chemistry - scripps research institutechemistry jeffery w. kelly, ph.d.* lita annenberg hazen...

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

D E P A R T M E N T O F

C H E M I S T R Y

S T A F F

K.C. Nicolaou, Ph.D.*ChairmanAline W. and L.S. Skaggs

Professor of ChemicalBiology

Darlene Shiley Chair inChemistry

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.**ProfessorEly R. Callaway, Jr., Chair

in ChemistryDirector, Worm Institute of

Research and Medicine

Gunnar Kaufmann, Ph.D.Assistant Professor of

Chemistry

Jeffery W. Kelly, Ph.D.*Lita Annenberg Hazen

Professor of Chemistry

RamanarayananKrishnamurthy, Ph.D.

Associate Professor

Lucas J. Leman, Ph.D.Assistant Professor of

Chemistry

Richard A. Lerner, M.D.***President, The Scripps

Research InstituteLita Annenberg Hazen

Professor ofImmunochemistry

Cecil H. and Ida M. GreenChair in Chemistry

Roy Periana, Ph.D.*****Professor

Evan T. Powers, Ph.D.Associate Professor of

Chemistry

Julius Rebek, Jr., Ph.D.*ProfessorDirector, The Skaggs Institute

for Chemical Biology

Edward Roberts, Ph.D.Professor

Floyd E. Romesberg, Ph.D.Associate Professor

William Roush, Ph.D.*****Professor

Peter G. Schultz, Ph.D.*ProfessorScripps Family Chair

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

(Andrew) Bin Zhou, Ph.D.Assistant Professor of

Immunochemistry

S E N I O R S C I E N T I S T

Luis Martinez, Ph.D.*****

S T A 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.

I N S T R U M E N T A T I O N /

S E R V I C E F A C I L I T I E S

Raj K. Chadha, Ph.D.Director, X-ray

Crystallography Facility

Dee H. Huang, Ph.D.Director, Nuclear Magnetic

Resonance Facility

Gary E. Siuzdak, Ph.D.Senior Director, Mass

Spectrometry Facility

S E N I O R R E S E A R C H

A S S O C I A T E

Suresh Pitram, Ph.D.

R E S E A R C H A S S O C I A T E S

Ramzey Abujarour, Ph.D.

Rajesh Ambasudhan, Ph.D.

Manuel Amorin Lopez, Ph.D.

Mark Ams, Ph.D.

Yoshio Ando, Ph.D.

Deepshikha Angrish, Ph.D.

Shinji Ashida, Ph.D.

Micahel Baksh, Ph.D.

Deboshri Banerjee, Ph.D.

Elizabeth Barrett, Ph.D.****

Roland Barth, Ph.D.*****

Clay Bennett, Ph.D.

Moritz Biskup, Ph.D.†

Universität KarlsruheKarlsruhe, Germany

Anthony Boitano, Ph.D.†

Genomics Institute of theNovartis Foundation

San Diego, California

Laure Bouchez, Ph.D.

Kristopher Boyle, Ph.D.

Christopher Burke, Ph.D

Antonio Burtoloso, Ph.D.†

University of Sao PauloSao Paulo, Brazil

Mark Bushey, Ph.D.†

Exxon, Inc.Union City, New Jersey

Darren Bykowski, Ph.D.*****

Petr Capek, Ph.D.

Katerina Capkova, Ph.D.

Arani Chanda, Ph.D.

Ke Chen, Ph.D.

Peng Chen, Ph.D.

Govardhan Cherukupalli,Ph.D.†

Epix PharmaceuticalsLexington, Massachusetts

Jodie Chin, Ph.D.

Srinivas Reddy Chirapu, Ph.D.

Chandramouli Chiruta, Ph.D.

Dong-Gyu Cho, Ph.D.

So-Hye Cho, Ph.D.

Sungwook Choi, Ph.D.

Joyanta Choudhury, Ph.D.

Sarwat Chowdhury, Ph.D.

Stepan Chuprakov, Ph.D.

C H E M I S T R Y 2 0 0 8 T H E S C R I P P S R E S E A R C H I N S T I T U T E 7 9

Petr Cigler, Ph.D.

T. Ryan Cirz†

AchaogenSouth San Francisco,

California

Scott Cockroft, Ph.D.†

University of EdinburghEdinburgh, Scotland

David Colby, Ph.D.†

Purdue UniversityWest Lafayette, Indiana

Kevin Cole, Ph.D.†

Eli Lilly and CompanyIndianapolis, Indiana

Christine Crane, Ph.D.

Matthew Cremeens, Ph.D.†

Gonzaga UniversityRedmond, Washington

Fernando Rodrigo PinachoCrisostomo, Ph.D.†

Burnham Institute forMedical Research

La Jolla, California

Jeffrey Culhane, Ph.D.

Stephen Dalby, Ph.D.

Etzer Darout, Ph.D.†

Pfizer Inc.Groton, Connecticut

Amy DeBaillie, Ph.D.†

Eli Lilly and CompanyIndianapolis, Indiana

Judith Denery, Ph.D.

Ross Denton, Ph.D.†

University of CambridgeCambridge, England

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.

David Edmonds, Ph.D.

Jem Efe, Ph.D.

Jan Elsner, Ph.D.†

Celgene PharmaceuticalsSan Diego, California

Daniel Ess, Ph.D.

Cyrine Ezzili, Ph.D.

Xingang Fang, Ph.D.

Simon Ficht, Ph.D.†

Sanofi-Aventis DeutschlandGmbH

Frankfurt, Germany

Joseph Rodolph Fotsing,Ph.D.†

Senomyx, Inc.San Diego, California

Bozena Frackowiak, Ph.D.†

Politechnika OpolskaOpole, Poland

Graeme Freestone, Ph.D.†

Metabasis Therapeutics, Inc.San Diego, California

Yu Fu, Ph.D.

Amelia Fuller, Ph.D.†

Santa Clara UniversitySanta Clara, California

Jianmin Gao, Ph.D.†

Boston CollegeChestnut Hill, Massachusetts

Haibo Ge, Ph.D.

Savvas Georgiades, Ph.D.****

Ola Ghoneim, Ph.D.†

Qatar UniversityDoha, Qatar

Nathan Gianneschi, Ph.D.†

University of CaliforniaSan Diego, California

Cristina Gil-Lamaignere,Ph.D.†

University Hospital NuestraSeñora de la Candelaria

Santa Cruz de Tenerife, Spain

Rodolfo Gonzalez, Ph.D.

Scott Grecian, Ph.D.

Neil Grimster, Ph.D.

Rajesh K. Grover, Ph.D.

Jan Grunewald, Ph.D.

Tanja Gulder, Ph.D.

Richard Guy, Ph.D.****

Masaki Handa, Ph.D.†

Sagami Chemical ResearchCenter

Ayase, Kanagawa, Japan

Yuanjun He, Ph.D.

Jason Hein, Ph.D.

Dube Henry, Ph.D.

Marcos Hernandez, Ph.D.

Par Holmberg, Ph.D.†

Memorial Sloan KetteringCancer Center

New York, New York

Wen-Xu Hong, Ph.D.

Zhangyong Hong, Ph.D.

Richard J. Hooley, Ph.D.†

University of CaliforniaRiverside, California

Tamara Hopkins, Ph.D.†

Boehringer IngelheimPharmaceuticals, Inc.

Ridgefield, Connecticut

Allen Horhota, Ph.D.

Tony Horneff, Ph.D.

Jun-Li Hou, Ph.D.

Claas Hovelmann, Ph.D.

Fang Hu, Ph.D.†

Department of MolecularBiology, Scripps Research

Xiaoyi Hu, Ph.D.

Zheng-Zheng Huang, Ph.D.†

DuPont Central Researchand Development

Wilmington, Delaware

Ben Hutchins, Ph.D.

Der-ren Hwang, Ph.D.†

Academia SinicaTaipei, Taiwan

Giltae Hwang, Ph.D.

Michael Jahnz, Ph.D.†

NOXXON Pharma AGBerlin, Germany

Rong Jiang, Ph.D.*****

Guo Jiantoa, Ph.D.

Hiroyuki Kakei, Ph.D.†

Takeda PharmaceuticalCompany Limited

Osaka, Japan

Jaroslaw Kalisiak, Ph.D.

Seiji Kamioka, Ph.D.

Moumita Kar, Ph.D.

Kwang Mi Kim, 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 ResearchInstitutes

Miaoli County, Taiwan

Jong Seok Lee, Ph.D.

Ki-Bum Lee, Ph.D.†

Rutgers UniversityPiscataway, New Jersey

Sejin Lee, Ph.D.†

SK Drug Development CenterDaejong, Korea

Alexandre Lemire, Ph.D.****

Edward Lemke, Ph.D.

Christophe Letondor,Ph.D.****

Chuang-Chuang Li, Ph.D.†

Peking UniversityPeking, China

Fangzheng Li, Ph.D.*****

8 0 C H E M I S T R Y 2 0 0 8 T H E S C R I P P S R E S E A R C H I N S T I T U T E

Hongming Li, Ph.D.†

Schering-PloughKenilworth, New Jersey

Ke Li, Ph.D.†



Pi-Hui Liang, Ph.D.†

Academia SinicaTaipei, Taiwan

Yeon-Hee Lim, Ph.D.†

Schering-PloughKenilworth, New Jersey

Tongxiang Lin, Ph.D.

Troy Lister, Ph.D.†

NovartisCambridge, Massachusetts

Christopher Liu, Ph.D.†

Cubix PharmaceuticalsLexington, Massachusetts

Wenshe Liu, Ph.D. †

Texas A&M UniversityCollege Station, Texas

Michael Luzung, Ph.D.

Utpal Majumder, Ph.D.

Sreeman Mamidyala, Ph.D.

Takeshi Masuda, Ph.D.

Michael Maue, Ph.D.†

Bayer CropScience AGMonheim, Germany

Alexander Mayorov, Ph.D.

Charles Melancon, Ph.D.

Lionel Moisan, Ph.D.†

CEAGif-Sur-Yvette, France

Ana Montero, Ph.D.****

Miguel Morales, Ph.D.

Adam Morgan, Ph.D.†

Concert Pharmaceuticals, Inc.Lexington, Massachusetts

Ting-Wei Mu, Ph.D.

S. Vasudeva Naidu, Ph.D.

Yuya Nakai, Ph.D.

Joonwoo Nam, Ph.D.†

CytRx CorporationSan Diego, California

Tae-Gyu Nam, Ph.D.

Andrew Nguyen, M.D., Ph.D.

Romain Noel, Ph.D.

George Nora, Ph.D.*****

Severin Odermatt, Ph.D.****

Christian Olsen, Ph.D.

Yazmin Osornio, Ph.D.****

Junguk Park, Ph.D.

Nitin Patil, Ph.D.

Johan Paulsson, Ph.D.

Richard Payne, Ph.D.†

University of SydneySydney, Australia

Xuemei Peng, Ph.D.****

Murali Peram Surakattula,Ph.D.†

CytRx CorporationSan Diego, California

Roshan Perera, Ph.D.†

University of TexasAustin, Texas

Ramulu Poddutoori, Ph.D.

Jonathan Pokorski, Ph.D.

Agustí Lledó Ponsati, Ph.D.

Daniela Radu, Ph.D.†



Ronald Rahaim, Ph.D.*****

Praveen Rao, Ph.D.†

University of WaterlooWaterloo, Ontario, Canada

Per Restorp, Ph.D.

Kimberly Reynolds, Ph.D.

Jin-Kyu Rhee, Ph.D.

Fatima Rivas, Ph.D.

Joshua Roth, Ph.D.*****

Troy Ryba, Ph.D.†

Broad Institute of MIT andHarvard

Cambridge, Massachusetts

Youngha Ryu, Ph.D.†

Texas Christian UniversityFort Worth, Texas

Catherine Saccavini, Ph.D.

Nicholas Salzameda, Ph.D.

Antonio Sanchez-Ruiz, Ph.D.

Yoshikazu Sasaki, Ph.D.

Stefan Schiller, Ph.D.

Niklas Schone, Ph.D.

Michael Schramm, Ph.D.†

California State University Long Beach, California

Young Jun Seo, Ph.D.

Edward Sessions, Jr., Ph.D.

Shigeki Seto, Ph.D.

Mary Jo Sever, Ph.D.

Alex Shaginian, Ph.D.†

Ardea BiosciencesSan Diego, California

David Shaw, Ph.D.

Weijun Shen, Ph.D.

Xiao Shengxiong, Ph.D.

Bingfeng Shi, Ph.D.

Yan Shi, Ph.D.

Hiroki Shigehisa, Ph.D.

Hiroyuki Shimamura, Ph.D.

Siddhartha Shenoy, Ph.D.

Ryan Simkovsky, Ph.D.

Chinnappan Sivasankar,Ph.D.****

Michael Smolinski, Ph.D.†

Kinex PharmaceuticalsBuffalo, New York

Xinyi Song, Ph.D.

Simon Stamm, Ph.D.

Sebastian Steiniger, Ph.D.

Antonia Stepan, Ph.D.

James Stover, Ph.D.

Bernhard Stump, Ph.D.

Shun Su, Ph.D.

Hui Kai Sun, Ph.D.*****

Shinobu Takizawa, Ph.D.

Adam Talbot, Ph.D.†

Institute of Chemical andEngineering Sciences

Jurong Island, Singapore

Annie Tam, Ph.D.

Yefeng Tang, Ph.D.

Mariola Tortosa, Ph.D.†

Instituto de Quimica Organica,CSIC

Madrid, Spain

Craig Turner, Ph.D.****

Matthew Tremblay, Ph.D.

Vincent Trepanier, Ph.D.†



Jonathan Tripp, Ph.D.

Meng-Lin Tsao, Ph.D.†

University of California Merced, California

Andrew Udit, Ph.D.

Taiki Umezawa, Ph.D.†

Hokkaido UniversitySapporo, Japan

Kenji Usui, Ph.D.†

Tokyo Institute of TechnologyTokyo, Japan

Carlos Valdez, Ph.D.†

Rigel Pharmaceuticals, Inc.South San Francisco,

California

Punna Venkateshwarlu, Ph.D.

Feng Wang, Ph.D.

Jian Wang, Ph.D.


Jiangyun Wang, Ph.D.†

Institute of BiophysicsBeijing, 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.†



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 ScienceTokyo, Japan

Ura Yasuyuki, Ph.D.†

Nara Women’s UniversityNara, Japan

Yan Yin, Ph.D.

Ian Young, Ph.D.

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

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 A T O R S

Keisuke Fukuchi, Ph.D.Sankyo Co., Ltd.Tokyo, Japan

Christine Hernandez, Ph.D.†

University of PhilippinesDiliman, Philippines

(Edmond) Shie-Liang Hsieh,Ph.D.

National Yang-Ming UniversityTaipei, 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 theNegev

Be’er Sheva, Israel

Takayoshi Suzuki, Ph.D.†

Nagoya City UniversityNagoya, Japan

Yoshiyuki Yoneda, Ph.D.†

Daiichi Pharmaceutical Co.,Ltd.

Tokyo, Japan

S C I E N T I F I C A S S O C I A T E

Jon Ashley

* Joint appointment in TheSkaggs Institute for ChemicalBiology

** Joint appointments in TheSkaggs Institute for ChemicalBiology and the Department ofImmunology and MicrobialScience

*** Joint appointments in TheSkaggs Institute for ChemicalBiology and the Department ofMolecular Biology

**** Appointment completed

***** Scripps Florida

† Appointment completed; newlocation shown


Chairman’s Overview

As the “central science,” chemistry stands betweenbiology and medicine and between physics andmaterials science and provides the crucial bridge

for drug discovery anddevelopment. But chem-istry has a much moreprofound and useful rolein science and society. Itis the discipline that con-tinually creates the myriadof new materials that weall encounter in our every-day lives: pharmaceuti-cals, high-tech materials,polymers and plastics,insecticides and pesticides,fabrics and cosmetics, fertilizers, and vitamins—basicallyeverything we can touch, feel, and smell.

Chemistry at Scripps Research focuses on chemicalsynthesis and chemical biology, the areas most relevantto biomedical research and materials science. The mem-bers of our faculty are distinguished teacher-scholars whomaintain highly visible and independent research programsin areas as diverse as biological and chemical catalysis,synthesis of natural products, combinatorial chemistry,molecular design, supramolecular chemistry, chemicalevolution, materials science, and chemical biology. Thechemistry graduate program attracts some of the best-qualified candidates from the United States and abroad.Our major research facilities, under the direction of DeeH. Huang (nuclear magnetic resonance), Gary Siuzdak(mass spectrometry), and Raj Chadha (x-ray crystallog-raphy), are second to none and continue to provide cru-cial support to our research programs. In addition, theMabel and Arnold Beckman Center for the ChemicalSciences constantly receives high praise from visitorsfrom around the world for its architectural design andoperational aspects, both highly conducive to research.

Research in the Department of Chemistry goes onunabated, establishing international visibility and attract-ing attention, as evidenced by numerous lecture invita-tions, visits by outside scholars, and headline news inthe media. As of 2007, the Institute for Scientific Infor-mation ranked 2 members of the department as highlycited researchers (in the top 100 worldwide).

Richard Lerner and his group continue their researchon antibodies, chemical synthesis, and the biological

role of polyoxygen species. Scientists in Albert Eschen-moser’s group continue to work on the chemical etiol-ogy of nucleic acids and the origins of life.

Barry Sharpless and his group persist in their endeav-ors to discover and develop better catalysts for organicsynthesis and to construct, through innovative chemistryand biology, libraries of novel compounds for biologicalscreening. Their click chemistry, which has had a majorimpact in many areas of the molecular sciences, con-tinues to be an important focus of their research.

Members of my own group continue to explore chem-ical synthesis and chemical biology, with a focus on thetotal 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 recep-tors, including molecules that bind neurotransmitters andmembrane components, for studies in molecular recog-nition. Larger host receptors can surround 3 or moremolecular guests and act as chambers in which thechemical reactions of the guests are accelerated. Scien-tists in the group also synthesize small molecules that actas protein helix mimetics for pharmaceutical applications.

Peter Schultz and researchers in his laboratory areexpanding the number of genetically encoded amino acidsto include fluorescent, photocaged, metal-binding, chemi-cally reactive, and posttranslationally modified aminoacids. These scientists have also adapted this technol-ogy to mammalian cells and are applying these tools inbasic and applied problems in cell biology. In addition,members of the group have used cell-based screens toidentify small molecules that selectively differentiateand expand embryonic and adult stem cells and repro-gram lineage-committed cells, as well as novel genesand small molecules that affect a number of physiologicand disease processes.

Chi-Huey Wong and his group further advance thefields of chemoenzymatic organic synthesis, chemicalglycobiology, and the development of enzyme inhibitors.A new strategy for the synthesis of glycoproteins basedon sugar-assisted glycopeptide ligation has been developed.The programmable 1-pot synthesis of oligosaccharidesdeveloped by this group has been further used in theassembly of glycoarrays for study of saccharides thatbind to proteins. Members of this group also developednew probes to study posttranslational glycosylation andidentify glycoprotein biomarkers associated with cancer.

Researchers in Dale Boger’s laboratory continue theirwork on chemical synthesis; combinatorial chemistry; het-


K.C. Nicolaou, Ph.D.

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 researchgrounded on organic chemistry as applied to specific bio-logical systems. The targeted programs span a wide rangeof interests, from immunopharmacotherapy to biologicaland chemical warfare agents to filarial infections suchas “river blindness” to quorum sensing in bacteria andnew cancer therapeutic strategies. Recent achievementsinclude in vivo detection of botulinum neurotoxin antag-onists, the development of peptides and antibodies asdrug delivery modules that home to cancer cells andactive vaccines for nicotine addiction and obesity thatare now in preclinical trials.

M. Reza Ghadiri and his group are making importantcontributions in the design and study of a new generationof antimicrobial agents, based on self-assembling peptidenanotube architecture, to combat multidrug resistantinfections. In addition, members of the group continueto make novel contributions in several ongoing basicresearch endeavors, such as designing biosensors, devel-oping molecular computation, designing self-reproducingsystems, understanding the origins of life, and creatingemergent chemical systems.

M.G. Finn and his group have pioneered the use ofvirus particles as chemical reagents and building blocksfor nanochemical structures. This effort is directed towardthe development of new diagnostics for disease and cata-lysts for organic reactions. Members of Dr. Finn’s labo-ratory also develop and investigate new organic andorganometallic reactions and use these processes tosynthesize biologically active compounds.

Jeff Kelly and his group are exploring the interfacebetween the chemistry, biology, and pathobiology ofproteome maintenance. The aim of their projects is tounderstand the physical and biological basis of proteinfolding and the competitive misfolding and aggregationprocesses that lead to age-associated neurodegenerativediseases. Information on proteome maintenance is usedto develop new small-molecule therapeutic strategies for avariety of diseases, including neurodegenerative diseases.

Anita Wentworth and the researchers in her group areinvestigating the chemical basis of complex disease statesand are synthesizing peptide- and small molecule–basedtherapeutic agents. These scientists focus on diseasestates in which inflammatory and reactive oxygen spe-cies are prominent, such as atherosclerosis, Alzheimer’sdisease, and other diseases of ageing.

Researchers in Floyd Romesberg’s laboratory are usingdiverse techniques ranging from bioorganic and biophysi-cal chemistry to bacterial and yeast genetics to under-stand and manipulate the process of evolution. Majorefforts include designing unnatural base pairs and usingdirected evolution of DNA polymerases to efficiently syn-thesize unnatural DNA containing the base pairs, usingspectroscopy to understand biological function and howit evolves, and understanding how induced and adap-tive mutations contribute to evolution in eukaryotic andprokaryotic cells.

Phil Baran and his group are interested in how thegeneral challenge of chemoselectivity in organic chemistrycan be answered through the auspices of total synthe-sis. He and his coworkers have developed extremelyconcise chemical solutions to the synthetic challengesposed by numerous families of natural products. Thesesyntheses systematically tackle the issue of chemose-lectivity and are characterized by striking brevity, newbiosynthetic postulates, the invention of new methods,and a minimum use or complete absence of protectinggroups and superfluous oxidation state manipulations.

The Frontiers in Chemistry Lecturers (19th AnnualSymposium) for the 2007–2008 academic year wereM. Christina White, University of Illinois; Ben L. Feringa,University of Groningen, the Netherlands; Ian Paterson,Cambridge University; and Harry Noller, University ofCalifornia, Santa Cruz. In addition, we enjoyed hostingthe following professors: Samir Zard, Ecole Polytech-nique, 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.


INVESTIGATORS’ REPORTS

Practical Total Synthesis ofNatural 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

From penicillin to paclitaxel (Taxol), natural prod-ucts have an unparalleled track record in the bet-terment of human health. In fact, 9 of the top 20

best-selling drugs were either inspired by or derived fromnatural products. Even the best-selling drug of all time,atorvastatin (Lipitor), was based on a natural productlead. Total synthesis, the art and science of recreatingthese entities in the laboratory, invariably leads to funda-mental discoveries in chemistry, biology, and medicine.

We focus on solving interesting challenges in thetotal synthesis of natural products and on bridging gapsin synthetic capabilities by inventing new reactions.Through judicious target selection and creative retrosyn-thetic analyses, total synthesis becomes an engine fordiscovery that drives the field of organic chemistry tonew levels of sophistication and practicality. Syntheticorganic chemistry requires tremendous ingenuity, artis-tic taste, experimental acumen, persistence, and char-acter. Not surprisingly, drug development relies on theexpertise of researchers who have these characteristics.Although we focus entirely on educating students infundamental chemistry, we also collaborate with expertbiologists to explore the medicinal potential of newlysynthesized natural products and the products’ analogs.

Recently completed total syntheses (Fig. 1) include(1) the anticancer agent stephacidin; (2) the antibac-terial agents ageliferin and axinellamine; (3) membersof the bioactive fischerindole, hapalindole, and welwitin-dolinone indole alkaloid family; (4) the anticancer agenthaouamine A; and (5) the structurally exotic marinealkaloid chartelline C. Current natural product targets(Fig. 2) include stylissadine A, sarcodonin, and vinigrol;the neuroactive agent psychotrimine; and the potentangiogenic agent cortistatin A.

PUBLICATIONSBurns, 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, radical-mediated C-H functionalization. J. Am. Chem. Soc. 130:7247, 2008.

Grube, A., Immel, S., Baran, P.S., Köck, M. Massadine chloride: a biosyntheticprecursor 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.


F i g . 1 . Recently completed total syntheses.

O’Malley, D.P., Yamaguchi, J., Young, I.S., Seiple, I.B., Baran, P.S. Total synthesisof (±)-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 tocarbonyl 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 (+)-corti-statin 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 BioorganicChemistryD.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

The research interests of our group include the totalsynthesis of natural products, development of newsynthetic methods, heterocyclic chemistry, bioor-

ganic and medicinal chemistry, the study of DNA-agentinteractions, and the chemistry of antitumor antibiotics.We place a special emphasis on investigations to definethe structure-function relationships of natural or designedorganic agents.

S Y N T H E T I C M E T H O D S

Central to much of our work are investigations todevelop and apply the hetero Diels-Alder reaction, includ-ing the use of heterocyclic and acyclic azadienes (Fig. 1),the thermal reactions of cyclopropenone ketals, inter-

molecular and intramolecular acyl radical–alkene additionreactions, medium- and large-ring cyclization technol-ogy, and solution-phase combinatorial chemistry. Ineach instance, the development of the methods repre-sents the investigation of chemistry projected as a keyelement in the synthesis of a natural or designed agent.

T O T A L S Y N T H E S I S O F N A T U R A L P R O D U C T S

Efforts are under way on the total synthesis of anumber of natural products that constitute agents inwhich we have a specific interest. Representative agentscurrently under study include (+)-CC-1065 and func-tional analogs; the duocarmycin class of antitumorantibiotics, including yatakemycin; tropoloalkaloids;prodigiosin and roseophilin; the deoxybouvardin andRA-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 cyto-statin; the combretastatins; storniamide A; phomazarin;ningalins; lamellarin O; lukianol A; piericidins; notha-podytine and mappicine; rubrolone; vindoline; and vin-blastine (Figs. 2 and 3).B I O O R G A N I C C H E M I S T R Y

The agents listed in the previous paragraph wereselected on the basis of their properties; in manyinstances, they are agents related by a projected prop-erty. For example, (+)-CC-1065, the duocarmycins,and yatakemycin are antitumor antibiotics and relatedsequence-selective DNA minor groove alkylating agents.Representative of such efforts, studies to determine thestructural features of yatakemycin and the duocarmycinsthat contribute to the sequence-selective DNA alkylationproperties of these agents have resulted in the identifi-


F i g . 2 . Recent natural product targets.

F i g . 1 . N-Sulfonyl-1-aza-1,3-butadiene Diels-Alder reaction.

cation of a unique source of catalysis for the DNA alky-lation reaction. Efforts are under way to develop DNAcross-linking agents of a predefined cross-link, to fur-

ther understand the nature of the noncovalent and cova-lent interactions between agents and DNA, and to applythis understanding to the de novo design of DNA-bindingand DNA-effector agents. Techniques for the evaluationof the agent-DNA binding and alkylation properties, col-


Fig. 3. Additional recent total syntheses.

F i g . 2 . Recent total syntheses.

laborative efforts in securing biological data, nuclearmagnetic resonance structures of DNA-agent complexes,molecular modeling, and studies of DNA-agent interac-tions are integral parts of the program.

Additional ongoing studies include efforts to definethe fundamental basis of the DNA-binding or cleavageproperties of bleomycin A2, sandramycin, and the luzo-peptins; to design inhibitors of the folate-dependentenzymes glycinamide ribonucleotide transformylase andaminoimidazole carboxamide ribonucleotide transfor-mylase as potential antineoplastic agents; to establishthe chemical and biological characteristics responsiblefor the sleep-inducing properties of the endogenouslipid oleamide; to inhibit tumor growth through inhibitionof angiogenesis; to inhibit aberrant gene transcriptionassociated with cancer; and to control intracellular sig-nal transduction through the discovery of antagonistsor agonists that affect protein-protein interactions, includ-ing receptor dimerization.

PUBLICATIONSEubanks, L.M., Hixon, M.S., Jin, W., Hong, S., Clancy, C.M., Tepp, W.H., Bald-win, 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 uncoveredthrough high-throughput identification of botulinum neurotoxin A antagonists [pub-lished 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., Cra-vatt, B.F., Boger, D.L. Structure-activity relationships of the α-ketooxazole inhibi-tors of fatty acid amide hydrolase. J. Med. Chem. 50:3359, 2007.

Ishikawa, H., Boger, D.L. Total synthesis of (–)- and ent-(+)-4-desacetoxy-5-desethylvindoline. 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 analoguessubject 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 A2aglycon: 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 andselective α-ketoheterocycle-based inhibitors of the anandamide and oleamidecatabolizing 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 andthe 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 DNAalkylation 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, evalua-tion, and crystal structures of transition state analogue inhibitors of inosinemonophosphate cyclohydrolase. J. Biol. Chem. 282:13033, 2007.

Phage Escape for the Predictionof Protein EvolutionT.J. Dickerson, J. Denery, L. Eubanks, A. Hoyt, K.D. Janda,A. Moreno, Y. Nakai, A. Nguyen, A. Nunes, A. Rohrbach, C. Saccavini

The relationship between host and pathogen is ina constant state of flux, with each side continuallyevolving in a struggle to maximize the chance for

survival. A plethora of defensive systems has evolved tocounteract and/or eliminate invading pathogens. Thesesystems exert pressure upon the pathogen, leading tomechanisms that combat the host, including immuneevasion and drug resistance. Subsequently, the hostmust counter these improved pathogens with a selectedadaptive immune response. This cycle can continueindefinitely, with pathogen and host counteracting thesurvival strategy of each other.

From a structural point of view, the contest is usu-ally played out between the relatively unstructured proteinloops of both systems, where generation of 3-dimen-sional structural diversity of these parts of the proteindoes not affect overall protein function. The accessibleprotein-sequence diversity of both pathogen and hostimmune system is exceedingly expansive, thereby mak-ing an a priori analysis of where the mutation can occurand how antibodies subsequently respond an extremelydifficult challenge. However, adaptive immunity is inher-ently a reactive system and cannot operate in a proactivemode; challenge from an exogenous antigen is requiredbefore a response is made. Thus, despite the seeminglylimitless diversity that can be accessed by the mamma-lian immune system, only a small part of this theoreticaldiversity is present at any given time. Only after a spe-cific challenge is the full extent of the immune repertoirebrought to bear upon an invading pathogen. A techniquethat could be used to predict the advance of evolvinghuman disease and predetermine suitable therapeuticstrategies before a pathogen becomes a public healththreat would be valuable both for understanding patho-gen evolution and for transforming drug discovery into aprocess that predetermines therapeutic strategies beforethe onset of epidemics or pandemics.

Recently, we developed a technology termed “phageescape” that allows a preemptive determination of theevolutionary pathways used by the causative organismsof specific diseases. This technology has been appliedto the evolution of the hemagglutinin viral surface pro-


tein from the highly pathogenic avian influenza virusH5N1 (“bird flu”). During the past year, we success-fully prepared all of the experimental tools needed toperform a “checkmate analysis” of influenza virus hemag-glutinin type 5. Currently, we are generating a map ofthe evolution of this protein (Fig. 1).

PUBLICATIONSBrogan, A.P., Dickerson, T.J., Janda, K.D. Nornicotine-organocatalyzed aqueousreduction of α,β-unsaturated aldehydes. Chem. Commun. (Camb.) Issue 46:4952,2007.

Capková, K., Yoneda, Y., Dickerson, T.J., Janda, K.D. Synthesis and structure-activity relationships of second-generation hydroxamate botulinum neurotoxin Aprotease 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 molecu-lar 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 cocaineimmunopharmacotherapy. Bioorg. Med. Chem. Lett. 17:4280, 2007.

Lowery, C.A., Dickerson, T.J., Janda, K.D. Interspecies and interkingdom commu-nication 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 bymethamphetamine glycation products chemically links chronic drug abuse and car-diovascular 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 thebotulinum neurotoxin: using chemistry and biology to modulate the most deadlypoison. 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 adventitiousbinding of xylose to the quorum-sensing protein SdiA of Escherichia coli. Bioorg.Med. Chem. Lett. 17:6202, 2007.

Chemical and FunctionalGenomic Approaches to StemCell Biology and RegenerativeMedicine

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

Recent advances in stem cell biology may makepossible new approaches for the treatment of anumber of diseases, including cardiovascular

disease, neurodegenerative disease, musculoskeletaldisease, diabetes, and cancer. These approaches couldinvolve cell replacement therapy and/or drug treatmentto stimulate the body’s own regenerative capabilitiesby promoting survival, migration/homing, proliferation,and differentiation of endogenous stem/progenitor cells.However, such approaches will require identification ofrenewable cell sources of engraftable functional cells, animproved ability to manipulate proliferation and differ-entiation of the cells, and a better understanding ofthe signaling pathways that control the fate of the cells.

Equipped with a high-throughput screening platformand large arrayed molecular libraries—combinatorialchemical libraries, genome-scale cDNA libraries (forgain of function), and small interfering RNA libraries(for loss of function)—we are developing and integratingchemical and functional genomic tools to study stemcell biology and regeneration. We screen these librariesto identify and further characterize small molecules andgenes that can control stem cell fate in various systems,including (1) self-renewal regulation of embryonic andadult stem cells; (2) directed and step-wise differenti-ation of embryonic stem cells toward neuronal, cardiac,and pancreatic lineages; (3) directed neuronal differ-entiation and subtype neuronal specification of humanand rodent neural stem cells; (4) cellular plasticity andreprogramming of lineage-restricted somatic cells tomore primitive precursor cells; (5) functional prolifera-tion of cardiomyocytes and pancreatic beta cells fromadults; (6) developmental signaling pathways and epi-genetic mechanisms (histone and DNA de/methylation);and (7) development of new technologies for stem cellderivation and gene targeting.

In addition, we are characterizing the molecularmechanism of these identified small molecules and


F i g . 1 . Work flow for using phage escape technology to identify

escape mutants. In an immunologic checkmate analysis, phage-

bound protein variants (escape mutant, right middle) are able to

escape a known collection of neutralizing antibodies (top center).

genes by using various approaches, including detailedinvestigations of structure-activity relationship, affinitychromatography for target identification, transcriptomeprofiling, proteomics analysis, chemical/genetic epistasis,and biochemical and functional assays in vitro and invivo. So far, we have identified and are characterizingfunctional small molecules and/or genes in each of thepreviously mentioned distinct biological processes thatinvolve regulation of stem/progenitor cells.

More recent examples include identification andcharacterization of distinct small molecules for self-renewal of human embryonic stem cells and clonalexpansion/survival; dopaminergic neuron specificationfrom mouse embryonic stem cells; derivation of ratembryonic stem cells; reprogramming of somatic cellsto a pluripotent state; definitive endoderm and pancre-atic induction; chemically defined monolayer conditionsfor self-renewal of embryonic stem cells and their directeddifferentiation to cardiac lineages; proliferation of humanbeta cells; and regulating Wnt signaling. These studiesmay ultimately facilitate the therapeutic application ofstem cells and the development of small-molecule drugsto stimulate tissue and organ regeneration in vivo.

PUBLICATIONSShi, Y., Do, J.-T., Desponts, C., Hahm, H.-S., Schöler, H.R., Ding, S. A combinedchemical 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 regenerativemedicine. Nature 453:338, 2008.

Chemical Etiology of NucleicAcid StructureA. Eschenmoser, R. Krishnamurthy, G.K. Mittapalli, R.R. Kondreddi, Y. Osornio, V.S. Naidu

In the general context of our project to map the land-scape of potentially primordial informational oligomersystems, we have been working during the past year

on the following topics.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 – T A G G E D

2 ′g 3 ′ - P H O S P H O D I E S T E R – L I N K E D G L Y C E R I C A C I D

B A C K B O N E S

We have undertaken the synthesis and study of thebase-pairing properties of oligomers derived from a2′,3′-phosphodiester–linked glyceric acid backbone thathas 2,4-disubsituted 5-aminopyrimidines, attached tothe carboxyl group of glyceric acid via an amide bondat the 5-amino group, as recognition elements (Fig. 1).

The structure of this oligomeric system is based on astructural simplification of the oligonucleotides contain-ing lyxopyranosyl (2′g4′)– and threofuranosyl (2′g3′)–linked phosphodiester backbones, which we studiedpreviously. Among the oligomer systems depicted in Fig-ure 1, the nucleic acid derived from the glycerol backboneis not considered to be a potentially prebiotic system, incontrast to the oligomer system derived from glyceric acidand tagged via amide bonds with 5-aminopyrimidines.

We have completed the synthesis of such a glyc-eric acid–derived oligomer containing six 5-aminouracilunits (6-mer) and have studied its base-pairing prop-erties 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

with the corresponding poly(rA), and even occurred withα-L-threofuranosyl-(3′g2′) nucleic acid. We are expand-ing the study to include the complementary partnerstrand tagged with 2,4,5-triaminopyrimidine and haveexplored different pathways for synthesizing the suit-ably protected building blocks necessary for the auto-mated oligonucleotide synthesis.


F i g . 1 . Structural simplification of α-L-threofuranosyl-(3′g2′)nucleic acid, which was inspired by studies on (3′g4′)-lyxopyra-

nosyl nucleic acid, gives rise to acyclic informational oligomeric

systems. Two examples are shown: glycerol nucleic acid and glyc-

eric acid nucleic acid.

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.

S Y N T H E S I S O F O L I G O D I P E P T I D E S O F

A S P A 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 T A G G E D

W I T H O R O T I C A C I D

In our search for alternative heterocycles that wouldbe potentially prebiotic and offer opportunities forbecoming chemoselectively attached to a backbone,we considered orotic acid as a candidate. Orotic acidand its 5-substituted derivatives have been identifiedas products from the hydrolysis of polymeric materialformed from hydrogen cyanide. In addition, the involve-ment of orotic acid as a nucleobase in the biosyntheticassembly of pyrimidine nucleotides justifies and war-rants exploration of its base-pairing properties. We aresynthesizing the necessary building blocks for the syn-thesis and study of the base-pairing properties of olig-omers consisting of aspartyl-3-aminoalanine dipeptideunits tagged with orotic acid (Fig. 3). The choice of the

oligodipeptide backbone on which orotic acid would beattached was influenced by the results of our previousstudies. Because the carboxyl group is now on theheterocycle, amide bond–mediated tagging of the car-boxyl group of orotic acid requires a 3-aminoalanineas 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 L Y O X Y L A T E A N D

D I H Y D R O X Y F U M A R A T E

A research project such as mapping the landscapeof potentially primordial informational oligomer systemseventually demands the conception of, and the commit-ment to, a detailed chemical scenario for the type oforganic chemistry that is supposed to have led to sucholigomers under primordial conditions. Figure 4 depictsthe chemical nature of the scenario we have decided tostudy experimentally. In the reaction cycle shown, gly-oxylate would autocatalytically convert itself into itsdimer dihydroxyfumarate. Dihydroxyfumarate is a knowncompound that we postulate can act as a common start-ing material for a large variety of biomolecules, such assugars, α-amino acids, and pyrimidines, and for otherorganics of etiologic interest by reactions that are essen-tially unexplored thus far but are deemed compatible

with the constraints of a primordial chemistry. We areconducting exploratory studies for assessing the chem-istry of selected intermediates postulated to be formedfrom the chemistry of glyoxylate and dihydroxyfumarate.Some of the preliminary results are encouraging.

PUBLICATIONSEschenmoser, A. On a hypothetical generational relationship between HCN andconstituents of the reductive citric acid cycle. Chem. Biodivers. 4:554, 2007.

Eschenmoser, A. The search for the chemistry of life’s origin. Tetrahedron63:12821, 2007.

Koch, K., Schweizer, B., Eschenmoser. A. Reactions of the HCN-tetramer withaldehydes. Chem. Biodivers. 4:541, 2007.

Organic, Medicinal, andBiological ChemistryM.G. Finn, A. Accurso, S. Brown, S.-H. Cho, V. Hong, J. Lau,

S. Lee, Y.-H. Lim, S. Presolski

In addition to our work on biological polyvalency andimmunology with engineered virus particles, sup-ported by the Skaggs Institute for Chemical Biology,

we focus on the development of catalysts and the syn-thesis of biologically useful structures. Two of these pro-jects are described in the following sections.

C O P P E R - C A T A L Y Z E D A Z I D E - A L K Y N E C L I C K

C H E M I S T R Y

We have continued our development of new cata-lysts and conditions for the copper-catalyzed azide-


F i g . 3 . Orotic acid as a recognition element. Also shown is the

oligodipeptide tagged with orotic acid.

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.

alkyne cycloaddition reaction, which has become a prin-cipal example of click chemistry in the synthesis of pos-sible drugs, dendrimers, polymers, and functionalizedsurfaces in laboratories around the world. In the pastyear, using an active but highly air-sensitive catalyst, wedeveloped a convenient electrochemical protocol forperforming bioconjugations. This procedure enablesinvestigators who lack sophisticated inert-atmosphereequipment to perform the reaction under demandingconditions. We have also discovered new derivatives ofthe (benzimidazole-methyl)amine ligands reported lastyear, which accelerate the copper-catalyzed azide-alkynecycloaddition reaction to a remarkable degree. A com-prehensive picture is rapidly emerging of the types ofligands effective under the diverse conditions in whichthis cycloaddition reaction is applied.

An important application of click chemistry is thesynthesis and modification of polymeric materials. Wefound that metal adhesives can be formed by the simpleapplication of mixtures of polyvalent azide and alkynecompounds to copper-containing surfaces. By incorporat-ing amino groups to help speed the click reaction, andflexible cross-linking molecules to protect against stressfracturing in the resulting polymers, greatly improvedadhesives have been created. Figure 1 illustrates the

strength of one of these formulations, which has poten-tial in such applications as protective coatings, electri-cally 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 P A T I T I S B

V I R U S E S : M I S D I R E C T I N G P R O T E I N - P R O T E I N

I N T E R A C T I O N S

Modulation of protein-protein contacts by smallmolecules is an attractive strategy for the developmentof biologically active compounds. In many instances,the target interprotein interaction covers a substantiallandscape with high thermodynamic stability. Virusparticles rely on the assembly of protein subunits thatengage in well-defined protein-protein interactions.However, these interactions are necessarily weak untilthe late stages of assembly; such cooperativity is nec-essary to ensure that protein is efficiently used in themultistep construction process.

Viral capsid intermediates are therefore a class ofprotein-protein interface targets that may be easier forsmall molecules to affect. We have investigated this pos-sibility for hepatitis B virus (HBV), a pathogen thatinfects 400 million people worldwide. The antiviral activ-ity of the heteroaryldihydropyrimidine class of com-pounds has been known for several years. We found thattheir mechanism of action is the distortion, rather thanthe inhibition, of the protein assembly process. In a typeof molecular jujitsu, these small molecules use thenatural interactions of the capsid proteins against thevirus, by increasing the energy of protein subunit associ-ation and the rate of subunit aggregation. This increasecauses the viral proteins to assemble in error-pronefashion, forming large and irregular structures ratherthan the symmetric particles necessary for the properfunction of the virus (Fig. 2).

By making many heteroaryldihydropyrimidine ana-logs, we found compounds that nucleate the misincor-poration of hundreds of protein molecules at a time,allowing these compounds to be highly effective inhib-itors in cell culture. We continue to improve the per-formance of these structures and test the best analogsfor activity against HBV in animal models. Further-more, we think that assembly misdirection of this kindis a general antiviral strategy. By attacking the rela-tively easy target of protein-protein interactions pre-sent in early virus assembly intermediates, selectiveagents can be developed in a new and effective way.

PUBLICATIONSDestito, G., Yeh, R., Rae, C.S., Finn, M.G., Manchester, M. Folic acid-mediated tar-geting of cowpea mosaic virus particles to tumor cells. Chem. Biol. 14:1152, 2007.


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.

Hong, V., Udit, A.K., Evans, R.A., Finn, M.G. Electrochemically protectedcopper(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-virusconstruction 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 ona viral scaffold. Chembiochem 8:1455, 2007.

Liu, Y., Díaz, D.D., Accurso, A.A., Sharpless, K.B., Fokin, V.V., Finn, M.G. Clickchemistry in materials synthesis, III: metal-adhesive polymers from Cu(I)-catalyzedazide-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 ofcarbohydrate 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 dothybrids. 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-acceler-ated 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. Benzimidazoleand 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., Lan-der, G., Quispe, J., Potter, C.S., Carragher, B., Tirrell, D.A., Finn, M.G. Unnaturalamino 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 proteinstructural biology. J. Am. Chem. Soc. 130:7357, 2008.

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 mem-brane proteins. Angew. Chem. Int. Ed. 46:7023, 2007.

Organometallic Catalysis inSynthesis, Bioorganic Chemistry,and Materials ResearchV.V. Fokin, A. Chanda, S. Chuprakov, J. Fotsing, S. Grecian,

T. Horneff, L. Krasnova, S.-W. Kwok, J. Raushel, T. Weide

Our goals are to discover new reactions and todevelop their practical applications in organicsynthesis, chemical biology, and materials sci-

ence. Transformations catalyzed by transition metalsare central to our research. With many variables foroptimization, these reactions have great potential tobecome useful on both laboratory and industrial scales.Although we often use automation techniques to screenextensive sets of catalysts, ligands, and conditions(sometime on the basis of just a hunch that “it shouldwork”), mechanistic investigations of the initial reactivityare prominent in our research. Analysis of reaction kinet-ics, in situ infrared monitoring, reaction heat flowcalorimetry, and other physicochemical methods areroutinely used to understand the mechanistic under-pinnings of the processes under investigation. Compu-tational studies of reactive intermediates and mechanisticpathways compliment these experimental techniques.Equipped with reactions that proceed reliably with arange of readily available starting materials and areexperimentally simple to perform, we endeavor toassemble molecules with interesting and useful func-tion, ranging from new biologically active small mole-cules to macromolecular scaffolds such as dendrimersfor studying multivalency effects and polymers for usein coatings and adhesives.

We have focused on metal-catalyzed reactions ofacetylenes because such processes can reliably producea plethora of heterocyclic systems. Copper(I)- and ruthe-nium(II)-catalyzed reactions of acetylenes with 1,3-dipolesdeveloped in our laboratories are shown in Figure 1.Reactions of alkynes with organic azides take a specialplace in the arsenal of catalytic dipolar cycloadditionswe use. Although azides are energetic compounds, theyare quite inert: stable to water, oxygen, most functionalgroups found in biological molecules, and many of thecommon organic reagents and conditions. With acety-


F i g . 2 . Negative-stain electron micrographs of the assembly prod-

ucts of HBV capsid protein induced by different heteroaryldihydropy-

rimidine derivatives. HBV capsids are typically 35 nm in diameter;

the structures shown here are many times that size.

lenes, organic azides produce 1,2,3-triazoles, exception-ally stable 5-membered heterocycles. These reactions,however, are very slow without a catalyst.

The copper- and ruthenium-catalyzed cycloaddi-tions of azides and alkynes provide ready access to1,2,3-triazoles of various substitution patterns. Thecopper-catalyzed variant has become the premier clickreaction and is used in diverse applications by chemistsacross the world in organic synthesis, medicinal chem-istry, chemical biology, and materials science. In addi-tion to its exceptional reliability and tolerance to a widerange of functional groups, the reaction has providedvaluable insights into the unique and, until recently,unexplored reactivity patterns of organic azides andin situ generated copper acetylides. Indeed, catalyticsyntheses of isoxazoles and pyrazoles are the recentadditions to the family of such reactions. The ruthe-nium-catalyzed reaction (Fig. 1) enables “fusion” oforganic azides and both terminal and internal alkyneswith a complementary regioselectivity and appears toproceed through a completely different mechanism.

This past year, we discovered the exquisite cata-lytic activity of pentamethylcyclopentadienyl ruthenium(II)chloride complexes in reactions of alkynes with nitrileoxides. These cycloadditions result in facile formationof 3,4-disubstituted and fully substituted isoxazoles.Until now, no general methods for regioselective syn-thesis of these heterocycles were available. When ther-mal cycloadditions of nitrile oxides with alkynes aresuccessful, they favor the formation of the 3,5-disub-stituted isomers. Furthermore, examples of reactivepartners for nitrile oxides are limited to a handful ofhighly activated, electron-deficient alkynes. Unactivatedor sterically hindered acetylenes usually do not react

at all. In contrast, in the presence of ruthenium com-plexes, nitrile oxides and both terminal and internalalkynes are “fused” within minutes to hours at roomtemperature. In addition to the immediate practical ben-efits, this transformation suggests that different dipolescan be activated and engaged in catalysis by rutheniumcomplexes. Such processes are being investigated.

Both copper- and ruthenium-catalyzed cycloaddi-tions and their applications in the synthesis of biologi-cally active compounds and novel materials have beenthe subject of our intense attention during the past year.We have investigated the mechanism of these processesand have endeavored to develop new ligands to makethe reactions more efficient. We have used the reactionsto synthesize libraries of compounds for HIV proteaseinhibition, in collaboration with J.H. Elder and A.J. Olson,Department of Molecular Biology; nicotinic receptoragonists and antagonists, in studies with P. Taylor,University of California, San Diego; metallo-β-lactamaseinhibitors, in collaboration with P. Hodder, TranslationalResearch Institute; and polymeric materials and den-dritic constructs for polyvalent display and imagingapplications (Fig. 2).


F i g . 1 . Catalytic syntheses of heterocycles from alkynes and

1,3-dipolar species.

F i g . 2 . Small molecules synthesized by using azide-alkyne

cycloadditions.

PUBLICATIONSBoren, 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 mecha-nism. J. Am. Chem. Soc. 130:8923, 2008.

Finn, M.G., Kolb, H.C., Fokin, V.V., Sharpless, K.B. Concept and applications ofclick 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 mate-rials 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-1,2,3-triazoles. Org. Lett. 10:3171, 2008.


Rasmussen, L.K., Boren, B.C., Fokin, V.V. Ruthenium-catalyzed cycloaddition ofaryl azides and alkynes. Org. Lett. 9:5337, 2007.

Rodionov, V.O., Presolski, S.I., Díaz, D.D., Fokin, V.V., Finn, M.G. Ligand-acceler-ated 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., Drock-enmuller, E., Hawker, C.J. Role of architecture and molecular weight in the forma-tion of tailor-made ultrathin multilayers using dendritic macromolecules and clickchemistry. J. Polym. Sci. A Polym. Chem. 45:2835, 2007.

Yoo, E.J., Ahlquist, M., Bae, I., Sharpless, K.B., Fokin, V.V., Chang, S. Mechanisticstudies on the Cu-catalyzed three-component reactions of sulfonyl azides, 1-alkynesand amines, alcohols, or water: dichotomy via a common pathway J. Org. Chem.73:5520, 2008.

Design of Functional Synthetic SystemsM.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

We are engaged in multidisciplinary researchto uncover new chemical and biochemicalapproaches for the design of functional molec-

ular, supramolecular, and complex self-organized sys-tems. Our efforts span disciplines ranging from syntheticorganic, bioorganic, and physical organic chemistry tonanotechnology, biophysics, enzymology, and molecularbiology. Current research includes the design of syntheticpeptide catalysts, antimicrobial self-assembling peptidenanotubes, semisynthetic allosteric enzymes, self-repli-cating molecular systems and emergent networks, sin-gle-molecule DNA sensing, molecular computation, andprebiotic chemistry.A N T I M I C R O B I A L P E P T I D E N A N O T U B E S

We have shown that appropriately designed cyclicpeptide subunits can self-assemble through hydrogen

bond–directed ring stacking into open-ended hollowtubular structures that have marked antibacterial andantiviral activities in vitro. The effectiveness of thisnovel supramolecular class of bioactive species asselective antibacterial agents was highlighted by thehigh efficacy of one of these antimicrobials againstlethal methicillin-resistant Staphylococcus aureus infec-tions in mice. Currently, we are exploring rational designof cyclic glycopeptides and selections from combinato-rial libraries to discover novel antiviral supramolecularcompounds (Fig. 1).

D E S I G N O F S I G N A L S E L F - A M P L I F Y I N G D N A S E N S O R S

We constructed a novel sequence-specific DNA detec-tion system based on rationally designed semisyntheticenzymes. The system is composed of covalently asso-ciated inhibitor-DNA-enzyme modules that function viaDNA hybridization–triggered allosteric enzyme activa-tion and signal amplification through substrate turnover(Fig. 2). The functional capacity of the system is high-

lighted by the sequence-specific detection of approxi-mately 10 fmol of DNA in less than 3 minutes underphysiologic conditions. Our studies suggest that ratio-


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.

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.

nally designed intrasterically regulated enzymes may bea promising new class of reagents for highly sensitive,rapid, and 1-step detection of label-free DNA sequencesthat does not depend on polymerase chain reactions.S I N G L E - M O L E C U L E D N A S E Q U E N C I N G

We are interested in the study of matter at the levelof single molecules. For these studies we use the trans-membrane protein α-hemolysin as a rapid and highlysensitive sensor element for stochastic analysis of themolecules lodged or trapped inside the protein pore;the analysis relies on detecting the perturbations in theconductance levels produced in the ion channel in thenative protein. Using this technique, we developed anapproach by which a single-stranded DNA molecule canbe trapped in a specific configuration inside an α-hemo-lysin channel, manipulated, and studied with high sensi-tivity at the single-molecule level. We have been ableto detect up to 9 consecutive DNA polymerase–catalyzedsingle-nucleotide primer extensions (Fig. 3) with high

sensitivity and spatial resolution (≤2.4 Å). The single-base resolution of this approach and the ability to con-trol the passage of DNA in single-base steps satisfy the2 minimal requirements of a nanopore-based sequenc-ing device.C O M P L E X S Y N T H E T I C N E T W O R K S

Living cells use complex networks of evolutionarilyselected biomolecular interactions and chemical trans-formations to process multiple extracellular input sig-nals rapidly and simultaneously. We are interested inunderstanding and experimentally modeling the orga-nizational and functional properties of biological net-works. We have developed a general strategy for thedesign and construction of self-organized synthetic pep-tide networks based on the sequence-selective auto-catalytic and cross-catalytic template-directed coiledcoil peptide fragment condensation reactions in aque-

ous solutions. The synthetic networks have some of thebasic architectural and dynamic features of the livingnetworks, reorganize in response to changes in environ-mental conditions and inputs (Fig. 4), and perform basic

Boolean logic functions. We suggest that the abilityto rationally construct predictable chemical circuitrymight be useful in advancing the modeling and betterunderstanding of some of the basic dynamic informa-tion-processing characteristics of the more complex cel-lular networks.M O L E C U L A R C O M P U T A T I O N

A fundamental goal of computing is to reproducein a molecular setting the familiar properties of micro-electronics, such as digital logic, component modularity,and hierarchical design capacity. In this regard, signif-icant advances have been made in the design of molec-ular logic gates by using small-molecule and rotaxanecomplexes, deoxyribozymes, enzymatic biochemicalnetworks, peptide networks, and other systems. How-ever, the molecular logic gates must be integrated intomore complex networks in which outputs from each gatecan serve as inputs to downstream gates.

We recently described the construction of a basis setof DNA-based logic gates (AND, OR, AND-NOT) capableof communicating with one another. These gates wererewired into a higher-order circuit that enforces a netXOR (Exclusive OR) Boolean behavior (Fig. 5), showingthat the components can be modularly recombined toimplement novel logic processing. Our results supportthe notion that with a basis set of only a few logic gatesand within the limits imposed by the availability ofuniquely addressable oligonucleotide sequences, designof molecular circuits capable of performing a large vari-ety of digital logic operations might be within reach.


F i g . 4 . Adaptive reorganization in a synthetic peptide network. The

graph structure or wiring of a synthetic peptide network responds dra-

matically to changes in the environmental stimuli (pH or salt content).

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.

P R E B I O T I C C H E M I S T R Y

The emergence of a polymer that could store geneticinformation, replicate, and exhibit phenotypic propertiessubject to selective environmental pressures marked acrucial stage in the transition from the prebiotic worldto biology; however, the nature of such a polymer remainsunresolved. We have discovered an oligomer family thatquickly and efficiently self-assembles via reversible cova-lent anchoring of nucleobase recognition units onto simplepeptide backbones. The resulting oligomers specificallyself-pair and cross-pair with complementary strandsof RNA and DNA in Watson-Crick fashion. Moreover,the oligomers undergo dynamic component exchange,template-directed assembly processes, and dynamicsequence modification in response to changing selec-tive pressures. Such oligomers could therefore haveparticipated in a number of processes that would beadvantageous for primordial genetic systems, such asdynamic sequence repair and adaptation.

PUBLICATIONSCockroft, S.L., Chu, J., Amorin, M., Ghadiri, M.R. A single-molecule nanoporedevice 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 fromimmobilized 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 pro-grammable 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 inde novo designed coiled coil peptides via rational active site engineering. J. Am.Chem. Soc. 129:2959, 2007.

A Merging of Chemistry and BiologyK.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,

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

During the past year, we used various applicationsof organic chemistry to address biological prob-lems. Representative examples of our results

are given for 3 research programs: inhibition of bacte-rial virulence via the disruption of bacterial communi-cation, discovery of a link between drug abuse andcardiovascular disease, and selection and characteri-zation of human neutralizing antibodies against Bacillusanthracis toxin.

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 A T 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 A T I O N

The ability of microorganisms to coordinate theirgene expression according to population density hasbeen termed quorum sensing. This chemical exchangeof information among single-cell organisms is mediatedby secreted signaling molecules termed autoinducers.Important biological and clinical aspects of quorumsensing include the regulation of bacterial virulencefactors and the formation of biofilms; hence, inhibi-tion of signaling associated with quorum sensing couldprovide a promising new strategy for the attenuationof bacterial infections. Indeed, analogs of autoinducershave been used as small-molecule antagonists in sev-eral quorum-sensing circuits as a means of signalinginterference. Alternatively, we have pioneered an anti-body-based strategy to combat quorum sensing throughdisruption of signal transmission.

Recently, we applied our antibody-based technologyto the interference of the quorum-sensing circuits ofStaphylococcus aureus. This microorganism is the mostcommon cause of hospital-acquired infections, includingdiseases ranging from skin infections and food poisoningto life-threatening nosocomial infections. The increasingresistance of S aureus isolates to glycopeptide antibi-otics, most prominently vancomycin, is a major concernin intensive care units, and an alternative strategy tocombat this pathogen is urgently required.


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.

Staphylococcus aureus uses a set of 4 cyclic autoin-ducing peptides (AIP-1–AIP-4) to regulate its quorum-sensing machinery, which is responsible for orchestratingthe expression of virulence genes. Thus, inhibition of theS aureus system would result in decreased pathogenic-ity. We generated a monoclonal antibody, AP4-24H11,to sequester AIP-4 (Fig. 1). This antibody was elicited

against a rationally designed hapten (AP4, Fig. 1) andefficiently interfered with the quorum sensing of S aureusin vitro, as determined by real-time polymerase chainreaction analysis and inactivation of AP4-24H11 by syn-thetic AIP-4. Importantly, AP4-24H11 suppressed bothS aureus–induced dermal injury in a mouse model ofabscess formation in vivo and provided complete pro-tection against a lethal S aureus challenge. Thesefindings provide a strong foundation for further investi-gations of immunopharmacotherapy as treatment ofbacterial infections in which quorum sensing controlsthe 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 T A M I N E

U S E A N D C A R D I O V A S C U L A R D I S E A S E

The rapid spread of methamphetamine abuse acrossthe United States is as alarming as the propensity ofthe drug to induce severe addiction and the health-related consequences of addiction. Whereas before2001 methamphetamine use occurred predominantlyin the western United States, its use now is extendingrapidly throughout the United States and across differ-ent ethnic groups. The threat that methamphetaminenow poses to society underscores the need to morethoroughly examine the ramifications of chronic meth-amphetamine abuse.

In addition to causing severe dopaminergic neuro-toxic effects, chronic methamphetamine self-administra-tion induces increasing drug tolerance that correlates withescalating intake. Although the molecular mechanismbehind pharmacologic tolerance is not fully elucidated,we hypothesized that methamphetamine covalentlymodifies endogenous proteins in a process known asglycation (Fig. 2) before reaching the brain and medi-

ating its well-characterized stimulant effects. Glycationreactions, collectively termed the Maillard reaction,have been studied for decades in the food industry inthe development of flavor and color; however, Maillardproducts can also assume a biologically hazardous rolewhen synthesized in vivo. Acquiring the ability to cross-link proteins, these irreversible reaction products, termedadvanced glycation end products (AGEs), have gainednotoriety for their participation in a range of patho-logic changes.

Protein glycation by methamphetamine induces animmune response against these modified proteins, whichcould lead to sequestration of drug and, ultimately, thedevelopment of tolerance. We have shown that this drug-dependent glycation mechanism is operative in vivo. Wedetected antibodies against methamphetamine-derivedAGEs in rats that chronically self-administered the drug,and we noted a direct relationship between the level ofmethamphetamine intake and the respective antibodytiters against methamphetamine-glycated proteins.

Additionally, we detected increased levels of proin-flammatory and other cytokine molecules, particularlyvascular endothelial growth factor. AGE-associated upreg-


F i g . 2 . Reaction scheme of methamphetamine protein glycation

as initiated by glucose and methamphetamine.

F i g . 1 . Structure of the S aureus autoinducer AIP-4 and AP4

hapten used to generate the quorum-quenching antibody AP4-24H11.

ulation of this growth factor has been associated withthe onset of heart disease, but these effects had notbeen previously associated with methamphetamine-derived AGEs. Because AGEs can alter protein functionin vivo and participate in various diseases, metham-phetamine-derived AGEs provide an unrecognizedmolecular mechanism for the development of vasculi-tis and other cardiovascular maladies with high inci-dence in chronic methamphetamine users.H U M A N N E U T R A L I Z I N G A N T I B O D I E S A G A I N S T

A N T H R A X T O X I N

A less-than-adequate therapeutic plan for the treat-ment of anthrax in the 2001 bioterrorism attacks hashighlighted the importance of developing alternative orcomplementary therapeutic approaches for biothreatagents. Vaccination against B anthracis for protectionagainst anthrax has been known for more than a cen-tury. However, the prolonged vaccination schedules andinduction times required for an immune response areserious drawbacks, because the therapeutic window fortreatment of anyone exposed to a deliberate release of B anthracis is limited. Alternatively, recently developedantibiotic prophylaxis for the treatment of personsexposed to anthrax, although important, would also beof lesser value if the infection were caused by an antibi-otic-resistant strain.

Passive immunization has provided an attractiveavenue as a treatment both before and after exposureto B anthracis. Indeed, in many animal studies, passivetransfer of antiserum successfully provided protectionagainst anthrax. Furthermore, passive immunizationcould have advantages over active vaccination andantibiotic treatments via few toxic effects, high speci-ficity, the capability for stockpiling large quantities ofthe antiserum, and immediate protection against abiological attack.

Using a phage-displayed human single-chain vari-able fragment (scFv) antibody library, we selected andcharacterized several human monoclonal neutralizingantibodies against the toxin of B anthracis. In total,15 clones with distinct sequences and high specificityfor the protective antigen region of the anthrax toxin(Fig. 3) were analyzed by using biophysical and cell-based cytotoxicity assays. From this panel of antibod-ies, a set of neutralizing antibodies was identified, andthe potency of protection was established by using amacrophage cytotoxicity assay. Among the neutralizingantibodies identified, 1 clone had excellent affinity forthe protective antigen region of the anthrax toxin and

provided superior protection from lethal toxin in thecell cytotoxicity assay. Our results add to the ever-grow-ing arsenal of immunologic and functional analysis ofmonoclonal antibodies to the exotoxins of anthrax. Inaddition, the antibodies may be new candidates forprophylactic and therapeutic agents.

PUBLICATIONSBrogan, A.P., Dickerson, T.J., Janda, K.D. Nornicotine-organocatalyzed aqueousreduction of α,β-unsaturated aldehydes. Chem. Commun. (Camb.) Issue 46:4952,2007.

Capková, K., Yoneda, Y., Dickerson, T.J., Janda, K.D. Synthesis and structure-activity relationships of second-generation hydroxamate botulinum neurotoxin Aprotease 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 anti-body-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 cocaineimmunopharmacotherapy. Bioorg. Med. Chem. Lett. 17:4280, 2007.

Kaufmann, G.F., Park, J., Janda, K.D. Bacterial quorum sensing: a new target foranti-infective immunotherapy. Expert Opin. Biol. Ther. 8:719, 2008.

Kaufmann, G.F., Park, J., Mee, J.M., Ulevitch, R.J., Janda, K.D. The quorumquenching antibody RS2-1G9 protects macrophages from the cytotoxic effects ofthe Pseudomonas aeruginosa quorum sensing signalling molecule N-3-oxo-dode-canoyl-homoserine lactone. Mol. Immunol. 45:2710, 2008.

Lowery, C.A., Dickerson, T.J., Janda, K.D. Interspecies and interkingdom commu-nication 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., Mei-jler, M.M., Ulevitch, R.J., Janda, K.D. Infection control by antibody disruption ofbacterial quorum sensing signaling. Chem. Biol. 14:1119, 2007.

Park, J., Kaufmann, G.F., Bowen, J.P., Arbiser, J.L., Janda, K.D. Solenopsin A, avenom alkaloid from the fire ant Solenopsis invicta, inhibits quorum-sensing sig-naling in Pseudomonas aeruginosa. J. Infect. Dis. 198:1198, 2008.


F i g . 3 . Targeting of the protective antigen (PA) region of B

anthracis toxin by human monoclonal antibodies.

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 factortype 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 bymethamphetamine glycation products chemically links chronic drug abuse and car-diovascular 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 thebotulinum neurotoxin: using chemistry and biology to modulate the most deadlypoison. 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 formonitoring 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 pro-drug 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 Bacillusanthracis and Clostridium botulinum. Expert Rev. Vaccines 7:55, 2008.

Zhou, B., Carney, C., Janda, K.D. Selection and characterization of human anti-bodies 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. Delineat-ing the susceptibility of botulinum neurotoxins to denaturation through thermaleffects. FEBS Lett. 582:1526, 2008.

Zhou, H., Zhou, B., Ma, H., Carney, C., Janda, K.D. Selection and characteriza-tion of human monoclonal antibodies against Abrin by phage display. Bioorg. Med.Chem. Lett. 17:5690, 2007.

Maintaining the Proteome toAmeliorate Human DiseaseJ.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

Maintenance of the proteome (proteostasis) bothinside and outside human cells is essentialfor development, reproduction, and success-

ful aging. Deficiencies in proteostasis lead to manymetabolic, oncologic, neurodegenerative, and cardio-vascular diseases. Understanding the mechanisms ofproteostasis, especially defects in the pathways of theproteostasis network that occur with aging, enables thedesign of new therapeutic strategies to ameliorate age-onset protein misfolding diseases, a main goal of ourresearch. We use animal and cell-based disease modelsand biophysical approaches in combination with medi-cinal chemistry and structure-based drug design. Ourcollaborators, W.E. Balch, Department of Cell Biology;J. Buxbaum, Department of Molecular and Experimental

Medicine; J.R. Yates, Department of Chemical Physiol-ogy; E. Masliah, University of California, San Diego; andA. Dillin, the Salk Institute for Biological Studies, LaJolla, California, play an essential role in our multidisci-plinary approach.A M E L I O R A T I O N O F L Y 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-functiondiseases often caused by a mutation in one of thelysosomal enzymes, which results in excessive mis-folding of the enzyme within the endoplasmic reticu-lum and cytosolic degradation instead of proper foldingand trafficking of the enzyme to the lysosome. In 2separate studies, we found that the innate proteosta-sis capacity of a cell can be enhanced with small mol-ecules termed proteostasis regulators to fold mutatedenzymes that would otherwise misfold and be degraded,resulting in increased trafficking of the mutated enzymeto the lysosome and increased function. In the first study,we found that diltiazem and verapamil, L-type calciumchannel blocker drugs approved by the Food and DrugAdministration, increased folding capacity in the endo-plasmic reticulum, trafficking, and activity of mutantlysosomal enzymes associated with 3 distinct lysosomalstorage diseases: Gaucher disease, α-mannosidosis,and type IIIA mucopolysaccharidosis. These compoundslikely act by calcium ion–mediated enhancement ofendoplasmic reticulum lumenal chaperone function. Inthe second study, we discovered that 2 proteostasisregulators partially restored folding, trafficking, and func-tion of mutant enzymes in Gaucher and Tay-Sachs celllines by activating the unfolded protein response, asignaling pathway that influences proteostasis in thesecretory pathway. Moreover, we found that the com-bination of a proteostasis regulator and a pharmaco-logic chaperone, a chemical that binds directly to agiven enzyme and thereby stabilizes the enzyme, syn-ergistically restored enzyme function, because of theirdistinct mechanisms of action.U N D E R S T A 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

D I S E A S E

We are interested in understanding the molecularand mechanistic basis for the age-onset nature of Alz-heimer’s disease. Genetic and biochemical evidenceimplicates aggregation of amyloid β-peptide (Aβ), enabledby an age-onset decrease in proteostatic capacity, asthe cause of neurodegeneration in this disease; how-ever, precise identification of the toxic structure andthe mechanism of neurotoxic effects remain elusive. Inpatients with Alzheimer’s disease, the correlation between

1 0 0 C H E M I S T R Y 2 0 0 8 T H E S C R I P P S R E S E A R C H I N S T I T U T E

disease severity and the concentration of sphericalaggregates, annular structures, protofibrils and othersoluble oligomeric species is better than the correla-tion between disease severity and the concentrationof fibrillar amyloid.

Previously, we showed that mutating the phenylal-anine 19–phenylalanine 20 backbone amide bond toan E-olefin bond allows the formation of spherical aggre-gates to the exclusion of fibrils. In a more extensiveamide-to-ester mutagenesis scan through the hydropho-bic core (residues 17–21) of Aβ 1-40, we compared themutants with wild-type Aβ 1-40 and the E-olefin Aβ1-40 mutant. Even though the E-olefin mutant, theamide-to-ester mutant, and wild-type Aβ 1-40 formaggregates of different morphologies, all 3 types of aggre-gates were similarly toxic to PC12 neuronal cells. Thisfinding suggests that a common, but low-abundance,aggregate morphology mediates toxic effects or that sev-eral different aggregate morphologies are similarly toxic.O X I D I Z E D M E T A 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 A T I O N

One of the central mysteries of Alzheimer’s diseaseis how Aβ forms amyloid in vivo when both thermody-namic and kinetic barriers against aggregation exist.We propose that covalent modification of Aβ by small-molecule oxidation products can explain, at least in part,the ability of Aβ to form amyloid at physiologic concen-trations and thus place a load on the proteostasis net-work. Using Aβ conjugates site-specifically modifiedwith a cholesterol aldehyde at aspartic acid 1, lysine16, or lysine 28, we found that modification loweredthe critical concentration for aggregation into the nano-molar range, within the physiologic concentration rangeof Aβ, and dramatically increased the rate of aggrega-tion. Aβ modified at lysine 16 formed amorphous aggre-gates fastest and at the lowest concentrations (within2 hours at 20 nM).

The same cholesterol aldehyde is found in humanatherosclerotic lesions and rapidly promotes apolipopro-tein C-II amyloid formation in vitro. Thus, enhance-ment of amyloid formation by oxidized metabolitesappears to be common to several diseases and suggeststhat strategies to prevent such modification may havetherapeutic potential for a spectrum of human diseases.T R E A T 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 misassembleinto extracellular aggregates. The rate-limiting step inamyloid formation by transthyretin is the dissociation

of the tetramer. We have developed kinetic stabilizers ofthe tetrameric structure of transthyretin that have novelchemistries and mechanisms of actions. To improveinhibitors of transthyretin amyloidogenesis, we are opti-mizing each of the 3 substructural elements that makeup a typical inhibitor: the 2 aryl rings and the linkerthat joins the rings. We evaluated structural modifica-tions to the aryl ring 1 by screening a library of 2-aryl-benzoxazoles that have thyroid hormone–like arylsubstituents on the 2-aryl ring. The 3,4,5-substitutedthyroxine-like aryl ring appears to be the optimal solu-tion for the structure of aryl ring 1. In addition, wesynthesized 40 bisaryl compounds to optimize the struc-ture of the linker. We found that direct connection ofthe 2 aryls, or linkage through nonpolar E-olefin or–CH2CH2– substructures, generates the most potentand selective inhibitors of transthyretin amyloidogenesis.Five high-resolution (1.4–1.8 Å) x-ray crystallogra-phy structures of transthyretin reveal that the 3,5-dimethyl-4-hydroxyphenyl ring preferentially occupiesthe inner cavity of the thyroxine-binding site and thatthe 3,5-dibromo-4-hydroxyphenyl aryl prefers the outercavity because the phenol is deprotonated with flank-ing electron withdrawing group substitution. A studyto optimize the remaining aryl ring is well under way.

Tetramers of transthyretin can also be kineticallystabilized by trans-suppression, as we showed previ-ously with T119M transthyretin subunit incorporationinto the tetramer, which stabilizes heterotetramerscontaining T119M and V30M transthyretin subunits.In an analogous manner, heterotetramers composedof murine transthyretin and human transthyretin sub-units are kinetically stable and nonamyloidogenic.This information is important for evaluating trans-genic models of human transthyretin amyloidosis inwhich the transgenic animals have a low copy numberof the mutant amyloidogenic human transthyretin gene.U N D E R S T A N D I N G A N D A M E L I O R A T I N G G E L S O L I N

A M Y L O I D O S I S

Gelsolin amyloid disease is another age-onset degen-erative malady linked to protein aggregation that isthought to be due to an age-associated decline inproteostasis. A mutation in gelsolin (D187N) leads toaberrant folding and cleavage by furin within the Golgiapparatus during trafficking. Subsequent cleavage of thegelsolin fragment by the matrix metalloprotease MT1-MMP outside the cell results in 5- and 8-kD fragmentsof gelsolin, which deposit as amyloid in the extracellu-lar matrix. In collaboration with W.E. Balch, Depart-

C H E M I S T R Y 2 0 0 8 T H E S C R I P P S R E S E A R C H I N S T I T U T E 1 0 1

ment of Cell Biology, we have developed 2 transgenicmouse models of human D187N gelsolin amyloidosisthat recapitulate the aberrant endoproteolytic cascadeand the aging-associated decline in proteostasis thatresult in extracellular amyloidogenesis in humans.

PUBLICATIONSBalch, W.E., Morimoto, R.I., Dillin, A., Kelly, J.W. Adapting proteostasis for dis-ease intervention. Science 319:916, 2008.

Bieschke, J., Siegel, S.J., Fu, J., Kelly, J.W. Alzheimer’s Aβ peptides containingan isostructural backbone mutation afford distinct aggregate morphologies but anal-ogous cytotoxicity: evidence for a common low-abundance toxic structure(s)? Bio-chemistry 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 bac-teria to humans. Trends Biochem. Sci. 32:217, 2007.

Jäger, M., Dendle, M., Kelly, J.W. A cross-strand Trp-Trp pair stabilizes a WWdomain at the expense of function. Protein Sci. 16:2306 2007.

Jäger, M., Nguyen, H., Dendle, M., Gruebele, M., Kelly, J.W. Influence of hPin1WW N-terminal domain boundaries on function, protein stability, and folding. Pro-tein Sci. 16:1495, 2007.

Johnson, S.M., Connelly, S., Wilson, I.A., Kelly, J.W. Biochemical and structuralevaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogene-sis inhibitors. J. Med. Chem. 51:260, 2008.

Kelly, J.W. Compromised cellular folding fidelity results in numerous clinicallyimportant diseases. Nature 446:112, 2007.

Liu, F., Du, D., Fuller, A.A., Davoren, J.E., Wipf, P., Kelly, J.W., Gruebele, M. Anexperimental survey of the transition between two-state and downhill protein fold-ing 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 homeo-stasis in three distinct lysosomal storage disease cell lines by altering calciumhomeostasis. 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 amy-loidogenic prostatic acidic phosphatase fragments dramatically enhance HIV infec-tion. Cell 131:1059, 2007.

Reixach, N., Foss, T.R., Santelli, E., Pascual, J., Kelly, J.W. Human-murine trans-thyretin heterotetramers are kinetically stable and non-amyloidogenic: a lesson inthe 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 metabolitesfound in human atherosclerotic lesions promote apolipoprotein C-II amyloid fibrilformation. Biochemistry 46:5552, 2007.

Wiseman, R.L., Koulov, A., Powers, E.T., Kelly, J.W., Balch, W.E. Protein energeticsin 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 adaptablestandard 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 treatGaucher disease. FEBS Lett. 274:4944, 2007.

Total Synthesis, New SyntheticTechnologies, and ChemicalBiologyK.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

We focus on the total synthesis of natural prod-ucts, the discovery and development of newsynthetic technologies, and chemical biology.

Naturally occurring substances are selected as synthetictargets for their novel molecular architectures, importantbiological properties, and interesting mechanisms ofaction. The projects are designed to optimize the oppor-tunities for discovery and invention in the areas ofchemistry, biology, and medicine. The natural productsthiostrepton, azaspiracid-1–azaspiracid-3, abyssomycin C,the bisanthraquinones and the marinomycins exemplifythis philosophy. Current projects include studies on theantibiotics nocathiacin I, platensimycin, and platencin;the antitumor agents lomaiviticins A and B and unciala-mycin; the anti-HIV agent biyouyanagin A; and themarine biotoxin maitotoxin (Fig. 1).

In addition, we are developing synthetic technolo-gies and strategies for chemical synthesis and chemicalbiology studies. Our overall aims are to advance theart and science of chemical synthesis and to developenabling technologies for biology and medicine whilemaximizing educational opportunities and training ofyoung men and women in chemistry.

PUBLICATIONSNicolaou, K.C., Chen, J.S., Zhang, H., Montero, A. Asymmetric synthesis and bio-logical 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. Chemicalsynthesis of the GHIJK ring system and further experimental support for the origi-nally 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, Band 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 synthesisof thiopeptide antibiotics GE2270A, GE2270T, and GE2270C1. Chem. Asian J.3:413, 2008.


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 theGHIJKLMNO 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 synthesisof 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 strat-egy 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 ofkinamycins C, F, and J. J. Am. Chem. Soc. 129:10356, 2007.

Nicolaou, K.C., Lister, T., Denton, R.M., Gelin, C.F. Cascade reactions involvingformal [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 ofthe “left-domain” of haplophytine. Angew. Chem. Int. Ed. 46:4715, 2007.

Nicolaou, K.C., Ortiz, A., Denton, R.M. Metathesis reactions in the synthesis ofcomplex molecules. Chem. Today 25:70, 2007.

Nicolaou, K.C., Pappo, D., Tsang, K.Y., Gibe, R., Chen, D.Y.-K. A chiral poolbased 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 synthesisof the originally proposed and revised structures of palmerolide A and isomersthereof. J. Am. Chem. Soc. 130:3633, 2008.

Nicolaou, K.C., Tang, Y., Wang, J., Stepan, A.F., Li, A., Montero, A. Total synthe-sis 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.

Nicolaou, K.C., Wang, J., Tang, Y. Synthesis of the sporolide ring frameworkthrough a cascade sequence involving an intramolecular [4+2] cycloaddition reac-tion 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 ofazaspiracid-1. Cell. Physiol. Biochem. 20:957, 2007.

Vilariño, N., Nicolaou, K.C., Frederick, M.O., Vieytes, M.R., Botana, L.M. Irre-versible cytoskeletal disarrangement is independent of caspase activation during invitro azaspiracid toxicity in human neuroblastoma cells. Biochem. Pharmacol.74:327, 2007.

Translational Chemistry and MedicineE. 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

Introduction of new medicines is crucial to preservehuman health. We are dedicated to the pursuit ofnew and better therapies and continually challenge

the frontiers of drug discovery. As we take major sci-entific steps toward the future, we move to a moreknowledge-based drug discovery. Our goal is to gener-ate high-quality clinical candidates as new medicinesin therapeutic areas such as epilepsy/seizures, neuro-pathic pain, autoimmune diseases (e.g., multiple scle-rosis), and developmental disorders (e.g., autism).T R E A T 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 stateof the CNS is caused by an imbalance between inhibi-tory and excitatory neurotransmission. Current epilepsytherapy focuses on the modulation of the classicalneurotransmitters glutamate and γ-aminobutyric acid.The neuropeptide galanin antagonizes excitatory gluta-minergic neurotransmission in the hippocampus, sug-gesting that galanin may have a role in seizure activity.

In collaboration with T. Bartfai and X. Lu, Molecu-lar and Integrative Neurosciences Department, we haveidentified new nonpeptidic ligands for the galanin recep-tors GalR1 and GalR2. This set of small, druglike mol-ecules can displace the peptide galanin from its proteinbinding site. Selectivity and potency of these initial molec-ular starting points are being optimized.D U A L O P I O I D A G O N I S T S – C H O L E C Y S T O K I N I N

A N T A G O N I S T S F O R T R E A T M E N T O F C H R O N I C A N D

N E U R O P A T H I C P A I N

Nociception, or the perception of pain, and its mod-ulation depend on the interaction of many endogenous


F i g . 1 . Selected target molecules.

neurotransmitters in the spinal cord. The interactionof endogenous peptides such as cholecystokinin withexogenously administered opioids markedly alters activ-ity in acute and chronic pain states. This interactionmay lead to the development of novel medications thatare more effective and safer than currently availableopioids alone.

Molecules with the property of being both opioidagonists and cholecystokinin antagonists would be usefulin conditions in which the effectiveness of opioids isreduced, as in the development of tolerance to opioidpain relievers in chronic pain (e.g., in pain caused bycancers) and in neuropathic pain conditions in whichopioids are ineffective. Thus, because of the prevention(or reversal) of tolerance, additionally physical depen-dence on opioids might be diminished or inhibited. Theadvantages of developing a single compound with dualopioid agonist–cholecystokinin antagonist activity ratherthan a combination of an opioid agonist taken with aseparate cholecystokinin antagonist are clear. Develop-ment of a single compound involves only a single setof parameters, such as toxicology, pharmacokinetics,and formulation, rather than 2 independent and oftenunrelated sets of data.

In collaboration with F. Porreca and J. Lai, Univer-

sity 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-

lecystokinin receptors 1/(A) and 2/(B) are all members

of this subclass of G protein–coupled receptors (Fig. 1).

I M M U N O M O D U L A T I N G C O M P O U N D S F O R T H E

T R E A T M E N T O F M U L T I P L E S C L E R O S I S

Sphingosine 1-phosphate (S1P) is an endogenousmediator that functions both as an intracellular messen-ger and as an extracellular signaling molecule. S1P isinvolved in a number of processes, including vascularstabilization, cardiac development, and cancer angiogen-esis. Extracellularly, S1P elicits its biological effectsthrough a family of G protein–coupled receptors thatbind to the S1P1–S1P5 subtypes of S1P. Activation ofthe receptor for S1P1 had effects in multiple sclerosisand organ transplantation via the immunosuppressionevoked by the nonselective S1P agonist FTY720, whichis currently in clinical trials. It was speculated that theeffects on heart rate and lung function in clinical trailswith FTY720 were due to agonism of S1P3.

In collaboration with H. Rosen and his group, Depart-ment of Immunology ane Microbial Science, we haveidentified novel small-molecule compounds that areexquisitely selective for S1P1, are stable, are orallyactive, and penetrate into the CNS (Table 1). Thesecompounds are expected to be useful in disease statessuch as multiple sclerosis.


F i g . 1 . Opioid agonist–cholecystokinin antagonist hybrids.

T a b l e 1 . Agonists of sphingosine 1-phosphates.

Compound Total polar surface area, Å2

Molecularweight, kD

CalculatedlogP

EC50, nM

Sphingosine 1-phosphate 1

Sphingosine 1-phosphate 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


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




CYM5442 84.7 409.5 3.7 1.1 Not applicable


CYM5457 84.7 397 3.4 3.4 692



CYM5386 46.3 387 5.2 4.5 1300

I L - 6 A N T A G O N I S T S F O R T R E A T M E N T O F

I N F L A M M A T O R Y D I S E A S E S

Clinical studies have provided strong evidence thatspecific blockade of IL-6–regulated signaling pathwaysis a validated approach for treatment of inflammatorydiseases. Currently, only monoclonal antibodies areavailable to block the actions of IL-6. Using the human-ized antibody tocilizumab to block the actions of IL-6has been therapeutically effective in patients with rheu-matoid arthritis, systemic juvenile idiopathic arthritis,and Crohn’s disease. Tocilizumab is in phase 1 clinicaltrials in the United States and in phase 2 clinical trialsin France for the treatment of multiple myeloma.

However, serious adverse effects with tocilizumabhave been reported, including a death and allergic pneu-monitis. Increases in the levels of serum lipids, liverfunction abnormalities, and reduction in white bloodcell count occurred in substantial numbers of patients.In collaboration with Dr. Porreca, we have identifiedsmall, orally active IL-6 receptor antagonists that arebeing modified for affinity and druglike properties. Onceoptimized, these antagonists should have fewer adverseside effects than does tocilizumab.

PUBLICATIONSLum, 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. 2,5-Diaminopyrimidines and 3,5-disubstituted azapurines as inhibitors of glycogen syn-thase 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 inhibi-tors. Bioorg. Med. Chem. Lett. 18:1772, 2008.

Chemical, Biological, andBiophysical Approaches toUnderstanding EvolutionF.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

The molecules of biology are unique because theyhave been evolved for function. We use multi-disciplinary methods in conjunction with chemi-

cal biological principles to develop unique approachesto understanding evolution.

I N C R E A S I N G T H E C H E M I C A L A N D G E N E T I C

P O T E N T I A L O F D N A

We are interested in increasing the informationpotential of DNA by expanding the genetic alphabetwith a third base pair composed of unnatural nucleo-bases. Using hydrophobicity, polarity, shape comple-mentarity, and hydrogen bonding, we have developednovel unnatural base pairs, including several that arereplicable in vitro. More recently, we screened more than3600 unnatural nucleotides and identified a base pairthat, after optimization, is replicated with an efficiencyclose to that of natural DNA synthesis.

Nature developed the natural genetic code, not onlyby optimizing DNA and RNA but also by evolving thepolymerases that synthesize these nucleic acids. Wedeveloped an activity-based selection system (Fig. 1)

to evolve polymerases for any desired function. Usingthis system, we have already evolved polymerases witha variety of novel functions, including the synthesis ofDNA containing one of the unnatural base pairs. We areoptimizing these polymerases and evolving new ones.R E E N G I N E E R I N G A N C I E N T A N T I B I O T I C S

Because of the potential for cross-resistance, a greatneed exists for new antibiotics, especially ones that actvia novel mechanisms. Although medicinal chemistshave successfully reengineered already validated antibi-otic scaffolds that were compromised by resistance, theidentification of novel synthetic (nonnatural) scaffolds


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

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.

has been extraordinarily challenging. Thus, natural prod-ucts that might be candidates for antibiotics, perhapseven products that do not appear to still be activebecause of cross-resistance, warrant a careful examina-tion. Using modern tools of synthesis and chemical biol-ogy, we might be able to determine why the productslost activity and perhaps use this knowledge to reengi-neer them to again be potent, broad-spectrum antibiotics.

Arylomycins are a series of biphenyl-linked macro-cyclic lipopeptide natural products that inhibit the essen-tial bacterial signal peptidase I (SPase) in vitro but havelow potency and a narrow spectrum as antibiotics. Aftersome initial interest, these natural products were aban-doned by the pharmaceutical industry because of theirinsufficient potency, which presumably was due to theresistance that developed during their use in bacterialwarfare over eons of time. We recently reported the firsttotal synthesis of a member of this class of natural prod-ucts: arylomycin A2.

With large quantities of arylomycin A2 in hand, weevaluated it against a wide variety of bacteria and dis-covered that it is extremely potent against the importanthuman pathogen Staphylococcus epidermidis. By usingUV light to create mutants of S epidermidis, we discov-ered that resistance evolved via the introduction of aproline residue into a conserved region of the SPase sub-strate-binding site. Sequence analysis of other bacterialSPases revealed that all bacteria with natural resistanceto arylomycin A2 already had the "resistance-conferring"proline, and we found that when this proline is removedgenetically, important pathogens such as Escherichiacoli and Staphylococcus aureus become sensitive toarylomycin A2. Sequence analysis also indicated addi-tional pathogens predicted to be sensitive, including thegram-positive pathogens Streptococcus pyogenes andStreptococcus pneumoniae and the gram-negative path-ogens Helicobacter pylori and Chlamydia trachomatis.

These data suggest that if the arylomycins can bereengineered to bind SPase regardless of the resistance-conferring proline, they will again be potent, broad-spectrum antibiotics. Currently, we are characterizingthe mechanism of arylomycin resistance to determinehow to reengineer these natural products for potencyand for the design, synthesis, and characterization ofpotentially active derivatives.E V O L U T I O N O F P R O T E I N D Y N A M I C S

Molecular recognition underlies almost all of a pro-tein’s biological functions. Nowhere is the evolution ofmolecular recognition more impressive than within the

immune response; antibodies are evolved within a mat-ter of days to selectively recognize almost any foreignmolecule. Antibodies are also remarkable because dif-ferent intermediates can be isolated during their evo-lution. We use ultrafast nonlinear optical and nuclearmagnetic resonance spectroscopy to characterize theseintermediates. We have generated a comprehensive viewover all timescales, from femtoseconds to seconds, ofhow antibodies are evolved for molecular recognition.

Finally, the products of evolution are molecules withunique vibrational dynamics. The study of vibrationaldynamics in proteins and nucleic acids has been lim-ited by spectral complexity, but selective deuterationof a protein or a nucleic acid results in a carbon-deu-terium oscillator that absorbs light in an otherwisetransparent region of the infrared spectrum. The syn-thesis of selectively deuterated proteins has providedus with a residue-specific probe of flexibility, function,and folding. Previously, we focused on the biologicalredox activities of cytochrome c. More recently, we havefocused on the protein recognition module SH3 and theenzyme dihydrofolate reductase.

PUBLICATIONSCirz, 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 Staphylococcusaureus to the antibiotic ciprofloxacin. J. Bacteriol. 189:531, 2007.

Heideker, J., Lis, E.T., Romesberg, F.E. Phosphatases, DNA damage checkpointsand checkpoint deactivation. Cell Cycle 6:3058, 2007.

Hwang, G.T., Leconte, A.M., Romesberg, F.E. Polymerase recognition and stabilityof 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., Geier-stanger, B.H., Wemmer, D.E., Schultz, P.G., Spraggon, G., Romesberg, F.E.Efforts toward expansion of the genetic alphabet: structure and replication ofunnatural base pairs. J. Am. Chem. Soc. 129:10466, 2007.

Roberts, T.C., Smith, P.A., Cirz, R.T., Romesberg, F.E. Structural and biologicalanalysis of synthetic arylomycin A2. J. Am. Chem. Soc. 129:15830, 2007.

Smith, P.A., Romesberg, F.E. Combating bacteria and drug resistance by inhibitingmechanisms of persistence and adaptation. Nat. Chem. Biol. 3:549, 2007.


Synthesis of Natural Products,Development of SyntheticMethods, and MedicinalChemistryW.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

Our research has 2 major themes. One is the totalsynthesis of structurally complex, biologicallyactive natural products such as those shown

in Figure 1. In each of these syntheses, we emphasizethe discovery, development, and/or illustration of newreactions and methods for achieving high levels of stere-ochemical control. These efforts are pursued in parallelwith reaction design, stereochemical studies, and thedevelopment of new synthetic methods. We are particu-larly interested in stereochemical aspects of intramolecu-lar and transannular Diels-Alder reactions, developmentof methods for the diastereoselective and enantioselec-tive reactions of allylmetal compounds with carbonylcompounds, and nucleophilic phosphine-catalyzedorganic reactions.

Recent research has included stereochemical studiesof transannular Diels-Alder reactions used in total syn-theses of spinosyn A and superstolide A and develop-ment of new versions of the double allylboration reactionsof aldehydes with γ-boryl-substituted allylboranes forstereocontrolled synthesis of 1,5-ene-diols, which arebeing used in several ongoing syntheses, including thoseof tetrafibricin, apoptolidin A, and peloruside. In addition,we have synthesized highly substituted tetrahydrofuransvia [3+2]-annulation reactions of highly functionalizedallylsilanes; this chemistry was recently applied to totalsyntheses of 10-hydroxytrilobacin and 3 stereoisomers.We have also developed phosphine-mediated organocat-alytic reactions, and we recently completed the totalsynthesis of tedanolide.

Our second area of major interest involves problemsin bioorganic chemistry and medicinal chemistry. Onelong-term project is the design and synthesis of inhibi-tors of cysteine proteases isolated from tropical para-sites, such as Trypanosoma cruzi, the causative agentof Chagas’ disease, and Plasmodium falciparum, the

most virulent of the malaria parasites. This research isperformed in collaboration with colleagues at the Uni-versity of California, San Francisco. In collaboration withS. Reed, University of California, San Diego, we havedeveloped a cysteine protease inhibitor with remarkableability to prevent Entamoeba histolytica from invadinghuman intestinal tissue. Optimization of this inhibitor


F i g . 1 . Structures of recently synthesized natural products.

for in vivo applications is in progress. New projectsinvolve discovery of small molecules that affect cancerand other disease-related biochemical targets (e.g.,nuclear hormone receptors), studies of structure-activityrelationships, and optimization of the pharmacologicprofile of certain natural products.

PUBLICATIONSChen, Y.-T., Lira, R., Hansell, E., McKerrow, J.H., Roush, W.R. Synthesis ofmacrocyclic 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 thesynthesis 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 apoptolidinA, 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 protec-tion 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) pyran-containing 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-hydroxyal-lylsilanes via a chiral (Z)-γ-silylallylboronate. Org. Lett. 9:4315, 2007.

Methot, J.L., Roush, W.R. Applications of tricoordinated phosphorus compounds inorganic catalysis. In: Organophosphorus Compounds. Trost, B.M. (Ed.). ThiemeChemistry, New York, in press. Vol. 42 in Science of Synthesis.

Roth, J., Madoux, F., Hodder, P., Roush, W.R. Synthesis of small molecule inhibi-tors of the orphan nuclear receptor steroidogenic factor-1 (NR5A1) based on iso-quinolinone 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 keytransannular Diels-Alder reaction. J. Org. Chem. 73:1818, 2008.

Biological ChemistryP.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

Although chemists are remarkably adept at thesynthesis of molecular structure, they are farless sophisticated in designing and synthesizing

molecules with defined biological or chemical functions.Nature, on the other hand, has produced an array ofmolecules with remarkably complex functions, rangingfrom photosynthesis and signal transduction to molec-

ular recognition and catalysis. Our aim is to combinethe synthetic strategies and biological processes ofNature with the tools and principles of chemistry tocreate new molecules with novel chemical and bio-logical functions. By studying the properties of theresulting molecules, we can gain new insights intothe molecular mechanisms of complex biological andchemical systems.

For example, we have shown that the tremendouscombinatorial diversity of the immune response can bechemically reprogrammed to generate selective enzyme-like catalysts. We have developed antibodies that cat-alyze a wide array of chemical and biological reactions,from acyl transfer to redox reactions. Characterizationof the structure and mechanisms of these catalyticantibodies has led to important new insights into themechanisms of biological catalysis. In addition, thedetailed characterization of the properties and struc-tures of germ-line and affinity-matured antibodies hasrevealed fundamental new aspects of the evolution ofbinding and catalytic function, in particular, the roleof structural plasticity in the immune response. Mostrecently, we have focused on in vitro evolution methodsthat involve the development of novel chemical screensand selections for identifying metalloantibodies withproteolytic activity.

In addition, we are extending this combinatorialapproach to many other problems, including the gen-eration of novel cellular reporters, the ab initio evolu-tion of novel protein domains, and the synthesis ofstructure-based combinatorial libraries of small hetero-cycles. The libraries of small heterocycles are beingused in conjunction with novel cellular and organismalscreens to identify molecules that modulate the activ-ity of important proteins involved in such cellular pro-cesses as differentiation, proliferation, and signaling.Indeed, we have identified molecules that control adultand embryonic stem cell differentiation and stem cellself-renewal and that reprogram lineage-committed cellsto alternative cell fates. We are using x-ray crystallo-graphic and biochemical studies, together with mRNAprofiling technology and genetic complementation, tocharacterize the mode of action of these compounds andto study their effects on cellular processes and in ani-mal models of regeneration. More recently, we extendedsuch studies to a variety of genetic and neglected dis-eases (e.g., malaria, type 1 diabetes, spinal muscularatrophy, sickle cell anemia). We are also developingand applying modern genomics tools (e.g., cell-basedphenotypic screens of arrayed genomic cDNA and short


interfering RNA libraries) and proteomics tools (massspectrometric phosphoprotein profiling) to a variety ofsignificant biomedical problems in cancer biology, neu-rodegenerative disease, and virology. In addition, we areinvestigating the role and regulation of noncoding RNAs.

We have also developed a general biosyntheticmethod that makes it possible to site specifically incor-porate unnatural amino acids into proteins in vitro andin vivo. Using this method, we effectively expanded thegenetic code of living organisms by adding new com-ponents to the existing biosynthetic machinery. Wehave genetically encoded amino acids with novel spec-troscopic and chemical properties (e.g., metal-binding,sulfated, fluorescent, photocross-linking, and photoisom-erizable) in response to unique 3- and 4-base codons.These amino acids are being used to explore proteinstructure and function both in vitro and in vivo, createnovel therapeutic agents and biomaterials, and evolveproteins with novel properties. This approach has beendeveloped for Escherichia coli, yeast, and mammaliancells, and we are now extending it to multicellular organ-isms. Our results have removed a billion-year constraintimposed by the genetic code on the ability to chemicallymanipulate the structures of proteins during translation.

PUBLICATIONSGalkin, 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 effica-cious 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 genomicscreen 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 recognitionof 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 ana-logue of phosphotyrosine in Escherichia coli. ACS Chem. Biol. 2:474, 2007.

Wang, J., Schiller, S., Schultz, P.G. A biosynthetic route to dehydroalanine-con-taining proteins. Angew. Chem. Int. Ed. 46:6849, 2007.

Xie, J., Liu, W., Schultz, P.G. A genetically encoded bidentate, metal-bindingamino 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 replica-tion 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. Con-trol 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 tothe genetic code of bacteria. Angew. Chem. Int. Ed. 47:722, 2008.

Click Chemistry and Biological ActivityK.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

The driving forces in our research are the discov-ery and understanding of chemical reactivity, theharbingers of new discoveries in chemistry. Our

goal is to develop chemical transformations that enablescientists to rapidly synthesize diverse compounds withdesired properties; after all, it is the function of mole-cules that matters. The nature of the building blocksand the speed with which synthesis, screening for thedesired function, and lead optimization can be performedare determining factors in the search for new com-pounds, whether the new entities are drugs, better plas-tics, or dyes. The greater the variety of scaffolds andfunctional groups that can be used in the rapid construc-tion of candidate compounds, the more likely it is thatnew and useful function will be discovered. Becauseof the enormous number of compounds to explore (thenumber of small druglike molecules may be as highas 1064), the size of a given collection becomes muchless important than the ability to rapidly probe the col-lection for a desired activity.

Several years ago, we proposed a minimalisticapproach to synthesis that relies solely on the bestreactions for assembly of new molecules. Inspired bythe natural synthesis of the myriad functional mole-cules (nucleic acids, proteins, and carbohydrates) fromjust a handful of building blocks, we devised a fast,reliable, and highly modular style of organic synthesis,which we termed click chemistry. Click reactions fulfillthe most stringent criteria of usefulness and convenience(Fig. 1); they are highly energetically driven, and the


F i g . 1 . Click chemistry: molecular diversity from a handful of

near-perfect reactions.

majority of them form carbon-heteroatom bonds. Thereactions produce only the expected products and workregardless of which functional groups are present in thestarting materials. Naturally, the number of reactions thatmeet these criteria is limited, but we contend that awide variety of interesting and useful molecules canbe easily made by using click chemistry and that thechances for achieving desirable biological activity withsuch compounds are at least as good as chances withthe traditional target-guided approach.

Recently, we realized that olefins are probably themost attractive starting molecules available to syntheticorganic chemists. Olefins are readily accessible in largequantities and in many varieties, and processes for theirselective oxidation provide convenient access to electro-philic intermediates such as epoxides, aziridines, aziri-dinium ions, and cyclic sulfates. These electrophilicintermediates are ideal for introduction of reactive “hotspots,” such as azides and acetylenes, that can beused for the assembly of final structures via 1,3-dipo-lar 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 wedeveloped with V.V. Fokin, Department of Chemistry,take a prominent place in click reactions. These trans-formations enable reliable assembly of complex mole-cules by means of the 1,2,3-triazole heterocycle.

Although both alkynes and azides are highly ener-getic, they are quite unreactive to a broad range ofreagents, solvents, and other common functional groups.This inertness allows clean sequential transformationsof broad scope without the need for protecting groups,even if the reactions are performed in aqueous solventin the presence of atmospheric oxygen. The 1,2,3-tria-zoles have advantageous properties of high chemicalstability (in general, they are inert to severe hydrolytic,oxidizing, and reducing conditions, even at high tem-peratures), strong dipole moment, presence of aromaticgroups, and the ability to accept hydrogen bonds. Thus,they can interact productively in several ways withbiological molecules. For example, 1,2,3-triazoles canreplace the amide bond in peptides, preventing prote-olytic degradation of the peptides.

Our focus on this powerful and underappreciatedclass of azoles led us back to the simple parent triazole(C2H3N3), which in solution is a rapidly equilibratingmixture of 2 tautomers (Fig. 2). The physical propertiesof the NH-triazole struck us as highly unusual and are, in

fact, much like those of water. These properties includeits weak acid-base character, high proton conductivity,and a liquid range spanning nearly 200 degrees. In addi-tion, the NH-1,2,3-triazole is stable: it is insensitive toimpact, friction, rapid heating, and even detonation.We studied the Michael reaction of NH-triazole withα,β-unsaturated ketones. The 1H-1,2,3-triazolyl-ketoneswere selectively generated when the triazole was com-bined with a variety of enones under solvent-free con-ditions. The use of aprotic solvents with a catalyticbase gave the corresponding 2H-regioisomers. Together,these 2 protocols provide direct access to either the N1-or N2-substituted 1,3-triazolyl ketone regioisomers.

PUBLICATIONSFinn, M.G., Kolb, H.C., Fokin, V.V., Sharpless, K.B. Concept and applications ofclick 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 mate-rials 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-1,2,3-triazoles. Org. Lett. 10:3171, 2008.

Kwok, S.-W., Hein, J.E., Fokin, V.V., Sharpless, K.B. Regioselective synthesis ofeither 1H- or 2H-1,2,3-triazoles via Michael addition to α,β-unsaturated ketones.Heterocycles 76:1141, 2008.


Radic, Z., Manetsch, R., Fournier, D., Sharpless, K.B., Taylor, P. Probing gorgedimensions 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-anhy-droerythromycin 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.


F i g . 2 . Michael additions of NH-triazole.

Vestberg, R., Malkoch, M., Kade, M., Wu, P., Fokin, V.V., Sharpless, K.B., Drock-enmuller, E., Hawker, C.J. Role of architecture and molecular weight in the forma-tion of tailor-made ultrathin multilayers using dendritic macromolecules and clickchemistry. J. Polym. Sci. A Polym. Chem. 45:2835, 2007.

Yoo, E.J., Ahlquist, M., Bae, I., Sharpless, K.B., Fokin, V.V., Chang, S. Mechanisticstudies on the Cu-catalyzed three-component reactions of sulfonyl azides, 1-alkynesand amines, alcohols, or water: dichotomy via a common pathway. J. Org. Chem.73:5520, 2008.

Chemistry, Biology, andInflammatory DiseaseP. 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

Our research is interdisciplinary and involvesaspects of bioorganic, biophysical, physicalorganic, synthetic, and analytical chemistry cou-

pled with biochemical techniques, cell-based assays,and animal models. We are interested in uncoveringnew mechanisms of disease in major conditions suchas atherosclerosis, neurodegenerative diseases, ischemia-reperfusion injury, macular degeneration, cancer, andinfectious diseases.A N T I B O D Y - C A T A L Y Z E D W A T E R O X I D A T I O N P A T H W A Y

Our discovery that all antibody molecules can cat-alyze the reaction between singlet oxygen and water togive hydrogen peroxide is causing a revision of the ideathat antibodies are only an adapter molecule withinthe immune system, linking recognition and killing offoreign pathogens. We are exploring both the chemicaland biological aspects of this pathway, and new insightsinto how the pathway plays a role in immune defenseand inflammatory damage are emerging.

We are searching for the active site for the anti-body-catalyzed water oxidation pathway within the anti-body structure. We have cloned and expressed solubleindividual domains (VHVL, CH1CL, VH, VL, CH1, CL) ofthe murine Fab 4C6. All of the domains can generatehydrogen peroxide when presented with singlet dioxy-gen, suggesting that the driving force is related to theimmunoglobulin fold of the whole antibody.I N F L A M M A T O R Y A L D E H D Y E S A N D P R O T E I N

M I S F O L D I N G

We have shown that the inflammation-derived cho-lesterol seco-sterols atheronal-A and atheronal-B triggera deformation in the secondary structure of the normallyfolded low-density lipoprotein apoB-100 into a proamy-loidogenic form. In collaboration with J.W. Kelly and

his group, Department of Chemistry, we extended thismodel and showed that these cholesterol seco-sterolsalso trigger the misfolding of amyloid β-peptide1-40,leading to formation of fibrils similar to those observedin patients with Alzheimer ’s disease. Using mutatedsynthetic sequences of amyloid β-peptide1-40, we foundthat the accelerated aggregation of this protein onlyoccurs when only lysine 16, not lysine 28 or the N-termi-nal amino group of aspartic acid 1, of the sequenceis modified. More recently, in studies of inflammatoryaldehyde–initiated misfolding of antibody light chains(Bence-Jones proteins), we found that different aldehydescan trigger different forms of aggregation in differentproteins. Thus, we have shown that the cholesterolseco-sterols atheronal-A and atheronal-B accelerate anamorphous form of aggregation, whereas 4-hydroxynone-nal induced an amyloid form of aggregation of both λand κ light chains (Fig. 1).

Epidemiologic and clinical evidence point to anincreased risk of cancer when linked with chronic inflam-mation, in a process thought to involve the establishmentof a local inflammatory microenvironment conducive tothe development of neoplasia. However, because of thecomplex interrelationships between the 2 conditions, theprecise molecular and cellular mechanisms that underpinthis relationship remain largely unresolved.

We found that the inflammation-derived cholesterol5,6-seco-sterol aldehydes atheronal-A and atheronal-Bcause a loss of function of wild-type tumor suppressorprotein p53, the so-called guardian of the genome, ina process that involves p53 misfolding and amyloidogen-


F i g . 1 . Electron micrograph of fibrillar aggregation of antibody

light chains induced by cholesterol seco-sterol and 4-hydroxynone-

nal (shown in white).

esis. Atheronal-A and atheronal-B, but not the aldehy-des 4-hydroxynonenal and 4-hydroxyhexenal derivedfrom polyunsaturated fatty acids, induce misfolding ofwild-type p53 into an amyloidogenic form that bindsthioflavin T and Congo red dye but cannot bind to aconsensus DNA sequence (Fig. 2). Treatment of lung car-

cinoma cells expressing wild-type p53 with atheronal-Aand atheronal-B leads to dysfunctional p53, as deter-mined by analysis of extracted nuclear protein and tran-scription activation of p21.

Our results reveal a hitherto unknown chemical linkbetween inflammation and cancer and expand the alreadypivotal role of p53 dysfunction in the risk for cancer. Theincreasing generality and specificity of aldehyde-initiatedprotein misfolding suggests that inflammatory aldehydesand their posttranslational modification of amyloido-genic peptides may be the chemical link between theknown associations of inflammation, oxidative damage,and various misfolding diseases.I N T E R A C T I O N B E T W E E N P R O T O Z O A N J - B I N D I N G

P R O T E I N 1 A N D G L Y C O S Y L A T E D D N A

Current treatments of parasitic infections such asleishmaniasis (cutaneous or visceral, Leishmania

species), African trypanosomiasis (sleeping sickness,Trypanosoma brucei), and American trypanosomiasis(Chagas’ disease, Trypanosoma cruzi) have limitedeffectiveness, thereby increasing drug resistance andinherent toxic effects of the drugs. Thus, an elucida-tion of new parasite-specific biological targets for ther-apeutic agents is needed. In this regard, the discoverythat DNA from members of the order Kinetoplastida,but not other eukaryotes, contains an unusual modi-fied base, β-D-glucosyl(hydroxymethyl)uracil, calledbase J, was a breakthrough. Extracts of several kineto-plastids contain a J-binding protein (JBP) that spe-cifically binds to J-containing duplex DNA. JBP-1 isessential in Leishmania.

As a drug target, JBP has merit. The protein shareslittle homology with other proteins in the Protein DataBank, and it has a unique ligand, J-DNA containingtelomeric stretches of double-stranded DNA, that doesnot occur in other eukaryotes. However, a preliminaryhigh-throughput screen, focused on disrupting bindingbetween JBP-1 and J-DNA, with a library of compoundsconsisting of all the major drug pharmacophoric groupshas revealed no compounds of interest.

In parallel, we have studied the molecular recogni-tion that underlies JBP-1 recognition of glycosylatedDNA. In collaboration with D.P. Millar and D.A. Case,Department of Molecular Biology, we found that JBP-1interacts with the J-containing DNA only when a criti-cal conformation of the glucose within the major grooveis established. More recently, we discovered that lowmicromolar concentrations of the DNA intercalatorsdaunorubicin and mitoxantrone disrupt the binding ofJBP-1 with duplex DNA containing J-DNA. Modelingsuggests that DNA binding of the intercalators leadsto distortion, which leads to disruption of the edge-onconformation of the glucose within the major grooveof the DNA.

PUBLICATIONSGrover, 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 antibodylight chain amyloid and amorphous aggregation. Biochemistry 47:7695, 2008.

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 producesnon-self antigens for a broadly neutralising, HIV-1 specific antibody. J. Mol. Biol.372:16, 2007.

Scheinost, J.C., Boldt, G.E., Wentworth, P., Jr. Protein misfolding diseases. In:Encyclopedia of Chemical Biology, Wiley Blackwell, New York, in press.


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.

Scheinost, J.C., Wang, H., Boldt, G.E., Offer, J., Wentworth, P., Jr. Cholesterol seco-sterol-induced aggregation of methylated amyloid-β peptides, insights into aldehyde-initiated 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. Interactionof 2-N,N-dimethylamino-1,3,4-thiadiazole-5-methylsulfonamide with 12 mamma-lian isoforms: kinetic and x-ray crystallographic studies. Bioorg. Med. Chem. Lett.18:999, 2008.

Wentworth, P., Jr., Witter, D. Antibody-catalyzed water-oxidation pathway. PureAppl. Chem. 80:1849, 2008.

Bioorganic and SyntheticChemistryC.-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

We develop new chemical and enzymatic strate-gies for synthesis of bioactive small mole-cules and biomolecules. We use the methods

to probe carbohydrate-mediated recognition events impor-tant in cancer, bacterial infections, and viral infections,including HIV disease and influenza.S Y N T H E T I C M E T H O D S

We have developed new methods for sugar-assistedligation of glycopeptides for synthesis of homogenousglycoproteins. We have used the methods in conjunc-tion with enzymatic glycosylation techniques to assemblecomplex glycopeptides by chemical synthesis, and weare optimizing the techniques to achieve the total syn-thesis of therapeutic glycoproteins. Glycoproteins areexpressed in vivo as complex mixtures of glycoforms,a situation that hinders efforts to study the role of gly-cosylation in protein folding, stability, and function. Bysynthesizing pure glycoforms, we can characterize inmolecular detail the effects of glycans on protein function.

Using chemical techniques such as programmable1-pot oligosaccharide synthesis, as well as enzymaticsynthesis, we create glycoarrays on glass slides for high-throughput quantitative analysis of protein-carbohydrateinteractions. These arrays are being used to study thebinding specificity of carbohydrate-binding receptors andantibodies. We have applied aldolases, glycosyltrans-ferases, glycosidases, and other enzymes to developpractical new methods of synthesizing molecules suchas iminocyclitols, which are inhibitors of glycosidasesand other enzymes, and glycopeptides, and other glyco-conjugates. Using directed evolution, we are evolving

these enzymes to catalyze new reactions and synthesizenew molecules of pharmaceutical relevance.

C A R B O H Y D R A T E - M E D I A T E D R E C O G N I T I O N I N

D I S E A S E

We are using our synthetic methods to discoverinhibitors and therapeutic agents in several diseasesrelated to carbohydrates. Current targets include bac-terial transglycosidase, sulfatases, and glycoprocessingenzymes involved in the biosynthesis of carbohydratesthat mediate cancer metastasis, inflammation, and viralinfection. Enzymatically synthesized iminocyclitols arebeing investigated as treatments for osteoarthritis andGaucher disease. Inspired by the broadly neutralizinganti-HIV antibody 2G12, which recognizes a densearray of oligomannose displayed on HIV gp120, weare designing dendrimeric oligomannose structuresfor development of an HIV vaccine. In collaboration withD.R. Burton, Department of Immunology, we are test-ing the immunogenicity of these constructs. We havedesigned glycolipid ligands for CD1, which activatenatural killer T cells and are a promising new immuno-therapeutic approach for treatment of bacterial and viralinfections and cancer. They may also be useful asadjuvants in vaccine development.

G L Y 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 L Y C O B I O L O G Y

Using metabolic oligosaccharide engineering, wehave developed methods for incorporating tagged sug-ars into glycans expressed on mammalian cells. Theengineered glycans can be labeled with a variety ofmolecules by using click chemistry. One application isglycan-specific fluorescent labeling, which is used forfluorescent imaging to compare glycosylation patternsof different cells, such as normal vs cancer cells orcancer cells vs cancer stem cells. We found that pro-tein fucosylation and sialylation are both elevated incancer 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 bio-chemical incorporation of the sugars into cellular gly-coproteins, click chemistry is used to attach a handlefor purification of tagged proteins. Mass spectrometricproteomic methods are then used to identify proteinsthat are differentially glycosylated. We are using GIDmapto identify proteins that are aberrantly glycosylated indifferent stages of cancer. These cancer-associated gly-coproteins may be useful as biomarkers for diagnosticsor as targets for therapeutic intervention.


PUBLICATIONSBennett, C.S., Dean, S.M., Payne, R.J., Ficht, S., Brik, A., Wong, C.-H. Sugar-assisted 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 peptideand 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-triazoleazide-alkyne click compound is a potent inhibitor of a multidrug-resistant HIV-1protease variant. J. Med. Chem. 51:6263, 2008.

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 toactivate 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 interac-tion 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: biologicaland 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-basedenzyme 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 gly-copeptide 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.

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 oligoman-nose 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 gly-cosyltransferase inhibitors. In: Iminosugars: From Synthesis to Therapeutic Applica-tions. 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, andOrganometallic and Synthetic MethodsJ.-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 V A T I O N

C A T A L Y Z E D B Y P A L L A D I U M ( I I ) – A M I N O A C I D

C O M P L E X E S

Although cleavage of inert carbon-hydrogen bondsby transition metals has been extensively studied,exploitation of this reactivity for regioselective

and enantioselective catalytic reactions of syntheticallyuseful chemical substances is still at its infant stage.The 2 major challenges are the development of practi-cal catalysis and the modulation of regioselectivity andstereoselectivity by external ligands. We recently madea number of discoveries that offer promising solutionsto these problems (Fig. 1).

We discovered the first palladium(II)/palladium(0)catalytic system to couple both sp2 and sp3 carbon-hydro-gen bonds with organotin and organoboron reagents. Wealso established conditions to use air as the stoichiomet-ric oxidant. We further discovered that mono-N-protectedamino acids are suitable ligands for enantioselectivecarbon-hydrogen activation reactions. We think thatthe α-chirality of amino acids is relayed to the mono-protected nitrogen center that coordinates with the


F i g . 1 . GIDmap glycoproteomic analysis via metabolic oligosac-

charide engineering.

F i g . 1 . Enantioselective carbon-hydrogen activation/carbon-car-

bon coupling reactions.

metal center and controls stereoselectivity at the car-bon-hydrogen activation step. We are extending thisenzymelike chiral recognition to sp3 carbon centersattached to 2 prochiral carbon-hydrogen bonds (CH2groups) and are expanding the functional groups tobroadly useful carboxyl and amine groups.R E A G E N T - C O N T R O L L E D M O N 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 V A T I O N : C O N S T R U C T I O N

O F D E M A N D I N G 1 , 2 , 3 - S U B S T I T U T E D A R E N E S

To improve the practicality of carbon-hydrogenactivation reactions, we have focused on inventing newapproaches to activate carbon-hydrogen bonds inabundant substrates containing broadly useful func-tional groups such carboxyl, amino, and hydroxyl groups.We recently discovered that table salt promotes carbon-hydrogen activation in arene and aliphatic carboxylicacids. This reactivity led to the development of arylationand halogenation of inert carbon-hydrogen bonds in car-boxylic acids (Fig. 2). The replacement of table salt with

bulkier tetraalkylammonium chloride salts markedlyimproved the monoselectivity of ortho-carbon-hydrogenfunctionalization. These reactions offer a solution to thewell-known challenge in accessing 1,2,3-substitutedarenes in medicinal chemistry and synthesis.F U N C T I O N A L I Z A T I O N O F B I O L O G I C A L L Y A C T I V E

N A T 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 V A T I O N

Using carbon-hydrogen activation/carbon-carboncoupling reactions, we hope to rapidly access diversi-fied structures that are analogous to biologically activecompounds yet difficult to synthesize by using conven-tional methods. Dehydroabietic acid is a natural prod-uct identified as an efficient opener of BK ion channels.Compounds with such activity could lead to usefultreatments for diseases such as acute stroke, epilepsy,and asthma. Typically, diversification of such structuresis difficult because of the lack of reactive sites onthese molecules other than the carboxylic acid moiety,which is essential for biological activity of the mole-cule. Masking the carboxylic acid as the hydroxamicacid allows for functionalization at the methyl carbon-

hydrogen bond, yielding a novel class of analogs in gramquantities for biological studies (Fig. 3).

V E R S A T 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

A R Y L E T H Y L A M I N E S V I A C A R B O N - H Y D R O G E N

A C T I V A T I O N

Heterocycle synthesis is a core technology in med-icinal chemistry. Using various amino groups to directcarbon-hydrogen activation, we are developing novelsynthetic disconnections through amination of carbon-hydrogen bonds. These reactions are either comple-mentary to current methods or allow rapid access tounique heterocyclic structures from readily availablechemicals (Fig. 4).

PUBLICATIONSGiri, R., Maugel, N.L., Foxman, B.M., Yu, J.Q. Dehydrogenation of inert alkylgroups via remote C-H activation: converting a propyl group into a π-allylic com-plex. 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 insimple carboxylic acids. J. Am. Chem. Soc. 129:3510, 2007.

Giri, R., Yu, J.Q. Iodine monoacetate as a reagent. In: Encyclopedia of Reagentsfor Organic Synthesis, 2nd ed. Paquette, L.A., et al. (Eds.). Wiley Blackwell, Hobo-ken, 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. Issue2:204, 2008.

Li, J.J., Giri, R., Yu, J.Q. Remote C-H bond functionalization reveals the distance-dependent isotope effect. Tetrahedron 64:6979, 2008.

Mei, T.S., Giri, R., Maugel, N., Yu, J.Q. PdII-catalyzed monoselective ortho halo-genation of C-H bonds assisted by counter cations: a complimentary method todirected ortho lithiation. Angew. Chem. Int. Ed. 47:5215, 2008.

Shi, B.F., Maugel, N., Zhang, Y.H., Yu, J.Q. PdII-catalyzed enantioselective activa-tion of C(sp2)-H and C(sp3)-H bonds using monoprotected amino acids as chiralligands. Angew. Chem. Int. Ed. 47:4882, 2008.

Wang, D.H., Wasa, M., Giri, R., Yu, J.Q. Pd(II)-catalyzed cross-coupling of sp3 C-H bonds with sp2 and sp3 boronic acids using air as the oxidant. J. Am. Chem.Soc. 130:7190, 2008.


F i g . 2 . Reagent-controlled selective halogenation of ortho-car-

bon-hydrogen bonds.

F i g . 3 . Carbon-hydrogen functionalization of natural products.

F i g . 4 . Heterocycle synthesis via carbon-hydrogen activation.

chemistry - scripps research institutechemistry jeffery w. kelly, ph.d.* lita annenberg hazen...

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