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 0841232385, 9780841232389

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1257

ACS SYMPOSIUM SERIES 1257

CHENG ET AL.

Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1

Volume 1

Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel

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X X Y

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HN H. N. Ch Cheng, CCynthia hi AA. M Maryanoff, ff Bradley D. Miller, and Diane Grob Schmidt

Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1

ACS SYMPOSIUM SERIES 1257

Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 H. N. Cheng, Editor U.S. Department of Agriculture, New Orleans, Lousiana

Cynthia A. Maryanoff, Editor Baruch S. Blumberg Institute, Doylestown, Pennsylvania

Bradley D. Miller, Editor ACS International Activities, Washington, DC

Diane Grob Schmidt, Editor University of Cincinnati, Cincinnati, Ohio

Sponsored by the ACS Division of Organic Chemistry

American Chemical Society, Washington, DC Distributed in print by Oxford University Press

Library of Congress Cataloging-in-Publication Data Names: Cheng, H. N., editor. | American Chemical Society. Division of Organic Chemistry. Title: Stereochemistry and global connectivity : the legacy of Ernest L. Eliel Volume 1 / H.N. Cheng, editor (U.S. Department of Agriculture, New Orleans, Lousiana) [and three others] ; sponsored by the ACS Division of Organic Chemistry. Description: Washington, DC : American Chemical Society, [2017]- | Series: ACS symposium series ; 1257 | Includes bibliographical references and index. Identifiers: LCCN 2017045040 (print) | LCCN 2017050615 (ebook) | ISBN 9780841232372 (ebook) | ISBN 9780841232389 (v. 1) Subjects: LCSH: Stereochemistry. | Communication in science. | Conformational analysis. | Eliel, Ernest L. (Ernest Ludwig), 1921-2008. Classification: LCC QD481 (ebook) | LCC QD481 .S7574 2017 (print) | DDC 547/.1223--dc23 LC record available at https://lccn.loc.gov/2017045040

The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48n1984. Copyright © 2017 American Chemical Society Distributed in print by Oxford University Press All Rights Reserved. Reprographic copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Act is allowed for internal use only, provided that a per-chapter fee of $40.25 plus $0.75 per page is paid to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. Republication or reproduction for sale of pages in this book is permitted only under license from ACS. Direct these and other permission requests to ACS Copyright Office, Publications Division, 1155 16th Street, N.W., Washington, DC 20036. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

Foreword The ACS Symposium Series was first published in 1974 to provide a mechanism for publishing symposia quickly in book form. The purpose of the series is to publish timely, comprehensive books developed from the ACS sponsored symposia based on current scientific research. Occasionally, books are developed from symposia sponsored by other organizations when the topic is of keen interest to the chemistry audience. Before agreeing to publish a book, the proposed table of contents is reviewed for appropriate and comprehensive coverage and for interest to the audience. Some papers may be excluded to better focus the book; others may be added to provide comprehensiveness. When appropriate, overview or introductory chapters are added. Drafts of chapters are peer-reviewed prior to final acceptance or rejection, and manuscripts are prepared in camera-ready format. As a rule, only original research papers and original review papers are included in the volumes. Verbatim reproductions of previous published papers are not accepted.

ACS Books Department

Contents Foreword .......................................................................................................................... ix In Memory of Ernest Eliel .............................................................................................. xi Preface ............................................................................................................................ xiii 1.

Stereochemistry and Global Connectivity: An Overview .................................... 1 H. N. Cheng, Cynthia A. Maryanoff, Bradley D. Miller, and Diane Grob Schmidt

Tribute to Ernest Eliel 2.

Ernest L. Eliel as “Hidden Advisor” .................................................................... 13 Jeffrey I. Seeman

3.

Science and Knowledge in the Service of Humanity: The Example of Ernest Eliel .......................................................................................................................... 49 Luis A. Montero Cabrera

Global Connectivity 4.

Educational Outreach Activities between the ACS Division of Chemical Education’s International Activities Committee and the Sociedad Cubana de Química .............................................................................................................. 57 Charles H. Atwood and Luis Alberto Montero-Cabrera

5.

Ernest Eliel Workshop – US and Cuba Collaboration in Chemistry Education and Neglected Disease Drug Discovery .............................................. 63 W. L. Scott, J. G. Samaritoni, M. J. O’Donnell, A. B. Dounay, A. A. Fuller, P. S. Dave, J. M. Sanchez, D. G. Tiano, and D. G. Rivera

6.

An Update on International Activities at the ACS .............................................. 95 Bradley D. Miller

Carbohydrates 7.

Saccharide Structure and Reactivity Interrogated with Stable Isotopes ........ 105 Wenhui Zhang, Reagan Meredith, Mi-Kyung Yoon, Ian Carmichael, and Anthony S. Serianni

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8.

Synthesis, X-ray Crystallographic and Computational Analysis of 2,3-Dideoxy-α- and β-D-erythro-Hexopyranosyl Cyanides: Anomeric Effect of the Cyano Group .............................................................................................. 155 Madeline Rotella, Mark Bezpalko, Nicholas Piro, Nicholas Lazzara, Scott Kassel, Deanna Zubris, and Robert Giuliano

9.

Hydration-Mediated Effects of Saccharide Stereochemistry on Protein Heat Stability ................................................................................................................. 171 Renata Kisiliak and Yoav D. Livney

10. Soluble Polymer Containing an N-Methyl-D-glucamine Ligand for the Removal of Pollutant Oxy-Anions from Water ................................................. 197 Bernabé L. Rivas and Julio Sánchez Editors’ Biographies .................................................................................................... 213

Indexes Author Index ................................................................................................................ 217 Subject Index ................................................................................................................ 219

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Foreword A major strength of American Chemical Society (ACS) is the large number of volunteers who help to grow and sustain the organization, from local sections to technical divisions, from regional to national meetings, from task forces to national committees, and from conducting research to writing and reviewing manuscripts for journals. Some of them spend literally thousands of hours on behalf of ACS and the global chemistry enterprise, helping students or fellow scientists, organizing meetings and symposia, reaching out to the local communities, publicizing the benefits of science, and connecting with global colleagues and partners. One of the people who excelled in these efforts was the late Prof. Ernest L. Eliel. For many years he taught at the University of Notre Dame and the University of North Carolina and was an acknowledged leader in organic stereochemistry and conformational analysis. He was also a leader at ACS, serving as ACS President in 1992 and Chair of ACS Board of Directors in 1987-89. He initiated several international activities, particularly with respect to Latin America. I had worked with him on many occasions, and he was always courteous, judicious, analytical, and thoughtful. Unfortunately Prof. Eliel left us in 2008. As ACS Immediate Past President in 2016, it was my pleasure to be associated with a symposium organized by Dr. Cynthia Maryanoff and me at the ACS National Meeting in Philadelphia in honor of Prof. Eliel. Because of the success of the symposium, Dr. Maryanoff and I decided to team up with Dr. H. N. Cheng and Dr. Bradley Miller to edit a book highlighting stereochemistry and global connectivity, which represented two of the key legacies of Prof. Eliel. We were very pleased that so many of our academic colleagues were willing to participate in this project in honor of Prof. Eliel. Thanks are due to all of the authors and my co-editors for their wonderful efforts. Because stereochemistry is a fundamental chemistry concept, ongoing research is carried out in different subfields of chemistry (such as organic, medicinal, carbohydrates, polymers), using various analytical techniques (such as NMR, X-ray crystallography, and circular dichroism). The two volumes of this book contain many research papers that represent cutting-edge research in all the above areas. Because chemistry is now a world-wide enterprise, global connectivity is important to chemistry practitioners, and the chapters on international activities should be of great interest as well. Hopefully the readers will find these two book volumes useful to them in their research or professional work, and the example of Prof. Eliel will motivate them to volunteer their time and talent to ACS and the global chemistry enterprise!

Diane Grob Schmidt 2015 ACS President ix

In Memory of Ernest Eliel

(Photo Credit: Reproduced from Chem. & Eng. News, 2008, 86(14), 62-67. Peter Cutts Photography)

Ernest Ludwig Eliel was an eminent organic chemist known for his seminal contributions to organic stereochemistry and conformational analysis. He was born in Cologne, Germany and moved to Scotland, then Canada, then Cuba. He received his B.S. from the University of Havana in 1946. He moved to the USA in 1946 and obtained his PhD at the University of Illinois in two years with eight publications that resulted from his work. He taught at the University of Notre Dame in 1948-1972 and then moved to the University of North Carolina at Chapel Hill. He was well-known as an outstanding teacher and researcher. He died in Chapel Hill in 2008. Eliel was active in the ACS and served in numerous capacities. He was Chair of the ACS Board of Directors in 1987-89 and ACS President in 1992. His scientific contributions and professional involvements were widely recognized. Among his awards and recognition were membership of the National Academy of Sciences (NAS) (1972), the George C. Pimentel Award in Chemical Education xi

(1995), the ACS Priestley Medal (1996) and the NAS Award for Chemistry in Service to Society (1997). We dedicate this book to the memory of his contributions to chemistry and his efforts to connect scientists worldwide through its practice.

xii

Preface The two volumes of this book are partially based on a symposium held at the ACS Fall National Meeting in Philadelphia in August 2016 on “Connectivity and the Global Reach of Chemistry: Honoring the Life and Scientific Contributions of Ernest L. Eliel.” The symposium speakers consisted of friends and former colleagues of Prof. Eliel, and they provided reminiscences of Eliel as well as updates of their own research work in honor of Eliel. The symposium was well-received and encouraged us to edit this two-volume symposium book. In addition to the symposium speakers, we also invited several other colleagues in the global chemistry enterprise to contribute papers related to stereochemistry and global connectivity. Prof. Eliel was well-known scientifically for his work on organic stereochemistry and conformational analysis. His book, Stereochemistry of Carbon Compounds, was a classic that had a profound influence on the field. He was also active in the ACS and served as Chair of the ACS Board of Directors in 1987-89 and ACS President in 1992. As an ACS leader, he recognized the importance of global connectivity and promoted the strengthening of international activities in chemistry. He started several valuable programs that facilitated international exchange, particularly those that related to Cuba, Mexico and the rest of Latin America. In view of Prof. Eliel’s outstanding contributions to chemistry and its global practice, we are featuring two topics in this book that reflect his legacy: stereochemistry and global connectivity. Thus, the aim of this book is to recognize the contributions of Prof. Eliel and to highlight the latest developments in stereochemistry and global connectivity so that younger people who may not know Eliel can benefit from the information given in the chapters of this book. A total of 22 chapters (in two volumes) are included in this book. In this volume, 10 chapters are included. They are grouped into three sections: 1) Tribute to Ernest Eliel 2) Global Connectivity 3) Carbohydrates Chapter 1 is an overview that summarizes the contents of all other chapters and also provides the background for the topics covered in the book. The second volume of this book comprises two additional sections, one on Stereochemistry of Organic Compounds, and another on NMR Applications. This book is targeted to all chemists and chemical engineers, particularly those with an interest in stereochemistry or international relations. Since xiii

stereochemistry is an important basic concept for chemistry, biochemistry, polymer science, and other related areas, hopefully many chemistry professionals and students find the chapters useful. Moreover, the chemistry enterprise is becoming increasingly globalized. Thus, global connectivity and networking may be of interest as well. This book is also suitable as a reference work for libraries. We appreciate the efforts of the authors who took time to prepare their manuscripts and the reviewers for their cooperation during the peer review process. We also thank Arlene Furman, Tracey Glazener, Elizabeth Hernandez, Tara Urban, and their colleagues at ACS Books for their patient and effective handling of the manuscripts. The center photograph on the book cover first appeared on the cover of Chem. Eng. News 1994 73 (20). Photo credit: Peter Cutts.

H. N. Cheng USDA – Agricultural Research Service Southern Regional Research Center New Orleans, LA 70124, USA

Cynthia A. Maryanoff Baruch S. Blumberg Institute Drug Discovery and Development Doylestown, PA 18902, USA

Bradley D. Miller ACS International Activities External Affairs & Communications Office of the Secretary and General Counsel American Chemical Society Washington, DC 20036, USA

Diane Grob Schmidt Department of Chemistry University of Cincinnati Cincinnati, OH 45221, USA

xiv

Chapter 1

Stereochemistry and Global Connectivity: An Overview H. N. Cheng,1,* Cynthia A. Maryanoff,2,* Bradley D. Miller,3,* and Diane Grob Schmidt4,* 1USDA

Agricultural Research Service, Southern Regional Research Center, New Orleans, Louisiana 70124, United States 2P. O. Box 339, Holicong, Pennsylvania 18928, United States 3ACS International Activities, External Affairs & Communications, Office of the Secretary and General Counsel, American Chemical Society, Washington, DC 20036, United States 4Department of Chemistry, University of Cincinnati, 301 Clifton Ct., Cincinnati, Ohio 45221, United States *E-mail: [email protected]; [email protected]; [email protected]; [email protected]

Stereochemistry is a fundamental concept in chemistry with relevance to all branches of chemistry, and all practicing chemists should have a working knowledge of this topic. Likewise, global awareness and connectivity should be of interest to chemistry professionals and students alike because the chemistry enterprise is becoming increasingly internationalized. These topics are highly significant, with many ongoing activities, both at research and professional levels, as exemplified by the chapters of this book. This article provides an overview and pays a particular tribute to the late Prof. Ernest Eliel, who excelled in both topics and was an exemplar for future scientists.

Introduction Stereochemistry is a fundamental property of many chemical substances. Most chemists and chemical engineers have been exposed to stereochemistry in © 2017 American Chemical Society

organic chemistry classes, where the terms cis-trans isomerism, stereoisomers, enantiomers, chirality, enantiomeric excess, and conformational analysis are taught (1–3). The same concepts are also highly relevant in other areas, such as biology, biochemistry, inorganic chemistry, and polymer science. As examples, most natural materials, e.g., amino acids, carbohydrates, nucleic acids, and fatty acids have inherent stereochemistry. Cellular and enzymatic transformations involving these materials often preserve the stereochemistry of the starting materials. Bioactive compounds (e.g., pharmaceuticals and agro-chemicals) are also sometimes enantiomerically enriched in order to achieve higher efficacy. Thus, asymmetric synthesis is a major endeavor in organic chemistry (4, 5). Moreover, stereochemistry is also important in polymer chemistry. Most biopolymers, e.g., polypeptides, polysaccharides, and polynucleotides, are enantiomerically enriched. Synthetic stereoregular polymers have been found to have useful properties, and their successful synthesis and commercialization have important economic consequences (6, 7). Likewise, stereochemistry is important in inorganic chemistry (8). Many inorganic and organometallic compounds are being used for organic asymmetric catalysis or stereospecific polymerization (7–9). One of the leading scientists who had left a major impact on stereochemistry was the late Prof. Ernst L. Eliel, who taught at Notre Dame University (1948-1972) and University of North Carolina (1972-2008). A major emphasis of his work was the stereochemistry and conformational analysis of organic molecules, including derivatives of cyclohexane and saturated heterocyclic rings (10, 11). His 1962 textbook, Stereochemistry of Carbon Compounds, has influenced generations of organic chemists (12). Prof. Eliel was also a proponent of global connectivity. As a leader in the American Chemical Society (ACS), he started programs that brought chemical scientists from Latin American and Central Europe to U.S. laboratories for short sabbaticals. He was a founding member of the U.S.-Mexico Foundation for Science. He was one of the co-organizers of three U.S.-Taiwan symposia. He initiated “Global Instrument Partners” in the early 2000s to help Latin American chemists gain access to advanced analytical instrumentation. Above all, he reached out to Cuba and initiated collaborative programs. Prof. Eliel had correctly assessed the global potential of the chemistry enterprise. Today, we are observing the increasing trend of globalization in a number of areas, such as trade, business, culture, and science (13–16). For a business, global connections can potentially help innovation through increased speed of R&D, decreased cost, access to a greater talent pool, greater responsiveness to local markets and needs, and shared risks; for a scientist, global connectivity has the benefit of increased collaboration opportunities, use of international facilities, access to global talent, and cultural enrichment (17). An overview is provided in this article of the two topics wherein Prof. Eliel had made notable contributions: stereochemistry and global connectivity. Some readers may wonder about the possible relationship between these two topics. As we know, stereochemistry deals with the spatial arrangements of atoms and molecules and the effects of these arrangements on their reactions or properties. Global connectivity deals with the spatial arrangements of people and the effects 2

of these arrangements on their interactions or productivity. Thus, in a heuristic and strategic manner, these two topics are related.

Tribute to Eliel Because of his prominence, biographies have been written about Eliel (10, 11). In his chapter (18), Seeman undertook the herculean task of summarizing Eliel’s scientific career, which covered a broad range of topics, ranging from organic synthesis to stereochemistry to natural product chemistry. In addition to his outstanding scientific and professional contributions, he was also a mentor to many other scientists and a warm and helpful friend. Seeman reminisced upon his long-term association with Eliel and provided first-hand information on Eliel as his own “hidden advisor” and friend. In another chapter (19), Montero recounted the efforts by Eliel in 1993 to communicate with Cuba and the Cuban Society of Chemistry (SCQ). Despite political difficulties, these communications resulted in mutually beneficial scientific interactions. Montero indicated that Eliel provided a good example of scientific statesmanship that placed human and scientific values above social, political and economic differences. These efforts have contributed to the promising inter-society relationship between ACS and SCQ today.

Global Connectivity As noted in the Introduction, Eliel actively promoted international activities when he was in the leadership position at ACS. He was especially partial to Cuba, because he went to school there and graduated with his chemistry degree from the University of Havana. It is therefore fitting to include two chapters in this book that report on current educational collaborations among Cuban and American scientists (20, 21) and an update on ACS international activities (22). In 2015 Presidents Barack Obama and Raúl Castro started normalizing relations between the U.S. and Cuba, thereby permitting a Cuban delegation to attend the 2015 Boston ACS meeting. In their chapter (20), Attwood and Montero described a meeting of representatives from SCQ and the ACS Division of Chemical Education International Activities Committee at Boston, that led to the attendance of four ACS educators at the 2016 Simposio Internacional de Química in Cayo Santa Maria, Cuba as well as a forthcoming workshop at the University of Utah. In a separate development, Scott et al reported (21) on a U.S.-Cuban collaborative workshop that took place at the University of Havana in October 2016. The one-week workshop focused on neglected disease drug discovery and entailed both Cuban hosts and professors and students from three U.S. institutions – Indiana University-Purdue University Indianapolis (IUPUI), Santa Clara University and Colorado College. The students learnt to use IUPUI’s Distributed Drug Discovery (D3) synthetic procedures. The workshop led to improved cross-cultural understanding and enhanced training for the students involved. 3

In his chapter (22), Miller (Director of ACS International Activities) indicated that his office is working closely with the ACS Committee on International Activities and others to organize a large number of systematic and impactful activities in order to advance the global chemistry enterprise. These include ACS global alliances, international chapters, science and human rights, ACS International CenterTM, ACS-Pittcon collaboration, ethics workshops, and Global Innovation Imperatives.

Stereochemistry of Organic Compounds Although the basic concepts of stereochemistry have been well known for many years, there are still many nuances and details that need to be elucidated. An example was the discovery of a strong S-C-P anomeric effect in 2-diphenylphosphinoyl-1,3-dithiane, where a suitable interpretation was pending. In their chapter (23), Juaristi and Notario confirmed the anomeric effect using computational chemistry and found fluorine to be a good lone pair electron donor towards geminal sigma bonds. Another example was given by Bailey and Lambert (24) on the conformational behavior of 5-phenyl-1,3-dioxanes that bore remote substituents. They showed how a non-classical CH…O hydrogen bond might be tuned in response to the electron-withdrawing or electron-donating ability of substituents positioned remotely on the aryl ring: electron-withdrawing substituents decreased the conformational energy of the phenyl group while electron-donating substituents increased the conformational energy of the group. In their chapter (25), Soai and Matsumoto explored asymmetric autocatalysis, where a chiral product acted as the asymmetric catalyst for its own production. Pyrimidyl alkanol was found to be a highly efficient asymmetric autocatalyst in the enantioselective addition of diisopropyl zinc to pyrimidine-5-carbaldehyde to produce more of itself. By using asymmetric autocatalysis in a clever way, spontaneous absolute asymmetric synthesis was achieved without the intervention of any added enantiomeric material. Rivera and Paixao (26) described a recent international endeavor that combined multicomponent reactions (MCRs) with highly stereoselective organocatalysis for the synthesis of enantiomerically pure compounds. The reaction sequences comprised the asymmetric aminocatalytic functionalization of α,β-unsaturated aldehydes followed by isocyanide-MCRs with such oxo-components as enantiomeric inputs. The method provided a convergent and stereoselective way of producing natural product-like compounds such as hydroquinolines, chromenes, epoxy- and depsi-peptides. Magriotis (27) reviewed the successful approaches toward the total synthesis of ecteinascidin-743 that was isolated from the Caribbean tunicate Ecteinascidia turbinate. It is an effective anti-tumor drug approved in the EU and the US. He hopes that one or a combination of the synthetic approaches that have been developed would be adopted in the commercial production of this material. Loeb et al (28) optimized the yield of the condensation reaction between phenanthroline-5,6-diones and ethylenediamine through the combined use of 4

theory and experimentation. They discovered the formation of a “non-aromatic” intermediate to be the cause for the lower yield. Characterization of this intermediate permitted them to facilitate its conversion to the desired product and obtain close to quantitative yield for the reaction. Electronic circular dichroism (ECD) is a valuable tool to study the unknown absolute configuration of an optically active molecule, but it is sensitive to solvent effects. Robinson et al (29) studied the solvent effects on the ECD spectrum of the compound 3,3′-dibromo-1,1′-bi-2-naphthol. They used computational chemistry (PCM model) and obtained results that were quite close to those observed experimentally.

Carbohydrates The carbohydrate molecules contain chiral centers and they are rich in stereochemistry. In their chapter (30), Serianni et al showed a large number of high-impact problems solved through the combination of NMR, isotopic enrichment, and computational methods. Examples included the use of stable isotopes to detect and quantify the cyclic and acyclic forms of reducing sugars in solution and to investigate relationships between saccharide structure, conformation and the kinetics of anomerization. Other examples included cis-trans isomerization of the N-acetyl side-chains, conformational analysis, and mechanistic studies. Giuliano et al (31) synthesized 2,3-dideoxy glycosyl cyanides using the Ferrier reaction of glycals with trimethylsilyl cyanide and Lewis acid catalysts, chemoselective reduction of the double bond in the products, and deacylation. X-ray crystallographic analysis of one of the products revealed features consistent with the anomeric effect of the cyano group, similar to other glycosyl cyanides. The results were confirmed with computational studies. Kisiliak and Livney (32) studied the role of sugars in enhancing the thermal stability of globular proteins. They chose stereochemically different mono- and di-saccharides and found rising protein denaturation temperature with increasing sugar concentration. No binding was found between the sugars and the protein. Sugars affected protein mainly indirectly via the water. The extent of thermal protection conferred to the protein correlated with the hydration number of the sugar within each group of isomers. Rivas and Sanchez (33) synthesized the water-soluble polymer, poly(glycidyl methacrylate-N-methyl-D-glucamine) and used it to remove arsenate, chromate and borate in water as a function of pH, polymer concentration, and presence of interfering ions. Thus, this polymer in combination with ultrafiltration might be regarded as a new separation system.

NMR Applications NMR is a versatile method that can be used to study stereochemistry. One of the techniques developed was rapid-injection NMR (RI-NMR), devised by McGarrity (34) and used extensively by Eliel et al (35–37). In their chapter, 5

Thomas and Denmark (38) provided an authoritative review of RI-NMR, including the background, the design of the setup, and many applications of RI-NMR in organic chemistry. Dybowski (39) provided an excellent review of solid state NMR and many examples of its applications, including studies of orientation in polymer materials, identification of species at catalyst surfaces, estimates of porosity in porous materials, and chemical changes in art masterworks. In many artworks slow reactions between free fatty acids and pigment-derived ions produce soaps that cause the appearance of protrusions (soap aggregates), clear spots in paintings, and crazes. Through a combination of 13C, 207Pb and 119Sn NMR, he was able to gain a better understanding of the chemistry involved. For many years Tonelli has focused on polymer conformation to explain structure/property correlations, particularly 13C NMR and polymer microstructures. In his chapter (40), he reviewed the use of the conformationally sensitive γ-effects to assign NMR spectra of polymers in solutions and melts. He also discussed the studies of conformations in solid polymers. Silk is a natural protein fiber that exhibits outstanding end-use properties. Asakura and his group have used NMR in ingenious ways to study the structure and conformation of silk fibroins. In their chapter (41), they reviewed their work relating to the silk fibroins stored in Bombyx mori and Samia cynthia ricini silkworms. Detailed information on the helix or coil conformation of different structural segments was obtained, and a good understanding of the strength of the silk fiber was achieved. In the last chapter of the book, Biswas, et al (42) used biodiesel as a renewable feedstock for new polymers. They introduced the epoxide functionality into biodiesel and converted it into homopolymers (and copolymers) through cationic polymerization with fluorosulfonic acid. Because of the stereochemistry involved, both linear and cyclic products were found. NMR was used to determined polymer structures and reaction mechanisms.

Conclusions This article provides a synopsis of the papers given in both volumes of this book, providing a glimpse of the advances and recent developments in stereochemistry and global connectivity. The authors hope that this article may give the readers a better appreciation and more in-depth knowledge of these two topics. Since Prof. Eliel had made important contributions to these topics in his long and illustrious career, these two topics serve as part of his legacy to us. It is fitting therefore to report on the progress in these areas as we honor his memory.

Acknowledgments The authors thank the authors of the book chapters for their contributions to this book and Dr. K. T. Klasson for helpful comments. Mention of trade names 6

or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

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Kalsi, P. S. Stereochemistry Conformation and Mechanism, 9th ed.; New Age International: New Delhi, India, 2017. Topics in Stereochemistry; Denmark, S. E., Siegel, J., Eds.; Wiley: Hoboken, NJ, 2006; Vol. 25. Morris, D. G. Stereochemistry; Royal Society of Chemistry: Cambridge, U.K., 2001. Gawley, R. E.; Aube, J. Principles of Asymmetric Synthesis, 2nd ed.; Elsevier: Oxford, U.K., 2012. Stereoselective Synthesis of Drugs and Natural Products; Andrushko, V., Andrushko, N., Eds.; Wiley: Hoboken, NJ, 2013. Baugh, L. S.; Canich, J. M. Stereoselective Polymerization with Single-Site Catalysts; CRC Press: Boca Raton, FL, 2008. Polymeric Chiral Catalyst Design and Chiral Polymer Synthesis; Itsuno, S., Ed.; Wiley: Hoboken, NJ, 2011. Kepert, D. L. Inorganic Stereochemistry; Springer-Verlag: Berlin, Germany, 1982. Steinborn, D. Fundamentals of Organometallic Catalysis; Wiley-VCH: Weinheim, Germany, 2011. Ernest L. Eliel. Wikipedia. https://en.wikipedia.org/wiki/Ernest_L._Eliel (accessed May 21, 2017). Seeman, J. I. Ernest Eliel – A Biographical Memoir; National Academy of Science: 2014. http://www.nasonline.org/publications/biographicalmemoirs/memoir-pdfs/eliel-ernest.pdf (accessed May 21, 2017). Eliel, E. L. Stereochemistry of Carbon Compounds; McGraw-Hill: New York, NY, 1962. The National Academies Committee on Prospering in the Global Economy of the 21st Century. Rising Above the Gathering Storm; National Academic Press: Washington, DC, 2007. Regions, Globalization, and the Knowledge-Based Economy; Dunning, J. H., Ed.; Oxford University Press: Oxford, U.K., 2002. Jobs, Collaborations, and Women Leaders of the Global Chemistry Enterprise; Cheng, H. N., Miller, B. D., Wu, M. L., Eds.; ACS Symposium Series 1195; American Chemical Society: Washington, DC, 2015. Chemistry without Borders: Careers, Research and Entrepreneurship; Cheng, H. N., Rimando, A. M., Miller, B. D., Schmidt, D. G., Eds.; ACS Symposium Series 1219; American Chemical Society: Washington, DC, 2016. Cheng, H. N. Innovations from International Collaborations. Chem. Eng. News 2014, 92 (50), 33. 7

18. Seeman, J. Ernest Eliel as “Hidden Advisor”. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1; ACS Symposium Series 1257; American Chemical Society: Washington, DC, 2017; Chapter 2. 19. Montero-Cabrera, L. A. Science and Knowledge in the Service of Humanity: The Example of Ernest Eliel. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1; ACS Symposium Series 1257; American Chemical Society: Washington, DC, 2017; Chapter 3. 20. Atwood, C. H.; Montero-Cabrera, L. A. Educational Outreach Activities between the ACS Division of Chemical Education’s International Activities Committee and the Sociedad Cubana de Química. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1; ACS Symposium Series 1257; American Chemical Society: Washington, DC, 2017; Chapter 4. 21. Scott, W. L.; Samaritoni, J. G.; O’Donnell, M. J.; Dounay, A. B.; Fuller, A. A.; Dave, P.; Sanchez, J. M.; Tiano, D. G.; Rivera, D. G. Ernest Eliel Workshop – US and Cuba Collaboration in Chemistry Education and Neglected Disease Drug Discovery. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1; ACS Symposium Series 1257; American Chemical Society: Washington, DC, 2017; Chapter 5. 22. Miller, B. D. An Update on International Activities at the ACS. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1; ACS Symposium Series 1257; American Chemical Society: Washington, DC, 2017; Chapter 6. 23. Juaristi, E.; Notario, R. Theoretical Evidence for the Relevance of n(S) → σ*(C-P), σ*(C-S) → σ*(C-P), and n(F) → σ*(C-X) (X = H, C, O, S) Stereoelectronic Interactions. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2; ACS Symposium Series 1258; American Chemical Society: Washington, DC, 2017; Chapter 1. 24. Bailey, W. F.; Lambert, K. M. The Importance of Electrostatic Interactions on the Conformational Behavior of Substituted 1,3-Dioxanes: The Case of 5-Phenyl-1,3-dioxane. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2; ACS Symposium Series 1258; American Chemical Society: Washington, DC, 2017; Chapter 2. 25. Soai, K.; Matsumoto, A. Asymmetric autocatalysis and the origin of homochirality. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2; ACS Symposium Series 1258; American Chemical Society: Washington, DC, 2017; Chapter 3. 26. Rivera, D. G.; Paixão, M. W. Interplay between Organocatalysis and Multicomponent Reactions in Stereoselective Synthesis. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2; ACS Symposium Series 1258; American Chemical Society: Washington, DC, 2017; Chapter 4. 27. Magriotis, P. A. Synthetic Approaches to the Stereochemically Complex Antitumor Drug Ecteinascidin-743: A Marine Natural Product by the Name Yondelis® or Trabectidin. In Stereochemistry and Global Connectivity: The 8

28.

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30.

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Legacy of Ernest L. Eliel Volume 2; ACS Symposium Series 1258; American Chemical Society: Washington, DC, 2017; Chapter 5. Sanhueza, L.; Cortés, D.; González, I.; Loeb, B. Condensation Reaction between Phenanthroline-5,6-diones and Ethylenediamine and its Optimization through Dialogue between Theory and Experiment. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2; ACS Symposium Series 1258; American Chemical Society: Washington, DC, 2017; Chapter 6. Robinson de Souza, A.; Ximenes, V. F.; Morgon, N. H. Solvent Effects on Electronic Circular Dichroism Spectra. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2; ACS Symposium Series 1258; American Chemical Society: Washington, DC, 2017; Chapter 7. Zhang, W.; Meredith, R.; Yoon, M.; Carmichael, I.; Serianni, A. S. Saccharide Structure and Reactivity Interrogated with Stable Isotopes. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1; ACS Symposium Series 1257; American Chemical Society: Washington, DC, 2017; Chapter 7. Rotella, M.; Bezpalko, M.; Piro, N.; Lazzara, N.; Kassel, S.; Zubris, D.; Giuliano, R. Synthesis, X-ray Crystallographic and Computational Analysis of 2,3-Dideoxy-α/β-erythro-hexopyranosyl Cyanides. Anomeric Effect of the Cyano Group. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1; ACS Symposium Series 1257; American Chemical Society: Washington, DC, 2017; Chapter 8. Kisiliak, R.; Livney, Y. D. Sugar Stereochemistry Effects on Water Structure and on Protein Stability: the Templating Concept. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1; ACS Symposium Series 1257; American Chemical Society: Washington, DC, 2017; Chapter 9. Rivas, B. L.; Sanchez, J. Soluble Polymer Containing a N-Methyl-DGlucamine Ligand for the Removal of Pollutant Oxy-anions from Water. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1; ACS Symposium Series 1257; American Chemical Society: Washington, DC, 2017; Chapter 10. McGarrity, J. F.; Prodolliet, J.; Smyth, T. Rapid injection NMR: a simple technique for the observation of reactive intermediates. Org. Magn. Reson. 1981, 17, 59–65. Frye, S. V.; Eliel, E. L.; Cloux, R. Rapid-Injection Nuclear Magnetic Resonance Investigation of the Reactivity of α- and β-Alkoxy Ketones with Dimethylmagnesium: Kinetic Evidence for Chelation. J. Am. Chem. Soc. 1987, 109, 1862–1863. Chen, X.; Hortelano, E. R.; Eliel, E. L.; Frye, S. V. Are chelates truly intermediates in Cram’s chelate rule? J. Am. Chem. Soc. 1990, 112, 6130–6131. Chen, X.; Hortelano, E. R.; Eliel, E. L.; Frye, S. V. Chelates as intermediates in nucleophilic additions to alkoxy ketones according to Cram’s rule (cyclic model). J. Am. Chem. Soc. 1992, 114, 1778–1784. 9

38. Thomas, A. A.; Denmark, S. E. Ernest L. Eliel, a Physical Organic Chemist with the Right Tool for the Job: Rapid Injection Nuclear Magnetic Resonance. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2; ACS Symposium Series 1258; American Chemical Society: Washington, DC, 2017; Chapter 8. 39. Dybowski, C. Characterization of Materials with NMR Spectroscopy. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2; ACS Symposium Series 1258; American Chemical Society: Washington, DC, 2017; Chapter 9. 40. Tonelli, A. E. From NMR Spectra to Molecular Structures and Conformation. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2; ACS Symposium Series 1258; American Chemical Society: Washington, DC, 2017; Chapter 10. 41. Asakura, T.; Suzuki, Y.; Nishimura, A. Solution NMR Structure and Conformation of Silk Fibroins stored in Bombyx mori and Samia cynthia ricini Silkworms. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2; ACS Symposium Series 1258; American Chemical Society: Washington, DC, 2017; Chapter 11. 42. Biswas, A.; Liu, Z.; Furtado, R.; Alves, C. R.; Cheng, H. N. Novel Polymeric Products derived from Biodiesel. In Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2; ACS Symposium Series 1258; American Chemical Society: Washington, DC, 2017; Chapter 12.

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Tribute to Ernest Eliel

Chapter 2

Ernest L. Eliel as “Hidden Advisor” Jeffrey I. Seeman* Department of Chemistry, University of Richmond, Richmond, Virginia 23173, United States *E-mail: [email protected]

Ernest L. Eliel was a titan in the global chemical enterprise. In addition to his enormous scientific and professional contributions, he was also a mentor to many other scientists and a warm and helpful friend. In this tribute, the author summarizes the highlights of Eliel’s scientific achievements. He also describes Eliel the man. He then reminisces upon his long-term association with Eliel and provides first-hand description of Eliel as his own “hidden advisor” and friend.

Introduction In 2000, 25 eminent chemists wrote essays in a memorial volume dedicated to Derek H. R. Barton, who had died just two years previously. That book entitled The Bartonian Legacy (1) was fittingly published by Imperial College Press, where Barton had received his Ph.D. in 1940, and where he taught for over 20 years, from 1957 to 1978. One of the chapters in that book was written by Ernest L. Eliel. That chapter is entitled, Derek Barton as “Hidden Advisor” (1). Eliel began his chapter as follows,

“Most scientists, when you ask them who most influenced their early careers, will give you the names of their Ph.D. advisor, their postdoctoral supervisor and, if they did an undergraduate thesis, perhaps their undergraduate mentor. However, the persons who have had the greatest influence on my career are two Nobel laureates with whom I was never officially associated: D. H. R. Barton and V. Prelog. But whereas I © 2017 American Chemical Society

have spent considerable amount of time with Prelog – six weeks when he was a Reilly lecturer at the University of Notre Dame in 1950 and a whole year when I was at the ETH in Zürich on a sabbatical leave in 1967-68 – I have never spent more than a day at a time with Derek Barton. Nevertheless he is largely responsible for the development of the directions of my research” (1).

Well, Ernest was most certainly one of this author’s hidden advisors. And he most certainly was either a visible or hidden advisor to all those who have contributed to the symposium, Connectivity and the Global Reach of Chemistry: Honoring the Life and Scientific Contributions of Ernest L. Eliel, held on August 22, 2016, at the 252nd ACS National Meeting in Philadelphia (Figure 1 and its predecessor, Figure 2). This chapter and many other chapters in this volume (2) stem directly from that symposium. Indeed, this volume is testament to the many others who so much valued the man who was Ernest L. Eliel that they requested that they, too, could participate in Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel. This book is edited by H. N. Cheng, Cynthia Maryanoff, Bradley D. Miller, and Diane Grob Schmidt. Grob Schmidt was the 2015 ACS President; she, Cheng and Maryanoff all served in the ACS governance with Ernest for many years. Cheng and Maryanoff have also served in the ACS International Activities Committee. Miller is Director, ACS International Activities, and worked closely with Ernest in his lifelong endeavor to improve the lot of chemists in developing countries. In this chapter I shall reveal Ernest as my hidden advisor. In addition, the editors of this volume also asked me to summarize Ernest’s scientific career. I am delighted to do so. I shall not discuss the details of his activities with the American Chemical Society (former ACS President, Chair of the Board of Directors, and many other positions) nor his many years of activities on behalf of chemists in developing countries, primarily though not exclusively in Latin America. Interested readers may look up one reference written earlier by me (3) and one by Ernest himself (4) that cover these topics. For those who wish to learn more about Ernest Eliel, I first and foremost recommend his own full length autobiography published in 1990, From Cologne to Chapel Hill (5) (Figure 3), as well as his four books (6–9), his short booklet Science & Serendipity (10), his Official [Campaign for ACS President] Statement printed in Chemical and Engineering News (C&EN) (11), and his many articles in the Journal of Chemical Education (12–21) on the topics he loved so dearly: stereochemistry and conformational analysis. One can also read an interview of him conducted by Istvan Hargittai (22) and Rudy Baum’s three-page C&EN biographical article announcing Eliel’s receipt of the Priestley Medal (23). Eliel was also co-editor of the first 21 volumes in the series Topics in Stereochemistry.

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Figure 1. Five of the participants in the August 22, 2016 symposium on Connectivity and the Global Reach of Chemistry: Honoring the Life and Scientific Contributions of Ernest L. Eliel. (L to R) Kenso Soai, William Bailey, Jeffrey Seeman, Eusebio Juaristi, and Anthony Serianni. Photograph courtesy of J. I. Seeman.

Figure 2. An earlier photograph of several of several individuals shown in Figure 1. During a swimming adventure at the 1980 Gordon Conference on Stereochemistry. (L to R) Fritz Vierhapper, Kenso Soai, William Bailey, Eliel, and Eusebio Juaristi. Curiously enough but perhaps not randomly, the order (left to right) of Soai, Bailey, and Juaristi has spontaneously repeated itself in Figure 1, 36 years later. Photograph courtesy of K. Soai.

15

Figure 3. Cover of Eliel’s autobiography published in 1990 by the American Chemical Society in the series Profiles, Pathways and Dreams (5). And there is more documentation. In 1972 Notre Dame Magazine published an article by Ernest entitled The Importance of Being Ernest (24). I’ve written several biographical essays on Ernest since the publication of his autobiography. In 2002 I published a biographical essay that covered Ernest’s scientific achievements, his life experiences, and his philosophies (25). In 2009 I reviewed Ernest’s contributions to a cause that was most close to his heart: his work on behalf of chemists and chemistry in the underdeveloped countries of Latin America (3). In 2014, following Ernest’s death and at the invitation of the National Academy of Sciences, I wrote a comprehensive biography of Ernest for the Biographical Memoirs of the National Academy of Sciences (26).

Ernest Eliel, the Scientist: The First Steps An Early Interest in Stereochemistry Ernest’s independent research began at the University of Notre Dame in the late 1940s. This work was a harbinger of what would be his research focus for the next 40 years: stereochemistry and synthesis. Knowledgeable readers – those of you who knew or know of Ernest– likely have sat up straight in your chairs and reflectively thought, “What is Seeman saying? Eliel was not a synthetic organic chemist.” Here is the story. You judge for yourself. Shortly after World War II, the stable and radioactive isotopes of hydrogen and carbon were beginning to become commercially available. These would almost immediately become tools that organic chemists would seize upon to investigate the simplest aspects of chemical structure and the most complex concepts of reaction mechanism and biosynthesis. Eliel was one of those 16

early pioneers to use 2H (deuterium). In 1949 Eliel wanted to determine if the one-neutron difference between 1H and 2H would be sufficient such that a compound of structure having a carbon atom to which is attached four different groups – two of which were 1H and 2H – would have a nonzero optical rotation. In essence, Eliel was to prepare a compound R1R2C1H2H = R1R2CHD in nonracemic form. He chose as his target C6H5CHDCH3 (2) and the related 4-substituted derivatives 3 and 4 (27). The syntheses of homochiral 2 and its enantiomer were rather straight-forward (Scheme 1). Racemic α-phenethylalcohol was converted to the half ester of phthalic acid, which was resolved via the brucine salt. Reaction of (-)-α-phenethyl chloride with lithium aluminum deuteride/lithium deuteride led to 2. Friedel-Crafts acylation led to 3, and ultimately to crystalline oxime 4, which was prepared and purified via multiple crystallizations to obtain an ultra-pure 4 necessary to establish the absence of an optically active impurity (Scheme 1). The substantial and non-zero rotations of 2 – 4 established that “a compound of the type R1R2CHD is capable of rotating the plane of polarized light” (27). Optical rotatory measurements on 2 prepared by other methods by Ronald L. Eisenbaumer and Harry S. Mosher in 1979 showed that Eliel’s reported values of his 2 “agree[d] well with ours both in sign and magnitude” (28).

Scheme 1. Eliel’s scheme for obtaining non-racemic compounds of the general structure R1CHDR2 (27). In 1949, Eliel did not know the absolute configuration of these compounds nor did he know their enantiomeric excess. This was determined 30 years after Eliel’s publication by Eisenbaumer and Mosher (28).

This early contribution of Eliel’s is noteworthy for another reason. The title of his paper is The Reduction of Optically Active Phenylmethylcarbinyl Chloride with Lithium Aluminum Deuteride (27). This title ignores the primary objective of Eliel’s research project, though it does highlight another important – as of 1949 – observation, the Walden inversion-stereochemical pathway in the reaction of secondary chlorides with lithium aluminum hydride/lithium hydride. Why Eliel chose this title over another such as Demonstration that Compounds of Type R1R2CHD are Capable of Rotating the Plane of Polarized Light is lost to history. Both results, however, speak to Ernest’s career-long interest in stereochemistry. In the same time period, Eliel began a program to synthesize yohimbine, an indole alkaloid derived from the bark of the Pausinystalla yohimbe tree found in western and central Africa. Of course, this effort was doomed to failure, in large measure because the synthetic technology needed to synthesize yohimbine was some time in the future. The other reason was that Eliel, not being trained as a synthetic chemist, had bitten off too much for him to chew. It was not until 1969, 17

twenty years later, that the first total synthesis of yohimbine was reported in full detail by Eugene van Tamelen and his group, then at the University of Wisconsin (29).

Ironically, one of the co-authors of van Tamelen’s yohimbine papers was Albert W. Burgstahler, who had been an undergraduate in Eliel’s laboratory at Notre Dame. Van Tamelen states in his 1969 full paper that he began this work in 1954 and it was completed in 1958. Why van Tamelen needed a decade from his 1958 communication (30) to full paper (29) is not evident and may be lost to history. By an interesting coincidence of timing, in the issue of the Journal of the American Chemical Society in which this Eliel paper’s The Reduction of Optically Active Phenylmethylcarbinyl Chloride with Lithium Aluminum Deuteride appeared, John D. Roberts published the syntheses of cyclobutanone, cyclobutanol, cyclobutene and cyclobutane (31), Donald J. Cram published the first paper in his multifold series of papers entitled Studies in Stereochemistry (32), Carl Djerassi published the preparation of several desoxycorticosterone analogues (33), and Russell Marker published the preparation of cortisone-like compounds from steroidal sapogenines (34). As Eliel himself related in his autobiography, his first years at Notre Dame were not outstandingly productive. Indeed, Eliel stated that “the academic year 1952—1953 may have marked the nadir in my scientific career.” Nonetheless, he was somewhat productive, Notre Dame was not then a powerhouse in chemistry, and thus he was promoted to assistant professor in 1950 and associate professor with tenure in 1953. The yohimbine project and several others were put to bed, never to arise, but certain events would shortly and compactly occur and would form the basis for the rest of Eliel’s research career. During 1950-1953, Eliel experienced several intellectual stimuli that would meld together in his mind and pivotally shape his scientific career. In these years, he got to interact closely with Barton and Vladimir Prelog and become infused with the latest ideas of stereochemistry and conformational analysis. Barton published his Nobel Prize-propelling paper on conformational analysis in 1950 (35). Eliel was also receiving reports from Burgstahler, who as a graduate student at Harvard, had listened attentively to Barton’s lectures at Harvard. Barton was filling in for Robert B. Woodward, who was on sabbatical leave (though he remained physically in Cambridge) in 1949-1950. Shortly after Eliel heard Barton’s “electrifying lecture” at Notre Dame on conformational analysis, he (Eliel) “was intrigued by an article by [John] Read [and William J. Grubb] (36) concerning the nitrobenzolylation of the four 18

diastereomeric menthols and especially by the fact that neoisomenthol (3) reacted three-times faster than neomenthol [4]” (25) (Scheme 2). This observation led Eliel to write his first paper (1953) on the relationship between the conformational properties of compounds and their chemical reactivity.

Scheme 2. Neoisomenthol (7), which exists in two conformations of relatively equal composition, reacts three times faster than neomenthol (8) which exists primarily in the conformation 8a in which the two alkyl groups are equatorial and the hydroxyl group is axial. Relative rates of reaction data are from Read and Grubb (36). See the text for Eliel’s explanation of these results and literature references. Eliel explained the relative rates of acylation reported by Read and Grubb (36) on the basis of (a) an estimate of the relative concentrations of conformations “e” and “a” of the four menthol isomers 5-8 (Scheme 2), and (b) the principle, first 19

enunciated by Barton in 1950 (35), that acylation of equatorial hydroxyl groups would proceed faster than acylation of axial (then called “polar”) hydroxyl groups. Eliel’s explanation was slightly refined in 1983 in discussions with this author, as reported in footnote 278 of a Chemical Reviews publication (37). In his 1953 paper (38), Eliel understood and explained that for compounds that exist in multiple conformations, one must consider the proportion of each reactive conformation and the reaction rate constant for each conformation. The quantitative explanation of these ideas would follow within several years and is discussed below.

John Read – Possibly One of Ernest Eliel’s Hidden (though “By Literature Only”) Advisors In his 1953 paper Eliel cites John Read’s kinetic results (Scheme 2) as stimulating his interest in the role of conformations on chemical reactivity. Almost certainly, Read’s publications influenced Eliel far more than Eliel has indicated, not to say that Eliel was hiding anything. Read’s influence, like the influence of many individuals on each of us, simply often went unspoken. Perhaps Read was a hidden advisor for Eliel. What is the connection between John Read and Eliel, other than Eliel’s use (38) of Read’s menthol results (36) summarized in Scheme 2? When Eliel began his research on the preparation of compounds of type R1R2CHD in the late 1940s, his literature search surely included the preparation and optical resolution of compounds of type R1R2 R3R4C where none of the substituents contain a carbon atom. In 1914 Read and his mentor, William Jackson Pope, reported,

“Amongst the most fundamental stereochemical problems that have hitherto remained unsolved, the question of determining the greatest degree of molecular simplicity which is compatible with the persistence of optical activity stands out as prominent. No optically active substance the molecule of which contains less than three carbon atoms has up to the present been satisfactorily characterized. . . In the present paper we describe the preparation of the externally compensated chloroiodomethanesulphonic acid, CHClI•SO3H [9], and show that this substance can be resolved into optically active components . . . ” (39).

Indeed, Read’s research, stereochemistry and optical activity began even earlier than 1914. In 1909 William Henry Perkin, Jr., Otto Wallach, and Pope published a paper entitled Optically Active Substances Containing no Asymmetric Atom. 20

1-Methylcyclohexylidene-4-acetic acid [10] (40). This paper ended with the statement, “Our thanks are due to Dr. John Read for the care with which he has carried out much of the experimental work involved in the present paper” (40). The Perkin, Wallach and Pope (and Read) paper began with the statement, “A perusal of van’t Hoff’s early work on optical activity makes it clear that he adopted the view, first advanced by Pasteur, that the optical activity of amorphous substances is due to enantiomorphism of molecular configuration. . . The optical activity is, in fact, not, as is still sometimes stated, due to the presence of an asymmetric carbon atom, but originates in the enantiomorphous molecular configuration. . . No case has, in fact, been experimentally realized of a substance exhibiting optical activity in the amorphous state and containing no asymmetric carbon (nitrogen, Sulphur, selenium tin, or silicon) atom. . . it appeared desirable to attempt, by modern synthetic methods, the preparation of such compounds. . . therefore [we] synthesized the 1-methyl-cyclohexylidene-4-acetic acid” (10) (40).

That paper also reported that “By a curious coincidence, Marckwald and Meth were at the same time engaged upon the synthesis of the same compound for the same purpose…” (28, 40). However, Marckwald and Meth actually prepared isomers of 10, namely 11. The racemix mixture 11 could be resolved into its optically active enantiomers, its optical activity was due to the presence of a chiral atom, C4. This is an early example in organic chemistry of what Robert K. Merton called “multiple simultaneous independent discoveries” (41). However, in this instance, Perkin, Pope, Wallach and Read’s compound was, indeed, 10 while Marckwald and Meth’s compound was 11, all of which are chiral. 21

Thus, the research of the very early stereochemist John Read (1884 – 1963) (42) served as the basis for much of Eliel’s early work in the field. Like Eliel, Read contributed to a wide range of activities: he authored a number of textbooks and lectured and wrote on the history of chemistry. In 1959, Read was the fourth recipient of the Dexter Award for history of chemistry given by the Division of History of Chemistry of the American Chemical Society – a still active award program, though now called the HIST Award for Outstanding Achievement in the History of Chemistry. Read’s award lecture appeared in the Journal of Chemical Education in 1960 (43), and Read appeared on the cover of the March 1960 issue of that journal.

Ernest Eliel, the Scientist: From Stereochemistry to Synthesis Having a qualitative understanding of the role of conformation on chemical reactivity was a major step forward in organic chemistry. The next step was a quantitative theory. Eliel jumped immediately into that challenge. The simplest set of reactions that describes the role of conformational multiplicity on chemical reactivity is shown in equation 1. First, consider a molecule that exists in two conformations (A2 and A3) (equation 1). The following discussion holds for molecules that exist in more than two conformations; the equations are just a bit more complex. One major question was, and is, what is the equilibrium distribution of the conformations, K? Today there are many spectroscopic tools which could solve this problem, even more than the abundance discussed in Eliel and Wilen’s massive 1994 book on the Stereochemistry of Organic Compounds (7). But in the early 1950s there was no known method to determine the equilibrium distribution of the conformations, K, in equation 1. Well, that’s not exactly true. There was a method, or at least there was a method that had been used. In the early 1950s a number of reports asserted that the equilibrium distribution K was equal to the observed or product ratio in a reaction described by equation 2 (whether first order or second order, taking into consideration a reagent). For example, in alkylation of various tropane derivatives (equation 5), the literature wrongly concluded that the major conformation was that from which the minor product was alkylated. David Y. Curtin and Louis P. Hammett shortly thereafter explained that “the ratio of products [(equations 4 and 5)] depends only on the difference in [free] energy of the two transition states [leading to product, equation 3], as postulated by Curtin and Hammett” and as taught by Eliel in 1962 (6). This statement is true provided the rates of reaction are much faster (44) than the rates of conformational interchange. 22

This teaching of the Curtin-Hammett principle immediately and successfully discounted the previous attempts to equate equilibrium distributions to product distributions, or the converse (equation 1). However, it was not until thirty years later that an alternative but equivalent formulation of the Curtin-Hammett principle (equation 4) was proposed and used to fully characterize the kinetic system shown by equations 2, 5 and 6 (45). Eliel included this important update in his 1994 book (7).

Back to the early 1950s. With the recognition that there was no obligatory 1:1 relationship between the equilibrium distribution K and the product ratio in equation 2 chemistry, a new method of conformational analysis was required. Into the fray jumped Eliel and, quite independently and simultaneously, the great physical organic chemist Saul Winstein. Both Eliel and Winstein independently proposed the Kinetic Method of Conformational Analysis. At the time, one of 23

Derek Barton’s former graduate students Ned Holness was a postdoctoral student with Winstein from the beginning of 1952 to mid-1954. Holness was well-versed in conformational analysis, and it was quite natural that Winstein would make his sole contribution to the field of conformational analysis with Holness. Eliel and R. S. Ro (46), as well as Winstein and Holness (47), proposed a clever reaction model to estimate the equilibrium distribution K in equation 1. It was not by coincidence that they both focused their attention to equation 2 chemistry because that was and is, as mentioned above, the simplest example of the role of conformations on chemical reactivity. Winstein and Holness derived equation 7 from the kinetics of equation 2. Eliel and Ro derived equation 8 from the kinetics of equation 2. Equations 7 and 8 can readily be shown to be equivalent. Both Winstein and Holness (47) and Eliel and Ro (46) made the following conceptual leap: Both groups imagined that having a conformationally-fixed analogue of A2 could be a model for A2; and that a conformationally-fixed analogue of A3 could be a model for A3. If k′21 could be measured and used as an equivalent for k21; and if k′34 could be measured and used as an equivalent for k34 (equation 9), then substituting those numbers into equation 8 and determining kobs for the parent reaction, it followed that K could then be calculated. Or, as Winstein and Holness said,

“Considering equation [7] further, we see that one could solve for n2 and n3 whose sum is unity, if k21 and k34 could be estimated from other sources. In this way, rate measurements could be made the basis of a quantitative method of conformational analysis” (47).

24

Eliel and Ro (46) calculated the equilibrium distribution of cyclohexyl tosylate (12a ⇌ 12e) to be ca. 3.3 using the solvolyses of cis-4-tert-butylcyclohexyl tosylate (13) and trans-4-tert-butylcyclohexyl tosylate (14) as models for the unsubstituted cyclohexyl tosylate (12, see equations 11a and 11b as models for equation 10).

This clever multiple simultaneous discovery by Eliel and Winstein unfortunately suffered from a fatal flaw. tert-Butyl substituents were very soon thereafter found to significantly alter the conformational properties of the cyclohexane rings to which they were attached, thereby rendering their use as models for the unsubstituted cyclohexane ring invalid (48–51). That this just-discussed kinetic method of conformation analysis failed to meet its objective should not distract from the fact that equation 7 remains a valid kinetic expression describing equation 2. Indeed, the combination of equation 4 and equation 7 has proven to be a very important and useful device to determine the reaction rate constants of the two conformations in systems described by equation 2. In 1980, Seeman et al. determined the overall methylation rate constants kobs and the equilibrium distributions K for a variety of nicotine analogues shown in equation 6 and, using equations 4 and 7, calculated the individual reaction rate constants of the two conformations (44). In 1957, Eliel and Ro published (52) what Eliel termed the Equilibrium Method of Conformational Analysis. This method is illustrated in equation 12 and certainly establishes the equilibrium distribution for the model system. However, to equate K for the system shown in equation 12 to that for K for the unsubstituted cyclohexanol again relies on the assumption that the tert-butyl substituent does not cause structural and other consequential distortions.

25

Following a 1958 fall semester sabbatical in John D. Roberts’s laboratory at Caltech, Eliel recognized that the Winstein-Holness equation 7 could be adapted to properties other than reaction rate constants, namely NMR parameters (equation 13). Thus, in 1959 Eliel published (53) a Citation Classic (54) entitled Conformational Equilibria by Nuclear Magnetic Resonance Spectroscopy. Thirty years later (7), Eliel discussed the strengths and weaknesses of this NMR method, including the use of low temperature NMR to freeze the individual conformations and then measure relevant chemical shifts and coupling constants, the temperature dependence of chemical shifts and the relative insensitivity of coupling constants to temperature. Conformational energies, originally referred to as A-factors, of many common and less common substituents have been determined using these methods, often by Eliel, two pages of which are tabulated in Eliel’s reference text (7).

In the 1960s and 1970s Eliel’s group at Notre Dame (Figure 4) and then at the University of North Carolina studied the conformational properties of a wide range of saturated heterocyclic compounds, see Scheme 3.

Scheme 3. A summary of various saturated heterocycles whose conformational analysis was studied by the Eliel group in the 1960s (55–59). 26

Figure 4. In front of the chemistry building, Notre Dame, ca. 1960. Photograph courtesy of the Eliel family. In 1970 Franz Nader examined the Grignard reaction with several conformationally fixed ortho esters (Scheme 4) (60, 61). The axially-substituted ortho ester 15 reacted to furnish nearly all axially-substituted 2-alkyl-1,3-dioxanes (16) while the equatorially-substituted ortho ester 17 was largely unreactive. This was the entry point for Eliel’s enantioselective syntheses. Shortly thereafter, Armando Hartmann examined the Corey-Seebach reaction of 1,3-dithaines with n-butyllithium, but with conformationally-fixed substrates (Scheme 5) (62, 63). According to Eliel, “the results proved startling . . . leading to enzymelike stereoselectivity [which] might be harnessed to a highly stereoselective (asymmetric) synthesis” (5). And in ensuing years, ‘Eliel-the- physical organic chemist’ transformed himself to ‘Eliel-the-natural products synthetic chemist’! The key chiral template in the asymmetric synthesis of secondary and tertiary α-hydroxy aldehydes and the derived acids and glycols in greater than 90% enantiomeric excess is the benzoxathiin 22 was first prepared by Lynch in 1984 (Scheme 6) (64). In recognition of the utility of this reagent, an Organic Synthesis preparation is now available (65, 66). An example of the use of this chiral template was achieved by Frey in the synthesis of the enantiomers of mevalolactone (Scheme 7) via the common intermediate, the diol 23 (67). Scheme 8 illustrates a variety of the compounds synthesized by the Eliel group using enantioselective methodologies. 27

Scheme 4. First observation of stereospecific reactivity in the saturated heterocyclics in Eliel’s research program (60, 61).

Scheme 5. Stereoselective reactions (62, 63) leading subsequently to enantioselective syntheses.

Scheme 6. Preparation of Eliel’s chiral template (64), now an Organic Synthesis preparation (65, 66).

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Scheme 7. Synthesis of the enantiomers of mevalolactone in very high enantiomeric excess by Frye and Eliel (67).

As the story of Ernest Eliel’s research career draws to a close, it is important to recognize the evolution of Eliel’s research program. In the earliest days of his independent career, he studied the relationship between conformations and chemical reactivity. He then examined a wide range of carbocyclic and heterocyclic compounds and identified their conformational preferences; and lastly, he combined all this knowledge to perform enantioselective syntheses (Figure 5).

Ernest Eliel, the Man A Citizen of the Global Chemical Community The assignment given to me by the editors of this volume was to cover Ernest’s science and my own relationship with him. So, as stated above, I shall not discuss the details of his extensive activities with the American Chemical Society nor his many years of activities on behalf of chemists in undeveloped countries. Brad Miller has contributed a chapter in this volume on Ernest’s activities with the ACS Office of International Activities.

29

Scheme 8. Examples of compounds synthesized by the Eliel group using the enantioselective methodologies illustrated in the previous three schemes.

I shall not discuss Ernest’s contributions as a teacher at Notre Dame or at the University of North Carolina except to say that his graduate students and postdoctoral students have very strong positive feelings about their former mentor. One example is illustrative. As required by the ACS, I sent a request for permission to Susan L. Morris-Natschke to include a photograph of her in this chapter. Morris-Natschke, now a research professor at UNC’s Eshelman School of Pharmacy, immediately responded in the affirmative and wrote a very lovely remembrance of Ernest in her email to me. An excerpt of this remembrance follows: I can remember Professor Eliel reducing a whole page from my dissertation draft down to essentially one paragraph. But his comments in the margin were not negative at all. Instead, they were extremely positive and just mentioned how his reductions made the point much clearer. This experience was definitely a lesson to remember in science and in life as well. –Susan L. Morris-Natschke, 2017 30

Figure 5. Celebrating a high enantiomeric excess at his 58th birthday with his research group, December 28, 1979. At the far left is Joseph Lynch. Looking at Lynch is Boyd Keys. At the far right is Muthiah Manoharan. Just behind Manoharan, only a portion of her face appearing, is Joy Carter, Eliel’s administrative assistant. To the left of Carter, also partially hidden, is Susan Morris-Natschke. Photograph courtesy of the Eliel Family and K. Soai.

I shall also not dwell on Ernest’s important textbooks, except to say that some chemists believe that they are the most important contribution Ernest made to chemistry. How did Ernest feel about his textbooks? He wrote once to me,

“They are part of my educational function. I am very proud of the fact that some 100,000 chemists from all over the world have read my 1962 book (6); many have expressed their appreciation to me personally. (Just tonight, at a Jewish Federation affair, I met a man who had just retired from IBM (!) who told me, when I was introduced to him, that ‘I was his hero’ in as much as he had studied from my book in graduate school at MIT in the late 1960’s)” (68).

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Ernest the Athlete In 2001, at the age of 79, Ernest wrote to me: “I swim 500m (10 laps) every day at lunch time (unless I am sick or have an overriding commitment which is rare). It used to take me 20 minutes but now that I am getting older it takes 25. Incidentally I was a fair tennis player as a teenager, but lost that skill during emigration when I had no chance to play. (Cuba was too hot.) When I took it up with Eva again in 1949, I was no longer any good, got disgusted and gave it up” (68). One of the reviewers of this chapter wrote, “I can provide corroboration on the swimming prowess because I was often in the same pool and getting clobbered despite being over 40 years younger!” Eva (Figure 6) told me that she continued to play tennis well into her 80s. She was an accomplished and talented individual. For many years, Eva was a classic music announcer/host of her program with WUNC, the national public radio station in Chapel Hill. She died on March 23, 2013, in Chapel Hill at the age of 89, not quite five years after Ernest.

Figure 6. Ernest and Eva after a luncheon with the author at an ACS National Meeting, 1996. Photograph courtesy of J. I. Seeman.

32

A Hint of Sadness In Ernest’s autobiography, buried in the middle of a paragraph on page 72, are several telling and even sad sentences. Of course, I mean that these words reveal a sadness of Ernest’s. He wrote that in the 1960s, he was “quite happy at Notre Dame . . . By the early 1970s, the situation had changed . . . the ecumenism that had developed so mightily under Pope John XXIII was beginning to abate, and the University of Notre became preoccupied once again with its Catholic character and that of its faculty. An endowed professorship had been established in the Chemistry Department, but it was not offered to me. In the summer of 1971, in response to a second offer, I accepted a W. R. Kenan, Jr., professorship at the University of North Carolina, with the move to be effective on July 1, 1972. In April 1972, I was elected a member of the National Academy of Sciences. Although I was, of course, very happy about this singular honor, my colleagues at Notre Dame, for obvious reasons, viewed it with mixed emotions” (5). Ernest was not a man to bemoan his travails nor life’s unfairness. In this essay, I have not recounted his escape in 1938 as a 16-year-old from Nazi Germany to Scotland just months before Kristallnacht; his separation from his parents, who fled to Palestine, and from his two brothers, one of whom lived in the UK and the other in Brazil during the war; his time in internment camps near Liverpool, on the Isle of Man and ultimately in Canada; and his five years abandonment in Havana, where he learned Spanish, worked in the pharmaceutical laboratory of George Rosenkranz (subsequently, famous for his scientific leadership of Syntex in Mexico City) and received an undergraduate degree from the University of Havana. Only in 1946 did Ernest arrive in the United States, again as a refugee. His optimism and resilence are illustrated by one of his principles of life, when dealing with such life events, as follows: “A very wise friend told me that ‘wherever you are, act as though you were going to spend the rest of your life there’ ” (26). For more of Ernest’s life story including details of his childhood and time in Scotland and Cuba, see his autobiography From Cologne to Chapel Hill (5), a biographical memoir written for the National Academy of Sciences (26) and other papers (3, 24, 25).

Eliel’s “Happiest Moments in Life, in Science” I interviewed Eliel several times for my biographical paper Ernest L. Eliel: A Life of Purpose, Determination, and Integrity (25), which was included in a February 2002 special issue of the journal Chirality (69) to honor his receipt of the 1996 Chirality Medal bestowed by the Società Chimica Italiana and, by happy 33

coincidence, his 80th birthday. Several quotes from those interviews were not included in that 2002 article. Here’s one: JIS: “Ernest, what were the happiest moments in your life, in science?” ELE: “One happy moment was when I arrived in this country, after waiting almost nine years. Another was when I finished my Ph.D. and got my first job. Another was when I got married. Another was when I was promoted to full professor at Notre Dame. Interestingly not when I got tenure, because I never doubted that would happen. But when I was promoted to full professor, I felt I was somehow part of the establishment. When I became a member of the National Academy and when I got my first honorary degree, at Duke in 1983 [Figure 7]. The Jefferson Award of UNC, for service to the University. Then, of course, when I got the Priestley Medal [Figure 8], when I was elected president of the ACS . . . ” (70).

Figure 7. After receipt of the D.Sc. degree at Duke University, May 8, 1983. Photograph courtesy Eliel family.

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Figure 8. The cover of Chemical & Engineering News on the occasion of his receipt of the Priestley Medal. Photograph courtesy of the American Chemical Society. Ernest’s was a life full of opportunities seized, adventures lived, achievements recorded, and awards received. He also faced and overcame life-threatening dangers and mountainous obstacles.

Ernest’s Interest in “Hidden Advisors” Ernest was fascinated by the hidden aspects of the sociology of science. Here are two instances separated by 30 years. First, on January 9, 1970, at the age of 48, Ernest wrote to the eminent R. B. Woodward and asked if Woodward’s close 35

friend and associate Derek Barton had served as Woodward’s “hidden coworker in some of the beautiful syntheses you have accomplished?” (71)See Figure 9.

Figure 9. In his January 9, 1970, letter to R. B. Woodward (71), Eliel asks if Woodward considered Barton to be his “hidden coworker” in some of his syntheses. Eliel refers to a “reprint,” almost certainly Eliel’s essay published in the November 7, 1969, issue of Science discussing Barton’s receipt of the 1969 Nobel Prize in Chemistry (71). In that essay, Eliel refers Barton’s “principle of conformational stereochemical control used by R. B. Woodward in his total synthesis of reserpine” (72). Letter courtesy of the Eliel family. Second, as noted at the top of this essay, shortly after Derek Barton’s death in 2000, Eliel wrote that Barton was his (Eliel’s) “hidden advisor” (1).

Ernest as My “Hidden Advisor” The invitation to contribute a lecture to the symposium honoring Ernest Eliel and to write a chapter based on that lecture came with a dual request from the symposium organizers and volume editors: that I describe Ernest’s scientific 36

achievements as well as share my own personal relationship with him. I know full well that Ernest would have been embarrassed by what I am now to write and you to read. But even if he were still alive – Oh! so much I wish! – I would write no differently, except perhaps that I would have more items to include in the following pieces of advice I received from Ernest. Here is my rather abbreviated story about my friend (Figure 10) and about Ernest Eliel as My “Hidden Advisor.”

Figure 10. Ernest and the author at a Virginia plantation just east of Richmond, Virginia, ca. 1996. Photograph courtesy of J. I. Seeman. I got to know Ernest during the research for and writing of my 1983 review article entitled Effect of Conformational Change on Reactivity in Organic Chemistry. Evaluations, Applications, and Extensions of Curtin-Hammett/Winstein-Holness Kinetics in the early 1980s. Inexplicably missing from that title are the words “and a Historical Overview,” for indeed, the last section of that review is a 12-page historical perspective on the development of the Curtin-Hammett principle and the Winstein-Holness (Eliel-Ro) equation. Ernest not only participated in providing information and quotes about his own participation in the development of conformational analysis, but it was he who counseled me on where my review article should be published and it was he who introduced me to the then editor of Chemical Reviews, Anthony Trozzolo. The publication of my article (37) in Chemical Reviews was a milestone in many ways and a turning point for me and my career. First, Chemical Reviews had 37

not previously included a historical section in their articles, but editor Anthony Trozollo showed no hesitancy – quite the reverse. I acknowledge Tony and Joe Bunnett, who first initiated that article, with much gratitude. Second, over the course of the following years and even up to 2016, folks have commented to me on that article. Less frequently, they have complimented me on the detailed kinetic analyses: it is the history section that they mostly extoll. That publication and those compliments led me to my career as a historian of chemistry. I recognized that within the community of chemists, there was an eager audience for the history of their science and for a discussion of the human side of chemistry. I was eager to contribute to that knowledge and that discussion. At about the same time, I engaged Ernest in a conversation about my future, that is, my future as an academic. At the time, I had been in industry for a decade and was eager, or so I thought, to move into academia. The very same Trozzolo had moved from industry, Bell Telephone Laboratories, to Notre Dame just a few years after Ernest had moved from Notre Dame to the University of North Carolina. Ernest advised that I should consider writing a book, for it was his 1962 book Stereochemistry of Organic Compounds which, Ernest thought, had really brought him enormous recognition (Figure 11). That suggestion immediately connected with another project I was considering.

Figure 11. Eliel holding what is likely to have been the manuscript of his 1962 book Stereochemistry of Carbon Compounds (6) at the Notre Dame post office, 1960. Photograph courtesy of the Eliel family.

38

In my early 1980s fantasy of being an academic, I had envisioned teaching a course that focused on the achievements of a handful of renowned chemists. My idea was to select one key paper for every decade of each chemist’s career, discuss how each paper was important at the time of its publication and how each paper was important even years later. I would then reveal how one paper led to the next and so on, thereby illustrating the development of each chemist’s career as a whole. But the final coup would be that, when all the lessons were completed, the course would reveal the growth of organic chemistry as a field. Ernest’s suggestion in 1984 that I write a book was timely. I would write the story of 20 or 25 chemists for the course I had imagined. As this idea took root, I realized that it ought not be my task to identify the six or so seminal publications of these chemists. I would ask the chemists to select their own papers. It was just a short jump to realize that these should be autobiographical rather than biographical chapters. Eventually, the one volume coffee table book of autobiographical chapters became the 20-volume series of autobiographies called Profiles, Pathways and Dreams. One of the volumes is Ernest’s (6). Ernest’s next major “hidden advisory role” was caused by a most alarming and unanticipated crisis. In November or December 1989, Vladimir Prelog informed me that David Ginsburg’s translation of the manuscript for his (Prelog’s) Profiles autobiography was unacceptable. Ginsburg had suffered a stroke and thus was severely limited in his translational abilities. I asked Prelog to send me the translation and the original in German for Ernest’s review. Why Ernest? Because Ernest and Prelog were close friends. Because Ernest was fluent in German. Because Ernest was a physical organic chemist with a specialization in stereochemistry, as was Prelog. And because Ernest was familiar with the Profiles project, being an author himself. Ernest confirmed that the translation was lacking. But he steadfastly refused to re-translate Prelog’s manuscript, for he was too busy writing his own autobiography and he was in the midst of being Chair of the Board of Directors of the American Chemical Society. My gloom was only brief. Ernest immediately recommended another person to perform the translation, Otto Theodor Benfey (Figure 12). Ted Benfey is also fluent in German, for Ted, like Ernest, is a native German-speaker who had escaped Nazi Germany as a youth. Benfey is also a Ph.D. organic chemist – a student of Christopher Ingold of the Cahn-Ingold-Prelog (CIP) rule. And Benfey is a historian of chemistry, a former editor himself (of the ACS journal Chemistry), and a translator of scientific manuscripts from German (and French) to English. And, quite fortunately for Prelog, the readers of the Profiles volumes, and for me, Benfey immediately agreed to and did retranslate Prelog’s manuscript. Thus began my relationship and special friendship with Ted Benfey which continues happily to this day. Indeed, at the same ACS National Meeting which hosted the Eliel symposium, I organized a symposium held within the domain of the Division of History of Chemistry honoring the 90th birthday of Ted Benfey.

39

Figure 12. Ted Benfey, Greensboro, NC, 2015. Photograph courtesy of J. I. Seeman.

Next on my short list of memorable moments of Eliel’s “hidden advisorships” with me is his receipt of the Priestley Medal in 1996. Ernest was surely one of the most deserving of Priestley Medalists, having contributed to science (see above), to education (his many textbooks and his 10 articles in the Journal of Chemical Education), to professional societies (he was President and Chair of the Board of Directors of the American Chemical Society in addition to holding many other positions; he was also Vice Chair and then Chair of the Council of Scientific Society Presidents), and to the worldwide profession of chemistry (his many activities on behalf of chemists from undeveloped countries). Actually, it was not his receipt of the Priestley Medal that was a pedagogical moment for me. Rather, it was his request that I review his notes and drafts of his Priestley Medal address that demonstrated, in a very personal fashion, that no matter how prestigious one is, one can always learn and benefit from the knowledge and advice of others. Years later, when I was archiving Ernest’s files for the Chemical Heritage Foundation, I found the notes I had sent to him on that occasion and was reminded how helpful those ideas were to him. (I also remembered sitting at his table at the ACS Awards Banquet, with Eva and his daughters, Carol and Ruth ant their husbands. With us was another man – also an author in this volume – who considers Ernest his non-hidden advisor, Eusebio Juaristi [see Figures 1 – 2]). Ernest influenced me in many ways. I remember a phone call, one spring, when I inquired whether he had any students who might be interested in a summer position in my laboratory. After a pleasant chat, Ernest then read back a perfect announcement that he had written during our conversation. That is my model for efficiency. I remember many UNC-Duke University basketball games where this otherwise subdued Ernest turned into a loudly cheering basketball fan. 40

I remember attending a 65th birthday symposium held at UNC for Ernest. The after-dinner speaker, an eminent chemist from the mid-West, referred to Ernest as “Ernie.” My suspicions were well founded. When I asked Ernest about the name “Ernie,” he grimaced as if he had just been forced to drink the most ugly tasting cough medicine. I remember his tales of swimming 10 laps every afternoon but also enjoying, as a reward, a very sugary Danish – much to the chagrin of his wife, Eva. Indeed, this classically educated, classically-styled man could simultaneously be known as a “bulldog” by his professional colleagues yet meekened by Eva. Ernest had a lot of patience with me as an editor of his autobiography. But as the “bulldog” nickname suggests, he could easily and rapidly draw a line in the sand. He could be testy, too. As an editor, I had experienced others reacting negatively when I asked for more and more from them but not Ernest. While collecting “data” for my Chirality biography of Ernest (25), I asked more and more questions. Toward the end, Ernest wrote in an email, “This is not how we had agreed to play the game. You asked for ‘spontaneous answers’ [to your questions] and now you are asking for more on the same subjects! There seems to be a contradiction there! In any case, I cannot deal with a geometric progression of e-mails! However, I gather you will have a chance to follow up personally” (68). (Ernest was surely referring to his and Eva’s annual visit with me in Richmond, on their way north to the annual spring meeting of the National Academy of Sciences.) When I interviewed him for the Chirality paper, I asked, “Do you feel any letdown, no longer being ACS President or on the Board of Directors?” He answered, “No. I am reconciled to getting older and less involved (and I am still quite involved with ACS). It is only when I get mad at an action of the ACS Board that I wished I were still a member!” (73) This is a model for aging gracefully. Yes, I have many memories of Ernest L. Eliel. I think of him often, and in writing this essay, I am reminded of how much of him remains within me. Of course, role models are not reproductions but rather adoptions and incorporations of the best we see, as defined by its relevance to oneself. I wonder how many others consider Ernest their Hidden Advisor. I suspect many.

Our Last Time Together In June 2008 the Division of Organic Chemistry of the ACS held the 32nd Reaction Mechanism Conference at the University of North Carolina at Chapel Hill. Malcolm Forbes, one of the meeting’s organizers, asked if I would give a 41

biographical lecture about Ernest at a session honoring him. By then, Ernest was too ill to attend. I joined many of his former students at that somewhat somber event, Friday, June 27th. I stayed only one day. My talk was at 8:30 am, and I left shortly after lunch. I had been visiting Ernest frequently over those past several years, witnessing his steady decline caused by an undiagnosed neuromuscular disease related to Parkinson’s. Previously, I would visit Ernest and Eva at their home. This time, he was in the nursing home section of the senior living community where Eva and he had relocated from their comfortable Chapel Hill home several years earlier. I wrote the following mini-essay that evening, shortly after I arrived home in Richmond, a three hour drive from Chapel Hill. Once more, Ernest (December 28, 1921 – September 18, 2008) was a role model for me, demonstrating how to say goodbye in peace and with friendship. He died shortly thereafter.

A Parting I hold on to things. Like friends. And, as a matter of principle, I don’t like partings. I much prefer shalom. “Shalom” means hello, goodbye, peace and harmony, all rolled up into a package of six letters. Well, six letters in English. I guess there are times when friendships . . . acquaintances . . . simply and slowly disappear. In those instances, they are often without even a goodbye. The last time was not anticipated to be the last time, it just was. Some time later, we recognize it for what it was, for what it is. And so, this brings me back to today, this afternoon, just a while ago and 200 miles away. I was sitting with Ernest and Eva, friends for 30 years. Ernest is a special man and a special scientist. Ernest has been a special friend. He and I have been many places together. We have been serious together. We have had fun together. We have shared meals all around the country. I can hardly remember all the places. Together, we saw quite a number of UNC-Duke basketball games, rising in unison at the climax of many a UNC near-halftime resurrection. This, from a classic German scholar who really wasn’t all that interested in basketball! He and Eva stayed with me in Richmond many times, and of course, I stayed with them in Chapel Hill. Good friends. Ernest also has had a major effect on my professional career as he has on the careers of my others. I can tell you more about that, but for now, just believe me. And I mean major. Today, I thanked him for so much. I asked him, had I thanked him before. We were both certain that I had. But I just wanted to cover the territory again. Compulsively, just in case. Actually, that wasn’t really the reason. There was just so little more to say. I wanted to stay longer. I wanted to leave immediately. I didn’t know what I wanted. So, for about 30 minutes, I tried to leave. Eva kept on interrupting my goodbye with one seemingly irrelevant story or another, as if she were trying to keep me there. 42

Finally, I was up and gave him a hug. I walked around to his other side, around his bed, and gave him another hug. The light just did not shine in his eyes anymore. They did, the last time I was with him, just a few weeks earlier. Not this visit, not once. He said, “It is a parting.” I leaned over, “What did you say?” knowing exactly what he had said. “A parting,” he repeated. I wept as I left.

Acknowledgments The author thanks the organizers of the Eliel symposium (Cynthia Maryanoff and Diane Grob Schmidt) and the editors of this volume (Maryanoff and Grob Schmidt along with H. N. Cheng and Bradley D. Miller) for their kindness in inviting me to participate in these events. The author also thanks Cheng and two reviewers for their help in preparing this chapter for publication and the photographers/owners of the various photographs reproduced and cited herein for their permission to include their photographs. Finally, the author salutes the lives and accomplishments of Ernest and Eva Eliel and thanks their daughters Carol and Ruth for their encouragement and cooperation.

Dedication This chapter is dedicated to Joseph Gal, an expert in the areas of stereochemistry, chirality, and history of chemistry as was Ernest Eliel, on the occasion of Gal’s 75th birthday.

References 1. 2.

3. 4. 5. 6. 7. 8.

Eliel, E. L. Derek Barton as ‘Hidden Advisor’. In The Bartonian Legacy; Scott, A. I., Potier, P., Eds.; Imperial College Press: London, 2000; pp 16−22. Stereochemistry and Global Connectivity: The Legacy of Ernest Eliel; Cheng, H. N., Maryanoff, C. A., Miller, B. D., Schmidt, D. G., Eds.; ACS Symposium Series; American Chemical Society: Washington, DC, 2017. Seeman, J. I. A Debt Repaid. Ernest L. Eliel’s Life Made Possible by Five Years in Latin America. J. Mex. Chem. Soc. 2009, 53, 78–92. Eliel, E. L. ACS Comment. Transnational Aspects of Chemistry. Chem. Eng. News 1992, 70, 31–34. Eliel, E. L. From Cologne to Chapel Hill. In Profiles, Pathways and Dreams; Seeman, J. I., Ed.; American Chemical Society: Washington, DC, 1990. Eliel, E. L. Stereochemistry of Carbon Compounds; McGraw-Hill: New York, 1962. Eliel, E. L.; Wilen, S. H. Stereochemistry of Organic Compounds; John Wiley & Sons: New York, 1994. Eliel, E. L.; Wilen, S. H.; Doyle, M. P. Basic Organic Stereochemistry; John Wiley & Sons: New York, 2002. 43

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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29.

Eliel, E. L.; Allinger, N. L.; Angyal, S. J.; Morrison, G. A. Conformational Analysis; John Wiley & Sons: New York, 1967. Eliel, E. L. Science & Serendipity. The Importance of Basic Research; American Chemical Society: Washington, DC, 1992. Eliel, E. L. Official Statements by Election Candidates. Chem. Eng. News 1986, 64, 26–47. Eliel, E. L. Chromatographic Adsorption. J. Chem. Educ. 1944, 21, 583–588. Eliel, E. L.; Prosser, T. J.; Young, G. W. Use of Mass Spectrometry in Organic Analysis. J. Chem. Educ. 1957, 34, 727–727. Eliel, E. L. Conformational Analysis in Mobile Systems. J. Chem. Educ. 1960, 37, 126–133. Eliel, E. L. Teaching Organic Stereochemistry. J. Chem. Educ. 1964, 41, 73–76. Eliel, E. L. Recent Advances in Stereochemical Nomenclature. J. Chem. Educ. 1971, 48, 163–167. Eliel, E. L. Conformational Analysis - The Last 25 Years. J. Chem. Educ. 1975, 52, 762–767. Eliel, E. L. Stereochemical Non-Equivalence of Ligands and Faces (Heterotopicity). J. Chem. Educ. 1980, 57, 52–55. Eliel, E. L. The R/S System: A Method for Assignment and Some Recent Modifications. J. Chem. Educ. 1985, 62, 223–224. Gallego, M. T.; Brunet, E.; Garcia Ruano, L. L.; Eliel, E. L. Diastereospecific Synthesis of cis- and trans-2,3-Dimethyl-1,4-Thiamorpholines: An Advanced Organic Synthesis and NMR Project. J. Chem. Educ. 1991, 68, 517–520. Eliel, E. L.; Engelsman, J. J. The Heats of Combustion of Gaseous Cyclotetradecane and trans-Stilbene - A Tale of Long-Standing Confusion. J. Chem. Educ. 1996, 73, 903–905. Hargittai, I. Ernest L. Eliel. Interview. Chem. Intell. 1998, 4, 4–11. Baum, R. M. Ernest L. Eliel to Receive 1996 Priestley Medal. Chem. Eng. News 1995, 73, 37–39. Horiszny, J. The Importance of Being Ernest. Notre Dame Magazine 1972 (April), 41–45. Seeman, J. I. Ernest L. Eliel: A Life of Purpose, Determination, and Integrity. Chirality 2002, 14, 98–109. Seeman, J. I. Ernest L. Eliel, 1921-2008; National Academy of Sciences: Washington, DC, 2014; p 1−31. Eliel, E. L. The Reduction of Optically Active Phenylmethylcarbinyl Chloride with Lithium Aluminum Deuteride. J. Am. Chem. Soc. 1949, 71, 3970–3972. Eisenbaumer, R. L.; Moser, H. S. Enantiomerically Pure (R)-(+)-2Phenylethanol-2-d and -1,1,2-d3, and (S)-(+)-1-Phenylethane-1-d, -1,2-d2, -1,2,2-d3, and -1,2,2,2-d4. J. Org. Chem. 1979, 44, 600–604. van Tamelen, E. E.; Shamma, M.; Burgstahler, A. W.; Wolinsky, J.; Tamm, R.; Aldrich, P. E. Total Synthesis of Yohimbine. J. Am. Chem. Soc. 1969, 91, 7315–7333. 44

30. van Tamelen, E.; Shamma, M.; Burgstahler, A.; Wolinsky, J.; Tamm, R.; Aldrich, P. The Total Synthesis of Yohimbine. J. Am. Chem. Soc. 1958, 80, 5006–5007. 31. Roberts, J. D.; Sauer, C. W. Small-Ring Compounds. III. Synthesis of Cyclobutanone, Cyclobutanol, Cyclobutene and Cyclobutane. J. Am. Chem. Soc. 1949, 71, 3925–3929. 32. Cram, D. J. Studies in Stereochemistry. I. The Stereospecific Wagner-Meerwein Rearrangement of the Isomers of 3-Phenyl-2-Butanol. J. Am. Chem. Soc. 1949, 71, 3863–3870. 33. Djerassi, C.; Scholz, C. R. The Preparation of Two Aromatic Analogs of Desoxycorticosterone Acetate. J. Am. Chem. Soc. 1949, 71, 3962–3966. 34. Marker, R. E. Steroidal Sapogenins. 174. 17-Hydroxy-20-Ketopregnanes from Steroidal Sapogenins. J. Am. Chem. Soc. 1949, 71, 4149–4151. 35. Barton, D. H. R. The Conformation of the Steroid Nucelus. Experientia 1950, VI, 316–320. 36. Read, J.; Grubb, W. J. Researches in the Menthone Series. Part XIII. Relative Molecular Configurations of the Menthols and Menthylamines. J. Chem. Soc. 1934, 1779–1783. 37. Seeman, J. I. Effect of Conformational Change on Reactivity in Organic Chemistry. Evaluations, Applications, and Extensions of Curtin-Hammett/ Winstein-Holness Kinetics. Chem. Rev. 1983, 83, 83–134. 38. Eliel, E. L. The Origin of Steric Hindrance in Cyclohexane Derivatives. Experientia 1953, 9, 91–93. 39. Pope, W. J.; Read, J. LXXXIII. -- The Optical Activity of Compounds of Simple Molecular Constitution. Ammonium D- and L-Chloroidomethanesulphonates. J. Chem. Soc. 1914, 811–821. 40. Perkin, W. H.; Pope, W. J.; Wallach, O. Optically Active Substances Containing No Asymmetric Atom. 1-Methylcyclohexylidene-4-acetic Acid. J. Chem. Soc. 1909, 1789–1802. 41. Merton, R. K. Singletons and Multiples in Scientific Discovery: A Chapter in the Sociology of Science. Proc. Am. Philos. Soc. 1961, 105, 470–486. 42. Hirst, E. L. John Read. 1884-1963. Biogr. Mem. Fellows R. Soc. 1963, 9, 236–260. 43. Read, J. Science, Literature, and Human Thought. J. Chem. Educ. 1960, 37, 110–117. 44. Seeman, J. I.; Farone, W. A. Analytical Solution to the Curtin-Hammett/ Winstein-Holness Kinetic System. J. Org. Chem. 1978, 43, 1854–1864. 45. Seeman, J. I.; Secor, H. V.; Hartung, H.; Galzerano, R. Steric Effects in Conformationally Mobile Systems. The Iodomethylation of 1-Methyl2-Arylpyrrolidines Related to Nicotine. J. Am. Chem. Soc. 1980, 102, 7741–7747. 46. Eliel, E. L.; Ro, R. S. Conformational Effects in SN2 Reactions. Chem. Ind. (London) 1956, 251–252. 47. Winstein, S.; Holness, N. J. Neighboring Carbon and Hydrogen. XIX. tButylcyclohexyl Derivatives. Quantitative Conformational Analysis. J. Am. Chem. Soc. 1955, 77, 5562–5578. 45

48. Kwart, H.; Takeshita, T. Evaluation of the Relative Importance of ChargeDipole Interactions and Steric Strain Acceleration in Conformationally Mobile Systems. J. Am. Chem. Soc. 1964, 86, 1161–1166. 49. Mateos, J. L.; Perez, C.; Kwart, H. Direct Evidence of Limitations in the Applicability of the Kinetic Method of Conformational Analysis. J. Chem. Soc., Chem. Commun. 1967, 125–127. 50. Eliel, E. L.; Biros, F. J. Conformational Analysis. XII. Acetylation Rates of Substituted Cyclohexanols. The Kinetic Method of Conformational Analysis. J. Am. Chem. Soc. 1966, 88, 3334–3343. 51. McKenna, J. Conformational Analysis by Kinetic Methods: A Critique: Theory and Experimental Development of Procedures Based on Very Fast Chemical Reactions. Tetrahedron 1974, 30, 1555–1562. 52. Eliel, E. L.; Ro, R. S. Conformational Analysis. III. Epimerization Equilibriua of Alkylcyclohexanols. J. Am. Chem. Soc. 1957, 79, 5992–5994. 53. Eliel, E. L. Conformational Equilibria by Nuclear Magnetic Resonance Spectroscopy. Chem. Ind. (London) 1959, 568. 54. Eliel, E. L. This Week’s Citation Classic: Eliel, E. L. Conformational Equilibria by Nuclear Magnetic Resonance Spectroscopy. Chem. Ind. 1959 (18), 568. Current Contents 1982 (October 24), 22. 55. Willy, W. E.; Binsch, G.; Eliel, E. L. Conformational Analysis. XXIII. 1,3Dioxolanes. J. Am. Chem. Soc. 1970, 92, 5394–5402. 56. Eliel, E. L.; Hutchins, R. O. Conformational Analysis. XVIII. 1,3-Dithianes. Conformational Preferences of Alkyl Substituents and the Chair-Boat Energy Difference. J. Am. Chem. Soc. 1969, 91, 2703–2715. 57. Eliel, E. L.; Giza, C. A. Conformational Analysis. XVII. 2-Alkoxy- and 2Alkylthiotetrahydropyrans and 2-Alkoxy-1,3-Dioxanes. Anomeric Effect. J. Org. Chem. 1968, 33, 3754–3758. 58. Eliel, E. L.; Knoeber, M. C., Sr. The “Size” of a Lone Pair of Electrons. Evidence for an Axial t-Butyl Group. J. Am. Chem. Soc. 1966, 88, 5347–5349. 59. Eliel, E. L.; Knoeber, M. C., Sr. Conformational Analysis. XVI. 1,3-Dioxanes. J. Am. Chem. Soc. 1968, 90, 3444–3458. 60. Eliel, E. L.; Nader, F. W. Conformational Analysis. XX. The Stereochemistry of Reaction of Grignard Reagents with Ortho Esters. Synthesis of 1,3-dioxanes with Axial Substituents at C-2. J. Am. Chem. Soc. 1970, 92, 584–590. 61. Eliel, E. L.; Nader, F. W. Stereochemistry of the Reaction of Grignard Reagents with Ortho Esters. A Case of Orbital Overlap Control Synthesis of Unstable Polyalkyl-1,3-Dioxanes. J. Am. Chem. Soc. 1969, 91, 536–538. 62. Hartmann, A. A.; Eliel, E. L. Protonation and Methylation of Conformationally Fixed 2-Lithio-1,3-dithianes. Some Reactions of Remarkable Stereoselectivity. J. Am. Chem. Soc. 1971, 93, 2572–2573. 63. Eliel, E. L.; Hartmann, A. A.; Abatjoglou, A. G. Organosulfur Chemistry. Ii. Highly Stereoselective Reactions of 1,3-Dithianes. “Contrathermodynamic” Formation of Unstable Diastereoisomers. J. Am. Chem. Soc. 1974, 96, 1807–1816. 46

64. Lynch, J. E.; Eliel, E. L. Asymmetric Syntheses Based on 1,3-Oxathianes. 2. Synthesis of Tertiary a-Hydroxy Aldehydes, a-Hydroxy Acids, Glycols (RR′C(OH)CH2OH) and Carbinols (RR′C(OH)CH3) in High Yield. J. Am. Chem. Soc. 1984, 106, 2943–2948. 65. Eliel, E. L.; Lynch, J. E.; Kume, F.; Frye, S. V. Chiral 1,3-Oxathiane from (+)Pulegone: Hexahydro-4,4,7-Trimethyl-4H-1,3-Benzoxathiin. Org. Synth. 1987, 65, 215–219. 66. Eliel, E. L.; Lynch, J. E.; Kume, F.; Frye, S. V. Chiral 1,3-Oxathiane from (+)Pulegone: Hexahydro-4,4,7-Trimethyl-4H-1,3-Benzoxathiin. Org. Synth. 1993, 8, 302–306. 67. Frye, S. V.; Eliel, E. L. Nonenzymatic Synthesis of (R)-(-)- and (S)-(+)-Mevalolactone in High Enantiomeric Purity. J. Org. Chem. 1985, 50, 3402–3404. 68. Eliel, E. L. Email to Seeman, J. I., Chapel Hill, NC, January 15, 2001. 69. Berova, N.; Bailey, W. F. Editor’;s Note: Special Issue Honoring Professor Ernest Eliel. Chirality 2002, 14, 97. 70. Eliel, E. L. Interview with Seeman, J. I., Richmond, VA, 2001. 71. Eliel, E. L. Letter to Woodward, R. B., Notre Dame, IN, January 9, 1970. 72. Klein, L. R.; Eliel, E. L.; Goldberger, M. L. Nobel Laureates in Economics, Chemistry, and Physics. Science 1969, 166, 715–722. 73. Eliel, E. L. Email to Seeman, J. I., Chapel Hill, NC, January 18, 2001.

47

Chapter 3

Science and Knowledge in the Service of Humanity: The Example of Ernest Eliel Luis A. Montero Cabrera* Facultad de Química, Universidad de La Habana, 10400 Havana, Cuba *E-mail: [email protected]

A description of the efforts of Prof. Ernest Eliel to communicate with Cuba and the Cuban Society of Chemistry and the subsequent developments is described. Despite political difficulties, these communications resulted in mutually beneficial scientific interactions. Prof. Eliel provided a good example of scientific statesmanship that placed human and scientific values above social, political and economic differences. These efforts contributed to the excellent and promising inter-society relationship existing nowadays.

In 1993, I had the privilege of holding a couple of long-distance telephone conversations in Spanish from Madrid with a prominent American scientist. The contact was made because the scientist had made known to his biographers and in a press interview that he got his chemistry degree from the University of Havana. The information was unexpected for me and actually astonished me because science was not in good shape in Cuba (1) before 1959. That scientist was at that time in a senior management position at the well-known and (for me) much admired American Chemical Society (ACS), one of largest professional organizations in the world. The phone partner was Prof. Ernest Eliel, a well-known organic chemist and guru of organic stereochemistry. Much of what is being exploited today of biologically active molecules could be understood from the early teachings of Prof. Eliel.

© 2017 American Chemical Society

The background in which this conversation took place was singular from the current perspective in the 21st century. For a chemist from Cuba where political reports and news media in the past 30 years portrayed everything from the U.S. as suspect and even hostile, the conversation could be considered particularly “risky.” However, it was a visionary dialogue between a representative of the scientific community of the U.S., the most powerful country in the world, and a poor Cuban scientist enjoying a sabbatical year in Spain at a time when everything in Cuba was precarious because of the deep economic crisis in Cuba in the 1990’s. At the time the support of the socio-polical process that underlay Cuban science was widely questioned. The U.S. was for many the “winner” of something that future generations will likely see as a big waste in history: the so-called “cold war.” It had divided the world into blocks where each party saw the same reality in a very different way according to their political philosophy and national interests. In those post-Cold War years, our country was trying to survive after the ideological and economic disaster of losing the vast majority of its external ties and references. The same people from Eastern Europe who a few years before showed themselves to be in solidarity with us according the “proletarian internationalism” suddenly changed their faces and told us that this was of no use. Suddenly we became a “rara avis” on the American continent. Many people bet that the only thing that could make progress was the “law of the jungle” in both the economic and the social spheres. Some looked at us with pity, others with the insane joy of those who contemplated the killing of the hated. Fortunately, many still treated us with respect. Even today I did not know Prof. Eliel’s political inclinations. I do not talk about political philosophy with most of my friends and collaborators in Cuba and abroad. They know mine through references and (I guess) some by pure intuition. Scientists who wish to work together for the benefit of knowledge and humanity avoid potentially divisive conversations on political and religious topics. In so doing, we avoid disagreements that can affect that common cause. The 1993 conversation with Prof. Eliel had been facilitated by another friend from the U.S. who is a scientist well known for the originality of his contributions: Prof. Roald Hoffmann, also a good friend of Cuba. At that time I was a vice president of the Cuban Society of Chemistry (SCQ). Unfortunately, SCQ was then essentially paralyzed in its operation and only existed in name. The direct and indirect results of these constructive telephone conversations can be felt today. We hope they will project into the future with the strength that is reinforced by the natural friendship and mutual benefits of the Cuban and U.S. chemical communities. In 1993 for the first time, a communication was established. From my part, I gave up instinctively many prejudices regarding the “dialogue with the enemy.” Everything indicated that the intention of Prof. Eliel was humane and altruistic. He wanted to embrace a chemical community that was geographically and historically close to the U.S., and to which he was sentimentally attached. It should be noted that these efforts would not improve his career; they were motivated only by a greatness of soul. Subsequently and during the rest of his life, Prof. Eliel and I, together with many colleagues in Cuba, sponsored a number of successful activities, some of them conducted during periods of worsening hostility between the U.S. and Cuba’s 50

governments. Other well-known Cuban chemists also established professional relations with him, and the ACS at his request invited a relatively large delegation of us to the Congress of Chemical Societies of North America in Cancun, Mexico, in 1997. Numerous delegations of North American chemists also participated in the activities of the SCQ in our country beginning in 2000, when SCQ was revived. Yet, Eliel’s efforts to place Cuba on the world scientific scene had not begun with the 1993 conversation mentioned earlier and the subsequent activities. Already in 1944, as an undergraduate student from the University of Havana, Eliel had published the first Cuban article in an ACS journal (2). It was probably also the first Cuban article in an international scientific journal during the 20th century. It may be interesting to know that, according to the convention of Cuban higher education before the Reformation of 1962 (3), graduates in basic sciences received the title of “Doctor” even though the training received was essentially comparable to the B.S. at the United States. Probably Ernest Eliel graduated with something like “Doctor of Physical and Chemical Sciences.” A very high proportion of these graduates only found employment in high school education in Cuba at that time. That degree became an inconvenience for Eliel when he decided to get his PhD in the U.S. As Eliel mentioned somewhere, his university had already consecrated him as a “Doctor”, so why did he need to study towards a PhD? Certainly his achievements in Cuba both scientifically and professionally deserved to be recognized. During the afternoon on October 20, 2004, a very remarkable event occurred in the historic “Aula Magna” of the University of Havana. At the request of the Cuban chemical community, particularly by the Faculty of Chemistry, Ernest Eliel’s Alma Mater awarded him a “Doctor Honoris Causa.” It was then a very rare opportunity in which the flags of the United States and Cuba were displayed together in a public and solemn act in Havana. Prof. Eliel gave a very moving speech (Figure 1). It was a tribute to knowledge and goodwill among our peoples, as it should be. It is worth mentioning that this occurred during a renewed hostile political environment between the two countries. The U.S. administration at the time was not friendly to Cuba and ignored or despised Cubans who live in Cuba. That year they issued an official document (4) that contained the following paragraph: “Large sums [of money] have also been directed to activities such as biotechnology centers and biosciences not appropriate in magnitude and expenditures for a fundamentally poor nation, and they have not been justified financially. In fact, this sector continues to receive strong investments independently of the cuts in other sectors of the economy, one of which is that of basic infrastructures. Investments in the biotechnology sector have not resulted in a basic internal capital flow, and have led to questions about the type of activities being undertaken.” The content of the above paragraph merits reflection, but what I have pointed out in italics is particularly striking. It declared that high-level science is not for us, that it is not for those “fundamentally poor” to do it. And that happened in the same year in which we honored in Cuba a successful common son for his scientific achievements and friendship. 51

Figure 1. Prof. Eliel speaking at the Univ. of Havana on the occasion of the “Doctor Honoris Causa” Award. (courtesy of the Eliel family). Fortunately, the great northern nation is also rich in people of good will. Science and knowledge are vehicles of friendship and humanity. What has happened in 2015 and 2016 in terms of rapprochement between the Cuban and American chemical communities is worthy of a phrase from President Obama: “We are all Americans.” America, with poorer and richer nations, from North to South, contains plenty of potential to provide all its peoples with a decent and respectable life and welfare. The actions of ACS and media have been influential in publicizing SCQ and Cuban chemistry in the northern country. A series of “Eliel Symposia” have been programmed in both countries where the main objective is scientific communication and educational exchange. A detailed account of these symposia would be outside the scope of this article. Suffice it to say that our mutual interest in diversity and inclusivity, the establishment of personal relationships, friendship and mutual help should serve as good foundations for future relations between Cuban and the U.S. scientific societies. Prof. Eliel and his efforts represented the 52

best manifestation of humanity and left a great legacy for people in both countries in the future. The joint Cuban-U.S. activities will be a result of the actions he undertook when very few predicted that those could happen in this way. Honor to whom honor deserves: Let’s toast to the memory of Prof. Ernest Eliel and his Cuban initiative!

References 1.

2. 3. 4.

Truslow, F. A.; Armstrong, W. J.; Benton, H. A.; Lorenzana, J. C.; Funkhouser, R. L.; Glaessner, P. J. W.; Godwin, F. W.; Lees, M. B.; Mather, W. B.; Pajunen, P.; Russell, E. W.; Shrewsbury, C. L.; Staley, E.; Swerling, B. C.; Wijdenes, S. H. J.; Williams, C.; Wood, N. H. Report on CUBA. Findings and Recommendations; International Bank for Reconstruction and Development: Washington, DC, 1951. Eliel, E. L. Chromatographic adsorption. J. Chem. Educ. 1944, 21 (12), 583–588. La Reforma de la Enseñanza Superior en Cuba; Consejo Superior de Universidades: La Habana, 1962, p 115. Powell, C. L. Commission for Assistance to a Free Cuba; U.S. State Department: Washington, DC, 2004.

53

Global Connectivity

Chapter 4

Educational Outreach Activities between the ACS Division of Chemical Education’s International Activities Committee and the Sociedad Cubana de Química Charles H. Atwood*,1 and Luis Alberto Montero-Cabrera*,2 1Department

of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112, United States 2Facultad de Química, Universidad de La Habana, Havana, 10400, Cuba *E-mail: [email protected]; [email protected]

Ernest L. Eliel, in his capacity as ACS president, initiated contacts with the Sociedad Cubana de Química in the early 1990’s and 2000’s. Using those initial contacts as a springboard, several ACS presidents, especially Marinda L. Wu and Diane Grob Schmidt, continued to improve relationships between the two societies. In 2015 Presidents Raúl Castro and Barack Obama started normalizing relations between the two countries, permitting a Cuban delegation to attend the 2015 Boston ACS meeting. At this meeting representatives from the International Activities Committee of the Division of Chemical Education met with the Cuban delegation to discuss possible collaborations. This led to the attendance of four ACS educators at the 2016 Simposio Internacional de Química in Cayo Santa Maria, Cuba as well as a forthcoming workshop at the University of Utah.

Introduction Professional chemists and their affiliated chemistry societies are used to communication, cooperation, and collaboration with fellow scientists in other countries. Even in cases where the nations are involved in long standing © 2017 American Chemical Society

disagreements, discussion and exchange are always better than distance and isolation. Regrettably, the Cuban and US chemical communities were isolated from each other for a long time because of a prolonged and frequently hostile political confrontation between the two countries. Furthermore, the few and feeble contact points that had been made had essentially no hope of continuation. The situation created barriers to knowledge exchange and human contact that few were interested in overcoming. Ernest L. Eliel, a graduate of the University of Havana and a former ACS president, was interested in bridging the divide between our two nations. In the early 1990’s he and Luis Alberto Montero-Cabrera, in their capacities as leaders of both the American (ACS) and Cuban (SCQ) Chemical Societies, began to discuss mutual chemical education interactions (1). Even though the relationship between the countries was contentious at times, some education exchanges occurred over the late 1990’s and into the early 2000’s. In acknowledgement of Dr. Eliel’s efforts, he was awarded a Dr. Honoris Causa in 2004 by the University of Havana. In August 2013 Professor Montero-Cabrera became president of the SCQ. Later that year at the Istanbul IUPAC meeting he contacted Dr. Marinda Wu, ACS president at the time, to renew educational contacts between the US and Cuba. Because of scheduling conflicts, Dr. Wu was unable to accept an invitation to visit Cuba. However, the momentum was beginning to build. In October 2014 at the Latin American Chemical Congress (CLAQ) meeting in Peru, Professor Daniel García, at that time a member of the SCQ board, contacted Dr. Bradley Miller, Director of the ACS Office of International Activities (OIA) regarding further collaborations. Dr. Miller relayed an invitation from the ACS for a Cuban delegation to attend the Boston ACS meeting in August 2015. Fortuitously, in December 2014 presidents Raúl Castro and Barack Obama announced the beginning of a relations normalization process between the two countries. At the Boston ACS meeting, four SCQ Board members met Dr. Diane Grob Schmidt, ACS President for 2015, with the intent to seize the initiative established by the Castro-Obama announcement. Included with the ACS announcement of the Cuban delegation’s presence at the meeting was an invitation for ACS Divisions to reach out to the Cuban chemists. Dr. Grob Schmidt and other ACS executives later visited Havana for the SCQ congress in October 2015. Several new and important proposals for further ACS-SCQ collaboration were made in this new era of improving relations between the two nations. One ACS group that heeded the call to reach out to the SCQ was the International Activities Committee (IAC) of the ACS Division of Chemical Education (DivCHED). The DivCHED IAC asked to meet with the Cuban delegation at the Boston ACS meeting. Committee members Resa Kelly, Conrad Bergo, and Lourdes Echegoyen met with the Cuban delegation to discuss areas of possible collaboration. Discussions revealed that the Cuban chemists were especially interested in improving Cuban high school teaching as well as understanding the impact chemical education research has had on university teaching in the US. Based on that information the DivCHED IAC established several goals to interact and collaborate with the SCQ. It was decided that the DivCHED IAC should send a delegation to Cuba to see how teaching there differs from teaching in the US. Plans were also made to hold a workshop in the US 58

for several Cuban chemists. Another goal was for DivCHED memebers to give professional presentations in Cuba.

Initial Efforts Soon after the Boston ACS meeting, ACS President Diane Grob Schmidt announced that the 2015-2016 ACS Innovative Projects Grants would entertain requests that emphasized ACS-SCQ interactions. Based on this information Resa Kelly, Charles Atwood, and Joel Harris, incoming chair of the ACS Analytical Division (ANYL), submitted Global Improvement Grants (GIG) and Innovative Project Grants (IPG) to the ACS seeking funds to initiate contacts between the ACS and SCQ educational and analytical chemistry communities. Both grants were subsequently funded, permitting Atwood and Kelly to travel to Cuba in June 2016 (GIG) and lay the groundwork for an ACS-SCQ workshop in Salt Lake City in April 2017. Discussions between Luis Alberto Montero-Cabrera and Charles Atwood ensued from January to April 2016 regarding possible dates for a DivCHED IAC visit to Cuba. In mid-April the SCQ issued an invitation for the DivCHED to send several educators to the 2016 Simposio Internacional Quimica in Cayo Santa Maria, Cuba, to be held from June 7th – 10th, 2016. It was also decided to make this an Ernest L. Eliel memorial event. Atwood then sought out several speakers for the symposium, finally deciding on Norbert Pienta, Resa Kelly, Thomas Bussey, and himself. In parallel, discussions between the SCQ, ACS ANYL, and DivCHED IAC took place to decide details on the Salt Lake City workshop. The workshop was planned for April 6th-8th, 2017 so that it will occur soon after the forthcoming ACS meeting in San Francisco, CA. Our logic was that some Cuban chemists could attend the ACS meeting and then fly to Salt Lake City. The University of Utah Chemistry Department was chosen for the venue. The SCQ agreed to fly eight to ten chemists from Havana to Miami, using IPG funds to get them from Miami to Salt Lake City, provide room and board, and pay for any US workshop participants to attend. It was also decided that several aspects of analytical chemistry as well as an overview of US chemical education activities would be the main topics of the workshop. Another area of emphasis will be high school education, as this is especially important to the SCQ.

2016 Simposio Internacional de Química The symposium (2) began with a plenary lecture from SCQ President Montero-Cabrera on “Science, Technology and Innovation in Cuba: the Current Situation and a Desirable Evolution.” He drew a depiction of the current state of Cuban basic sciences, the problems and perspectives. That was followed by Pienta, Editor of the Journal of Chemical Education, who gave a plenary lecture on “Chemistry Education: the Roles of Evidence-based Research and the Journal of Chemical Education.” Afterwards, symposium participants went to a smaller 59

venue to start the Eliel portion of the symposium. Speakers and presentation titles are given below in Table 1.

Table 1. Program for the Ernest Eliel Session at the 2016 Simposio Internacional de Química Presentation

Speaker

Affiliation

Leslie YáñezGonzalez

University of Havana

Homage to Ernest Eliel

Charles Atwood

University of Utah

Outreach Opportunities to the Cuban Chemical Society from the International Activities Committee of the American Chemical Society’s Division of Chemical Education

Margarita Villanueva

University of Havana

The Transition from Secondary School to University: Difficulties and Challenges

Resa Kelly

San Jose State University

The Most Important Aspects of Chemistry Teaching, Where There Could Be Collaboration Between Cuba and The US

Dolores Torres and Grecia Garcia

University of Havana

The Use of Information and Communication Technologies in Teaching Chemistry. Use and Abuse of the Same

Rebeca Vega and Oneyda Fernández

University of Havana

Motivation for Studying Chemistry

Thomas Bussey

University of California – San Diego

Examining Instructors’ and Students’ Perceptions of Active Learning

Hassan Martínez Hung

Eastern University, Cuba

Introducing Real Situations into Chemistry Teaching

Venera Jouraeva

Cazenovia College, USA

Adding Fun to Teaching Chemistry

Manuel Alvarez Prieto

University of Havana

Why the Inclusion of Metrology is Important to Chemistry and Laboratory Management in Teaching Undergraduate Analytical Chemistry

Javier Ernesto Vilasó Cadre

Eastern University, Cuba

Pedagogical Design for Integrating Chemistry and Environmental Management Through Research and Action in Real Scenarios

Yolanda Zoe Rodríguez Rivero

Central University of Las Villas

Teaching General Chemistry Using Virtual Laboratories

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Interactions between educators from the two countries were extensive, collegial and meaningful. Of significant importance was the recognition that educational issues in both nations were very similar. Many plans for future interactions were discussed. On Friday, June 10th the four ACS representatives left Cayo Santamaria for a 250-mile drive to Havana in a 1954 Chevrolet Belair station wagon. That evening we had dinner with Luis Alberto Montero-Cabrera, Manuel Alvarez Prieto and their spouses in downtown Havana. More discussions ensued, resulting in a plan for a contingent of ACS folks to return to Havana in early 2017 prior to the April workshop.

Future ACS-SCQ Interactions In February 2017, Atwood, Kelly, and Harris journeyed to Havana with these specific goals in mind: 1) Interact with Cuban chemical educators and analytical chemists to understand the present situations in both countries. 2) View analytical and educational facilities at the University of Havana. 3) Interact with Havana high school teachers. 4) View the present high school facilities in Havana. Margarita Villanueve and Leslie Yáñez Gonzalez agreed to host our small contingent. Once we completed the Havana journey in February, Harris, Kelly, and Atwood (in conjunction with SCQ members) began planning for the April workshop. Some important discussion points for both nations included what modern instrumentation could benefit research, what educational issues overlap in both countries, how analytical chemistry education is the same and different in both nations, what collaborative research can be done in Cuba and the US, teaching laboratory instrumentation in both countries, how we can improve high school education in both nations, and what future interactions between the ACS and SCQ could advance our initial efforts?

Conclusion Chemistry is the central science linking scientific disciplines studied by citizens of both countries. Consequently, chemical education is essential for human development in both the United States and Cuba. As decades of estrangement have been rapidly forgotten by good will and friendship during the last few months of interactions, improved SCQ and ACS actions can prove valuable for the futures of both countries. Several common initiatives should be pursued. Some examples are periodic seminars, summer courses, visiting scholarships, projects for lab and equipment investment and sharing, plans to promote scientific interests among minorities and isolated communities are a few of the potential benefits of this improving mutual trust which Ernest Eliel initiated in the 1990’s. Both the SCQ and the ACS must continue their pursuit of the educational and scientific interests our two nations share.

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References 1.

2.

Montero-Cabrera, L. A. Science and Knowledge in the Service of Humanity: The Example of Ernest Eliel. Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1; ACS Symposium Series 1257; American Chemical Society: Washington, DC, 2017; Chapter 3 (this volume). 2016 Simposio Internacional de Química. http://siq.uclv.edu.cu/ (accessed January 29, 2017).

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Chapter 5

Ernest Eliel Workshop – US and Cuba Collaboration in Chemistry Education and Neglected Disease Drug Discovery W. L. Scott,*,1 J. G. Samaritoni,1 M. J. O’Donnell,1 A. B. Dounay,2 A. A. Fuller,3 P. S. Dave,1 J. M. Sanchez,1 D. G. Tiano,3 and D. G. Rivera4 1Department of Chemistry and Chemical Biology, Indiana University Purdue University Indianapolis, 402 N. Blackford Street, Indianapolis, Indiana 46202-3274, United States 2Department of Chemistry and Biochemistry, Colorado College, 14 East Cache La Poudre Street, Colorado Springs, Colorado 80903, United States 3Department of Chemistry & Biochemistry, Santa Clara University, 500 El Camino Real, Santa Clara, California 95053, United States 4University of Havana, Zapata y G, 10400, La Habana, Cuba *E-mail: [email protected]

This chapter describes the “Ernest Eliel Workshop – US and Cuba Collaboration in Chemistry Education and Neglected Disease Drug Discovery” carried out at the University of Havana in October 2016. Through lectures and laboratories Cuban students were educated in chemistry and biology while they applied their understanding to addressing a serious humanitarian challenge – discovering drugs for neglected diseases. The one-week workshop was conducted by both Cuban hosts and professors and students from three US institutions – Indiana University Purdue University Indianapolis (IUPUI), Santa Clara University and Colorado College. The workshop lectures ranged from macroscopic overviews of the drug discovery process, both scientific and economic, to more detailed presentations of the skills and scientific understanding required to carry out discovery research effectively. The lab portion utilized one of IUPUI’s Distributed Drug Discovery (D3) synthetic procedures. It was solid-phase based and enabled

© 2017 American Chemical Society

students to make 22 compounds which were then submitted for antimicrobial testing. As student education was taking place the Cuban-US collaborative nature of the workshop led to deeper scientific and cross-cultural understanding. These connections are certain to bear fruit in the scientific and educational work we are planning.

Introduction It is both an honor and pleasure to provide a chapter for this book commemorating Ernest Eliel. Whether we know it or not, all synthetic and computational chemists have been affected by his life and work. One of us (WS) remembers this personally: “My Ph.D. advisor, Professor David A. Evans was immersed, at the beginning of his career at UCLA, in the physical organic chemistry milieu of Saul Winstein and Don Cram. Evans introduced me to Eliel’s “Stereochemistry of Carbon Compounds” and with Eliel’s compendium of stereochemical insight and Evans’ mentoring I became 3-dimensional in how I understood and predicted a molecule’s chemical and biological properties.” Professor Eliel continues to impact my life. His personal connection to the University of Havana and his passion for education was honored at a symposium I attended on US-Cuba collaboration at the 2015 ACS National Meeting in Boston, and it was there that I met Professor Daniel Garcia Rivera from the University of Havana. As we talked it was immediately clear that our shared expertise in solid-phase synthesis of biomimetic molecules with drug discovery potential, coupled with a passion for education, could be fertile ground for a US-Cuba collaboration. That meeting with Daniel gave rise to a successful application to the ACS for the Global Innovation Grant that supported our “Ernest Eliel Workshop – US and Cuba Collaboration in Chemistry Education and Neglected Disease Drug Discovery”. In the fall of 2016 we carried out the one-week workshop at the University of Havana. This chapter describes the workshop, the expanding collaborations it catalyzed, and the future it promises. We again think of Professor Eliel - his scientific accomplishments, passion for education, and Cuban connection - and believe his spirit is still guiding and encouraging us.

Foundation for Cuban Workshop: Distributed Drug Discovery (D3) At the core of the workshop is the Distributed Drug Discovery (D3) program created at IUPUI in 2003 (1–6). D3 seeks to educate students in chemistry and biology while they apply their learned skills and understanding to helping solve a serious humanitarian challenge – discovering drugs for neglected diseases, which we define broadly as diseases that lack the financial incentives supporting a traditional drug discovery process. The distributed power of D3 is possible when the large problem of drug discovery is broken into smaller challenges implemented at multiple schools across the world. Students are educated in 64

chemistry and biology while simultaneously enlisting their distributed and combined efforts in the larger drug discovery goal. Key challenges for D3 are: •











Making available simple, inexpensive, powerful and reproducible chemistry laboratory procedures and equipment that allows distribution of D3 problem-solving syntheses to schools with limited resources. Enumerating large virtual catalogs of molecules readily accessible by D3 procedures and finding computational experts to help select molecules to make for a given disease target. Enlisting biological testing resources that are relevant to neglected diseases, either student-run or freely available, and providing relevant biological activity data. Implementing and coordinating the neglected disease drug discovery research component, both laboratory and theoretical, in a motivational educational framework. Managing all the components: tracking molecules made physically and computationally, arranging for their biological screening, and recording, analyzing and sharing data. Building and nourishing international collaborations and understanding among schools, researchers, educators and students.

D3 has already made significant progress addressing these challenges. The particular disease target for this Cuban workshop was drug resistant microbes. In this context we sought to extend the scope of D3, learn more about its current strengths and limitations, and use this understanding to improve future workshops and the global coordination and implementation of D3. In the process we hoped to build scientific and personal connections between Cuban and US chemists.

Workshop Overview IUPUI has conducted workshops based on D3 at multiple sites in the US (7–9) as well as in Russia, Poland and Spain (10). These experiences prepared us for the Cuban workshop. Participants The “Ernest Eliel Workshop – US and Cuba Collaboration in Chemistry Education and Neglected Disease Drug Discovery” was conducted at the University of Havana the week of October 17-21, 2016 (11). US participants were Professors William Scott (WS) from Indiana University-Purdue University Indianapolis, Indiana (IUPUI); Amelia Fuller (AF) from Santa Clara University (Santa Clara, California); Amy Dounay (AD) from Colorado College (Colorado Springs, Colorado), and three undergraduates – Priya Dave (PD) and Juan Sanchez (JS) (both fluent in Spanish) from IUPUI, and Daniel Tiano (DT) from Santa Clara University. Cuban participants were host Professor Daniel Garcia 65

Rivera (DR) and 27 University of Havana students: 17 undergraduates, 6 masters, 3 Ph.Ds. and one postdoc (Figure 1).

Figure 1. Cuba Workshop Participants. (see color insert) Back in Indianapolis Drs. Marty O’Donnell (MO) and Geno Samaritoni (GS) provided essential educational and practical resources as they helped coordinate the workshop from a distance. Daily Program Each day the workshop began at 9 AM and ended by 5 PM. Over the course of the week there were 11 lectures by US professors, an active learning session, 4 presentations by Cuban students, a joint US/Cuban poster session and laboratory work which took place during a 2½ to 3 hr period each of three afternoons. There was time set aside for US and Cuban colleagues to discuss topics of mutual interest and propose future collaborations. Our days were quite busy, however it was not all work. Each day we socialized during a 2-hour mid-day break, at dinner, and after hours (Figure 2). These times provided a welcome and balanced opportunity to build on the trust and comradery generated during our intense shared scientific work and to learn about each other’s personal lives and culture in an unselfconscious and relaxed manner.

Lecture Component of the Workshop The workshop lectures ranged from macroscopic overviews of the drug discovery process, both scientific and economic, to more detailed presentations of the skills and scientific understanding required to effectively carry out drug discovery research. At the macroscopic level we sought to give students a balanced view of the strengths and limitations of drug discovery through pharmaceutical research. This industry has given us life-saving drugs for bacterial infections, diabetes, cancer, 66

HIV/AIDS and many other serious diseases. At the same time, drug discovery research for neglected diseases has languished because profit-driven research is not supported by small or poor patient populations.

Figure 2. Lunch Between Lectures and Lab. (see color insert)

We proposed the D3 concept and program as a way to address this economic limitation by having students participate in drug discovery (in this case antimicrobial agents) as they learn the fundamental scientific disciplines behind the process. From the general science level down to specific and applied detail they learn the scientific principles and procedures that enable them, in the lab portion of the workshop week, to immediately and meaningfully participate in drug discovery for a humanitarian cause.

Lectures at a Macro Level Lecture 1. “Short Course in Drug Discovery” (WS) In the first lecture WS covered general aspects of the drug discovery process. He provided an historical understanding of how drugs are discovered from natural products (modified or unmodified, often based on observations from indigenous scientists) to more contemporary computationally-driven drug discovery (Figure 3). Drugs were discussed whose origins could be traced back to a farmer’s observation of hemorrhaging cows (Warfarin as an anticoagulant) or a blood pressure lowering peptide in snake venom (Captopril to treat hypertension). At the other extreme, the discovery process for life saving HIV protease inhibitors was linked to a more intensive “rational” and computationally-based drug discovery approach. 67

Figure 3. Lecture Presentation (see color insert)

Lecture 2. “Neglected Tropical Disease Research at Colorado College: Design and Synthesis of New Drugs for African Sleeping Sickness” (AD) In the second lecture AD, who worked as a medicinal chemist at Pfizer prior to joining the faculty at Colorado College, gave an informed perspective on the challenges and economic realities of commercial drug discovery. She did this in an introduction to her talk, explaining how the high cost of commercial drug discovery causes many tropical diseases to be neglected by the pharmaceutical industry. As a potential solution to this problem AD described how her research students at Colorado College are addressing, in their hands-on research and as part of their educational training, this need for drugs to treat African sleeping sickness.

Lecture 3. The Distributed Drug Discovery (D3) Program (WS) WS gave a general lecture on the Distributed Drug Discovery (D3) program, which is at the heart of both the educational and lab portion of this workshop. D3 educates student scientists in drug discovery, applied synthesis and biological evaluation while connecting them to critical humanitarian drug discovery needs. As they learn theory and practice they become part of a large, internationally distributed research collaboration for the discovery of drugs to treat neglected diseases. D3 currently teams students at the Medical University of Lublin (Poland) and four sites in the United States: Colorado College, Goshen College, Santa Clara University, and IUPUI. With this workshop students and professors at the University of Havana (Cuba) became part of this international network. WS discussed three essential requirements for a D3 laboratory: 1) Powerful, reproducible synthetic procedures to enable the synthesis of large numbers of potential drug molecules. 2) Simple, inexpensive equipment to carry them out. 3) Large D3 virtual catalogs of combinatorially enumerated molecules accessible by these student-run D3 procedures. 68

All the current D3 procedures are carried out by solid-phase combinatorial chemistry using fundamental reactions developed in solution (12, 13) and on solid-phase (3–6, 14–32). The solid-phase nature enables multi-step syntheses to be successfully carried out with simple equipment on a micromole scale. The student or student teams can then select target molecules from these catalogs and synthesize, using the D3 procedures and equipment, 6 unique molecules at a time. In this fashion students distributed globally can synthesize large numbers of new molecules for evaluation against neglected disease targets. WS discussed each of these requirements in more detail.

1. Powerful, Reproducible D3 Synthetic Procedures Nine D3 protocols are now in place (Scheme 1). D3 Labs 1 (4), 3 (5) and 9 (33) have been published.

Scheme 1. Nine D3 Protocols. 69

2. Simple, Inexpensive Equipment To Carry Out the Syntheses Students execute all D3 procedures using the simple Bill-Board equipment (34) shown in Figure 4. This equipment facilitates carrying out six simultaneous, separate solid-phase reaction sequences in a combinatorial grid.

Figure 4. Bill-Board Equipment with Vessels, Drain Tray and Collection Rack. With the US-Cuba embargo still in place, it wasn’t simple to make sure the necessary equipment and reagents were in place for the start of the workshop. First we made sure it was legal to transport to Cuba the Bill-Boards which would be our essential synthesis equipment. Fortunately, recent modifications to the US Export Administration Regulations had a license exemption category for certain items supporting the Cuban people entitled “License Exception Support for the Cuban People (SCP)”. Our Bill-Board equipment, which was purchased from personal funds and donated to Professor Garcia Rivera for his students’ use, fell into that category. There were no readily identified commercial services to ship them. So we carefully documented the Bill-Boards’ purpose and source, packed them in our luggage, and took them with us as we traveled - Greyhound bus to Chicago, plane to Miami, charter to Havana. As for needed reagents, DR arranged for those not already available in Cuba to be shipped from Germany, where he was doing summer research.

3. Large D3 Virtual Catalogs From available reagents and any of the nine combinatorial solid-phase procedures listed in Scheme 1, large virtual catalogs can be enumerated. For D3 Lab 1 we constructed a 24,416 member virtual catalog of molecules realistically accessible by student synthesis. We are in the process of enumerating virtual D3 catalogs based on all the other procedures. These catalogs can serve as raw material for computational chemists to select molecules for students to make as potential drug candidates for neglected diseases. While the generic structures 1 and 9, from D3 Lab 1 and 9 respectively (Scheme 1), may appear quite uninteresting, compounds 1a and 1b (Figure 5) show they can be quite complex, and compounds 1c and 1d, 9a and 9b, confirm that molecules present in virtual catalogs can be reproducibly made by students using these procedures in the US, Poland, Russia and Spain (D3 Lab 1) (4) or at IUPUI and Santa Clara University (D3 Lab 9). 70

Figure 5. Representative Molecules, from D3 Labs 1 and 9, Replicated by Separate Students.

Lecture 4. “Synthesis and Study of Peptoids and Related Peptidomimetics” (AF) AF was D3’s first external collaborator. In this lecture she relayed to our Cuban colleagues her extensive experience in the peptoid field, convincing workshop participants that peptoids, N-substituted glycine oligomers, offer an exciting scaffold to adapt to D3 (35). Peptoids are commonly prepared on solid support, and readily accessible primary amines introduce variable functionality (36). AF highlighted this robust and efficient synthesis, along with applications of peptoids from her own lab and from the work of others. She detailed use of peptoids as mimics of bioactive peptides (37, 38) and as materials (39), and described how large libraries of peptoids have been prepared and subjected to high-throughput screening campaigns to identify novel ligands for varied target proteins (40). The lecture also detailed her own work investigating structural features of peptoids (41–43), and on new efforts, including those from her lab, to diversify the peptoid scaffold by making modifications to the oligoamide backbone (44–46). In addition to their many interesting applications, peptoids seemed of particular interest to this audience because of their relevance to the work of DR’s lab, whose targets, including N-alkylated peptides, bear some structural similarities. Lectures on More Detailed and Applied Work Lecture 5. “Implementation of the D3 Lab at Colorado College” (AD) AD’s presentation highlighted the successful incorporation of D3 Lab 2 into the “Organic 2” course at Colorado College. Additionally, AD described her investigations toward a number of new extensions to the D3 platform. For example, in an effort to incorporate more green chemistry principles into the D3 learning experience, she created a research project assignment for her advanced organic synthesis course for majors. This assignment required students to evaluate the D3 Lab 2 protocol using the 12 Principles of Green Chemistry (47, 48), propose a greener method, and then develop and optimize the new method in the 71

laboratory. She is currently following up on promising preliminary results from these student projects in order to identify greener methods for broader utilization across the D3 network.

Active Learning session: “Design of a Combinatorial Library of N-Acylated Amino Acids” (AD) AD has also developed a new pre-lab exercise at Colorado College to introduce students to the process of hypothesis-driven drug design. Following a talk describing this activity she conducted an active learning session with the student workshop participants. In this activity, students evaluated existing antimicrobial data for a series of amino acid derivatives accessible by D3 Lab 2 (which they were about to perform). Using the provided data, the students developed structure-activity relationship (SAR) hypotheses and proposed the next set of new analogs to be synthesized in the lab. Working first with a partner, and later with a larger assigned “medicinal chemistry team,” students had the opportunity to propose new design ideas, convince other team members of the ideas’ merits, and negotiate toward a final team proposal (Figure 6). The students were highly engaged in this exercise, which was intended to simulate a real-life medicinal chemistry team situation in which chemists must work together to prioritize ideas and agree upon a small set of high-priority synthetic targets.

Figure 6. Student Team Deciding which New Molecules to Make. (see color insert)

Lecture 6. “Combinatorial Synthesis of Aromatic Oligoamides” (AF) At an applied laboratory level AF described the implementation of her new D3 Lab 9 “peptoid” lab developed at Santa Clara University (Scheme 2). 72

Scheme 2. New D3 Lab 9: "Peptoid Inspired" Lab Developed at Santa Clara U. Demonstrating that the D3 laboratory practices can be readily adapted to employ new chemistry, AF detailed the laboratory she teaches at Santa Clara University. In doing so, she also shared some photos and information about Santa Clara University and its students. This introduction prompted interest and amazement from the Cuban participants—in particular, they highlighted that they had never before seen a University affiliated with a church, indeed with a campus centered around it! But outside of the church and in this lab, AF’s students prepare combinatorial arrays of aromatic oligoamides. Students in the lab share with AF a long-term goal of identifying biological activities for this scaffold, which is structurally similar to other bioactive molecules. AF described how students used the Bill-Board apparatus to carry out the D3 Lab 9 six-step synthetic sequence and detailed synthetic results from a recent iteration of the lab course (33). As part of the D3 validation process 12 of the molecules her students made were independently replicated by a student at IUPUI using her published procedure. Lastly, she concluded her talk with short presentations on her ongoing efforts to develop new D3-compatible lab procedures to prepare additional molecules with medicinal potential.

Lecture 7. “D3 Pseudomonas aeruginosa (PA) Biofilm Project” (WS) In this lecture WS reported on the first D3 coupled chemistry/biology project (23, 31). It incorporated both D3 synthesis and D3 biological evaluation in student work that identified several potent inhibitors of P. aeruginosa (PA) growth. PA is a major cause of debilitating and deadly infections in patients with the orphan disease cystic fibrosis. Using simple, inexpensive, reproducible and powerful synthetic procedures, students made large numbers of unique unnatural amino acids in their second semester organic chemistry lab. In a subsequent microbiology lab course they tested these compounds against PA. Through this distributed process students identified several potent inhibitors of 73

PA. The active compounds are racemic, unnatural analogs of phenylalanine and its carboxyl derivatives. In follow-up studies individual enantiomers of three of the active racemic compounds were separated by chiral chromatography. Biological evaluation showed that in each case the biological activity resided almost exclusively in a single enantiomer. Literature precedent indicates these active compounds are functioning as antimetabolites, with implications for potential toxicity in humans. In subsequent undergraduate lab work we are further modifying the active molecules to seek derivatives that will have a greater therapeutic ratio.

Lectures Directly Related to Workshop Lab Lecture 8. “Solid-Phase Synthesis and Combinatorial Chemistry” (WS) Here WS discussed the chemistry and methodology that enables the molecules to be synthesized in the lab portion. Solid-phase synthesis (SPS) and combinatorial chemistry are crucial components of D3 laboratories because together they enable the efficient synthesis, on a small scale (typically 50 micromoles), of multiple compounds from a limited set of reagents - and with little loss of material. WS illustrated the essential SPS procedural steps and methodology with an example of the solid-phase synthesis of a tripeptide. This example also served to illustrate the power of combinatorial chemistry since 8,000 tripeptides could be made using SPS with access to 20 different amino acids at each position of the tripeptide (20 x 20 x 20 = 8,000). Students were instructed on how universal and fundamental the combinatorial synthetic process is in nature: millions of DNA sequences and proteins available from just 24 starting materials (“reagents”) - 4 nucleotides and 20 amino acids. They were now prepared to do in the lab their own combinatorial solid-phase organic syntheses based on the synthetic sequence of D3 Lab 2 (Scheme 3).

Scheme 3. D3 Lab 2: The Synthesis of N-Acylated Natural α-Amino Acids.

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Lecture 9. “D3 Lab 2: Reaction Mechanisms for Fmoc-Deprotection, N-Acylation and Resin Cleavage” (MO) Lecture 9 presented an opportunity to demonstrate distance learning aspects of D3. Electronically and in his physical absence, MO presented an audio-visual lecture (49) on the detailed mechanisms involved in each of the transformations conducted in the D3 Lab 2 synthetic procedure. Reaction mechanisms are an important aspect of the D3 program. They play a crucial role in explaining how known reactions proceed and rationalizing how unexpected by-products are formed. The D3 Lab 2 mechanisms tutorial illustrated each step of a reaction mechanism in dynamic “layered” audio-videos. It covered arrow-pushing, acid-based chemistry in terms of pKa’s and reaction equilibria, Le Chatelier’s Principle, reagent structures/acronyms, functional groups, nucleophiles/bases and electrophiles/acids, electronegativity, resonance, stereochemistry, racemization, etc. In his audio/visual lecture seven total ChemDraw schemes were discussed for the D3-2 laboratory (approximately 30 minutes total): 1) Synthesis Overview; 2) Arrow-Pushing and Acid-Base Chemistry; 3) Fmoc Deprotection; 4) and 5) N-Acylation; 6) and 7) Cleavage. Audio/video of the step-by-step progress of each reaction was presented. This and other audio-visual tutorials have been well received by students and instructors. They can be shown during lectures or, at IUPUI, as an out-ofclass assignment following a more general AV tutorial covering: Introduction to Research in the Lab; Choosing a Class of Targets; Background References; Amino Acids and Peptides as Drugs; Bill-Board Setup and Reaction Scheme; Individual ChemDraw Assignment (structures, SMILES, etc.) for each student team’s (13 students) Bill-Board; Reaction Inputs; Final Products (Fall 2016: 44 unique products unique products in 120 total reactions); Acknowledgements. Students can stop the tutorial at any point and replay it for a better understanding of the material. As demonstrated in this Cuba Workshop, with the link (49) given for this lecture, Audio-Visual Tutorials can be transported without the instructor.

Lecture 10. Compound Enumeration, Computational Analysis and “Teach-Discover-Treat” (WS) There was a brief lecture on compound enumeration of D3 virtual catalogs, computational analysis, and its application to searching these catalogs. After discussing the nature of some of the computational algorithms used in modeling work WS highlighted an ACS open-access computational project “Teach-Discover-Treat (TDT).” TDT fits well to a D3 model. In this ACS Computational Division initiative the computational community is challenged to develop computational models for neglected diseases. They should use freely available software tools and develop tutorials and models appropriate for educational purposes. The D3 community is closely following the progress of TDT, as linking it to D3 virtual catalogs could leverage its power by coupling 75

the educational and research capabilities of D3 synthesis to TDT computational analysis.

Lecture 11. D3 Collaboration with the Community for Open Antimicrobial Drug Discovery (CO-ADD) (WS) This final lecture discussed the variety of ways by which molecules that students make can be evaluated for biological activity: 1) They can be tested by students as part of regular course work. For example, IUPUI’s microbiology students currently test molecules, made by IUPUI chemistry students, for their ability to inhibit the growth of Pseudomonas aeruginosa, a bacteria especially harmful to patients with cystic fibrosis (31). 2) They can be tested by academic collaborators (D3 collaborators are doing this in collaboration with microbiologists in Lublin, Poland). 3) Use commercial testing services (too expensive). 4) Use Open-Access resources a) Governmental (e.g. National Institutes of Health). b) Industrial (e.g. Lilly’s Open Innovation Drug Discovery program [“OIDD”]). c) Academic (e.g. Community for Open Antimicrobial Drug Discovery [“CO-ADD”]). Lecture 11 focused on that final test resource, CO-ADD, since it will be the testing site for compounds made by the Cuban students in their workshop lab. As stated on their website “CO-ADD is a not-for-profit initiative led by academics at The University of Queensland. Our goal is to screen compounds for antimicrobial activity for academic research groups for free. We aim to help researchers worldwide to find new, diverse compounds to combat drug-resistant infections.” CO-ADD screens compounds against the key “ESKAPE” pathogens, E. coli, K. pneumoniae, A. baumannii, P. aeruginosa, S. aureus (MRSA), as well as the fungi C. neoformans and C. albicans. (The hazardous nature of many of these pathogens precludes developing simple assays that can be safely conducted in a regular undergraduate microbiology lab). D3 has taken advantage of CO-ADD resources in the past and the Cuban students were told that we planned to send to CO-ADD duplicate lots of all 22 compounds made by them in this workshop.

Lab Component of the Workshop In the lab phase of this Cuban workshop students (Figure 7) utilized the D3 Lab 2 procedure and Bill-Board equipment (shown in Scheme 3 and Figure 4) to synthesize many new acylated natural amino acids 4 to be tested as potential antimicrobial agents. 76

Figure 7. Lab Students and TAs. (see color insert)

As discussed earlier, two of the essential requirements for a D3 laboratory are: 1) Powerful, reproducible synthetic procedures to enable the synthesis of large numbers of potential drug molecules and 2) simple, inexpensive equipment to carry them out. All of the current D3 procedures are based on solid-phase combinatorial chemistry. The solid-phase nature enables multi-step syntheses to be successfully carried out with simple equipment on a micromole scale. The combinatorial aspect allows the creation of large catalogs of virtual molecules accessible by these D3 procedures. The students or student teams can then select target molecules from these catalogs and synthesize, using D3 procedures and equipment, 6 unique molecules at a time. Training of Our Undergraduate TAs To Lead the Laboratory Portion of the Workshop Two of our teaching assistants, PD and JS, in addition to being fluent in the Spanish language, were given extensive training by GS in D3 Lab 2 procedures at IUPUI prior to the workshop. Beginning in the summer months they personally completed the synthetic sequence two times and verified and optimized the chromatographic purification procedure for the control compound. In addition, PD and JS provided a complete translation of the laboratory procedure into Spanish, and the translated version was then successfully “beta-tested” by an independent IUPUI student who was also fluent in Spanish. JS and PD were each given the experience of preparing and distributing an isopycnic (neutral buoyancy) suspension of the starting resin, a prerequisite for even resin distribution to the individual reaction vessels, and they were provided with 1) a complete inventory of chemicals/equipment/supplies required for the workshop, 2) a complete set of reagent calculations, 3) a suggested order of tasks, 4) a detailed plan of multiple reagent preparation/distribution, 5) a sheet of adhesive, printed labels for the reagent vials, 6) an isopycnic preparation/distribution procedure complete with a photograph of the setup and 7) a compilation of key pre-lab remarks and suggestions for each session along with a listing of supplies/reagents/solvents needed for each session. 77

Our third teaching assistant, DT, was a student in AF’s D3 laboratory course at SCU and has since continued research in her independent laboratory optimizing procedures for new D3-compatible scaffolds. In addition, DT has served as an undergraduate TA for several organic chemistry labs at SCU. As such, he had extensive experience both with leading novice chemists in the lab and conducting solid-phase synthesis experiments. D3 Lab Procedures and Layout For the Ernest Eliel Cuban Workshop we chose to utilize D3 Lab 2 because this lab involves the fewest number of steps of all our D3 procedures, makes use of readily available reagents, and teaches all the basic skills of solid-phase synthesis while students produce members of an important class of molecules with documented drug potential. The 24 students participating in the lab were assigned, in teams of two or three, to each of the 10 Bill-Boards. The combinatorial nature of D3 Lab 2 (Scheme 3) was carried out with two unique amino acids providing R1 in Row A or B, and three unique carboxylic acids as acylating agents R2 in Columns 1, 2 or 3 of the 2 x 3 grid of the Bill-Board. This affords six unique compounds in each Bill-Board. Reproducibility is a key requirement in the D3 program. This was demonstrated in two ways in the workshop laboratory: 1) As a control, every Bill-Board (and team) was assigned the same amino acids (phenylalanine = R1 in Row A, and tyrosine [protected as the t-butyl ether)] = R1 in Row B) and carboxylic acid (4-fluorobenzoic acid = R2) in Column 1. This meant that everyone should have synthesized phenylalanine 4-fluorobenzamide in position A1 and tyrosine 4-fluorobenzamide in position B1. Thus A1 and B1 are the “controls” (we already knew they would work) and can be used to guarantee the students obtain products (and satisfaction) if they properly carry out the synthetic procedures. 2) Throughout the lab different carboxylic acids R2 were used in columns 2 & 3, but each Bill-Board based on these different carboxylic acids was replicated by another Bill-Board team. In this way the four unique products produced in positions A2, A3 and B2, B3 are also replicated (actually duplicated in this case). For example, layouts illustrating replication at the control level of A1 and B1, and new derivatives in A2, A3, B2 B3 for replicated Bill-Boards 1 & 2, and 3 & 4, are shown in Figure 8.

Figure 8. Bill-Board Layout for Replicated Teams 1 & 2, and Replicated Teams 3 & 4. 78

This pattern of replication was done throughout the lab for rest of the remaining Bill-Board teams: 5 & 6; 7 & 8; 9 & 10, leading to ten lots of replicated compounds 1 (in every A1) and 4 (in every B1), and 2 lots each of the 20 new compounds in the other four Bill-Board positions A2, A3, B2 and B3. The total of 22 unique compounds made by the students are shown in Figure 9.

Figure 9. 22 Structurally Unique Compounds Made in Cuba Workshop.

Carrying Out the Lab Work Prior to the first lab the US student teaching assistants (TAs: PD, JS, and DT) distributed to each of the six reaction vessels in the ten Bill-Boards the resin-bound Fmoc amino acids phenylalanine and tyrosine (protected with a t-butyl group) in Rows A and B respectively. The first day the Cuban students carried out the deprotection (Fmoc removal) of the resin bound amino acids in Rows A and B, washed the resins, and added the assigned carboxylic acids (in previously prepared stock solutions containing catalyst hydroxybenzotriazole) to the appropriate columns, followed by coupling agent diisopropylcarbodiimide to all reaction vessels. For all these steps students followed a carefully written D3 Lab 2 procedure which had been extensively tested and verified by students at IUPUI and other schools in other global locations (Figure 10). In advance, the Cuban students were given the English version of the D3 Lab 2 procedure as well as a version PD and JS had translated into Spanish. 79

Figure 10. Carrying out D3 procedure (see color insert) The second day students extensively washed the resin in each of the six Bill-Board vessels and then cleaved the products from the resin with trifluoroacetic acid (TFA), draining the cleaved products into the individually tared and labeled product collection vials. They removed a 0.1 mL sample of product containing cleavage solution and placed it in an HPLC vial, completing their work for the second lab day. The TFA was evaporated from the 60 HPLC vials (10 Bill-Boards, 6 samples each) which were placed in a box and returned to IUPUI. GS subsequently analyzed the quality all 60 samples by LC/MS. The TFA/dichloromethane in the ten A1 collection vials from the 10 Bill-Board teams was evaporated overnight to provide samples, from all ten teams, of A1 for chromatographic purification the next day. The third afternoon students weighed the A1 collection vial from their BillBoard to calculate crude yields. They then chromatographed A1 on a simple 500 mg cyanosilica column, using a step-wise gradient eluent and collecting 1-2 mL fractions. The collected fractions were analyzed by TLC on a cyanosilica TLC plate and fractions containing only a single spot were combined into a tared vial. Typically the total volume of eluent was ~10 mL and product was collected in 1-3 fractions. These combined fractions were evaporated after the workshop was completed, weights recorded, and samples sent to Germany for NMR analysis. Post Workshop Follow-Up and Results 1) All 60 reaction products were analyzed by LC/MS at IUPUI. Results were sent back to Cuba for students to record and discuss. The crude A1 controls (10 samples from the 10 teams) had an average purity of 82% (range: 59-97%). For most of the 60 samples the crude purity was >70%. DMF was sometimes observed as a contaminant. It was present at 15-66% in eleven of the 60 samples, probably the result of incomplete washing of resin prior to the cleavage step. Reproducible purity among duplicate Bill-Boards was found to be variable, again often a function of contamination by DMF. 2) All product vials were evaporated to a film and weighed to calculate and record crude yield. 3) 1 mg samples of replicate lots of all 22 unique compounds were transferred to vials for CO-ADD antimicrobial testing. The vials used 80

were barcoded and were requested by GS, in advance, from CO-ADD. Prior to submission of the samples, an official CO-ADD spreadsheet was completed by GS and sent to CO-ADD. Once the spreadsheet was received by CO-ADD, Australian importation papers were provided by CO-ADD and were included with the samples. We are all eagerly awaiting the test results. 4) All ten A1 control products purified by the student teams were sent to Germany for NMR analysis. The spectrum obtained for one of them is shown in Figure 11 and is representative of the ten samples.

Figure 11. 1H Spectra of a Sample of A1 Purified by Students in Team 1 (Integration Cut Off for Display).

Assessment Goals The overall goal of this D3-based workshop was to integrate global chemistry education and drug discovery research into the University of Havana’s undergraduate instructional labs, exposing students to a compelling application of their learning as they prepare and test new compounds with drug potential against neglected diseases. The workshop also sought to build collaborations between Cuban and US scientists. In particular, D3 could benefit from the shared expertise of Professors Scott, O’Donnell, Fuller, Dounay and Rivera in education, drug discovery, combinatorial chemistry, solid-phase synthesis, and other synthetic approaches to bioactive molecules. These collaborations would further D3’s goal of finding drug leads for neglected diseases while building international partnerships in education and research. We hoped the common drug discovery goal would provide a natural environment to share and celebrate not just science and education, but also the cultural aspects of our unique backgrounds, both at an individual and societal level. How did we do? Scientific Exchange 1.

Eleven lectures and an active learning session were given by the faculty. Four Cuban students each gave 30 minute talks on their research. 81

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Students successfully completed a three-step solid-phase synthesis. Each student team made six different molecules as they simultaneously carried out this sequence in six separate reaction vessels. With extensive replication, a total of 22 unique molecules were made and all of them were characterized by LC/MS. A subset was purified by chromatography and characterized by 1H NMR. Replicate samples of the crude products (almost all > 70% pure by LC/MS) were submitted to CO-ADD for evaluation of biological activity. Three hours were dedicated to more informal discussions of ongoing work and to developing ideas for possible future research connections. This time included proposals made by Cuban students to articulate innovative ideas that could be enabled by the D3 technology. A poster session was held in which Cuban and US students shared their research with one another and with faculty (Figure 12).

Figure 12. Student Poster Presentation. (see color insert)

Chemical Education: Dissemination of Knowledge and Equipment Curricular materials and equipment were demonstrated through the course of this workshop, and everything was left with the Cuban faculty and students for their future use. Specific materials shared included video lectures, written laboratory procedures (in English and Spanish), active learning library design exercises, a majority of physical equipment needed for the laboratory experiments, including the Bill-Board apparatus (quantity = 12), and surveys of student learning outcomes. Through the lectures, WS, AD, and AF conveyed three different applications of the D3 approach as it is implemented at their respective institutions. MO, in his absence, presented an audio/visual lectures teaching the mechanistic details of the steps in the D3 lab procedure. Through these presentations, the US faculty highlighted that the D3 laboratory idea can be tailored to the unique needs and strengths of a given institution. Moreover, this program welcomes innovations from new collaborators. Three US students (two of whom were fluent in Spanish) served as instructors for the laboratory portion of the workshop. They worked closely with Cuban students to introduce them to the use of the equipment and guided them through the procedures. Their familiarity with the course material as well as with the student perspective provided a rich dimension to the workshop. 82

Cultural Exchange: Ongoing Relationships, Planned Actions Rich relationships developed over the course of the week among students and faculty participating in the workshop. Activities outside of the workshop (e.g., at lunch, US-Cuban student outings) promoted candid interactions. Students exchanged email and Facebook contact information and intend to keep in touch with one another via social media. Faculty intend to continue ongoing research and educational collaborations via email, phone, and Skype.

Cuban Student Perspectives and Reflections We surveyed the Cuban students with several questions. Responses in Spanish were translated into English. Here is a sampling of Cuban students’ reflections and perspectives prompted by these questions. Please comment on how your understanding of the subject has changed as a result of this workshop. •



“With this workshop, I was able to better understand the complete process [of drug discovery], from the onset of an idea, to the medicine getting put into the market.” “I had never approached solid phase synthesis because it was not related to my research. However, though this workshop, I have been able to understand why organic reactions are completed in the solid phase and the concept of combinatorial chemistry, as well as the importance of curing neglected diseases. I can better understand the process of conducting chemical reactions.”

Please comment on what skills you have gained as a result of this class. •

“Skills in working with the Bill-Board and combinatorial synthesis.”

Please comment on how this class has changed your attitudes toward chemistry. • • •



“It [The workshop] has motivated me to take additional chemistry courses and expand my knowledge of solid phase synthesis.” “This workshop has increased my interest in chemistry because of its application to combating 3rd world diseases.” “This workshop showed me how chemistry can be very useful in helping humanity. It has motivated me to do research in the field of neglected disease.” “It [The workshop] has motivated me to take additional chemistry courses and expand my knowledge of solid phase synthesis.”

What will you carry with you into other classes or other aspects of your life? 83

• •

“The use of the Bill-Board and the incentive of discovering new medications.” “A new vision. Because this is an innovative project, we can take part of a big research project.”

Please comment on how the instructional approach to this workshop helped your learning. • •

“The exchange of students and professors was very important and beneficial.” “This workshop was help to improve my ideas about drug discovery of neglected disease and in the application of combinatorial chemistry on solid phase.”

Please comment on how the workshop activities helped your learning. • •



“It helped improve my understanding of English and understanding of peptide synthesis.” “By listening to the presentations I could better understand solid phase reactions and the experimental work allowed me to put in practice the knowledge I acquired.” “The best is working in groups, because we can learn from each others. Other really good thing is that professors were at the lab together with students and instructors.”

We also conducted post-workshop surveys soliciting student impressions, rated on a scale of 1 to 5, of the efficacy of selected workshop elements on their learning. Some of the most strongly consistent responses (>4.5, N=20, standard deviation from .28 to .87. Scale: 5 = great gain; 4 = good gain; 3 = moderate gain; 2 = a little gain; 1 = no gain.) indicated an increase in: • • • • • •

Understanding why it is challenging to cure "neglected" diseases. Understanding the conceptual application of combinatorial chemistry. Understanding how studies done in this workshop address real world issues. Ability to integrate information from lectures to understand new reaction mechanisms in the laboratory. Enthusiasm for chemistry. Confidence they could contribute to chemistry research.

Students said their interactions with other participants, together with the experimental work, greatly assisted learning. It was gratifying that student responses were overwhelmingly and uniformly positive. Although we recognize that self-reported information on student learning does not represent the most accurate way to evaluate student learning outcomes, we are encouraged by the positive responses in this initial workshop endeavor. Here are two samples of Cuban students’ reflections on their workshop experience: 84

“The symposium of the D3 project was an exceptional experience, where we learned a lot about neglected diseases and the actual way to develop drugs for treating them. We had the opportunity of sharing knowledge and opinions with students from Colorado Collage, IUPUI and Santa Clara University. I think it is remarkable what they are doing in this project, paying attention to such neglected diseases, which are the cause of millions of deaths in the word. I hope other universities join to this project, as University of Havana have done.” (Dayan Viera Barredo, Undergraduate student of Chemistry, 4th year) “The D3 workshop was a great opportunity for the exchange of science and culture between the two countries. During that week, I learned about not only combinatorial chemistry and how to use a billboard instrument, but also I learned about American people.” (Ana C. Rodríguez Humpierre Undergraduate student of Chemistry, 5th year) US Student Perspectives and Reflections The three US students that accompanied us, PD and JS from IUPUI, and DT from Santa Clara University (Figure 13), provided a crucial resource technically, personally and culturally - for the successful implementation of the Cuban workshop.

Figure 13. USA Student TAs Relaxing (see color insert) Their student status, role as teaching assistants with expertise in D3 laboratory procedures (coupled with their ability to communicate in Spanish) enabled them to make strong links scientifically and personally to Cuban students and their culture. It was a life changing experience for them. What follows, in their own words, are reflections from each of them regarding their workshop experience:

Priya Dave (IUPUI) Like many other Americans, my views on Cuba before visiting had been shaped by the media and from what I had studied in history textbooks. Cuba supposedly was an impoverished country without access to proper resources. Going to Cuba as an American, I felt a little uneasy when thinking about the tense political backdrop that seemed to overshadow our government’s relations 85

- I couldn’t help but think about what the Cuban students would think about Americans. What were they taught in their history textbooks and what did their news channels broadcast? Our first full day in Havana I quickly learned my perspective on Cuba was incorrect in many ways. Cuba was far from a third world country I had imagined – it had an almost non-existent unemployment rate, incredible education system, and not to mention low crime rate. It was refreshing to see the lack of American influence. There were no McDonald’s or Starbucks, or name brand hotels crowding the streets of the city. Instead the restaurants, architecture, and cars lining the roads were of their own distinct style. The first day, as we American students walked into the chemistry lab, we couldn’t help but notice all of the differences. Most striking was that there were only two small fume hoods in the center of the laboratory and the rest of the benches were empty spaces. A common theme throughout the week was working with what we had. And that is exactly what we did - we improvised. The Cuban students were already used to doing this. In fact, at any given step, the students would be thinking about how they could alter the procedure or if there wasn’t enough equipment, what to use instead. It was incredible to witness how they worked, using the procedure as a template instead of following it word by word. I could tell that all students had a solid understanding and passion for chemistry! Preparing for the Cuban lab began several months before embarking. As part of my Spanish service learning class, I began translation of an introductory video from English to Spanish. Afterwards, Juan and I together split translation of the actual chemistry procedure. Translation took hours and it was a challenging process! We even had to alter some of the script’s content to better suit a Cuban audience. Preparation also involved running several trials of the D3 procedure, synthesizing many novel potential drugs, and learning about the process of drug discovery. Additionally, after translation of the script, two students familiar with Spanish came in to test the procedure, which was ultimately successful! Overall, the most incredible aspect about travelling abroad with Distributed Drug Discovery was being able to connect with students. As a Spanish student, I had studied abroad with American university students for short periods of time, but there was nothing quite like the insights we were able to make through meeting and working with students from another culture. The students opened up their worlds to us. We got a flavor of their lives. They took us to their favorite outdoor pizza shop, and a whimsical local ice cream parlor. We went to a quiet, serene beach and explored the best views of the city! Ultimately, they were just as fascinated about our way of life in the U.S. as we were about Cuba. As a humanities major, I recall when I told the students, “I’m studying humanities, but after that I will go to medical school.” They looked at me in shock and multiple students asked me, “Can you really do that?” In Cuba, right out of high school students pick a career. I was just as fascinated by the fact that in Cuba all students get free education. In the United States, many more people would take advantage of a college education if it were free! But in fact, Cuba presents the contrary. The reality is that even with a degree in a lucrative field, such as medicine or chemistry, 86

students do not get paid for the amount they work. Many Cubans find it more practical to work in the tourism industry, giving tours of the city or working in the restaurants instead. If I had one word to describe Cuba, it would be genuine. The students were genuinely passionate about what they were doing. They were extremely inviting, ensuring that we were always having a good time. They spent their time with us each night, showing us the city. The experience left me with a new perspective on Cuba and a great appreciation for the Cuban values, culture, and people.

Juan Sanchez (IUPUI) I was busy enough preparing for the trip that I did not have time to form expectations about it, so I just decided to go with the flow and let every bit of this experience surprise me. I had prepared over the summer by learning all the steps of the procedure and how to use the equipment, so by the time the trip came around I was confident that everything would go well. Geno did an excellent job of labeling the equipment we were going to use. This made the preparations of the reagents for the workshop efficient, and gave us time to solve other issues in the setup of the lab at the University of Havana. It was an enjoyable challenge to use my language skills to translate the procedure from English to Spanish. We had worked long, hard hours to complete a procedure that had clear instructions in the students’ native language about how to proceed in the lab. I wanted to hear the opinions of the students regarding the translated procedure. The laboratory facility that we used at University of Havana was by no means state of the art. However, it was inspiring to see how the students and professors had the talent to look around the lab and create the best conditions possible for us to set up the lab for the workshop. I will never forget the moment when I realized that all of the students and professional chemists we had met decided to become chemists because it was what they truly felt passionate about. In Cuba many professions are not well compensated, but many people choose them over working in tourism where they can earn much more, because their genuine passion for what they do is worth more to them than money. Even though they lack the equipment and facilities that we take for granted, they still are able to do the lab work and learn, and they still become great chemists. That week we connected so well with the students in part because they were open and friendly, but also because they shared our passion for learning no matter the monetary compensation. One of the most impactful things I saw in this trip was the genuine love that all the students have for chemistry. All the time that they spend studying chemistry is a labor of love, and not for the money. It was upsetting to realize that there are passionate and driven students in Cuba but they do not have the resources to reach their full potential. A lot of their work is delayed because they send samples outside the country to be analyzed, whereas we have the facilities, such as an LC/ MS and NMR, that enable us to get results in a few hours. Having the opportunity to interact with the students made me hope for a normalized relationship between 87

our two countries. I hope that we can find ways to have future collaborations with passionate Cuban scientists who are also fully equipped to do the work to the best of their potential. Havana was beautiful, but its beauty was enhanced by the wonderful professors and students who hosted us. They were generous enough with their time to show us their daily lives and tour us around the city. Many friendships were made the week we were there. We are already planning a trip to visit them in the near future. I would love to visit my new friends and also see other parts of Cuba.

Daniel Tiano (Santa Clara University) As a student who regrettably decided not to study abroad when I had the opportunity, one can imagine the surprise and joy I felt when I was offered an opportunity to participate in the Ernest Eliel U.S.-Cuba Collaborative Workshop. My love for teaching and desire to do something meaningful in the world made it an easy choice to go. When I arrived at the hotel on the first day, I admittedly felt a little nervous about the workshop. What if my Spanish accent is too horrendous to understand? What if things don’t go smoothly in lab? How will I be received by the University of Havana Chemistry Department? My apprehensions were immediately laid to rest after the first day of the workshop. I remember having friendly, relaxed conversations with the Cuban students/faculty from the get-go; one of the first conversations I had with one of them was about American and Cuban television. The Cuban students/faculty continued to shower me with hospitality throughout the week. I had dinner at the department chair’s house twice, went to lunch with the students/faculty nearly every day, and even went on a late-night adventure with them to Christ of Havana! In addition to the casual outings and activities, I was also able to talk with the Cubans on a deeper level about things like how they perceive America vs. individual Americans, their thoughts on the relationship between the U.S. and Cuba, and what they thought about their country’s leadership. Of all the ways this trip shaped my world view, the one I value most is the exchange of opinions and ideas I was able to participate in. Believe it or not, I was able to do some actual work in addition to all the fun I had! While the American professors gave lectures and prepared the Cubans for the lab, the other American students and I prepared the lab materials and oversaw the procedure as TA’s. We had all done a variation of the procedure before, which allowed us to troubleshoot and catch mistakes before they happened. One of the things that surprised me the most about the whole trip was how little the Cubans had to work with in the lab equipment-wise. Each person got one or two pairs of gloves to use for the entire day (entire week). There was only one working hood, which forced twenty-something students to work with volatile chemicals in the open lab. There were unsecured air tanks in the corner of the lab. There weren’t any hotplates, so we heated a sample using a propped-up hair dryer. The way the Cubans made do with their limited resources was eye-opening and humbling, 88

especially coming from a place where I can use several pairs of gloves in one lab session without thinking twice. Needless to say, I would miss another week of school in a heartbeat to go to Cuba again.

Plans for Future Collaborations Research Collaborations We had discussions during the day and after hours regarding possible future collaborations among US faculty and between US and Cuban faculty and students. Some specific project ideas are summarized below. Two proposals emerged from the laboratory of DR that build on their expertise in multi-component reactions. One proposed access to many analogs of metallopeptidase inhibitors, and the other to multiple analogs of putative angiotensin converting enzyme (“ACE”) inhibitors. Cuban students will work to develop robust and reproducible procedures for these experiments that can be applied in other laboratories. Once they have developed these, students and faculty at US institutions, including SCU, IUPUI, and CC, will replicate the results to validate the procedures and make new analogs. WS and MO proposed a new D3 lab that would merge their published solid-phase unnatural amino acid synthesis procedure with DR’s published Ugi functionalization of resin-bound amino acids. This would be optimized and reproducibly validated in a joint IUPUI/University of Havana collaboration, possible carried out at IUPUI by a student on a short term exchange from the University of Havana. AD had presented ongoing work exploring greener approaches for chemistry currently utilized by D3, including comparisons of solution phase and solid phase synthesis techniques and explorations of replacing more hazardous and expensive solvents with less expensive and milder options. To enable cross-site replication of promising procedures and undertake new initiatives on this front, AF will incorporate similar projects into SCU laboratory courses. Discussions will continue between DR and AD centered in particular on sharing more ideas about their common interests in developing anti-parasitic compounds. All US faculty reiterated their willingness to serve as a resource for cross-site replications and validations of procedures. WS invited DR to visit the United States after the workshop. In November of 2016 DR visited the campuses of both IUPUI and Notre Dame for discussions and talks with scientists, students, and officials connected to offices of international activities. He gave an invited lecture on his work to scientists at Eli Lilly but was unable to have exchanges with Lilly scientists because of the ongoing Cuban embargo. In addition to the science and educational exchanges he had while in the US DR was present to witness the United States vote on its next president. After the election he traveled to the nation’s capital in Washington DC to have discussions with officials of the American Chemical Society. 89

Distributed Drug Discovery Dissemination The faculty expressed a common interest in identifying strategies to disseminate the central ideas of D3 more broadly and to engage more collaborators globally. US faculty will publicize the participation of students and faculty in this workshop within their institutions and, when possible, through local media channels. The workshop faculty are outlining a strategy for enhancing the visibility of the D3 program within the broader scientific community, including publications and presentations at conferences.

Final Thoughts As we worked together conducting the “Ernest Eliel Workshop – US and Cuba Collaboration in Chemistry Education and Neglected Disease Drug Discovery” we gained a richer understanding of why chemistry is often referred to as “The Central Science”. Over a one week period we educated students in fundamental chemistry knowledge and skills and showed them how essential this understanding is to addressing the critical need for drugs to treat neglected diseases. While they learned about the central role of chemistry in the complex of disciplines required for drug discovery they took part in that process by making many new molecules that are being tested for their potential as antimicrobial agents. In addition, as we built trust and comradery working together on shared scientific goals we learned about each other and our respective cultures in a natural and unselfconscious way. We saw chemistry as a “Central Science” that went beyond its role in scientific research and discovery to being a universal medium for breaking down geographic and cultural boundaries. We believe if Ernest Eliel could have witnessed Cuban and United States educators and students working together in this workshop, named in his honor, he would have seen his legacy in operation and had a broad smile on his face!

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Scott, W. L. Distributed Drug Discovery. Department of Biochemistry, Seminar, Jagiellonian University, Krakow, Poland, April 7, 2003. O’Donnell, M. J.; Scott, W. L. Distributed Discovery in the Undergraduate Organic Lab: Combinatorial Solid-Phase Synthesis of Amino Acid Derivatives. 18th Biennial Conference on Chemical Education (BCCE); Symposium (Invited): “Combinatorial Chemistry in the Undergraduate Curriculum,” Iowa State University; Ames, IA; July 21, 2004. Scott, W. L.; O’Donnell, M. J. Distributed Drug Discovery, Part 1: Linking Academia and Combinatorial Chemistry to Find Drugs for Developing World Diseases. J. Comb. Chem. 2009, 11, 3–13. Scott, W. L.; Alsina, J.; Audu, C. O.; Babaev, E.; Cook, L.; Dage, J. L.; Goodwin, L. A.; Martynow, J. G.; Matosiuk, D.; Royo, M.; Smith, J. G.; Strong, A. T.; Wickizer, K.; Woerly, E. M.; Zhou, Z.; O’Donnell, M. J. Distributed Drug Discovery, Part 2: Global Rehearsal of Alkylating Agents 90

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for the Synthesis of Resin-Bound Unnatural Amino Acids and Virtual D3 Catalog Construction. J. Comb. Chem. 2009, 11, 14–33. Scott, W. L.; Audu, C. O.; Dage, J. L.; Goodwin, L. A.; Martynow, J. G.; Platt, L. K; Smith, J. G.; Strong, A. T.; Wickizer, K.; Woerly, E. M.; O’Donnell, M. J. Distributed Drug Discovery, Part 3: Using D3 Methodology to Synthesize Analogs of an Anti-Melanoma Compound. J. Comb. Chem. 2009, 11, 34–43. Scott, W. L.; Denton, R. E.; Marrs, K. A.; Durrant, J. D.; Samaritoni, J. G.; Abraham, M. M.; Brown, S. P.; Carnahan, J. M.; Fischer, L. G.; Glos, C. E.; Sempsrott, P. J. Distributed Drug Discovery: Advancing Chemical Education through Contextualized Combinatorial Solid-Phase Organic Laboratories. J. Chem. Educ. 2015, 92, 819–826. Scott, W. L.; O’Donnell, M. J. Solid Phase Synthesis and an Introduction to Combinatorial Chemistry; NSF Workshop; Ketcha, D. M., Taylor, R. T.; Miami University, Miami, OH, July 27−August 1, 2003. Scott, W. L.; Denton, R. E.; Samaritoni, J. G.; Marrs, K. A.; O’Donnell, M. J. International Distributed Drug Discovery (D3) Workshop; Indiana University Purdue University Indianapolis, July 22−26, 2013. O’Donnell, M. J.; Durrant, J. D.; Denton, R. E.; Marrs, K. A.; Samaritoni, J. G.; Scott, W. L. A Sophomore Organic Lab Research Experience in the Context of Distributed Drug Discovery (D3) for Neglected Diseases; BCCE – Biennial Conference on Chemical Education (BCCE), Workshop, Grand Valley State University, MI, August 3−7, 2014. In 2004 WLS conducted other one-week workshops at the University of Barcelona (Spain), Medical University of Lublin (Poland) and Moscow State University (Russia). Scott, W. L.; Samaritoni, J. G.; O’Donnell, M. J.; Dounay, A. B.; Fuller, A. A.; Dave, P.; Sanchez, J. M.; Tiano, D. G.; Rivera, D. G. Ernest Eliel Workshop – US and Cuba Collaboration in Chemistry Education and Neglected Disease Drug Discovery, University of Havana, Havana, Cuba, October 17−21, 2016. O’Donnell, M. J. The Preparation of Optically Active a-Amino Acids from the Benzophenone Imines of Glycine Derivatives. Aldrichim. Acta 2001, 34, 3–15. O’Donnell, M. J. The Enantioselective Synthesis of a-Amino Acids by PhaseTransfer Catalysis with Achiral Schiff Base Esters. Acc. Chem. Res. 2004, 37, 506–17. O’Donnell, M. J.; Zhou, C.; Scott, W. L. Solid-Phase Unnatural Peptide Synthesis (UPS). J. Am. Chem. Soc. 1996, 118, 6070–6071. Scott, W. L.; Martynow, J. G.; Huffman, J. C.; O’Donnell, M. J. Solid-Phase Synthesis of Multiple Classes of Peptidomimetics from Versatile Resin-Bound Aldehyde Intermediates. J. Am. Chem. Soc. 2007, 129, 7077–7088. Scott, W. L.; Zhou, Z.; Martynow, J. G.; O’Donnell, M. J. Solid-Phase Synthesis of Amino- and Carboxyl-Functionalized Unnatural -Amino Acid Amides. Org. Lett. 2009, 16, 3558–3561. 91

17. Samaritoni, J. G.; Copes, A. T.; Crews, D. K.; Glos, C.; Thompson, A. L.; Wilson, C.; O’Donnell, M. J. Unexpected Hydrolytic Instability of N-Acylated Amino Acid Amides and Peptides. J. Org. Chem. 2014, 79, 3140–3151. 18. Scott, W. L.; Brown, S. P.; Audu, C.; Samaritoni, J. G.; Sempsrott, P. J.; Strong, A. T.; Zhou, Z.; O’Donnell, M. J. Distributed drug discovery: Unnatural amino acid amides. 238th ACS National Meeting, Washington, DC, August 16, 2009, Poster, ORGN 455. 19. Scott, W. Distributed Drug Discovery (D3): Linking basic research and education to find drug leads for neglected diseases. 240th ACS National Meeting, Boston, MA, August 23, 2010, Oral, BMGT 16. 20. Scott, W. L.; O’Donnell, M. J. Distributed drug discovery (D3): Enabling computational and synthetic chemists to work together to educate students while discovering drugleads for neglected diseases. 243rd ACS National Meeting, San Diego, CA, March 25, 2012, Oral, COMP 91. 21. Crews, D. K.; Samaritoni, J. G.; Scott, W. L.; O’Donnell, M. J. Solid phase synthesis of Nacylated amino acid amides using Rink resin. 245th ACS National Meeting, New Orleans, LA, April 7, 2013, Poster, CHED 957. 22. Scott, W. L.; Anderson, G. G.; Denton, R. E.; Harper, R. W.; Marrs, K. A.; Samaritoni, J. G.; O’Donnell, M. J. Distributed Drug Discovery (D3): Advancing chemical education through contextualized organic laboratories. 246th ACS National Meeting, Indianapolis, IN, September 10, 2013, Oral, CHED 344. 23. O’Donnell, M. J.; Abraham, M. M.; Anderson, G. G.; Carnahan, J. M.; Coffey, B. M.; Denton, R. E.; Harper, R. W.; LaCombe, J. M.; Marrs, K. A.; Samaritoni, J. G.; Scott, W. L. Distributed Drug Discovery (D3): Contextualized educational laboratory program in chemistry and biology toward inhibiting biofilm formation related to cystic fibrosis. 246th ACS National Meeting, Indianapolis, IN, September 11, 2013, Oral, CHED 397. 24. Denton, R. E.; Abraham, M. M.; Callahan, C. A.; Cankarova, N.; Carnahan, J. M.; Cerninova, V.; Copes, A.; Fortunak, J. M.; Harper, R. W.; Popiolek, L.; Samaritoni, J. G.; Soural, M.; Thompson, R.; Tomanova, M.; O’Donnell, M. J.; Scott, W. L. Distributed Drug Discovery (D3): Developing national and international distributed educational processes and standards in the context of identifying anti-malarial lead compounds. 246th ACS National Meeting, Indianapolis, IN, September 11, 2013, Oral, CHED: 398. 25. Scott, W. L.; Denton, R. E.; Harper, R. W.; Samaritoni, J. G.; O’Donnell, M. J. Distributed drug discovery (D3): Virtual D3 biomimetic catalogs, tested molecules, hit follow-up, drugs. 246th ACS National Meeting, Indianapolis, IN, September 8, 2013, Oral, MEDI 20. 26. Abraham, M. M.; LaCombe, J. M.; Anderson, G. G.; Carnahan, J. M.; Coffey, B. M.; Denton, R. E.; Harper, R. W.; Marrs, K. A.; Samaritoni, J. G.; Scott, W. L.; O’Donnell, M. J. Distributed drug discovery (D3): Successful integration of D3 components. Undergraduate computational, synthetic, and biological evaluation of phenylalanine derivatives as potential biofilm inhibitors. 246th ACS National Meeting, Indianapolis, IN, September 9, 2013, Poster, CHED 195. 92

27. Carnahan, J. M.; Samaritoni, J. G.; Crews, D. K.; Krchnak, V.; Lawrence, B. M.; Scott, W. L.; O’Donnell, M. J. Distributed drug discovery (D3): Saponification of N-acylated L-phenylalanine from Wang or Merrifield resin. Assessment of cleavage efficiency and epimerization. 246th ACS National Meeting, Indianapolis, IN, September 9, 2013, Poster, CHED 273. 28. Fischer, L. G.; Glos, C. E.; Lopez, D.; Samaritoni, J. G.; Stickney, K. W.; O’Donnell, M. J.; Scott, W. L. Distributed drug discovery (D3): Facilitating research and collaboration through an undergraduate combinatorial chemistry course that led to the discovery and follow up of a molecule affecting the oxytocin receptor. 246th ACS National Meeting, Indianapolis, IN, September 8, 2013, Poster, CHED 72. 29. Samaritoni, J. G.; Crews, D.; Glos, C. E.; Thompson, A. L.; Wilson, C.; Martin J. O’Donnell, M. J.; William L. Scott, W. L. Unexpected hydrolytic instability of N-acylated amino acid amides and peptides. 246th ACS National Meeting, Indianapolis, IN, September 11, 2013, Poster, ORGN 545. 30. Pruett, C. H.; Collins, A. D.; Scott, W. L.; O’Donnell, M. J.; Hopkins R. B. Application of distributed drug discovery methodology to the synthesis of some analogs of antimalarial screen hits. 247th ACS National Meeting, Dallas, TX, March 17, 2014, Poster, CHED 935. 31. Scott, W. L.; Samaritoni, J. G.; Anderson, G.; Marrs, K.; Colglazier, S.; Hitchens, J.; Burris, S.; Ware, M.; O’Donnell, M. J. Distributed drug discovery (D3) in action: Finding inhibitors of P. aeruginosa. 252nd ACS National Meeting, Philadelphia, PA, August 24, 2016, Oral, MEDI 275. 32. Scott, W. L. Samaritoni, J. G.; Popiolek, L.; Dounay, A.; Schirch, D.; Garcia Rivera, D.; Biernasiuk, A.; Malm, A.; O’Donnell, M. J. Distributed drug discovery (D3) update: First global student collaboration in neglected disease discovery. 252nd ACS National Meeting, Philadelphia, PA, August 24, 2016, Oral, CHEM 412. 33. Fuller, A. A. Combinatorial Solid-Phase Synthesis of Aromatic Oligoamides: A Research-Based Laboratory Module for Undergraduate Organic Chemistry. J. Chem. Educ. 2016, 93, 953–957. 34. Polypropylene Bill-Board, drain trays, collection trays and other associated supplies from Chemglass: Catalog #CG-1869. http://www.chemglass.com/ pages/billboards.asp (accessed April 20, 2017). 35. For a recent review, see: Sun, J.; Zuckermann, R. N. Peptoid Polymers: A Highly Designable Bioinspired Material. ACS Nano 2013, 7, 4715–4732. 36. Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. Efficient Method for the Preparation of Peptoids [Oligo(N-Substituted Glycines)] by Submonomer Solid-Phase Synthesis. J. Am. Chem. Soc. 1992, 114, 10646–10647. 37. Huang, M. L.; Benson, M. A.; Shin, S. B. Y.; Torres, V. J.; Kirshenbaum, K. Amphiphilic Cyclic Peptoids that Exhibit Antimicrobial Activity by Disrupting Staphylococcus aureus Membranes. Eur. J. Org. Chem. 2013, 3560–3566.

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38. Lee, B.-C.; Chu, T. K.; Dill, K. A.; Zuckermann, R. N. Biomimetic Nanostructures: Creating a High-Affinity Zinc-Binding Site in a Folded Nonbiological Polymer. J. Am. Chem. Soc. 2008, 130, 8847–8855. 39. Robertson, E. J.; Battigelli, A.; Proulx, C.; Mannige, R. V.; Haxton, T. K.; Yun, L.; Whitelam, S.; Zuckermann, R. N. Design, Synthesis, Assembly, and Engineering of Peptoid Nanosheets. Acc. Chem. Res. 2016, 49, 379–389. 40. Alluri, P. G.; Reddy, M. M.; Bachhawat-Sikder, K.; Olivos, H. J.; Kodadek, T. Isolation of Protein Ligands from Large Peptoid Libraries. J. Am. Chem. Soc. 2003, 125, 13995–14004. 41. Fuller, A. A.; Yurash, B. A.; Schaumann, E. N.; Seidl, F. J. Self-Association of Water-Soluble Peptoids Comprising (S)-N-1-(Naphthylethyl)glycine Residues. Org. Lett. 2013, 15, 5118–5121. 42. Fuller, A. A.; Holmes, C. A.; Seidl, F. J. A Fluorescent Peptoid pH-Sensor. Biopolymers (Peptide Science) 2013, 100, 380–386. 43. Fuller, A. A.; Seidl, F. J.; Bruno, P. A.; Plescia, M. A.; Palla, K. S. Use of the Environmentally Sensitive Fluorophore 4-N,N-Dimethylamino-1,8naphthalimide to Study Peptoid Helix Structures. Biopolymers (Peptide Science) 2011, 96, 627–638. 44. Hjelmgaard, T.; Faure, S.; De Santis, E.; Staerk, D.; Alexander, B. D.; Edwards, A. A.; Taillefumier, C.; Nielsen, J. Improved Solid-Phase Synthesis and Study of Arylopeptoids with Conformation-Directing Side Chains. Tetrahedron 2012, 68, 4444–4454. 45. Aditya, A.; Kodadek, T. Incorporation of Heterocycles into the Backbone of Peptoids to Generate Diverse Peptoid-Inspired One Bead One Compound Libraries. ACS Comb. Sci. 2012, 14, 164–169. 46. Kodadek, T.; McEnaney, P. J. Towards Vast Libraries of Scaffold-Diverse, Conformationally Constrained Oligomers. Chem. Commun. 2016, 52, 6038–6059. 47. Anastas, P. T.; Warner, J. C. Green Chemistry: Theory and Practice; Oxford University Press: New York, 1998; p 30. 48. https://www.acs.org/content/acs/en/greenchemistry/what-is-greenchemistry/principles/12-principles-of-green-chemistry.html (accessed April 20, 2017). 49. O’Donnell, M. J. D3 Lab 2: Reaction Mechanisms for Fmoc-Deprotection, N-Acylation and Resin Cleavage. https://iu.mediaspace.kaltura.com/ media/D3+Deprotect+Acylate+Cleave+Mech+Tutorial+O%27Donnell/ 1_k0tzmfv7 (accessed: April 20, 2017).

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Chapter 6

An Update on International Activities at the ACS Bradley D. Miller* Office of International Activities, American Chemical Society, 1155 Sixteenth Street, N.W., Washington, DC 20036, United States *E-mail: [email protected]

ACS Office of International Activities aims to build global networks for ACS members that enable the broader chemistry community to engage in international research and education, especially targeting areas where chemistry provides solutions to global challenges. The Office works closely with the ACS Committee on International Activities and others to organize a large number of systematic and impactful activities in order to advance the global chemistry enterprise. Some of the activities summarized here include ACS global alliances, international chapters, science and human rights, ACS International Center, ACS-Pittcon collaboration, ethics workshops and Global Innovation Imperatives.

Introduction Virtually three decades ago, the American Chemical Society (ACS) amended its constitution to include a provision to guide its global presence and activities. Article II, Section 3 establishes that ACS shall “cooperate with scientists internationally and shall be concerned with the worldwide application of chemistry to the needs of humanity.” It is under these principles that ACS International Activities operates today. In 1977, the ACS Board of Directors established the Office of International Activities (OIA) for the following purposes.

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To administer cooperative international projects in chemical education, research, and development funded by government agencies and other organizations. To handle member service projects relating to international matters. To provide liaison for international matters within the ACS and between the ACS and other organizations. To serve as staff liaison to the Committee on International Activities and to assist it in implementing programs for which it has secured funding.

OIA’s first director was Mr. Gordon H. Bixler, former editor-in-chief of C&EN. As director, he shaped the Society’s early exchange and research collaboration programs with the Soviet Union, China, India and Egypt. Dr. John M. Malin served as OIA Director for 18 years and was instrumental in the establishment of the ACS International Initiatives Program, Project Bookshare, ACS Environmental Chemistry Workshops in Eastern Europe and the Former Soviet Union, ACS exchanges with Latin America, and early chemistry collaboration with Cuba. From 2005-2007, Dr. Tamara Nameroff served as OIA Director and furthered the German –American Frontiers of Chemistry Program (which later became Transatlantic Frontiers of Chemistry) and ACS sustainable science-based interactions with chemistry communities in Europe, Asia and Africa. For the past 10 years, Dr. Bradley Miller has been ACS Director of International Activities, notably leading ACS contributions to the 2011 International Year of Chemistry, the development of international research experiences for undergraduate students, global research collaboration, training, and science diplomacy programs, and growing ACS International Chapters establishment and ACS Alliances. The ACS Office of International Activities has carried out its functions and service to member volunteers in several divisions of the Society, including Office of the Secretary, the Education Division, Membership and Scientific Advancement, and most recently in the newly created ACS External Affairs and Communications of the Office of the Secretary and General Counsel. In this new context, ACS International Activities is responsible for building international recognition of, and collaboration with, ACS through alliance partners, international conferences, professional exchange/research programs and global networks to foster international research and scientific exchange. IA continues to develop activities which are science driven and focused on advancing the interests and priorities of ACS member-volunteers with global interests.

ACS Global Alliances ACS believes that chemistry’s contributions toward global concerns, such as education, environment, and health and safety, should be extensive. In order to make significant progress on these issues, ACS partners with organizations around the globe to leverage our resources and capabilities along with those of 96

our partners. International alliances and partnerships support the ACS Strategic Plan by engaging members and scientific professionals to advance the Society’s mission and address, through chemistry, many challenges facing the world. With well-defined key performance indicators, ACS currently maintains international alliances with the following organizations (1). • • • • • • • • • •

Chinese Chemical Society (CCS) Chemical Research Society of India (CRSI) French Chemical Society (SCF) German Chemical Society (GDCh) Federation of Asian Chemical Societies (FACS) South African Chemical Institute (SACI) Latin American Federation of Chemical Associations (FLAQ) Canadian Society for Chemistry (CSC) Mexican Chemical Society (SQM) European Chemical Sciences (EuCheMS)

International Chemical Sciences Chapters ACS works to advance the field of chemistry around the world. Scientists outside the United States have formed International Chemical Sciences Chapters (ICSCs) to allow chemists within a geographic area to connect with one another, as well as with ACS members around the world. More than 27,000 ACS members are located outside of the United States. International Chemical Sciences Chapters provide a means for these ACS members to socialize, exchange technical information, and gain international recognition. ACS Bylaws provide for the establishment of overseas chapters of the American Chemical Society.With the ACS Board approving three new ACS Chapters at its December 2016 meeting, the number of ACS Chapters now stands at 19, with the additions of Greater Beijing / Tianjin, Southwest China and Iraq. Other active chapters include the following (2). • • • • • • • • • • • • •

Australia Brazil Hong Kong Hungary India Malaysia Nigeria Peru Romania Saudi Arabia Shanghai South Africa South Korea 97

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Taiwan Thailand United Arab Emirates

ACS Science and Human Rights In 1978, ACS began to investigate cases of alleged violations of the scientific freedom or human rights of chemists and chemical engineers in other countries. An ACS International Activities Committee (IAC) subcommittee investigated such cases in Argentina, China, Czechoslovakia, Iran, Korea, the Philippines, Russia, the Soviet Union and Turkey. The IAC Subcommittee on Scientific Freedom and Human Rights, led by Prof. Zafra Lerman, organized these investigations and ACS response to human rights abridgements. They also organized symposia at ACS national meetings. Individuals honored at these events were Yuri Tornapolsky (USSR), Feng Li Zhi (China), Vil Mirzayanoff (Russia) and Aleksandr Nikitin (Russia). In 2010 the ACS Board of Directors revised the procedures for the Society’s response to questions of human rights and scientific mobility. Participation in this updated process is open to any ACS member, committee, technical division, local section, chapter and external human rights groups and networks to bring to the ACS Board’s attention reports of violations of human rights and scientific mobility related to chemists and practitioners in allied sciences. ACS International Activities is tasked with working with the IAC regional subcommittees, ACS members, U.S. Department of State, foreign embassies and other professional societies and organizations active in human rights to monitor and seek information on reported threats to human rights of practitioners in chemistry and closely allied science and engineering areas. In 2011, OIA, in conjunction with AAAS Coalition’s Welfare of Scientists’ Working Group, developed a human rights best practices primer, which focuses on equipping scientific and engineering societies, as well as other scientifically oriented organizations, with the tools to effectively develop processes and procedures to address human rights issues, particularly responding to allegations of human rights violations. ACS International Activities also organizes and delivers webinars in the ACS Science and Human Rights Webinar Series, which explores the connection between science, specifically chemistry, and human rights. Webinars in this series have included the Malta Conferences, SESAME, Scholars at Risk, and OPCW, winner of the 2013 Nobel Peace Prize. OIA continues to further cultivation the Society’s science and human rights networks and their activation, including Scholars At Risk, AAAS Human Rights Coalition (including ACS participation / leadership on the Coalition Steering Committee and the Welfare of Scientists Working Group), NEAR, Amnesty International, Human Rights Watch, the National Academies Committee on Human Rights, the State Department’s Office of Multilateral and Global Affairs, in the Bureau of Democracy, Human Rights and Labor, Physicians for Human Rights, and the UN Office of the High Commissioner for Human Rights (OHCHR) 98

and our sister societies. Over the years, many of the cases ACS has assisted with were initiated from these groups. In an effort to increase member engagement, ACS Science and Human Rights has established an alert network to notify members of changes to ACS monitored human rights cases. Over 200 ACS members have registered for this email list and receive links to partner petitions, notifications for ACS science & human rights events such as webinars and symposia, and other relevant information. ACS Science and Human Rights also maintains relationships with UNESCO, Scholars at Risk,

ACS International Center In 2009, then-president elect Joe Francisco appointed a task force and working group to brainstorm the concept and viability and to set plans for establishing an ACS International Center (IC). The IC was envisioned to serve as a mechanism to encourage U.S. talent to go abroad to engage in international research experiences, learn about innovation in the chemical enterprise, and transfer the knowledge into the U.S. marketplace. At the same time, the Center could sponsor topflight international talent to come to the U.S. After analyzing the results of informant interviews with academic and industrial chemists active in international exchange, reviewing trends in the S&T exchange literature and considering models for existing international centers in the US and abroad, IC planners found the concept of an ACS International Center workable, its activity needed to be science driven and characterized by a clearinghouse / network development function; its success judged by productive partnerships and alliances, research collaborations, transnational journal co-authorship and proposal development, and sustained student and researcher exchange. The ACS International Center encourages, engages and support international exchange of scientific information at all levels. Authorized by the Board’s Committee of Budget and Finance in 2012, the ACS International Center (IC) aims to facilitate the exchange of resources and opportunities for the globally curious STEM practitioner. The ACS International Center has information on nearly 600 programs across 16 geographic regions (including one for ‘global’) and six career experience levels. To assist in connecting the ACS community with global providers, the IC currently works with 29 organizations that serve as ACS International Center Affiliates. Affiliates work with the IC to promote their opportunities and resources and participate in the community by organizing webinars or other outreach events. The International Center hosts quarterly webinar series dedicated to providing the STEM community with information relevant to upcoming opportunities and trends in global education. The ACS International Center (IC) closed 2016 with a strong showing of traffic and unique visitors to the site. The IC completed the year with 36,521 unique visitors, 42,444 daily unique visitors and 110,805 page views. All figures exceeded established targets for 2016. The site also now features videos from industry leaders on 99

the important role of international science mobility and collaboration as well as article from leading science journals such as Nature and other resources (3).

ACS-Pittcon Collaboration With support from the Society for Analytical Chemists of Pittsburgh (SACP), the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (PITTCON) and the Wallace H. Coulter Foundation, ACS International Activities works with Pittcon to organize delegations of early-career scientists to attend PITTCON meetings. The regions are selected each year through application peer review by IAC. The program recently celebrated its 20th anniversary.

ACS U.S. Department of State Workshops on Ethics With support from the US Department of State, OIA plans, organizes and implements Global Chemists’ Code of Ethics (GCCoE) workshops and institutes worldwide. Using its network of leaders in chemistry around the world, ACS works with the Federation of Asian Chemists (FACS), OPCW, PNNL, Sandia, partner chemical societies and Cooperative Threat Reduction priority nation representatives to organize these events (4). In Malaysia, Morocco, Kenya ACS International Activities organizes five-day training programs for participants from multiple countries in in the region in a variety of presentations and activities. The participants are trained in the background and intricacies of the GCCE as well as topics related to the Code, including Safety and Security, Publishing, and Communicating Science. Following the event, participants conduct a workshop/event in their home institution using the presentation materials from the initial workshop. They are encouraged to make form and content modifications if necessary to cater to the needs of their audience. To help with the delivery of this event, modest grants are competitively awarded.

Global Innovation Imperatives (Gii) Global Innovation Imperatives (Gii) is an ACS International Activities program that was originally started and implemented in collaboration with the U.K.-based Society of Chemical Industry. The Gii program fosters creative solutions to imperatives of global significance (e.g., clean water, food and health, etc.) (5). Gii’s goal is to stimulate significant action among ACS members, who include: • • • •

Innovation leaders Business executives Academia/education leaders Multinational businesses 100

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Non-government organizations Governments

The 2016 Global Innovation Imperative project’s white paper was recently completed and is titled “Green Chemistry Experiments for Remote Locations.” The 2016 project was submitted in partnership with the ACS International Chemical Sciences Chapter in Brazil and focused on green chemistry in rural areas. The event was held in the beginning of November 2016 in Belem, Brazil. A final draft of the 2016 Brazil Gii White Paper, as well as others from earlier programs, is available at https://www.acs.org/intrernational.

ACS International Activities’ Interactions with Cuba Since 1998 when the first delegation of twelve ACS members and staff visited Cuba to as recently as April 2017, ACS International Activities has cultivated a robust and sustained relationship with the chemistry community in Cuba, despite impediments to scientific mobility and exchange. These efforts were inspired by Professor Ernest Eliel and his volunteer service and leadership to the ACS and have culminated in contributions to the literature (6, 7) as well as elements of abiding trust and cooperation between and among chemists, chemical engineers and chemistry educators in both countries.

Lessons Learned With its many years of service, ACS International Activities works to amplify ACS member-volunteer global networks, communities and interests. It works with the ACS Board of Directors and Committee on International Activities to apply and build international recognition of the ACS, its brand and the Society’s relevant programs and activities—finding creative ways to apply ACS services to local needs around the world - targeting areas where chemistry provides solutions to global challenges. Successful programs are those which are driven by and remain true to the Society’s scientific mission and principles. As ACS International Activities – with guidance and direction from the ACS Committee on International Activities – refines and elaborates its global efforts in the decades to come, we are indebted to and inspired by the volunteer contribution of time, talent and energy of many, many ACS members with global interests. Among them is Professor Ernest Eliel who motivated us in our international pursuits to work hard with honesty, be resilient and never succumb to failure (8).

References 1. 2.

ACS Global Alliances. https://www.acs.org/content/acs/en/global/ international/alliances.htmlACS (accessed on April 29, 2017). International Chapters. https://www.acs.org/content/acs/en/global/ international/chapters.html (accessed on April 29, 2017). 101

3. 4.

5.

6.

7. 8.

ACS International Center. https://www.acs.org/content/acs/en/global/ international/international-center.html (accessed on April 29, 2017). ACS Science and Human Rights and Global Chemists Code of Ethics. https://www.acs.org/content/acs/en/global/international/science-andhuman-rights.html (accessed on April 29, 2017). Global Innovations Imperatives. https://www.acs.org/content/acs/en/global/ international/gii.html?_ga=1.17006905.1607391587.1467043829 (accessed on April 29, 2017). Chemistry without Borders: Careers, Research and Entrepreneurship; Cheng, H. N., Rimando, A. M., Miller, B. D., Schmidt, D. G., Eds.; ACS Symposium Series 1219; American Chemical Society: Washington, DC, 2016. Cheng, H. N.; Rimando, A. M.; Miller, B. D.; Schmidt, D. G. Chemistry without Borders: An Overview. ACS Symp Ser. 2016, 1219, 1–13. Eliel, E. L. From Cologne to Chapel Hill; American Chemical Society: Washington, DC, 1990.

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Carbohydrates

Chapter 7

Saccharide Structure and Reactivity Interrogated with Stable Isotopes Wenhui Zhang, Reagan Meredith, Mi-Kyung Yoon, Ian Carmichael, and Anthony S. Serianni* Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556-5670, United States *E-mail: [email protected]

Several topics in saccharide chemistry and biochemistry, which were impacted by the work of Ernest Eliel and his contemporaries, are reviewed. We show how stable isotopic enrichment, NMR spectroscopy, and modern computational methods have been applied synergistically to reveal subtle and sometimes unexpected properties of saccharides in solution. Examples include the use of stable isotopes to detect and quantify the cyclic and acyclic forms of reducing sugars in solution, and to investigate relationships between saccharide structure, conformation and the kinetics of anomerization. Thermodynamic and kinetics studies of cis-trans isomerization of the N-acetyl side-chains of saccharides are enabled by selective 13C-enrichment and saturation-transfer NMR methods. Redundant NMR spin-couplings sensitive to the same molecular torsion angle can be interpreted collectively to derive conformational populations of flexible fragments such as O-acetyl side-chains and O-glycosidic linkages. NMR studies of saccharide chemical transformations using stable isotopes reveal stereospecific skeletal rearrangements such as C1–C2 transposition that defied prior detection, opening the opportunity to develop new catalysts and/or better understand catalytic mechanisms of chemical and biochemical processes involving saccharides.

© 2017 American Chemical Society

Introduction Carbohydrates provide a unique and expansive playground on which to investigate the intra- and intermolecular forces that dictate conformational equilibria and dynamics of molecules in solution. This opportunity evolves from the enormous structural diversity of saccharides with respect to carbon scaffold, configuration and substitution (1). This playground is particularly appealing because saccharides are biologically important molecules found in vivo in different degrees of polymerization (i.e., monosaccharides, oligosaccharides or polysaccharides) and in different modes of molecular conjugation (i.e., free in solution or appended to proteins, lipids and other biomolecules) (2, 3). This biological relevance provides a compelling argument to investigate saccharide structure, which plays key roles in determining many important biological functions and processes, including diseases such as diabetes and cancer. Unraveling the relationships between saccharide structure and their chemical and biological functions cannot be achieved, however, by restricting studies to only those saccharides found in biological systems. Such an approach, while efficient from a biological perspective, samples only a fraction of the total structural space, space that arguably must be sampled generously in order to derive reliable relationships between saccharide covalent structure and higher-order structural features such as conformational equilibria and dynamics. The term “structure” is hierarchical (Scheme 1). In its simplest definition, it describes the atoms comprising the saccharide and the covalent bonds between them. Higher-order definitions include the absolute configuration of their constituent chiral carbons, the available conformational options (conformational equilibria), and the kinetics of exchange between accessible conformational states (dynamics). These features are influenced by solvation, be it by simple solvent molecules like water or by functional groups present in the binding site of a biological receptor. If the saccharide contains ionizable functionality, solution pH may influence some or all of these properties (4, 5).

Scheme 1. Hierarchies of molecular structure 106

The enormous scientific achievements of Ernest Eliel in the field of organic stereochemistry benefitted from complementary studies of saccharides. Indeed, the book entitled Conformational Analysis by Eliel, Allinger, Angyal and Morrison, published in 1965 (6), testifies to this fact, wherein many of the stereochemical principles articulated by Eliel from his studies of general organic systems were applied, tested and refined with the use of saccharides. The inclusion of Stephen Angyal as a coauthor of this seminal book was no accident; Eliel realized the central role of saccharides in confirming and amplifying the principles of stereochemistry (7–9) that he had worked to develop. Researchers who have built upon the solid foundation provided by Eliel’s seminal studies have benefitted from research tools and methods that were unavailable, perhaps unimaginable, in the mid-20th century. These tools include, among others, very high field superconducting magnets (>14 Tesla) in NMR spectroscopy to improve spectral dispersion and sensitivity (10), multi-dimensional NMR data collection to resolve and assign complex 1H NMR spectra (11), polarization transfer methods to increase NMR sensitivity and selectivity (12, 13), and routine access to highly enriched and pure stable isotopes such as 13C, 15N and 17,18O on large scales and at reasonable cost (14). The timely convergence of these tools enabled modern NMR structural studies of complex molecules having molecular weights in excess of 50 kD, a remarkable development considering that 1H NMR spectrometers at 60–90 MHz (1.41 Tesla) using permanent magnets and operating in continuous wave modes were just coming of age in the 1960’s when Eliel was conducting his research. In this chapter, several topics pertinent to the field of saccharide chemistry and biochemistry are discussed which were impacted by early work of Eliel and his contemporaries. We show how the interplay of isotopic enrichment and modern NMR methods, coupled to modern computational methods, has been used to reveal subtle and surprising properties of these important biomolecules, including unusual skeletal rearrangements.

Saccharide Anomerization The spontaneous ring-opening and ring-closing of aldoses and ketoses in solution is known as anomerization (15, 16). This process involves the acyclic aldehydo and keto forms of reducing saccharides as central intermediates (Scheme 2). The types and distributions of cyclic forms produced depend on aldose and ketose structure; typically only five- (furanose) and six -membered (pyranose) rings form, since larger and smaller rings have unfavorable enthalpies and/or entropies of activation (17). In addition to ring-opening and ring-closing, the acyclic carbonyl forms of aldoses and ketoses can also react with solvent water to give acyclic hydrate forms (gem-diols) (Scheme 2).

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Scheme 2. General scheme showing exchange between cyclic and acyclic forms of an aldose in solution during anomerization

Modern experimental measurements of anomerization equilibria are commonly made by NMR spectroscopy, and 13C NMR in conjunction with selective 13C-enrichment at anomeric carbons provides a superior approach to making these determinations (18–20). This application is illustrated in Figure 1, which shows a 13C{1H} NMR spectrum (150 MHz) obtained on an aqueous (2H2O) solution of D-[1-13C]mannose (1) in which six monomeric forms are detected in equilibrium.

Since 1 contains 99 atom-% 13C isotope at C1, the detection of the labeled carbons is ~100 times greater than for the remaining natural abundance carbons. Signals from the weak natural carbons can be observed between 60–80 ppm. The intense signals at ~95 ppm arise from the labeled C1 carbons of the dominant pyranose forms, while the furanose C1 signals appear slightly downfield of the pyranose C1 signals. The very weak signal observed at ~205 ppm arises from labeled C1 of the acyclic aldehyde form, while that at ~91 ppm arises from labeled C1 of the acyclic hydrate form (Tables 1 and 2). Integration of the C1 signals gives the following percentages of forms in solution at 30 °C: α-pyranose, 66.24 ± 0.05%; β-pyranose, 32.85 ± 0.06%; α-furanose, 0.64 ± 0.04%; β-furanose, 0.25 ± 0.04%; 108

aldehyde, 0.0044 ± 0.0004%; hydrate, 0.022 ± 0.001% (18). The large chemical shift dispersion of the C1 signals makes 13C NMR highly suitable for the detection of cyclic and acyclic forms of reducing saccharides in solution. When the reducing saccharide is a ketose, 13C NMR provides the only reliable means to determine anomeric equilibria, since these molecules lack anomeric hydrogens.

Figure 1. 13C{1H} NMR spectrum of D-[1-13C]mannose (1) in 2H2O, showing the assignment of the labeled C1 signals from the six monomeric forms in solution (α/β-pyranoses, α/β furanoses, and the acyclic aldehyde and hydrate forms). The weak signals between 60–80 ppm arise from the natural abundance C2–C6 carbons in the six forms. Data were taken from ref. (18). A source of error in the measurements shown in Figure 1 is the potential for signal mis-assignment, especially that of the acyclic hydrate form. This problem can be partly addressed by measuring the NMR J-coupling between C1 and its directly attached hydrogen (1JC1,H1) (Table 1). These 1JCH values are sensitive to structure near the C1 carbon, and their values, in addition to C1 chemical shift, can be used to make signal assignments. An example of this application is shown in Figure 2, which shows the C1 carbon signal of the hydrate form of 1 when its directly attached hydrogen (and other hydrogens two- and three-bonds removed from C1) are decoupled or coupled to the C1 carbon. The large splitting (164.2 Hz) is attributed to the one-bond 1JC1,H1. 1JC1,H1 values of 164 – 165 Hz are typically observed for hydrate forms, and 178 – 183 Hz for aldehyde forms (Table 1); significant deviations from these values constitute evidence that the assignment may be incorrect. 109

Table 1. C1 Chemical Shifts and 1JC1,H1 Values for the Cyclic and Acyclic Forms of D-[1-13C]Aldopentoses

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Table 2.

13C

Chemical Shiftsa of C1 in the Cyclic and Acyclic Forms of D-[1-13C]Aldohexoses in Aqueous Solutionb

Figure 2. Appearance of the C1 signal of the hydrate form of 1 in 13C{1H} NMR spectra obtained with (A) and without (B) broadband 1H-decoupling. The signal in (B) is split by the large 1JC1,H1 (164.2 Hz) characteristic of hydrate forms. In this case, additional splittings are not observed, indicating that 2JC1,H2 and 3JC1,H3 values are probably small or zero. Data were taken from ref. (18).

13C{1H}

NMR spectra such as that shown in Figure 1 provide equilibrium constants for the component equilibria of aldose anomerization through signal integration, provided that the data were acquired under conditions that allow accurate quantitative analysis (18, 19). The results of studies of aldopentoses and aldohexoses are summarized in Tables 1–4, which list the C1 chemical 111

shifts of the various forms, 1JC1,H1 values, and percentages in solution, for the cyclic and acyclic forms of aldopentoses and aldohexoses in aqueous solution. A comparison of the percentages of acyclic forms of these aldoses is shown in Figure 3. Aldehydic content ranges from 0.0032 – 0.094% in solution, with solutions of allose and glucose containing the smallest percentages and those of ribose and idose the largest. Hydrate percentages range from 0.0059 – 0.7%, with solutions of allose, glucose and mannose containing very small percentages, and solutions of idose containing the largest (0.7 %; data not shown in Figure 3B).

Figure 3. Percentages of aldehyde (A) and hydrate (B) forms in aqueous solutions of aldopentoses and aldohexoses. Data were taken from Tables 3 and 4. The percentage of hydrate form for idose (0.7%) is not plotted in (B). Al = allose; Ar = arabinose; At = altrose; Ga = galactose; Gl = glucose; Gu = gulose; Id = idose; Ly = lyxose; Ma = mannose; Ri = ribose; Ta = talose; Xy = xylose. Data were taken from refs. (18) and (19).

In some cases, anomerization equilibria include other acyclic forms in addition to the carbonyl and hydrate forms. This behavior is displayed by the biologically important α-ketoacid, N-acetyl-neuraminic acid (2) (Scheme 3). The partial 13C{1H} NMR spectrum of [2-13C]2 at pH 2 and 25 °C is shown in Figure 4 (21). Labeled C2 signals arising from the pyranose forms of 2 appear 112

at ~96 ppm. The β-pyranose (2βp) is most preferred (91.2 %), followed by the α-pyranose (2αp) at 5.8% (Scheme 3). The weak signal at ~94 ppm arises from the acyclic hydrate form (2h) (1.9%; Scheme 3). The spectral region between 140 – 200 ppm contains signals arising from the acyclic keto form (2k) (198 ppm; 0.7%; Scheme 3) and, unexpectedly, the acyclic enol form (2e) (143 ppm; 0.5%; Scheme 3). Natural abundance C1 (COOH) signals from 2αp and 2βp also appear in this region (these signals are split by the one-bond 1JC1,C2), as do the signals arising from the amide carbons in both pyranoses.

Figure 4. Partial 13C{1H} NMR spectrum (150 MHz) of [2-13C]2 in 95/5 v/v 1H2O/2H2O at pH 2 and 25 °C. (A) Labeled C2 signals for the 2αp, 2βp and 2h forms (Scheme 3). (B) Carboxyl region showing signals arising from the labeled C2 carbons of 2k and 2e. Unlabeled carboxyl C1 carbons and N-acetyl carbonyl (COam) carbons in 2αp and 2βp appear at ~175 ppm. Data were taken from ref. (21). 113

Scheme 3. Anomerization of Neu5Ac (2), and percentages of forms in aqueous solution at pH 2.0. Data were taken from ref. (21).

Solution conditions affect anomerization equilibria, especially temperature and pH. For example, increasing the temperature of aqueous solutions of D-[113C]threose (3) exerts little, if any, effect on the percentages of furanose forms, but the percentages of the acyclic hydrate and aldehyde forms decrease and increase, respectively, with increasing solution temperature (Figure 5) (22).

In contrast, the ketopentose, D-threo-pentulose (D-xylulose) (4), anomerizes to potentially give solutions containing two cyclic ketofuranoses and two acyclic forms (Scheme 4), but the acyclic hydrate form cannot be detected by 13C NMR even when 4 is labeled with 13C at C2 (23). The percentages of the three forms depend on solution temperature as shown in Figure 6. As observed for 3, the percentage of acyclic carbonyl form increases appreciably with increasing temperature, at the expense of the β-ketofuranose. Compared to 3, solutions of 4 contain much more acyclic carbonyl form (2.4% for 3 vs 24% for 4 at 50 °C). 114

Figure 5. Percentages of cyclic (A) and acyclic (B) forms of D-[1-13C]threose (3) in aqueous solution (2H2O, 0.1 M tetrose, 50 mM Na-acetate, p2H 5.0) at different temperatures. (A) Filled circles, α-furanose; open circles, β-furanose. (B) Filled squares, aldehyde; open squares, hydrate. The sizes of the symbols provide estimates of the errors in each data point. Data were taken from ref. (22).

The kinetics for each component equilibrium in aldose and ketose anomerization is obtainable from NMR spectra of anomerizing systems at chemical equilibrium. Since the acyclic carbonyl forms of aldoses and ketoses are the presumed obligatory intermediates in the exchange of cyclic forms and the formation of hydrates, selective saturation of the well-resolved carbonyl carbon signals (or aldehydic hydrogens) results in the transfer of saturation to corresponding signals arising from the cyclic and acyclic hydrate forms due to chemical exchange (22, 24). The resulting rate of loss in signal intensity is determined by the ring-opening rate constants, kopen, and the spin-lattice relaxation times of the signals. This application of saturation-transfer NMR spectroscopy (25–27) is illustrated for the anomerization of D-[1-13C]erythrose (5), whose anomerization equilibrium is shown in Scheme 5. Note the significantly higher percentage of acyclic aldehyde and hydrate forms of 5 compared to systems in which pyranosyl rings can form (Tables 3 and 4). For 5, only furanoses form upon ring closure of the acyclic aldehyde. If 5 is enriched with 13C at C1, three 115

signals are observed in the anomeric carbon region (Figure 7A): α-furanose (αf), β-furanose (βf) and hydrate (h). The C1 signal of the acyclic aldehyde (not shown) is observed at ~205 ppm. Saturation of the aldehyde C1 signal for increasing amounts of time causes significant loss of signal intensity for C1 of the αf and βf forms (Figure 7, B and C). Linearizing the data (Figure 7D) allows kopen values for cyclic forms, and kdehydration for the hydrate (not shown), to be determined. Determinations of the individual Keq values for the component equilibria in Scheme 5 allow kclose and khydration values to be calculated, thereby providing complete characterization of the anomerization kinetics under a specific set of solution conditions. This method is generally appropriate to measure rate constants in the range 0.05 – 10 s-1; values >10 s-1 are obtained from quantitative treatments of line-broadening in the presence of chemical exchange (Gutowsky-Holm treatment) (27–31).

Scheme 4. Anomerization of D-[2-13C]xylulose (4), showing percentages of forms in solution determined by 13C NMR (0.3 M ketose, 85/15 v/v 1H2O/2H2O, 50 mM Na-acetate buffer, pH 4.0, 26 °C). Data were taken from ref. (23).

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Figure 6. Percentages of cyclic and acyclic forms of D-[2-13C]threo-pentulose (D-[2-13C]xylulose) (4) in aqueous solution (see solution conditions in Scheme 4) at different temperatures. (A) β-furanose. (B) open squares, keto; filled circles, α-furanose. The sizes of the symbols provide estimates of the errors in each data point. Data were taken from ref. (23). The effect of phosphate group ionization on the anomerization kinetics of pentose 5-phosphates is shown in Figure 8 for D-[1-13C]ribose 5-phosphate (R5P) (6) (24).

This system is similar to that shown in Scheme 5 for 5 in that only two cyclic furanose and two acyclic forms of R5P are possible in solution. The effect of phosphate differs for both anomers, with the α-furanose more prone to ring-opening than the β-furanose at all solution pH values studied. Saturation-transfer experiments were conducted to measure kopen values at pH 2.3 and 4.0, and line-broadening experiments were conducted to make kopen measurements at the remaining pH values. In general, the presence of phosphate in the saccharide increases anomerization rate constants relative to the same 117

molecule devoid of phosphate, suggesting a potential role for intramolecular catalysis in the anomerization of phosphorylated sugars in vivo (24).

Scheme 5. Anomerization of D-erythrose (5), showing percentages of forms in 2H2O solution at 60o determined by 1H NMR. Data were taken from ref. (28).

Table 3. Percentages of Cyclic and Acyclic Forms of D-[1-13C]Aldopentoses in Aqueous Solutiona

118

Table 4. Percentages of Cyclic and Acyclic Forms of D-[1-13C]Aldohexoses in Aqueous Solutiona

Kinetic studies of anomerizing systems involving pyranosyl rings have also been reported, and data for the aldohexose, D-[1-13C]talose (7), are summarized in Scheme 6. From a thermodynamic perspective, this system is similar to that of D-mannose (1) (Figure 1), with pyranose forms dominating over furanose forms. Under the solution conditions indicated, kopen values range from 0.004 – 0.04 s-1, and kclose values range from 3 – 43 s-1. Interconversions of talopyranoses with the acyclic aldehyde occur more slowly compared to corresponding furanose interconversions. Thus, while talofuranoses are not favored thermodynamically, they are favored kinetically (32).

119

Figure 7. 13C Saturation transfer experiment conducted on D-[1-13C]erythrose (5) (0.1 M in 2H2O, 50 mM Na-acetate buffer at p2H 5.0) and 55 °C. (A and B) Partial 13C{1H} NMR spectra of 5 showing signals arising from C1 of the α- and β-furanoses (αf, βf) and hydrate (h) forms in the absence (B) and presence (B) of saturation (15 s) at C1 of the acyclic aldehyde form. (C) Plot of signal intensity vs saturation time, showing different rates of decay of the signals for αf (open circles) and βf (closed circles) forms. (D) Semilog plot of the data in (C) for αf, from which a kopen value of 0.40 s-1 is obtained; treatment of the data for βf gives a kopen of 0.19 s-1. Under these solution conditions, the effect of saturation on the C1 signal of the hydrate form is small, and only an upper limit of 80% yield, each containing one more carbon than the starting aldose (chain extension). The C2-epimeric products are purified by chromatography (60). If the hydrogenolysis is conducted with 2H2 gas in 2H2O solvent, the product [1-13C]aldoses will also contain 2H at C1 (Scheme 19) (59, 61). These [1-13C]-labeled aldoses can then be subjected to MCE to transfer the 13C and/or 2H to C2 of the C2-epimeric products. CR and MCE reactions have been effectively integrated into synthetic reaction pathways to provide access a wide range of selectively, multiply and/or uniformly labeled saccharides and their derivatives (e.g., nucleosides) (Scheme 20) (62, 63).

Scheme 19. Introduction of carbon (blue C), hydrogen (red H) and oxygen (green O) isotopes at C1 and/or C2 of aldoses through solvent exchange and cyanohydrin reduction (CR) (see color insert)

Scheme 20. Synthetic routes showing the integration of cyanohydrin reduction (CR) and molybdate-catalyzed epimerization (MCE) in the synthesis of singlyand doubly-13C-labeled aldopentoses and aldohexoses, and 13C-labeled nucleosides, from D-erythrose (see color insert) 144

Inspection of the bimolybdate complexes shown in Scheme 18 shows that the space enveloping H1 of the aldose reactant is largely unobstructed, such that replacement with a larger R-group (to give a 2-ketose reactant) should be possible without affecting reactivity. This expectation is realized in practice. Studies show that MCE interconverts 2-ketoses with 2-C-substituted aldoses with high stereospecificity, providing a convenient route to branched-chain aldoses (64–66). Two examples of this application are shown in Scheme 21. Reaction B demonstrates the high tolerance of the reaction to relatively bulky R-groups appended to C2 of the 2-ketose reactant.

Scheme 21. Two reactions (A) and (B) showing the application of molybdate-catalyzed rearrangement (MCE) to interconvert 2-C-substituted D-erythroses with 2-ketoses B. Molybdate-Catalyzed Conversion of Osones to Aldonates The C1–C2 transposition that accompanies MCE can be informally viewed as an internal redox process wherein the oxidation states of C1 and C2 are exchanged during the transformation. This mental construct for the reaction leads to the expectation that aldonates should be produced when 1,2-dicarbonyl sugars such as D-arabino-hexos-2-ulose (D-glucosone) (30) are used as reactants. Recent unpublished work from this laboratory indicates that the reaction of [1-13C]30 with molybdate at 90 °C gives D-[2-13C]gluconate (31) and D-[2-13C]mannonate (32) in a 85/15 ratio (Scheme 22). Osone 30 presumably binds bimolybdate in its dihydrate form to satisfy the hydroxyl group requirements discussed above. By analogy to the complexes that form with aldoses (Scheme 18), two different complexes with 30 are possible. One complex gives D-[2-13C]31, and the other D-[2-13C]32. Unlike the aldose reactions, however, the reaction with 30 is not reversible; the aldonates apparently cannot be converted to the osone, and thus an aldonate cannot be used to generate its C2-epimer. The negatively charged aldonates do not form bimolybdate complexes, presumably because of electrostatic repulsion (both partners are negatively charged). Since the reaction 145

is irreversible, the ratio of C2-epimeric aldonates is not determined by their relative stabilities, but rather by the relative stabilities of the two bimolybdate complexes (binding phase) and/or the relative catalytic efficiencies of the two complexes (catalytic phase). The rates of release of aldonate products from their complexes are assumed to be identical. It is interesting to note that, by analogy to the 2-ketose reactants shown in Scheme 21, 2,3-dicarbonyl sugars in their acyclic dihydrate forms should also form productive bimolydate complexes, leading to branched-chain aldonates (Scheme 23). This potential transformation, however, remains to be tested in the laboratory.

Scheme 22. 13C-Labeled products generated from the reaction of D-[1-13C]glucosone (30) with molybdate, observed by 13C NMR

Scheme 23. Hypothetical reaction of a 2,3-dicarbonyl sugar with bimolybdate to give branched-chain aldonates; this transformation remains to be tested in the laboratory.

C. Phosphate-Mediated Conversion of Osones to 2-Ketoses As discussed above, osones are reactive substrates in molybdate-catalyzed reactions where C1–C2 transposition occurs to give a pair of C2-epimeric aldonates. Recent work has shown, however, that this type of transposition in osones is not confined to molybdate-mediated reactions. Prior work has shown that D-glucosone (30) undergoes spontaneous degradation in dilute phosphate buffer at pH 7.4 and 37 °C to give D-ribulose (8) (Scheme 24) (67). Recent NMR studies conducted with D-[2-13C]glucosone (30) confirm this behavior, with unlabeled formate and D-[1-13C]ribulose observed as the major degradation products (68). The reaction pathway presumably 146

involves the formation of 2,3-enediol and 1,3-dicarbonyl intermediates, the latter undergoing attack at C1 by OH- with subsequent C1–C2 bond cleavage and protonation to give the 2-ketopentose and formate.

Scheme 24. Degradation of D-[2-13C]glucosone (30) to give D-[1-13C]ribulose (8) and unlabeled formate. The pathway presumably involves 2,3-enediol and 1,3-dicarbonyl sugars as intermediates.

Additional studies of this degradation pathway using other 13C-isotopomers of 30, however, indicated that the pathway shown in Scheme 24 is incomplete, and that, surprisingly, C1–C2 transposition also occurs during degradation. Initial indications of this transposition were found in the reaction shown in Scheme 24 in that a small amount of [13C]formate was observed by 13C NMR in the reaction mixture even though the mechanism shown does not explain its formation. A more definitive experiment was conducted in which D-[1,3-13C2]glucosone (30) was used as the substrate for degradation. Under these reaction conditions, the detection of D-[1,2-13C2]ribulose (33) in the reaction mixture would constitute clear evidence that C1–C2 transposition occurred during degradation. The 13C{1H} NMR spectrum of the products of this reaction is shown in Figure 17. These data show that most of the D-[1,3-13C2]30 degrades as shown in Scheme 24, giving D-[2-13C]8 and H13COO- as the primary end-products. However, closer inspection of the C2 signals arising from D-[2-13C]8 reveals weak satellites on each signal. The upfield region of the spectrum contains the C1 signals arising from each of the three forms of D-[1,2-13C2]8 present in solution (keto and two furanose forms). Each of these signals is split by one-bond 13C-13C J-couplings that are identical to those measured in authentic D-ribulose (23) and to the splittings measured from the C2 satellites (αf, 51.8 Hz; βf = 51.3 Hz; keto, 41.5 Hz). These and other lines of evidence indicate that during the degradation of 30, most of the carbon (~90%) flows down the pathway involving direct C1–C2 bond cleavage to give 8 and formate. However, approximately 10% of 30 undergoes C1–C2 transposition during degradation (Scheme 25). Potential mechanisms for this transposition involve inorganic phosphate as a catalyst in the initial formation of a 1,3-dicarbonyl cyclic phosphate intermediate (Scheme 26) and subsequently as a tether during C1–C2 transposition (68). It is noteworthy that arsenate appears to substitute for Pi in these reactions (68). 147

Figure 17. 13C{1H} NMR spectrum of a reaction mixture from the degradation of D-[1,3-13C2]30 (100 mM NaPi, pH 7.5, 37 °C) after 20 days. (A) Full spectrum showing three signals “a” from D-[2-13C]8 (keto and two furanose forms), and H13COO– (signal “b”). (B) The anomeric carbon region of (A) showing the furanose C2 signals of D-[2-13C]8 (signals “a”), the furanose C2 signals from D-[1,2-13C2]8 which appear as satellites on both “a” signals, and unreacted D-[1,3-13C2]30 (signals “b”). (C) Upfield region of (A) showing the C1 signals from D-[1,3-13C2]8 (three signals “a” each split by 1JC1,C2, [2-13C]glycolate (signal b) and an unidentified intermediate (signal “c”). Data were taken from ref. (68).

The preceding discussion serves to illustrate that C1–C2 transposition may be a more common skeletal rearrangement in saccharides than currently appreciated. These rearrangements are remarkable, but their detection requires the use of 13C-labeling in conjunction with NMR and other analytical methods to determine the fates of individual carbons during the reaction. In the original studies of molybdate-catalyzed C2-epimerization of aldoses (54–56), and of glucosone degradation (67), 13C-labeling was not employed, leading to erroneous or incomplete mechanisms for these reactions. It is interesting to note that the transfer of two-carbon fragments is a common occurrence in saccharide metabolism. For example, the coenzyme, thiamine pyrophosphate, promotes reactions catalyzed by the pentose phosphate pathway enzyme, transketolase, wherein the coenzyme functions as a carrier of a negatively charged acylium anion formed from the C1–C2 fragment of a 2-ketose, with the inherently unstable anion resonance-stabilized when covalently attached to the coenzyme (69–71). In principle, this carrier might also enable C1–C2 exchange during the two-carbon exchange as shown in Scheme 27, although, like the glucosone degradation pathway, only a small percentage of the catalytic cycles may follow this pathway. Studies with 13C-labeled substrates would be needed to test this possibility. 148

Scheme 25. Reaction partitioning observed during the degradation of D-[1,3-13C2]glucosone (30) in phosphate buffer (see color insert)

Scheme 26. Proposed formation of phosphate complexes during the degradation of D-glucosone (30), showing its conversion to a phosphorylated 1,3-dicarbonyl intermediate (68) (see color insert)

Scheme 27. A potential (untested) mechanism for C1–C2 transposition in 2-(1,2-dihydroxyethyl)-TPP during two-carbon (acylium anion) transfer catalyzed by transketolases. Formation of the cyclopropanediol intermediate may not be favorable. 149

Molybdenum-catalyzed skeletal rearrangements mimic enzyme-catalyzed reactions in their simplicity and high stereospecificity. Whether enzymes have evolved to exploit the inherent catalytic properties of molybdate in this fashion remains to be determined, as is the potential role of molybdate in chemical evolution. Other elements of the Periodic Table that lie in the vicinity of molybdenum have not shown an ability to catalyze C1–C2 transposition in aldoses. The one element that has not yet been tested is technetium, whose oxides have solution properties similar to those of molybdate (72), but whose rarity and radioactivity thus far have discouraged studies of its reactivity.

Concluding Remarks As discussed in the foregoing paragraphs, studies of the structures and reactivities of saccharides are enabled and/or strengthened when isotopically labeled substrates, especially 13C-labeled, are used to increase the information content of laboratory experiments. We have shown how these isotopes can be used to detect and quantify the cyclic and acyclic forms of reducing saccharides in solution and to investigate relationships between saccharide structure, conformation and the kinetics of tautomer exchange. With the use of 13C-labeled compounds, redundant NMR spin-couplings sensitive to the same molecular torsion angle can be interpreted collectively to derive conformational models of flexible fragments with minimal input from theory. The latter development provides needed experimental validation of conformational predictions derived from computational methods, especially MD simulations. Finally, studies of chemical reactivity using stable isotopes reveal remarkable skeletal rearrangements in saccharides that have defied detection, opening the opportunity to develop new catalysts and/or better understand catalytic mechanisms in chemical and biochemical systems. When Ernest Eliel and his contemporaries founded the field of stereochemistry, they established the fundamental principles of stereochemical analysis and of stereochemical control of chemical reactivity (6–9, 73). In the fifty or so years since Eliel’s pioneering work was conducted, enormous progress in isotope labeling and in analytical methods have provided new opportunities to test these fundamental principles and to extend their applications to increasingly more complex systems, including saccharides. It is safe to say that, fifty years hence, investigators looking back on work now being done will make the same claims, namely, fundamental principles remain so, but new tools and methodologies allow the discovery of new ways to exploit them.

Acknowledgments A.S. is indebted to many talented Notre Dame undergraduates, graduate students, postdocs and visiting scholars who conducted the studies discussed herein over a time period spanning more than thirty years. A.S. would also like to thank the National Institutes of Health and the National Science Foundation for their generous financial support over the same time period, with particular 150

attribution given to current funding from NSF (CHE 1402744) and to continued material and intellectual support provided by Omicron Biochemicals, Inc. I.C. thanks the Department of Energy Office of Science, Office of Basic Energy Sciences, for financial support of the Notre Dame Radiation Laboratory (NDRL) under award number DE-FC02-04ER15533. This is document number NDRL 5169.

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153

Chapter 8

Synthesis, X-ray Crystallographic and Computational Analysis of 2,3-Dideoxy-αand β-D-erythro-Hexopyranosyl Cyanides: Anomeric Effect of the Cyano Group Madeline Rotella, Mark Bezpalko, Nicholas Piro, Nicholas Lazzara, Scott Kassel, Deanna Zubris, and Robert Giuliano* Department of Chemistry, Villanova University, Villanova, Pennsylvania 19085, United States *E-mail: [email protected]

The synthesis of two 2,3-dideoxy glycosyl cyanides was carried out by the Ferrier reaction of O-peracetylated D-glucal with trimethylsilyl cyanide and Lewis acid catalysts, chemoselective reduction of the double bond in the products, and deacylation. X-ray crystallographic analysis of the products revealed features consistent with the anomeric effect of the cyano group, similar to other glycosyl cyanides that are included for comparison. Computational studies were carried out to evaluate the energies and geometrical parameters of conformations in which the cyano group is axial and equatorial in both model and synthesized compounds.

Introduction The anomeric effect is generalized in the iconic text by Eliel, Wilen, and Mander as the preference for a gauche conformation in structural units of the type R-O-C-X where X is an electronegative group such as halogen (1). An unshared pair of electrons on the oxygen is antiperiplanar to X in the more stable conformation (Figure 1).

© 2017 American Chemical Society

Figure 1. Generalized anomeric effect.

For a simple structure such as chloromethyl methyl ether the gauche conformation is more stable by approximately 2 kcal/mol (2). The term “anomeric effect” has its origin in carbohydrate chemistry, where a 2:1 α/β equilibrium ratio (preference for the axial methoxy group) in methyl α- and β-D-glucopyranosides was first noted by Edward and later termed the “anomeric effect” by Lemieux (3, 4). Studies of equilibria of acetylated aldohexopyranoses by Lemieux and Chü revealed preferences for the α anomers (axial OAc) of -0.94 and -1.48 kcal/mol respectively for peracylated xylo and lyxo-pyranoses (4). While not limited to carbohydrates, it is in the structure and reactions of glycosides that the anomeric effect has been most widely studied, although to this day the exact nature of its origins is still not completely understood. Contributions from electrostatic interactions (dipole-dipole) or hyperconjugative effects (donor-acceptor electron delocalization) involving non-bonded heteroatom lone pairs are the most widely cited factors (5, 6). Accompanying the hyperconjugative effects would be a lengthening of the C-1-C-X bond in the anomer in which the electronegative group is axial and a shortening of the O-5-C-1 bond relative to the anomer in which the electronegative group is equatorial. Hyperconjugative effects were not shown to be responsible for the anomeric effect in a computational study that suggested that electrostatic interactions and Pauli repulsions dominate the conformational preference in substituted tetrahydropyrans (7). The anomeric effect in carbohydrates has been most widely studied where the anomeric substituent is a halogen or the oxygen of a free sugar or glycoside. The focus of the study presented here is on the anomeric effect of the cyano group in hexopyranosyl cyanides. Values for the anomeric effect of the cyano group in systems such as those in Figure 1, in cyclohexane, oxane, and in carbohydrates have been reported based on computational, equilibration and NMR studies. Ab-Initio studies at the 6-31G* level of theory of systems of the type R-O-C-X where R = methyl and X = CN indicated an energy difference between gauche and trans conformations of approximately 1.43 kcal/mol (8). The energy differences between gauche and trans conformations of 3-hydroxypropanenitrile are 0.65 and 1.77 kcal/mol depending on the H-O-C-C dihedral angle (9). In an equilibration study of tetra-O-acety-1-bromo-D-glucopyranosyl cyanides and their analogs in the D-galacto series, the anomeric effect of the cyano group was estimated to be 2.42 kcal/mol and 1.95 kcal/mol, respectively, in the gluco- and galacto- series (10). Variable temperature NMR studies of cyanocyclohexane and cyanotetrahydropyran gave ring inversion ΔH° (ax→eq) values of -0.18 and +0.36 156

kcal/mol (11). In a study of conformational preferences of 2-cyano derivatives of oxane, thiane, and selenane using natural bond order (NBO) analysis with the B3LYP/6-311+G(3df,2p) wave functions, stabilization of the 2-cyanooxane conformation with the axial cyano group was found (ΔG173(solv) = 0.57 kcal/mol; solv = CCl4) along with expected lengthening of the C-CN bond and shortening of the O-C(CN) bond in the conformation in which the cyano group is axial (12). In addition to the nO−σ*C-CN interaction (HCAE) these authors also propose an nO−π*CN through-space interaction as contributors to the greater stability of the axial CN conformation. In a study published in 2013, the values of ΔE (anomeric) for 2-cyanooxane was found to be in the range 0.96 – 1.22 kcal/mol depending on the basis set and computational method; the origin of the stabilization was found to correlate with exchange components and not electrostatic effects (13). The origin of the anomeric effect in these heterocycles was also probed in a different study reported in 2016 that ascribed the stabilization of conformations with axial cyano groups to “cooperative and uncooperative impacts” of the hyperconjugative effect, Pauli exchange-type repulsions, and dipole-dipole interactions, but not nO−π*CN stabilization. In this report, the LC-ωPBE/6-311+G** calculated ΔG value for equatorial vs axial conformations of 2-cyanooxane was 0.94 kcal/mol (14). While there is a lack of general agreement on the origins and magnitude of the anomeric effect, the observed preference for conformations in which an electronegative group occupies an axial position has dramatic effects on both carbohydrate structure and reactivity. We have encountered these effects in our laboratory in research on phytotoxin synthesis based on carbohydrates that contain a cyano group at the anomeric position. The synthesis, structural studies, and computational analysis of these glycosyl cyanides are the focus of this article.

Results and Discussion Glycosyl cyanides have been used in the synthesis of glycosyl amino acids (15–17), formyl (18) and carboxyglycosides (19), heterocyclic C-glycosides (20) and nucleosides (21), exo-glycals (22), and aminomethyl glycosides (23). Additional applications of glycosyl cyanides in the synthesis of heterocycles have been described in a recent review (24). During the course of our studies of the synthesis of the phytotoxin diplopyrone from carbohydrates, we utilized glycosyl cyanides that were readily prepared by the addition of trimethylsilyl cyanide to glycals in the presence of boron trifluoride diethyl ether (Scheme 1). The procedure of Grynkiewicz and BeMiller used for the preparation of 2 gave exclusively the α-anomer (25). Other procedures for the synthesis of glycosyl cyanides also favor the formation of the α-anomer, with moderate to excellent stereoselectivities for most glycal substrates (26, 27). The preference for α-anomers in Ferrier I reactions such as these has been ascribed to the anomeric effect and conformational effects in the glycal substrate (28). Selective reduction of the alkene in 2 gave diacetate 3, which was deacylated to give diol nitrile 4 as a solid that was recrystallized from chloroform. 157

Scheme 1. Synthesis of 2,3-dideoxy-α-D-erythro-hexopyranosyl cyanide (4).

For the synthesis of β-cyano glycoside 7, we used the procedure of Ghosh and coworkers, which was reported to give a 5:2 ratio of α/β anomers 2 and 5 (Scheme 2) (26). In our hands the yield was 64% and the α/β anomeric ratio was 3:2. Optimal conditions for reduction of the alkene required prior sonication of the suspension of alkene in methanol, and the use of 1% by weight of catalyst (10% Pd/C). Heavier catalyst loadings resulted in faster reaction times but also the formation of products from reduction of the nitrile group. The β-glycosyl cyanide 6 was separated and deacylated with p-toluenesulfonic acid in refluxing methanol or with catalytic sodium methoxide in methanol (Zemplén method) at -20 °C to give 7, which was recrystallized from ethyl acetate. Caution had to be observed in deacylations with sodium methoxide in order to avoid epimerization and also the formation of imidate ester side products (20). These transformations allowed the selective manipulation of reducible functional groups in the glycosyl cyanides (29).

Scheme 2. Synthesis of 2,3-dideoxy-α-D-erythro-hexopyranosyl cyanide (7).

158

The x-ray crystallographic analyses of 2,3-dideoxy-α- and β-D-erythrohexopyranosyl cyanides 4 and 7 were carried out and tables of atomic coordinates, bond lengths, bond angles, and other data are available through the Cambridge Crystallographic Data Center (CCDC) (30). ORTEP diagrams of (Figure 2) reveal that both adopt a chair conformation with axial (4) and equatorial (7) cyano groups. Also noteworthy is the gg conformation adopted at the C5-C-6 bond. The C-4-C-5-C-6-O-6 torsion angles are 48. 3° (4) and 63.7° (7) and the O-1-C-5-C-6-O-6 torsion angles are 73.7° (4) and 58.38° (7).

Figure 2. ORTEP diagrams of 4 and 7. Bond distances for C-1-CN and O-5-C-1 obtained from our crystallographic and computational data for 4 and 7 are shown in Figure 3 along with references for crystallographic data reported in the literature (31–34). Calculated bond length values for compounds 4 and 7 are shown beneath those obtained from crystallographic data. The trends that emerge from comparisons of these bond distances are the expected lengthening of the C-1-CN bond in the α-anomers and shortening of O-5-C-1 bond, and vice versa in the β-anomers of the glycosyl cyanides. The trend seems to hold across various configurations in the pyranosides and also in the presence (or absence) of functionality at other ring carbons. Side-by-side comparisons of data for 12 and 13 and 14 and 15 are valuable in that the only changes in structure are the anomeric configurations. Compound 8, previously synthesized in our laboratory, also exhibits lengthening of the C-1-CN compared to what is observed for β-glycosides 7, 10, 11, and 13. Computational analyses were carried out to in an effort to determine the magnitude of the anomeric effect in glycosyl nitriles 4 and 7, and to compare calculated bond lengths with those obtained from crystallographic data. Structures that were analyzed computationally are shown in Figure 4. Calculated bond lengths and conformational energy differences were determined using B3LYP/6-31+G* in the gas phase and in solvent (water), and using B3LYP/6-311++G** in the gas phase. Results are shown in Tables 1 – 4. The methods used and model compounds chosen are similar to those used by Tvaroška and coworkers in their calculations of the anomeric effects of peroxy and hydroperoxy groups (35). For 2-cyanooxanes 18 and 19, the calculated standard free energy difference is 0.61 kcal/mol, favoring the axial CN group in 159

19 (gas phase, B3LYP/6-311++G**) as shown in Table 2. This value is close to that reported by Sorensen and coworkers (12) (0.57 kcal/mol, CCl4 as solvent, ε = 2.228, MP2/6-311+G(2df,2p) but less than that reported by Ghanbarpour and Nori-Shargh (14) (0.94 kcal/mol, gas phase, LC-ωPBE/6-311+G**) and da Silva and coworkers (13) (0.96 – 1.22 kcal/mol for HF/6-31G(d,p), MP2, and CCSD(T) methods. Our calculated value for ΔG° of -0.72 kcal/mol for cyanocyclohexanes 20 and 21 (gas phase, B3LYP/6-311++G**) is larger than that determined by NMR techniques (-0.2 kcal/mol) (36). The gas-phase ΔG° value is 0.05 kcal/mol at the B3LYP/6-311++G** level with only a slight preference for 4 over 7 (Table 2).

Figure 3. Selected bond distances in glycosyl cyanides from crystallographic data and computational analysis (gg conformer of 4 and 7; B3LYP/6-311++G**, gas phase).

160

Figure 4. Structures analyzed computationally in this work.

The enthalpies and Gibbs free energies calculated for 4 and 7 were found to be highly dependent on both the C-5-C-6 and C-6-O-6 conformations, which may result in a smaller than expected calculated anomeric effect for these cyano sugars. As shown in Table 4, the value for ΔG° in 4 (axial CN) increases from 0.20 to 0.57 to 0.59 kcal/mol in going from the tg, to gg, to gt conformations, while in 7 (equatorial CN) the progression is from 0.00 to 0.10 to 0.64 kcal/mol in going from gg to tg to gt. The crystallographic analyses of 4 and 7 indicate that gg conformations are adopted for both in the solid state. The calculated energy differences probed by systematically changing the O-1-C-5-C-6-O-6 and C-5-C6-O-6-H torsion angles reveal a large influence of these parameters on compound stability in both the glycosyl cyanides and their carbocyclic analogs. As a result, it is difficult to separate and quantify the relatively small stabilization due to the anomeric effect of the CN group in 4 and 7. In a study of C-C and C-O bond conformations of hydroxylmethyl groups in carbohydrates and their J-couplings, Serianni and coworkers (37) noted that the introduction of a hydroxyl group at C-4 in 22 affected the relative stabilities of rotamers in 23 and 24 with gg and gt being the most stable in 23 and tg being the most stable in 24 (Figure 5). Bond lengths were determined computationally for the C-1-CN and O-5-C1 bonds. The calculated bond lengths reported in Figure 3 for the C-1-CN and O-5-C-1 bonds in 4 and 7 are based on the gg conformation, (Table 3 and Figure 6, B3LYP/6-311++G**, gas phase). In α-anomer 4, the calculations show that the C-1-CN bond is lengthened over what it is in the β-anomer 7, as expected. A slight shortening of the O-5-C-1 bond in 4 is also observed.

161

Table 1. The B3LYP/6-31+G* calculated geometrical parameters (r and Δr in Å) and the relative enthalpy and Gibbs free energy (ΔH° and ΔG° in kcal mol−1) in the gas-phase and in solution (water as solvent) for 4 vs 7 and 17 vs 16. Each structure is represented as the gt conformer of the –CH2OH substituent.

a

Axial(4)

Equatorial(7)

Δ(Eq−Ax)

H°gas

−553.574478a

−553.573885a

0.34b

G°gas

−553.619849a

−553.619651a

0.12b

r(C1-CN)gas

1.487c

1.473c

−0.014c

r(C1-O5)gas

1.421c

1.425c

0.004c

H°solv

−553.592738a

−553.591777a

0.61b

G°solv

−553.638355a

−553.637770a

0.37b

r(C1-CN)solv

1.493c

1.476c

−0.017c

r(C1-O5)solv

1.426c

1.429c

0.003c

Axial(17)

Equatorial(16)

Δ(Eq−Ax)

H°gas

−517.656098a

−517.654200a

1.19b

G°gas

−517.702172a

−517.700546a

1.02b

r(C1-CN)gas

1.473c

1.470c

−0.003c

r(C1-O5)gas

1.548c

1.547c

−0.001c

H°solv

−517.672237a

−517.671142a

0.69b

G°solv

−517.718283a

−517.717533a

0.47b

r(C1-CN)solv

1.475c

1.471c

−0.004c

r(C1-O5)solv

1.549c

1.548c

−0.001c

H° and G° in Hartrees. Δr(Eq−Ax) in Å.

b

ΔH°(Eq−Ax) and ΔG°(Eq−Ax) in kcal mol−1.

162

c

r and

Table 2. The B3LYP/6-311++G** gas-phase calculated geometrical parameters (r and Δr in Å) and the relative enthalpy and Gibbs free energy (ΔH° and ΔG° in kcal mol−1) for 4 vs 7, 17 vs 16, 19 vs 18, and 21 vs 20, with a gt conformer of the –CH2OH substituent.

a

Axial(4)

Equatorial(7)

Δ(Eq−Ax)

H°gas

−553.727387a

−553.726822a

0.35b

G°gas

−553.772682a

−553.772609a

0.05b

r(C1-CN)

1.482c

1.468c

−0.014c

r(C1-O5)

1.420c

1.424c

0.004c

Axial(17)

Equatorial(16)

Δ(Eq−Ax)

H°gas

−517.798773a

−517.796915a

1.17b

G°gas

−517.844797a

−517.843458a

0.84b

r(C1-CN)

1.468c

1.465c

−0.003c

r(C1-O5)

1.546c

1.545c

−0.001c

Axial(19)

Equatorial(18)

Δ(Eq−Ax)

H°gas

−363.958084a

−363.95698a

0.69b

G°gas

−363.996597a

−363.995620a

0.61b

r(C1-CN)

1.484c

1.469c

−0.015c

r(C1-O5)

1.420c

1.421c

0.001c

Axial(21)

Equatorial(20)

Δ(Eq−Ax)

H°gas

−328.033622a

−328.072602a

−0.53b

G°gas

−328.034462a

−328.073746a

−0.72b

r(C1-CN)

1.468c

1.464c

−0.004c

r(C1-O5)

1.550c

1.548c

−0.002c

H° and G° in Hartrees. Δr(Eq−Ax) in Å.

b

ΔH°(Eq−Ax) and ΔG°(Eq−Ax) in kcal mol−1.

163

c

r and

Table 3. The B3LYP/6-311++G** gas-phase calculated geometrical parameters (r and Δr in Å) for 4, 7, 17, and 16, with tg, gg, and gt conformers of the –CH2OH substituent defined by dihedral angles Φ O1(C)-C5-C6-O6 and φ C5-C6-O6-H.

164

Axial(4)

Equatorial(7)

Δ(Eq−Ax)

Axial(17)

Equatorial(16)

Δ(Eq−Ax)

Φ(°) tg gg gt

168.01 −79.82 63.33

178.93 −66.93 56.90

10.92 12.82 −6.43

169.62 −60.29 60.12

176.69 −77.77 61.41

7.07 −17.48 1.29

φ(°) tg gg gt

280.56 60.46 303.43

178.45 58.55 304.53

−102.11 −1.91 1.1

287.38 −60.29 60.12

176.69 −77.77 61.41

−110.69 −17.48 1.29

r(C1-CN)(Å) tg gg gt

1.482 1.483 1.482

1.468 1.468 1.468

−0.014 −0.015 −0.014

1.468 1.468 1.468

1.465 1.469 1.465

−0.003 0.001 −0.003

r(C1-O-5)(Å) tg gg gt

1.422 1.420 1.420

1.424 1.424 1.424

0.002 0.004 0.004

1.545 1.547 1.546

1.545 1.549 1.545

0.000 0.002 −0.001

Table 4. The B3LYP/6-311++G** gas-phase calculated relative enthalpy and Gibbs free energy (bΔH° and ΔG° relative to lowest energy conformer (4 vs 7, 16 vs 17) in kcal mol-1) with tg, gg, and gt conformers of the –CH2OH substituent defined by dihedral angles Φ O1(C)-C5-C6-O6 and φ C5-C6-O6-H (see Table 3). ΔH°

ΔH°

H°gas tg gg gt

Axial(4) −553.728276a −553.727435a −553.727387a

−0.08b 0.45b 0.48b

Axial(17) −517.801381a −517.799395a −517.798773a

0.53b 1.77b 2.16b

H°gas tg gg gt

Equatorial(7) −553.727988a −553.728151a −553.726822a

0.10b 0.00b 0.83b

Equatorial(16) −517.802220a −517.799162a −517.796915a

0.00b 1.92b 3.33b

ΔG°

a

ΔG°

G°gas tg gg gt

Axial(4) −553.773312a −553.772722a −553.772682a

0.20b 0.57b 0.59b

Axial(17) −517.846666a −517.844998a −517.844797a

0.69b 1.73b 1.86b

G°gas tg gg gt

Equatorial(7) −553.773477a −553.773629a −553.772609a

0.10b 0.00b 0.64b

Equatorial(16) −517.847762a −517.845373a −517.843458a

0.00b 1.50b 2.70b

H° and G° in Hartrees.

b

ΔH°(Eq−Ax) and ΔG°(Eq−Ax) in kcal mol−1.

Figure 5. Model compounds studied by Serianni et al. (Reproduced from reference (38). Copyright 2004, ACS).

Figure 6. Illustration of tg, gg, and gt conformers of the –CH2OH substituent for 7.

165

In summary, these calculations and crystallographic analyses support the expected differences in bond lengths that are consistent with the classical anomeric effect in glycosyl cyanides. The anomeric effect of the cyano group in such compounds, while not new to this report, is confirmed by our studies that also revealed larger than expected effects of C-5-CH2OH and C-6-O-H conformations on the stability of carbohydrates in which the cyano group is axial or equatorial.

Experimental General Procedures Melting points were recorded on a Thomas-Hoover apparatus and they are uncorrected. Thin-layer chromatography was carried out on aluminum foil-backed silica gel plates (EMD) coated with a fluorescent indicator. Plates were developed with cerium molybdate stain. Flash chromatography was carried out using 230-400 mesh silica gel. NMR spectra were recorded on a Varian (Agilent) Mercury 300 Plus spectrometer in CDCl3 or CD3OD for 1H NMR at 300.0 MHz, tetramethysilane reference, δ = 0.0 ppm, and, 13C NMR 70.0 MHz, CDCl3 reference, δ = 77.0 ppm. Spectral assignments were confirmed using DEPT, 13C detected HETCOR, and gHMBC experiments. Crystallography Experimental procedures for the crystallographic analysis of 4 and 7 are included in supplementary data along with tables of crystal data and structure refinement, thermal parameters, bond lengths, bond angles, and torsion angles. These data have been deposited with the Cambridge Crystallographic Data Centre (30). Computational Methods Crystallographic .cif data files for 7 were used as inputs and structurally modified as needed for HF/6–31G* geometry optimizations for all structures shown in Figure 4. Subsequent B3LYP/6-31+G* optimized geometries were obtained with analytic harmonic frequencies examined to determine the nature of the stationary points observed; calculations were conducted both in the gas-phase and in water as a continuum dielectric. Gas-phase geometries were then optimized for varied dihedral angles Φ O1(C)-C5-C6-O6 and φ C5-C6-O6-H at the B3LYP/6-311++G** level of theory using Spartan10. Relative energies (H° and G°) are reported for the B3LYP optimized structures and include zero-point energy corrections. 4,6-Di-O-acetyl-2,3-dideoxy-α-D-erythro-hexopyranosyl Cyanide 3 To a stirring solution of 2 (25) (1.664 g, 7 mmol) in methanol (30 mL) was added 10% Pd/C (1.7 mg, 1% by wt.). Dissolution of 2 in methanol required sonication for approximately 40 min (prior to adding catalyst). The mixture 166

was stirred under hydrogen at atmospheric pressure (balloon) and progress was monitored by 1H NMR. The reaction could also be carried out using a Burrell wrist-action shaker or a Parr apparatus (30 psi). Complete reduction typically occurred after 1-2 days. The reaction mixture was filtered through Celite using an additional portion of methanol (10 mL) and the filtrate was concentrated under reduced pressure to give 1.36 g (81%) of 3 as an oil: Rf 0.3 (40% ethyl acetate-hexanes), [α]D +56.2 (c, 1.0, dichloromethane, lit (25) [α]D +37, 1H NMR (300 MHz, CDCl3) δ 4.85 (d, 1H, J1,2 = 5.4, H-1), 4.70 (ddd, 1H, J = 4.8, 10.5, 10.6, H-4), 4.28 (dd, 1H, J5,6 or J5,6′ = 4.8, J6,6′ = 12.3 H-6 or H-6′), 4.15 (dd, 1H, J5,6 or J5,6′ = 2.4, J6,6′ = 12.3 H-6 or 6′), 3.94 (m, 1H, H-5), 2.3-1.8, (m, 4H, H-2,2′, H-3,3′), 2.10 (s, 3H, CH3), 2.08 (s, 3H, CH3); 13C NMR (75.4 MHz, CDCl3) δ 170.7, 169.8 (C=O), 116.6 (CN), 74.3, 66.3, 64.2, 62.3, 27.6, 25.4, 20.9, 20.7. The 1HNMR spectrum of 3 (300 MHz) matched that reported (200 MHz) (25). HRMS calcd for C1H16NO5[M + H]+: 242.1028. Found: 242.1026. 2,3-Dideoxy-α-D-erythro-hexopyranosyl Cyanide 4 To a stirring solution of diacetyl derivative 3 (205 mg, 0.85 mmol) in anhydrous methanol (20 mL) at -20 °C was added drops of commericially available 25% sodium methoxide/methanol until the solution remained basic (approximately 0.25 mL total). Starting material was consumed after 2.5 h as evidenced by TLC (9:1) chloroform methanol. A scoop of Dowex 50H+ resin was added and after stirring briefly the mixture was filtered and concentrated. Purification by flash chromatography (38) gave 4 (92, mg, 69%) as a solid: Rf 0.17 (9:1 CHCl3-CH3OH), mp 78-80 °C, [α]D + 66 (c, 1.2, methanol), 1H NMR (300 MHz, CD3OD) δ 4.95 (m, 1H, H-1), 4.86 (bs, 2H, OH), 3.85 (dd, 1H, J = 1.8, 12.3, H-4), 3.67 (dd, 1H, J = 4.5, 13.5, H-5), 3.45 (m, 2H, H-6,6′), 2.07 – 1.63 (m, 4H, H-2,2′, H-3,3′); 13C NMR (75.4 MHz, CD3OD) δ 117.2 (CN), 79.7 (C-1), 64.54, 63.8, 61.1, 28.4, 27.7. Recrystallization from chloroform gave a sample for x-ray analysis. HRMS calcd for C7H11NO3Na [M + Na]+: 180.0641. Found: 180.0637 Deacetylation of 3 was also carried out by acid-catalyzed methanolysis. A mixture of 3 (184 mg, 0.76 mmol) and p-TsOH (7.3 mg, 5 mol%, dried at 40 °C under 25 mm vacuum for 24 h) and anhydrous methanol (8 mL) was stirred under reflux for 10 h. TLC (9:1 chloroform/methanol) showed only traces of starting material and product. Barium carbonate was added and after stirring 5 min the mixture was filtered through a pad of Celite and concentrated under reduced pressure to a residue that was purified by flash chromatography to give 62 mg (52%) of 4. 4,6-Di-O-acetyl-2,3-dideoxy-β-D-erythro-hexopyranosyl Cyanide 6 To a stirring solution of a mixture of 2 and 5 (26) (0.72 g 2.88 mmol) in methanol (15 mL) sonicated for approximately 40 min was added 10% Pd/C (7.6 mg) and the mixture was. The mixture was shaken in a Burrell wrist-action shaker under hydrogen at atmospheric pressure (balloon) overnight, filtered through Celite using an additional portion of methanol (10 mL) and the filtrate 167

was concentrated under reduced pressure. Separation of product mixture was carried out by flash chromatography (30% ethyl acetate/hexanes) to give 184 mg (26%) of α-anomer 3 and 172 mg (25%) of β-anomer 6 as oils. Compound 6 had: Rf 0.24 (40% ethyl acetate-hexanes), [α]D +45 (c, 1.2, chlorororm), 1H NMR (300 MHz, CDCl3) δ 4.69 (ddd, 1H, J = 4.8, 10.5, 10.2, H-4), 4.26 (dd, 1H, J1,2a = 10.5, J1,2e = 3.3, H-1), 4.16 (m, 2H, H-6,6′), 3.56 (m, 1H, H-5), 2.4-1.5, (m, 4H, H-2,2′, H-3,3′), 2.06 (s, 3H, CH3), 2.01 (s, 3H, CH3); 13C NMR (75.4 MHz, CDCl3) δ 170.7, 169.78 (C=O), 116.9 (CN), 76.6, 66.2, 65.4, 62.6, 29.0, 28.2, 20.9, 20.8. HRMS calcd for C1H16NO5[M + H]+: 242.1028. Found: 242.1028. 2,3-Dideoxy-β-D-erythro-hexopyranosyl Cyanide 7 To a stirring solution of diacetyl derivative 6 (172 mg, 0.71 mmol) in anhydrous methanol (18 mL) at -20 °C was added a few drops of commericially available 25% sodium methoxide/methanol until the solution remained basic (approximately 0.2 mL total). Starting material was consumed after 2 h as evidenced by TLC (9:1) chloroform methanol. A scoop of Dowex 50H+ resin was added and after stirring briefly the mixture was filtered and concentrated. Purification by flash chromatography gave 7 (81, mg, 73%) as a solid: Rf 0. 13 (9:1 chloroform-methanol), mp 138-139.5 °C, [α]D + 78.9 (c, 1.0, methanol), 1H NMR (300 MHz, CD3OD) δ 4.90 (bs, 2H, OH), 4.40 (dd, 1H, J = 11.7, 2.4, H-4), 3.84 (dd, 1H, J1,2a = 12.3, J1,2e = 2.1, H-1), 3.65 (dd, 1H, J = 6.3, 12.3, H-6 or H-6′), 3.41 (ddd, 1H, H-6 or H-6′), 3.19 (m, 1H, H-5), 2.20 – 1.40 (m, 4H, H-2,2′, H-3,3′); 13C NMR (75.4 MHz, CD3OD) δ 117.9 (CN), 83.6 (C-1), 64.9, 64.4, 61.3, 31.1, 29.5. Recrystallization from ethyl acetate gave a sample for x-ray analysis. HRMS calcd for C6H11O3 [M - CN]+: 131.0708. Found: 131.0705

Acknowledgments and Dedication The authors thank the Chemistry Department of Villanova University for financial support. The honor of being included in this volume is particularly meaningful for us as two of our former faculty members knew Ernest Eliel very well. Jose R. de la Vega obtained his doctorate from the Universidad de la Habana in Cuba and was a faculty member at Villanova from 1961-1998. Walter W. Zajac, Jr. was a faculty member at Villanova from 1959-2000. Jose, a physical chemist, and Walter, an organic chemist, both spoke very fondly of Ernest Eliel as a scientist and educator.

References 1. 2. 3.

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33. Kini, G. D.; Petrie, C. R.; Hennen, W. J.; Dalley, N. K.; Wilson, B. E.; Robins, R. K. Carbohydr. Res. 1987, 159, 81–94. 34. Somsák, L.; Czifrák, K.; Deim, T.; Szilágyi, L.; Bényei, A. Tetrahedron: Asymmetry 2001, 12, 731–736. 35. Kośnik, W.; Bocian, W.; Chmielewski, M.; Tvaroška, I. Carbohydr. Res. 2008, 343, 1463–1472. 36. Eliel, E. L.; Wilen, S. H; Mander, L. N. Stereochemistry of Organic Compounds; John Wiley and Sons: New York, 1994; pp 696−697. 37. Thibaudeau, C.; Stenutz, R.; Hertz, B.; Klepach, T.; Zhao, S.; Wu, Q.; Carmichail, I.; Serianni, A. S. J. Am. Chem. Soc. 2004, 126, 15668–15685. 38. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923–2925.

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Chapter 9

Hydration-Mediated Effects of Saccharide Stereochemistry on Protein Heat Stability Renata Kisiliak and Yoav D. Livney* Biotechnology & Food Engineering, Technion, Israel Institute of Technology, Haifa, 3200000, Israel *E-mail: [email protected]

Thermal stability of globular proteins is important in biotechnological, food and pharmaceutical applications. Sugars provide significant protection against thermal denaturation of globular proteins, but the mechanisms of this protection are still incompletely understood, particularly the role of sugar stereochemistry in its effectiveness as a protein stabilizer. To shed new light on this important problem, we systematically studied isomeric sugars within groups of mono- and di-saccharides, and investigated the effect of stereochemistry on sugar hydration, and its impact on protein stability. No binding was found between the sugars and the protein, supporting our hypothesis that sugars affect proteins mainly indirectly via the water. Protein denaturation temperature increased with increasing sugar concentration in the following order of efficacy: Monosaccharides: galactose > glucose > mannose; Disaccharides: trehalose > cellobiose > maltose. Our main finding is that the extent of thermal protection conferred to the protein correlates with the hydration number (nh) of the sugar within each group of isomers.

Introduction Low molecular-weight carbohydrates (also called osmolytes) are known to confer protective effect against deterioration of biological molecules in living © 2017 American Chemical Society

organisms under stressed conditions, such as high temperature, drying or freezing. As a result of these well-known phenomena, numerous studies have been carried out in this field to reveal the mechanisms of the stabilization, and to apply this protective effect, e.g. in biological material preservation during processing and storage. Globular proteins, for example, provide many important functional and physicochemical properties to food products, by catalyzing reactions, absorbing to the oil or air surface and stabilizing emulsions and foams, forming gels and more (1–3). The functionality of such proteins is strictly dependent on their structure and stability, which are affected by various parameters, such as temperature, pressure, humidity (during processing and storage), and the presence of co-solutes, like sugars or salts. In food and pharmaceutics production, thermal processes like heating, drying, or freezing play a major technological role, serving various purposes. Heating modifies the structure of proteins and their functional properties in solutions, affecting solubility, viscosity, gelation and surface properties. Adding co-solutes that can be either ionic (4) or non-ionic (5) is an effective way to protect from denaturation, or to solubilize proteins, thereby controlling protein’s functionality. The influence of low-molecular weight solutes on proteins has been studied intensively using different techniques, including NMR (6–9), Raman and IR spectroscopy (6, 10–15), density and sound velocity measurements (16–22), circular dichroism (6), fluorescence spectroscopy (23), light scattering (11), calorimetry (6, 17, 18, 24, 25) and molecular dynamics simulations (22, 26–31). Despite the fact that stabilizing effects of osmolytes are being investigated for about a century, this issue still remains a matter of debate. A general mechanism of protein stabilization by osmolytes in aqueous solutions suggested in literature is known as “The preferential interaction theory”, by Timasheff and co-workers (32–35). This classical view focusses on the relative interactions of the protein with water vs. the protein with the cosolute: co-solutes which are preferentially adsorbed onto the protein, solubilize and denature it, while those which lead to the preferential hydration of the protein, tend to protect the globular native state of proteins in aqueous solutions. On the basis of extensive studies Timasheff and his colleagues had conducted for more than half a century, he postulated that kosmotropic osmolytes are preferentially excluded from the protein surface, whereas water molecules accumulate near its surface. Thus, sugars (known noionic kosmotropes) lead to preferential hydration of the protein, i.e., at equilibrium, there is a higher water concentration near the protein compared to the bulk (19, 33, 36). To sustain this sugar concentration gradient, there is a need for energy investment, so the resultant state of the solution is energetically higher (and less favorable), than the state in the absence of the sugar. As a consequence, a preferentially excluded solute drives the equilibrium between the native and denatured states of the protein toward the native state – a state of the protein that has lower surface area exposed to water than the denatured state. This is well demonstrated by earlier works that showed that kosmotropic osmolytes decreased the apparent specific volume and adiabatic compressibility of several native proteins, which proved that they became more compact (33, 37–39). Concordingly, O’Connor et al. (40) measured the change in free energy of RNase and α-lactalbumin in the presence of sugars, Barreca et al. (41) found 172

the consequently expected changes in the hydrodynamic volume of BSA during heating in the absence and presence of trehalose, and Saunders et al. (41) showed by CD technique that isothermal titration of polyol osmolytes into thermally- or acid-unfolded protein causes it to fold. However, the preferential interaction theory focusses on the relative strength of interactions with the protein by the solvent and the cosolvent, while in some cases understanding the interactions between the solvent and co-solvent is the key to explaining protein stability in that solvent-cosolvent system. Consequently, the theory cannot fully explain, for example, why different sugars (and in particular, different stereoisomers of the sugar molecule) increase protein thermostability to different extents. Earlier works already reported that different carbohydrates differ in their hydration properties in binary aqueous solutions (7, 8, 16, 42, 43) differences that were expressed in terms of nh, isentropic compressibility, partial molar expansibility and partial molar volume. Newer works based on advanced techniques that analyze the system on a molecular or atomic level, also stress the importance of investigating sugar-water interactions for the understanding more complex biological systems (14, 15, 26, 40, 44, 45). Thus, sugar properties in water are important contributors to the preferential hydration of proteins in solutions, playing an important role in the explanation of protein stabilization. Therefore, in comparing sugars stabilizing effect of a protein under study, it may be preferable to compare sugars that differ in only one hydration-related property at a time, in this case- sugar stereochemistry. In our group, Shpigelman et al. (46, 47) have found a strong correlation between the hydration numbers of four isomeric sugars, galactose, glucose mannose and talose in aqueous solutions, and the extent of cloud-point depression of a synthetic polymer, poly-N-isopropylacrylamide (PNIPA), which served as a model for certain attributes of a protein. The main effect of either rising sugar concentration, or rising nh, was a shift of the coil-to-globule transition of PNIPA to lower temperatures. I.e., the more the stereochemical structure of a sugar promotes its higher hydration, the more it would promote the globular state of the polymer in its presence. We expected that a similar correlation would be observed in case of a protein dissolved in sugar solution, i.e. sugar will promote the protein’s globular state, thus retard its unfolding induced by high temperatures, and this stabilization effect will be enhanced with both sugar concentration, and the nh of the sugar. Molecular structures of the three monosaccharides used in the above work are represented in Figure 1.

Figure 1. Isomeric monosaccharides (aldohexopyranoses). From left to right: α - D(+)mannose, glucose, and galactose. 173

Thus, in the present study, we have chosen to work with β-lactoglobulin (β-lg). Bovine β-lg is a major whey protein that is used as a food ingredient and its secondary and tertiary structures are well defined. According to X-ray crystallography (48), the structure of a β-lg monomer consists of nine anti-parallel β-strands, eight of which wrap around to create a conical barrel or calyx. The 9th strand participates in the formation of a protein dimer (at neutral pH), and finally, there is a 3-turn α-helix on the outer surface of the calyx (48). It’s secondary structure consists of approximately 50% β-sheet, 15% α-helix, 20% turns and 15% random coil (10). The process of thermal denaturation of β-lg is complex and has been studied quite extensively. According to literature (48), this protein appears to denature first through dissociation of the dimer (at neutral pH), then, at higher temperatures unfolding of the alpha-helix commences, which reveals a free thiol that acts as the initiator of a sulfhydryl-disulfide interchange chain reaction, eventually leading to covalent aggregation, in addition to hydrophobic interactions-based aggregation. In this paper we report an experimental study aimed at shedding light on the mechanism of protein thermal stabilization by low molecular-weight sugars. For this purpose, we performed thermodynamic characterization of the possible sugar-β-lg interactions, using Isothermal Titration Calorimetry (ITC). We also determined a quantitative relationship between the hydration numbers of sugar isomers (mono- and di-saccharides) and their effect on thermal stability of proteins, utilizing microcalorimetry (DSC) and Fourier-Transform Infra Red (FTIR) spectroscopy techniques.

Experimental Section Materials Bovine β-lg was obtained from Davisco Foods International (Le Sueur, MN, USA), dialyzed against double de-ionized water and freeze-dried. Sugars were purchased from Fluka and Sigma Aldrich, and stored in a desiccator at room temperature. All solutions were made by weight in double de-ionized water to which 0.02% (w/v) sodium azide was added as a preservative.

Methods Isothermal Titration Calorimetry (ITC) Titration experiments were performed using a VP-ITC instrument (Micro-Cal Inc., Northampton, MA, USA). Mannose, glucose and galactose solutions at initial concentrations of 0.5 molal (0.46 M at 25°C) were injected into the sample cell (approximate volume 1.47 ml) which was filled with 1mM aqueous β-lg solution. As blanks, sugar solutions were injected into the sample cell, which was filled with water only, and also water was injected into the protein solution. These 174

two blanks were subtracted from the sample titration curves to eliminate heat of dilution effects, so that only the protein-sugar interactions would be accounted for. In the urea experiment - its concentration was 0.4 M. Several important injection parameters were as following: injection volume 9 μL, number of injections 30, equilibration time between injections 240 sec, stirring speed 307 rpm and cell temperature 25°C.

Differential Scanning Calorimetry (DSC) DSC measurements were performed with VP-DSC microcalorimeter (Micro-Cal Inc., Northampton, MA, USA). Sugar solution concentrations ranged from 0.25 to 1.0 molal for monosaccharides and from 0.05 to 0.2 molal for disaccharides. Concentration differences between mono- and di-saccharides originated from cellobiose limited solubility (relative to other two isomers). β-lg was dissolved at a constant concentration of 2 mg/ml in the sugar solutions and stirred over night at room temperature for complete dissolution. Degassed protein solutions were scanned in the DSC against the respective sugar solutions as references. Solutions were heated from 50°C to 100°C at a heating rate of 1°C/min. The temperature of the transition peak maximum in the thermogram (Td) was assigned to the denaturation temperature of the protein. Raw thermograms were analyzed by Micro-Cal Origin® software.

Fourier Transform Infra Red (FTIR) Spectroscopy To measure the effect of sugars on the conformation of a protein during its thermal treatment, a chemometric method based on a multivariate Partial Least Squares (PLS) analysis was used. This method correlates several spectral parameters with the reference values of a calibration set of spectra, thus providing a higher degree of precision (10, 49, 50). Calibration samples were prepared by mixing known amounts of two mother solutions: a solution of native β-lg at a concentration of 5 mg/ml and a solution of the denatured protein, which was obtained by incubating the native protein solution at 100°C for 5 minutes and then cooling rapidly. β-lg solution was filtered through 0.22 micron Millipore filter and its concentration after filtration was determined by measuring absorbance at 278 nm and using extinction coefficient of 0.96 L*cm1*gr-1. Thus, its final concentration was 4.2 mg/ml. This way, different contents of native and denatured structures were obtained in one sample. The content of native structure in the calibration samples ranged between 0% (only denatured solution, assuming complete denaturation occurred during the heat treatment) and 100% (only native β -lg solution, assuming the protein used was purely native, and had undergone no heat treatment). These assumptions are only approximately correct, but for the purpose of comparing the effects of sugars, only relative values are required, therefore these assumptions were sufficiently valid. The compositions prepared were at intervals of 10%. Calibration measurements were done at 25°C, in duplicates. 175

Experimental samples comprised solutions of β-lg in different sugars (each at several concentrations), which underwent the same thermal treatment (100°C, 5 min). The FTIR instrument used (Tensor 27, Bruker Optics, MA, USA) was equipped with CaF2 windows sample cell of 7 micrometer path length, and a MCT detector which was cooled with liquid nitrogen. Additionally, the instrument was purged with nitrogen gas during the experiments to eliminate water vapor. For each spectrum, a 60-scans interferogram was collected at a single beam mode and at 4 cm-1 resolution. A first derivative with 9 smoothing points was applied on protein spectra. Measurements were performed at constant and controlled temperature of 25°C.

Results and Discussion The main aim of the project was to determine a quantitative relationship between the increase in thermal stability of a protein and the stereochemistrydependent hydration number (nh) of sugar isomers. We hypothesized that the protective effect of a sugar on a protein increases with sugar hydration, i.e. the larger the nh, the more kosmotropic the sugar, and the stronger its protective effect on the protein’s globular conformation during heating. As a preceding step, we aimed at measuring the thermodynamics of possible binding interactions between the sugars and the protein macromolecule, to determine whether there is any direct interaction occurring between them. Our hypothesis was that there is no significant binding, and the effects of sugars are mainly mediated via their hydration. We used ITC to examine this hypothesis.

ITC Study - Measurement of Interactions between Sugar and β-lg Raw ITC plots of titrations of glucose and urea are presented in Figure 2. Urea was studied for comparison, as a known chaotropic cosolute (in contrast with the kosmotropic behavior of sugars). As can be seen, titrations of glucose solution into the protein solution, Figure 2(a) or into water, Figure 2(b), were exothermic. Titration of water into the protein solution, Figure 2(c), was expectedly negligible compared to other titrations. Urea, on the other hand, gave endothermic peaks when diluted in water, or added into the protein solution, Figure 2(d) and (e), respectively. Raw ITC results of galactose and mannose isomers were similar to those of glucose. It is interesting to compare sugars’ titration results with those of urea, a nonionic protein denaturant. When observing ITC curves of the three isomeric monosaccharides in Figure 3, after blank subtractions, sugars gave slightly endothermic and rather constant heat of interaction with the protein. Urea, in contrast, gave an exothermic heat of interaction, and its titration curve showed a “Langmuir-like” binding curve, rising towards saturation, in line with the known 176

fact that urea shows preferential adsorption to proteins. It can be seen that overall there is no measurable attractive interaction between the sugars and the protein and the net enthalpies of this interaction are positive and very close to zero. Moreover, we found no significant differences between the titration curves of the three monosaccharides (experiments were repeated at least twice and also at 1 molal of the sugars). Finally, no binding model could be fit to the titration curves of the sugars shown in Figure 3. The exothermic binding curve of urea to β-lg suggests of weakly attractive interactions, probably by hydrogen bonding. Furthermore, we managed to fit the urea data to a one-type-of-sites binding model with the binding constant in the order of 102 M-1, and enthalpy of -4 cal/mol, Figure3, right. It has to be stressed however, that unlike the ordinary binding with typical binding constants of 103 to 106 M-1, interactions of non-ionic co-solutes with proteins might have to be treated differently. According to Schelmann’s review (51), typical interaction constants in this case are of the order of 0.01 to approximately 1 M-1. For example, average binding constant of urea with T4 lysozyme was reportedly ~1.2 M-1. Conversely, for sugars, interaction constants with the protein are slightly less than unity. Moreover, binding constants greater than 1, indicate a favorable binding, whereas values less than 1 (which are characteristic to kosmotropic osmolytes) indicate preferential hydration of the protein.

Figure 2. Raw ITC plots of sugar and urea titrations into β-lg solution. (a) Titration of glucose into β-lg solution, (b) glucose into water, and (c) water into β-lg; (d) titration of urea into β-lg solution, (e) urea into water.

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Figure 3. Interaction curves of β-lg with isomeric monosaccharides or with urea. β-lg was titrated with (●)-urea, (■)-glucose, (○)-galactose, and (×)-mannose.

The interaction of proteins with co-solvent components is a balance between excluded volume (i.e. bulky cosolutes reduce the mixing entropy, hence are worse cosolvents for the protein compared to water) and weak binding (attractive interactions, which lead to a more negative mixing enthalpy with the protein). It is the sum of these two effects that controls the final behavior of a protein in solution; when the dominant effect is the binding, the co-solute is a denaturant. When the dominant effect is the excluded volume, the co-solute is a stabilizing osmolyte (as in case of saccharides). It is important to note that the excluded volume of sugars, due to their more bulky structure, compared to water, is enhanced by the fact they are relatively strongly hydrated (22), hence behave as if they are in fact even more bulky, leading to a more pronounced, and stereochemistry-dependent, excluded volume effect. To rule out the possibility, that the lack of binding observed at 25°C was coincidental, and that entropically-driven binding is actually possible at other temperatures, we performed ITC measurements of β-lg with glucose or urea 178

as a titrant at three different temperatures. By carrying out ITC experiments at different temperatures, it is also possible to shed more light on the type of interactions between the protein and the osmolyte (52, 53). Figure 4 shows titration curves for glucose at 3 different temperatures. These curves do not show any significant dependence on temperature, and no binding model could be fit at any of the temperatures. More pronounced differences were observed between the interaction curves of urea at different temperatures, though these differences were quite small.

Figure 4. titration curves of β-lg with glucose or urea at different temperatures. Titrations with glucose are marked by solid markers, and titrations with urea – by open markers. Titration temperatures were (■,□) 25°C, (●,○) 30°C, (▲,4) 35°C 179

Consequently, our results, which show no binding of the sugars to the protein, support our hypothesis that the stabilizing effect of sugars must be exerted indirectly, via the water. We hypothesize that the more hydrated a sugar isomer is, the larger its excluded volume effect is expected to be, hence the worse a co-solvent it would be for the protein.

DSC Study - The Effect of Sugars on the Denaturation Temperature of β-lg

To investigate the influence of sugar stereochemistry on the protection against β-lg denaturation we used DSC technique in which the denaturation temperature, Td, of a protein in various solutions can be accurately determined. There are many reports of β-lg thermal denaturation in the presence of different solutes. For example, Matheus et al. (13) reported denaturation temperature of 72.0°C of β-lg (5 mg/ml, in a pH 7.2 phosphate buffered saline) as measured by DSC at a heating rate of 1°C per minute. Burova et al. (24) described a transition temperature of 80°C of 2.24 mg β-lg/ml (dissolved in a pH 6.6 phosphate buffer) and Boye et al. (54) found that increasing glucose concentration from 100 to 500 g/l caused an increase of 6.7°C in denaturation temperature of β-lg (i.e. from 78.3 to 85.0°C). It is worth noting that according to literature, the denaturation temperature of β-lg is independent of protein concentration in the range measured (0.1 – 3 mg/ml) (24). The concentration of protein used in our DSC measurements, 2 mg/ml, was within this range. The denaturation of a protein exposes a large surface area and reveals apolar groups to the solvent, as a result of unfolding. This exposure of peptide groups may change the interaction of a protein with the solvent. Cosolutes which have more favorable interactions with the protein at its unfolded state may induce its unfolding, and vice versa (5). While observing the change in specific heat, using a differential scanning calorimeter (DSC), as a protein solution is heated, the endothermic peak observed due to this exposure of less polar groups indicates its denaturation. The temperature of the maximum point in this curve has been referred to here as the denaturation temperature (Td). We carried out DSC measurements β-lg in pure water, and in rising concentrations of the three aldohexoses under study. The difference between Td in a sugar solution and that in water was defined ΔTd. The results are presented in Figure 5. Figure 5 shows a close-to-linear dependence of ΔTd on sugar concentration. The slope of the linear fit was referred to as Kd, and it describes the strength of thermal stabilization conferred to a protein by the sugar. The inset of Figure 5 shows thermograms of aqueous solutions of β-lg in the presence of increasing concentrations of the monosaccharide galactose. It can be seen, that increasing galactose concentration shifts the endothermic peak of a protein denaturation to higher temperatures. Thus, transition temperature of β-lg increased from 82.9°C in the absence of sugar to 87.2°C in the presence of 0.5 molal galactose and to 89.7°C in the presence of 1 molal of the sugar. 180

Figure 5. ΔTd vs. monosaccharide concentration ,(■)-galactose, (♦)-glucose, (▲)-mannose. The slope of the linear fit, termed Kd, describes the strength of thermal stabilization conferred to a protein by the sugar. Inset: DSC denaturation thermograms of β-lg in the absence, and in the presence of rising galactose concentrations. Td values are marked above the peaks.

When comparing the three sugar isomers, in terms of Kd, we see that galactose was most effective in protecting the protein against thermal denaturation, followed by glucose, and mannose was least protective. We hypothesized (22) that the protein-solvent-quality of each sugar stereoisomer is related to the hydration of the sugar, and may be quantified in terms of the sugar’s nh. Fig 6 plots Kd as a function of aldohexose nh. This correlation is in the same order, but in the opposite direction compared to correlation Shpigelman et al. (47, 55, 56) found with PNIPA and these three isomeric aldohexoses, and also with talose (47). In these studies we found that the higher the nh, the more negative the slope (Km). Similarly, in another study from our group (Manukovsky et al. (57)), we found the same order of effect of the three isomers studied here, on the deswelling of PNIPA gels, and on sugar partition between the inside and outside of the gel. 181

Figure 6. Kd as a function of aldohexose hydration number (nh). Results are based on triplicates, error bars represent standard error.

The proposed explanation for the opposite trend in case of a protein compared to PNIPA is as follows: PNIPA presents LCST behavior, i.e. it goes through a coilto-globule transition upon heating in aqueous solution, so the soluting-out effect of the sugars causes a downward shift of this phase transition temperature. However, when a globular protein is heated, it behaves according to a UCST pattern, and goes from a “globular” to an “unfolded” conformation. In this case, the effect of the sugar is reversed, as the fact it is a worse cosolvent for the protein, means that it promotes the globular over the coil conformation, i.e. it shifts the transition to higher temperatures. Therefore, in both cases, the more hydrated the sugar, the worse a co-solvent it is for the polymer, and the more it promotes the globular conformation of the polymer. In the case of the globular protein, this means better protection of the protein from thermal denaturation/unfolding. We have recently proposed the “templating” effect (22) to explain the effects of different sugar stereoisomers on water structure and on polymers in the ternary solution. We have shown that sugars behave as nonionic kosmotropes thanks to the fact that they form stronger hydrogen bonds with water compared to waterwater bonds (22). We have shown that the better a sugar isomer fits into the ideal tetrahedral structure of water, as embodied in hexagonal ice, the better a template it forms for cooperative hydration, hence the higher its nh in binary (sugar-water) solutions, and the better protection it provides to the protein in a ternary solution (22). 182

Figure 7. Kd values vs. the average number of equatorial OH groups of the isomeric sugars.

Furthermore, the different influence of sugar isomers can be explained in terms of mixing entropy, or in terms of the "excluded volume effect" mentioned above. The hydrated sugar resembles larger solvent molecules, and from entropic considerations, according to the lattice model, larger solvent molecules have lower mixing entropy with the polymer, and they take up a larger volume from which the polymer is excluded. Consequently, they form worse co-solvents for the polymer, causing it to compact, thereby maximizing intra-polymer interactions, and minimizing polymer solvent interactions, thereby stabilizing the native globular conformation. The excluded volume effect may also explain the increasing “soluting out” effect with increasing sugar concentration, and with rising sugar molecular weight. An important enthalpic effect originates from the fact that in both proteins and saccharides the most common functional groups (amides in proteins, and hydroxyls in saccharides) prefer accepting hydrogens in a hydrogen bond, than donating ones. Water, on the contrary, is equally able to donate and to accept hydrogens in H-bonds. Therefore, a sugar is a worse cosolvent for the protein than water, as instead of offering hydrogens to the protein, it competes with the protein on the available water hydrogens. Another explanation suggested in the literature for the effects of different sugars (including sugar isomers) on water structure and on protein stability, is 183

the number of equatorial hydroxyls (6–8, 58) in the sugar molecules, as they are considered more solvent accessible. However, it is unlikely that a single structural property of a sugar would dictate its effect on water-structure or its protein stabilizing effects. Herein, we have observed no correlation between the Kd values and the average number of equatorial hydroxyls of the isomeric sugars, Figure 7. In contrast, the hydration number, nh, is a comprehensive consequence of many structural aspects of the sugars. In a similar manner, we studied the isomeric disaccharides maltose, cellobiose and trehalose, which are all di-glucoses, only differing in the glucoside bond position and configuration (maltose- α1-4; cellobiose β1-4, and trehalose α1-1a). As in the case of the monosaccharides, there were also positive ΔTm values in the presence of each of the three disaccharides (an example is given for cellobiose isomer in Figure 8). In this case, there also seemed to be a positive correlation between Kd and nh, (Figure 9), though not all differences were statistically significant, apparently due to the large stereosimilarity, as they are all glucose dimers. Secondly, the molal concentrations of the disaccharides used were much smaller than those of the monosaccharides (due to the low solubility of cellobiose). The lower concentrations used have probably also decreased the significance of the differences, keeping in mind that the stabilizing effect of sugars is mostly manifested at high concentrations (1).

Figure 8. ΔTd as a function of concentration of the disaccharide cellobiose.

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Figure 9 shows that trehalose was found here to be a significantly stronger stabilizer of β-lg than maltose. Cellobiose showed a slightly (though not significantly) lower protective effect than trehalose. The lowest hydration number observed by maltose apparently results from its α1-4 “folded” configuration which is responsible for the high prevalence of an intramolecular O2-O3′ hydrogen bond (59), resulting in fewer H-bonds with water, hence lower hydration number.

Figure 9. Kd as a function of disaccharide hydration number. Results are based on triplicates, error bars represent standard error.

FTIR Study - The Effect of Sugars on the Native Structure of β-lg after Heating To elucidate the influence of sugars on β-lg conformation during thermal treatment, FTIR spectroscopy, combined with chemometric analysis based on partial least squares (PLS) regression, were utilized. Figure 10(a) presents infra red spectra of β-lg, showing amide I and II peaaks, and Figure 10(b) shows its magnified amide I peak. Amide I and II peak positions are at 1636.8 and 1550.1 cm-1, respectively. Similar positions are reported by Wang et al. (10) for β-lg in aqueous solution where amide I was observed at 1628 cm-1 and amide II, at 1558 cm-1. Allain et al. (12) measured amide I of β-lg at 1635 cm-1, (experiments were performed at neutral pH).

185

Figure 10. Infra red spectra of native and denatured β-lg. (a) amide I and amide II, (b) amide I, (c) first-derivative amide I and amide II.

According to Figure 10(b), it can be seen that there is a small shift in the Amide I peak of the denatured β-lg compared to the native protein. It is important to note that the term “denatured” protein refers to an ensemble of conformational states when thermal transition is complete. In practice, a denatured protein is not necessarily equivalent to the completely unfolded protein, which is an idealized state referring to the polypeptide chain in its fully solvated conformation. We preferred the term “denatured” only to indicate the difference As was described in detail in the experimental section, a calibration curve for the quantitative analysis was based on samples of known native contents. Their spectra are presented in Figure 11(a) and (b). A calibration curve in the inset of Figure 11(c) is based on 20 points and has a correlation coefficient of 0.98. The optimal number of ranks (or the principle components, PCs) was 1 (this number is reasonable for our two-component system composed of native and denatured states). When analyzing plots of R2 and RMSE of a model versus number of PCs (not shown), it was verified that higher number of PCs could only give slightly better results of R2 and RMSE, although the improvement was not significant and so the lower number of PCs was chosen to avoid overfitting.

186

Figure 11. Infra red spectra of the calibration protein samples, (a) amide I and II, (b) amide I, (c) first derivative amide I, (d) calibration curve for the quantitative evaluation of a protein conformational change. X axis represents the actual values of native protein percent (% of native protein in the mixture of native and denatured), and Y axis represents predicted values by the model, based on the chemometric spectral analysis.

When we plotted the content of native conformation vs. sugar concentration, a linear regression was obtained. In Figure 12, an example of galactose is given, while the other two isomers gave similar results. The slope of the regression line of each graph was defined Kdf and used to indicate the strength of thermal stabilization by the sugar, as measured by FTIR (analogously to Kd obtained by DSC). When Kdf values in the presence of the different sugars were plotted as a function of sugar hydration number (Figure 13), the following results were obtained. This positive trend is in agreement with the respective positive correlation obtained by the DSC, though it can be seen that glucose did not differ significantly in the strength of thermal stabilization from mannose or from galactose, but the two extreme sugars, mannose and galactose, were statistically different. Thus, conformational results also support our hypothesis and indicate that the more hydrated the sugar is, the stronger its effect on protein stabilization of the native conformation. 187

Figure 12. β-lg native conformation content as a function of sugar concentration, by FTIR. To enable a more detailed investigation of the β-lg structural changes after heat treatment, a second-derivative of the infra red spectra was used. This is a resolution-enhancement techniques that permit a separation of the absorbance band into its components. Thus, absorbance band in the original spectrum appears as negative sub-bands (with minima peaks) in the respective second-derivative spectrum. Second derivative of the native amide I at the region of 1600-1700 cm-1, in Figure 14 (Line marked with empty arrows), resolved main structural elements that according to the literature can be assigned to the following secondary structures; 1656.8 cm-1 for α-helix, 1631.8 cm-1 for antiparallel β-sheet structure and 1686.9 cm-1 for intermolecular hydrogen-bonded β-structure (12, 54). As for the spectrum of β-lg after thermal treatment (Figure 14, line marked with solid black arrows), it can be seen that bands that corresponded to β-structures in the native protein, decreased in intensity. This trend is in line with the one reported by Boye et al. (54) who studied β-lg during heating in a transmission cell and reported the loss of these structures during the process. Also, a band that was attributed to α-helix (at 1656 cm-1) decreased in intensity and its shift to lower wavenumbers may indicate the appearance of unordered segments in the denatured protein. In addition, a new band at approximately 1618 cm-1 is due to intermolecular β-sheets that result from the process of aggregation (12). 188

Figure 13. Kdf as a function of sugar hydration number.

Figure 14. Second-derivative spectra of β-lg. Empty arrows point to the line representing the native protein, solid black arrows point to the line representing the protein heated in water only, and the other lines (grey, no arrows) – protein heated in the presence of various galactose concentrations. 189

Interestingly, spectra of β-lg that was heated in the presence of various galactose concentrations – their bands lie approximately between the native and the denatured proteins’ corresponding bands, Figure 14 (grey lines, no arrows). This result shows that sugar hinders the conformational changes in the protein induced by heating. Thus, there are milder changes in the bands attributed to β- and α-structures compared to the corresponding bands in the heated protein spectrum in water only. Thus, by monitoring conformational changes in the protein, we have shown that sugars hinder the process of unfolding and stabilize the native secondary structure and conformation.

Conclusions Based on ITC measurements, we conclude that the effect of carbohydrates on a protein is apparently not through binding to the protein, but indirectly – via the water. The exothermic heat of sugar dilution in water is an indication that the interactions of sugars with water are stronger than water-water bonds, as we have recently found strong evidence for by MD simulations (22), which is the main reason sugars are kosmotropes. We further conclude that the endothermic mixing of sugars with the protein supports our hypothesis and indicates that sugar solution is not a favorable solvent for the protein. We believe, this observation forms the basis for the sugar stabilization of a native form of a protein. We found that the hydration number of a sugar (determined based on ultra-accurate density and sound velocity measurements) can predict its stabilizing strength of a protein against thermal stress in aqueous solutions. This emphasizes the importance of sugar stereochemistry on hydration properties of different sugars in explaining their different kosmotropic effects on globular proteins. The hydration of a nonionic kosmotropic cosolute, like sugar, is an important component of the mechanism of its stabilizing effect on the protein in aqueous systems. Different orientations of hydroxyl groups in a sugar stereoisomer form a better or a worse template, respectively leading to more or less cooperative hydration, and consequently lead to higher or lower hydration number (the number of water molecules in the sugar-water cluster). The more hydrated the sugar is, the worse a cosolvent it is for the protein compared to water, mainly due to the large size of the hydrated sugar (that causes lower mixing entropy with the protein, and to an excluded volume effect, which is even stronger for higher molecular weight saccharides). The larger the hydration number, the worse a cosolvent the sugar is, thus the more it favors the compact globular state of the protein. This leads to shifting the denaturation temperature to higher values. This conclusion is also supported by FTIR protein secondary structure change measurements, i.e., the larger the hydration number, the higher the residual content of the native secondary structure of a protein which was thermally stressed in the presence of the sugar. Thus, when comparing isomeric sugars, the better a template a sugar is for cooperative hydration, the higher the hydration number, and the stronger its protective effect on the protein. 190

Also for different glucose dimers, a correlation was found between their hydration number and their protective effect against protein thermal denaturation.

Acknowledgments This work was supported by ISF, the Israel Science Foundation Grant No. 1270/05. We thank Prof. Yuval Shoham for the use of his DSC and ITC equipment, Asst. Prof. Avi Shpigelman for his help with the FTIR, and Dr. Irina Portnaya for her help with the DSC and ITC.

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Chapter 10

Soluble Polymer Containing an N-Methyl-D-glucamine Ligand for the Removal of Pollutant Oxy-Anions from Water Bernabé L. Rivas*,1 and Julio Sánchez2 1Polymer

Department, Faculty of Chemistry, University of Concepción, Casilla 160-C, Concepción, Chile 2Departamento de Ciencias del Ambiente, Facultad de Química y Biología, Universidad de Santiago de Chile, USACH, Casilla 40, Correo 33, Santiago, Chile *E-mail: [email protected]

The ability of a water-soluble poly(glycidyl methacrylate-Nmethyl-D-glucamine) polymer, P(GM-NMG), to interact with and remove arsenate, chromate, and borate from water was evaluated. The monomer GM-NMG was prepared in the laboratory and subsequently polymerized. The polymer was purified, characterized, and used as a sorbent of anions by polymerenhanced ultrafiltration. Removal experiments were conducted as a function of pH, polymer concentration, and effect of interfering ions. In addition, the maximum retention capacity of P(GM-NMG) was studied using an enrichment method. The results demonstrated that P(GM-NMG) can remove arsenate and chromate at acidic pHs and borate at basic pHs. Moreover, an increase in the concentration of P(GM-NMG) was determined to produce a higher removal of arsenic, but the retention reached a critical point in the case of chromium and boron. The presence of interfering ions slightly decreased the removal of arsenic and chromium, but not borate. The maximum retention capacity was 46.0 mg As/g polymer, 21.0 mg Cr/g polymer and 12.4 mg B/g polymer.

© 2017 American Chemical Society

Introduction Today, a variety of pollutants exist in aqueous environments that can produce serious health effects in plants, animals and humans. These contaminants are of organic or inorganic origin, and their increased presence in aqueous systems is caused by an increase in the disposal of chemical, agricultural, animal, and industrial products. Among the environmental pollutants, metal ions (cations and anions) have recently gained significant attention because of their toxicity. Some of these ions are found as oxy-anions in aqueous media. For example, arsenate, chromate and borate are oxy-anions in aqueous solution and their speciation depends on pH and concentration (1–3). Arsenic is a very toxic element that is generated by natural processes as well as by anthropogenic activities (4). Arsenic species are present in natural waters primarily as arsenate, As(V), and arsenite, As(III). The maximum permissible concentration for arsenic in drinking water accepted by the European Union and by the World Health Organization (WHO) is 10 µg/L (5). Chromium species are commonly present in the wastewater of industries such as metal plating, paints, and pigments, leather tanning, textile dyeing, printing inks and additives for wood preservation (6, 7). Chromium in aqueous media can exist as ions with trivalent and hexavalent oxidation states, with Cr(VI) existing as an oxy-anion and Cr(III) existing as the single form of a metal cation in solution (8–10). Currently, the WHO guidelines give a provisional value for total chromium of 50 µg/ L (11). Boron is widely distributed in the environment, and the presence of this element is of concern to the scientific community due to its effect on living organisms, especially plants (12). A low concentration of boron is necessary for the growth of plants but an excessive amount can be toxic. Boron is an essential element in the human diet, but the specific biochemical function has not yet been identified (13, 14). Excessive intake of boron can cause nausea, headache, diarrhea, kidney damage, and even death from circulatory collapse. The WHO had originally suggested a maximum permissible limit of 0.5 mg boron/L in drinking water; however, this value was recently revised and a new limit was set at 2.4 mg/L (3). Several technologies are used to remove arsenic, chromium and boron from water that employ membranes or polymeric materials, ion-exchange resins and fibers and water-soluble polymers (15). In general, amino and quaternary ammonium functional groups are present on the adsorbent surface, and the chromium adsorption mechanisms are electrostatic interaction and/or ion exchange (16). In addition, the strong basic nature of the quaternary ammonium groups of the functionalized adsorbents imparts high adsorption capacities over a wide range of pHs. The ligand N-methyl-D-glucamine (NMG) contains a tertiary amine and a sorbitol moiety. Ion exchangers containing NMG groups were originally dedicated to boron removal (17–19); however, they can also be used to retain As(V) (20, 21) and other oxy-anions such as Cr(VI), although the sorption mechanism of chromate ionsinvolves both electrostatic interaction with the protonated amino group and the reduction of Cr(VI) to Cr(III) (22). 198

Among the new materials for the removal of arsenic, chromium and boron, water-soluble polymers have been developed and applied in combination with ultrafiltration membranes in a polymer-enhanced ultrafiltration (PEUF) technique (1–3). PEUF is a hybrid method of membrane separation in which a water-soluble polymer and pollutant ion are in contact on the feed side of a filtration system. The water-soluble polymer interacts with the target ions, allowing the ions to bind to the polymer and form polymer-ion macromolecules that are then retained primarily by a size-exclusion mechanism. The unbound ionic species have smaller diameters than the membrane cut-off diameter and pass through with the permeate (15). The present study involves the synthesis and characterization of the water-soluble polymer poly(glycidylmethacrylate-N-methyl-D-glucamine), P(GM-NMG), and the removal of arsenate, chromate and borate as a function of pH, polymer concentration, and presence of interfering ions. In addition, the maximum retention capacity of P(GM-NMG) was studied using an enrichment method.

Experimental Section Synthesis of P(GM-NMG) To obtain P(GM-NMG), the monomer was first prepared and then polymerized. The synthesis of GM-NMG monomer was performed in a three-neck round bottom flask containing 60 mL of an N-methyl-D-glucamine (NMG, Sigma–Aldrich) aqueous solution (0.83 mol/L). Next, 6.8 mL (50 mmol) of glycidyl methacrylate (GM, Fluka) was slowly added to the NMG solution. The reaction was vigorously stirred at 70 °C for 5 h. Upon completion of the reaction, only one aqueous phase was observed. The product of the reaction was washed with diethyl ether to remove excess GM (15). The aqueous solution containing the GMA–NMG monomer was further polymerized in situ by radical polymerization. The monomer was transferred to a polymerization Schlenk tube, and the initiator of the polymerization, ammonium persulfate (Sigma–Aldrich), was added at a concentration of 1 mol %. The solution was degassed with N2 for 20 min, and the Schlenk tube was placed in a thermoregulated bath at 70 °C for 24 h under an N2 atmosphere. The synthetic route is depicted in Figure 1.

Purification by Ultrafiltration Membranes After the polymerization reaction, the polymer solution was diafiltered with twice-distilled water using a regenerated cellulose membrane with a molecular mass cut-off of 50 kDa (Millipore). The solution containing the polymeric fraction larger than 50 kDa was freeze-dried and used for analysis and removal of oxyanions. 199

Figure 1. Preparation of A) monomer GM-NMG and B) polymer P(GM-NMG) Characterization of P(GM-NMG) The polymeric fraction was then characterized by potentiometric titration, FTIR, and 1H NMR spectroscopy. In the potentiometric titration, 10 mL aliquots of 5 mmol/L P(GM-NMG) were titrated with an aqueous solution of 5 mmol/ L HNO3. The aqueous solution of HNO3 was prepared from Titrisol (Merck). Potentiometric measurements were made using a pH meter (330 Inolab WTW). FTIR spectra were obtained with a Nexus Nicollet spectrometer. For the FTIR analysis, 1 mg of sample was combined with 100 mg KBr. For the 1H NMR analysis, the polymer was dissolved in D2O and the NMR spectra were recorded on a Bruker AC 250 spectrometer (15). To determine the molecular weights of polymer fractions, the size exclusion chromatography (SEC)was used. The equipment was HPLC Flexar 200 with columns PL-AQUAGEL-OH. The polymer sample was first filtered (filter MCM de 0.45μm) and then 20 μLwere injected. The cuantification of molecular weight was carried out using TC-SEC andTurboCHROME software. Polymer-Enhanced Ultrafiltration The main components of the ultrafiltration system were a filtration unit (Amicon 8050 stirred cell of 50 mL volume), an ultrafiltration membrane filter with a 10 kDa molecular weight cut-off, a reservoir, and a pressure source, e.g., pressurized nitrogen gas (23). The oxy-anionic solutions of arsenate, chromate 200

and borate were prepared from Na2HAsO4 7H2O (Merck), K2CrO4 (Merck) and boric acid (Merck), respectively. PEUF was operated using two methods: washing and enrichment.

Washing Method For the washing method, an aqueous sample containing the water-soluble polymer and the oxy-anions to be separated was placed in an ultrafiltration cell at a given pH. The solution was brought to a total volume of 20 mL, and the pH was adjusted by the addition of 0.1 mol/L NaOH or 0.1 mol/L HNO3 (Merck). The experiments were conducted at pH levels of 3, 6, and 9. In the case of boron, the removal was also studied at pH 10. The pH was measured by a pH meter (H. Jurgen and Co., Germany). The polymer was allowed to be in contact with the oxy-anions for 10 min. The washing water in the reservoir was adjusted to the same pH of the cell solution. Filtration experiments were performed under a total pressure between 1 to 3.5 bars with a regenerated cellulose ultrafiltration membrane with an exclusion range of 10 kDa. The total volume in the cell was kept constant during the diafiltration experiments. A blank experiment was also performed without the water-soluble polymer. The effect of various interfering ions was studied using the following salts: NaCl (Merck), Na2SO4 (Fluka), and KH2PO4 (Merck). To systematically study the polymer interactions with oxy-anions in the solution using the PEUF technique via the washing method, the retention (R %) value was calculated, which is defined as the fraction of oxy-anions remaining in the cell (Equation 1).

where [oxy-anions cell] is the quantity of oxy-anions that were retained in the cell and [oxy-anions i] is the initial quantity of oxy-anions in the feed (As, Cr or B).

Enrichment Method The second method presented in this work is the enrichment method, which determines the maximum retention capacity (MRC) of the water-soluble P(GMNMG) polymer. The solution containing oxyanions was passed from the reservoir through the ultrafiltration cell that was filled with the polymer solution. The cell and reservoir solutions were adjusted to the same pH. The maximum retention capacity of the water-soluble polymer was determined by the enrichment method and is defined as follows (see equation 2):

where Pm is the amount of polymer (g), M is the initial concentration of As, Cr or B (mg/L), and V is the volume of permeate (L) that passed through the membrane. The maximum retention capacity of oxy-anions was calculated when 200 mL of permeate was collected. 201

The concentration of arsenic and chromium in the filtrate was measured by atomic absorption spectroscopy using a UnicamSolaar spectrometer (15). The concentration of boron in the filtrate was measured by the azomethine-H method (24).

Results and Discussion Characterization of P(GM-NMG) The water-soluble P(GM-NMG) polymer was characterized by the following techniques: potentiometric titration, FTIR, and 1H NMR spectroscopy. The potentiometric titration was carried out in duplicate and the curves are shown in Figure 2a. The pKa of the polymer was determined from the curve to be 6.19. The equivalence point was reached at pH 5.0, indicating that the aminated polymer was protonated below pH 5.0. These experiments revealed a decrease in the basicity of the glucamine group (pKa of NMG = 9.6). This decrease can be explained by the inductive effect of the hydroxyl groups that were closest to the tertiary amine (25).

Figure 2. a) Potentiometric titration, b) FTIR spectrum and c) 1H NMR spectrum of P(GM–NMG). Reproduced with permission from reference (15). Copyright 2017 Elsevier. 202

Figure 2b depicts the FTIR spectrum of P(GMA–NMG). The characteristic absorption bands associated with the methacrylate and NMG groups are present in the spectrum. In the case of NMG, the vibrational bands overlap at 1077 cm-1 (ν(C-O)), 1035 cm-1 (ν(C-N)) and 1174 cm-1 (ν(CH-OH)). The vibrational bands of GMA appear at 1719 cm-1 and 1563 cm-1 for ν(C=O) and ν(CO-O), respectively. In addition, the vibrations associated with the epoxy ring (1270 cm-1 and 847 cm1) were not present in the spectrum, suggesting that N-methyl-D-glucamine was attached to the precursor through opening of the epoxy ring (15). Figure 2c illustrates the 1H NMR spectrum of P(GM–NMG). The absence of a signal associated with the double bond of the monomer, which often appears between 5.0 and 6.5 ppm, provides evidence that polymerization of the vinyl group occurred. The proton signals of the main chain and NMG ligand of the polymer are interpreted in Figure 2c. Table 1 shows the molecular weights (Mw and Mn) of P(GM-NMG). As expected, free radical polymerization is capable of producing high molecular weights in relatively short reaction times. However, this type of polymerization produces a broad molecular weight distribution so the polydispersity is expected to be high. But in the case of P (GM-NMG), a relatively low polydispersity is observed. It is suggested that this result is due to the fact that the lower molecular weight polymer chains were removed when the polymer solution was fractionated.

Table 1. Molecular weightof P(GM–NMG) Polymer

Mw (Da)

Mn (Da)

Mw/Mn

P(GM–NMG)

974260

840030

1.16

Removal of Oxyanions by Polymer-Enhanced Ultrafiltration The removal of arsenic, chromium, and boron was performed via the washing method of polymer-enhanced ultrafiltration, PEUF, in separate experiments. In these experiments, we used a regenerated cellulose ultrafiltration membrane of 10 kDa. The applied pressure varied between 1 to 3.5 bar (1–3). The concentration of the polymer was constant during the ultrafiltration and the concentration of metal ion in the feed was 30 mg/L for arsenic, 30 mg/L for chromium and 2 mg/L for boron. The total volume of filtrate was 100 mL. In this section, we analyze the effect of pH, amount of polymer and presence of interfering ions on the removal capacity of the polymer.

Effect of pH on the Removal of As, Cr, and B The effect of pH on the removal of arsenate, chromate and borate was studied over a wide pH range from acidic to basic. The pH was previously adjusted in both the ultrafiltration cell and the water reservoir. 203

The results revealed that the retention of arsenic and chromium increased as the pH decreased. In the case of arsenic, when the pH was 3.0, the retention was 82.4%. At pH 3.5, 4.0 and 4.5, the retention percentages of arsenic were 50%, 35% and 14%, respectively (15). Considering the titration curve, it is possible to correlate the number of mole of protonated amine (−NH+) that were present in the feed solution with the amount of arsenic species that was removed.This observation indicated that the protonated amine and the retention capacity decreased gradually and that the retention occurred primarily through an ion-exchange mechanism on the protonated amine. However, the retention ability could not be confirmed to result purely from the amine function, as it is also known that the presence of polyol can improve the interaction with oxyanions (26). Finally, at pH values above the determined pKa (between 6.5 and 8.0), the retention of arsenic was negligible (see Figure 3). The ability of P(GM–NMG) to remove chromium was also higher at acidic pHs. Chromium is present as an oxy-anion over a wide range of pH values. The polymer removed 60% to 40% between pHs 3.0 and 4.5 (see Figure 3). The retention of chromium could result from the presence of protonated amine, therefore the interaction could be electrostatic. However, studies with resins containing NMG groups revealed that the interaction between NMG and chromium is not only electrostatic, and complexation of the reduced chromium and NMG can occur (22). Santander et al. observed in FTIR spectrum (1100–1000 cm−1) some difference in the alcohol signal (before and after chromium sorption) due to interaction of the functional groups of the polymer and metal ions. Considering the appearance of a new signal at 1728 cm−1, which indicates the presence of ketone groups, is concluded that the mechanism corresponds to the interaction of the chromium with the 1,3 cis-diol groups and the following reduction of the metal from Cr(VI) to Cr(III) forming a complex and the consequent oxidation of the resin forming the ketone group (22). In the case of boron, the results showed opposite behavior compared to arsenic and chromium. The retention of boron was highest at basic pHs. This can be explained by the chemistry of boron in aqueous media. The borate anion B(OH)4dominates at higher pH values, whereas nonionized boric acid B(OH)3 dominates at lower pHs (3). Polymers with hydroxyl groups are capable of interacting with the borate anion. The presence of borate ions is enhanced in basic media, and the chelation of borate with -OH functional groups is therefore favored at higher pH values (13, 27). Literature describes that NMG groups capture boron through a covalent attachment and formation of an internal coordination complex.Itwas also reported that boron liberating hydrolysis is relatively easy at pH less than 1.0 and therefore, relatively high acid concentrations are required for the complete and rapid elution of the boric acid from boron selective polymer (13). The boron retention capacity of P(GM-NMG) was 60% at pH 10 (see Figure 3) compared with other aminated polymers in the same experimental conditions (3).

204

Figure 3. Removal profiles of As, Cr, and B as a function of pH.

205

Influence of Polymer Concentration on the Removal of As, Cr, and B The oxy-anion removal was optimized by varying the polymer concentration and polymer:metal molar ratio at pH 3 for arsenic and chromium and at pH 10 for boron. The polymer:metal molar ratio consider the repeating unit of the polymer (monomer).The results for arsenic retention are presented in Figure 4a. The results revealed that when the polymer concentration increased, the retention capacity of arsenic also increased. This result was likely due to the quantity of functional groups that were available for binding arsenic. Several studies in the literature demonstrated that the retention of arsenic does not follow a linear dependence on the polymer concentration, and the effect of conformational changes of the polymer should be considered (23). P(GM–NMG) was successfully used even at high concentrations, obtaining retentions of As(V) greater than 80% without creating fouling problems during ultrafiltration. In the case of chromium and boron, increasing the amount of polymer was also observed to increase the removal capacity to a maximum. However, an excess of polymer can potentially induce changes in the correlative positions of complementary reactive sites, which might result from the rearrangement of charge to form densely packed coils, thereby decreasing electrostatic interactions and the removal capacity (28). The experimental data indicated that the optimum polymer:chromium molar ratio was 70:1, which gave a removal of 60% (see Figure 4b). Conversely, the optimum polymer:boron molar ratio was 40:1, which gave a maximum removal of 60% (see Figure 4c).

Effect of Competitive Anions on the Removal of As, Cr, and B To determine the selectivity of P(GM–NMG) towards As, Cr, and B, experiments were performed in the presence of interfering divalent and monovalent anions, such as sulfate and chloride. The oxy-anion removal was performed at the optimum polymer:metal molar ratio at pH 3 for arsenic and chromium and at pH 10 for boron. The removal of arsenate was observed to decrease in the presence of chloride and sulfate. The results demonstrated that the arsenic retention decreased from 82.4% to 62.3% when the chloride concentration was 280 mg/L (see Figure 5). When sulfate ions were present in the feed solution (230 mg/L), the removal of arsenic decreased to 58%. Similar behavior was observed for chromate removal. The retention of chromium decreased from 64% to 47% and 41% for a chloride concentration of 280 mg/L and a sulfate concentration of 230 mg/L, respectively. The effect of added salts on the arsenic and chromium binding by the P(GM–NMG) polymer can be understood as competition for binding sites on the polymer. The affinity of anions for binding to the polymer is similar to the behavior that is observed for an ion-exchange resin that contains ammonium groups for the removal of arsenic through an ion-exchange process (15). Generally, divalent anions bind more strongly to the charged sites of the polymer and also more effectively compress the electrical double layer around the polymer than monovalent anions (29). 206

Figure 4. Removal profiles of a) As, b) Cr, and c) B as a function of polymer:metal molar ratio.

207

Figure 5. Removal profiles of As, Cr and B in the presence of interfering ions.

Table 2. Maximum retention capacity of As, Cr, and B by P(GM-NMG) determined by the enrichment method. Pollutant

MRC

As

46.0 mg As/g polymer

Cr

21.0 mg Cr/g polymer

B

12.4 mg B/g-polymer

In the case of boron, the results revealed that the retention did not decrease with the presence of interfering ions. The removal capacity of boron at pH 10 was 60% in the absence of interfering salts (using 2 mg/L of boron in the feed). When 200 mg/L of chloride or 200 mg/L of sulfate was added to the feed, the removal capacity of boron was 62% and 64%, respectively (3). The ligand Nmethyl-D-glucamine (NMG) is an efficient boron-chelating group. This ligand has been incorporated into different matrices to study borate removal from aqueous environments (18, 30, 31). Maximum Retention Capacity of P(GM-NMG) The maximum retention capacity (MRC) of arsenate, chromate and borate by P(GM-NMG) was determined using the enrichment method. This method consists of adding the maximum concentration of arsenate to the polymer solution so that the polymer can bind oxy-anions to reach saturation. This method is similar to the column method in solid-liquid separations (23). The experiments were conducted at the optimum conditions that were previously determined from the washing method. The results showed similar behavior for the retention capacities 208

that were observed in the washing method. At optimum conditions, P(GM-NMG) has affinities and therefore retains oxy-anions in the following order: arsenic > chromium > boron. The MRC values are summarized in Table 2.

Conclusions Water-soluble P(GM-NMG) was prepared by free radical polymerization following an easy two-step synthetic route: monomer preparation and in situ polymerization. The polymer was purified and characterized by potentiometric titration to determine the pKa of the polymer. The structure of the polymer was corroborated by FTIR and 1H NMR spectroscopies. The P(GM-NMG) polymer was used as a sorbent for arsenic, chromium and boron in polymer enhanced ultrafiltration in which the pH, polymer concentration and presence on interfering ions were varied. The results demonstrated that P(GM-NMG) can remove arsenate and chromate at acidic pHs and borate at basic pHs. Moreover, an increase in the concentration of P(GM-NMG) was determined to increase the removal of arsenic, but the retention reached a critical point in the case of chromium and boron. Interfering ions slightly decreased the removal of arsenic and chromium but not borate. The maximum retention capacity of P(GM-NMG) was 46.0 mg As/g polymer, 21.0 mg Cr/ g polymer and 12.4 mg B/g polymer. These results are promising for the development of separation systems based on P(GM-NMG) and ultrafiltration membranes.

Acknowledgments The authors thank FONDECYT (Grants No 11501510 and No 11140324).JS thanks to Proyecto DICYT, code 021741SP, Vicerrectoría de Investigación, Desarrollo e Innovación, USACH.

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Note As President of the Chilean Chemical Society and President of the Latin American Federation of Chemical Associations, FLAQ, during the period 1991-1998, I had the opportunityand great pleasure to know and share with Professor Ernest Eliel. 210

Professor Eliel played an important role in stimulating and strengthening the development of chemistry in different countries of Latin America. He always had the initiative to do it and frequently participated in local and international meetings. For all of his contributions to Chilean chemistry, Professor Eliel was recognized as an Honorary Member of the Chilean Chemical Society.

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Editors’ Biographies H. N. Cheng H. N. Cheng (Ph.D., University of Illinois) is currently a research chemist at Southern Regional Research Center of the U.S. Department of Agriculture in New Orleans, where he works on projects involving improved utilization of commodity agricultural materials, green chemistry, and polymer reactions. Prior to 2009 he worked for Hercules Incorporated where he was involved at various times with new product development, team and project leadership, new business evaluation, pioneering research, and supervision of analytical research. Over the years, his research interests have included green polymer chemistry, biocatalysis and enzymatic reactions, pulp and paper chemistry, functional foods, polymer characterization, and NMR spectroscopy. He is an ACS Fellow and a POLY Fellow and has authored or co-authored 230 papers, 25 patent publications, co-edited 16 books, and organized or co-organized 30 symposia at national ACS meetings since 2000. He is active in the ACS and serves in various capacities at national, division, and local levels.

Cynthia A. Maryanoff Cynthia A. Maryanoff (Ph.D., Princeton) is Foundation Distinguished Professor at the Baruch S. Blumberg Institute, in Doylestown, PA. She began her career in 1977 at Smith Kline & French Laboratories and joined Johnson & Johnson in 1981. She advanced through various Johnson & Johnson pharmaceutical units to the highest scientific position in the company. She retired from J&J in 2013. Her publication record includes more than 100 scientific papers, several books, and 67 U.S. or European patents. The more than 1,000 drug candidates she has been involved in developing include the anti-epileptic Topamax, and an atypical analgesic, Ultram/Tramadol, for treating pain, and the drug-eluting stent CYPHER. She has been active in ACS and served in many positions including national committees and division leadership. She organized and chaired 30 award symposia and organized or co-chaired 15 other symposia at national meetings. She is the recipient of numerous awards, including ACS Fellow, AAAS Fellow, the ACS Garvan–Olin Medal, the Earle B. Barnes Award for Leadership in Chemical Research Management, the Henry F. Whalen, Jr. Award for Business Development, and the Perkin Medal for outstanding work in applied chemistry from SCI.

© 2017 American Chemical Society

Bradley D. Miller Bradley D. Miller (Ph.D., University of Arizona) is the Director of ACS International Activities. He has worked for ACS since 1999, developing programs, products, and services to advance chemical sciences through collaborations in Africa, Asia, Europe, Latin America and the Middle East. He works with ACS staff and different governance units to create opportunities for chemistry to address global challenges through in-person and web-based scientific network development, research collaborations, and educational exchange. Miller serves on the U.S. National Commission for UNESCO and in 2009 was appointed to co-chair the ACS 2011 International Year of Chemistry Staff Working Group. He is also the long-time ACS staff liaison to the ACS International Activities Committee. A world traveler and an internationalist, he speaks English, French, Spanish and Portuguese.

Diane Grob Schmidt Diane Grob Schmidt (Ph.D., University of Cincinnati), the 2015 ACS President, was an R&D Executive at Procter & Gamble, where she served as section head for 17 years. Her P&G career covered 1981-2014. She is currently an Adjunct Professor in the Department of Chemistry at the University of Cincinnati. She holds a number of patents and played key roles in such brands as Tide®, Head & Shoulders®, Pert Plus® and Safeguard®. She has received many awards, including ACS Fellow, AAAS Fellow, National Academy of Inventors Fellow, ACS national Henry Hill Award, and Distinguished Scientist of Cincinnati from the Engineers and Scientists of Cincinnati (first woman so honored). She has served on the editorial boards of Chemical & Engineering News, the Journal of the Society of Cosmetic Chemists and the Journal of Chemical Health & Safety. She has been an ACS member for many years and held a wide variety of ACS positions, including three consecutive terms on the Board of Directors. As 2015 ACS President, her presidential theme was “Inspiring and Innovating for Tomorrow.” Her legacy as ACS President includes: championing U.S. and Global Grand Challenges [Nanotechnology, Energy, Neuroscience/BRAIN Initiative] via impactful programming, establishment of the American Association of Chemistry Teachers, and a focus on industry and ACS members.

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Indexes

Author Index Atwood, C., 57 Bezpalko, M., 155 Carmichael, I., 105 Cheng, H., xiii, 1 Dave, P., 63 Dounay, A., 63 Fuller, A., 63 Giuliano, R., 155 Kassel, S., 155 Kisiliak, R., 171 Lazzara, N., 155 Livney, Y., 171 Maryanoff, C., xiii, 1 Meredith, R., 105 Miller, B., xiii, 1, 95 Montero Cabrera, L., 49 Montero-Cabrera, L., 57

O’Donnell, M., 63 Piro, N., 155 Rivas, B., 197 Rivera, D., 63 Rotella, M., 155 Samaritoni, J., 63 Sanchez, J., 63 Sánchez, J., 197 Schmidt, D., xiii, 1 Scott, W., 63 Seeman, J., 13 Serianni, A., 105 Tiano, D., 63 Yoon, M., 105 Zhang, W., 105 Zubris, D., 155

217

Subject Index A

tg, gg, and gt conformers, illustration, 165f

ACS, update on international activities ACS global alliances, 96 ACS International Activities’ interactions with Cuba, 101 ACS international center, 99 ACS-Pittcon collaboration, 100 ACS science and human rights, 98 Global Innovation Imperatives (Gii), 100 International Chemical Sciences Chapters, 97 introduction, 95 lessons learned, 101 U.S. Department of State workshops on ethics, 100

C Cyano group, anomeric effect experimental computational methods, 166 crystallography, 166 2,3-Dideoxy-α-D-erythrohexopyranosyl cyanide 4, 167 2,3-Dideoxy-α-D-erythrohexopyranosyl cyanide 7, 168 general procedures, 166 introduction, 155 generalized anomeric effect, 156f results and discussion, 157 B3LYP/6-31+G, 162t B3LYP/6-311++G, 163t B3LYP/6-311++G gas-phase calculated geometrical parameters, 164t B3LYP/6-311++G gas-phase calculated relative enthalpy, 165t 2,3-dideoxy-α-D-erythrohexopyranosyl cyanide, 158s 2,3-dideoxy-α-D-erythrohexopyranosyl cyanide, synthesis, 158s glycosyl cyanides, selected bond distances, 160f ORTEP diagrams, 159f Serianni et al, model compounds studied, 165f structures analyzed, 161f

E Educational outreach activities conclusion, 61 future ACS-SCQ interactions, 61 initial efforts, 59 introduction, 57 2016 Simposio Internacional de Química, 59 Ernest Eliel session, program, 60t Eliel, Ernest L. Eliel, Ernest, the man celebrating a high enantiomeric excess, 31f compounds synthesized by the Eliel group, examples, 30s Ernest and Eva after a luncheon, 32f global chemical community, citizen, 29 Eliel, Ernest, the scientist neoisomenthol, 19s obtaining non-racemic compounds, Eliel’s scheme, 17s stereochemistry, early interest, 16 Tamelen’s yohimbine papers, 18 Eliel’s happiest moments in life, in science, 33 after receipt of the D.Sc. degree at Duke University, 34f Chemical & Engineering News, cover, 35f Ernest as my hidden advisor, 36 Benfey, Ted, 40f Eliel holding manuscript, 38f hidden advisory role, 39 Virginia plantation, Ernest and the author, 37f hidden advisors, Ernest’s interest, 35 Woodward, R. B., in his January 9, 1970 letter, 36f introduction, 13 August 22, 2016 symposium, participants, 15f Eliel’s autobiography, cover, 16f several individuals, earlier photograph, 15f our last time together, 41

219

parting, 42 Read, John, 20 chemistry building, Notre Dame, 27f Eliel’s chiral template, preparation, 28s enantiomers of mevalolactone, synthesis, 29s from stereochemistry to synthesis, 22 stereoselective reactions, 28s stereospecific reactivity, first observation, 28s various saturated heterocycles, summary, 26s Ernest Eliel Workshop: US and Cuba Collaboration in Chemistry Education and Neglected Disease Drug Discovery, 63 assessment Cuban student perspectives and reflections, 83 dissemination of knowledge and equipment, chemical education, 82 goals, 81 non-existent unemployment rate, Cuba, 86 Sanchez, Juan (IUPUI), 87 scientific exchange, 81 student poster presentation, 82f Tiano, Daniel (Santa Clara University), 88 USA student TAs relaxing, 85f US student perspectives and reflections, 85 final thoughts, 90 future collaborations, plans distributed drug discovery dissemination, 90 research collaborations, 89 introduction Cuban Workshop, foundation, 64 workshop, lab component, 76 carrying out the D3 procedure, 80f carrying out the lab work, 79 lab students and TAs, 77f replicated teams, bill-board layout, 78f sample of A1 purified by students, 1H spectra, 81f 22 structurally unique compounds, 79f undergraduate TAs, training, 77 workshop, lecture component, 66 bill-board equipment, 70f D3 virtual catalogs, lecture on compound enumeration, 75 lecture presentation, 68f lectures at a macro level, 67

lunch between lectures and lab, 67f more detailed and applied work, lectures, 71 N-acylated natural α-amino acids, synthesis, 74s new D3 lab 9, 73s new molecules to make, student team, 72f nine D3 protocols, 69s representative molecules, 71f workshop overview Cuba Workshop participants, 66f daily program, 66 participants, 65

S Saccharide stereochemistry, hydration-mediated effects conclusions, 190 experimental section differential scanning calorimetry (DSC), 175 fourier transform infra red (FTIR) spectroscopy, 175 materials, 174 methods, 174 introduction, 171 isomeric monosaccharides, 173f results and discussion, 176 aldohexose hydration number, Kd as a function, 182f β-lg, second-derivative spectra, 189f β-lg native conformation content, 188f β-lg with isomeric monosaccharides, interaction curves, 178f β-lg with isomeric monosaccharides, titration curves, 179f calibration protein samples, infra red spectra, 187f ΔTd vs. monosaccharide concentration, 181f disaccharide cellobiose, ΔTd as a function of concentration, 184f disaccharide hydration number, Kd as a function, 185f DSC study, 180 equatorial OH groups, Kd values vs. the average number, 183f FTIR study, 185 native and denatured β-lg, infra red spectra, 186f sugar and urea titrations, raw ITC plots, 177f

220

sugar hydration number, Kdf as a function, 189f Service of humanity, science and knowledge, 49 Prof. Eliel, 52f Stable isotopes, saccharide structure and reactivity, 105 concluding remarks, 150 introduction, 106 molecular structure, hierarchies, 106s saccharide anomerization, 107 aldehyde and hydrate forms in aqueous solutions, percentages, 112f anomerization equilibria and kinetics, 121s aqueous solution, percentages, 118t C1 chemical shifts, 110t C1 in the cyclic and acyclic forms, 13C chemical shiftsa, 111t 13C Saturation transfer experiment, 120f C1 signal of the hydrate form, appearance, 111f cyclic and acyclic forms, percentages, 117f cyclic and acyclic forms of D-[1-13C]threose in aqueous solution, percentages, 115f D-[1-13C]aldohexoses, percentages, 119t D-[1-13C]mannose, 13C{1H} NMR spectrum, 109f D-[2-13C]xylulose, anomerization, 116s D-erythrose, anomerization, 118s exchange between cyclic and acyclic forms, general scheme, 108s Neu5Ac, anomerization, 114s partial 13C{1H} NMR spectrum, 113f pH dependencies, 121f saccharides, stereospecific carbon skeletal rearrangements, 142 bimolybdate complexes, interconversion, 143s C1 and/or C2 of aldoses, introduction, 144s C1-C2 transposition, potential (untested) mechanism, 149s 13C-labeled products, 146s cyanohydrin reduction, synthetic routes, 144s D-[2-13C]glucosone, degradation, 147s degradation of D-[1,3-13C2]glucosone, reaction partitioning observed, 149s

221

2,3-dicarbonyl sugar, hypothetical reaction, 146s phosphate complexes, proposed formation, 149s reaction mixture, 13C{1H} NMR spectrum, 148f two reactions, 145s saccharide structure and anomerization kinetics, relationships, 122 N-formyl and N-acetyl side-chains, 133 aldofuranose ring, proposed anchimeric mechanism, 123s anomerization, unidirectional rate constants, 125t anomerization equilibria in furanose ring systems, Thorpe-Ingold effects, 127t anomerization rate constants, Thorpe-Ingold effects, 128t O-acetyl side-chains, 139 carbonyl carbon signal intensity, plots, 138f carbonyl signals, 13C{1H} NMR spectrum, 136f carboxyl group, 130 13C-labeled mono-O-acetylated D-glucopyranoses, 141s C3–O3 fragment, Newman projection, 142s common side-chains, structures, 132s C4-substituted aldofuranose rings, structures, 122s CTI rate constants, 139f equilibrium constants, 135f experimental J-couplings, 139 furanose ring deoxygenation, effects, 126 1H NMR spectrum, 134f intramolecular catalysis, possible modes, 131s intramolecular catalysis, proposed mechanism, 129s 3JH2,NH, dependencies on, 137f ketopentoses, 124 kopen, plots, 127f Newman projection, 136s N-formyl and N-acetyl side-chains, structural properties, 132s O-acetyl side-chains, aminosugars and conformational properties, 131 penturonic acids, ring-opening rate constants, 131t preferred ring conformations, 128s probability distributions, 141f ring-opening rate constants, 129t

P(GM-NMG), synthesis, 199 polymer-enhanced ultrafiltration, 200 ultrafiltration membranes, purification, 199 washing method, 201 introduction, 198 results and discussion As, Cr, and B as a function of pH, removal profiles, 205f As, Cr, and B in the presence of interfering ions, removal profiles, 208f maximum retention capacity, 208t P(GM-NMG), boron retention capacity, 204 P(GM-NMG), characterization, 202 P(GM-NMG), molecular weight, 203t polymer concentration, influence, 206 polymer-enhanced ultrafiltration, removal of oxyanions, 203 polymer:metal molar ratio, removal profiles, 207f potentiometric titration, 202f

several aldotetroses and C5-modified aldopentoses, ring-opening rate constants, 123t Stereochemistry and global connectivity carbohydrates, 5 conclusions, 6 global connectivity, 3 introduction, 1 NMR applications, 5 organic compounds, stereochemistry, 4 tribute to Eliel, 3

W Water, removal of pollutant oxy-anions, 197 conclusions, 209 experimental section enrichment method, 201 monomer GM-NMG and polymer P(GM-NMG), preparation, 200f P(GM-NMG), characterization, 200

222