AsiaChem | Chemistry in Japan | December 2021 Volume 2 Issue 1

Chemistry in Japan › The Chemical Society of Japan: Striving for Chemical Sciences and Technology for a Sustainable Human Society, p6 › Peptide Cyclization Methodologies Amenable to in Vitro Display, p12 › Supramolecular Polymerization: Personal History and Outlook Towards a Sustainable Future, p20 › “Think Globally, Act Locally” An interview with Prof. Ryōji Noyori, p88 › A History of Chemistry in Japan, 1820-1955, p104 › Science Diplomacy: where chemistry is crucial, p114 December 2021 Volume 2 Issue 1

FederationMembers: The Royal Australian Chemical Institute Bangladesh Chemical Society Brunei Chemical Society Cambodian Chemical Society Institute of Chemistry, Ceylon Chinese Chemical Society Hong Kong Chemical Society Chemical Research Society of India Indian Chemical Society Himpunan Kimia Indonesia Iraqi Chemists Union Israel Chemical Society Chemical Society of Japan Jordanian Chemical Society Korean Chemical Society Kuwaiti Chemical Society Institiut Kimia Malaysia Mongolian Chemical Society Nepal Chemical Society New Zealand Institute of Chemistry Chemical Society of Pakistan The Institute of Chemists PNG Integrated Chemists of the Philippines Mendeleev Russian Chemical Society Saudi Chemical Society Singapore National Institute of Chemistry Chemical Society of the South Pacific Chemical Society Located in Taipei, China Chemical Society of Thailand Chemical Society of Timor-Leste Turkish Chemical Society (TCS) Chemical Society of Vietnam TheFederationof AsianChemical Societies (FACS) includes 32 chemical societies of countries and territories in the AsiaPacific whose membership consists of individual qualified chemists. For forty years the FACS has been fostering the development of chemistry in the Asia-Pacific region. Membership Membership of the Federation is open to all not-for-profit chemical societies whose membership consists largely of individual qualified chemists and which are national professional chemical societies of countries and territories in the Asia-Pacific. Individual membership is open to individual chemists from countries and territories that have societies within the Federation. ACES FACS is a supporting organization of the Asian Chemical Editorial Society (ACES) journals. ACES was founded in 2005 and is an organization of 13 major chemical societies in the Asia-Pacific region committed to scientific excellence, publishing ethics, and the highest standards in publication. Promoting the advancement and appreciation of chemistry and the interests of Asia-Pacific professional chemists

ABSTRACT CALL: · Deadline for Oral Communications Presenters: 11th March, 2022 · Notification of Oral Communications Acceptance: 29th April, 2022 · Deadline for Poster Communications Presenters: 29th june, 2022 · Notification of Poster Communications Acceptance: 3st June, 2022 · Deadline for Student Grant Application: 29th April, 2022 REGISTRATION: · Standard Registration deadline: 17th June, 2022 · Late registration deadline: 5th August, 2022 PLENARY LECTURERS Cristina Nevado (Organic Synthesis/Medicinal Chemistry) University of Zurich, Switzerland Hanadi Sleiman (Chemistry and Biology) McGill University, Canada Joanna Aizenberg (Materials) Harvard University, USA João Rocha (Materials and Solids) University of Aveiro, Portugal Lutz Ackermann (Catalysis) University of Göttingen, Germany Nicola Armaroli (Energy and Sustainability) National Research Council, Italy Takuzo Aida (Polymer and Supramolecular Chemistry) The University of Tokyo, Japan Stay connected Innovate to Build Discover the future of Chemistry Design: Atelier João Borges

4 | December 2021 20 42 104 80 114 AsiaChem is produced biannually on behalf of the Federation of Asian Chemical Societies by: Publisher: Israel Chemical Society Editor-in-Chief: Prof. Ehud Keinan Marketing: Ms. Tali Lidor Layout & Design: Little Wing Designs, UK Printing: Gestelit Digital Ltd., Haifa, Israel Communications Director: Prof. Ehud Keinan Editorial Chemistry in Japan ���������������������������������������5 Ehud Keinan Essay Science Diplomacy: Where Chemistry is Crucial . . . . . . . . . . . . . 114 John M Webb, Thomas H Spurling, and Gregory W Simpson Science Frontiers C O N T E N T S AsiaChem December 2021 Volume 2, Issue 1 The Chemical Society of Japan . . . . . 6 Mitsuo Sawamoto (The Chemical Society of Japan) Ribosomal Synthesis of Nonstandard Peptides . . . . . . . . 12 Hiroaki Suga and Ata Abbas (University of Tokyo) Supramolecular Polymerization: Personal History and Outlook Towards a Sustainable Future . . . . . . . . . 20 Takuzo Aida and Kiyoshi Morishita (University of Tokyo) Cooperative Catalysis in Organic Synthesis . . . . . . . . . . 26 Yoshiaki Nakao (Kyoto University) New Insights into Bond Homolysis Process and Discovery of Novel Bonding System (C–π–C) by Generating Long-lived Singlet Diradicals . . . . . . . . . . 32 Manabu Abe, Zhe Wang, and Rikuo Akisaka (Hiroshima University) From Structural to Functional Materials: a Green Way to Produce Functional Biopolymers Based on Polypeptides . . 42 Kousuke Tsuchiya and Keiji Numata (Kyoto University) Nanoporous Chemical Plants: MOFs as Polymer Manufacturers . . . . 48 Takashi Uemura and Keat Beamsley (University of Tokyo) Reactivity Prediction Through Quantum Chemical Calculations . . . . 56 Satoshi Maeda, et al. (Hokkaido University) Therapeutic In Vivo Synthetic Chemistry by Glycosylated Artificial Metalloenyzmes for Innovative Biomedical Modality . . . . . 64 Katsunori Tanaka and Tsung-Che Chang (Tokyo Institute of Technology) Pillar-Shaped Macrocyclic Hosts Pillar[n]arenes: From Simple Receptors to Supramolecular Assemblies . . . . . . 72 Tomoki Ogoshi (Kyoto University) Two-Electrode Solar Water Splitting Permitting H2 Separation at a Dark Cathode ���������������������������������������������80 Hironobu Ozawa and Ken Sakai (Kyushu University) Interview Ryōji Noyori (Nagoya University) ������������������������������������������������������� 88 Ehud Keinan (Israel) Tête-à-tête with Eiichi Nakamura ������������������������������������������������������ 96 Ehud Keinan (Israel) History A History of Chemistry in Japan, 1820-1955 . . . . . . . . . 104 Yoshiyuki Kikuchi and Yona Siderer On the Cover This issue focuses on Chemistry in Japan through scientific articles, essays, interviews, a historical account, and the story of the Chemical Society of Japan, all highlighting both past and current scientific accomplishments of Japan’s chemistry community. 6 48 26 December 2021 | 5 Chemistry in Japan Dear Reader, I am happy to present you with the December 2021 edition of our AsiaChem magazine, which echoes the Federation of Asian Chemical Societies (FACS). Concluding from the success of the previous issue, I am sure that the new one will attract even greater attention worldwide. This issue starts a tradition of unique coverage of chemistry in specific member countries within the FACS expanse. As you can see from the wealth of topics covered by this issue, the decision to focus on one country is well justified. Japan has always been a science powerhouse, as reflected by the fact that it is the top Asian country based on Nobel and Wolf Prize records. Remarkably, of the 29 Japanese Nobel Prize Laureates, 21 received the prize since 2000. The rapidly increasing trend of awarding Asian scientists with major prizes parallels other trends. First, the center of gravity of the global scientific activity follows the apparent shift of the world economy from North America and Europe to Asia. Second, Asian countries notoriously known for their brain drain have become increasingly attractive to their scientists, thus, shifting the balance between brain drain and brain gain. And Nobel Prize Laureate Yuan-Tseh Lee has proposed to replace the term “brain drain” with “brain circulation” ( Asian scientists are increasingly taking leadership positions in meeting the global challenges of health and climate, which have recently gained much public attention. Nevertheless, the other challenges, including sustainable energy, water quality, the dwindling raw materials, food problems, and waste management, are no less significant. The common denominator of all global challenges is their chemical nature. Although politicians and governments cannot solve these problems, they still enhance media and public awareness, thus creating lucrative opportunities for science and technology. Undoubtedly, chemists will take a dominant role in these efforts, and Asian chemists of all disciplines will continue working together across political borders and cultural barriers to secure a better world for the next generations: newsletters/chemistry-in-europe-2021-4/ This issue comprises a broad variety of articles on cutting-edge science, history, essays, and interviews, serving a wide readership worldwide. The group of scientists represents the Japanese academic landscape regarding age and scientific interest. Mitsuo Sawamoto, Executive Director of the Chemical Society of Japan (CSJ), provides a concise overview and brief history of the CSJ, its missions, activities, and future goals. Hiroaki Suga and Ata Abbas of the University of Tokyo describe their innovative peptide cyclization methodologies amenable to in vitro display. Takuzo Aida and Kiyoshi Morishita of the University of Tokyo talk about supramolecular polymerization from a perspective of personal history and a sustainable future. Yoshiaki Nakao of Kyoto University describes cooperative catalysis for organic synthesis. Manabu Abe, Zhe Wang, and Rikuo Akisaka of Hiroshima University provide new Insights into the bond homolysis process and the discovery of a novel bonding system (C–π–C). Keiji Numata and Kousuke Tsuchiya of Kyoto University describe a structural to functional materials journey, proposing a green way to produce functional biopolymers based on polypeptides. Takashi Uemura and Keat Beamsley of the University of Tokyo describe a novel opportunity of using MOFs as means for polymer manufacturing. Satoshi Maeda and colleagues of Hokkaido University predict chemical reactivity through quantum chemical calculations. Katsunori Tanaka and TsungChe Chang of the Tokyo Institute of Technology describe in vivo synthetic chemistry using glycosylated artificial metalloenzymes. Tomoki Ogoshi of Kyoto University, the discoverer of the pillar[n]arene macrocycles, reviews their properties from simple receptors to supramolecular assemblies. Ken Sakai and Hironobu Ozawa of Kyushu University describe a two-electrode solar water splitting permitting hydrogen gas separation at a dark cathode. I had the pleasure of interviewing Ryōji Noyori of Nagoya University, 2001 Nobel Prize Laureate, learning about his exciting career and unique views on science and education. My conversation (tête-à-tête) with Eiichi Nakamura of the University of Tokyo revealed a leading scientist and musician’s life experience and aspirations. Yoshiyuki Kikuchi of the Aichi Prefectural University and Yona Siderer of the university of Jerusalem provide a fascinating account of the history of chemistry in Japan during 1820-1955. Three Australian scientists, John M Webb, Thomas H Spurling, and GregoryWSimpson, conclude this issue, discussing science diplomacy, where chemistry is crucial. I wish to thank all these authors for opening a wide window to Japanese science and technology. Special thanks go to the graphics designer, Catharine Snell of Little Wing Designs (UK), for her contributions to the magazine’s layout and unique character. Enjoy your reading! Ehud Keinan Technion – Israel Institute of Technology President, Israel Chemical Society IUPAC, Vice President and President-elect AsiaChem, Editor-in-Chief FACS Communications Director

6 | December 2021 The Chemical Society of Striving for Chemical Sciences and for a Sustainable Human Society Mitsuo Sawamoto Executive Director, The Born in Japan (1951), he received his B.Sc. (1974), M.Sc. (1976), and Ph.D. (1979) in polymer chemistry from Kyoto University. After postdoctoral research at the University of Akron, USA (1980–81), he joined the Department of Polymer Chemistry, Kyoto University. In 2017, upon retiring from Kyoto, he joined the Frontier Research Institute at Chubu University. He serves as an Executive Program Director at the Japan Science and Technology Agency (JST), Member of the Science Council of Japan (SCJ), Executive Director of the Chemical Society of Japan, and Chair of the International Organizing Committee Pacifichem 2021. He has published over 540 research papers, 50 reviews and book chapters, and 46 patents (with nearly 25,000 citations and an h-index of 71) in the areas of precision cationic and radical polymerizations, metal polymerization catalysts, precision synthesis of designed functional polymers, and sequence regulation in chain-growth polymerization. His long list of Awards and Honors includes the Arthur K. Doolittle Award of the ACS, the Macro Group UK Medal, the SPSJ Award for Outstanding Achievement in Polymer Science and Technology, the NIMS Award on Strong Future of Soft Materials, the 2015 Medal of Honor with Purple Ribbon (presented by Emperor Akihito and Prime Minister Shinzo Abe, Japan), the Alexander von Humboldt Research Award, and the Benjamin Franklin Medal in Chemistry (USA). Brief History The Chemical Society of Japan (CSJ), with a long history extending over 140 years and a membership of ca. 24,000, is one of the world’s largest, most active, and internationally recognized societies in chemistry. The history of CSJ dates back to 1878 (just ten years after the Meiji Restoration, where Japan was reborn), when about twenty motivated and enthusiastic young scholars launched a small organization, the Chemical Society, in Tokyo for the advancement of chemistry. In the following year the embryonic society was renamed The Tokyo Chemical Society and eventually the current name, The Chemical Society of Japan, in 1921. In 1948, shortly after the World War II, the then CSJ merged with the Society of Chemical Industry, founded in 1898, into an integrated organization with the same name: ”The Chemical Society of Japan”. The integration was in part symbolic in defining the renewed CSJ’s perspective: CSJ consists of comparable numbers of individual members from both academia and industry along with supporting company affiliates; its activities cover virtually all segments of pure and applied chemistry along with diverse interdisciplinary areas now extended to physics, biology, medicine, materials, and advanced technology. Since 2011 CSJ is a public interest incorporated association, a nonprofit tax-exempt organization legitimately certified and under the jurisdiction of the Japanese Cabinet. December 2021 | 7 Japan: d Technology three unprecedented trends: globalization, digitalization (or AI proliferation), and socialization. All three have the potential to rapidly overturn existing paradigms. Also, as we prepare for the challenges to come, we must continue to stay focused on the existing global challenges before us, namely global climate change, marine plastics, food and water shortages, and the yet uncontained COVID-19 pandemic. “As CSJ President, I believe that the vital missions of chemistry and our Chemical Society are to respond quickly and sensitively to these fast-developing trends and thereby to provide solutions for global challenges. In retrospect, the Japanese chemical industry’s bitter history in the 20th Century as a major aerial and oceanic polluter resulted in invaluable lessons learned. The industry, in turn, applied its knowledge and technology thus acquired to develop strategies to prevent environmental hazards, eventually transitioned to a solution provider, and has successfully played active roles in multiple environmental improvement efforts, including the revitalizing crystal-clear blue oceans, firefly-living fresh water, and clean air. I believe that, through the power of chemistry, the industry as solution provider will be at the forefront to find solutions to all worldwide challenges and thereby to establish a sustainable society.” Organization The CSJ membership, total ca. 24,000 as of 2020, includes individual regular members in academia (ca. 9600) and industry (ca. 3700), student members (ca. 4300), teachers (ca. 1550), supporting company affiliates (420+), and institutional members Missions The prime mission of CSJ is to promote chemical sciences and technology in collaboration with other domestic and global chemistry-related societies and associations. Above all, the overriding objective is thus to contribute to the betterment of human life. The recently redefined CSJ mission statement goes: The Chemical Society of Japan, with diverse members in academia, industry, and government, will internationally play leading roles in promoting the progress in state-of-the-art fundamental research and the implementation of developments in chemical science and technology, and will thereby contribute to building a sustainable human society. In his inaugural address in June 2020, CSJ President Yoshimitsu Kobayashi said in excerpt: “The global society currently faces By Mitsuo Sawamoto

8 | December 2021 (such as libraries ; ca. 360). International membership grows steadily, particularly fromChina, Korea, and other Asian countries. The CSJ Office operates by the Board of Directors and the Secretariat: The Board of Directors is the second highest decision-making, management organization, under the CSJ General Assembly, and consists of President, Senior Vice President, four Vice Presidents, Executive Di rector, General Secretar y, 19 Directors, and four Auditors. The CSJ President, serving a two-year term, is elected by online general election by all the regular members; the position alternates for two consecutive terms from academia and the following third term from industry. The current President for fiscal 2020-2022 is Dr. Yoshimitsu Kobayashi (Figure 1), the Mitsubishi Chemical Holdings, and the President-Elect is Professor Hiroaki Suga, the University of Tokyo. Figure 1. CSJ President: Dr. Yoshimitsu Kobayashi, Mitsubishi Chemical Holdings Organizationally, the CSJ comprises of a Secretariat (Headquarters), Departments, and Regional Sections. The CSJ Secretariat consists of about 20 staff members, under Executive Director, Secretary General, and three Managers who work in three Sections (General Af fairs; Projects, Meetings, and International Exchange; and Publications and Information) related to the Departments described below; the General Affairs Section also deals with the Society’s finance. The CSJ Headquarters is located in a central academic area (Ochanomizu) of metropolitan Tokyo in the Chemistry Hall, a CSJ-owned building inaugurated in 1991 by members’ contributions and wholly renovated just last year in 2020 (Figure 2). The Departments are CSJ’s functional organizations: General Af fairs, Research Exchange, Publications and Information, Academia-Industry Exchange, and Education and Public Relations). In accordance with their functions, these Departments hold total about 30 Committees, such as Membership, Award Selection, International Exchange, Journal Publication, Research Promotion, etc. In addition to these Depar tments and Committees, since 2018 the CSJ comprises 21 Divisions for virtually all specific fields in chemistry, including analytical, inorganic, organic, macromolecular, and others. In parallel with Divisions are five Topical Groups, self-suppor ting research organizations currently focused on Colloids and Interfacial Chemistry, Chemo-informatics, Biofunctional Chemistry, Biotechnology, and Organic Crystals. Seven Regional Sections cover local CSJ activities geographically extended all over Japan. The Kanto Section (metropolitan Tokyo and its vicinity) is the largest with ca. 50 % of all CSJ membership; the Kinki (Osaka, Kyoto, Kobe, and vicinity) and the Tokai (centered in Nagoya) Sections are the second and the third largest Sections, respectively. Each Regional Section runs a variety of activities including local membership promotion, regional symposia, and outreach events for young potential chemists (children and school pupils) and the general public in the region. The annual operating budget of CSJ in fiscal 2020 is about one billion Japanese yen (JPY) or nine million US dollars (USD), where the primary revenue comes from the membership fees (regular, student, and company affiliate fees). Activities Meetings. The CSJ holds two annual meetings: the CSJ Spring Annual Meeting in March and the CSJ Chemistry Festa in October. The Spring Meeting (Figure 3), perhaps one of the most important CSJ activities, usually involves ca. 8,000 participants and over 6,000 oral and poster papers presented in general sessions and special symposia, expositions, and Figure 2. The Chemistry Building: The CSJ Headquarters and offices December 2021 | 9 Figure 3. CSJ Spring Annual Meeting: Lectures and poster sessions

10 | December 2021 public outreach events, where the General Assembly, Presidential lecture, and the Award Presentation Ceremony are also held. In contrast, the Chemistry Festa focuses on academia-industry exchange and collaboration, where a majority of the organizing committee members accordingly come from the industry. Coupled with carefully selected special topic symposia, exhibitions, and human networking events, the Festa provides excellent opportunities for industry-academia collaboration and for student job-hunting and recruiting. Along with the two annual nationwide meetings, the headquarter Departments and the Regional Sections organize a variety of symposia and workshops throughout a year. Publications. CSJ actively and internationally publishes two journals, two societal organs, and books (Figure 4). The two peer-reviewed journals are monthly published online in English. Bulletin of the Chemical Society of Japan (BCSJ), launched in 1926, publishes original articles, reviews, and accounts, in total ca. 200 papers per annum, with an impact factor 5.448 as of 2020, which steadily rising. Chemistry Letters (CL or ChemLett), launched in 1972, is for rapid current-awareness communications and short reviews, monthly with ca. 400 papers a year, with impact factor 1.389 as of 2020. The two organs, both primarily in Japanese and partially electronic, are windows to its members. Kagaku to Kogyo (Chemistry and Industry) is the CSJ’s primary monthly organ delivered online and by mail, and free of charge to all themembers. It features hot-topics accounts, Regional Section and CSJ Division reports, meeting announcements, messages to the members, and help-wanted advertisements. Kagaku to Kyoiku (Chemistry and Education), as its title implies, is primarily directed to school teachers and those who interested in chemistry education. It focuses on fundamental topics in chemistry (such as the IUPAC-authorized periodic table and atomic weights, SI units, etc.), new experiment programs developed by the members, and reviews. In addition, The Chemical Record and Chemistry: An Asian Journal are joint publications with Wiley-VCH, covering more or less personal research accounts. With several overseas chemistry-related societies, the CSJ has recently joined publishing a so-called preprint journal, ChemRxiv™, to follow a current trend of non-peer-reviewed online publication for the rapid exchange of ever proliferating research information. The CSJ also publishes books, such as Kagaku Binran (Chemistry Handbook), an authoritative compilation of chemistry data), and CSJ Current Reviews, a series of monographs covering hot topics, now in about 50 volumes. Awards and Research Grants. For recognition of members’ achievements and societal service, The Chemical Society annually presents ten awards, including the Award of the Chemical Society of Japan (the highest honor of research achievement), the Award for Creative Work, the Award for Young Chemists, the Award for Technical Development, the Award for Outstanding Young Women Chemists, and the Award for Chemical Education. Based on the private endowment by the 2019 Nobel Prize in Chemistry laureate, the Akira Yoshino Research Program provides a Figure 4. CSJ publications: Journals, organs, and books: (first row from left) Bulletin of the Chemical Society of Japan, Chemistry Letters, Kagaku to Kogyo, Kagaku to Kyoiku; (second row from left) The Chemical Record, Chemistry: an Asian Journal, Kagaku Binran, CSJ Current Review. December 2021 | 11 funding to a selected proposal on the topics annually specified by the donor, such as novel materials for lithium-ion batteries. In 2021, based on another private legacy endowment fund, CSJ has set a brand-new award, the Saburo Nagakura Award named after the donor, an honorary Society member, to recognize and promote a promising researcher either in academia or industry with original, creative, and novel research, development, and/or education. For the first time for the Society, the award presents a non-restricted cash prize of 10 million Japanese yen (ca. 100 thousand USD) to a single recipient a year to be selected from the awardees of the afore-mentioned CSJ Awards except for the Award of the Chemical Society of Japan. International Exchange. Quite naturally, the CSJ actively commits to international exchange activities (Figure 5) in collaboration with the chemistry-related societies and organizations worldwide, including the American Chemical Society (ACS), the Canadian Society for Chemistry (CSC), the Chinese Chemical Society (CCS), the Chemical Research Society of India (CRSI), the Chemical Society Located in Taipei (CSLT), German Chemical Society (GDCh), the Israel Chemical Society, (ICS), Korean Chemical Society (KCS), the New Zealand Institute of Chemistry (NZIC), the Royal Australian Chemical Institute (RACI), the Royal Society of Chemistry (RSC), and many others. The Japanese Chemical Society is an active member of international chemistry organizations, such as the International Pure and Applied Chemistry (IUPAC) and the Federation of Asian Chemical Societies (FACS) (Figure 5). The CSJ, ACS, and CSC are the three founding societies of the International Chemical Congress of Pacific Basin Societies (Pacifichem). This Congress, held in every five years in Honolulu, HA, USA, is perhaps one of the largest and most comprehensive chemistry congresses with over 15,000 participants, co-organized by the seven Pacific Rim chemical societies (the founding members with CCS, KCS, NZIC, and RACI). Another interesting activity is the Chemical Sciences and Society Summit (CS3), a series of symposia jointly held by pairs of a chemical society and a funding agency in China, Germany, Japan, UK, and USA; the Japanese pair consists of CSJ and the Japan Science and Technology Agency (JST). Every 2-3 years CS3 provides a forum to discuss topics important for chemistry relative to the world society, such as sustainability, environment, climate change, water, etc., and the next meeting will be hosted by CSJ and JST. With the Korean (KCS) and the Taipei (CSLT) partners, the CSJ holds a bilateral exchange agreement. Alternatingly every year, one partner society invites the president and/or younger chemists of the other to its annual meeting for lectures and human networking. Outreach. To foster next generation chemists and strengthen the relationship with the general public, the CSJ Headquarters and the Regional Sections regularly hold outreach events open to the public and particularly to school children and pupils. Of particular interest is the “I-Love-Chemistry Club” meeting, featuring chemistry experiments for kids, exhibitions, and Q&A sessions. To the delight of the CSJ members, juvenile participants show intense curiosity in chemical science and ask tough questions that often puzzle the instructors. For example, they may ask “Why does an orange-flavored jelly that looks a soft solid soon melt in our mouth and taste sweet?” To answer such questions, instrutors cannot use any technical terms, however commonly used by professional chemists, such as hydrogel and hydrogen bonding. Future Perspective The CSJ has been consistently active and steadily growing in promoting the progress in chemical science and technology. Its activities have been expanding in scope to encompass not only chemistry per se but a wide variety of related fields as biology, physics, medicine, pharmacy, and materials science. As stated above in the CSJ’s missions, Chemistry for Sustainable Society and the World is an eminently important mission. As an expert group of professionals in molecules, substance transformation, materials creation, and process innovation, the Chemical Society of Japan has decided to meet the global challenges with concrete, viable, and implementable solutions, including sustainability, resilience, energy demand, food and water supply, global warming, and preserving the environment. ◆ Figure 5.CSJ’s international activities

12 | December 2021 Hiroaki Suga Hiroaki Suga is a Professor of the Department of Chemistry, Graduate School of Science in the University of Tokyo. He received Ph.D. at MIT (1994) followed by post-doctoral fellow in MGH (1997). He was Assistant and tenured Associate Professor in the State University of New York, University at Buffalo (1997–2003) and Professor in the Research Center for Advanced Science and Technology in the University of Tokyo (2003–2010). Since 2010, he has the present position. He is the recipient of Akabori Memorial Award 2014, Max-Bergmann Medal 2016, Nagoya Medal Silver 2017, Vincent du Vigneaud Award 2019, Bohlmann Lecture 2019 and The Research Award of the Alexander von Humboldt Foundation 2020. He is also a co-founder of PeptiDream and MiraBiologics in Japan. Ata Abbas Ata Abbas was born and grew up in India. After receiving his MSc (organic chemistry) from Aligarh Muslim University, India, he worked for a pharmaceutical company for some time. He later went on to receive his PhD from Nanyang Technological University, Singapore in 2015. Currently he is a post-doctoral researcher in Suga lab at The University of Tokyo where his interests are new chemical reactions to diversify genetically encoded macrocyclic peptide libraries and RaPID mRNA display. He is particularly passionate about mild, water based chemistries that are applicable to biological systems. Display technology platforms offer their own unique set of challenges for chemical transformations, at the heart of which lies peptide macrocyclization. The amenable reactions for peptide macrocyclization on this platform need to meet a number of criteria like high reactivity, selectivity, mild conditions, irreversibility and in many cases, a unique requirement to be assimilated into the translation machinery. Skillful utilization of these reactions has led to the formation of huge macrocyclic peptide libraries with varied linkages and topographies which have in turn led to the discovery of a number of hits for purposes such as drug discovery and others. Herein, we review those reactions which have mainly been applied in mRNA and phage display and discuss their technical characteristics and significance. GENETICALLY ENCODED LIBRARIES of peptides are an inexhaustible repertoire of therapeutic entities. They, however, generally work better when cyclized. Cyclic peptides are known to have two major advantages over their linear counterparts. Firstly, they are more resistant to proteases1 and hence have longer half-lives and better bioavailability2 for application as drugs etc. Secondly, they are more compact, have lesser degrees of freedom and fewer available conformations due to which they bind more tightly to the target protein by saving on entropy cost.3 Moreover, they are indicated to possibly have better cell permeability than their linear counterparts. The development of methodologies applicable to peptide cyclization under mild conditions constitutes an important and active area of research. Such methodologies must fulfil the requirement of application to not only diverse sequences but Peptide Cyclization Methodologies Amenable to in Vitro Display By Hiroaki Suga and Ata Abbas December 2021 | 13 also structures consisting of one, two, three or even more cyclic motifs. Cyclization reactions become more complicated and challenging due to the presence of various reactive sidechains on proteinogenic amino acids. Even though there are various techniques for chemical synthesis of cyclic peptides on solid support based on traditional protection-deprotection chemistries4 and/or metal-catalyzed reactions,5 most of these reactions are not suitable for the use on display platforms because of the following reasons: they must be compatible to physiological-like conditions (e.g. at near-neutral pH) and high chemoselective to the aiming functional groups. This review deals with techniques of peptide cyclization as applied to in vitro display techniques, represented by the phage and mRNA displays. Challenges Display technologies6, 7 rely on the translation machinery consisting of ribosome, protein translation factors, various enzymes including aminoacyl-tRNA synthetases, amino acids, tRNAs, mRNAs, energy sources, and others. Thus, the cyclization chemistry needs to selectively work for the aimed peptides in the presence of all these bio- and small-molecules. Even a harder challenge is that their chemistry must efficiently take place regardless of peptide sequences originating from huge mRNA libraries and vast tertiary structures originating from the diverse peptide sequences. For the phage display, a classical and general method for generating cyclic peptides is disulfide bond formation via two cysteine (Cys) residues. This is simply because their genotype of mRNA or DNA sequence is packaged in the bacteriophage, the easiest way to cyclize the peptide sequences is to use the naturally occurring crosslinking bond(s) of disulfide. However, disulfide bond is a reducible bond, and therefore in consideration for physiological conditions this bond is not necessarily ideal for drug use. Even though such a disulfide bond can be elaborated to an alternative bond, but in such a case the activity of the parental peptide is often diminished. Thus, it is important to develop an alternative approach to produce macrocyclic peptides closed by a more physiologically stable bond from the initial library. For the mRNA display, the respective peptides are directly attached to the genotype sequences of mRNA via puromycin molecule. Occasionally, the mRNA sequence is reverse transcribed to cDNA sequence forming the noncovalent annealing pair. This means that the peptide-mRNA/cDNA fusion contains not only the peptide motif but also ‘naked’ nucleic acids, and thereby the chemistry for cyclization is even more challenging than the phage case, where the cyclization must take place without unwanted reactions with sidechains of peptide nor with nucleic acid’s nucleobases/phosphates. Cyclization strategies Traditionally, peptide cyclization has been categorized as taking place between two ends of the peptide (head-to-end), two sidechains (sidechainto-sidechain) or one end to a sidechain (head-tosidechain and sidechain-to-end). However, for the sake of this review which deals mainly with those methods applied to display technologies, we will broadly categorize the strategies in two, i.e., cyclization without using genetic code manipulation and cyclization using genetic code manipulation. Cyclization via chemical crosslinking This strategy usually takes advantage of inherent reactivity of a native amino acid side chain and an external organic motif. Majority of groups have exploited nucleophilicity of thiol groups of

14 | December 2021 Cys or amino groups of lysine (Lys) ε-sidechain (or occasionally N-terminus), which are present at fixed positions in the translated peptide that conjugate with a small organic motif added after translation. Thus, this strategy has been applied for the majority of phage display works. Thioether bond formation This has been a popular strategy due to its simplicity and the ability to yield macrocyclic peptides with more than one loop. Cys thiols at fixed positions react with organohalides forming thioether bonds in a SN2 reaction. The libraries have vast diversities consisting of proteinogenic amino acids only. Using bis/tris/tetrakis (bromomethyl) benzenes Inspired by some naturally occurring peptides with multiple fused rings and loops and having interesting biological activities,8-10 many groups have tried to develop methods to create peptide libraries having similar topologies. Beginning of this was the report of Timmerman (Figure 1) that treating di-, tri-, and tetra-Cys containing peptides with bis-, tris-, and tetrakis(bromomethyl) benzene derivatives in aqueous ACN results in fast, one-step chemical synthesis of single-, double-, and triple loop peptides.11 In 2009, this reaction was later utilized by Winter et al.12 to produce bicyclic peptide libraries for phage display. They designed peptide libraries with three reactive Cys residues, each separated by several random amino acids and conjugated with tris(bromomethyl)benzene (TBMB) in aqueous solvents (Figure 2A). The conjugation reaction however posed several challenges including cross reactivity of TBMB with the disulfide bridges D1 and D2 domain in the phage PIII and a loss in phage infectivity due, probably, to the crosslinking of the phage coat protein through lysine side chains. The problems were, however, solved by using a disulfide free gene-3-protein phage and using low concentration of TBMB. The phage display selection was successfully carried out to find an inhibitor ligand to human plasma kallikrein. This elegant approach represents that the appropriate engineering of the phage system allows to control selective crosslinking of Cys residues only appeared in the random library of displayed peptides. In 2012, Szostak group also utilized a similar strategy to cyclize highly modified peptides having two flanking cysteine residues using dibromoxylene.13 The peptide libraries having several non-proteinogenic amino acids were used for in vitro selection based on mRNA display against the target protease thrombin with successful isolation of binders with low nanomolar affinity. Using perfluoroarenes Perfluoroarenes react with a reactive thiol in peptide via nucleophilic aromatic substitution reaction SNAr, which has been used extensively for polymer arylation and bioconjugation.14-18 Derda et al.19 used decafluoro-diphenylsulfone (DFS) to crosslink Cys thiols yielding cyclic peptides in one of the fastest Cys conjugation reactions (Figure 2B). They improved the previously reported SNAr reagents such as 1-chloro-2,4-dinitrobenzene,20 perfluorobenzene21, 22 and perfluorobiphenyl22 which show low reactivity and poor solubility in aqueous systems. The group has demonstrated this reaction to be biocompatible and faster than most Cys conjugation reactions with the reaction rates up to 180 M-1S-1, although the rate is largely sequence dependent; e.g. positively charged residues such as arginine accelerated it while negatively charged aspartate supressed the rate. This unique reaction is fairly selective for Cys, but with large excess and prolonged exposure to DFS showed some cross-reactivity with amine groups. As for applicability of the reaction in phage display, a clone of M13 phage could be 60–70% modified with DFS in 5% DMF as cosolvent. The modification efficiency was decreased to 35% when a whole library containing 109 peptides was used. Interestingly, the crosslinked peptides generally exhibit higher oxidative resistance compared with the traditional α,α’-dibromo-meta-xylene. Using Dichloro-oxime In 2015, Dawson et al. reported side chain linking of cysteine or homocysteine thiols using dichloroacetone (DCA) to give stapled (macrocyclic) peptide with an acetone bridge.23 This linking not only stabilized the secondary structure of the peptides but also provided a ketone moiety to link various molecular tags through oxime ligation. Building further on this concept, Derda et al. used pre-formed dichloro-oxime (DCO) derivatives (Figure 2C) to cyclize phage displayed glycopeptide libraries.24 Reaction went on to completion giving approximately C C S S C C HS SH Br Br C C C Br Br Br C C C S S S HS SH SH C C C C S S S S C C C C SH HS SH HS A B C Br Br Br Br F F S F S F F O O F F S F F F S F F F F O O F F F F Br Br Br Cl Cl N R S S N R C C C S S S C C SH HS C SH C SH HS C C C C C R = biotin mono-glycoside A B C H N H N H N H N Figure 1. Formation of peptide loops by reacting Cys-containing peptides with di-, tri-, or tetra-Cys reacting to bis-, tris-, or tetrakis-(bromomethyl)benzene as a crosslinking agent. Figure 2. Examples of peptide cyclizations using bromomethyl benzenes, amenable to display technologies. (A) Bicyclic peptide library using 1,3,5-tris(bromomethyl) benzene reported by Winter group. (B) Decafluoro-diphenylsulfone (DFS) cyclizaton and (C) Dichloro-oxime cyclization reported by Derda group. December 2021 | 15 85% adduct in 3 hrs with a rate constant of 1.1 M-1S-1. Interestingly, it was found, unlike the reports of Heinis and Winter,12 that DCO modification did not result in losing phage infectivity and more than 80% of phage remained viable after modification. This suggests that crosslinking of phage coat protein is negligible with DCO. Amide bond formation In one of the first reports, Robert et al. reported a general route for post-translational cyclization of mRNA display libraries by treating translated peptide with disuccinimidyl glutarate (DSG) at pH 8.25 DSG reacted near-quantitively with N-terminal amine and an internal Lys ε-amino group crosslinked via two amide bonds. The same group then demonstrated mRNA display of DSG-linked library against Gαi1, successfully discovering a strong cyclic peptide binder with Kd = 2.1 nM. 26 Disufide-rich loop formation Disulfide bond formation was one of the first approaches developed to cyclize linear peptides displayed on phage but due to the instability of disulfide bond in reducing cellular environment, this approach finds little practical value for in vivo applications. However, plant-based cyclotides are a unique class of peptides having multiple loops in the form of cysteine knots. Their remarkable thermal and proteolytic stability and a wide range of biological activities make them ideal macrocycles to be screened as ligands for target proteins. There are several reports of selection of cyclotides with novel function using in vitro displays.27-30 As a recent demonstration, Wenyu et al. reported mRNA-display of a cyclotide library derived from Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), in which two loops, 1 and 5, were randomized. The selection campaign against human Factor XIIa (hFXIIa) successfully yielded an extraordinary potent and selective variant, referred to as MCoFx1, giving Ki of 0.37 nM to hFXIIa that is greater than three orders of magnitude selective over trypsin and other related proteases.31 Cyclization using genetic code reprogramming Genetic code reprogramming is a powerful technique which enables incorporation of nonproteinogenic amino acids in translated polypeptides via codon reassignment32 or expansion.33, 34 The technique has evolved and matured over the years (for recent reviews see these references35, 36) in which task of reprogramming is achieved through a combination of an Escherichia coli reconstituted cell-free translation system and pre-aminoacylated tRNA with various nonproteinogenic amino acids facilitated by flexizymes. This system, referred to as FIT (Flexible In-vitro Translation), enables for devising many unique methods for macrocyclization of peptides discussed in the following sections. Thioether Bond Formation Thioether bond formation by nucleophilic substitution Unlike the aforementioned strategy of adding an external organic moiety with multiple halogens, this strategy results in the formation of one thioether bond per cycle. The halo part is incorporated at the initiator position or at a suitable side chain through genetic code reprogramming.37 An intramolecular substitution reaction by a downstream Cys thiol results in the formation of a physiologically stable thioether linkage. Suga group has explored, evolved and exploited this technique thoroughly, resulting in a number of interesting macrocyclic libraries and successful selections against various targets (for recent representative examples see references37-45). In 2008, Goto et al. have used a methioninedepleted FIT system where the initiation codon AUG becomes vacant, and engineered the initiation event. To this sytem is added an aminoacyltRNAfMet CAU charged with N-chloroacetylated amino acid, such as tryptophan (ClAc-Trp) or tyrosine (ClAc-Tyr), prepared by a flexizyme (eFx).37 The ClAc-Trp-tRNAfMet CAU was set as an initiator, for example, for the peptide expression, ribosome elongates amino acids starting from the ClAc-Trp according to mRNA template sequence, followed by a Cys residue at a downstream position. When the peptide synthesis is completed, the Cys thiol spontaneously reacts with the ClAc group to yield a thioether linked macrocyclic peptide (Figure 3A). It should be noted that other haloAc group, such as BrAc and IAc, yielded many byproducts originating from adducts of thiols present in the translation system, e.g. mercaptoethanol, DTT, and Cys. Thus, the ClAc group was the perfect reactivity toward the Cys thiol in peptide chain that effectively promotes the desired intramolecular reaction over undesired intermolecular reaction. This strategy has been applied to constructing mass libraries (over trillion members) of thioether macrocycles in combination with genetic code reprogramming for the incorporation of exotic amino acids46-48 including N-methyl-L-amino acids49,50, D-amino acids51-53, and β-amino acids54,55, etc. Suga group has integrated this strategy with mRNA display, referred to as RaPID (Random nonstandard Peptides Integrated Discovery) system, and enabled the ‘rapid’ discovery of various potent macrocycles56 against extracellular and intracellular proteins and has reported more than 35 successful selection outcomes with a range of low nM to pM KD values in the period of a decade.57-84 D W C C L/D W HS N H Cl O Pu mRNA•cDNA C Pu mRNA•cDNA N H S O L/D W HS N H Cl O C C C HS SH C C C C HS D W N H S O S S S Br Br Br A B C fM P D V C Cab F W K Y NH Cl O HS T P D V C F W K Y C S S E Urotensin-II fM P D V C Cab F W K Y NH O S Urotensin-II analog D Y HS O Br C D C GryA Y D C O S Figure 3. Ribosomal synthesis of macrocycles closed by a thioether bond via nucleophilic substitution. (A) N-terminal ClAc-Trp installed by the genetic code reprogramming reacts with a downstream Cys. (B) Tricyclic peptide synthesis of the intramolecular N-terminal ClAc with the second downstream Cys in the combination with TBMB that crosslinks three remaining Cys residues. (C) The ClAc group on the sidechain of Cab installed by the genetic code reprogramming reacts with a downstream Cys. The sequence represents a sequence of human urotensin II. (D) Macrocyclization using thioether bond formation by intramolecular reaction between non-proteinogenic amino acid O2beY and Cys inside living bacterial cells via intein-based protein splicing.

16 | December 2021 Interestingly, the N-terminal ClAc group reacts with Cys thiol at almost any position, except for Cys at the adjacent downstream position to ClAc-initiator (i.e. at the second position). This is simply because Cys cannot sterically reach to the ClAc group. Thus, when there is a Cys residue at the second position, arbitrary sequence and length of peptide followed by a downstream Cys residue, the latter Cys thiol (generally the second Cys residue) selectively reacts with the N-terminal ClAc group to form thioether-macrocycle. This fact has allowed to build a strategy for ribosomal synthesis of tricyclic peptides (Figure 3B). In this scheme, a peptide contains a total of four Cys residues, where ClAc-Trp is followed by Cys and then the rest of peptide sequence has three Cys residues at various position. The second Cys spontaneously reacts with the N-ClAc group to afford a monocycle. Then, the treatment of TBMB crosslinks the remaining three Cys residues to form a topologically complex tricyclic peptide. This ClAc thioether strategy can be expanded to inter-sidechain cyclization by incorporating an Nγ-ClAc-α,γ-diaminobutylic acid (ClAc-Cab).85 Again, a downstream cysteine thiol reacts with the ClAc group to afford a macrocycle closed by the thioether bond. Application of this methodology was demonstrated by translating a known biologically active peptide human urotensin II which is a potent vasoconstrictor. Single disulfide bond between cysteine residues at position 5 and 10 was replaced with a thioether bridge between Cab at position 5 and a cysteine at position 10 (Figure 3C). The resulting peptide was shown to retain biological activity and remarkable stability towards proteinase K under reducing conditions.85 In 2014, Fasan et al. developed a strategy of producing thioether linked macrocyclic peptides inside living bacterial cells (E.coli) which can be utilized on phage display platform (Figure 3D).86, 87 In order to supress cross reactivity with many other nucleophiles in the cellular environment, they ribosomally incorporated a rather slow reacting nonproteinogenic amino acid (O-(2bromoethyl)-tyrosine) termed O2beY. For proteolytic release of the cyclized peptide, they also incorporated an intein-based protein splicing element. Both features combined together, resulted in ribosomal production of a linear precursor peptide having a cysteine reactive nonproteinogenic amino acid O2beY and an intein splicing element. Remarkably, another cysteine present in the intein element did not show any reactivity towards cyclization reaction due to being partially buried within the active site. Yet, the practice of this approach for the disovery of de novo macrocyclic peptides has not been reported. Michael Addition Nucleophilicity of thiolate can also be exploited in Michael type addition reactions to yield thioether linkage. In fact, many biologically active natural lanthipeptides utilize this strategy for cyclization. For such ribosomally synthesized and posttranslationally modified peptides, dehydratase enzymes recognize the N-terminus of the precursor leader peptide and convert serine and threonine residues in the core peptide to dehydroalanine (Dha) and dehydrobutyrine (Dhb) respectively. The α,β-unsaturated moieties in Dha and Dhb acts as the electrophile where enzyme assisted Michael addition reaction by cysteine thiol generates a thioether linkage. The most extreme case observed in natural products is biosynthesis of nisin. Inspired by this chemistry, Goto et al.88 used genetic code reprogramming to incorporate vinylglycine in translated peptides which was isomerized to dehydrobutyrine by simply heating the peptide at 95˚C for 30 minutes. This was followed by spontaneous Michael addition by a cysteine thiol to give methyllanthionine containing macrocyclic peptide. They later demonstrated the applicability of this reaction by synthesizing two ring segments of the natural bioactive peptide nisin (Figure 4). Due to high temprature requirement of this cyclization step, this approach is inapplicable to the display system; therefore, a better alternative approach is needed. Oxidative Coupling Genetic code reprogramming allows for incorporation of various nonproteinogenic amino acids including those with orthogonal reactive handles to accomplish click type ligation (vide infra). A practically useful application of this methodology was incorporation of benzylamine and 5-hydroxyindole.89 These functional groups are known to react instantly under oxidative conditions to yield a fluorescent heterocyclic moiety. This methodology (Figure 5), although not used for display technology yet, seems to offer immense practical utility and potential for application in display-based selection. Azide-Alkyne Coupling Copper catalyzed Azide-Alkyne Click (CuAAC) reaction90, 91 needs no introduction and remains one of the most versatile and practically useful bioconjugation reaction (for some reviews see92-97). It has been exploited widely for peptide cyclization in solid phase98 and solution phase peptide synthesis.99-101 Its underutilization in macrocyclization of peptides for display technologies, however, is, in part, due to the lack of compatibility with nucleotides102-104 (with RNA in particular). RNA is susceptible to oxidation and degrades quickly in presence of Cu in aqueous medium.105 Use of acetonitrile as cosolvent, Cu stabilizing ligands and degassing buffer solutions are some of the ways to prevent mRNA degradation when using CuAAC reaction. Additionally, since double incorporation of both azide and alkyne bearing unnatural amino acids is rather tedious and low yielding, the use of this strategy for preparing monocyclic peptide F NH2 HN O HO N H F N H O N O NH WOH 0.5 mM K3Fe(CN)6 pH8, RT, 5 min WOH C fM H N O N H G A L M G N G G C G A L M G N G fM G H N O N H fM G G A L M G N G C HN O NH S HS HS 95°C Figure 4. Benzyl amine and hydroxyindole incorporated in translated peptides react rapidly under oxidative conditions, yielding a unique fluorogenic aromatic linkage. Figure 5. Cyclization via Michaels addition. Model peptide with vinylglycine isomerising to dehydrobutyrine on heating to 95˚C and subsequent intramolecular Michael addition by cysteine thiol to give the macrocycle.