T., Yamaguchi, M., Kakugawa, K., Harada, S., Hamada, Y.: Enantioselective total synthesis of (+)-gephyrotoxin 287C. Adv. Synth. Catal. 357, 2547–2555 (2015) 14. Piccichè, M., Pinto, A., Griera, R., Bosch, J., Amat, M.: Enantioselective total synthesis of (+)-gephyrotoxin 287C. Org. Lett. 19, 6654–6657 (2017) 15. White, J.M., Tunoori, A.R., Georg, G.I.: A novel and expeditious reduction of tertiary amides to aldehydes using Cp2 Zr(H)Cl. J. Am. Chem. Soc. 122, 11995–11996 (2000) 16. Yoritate, M., Meguro, T., Matsuo, N., Shirokane, K., Sato, T., Chida, N.: Two-step synthesis of multi-substituted amines by using an N-methoxy group as a reactivity control element. Chem. Eur. J. 20, 8210–8216 (2014) 17. Ando, K., Oishi, T., Hirama, M., Ohno, H., Ibuka, T.: Z-Selective Horner-Wadsworth-Emmons reaction of ethyl (diarylphosphono)acetates using sodium iodide and DBU. J. Org. Chem. 65, 4745–4749 (2000) 18. Shirokane, K., Kurosaki, Y., Sato, T., Chida, N.: A direct entry to substituted N-methoxyamines from N-methoxyamides via N-oxyiminium Ions. Angew. Chem. Int. Ed. 49, 6369–6372 (2010) 19. Ireland, R.E., Norbeck, D.W.: Application of the Swern oxidation to the manipulation of highly reactive carbonyl compounds. J. Org. Chem. 50, 2198–2200 (1985) 20. Song, J.I., An, D.K.: New method for synthesis of aldehydes from esters by sodium diisobutylt-butoxyaluminum hydride. Chem. Lett. 36, 886–887 (2007) 21. For a recent selected review on Stemona alkaloids, see: Pilli, R.A., Rosso, G.B., de Oliveira, M.C.F.: The chemistry of Stemona alkaloids: an update. Nat. Prod. Rep. 27, 1908–1937 (2010) 22. Lin, W.-H., Ye, Y., Xu, R.-S.: Chemical studies on new Stemona alkaloids, IV. Studies on new alkaloids from Stemona tuberosa. J. Nat. Prod. 55, 571–576 23. Yang, X.-Z., Zhu, J.-Y., Tang, C.-P., Ke, C.-Q., Lin, G., Cheng, T.-Y., Rudd, J.A., Ye, Y.: Alkaloids from roots of Stemona sessilifolia and their antitussive activities. Planta Med. 75, 174–177 (2009) 24. Irie, H., Harada, H., Ohno, K., Mizutani, T., Uyeo, S.: The structure of the alkaloid protostemonine. Chem. Commun., 268–269 (1970) 25. Lin, L.-G., Dien, P.-H., Tang, C.-P., Ke, C.-Q., Yang, X.-Z., Ye, Y.: Alkaloids from the roots of Stemona cochinchinensis. Helv. Chim. Acta 90, 2167–2175 (2007) 26. Wu, Y.-X., He, H.-Q., Nie, Y.-J., Ding, Y.-H., Sun, L., Qian, F.: Protostemonine effectively attenuates lipopolysaccharide-induced acute lung injury in mice. Acta Pharmcol. Sin. 39, 85–96 (2018)

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27. Takahashi, Y., Sato, T., Chida, N.: Iridium-catalyzed reductive nucleophilic addition to tertiary amides. Chem. Lett. 48, 1138–1141 (2019) 28. Gregory, A.W., Chambers, A., Hawkins, A., Jakubec, P., Dixon, D.J.: Iridium-catalyzed reductive nitro-Mannich cyclization. Chem. Eur. J. 21, 111–114 (2015) 29. Huang, P.-Q., Ou, W., Han, F.: Chemoselective reductive alkynylation of tertiary amides by Ir and Cu(I) bis-metal sequential catalysis. Chem. Commun. 52, 11967–11970 (2016) 30. Yoritate, M., Takahashi, Y., Tajima, H., Ogihara, C., Yokoyama, T., Soda, Y., Oishi, T., Sato, T., Chida, N.: Unified total synthesis of stemoamide-type alkaloids by chemoselective assembly of five-membered building blocks. J. Am. Chem. Soc. 139, 18386–18391 (2017) 31. Soda, Y., Sugiyama, Y., Yoritate, M., Tajima, H., Shibuya, K., Ogihara, C., Oishi, T., Sato, T., Chida, N.: Org. Lett. 22, 7502–7507 (2020) 32. Motoyama, Y., Aoki, M., Takaoka, N., Aoto, R., Nagashima, H.: Highly efficient synthesis of aldenamines from carboxamides by iridium-catalyzed silane-reduction/dehydration under mild conditions. Chem. Commun., 1574–1576 (2009) 33. Brito, G.A., Pirovani, R.V.: Stemoamide: total and formal synthesis. A Review. Org. Prep. Proced. Int. 50, 245–259 (2018) 34. Trost, B.M., Weiss, A.H., von Wangelin, A.J.: Dinuclear Zn-catalyzed asymmetric alkynylation of unsaturated aldehydes. J. Am. Chem. Soc. 128, 8–9 (2006) 35. Sajiki, H., Mori, S., Ohkubo, T., Ikawa, T., Kume, A., Maegawa, T., Monguchi, Y.: Partial hydrogenation of alkynes to cis-olefins by using a novel Pd0 –polyethyleneimine catalyst. Chem. Eur. J. 14, 5109–5111 (2008) 36. Pietta, P.G., Cavallo, P., Marshall, G.R.: 2,4-Dimethoxybenzyl as a protecting group for glutamine and asparagine in peptide synthesis. J. Org. Chem. 36, 3966–3970 (1971) 37. Kohno, Y., Narasaka, K.: Synthesis of (±)-stemonamide by the application of oxidative coupling reactions of Stannyl compounds with silyl enol ethers. Bull. Chem. Soc. Jpn. 69, 2063–2070 (1996)

Chapter 14

Equilibrium-Controlled Stereoselective Sequential Cyclizations Enabled Concise Total Synthesis of Complex Indole Alkaloid, Tronocarpine Atsushi Nakayama Abstract Tronocarpine is a pentacyclic monoterpenoid indole alkaloid, isolated from Tabernaemontana corymbosa in 2000 by Kam and coworkers. Although several synthetic studies of this molecule have been reported, no total synthesis has been reported until the end of 2019. The first asymmetric total synthesis of tronocarpine was reported by Han and coworkers in twenty steps from tryptamine, utilizing a catalytic asymmetric Michael/aldol cascade reaction. At about the same time, we, too, succeeded in carrying out the racemic total synthesis of tronocarpine and reported our achievement in the beginning of 2021. Our investigations set the equilibriumcontrolled stereoselective tandem cyclization as a key reaction. The designed tandem cyclization, with a chain-like substrate including an indole ring, all-carbon units, and functional groups, was realized to construct the pentacyclic skeleton of tronocarpine in a one-pot operation. Thereby, we reduced the number of synthetic steps significantly, achieving the concise total synthesis of tronocarpine in nine steps from commercially available reagents. This synthetic strategy resulted in the construction of an azabicyclo[3.3.1]nonane core—a key skeleton of tronocarpine—which could then be applied to the total synthesis of other chippiine–dippinine-type alkaloids. We also succeeded in achieving the enzymatic optical resolution, to obtain chiral products of the thus-developed synthetic routes. Keywords Tronocarpine · Monoterpenoid indole alkaloid · Tandem cyclization · Total synthesis

14.1 Introduction Many monoterpene indole alkaloids, which have fascinating molecular structures and unique biological properties, have been reported to date [1–7]. They have attracted the attention of researchers from a wide range of fields, including natural product A. Nakayama (B) Graduate School of Science, Osaka Metropolitan University, 3-3-138 Sugimoto Sumiyoshi-ku, Osaka 558-8585, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_14

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A. Nakayama O

4

H NH

MeO

NH 22

O

N H OH

H

Tronocarpine (1)

O NH H

21

21

MeO

N H

CO2Me OH

H

Chippiine

N H

CO2Me OH

H

Dippinine B

Fig. 14.1 Structures of chippiine–dippinine-type alkaloids

chemistry. These monoterpenoid indole alkaloids have great potential to be lead molecules for the development of new drugs [8, 9]. Many researchers in the field of natural product chemistry have conducted isolation studies with many plants. Among them, much research on the constituents of the genus Tabernaemontana, Apocynaceae, has been investigated [10–27]. Among the monoterpenoid indole alkaloids that display biological activity, the chippiine–dippinine-type indole alkaloids (polycyclic alkaloids of the post-iboga type) have attracted considerable interest from synthetic organic chemists due to their highly fused architecture and biological activity (Fig. 14.1) [28–34]. However, despite great efforts by synthetic organic chemists, over decades, to synthesize such natural products (alkaloids), only a few successful total syntheses have been reported [35–41]. This chapter describes our recent success in the total synthesis of tronocarpine (1), a pentacyclic chippiine–dippinine-type alkaloid, incorporating a seven-membered lactam [40]. The key feature of our synthesis is the effective construction of an azabicyclo[3.3.1]nonane core, a key structural motif of chippiine–dippinine-type alkaloids, utilizing equilibrium-controlled stereoselective tandem cyclization.

14.2 Tronocarpine Tronocarpine (1) was first isolated from the stem bark of Tabernaemontana corymbosa by Kam and coworkers, in 2000, after which its structure was elucidated by various NMR analysis techniques (1D-, 2D-, and NOE experiments) (Fig. 14.2) [42]. These investigations revealed that it had the following structural characteristics: (1) a unique [6.5.6.6.7]pentacyclic skeleton, (2) a seven-membered lactam, (3) an azabicyclo[3.3.1]nonane core, (4) three asymmetric centers, including a quaternary carbon center (C3, carbon numbering is provided in Ref. 42) adjacent to the C2 position of the indole, (5) an α,β-unsaturated ketone moiety, and (6) a hemiaminal moiety. It was also observed that the skeleton of 1 differed from that of other chippiine–dippininetype alkaloids in terms of bond connection. 1 contains a seven-membered lactam, including N4 and C22 connecting to the C3 quaternary carbon center, while most alkaloids in this family have a bond connection between N4 and C21.

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5 4

10

7 1

N

12

O

2 14 17

[6.5.6.6.7]Pentacyclic skeleton

NH

3 22

16

HO

O

NH

15

21 20

19

18

N H OH

O

Seven-membered lactam

O H

Azabicyclo[3.3.1]nonane core 3 stereogenic centers containing quaternary carbon center , -Unsaturated ketone Hemiaminal moiety

Tronocarpine (1)

Fig. 14.2 Structural characteristics of tronocarpine (1)

It should be noted here the absolute stereochemistry of natural (+)-1 was not determined and the biological activity of 1 remains unknown.

14.3 First Total Synthesis of (+)-Tronocarpine by Hans and Coworkers As described above, 1 is an attractive target for synthetic communities and several efforts to synthesize 1 have been reported [43–49]. Most synthetic strategies attempt to construct the key structure of 1 in an early stage of synthesis, succeeding in the construction of the tetra- or pentacyclic skeleton. However, most lack the carbon unit and functionalities from 1, in particular, the hemiaminal moiety and α,β-unsaturated ketone, and it was difficult to install them later in the synthesis. Nevertheless, Han and coworkers finally succeeded in the first asymmetric total synthesis of (+)-1 from tryptamine in the 20 longest linear steps. They then determined the absolute stereochemistry of 1 by single-crystal X-ray analysis [37]. Their key reaction was an asymmetric Michael/aldol cascade reaction in the presence of a chiral phase-transfer catalyst 2, developed by them, to construct the key azabicyclo[3.3.1]nonane core with excellent selectivity (67%, 93% ee; 54%, 98.5% ee after one crystallization). This powerful reaction afforded a tetracyclic derivative 4 having a nitrile moiety, instead of an ester moiety, at the C3 position from indole derivative 3 prepared from tryptamine in five steps (Scheme 14.1). Subsequently, the resultant tetracyclic compound 4 containing a hydroxy group at C15 was converted to the tetracyclic seven-membered lactam 5 through a five-step sequence, after which the sequential oxidation steps and the introduction of iodide at the α-position of an α,β-unsaturated ketone afforded the iodide 6. Next, the Sonogashira coupling reaction with trimethylsilyl acetylene, with the introduction of two-carbon units, afforded a pentacyclic derivative 7 having an all-carbon unit. Subsequent reaction sequences, including deprotection, removal of the extra oxygen functionality at C15, reduction, deprotection, and conversion of acetylene to a ketone in the oxymercuration reaction under acidic conditions, finally gave (+)-1. Later, they improved their first-generation synthesis of 1 and, in 2020, reported the second-generation synthesis [38]. Their success in terms of reducing the number of

298

A. Nakayama

HO N

1. N2H4 NPhth 2. ClCO 2Me 3. Dess-Martin ox. 4. Raney Ni CN 5. Dess-Martin ox.

H N

NPhth

NO2 2 (20 mol%) acrolein, KOH aq.

CN

3

N

N

Et2O/CHCl3 67%, 93% ee (54%, 98.5% ee 3 (5 steps from tryptamine) after one crystallization)

O

O

17

2nd generation: OEt

15

4 OH (dr = 1:1)

Sn(n-Bu)3

2nd generation: No protection 4

N CO2Me O

N

N CO2Me

1. Pd(OAc)2 Cu(OAc)2 2. I2, DMAP

O

N O

5

O

6 N CO2Me O

N O O 7

15

TMS

O I

82%

O

1. CeCl3 then NaBH4 2. NaH, CS2, MeI 3. AIBN, TMS3SiH 4. LiEt3BH 5. TBAF 6. H2SO4, H2O Hg(OAc)2

TMS Pd(PPh3)2Cl2 CuI, i-Pr2NH

NH 3

N HO

16

O

17

O (+)-Tronocarpine (1) = (3S,16S,17R)-tronocarpine (20 steps from tryptamine)

Scheme 14.1 Han’s 1st-generation synthesis of (+)-tronocarpine (1)

synthetic steps from 20 to 15 is attributed to avoiding the protection/deprotection steps of the nitrogen (N4). They changed the two-carbon units from trimethyl acetylene to ethoxyvinylstannate, and thus made the conversion more efficient in the final stages of synthesis. In both synthetic routes, they introduced two-carbon units in the late stage of synthesis to avoid unexpected side reactions.

14 Equilibrium-Controlled Stereoselective Sequential Cyclizations …

299

14.4 Synthetic Design for the Construction of the Azabicyclo[3.3.1]nonane Core by Equilibrium-Controlled Stereoselective Tandem Cyclization As Han and coworkers demonstrated above, their strategy of constructing the important carbon skeleton in advance and then introducing the C2 unit and all other functionalities later is reasonable. On the other hand, if building the complex architecture can be achieved from the chain-like substrate having the required all-carbon unit and versatile functional groups simultaneously, the synthetic scheme should become simpler and more efficient because the extra conversion sequence in the later stage is now avoided. Therefore, to realize our concise total synthesis of 1 based on the above concept, we designed the following synthetic strategy (Scheme 14.2). In the synthesis of chippiine–dippinine-type alkaloids, the most important and difficult challenge is how to construct the azabicyclo[3.3.1]nonane core skeleton stereoselectively and shortly. To overcome this difficulty, we planned to conduct tandem cyclization involving the equilibrium. We aimed to achieve this by setting

Minimum transformations

O

N3 H

NH

N O

N H

N H

OH Tronocarpine (1)

H

O

11 (one-pot from 8)

3

O 10H azabicyclo[3.3.1]nonane core formation

17

MeO

N3

H O

OH

21

3

MeO2C

CHO

8 (having all carbon units of tronocarpine)

CO2Me

N3 21

N H 17 MeO2C

OH N

N3 CO2Me

OH O

H

N H

N H MeO

17

CO2Me

H

X

O

trans-9

cis-9

O

3

All stereocenters would be controlled by the stereochemistry of C17 by ustilizing the equilibriums between intermediates.

Scheme 14.2 Synthetic plan of 1 utilizing the equilibrium-controlled stereoselective tandem cyclization method to construct the azabicyclo[3.3.1]nonane core

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up suitable equilibrium reactions and conditions. Then, if we could transform the designed substrate to a thermodynamically stable compound, each bond formation could be conducted in a one-pot operation. Thus, the desired tronocarpine-like skeleton would be obtained in short steps. First, we envisioned that if α,β-unsaturated aldehyde 8 (or its synthetic equivalent) having all-carbon units of 1 could be synthesized, a tandem cyclization involving the intramolecular aldol reaction and the subsequent six-membered lactam formation would proceed favorably and rapidly, thereby affording an azabicyclo[3.3.1]nonane core. Upon the first nucleophilic attack from C3 to the aldehyde, the resultant stereochemistry of the C3 position might not be controlled. As a result, trans-9 and cis-9 would be generated, after which the subsequent formation of a six-membered lactam would then proceed in one pot with only cis-9, because of the short distance between N1 and the ester moiety at the C17 position, to afford tetracyclic compound 10. On the other hand, the trans-9 cannot form the six-membered lactam, and return to 8 by the retro aldol reaction in the reaction system. The returned starting material 8 would then participate in the aldol reaction once again. As a result, all material would be converted to 10 by the equilibrium existing in this process. It is noteworthy that the resultant stereochemistries of the azabicyclo[3.3.1]nonane core including the quaternary carbon center C3 after this tandem cyclization would be controlled by the C17 position. During these sequential reactions, epimerization of C17 stereochemistry under basic conditions should be avoided. Then, azide reduction would proceed to give the seven-membered lactam formation between N4 and the ester at the C3 position, thus affording a functionalized pentacyclic compound 11 in a one-pot operation. Subsequently, a minimum number of transformations would be required to convert 11 to 1 because 11 already has suitable functional groups for conversion of the desired moieties. Therefore, this strategy would readily yield derivatives having the azabicyclo[3.3.1]nonane core and, furthermore, can be expected to be applied to synthesize other chippiine–dippinine-type alkaloids.

14.5 Concise Total Synthesis of (Rac)-Tronocarpine We then carried out our proposed synthetic procedure for tandem cyclization to verify whether the synthetic strategy described above works. We decided to set βmethoxy aldehyde 21 as the equivalent of the cyclization precursor 8 because we were concerned about the potential instability of α,β-unsaturated aldehyde (Scheme 14.3). The coupling reaction between the 1,3-dicarbonyl derivative 12 and α,β-unsaturated ester 13 proceeded in the presence of tributylphosphine [50] to afford triester derivative 14. The subsequent indole formation by Pd-catalyzed hydrogenation was conducted in a one-pot operation, affording indole triester 15. Then a C5–C6 twocarbon unit including a nitrogen atom was introduced to the C3 position of the indole moiety. 15 was reacted with bromoacetaldehyde dimethyl acetal (16) in the presence of trifluoroacetic acid (TFA) to give a bromide derivative in 52% yield [51], followed by the nucleophilic substitution reaction with sodium azide to afford azide triester

14 Equilibrium-Controlled Stereoselective Sequential Cyclizations …

301

17 in excellent yield. Selective reduction of an ester (C21) was achieved by treatment with diisobutylaluminium hydride (DIBAL) at –78 °C, affording aldehyde 18, along with 30% recovery of 17. We chose the Mukaiyama aldol reaction with a silyl enol ether for introduction of a C18–C19 two-carbon unit to avoid the undesired intramolecular aldol reaction between C3 and aldehyde (C21). Treatment of 18 with tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf) and diisopropylethyl amine (DIPEA) at –78 °C afforded the corresponding TBS silyl enol ether 19. After confirming that the reaction had run to completion (TLC monitoring), an excess of dimethyl acetal and additional TBSOTf CO2Me

O NO2

CO2Me

P(n-Bu)3 (10 mol%)

+

12

O NO2

MeCN, rt

CO2Me

MeO2C 13

14 MeO

16 Pd/C H2 (1 atm)

CO2Me N H MeO2C

MeOH, rt 52%

CO2Me

Br

N3

OMe

1) TFA, Et3SiH, 16 CH2Cl2, rt, 56%

CO2Me N H MeO2C

2) NaN3, DMF

52%

CO2Me 21 CHO

TBSOTf DIPEA THF

OMe

OTBS 19 N3

N3

N3 CO2Me MeO

OMe

CO2Me N H MeO2C

18

N

then TBSOTf

N3

N3

N H MeO2C

CHO OMe

21 CO2Me

17

15 (dr = 1:1)

DIBAL THF

CO2Me

CO2Me

+

N H MeO2C

CO2Me + OMe OMe OMe

3

N H MeO2C

CO2Me CHO

17

20

19

OMe

CO2Me 20A

20B

21 (8 diastereomers mixture)

Acetone, rt, 86%

Scheme 14.3 Preparation of the tandem cyclization precursor 21

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A. Nakayama

were added in one pot. Then, the reaction mixture was warmed to room temperature. The Mukaiyama aldol reaction proceeded rapidly and the desired tandem cyclization precursor 21 was obtained as an inseparable racemic eight diastereomeric mixture in 35% yield, with accompanying overreacted compounds 20A and 20B in a total yield of 25%. To our delight, we could convert these by-products to the desired 21 by treatment with Amberlyst®15 in acetone. Transacetalization of 20A and 20B proceeded well; we finally obtained 21 in 57% combined yield from the starting material 18. Thus, we prepared the tandem cyclization precursor 21 in five steps from 12 and 13. Having the precursor of the key cyclization reaction in hand, we then attempted to carry out the tandem cyclization with substrate 21 having all-carbon units (Scheme 14.4). After several investigations, we realized the desired tandem cyclization by controlling the reaction conditions, both the basic and reductive conditions, in one pot. This reaction involved the following: (1) β-elimination to form an α,β-unsaturated aldehyde, (2) an aldol/retro aldol reaction, (3) six-membered lactam formation, (4) azide reduction, and (5) seven-membered lactam formation. Thereby, we obtained the desired pentacyclic compound 11 with a tronocarpine skeleton in 70% yield as an inseparable mixture of C19–C20 olefin isomers (E:Z = 10:1). Interestingly, after the reaction, all diastereomers of 21 converged into one diastereomer of 11. The structure of the pentacyclic product 11, including its stereochemistry, was confirmed by 2D-NMRs (COSY, HMQC, HMBC, NOESY).

Equilibrium-controlled tandem cyclization

Na2CO3 then then Lindlar catalyst, H2 (1 atm), rt then Na2CO3

N3 3

N H MeO2C

CO2Me

N

CHO 17

20

19

70%

OMe

21 (8 diastereomers mixture) (6 steps)

O OH

H

(rac)-Tronocarpine (1)

11 (E:Z = 10:1)

[6-5-7-6-6]-ring formation Quaternary stereocenter

OH NH

DMP CH2Cl2, rt then (+)-CSA THF, H2O rt, 89%

N H H HO 22

Tf2O (CH2Cl)2 NaBH4 THF H2O

H N

OH O

H

O

one pot

O

N H

H H 21N

O

OH

H

19

N O

N 61% O

H

one pot

(9 steps)

Scheme 14.4 Synthesis of a pentacyclic compound 11 having all-carbon units by the equilibriumcontrolled tandem cyclization and completion of the total synthesis of tronocarpine (1)

14 Equilibrium-Controlled Stereoselective Sequential Cyclizations …

303

Details of this tandem reaction are as follows (Scheme 14.5). First, the tandem cyclization is initiated by the formation of α,β-unsaturated aldehyde by β-elimination of the methoxy group at C19 of 21. At the outset of our investigation, we were concerned that the intramolecular aldol reaction without β-elimination would occur simultaneously. However, after careful observation, we discovered that the intramolecular aldol reaction is suppressed around 0 °C, causing the β-elimination to proceed well at that temperature. We confirmed the stereochemistry of the olefin as a Z isomer by the isolation of 8. After confirming the completion of the β-elimination by TLC, warming the reaction mixture to 40 °C accelerated the intramolecular aldol reaction. As we expected, since the stereochemistry at the C3 position could not be controlled, cis-9 and trans-9 were obtained as a 1:1 diastereomeric mixture. The stereochemistry at the C21 hydroxy group was also not controlled. We confirmed an equilibrium between cis-9 and trans-9 by the retro aldol reaction, and the regeneration of trans-9 to 8, under the reaction conditions. On the other hand, the six-membered lactamization with cis-9 generated tetracyclic compounds 10A and 10B, which are epimers at the C21 position. We isolated 10A and 10B, respectively, and determined the production ratio as 1:1. Investigations revealed that while the C19–C20 olefin stereochemistry of 10A was determined to be about a 3:1 E:Z mixture, 10B was a single isomer. Moreover, since equilibrium between 10A and 10B existed, treatment of 10B under the tandem cyclization conditions afforded a 1:1 mixture of 10A and 10B. Therefore, as we have described thus far, we have developed an efficient method for obtaining compounds having the azabicyclo[3.3.1]nonane core. Next, we reduced azides by hydrogenation using a Lindlar catalyst under hydrogen. The reduction was rapidly completed, after which azides were converted to primary amines to afford amines 23A and 23B. It is noteworthy that the C19–C20 double bond was reduced when Pd/C or Pd(OH)2 was used. Even with the Lindlar catalyst, the double bond was also reduced when the reaction time was prolonged under hydrogen. Thus, it is important to switch from a hydrogen atmosphere to an argon atmosphere immediately after the hydrogenation was completed. Then, the subsequent addition of Na2 CO3 [52] to the reaction system accelerated the formation of a seven-membered lactam. Notably, although we should have observed two isomers 11 and 11' (the epimers at the C21 hydroxy group), only 11 was obtained under this one-pot tandem cyclization procedure. This experimental result could then be used to calculate, and compare the thermodynamic stability of 11 and 11'. To this end, the density functional theory calculation was performed at the B3LYP/6-31g(d,p) level. Results indicated that 11 is more stable than 11' by 1.4 kcal/mol, which is in good agreement with experimental observations. The mechanism included the formation of the seven-membered lactam from tetracyclic compound 23B to afford the pentacyclic compound 11', as well as 11. However, 11' was converted to the more stable compound 11 via the tetracyclic compound 24 by the retro aldol reaction under the basic conditions. We then isolated 10B, converted it to 11' via 23B under neutral conditions, and confirmed that the pentacyclic compound 11' was rapidly converted to 11 (without observing 24). Therefore, the E/Z ratio of

304

A. Nakayama aldol/retro aldol reaction N3

H O

21

-elimination

3

MeO2C

N H

17

N3

N3 3

N H MeO2C

17

CO2Me

Na2CO3

CHO

MeOH

20

19

OMe

3

N H MeO2C

21 (8 diastereomers mixture)

H

MeO

X

O

trans-9

CO2Me

+

N3 OH

CHO 20

21 19

17

8

N H MeO

3

17

CO2Me

H

O cis-9

lactamization

N3

N3

19 20

19

H 21

OH

20

Na2CO3

21

CO2Me

N

O 10B (E only)

10A (E:Z = 3:1) Lidlar cat., H2 (1 atm)

CO2Me

N

H

O

20

NH2 19

H OH

20

Na2CO3

CO2Me

N

lactamization H H N

N

CO2Me

O 23B

23A Na2CO3

1.4 kca/mol more stable than

N O

OH CHO

H 24

aldol/retro aldol reaction

Scheme 14.5 Details of the tandem cyclization with substrate 21

H

Na2CO3 H H O N H

NH

OH O

H O 11 (E:Z = 10:1)

OH H

N

H

O

H

Lidlar cat., H2 (1 atm)

azide reduction NH2

19

OH H

O

N O

H

14 Equilibrium-Controlled Stereoselective Sequential Cyclizations …

305

11 was considered to be correlated with the E/Z ratio of compounds 10A and 10B in the reaction mixture. Moreover, while the E/Z ratio of 10A was 3:1 from the intramolecular aldol reaction results, 10B was only the E-isomer between the C19– C20 double bond. The final E/Z ratio is dependent on the conversion from 10A and 10B to 23A and 23B via equilibrium between 10A and 10B, 23A and 23B, or both. Finally, isomerization of the olefin, which probably occurs during the equilibriumcontrolled tandem cyclization by the repeated addition/elimination of MeOH from the α,β-unsaturated aldehyde moiety generated from each intermediate, was realized. The final E/Z ratio of 11 depended on the reaction conditions and times. Moreover, deuterium experiments with MeOD were also conducted for this tandem cyclization. As a result, C17 position was not deuterated. Thus, no epimerization occurred at C17 position with this cyclization precursor 21. To summarize, in this tandem cyclization, the equilibrium existing in each reaction controls the stereochemistry and ultimately gives the pentacyclic skeleton of tronocarpine (1) with high stereoselectivity. As the key skeleton of tronocarpine (1) was successfully constructed in short steps, we converted the pentacyclic compound 11 to 1 by the sequential functional group transformation (Scheme 14.4). Compound 11 already had an allylic hydroxy group at C21, and treatment of 11 with bis(trifluoromethane)sulfonimide, gave a 1,3-rearrangement of the hydroxy group from the C21 position to the C19 position. Then, upon the addition of Na2 CO3 for neutralization, THF, and H2 O in one pot, the excess sodium borohydride reduced the six-membered lactam to the primary alcohol, affording diol 22. Finally, the total synthesis of (rac)-tronocarpine (1) was successfully accomplished by sequential Dess–Martin oxidation and acidic treatment with (+)-camphorsulfonic acid. All spectral data of our synthetic 1 were in good agreement with those of Han’s reported data [37] for 1. This total synthesis also enabled us to obtain 1 in nine steps from commercial reagents.

14.6 Attempts to Obtain an Optical Tronocarpine We turned our attention to obtaining an optically active 1, as the optical separation of the racemic compound is a direct method to obtain both enantiomers simultaneously. Thus, we screened the available chiral columns and conditions for good separation. Fortunately, we established good conditions for the separation with a Chiralcel ODH column (see Ref. 41 for details), and obtained optically pure (+)-1 ([α]28 D + 277, c 0.167, CH2 Cl2 ) and (–)-1 ([α]28 D –282, c 0.153, CH2 Cl2 ). We also attempted to develop the asymmetric synthetic route toward 1. As described in Sect. 14.4, the stereochemistry of the azabicyclo[3.3.1]nonane core of 1 constructed by the tandem cyclization is controlled by the stereochemistry of C17. In other words, if we could prepare a substrate having an enantioenriched carbon center at the C17 position, the subsequent tandem cyclization should afford the chiral pentacyclic product 11. Therefore, following this idea, we decided to prepare a chiral triester 14 (Scheme 14.6a). First, we tried an asymmetric conjugate addition between 12 and 13 with a chiral phosphine 25 or a chiral amine catalyst 26

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A. Nakayama

(a) Chiral phosphine (25) or

CO2Me +

O NO2

CO2Me Chiral amine (26)

MeO2C

12

CO2Me 3

X

O NO2

17

CO2Me

CO2Me

14

13 Et N

Ph Ph

O

P

P

N Ph Ph 25

26

OH

(b) 3

N H MeO2C

17

(rac)-15 (dr = 1:1)

CO2Me

Novozyme 435 (200 w/w%)

CO2Me MeCN 0.1 M phosphate buffer

3

N H MeO2C **

CO2Me CO2Me

17

(43%, 35% ee)

** : stereochemistry not determined

3

+

N H MeO2C **

CO2Me CO2R

17

27 (R = H) (25%) TMSCHN2 MeOH/toluene rt (+)-15 (R = Me) (87%, 81% ee)

Scheme 14.6 a Trial for the asymmetric conjugate addition. b Enzymatic kinetic resolution with triester 15 to obtain the chiral 15

instead of tributylphosphine. However, these reactions did not proceed—no products were obtained. We found that tributylphosphine proves to be a good catalyst for this Michael reaction using these substrates. Finally, we attempted an enzymatic kinetic resolution of racemic triester 15 accompanied by selective hydrolysis (Scheme 14.6b). As a result of screening the enzymes, Amano lipase PS-C hydrolyzed an ester, affording the corresponding carboxylic acid 27; the triester 15 was also recovered. Then 27 was converted to methyl ester 15. The isolated yield and enantiomeric excess of these products ((–)15 and (+)-15) were determined to be 12%, 31% ee, and 68%, 32% ee, respectively. The absolute stereochemistry at the C17 position was not determined. After several investigations, we found that with Novozyme 435, we obtained the desired kinetic resolution: (–)-15 and (+)-15. We finally obtained the enantioenriched (+)-15 with good selectivity (81% ee) when acetonitrile was used as a cosolvent.

14 Equilibrium-Controlled Stereoselective Sequential Cyclizations …

307

14.7 Conclusion and Perspective In this chapter, we have presented the details of our recent concise total synthesis of the chippiine–dippinine-type alkaloid, tronocarpine (1). The precisely controlled tandem cyclization involving equilibrium with a chain-like substrate, which has allcarbon units and “foothold” functional groups, effectively enabled the construction of the azabicyclo[3.3.1]nonane skeleton. This fundamental skeleton of chippiine– dippinine-type alkaloids can be used to conduct the total synthesis of this type of natural product. Moreover, the reductive conditions afforded the seven-membered lactam, a characteristic moiety of 1, in one pot. Thus, we succeeded in obtaining the tronocarpine-like pentacyclic compound in short steps. This synthetic method enabled us to prepare 1, and some key intermediates, reasonably rapidly and in large quantities. Moreover, we also developed a rare enzymatic optical separation of the triester compound and obtained an optically active compound at C17. This method enables us to conduct the asymmetric total synthesis of 1 and related compounds. Needless to say, the development of efficient synthetic routes to bioactive natural products is very important. It is then important to evaluate their potential as lead compounds toward drugs. In the past, evaluation of the biological activities of natural products was often limited to in vitro studies; however, in recent years, even structurally complex molecules are increasingly being tested in vivo by a lot of short-step total synthesis. Tandem cyclization is one of the most effective methods to achieve the shortstep total synthesis. One advantage of setting up the equilibrium in the reaction system is that a thermodynamically stable skeleton can be predominantly constructed. Furthermore, tandem reactions reduce the total number of reaction steps, enabling researchers to obtain samples immediately. We have shown that the equilibriumcontrolled tandem cyclization is useful for developing an efficient synthetic route for a complex natural product and can deliver it reasonably rapidly and in large quantities. In the near future, we will pursue the potential for drug development of chippiine–dippinine-type alkaloids including 1.

References 1. Saxton, J.E. (ed.): The Chemistry of Heterocyclic Compounds, Indoles Part 4, The Monoterpenoid Indole Alkaloids. Wiley, New York (1983) 2. Saxton, J.E. (ed.): The Chemistry of Heterocyclic Compounds, Supplement to Volume 25, Part 4, The Monoterpenoid Indole Alkaloids. Wiley, New York (1994) 3. Saxton, J.E.: Recent progress in the chemistry of the monoterpenoid indole alkaloids. Nat. Prod. Rep. 14, 559–590 (1997) 4. Kam, T.-S.: Alkaloids from Malaysian flora. In: Alkaloids: Chemical and Biological Perspectives, vol. 14, pp. 284–435. Pergamon, Amsterdam (1999). 5. Kam, T.-S., Choo, Y.-M.: Bisindole alkaloids. In: The Alkaloids, vol. 63, pp. 181–337. Academic Press, Amsterdam (2006). 6. Buckingham, J., Baggaley, K.H., Roberts, A.D., Szabo, L.F.:´ Dictionary of alkaloids, 2nd ed., pp. lvii−lxvi. Boca Raton, CRC Press (2010).

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Part IV

New Approach for Drug Discovery Using Natural Products

Chapter 15

High-Throughput Searches for Natural Products as Aggregation Modulators of Amyloidogenic Proteins Kazuma Murakami

Abstract The aggregation of amyloidogenic proteins is a pathological hallmark of various neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. In particular, the oligomeric assembly of amyloids is responsible for neuronal dysfunction and memory loss. Thus, inhibition of amyloid aggregation is a research target for the development of therapeutic strategies against these diseases. Despite the intensive search for new medicinal seeds from natural products that target oligomers having diverse conformations and molecular sizes and the clinical trials that have been performed over the last decades, efforts in this direction have been unsuccessful so far. To develop oligomer-specific drugs with few adverse effects, we recently developed two unique methods for searching aggregation modulators of amyloid β, one of the causing agents of Alzheimer’s disease. This chapter reviews the latest development of high-throughput searches for aggregation inhibitors using natural product libraries, including our own results, and discusses their therapeutic potential for clinical applications. Keywords Amyloid β · Aggregation · Natural product · High-throughput screening · Triterpenoid · Flavonoid

15.1 Aggregation of Amyloidogenic Proteins and Neurodegeneration The accumulation of amyloidogenic proteins is involved in neurodegenerative diseases, as exemplified by amyloid β-protein (Aβ) and tau protein for Alzheimer’s disease (AD), and α-synuclein (αSyn) for Parkinson’s disease [1–3]. Such diseaserelated amyloids are prone to self-assembly into matured amyloid fibrils having crossβ-sheet structures through metastable intermediates called oligomers [4], causing neurotoxicity (Fig. 15.1). AD is the first cause of 60–70% of the cases of dementia, K. Murakami (B) Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-Ku, Kyoto 606-8502, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_15

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and its prevalence is increasing in our aging societies. Consequently, the social and economic impact of AD has stimulated intensive research efforts in the chemical field to find a treatment. Aggregation of Aβ is thought to be a cause or a worsening factor in AD progression [5, 6]. However, AD is a multifactorial disorder that can be affected by aggregation of other pathological proteins such as tau [7], αSyn [8, 9], or islet amyloid polypeptide (IAPP) [10], which is an amyloid involved in the progression of type 2 diabetes mellitus. In vitro, cellular, and animal experimental studies have revealed the occurrence of several types of molecular interactions among the different amyloids, including cross-seeding of aggregates in one protein initiating misaggregation of another. Aβ is believed to serve as the center of the cross-seeding of aggregation [11–13] and neurodegeneration [14] (i.e., Aβ coaggregates with tau, αSyn, and IAPP). Thus, Aβ aggregates are major targets in drug development and research in the diagnostics and therapeutics of neurodegenerative diseases. Aβ, which is generated from the Aβ-protein precursor by promiscuous proteases, is typically composed of 39–43 amino acids residues, more commonly of 40 or 42 amino acids (Aβ42 and Aβ40) [15]. Aβ oligomers having a wide range of molecular sizes commonly act as neuronal toxins because they lead to synaptic dysfunction and neuronal loss via oxidative stress, apoptosis, and inflammation [16, 17]. In this context, “oligomer” is an ambiguous term used to refer to water-soluble aggregates ranging from dimers to hundreds of monomers. Aβ42 aggregates faster to form high-molecular-weight oligomers and protofibrils (mainly 12–24-mer) and is more neurotoxic than Aβ40, and the oligomers of both isoforms are more neurotoxic than the corresponding fibrils. Thus, Aβ42 oligomers are a primary toxin in the etiology of AD [18]. In 1993, the in vitro mechanism of Aβ aggregation was first explained using a seeding model based on the one-dimensional crystallization of proteins [19]. Thereafter, uniform stacking models were reported, such as a nucleation-dependent

Fig. 15.1 Schematic diagram of the aggregation of amyloidogenic proteins. Naturally unstructured monomeric amyloids (Aβ, αSyn, or tau) undergo partial folding under pathological conditions, initiating the self-assembly into low-molecular-weight oligomers in the nucleation phase, followed by the formation of quasistable high-molecular-weight oligomers and fibrillization in the elongation phase

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polymerization model [20] and a template-dependent dock-lock model [21], among which the former, which comprises a two-phase process of nucleation and elongation, is particularly useful (Fig. 15.2). The nucleation phase involves the rate-limiting formation of low-molecular-weight oligomers (called “nuclei,” 2–6-mer), which is associated with a lag time. In the subsequent elongation phase, each nucleus associates with monomers by acting as a template for polymerization, followed by a plateau region where the monomeric concentration reaches equilibrium. Given that most neurodegenerative diseases show overlapping clinical symptoms, this model would require modification to take the heterogeneous seeds into account [22]. In this context, the screening of natural products that prevent aggregation and the associated neurotoxicity of Aβ has been the focus of intensive research, as reviewed elsewhere [23–25]. In the last 10 years, the introduction of large chemical libraries developed by the pharmaceutical industries and nonprofit research institutes has facilitated the development of high-throughput screening (HTS) methods, which are used in combination with computational methods and LC–MS-based metabolomic approaches to identify antiaggregative small molecules. The following sections will cover the use of these searching methodologies for searching aggregation modulators in pursuit of new drug candidates.

Fig. 15.2 Nucleation-dependent polymerization as an aggregation mechanism of Aβ. The elongation phase is considered to involve an off-pathway to quasistable high-molecular-weight oligomers and an on-pathway to fibrils

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Fig. 15.3 Structure of fluorescent probes (Th-T, Congo Red, and PiB) of amyloid aggregates

15.2 Fluorescent Probes of Amyloid Aggregates Amyloid aggregation can be monitored using fluorescent probes, which show an increased fluorescence upon binding to the cross-β-sheet structures of fibrils. Since the initial report by Naiki et al. [26] and Levine et al. [27] on amyloid detection using Th-T, other probes such as Th-T derivatives and Congo Red derivatives (Fig. 15.3) have been developed for the analysis of the structure and interaction of aggregates and the aggregation kinetics [28]. Despite being originally developed for the purpose of Aβ detection, Th-T and Congo Red have also been applied to other amyloidogenic proteins, including tau, αSyn, prions, SOD1, and TDP-43 [28], which suggests that these probes are sensitive to cross-β-sheet structures in general. Some of these probes have been applied to the staining of amyloid depositions in vivo and ex vivo in the brains of humans and animals, contributing to the diagnostics of neurodegenerative diseases. For instance, Pittsburgh compound B (PiB), a radioactive 11 C-labeled derivative of Th-T, has been used in positron emission tomography (PET) scanning to image Aβ plaques in AD brains since the first study on humans in 2004 [29]. Although most amyloid probes are small molecules, nucleic acid ligands can be also used to detect amyloid aggregates. Rahimi et al. produced RNA aptamers, whose binding was dependent on fibrillogenesis, for the detection of β-sheet structures with higher sensitivity than Th-T [30]. Since the application of antibodies as probes for amyloid aggregates has been reviewed elsewhere [31], this chapter focuses on the use of Th-T as an amyloid probe.

15.3 HTS of Natural Products That Modulate Amyloid Aggregation The epidemiological and clinical studies on traditional medicines and dietary habits for AD onset [32] have prompted a huge interest in natural products as a chemical source of AD-targeting drugs [33, 34]. Natural products can be examined via in vitro HTS alone and in combination with computational studies [35–37], and the potentially useful molecules are then selected as a starting point to rationally design derivatives with further biological activities. However, the search for inhibitors of Aβ aggregation using large chemical libraries is heavily reliant on serendipity, which is not conducive to obtain a sufficiently diverse array of chemicals as drug candidates

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to enter clinical trials. Thus, the identification of Aβ modulators that match certain established criteria in HTS using large chemical libraries is essential. On the basis of the fact that mature amyloid fibrils have been suggested as a reservoir of toxic oligomeric species, some screening studies have identified compounds that reduce the Aβ42-induced cytotoxicity by promoting Aβ42 fibril formation [38], revealing fibril-binding compounds as potential candidates for AD therapeutics. Eisenberg and colleagues discovered the formation of cocrystal structures of fibrillar β-sheets in Aβ16-21 fragments with the dye Orange G [39] and identified a specific pattern of hydrogen bonds and apolar interactions between Aβ and the dye ligand. It should be noted that the fibril structure can be stabilized in the fibrilbound molecule by rationally modulating the ligand structure to generate a tight and low energy interface. On the basis of the pharmacophore of the ligand binding site to Aβ, these authors screened ~18,000 natural products from the Cambridge Structure Database (http://www.ccdc.cam.ac.uk) and the Zinc Database of purchasable compounds (http://zinc.docking.org/) [40] that bound to Aβ fibrils and stabilized the complex structure. Coupled with the RosettaLigand program [41], the computational structure-based docking procedure identified binders of amyloid fibers (BAFs) that reduced Aβ42-induced neurotoxicity against mammalian cells such as HeLa and PC-12 by up to 90% [42]. Structure–activity relationship studies of BAFs and their derivatives suggested that ligand binding to Aβ42 could increase the stability of the fibril structure and decrease the cytotoxicity, possibly by shifting the structural equilibrium of Aβ42 from oligomers to fibrils. The repeated refinement of the pharmacophore model led to eight active compounds (BAF1, 4, 8, 11, 12, 26, 30, and 31) with diverse chemical backbones (Fig. 15.4a). Using a library of more than 1,800 compounds from Pilot Prestwick, Navigator Pathways, and Maybridge libraries from the Center of Chemical Genomics in the

Fig. 15.4 Structure of (a) BAF1, 4, 8, 11, 12, 26, 30, 31 and (b) BF-3, AQ-4, THQ-1, DHQ-1, DHQ-2

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University of Michigan High-Throughput Screening core facility [43], Ramamoothy and colleagues conducted an HTS based on the criteria of modulating the membraneassisted Aβ40 aggregation, which has been associated with neurotoxicity. Aβ causes a detergent-like disruption of the cellular membrane by forming a pore [44]. The screening was conducted using Aβ40 in the presence of large unilamellar vesicles composed of a 7:3 mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphol, which mimics the charge distribution of cell membranes [45]. First, the HTS gave 40 compounds, which were then subjected to a concentration response curve titration screening, leading to 21 hit compounds with structural similarities. Most of these compounds exhibit planarity with fused rings, hydrogen bonding sites, a lack of free rotation among some of the aromatic groups, and functional groups such as anthraquinone, benzenediol, benzofuran, tetrahydroquinoline, thiourea, and dihydroquinazoline. After an additional selection to avoid false positive activity, five compounds were selected for further biophysical and biological studies to elucidate the molecular dynamics and interaction with Aβ40 in the membrane-mimicking environment: wedelolactone (BF-3), hypocrellin A (AQ-4), 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxy1,2,3,4-tetrahydroisoquinoline hydrochloride (THQ-1), 2-(1,3-benzodioxol-5yl)-6-nitro-1,2,3,4-tetrahydroquinazolin-4-one (DHQ-1), and 6-nitro-2-(3,4,5trimethoxyphenyl)-2,3-dihydro-1H-quinazolin-4-one (DHQ-2) (Fig. 15.4b). These compounds perturb the morphological structures and the secondary structure of Aβ40 aggregates via direct binding, resulting in a reduction of membrane disruption. The molecular analysis of the interaction of natural products with membrane-catalyzed Aβ aggregates that result in membrane disruption could pave the way for the development of drugs against AD. In contrast to Aβ, the search for aggregation modulators of tau (441 amino acids) and αSyn (140 amino acids) via HTS is not straightforward because performing reproducible aggregation experiments for both proteins is challenging, and lot-tolot differences among laboratories are large. Crowe and colleagues proposed HTS methods for ~ 51,000 compounds using a heparin-induced fibrillization of tau [46]. Two 2,3-di(furan-2-yl)-quinoxalines and members of the pyrimidotriazine, benzofuran, porphyrin, and anthraquinone families were found to inhibit tau fibrillization. Otzen and colleagues developed HTS methods using SDS-stimulated αSyn aggregation combined with Förster resonance energy transfer between terbium ion and Th-T fluorescein, fulfilling the reproducible detection of the initial stages in αSyn aggregation [47]. Their screening of 746,000 compounds led to 58 hits that inhibited αSyn aggregation and prevented membrane permeabilization by αSyn oligomers in cellula. The most effective inhibitors were derivatives of (4-hydroxynaphthalen-1-yl) sulfonamide.

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15.4 Searching for Natural Products That Delay the Nucleation Phase and Promote the Elongation Phase of Aβ Aggregation In Japan, Osada (RIKEN) has founded the Natural Products Depository (NPDepo) as a public chemical bank including natural products and their derivatives extracted from the research of natural product chemists and synthetic chemists [48]. On the basis of a nucleation-dependent polymerization model [20], our group proposed a classification of compounds into nine groups (groups A–I) contingent on their ability to affect the nucleation or elongation phases of Aβ [49] (Fig. 15.5). In the pursuit of anti-AD drugs that delayed the nucleation phase and promoted the elongation phase (group C), we screened 480 natural products from the NPDepo, which consisted of 80 known biologically active compounds, named as the authentic library (http:// www.cbrg.riken.jp/npedia/?LANG=en), and 400 compounds comprising the pilot library [50]. The authentic library is composed of medicines on the market and is used for drug repositioning. Meanwhile, the pilot library serves as an initial test for compounds with standard scaffolds having canonical structures, and then Ward’s method is applied for hierarchical cluster analysis using Tanimoto coefficient for the purpose of gauging similarities and differences in the selected compounds. The categorization was performed by constructing a two-dimensional color heat map (Fig. 15.6a). Figure 15.6b displays the six compounds categorized as group

Fig. 15.5 Classification of amyloid modulators on the basis of the nucleation and elongation phases. In the nucleation phase, only the on-pathway route is indicated

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C from the authentic library: naloxone hydrochloride, streptozocin, sulfamerazine, warfarin, siccanin, and polyoxin D, all of which were first identified as inhibitors of Aβ42 aggregation. Moreover, the nine compounds of group C from the pilot library with unidentified biological activity are shown in Fig. 15.6c. The promotion of Aβ42 fibril elongation upon treatment with total 15 compounds of group C was confirmed by conducting a transmission electron microscopy (TEM) analysis for the Aβ42 aggregates. Having confirmed the delayed nucleation via Th-T assay, the effect on Aβ42 oligomerization was investigated by dot blotting using the commercially available antioligomer antibody A11 [51], finding that the oligomer signals decreased in the presence of the 15 compounds of group C. Furthermore, an MTT cell toxicity test using mouse neuroblastoma Neuro-2a cells revealed that 12 of the 15 compounds (80%) of group C significantly reduced the Aβ42-induced neurotoxicity. We substantiated that the results of the Th-T screening regarding the properties of the compounds of group C were in good agreement with those of the TEM and dot blotting analyses. To obtain further insight into the interaction of compounds of group C with Aβ42, we performed an electrophoretic mobility shift assay (EMSA) using native PAGE and found that 4 of the 15 compounds induced the mobility of Aβ42. Because the four compounds have cyclohexanone (3-g5), 2-pyrone (warfarin and 4-a8), or 4pyrone (3-a9 and 5-a9), these could form covalent bonding such as a Michael adduct or Schiff base with the Lys residues of Aβ42 as a plausible interaction mechanism (Fig. 15.6b, c; See also Sect. 15.6). Considering oligomeropathy as an etiology of AD [17, 18, 52, 53], these findings support suppressing the nucleation and enhancing a less-toxic fibrillization as promising approaches for anti-AD drug development and suggest warfarin as a drug repositioning candidate.

15.5 Differential Activity Searching for Aβ Aggregation Inhibitors Using LC–MS Combined with Principal Component Analysis (PCA) Some formulations of Kampo medicine, such as Yokukansan and Chotosan, have been conventionally used to combat dementia [54, 55]. In particular, a medicinal herb known as “shoyaku” in Japan has shown potential as a natural source of antiAD drugs. A combination of multiple compounds, rather than singular compounds, is more likely responsible for the complex biological functions of shoyaku, which hinders the elucidation of the structure and mechanism of each constituent involved in the anti-Aβ42 aggregation activity. In fact, analytical studies on the isolation and structural determination of bioactive substances of seven shoyaku derivatives found in Yokukansan showed that the main components in each shoyaku compound are not always responsible for the biological activities [54]. For example, indole alkaloids such as isorhynchophylline and hirsutine were identified as major compounds in Uncaria rhynchophylla (Chotoko, a shoyaku of Yokukansan) does not inhibit the

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Fig. 15.6 a Antiaggregation profiles of compounds from the authentic library with classification of compounds with color-coded heat maps (left), with the intensity increasing from blue (low intensity) to red (high intensity) (middle). Pie chart of 480 compounds categorized into nine groups is also shown (right). b Structures of hit compounds of group C from the authentic library. c Structures of hit compounds of group C from the pilot library. In (B,C), the structural moieties as potential Michael acceptors are shown in a circle

aggregation of Aβ42 [56]; instead, uncarinic acids A–D were found to be inhibitory even as minor compounds [57]. Since the isolation of the ingredients of shoyaku to identify the bioactive natural products is complicated and time-consuming, we screened 46 shoyaku searching for differences in their inhibitory activities against Aβ42 aggregation using LC–MS-based metabolomics coupled with PCA [58]. First, we conducted the screening of 46 shoyaku extracts from 18 plants. These shoyaku were categorized into three groups: (1) those having different activities depending on the plant parts (Chinese wolfberry, plantain, Poria cocos, dandelion, Japanese red elder, Chinese honey locust, cinnamon, lotus, and bitter orange),

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(2) those with activity from the same plant parts (styphnolobium, magnolia officinalis, senna, and kudzu), and (3) an inactive group (Japanese snake gourd, American ginseng, tachibana orange, notoginseng, and eucommia). In particular, we focused on five extracts (Kakou, Kayou, Gusetsu, Rensu, and Renbou) from lotus because their inhibitory activities were considerable different depending on the part of the plant from which they were extracted (i.e., petiole, leaf, root node, stamen, and receptacle, respectively). It should be noted that Kayou (Nelumbinis folium), Gusetsu (Nelumbinis rhizomatis nodus), Rensu (Nelumbinis nelumbinis), and Renbou (Nelumbo nucifera), which were obtained from the leaf, the root node, the stamen, and the receptacle, respectively, largely inhibit Aβ42 aggregation, while the inhibitory activity of Kakou (Nelumbinis petiolus) from the petiole is relatively slow and weak. Then, a comparison between the components of active shoyaku with those of inactive shoyaku allowed identifying the antiaggregative compounds. We then subjected the five shoyaku from lotus, among which Renbou was found to be the most active and Kakou the least active, to LC–MS analysis and used the obtained retention time, signal intensity, and molecular weight of the analytes for the subsequent PCA (Fig. 15.7a), which allowed us to visualize similarities and differences with minimal loss of structural and physicochemical information [59, 60]. As can be extracted from the score plot in the positive and negative modes, Kayou, Rensu, and Renbou were more likely to have identical active compounds than Gusetsu. Then, the corresponding loading plot and analysis of clustered patterns afforded 29 natural products including flavonoids and terpenoids as active compound candidates in lotus-derived shoyaku. To validate the result, we conducted a Th-T assay on 12 compounds (Fig. 15.7b) that showed strong MS intensities and were commercial available. These compounds were classified into three groups: (I) a group showing strong inhibition (datiscetin, kaempferol, morin, robinetin, and quercetin), (II) a group with moderate inhibition (myricitrin, quercetin-3-O-glucuronide, and quercetin-3-O-glucoside), and (III) a group without inhibitory activity (curcumol, kaempferol-3-O-glucuronide, and kaempferol-3-O-glucoside). Fisetin was excluded from this classification because its inherent potent fluorescence interfered Th-T fluorescence. To the best of our knowledge, this was the first study to propose lotus-derived shoyaku as a potential drug for AD treatment. Considering the various pharmacological benefits of lotus [61], lotus-derived shoyaku is expected to find application in the treatment of AD, second only to Yokukansan and Chotosan. Metabolomics coupled with a statistical procedure has offered a new methodology based on activity differences for the exhaustive analysis of herbal extracts with predictable reliability and efficacy [62].

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Fig. 15.7 a Score plot (left) and loading plot (right) of the compounds in Kakou, Kayou, Gusetsu, Rensu, and Renbou based on the LC–MS data obtained in positive mode. QC = quality control. In the score plot, data points that have similarities are plotted at the center or close to each other, whereas data points having differences are plotted far from the center. PC1 is the axis showing the largest variance (first principal component) of the data in the orthogonal coordinate axes. PC2 is the axis perpendicular to PC1 and indicates the second-largest variance in the data. Each plot in the loading plot generated from the score plot represents each unique molecule for each sample group (shoyaku) to distinguish these sample groups. b Structure of candidate compounds as active compounds from lotus-derived shoyaku extracts according to PCA. GlcA = glucuronide, Glc = glucoside. The structural moieties as potential Michael acceptors are shown in a circle

15.6 Inhibitory Mechanism of Aβ Aggregation by Natural Products For the development of antiamyloid drugs, the molecular understanding of the specific interaction between natural product inhibitors and amyloid aggregates and the resulting biomolecular process that is linked to neurotoxicity is indispensable.

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Natural product inhibitors have been found to form noncovalent interactions such as π/π or CH/π interactions, hydrogen bonding, and salt bridge with one or multiple aggregation stages of amyloid (Fig. 15.8a). These small compounds exhibit planar or flat structures comprising a conjugated system such as aromatic rings and α,βunsaturated carbonyl groups. In fact, most fluorescent probes of Aβ aggregates such as Th-T present these structural features. Curcumin, which inhibits aggregation of Aβ, αSyn, and tau, is a representative of this class of compounds [63]. Curcumin harbors an α,β-unsaturated β-diketone and two aromatic O-methoxy-phenolic groups, undergoing diketo/keto–enol tautomerism. Although the α,β-unsaturated carbonyl group can serve as a good Michael acceptor, several solid-state NMR spectroscopic studies indicated that curcumin interacts noncovalently with Aβ42 fibrils through an aromatic interaction with His and Phe residues in the N-terminal region [64, 65], and the methoxy or hydroxy groups of curcumin also participate in the association with the Aβ42 fibrils via conjugation [64]. Similarly, Wanker and colleagues revealed that (–)-epigallochatechin gallate (EGCG) inhibits Aβ42 aggregation by redirecting amyloid aggregation into nontoxic, off-pathway, highly stable oligomers [66]. They also suggested that EGCG interacts via noncovalent bonding with αSyn [66]. Our group demonstrated that noncatechol-type flavonoids (morin and datiscetin) inhibit both the elongation and nucleation phases of Aβ42 aggregation by targeting His13,14 residues, resulting in the inhibition of Aβ42 aggregation. The position and number of hydroxyl groups on the B-ring of noncatechol-type flavonoids could be one of the determining factors for their antiaggregative activities [67]. In the course of our investigations on the properties of uncarinic acid C and asiatic acid, which are ursane-type triterpenoids, we unveiled the role of the carboxylic acid moiety that forms a salt bridge with the side chains of Lys16,28 (Fig. 15.8b) [68]. To clarify the effect of this interaction on the oligomerization (early aggregation) of Aβ42, Aβ42 treated with each compound was subjected to ion mobility-mass spectrometry (IM-MS) analysis combined with native ionization techniques. Given the inherent tendency of native oligomers to form structurally heterogeneous assemblies, IM-MS is a powerful tool to measure the molecular sizes and drift time without using organic solvents that may disrupt the noncovalent interactions. We found that the formation of a salt bridge with Aβ42 prevents dimer and trimer formation, suppressing larger oligomerization. In contrast, the corresponding congeners without carboxy groups (α-amylin) do not exhibit such a behavior. Further comparison of rhein with chrisophanic acid as anthraquinoid derivatives confirmed the contribution of the salt bridge formation to the inhibition of Aβ42 aggregation. Covalent bonding, mainly via Michael addition or Schiff base formation, is the most potent interaction through which antiaggregation compounds exert their action [69]. Fink and colleagues first evidenced the Schiff base formation between baicalein and αSyn by conducting MS measurements (Fig. 15.8c) [70]. Also on the basis of an MS analysis, Kelly and colleagues suggested the formation of covalent bonding between EGCG and various amyloids, although convincing evidence was not provided [71]. Our group demonstrated that catechol-containing flavonoids such as (+)-taxifolin form Michael adducts with the Lys16,28 side chains of Aβ42 through flavonoid autoxidation [72]. (–)-Apomorphine, a dehydrated compound of

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Fig. 15.8 a Two possible (noncovalent and covalent) interactions of aggregation modulators and the oxidation state–dependent binary mode of (–)-apomorphine. b Salt bridge of uncarinic acid C with Aβ42. c Schiff base structure of baicalein with αSyn. d Michael adduct of warfarin with Aβ42

morphine, is prone to autoxidation due to the presence of a conjugated system and a phenol moiety. Consequently, (–)-apomorphine forms a Michael adduct with the Lys16,28 residue of Aβ42 before producing an unstable o-quinone form. Further autoxidation furnishes phenanthrene moieties, which suppress Aβ42 aggregation. In contrast, its inhibitory activity was found to decrease under reducing conditions using tris(2-carboxyethyl)phosphine. Intriguingly, an LC–MS/MS analysis coupled with NMR suggested that the target of (–)-apomorphine could be Lys16,28 in Aβ42, and then the extension of the conjugated system in (–)-apomorphine upon autoxidation can promote its planarity with the increase in the inhibitory activity, resulting in the oxidation state–dependent binary mode of (–)-apomorphine (Fig. 15.8a) [73]. Collaborative studies with Asai (Tohoku University) allowed identifying biosynthetic fungal decalin-containing diterpenoid pyrones as inhibitors of nucleation and elongation through Michael addition involving the 4-pyrone moiety. The compounds

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of group C described in Sect. 15.4 also contain a Michael acceptor moiety, such as cyclohexanone (3-g5), 2-pyrone (warfarin and 4-a8), and 4-pyrone (3-a9 and 5-a9) (Fig. 15.6b, c) [50]. In particular, the adduct formation between Aβ42 and warfarin was observed and postulated as responsible for the suppression of Aβ42-induced neurotoxicity (Fig. 15.8d). Most of the compounds with strong inhibition identified by PCA-based LC–MS analysis are flavonoid, in which 4-pyrones are fused with a benzene ring, also known as chromone (Fig. 15.7b). In contrast, the hit compounds selected by other criteria do not have such structural features (Fig. 15.4). Michael acceptors targeting nucleophilic amino acids (e.g., Lys, His, Arg, and Cys) are one of the most studied compounds in the investigation of the role of protein– ligand interactions in biological functions. Given the critical role of Lys16,28 in Aβ42 aggregation [74], Lys-specific inhibitors are promising candidates as anti-AD drugs. For example, Bitan and colleagues reported that tweezer-shape Lys-specific inhibitors are broad-spectrum inhibitors of aggregation and cytotoxicity of amyloidogenic proteins [75, 76]. However, particular attention should be paid to the off-target and adverse effects of Lys residues in physiological proteins on biological functions. Widespread off-target Lys binding might affect the bioavailability of irreversible inhibitors [77].

15.7 Conclusions and Future Perspectives In this chapter, the classification of natural products in nine groups according to the modulation of the nucleation and elongation phases of Aβ42 aggregation and the screening of natural products based on activity differences were described as efficient screening methods for aggregation modulators of amyloidogenic proteins. In the former method, the search for compounds that delay toxic oligomer formation and promote less-toxic fibril formation offers promising scaffolds as anti-AD drugs. In the latter approach, metabolomics coupled with highly sensitive and accurate spectroscopic techniques was demonstrated as a powerful strategy for the exhaustive analysis of large chemical libraries with predictable reliability and efficacy, which was applied to the identification of antiaggregative natural products by making a correlation between shoyaku extracts from different parts of the same plant and the biological activity. Recent NMR-based approaches may also help accelerate the structure determination of each component in shoyaku [78]. Moreover, the largest chemical libraries in the world, such as the Molecular Library and Imaging Program in the NIH (https://commonfund.nih.gov/molecularlibraries/index) and the Compound Management and Screening Center in the Max Planck Institute of Molecular Physiology (http://comas.mpi-dortmund.mpg.de/), will further facilitate HTS in terms of quantity and quality. Further progress in the biomedical application of natural products requires addressing the following issues: First, the identification and functional analysis of natural products and their metabolites are major impediments for medical development. This challenge can

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be addressed by in vivo proteome and transcriptome analysis for the evaluation of the biological activities and pharmacokinetics. Machine learning could also provide useful insights into the structural features responsible for the antiaggregation properties and the underlying mechanism. Furthermore, synthetic strategies toward improved analogs of natural products with biological activities, minimization of the natural products that match druglikeness (e.g. rule of five), and the introduction of other functional scaffolds should be explored. Second, limitations and depletion of natural resources are long-standing problems in natural product chemistry. To overcome this issue, the use of biosynthetic intermediates of pseudo-natural fungal products identified by bioinformatic and genome mining techniques is a promising solution. Recently, the “microbiome molecules” produced in the human microbiota have attracted attention as unique compounds interacting with the cellular machinery and affecting human health [79– 81]. For example, colibactin, a microbial genotoxin, can crosslink with the human genome, leading to an emergency signal and prophage induction [82]. As genes can be predicted to encode compounds with antibiotic properties, humimycin A, which is secreted by human microbiota Rhodococcus equi and Rhodococcus erythropolis, exhibits an antibiotic effect against a strain of methicillin-resistant Staphylococcus aureus (Fig. 15.9) [83]. These unique activities of microbiome molecules will open horizons for the development of microbiome-based therapeutics and diagnostic biomarkers. Overall, the utilization of diversified natural product libraries and their unique spectroscopic analysis for the investigation of drug seeds will be a new trend in the field of natural product chemistry in the near future.

Fig. 15.9 Structure of colibactin 788 and humimycin A as microbiome molecules

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Acknowledgements This study was supported in part by JSPS KAKENHI (Grant 16H06194 and Grant 20K05849 to K.M.) and The Naito Foundation (to K.M.). I am grateful to Prof. Kazuhiro Irie at the Graduate School of Agriculture, Kyoto University for his enthusiastic encouragement and guidance, and to Dr. Mizuho Hanaki and Ms. Shiori Horii at the Graduate School of Agriculture, Kyoto University, for performing the experiments. I acknowledge the Chemical Resource Development Research Unit, Technology Platform Division, RIKEN Center for Sustainable Resource Science (CSRS), for providing NPDepo, and Dr. Hiroki Gunji in Alps-Pharmaceutical Industry Co., Ltd. for providing a shoyaku library. I would like to thank Enago (www.enago.jp) for the English language review.

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Chapter 16

Discovery of Natural Product Analogues with Altered Activities by a High-Throughput Strategy Hiroaki Itoh

Abstract To exploit natural products as a source of biologically active compounds, accelerating the discovery of analogues that exhibit more potent and modulated biological activities is essential. In this chapter, two specific examples of a one-beadone-compound (OBOC)-based functional enhancement and modulation of peptidic natural products are demonstrated. As structurally distinct natural product scaffolds, macrocyclic lysocin E and linear gramicidin A were adopted. The combination of solid-phase synthesis with a split-and-mix approach, microscale functional evaluation, and structure determination based on tandem mass spectrometry led to the successful discovery of natural product analogues that exhibited enhanced and altered functions. These results exemplify the potential of this strategy for accelerating the structure–activity relationship study and structural optimization of natural products. Keywords Solid-phase total synthesis · One-bead-one-compound · Peptidic natural products · Antibiotic · Biological membrane

16.1 Introduction As living organisms have evolved, natural products, such as secondary metabolites, have been structurally optimized for biological defense and to compete with other individuals through interactions with biomacromolecules. The uniqueness of structures and functions illustrates the irreplaceable value of natural products for learning and evolving in the design of biologically active molecules [1–3]. However, for utilizing natural products, such as medicines, agrochemicals, and other biological tools, it is often necessary to finely tune the activities and physicochemical properties that are enabled by structural modification based on chemical synthesis. The possible chemical synthesis strategy for obtaining structurally-modified natural products can be primarily classified into the following parts: semisynthetic H. Itoh (B) Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-0033, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_16

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derivatization and total synthesis. The former strategy can be generally conducted by chemoselective reactions or partial degradation of natural products. In this case, complex scaffolds of natural products can be directly utilized, whereas limitations intrinsically exist in the modification of the core structures. In contrast, the latter strategy is suitable for changing the deep-seated structures of parent natural products [4–8]. From this aspect, the total synthesis-based strategy can contribute to generating a unique set of natural product analogues that exhibit large structural variations. It is also important to increase the number of synthetic analogues that are submitted to functional evaluations because investigating the densely populated chemical space around the natural products could enhance the chance of discovering analogues that exert ideal functions. However, performing one-by-one total synthesis of natural product analogues is generally time-consuming, and the process often becomes impractical due to the necessity of many synthetic resources. Based on these facts, most of the chemical space around natural products remains unexplored. Conversely, overcoming the limitation of synthesis processes should significantly accelerate seed and lead discovery for applications. It is thus necessary to develop an efficient total synthesis-based strategy for enabling the generation and evaluation of various natural product analogues with high throughput. To address the above fundamental issues, we devised a one-bead-one-compound (OBOC) strategy for the generation and functional evaluation of analogues of structurally complex peptidic natural products. The OBOC approach was originally introduced by Lam for the solid-phase construction of small peptide libraries [9–11]. However, the applications of this bead-based approach to complex natural products have been very limited [12, 13]. Basically, the OBOC strategy comprises the following four features (Fig. 16.1): (i) Because the on-bead structural randomization occurs by the split-and-mix approach, the analogues are constructed by solidphase synthetic methods. In this manner, randomization of monomer components can readily generate thousands of natural product analogues. (ii) Because small resin beads are used for solid support, one bead carries one specific analogue on a microgram scale. (iii) We cannot identify the structure of each bead-bound analogue during the synthesis because analogues are randomly generated in the split-andmix approach. To identify the structure carried by each bead, microscale structure determination is needed. In our study, tandem mass spectrometry (MS/MS) analysis was utilized [14]. (iv) In the OBOC approach, the activity of each analogue can be assessed after separating each bead. Importantly, both on-bead and in-solution assays are applicable, which facilitates the investigation of multimodal molecular functions. In this chapter, specific examples of the OBOC-based functional enhancement and modulation of peptidic natural products are described. As two different starting structural scaffolds, the membrane-targeting antibacterial natural products lysocin E (1) [15] and gramicidin A (2) [16] were adopted (Fig. 16.2).

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Fig. 16.1 OBOC-based strategy for the synthesis and evaluation of diverted peptidic natural products

16.2 Lysocin E Lysocin E (1, Fig. 16.2) is an antibacterial peptidic natural product derived from Lysobacter sp [17]. The 37-membered macrocyclic core of 1 consists of 12 amino acid residues with an N-methyl amide group and an ester linkage. Compound 1 exerts highly potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) through rapid bacteriolysis in the presence of serum [18]. In addition, 1 has a greater therapeutic effect on MRSA-infected mice [50% effective dose (ED50 ) = 0.4 mg/kg] than that of vancomycin, which is clinically used for treating MRSA infections. Importantly, 1 is the first molecule reported to exert antibacterial activity through complexation with bacterial menaquinone (MK), while multiple natural products have been reported to target MK since the discovery of 1 [19–21]. Compound 1 has also been reported to interact with lipid II, a precursor of the bacterial cell wall [22]. The target isoprenoid quinone MK and its reduced form menahydroquinone (MKH) are essential for electron transport chains that are located in the bacterial cell membrane [23, 24]. The complexation between 1 and MK disrupts bacterial membrane integrity as an initial step, which eventually causes bacteriolysis. Since such a mechanism of action is distinct from those of any clinically used antibacterial agents, 1 is a promising structural platform for developing new antibiotics against multidrug-resistant bacterial infections. In our previous structure–activity relationship (SAR) study of 1, the 15 analogues of 1 were synthesized and functionally assessed to decipher the potential roles of the side chains for exerting the MK-dependent antibacterial activity [25, 26]. The

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Fig. 16.2 Structures of lysocin E (1) and gramicidin A (2)

analysis found that the N-methyl amide group in the macrocycle and three possible interactions of side chains likely play important roles in the MK recognition by 1 and the resulting membrane disruption as follows (Fig. 16.3): the hydrophobic interaction of the (R)-3-hydroxy-5-methylhexanamide moiety attached to Thr-1 with the lipid chains, electrostatic interaction of the cationic protonated guanidines at Arg-2/7 with anionic phospholipid polar heads of the bacterial membrane components [27, 28], and the aromatic-aromatic interaction between the electron-rich indole ring of Trp10 and the phenyl ring of N-Me-Phe-5 with the electron-deficient naphthoquinone

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ring of MK [29, 30]. The SAR analysis also revealed that the hydroxy group at the 5methylhexanamide moiety and the carboxylic acid of Glu-8 are likely not responsible for the antibacterial activity of 1. Notably, compared to parent 1, all 15 synthesized analogues were less potent, implying that it is highly challenging to enhance the activity of the natural product.

Fig. 16.3 Potential side-chain functional groups of 1 that are responsible for the disruption of the MK-containing membrane

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16.3 Strategy Design for Discovering More Potent Analogues of Lysocin E To overcome the difficulty of discovering more potent analogues of natural products, we envisaged accelerating the processes of total synthesis of the analogues and their functional evaluation by a new OBOC-based strategy. Namely, we envisioned the activity enhancement of 1 by expeditious generation and evaluation of the bead-based library, which comprised thousands of analogues of 1. Upon constructing an analogue library, the randomization sites were selected to prevent changing biologically essential factors and to maximize the hit rate of the active analogues. First, the stereochemistry of the Cα positions of the component amino acids and achiral Gly-4 were maintained because the set of stereochemistries in the macrocycle could be essential for the bioactive conformation of 1. Second, (R)3-hydroxy-5-methylhexanamide, Arg-2/7, N-Me-Phe-5, and Trp-10 were unchanged because these moieties were found to be essential for exerting potent activity in the previous SAR study. Third, Glu-8 was retained because the side-chain carboxylic acid was utilized for anchoring the peptide to a bead. Fourth, ester-linked Thr-1/12 was retained because the yield of solid-phase esterification was presumably more sensitive to the substrate structures than that of amidation. Based on these considerations, Ser3, Leu-6, Gln-9, and Ile-11 were determined to be sites for structural randomization in split-and-mix synthesis (Fig. 16.4). When designing the library, the amino acid components were selected to diversify the physicochemical properties of the macrocycle. Specifically, hydrophobic, acidic, basic, hydroxy, amide, aromatic, and methyl groups were selected for random substitution of residues-3, -6, -9, and -11, which produced 2401 (74 ) compounds that were structurally varied through split-and-mix synthesis. Importantly, the set of seven amino acids was selected to have distinct mass numbers in each residue (e.g., N for residues-3, -6, and -11 instead of Q; O for residue-9 instead of K), which enabled all the analogues to have unique fragmentation patterns in MS/MS analysis. As a bead, TentaGel [31] macrobeads (MBs) were adopted because of their high capacity (3.5 nmol per 0.3 mm bead) and narrow size distribution, which are essential for comparing the biological assays of different compounds. Furthermore, the high swelling rate of the polyethylene glycol (PEG)-grafted polystyrene polymer in various solvents permits a variety of reaction conditions, leading to the high reactivity of the N-terminal amines of bead-linked peptides. For a linkage between the bead and macrocycle, an o-nitroveratryl linker [32, 33] was adopted because the acidstable and photocleavable nature of the linker permits both on-bead and in-solution experiments after the on-bead construction of peptides is complete. Namely, beadlinked protecting group-free macrocycles were used for the on-bead MK complexation assay. It was presumed that bead-linked 1 would maintain the ability for MK complexation because the esterification of Glu-8 showed no significant effect on MK recognition in a previous SAR study [26]. Upon the in-solution antibacterial assay and MS/MS sequencing analysis, bead-free peptides were readily generated by UV irradiation.

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Fig. 16.4 Design of the 1-based library. One-letter codes and molecular weight of the residues are displayed in parentheses

16.4 Construction of a Lysocin E-Based Library Construction of the analogue library of 1 started from Glu-8-loaded o-nitroveratrylTentaGel MBs (3, Fig. 16.5). To achieve both sufficient library coverage (95%) and high efficiency of assays, a threefold number of beads (7510 beads) was used for the synthesis [34]. Solid-phase chain elongation was carried out by

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sequential piperidine-mediated Fmoc removal and microwave-assisted condensation [35–37] of Nα -Fmoc-protected amino acids using O-(7-aza-1H-benzotriazol-1yl)-N,N,N ' ,N ' -tetramethyluronium hexafluorophosphate (HATU) and 1-hydroxy-7azabenzotriazole (HOAt) at 40 °C [38]. After the introduction of 4 to the sequence, the esterification of the Cβ -hydroxy group at Thr-1 was carried out on the solid support by activation of Fmoc-l-Thr(Ot-Bu)-OH using N,N ' -diisopropylcarbodiimide (DIC). To randomize the components of residues-3, -6, -9, and -11, the beads were split into 7 portions, each of which was condensed with a different Nα -Fmoc amino acid. After mixing all the beads, further chain elongation was continued. Split-and-mix synthesis thus produced bead-linked 12-mer linear peptides with structurally randomized residues-3, -6, -9, and -11 (5). For on-bead macrocyclization, the C-terminal allyl group was removed using Pd(PPh3 )4 with morpholine to liberate the free carboxylic acid as a macrocyclization precursor. Subsequently, the on-bead macrolactamization proceeded by the action of (benzotriazole-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP) [39]. Finally, acid-labile protecting groups for side chains (Boc, t-Bu, Pbf [40], and TBS) were removed by aqueous TFA, which successfully afforded 2401 bead-linked macrocyclic peptides.

16.5 Discovery of Lysocin E Analogues Showing Enhanced Antibacterial Activity After the construction of the bead-linked 2401 peptides, we next conducted an onbead MK complexation assay to screen the beads that exhibited high affinity for menaquinone-4 (MK-4) (Fig. 16.6). In this assay, the bead-linked macrocycles were incubated with MK-4 in MeOH to form the complex and were then dispensed into 96well microplates. After the unbound MK-4 was washed with MeOH, the bead-bound MK-4 was eluted by incubating the beads in n-BuOH at 50 °C. To quantify the very small amount of bead-derived MK-4, UV detection was found to be insufficient with respect to sensitivity. Alternatively, the amount of MK-4 was measured by fluorescence from the menahydroquinone-4 (MKH-4) that was produced by reduction using NaBH4 . The top 3% of 7510 beads (241 beads that exhibited fluorescence intensities over 1000 a.u.) were subjected to structure determination and an in-solution assay. Prior to the in-solution experiments, the o-nitroveratryl linker was cleaved by UV irradiation (365 nm) to afford bead-free peptides, which were first subjected to MS/MS-based structure determination. In general, MS/MS analysis of cyclic peptides provides a complex fragmentation pattern due to the multiple cleavable amide bonds in the macrocycle, which makes it difficult to perform structure determination. To enable facile interpretation of the MS/MS spectra, the ester linkage of the macrocycle was selectively hydrolyzed under basic conditions (1% NH3 , MeOH/H2 O) to produce the corresponding linear seco acid. Matrix-assisted laser desorption/ionization (MALDI) MS/MS analysis of the hydrolyzed seco acids revealed that the 241 beads carried 166 unique compounds. Importantly, parent 1

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Fig. 16.5 Construction and screening platform of the 1-based library

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Fig. 16.6 Dot plot of the MK-4 complexation assay and screening of 2401 peptides

was observed in this analysis, indicating that the set of synthesis and MK complexation assays were appropriate to discover the active compounds from the mixture of randomly generated compounds. Next, the antibacterial activities of the 166 compounds were evaluated using S. aureus Smith ATCC 13709. The application of 10% bovine calf serum (BCS)supplemented medium increased the sensitivity of this assay, which allowed us to identify the antibacterial activity using each of the one-bead-derived compounds. As a result, new 22 analogues (6–27) and parent 1 completely inhibited the growth inhibition of S. aureus, while 143 analogues showed no significant activity. The newly discovered analogues 6–27 were resynthesized on a several-milligram scale and subjected to assays to investigate their detailed biological activities (Fig. 16.7). The antibacterial activities of 6–16 against methicillin-susceptible S. aureus strain (MSSA1) were found to be equipotent or more potent [minimum inhibitory concentration (MIC): 0.015–0.0625 μg/mL] than that of 1 (MIC: 0.0625 μg/mL), while 17–27 were less potent than 1. Notably, among the analogues 6–16, 6, 7, and 8 exhibited fourfold more potent antibacterial activities (MIC: 0.015 μg/mL) than that of 1. To investigate whether analogues 6–16 have the same mode of action as parent 1 for exerting their potent antibacterial activities, membrane disruption activity was evaluated using MK-4-containing liposomes as a model lipid bilayer system. To mimic the bacterial membrane, large unilamellar vesicles (LUVs) doped with or without 1.25 mol% MK-4 [41, 42] were prepared using egg yolk phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG) [43]. In this system, the carboxyfluorescein encapsulated in LUVs was diluted, and its fluorescence was enhanced upon membrane disruption. The concentration dependency of the disruption activity was evaluated by comparison of the 50% effective concentration (EC50 ) values. The EC50 against two different types of LUVs clearly indicated that all the analogues showed smaller EC50 values against the LUVs comprising PC/PG/MK-4 compared with the EC50 against the LUVs comprising PC/PG. These results strongly

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Fig. 16.7 Structures and activities of the newly discovered analogues 6–16

suggested that 1 and 6–16 exert membrane-disrupting activities through the same MK-4-dependent mechanisms. The large set of SAR data obtained suggested for the first time the structural requirements of residues-3, -6, -9, and -11 for exerting their biological activity. Hydrophobic and bulky Leu (L) and Ile (I) were found to be the common components of residues-6 and -11, respectively, in parent 1 and all potent analogues 6–16. The clearcut structural uniformity among 12 compounds indicated that these hydrophobic and bulky amino acids are highly important for the potent antibacterial activities of 1 and may play a role in the interaction between the macrocycle and bacterial lipid tails and/or organization of the bioactive conformation of peptides [44]. In contrast, the Cβ -hydroxy group of residue-3 and primary amide of residue-9 in 1 were found to be less important for the activity because a wider variety of amino acids was found in analogues 6–16. Intriguingly, the structures of the most potent analogue 6 and parent 1 differed only in the presence or absence of the Cβ -hydroxy group at residue-3 (S

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for 1, A for 6). This observation indicates that the developed strategy was useful to decipher the effect of very minute structural change on the activity of parent 1, the prediction of which is highly difficult. Overall, the OBOC-based strategy was demonstrated to be effective for gaining more potent natural product analogues and for deciphering the structural factors that are responsible for the activity.

16.6 Gramicidin A Gramicidin A (2, Fig. 16.2) is a linear peptidic natural product that is biosynthesized by Bacillus brevis and has long been known as an antibiotic that exhibits broadspectrum antibacterial activity against Gram-positive bacteria [45, 46]. Although 2 is utilized as an ingredient of topical antibacterial agents [47, 48], the severe hemolytic activity [49] and the high toxicity against mammalian cells [50] of 2 have prevented its wider application, such as for systemic use. The most characteristic function of 2 is the monovalent cation-selective ionchannel-forming activity (H+ , Na+ , and K+ ) in a lipid bilayer. The amino acid sequence comprising 15 amino acids with d/l-alternating Cα -stereochemistries and an N-terminal formyl group and a C-terminal 2-aminoethanol structure has the ability to form an intramolecular hydrogen bond network, resulting in a helical structure with 6.3 residues per turn, which is designated β6.3 -helix (Fig. 16.8) [51–53]. In a lipid bilayer, the two helices of 2 are assembled at their N-terminal sides to form a nanotube that spans across the biological membrane and functions as a monovalent cation-selective channel [54, 55]. This ion channel activity of 2 is believed to induce the desired antibacterial activity along with undesirable hemolytic and cytotoxic activities. Fig. 16.8 Ion-channel-forming β6.3 -helical dimer of 2 (PDB ID: 1MAG)

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16.7 Strategy Design for Discovering Gramicidin A Analogues with Altered Activity Profiles With the aim of developing useful seeds for drugs and biological tools based on 2, structurally modifying 2 was necessary to maintain the desired antibacterial activity and alleviate the undesirable mammalian cytotoxicity and hemolytic activity. We approached this issue with an OBOC-based strategy, which enabled the synthesis and evaluation of thousands of analogues of 2. The analogue library was designed to maintain the β6.3 -helical conformation because the various biological activities of 2 are caused by its ion-channel-forming ability (Fig. 16.9). Thus, d/l-alternating Cα -chiralities, an N-terminal formyl group, a C-terminal 2-aminoethanol structures, and side chains of residues-1, -2, -9, -11, -13, and -15 [56–59] were unchanged due to the importance of these factors for forming the dimeric β6.3 -helix. Among the remaining 9 residues, we randomize all six d-configured residues (Leu-4, -10, -12, and -14/Val-6 and -8) to simplify the preparation of synthetic monomers. To modulate the biological functions of 1 by forming intra- and intermolecular hydrogen bonds with surrounding biomolecules, d-Thr (T) and d-N γ -methylaspargine (d-Asm, N' ) [60] were adopted as the monomers in addition to the original aliphatic d-Leu and d-Val for randomizing the six residues. T and N’ can presumably mimic the atomic arrangement of the Cβ - or Cγ -branched chains of V and L, and the Cβ - and N γ -methyl groups would increase the hydrophobicity of the helix surface to facilitate membrane insertion and ion channel formation. This design provided 4096 (46 ) analogues through split-and-mix synthesis.

16.8 Construction of the Gramicidin A-Based Library Fmoc-based solid-phase synthesis with the split-and-mix approach was applied to construct the analogue library (Fig. 16.10). Hydroxymethylbenzoic acid (HMBA) [61] was employed to anchor the peptide chain to the TentaGel MB. The stability of the HMBA ester under acidic conditions permits C-terminal ester-amide exchange using 2-aminoethanol after acid-mediated side-chain deprotection, which leads to relatively high purity of the crude material. To construct the library, 13584 beads were used, which is a threefold number of 4096 peptides, to provide 96% library coverage. In the synthesis, the nitrogen atom at the indole ring of l-Trp, the Cβ hydroxy group of d-Thr, and the N γ -methylamide group of d-Asm were protected by acid-labile Boc, t-Bu, and 2,4,6-trimethoxy benzyl (Tmb) groups, respectively, to suppress the side reaction and to maintain the condensation efficiency by reducing the undesired interchain interaction on a solid support [62, 63]. The construction of bead-linked 4096 peptides was commenced from Fmoc-lTrp(Boc)-loaded HMBA-TentaGel MBs (28). The d,l-alternating structures were elongated by sequential piperidine-mediated Fmoc removal and condensation of Nα -Fmoc amino acids by HOAt and HATU. To increase the condensation efficiency,

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Fig. 16.9 Design of the 2-based library. One-letter codes (N' represents N γ -methylaspargine, Asm) and molecular weight of the residues are displayed in parentheses

the reaction mixture was heated to 60 °C using microwave irradiation. Upon the introduction of residues-4, -6, -8, -10, -12, and -14 to the sequences, the beads were split into 4 portions, and each portion was condensed with the four different Nα Fmoc amino acids (V, L, T, and N' ) for structural randomization. After mixing all the beads, the next amino acid residue was introduced to furnish the whole amino acid sequence of 1. Then, the formylation of the N-terminal amine using p-nitrophenyl formate afforded bead-linked protected peptides. Finally, deprotecting the side chains by TFA with i-Pr3 SiH [64] and H2 O as cation scavengers produced protecting groupfree bead-linked peptides. After dispensing 13584 beads carrying 4096 analogues into 96-well microplates, each bead was treated with 2-aminoethanol in DMF to produce the library peptides via ester-amide exchange at the C-termini of beadlinked peptides. For the subsequent in-solution assays and structure determination, the crude materials were dissolved in DMSO to produce 13584 peptide solutions.

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Fig. 16.10 Construction and screening platform of the 2-based library

16.9 Discovery of Gramicidin A Analogues That Exhibit Altered Activities After constructing the library, we performed three types of microscale assays to screen the analogues that exhibited distinct activity from that of parent 2. Namely, 13584 peptide solutions were evaluated for their H+ /Na+ transport activity in LUVs, mammalian cytotoxicity against the P388 mouse leukemia cell line, and antibacterial activity against Gram-positive Streptococcus pyogenes (Fig. 16.11). First, the H+ /Na+ transport assay and the cytotoxicity assay were conducted using a single concentration of each peptide. In the H+ /Na+ transport assay, the pH-sensitive fluorescent dye pyranine [65] was encapsulated in LUVs consisting of PC and PG, and a pH gradient across the membrane was applied by adding NaOH. The degree of H+ /Na+ transport induced by a peptide was assessed by the change in the fluorescent emission from pyranine. In this assay, we screened 600 of 13584 peptides based on the peptides exhibit higher H+ /Na+ transport activities than that of parent 2. We then evaluated the mammalian cytotoxicity of 13584 peptides by a colorimetric assay [66]. The results revealed that 74 solutions (cell viability ≤20%: group A) exhibited relatively high cytotoxic activity, while 519 solutions (cell viability ≥30%: group B) showed less cytotoxicity. Next, the three concentrations of the peptides that belonged

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Fig. 16.11 Scatter plot of three microscale assays and screening of 4096 peptides

to both groups A and B were applied to assess the antibacterial activities against S. pyogenes. The potency of each peptide was assessed using fourfold serially diluted peptide solutions, and the peptides were classified into four groups (+++: 640-fold diluted solution was active, ++: 40-fold diluted solution was active, +: 16-fold diluted solution was active, -: 16-fold diluted solution was inactive). Finally, all the peptides in groups A and B were structurally determined by MS/MS analysis, revealing that groups A and B comprised 41 and 276 unique sequences, respectively. Of note, the structure determination revealed that four solutions in group A corresponded to parent 1, demonstrating the reliability of our screening system. Among the newly discovered 316 analogues, 10 analogues (A1, B0 1–B0 4, B1 1– B1 3, B2 1, and B2 2) were selected for investigating the detailed functions as follows. A1 was the sole compound that exhibited more potent antibacterial activity than that of parent 2 among the 40 analogues in group A. The 276 analogues of group B were classified into three subgroups, B0 , B1 , and B2 , according to the number of hydrogen-bonding residues T and N' and the frequency of appearance as antibacterial compounds. Then, representative analogues were selected from each subgroup. B0 1– B0 4 in subgroup B0 (the number of T and N' = 0) and B1 1–B1 3 in subgroup B1 (the number of T and N' = 1) were identified three times or more as antibacterial peptides and were not found in group A. B2 1 and B2 2 in subgroup B2 (the number of T and N' = 2) were identified three times or more. To verify the set of assays and investigate the detailed functions, the 10 selected analogues were individually prepared by solid-phase synthesis on a several-milligram scale and the activities were evaluated by using the following parameters: EC50 value

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of H+ /Na+ transport activity, 50% inhibitory concentration (IC50 ) of mammalian cytotoxicity against P388 cells, 10% hemolytic concentration (HC10 ), and MIC against S. pyogenes and Streptococcus pneumoniae. In the H+ /Na+ transport assay using LUVs, all 10 analogues displayed nanomolar-level EC50 values, which is consistent with the criteria of compound selection. In the other three assays, the analogues showed varied activities depending on the subgroups. The activity profiles of the most important analogues, A1, B0 1, and B1 2, are listed in Fig. 16.12. A1 possesses aliphatic amino acid V as residue-4 instead of the original L and showed more potent biological activities than that of parent 2 in all assays, including the eightfold and fourfold enhanced antibacterial activities against S. pneumoniae and S. pyogenes, respectively. B0 1, which possesses multiple substitutions of the original L with V at residues-4, -12, and -14, exhibited fourfold and twofold more potent antibacterial activities than that of 2 against S. pneumoniae and S. pyogenes, respectively, while exhibiting threefold weaker mammalian cytotoxicity and sevenfold weaker hemolytic activity. B1 2 bears one T instead of V at residue-8 and retained the nanomolar antibacterial activities of 2 (MIC: 8.3 nM and 67 nM against S. pneumoniae and S. pyogenes, respectively) yet showed 70-fold attenuated cytotoxicity (IC50 : 390 nM) and negligible hemolytic activity in the tested range of concentrations (HC10 : >3000 nM). The distinct activity profiles of A1, B0 1, and B1 2 clearly demonstrated that side-chain modification can modulate the biological activity of ion-channel-forming 2 and could be a useful structural basis for ion-channel-based anticancer and antibacterial agents.

Fig. 16.12 Structures and activities of 2 and newly discovered analogues A1, B0 1, and B1 2

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16.10 Conclusion In conclusion, we demonstrated two different examples for devising a new highthroughput strategy to chemically prepare and functionally evaluate thousands of analogues of membrane-targeting antibacterial peptidic natural products. The developed strategy integrates solid-phase synthesis with the OBOC-based approach for structural randomization, microscale assays for the selection of hit compounds, and MS/MS sequencing analyses for structural determination. First, the antibacterial natural product 1 that targets MK was adopted as the macrocyclic scaffold and a library comprising 2401 peptides was generated and evaluated. The potent antibacterial activities of 6, 7, and 8 were fourfold greater than that of 1, indicating that the combination of the on-bead MK complexation assay, in-solution antibacterial assay, and MS/MS analyses are useful for enhancing the original activity of 1. Second, the ion-channel-forming antibacterial natural product 2 was adopted as the linear scaffold and a library comprising 4096 peptides was generated and evaluated. In-solution microscale assays for evaluating the H+ /Na+ transport activity, mammalian cytotoxicity, and antibacterial activity revealed that the newly identified analogues B0 1 and B1 2 retained the antibacterial activity of 2 while showing significantly attenuated mammalian cytotoxicity and hemolytic activity. The results indicated that the system was useful to successfully alter the intrinsic function of 2. Both examples demonstrated that the developed strategy is useful for selecting promising natural product analogues with different activity profiles from the randomly generated analogues, which could accelerate the SAR study on natural products. The straightforward synthesis-evaluation strategy demonstrated in this chapter will help to explore the densely populated chemical space that comprises natural products and their analogues by total synthesis, which could maximize the potential of natural products. For 2, the number of analogues generated and evaluated in the present study was tenfold larger than the total number reported in the last 80 years from its discovery. Furthermore, the number of analogues of 1 generated and assessed by the OBOC strategy was 100-fold larger than that of the previously synthesized compounds. The author hopes that the developed strategy will facilitate the exploitation of the attractive structures and functions of natural products by combining the wisdom of nature with the creativity of human beings. Acknowledgements The author would like to deeply thank Prof. Masayuki Inoue (The University of Tokyo) for his support throughout the study. The author also gratefully acknowledges an inspiring and dedicated group of past and present coworkers whose names and contributions appear in the cited references.

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Chapter 17

Development of Novel Ligands That Modulate Innate-Like T Cells Shinsuke Inuki

Abstract Innate-like T cells function as a bridge between innate and acquired immunity and play an important role in the initial immune response toward infection. Natural killer T (NKT) cells or mucosal-associated invariant T (MAIT) cells are classified as innate-like T cells, which are activated by the T cell receptor recognition of ligands present on MHC-like molecules (CD1d, MR1). Activated NKT or MAIT cells mediate various immune responses via cytokine production. Through the modification of natural ligands using synthetic organic chemistry, the author has been elucidating the molecular recognition mechanisms of NKT or MAIT cell activation and developing chemical tools or lead compounds for drug discovery. In this chapter, our recent structure–activity relationship (SAR) studies for developing novel innate-like T cell modulators would be described. Keywords Innate-like T cell · NKT cell · MAIT cell · Glycolipid · Microbial antigen · Immunomodulator

17.1 Introduction The immune system consists of a complex network involving diverse cells such as T cells, B cells, or dendritic cells, which contribute to biological defense and homeostasis. Innate-like T cells are classed as a subset of T lymphocytes, which bridge innate and acquired immunity [1]. Representative innate-like T cells in humans include invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells. Unlike conventional T cells, these cells express a semi-invariant T cell receptor (TCR) and are restricted by conserved, monomorphic MHC-like molecules [cluster of differentiation 1d (CD1d) or MHC-related molecule 1 (MR1)] [2]. NKT and MAIT cells regulate multiple immune processes involved in various disorders as well as providing defense against infection, and thus, are promising candidates S. Inuki (B) Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshidashimoadachi-Cho, Sakyo-Ku, Kyoto 606-8501, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 H. Ishikawa and H. Takayama (eds.), New Tide of Natural Product Chemistry, https://doi.org/10.1007/978-981-99-1714-3_17

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Fig. 17.1 Activation of innate-like T cells (NKT and MAIT cells) by ligands bound to MHC-like molecules (CD1d and MR1)

for immunotherapies. These cells are activated in response to ligands (antigens) presented by MHC-like molecules on antigen-presenting cells (APC) (Fig. 17.1) [3]. Namely, ligands (antigens binding to MHC-like molecules) function as key modulators of innate-like T cells. Some endogenous and exogenous molecules have been identified as modulators, and many research groups are still exploring novel natural ligands. Additionally, based on these findings, structure–activity relationship (SAR) studies are also being vigorously conducted with aim of developing drug candidates or chemical tools. As part of an ongoing program toward the development of immunomodulators, the author is interested in the identification of novel ligands and the elucidation of the molecular recognition mechanisms of NKT or MAIT cell activation by the chemical modification of natural ligands.

17.2 Development of Novel NKT Cell Modulators 17.2.1 Background of NKT Cells iNKT cells are a subset of innate-like T cells expressing an invariant α chain TCR (Vα14–Jα18 in mice and Vα24-Jα18 in humans) [4]. iNKT cells are activated through the TCR recognition of glycolipids presented by the nonpolymorphic MHC class Ilike molecule CD1d protein. The activation of iNKT cells results in the induction of Th1 (e.g., IFN-γ, TNF), Th2 (e.g., IL-4, IL-13), and Th17 cytokines (e.g., IL17A). These cytokines can activate or suppress various immunological processes. For example, Th1 cytokines enhance cellular immune responses, associated with tumors [5, 6] and infection by intracellular bacteria and some viruses [7, 8]. Thus, the regulation of Th1 responses has gained attention as a potential target for a range of cancers and viral infections. Th2 cytokines modulate allergic reactions and infection to extracellular parasites, and are also involved in the amelioration of certain autoimmune diseases [e.g., inflammatory bowel diseases (IBD) and multiple sclerosis (MS)] [9, 10]. Th2-biased ligands are attractive candidates of pharmaceuticals for the treatment of IBD and MS. Th17 cytokines play an important role in protection against extracellular pathogens such as Streptococcus pneumoniae [11]. Therefore, the selective regulation of Th17 cytokine production is beneficial for developing

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vaccine adjuvants against pneumococcal infection. However, although individual cytokines induce independent immune responses, in some cases they interact positively and negatively with each other. For example, there is negative cross-regulation between Th1 and Th2 responses [12]. The Th1 and Th17 signaling pathways function synergistically, resulting in host defense against extracellular pathogens, but, occasionally, an imbalance between Th1 and Th17 signaling causes immunopathology [13]. These findings indicate that regulating cytokine production and/or balance is crucial for the development of effective immunotherapies. Thus, many studies have attempted to identify potent and/or selective NKT cell-regulated modulators [12]. One of the most potent NKT cell modulators is a glycolipid derived from a marine natural product, α-galactosyl ceramide (α-GalCer, KRN7000, Fig. 17.2) [14], which induces high levels of various cytokines. Extensive SAR studies of α-GalCer have been conducted, and several characteristic ligands have been found thus far, namely, 7DW8-5 [15], OCH [16], and a phytosphingosine-modified α-GalCer derivative [17]. 7DW8-5 is an α-GalCer analog with a terminal Ar group attached to an acyl chain, which promotes Th1 polarization. OCH is a Th2-biased α-GalCer derivative that contains a truncated sphingosine moiety. The phytosphingosine-modified α-GalCer variant induces the production of IL-4, a Th2 cytokine, compared with IFN-γ and IL-17, and was effective in the EAE model [17]. In addition to these modulators, the diverse modification of glycolipid ligands affects cytokine production levels and polarization of NKT cells [12]. However, there are still no clear guidelines for ligand design to control the activity and selectivity, and the underlying mechanism is not fully understood. Our group has studied the design and synthesis of novel glycolipid ligands, and the elucidation of NKT cell functions using these types of ligands.

His38

Methylene

A' pocket of CD1d O HO HO

OH

Ser28 O

Gln14

O NH

OH

HO O

C14H29 OH

N H Amide Ligand modification in the long fatty acyl chain

α-GalCer (KRN7000)

Fig. 17.2 Structure of α-GalCer with a saturated acyl chain (C26:0) (α-GalCer, KRN7000). Our ligand design strategy is based on the modification from methylene to an amide group in the long fatty acyl chain to allow the interaction between the polar residues and the ligands

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17.2.2 Ligand Design of Amide-Containing α-GalCer Analogues α-GalCer is composed of a sugar head, sphingosine, and an acyl chain component. The crystal structure of a ternary complex with CD1d-α-GalCer-TCR revealed that the sugar part is recognized by the TCR, and the lipid parts are accommodated with two large hydrophobic pockets: the A' pocket and F' pocket [18, 19]. The long alkyl chain of sphingosine binds to the F' pocket and the acyl chain moiety binds to the A' pocket of CD1d. Previous SAR studies of α-GalCer suggested that the binding ability of glycolipid ligands depends on their occupancy of the A' pocket, which may be associated with cytokine production. Indeed, global cytokine production is reduced when the acyl chain portion of the glycolipid is truncated, as in α-GalCer-C20:0 [20]. The A' pocket is a large hydrophobic groove composed of many hydrophobic residues, but contains a few hydrophilic residues, such as Gln14, Ser28, or His38 (Fig. 17.2) [19]. These residues are unique polar residues, located deep inside the hydrophobic binding pockets, which are expected to form a “water-shielded hydrogen bond” to ligands. When the water accessibility to a confined polar residue in the hydrophobic binding pockets is restricted, the hydrogen bond(s) between the receptor and ligand cannot be easily dissociated because simultaneous hydration by solvent water molecules is less likely to occur. Consequently, tight and long-lived receptor–ligand complexes can be formed [21, 22]. Recently, some groups have examined the behavior of “watershielded hydrogen bond”. Barril and co-workers reported that the formation of these hydrogen bonds between ligands and their receptors enhanced the kinetic stability of the complexes [23]. Additionally, some researchers have studied the dependence of hydrogen bond strength on the polarity of the surrounding environment. Kelly et al. found that the strength of hydrogen bonds in hydrophobic environments was increased compared with that of hydrogen bonds in polar environments [24]. Based on these findings, we focused on the polar residues, especially those in Gln14 and Ser28, in the A' pocket of CD1d, with the aim of designing CD1d ligands with functional groups that form a “water-shielded hydrogen bond” in an effort to identify novel NKT cell modulators [25]. An amide group was selected as a polar functional group to form the hydrogen bonds because it is easily inserted into the acyl chain and can potentially function as a hydrogen bond donor and acceptor. Thus, α-GalCer analogues 1a–e with an amide group in their long acyl chains were designed as shown in Fig. 17.3. Some research groups introduced several functional groups into the fatty acid chain of α-GalCer, and evaluated their activities [26–30]. Among them, Kim and co-workers focused on the hydrophilic residues in mouse CD1d (mCD1d) and designed truncated ω-hydroxy fatty acid-containing α-GalCer derivatives, revealing that the compound retained an activity comparable with that of α-GalCer [29].

17 Development of Novel Ligands That Modulate Innate-Like T Cells HO HO

OH

O

R

O NH

R=

OH

HO O

359

H N m

n

O

C14H29

1a-e: (m = 6-10, n = 10-14, m+n = 20)

OH R= 26 α-GalCer O 1a

N H H N

1b O O 1c

N H H N

1d O O N H

1e

Fig. 17.3 Structures of the α-GalCer analogues

17.2.3 Synthesis of α-GalCer Analogues The syntheses of α-GalCer analogues 1a–e are shown in Scheme 17.1 [25]. We prepared the key intermediate 2 starting from d-galactose and phytosphingosine according to a previously reported procedure [31]. The azide group in 2 was converted to a primary amine with PPh3 and H2 O, and the resulting product was coupled with the amino acid derivatives containing Cbz group using water-soluble carbodiimide (WSC·HCl) to provide the compounds 3a–e (m = 6–10). The Cbz and Bn groups of 3a–e were removed in the presence of Pd(OH)2 and a subsequent reaction with carboxylic acids and 4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) [32] led to the desired α-GalCer analogues (20–50% yields).

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Scheme 17.1 Synthesis of the α-GalCer analogues 1a–e

17.2.4 Evaluation of α-GalCer Analogues Next, we evaluated the binding potential of these α-GalCer analogues to CD1d proteins and characterized the functional NKT cell responses to these ligands using (1) an APC-free assay [25, 33–35] and (2) a binding assay using AlphaScreen [36]. The APC-free assay was performed with the mCD1d fusion protein bound to the culture plate. The CD1d protein was loaded with α-GalCer analogues and the resulting complex was presented to NKT hybridoma cells, which produce IL-2 upon ligand stimulation [37]. After incubation of the hybridoma with the ligandloaded mCD1d fusion proteins, IL-2 cytokine production levels were measured by an enzyme-linked immunosorbent assay (ELISA). As shown in Fig. 17.4a, derivatives 1a (m = 6) and 1b (m = 7) demonstrated IL-2 production comparable with α-GalCer (positive control), whereas analogue 1c (m = 8) increased cytokine production compared with α-GalCer. Furthermore, analogues 1d (m = 9) and 1e (m = 10) showed more potent activities. It should be noted that the insertion of only one amide group into the long fatty acyl chain markedly enhanced the levels of cytokine secretion. Next, a binding assay was conducted with the AlphaScreen system (Perkin Elmer Life Sciences), using streptavidin-coated donor beads, mCD1d-conjugated acceptor beads, and biotinylated phosphatidyl ethanolamine (Bio-PE) as tracer. In the absence of a competing ligand (analyte), Bio-PE binds to mCD1d, bringing the donor and acceptor beads closer together and generating a signal. In the presence of an analyte binding to mCD1d, the analyte competes with the tracer, and the alpha signal is reduced. As shown in Fig. 17.4b, analogues 1c (m = 8), 1d (m = 9), and 1e (m = 10) showed higher binding affinities compared to the derivatives 1a (m = 6) and 1b (m = 7), which correlated with the APC-free assay.

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Fig. 17.4 a APC-free assay for lipid binding to mCD1d with the indicated ligands (100 nM). IL-2 secretion induced by the analogues 1a–e, α-GalCer was used as a reference. The graphs show mean ± standard error for triplicate values, and the results shown were representative of two or three independent experiments. b AlphaScreen assay measuring the binding of α-GalCer and analogues 1a–e to mCD1d. IC50 was calculated with a sigmoid dose–response formula using GraphPad Prism. The figures represent a 95% confidential interval (95% CI)

Taken together, the results of the APC-free and binding assays revealed that the introduction of the amide increased the binding affinity to CD1d and the levels of cytokine secretion from the NKT hybridoma. Furthermore, the amide position of the acyl chain had a significant effect on activity, and in particular, the optimal position of the amide in the acyl chain was in the range of m = 9 and 10. These findings indicate that there are site-specific interactions between these amide groups and polar residues such as Gln14 or Ser28 in the A' pocket of CD1d.

17.2.5 MD Simulation of Complexes with mCD1d and α-GalCer Analogues To analyze the interactions between the amide groups and polar amino acid residues in the A' pocket of mCD1d, we conducted 20 ns MD simulations of the mCD1d complexes with the amide analogues 1a–e [25]. As shown in Fig. 17.5, we investigated the time-averaged number of hydrogen bonds formed between the amide group and the polar residues in the A' pocket, including the water-bridged hydrogen bonds. In the cases of analogues 1a and 1b, hydrogen bond formation between amide groups and Ser28 was not observed, and the amide groups occasionally interacted with

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Fig. 17.5 Number of time-averaged hydrogen bonds present between the amide group of 1a–e and the polar residues in the A' pocket. The vertical axis indicates the total numbers of direct and water-bridged hydrogen bonds

Gln14 and Tyr73, located closer to the entrance of the A' pocket. Derivative 1c interacted with Ser28 via an average of 0.24 hydrogen bonds, and most hydrogen bond interactions were with Gln14. Analogues 1d and 1e displayed similar interaction profiles, and these amide groups interacted extensively with Gln14 and Ser28. The interactions were predominantly water-bridged hydrogen bonds to Gln14 and direct hydrogen bonds to Ser28. The time-averaged number of hydrogen bonds formed by 1a–e corresponded well with the results of the APC-free and binding assays shown in Fig. 17.4, suggesting that hydrogen bond formation to polar residues in the A' pocket is primarily responsible for the interaction between ligands and CD1d protein.

17.2.6 Design of Th2 Selective Ligands As mentioned above, we found that the introduction of an amide group onto the acyl chains enhanced the binding activity to CD1d and the cytokine production levels from an NKT hybridoma. Based on these findings, we planned to develop Th2 selective ligands by modifying the acyl chain of α-GalCer [36]. The development of potent Th2 selective ligands has been difficult because of a shortage of guidelines for designing selective ligands. Several examples of Th2 selective ligands obtained by modifying acyl chains have been reported. α-GalCer derivatives with truncated (α-GalCer-C20:0) or polyunsaturated fatty acid chains (α-GalCer-C20:2) selectively promoted Th2 responses [20]. However, truncation of the acyl chain led to a decreased potency in inducing cytokine production. The activities of short acyl chain analogues were markedly reduced compared with α-GalCer-C26:0 (a non-selective potent ligand) [38, 39]. We hypothesized that the introduction of an amide into the acyl chain of α-GalCerC20:0 (a weak Th2 selective ligand, Fig. 17.6) [20] might improve the cytokine production activity while maintaining the selectivity. The structure of designed analogue 4 is shown in Fig. 17.6. The position of the amide was determined based on

17 Development of Novel Ligands That Modulate Innate-Like T Cells HO HO

OH

O

363

R

O NH

OH

HO O

C14H29 OH

R= 26 α-GalCer O 1e

N H 20

α-GalCer-C20:0 O N H

4

Fig. 17.6 Design of the Th2 selective α-GalCer analogues

the investigation of amide analogues 1a–e. Analogue 4 was prepared by a synthetic route identical to that described for the syntheses of the amide analogues 1a–e. The evaluation of analogues was performed by measuring the levels of cytokine production from mouse splenocytes to examine cytokine polarization (Th1 vs Th2 cytokine) through the CD1d-NKT cell system. Supernatant IFN-γ (Th1 cytokine) and IL-4 (Th2 cytokine) levels after 48 h of treatment with synthesized derivative 4 were quantified (Fig. 17.7). In addition to analogue 4, α-GalCer, α-GalCer-C20:0 and analogue 1e were also evaluated to investigate the effects of amide introduction and acyl chain length on selectivity. As shown in Fig. 17.7, IFN-γ and IL-4 production levels induced by amide analogue 1e were higher than or equal to that of α-GalCer, which indicated that analogue 1e was a non-selective ligand. α-GalCerC20:0 reduced IFN-γ and IL-4 production levels by more than 50-fold compared with α-GalCer, which was consistent with the results previously reported by Yu and Porcelli [20]. By contrast, 4 showed the significantly enhanced induction of IFN-γ and IL-4 compared with α-GalCer-C20:0. In terms of selectivity, 4 was found to be a Th2 selective ligand and exhibited 2.5-fold higher selectivity for IL-4 induction (IL4/IFN-γ > 2.5) compared with α-GalCer. A known Th2 ligand, OCH had selectivity for IL-4/IFN-γ = 2 in our assay (data not shown) [20]. Thus, we have developed a highly potent Th2-biased CD1d ligand containing modified lipid parts. We next investigated the property of the obtained ligand focusing on its effect on cellular localization. Porcelli et al. reported that α-GalCer-C10:0 and α-GalCerC20:2 ligands (Th2-biased ligands) were directly loaded onto cell-surface CD1d proteins and that the cell surface presentation rates of ligand-CD1d complexes were associated with Th2-biased responses [40]. To explain the ability of analogue 4 to induce a Th2-biased cytokine response, we observed the effects of ligands on cells

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Fig. 17.7 IFN-γ and IL-4 secretion by mouse splenocytes following stimulation by α-GalCer, C20:0 (α-GalCer-C20:0), or its analogues 1e and 4. The graphs show the mean ± SD of triplicate measurements, and the results shown are representative of at least three independent experiments. a IFN-γ secretion, b IL-4 secretion in the presence of analogues and α-GalCer as a control. c Relative ratio of cytokine production of analogues compared with α-GalCer, all at 1 nM

Fig. 17.8 Flow cytometry analysis of (a) α-GalCer, (b) analogue 1e, and (c) analogue 4. RBL.CD1d cells were incubated with α-GalCer (1 μM) and its analogues (100 nM) for durations ranging from 1 to 20 h. MFI of L363 staining at each time represented. L363 MFI is expressed as %MFI of the value measured after 20 h incubation. Each data point represents the mean ± SD of triplicate measurements, and the results shown were representative of at least three independent experiments

by flow cytometry analysis using RBL.CD1d cells expressing mCD1d and L363 (an antibody specific for the mCD1d-α-GalCer complex) [41]. Flow cytometry analysis of α-GalCer and the analogues 1e and 4 is shown in Fig. 17.8. After RBL.CD1d cells were cultured in the presence of α-GalCer or analogues for 1, 4, or 20 h, L363 cell surface staining was examined. All ligands displayed detectable presentation on

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the cell surface. The cell surface appearance kinetics of analogue 1e (non-selective ligand) was similar to that of α-GalCer: surface staining [median fluorescence intensity (MFI)] reached a maximum at 20 h. In contrast, L363 staining was observed at early time points, in the presence of analogue 4 (Th2 selective ligand). These results suggested that the appearance rates of ligand-CD1d complexes on the cell surface were involved in the Th2-biased responses, consistent with the model proposed by Porcelli [40]. These findings obtained using α-GalCer derivative 4 that induces high cytokine production could provide important insights into the mechanism of CD1d-mediated Th2-biased responses.

17.2.7 Summary of the Development of Novel NKT Cell Modulators To summarize, we focused on the polar amino acid residues deep inside the A' pocket of CD1d and designed α-GalCer analogues containing an amide group in the long fatty acyl chain of the glycolipid ligand. The insertion of an amide group at a suitable position (m = 9 or 10 methylene linkers) in the fatty acid chain increased its binding activity to CD1d and cytokine production from NKT hybridomas. MD simulation suggested that the interactions between the amide groups and the polar residues, such as Gln14 or Ser28, resulted in enhanced activity related to the formation of a hydrogen bond network. Further SAR studies led to the identification of a potent Th2 selective NKT cell modulator, which would be one of the most potent Th2-biasing ligands that retain the total cytokine induction levels, among the known ligands. Our analysis using the Th2 selective ligand revealed that the kinetics of ligandCD1d complex presentation on the cell surface correlated with its Th2 cytokinebias. Although not described here, we also successfully developed Th17 selective ligands [42, 43] or covalent modulators [44] by modifying the acyl chain of α-GalCer. These newly developed NKT cell modulators will contribute to the identification of pharmaceuticals/immune adjuvants and chemical probes involving the CD1d-NKT system.

17.3 SAR Studies of MAIT Cell Modulators 17.3.1 Background of MAIT Cells MAIT cells are innate-like T cells that are abundant in human tissues such as the gut, liver, as well as peripheral blood [45–47]. MAIT cells, like NKT cells, have a highly conserved TCR repertoire [48–50]. Upon infection with riboflavin-producing bacteria, such as Escherichia coli or Mycobacterium tuberculosis, MAIT cells are activated and produce inflammatory cytokines (e.g., INF-γ, TNF, and GM-CSF),

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which play an important role in biological defense against bacterial infection [51– 54]. MAIT cells are associated with immune responses in the early stages of infection. In addition, the involvement of MAIT cells in a variety of disorders, including cancer or autoimmune diseases has been reported [47, 55, 56]. Therefore, the modulation of MAIT cell activation has attracted significant attention in recent years as a therapeutic target for infectious diseases, cancer, or autoimmune disorders. MAIT cells are activated in response to antigens presented by MR1 on APC. Recently, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) 5a has been identified as an antigen (ligand) that forms a complex with MR1 and strongly activates MAIT cells (Scheme 17.2) [57, 58]. 5-OP-RU is an unstable intermediate resulting from Schiff base formation between 5-amino-6-D-ribitylaminouracil (5-ARU) 6a and methylglyoxal (Scheme 17.2). In a previous report, 5-OP-RU decomposed in water (t1/2 = 1.5 h, 37 °C) [59] because its iminocarbonyl moiety was readily hydrolyzed. Alternatively, as shown in Scheme 17.2, 7-methyl-8-D-ribityllumazine (RL-7-Me) 7a was formed via intramolecular condensation between the amino and carbonyl groups in 5-OP-RU [59]. In terms of the interaction of 5-OP-RU with MR1 and TCR, a Schiff base formation between the amino group of Lys43 and iminoglyoxal group of 5-OP-RU tightly binds 5-OP-RU to MR1. 5-OP-RU is mainly recognized by MAIT cells via an interaction between the ribityl group of 5-OP-RU and the TCR [57]. SAR studies of 5-OP-RU have been conducted using various approaches to investigate its molecular recognition by MR1 and MAIT-TCR or to improve its chemical stability [59–64]. For example, Mak et al. developed more stable ligands with an enone moiety instead of an iminocarbonyl group and found that the enonecontaining ligand-activated MAIT cells [59]. Lange et al. designed and synthesized a chemically stable ligand that released an active precursor that was a MAIT cell agonist via the enzymatic cleavage of a valine-citrulline-p-aminobenzyl carbamate linker [62].

OH OH HO

HO OH

4'

3'

HO

O

HN

H

H N

O

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H N

HN

O

HO N

O H 2O

N

O

NH

N

NH

H2N

OH

1'

HO HO

OH

OH

2'

H

O

NH

N O

O 5-A-RU (6a)

5-OP-RU (5a)

RL-7-Me (7a)

Scheme 17.2 Formation of 5-OP-RU and RL-7-Me from 5-A-RU and methylglyoxal

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17.3.2 Concept of SAR Studies Our group performed a SAR study of 5-OP-RU to examine its underlying molecular recognition by human MR1 and human MAIT-TCR and to obtain guidelines for identifying novel MAIT cell modulators [65]. In particular, we focused on the stereochemistries of the ribityl moiety in 5-OP-RU as a key functional group interacting with MR1 and MAIT-TCR. We designed, synthesized, and evaluated all stereoisomers of the ribityl group in 5-OP-RU. Furthermore, the bioactivities of 5-A-RU stereoisomers 6 and bicyclic RL-7-Me stereoisomers 7 for the MAIT-MR1 axis were investigated.

17.3.3 Synthesis of 5-OP-RU Analogues All stereoisomers of 5-A-RU 6a–h were prepared [65] according to the reported method (Scheme 17.3) [66]. The coupling between 6-chloro-5-nitrouracil 8 and the known amino alcohols 9 [67] gave nitrouracils 10, which were then converted to the desired 5-A-RU isomers 6 by a reduction of the nitro group. The preparation of each 5-OP-RU stereoisomer 5 was conducted from their precursor 6 and methylglyoxal in an aqueous buffer immediately prior to evaluation due to their instability. The RL7-Me stereoisomers 7, which were formed from 5 under aqueous conditions, were also synthesized [59].

17.3.4 Evaluation of 5-OP-RU Analogues 5-OP-RU 5a is a potent ligand that modulates MAIT cells but is unstable under physiological conditions [59]. This suggested that the chemical stabilities of 5-OP-RU isomers might be affected by differences in their stereochemistry. Awad et al. reported that subtle transformations of 5-OP-RU, such as deoxy analogues, had significant effects on their chemical stability [63]. Therefore, before assessing MAIT cell activation, the chemical stability of four diastereomers [5-OP-RU (5a) and its stereoisomers 5c, 5e, and 5g] prepared from the corresponding 5-A-RU stereoisomers was investigated [65]. Of note, the corresponding enantiomers (5b, 5d, 5f, and 5h, respectively) should have identical physicochemical properties. The incubation of 5-A-RU isomers 6 with methylglyoxal in D2 O led to the formation of 5-OP-RU isomers 5 and RL7-Me isomers 7, the amounts of which were monitored by 1 H NMR spectroscopy (Fig. 17.9). After reaching a maximum amount at 10 min, all four diastereomers 5 gradually degraded and disappeared within 2 h. Accordingly, the corresponding four RL-7-Me stereoisomers 7 accumulated over time with similar conversion rates. These results indicated that the formation and degradation kinetics of 5 and 7 were not influenced by the stereochemistry of the ribityl groups.

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Cl

NH2 9 KOH

R

EtOH H 2O

O2N

O

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R H N

NH

O2N O

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8

O NH

H 2N

H2 O 80 °C

O 10

H N

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Na2S2O4

O

10a (46%) 10e (55%)

6 (5-A-RU stereoisomers)

10b (50%) 10f (57%)

6a (63%) 6e (69%)

10c (40%) 10g (52%)

6b (70%) 6f (71%)

10d (46%) 10h (53%)

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OH

a: R =

e: R =

OH HO f: R =

HO

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OH

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OH

OH

h: R =

g: R = HO

OH

d: R = HO

HO

OH

HO

OH

c: R =

OH HO

HO

OH

b: R = HO

OH

OH

OH

OH HO

HO

HO

Scheme 17.3 Syntheses of 5-A-RU and RL-7-Me stereoisomers

Next, we investigated MAIT cell activation by stereoisomers in a co-culture assay using TG40 cells transfected with MAIT-TCR (TG40.MAIT-TCR) and human MR1expressing HeLa (HeLa.MR1) cells [65]. MR1 ligands such as 5-OP-RU are captured by MR1 on the APC, and MAIT cells recognize the complexes with MR1 and ligands through their TCR, resulting in the stimulation of MAIT cells [56]. The TG40.MAITTCR and HeLa.MR1 cells were co-incubated in the presence of the ligands. After 24 h of culture, we detected the surface expression of CD69, a T cell activation marker, using flow cytometry (Fig. 17.10a). Additionally, we measured the levels of IL-2 cytokine release by ELISA as an indicator of MAIT cell activation (Fig. 17.10b).

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Fig. 17.9 Formation kinetics of the 5-OP-RU and RL-7-Me stereoisomers. a Formation and degradation of 5-OP-RU 5a and its stereoisomers 5c, 5e, and 5g. b Formation of RL-7-Me 7a and its stereoisomers 7c, 7e, and 7g. The product formation was monitored by 1 H NMR analysis every 10 min after methylglyoxal (40% aqueous solution; 58 μL, 0.33 mmol) was added to a solution of each precursor 6a, 6c, 6e, and 6g (5.0 mg, 0.013 mmol) in D2 O (730 μL)

In both assays, the isomers 5b–5f and 5h induced considerably lower MAIT cell activation compared with 5-OP-RU (5a), whereas the 4' -OH epimer (5g) displayed potent activity equivalent to 5a in a dose-dependent manner. These results indicated that the stereochemistries of 2' - or 3' -OH groups are key to modulating MAIT cell activation. Awad et al. reported that, among the deoxy analogues of the ribityl group, the des-4' -OH analogue exhibited comparable agonistic activity with 5a, although analogues in the absence of the 2' - or 3' -OH groups were less active [63]. Unlike our findings, Braganza et al. reported that the stereoisomers 5b, 5c, 5e, and 5g exhibited activity comparable with 5-OP-RU 5a using mouse MR1 and MAIT-TCR [60]. This discrepancy is likely related to the differences in the TCRs used for the assay. Although the ligand binding domains of MR1 have high sequence identity (90%) between humans and mice [68], MAIT cell activation depends highly on the TCR class [69, 70]. As shown in Fig. 17.9, 5-OP-RU partially returns to the precursors 6 via hydrolysis, because of the instability of the iminocarbonyl moiety. Additionally, all isomers 5 gradually transformed into their thermodynamically stable lumazine forms 7. In these assays, several components including ribitylaminouracil-derived 5–7 and methylglyoxal co-exist during the experiment, which might affect MAIT cell activation [59]. Therefore, the compounds, methylglyoxal, 6 and 7 were also evaluated (Fig. 17.11) [65]. Methylglyoxal did not show activity in the co-culture assay using HeLa.MR1 and TG40.MAIT-TCR cells (data not shown). Next, we evaluated the 5-A-RU stereoisomers 6, which provided results that were similar to those of the 5-OP-RU stereoisomers 5. Only (2' S,3' S,4' R)-form 6a and (2' S,3' S,4' S)-form 6g exhibited agonistic activity for MAIT cell activation assessed by enhanced CD69 surface expression and IL-2 production. In contrast, no effects on the stimulation of MAIT cells were observed by treatment with the other stereoisomers. The SARs of

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Fig. 17.10 MAIT cell activation by the 5-OP-RU stereoisomers. a CD69 surface expression on TG40.MAIT-TCR cells using flow cytometry after 24 h incubation in co-culture with HeLa.MR1 cells in the presence of each ligand. b IL-2 production in the supernatant was measured by ELISA. The graphs show the mean ± SD of triplicate measurements, and the results shown are representative of at least three independent experiments. ND = not detected

the lumazine stereoisomers 7 were similar to those of the 5-A-RU stereoisomers 6 and the 5-OP-RU stereoisomers 5. (2' S,3' S,4' R)-form 7a and (2' S,3' S,4' S)-form 7g increased CD69 expression and IL-2 production, whereas the other stereoisomers 7b, 7c, and 7e showed no activity for MAIT cell activation. To summarize, all forms of 5–7 bearing (2' S,3' S)-stereochemistries functioned as agonists for MAIT cell activation, highlighting that the structure of the ribityl moiety is crucial for MAIT cell activation. Modification of the 4' -OH moiety might improve its recognition by human MAIT-TCR. Thus the further modification or conversion of the uracil and lumazine core structures might aid the development of potent ligands.

17.3.5 Binding Mode Analysis of 5-OP-RU Analogues Finally, the binding mode analysis of the 5-OP-RU stereoisomers to human MR1 (hMR1) and MAIT-TCR was performed using Molecular Operating Environment (MOE) (Fig. 17.12) [65]. We used the X-ray crystal structure of the MR1-ligandTCR ternary complex (PDB: 4L4V) as a template for the binding models. The uracil ring and iminoglyoxal moieties of all stereoisomers bound to the MR1 cleft in a similar conformation, consistent with the report of Braganza et al. [60]. In terms of the agonistic isomers 5a and 5g bearing common stereochemistries at the 2' OH and 3' -OH groups of the ribityl moiety, the 2' -OH and 3' -OH groups interacted with TCR Tyr95 and MR1 Arg94, respectively, via hydrogen bonds. In contrast, the 2' -OH or 3' -OH group of the non-activating isomers 5b–5f and 5h had a different orientation from that of 5a and 5g, possibly exacerbating hydrogen bond network formation in this region. The 4' -OH group of 5a formed a hydrogen bond with MR1 Arg94, whereas the corresponding 4' -OH group of epimer 5g formed a hydrogen bond with TCR Gly98 and MR1 Tyr152. The 5' -OH group of 5a and 5g interacted

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Fig. 17.11 MAIT-cell activation by 5-A-RU and RL-7-Me stereoisomers. a, b TG40.MAIT-TCR cell activation was detected as CD69 surface expression using flow cytometry. c, d IL-2 production was detected by ELISA after 24 h incubation in co-culture with HeLa.MR1 cells. The graphs show the mean ± SD of triplicate measurements, and the results shown are representative of at least three independent experiments. ND = not detected

with MR1 Tyr152 and MR1 Arg94, respectively, through hydrogen bond formation. These results indicate that the interactions of the ligands with TCR Tyr95, MR1 Arg94 and MR1 Tyr152 are critical for the activation of MAIT cells. The formation of a well-ordered hydrogen bond network within the ternary complex, likely to be 5a, could contribute to the potent activity of 5g.

17.3.6 Summary of SAR Studies on 5-OP-RU Analogues To investigate the effects of the chirality of analogues on MAIT cell activation, all stereoisomers of 5-OP-RU (5a) were designed and synthesized. We found that only the 4' -OH epimer 5g showed potent activity among the seven stereoisomers other than 5-OP-RU. These results revealed that the configuration of the 2' -OH and 3' -OH groups is important for agonistic activity, and further transformation of the 4' -OH moiety might improve the recognition of human MAIT-TCR. Additionally, the binding mode analysis suggested that the hydrogen bond formation between the ligands and Tyr95 of TCR and/or Arg94 and Tyr152 of MR1 would be a key

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Fig. 17.12 Binding mode analysis of 5-OP-RU 5a and its stereoisomer 5g using the MR1-ligandTCR ternary complexes (PDB: 4L4V) as a template. Interactions between MAIT-TCR (yellow), MR1 (pale green), and (a) 5a or (b) 5g with the distance (green)

interaction for the formation of ternary complexes, which is consistent with previous results using deoxy analogues [63]. Our docking study using the stereoisomer 5 suggested that a direct interaction between TCR Gly98 and ligand is a potential target for regulating their complex formation. The comparable bioactivities of the lumazine forms bearing the same stereochemistries suggested that the modification of the uracil core structure might be a promising approach for the identification of novel MAIT cell modulators. These SAR studies provide information for the design of novel MAIT cell modulators, contributing to the development of drug candidates or chemical tools.

17.4 Conclusion This chapter reviewed SAR studies aiming to develop novel NKT cell and MAIT cell modulators. In terms of developing NKT cell modulators, the simple modification of the long fatty acyl chain of glycolipid ligands, introducing an amide group, led to the enhancement of cytokine production. MD simulation suggested that the introduced amide group interacted with the confined polar residues (Gln14 and Ser28) in the hydrophobic lipid-binding pocket of CD1d. This approach was applied to identify a novel Th2 selective NKT cell modulator. Furthermore, an analysis using a Th2 selective ligand revealed that the appearance rates of ligand-CD1d complexes on the cell surface were involved in Th2-biased responses. The SAR studies of a MAIT cell modulator, 5-OP-RU, manifested the key interaction of ligands with MR1 and TCR. Furthermore, altering the core structure of 5-OP-RU was a guiding principle for obtaining highly active ligands. These findings provide useful guidelines for designing NKT and MAIT cell modulators, contributing for the development of pharmaceuticals/immune adjuvants, as

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well as chemical tools to elucidate molecular mechanism involving innate-like T cells. Acknowledgements The author would like to express his sincere and wholehearted appreciation to Professor Hiroaki Ohno (Graduate School of Pharmaceutical Sciences, Kyoto University) and Professor Yukari Fujimoto (Department of Chemistry, Faculty of Science and Technology, Keio University) for their kind guidance, constructive discussions and constant encouragement during this study. The author would like to thank Dr. Osamu Ichihara and Mr. Daisuke Yoshidome (Schrödinger K. K.) for their support with MD simulation, and Professor. Sho Yamasaki (Research Institute for Microbial Diseases, Osaka University) for his professional guidance on the MAIT cell activation assay. The author is grateful to all the colleagues of the Department of Bioorganic Medicinal Chemistry (Graduate School of Pharmaceutical Sciences, Kyoto University) and the laboratory of Biomolecular Chemistry (Department of Chemistry, Faculty of Science and Technology, Keio University) for their valuable comments and their assistance and cooperation in various experiments.

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