Chemistry
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Chemistry A chiral catalyst for enantioselective carbon-hydrogen activation. Work done in the laboratory of Jin-Quan Yu, Ph.D., associate professor. Jin-Quan Yu, Ph.D., Associate Professor CHEMISTRY DEPAR TMENT OF CHEMISTRY S TA F F K.C. Nicolaou, Ph.D.* Chairman Aline W. and L.S. Skaggs Professor of Chemical Biology Darlene Shiley Chair in Chemistry Dariush Ajami, Ph.D. Assistant Professor of Molecular Assembly Phil S. Baran, Ph.D. Professor Dale L. Boger, Ph.D.* Richard and Alice Cramer Professor of Chemistry Tobin J. Dickerson, Ph.D. Assistant Professor Albert Eschenmoser, Ph.D.* Professor Sheng Ding, Ph.D. Associate Professor M.G. Finn, Ph.D.* Professor Valery Fokin, Ph.D. Associate Professor M. Reza Ghadiri, Ph.D.* Professor William A. Greenberg, Ph.D. Assistant Professor of Chemistry Inkyu Hwang, Ph.D. Assistant Professor Kim D. Janda, Ph.D.** Professor Ely R. Callaway, Jr., Chair in Chemistry Director, Worm Institute of Research and Medicine Gunnar Kaufmann, Ph.D. Assistant Professor of Chemistry 2008 Jeffery W. Kelly, Ph.D.* Lita Annenberg Hazen Professor of Chemistry Ramanarayanan Krishnamurthy, Ph.D. Associate Professor Lucas J. Leman, Ph.D. Assistant Professor of Chemistry Richard A. Lerner, M.D.*** President, The Scripps Research Institute Lita Annenberg Hazen Professor of Immunochemistry Cecil H. and Ida M. Green Chair in Chemistry Roy Periana, Ph.D.***** Professor Evan T. Powers, Ph.D. Associate Professor of Chemistry Julius Rebek, Jr., Ph.D.* Professor Director, The Skaggs Institute for Chemical Biology Edward Roberts, Ph.D. Professor THE SCRIPPS RESEARCH INSTITUTE (Andrew) Bin Zhou, Ph.D. Assistant Professor of Immunochemistry 79 Deboshri Banerjee, Ph.D. Elizabeth Barrett, Ph.D.**** Roland Barth, Ph.D.***** SENIOR SCIENTIST Clay Bennett, Ph.D. Luis Martinez, Ph.D.***** Moritz Biskup, Ph.D. † Universität Karlsruhe Karlsruhe, Germany S TA F F S C I E N T I S T S Lisa Eubanks, Ph.D. Rajesh Grover, Ph.D. Sarah Hanson, Ph.D. Lubica Supekova, Ph.D. Wen Xiong, Ph.D. Anthony Boitano, Ph.D. † Genomics Institute of the Novartis Foundation San Diego, California Laure Bouchez, Ph.D. Kristopher Boyle, Ph.D. Christopher Burke, Ph.D SERVICE FACILITIES Antonio Burtoloso, Ph.D. † University of Sao Paulo Sao Paulo, Brazil Raj K. Chadha, Ph.D. Director, X-ray Crystallography Facility Mark Bushey, Ph.D. † Exxon, Inc. Union City, New Jersey Dee H. Huang, Ph.D. Director, Nuclear Magnetic Resonance Facility Darren Bykowski, Ph.D.***** Gary E. Siuzdak, Ph.D. Senior Director, Mass Spectrometry Facility Katerina Capkova, Ph.D. I N S T R U M E N TAT I O N / Floyd E. Romesberg, Ph.D. Associate Professor Petr Capek, Ph.D. Arani Chanda, Ph.D. Ke Chen, Ph.D. Peng Chen, Ph.D. William Roush, Ph.D.***** Professor SENIOR RESEARCH Peter G. Schultz, Ph.D.* Professor Scripps Family Chair Suresh Pitram, Ph.D. Govardhan Cherukupalli, Ph.D. † Epix Pharmaceuticals Lexington, Massachusetts R E S E A R C H A S S O C I AT E S Jodie Chin, Ph.D. Ramzey Abujarour, Ph.D. Srinivas Reddy Chirapu, Ph.D. Rajesh Ambasudhan, Ph.D. Chandramouli Chiruta, Ph.D. Manuel Amorin Lopez, Ph.D. Dong-Gyu Cho, Ph.D. Mark Ams, Ph.D. So-Hye Cho, Ph.D. Yoshio Ando, Ph.D. Sungwook Choi, Ph.D. Deepshikha Angrish, Ph.D. Joyanta Choudhury, Ph.D. Shinji Ashida, Ph.D. Sarwat Chowdhury, Ph.D. Micahel Baksh, Ph.D. Stepan Chuprakov, Ph.D. K. Barry Sharpless, Ph.D.* W.M. Keck Professor of Chemistry Anita D. Wentworth, Ph.D. Assistant Professor Paul Wentworth, Jr., Ph.D. Professor Chi-Huey Wong, Ph.D.* Professor of Chemistry Jin-Quan Yu, Ph.D. Associate Professor A S S O C I AT E 80 CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE Petr Cigler, Ph.D. David Edmonds, Ph.D. Neil Grimster, Ph.D. Giltae Hwang, Ph.D. T. Ryan Cirz † Achaogen South San Francisco, California Jem Efe, Ph.D. Rajesh K. Grover, Ph.D. Jan Elsner, Ph.D. † Celgene Pharmaceuticals San Diego, California Jan Grunewald, Ph.D. Michael Jahnz, Ph.D. † NOXXON Pharma AG Berlin, Germany Daniel Ess, Ph.D. Richard Guy, Ph.D.**** Ph.D. † Scott Cockroft, University of Edinburgh Edinburgh, Scotland Ph.D. † David Colby, Purdue University West Lafayette, Indiana Kevin Cole, Ph.D. † Eli Lilly and Company Indianapolis, Indiana Christine Crane, Ph.D. Matthew Cremeens, Ph.D. † Gonzaga University Redmond, Washington Fernando Rodrigo Pinacho Crisostomo, Ph.D. † Burnham Institute for Medical Research La Jolla, California Cyrine Ezzili, Ph.D. Xingang Fang, Ph.D. Simon Ficht, Ph.D. † Sanofi-Aventis Deutschland GmbH Frankfurt, Germany Joseph Rodolph Fotsing, Ph.D. † Senomyx, Inc. San Diego, California Bozena Frackowiak, Ph.D. † Politechnika Opolska Opole, Poland Etzer Darout, Pfizer Inc. Groton, Connecticut Ph.D. † Amy DeBaillie, Eli Lilly and Company Indianapolis, Indiana Graeme Freestone, Metabasis Therapeutics, Inc. San Diego, California Amelia Fuller, Ph.D. † Santa Clara University Santa Clara, California Jianmin Gao, Ph.D. † Boston College Chestnut Hill, Massachusetts Haibo Ge, Ph.D. Judith Denery, Ph.D. Ross Denton, Ph.D. † University of Cambridge Cambridge, England Savvas Georgiades, Ph.D.**** Ola Ghoneim, Ph.D. † Qatar University Doha, Qatar Caroline Desponts, Ph.D. Antonia Di Mola, Ph.D. Deguo Du, Ph.D. Anna Dubrovska, Ph.D. Viktoriya Dubrovskaya, Ph.D. Joshua Dunetz, Ph.D. † Pfizer Inc. Groton, Connecticut Kyle Eastman, Ph.D. Yuanjun He, Ph.D. Jason Hein, Ph.D. Dube Henry, Ph.D. Nathan Gianneschi, Ph.D. † University of California San Diego, California Cristina Gil-Lamaignere, Ph.D. † University Hospital Nuestra Señora de la Candelaria Santa Cruz de Tenerife, Spain Rodolfo Gonzalez, Ph.D. Scott Grecian, Ph.D. Rong Jiang, Ph.D.***** Guo Jiantoa, Ph.D. Hiroyuki Kakei, Ph.D. † Takeda Pharmaceutical Company Limited Osaka, Japan Jaroslaw Kalisiak, Ph.D. Seiji Kamioka, Ph.D. Moumita Kar, Ph.D. Marcos Hernandez, Ph.D. Kwang Mi Kim, Ph.D. Par Holmberg, Ph.D. † Memorial Sloan Kettering Cancer Center New York, New York Wen-Xu Hong, Ph.D. Yu Fu, Ph.D. Ph.D. † Masaki Handa, Ph.D. † Sagami Chemical Research Center Ayase, Kanagawa, Japan Ph.D. † Jeffrey Culhane, Ph.D. Stephen Dalby, Ph.D. Tanja Gulder, Ph.D. Zhangyong Hong, Ph.D. Richard J. Hooley, Ph.D. † University of California Riverside, California Tamara Hopkins, Ph.D. † Boehringer Ingelheim Pharmaceuticals, Inc. Ridgefield, Connecticut Allen Horhota, Ph.D. Tony Horneff, Ph.D. F. Scott Kimball, Ph.D. Jeremy Kister, Ph.D. Keith Korthals, Ph.D. Larisa Krasnova, Ph.D. Arkady Krasovskiy, Ph.D. Luke Lairson, Ph.D. Jae Wook Lee, Ph.D. Jinq-Chyi Lee, Ph.D. † National Health Research Institutes Miaoli County, Taiwan Jong Seok Lee, Ph.D. Claas Hovelmann, Ph.D. Ki-Bum Lee, Ph.D. † Rutgers University Piscataway, New Jersey Fang Hu, Ph.D. † Department of Molecular Biology, Scripps Research Sejin Lee, Ph.D. † SK Drug Development Center Daejong, Korea Xiaoyi Hu, Ph.D. Alexandre Lemire, Ph.D.**** Zheng-Zheng Huang, Ph.D. † DuPont Central Research and Development Wilmington, Delaware Edward Lemke, Ph.D. Ben Hutchins, Ph.D. Chuang-Chuang Li, Ph.D. † Peking University Peking, China Jun-Li Hou, Ph.D. Der-ren Hwang, Ph.D. † Academia Sinica Taipei, Taiwan Christophe Letondor, Ph.D.**** Fangzheng Li, Ph.D.***** CHEMISTRY 2008 Hongming Li, Ph.D. † Schering-Plough Kenilworth, New Jersey Joonwoo Nam, Ph.D. † CytRx Corporation San Diego, California Ke Li, Ph.D. † DuPont Central Research and Development Wilmington, Delaware Tae-Gyu Nam, Ph.D. THE SCRIPPS RESEARCH INSTITUTE Troy Ryba, Ph.D. † Broad Institute of MIT and Harvard Cambridge, Massachusetts Ph.D. † Sebastian Steiniger, Ph.D. Antonia Stepan, Ph.D. James Stover, Ph.D. Youngha Ryu, Texas Christian University Fort Worth, Texas Bernhard Stump, Ph.D. Catherine Saccavini, Ph.D. Hui Kai Sun, Ph.D.***** Severin Odermatt, Ph.D.**** Nicholas Salzameda, Ph.D. Shinobu Takizawa, Ph.D. Yeon-Hee Lim, Ph.D. † Schering-Plough Kenilworth, New Jersey Christian Olsen, Ph.D. Antonio Sanchez-Ruiz, Ph.D. Yazmin Osornio, Ph.D.**** Yoshikazu Sasaki, Ph.D. Tongxiang Lin, Ph.D. Junguk Park, Ph.D. Stefan Schiller, Ph.D. Adam Talbot, Ph.D. † Institute of Chemical and Engineering Sciences Jurong Island, Singapore Troy Lister, Ph.D. † Novartis Cambridge, Massachusetts Nitin Patil, Ph.D. Niklas Schone, Ph.D. Annie Tam, Ph.D. Yefeng Tang, Ph.D. Christopher Liu, Ph.D. † Cubix Pharmaceuticals Lexington, Massachusetts Richard Payne, Ph.D. † University of Sydney Sydney, Australia Michael Schramm, Ph.D. † California State University Long Beach, California Wenshe Liu, Ph.D. † Texas A&M University College Station, Texas Xuemei Peng, Ph.D.**** Edward Sessions, Jr., Ph.D. Murali Peram Surakattula, Ph.D. † CytRx Corporation San Diego, California Shigeki Seto, Ph.D. Pi-Hui Liang, Ph.D. † Academia Sinica Taipei, Taiwan Michael Luzung, Ph.D. Utpal Majumder, Ph.D. Sreeman Mamidyala, Ph.D. Andrew Nguyen, M.D., Ph.D. Romain Noel, Ph.D. George Nora, Ph.D.***** Johan Paulsson, Ph.D. Alexander Mayorov, Ph.D. Charles Melancon, Ph.D. Lionel Moisan, Ph.D. † CEA Gif-Sur-Yvette, France Ana Montero, Ph.D.**** Miguel Morales, Ph.D. Shun Su, Ph.D. Mariola Tortosa, Ph.D. † Instituto de Quimica Organica, CSIC Madrid, Spain Craig Turner, Ph.D.**** Roshan Perera, Ph.D. † University of Texas Austin, Texas Takeshi Masuda, Ph.D. Michael Maue, Ph.D. † Bayer CropScience AG Monheim, Germany Young Jun Seo, Ph.D. 81 Mary Jo Sever, Ph.D. Alex Shaginian, Ph.D. † Ardea Biosciences San Diego, California David Shaw, Ph.D. Ramulu Poddutoori, Ph.D. Weijun Shen, Ph.D. Jonathan Pokorski, Ph.D. Xiao Shengxiong, Ph.D. Agustí Lledó Ponsati, Ph.D. Bingfeng Shi, Ph.D. Daniela Radu, Ph.D. † DuPont Central Research and Development Wilmington, Delaware Yan Shi, Ph.D. Vincent Trepanier, Ph.D. † Institute of Chemical and Engineering Sciences Jurong Island, Singapore Jonathan Tripp, Ph.D. Meng-Lin Tsao, Ph.D. † University of California Merced, California Andrew Udit, Ph.D. Hiroki Shigehisa, Ph.D. Hiroyuki Shimamura, Ph.D. Ronald Rahaim, Ph.D.***** Siddhartha Shenoy, Ph.D. Praveen Rao, Ph.D. † University of Waterloo Waterloo, Ontario, Canada Matthew Tremblay, Ph.D. Ryan Simkovsky, Ph.D. Chinnappan Sivasankar, Ph.D.**** Taiki Umezawa, Ph.D. † Hokkaido University Sapporo, Japan Kenji Usui, Ph.D. † Tokyo Institute of Technology Tokyo, Japan Carlos Valdez, Ph.D. † Rigel Pharmaceuticals, Inc. South San Francisco, California Adam Morgan, Ph.D. † Concert Pharmaceuticals, Inc. Lexington, Massachusetts Per Restorp, Ph.D. Ting-Wei Mu, Ph.D. Jin-Kyu Rhee, Ph.D. Michael Smolinski, Ph.D. † Kinex Pharmaceuticals Buffalo, New York S. Vasudeva Naidu, Ph.D. Fatima Rivas, Ph.D. Xinyi Song, Ph.D. Feng Wang, Ph.D. Yuya Nakai, Ph.D. Joshua Roth, Ph.D.***** Simon Stamm, Ph.D. Jian Wang, Ph.D. Kimberly Reynolds, Ph.D. Punna Venkateshwarlu, Ph.D. 82 CHEMISTRY Jiangyun Wang, Ph.D. † Institute of Biophysics Beijing, China Lin Wang, Ph.D. Sheng-Kai Wang, Ph.D. Weidong Wang, Ph.D. Xisheng Wang, Ph.D. Yajuan Wang, Ph.D. Yuanhua Wang, Ph.D. Timo Weide, Ph.D. Albert Willis, Ph.D. † Pharmagra Labs, Inc. Brevard, North Carolina Tao Wu, Ph.D. † Institute of Chemical and Engineering Sciences Jurong Island, Singapore Heiko Wurdak, Ph.D. Jian Xie, Ph.D. Wen Xiong, Ph.D. Yue Xu, Ph.D. Junichiro Yamaguchi, Ph.D. Ryu Yamasaki, Ph.D. † Tokyo University of Science Tokyo, Japan Ura Yasuyuki, Ph.D. † Nara Women’s University Nara, Japan Yan Yin, Ph.D. 2008 Heyue Zhou, Ph.D. Hongyan Zhou, Ph.D. Shoutian Zhu, Ph.D. Joerg Zimmermann, Ph.D. V I S I T I N G I N V E S T I G AT O R S Keisuke Fukuchi, Ph.D. Sankyo Co., Ltd. Tokyo, Japan Christine Hernandez, Ph.D. † University of Philippines Diliman, Philippines (Edmond) Shie-Liang Hsieh, Ph.D. National Yang-Ming University Taipei, Taiwan Masakazu Imamura, Ph.D. † Astellas Pharma Inc. Tsukuba, Ibaraki, Japan Kuniyuki Kishikawa, Ph.D. † Kyowa Hakko Kogyo Co., Ltd. Sunto-gun, Shizuoka, Japan Michael Meijler, Ph.D. † Ben-Gurion University of the Negev Be’er Sheva, Israel Takayoshi Suzuki, Ph.D. † Nagoya City University Nagoya, Japan Yoshiyuki Yoneda, Ph.D. † Daiichi Pharmaceutical Co., Ltd. Tokyo, Japan Ian Young, Ph.D. S C I E N T I F I C A S S O C I AT E Zhanqian Yu, Ph.D. Xu Yuan, Ph.D. Weiqiang Zhan, Ph.D. Hongjun Zhang, Ph.D. Xuejun Zhang, Ph.D. Yanghui Zhang, Ph.D. Yingchao Zhang, Ph.D. † Hoffmann-La Roche, Inc. Nutley, New Jersey Jon Ashley THE SCRIPPS RESEARCH INSTITUTE * Joint appointment in The Skaggs Institute for Chemical Biology ** Joint appointments in The Skaggs Institute for Chemical Biology and the Department of Immunology and Microbial Science *** Joint appointments in The Skaggs Institute for Chemical Biology and the Department of Molecular Biology **** Appointment completed ***** Scripps Florida † Appointment completed; new location shown CHEMISTRY 2008 Chairman’s Overview s the “central science,” chemistry stands between biology and medicine and between physics and materials science and provides the crucial bridge for drug discovery and development. But chemistry has a much more profound and useful role in science and society. It is the discipline that continually creates the myriad of new materials that we all encounter in our everyday lives: pharmaceuticals, high-tech materials, polymers and plastics, K.C. Nicolaou, Ph.D. insecticides and pesticides, fabrics and cosmetics, fertilizers, and vitamins—basically everything we can touch, feel, and smell. Chemistry at Scripps Research focuses on chemical synthesis and chemical biology, the areas most relevant to biomedical research and materials science. The members of our faculty are distinguished teacher-scholars who maintain highly visible and independent research programs in areas as diverse as biological and chemical catalysis, synthesis of natural products, combinatorial chemistry, molecular design, supramolecular chemistry, chemical evolution, materials science, and chemical biology. The chemistry graduate program attracts some of the bestqualified candidates from the United States and abroad. Our major research facilities, under the direction of Dee H. Huang (nuclear magnetic resonance), Gary Siuzdak (mass spectrometry), and Raj Chadha (x-ray crystallography), are second to none and continue to provide crucial support to our research programs. In addition, the Mabel and Arnold Beckman Center for the Chemical Sciences constantly receives high praise from visitors from around the world for its architectural design and operational aspects, both highly conducive to research. Research in the Department of Chemistry goes on unabated, establishing international visibility and attracting attention, as evidenced by numerous lecture invitations, visits by outside scholars, and headline news in the media. As of 2007, the Institute for Scientific Information ranked 2 members of the department as highly cited researchers (in the top 100 worldwide). Richard Lerner and his group continue their research on antibodies, chemical synthesis, and the biological A THE SCRIPPS RESEARCH INSTITUTE 83 role of polyoxygen species. Scientists in Albert Eschenmoser’s group continue to work on the chemical etiology of nucleic acids and the origins of life. Barry Sharpless and his group persist in their endeavors to discover and develop better catalysts for organic synthesis and to construct, through innovative chemistry and biology, libraries of novel compounds for biological screening. Their click chemistry, which has had a major impact in many areas of the molecular sciences, continues to be an important focus of their research. Members of my own group continue to explore chemical synthesis and chemical biology, with a focus on the total synthesis of new anticancer agents, antibiotics, marine-derived neurotoxins, antimalarial compounds, and other bioactive natural and designed molecules. Julius Rebek and his group devise biomimetic receptors, including molecules that bind neurotransmitters and membrane components, for studies in molecular recognition. Larger host receptors can surround 3 or more molecular guests and act as chambers in which the chemical reactions of the guests are accelerated. Scientists in the group also synthesize small molecules that act as protein helix mimetics for pharmaceutical applications. Peter Schultz and researchers in his laboratory are expanding the number of genetically encoded amino acids to include fluorescent, photocaged, metal-binding, chemically reactive, and posttranslationally modified amino acids. These scientists have also adapted this technology to mammalian cells and are applying these tools in basic and applied problems in cell biology. In addition, members of the group have used cell-based screens to identify small molecules that selectively differentiate and expand embryonic and adult stem cells and reprogram lineage-committed cells, as well as novel genes and small molecules that affect a number of physiologic and disease processes. Chi-Huey Wong and his group further advance the fields of chemoenzymatic organic synthesis, chemical glycobiology, and the development of enzyme inhibitors. A new strategy for the synthesis of glycoproteins based on sugar-assisted glycopeptide ligation has been developed. The programmable 1-pot synthesis of oligosaccharides developed by this group has been further used in the assembly of glycoarrays for study of saccharides that bind to proteins. Members of this group also developed new probes to study posttranslational glycosylation and identify glycoprotein biomarkers associated with cancer. Researchers in Dale Boger’s laboratory continue their work on chemical synthesis; combinatorial chemistry; het- 84 CHEMISTRY 2008 erocycle synthesis; anticancer agents, such as vinblastine, cyclostatin, chlorofusion, and yatakemycin; and antibiotics, such as vancomycin, teicoplanin, and ramoplanin. Scientists in Kim Janda’s laboratory conduct research grounded on organic chemistry as applied to specific biological systems. The targeted programs span a wide range of interests, from immunopharmacotherapy to biological and chemical warfare agents to filarial infections such as “river blindness” to quorum sensing in bacteria and new cancer therapeutic strategies. Recent achievements include in vivo detection of botulinum neurotoxin antagonists, the development of peptides and antibodies as drug delivery modules that home to cancer cells and active vaccines for nicotine addiction and obesity that are now in preclinical trials. M. Reza Ghadiri and his group are making important contributions in the design and study of a new generation of antimicrobial agents, based on self-assembling peptide nanotube architecture, to combat multidrug resistant infections. In addition, members of the group continue to make novel contributions in several ongoing basic research endeavors, such as designing biosensors, developing molecular computation, designing self-reproducing systems, understanding the origins of life, and creating emergent chemical systems. M.G. Finn and his group have pioneered the use of virus particles as chemical reagents and building blocks for nanochemical structures. This effort is directed toward the development of new diagnostics for disease and catalysts for organic reactions. Members of Dr. Finn’s laboratory also develop and investigate new organic and organometallic reactions and use these processes to synthesize biologically active compounds. Jeff Kelly and his group are exploring the interface between the chemistry, biology, and pathobiology of proteome maintenance. The aim of their projects is to understand the physical and biological basis of protein folding and the competitive misfolding and aggregation processes that lead to age-associated neurodegenerative diseases. Information on proteome maintenance is used to develop new small-molecule therapeutic strategies for a variety of diseases, including neurodegenerative diseases. Anita Wentworth and the researchers in her group are investigating the chemical basis of complex disease states and are synthesizing peptide- and small molecule–based therapeutic agents. These scientists focus on disease states in which inflammatory and reactive oxygen species are prominent, such as atherosclerosis, Alzheimer’s disease, and other diseases of ageing. THE SCRIPPS RESEARCH INSTITUTE Researchers in Floyd Romesberg’s laboratory are using diverse techniques ranging from bioorganic and biophysical chemistry to bacterial and yeast genetics to understand and manipulate the process of evolution. Major efforts include designing unnatural base pairs and using directed evolution of DNA polymerases to efficiently synthesize unnatural DNA containing the base pairs, using spectroscopy to understand biological function and how it evolves, and understanding how induced and adaptive mutations contribute to evolution in eukaryotic and prokaryotic cells. Phil Baran and his group are interested in how the general challenge of chemoselectivity in organic chemistry can be answered through the auspices of total synthesis. He and his coworkers have developed extremely concise chemical solutions to the synthetic challenges posed by numerous families of natural products. These syntheses systematically tackle the issue of chemoselectivity and are characterized by striking brevity, new biosynthetic postulates, the invention of new methods, and a minimum use or complete absence of protecting groups and superfluous oxidation state manipulations. The Frontiers in Chemistry Lecturers (19th Annual Symposium) for the 2007–2008 academic year were M. Christina White, University of Illinois; Ben L. Feringa, University of Groningen, the Netherlands; Ian Paterson, Cambridge University; and Harry Noller, University of California, Santa Cruz. In addition, we enjoyed hosting the following professors: Samir Zard, Ecole Polytechnique, France, as the Bristol-Myers Squibb Lecturer; E.J. Corey, Harvard University, as the Pfizer Lecturer; and Robert Bergman, University of California, Berkeley, as the Novartis Lecturer. CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE 85 INVESTIGATORS’ R EPORTS Practical Total Synthesis of Natural Products P.S. Baran, S. Ashida, N.Z. Burns, K. Chen, M.P. DeMartino, K.J. Eastman, C.A. Guerrero, T. Gulder, B.D. Hafensteiner, Y. Ishihara, P.J. Krawczuk, C. Li, D.W. Lin, J.W. Lockner, M.R. Luzung, T.J. Maimone, T. Masuda, T.R. Newhouse, D.P. O’Malley, H. Renata, J.M. Richter, N. Schone, I.B. Seiple, R.A. Shenvi, J. Shi, H. Shigeshisa, S. Su, A.F. Voica, J. Yamaguchi, I.S. Young rom penicillin to paclitaxel (Taxol), natural products have an unparalleled track record in the betterment of human health. In fact, 9 of the top 20 best-selling drugs were either inspired by or derived from natural products. Even the best-selling drug of all time, atorvastatin (Lipitor), was based on a natural product lead. Total synthesis, the art and science of recreating these entities in the laboratory, invariably leads to fundamental discoveries in chemistry, biology, and medicine. We focus on solving interesting challenges in the total synthesis of natural products and on bridging gaps in synthetic capabilities by inventing new reactions. Through judicious target selection and creative retrosynthetic analyses, total synthesis becomes an engine for discovery that drives the field of organic chemistry to new levels of sophistication and practicality. Synthetic organic chemistry requires tremendous ingenuity, artistic taste, experimental acumen, persistence, and character. Not surprisingly, drug development relies on the expertise of researchers who have these characteristics. Although we focus entirely on educating students in fundamental chemistry, we also collaborate with expert biologists to explore the medicinal potential of newly synthesized natural products and the products’ analogs. Recently completed total syntheses (Fig. 1) include (1) the anticancer agent stephacidin; (2) the antibacterial agents ageliferin and axinellamine; (3) members of the bioactive fischerindole, hapalindole, and welwitindolinone indole alkaloid family; (4) the anticancer agent haouamine A; and (5) the structurally exotic marine alkaloid chartelline C. Current natural product targets (Fig. 2) include stylissadine A, sarcodonin, and vinigrol; the neuroactive agent psychotrimine; and the potent angiogenic agent cortistatin A. F F i g . 1 . Recently completed total syntheses. PUBLICATIONS Burns, N.Z., Baran, P.S. On the origin of the haouamine alkaloids. Angew. Chem. Int. Ed. 46:205, 2007. Chen, K., Richter, J.M., Baran, P.S. 1,3-Diol synthesis via controlled, radicalmediated C-H functionalization. J. Am. Chem. Soc. 130:7247, 2008. Grube, A., Immel, S., Baran, P.S., Köck, M. Massadine chloride: a biosynthetic precursor of massadine and stylissadine. Angew. Chem. Int. Ed. 46:6721, 2007. Köck, M., Grube, A., Seiple, I., Baran, P.S. The pursuit of palau’amine. Angew. Chem. Int. Ed. 46:6586, 2007. Maimone, T.J., Voica, A.F., Baran, P.S. A concise approach to vinigrol. Angew. Chem. Int. Ed. 47:3054, 2008. 86 CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE SYNTHETIC METHODS Central to much of our work are investigations to develop and apply the hetero Diels-Alder reaction, including the use of heterocyclic and acyclic azadienes (Fig. 1), the thermal reactions of cyclopropenone ketals, inter- F i g . 1 . N-Sulfonyl-1-aza-1,3-butadiene Diels-Alder reaction. F i g . 2 . Recent natural product targets. O’Malley, D.P., Yamaguchi, J., Young, I.S., Seiple, I.B., Baran, P.S. Total synthesis of (±)-axinellamines A and B. Angew. Chem. Int. Ed. 47:3581, 2008. Richter, J.M., Whitefield, B., Maimone, T.J., Lin, D.W., Castroviejo, P., Baran, P.S. Scope and mechanism of the direct indole and pyrrole couplings adjacent to carbonyl compounds: total synthesis of acremoauxin A and oxazinin 3. J. Am. Chem. Soc. 129:12857, 2007. Shenvi, R.A., Guerrero, C.A., Shi, J., Li, C.C., Baran, P.S. Synthesis of (+)-cortistatin A. J. Am. Chem. Soc. 130:7241, 2008. Yamaguchi, J., Seiple, I.B., Young, I.S., O’Malley, D.P., Maue, M., Baran, P.S. Synthesis of 1,9-dideoxy-pre-axinellamine. Angew. Chem. Int. Ed. 47:3578, 2008. Synthetic and Bioorganic Chemistry D.L. Boger, E. Anderson, S. Baraldi, K. Boyle, C. Burke, R. Clark, D. Colby, C. Crane, J. DeMartino, J. Elsner, C. Ezzili, J. Garfunkle, H. Ge, D. Hochstatter, I. Hwang, R. Jones, H. Kakei, D. Kato, F.S. Kimball, J. Lajiness, S. Lee, C. Liu, K. MacMillan, J. Nam, K. Otrubova, P. Patel, W. Robertson, Y. Sasaki, M. Schnermann, S. Seto, C. Slown, S. Stamm, J. Stover, S. Takizawa, A. Tam, P. Va, L. Whitby, J. Xie, A. Zuhl he research interests of our group include the total synthesis of natural products, development of new synthetic methods, heterocyclic chemistry, bioorganic and medicinal chemistry, the study of DNA-agent interactions, and the chemistry of antitumor antibiotics. We place a special emphasis on investigations to define the structure-function relationships of natural or designed organic agents. T molecular and intramolecular acyl radical–alkene addition reactions, medium- and large-ring cyclization technology, and solution-phase combinatorial chemistry. In each instance, the development of the methods represents the investigation of chemistry projected as a key element in the synthesis of a natural or designed agent. T O TA L S Y N T H E S I S O F N AT U R A L P R O D U C T S Efforts are under way on the total synthesis of a number of natural products that constitute agents in which we have a specific interest. Representative agents currently under study include (+)-CC-1065 and functional analogs; the duocarmycin class of antitumor antibiotics, including yatakemycin; tropoloalkaloids; prodigiosin and roseophilin; the deoxybouvardin and RA-I class of antitumor agents; vancomycin, teicoplanin, ristocetin, chloropeptins, and related agents; ramoplanin; the luzopeptins, quinoxapeptins, thiocoraline, BE-22179, and sandramycin; bleomycin A2 and functional analogs; HUN-7293; chlorofusin; CI-920 (fostriecin) and cytostatin; the combretastatins; storniamide A; phomazarin; ningalins; lamellarin O; lukianol A; piericidins; nothapodytine and mappicine; rubrolone; vindoline; and vinblastine (Figs. 2 and 3). BIOORGANIC CHEMISTRY The agents listed in the previous paragraph were selected on the basis of their properties; in many instances, they are agents related by a projected property. For example, (+)-CC-1065, the duocarmycins, and yatakemycin are antitumor antibiotics and related sequence-selective DNA minor groove alkylating agents. Representative of such efforts, studies to determine the structural features of yatakemycin and the duocarmycins that contribute to the sequence-selective DNA alkylation properties of these agents have resulted in the identifi- CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE 87 Fig. 3. Additional recent total syntheses. F i g . 2 . Recent total syntheses. cation of a unique source of catalysis for the DNA alkylation reaction. Efforts are under way to develop DNA cross-linking agents of a predefined cross-link, to fur- ther understand the nature of the noncovalent and covalent interactions between agents and DNA, and to apply this understanding to the de novo design of DNA-binding and DNA-effector agents. Techniques for the evaluation of the agent-DNA binding and alkylation properties, col- 88 CHEMISTRY 2008 laborative efforts in securing biological data, nuclear magnetic resonance structures of DNA-agent complexes, molecular modeling, and studies of DNA-agent interactions are integral parts of the program. Additional ongoing studies include efforts to define the fundamental basis of the DNA-binding or cleavage properties of bleomycin A2, sandramycin, and the luzopeptins; to design inhibitors of the folate-dependent enzymes glycinamide ribonucleotide transformylase and aminoimidazole carboxamide ribonucleotide transformylase as potential antineoplastic agents; to establish the chemical and biological characteristics responsible for the sleep-inducing properties of the endogenous lipid oleamide; to inhibit tumor growth through inhibition of angiogenesis; to inhibit aberrant gene transcription associated with cancer; and to control intracellular signal transduction through the discovery of antagonists or agonists that affect protein-protein interactions, including receptor dimerization. PUBLICATIONS Eubanks, L.M., Hixon, M.S., Jin, W., Hong, S., Clancy, C.M., Tepp, W.H., Baldwin, M.R., Malizio, C.J., Goodnough, M.C., Barbieri, J.T., Johnson, E.A., Boger, D.L., Dickerson, T.J., Janda, K.D. An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists [published correction appears in Proc. Natl. Acad. Sci. U. S. A. 104:6490, 2008]. Proc. Natl. Acad. Sci. U. S. A. 104:2602, 2007. Hardouin, C., Kelso, M.J., Romero, F.A., Rayl, T.J., Leung, D., Hwang, I., Cravatt, B.F., Boger, D.L. Structure-activity relationships of the α-ketooxazole inhibitors of fatty acid amide hydrolase. J. Med. Chem. 50:3359, 2007. Ishikawa, H., Boger, D.L. Total synthesis of (–)- and ent-(+)-4-desacetoxy-5desethylvindoline. Heterocycles 72:95, 2007. Jin, W., Trzupek, J.D., Rayl, T.J., Broward, M.A., Vielhauer, G.A., Weir, S.J., Hwang, I., Boger, D.L. A unique class of duocarmycin and CC-1065 analogues subject to reductive activation. J. Am. Chem. Soc. 129:15391, 2007. Lee, S.Y., Clark, R.C., Boger, D.L. Total synthesis, stereochemical reassignment, and absolute configuration of chlorofusin. J. Am. Chem. Soc. 129:9860, 2007. Nam, J., Shin, D., Rew, Y., Boger, D.L. Alanine scan of [L-Dap2]ramoplanin A2 aglycon: assessment of the importance of each residue. J. Am. Chem. Soc. 129:8747, 2007. Romero, F.A., Du, W., Hwang, I., Rayl, T.J., Kimball, F.S., Leung, D., Hoover, H.S., Apodaca, R.L., Breitenbucher, J.G., Cravatt, B.F., Boger, D.L. Potent and selective α-ketoheterocycle-based inhibitors of the anandamide and oleamide catabolizing enzyme, fatty acid amide hydrolase. J. Med. Chem. 50:1058, 2007. Tichenor, M.S., MacMillan, K.S., Stover, J.S., Wolkenberg, S.E., Pavani, M.G., Zanella, L., Zaid, A.N., Spalluto, G., Rayl, T.J., Hwang, I., Baraldi, P.G., Boger, D.L. Rational design, synthesis, and evaluation, of key analogues of CC-1065 and the duocarmycins. J. Am. Chem. Soc. 129:14092, 2007. Tichenor, M.S., MacMillan, K.S., Trzupek, J.D., Rayl, T.J., Hwang, I., Boger, D.L. Systematic exploration of the structural features of yatakemycin impacting DNA alkylation and biological activity. J. Am. Chem. Soc. 129:10858, 2007. Xu, L., Chong, Y., Hwang, I., D’Onofrio, A., Amore, K., Beardsley, G.P., Li, C., Olson, A.J., Boger, D.L., Wilson, I.A. Structure-based design, synthesis, evaluation, and crystal structures of transition state analogue inhibitors of inosine monophosphate cyclohydrolase. J. Biol. Chem. 282:13033, 2007. THE SCRIPPS RESEARCH INSTITUTE Phage Escape for the Prediction of Protein Evolution T.J. Dickerson, J. Denery, L. Eubanks, A. Hoyt, K.D. Janda, A. Moreno, Y. Nakai, A. Nguyen, A. Nunes, A. Rohrbach, C. Saccavini he relationship between host and pathogen is in a constant state of flux, with each side continually evolving in a struggle to maximize the chance for survival. A plethora of defensive systems has evolved to counteract and/or eliminate invading pathogens. These systems exert pressure upon the pathogen, leading to mechanisms that combat the host, including immune evasion and drug resistance. Subsequently, the host must counter these improved pathogens with a selected adaptive immune response. This cycle can continue indefinitely, with pathogen and host counteracting the survival strategy of each other. From a structural point of view, the contest is usually played out between the relatively unstructured protein loops of both systems, where generation of 3-dimensional structural diversity of these parts of the protein does not affect overall protein function. The accessible protein-sequence diversity of both pathogen and host immune system is exceedingly expansive, thereby making an a priori analysis of where the mutation can occur and how antibodies subsequently respond an extremely difficult challenge. However, adaptive immunity is inherently a reactive system and cannot operate in a proactive mode; challenge from an exogenous antigen is required before a response is made. Thus, despite the seemingly limitless diversity that can be accessed by the mammalian immune system, only a small part of this theoretical diversity is present at any given time. Only after a specific challenge is the full extent of the immune repertoire brought to bear upon an invading pathogen. A technique that could be used to predict the advance of evolving human disease and predetermine suitable therapeutic strategies before a pathogen becomes a public health threat would be valuable both for understanding pathogen evolution and for transforming drug discovery into a process that predetermines therapeutic strategies before the onset of epidemics or pandemics. Recently, we developed a technology termed “phage escape” that allows a preemptive determination of the evolutionary pathways used by the causative organisms of specific diseases. This technology has been applied to the evolution of the hemagglutinin viral surface pro- T CHEMISTRY 2008 tein from the highly pathogenic avian influenza virus H5N1 (“bird flu”). During the past year, we successfully prepared all of the experimental tools needed to perform a “checkmate analysis” of influenza virus hemagglutinin type 5. Currently, we are generating a map of the evolution of this protein (Fig. 1). THE SCRIPPS RESEARCH INSTITUTE 89 Chemical and Functional Genomic Approaches to Stem Cell Biology and Regenerative Medicine S. Ding, R. Abu-Jarour, R. Ambasudhan, R. Coleman, C. Desponts, J. Efe, H.S. Hahm, S. Hilcove, J. Hsu, W. Li, T. Lin, Y. Shi, W. Xiong, Y. Xu, X. Yuan, H. Zhou ecent advances in stem cell biology may make possible new approaches for the treatment of a number of diseases, including cardiovascular disease, neurodegenerative disease, musculoskeletal disease, diabetes, and cancer. These approaches could involve cell replacement therapy and/or drug treatment to stimulate the body’s own regenerative capabilities by promoting survival, migration/homing, proliferation, and differentiation of endogenous stem/progenitor cells. However, such approaches will require identification of renewable cell sources of engraftable functional cells, an improved ability to manipulate proliferation and differentiation of the cells, and a better understanding of the signaling pathways that control the fate of the cells. Equipped with a high-throughput screening platform and large arrayed molecular libraries—combinatorial chemical libraries, genome-scale cDNA libraries (for gain of function), and small interfering RNA libraries (for loss of function)—we are developing and integrating chemical and functional genomic tools to study stem cell biology and regeneration. We screen these libraries to identify and further characterize small molecules and genes that can control stem cell fate in various systems, including (1) self-renewal regulation of embryonic and adult stem cells; (2) directed and step-wise differentiation of embryonic stem cells toward neuronal, cardiac, and pancreatic lineages; (3) directed neuronal differentiation and subtype neuronal specification of human and rodent neural stem cells; (4) cellular plasticity and reprogramming of lineage-restricted somatic cells to more primitive precursor cells; (5) functional proliferation of cardiomyocytes and pancreatic beta cells from adults; (6) developmental signaling pathways and epigenetic mechanisms (histone and DNA de/methylation); and (7) development of new technologies for stem cell derivation and gene targeting. In addition, we are characterizing the molecular mechanism of these identified small molecules and R F i g . 1 . Work flow for using phage escape technology to identify escape mutants. In an immunologic checkmate analysis, phagebound protein variants (escape mutant, right middle) are able to escape a known collection of neutralizing antibodies (top center). PUBLICATIONS Brogan, A.P., Dickerson, T.J., Janda, K.D. Nornicotine-organocatalyzed aqueous reduction of α,β-unsaturated aldehydes. Chem. Commun. (Camb.) Issue 46:4952, 2007. Capková, K., Yoneda, Y., Dickerson, T.J., Janda, K.D. Synthesis and structureactivity relationships of second-generation hydroxamate botulinum neurotoxin A protease inhibitors. Bioorg. Med. Chem. Lett. 17, 6463, 2007. Dickerson, T.J., McKenzie, K.M., Hoyt, A.S., Wood, M.R., Janda, K.D., Brenner, S.B., Lerner, R.A. Phage escape libraries for checkmate analysis. Proc. Natl. Acad. Sci. U. S. A. 104:12703, 2007. Eubanks, L.M., Dickerson, T.J. Investigating novel therapeutic targets and molecular mechanisms to treat botulinum neurotoxin A intoxication. Future Microbiol. 2:677, 2007. Ino, A., Dickerson, T.J., Janda, K.D. Positional linker effects in haptens for cocaine immunopharmacotherapy. Bioorg. Med. Chem. Lett. 17:4280, 2007. Lowery, C.A., Dickerson, T.J., Janda, K.D. Interspecies and interkingdom communication mediated by bacterial quorum sensing. Chem. Soc. Rev. 37:1337, 2008. Treweek, J., Wee, S., Koob, G.F., Dickerson, T.J., Janda, K.D. Self-vaccination by methamphetamine glycation products chemically links chronic drug abuse and cardiovascular disease. Proc. Natl. Acad. Sci. U. S. A. 104:11580, 2007. Willis, B., Eubanks, L.M., Dickerson, T.J., Janda, K.D. The strange case of the botulinum neurotoxin: using chemistry and biology to modulate the most deadly poison. Angew. Chem. Int. Ed. 47:8360, 2008. Willis, B., Eubanks, L.M., Wood, M.R., Janda, K.D., Dickerson, T.J., Lerner, R.A. Biologically templated organic polymers with nanoscale order. Proc. Natl. Acad. Sci. U. S. A. 105:1416, 2008. Yao, Y., Dickerson, T.J., Hixon, M.S., Dyson, H.J. NMR detection of adventitious binding of xylose to the quorum-sensing protein SdiA of Escherichia coli. Bioorg. Med. Chem. Lett. 17:6202, 2007. 90 CHEMISTRY 2008 genes by using various approaches, including detailed investigations of structure-activity relationship, affinity chromatography for target identification, transcriptome profiling, proteomics analysis, chemical/genetic epistasis, and biochemical and functional assays in vitro and in vivo. So far, we have identified and are characterizing functional small molecules and/or genes in each of the previously mentioned distinct biological processes that involve regulation of stem/progenitor cells. More recent examples include identification and characterization of distinct small molecules for selfrenewal of human embryonic stem cells and clonal expansion/survival; dopaminergic neuron specification from mouse embryonic stem cells; derivation of rat embryonic stem cells; reprogramming of somatic cells to a pluripotent state; definitive endoderm and pancreatic induction; chemically defined monolayer conditions for self-renewal of embryonic stem cells and their directed differentiation to cardiac lineages; proliferation of human beta cells; and regulating Wnt signaling. These studies may ultimately facilitate the therapeutic application of stem cells and the development of small-molecule drugs to stimulate tissue and organ regeneration in vivo. PUBLICATIONS Shi, Y., Do, J.-T., Desponts, C., Hahm, H.-S., Schöler, H.R., Ding, S. A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell 2:525, 2008. Xu, Y., Shi, Y., Ding, S. A chemical approach to stem-cell biology and regenerative medicine. Nature 453:338, 2008. THE SCRIPPS RESEARCH INSTITUTE F i g . 1 . Structural simplification of α- L -threofuranosyl-(3′g2′) nucleic acid, which was inspired by studies on (3′g4′)-lyxopyranosyl nucleic acid, gives rise to acyclic informational oligomeric systems. Two examples are shown: glycerol nucleic acid and glyceric acid nucleic acid. The structure of this oligomeric system is based on a structural simplification of the oligonucleotides containing lyxopyranosyl (2′g4′)– and threofuranosyl (2′g3′)– linked phosphodiester backbones, which we studied previously. Among the oligomer systems depicted in Figure 1, the nucleic acid derived from the glycerol backbone is not considered to be a potentially prebiotic system, in contrast to the oligomer system derived from glyceric acid and tagged via amide bonds with 5-aminopyrimidines. We have completed the synthesis of such a glyceric acid–derived oligomer containing six 5-aminouracil units (6-mer) and have studied its base-pairing properties with DNA, RNA, and α-L-threofuranosyl-(3′g2′) nucleic acid. Base pairing was strong between the 6-mer and poly(dA) (Fig. 2), was somewhat weaker Chemical Etiology of Nucleic Acid Structure A. Eschenmoser, R. Krishnamurthy, G.K. Mittapalli, R.R. Kondreddi, Y. Osornio, V.S. Naidu n the general context of our project to map the landscape of potentially primordial informational oligomer systems, we have been working during the past year on the following topics. I 2 ′g 3 ′- P H O S P H O D I E S T E R – L I N K E D G LY C E R I C A C I D O L I G O M E R S B A S E D O N 5 - A M I N O P Y R I M I D I N E – TA G G E D BACKBONES We have undertaken the synthesis and study of the base-pairing properties of oligomers derived from a 2′,3′-phosphodiester–linked glyceric acid backbone that has 2,4-disubsituted 5-aminopyrimidines, attached to the carboxyl group of glyceric acid via an amide bond at the 5-amino group, as recognition elements (Fig. 1). F i g . 2 . UV (left) and circular dichroism (right) spectroscopic data for base pairing between 5-aminouracil–tagged 2-phosphoglycerate hexamer and DNA, poly(dA); c = 5+5 µM. Measurements were made in phosphate buffer. with the corresponding poly(rA), and even occurred with α-L-threofuranosyl-(3′g2′) nucleic acid. We are expanding the study to include the complementary partner strand tagged with 2,4,5-triaminopyrimidine and have explored different pathways for synthesizing the suitably protected building blocks necessary for the automated oligonucleotide synthesis. CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE 91 SYNTHESIS OF OLIGODIPEPTIDES OF A S PA R T Y L - 3 - A M I N O A L A N I N E D I P E P T I D E TA G G E D WITH OROTIC ACID In our search for alternative heterocycles that would be potentially prebiotic and offer opportunities for becoming chemoselectively attached to a backbone, we considered orotic acid as a candidate. Orotic acid and its 5-substituted derivatives have been identified as products from the hydrolysis of polymeric material formed from hydrogen cyanide. In addition, the involvement of orotic acid as a nucleobase in the biosynthetic assembly of pyrimidine nucleotides justifies and warrants exploration of its base-pairing properties. We are synthesizing the necessary building blocks for the synthesis and study of the base-pairing properties of oligomers consisting of aspartyl-3-aminoalanine dipeptide units tagged with orotic acid (Fig. 3). The choice of the F i g . 4 . Hypothetical autocatalytic cycle for the dimerization of glyoxylate to dihydroxyfumarate and the biomolecules to be derived from the constituents of that cycle. with the constraints of a primordial chemistry. We are conducting exploratory studies for assessing the chemistry of selected intermediates postulated to be formed from the chemistry of glyoxylate and dihydroxyfumarate. Some of the preliminary results are encouraging. F i g . 3 . Orotic acid as a recognition element. Also shown is the oligodipeptide tagged with orotic acid. oligodipeptide backbone on which orotic acid would be attached was influenced by the results of our previous studies. Because the carboxyl group is now on the heterocycle, amide bond–mediated tagging of the carboxyl group of orotic acid requires a 3-aminoalanine as a tagging unit. E X P L O R I N G T H E C H E M I S T R Y O F G LY O X Y L AT E A N D D I H Y D R O X Y F U M A R AT E A research project such as mapping the landscape of potentially primordial informational oligomer systems eventually demands the conception of, and the commitment to, a detailed chemical scenario for the type of organic chemistry that is supposed to have led to such oligomers under primordial conditions. Figure 4 depicts the chemical nature of the scenario we have decided to study experimentally. In the reaction cycle shown, glyoxylate would autocatalytically convert itself into its dimer dihydroxyfumarate. Dihydroxyfumarate is a known compound that we postulate can act as a common starting material for a large variety of biomolecules, such as sugars, α-amino acids, and pyrimidines, and for other organics of etiologic interest by reactions that are essentially unexplored thus far but are deemed compatible PUBLICATIONS Eschenmoser, A. On a hypothetical generational relationship between HCN and constituents of the reductive citric acid cycle. Chem. Biodivers. 4:554, 2007. Eschenmoser, A. The search for the chemistry of life’s origin. Tetrahedron 63:12821, 2007. Koch, K., Schweizer, B., Eschenmoser. A. Reactions of the HCN-tetramer with aldehydes. Chem. Biodivers. 4:541, 2007. Organic, Medicinal, and Biological Chemistry M.G. Finn, A. Accurso, S. Brown, S.-H. Cho, V. Hong, J. Lau, S. Lee, Y.-H. Lim, S. Presolski n addition to our work on biological polyvalency and immunology with engineered virus particles, supported by the Skaggs Institute for Chemical Biology, we focus on the development of catalysts and the synthesis of biologically useful structures. Two of these projects are described in the following sections. I C O P P E R - C ATA LY Z E D A Z I D E - A L K Y N E C L I C K CHEMISTRY We have continued our development of new catalysts and conditions for the copper-catalyzed azide- 92 CHEMISTRY 2008 alkyne cycloaddition reaction, which has become a principal example of click chemistry in the synthesis of possible drugs, dendrimers, polymers, and functionalized surfaces in laboratories around the world. In the past year, using an active but highly air-sensitive catalyst, we developed a convenient electrochemical protocol for performing bioconjugations. This procedure enables investigators who lack sophisticated inert-atmosphere equipment to perform the reaction under demanding conditions. We have also discovered new derivatives of the (benzimidazole-methyl)amine ligands reported last year, which accelerate the copper-catalyzed azide-alkyne cycloaddition reaction to a remarkable degree. A comprehensive picture is rapidly emerging of the types of ligands effective under the diverse conditions in which this cycloaddition reaction is applied. An important application of click chemistry is the synthesis and modification of polymeric materials. We found that metal adhesives can be formed by the simple application of mixtures of polyvalent azide and alkyne compounds to copper-containing surfaces. By incorporating amino groups to help speed the click reaction, and flexible cross-linking molecules to protect against stress fracturing in the resulting polymers, greatly improved adhesives have been created. Figure 1 illustrates the F i g . 1 . Graduate student Vu Hong sits on a 20-L container filled with water, supported by 2 copper plates glued together with an adhesive prepared by graduate student Adrian Accurso. THE SCRIPPS RESEARCH INSTITUTE strength of one of these formulations, which has potential in such applications as protective coatings, electrically conducting junctions, and antifouling agents. A G E N T S W I T H A C T I V I T Y A G A I N S T H E PAT I T I S B VIRUSES: MISDIRECTING PROTEIN-PROTEIN INTERACTIONS Modulation of protein-protein contacts by small molecules is an attractive strategy for the development of biologically active compounds. In many instances, the target interprotein interaction covers a substantial landscape with high thermodynamic stability. Virus particles rely on the assembly of protein subunits that engage in well-defined protein-protein interactions. However, these interactions are necessarily weak until the late stages of assembly; such cooperativity is necessary to ensure that protein is efficiently used in the multistep construction process. Viral capsid intermediates are therefore a class of protein-protein interface targets that may be easier for small molecules to affect. We have investigated this possibility for hepatitis B virus (HBV), a pathogen that infects 400 million people worldwide. The antiviral activity of the heteroaryldihydropyrimidine class of compounds has been known for several years. We found that their mechanism of action is the distortion, rather than the inhibition, of the protein assembly process. In a type of molecular jujitsu, these small molecules use the natural interactions of the capsid proteins against the virus, by increasing the energy of protein subunit association and the rate of subunit aggregation. This increase causes the viral proteins to assemble in error-prone fashion, forming large and irregular structures rather than the symmetric particles necessary for the proper function of the virus (Fig. 2). By making many heteroaryldihydropyrimidine analogs, we found compounds that nucleate the misincorporation of hundreds of protein molecules at a time, allowing these compounds to be highly effective inhibitors in cell culture. We continue to improve the performance of these structures and test the best analogs for activity against HBV in animal models. Furthermore, we think that assembly misdirection of this kind is a general antiviral strategy. By attacking the relatively easy target of protein-protein interactions present in early virus assembly intermediates, selective agents can be developed in a new and effective way. PUBLICATIONS Destito, G., Yeh, R., Rae, C.S., Finn, M.G., Manchester, M. Folic acid-mediated targeting of cowpea mosaic virus particles to tumor cells. Chem. Biol. 14:1152, 2007. CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE 93 Zhang, Q., Ma, X., Ward, A., Hong, W.-X., Jaakola, V.-P., Stevens, R.C., Finn, M.G., Chang, G. Designing facial amphiphiles for the stabilization of integral membrane proteins. Angew. Chem. Int. Ed. 46:7023, 2007. Organometallic Catalysis in Synthesis, Bioorganic Chemistry, and Materials Research V.V. Fokin, A. Chanda, S. Chuprakov, J. Fotsing, S. Grecian, T. Horneff, L. Krasnova, S.-W. Kwok, J. Raushel, T. Weide ur goals are to discover new reactions and to develop their practical applications in organic synthesis, chemical biology, and materials science. Transformations catalyzed by transition metals are central to our research. With many variables for optimization, these reactions have great potential to become useful on both laboratory and industrial scales. Although we often use automation techniques to screen extensive sets of catalysts, ligands, and conditions (sometime on the basis of just a hunch that “it should work”), mechanistic investigations of the initial reactivity are prominent in our research. Analysis of reaction kinetics, in situ infrared monitoring, reaction heat flow calorimetry, and other physicochemical methods are routinely used to understand the mechanistic underpinnings of the processes under investigation. Computational studies of reactive intermediates and mechanistic pathways compliment these experimental techniques. Equipped with reactions that proceed reliably with a range of readily available starting materials and are experimentally simple to perform, we endeavor to assemble molecules with interesting and useful function, ranging from new biologically active small molecules to macromolecular scaffolds such as dendrimers for studying multivalency effects and polymers for use in coatings and adhesives. We have focused on metal-catalyzed reactions of acetylenes because such processes can reliably produce a plethora of heterocyclic systems. Copper(I)- and ruthenium(II)-catalyzed reactions of acetylenes with 1,3-dipoles developed in our laboratories are shown in Figure 1. Reactions of alkynes with organic azides take a special place in the arsenal of catalytic dipolar cycloadditions we use. Although azides are energetic compounds, they are quite inert: stable to water, oxygen, most functional groups found in biological molecules, and many of the common organic reagents and conditions. With acety- O F i g . 2 . Negative-stain electron micrographs of the assembly prod- ucts of HBV capsid protein induced by different heteroaryldihydropyrimidine derivatives. HBV capsids are typically 35 nm in diameter; the structures shown here are many times that size. Hong, V., Udit, A.K., Evans, R.A., Finn, M.G. Electrochemically protected copper(I)-catalyzed azide-alkyne cycloaddition. Chembiochem 9:1481, 2008. Kaltgrad, E., O’Reilly, M.K., Liao, L., Han, S., Paulson, J., Finn, M.G. On-virus construction of polyvalent glycan ligands for cell-surface receptors. J. Am. Chem. Soc. 130:4578, 2008. Kaltgrad, E., Sen Gupta, S., Punna, S., Huang, C.-Y., Chang, A., Wong, C.-H. Finn, M.G., Blixt, O. Anti-carbohydrate antibodies elicited by polyvalent display on a viral scaffold. Chembiochem 8:1455, 2007. Liu, Y., Díaz, D.D., Accurso, A.A., Sharpless, K.B., Fokin, V.V., Finn, M.G. Click chemistry in materials synthesis, III: metal-adhesive polymers from Cu(I)-catalyzed azide-alkyne cycloaddition. J. Polym. Sci. A Polym. Chem. 45:5182, 2007. Miermont, A., Barnhill, H., Strable, E., Lu, X., Wall, K.A., Wang, Q., Finn, M.G., Huang, X. Cowpea mosaic virus capsid: a promising carrier for the development of carbohydrate based antitumor vaccines. Chem. Eur. J. 14:4939, 2008. Portney, N.G., Tseng, R.J., Destito, G., Strable, E., Yang, Y., Manchester, M., Finn, M.G., Ozkan, M. Microscale memory characteristics of virus-quantum dot hybrids. Appl. Phys. Lett. 90:214104, 2007. Prasuhn, D.E., Jr., Singh, P., Strable, E., Brown, S., Manchester, M., Finn, M.G. Plasma clearance of bacteriophage Qβ particles as a function of surface charge. J. Am. Chem. Soc. 130:1328, 2008. Rodionov, V.O., Presolski, S., Díaz, D.D., Fokin, V.V., Finn, M.G. Ligand-accelerated Cu-catalyzed azide-alkyne cycloaddition: a mechanistic report. J. Am. Chem. Soc. 129:12705, 2007. Rodionov, V.O., Presolski, S., Gardinier, S., Lim, Y.-H., Finn, M.G. Benzimidazole and related ligands for Cu-catalyzed azide-alkyne cycloaddition. J. Am. Chem. Soc. 129:12696, 2007. Strable, E., Prasuhn, D.E., Jr., Udit, A.K., Brown, S., Link, A.J., Ngo, J.T., Lander, G., Quispe, J., Potter, C.S., Carragher, B., Tirrell, D.A., Finn, M.G. Unnatural amino acid incorporation into virus-like particles. Bioconjug. Chem. 19:866, 2008. Zhang, Q., Horst, R., Geralt, M., Ma, X., Hong, W.-X., Finn, M.G., Stevens, R.C., Wüthrich, K. Microscale NMR screening of new detergents for membrane protein structural biology. J. Am. Chem. Soc. 130:7357, 2008. 94 CHEMISTRY F i g . 1 . Catalytic syntheses of heterocycles from alkynes and 1,3-dipolar species. lenes, organic azides produce 1,2,3-triazoles, exceptionally stable 5-membered heterocycles. These reactions, however, are very slow without a catalyst. The copper- and ruthenium-catalyzed cycloadditions of azides and alkynes provide ready access to 1,2,3-triazoles of various substitution patterns. The copper-catalyzed variant has become the premier click reaction and is used in diverse applications by chemists across the world in organic synthesis, medicinal chemistry, chemical biology, and materials science. In addition to its exceptional reliability and tolerance to a wide range of functional groups, the reaction has provided valuable insights into the unique and, until recently, unexplored reactivity patterns of organic azides and in situ generated copper acetylides. Indeed, catalytic syntheses of isoxazoles and pyrazoles are the recent additions to the family of such reactions. The ruthenium-catalyzed reaction (Fig. 1) enables “fusion” of organic azides and both terminal and internal alkynes with a complementary regioselectivity and appears to proceed through a completely different mechanism. This past year, we discovered the exquisite catalytic activity of pentamethylcyclopentadienyl ruthenium(II) chloride complexes in reactions of alkynes with nitrile oxides. These cycloadditions result in facile formation of 3,4-disubstituted and fully substituted isoxazoles. Until now, no general methods for regioselective synthesis of these heterocycles were available. When thermal cycloadditions of nitrile oxides with alkynes are successful, they favor the formation of the 3,5-disubstituted isomers. Furthermore, examples of reactive partners for nitrile oxides are limited to a handful of highly activated, electron-deficient alkynes. Unactivated or sterically hindered acetylenes usually do not react 2008 THE SCRIPPS RESEARCH INSTITUTE at all. In contrast, in the presence of ruthenium complexes, nitrile oxides and both terminal and internal alkynes are “fused” within minutes to hours at room temperature. In addition to the immediate practical benefits, this transformation suggests that different dipoles can be activated and engaged in catalysis by ruthenium complexes. Such processes are being investigated. Both copper- and ruthenium-catalyzed cycloadditions and their applications in the synthesis of biologically active compounds and novel materials have been the subject of our intense attention during the past year. We have investigated the mechanism of these processes and have endeavored to develop new ligands to make the reactions more efficient. We have used the reactions to synthesize libraries of compounds for HIV protease inhibition, in collaboration with J.H. Elder and A.J. Olson, Department of Molecular Biology; nicotinic receptor agonists and antagonists, in studies with P. Taylor, University of California, San Diego; metallo-β-lactamase inhibitors, in collaboration with P. Hodder, Translational Research Institute; and polymeric materials and dendritic constructs for polyvalent display and imaging applications (Fig. 2). F i g . 2 . Small molecules synthesized by using azide-alkyne cycloadditions. CHEMISTRY 2008 PUBLICATIONS Boren, B.C., Narayan, S., Rasmussen, L.K., Zhang, L., Zhao, H., Lin, Z., Jia, G., Fokin, V.V. Ruthenium-catalyzed azide-alkyne cycloaddition: scope and mechanism. J. Am. Chem. Soc. 130:8923, 2008. Finn, M.G., Kolb, H.C., Fokin, V.V., Sharpless, K.B. Concept and applications of click chemistry: from the standpoint of advocates. Kagaku to Kogyo 60:976, 2007. Fokin, V.V. Click imaging of biochemical processes in living systems. ACS Chem. Biol. 2:775, 2007. Hawker, C.J., Fokin, V.V., Finn, M.G., Sharpless, K.B. Bringing efficiency to materials synthesis: the philosophy of click chemistry. Aust. J. Chem. 60:381, 2007. Kalisiak, J., Sharpless, K.B., Fokin, V.V. Efficient synthesis of 2-substituted-1,2,3triazoles. Org. Lett. 10:3171, 2008. THE SCRIPPS RESEARCH INSTITUTE 95 bond–directed ring stacking into open-ended hollow tubular structures that have marked antibacterial and antiviral activities in vitro. The effectiveness of this novel supramolecular class of bioactive species as selective antibacterial agents was highlighted by the high efficacy of one of these antimicrobials against lethal methicillin-resistant Staphylococcus aureus infections in mice. Currently, we are exploring rational design of cyclic glycopeptides and selections from combinatorial libraries to discover novel antiviral supramolecular compounds (Fig. 1). Liu, Y., Díaz, D.D., Accurso, A.A., Sharpless, K.B., Fokin, V.V., Finn, M.G. Click chemistry in materials synthesis, III: metal-adhesive polymers from Cu(I)-catalyzed azide-alkyne cycloaddition. J. Polym. Sci. A Polym. Chem. 45:5182, 2007. Rasmussen, L.K., Boren, B.C., Fokin, V.V. Ruthenium-catalyzed cycloaddition of aryl azides and alkynes. Org. Lett. 9:5337, 2007. Rodionov, V.O., Presolski, S.I., Díaz, D.D., Fokin, V.V., Finn, M.G. Ligand-accelerated Cu-catalyzed azide-alkyne cycloaddition: a mechanistic report. J. Am. Chem. Soc. 129:12705, 2007. Vestberg, R., Malkoch, M., Kade, M., Wu, P., Fokin, V.V., Sharpless, K.B., Drockenmuller, E., Hawker, C.J. Role of architecture and molecular weight in the formation of tailor-made ultrathin multilayers using dendritic macromolecules and click chemistry. J. Polym. Sci. A Polym. Chem. 45:2835, 2007. Yoo, E.J., Ahlquist, M., Bae, I., Sharpless, K.B., Fokin, V.V., Chang, S. Mechanistic studies on the Cu-catalyzed three-component reactions of sulfonyl azides, 1-alkynes and amines, alcohols, or water: dichotomy via a common pathway J. Org. Chem. 73:5520, 2008. Design of Functional Synthetic Systems M.R. Ghadiri, M. Amorin, J. Beierle, A. Chavochi, J. Chu, B. Frezza, N. Gianneschi, L. Leman, A. Loutchnikov, A. Montero, C. Olsen, J. Picuri, D. Radu, Y. Ura e are engaged in multidisciplinary research to uncover new chemical and biochemical approaches for the design of functional molecular, supramolecular, and complex self-organized systems. Our efforts span disciplines ranging from synthetic organic, bioorganic, and physical organic chemistry to nanotechnology, biophysics, enzymology, and molecular biology. Current research includes the design of synthetic peptide catalysts, antimicrobial self-assembling peptide nanotubes, semisynthetic allosteric enzymes, self-replicating molecular systems and emergent networks, single-molecule DNA sensing, molecular computation, and prebiotic chemistry. W ANTIMICROBIAL PEPTIDE NANOTUBES We have shown that appropriately designed cyclic peptide subunits can self-assemble through hydrogen F i g . 1 . Antiviral agents based on self-assembling cyclic peptide nanotubes. Cyclic D ,L-α-peptides act on endosomal membranes to prevent the development of low pH in endocytic vesicles, arrest the escape of virions from the endosome, and abrogate adenovirus infection. DESIGN OF SIGNAL SELF-AMPLIFYING DNA SENSORS We constructed a novel sequence-specific DNA detection system based on rationally designed semisynthetic enzymes. The system is composed of covalently associated inhibitor-DNA-enzyme modules that function via DNA hybridization–triggered allosteric enzyme activation and signal amplification through substrate turnover (Fig. 2). The functional capacity of the system is high- F i g . 2 . Schematic representation of an intrasterically inactivated inhibitor-DNA-enzyme construct (left) and the DNA hybridization– triggered enzyme activation (right). The construct can be used to sense low concentrations of cDNA because of its built-in capacity for signal amplification via rapid substrate turnover. lighted by the sequence-specific detection of approximately 10 fmol of DNA in less than 3 minutes under physiologic conditions. Our studies suggest that ratio- 96 CHEMISTRY 2008 nally designed intrasterically regulated enzymes may be a promising new class of reagents for highly sensitive, rapid, and 1-step detection of label-free DNA sequences that does not depend on polymerase chain reactions. THE SCRIPPS RESEARCH INSTITUTE ous solutions. The synthetic networks have some of the basic architectural and dynamic features of the living networks, reorganize in response to changes in environmental conditions and inputs (Fig. 4), and perform basic SINGLE-MOLECULE DNA SEQUENCING We are interested in the study of matter at the level of single molecules. For these studies we use the transmembrane protein α-hemolysin as a rapid and highly sensitive sensor element for stochastic analysis of the molecules lodged or trapped inside the protein pore; the analysis relies on detecting the perturbations in the conductance levels produced in the ion channel in the native protein. Using this technique, we developed an approach by which a single-stranded DNA molecule can be trapped in a specific configuration inside an α-hemolysin channel, manipulated, and studied with high sensitivity at the single-molecule level. We have been able to detect up to 9 consecutive DNA polymerase–catalyzed single-nucleotide primer extensions (Fig. 3) with high F i g . 4 . Adaptive reorganization in a synthetic peptide network. The graph structure or wiring of a synthetic peptide network responds dramatically to changes in the environmental stimuli (pH or salt content). Boolean logic functions. We suggest that the ability to rationally construct predictable chemical circuitry might be useful in advancing the modeling and better understanding of some of the basic dynamic information-processing characteristics of the more complex cellular networks. M O L E C U L A R C O M P U TAT I O N F i g . 3 . Single-molecule monitoring of DNA polymerase–catalyzed single-nucleotide primer extensions with high sensitivity via an α-hemolysin–DNA–rotaxane device. sensitivity and spatial resolution (≤2.4 Å). The singlebase resolution of this approach and the ability to control the passage of DNA in single-base steps satisfy the 2 minimal requirements of a nanopore-based sequencing device. COMPLEX SYNTHETIC NETWORKS Living cells use complex networks of evolutionarily selected biomolecular interactions and chemical transformations to process multiple extracellular input signals rapidly and simultaneously. We are interested in understanding and experimentally modeling the organizational and functional properties of biological networks. We have developed a general strategy for the design and construction of self-organized synthetic peptide networks based on the sequence-selective autocatalytic and cross-catalytic template-directed coiled coil peptide fragment condensation reactions in aque- A fundamental goal of computing is to reproduce in a molecular setting the familiar properties of microelectronics, such as digital logic, component modularity, and hierarchical design capacity. In this regard, significant advances have been made in the design of molecular logic gates by using small-molecule and rotaxane complexes, deoxyribozymes, enzymatic biochemical networks, peptide networks, and other systems. However, the molecular logic gates must be integrated into more complex networks in which outputs from each gate can serve as inputs to downstream gates. We recently described the construction of a basis set of DNA-based logic gates (AND, OR, AND-NOT) capable of communicating with one another. These gates were rewired into a higher-order circuit that enforces a net XOR (Exclusive OR) Boolean behavior (Fig. 5), showing that the components can be modularly recombined to implement novel logic processing. Our results support the notion that with a basis set of only a few logic gates and within the limits imposed by the availability of uniquely addressable oligonucleotide sequences, design of molecular circuits capable of performing a large variety of digital logic operations might be within reach. CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE 97 A Merging of Chemistry and Biology K.D. Janda, J. Ashley, K. Capková, S. De Lamo Marin, J. Denery, T. Dickerson, A. Di Mola, B. Ellis, L. Eubanks, K. Fukuchi, C. Hernandez, G. Kaufmann, C. Lowery, F i g . 5 . A multilevel circuit built from OR, AND, and AND-NOT gates that performs a net XOR (Exclusive-OR) analysis on the inputs. PREBIOTIC CHEMISTRY The emergence of a polymer that could store genetic information, replicate, and exhibit phenotypic properties subject to selective environmental pressures marked a crucial stage in the transition from the prebiotic world to biology; however, the nature of such a polymer remains unresolved. We have discovered an oligomer family that quickly and efficiently self-assembles via reversible covalent anchoring of nucleobase recognition units onto simple peptide backbones. The resulting oligomers specifically self-pair and cross-pair with complementary strands of RNA and DNA in Watson-Crick fashion. Moreover, the oligomers undergo dynamic component exchange, template-directed assembly processes, and dynamic sequence modification in response to changing selective pressures. Such oligomers could therefore have participated in a number of processes that would be advantageous for primordial genetic systems, such as dynamic sequence repair and adaptation. PUBLICATIONS Cockroft, S.L., Chu, J., Amorin, M., Ghadiri, M.R. A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution. J. Am. Chem. Soc. 130:818, 2008. Frezza, B.M., Cockroft, S.L., Ghadiri, M.R. Modular multi-level circuits from immobilized DNA-based logic gates. J. Am. Chem. Soc. 129:14875, 2007. Gianneschi, N.C., Ghadiri, M.R. Design of molecular logic devices based on a programmable DNA-regulated semisynthetic enzyme. Angew. Chem. Int. Ed. 46:3955, 2007. Leman, L.J., Weinberger, D.A., Huang, Z.-Z., Wilcoxen, K.M., Ghadiri, M.R. Functional and mechanistic analyses of biomimetic aminoacyl transfer reactions in de novo designed coiled coil peptides via rational active site engineering. J. Am. Chem. Soc. 129:2959, 2007. S. Mahajan, A. Mayorov, G. McElhaney, J. Mee, A. Moreno, Y. Nakai, A. Nguyen, A. Nunes, J. Park, A. Rohrbach, C. Saccavini, N. Salzameda, S. Steiniger, J. Treweek, A. Willis, Y. Xu, Y. Yoneda, B. Zhou, H. Zhou uring the past year, we used various applications of organic chemistry to address biological problems. Representative examples of our results are given for 3 research programs: inhibition of bacterial virulence via the disruption of bacterial communication, discovery of a link between drug abuse and cardiovascular disease, and selection and characterization of human neutralizing antibodies against Bacillus anthracis toxin. D I N F E C T I O N C O N T R O L B Y A N T I B O D Y - M E D I AT E D I N T E R F E R E N C E W I T H B A C T E R I A L C O M M U N I C AT I O N The ability of microorganisms to coordinate their gene expression according to population density has been termed quorum sensing. This chemical exchange of information among single-cell organisms is mediated by secreted signaling molecules termed autoinducers. Important biological and clinical aspects of quorum sensing include the regulation of bacterial virulence factors and the formation of biofilms; hence, inhibition of signaling associated with quorum sensing could provide a promising new strategy for the attenuation of bacterial infections. Indeed, analogs of autoinducers have been used as small-molecule antagonists in several quorum-sensing circuits as a means of signaling interference. Alternatively, we have pioneered an antibody-based strategy to combat quorum sensing through disruption of signal transmission. Recently, we applied our antibody-based technology to the interference of the quorum-sensing circuits of Staphylococcus aureus. This microorganism is the most common cause of hospital-acquired infections, including diseases ranging from skin infections and food poisoning to life-threatening nosocomial infections. The increasing resistance of S aureus isolates to glycopeptide antibiotics, most prominently vancomycin, is a major concern in intensive care units, and an alternative strategy to combat this pathogen is urgently required. 98 CHEMISTRY 2008 Staphylococcus aureus uses a set of 4 cyclic autoinducing peptides (AIP-1–AIP-4) to regulate its quorumsensing machinery, which is responsible for orchestrating the expression of virulence genes. Thus, inhibition of the S aureus system would result in decreased pathogenicity. We generated a monoclonal antibody, AP4-24H11, to sequester AIP-4 (Fig. 1). This antibody was elicited THE SCRIPPS RESEARCH INSTITUTE In addition to causing severe dopaminergic neurotoxic effects, chronic methamphetamine self-administration induces increasing drug tolerance that correlates with escalating intake. Although the molecular mechanism behind pharmacologic tolerance is not fully elucidated, we hypothesized that methamphetamine covalently modifies endogenous proteins in a process known as glycation (Fig. 2) before reaching the brain and medi- F i g . 1 . Structure of the S aureus autoinducer AIP-4 and AP4 hapten used to generate the quorum-quenching antibody AP4-24H11. against a rationally designed hapten (AP4, Fig. 1) and efficiently interfered with the quorum sensing of S aureus in vitro, as determined by real-time polymerase chain reaction analysis and inactivation of AP4-24H11 by synthetic AIP-4. Importantly, AP4-24H11 suppressed both S aureus–induced dermal injury in a mouse model of abscess formation in vivo and provided complete protection against a lethal S aureus challenge. These findings provide a strong foundation for further investigations of immunopharmacotherapy as treatment of bacterial infections in which quorum sensing controls the expression of virulence factors. A L I N K B E T W E E N C H R O N I C M E T H A M P H E TA M I N E U S E A N D C A R D I O VA S C U L A R D I S E A S E The rapid spread of methamphetamine abuse across the United States is as alarming as the propensity of the drug to induce severe addiction and the healthrelated consequences of addiction. Whereas before 2001 methamphetamine use occurred predominantly in the western United States, its use now is extending rapidly throughout the United States and across different ethnic groups. The threat that methamphetamine now poses to society underscores the need to more thoroughly examine the ramifications of chronic methamphetamine abuse. F i g . 2 . Reaction scheme of methamphetamine protein glycation as initiated by glucose and methamphetamine. ating its well-characterized stimulant effects. Glycation reactions, collectively termed the Maillard reaction, have been studied for decades in the food industry in the development of flavor and color; however, Maillard products can also assume a biologically hazardous role when synthesized in vivo. Acquiring the ability to crosslink proteins, these irreversible reaction products, termed advanced glycation end products (AGEs), have gained notoriety for their participation in a range of pathologic changes. Protein glycation by methamphetamine induces an immune response against these modified proteins, which could lead to sequestration of drug and, ultimately, the development of tolerance. We have shown that this drugdependent glycation mechanism is operative in vivo. We detected antibodies against methamphetamine-derived AGEs in rats that chronically self-administered the drug, and we noted a direct relationship between the level of methamphetamine intake and the respective antibody titers against methamphetamine-glycated proteins. Additionally, we detected increased levels of proinflammatory and other cytokine molecules, particularly vascular endothelial growth factor. AGE-associated upreg- CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE 99 ulation of this growth factor has been associated with the onset of heart disease, but these effects had not been previously associated with methamphetaminederived AGEs. Because AGEs can alter protein function in vivo and participate in various diseases, methamphetamine-derived AGEs provide an unrecognized molecular mechanism for the development of vasculitis and other cardiovascular maladies with high incidence in chronic methamphetamine users. HUMAN NEUTRALIZING ANTIBODIES AGAINST ANTHRAX TOXIN A less-than-adequate therapeutic plan for the treatment of anthrax in the 2001 bioterrorism attacks has highlighted the importance of developing alternative or complementary therapeutic approaches for biothreat agents. Vaccination against B anthracis for protection against anthrax has been known for more than a century. However, the prolonged vaccination schedules and induction times required for an immune response are serious drawbacks, because the therapeutic window for treatment of anyone exposed to a deliberate release of B anthracis is limited. Alternatively, recently developed antibiotic prophylaxis for the treatment of persons exposed to anthrax, although important, would also be of lesser value if the infection were caused by an antibiotic-resistant strain. Passive immunization has provided an attractive avenue as a treatment both before and after exposure to B anthracis. Indeed, in many animal studies, passive transfer of antiserum successfully provided protection against anthrax. Furthermore, passive immunization could have advantages over active vaccination and antibiotic treatments via few toxic effects, high specificity, the capability for stockpiling large quantities of the antiserum, and immediate protection against a biological attack. Using a phage-displayed human single-chain variable fragment (scFv) antibody library, we selected and characterized several human monoclonal neutralizing antibodies against the toxin of B anthracis. In total, 15 clones with distinct sequences and high specificity for the protective antigen region of the anthrax toxin (Fig. 3) were analyzed by using biophysical and cellbased cytotoxicity assays. From this panel of antibodies, a set of neutralizing antibodies was identified, and the potency of protection was established by using a macrophage cytotoxicity assay. Among the neutralizing antibodies identified, 1 clone had excellent affinity for the protective antigen region of the anthrax toxin and F i g . 3 . Targeting of the protective antigen (PA) region of B anthracis toxin by human monoclonal antibodies. provided superior protection from lethal toxin in the cell cytotoxicity assay. Our results add to the ever-growing arsenal of immunologic and functional analysis of monoclonal antibodies to the exotoxins of anthrax. In addition, the antibodies may be new candidates for prophylactic and therapeutic agents. PUBLICATIONS Brogan, A.P., Dickerson, T.J., Janda, K.D. Nornicotine-organocatalyzed aqueous reduction of α,β-unsaturated aldehydes. Chem. Commun. (Camb.) Issue 46:4952, 2007. Capková, K., Yoneda, Y., Dickerson, T.J., Janda, K.D. Synthesis and structureactivity relationships of second-generation hydroxamate botulinum neurotoxin A protease inhibitors. Bioorg. Med. Chem. Lett. 17:6463, 2007. Debler, E.W., Kaufmann, G.F., Meijler, M.M., Heine, A., Mee, J.M., Pljevaljcic, G., Di Bilio, A.J., Schultz, P.G., Millar, D.P., Janda, K.D., Wilson, I.A., Gray, H.B., Lerner, R.A. Deeply inverted electron-hole recombination in a luminescent antibody-stilbene complex. Science 319:1232, 2008. Dickerson, T.J., McKenzie, K.M., Hoyt, A.S., Wood, M.R., Janda, K.D., Brenner, S., Lerner, R.A. Phage escape libraries for checkmate analysis. Proc. Natl. Acad. Sci. U. S. A. 104:12703, 2007. Ino, A., Dickerson, T.J., Janda, K.D. Positional linker effects in haptens for cocaine immunopharmacotherapy. Bioorg. Med. Chem. Lett. 17:4280, 2007. Kaufmann, G.F., Park, J., Janda, K.D. Bacterial quorum sensing: a new target for anti-infective immunotherapy. Expert Opin. Biol. Ther. 8:719, 2008. Kaufmann, G.F., Park, J., Mee, J.M., Ulevitch, R.J., Janda, K.D. The quorum quenching antibody RS2-1G9 protects macrophages from the cytotoxic effects of the Pseudomonas aeruginosa quorum sensing signalling molecule N-3-oxo-dodecanoyl-homoserine lactone. Mol. Immunol. 45:2710, 2008. Lowery, C.A., Dickerson, T.J., Janda, K.D. Interspecies and interkingdom communication mediated by bacterial quorum sensing. Chem. Soc. Rev. 37:1337, 2008. Park, J., Jagasia, R., Kaufmann, G.F., Mathison, J.C., Ruiz, D.I., Moss, J.A., Meijler, M.M., Ulevitch, R.J., Janda, K.D. Infection control by antibody disruption of bacterial quorum sensing signaling. Chem. Biol. 14:1119, 2007. Park, J., Kaufmann, G.F., Bowen, J.P., Arbiser, J.L., Janda, K.D. Solenopsin A, a venom alkaloid from the fire ant Solenopsis invicta, inhibits quorum-sensing signaling in Pseudomonas aeruginosa. J. Infect. Dis. 198:1198, 2008. 100 CHEMISTRY 2008 Richardson, H.N., Zhao, Y., Fekete, E.M., Funk, C.K., Wirsching, P., Janda, K.D., Zorrilla, E.P., Koob, G.F. MPZP: a novel small molecule corticotropin-releasing factor type 1 receptor (CRF1) antagonist. Pharmacol. Biochem. Behav. 88:497, 2008. Treweek, J., Wee, S., Koob, G.F., Dickerson, T.J., Janda, K.D. Self-vaccination by methamphetamine glycation products chemically links chronic drug abuse and cardiovascular disease. Proc. Natl. Acad. Sci. U. S. A. 104:11580, 2007. Willis, B., Eubanks, L.M., Dickerson, T.J., Janda, K.D. The strange case of the botulinum neurotoxin: using chemistry and biology to modulate the most deadly poison. Angew. Chem. Int. Ed. 47:8360, 2008. Willis, B., Eubanks, L.M., Wood, M.R., Janda, K.D., Dickerson, T.J., Lerner, R.A. Biologically templated organic polymers with nanoscale order. Proc. Natl. Acad. Sci. U. S. A. 105:1416, 2008. Xu, Y., Hixon, M.S., Dawson, P.E., Janda, K.D. Development of a FRET assay for monitoring of HIV gp41 core disruption. J. Org. Chem. 72:6700, 2007. Yoneda, Y., Steiniger, S.C., Capkova, K., Mee, J.M., Liu, Y., Kaufmann, G.F., Janda, K.D. A cell-penetrating peptidic GRP78 ligand for tumor cell-specific prodrug therapy. Bioorg. Med. Chem. Lett. 18:1632, 2008. Zarebski, L.M., Vaughan, K., Sidney, J., Peters, B., Grey, H., Janda, K.D., Casadevall, A., Sette, A. Analysis of epitope information related to Bacillus anthracis and Clostridium botulinum. Expert Rev. Vaccines 7:55, 2008. Zhou, B., Carney, C., Janda, K.D. Selection and characterization of human antibodies neutralizing Bacillus anthracis toxin. Bioorg. Med. Chem. 16:1903, 2008. Zhou, B., Pellett, S., Tepp, W.H., Zhou, H., Johnson, E.A., Janda, K.D. Delineating the susceptibility of botulinum neurotoxins to denaturation through thermal effects. FEBS Lett. 582:1526, 2008. Zhou, H., Zhou, B., Ma, H., Carney, C., Janda, K.D. Selection and characterization of human monoclonal antibodies against Abrin by phage display. Bioorg. Med. Chem. Lett. 17:5690, 2007. Maintaining the Proteome to Ameliorate Human Disease J.W. Kelly, S. Choi, E. Culyba, M.T.A. Dendle, D. Du, C. Fearns, A. Fuller, T.-W. Mu, A. Murray, D. Ong, J. Paulsson, E.T. Powers, P. Rao, M. Saure, R. Simkovsky, S. Siegel, J. Solomon, K. Usui, Y. Wang, I. Yonemoto, Z. Yu aintenance of the proteome (proteostasis) both inside and outside human cells is essential for development, reproduction, and successful aging. Deficiencies in proteostasis lead to many metabolic, oncologic, neurodegenerative, and cardiovascular diseases. Understanding the mechanisms of proteostasis, especially defects in the pathways of the proteostasis network that occur with aging, enables the design of new therapeutic strategies to ameliorate ageonset protein misfolding diseases, a main goal of our research. We use animal and cell-based disease models and biophysical approaches in combination with medicinal chemistry and structure-based drug design. Our collaborators, W.E. Balch, Department of Cell Biology; J. Buxbaum, Department of Molecular and Experimental M THE SCRIPPS RESEARCH INSTITUTE Medicine; J.R. Yates, Department of Chemical Physiology; E. Masliah, University of California, San Diego; and A. Dillin, the Salk Institute for Biological Studies, La Jolla, California, play an essential role in our multidisciplinary approach. A M E L I O R AT I O N O F LY S O S O M A L S T O R A G E D I S E A S E S Lysosomal storage diseases are loss-of-function diseases often caused by a mutation in one of the lysosomal enzymes, which results in excessive misfolding of the enzyme within the endoplasmic reticulum and cytosolic degradation instead of proper folding and trafficking of the enzyme to the lysosome. In 2 separate studies, we found that the innate proteostasis capacity of a cell can be enhanced with small molecules termed proteostasis regulators to fold mutated enzymes that would otherwise misfold and be degraded, resulting in increased trafficking of the mutated enzyme to the lysosome and increased function. In the first study, we found that diltiazem and verapamil, L-type calcium channel blocker drugs approved by the Food and Drug Administration, increased folding capacity in the endoplasmic reticulum, trafficking, and activity of mutant lysosomal enzymes associated with 3 distinct lysosomal storage diseases: Gaucher disease, α-mannosidosis, and type IIIA mucopolysaccharidosis. These compounds likely act by calcium ion–mediated enhancement of endoplasmic reticulum lumenal chaperone function. In the second study, we discovered that 2 proteostasis regulators partially restored folding, trafficking, and function of mutant enzymes in Gaucher and Tay-Sachs cell lines by activating the unfolded protein response, a signaling pathway that influences proteostasis in the secretory pathway. Moreover, we found that the combination of a proteostasis regulator and a pharmacologic chaperone, a chemical that binds directly to a given enzyme and thereby stabilizes the enzyme, synergistically restored enzyme function, because of their distinct mechanisms of action. U N D E R S TA N D I N G T H E E T I O L O G Y O F A L Z H E I M E R ’ S DISEASE We are interested in understanding the molecular and mechanistic basis for the age-onset nature of Alzheimer’s disease. Genetic and biochemical evidence implicates aggregation of amyloid β-peptide (Aβ), enabled by an age-onset decrease in proteostatic capacity, as the cause of neurodegeneration in this disease; however, precise identification of the toxic structure and the mechanism of neurotoxic effects remain elusive. In patients with Alzheimer’s disease, the correlation between CHEMISTRY 2008 disease severity and the concentration of spherical aggregates, annular structures, protofibrils and other soluble oligomeric species is better than the correlation between disease severity and the concentration of fibrillar amyloid. Previously, we showed that mutating the phenylalanine 19–phenylalanine 20 backbone amide bond to an E-olefin bond allows the formation of spherical aggregates to the exclusion of fibrils. In a more extensive amide-to-ester mutagenesis scan through the hydrophobic core (residues 17–21) of Aβ 1-40, we compared the mutants with wild-type Aβ 1-40 and the E-olefin Aβ 1-40 mutant. Even though the E-olefin mutant, the amide-to-ester mutant, and wild-type Aβ 1-40 form aggregates of different morphologies, all 3 types of aggregates were similarly toxic to PC12 neuronal cells. This finding suggests that a common, but low-abundance, aggregate morphology mediates toxic effects or that several different aggregate morphologies are similarly toxic. O X I D I Z E D M E TA B O L I T E E N H A N C E M E N T O F A M Y L O I D F O R M AT I O N One of the central mysteries of Alzheimer’s disease is how Aβ forms amyloid in vivo when both thermodynamic and kinetic barriers against aggregation exist. We propose that covalent modification of Aβ by smallmolecule oxidation products can explain, at least in part, the ability of Aβ to form amyloid at physiologic concentrations and thus place a load on the proteostasis network. Using Aβ conjugates site-specifically modified with a cholesterol aldehyde at aspartic acid 1, lysine 16, or lysine 28, we found that modification lowered the critical concentration for aggregation into the nanomolar range, within the physiologic concentration range of Aβ, and dramatically increased the rate of aggregation. Aβ modified at lysine 16 formed amorphous aggregates fastest and at the lowest concentrations (within 2 hours at 20 nM). The same cholesterol aldehyde is found in human atherosclerotic lesions and rapidly promotes apolipoprotein C -II amyloid formation in vitro. Thus, enhancement of amyloid formation by oxidized metabolites appears to be common to several diseases and suggests that strategies to prevent such modification may have therapeutic potential for a spectrum of human diseases. T R E AT I N G T R A N S T H Y R E T I N A M Y L O I D O G E N E S I S Transthyretin is 1 of 27 secreted human proteins, including amyloid, known to misfold and misassemble into extracellular aggregates. The rate-limiting step in amyloid formation by transthyretin is the dissociation THE SCRIPPS RESEARCH INSTITUTE 101 of the tetramer. We have developed kinetic stabilizers of the tetrameric structure of transthyretin that have novel chemistries and mechanisms of actions. To improve inhibitors of transthyretin amyloidogenesis, we are optimizing each of the 3 substructural elements that make up a typical inhibitor: the 2 aryl rings and the linker that joins the rings. We evaluated structural modifications to the aryl ring 1 by screening a library of 2-arylbenzoxazoles that have thyroid hormone–like aryl substituents on the 2-aryl ring. The 3,4,5-substituted thyroxine-like aryl ring appears to be the optimal solution for the structure of aryl ring 1. In addition, we synthesized 40 bisaryl compounds to optimize the structure of the linker. We found that direct connection of the 2 aryls, or linkage through nonpolar E-olefin or –CH 2 CH 2 – substructures, generates the most potent and selective inhibitors of transthyretin amyloidogenesis. Five high-resolution (1.4–1.8 Å) x-ray crystallography structures of transthyretin reveal that the 3,5dimethyl-4-hydroxyphenyl ring preferentially occupies the inner cavity of the thyroxine-binding site and that the 3,5-dibromo-4-hydroxyphenyl aryl prefers the outer cavity because the phenol is deprotonated with flanking electron withdrawing group substitution. A study to optimize the remaining aryl ring is well under way. Tetramers of transthyretin can also be kinetically stabilized by trans-suppression, as we showed previously with T119M transthyretin subunit incorporation into the tetramer, which stabilizes heterotetramers containing T119M and V30M transthyretin subunits. In an analogous manner, heterotetramers composed of murine transthyretin and human transthyretin subunits are kinetically stable and nonamyloidogenic. This information is important for evaluating transgenic models of human transthyretin amyloidosis in which the transgenic animals have a low copy number of the mutant amyloidogenic human transthyretin gene. U N D E R S TA N D I N G A N D A M E L I O R AT I N G G E L S O L I N AMYLOIDOSIS Gelsolin amyloid disease is another age-onset degenerative malady linked to protein aggregation that is thought to be due to an age-associated decline in proteostasis. A mutation in gelsolin (D187N) leads to aberrant folding and cleavage by furin within the Golgi apparatus during trafficking. Subsequent cleavage of the gelsolin fragment by the matrix metalloprotease MT1MMP outside the cell results in 5- and 8-kD fragments of gelsolin, which deposit as amyloid in the extracellular matrix. In collaboration with W.E. Balch, Depart- 102 CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE ment of Cell Biology, we have developed 2 transgenic mouse models of human D187N gelsolin amyloidosis that recapitulate the aberrant endoproteolytic cascade and the aging-associated decline in proteostasis that result in extracellular amyloidogenesis in humans. Total Synthesis, New Synthetic Technologies, and Chemical Biology PUBLICATIONS Balch, W.E., Morimoto, R.I., Dillin, A., Kelly, J.W. Adapting proteostasis for disease intervention. Science 319:916, 2008. K.C. Nicolaou, A. Agua, R. Aversa, W. Brenzovich, A. Burtoloso, J. Chen, K. Cole, S. Dalby, R. Denton, D. Edmonds, S. Ellery, A. Estrada, B. Fraga, M. Frederick, M. Freestone, C. Gelin, J. Goodwin-Tindall, M. Hesse, P. Huang, V. Jeso, M. Kar, A. Krasovskiy, A. Lemire, A. Li, H. Li, Y. Lim, T. Lister, N. Mainolfi, U. Majumder, C. Mathison, A. Morgan, A. Nold, A. Ortiz, N. Patil, B. Pratt, R. Reingruber, F. Rivas, A. Sanchez Ruiz, D. Sarlah, D. Shaw, A. Stepan, A. Talbot, Y. Tang, V. Trepanier, G. Tria, T. Umezawa, J. Wang, T. Wu, W. Zhan, H. Zhang Bieschke, J., Siegel, S.J., Fu, J., Kelly, J.W. Alzheimer’s Aβ peptides containing an isostructural backbone mutation afford distinct aggregate morphologies but analogous cytotoxicity: evidence for a common low-abundance toxic structure(s)? Biochemistry 47:50, 2008. Dillin, A., Kelly, J.W. The yin-yang of sirtuins. Science 317:461, 2007. Fowler, D.M., Koulov, A.V., Balch, W.E., Kelly, J.W. Functional amyloid: from bacteria to humans. Trends Biochem. Sci. 32:217, 2007. Jäger, M., Dendle, M., Kelly, J.W. A cross-strand Trp-Trp pair stabilizes a WW domain at the expense of function. Protein Sci. 16:2306 2007. Jäger, M., Nguyen, H., Dendle, M., Gruebele, M., Kelly, J.W. Influence of hPin1 WW N-terminal domain boundaries on function, protein stability, and folding. Protein Sci. 16:1495, 2007. Johnson, S.M., Connelly, S., Wilson, I.A., Kelly, J.W. Biochemical and structural evaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors. J. Med. Chem. 51:260, 2008. Kelly, J.W. Compromised cellular folding fidelity results in numerous clinically important diseases. Nature 446:112, 2007. Liu, F., Du, D., Fuller, A.A., Davoren, J.E., Wipf, P., Kelly, J.W., Gruebele, M. An experimental survey of the transition between two-state and downhill protein folding scenarios. Proc. Natl. Acad. Sci. U. S. A. 105:2369, 2008. Mu, T.-W., Fowler, D.M., Kelly, J.W. Partial restoration of mutant enzyme homeostasis in three distinct lysosomal storage disease cell lines by altering calcium homeostasis. PloS Biol. 6:e26, 2008. Münch, J., Rücker, E., Ständker, L., Adermann, K., Goffinet, C., Schindler, M., Wildum, S., Chinnadurai, R., Rajan, D., Specht, A., Giménez-Gallego, G., Sánchez, P.C., Fowler, D.M., Koulov, A., Kelly, J.W., Mothes, W., Grivel, J.C., Margolis, L., Keppler, O.T., Forssmann, W.G., Kirchhoff, F. Semen-derived amyloidogenic prostatic acidic phosphatase fragments dramatically enhance HIV infection. Cell 131:1059, 2007. Reixach, N., Foss, T.R., Santelli, E., Pascual, J., Kelly, J.W. Human-murine transthyretin heterotetramers are kinetically stable and non-amyloidogenic: a lesson in the generation of transgenic models of diseases involving oligomeric proteins. J. Biol. Chem. 283:2098, 2008. Stewart, C.R., Wilson, L.M., Zhang, Q., Pham, C.L.L., Waddington, L.J., Staples, M.K., Stapleton, D., Kelly, J.W., Howlett, G.J. Oxidized cholesterol metabolites found in human atherosclerotic lesions promote apolipoprotein C-II amyloid fibril formation. Biochemistry 46:5552, 2007. Wiseman, R.L., Koulov, A., Powers, E.T., Kelly, J.W., Balch, W.E. Protein energetics in maturation of the early secretory pathway. Curr. Opin. Cell Biol. 19:359, 2007. Wiseman, R.L., Powers, E.T., Buxbaum, J.N., Kelly, J.W., Balch, W.E. An adaptable standard for protein export from the endoplasmic reticulum. Cell 131:809, 2007. Yu, Z., Sawkar, A.R., Kelly, J.W. Pharmacologic chaperoning as a strategy to treat Gaucher disease. FEBS Lett. 274:4944, 2007. e focus on the total synthesis of natural products, the discovery and development of new synthetic technologies, and chemical biology. Naturally occurring substances are selected as synthetic targets for their novel molecular architectures, important biological properties, and interesting mechanisms of action. The projects are designed to optimize the opportunities for discovery and invention in the areas of chemistry, biology, and medicine. The natural products thiostrepton, azaspiracid-1–azaspiracid-3, abyssomycin C, the bisanthraquinones and the marinomycins exemplify this philosophy. Current projects include studies on the antibiotics nocathiacin I, platensimycin, and platencin; the antitumor agents lomaiviticins A and B and uncialamycin; the anti-HIV agent biyouyanagin A; and the marine biotoxin maitotoxin (Fig. 1). In addition, we are developing synthetic technologies and strategies for chemical synthesis and chemical biology studies. Our overall aims are to advance the art and science of chemical synthesis and to develop enabling technologies for biology and medicine while maximizing educational opportunities and training of young men and women in chemistry. W PUBLICATIONS Nicolaou, K.C., Chen, J.S., Zhang, H., Montero, A. Asymmetric synthesis and biological properties of uncialamycin and 26-epi-unicialamycin. Angew. Chem. Int. Ed. 47:185, 2008. Nicolaou, K.C., Cole, K.P., Frederick, M.O., Aversa, R.J., Denton, R.M. Chemical synthesis of the GHIJK ring system and further experimental support for the originally assigned structure of maitotoxin. Angew. Chem. Int. Ed. 46:8875, 2007. Nicolaou, K.C., Dethe, D.H., Chen, D.Y.-K. Total syntheses of amythiamicins A, B and C. Chem. Commun. (Camb.) Issue 23:2632, 2008. Nicolaou, K.C., Dethe, D.H., Leung, G.Y.C., Zou, B., Chen, D.Y.-K. Total synthesis of thiopeptide antibiotics GE2270A, GE2270T, and GE2270C1. Chem. Asian J. 3:413, 2008. CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE 103 Nicolaou, K.C., Wang, J., Tang, Y. Synthesis of the sporolide ring framework through a cascade sequence involving an intramolecular [4+2] cycloaddition reaction of an o-quinone. Angew. Chem. Int. Ed. 47:1432, 2008. Vale, C., Gómez-Limia, B., Nicolaou, K.C., Frederick, M.O., Vieytes, M.R., Botana, L.M. The c-Jun-N-terminal kinase is involved in the neurotoxic effect of azaspiracid-1. Cell. Physiol. Biochem. 20:957, 2007. Vilariño, N., Nicolaou, K.C., Frederick, M.O., Vieytes, M.R., Botana, L.M. Irreversible cytoskeletal disarrangement is independent of caspase activation during in vitro azaspiracid toxicity in human neuroblastoma cells. Biochem. Pharmacol. 74:327, 2007. Translational Chemistry and Medicine E. Roberts, G. Cherukupalli, C. Chiruta, O. Ghoneim, M. Guerrero, S. Park, X. Peng, F. Pinacho-Crisostomo, R. Poddutoori, K. Reynolds, M. Toussaint, M. Urbano, S. Velaparthi, Y. Wang F i g . 1 . Selected target molecules. Nicolaou, K.C., Frederick, M.O., Burtoloso, A.C.B., Denton, R.M., Rivas, F., Cole, K.P., Aversa, R.J., Gibe, R., Umezawa, T., Suzuki, T. Chemical synthesis of the GHIJKLMNO ring system of maitotoxin. J. Am. Chem. Soc. 130:7466, 2008. Nicolaou, K.C., Guduru, R., Sun, Y.-P., Banerji, B., Chen, D.Y.-K. Total synthesis of the originally proposed and revised structures of palmerolide A. Angew. Chem. Int. Ed. 46:5896, 2007. Nicolaou, K.C., Krasovskiy, A., Trépanier, V.É., Chen, D.Y.-K. An expedient strategy for the synthesis of tryptamines and other heterocycles. Angew. Chem. Int. Ed. 47:4217, 2008. Nicolaou, K.C., Li, H., Nold, A.L., Pappo, D., Lenzen, A. Total synthesis of kinamycins C, F, and J. J. Am. Chem. Soc. 129:10356, 2007. Nicolaou, K.C., Lister, T., Denton, R.M., Gelin, C.F. Cascade reactions involving formal [2+2] thermal cycloadditions: total synthesis of artochamins F, H, I, and J. Angew. Chem. Int. Ed. 46:7501, 2007. Nicolaou, K.C., Lister, T., Denton, R.M., Gelin, C.F. Total synthesis of artochamins F, H, I, and J through cascade reactions. Tetrahedron 64:4736, 2008. Nicolaou, K.C., Majumder, U., Philippe Roche, S., Chen, D.Y.-K. Construction of the “left-domain” of haplophytine. Angew. Chem. Int. Ed. 46:4715, 2007. Nicolaou, K.C., Ortiz, A., Denton, R.M. Metathesis reactions in the synthesis of complex molecules. Chem. Today 25:70, 2007. Nicolaou, K.C., Pappo, D., Tsang, K.Y., Gibe, R., Chen, D.Y.-K. A chiral pool based synthesis of platensimycin. Angew. Chem. Int. Ed. 47:944, 2008. Nicolaou, K.C., Sun, Y.-P., Guduru, R., Banerji, B., Chen, D.Y.-K. Total synthesis of the originally proposed and revised structures of palmerolide A and isomers thereof. J. Am. Chem. Soc. 130:3633, 2008. ntroduction of new medicines is crucial to preserve human health. We are dedicated to the pursuit of new and better therapies and continually challenge the frontiers of drug discovery. As we take major scientific steps toward the future, we move to a more knowledge-based drug discovery. Our goal is to generate high-quality clinical candidates as new medicines in therapeutic areas such as epilepsy/seizures, neuropathic pain, autoimmune diseases (e.g., multiple sclerosis), and developmental disorders (e.g., autism). I T R E AT M E N T O F N E U R O L O G I C D I S E A S E S Epilepsy is a disease in which a hyperexcited state of the CNS is caused by an imbalance between inhibitory and excitatory neurotransmission. Current epilepsy therapy focuses on the modulation of the classical neurotransmitters glutamate and γ-aminobutyric acid. The neuropeptide galanin antagonizes excitatory glutaminergic neurotransmission in the hippocampus, suggesting that galanin may have a role in seizure activity. In collaboration with T. Bartfai and X. Lu, Molecular and Integrative Neurosciences Department, we have identified new nonpeptidic ligands for the galanin receptors GalR1 and GalR2. This set of small, druglike molecules can displace the peptide galanin from its protein binding site. Selectivity and potency of these initial molecular starting points are being optimized. DUAL OPIOID AGONISTS–CHOLECYSTOKININ Nicolaou, K.C., Tang, Y., Wang, J., Stepan, A.F., Li, A., Montero, A. Total synthesis and antibacterial properties of carbaplatensimycin. J. Am. Chem. Soc. 129:14850, 2007. Nicolaou, K.C., Tria, G.S., Edmonds, D.J. Total synthesis of platencin. Angew. Chem. Int. Ed. 47:1780, 2008. A N TA G O N I S T S F O R T R E AT M E N T O F C H R O N I C A N D N E U R O PAT H I C PA I N Nociception, or the perception of pain, and its modulation depend on the interaction of many endogenous 104 CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE neurotransmitters in the spinal cord. The interaction of endogenous peptides such as cholecystokinin with exogenously administered opioids markedly alters activity in acute and chronic pain states. This interaction may lead to the development of novel medications that are more effective and safer than currently available opioids alone. Molecules with the property of being both opioid agonists and cholecystokinin antagonists would be useful in conditions in which the effectiveness of opioids is reduced, as in the development of tolerance to opioid pain relievers in chronic pain (e.g., in pain caused by cancers) and in neuropathic pain conditions in which opioids are ineffective. Thus, because of the prevention (or reversal) of tolerance, additionally physical dependence on opioids might be diminished or inhibited. The advantages of developing a single compound with dual opioid agonist–cholecystokinin antagonist activity rather than a combination of an opioid agonist taken with a separate cholecystokinin antagonist are clear. Development of a single compound involves only a single set of parameters, such as toxicology, pharmacokinetics, and formulation, rather than 2 independent and often unrelated sets of data. In collaboration with F. Porreca and J. Lai, University of Arizona, Tucson, we are using a limited set of molecular templates that have affinity across a wide range of type 1 G protein–coupled receptors to develop compounds with the required dual pharmacology. The 3 cloned opiate receptors (µ, δ, and κ) and the 2 cho- I M M U N O M O D U L AT I N G C O M P O U N D S F O R T H E lecystokinin receptors 1/(A) and 2/(B) are all members of this subclass of G protein–coupled receptors (Fig. 1). F i g . 1 . Opioid agonist–cholecystokinin antagonist hybrids. T R E AT M E N T O F M U LT I P L E S C L E R O S I S Sphingosine 1-phosphate (S1P) is an endogenous mediator that functions both as an intracellular messenger and as an extracellular signaling molecule. S1P is involved in a number of processes, including vascular stabilization, cardiac development, and cancer angiogenesis. Extracellularly, S1P elicits its biological effects through a family of G protein–coupled receptors that bind to the S1P1–S1P5 subtypes of S1P. Activation of the receptor for S1P1 had effects in multiple sclerosis and organ transplantation via the immunosuppression evoked by the nonselective S1P agonist FTY720, which is currently in clinical trials. It was speculated that the effects on heart rate and lung function in clinical trails with FTY720 were due to agonism of S1P3. In collaboration with H. Rosen and his group, Department of Immunology ane Microbial Science, we have identified novel small-molecule compounds that are exquisitely selective for S1P1, are stable, are orally active, and penetrate into the CNS (Table 1). These compounds are expected to be useful in disease states such as multiple sclerosis. T a b l e 1 . Agonists of sphingosine 1-phosphates. Compound Total polar Molecular Calculated surface area, Å2 weight, kD logP EC 50, nM Sphingosine 1phosphate 1 Sphingosine 1phosphate 3 CYM5313 64.4 349 4.5 0.52 823 CYM5326 64.4 351 4.4 0.2 529 CYM5327 64.4 349 4.5 3.0 Not applicable CYM5332 55.7 381 4.5 4.1 5200 CYM5357 64.4 351 4.4 3.1 1800 CYM5358 81.5 365 3.3 1.7 Not applicable CYM5380 72.6 354 3.6 0.8 774 CYM5389 72.6 340 3.5 4.2 606 CYM5390 72.6 340 3.5 7 1500 CYM5391 72.6 354 3.7 0.5 716 CYM5399 72.6 354 3.7 1.1 691 CYM5410 85.0 341 2.0 0.14 938 CYM5418 72.6 366 4.6 0.1 Not applicable CYM5422 64.8 361 4.5 1.24 Not applicable CYM5440 75.9 395 4.2 0.5 Not applicable CYM5442 84.7 409.5 3.7 1.1 Not applicable CYM5449 67.7 464 5.7 0.7 Not applicable CYM5457 84.7 397 3.4 3.4 692 CYM5464 75.9 409 4.6 0.84 Not applicable CYM5452 54.2 416 5.6 1.5 Not applicable CYM5386 46.3 387 5.2 4.5 1300 CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE 105 I L - 6 A N TA G O N I S T S F O R T R E AT M E N T O F INCREASING THE CHEMICAL AND GENETIC I N F L A M M AT O R Y D I S E A S E S POTENTIAL OF DNA Clinical studies have provided strong evidence that specific blockade of IL-6–regulated signaling pathways is a validated approach for treatment of inflammatory diseases. Currently, only monoclonal antibodies are available to block the actions of IL-6. Using the humanized antibody tocilizumab to block the actions of IL-6 has been therapeutically effective in patients with rheumatoid arthritis, systemic juvenile idiopathic arthritis, and Crohn’s disease. Tocilizumab is in phase 1 clinical trials in the United States and in phase 2 clinical trials in France for the treatment of multiple myeloma. However, serious adverse effects with tocilizumab have been reported, including a death and allergic pneumonitis. Increases in the levels of serum lipids, liver function abnormalities, and reduction in white blood cell count occurred in substantial numbers of patients. In collaboration with Dr. Porreca, we have identified small, orally active IL-6 receptor antagonists that are being modified for affinity and druglike properties. Once optimized, these antagonists should have fewer adverse side effects than does tocilizumab. We are interested in increasing the information potential of DNA by expanding the genetic alphabet with a third base pair composed of unnatural nucleobases. Using hydrophobicity, polarity, shape complementarity, and hydrogen bonding, we have developed novel unnatural base pairs, including several that are replicable in vitro. More recently, we screened more than 3600 unnatural nucleotides and identified a base pair that, after optimization, is replicated with an efficiency close to that of natural DNA synthesis. Nature developed the natural genetic code, not only by optimizing DNA and RNA but also by evolving the polymerases that synthesize these nucleic acids. We developed an activity-based selection system (Fig. 1) PUBLICATIONS Lum, C., Kahl, J., Kessler, L., Kucharski, J., Lundström, J., Miller, S., Nakanishi, H., Pei, Y., Pryor, K., Roberts, E., Sebo, L., Sullivan, R., Urban, J., Wang, Z. 2,5Diaminopyrimidines and 3,5-disubstituted azapurines as inhibitors of glycogen synthase kinase-3 (GSK-3). Bioorg. Med. Chem. Lett. 18:3578, 2008. Montalban, A.G., Boman, E., Chang, C.D., Ceide, S.C., Dahl, R., Dalesandro, D., Delaet, N.G., Erb, E., Ernst, J.T., Gibbs, A., Kahl, J., Kessler, L., Lundström, J., Miller, S., Nakanishi, H., Roberts, E., Saiah, E., Sullivan, R., Wang, Z., Larson, C.J. The design and synthesis of novel α-ketoamide-based p38 MAP kinase inhibitors. Bioorg. Med. Chem. Lett. 18:1772, 2008. F i g . 1 . Activity-based phage display selection system for evolv- ing polymerases with novel activity. Infection of phage (B) with the polymerase library (A) leads to production of phage particles that Chemical, Biological, and Biophysical Approaches to Understanding Evolution F.E. Romesberg, D.A. Bachovchin, P. Capek, J.K. Chin, R.T. Cirz, M.E. Cremeens, N. Gingles, Y. Hari, D.A. Harris, A. Horhota, G.T. Hwang, A.M. Leconte, E.T. Lis, S. Matsuda, B.A. O’Neill, M. Patel, M.E. Powers, T.C. Roberts, Y.J. Seo, P.A. Smith, M.C. Thielges, P. Weinkam, W. Yu, J. Zimmermann he molecules of biology are unique because they have been evolved for function. We use multidisciplinary methods in conjunction with chemical biological principles to develop unique approaches to understanding evolution. T display 0–1 copies of the polymerase and 3–5 copies of the acidic peptide. Phage particles are combined with DNA primer–template (C) and incubated with the desired nucleoside triphosphates. Active mutants are isolated (D) and characterized. to evolve polymerases for any desired function. Using this system, we have already evolved polymerases with a variety of novel functions, including the synthesis of DNA containing one of the unnatural base pairs. We are optimizing these polymerases and evolving new ones. REENGINEERING ANCIENT ANTIBIOTICS Because of the potential for cross-resistance, a great need exists for new antibiotics, especially ones that act via novel mechanisms. Although medicinal chemists have successfully reengineered already validated antibiotic scaffolds that were compromised by resistance, the identification of novel synthetic (nonnatural) scaffolds 106 CHEMISTRY 2008 has been extraordinarily challenging. Thus, natural products that might be candidates for antibiotics, perhaps even products that do not appear to still be active because of cross-resistance, warrant a careful examination. Using modern tools of synthesis and chemical biology, we might be able to determine why the products lost activity and perhaps use this knowledge to reengineer them to again be potent, broad-spectrum antibiotics. Arylomycins are a series of biphenyl-linked macrocyclic lipopeptide natural products that inhibit the essential bacterial signal peptidase I (SPase) in vitro but have low potency and a narrow spectrum as antibiotics. After some initial interest, these natural products were abandoned by the pharmaceutical industry because of their insufficient potency, which presumably was due to the resistance that developed during their use in bacterial warfare over eons of time. We recently reported the first total synthesis of a member of this class of natural products: arylomycin A2. With large quantities of arylomycin A2 in hand, we evaluated it against a wide variety of bacteria and discovered that it is extremely potent against the important human pathogen Staphylococcus epidermidis. By using UV light to create mutants of S epidermidis, we discovered that resistance evolved via the introduction of a proline residue into a conserved region of the SPase substrate-binding site. Sequence analysis of other bacterial SPases revealed that all bacteria with natural resistance to arylomycin A2 already had the "resistance-conferring" proline, and we found that when this proline is removed genetically, important pathogens such as Escherichia coli and Staphylococcus aureus become sensitive to arylomycin A2. Sequence analysis also indicated additional pathogens predicted to be sensitive, including the gram-positive pathogens Streptococcus pyogenes and Streptococcus pneumoniae and the gram-negative pathogens Helicobacter pylori and Chlamydia trachomatis. These data suggest that if the arylomycins can be reengineered to bind SPase regardless of the resistanceconferring proline, they will again be potent, broadspectrum antibiotics. Currently, we are characterizing the mechanism of arylomycin resistance to determine how to reengineer these natural products for potency and for the design, synthesis, and characterization of potentially active derivatives. EVOLUTION OF PROTEIN DYNAMICS Molecular recognition underlies almost all of a protein’s biological functions. Nowhere is the evolution of molecular recognition more impressive than within the THE SCRIPPS RESEARCH INSTITUTE immune response; antibodies are evolved within a matter of days to selectively recognize almost any foreign molecule. Antibodies are also remarkable because different intermediates can be isolated during their evolution. We use ultrafast nonlinear optical and nuclear magnetic resonance spectroscopy to characterize these intermediates. We have generated a comprehensive view over all timescales, from femtoseconds to seconds, of how antibodies are evolved for molecular recognition. Finally, the products of evolution are molecules with unique vibrational dynamics. The study of vibrational dynamics in proteins and nucleic acids has been limited by spectral complexity, but selective deuteration of a protein or a nucleic acid results in a carbon-deuterium oscillator that absorbs light in an otherwise transparent region of the infrared spectrum. The synthesis of selectively deuterated proteins has provided us with a residue-specific probe of flexibility, function, and folding. Previously, we focused on the biological redox activities of cytochrome c. More recently, we have focused on the protein recognition module SH3 and the enzyme dihydrofolate reductase. PUBLICATIONS Cirz, R.T., Jones, M.B., Gingles, N.A., Minogue, T.D., Jarrahi, B., Peterson, S.N., Romesberg, F.E. The complete and SOS-mediated response of Staphylococcus aureus to the antibiotic ciprofloxacin. J. Bacteriol. 189:531, 2007. Heideker, J., Lis, E.T., Romesberg, F.E. Phosphatases, DNA damage checkpoints and checkpoint deactivation. Cell Cycle 6:3058, 2007. Hwang, G.T., Leconte, A.M., Romesberg, F.E. Polymerase recognition and stability of fluoro-substituted pyridone nucleobase analogues. Chembiochem 8:1606, 2007. Matsuda, S., Fillo, J.D., Henry, A.A., Rai, P., Wilkens, S.J., Dwyer, T.J., Geierstanger, B.H., Wemmer, D.E., Schultz, P.G., Spraggon, G., Romesberg, F.E. Efforts toward expansion of the genetic alphabet: structure and replication of unnatural base pairs. J. Am. Chem. Soc. 129:10466, 2007. Roberts, T.C., Smith, P.A., Cirz, R.T., Romesberg, F.E. Structural and biological analysis of synthetic arylomycin A2. J. Am. Chem. Soc. 129:15830, 2007. Smith, P.A., Romesberg, F.E. Combating bacteria and drug resistance by inhibiting mechanisms of persistence and adaptation. Nat. Chem. Biol. 3:549, 2007. CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE 107 Synthesis of Natural Products, Development of Synthetic Methods, and Medicinal Chemistry W.R. Roush, R. Bates, D. Bykowski, M. Chen, E. Darout, A. DeBaillie, J. Dunetz, G. Halvorsen, M. Handa, J. Hicks, T. Hopkins, C.-W. Huh, F. Li, A. Legg, R. Lira, L. Martinez, C. Nguyen, G. Nora, R. Pragani, R. Rahaim, J. Roth, H. Sun, M. Tortosa, J. Whitaker, S. Winbush ur research has 2 major themes. One is the total synthesis of structurally complex, biologically active natural products such as those shown in Figure 1. In each of these syntheses, we emphasize the discovery, development, and/or illustration of new reactions and methods for achieving high levels of stereochemical control. These efforts are pursued in parallel with reaction design, stereochemical studies, and the development of new synthetic methods. We are particularly interested in stereochemical aspects of intramolecular and transannular Diels-Alder reactions, development of methods for the diastereoselective and enantioselective reactions of allylmetal compounds with carbonyl compounds, and nucleophilic phosphine-catalyzed organic reactions. Recent research has included stereochemical studies of transannular Diels-Alder reactions used in total syntheses of spinosyn A and superstolide A and development of new versions of the double allylboration reactions of aldehydes with γ-boryl-substituted allylboranes for stereocontrolled synthesis of 1,5-ene-diols, which are being used in several ongoing syntheses, including those of tetrafibricin, apoptolidin A, and peloruside. In addition, we have synthesized highly substituted tetrahydrofurans via [3+2]-annulation reactions of highly functionalized allylsilanes; this chemistry was recently applied to total syntheses of 10-hydroxytrilobacin and 3 stereoisomers. We have also developed phosphine-mediated organocatalytic reactions, and we recently completed the total synthesis of tedanolide. Our second area of major interest involves problems in bioorganic chemistry and medicinal chemistry. One long-term project is the design and synthesis of inhibitors of cysteine proteases isolated from tropical parasites, such as Trypanosoma cruzi, the causative agent of Chagas’ disease, and Plasmodium falciparum, the O F i g . 1 . Structures of recently synthesized natural products. most virulent of the malaria parasites. This research is performed in collaboration with colleagues at the University of California, San Francisco. In collaboration with S. Reed, University of California, San Diego, we have developed a cysteine protease inhibitor with remarkable ability to prevent Entamoeba histolytica from invading human intestinal tissue. Optimization of this inhibitor 108 CHEMISTRY 2008 for in vivo applications is in progress. New projects involve discovery of small molecules that affect cancer and other disease-related biochemical targets (e.g., nuclear hormone receptors), studies of structure-activity relationships, and optimization of the pharmacologic profile of certain natural products. PUBLICATIONS Chen, Y.-T., Lira, R., Hansell, E., McKerrow, J.H., Roush, W.R. Synthesis of macrocyclic trypanosomal cysteine protease inhibitors. Bioorg. Med. Chem. Lett. 18:5860, 2008. Dunetz, J., Roush, W.R. Concerning the synthesis of the tedanolide C(13)-C(23) fragment via an anti-aldol reaction. Org. Lett. 10:2059, 2008. Handa, M., Scheidt, K.A., Bossart, M., Zheng, N., Roush, W.R. Studies on the synthesis of apoptolidin A, I: synthesis of the C(1)-C(11) fragment. J. Org. Chem. 73:1031, 2008. Handa, M., Smith, W.J. III, Roush, W.R. Studies on the synthesis of apoptolidin A, II: synthesis of the disaccharide unit. J. Org. Chem. 73:1036, 2008. Hicks, J.C., Huh, C.W., Legg, A.D., Roush, W.R. Concerning the selective protection of (Z)-1,4-syn-ene-diols and (E)-1,5-anti-ene-diols as allylic triethylsilyl ethers. Org. Lett. 9:5621, 2007. Hicks, J.D., Roush, W.R. Synthesis of the C(26)-C(42) and C(43)-C(67) pyrancontaining fragments of amphidinol 3 via a common pyran intermediate. Org. Lett. 10:681, 2008. Lira, R., Roush, W.R. Enantio- and diastereoselective synthesis of syn-β-hydroxyallylsilanes via a chiral (Z)-γ-silylallylboronate. Org. Lett. 9:4315, 2007. Methot, J.L., Roush, W.R. Applications of tricoordinated phosphorus compounds in organic catalysis. In: Organophosphorus Compounds. Trost, B.M. (Ed.). Thieme Chemistry, New York, in press. Vol. 42 in Science of Synthesis. Roth, J., Madoux, F., Hodder, P., Roush, W.R. Synthesis of small molecule inhibitors of the orphan nuclear receptor steroidogenic factor-1 (NR5A1) based on isoquinolinone scaffolds. Bioorg. Med. Chem. Lett. 18:2628, 2008. Roush, W.R. Total synthesis of biologically active natural products. J. Am. Chem. Soc. 130:6654, 2008. Tortosa, M., Yakelis, N.A., Roush, W.R. Total synthesis of (+)-superstolide A. J. Am. Chem. Soc. 130:2722, 2008. Winbush, S.M., Mergott, D.J., Roush, W.R. Total synthesis of (–)-spinosyn A: examination of structural features that govern the stereoselectivity of the key transannular Diels-Alder reaction. J. Org. Chem. 73:1818, 2008. Biological Chemistry P.G. Schultz, E. Brustad, P. Chen, C. Dambacher, D. Groff, J. Grünewald, J. Guo, B. Hutchins, S. Kazane, H. Lee, J.-S. Lee, C. Liu, C. Lyssiotis, C. Melancon, J. Mills, R. Perera, F. Peters, S. Schiller, M. Sever, L. Supekova, T. Young lthough chemists are remarkably adept at the synthesis of molecular structure, they are far less sophisticated in designing and synthesizing molecules with defined biological or chemical functions. Nature, on the other hand, has produced an array of molecules with remarkably complex functions, ranging from photosynthesis and signal transduction to molec- A THE SCRIPPS RESEARCH INSTITUTE ular recognition and catalysis. Our aim is to combine the synthetic strategies and biological processes of Nature with the tools and principles of chemistry to create new molecules with novel chemical and biological functions. By studying the properties of the resulting molecules, we can gain new insights into the molecular mechanisms of complex biological and chemical systems. For example, we have shown that the tremendous combinatorial diversity of the immune response can be chemically reprogrammed to generate selective enzymelike catalysts. We have developed antibodies that catalyze a wide array of chemical and biological reactions, from acyl transfer to redox reactions. Characterization of the structure and mechanisms of these catalytic antibodies has led to important new insights into the mechanisms of biological catalysis. In addition, the detailed characterization of the properties and structures of germ-line and affinity-matured antibodies has revealed fundamental new aspects of the evolution of binding and catalytic function, in particular, the role of structural plasticity in the immune response. Most recently, we have focused on in vitro evolution methods that involve the development of novel chemical screens and selections for identifying metalloantibodies with proteolytic activity. In addition, we are extending this combinatorial approach to many other problems, including the generation of novel cellular reporters, the ab initio evolution of novel protein domains, and the synthesis of structure-based combinatorial libraries of small heterocycles. The libraries of small heterocycles are being used in conjunction with novel cellular and organismal screens to identify molecules that modulate the activity of important proteins involved in such cellular processes as differentiation, proliferation, and signaling. Indeed, we have identified molecules that control adult and embryonic stem cell differentiation and stem cell self-renewal and that reprogram lineage-committed cells to alternative cell fates. We are using x-ray crystallographic and biochemical studies, together with mRNA profiling technology and genetic complementation, to characterize the mode of action of these compounds and to study their effects on cellular processes and in animal models of regeneration. More recently, we extended such studies to a variety of genetic and neglected diseases (e.g., malaria, type 1 diabetes, spinal muscular atrophy, sickle cell anemia). We are also developing and applying modern genomics tools (e.g., cell-based phenotypic screens of arrayed genomic cDNA and short CHEMISTRY 2008 interfering RNA libraries) and proteomics tools (mass spectrometric phosphoprotein profiling) to a variety of significant biomedical problems in cancer biology, neurodegenerative disease, and virology. In addition, we are investigating the role and regulation of noncoding RNAs. We have also developed a general biosynthetic method that makes it possible to site specifically incorporate unnatural amino acids into proteins in vitro and in vivo. Using this method, we effectively expanded the genetic code of living organisms by adding new components to the existing biosynthetic machinery. We have genetically encoded amino acids with novel spectroscopic and chemical properties (e.g., metal-binding, sulfated, fluorescent, photocross-linking, and photoisomerizable) in response to unique 3- and 4-base codons. These amino acids are being used to explore protein structure and function both in vitro and in vivo, create novel therapeutic agents and biomaterials, and evolve proteins with novel properties. This approach has been developed for Escherichia coli, yeast, and mammalian cells, and we are now extending it to multicellular organisms. Our results have removed a billion-year constraint imposed by the genetic code on the ability to chemically manipulate the structures of proteins during translation. PUBLICATIONS Galkin, A.V., Melnick, J.S., Kim, S., Hood, T.L., Li, N., Li, L., Xia, G., Steensma, R., Chopiuk, G., Jiang, J., Wan, Y., Ding, P., Liu, F., Sun, F., Schultz, P.G., Gray, N.S., Warmuth, M. Identification of NVP-TAE684: a potent, selective. and efficacious inhibitor of NPM-ALK [published correction appears in Proc. Natl. Acad. Sci. U. S. A. 104:2025, 2007]. Proc. Natl. Acad. Sci. U. S. A. 104:270, 2007. Liu, Y., Kern, J.T., Walker, J. R. Johnson, J., Schultz, P.G., Luesch, H. A genomic screen for activators of the antioxidant response element. Proc. Natl. Acad. Sci. U. S. A. 104:5205, 2007. Gumireddy, K., Sun, F., Klein-Szanto, A.,J., Gibbins, J.M., Saunders, A., Schultz, P.G., Huang, Q. In vivo selection for metastasis promoting genes in the mouse. Proc. Natl. Acad. Sci. U. S. A. 104:6696, 2007. Liu, W., Alfonta, L., Mack, A.V., Schultz, P.G. Structural basis for the recognition of para-benzoyl-L-phenylalanine by evolved aminoacyl-tRNA synthetases. Angew. Chem. Int. Ed. 46:6073, 2007. Xie, J., Supekova, L., Schultz, P.G. A genetically encoded metabolically stable analogue of phosphotyrosine in Escherichia coli. ACS Chem. Biol. 2:474, 2007. THE SCRIPPS RESEARCH INSTITUTE Click Chemistry and Biological Activity K.B. Sharpless, J. Culhane, J. Fotsing, S. Grecian, N. Grimster, J. Hein, T. Horneff, J. Kalisiak, K. Korthals, S.-W. Kwok, S. Pitram, J. Raushel, B. Stump, J. Tripp, C. Valdez, T. Weide he driving forces in our research are the discovery and understanding of chemical reactivity, the harbingers of new discoveries in chemistry. Our goal is to develop chemical transformations that enable scientists to rapidly synthesize diverse compounds with desired properties; after all, it is the function of molecules that matters. The nature of the building blocks and the speed with which synthesis, screening for the desired function, and lead optimization can be performed are determining factors in the search for new compounds, whether the new entities are drugs, better plastics, or dyes. The greater the variety of scaffolds and functional groups that can be used in the rapid construction of candidate compounds, the more likely it is that new and useful function will be discovered. Because of the enormous number of compounds to explore (the number of small druglike molecules may be as high as 1064), the size of a given collection becomes much less important than the ability to rapidly probe the collection for a desired activity. Several years ago, we proposed a minimalistic approach to synthesis that relies solely on the best reactions for assembly of new molecules. Inspired by the natural synthesis of the myriad functional molecules (nucleic acids, proteins, and carbohydrates) from just a handful of building blocks, we devised a fast, reliable, and highly modular style of organic synthesis, which we termed click chemistry. Click reactions fulfill the most stringent criteria of usefulness and convenience (Fig. 1); they are highly energetically driven, and the T Wang, J., Schiller, S., Schultz, P.G. A biosynthetic route to dehydroalanine-containing proteins. Angew. Chem. Int. Ed. 46:6849, 2007. Xie, J., Liu, W., Schultz, P.G. A genetically encoded bidentate, metal-binding amino acid. Angew. Chem. Int. Ed. 46:9239, 2007. Supekova, L., Supek, F., Lee, J., Chen, S., Gray, N., Pezacki, J., Schlapbach, A., Schultz, P.G. Identification of human kinases involved in hepatitis C virus replication by small interference RNA library screening. J. Biol. Chem. 283:29, 2008. Lemke, E.A., Summerer, D., Geierstanger, B.H., Brittain, S.M., Schultz, P.G. Control of protein phosphorylation with a genetically encoded photocaged amino acid. Nat. Chem. Biol. 3:769, 2007. Guo, J., Wang, J., Anderson, J.C., Schultz, P.G. Addition of an α-hydroxy acid to the genetic code of bacteria. Angew. Chem. Int. Ed. 47:722, 2008. 109 F i g . 1 . Click chemistry: molecular diversity from a handful of near-perfect reactions. 110 CHEMISTRY majority of them form carbon-heteroatom bonds. The reactions produce only the expected products and work regardless of which functional groups are present in the starting materials. Naturally, the number of reactions that meet these criteria is limited, but we contend that a wide variety of interesting and useful molecules can be easily made by using click chemistry and that the chances for achieving desirable biological activity with such compounds are at least as good as chances with the traditional target-guided approach. Recently, we realized that olefins are probably the most attractive starting molecules available to synthetic organic chemists. Olefins are readily accessible in large quantities and in many varieties, and processes for their selective oxidation provide convenient access to electrophilic intermediates such as epoxides, aziridines, aziridinium ions, and cyclic sulfates. These electrophilic intermediates are ideal for introduction of reactive “hot spots,” such as azides and acetylenes, that can be used for the assembly of final structures via 1,3-dipolar cycloadditions. The 1,3-dipolar cycloaddition of azides and alkynes, most extensively studied by R. Huisgen in the 1960s, and the copper- and ruthenium-catalyzed variants we developed with V.V. Fokin, Department of Chemistry, take a prominent place in click reactions. These transformations enable reliable assembly of complex molecules by means of the 1,2,3-triazole heterocycle. Although both alkynes and azides are highly energetic, they are quite unreactive to a broad range of reagents, solvents, and other common functional groups. This inertness allows clean sequential transformations of broad scope without the need for protecting groups, even if the reactions are performed in aqueous solvent in the presence of atmospheric oxygen. The 1,2,3-triazoles have advantageous properties of high chemical stability (in general, they are inert to severe hydrolytic, oxidizing, and reducing conditions, even at high temperatures), strong dipole moment, presence of aromatic groups, and the ability to accept hydrogen bonds. Thus, they can interact productively in several ways with biological molecules. For example, 1,2,3-triazoles can replace the amide bond in peptides, preventing proteolytic degradation of the peptides. Our focus on this powerful and underappreciated class of azoles led us back to the simple parent triazole (C2H 3N 3), which in solution is a rapidly equilibrating mixture of 2 tautomers (Fig. 2). The physical properties of the NH-triazole struck us as highly unusual and are, in 2008 THE SCRIPPS RESEARCH INSTITUTE F i g . 2 . Michael additions of NH-triazole. fact, much like those of water. These properties include its weak acid-base character, high proton conductivity, and a liquid range spanning nearly 200 degrees. In addition, the NH-1,2,3-triazole is stable: it is insensitive to impact, friction, rapid heating, and even detonation. We studied the Michael reaction of NH-triazole with α,β-unsaturated ketones. The 1H-1,2,3-triazolyl-ketones were selectively generated when the triazole was combined with a variety of enones under solvent-free conditions. The use of aprotic solvents with a catalytic base gave the corresponding 2H-regioisomers. Together, these 2 protocols provide direct access to either the N1or N2-substituted 1,3-triazolyl ketone regioisomers. PUBLICATIONS Finn, M.G., Kolb, H.C., Fokin, V.V., Sharpless, K.B. Concept and applications of click chemistry from the standpoint of advocates. Kagaku to Kogyo 60:976, 2007. Hawker, C.J., Fokin, V.V., Finn, M.G., Sharpless, K.B. Bringing efficiency to materials synthesis: the philosophy of click chemistry. Aust. J. Chem. 60:381, 2007. Kalisiak, J., Sharpless, K.B., Fokin, V.V. Efficient synthesis of 2-substituted-1,2,3triazoles. Org. Lett. 10:3171, 2008. Kwok, S.-W., Hein, J.E., Fokin, V.V., Sharpless, K.B. Regioselective synthesis of either 1H- or 2H-1,2,3-triazoles via Michael addition to α,β-unsaturated ketones. Heterocycles 76:1141, 2008. Liu, Y., Díaz, D.D., Accurso, A.A., Sharpless, K.B., Fokin, V.V., Finn, M.G. Click chemistry in materials synthesis, III: metal-adhesive polymers from Cu(I)-catalyzed azide-alkyne cycloaddition. J. Polym. Sci. A Polym. Chem. 45:5182, 2007. Radić, Z., Manetsch, R., Fournier, D., Sharpless, K.B., Taylor, P. Probing gorge dimensions of cholinesterases by freeze-frame click chemistry. Chem. Biol. Interact. 175:161, 2008. Sugawara, A., Sunazuka, T., Hirose, T., Nagai, K., Yamaguchi, Y., Hanaki, H., Sharpless, K.B., Omura, S. Design and synthesis via click chemistry of 8,9-anhydroerythromycin A 6,9-hemiketal analogues with anti-MRSA and -VRE activity. Bioorg. Med. Chem. Lett. 17:6340, 2007. Van der Eycken, E., Sharpless, K.B. Click chemistry. QSAR Comb. Sci. 26:1115, 2007. CHEMISTRY 2008 Vestberg, R., Malkoch, M., Kade, M., Wu, P., Fokin, V.V., Sharpless, K.B., Drockenmuller, E., Hawker, C.J. Role of architecture and molecular weight in the formation of tailor-made ultrathin multilayers using dendritic macromolecules and click chemistry. J. Polym. Sci. A Polym. Chem. 45:2835, 2007. Yoo, E.J., Ahlquist, M., Bae, I., Sharpless, K.B., Fokin, V.V., Chang, S. Mechanistic studies on the Cu-catalyzed three-component reactions of sulfonyl azides, 1-alkynes and amines, alcohols, or water: dichotomy via a common pathway. J. Org. Chem. 73:5520, 2008. Chemistry, Biology, and Inflammatory Disease P. Wentworth, Jr., D. Angrish, J. Dambacher, V. Dubrovskaya, R.K. Grover, J. Nieva, M. Puga, B.D. Song, M.M.R. Peram, J.K. Rogel, S.R. Troseth, H. Wang, A.D. Wentworth ur research is interdisciplinary and involves aspects of bioorganic, biophysical, physical organic, synthetic, and analytical chemistry coupled with biochemical techniques, cell-based assays, and animal models. We are interested in uncovering new mechanisms of disease in major conditions such as atherosclerosis, neurodegenerative diseases, ischemiareperfusion injury, macular degeneration, cancer, and infectious diseases. O THE SCRIPPS RESEARCH INSTITUTE 111 his group, Department of Chemistry, we extended this model and showed that these cholesterol seco-sterols also trigger the misfolding of amyloid β-peptide 1-40 , leading to formation of fibrils similar to those observed in patients with Alzheimer ’s disease. Using mutated synthetic sequences of amyloid β-peptide1-40, we found that the accelerated aggregation of this protein only occurs when only lysine 16, not lysine 28 or the N-terminal amino group of aspartic acid 1, of the sequence is modified. More recently, in studies of inflammatory aldehyde–initiated misfolding of antibody light chains (Bence-Jones proteins), we found that different aldehydes can trigger different forms of aggregation in different proteins. Thus, we have shown that the cholesterol seco-sterols atheronal-A and atheronal-B accelerate an amorphous form of aggregation, whereas 4-hydroxynonenal induced an amyloid form of aggregation of both λ and κ light chains (Fig. 1). A N T I B O D Y - C ATA LY Z E D WAT E R O X I D AT I O N PAT H WAY Our discovery that all antibody molecules can catalyze the reaction between singlet oxygen and water to give hydrogen peroxide is causing a revision of the idea that antibodies are only an adapter molecule within the immune system, linking recognition and killing of foreign pathogens. We are exploring both the chemical and biological aspects of this pathway, and new insights into how the pathway plays a role in immune defense and inflammatory damage are emerging. We are searching for the active site for the antibody-catalyzed water oxidation pathway within the antibody structure. We have cloned and expressed soluble individual domains (VHVL, CH1CL, VH, VL, CH1, CL) of the murine Fab 4C6. All of the domains can generate hydrogen peroxide when presented with singlet dioxygen, suggesting that the driving force is related to the immunoglobulin fold of the whole antibody. I N F L A M M AT O R Y A L D E H D Y E S A N D P R O T E I N MISFOLDING We have shown that the inflammation-derived cholesterol seco-sterols atheronal-A and atheronal-B trigger a deformation in the secondary structure of the normally folded low-density lipoprotein apoB-100 into a proamyloidogenic form. In collaboration with J.W. Kelly and F i g . 1 . Electron micrograph of fibrillar aggregation of antibody light chains induced by cholesterol seco-sterol and 4-hydroxynonenal (shown in white). Epidemiologic and clinical evidence point to an increased risk of cancer when linked with chronic inflammation, in a process thought to involve the establishment of a local inflammatory microenvironment conducive to the development of neoplasia. However, because of the complex interrelationships between the 2 conditions, the precise molecular and cellular mechanisms that underpin this relationship remain largely unresolved. We found that the inflammation-derived cholesterol 5,6-seco-sterol aldehydes atheronal-A and atheronal-B cause a loss of function of wild-type tumor suppressor protein p53, the so-called guardian of the genome, in a process that involves p53 misfolding and amyloidogen- 112 CHEMISTRY 2008 esis. Atheronal-A and atheronal-B, but not the aldehydes 4-hydroxynonenal and 4-hydroxyhexenal derived from polyunsaturated fatty acids, induce misfolding of wild-type p53 into an amyloidogenic form that binds thioflavin T and Congo red dye but cannot bind to a consensus DNA sequence (Fig. 2). Treatment of lung car- F i g . 2 . Optical microscopy images (100X) obtained with normal (upper) and cross-polarized (lower) light of aggregates generated by incubation of hexahistidine-tagged native p53 with atheronal-A and stained with Congo red. cinoma cells expressing wild-type p53 with atheronal-A and atheronal-B leads to dysfunctional p53, as determined by analysis of extracted nuclear protein and transcription activation of p21. Our results reveal a hitherto unknown chemical link between inflammation and cancer and expand the already pivotal role of p53 dysfunction in the risk for cancer. The increasing generality and specificity of aldehyde-initiated protein misfolding suggests that inflammatory aldehydes and their posttranslational modification of amyloidogenic peptides may be the chemical link between the known associations of inflammation, oxidative damage, and various misfolding diseases. THE SCRIPPS RESEARCH INSTITUTE species), African trypanosomiasis (sleeping sickness, Trypanosoma brucei), and American trypanosomiasis (Chagas’ disease, Tr ypanosoma cruzi) have limited effectiveness, thereby increasing drug resistance and inherent toxic effects of the drugs. Thus, an elucidation of new parasite-specific biological targets for therapeutic agents is needed. In this regard, the discovery that DNA from members of the order Kinetoplastida, but not other eukaryotes, contains an unusual modified base, β- D -glucosyl(hydroxymethyl)uracil, called base J, was a breakthrough. Extracts of several kinetoplastids contain a J-binding protein (JBP) that specifically binds to J-containing duplex DNA. JBP-1 is essential in Leishmania. As a drug target, JBP has merit. The protein shares little homology with other proteins in the Protein Data Bank, and it has a unique ligand, J-DNA containing telomeric stretches of double-stranded DNA, that does not occur in other eukaryotes. However, a preliminary high-throughput screen, focused on disrupting binding between JBP-1 and J-DNA, with a library of compounds consisting of all the major drug pharmacophoric groups has revealed no compounds of interest. In parallel, we have studied the molecular recognition that underlies JBP-1 recognition of glycosylated DNA. In collaboration with D.P. Millar and D.A. Case, Department of Molecular Biology, we found that JBP-1 interacts with the J-containing DNA only when a critical conformation of the glucose within the major groove is established. More recently, we discovered that low micromolar concentrations of the DNA intercalators daunorubicin and mitoxantrone disrupt the binding of JBP-1 with duplex DNA containing J-DNA. Modeling suggests that DNA binding of the intercalators leads to distortion, which leads to disruption of the edge-on conformation of the glucose within the major groove of the DNA. PUBLICATIONS Grover, R.K., Wentworth, P., Jr. Emerging therapies for kinetoplastid diseases. Prog. Infect. Dis., in press. Nieva, J., Shafton, A., Altobell, L.J. III, Tripurenani, S., Rogel, J.K., Wentworth, A.D., Lerner, R.A., Wentworth, P., Jr. Inflammatory aldehydes accelerate antibody light chain amyloid and amorphous aggregation. Biochemistry 47:7695, 2008. P R O T E I N 1 A N D G LY C O S Y L AT E D D N A Scanlan, C.N., Ritchie, G.E., Baruah, K., Crispin, M.D., Harvey, D.J., Singer, B.B., Lucka, L., Wormald, M.R., Wentworth, P., Jr., Zitzmann, N., Rudd, P.M., Burton, D.R., Dwek, R.A. Inhibition of mammalian glycan biosynthesis produces non-self antigens for a broadly neutralising, HIV-1 specific antibody. J. Mol. Biol. 372:16, 2007. Current treatments of parasitic infections such as leishmaniasis (cutaneous or visceral, Leishmania Scheinost, J.C., Boldt, G.E., Wentworth, P., Jr. Protein misfolding diseases. In: Encyclopedia of Chemical Biology, Wiley Blackwell, New York, in press. INTERACTION BETWEEN PROTOZOAN J-BINDING CHEMISTRY 2008 Scheinost, J.C., Wang, H., Boldt, G.E., Offer, J., Wentworth, P., Jr. Cholesterol secosterol-induced aggregation of methylated amyloid-β peptides, insights into aldehydeinitiated fibrillization of amyloid-β. Angew. Chem. Int. Ed. 47:3919, 2008. Temperini, C., Cecchi, A., Boyle, N.A., Scozzafava, A., Cabeza, J.E., Wentworth, P., Jr., Blackburn, G.M., Supuran, C.T. Carbonic anhydrase inhibitors. Interaction of 2-N,N-dimethylamino-1,3,4-thiadiazole-5-methylsulfonamide with 12 mammalian isoforms: kinetic and x-ray crystallographic studies. Bioorg. Med. Chem. Lett. 18:999, 2008. Wentworth, P., Jr., Witter, D. Antibody-catalyzed water-oxidation pathway. Pure Appl. Chem. 80:1849, 2008. Bioorganic and Synthetic Chemistry C.-H. Wong, C. Bennett, S. Dean, S. Ficht, Y. Fu, W. Greenberg, R. Guy, S. Hanson, Z. Hong, D.-R. Hwang, M. Imamura, K. Kishikawa, J.-C. Lee, P.-H. Liang, L. Liu, T. Polat, S.-K. Wang, Y.-Y. Yang e develop new chemical and enzymatic strategies for synthesis of bioactive small molecules and biomolecules. We use the methods to probe carbohydrate-mediated recognition events important in cancer, bacterial infections, and viral infections, including HIV disease and influenza. W THE SCRIPPS RESEARCH INSTITUTE 113 these enzymes to catalyze new reactions and synthesize new molecules of pharmaceutical relevance. C A R B O H Y D R AT E - M E D I AT E D R E C O G N I T I O N I N DISEASE We are using our synthetic methods to discover inhibitors and therapeutic agents in several diseases related to carbohydrates. Current targets include bacterial transglycosidase, sulfatases, and glycoprocessing enzymes involved in the biosynthesis of carbohydrates that mediate cancer metastasis, inflammation, and viral infection. Enzymatically synthesized iminocyclitols are being investigated as treatments for osteoarthritis and Gaucher disease. Inspired by the broadly neutralizing anti-HIV antibody 2G12, which recognizes a dense array of oligomannose displayed on HIV gp120, we are designing dendrimeric oligomannose structures for development of an HIV vaccine. In collaboration with D.R. Burton, Department of Immunology, we are testing the immunogenicity of these constructs. We have designed glycolipid ligands for CD1, which activate natural killer T cells and are a promising new immunotherapeutic approach for treatment of bacterial and viral infections and cancer. They may also be useful as adjuvants in vaccine development. SYNTHETIC METHODS We have developed new methods for sugar-assisted ligation of glycopeptides for synthesis of homogenous glycoproteins. We have used the methods in conjunction with enzymatic glycosylation techniques to assemble complex glycopeptides by chemical synthesis, and we are optimizing the techniques to achieve the total synthesis of therapeutic glycoproteins. Glycoproteins are expressed in vivo as complex mixtures of glycoforms, a situation that hinders efforts to study the role of glycosylation in protein folding, stability, and function. By synthesizing pure glycoforms, we can characterize in molecular detail the effects of glycans on protein function. Using chemical techniques such as programmable 1-pot oligosaccharide synthesis, as well as enzymatic synthesis, we create glycoarrays on glass slides for highthroughput quantitative analysis of protein-carbohydrate interactions. These arrays are being used to study the binding specificity of carbohydrate-binding receptors and antibodies. We have applied aldolases, glycosyltransferases, glycosidases, and other enzymes to develop practical new methods of synthesizing molecules such as iminocyclitols, which are inhibitors of glycosidases and other enzymes, and glycopeptides, and other glycoconjugates. Using directed evolution, we are evolving G LY C O P R O T E O M I C S A N D M O L E C U L A R G LY C O B I O L O G Y Using metabolic oligosaccharide engineering, we have developed methods for incorporating tagged sugars into glycans expressed on mammalian cells. The engineered glycans can be labeled with a variety of molecules by using click chemistry. One application is glycan-specific fluorescent labeling, which is used for fluorescent imaging to compare glycosylation patterns of different cells, such as normal vs cancer cells or cancer cells vs cancer stem cells. We found that protein fucosylation and sialylation are both elevated in cancer cell lines. A second application of this chemistry is GIDmap, a new method for glycoproteomic analysis (Fig. 1). Whole cells are fed with tagged sugars, and after biochemical incorporation of the sugars into cellular glycoproteins, click chemistry is used to attach a handle for purification of tagged proteins. Mass spectrometric proteomic methods are then used to identify proteins that are differentially glycosylated. We are using GIDmap to identify proteins that are aberrantly glycosylated in different stages of cancer. These cancer-associated glycoproteins may be useful as biomarkers for diagnostics or as targets for therapeutic intervention. 114 CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE Wang, S.-K., Liang, P.-H., Astronomo, R.D., Hsu, T.-L., Hsieh, S.-L., Burton, D.R., Wong, C.-H. Targeting the carbohydrates on HIV-1: interaction of oligomannose dendrons with human monoclonal antibody 2G12 and DC-SIGN. Proc. Natl. Acad. Sci. U. S. A. 105:3690, 2008. Whalen, L.J., Greenberg, W.A., Mitchell, M.L., Wong, C.-H. Iminosugar-based glycosyltransferase inhibitors. In: Iminosugars: From Synthesis to Therapeutic Applications. Compain, P., Martin, O.R. (Eds.). Wiley-VCH, Hoboken, NJ, 2007, p. 153. Wu, D., Fujio, M., Wong, C.-H. Glycolipids as immunostimulating agents. Bioorg. Med. Chem. 16:1073, 2008. Carbon-Hydrogen Activation, Catalytic Reactions, and Organometallic and Synthetic Methods J.-Q. Yu, K.M. Engle, R. Giri, T.-S. Mei, B.-F. Shi, D.-H. Wang, M. Wasa, X.-S. Wang, Y.-H. Zhang E N A N T I O S E L E C T I V E C A R B O N - H Y D R O G E N A C T I VAT I O N C ATA LY Z E D B Y PA L L A D I U M ( I I ) – A M I N O A C I D F i g . 1 . GIDmap glycoproteomic analysis via metabolic oligosac- COMPLEXES charide engineering. lthough cleavage of inert carbon-hydrogen bonds by transition metals has been extensively studied, exploitation of this reactivity for regioselective and enantioselective catalytic reactions of synthetically useful chemical substances is still at its infant stage. The 2 major challenges are the development of practical catalysis and the modulation of regioselectivity and stereoselectivity by external ligands. We recently made a number of discoveries that offer promising solutions to these problems (Fig. 1). PUBLICATIONS Bennett, C.S., Dean, S.M., Payne, R.J., Ficht, S., Brik, A., Wong, C.-H. Sugarassisted glycopeptide ligation with complex oligosaccharides: scope and limitations. J. Am. Chem. Soc. 130:11945, 2008. Ficht, S., Payne, R.J., Guy, R.T., Wong, C.-H. Solid-phase synthesis of peptide and glycopeptide thioesters through side-chain-anchoring strategies. Chem. Eur. J. 14:3620, 2008. Giffin, M.J., Heaslet, H., Brik, A., Lin, Y.-C., Cauvi, G., Wong, C.-H., McRee, D.E., Elder, J.H., Stout, C.D., Torbett, B.E. A copper(I)-catalyzed 1,2,3-triazole azide-alkyne click compound is a potent inhibitor of a multidrug-resistant HIV-1 protease variant. J. Med. Chem. 51:6263, 2008. A Hanson, S.R., Greenberg, W.A., Wong C.-H. Probing glycans with the copper(I)catalyzed [3+2] azide-alkyne cycloaddition. QSAR Comb. Sci. 26:1243, 2007. Kinjo, Y., Pei, B., Bufali, S., Raju, R., Richardson, S.K., Imamura, M., Fujio, M., Wu, D., Khurana, A., Kawahara, K., Wong, C.-H., Howell, A.R., Seeberger, P.H., Kronenberg, M. Natural Sphingomonas glycolipids vary greatly in their ability to activate natural killer T cells. Chem. Biol. 15:654, 2008. Liang, P.-H., Imamura, M., Li, X., Wu, D., Fujio, M., Guy, R., Wu, B.-C., Tsuji, M., Wong, C.-H. Quantitative microarray analysis of intact glycolipid-CD1d interaction and correlation with cell-based cytokine production. J. Am. Chem. Soc. 130:12348, 2008. Liang, P.-H., Wu, C.-Y., Greenberg, W.A., Wong, C.-H. Glycan arrays: biological and medical applications. Curr. Opin. Chem. Biol. 12:86, 2008. Northen, T.R., Lee, J.-C., Hoang, L., Raymond, J., Hwang, D.-R., Yannone, S.M., Wong, C.-H., Siuzdak, G. A nanostructure-initiator mass spectrometry-based enzyme activity assay. Proc. Natl. Acad. Sci. U. S. A. 105:3678, 2008. Payne, R.J., Ficht, S., Greenberg, W.A., Wong, C.-H. Cysteine-free peptide and glycopeptide ligation by direct aminolysis. Angew. Chem. Int. Ed. 47:4411, 2008. Sugiyama, M., Hong, Z., Liang, P.-H., Whalen, L.J., Greenberg, W.A., Wong, C.H. D-Fructose-6-phosphate aldolase-catalyzed one-pot synthesis of iminocyclitols. J. Am. Chem. Soc. 129:14811, 2007. F i g . 1 . Enantioselective carbon-hydrogen activation/carbon-car- bon coupling reactions. We discovered the first palladium(II)/palladium(0) catalytic system to couple both sp2 and sp3 carbon-hydrogen bonds with organotin and organoboron reagents. We also established conditions to use air as the stoichiometric oxidant. We further discovered that mono-N-protected amino acids are suitable ligands for enantioselective carbon-hydrogen activation reactions. We think that the α-chirality of amino acids is relayed to the monoprotected nitrogen center that coordinates with the CHEMISTRY 2008 metal center and controls stereoselectivity at the carbon-hydrogen activation step. We are extending this enzymelike chiral recognition to sp 3 carbon centers attached to 2 prochiral carbon-hydrogen bonds (CH 2 groups) and are expanding the functional groups to broadly useful carboxyl and amine groups. THE SCRIPPS RESEARCH INSTITUTE 115 hydrogen bond, yielding a novel class of analogs in gram quantities for biological studies (Fig. 3). REAGENT-CONTROLLED MONOSELECTIVE C A R B O N - H Y D R O G E N A C T I VAT I O N : C O N S T R U C T I O N OF DEMANDING 1,2,3-SUBSTITUTED ARENES To improve the practicality of carbon-hydrogen activation reactions, we have focused on inventing new approaches to activate carbon-hydrogen bonds in abundant substrates containing broadly useful functional groups such carboxyl, amino, and hydroxyl groups. We recently discovered that table salt promotes carbonhydrogen activation in arene and aliphatic carboxylic acids. This reactivity led to the development of arylation and halogenation of inert carbon-hydrogen bonds in carboxylic acids (Fig. 2). The replacement of table salt with F i g . 3 . Carbon-hydrogen functionalization of natural products. V E R S AT I L E H E T E R O C Y C L E S Y N T H E S I S F R O M ARYLETHYLAMINES VIA CARBON-HYDROGEN A C T I VAT I O N Heterocycle synthesis is a core technology in medicinal chemistry. Using various amino groups to direct carbon-hydrogen activation, we are developing novel synthetic disconnections through amination of carbonhydrogen bonds. These reactions are either complementary to current methods or allow rapid access to unique heterocyclic structures from readily available chemicals (Fig. 4). F i g . 2 . Reagent-controlled selective halogenation of ortho-car- bon-hydrogen bonds. bulkier tetraalkylammonium chloride salts markedly improved the monoselectivity of ortho-carbon-hydrogen functionalization. These reactions offer a solution to the well-known challenge in accessing 1,2,3-substituted arenes in medicinal chemistry and synthesis. F U N C T I O N A L I Z AT I O N O F B I O L O G I C A L LY A C T I V E N AT U R A L P R O D U C T S V I A C A R B O N - H Y D R O G E N A C T I VAT I O N Using carbon-hydrogen activation/carbon-carbon coupling reactions, we hope to rapidly access diversified structures that are analogous to biologically active compounds yet difficult to synthesize by using conventional methods. Dehydroabietic acid is a natural product identified as an efficient opener of BK ion channels. Compounds with such activity could lead to useful treatments for diseases such as acute stroke, epilepsy, and asthma. Typically, diversification of such structures is difficult because of the lack of reactive sites on these molecules other than the carboxylic acid moiety, which is essential for biological activity of the molecule. Masking the carboxylic acid as the hydroxamic acid allows for functionalization at the methyl carbon- F i g . 4 . Heterocycle synthesis via carbon-hydrogen activation. PUBLICATIONS Giri, R., Maugel, N.L., Foxman, B.M., Yu, J.Q. Dehydrogenation of inert alkyl groups via remote C-H activation: converting a propyl group into a π-allylic complex. Organometallics 27:1667, 2008. Giri, R., Maugel, N.L., Li, J.J., Wang, D.H., Breazzano, S.P., Saunders, L.B., Yu, J.Q. Palladium-catalyzed methylation and arylation of sp2 and sp3 C-H bonds in simple carboxylic acids. J. Am. Chem. Soc. 129:3510, 2007. Giri, R., Yu, J.Q. Iodine monoacetate as a reagent. In: Encyclopedia of Reagents for Organic Synthesis, 2nd ed. Paquette, L.A., et al. (Eds.). Wiley Blackwell, Hoboken, NJ, in press. Huang, Y.Q., Shen, Z.L., Okamura, T.A., Wang, Y., Wang, X.F., Sun, W.Y., Yu, J.Q., Ueyama, N. Silver(I) complexes with oxazoline-containing tripodal ligands: structure variation via counter anions and reaction conditions. Dalton Trans. Issue 2:204, 2008. Li, J.J., Giri, R., Yu, J.Q. Remote C-H bond functionalization reveals the distancedependent isotope effect. Tetrahedron 64:6979, 2008. Mei, T.S., Giri, R., Maugel, N., Yu, J.Q. PdII-catalyzed monoselective ortho halogenation of C-H bonds assisted by counter cations: a complimentary method to directed ortho lithiation. Angew. Chem. Int. Ed. 47:5215, 2008. Shi, B.F., Maugel, N., Zhang, Y.H., Yu, J.Q. PdII-catalyzed enantioselective activation of C(sp2)-H and C(sp3)-H bonds using monoprotected amino acids as chiral ligands. Angew. Chem. Int. Ed. 47:4882, 2008. Wang, D.H., Wasa, M., Giri, R., Yu, J.Q. Pd(II)-catalyzed cross-coupling of sp3 CH bonds with sp2 and sp3 boronic acids using air as the oxidant. J. Am. Chem. Soc. 130:7190, 2008.
