Chemistry
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
M. Reza Ghadiri, Ph.D., Professor, Department of Chemistry
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
CHEMISTRY 2005
DEPAR TMENT OF
CHEMISTRY
Hartmuth Kolb, Ph.D.**
University of California
Los Angeles, California
Chi-Huey Wong, Ph.D.*
Ernest W. Hahn Professor
and Chair in Chemistry
Jung-Mo Ahn, Ph.D.**
University of Texas
Dallas, Texas
Ramanarayanan
Krishnamurthy, Ph.D.
Associate Professor
Andrew Bin Zhou, Ph.D.
Assistant Professor
Dariush Ajami, Ph.D.
Richard A. Lerner, M.D.****
President, Scripps Research
Lita Annenberg Hazen
Professor of
Immunochemistry
Cecil H. and Ida M. Green
Chair in Chemistry
S TA F F S C I E N T I S T S
63
S TA F F
K.C. Nicolaou, Ph.D.*
Chairman
Aline W. and L.S. Skaggs
Professor of Chemical Biology
Darlene Shiley Chair in
Chemistry
Phil Baran, Ph.D.
Assistant Professor
Dale L. Boger, Ph.D.*
Richard and Alice Cramer
Professor of Chemistry
Bruce Clapham, Ph.D.**
Abbott Laboratories
Abbott Park, Illinois
Tobin Dickerson, Ph.D.
Assistant Professor
Albert Eschenmoser, Ph.D.*
Professor
Sheng Ding, Ph.D.
Assistant Professor
M.G. Finn, Ph.D.*
Associate Professor
Valery Fokin, Ph.D.
Assistant Professor
Lital Alfonta, Ph.D.
Byeong D. Song, Ph.D.
Toru Amaya, Ph.D.**
Osaka University
Osaka, Japan
Lubica Supekova, Ph.D.
Rajesh Ambasudhan, Ph.D.
I N S T R U M E N TAT I O N /
Masayuki Matsushita, Ph.D.
Assistant Professor
Evan Powers, Ph.D.
Assistant Professor
Julius Rebek, Jr., Ph.D.*
Professor
Director, The Skaggs Institute
for Chemical Biology
Ed Roberts, Ph.D.
Professor
SERVICE FACILITIES
Stellios Arseniyadis, Ph.D.
Raj K. Chadha, Ph.D.
Director, X-Ray Crystallography
Facility
Gonen Ashkenasy, Ph.D.
Dee H. Huang, Ph.D.
Director, Nuclear Magnetic
Resonance Facility
Masato Atsumi, Ph.D.
Gary E. Siuzdak, Ph.D.
Director, Mass Spectrometry
Facility
Christoph Behrens, Ph.D.
Floyd E. Romesberg, Ph.D.
Assistant Professor
SENIOR RESEARCH
Peter G. Schultz, Ph.D.*
Professor
Scripps Family Chair
M. Reza Ghadiri, Ph.D.*
Professor
K. Barry Sharpless, Ph.D.*
W.M. Keck Professor of
Chemistry
Inkyu Hwang, Ph.D.
Assistant Professor
Anita Wentworth, Ph.D.
Assistant Professor
Kim D. Janda, Ph.D.***
Professor
Ely R. Callaway, Jr., Chair in
Chemistry
Paul Wentworth, Jr., Ph.D.
Professor
Narendra B. Ambhaikar,
Ph.D.
A S S O C I AT E S
Ashraf Brik, Ph.D.
Yanping Chen, Ph.D.
Tobin Dickerson, Ph.D.
Michael Meijler, Ph.D.
Nurit Ashkenasy, Ph.D.
Elizabeth Barrett, Ph.D.
Clay Bennett, Ph.D.
Michael Best, Ph.D.**
University of Tennessee
Knoxville, Tennessee
Jan Bieschke, Ph.D.
Babu Boga, Ph.D.
Anthony Boitano, Ph.D.
Brant Boren, Ph.D.
Daryl Bosco, Ph.D.
Jeffery W. Kelly, Ph.D.*
Vice President, Academic
Affairs
Dean, Kellogg School of
Science and Technology
Lita Annenberg Hazen
Professor of Chemistry
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Peter Wirsching, Ph.D.*****
R E S E A R C H A S S O C I AT E S
Ramzey Abujarour, Ph.D.
Mohua Bose, Ph.D.**
Stanford University
Stanford, California
S E C T I O N C O V E R F O R T H E D E P A R T M E N T O F C H E M I S T R Y : A single rotaxane struc-
ture created by threading and capturing a DNA-poly(ethylene glycol) strand inside an α-hemolysin
transmembrane pore protein. This supramolecular system constitutes the basis of a research program
to design a rapid pore-mediated single-molecule DNA-sequencing technology. Work done in the laboratory of M. Reza Ghadiri, Ph.D.
64 CHEMISTRY 2005
Patrick Braun, Ph.D.**
University of Minnesota
Minneapolis, Minnesota
Antonella Converso, Ph.D.
Rebecca Fraser, Ph.D.
Zhangyong Hong, Ph.D.
Jeromy Cottell, Ph.D.
Graeme Freestone, Ph.D.
Richard Hookey, Ph.D.
Andy Brogan, Ph.D.
James Crawford, Ph.D.
Yanwen Fu, Ph.D.
Daniel Horne, Ph.D.
Adrian Brunkhorst, Ph.D.
Matthew Cremeens, Ph.D.
Jim Fuchs, Ph.D.
Paul Bulger, Ph.D.
Ashton Cropp, Ph.D.**
University of Maryland
College Park, Maryland
Carmen Galan, Ph.D.**
Massachusetts Institute of
Technology
Cambridge, Massachusetts
Che-Chang (Jeff) Hsu, Ph.D.**
EMD Biosciences, Inc.
San Diego, California
Kevin Bunker, Ph.D.
Mark Bushey, Ph.D.
Sara Butterfield, Ph.D.
Edelmira Cabezas, Ph.D.**
Intel Corp.
Santa Clara, California
Francesco De Riccardis,
Ph.D.**
Università di Salerno
Baronissi (SA), Italy
Jianmin Gao, Ph.D.
Tsui-Ling Hsu, Ph.D.
Qihong Huang, Ph.D.**
Wistar Institute
Philadelphia, Pennsylvania
Mu-yun Gao, Ph.D.
Zheng-Zheng Huang, Ph.D.
Konstantinos Dellios, Ph.D.**
Chemistry Laboratory of the
Government
Larisa, Greece
Nathan Gianneschi, Ph.D.
Amy Hurshman, Ph.D.
David Diaz-Diaz, Ph.D.
Arnaud Gissot, Ph.D.**
Université Victor Segalen
Bordeaux II
Bordeaux, France
Christine Dierks, Ph.D.
Rajesh Grover, Ph.D.
Hayato Ishikawa, Ph.D.
Ross Denton, Ph.D.
Shen Gu, Ph.D.*****
Tetsuo Iwasawa, Ph.D.
Romyr Dominque, Ph.D.**
Hoffmann-La Roche, Inc.
Nutley, New Jersey
Sayam Sen Gupta, Ph.D.
Michael Jahnz, Ph.D.
Clemens Haas, Ph.D.
Wei Jin, Ph.D.
Song Byeong Doo, Ph.D.
Young Wan Ham, Ph.D.**
Molecular Therapeutics, Inc.
Ann Arbor, Michigan
Florian Kaiser*****
Akiyuki Hamasaki, Ph.D.
Gyungyoun Kim, Ph.D.
David Edmonds, Ph.D.
Wooseok Han, Ph.D.
Greg Elliott, Ph.D.
Nile Emre, Ph.D.
Christophe Hardouin, Ph.D.**
Oril Industry, SA
Bolbec, France
Sang Jick Kim, Ph.D.**
Korean Research Institute of
Bioscience and Biotechnology
Taejon, Korea
Simon Eppacher, Ph.D.
Frank Hauke, Ph.D.
Youhoon Chong, Ph.D.**
Konkuk University
Seoul, Korea
Lisa Eubanks, Ph.D.
Mark Hixon, Ph.D.
Raffaella Faraoni, Ph.D.
Benoit Colasson, Ph.D.**
University of Pavia
Pavia, Italy
Laura Flatauer, Ph.D.
Rebecca Holmberg, Ph.D.**
Ionian Technologies, Inc.
Upland, California
Alexandre Carella, Ph.D.
Giacomo Carenzi, Ph.D.**
Nerviano Medical Sciences
Nerviana, Italy
Michael Cassidy, Ph.D.
Aileen Chang, Ph.D.**
Kresge Library
Scripps Research
Shuo Chen, Ph.D.
Heng Cheng, Ph.D.**
FibroGen, Inc.
South San Francisco, California
Jodie Chin, Ph.D.
Charles Cho, Ph.D.
Younggi Choi, Ph.D.**
Boehringer Ingelheim
Pharmaceuticals, Inc.
Ridgefield, Connecticut
Kevin Cole, Ph.D.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Wu Du, Ph.D.**
Merck Research Laboratories
Rahway, New Jersey
Der-Ren Hwang, , Ph.D.
Giltae Hwang, Ph.D.
Michael Kelso, Ph.D.
Yoonkyung Kim, Ph.D.*****
James Fletcher, Ph.D.**
Creighton University
Omaha, Nebraska
Sukwon Hong, Ph.D.
Wang Hong, Ph.D.
Theocharis Koftis**
Pharmathen Pharmaceuticals
Thessaloniki, Greece
Ravinder Reddy Kondreddi,
Ph.D.
Antoni Krasinski, Ph.D.**
ChemoCentryx, Inc.
Mountain View, California
CHEMISTRY 2005
65
Andreas Krebs, Ph.D.**
BASF
Ludwigshafen, Germany
Tao Tao Ling**
Nereus Pharmaceuticals, Inc.
San Diego, California
Robert Milburn, Ph.D.
Tülay Polat, Ph.D.
Kyung-Hoon Min, Ph.D.
Sreenivas Punna, Ph.D.
Grover Rajesh Kumar, Ph.D.
Jun Liu, Ph.D.
Christos A. Mitsos, Ph.D.
Jane Kuzelka, Ph.D.
Junjie Liu, Ph.D.**
AMSI
San Diego, California
Gopi Kumar Mittapalli, Ph.D.
Longwu Qi, Ph.D.**
Prolexys Pharmaceuticals, Inc.
Salt Lake City, Utah
Lionel Moisan, Ph.D.
Daniela Radu, Ph.D.
Lei Liu, Ph.D.
Ann Montero, Ph.D.
Nicole Rahe, Ph.D.
Wenshe Liu, Ph.D.
Shai Rahimipour, Ph.D.
Surakattula Murali Mohan
Reddy, Ph.D.
Carolina Martinez Lamenca,
Ph.D.
Eltepu Laxman, Ph.D.**
MediVas, LLC
San Diego, California
Ying (Cindy) Liu, Ph.D.
Yasutaka Morita, Ph.D.**
Kinki University
Fukuoka, Japan
Byong Se Lee, Ph.D.**
Asian Medical Center
Seoul, Korea
Dimitrios Lizos, Ph.D.
Mridul Mukherji, Ph.D.
Hing Ken Lee, Ph.D.*****
Jon Loren, Ph.D.**
Ligand Pharmaceuticals, Inc.
San Diego, California
Oscar Munoz, Ph.D.**
Universidad Veracruzana
Veracruz, México
Jinq-Chyi Lee, Ph.D.
Jongkook Lee, Ph.D.
Hendrick Luesch, Ph.D.**
University of Florida
Gainesville, Florida
Ki-Bum Lee, Ph.D.
Hongzheng (Eric) Ma, Ph.D.
Lac Lee, Ph.D**
Novartis Institutes for
Biomedical Research Inc.
Cambridge, Massachusetts
Sang Hyeup Lee, Ph.D.**
Korean Research Institute of
Bioscience and Biotechnology
Taejon, Korea
Sunil Mandal, Ph.D.**
Acenta Discovery, Inc.
Tucson, Arizona
Nello Mainolfi, Ph.D.
Roman Manetsch, Ph.D.
Enrique Mann, Ph.D.
Sanghyup Lee, Ph.D.
Andrew Myles, Ph.D.
Joonwoo Nam, Ph.D.
Sridhar Narayan, Ph.D.
Daniel Nicoletti, Ph.D.
Alain Noncovich, Ph.D.
Yasuo Norikane, Ph.D.
Mehdi Numa, Ph.D.
Barun Okram, Ph.D.
Dalit Rechavi-Robinson, Ph.D.
Yosup Rew, Ph.D.**
Amgen, Inc.
Thousand Oaks, California
Sebastien Richeter, Ph.D.**
Université Montpellier II
Montpellier, France
Stefanie Roeper, Ph.D.
F. Anthony Romero, Ph.D.
Youngha Ryu, Ph.D.
Riccardo Salvio, Ph.D.
Pradip Sasmal**
Dr. Reddy’s Laboratories, Ltd.
Hyderabad, India
Felix Marr, Ph.D.
Chunmei Li, Ph.D. **
Stephen F. Austin State
University
Nacogdoches, Texas
Ke Li, Ph.D.
Yongkai Li, Ph.D.**
Ligand Pharmaceuticals, Inc.
San Diego, California
Antonietta M. Lillo, Ph.D.**
Los Alamos National
Laboratory
Los Alamos, New Mexico
Yeon-Hee Lim, Ph.D.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Hideki Onagi, Ph.D.*****
Yazmin Osornio, Ph.D.
Alessandro Scarso, Ph.D.**
Università Cà Foscari di Venezia
Venice, Italy
Charles Papageorgiou, Ph.D.
Patrick Schanen, Ph.D.
Laxman Pasunoori, Ph.D.
Stefan Schiller, Ph.D.
Andrew McPherson, Ph.D.**
TargeGen, Inc.
San Diego, California
Goran Petrovic, Ph.D.
Edoardo Menozzi, Ph.D.
Steven Pfeiffer, Ph.D.**
Gilead
Foster City, California
Daniel Schlawe, Ph.D.**
Syncom BV
Groningen, the Netherlands
Shigeo Matsuda, Ph.D.
Laura McAllister, Ph.D.
Kathleen McKenzie, Ph.D.
Sergio Meth, Ph.D.**
Federal University of Rio de
Janeiro
Rio de Janeiro, Brazil
Jared Piper, Ph.D.
Suresh Pitram, Ph.D.
Michael Schramm, Ph.D.
Akira Shigenaga, Ph.D.**
Tokushima University
Tokushima, Japan
66 CHEMISTRY 2005
Dongwoo Shin, Ph.D.**
Samsung
Seoul, Korea
Gregory Watt, Ph.D.**
Nature Chemical Biology
Cambridge, Massachusetts
Junhwa Shin, Ph.D.
Lisa Whalen, Ph.D.
Sebastian Steiniger, Ph.D.
Matthew Whiting, Ph.D.
Xiuwen Zhu, Ph.D.
Joerg Zimmermann, Ph.D.
Makoto Yamashita, Ph.D.
Takeda Chemical Industries,
Ltd.
Osaka, Japan
V I S I T I N G I N V E S T I G AT O R S
Shula Stokols, Ph.D.
Aarron Willingham, Ph.D.
Ji Young Suk, Ph.D.
R. Luke Wiseman, Ph.D.
Daniel Summerer, Ph.D.
Xiowen Sun**
Lanzhou University
Lanzhou, China
Leo Takaoka, Ph.D.
Margarita Wuchrer, Ph.D.
Hui Xiong, Ph.D.**
University of Pennsylvania
Philadelphia, Pennsylvania
Trond Vidar Hansen, Ph.D.**
University of Oslo
Oslo, Norway
Wen Xiong, Ph.D.
Bumpei Hatano, Ph.D.**
Yamagata University
Yonezawa, Japan
Chung-Yi Wu, Ph.D.
Masamichi Yamanaka, Ph.D.**
Shizuoka University
Shizuoka, Japan
Eric Tippmann, Ph.D.
Ryu Yamasaki, Ph.D.
Laurent Trembleau, Ph.D.**
University of Aberdeen
Aberdeen, Scotland
Shuyuan Yao, Ph.D.
Meng-Lin Tsao, Ph.D.
Yong Sik Yoo, Ph.D.
Craig Turner, Ph.D.
Ninghui Yu, Ph.D.**
Serono, Inc.
Rockland, Massachusetts
Elke Ullrich, Ph.D.**
Syncom BV
Groningen, the Netherlands
Jan Van Maarseveen,**
Universiteit van Amsterdam
Amsterdam, the Netherlands
Juraj Velcicky, Ph.D.
David Vodak, Ph.D.**
Bend Research, Inc.
Bend, Oregon
Robert M. Yeh, Ph.D.
Tomoyasu Hirose, Ph.D.**
The Kitasato Institute
Tokyo, Japan
Jiayu Liao, Ph.D.
Genomics Institute of the
Novartis Research Foundation
San Diego, California
Jie Li, Ph,D.*****
Dawei Yue, Ph.D.
Huaqiang Zeng, Ph.D.
Qing-Hai Zhang, Ph.D.**
Department of Molecular
Biology
Scripps Research
Masayuki Oda, Ph.D.
Kyoto Prefectural University
Kyoto, Japan
Masaaki Sawa, Ph.D.
Dainippon Pharmaceuticals
Co., Ltd.
Osaka, Japan
Alexandro Volonterio, Ph.D.
Yuanxiang Zhao, Ph.D.
Masakazu Sugiyama, Ph.D.
Ajinomoto Co., Inc.
Kawasaki-shi, Japan
Jiangyun Wang, Ph.D.
LianXing Zheng, Ph.D.**
Models of Disease Center
Cambridge, Massachusetts
Shin-Ichi Takanashi, Ph.D.
Mitsubishi Pharma Corporation
Osaka, Japan
Xiaolong Wang, Ph.D.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Qisheng Zhang, Ph.D.
Suresh Mahajan, Ph.D.
* Joint appointment in The Skaggs
Institute for Chemical Biology
** Appointment completed; new
location shown
*** Joint appointments in The Skaggs
Institute for Chemical Biology
and the Department of
Immunology
**** Joint appointments in The Skaggs
Institute for Chemical Biology
and the Department of Molecular
Biology
***** Appointment completed
Jiyong Hong, Ph.D.
Genomics Institute of the
Novartis Research Foundation
San Diego, California
Zhanqian Yu, Ph.D.
Felix Zelder, Ph.D.
Jon Ashley
Maria-Teresa Dendle
Masakazu Fujio, Ph.D.
Mitsubishi Pharma Corporation
Yokohama, Japan
Wenjun Tang**
Boehringer Ingelheim
Pharmaceuticals, Inc.
Ridgefield, Connecticut
Jim Turner, Ph.D.
S C I E N T I F I C A S S O C I AT E S
Luda Bazhenova, M.D.
Moores Cancer Center
La Jolla, California
CHEMISTRY 2005 67
K.C. Nicolaou, Ph.D.
Chairman’s Overview
s the “central science,” chemistry stands between
biology and medicine and between physics and
materials science and provides the crucial bridge
for drug discovery and development. But chemistry has
a much more profound and useful role in science and
society. It is the discipline that continually creates the
myriad of new materials that we all encounter in our
everyday lives: pharmaceuticals, high-tech materials,
polymers and plastics, insecticides and pesticides, fabrics and cosmetics, fertilizers, and vitamins—basically
everything we can touch, feel, and smell.
Chemists at Scripps Research focus on chemical
synthesis and chemical biology, the most relevant areas
to biomedical research and materials science. The members of our faculty are distinguished teacher-scholars
who maintain highly visible and independent research
programs in areas as diverse as biological and chemical
catalysis, synthesis of natural products, combinatorial
chemistry, molecular design, supramolecular chemistry,
chemical evolution, materials science, and chemical
biology. The chemistry graduate program attracts some
of the best-qualified candidates from the United States
and abroad. Our major research facilities, under the
direction of Dee H. Huang (nuclear magnetic resonance),
Gary Siuzdak (mass spectrometry), and Raj Chadha
A
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
(x-ray crystallography), are second to none and continue to provide crucial support to our research programs. In addition, the Mabel and Arnold Beckman
Center for the Chemical Sciences constantly receives
high praise from visitors from around the world for its
architectural design and operational aspects, both
highly conducive to research.
Research in the Department of Chemistry goes on
unabated, establishing international visibility and attracting attention as evidenced by numerous lecture invitations, visits by outside scholars, and headline news in
the media. As of 2004, the Institute for Scientific Information ranked 4 members of the department as highly
cited researchers (in the top 100 worldwide); 2 of the
4 are among the top 35.
Dr. Lerner and his group continue to make advances
in catalytic antibodies, with new antibodies that catalyze important synthetic and biological reactions and
novel applications in chemical synthesis. The research
of the group recently was expanded to include the fundamental chemistry of polyoxygen species. Members of
the Sharpless group continue endeavors to discover and
develop better catalysts for organic synthesis and to construct, through innovative chemistry and biology, libraries
of novel compounds for biological screening.
Scientists in the La Jolla–based Eschenmoser group
advance in experimental studies on the chemical etiology
of nucleic acid structure by investigating nucleic acid
alternatives that have novel backbone structures unrelated
to the canonical phosphodiester-based oligonucleotide
systems. Members of my group continue explorations of
chemical synthesis and chemical biology, focusing on the
total synthesis of new anticancer agents, antibiotics,
marine-derived neurotoxins, antimalarial compounds,
antifeedant agents, other biologically active natural products, solid-phase synthesis, and combinatorial chemistry.
Members of the Rebek group devise biomimetic
receptors for studies in molecular recognition. These
include molecules that bind neurotransmitters and membrane components. Larger host receptors can surround
3 or more molecular guests and act as chambers where
the chemical reactions of the guests are accelerated.
Scientists in the Schultz laboratory continue to expand
the genetic code. Using unique triplet and quadruplet
codons, they have genetically encoded more than 30
novel amino acids in bacteria, yeasts, and mammalian
cells. Dr. Wong and his group further advance the fields
of chemoenzymatic organic synthesis, chemical glycobiology, and the development of enzyme inhibitors. A new
68 CHEMISTRY 2005
strategy for the synthesis of glycoproteins has been developed. The programmable 1-pot synthesis of oligosaccharides developed by this group has been further used
in the assembly of glycoarrays in microtiter plates for
study of saccharides and aminoglycosides that bind to
proteins and RNA, respectively. This group also developed new inhibitors of glycosyltransferases, sulfotransferases, and the HIV protease.
Members of the Boger group continue their work on
chemical synthesis; combinatorial chemistry; heterocycle
synthesis; anticancer agents, such as fostriecin and yatakemycin; and antibiotics, such as vancomycin, teicoplanin,
and ramoplanin. Scientists in the Janda laboratory focus
on the impact of organic chemistry in specific biological
systems. Their targeted programs span a wide range of
interests, from drug addiction to biological and chemical
warfare agents to catalytic antibodies to combinatorial
chemistry. Their recent achievements include the discovery that a secondary nicotine metabolite can inhibit
the formation of the fibrils characteristic of Alzheimer’s
disease, the biological validation of a common quorumsensing molecule, and a high-throughput assay based on
a blue fluorescent antibody sensor.
Dr. Ghadiri and members of his laboratory are making significant contributions in the design and study of
a new generation of antimicrobial agents, based on selfassembling peptide nanotube architecture, to combat
multidrug-resistant infections. In addition, they continue
to make novel contributions in several ongoing basic
research endeavors, such as design of biosensors, molecular computation, design of self-reproducing systems,
understanding the origins of life, and design of emergent chemical systems.
Dr. Finn and his group have pioneered the use of
virus particles as chemical reagents and building blocks
for nanochemical structures. This effort is directed toward
the development of new diagnostics for disease and catalysts for organic reactions. Members of the Finn laboratory
also develop and investigate new organic and organometallic reactions and use these processes to synthesize
biologically active compounds.
Research by members of the Kelly group emphasizes
the role of protein conformational changes in neurodegenerative disease and the alteration of these processes
through the design and synthesis of small molecules.
These scientists also take advantage of the power of chemistry and biology to study β-sheet folding. An emerging
interest is self-assembling biomaterials made from peptides and proteins.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Researchers in the Romesberg laboratory are using
diverse techniques ranging from bioorganic and biophysical chemistry to bacterial and yeast genetics to understand and manipulate evolution. Major efforts include
the design of unnatural base pairs and the directed
evolution of DNA polymerases to efficiently synthesize
unnatural DNA containing the base pairs; using spectroscopy to understand biological function and how it
evolves; and understanding how induced and adaptive
mutations contribute to evolution in eukaryotic and
prokaryotic cells.
Dr. Baran and his group have made extraordinary contributions in synthetic organic chemistry. In only 2 years,
they have invented practical chemical solutions to several
long-standing synthetic challenges of great interest, such
as the biologically active natural products pyrrole-imidazole
alkaloids, stephacidins, welwitindolinones, and chartellines.
The Frontiers in Chemistry Lecturers (17th Annual
Symposium) for the 2004–2005 academic year were
Carolyn Bertozzi, University of California, Berkeley; EiEichi Negishi, Purdue University; Thomas Steitz, Yale
University; and David Liu, Harvard University. Jean-Marie
Lehn, ISIS, Université Louis Pasteur, Strasbourg, and
Collège de France, Paris, also visited Scripps this year
as the 2004 Merck lecturer.
CHEMISTRY 2005
INVESTIGATORS’ R EPORTS
Practical Total Synthesis of
Natural Products
P.S. Baran, N.B. Ambhaikar, C.A. Mitsos, K. Li, R.A. Shenvi,
D.P. O’Malley, N.Z. Burns, M.P. DeMartino, C.A. Guerrero,
B.D. Hafensteiner, D.W. Lin, J.M. Richter
rom penicillin to paclitaxel (Taxol), natural products have an unparalleled track record in the
betterment of human health. In fact, 9 of the top
20 best-selling drugs were either inspired by or derived
from natural products. Even the best-selling drug of all
time, atorvastatin (Lipitor), was based on a natural
product lead. Total synthesis, the art and science of
recreating these entities in the laboratory, invariably
leads to fundamental discoveries in chemistry, biology,
and medicine.
We focus on solving interesting challenges in the
total synthesis of natural products and on bridging gaps
in synthetic capabilities by inventing new reactions.
Through judicious target selection and creative retrosynthetic analyses, total synthesis becomes an engine for
discovery that drives organic chemistry to new levels
of sophistication and practicality. Synthetic organic
chemistry requires tremendous ingenuity, artistic taste,
experimental acumen, persistence, and character. Not
surprisingly, drug development relies on the expertise
of researchers who have these characteristics. Although
we focus entirely on educating students in fundamental chemistry, we also collaborate with expert biologists
to explore the medicinal potential of newly synthesized
natural products and the products’ analogs.
Recently completed total syntheses (Fig. 1) include
the anticancer agents stephacidins A and B and avrainvillamide, the antibacterial agents sceptrin and ageliferin, and several members of the bioactive fischerindole
and hapalindole indole alkaloid family. Current natural
product targets (Fig. 2) include chartelline C, welwitindolinone A, haouamine A, strictamine, axinellamine,
and sarcodonin.
F
PUBLICATIONS
Baran, P.S., Guerrero, C.A., Ambhaikar, N.B., Hafensteiner, B.D. Short, enantioselective total synthesis of stephacidin A. Angew. Chem. Int. Ed. 44:606, 2005.
Baran, P.S., Guerrero, C.A., Hafensteiner, B.D., Ambhaikar, N.B. Total synthesis of
avrainvillamide (CJ-17,665) and stephacidin B. Angew. Chem. Int. Ed. 44:3892, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
F i g . 1 . Recently completed total syntheses.
69
70 CHEMISTRY 2005
bioorganic and medicinal chemistry, the study of DNAagent interactions, and the chemistry of antitumor
antibiotics. We place a special emphasis on investigations to define the structure-function relationships of
natural or designed organic agents.
SYNTHETIC METHODS
Central to much of our work are investigations to
develop and apply the hetero Diels-Alder reaction,
including the use of heterocyclic and acyclic azadienes
(Fig. 1), the thermal reactions of cyclopropenone ketals,
intermolecular and intramolecular acyl radical–alkene
addition reactions, medium- and large-ring cyclization
technology, and solution-phase combinatorial chemistry.
In each instance, the development of the methods represents the investigation of chemistry projected as a key
element in the synthesis of a natural or designed agent.
F i g . 1 . N-Sulfonyl-1-aza-1,3-butadiene Diels-Alder reaction.
F i g . 2 . Ongoing natural product total syntheses.
Baran, P.S., Richter, J.M., Lin, D.W. Direct coupling of pyrroles with carbonyl
compounds: short, enantioselective synthesis of (S)-ketorolac. Angew. Chem. Int.
Ed. 44:609, 2005.
Baran, P.S., Shenvi, R.A., Mitsos, C.A. A remarkable ring contraction en route to
the chartelline alkaloids. Angew. Chem. Int. Ed. 44:3714, 2005.
Synthetic and
Bioorganic Chemistry
D.L. Boger, S.B. Boga, K. Bunker, K. Capps, H. Cheng,
Y. Choi, Y. Chong, R. Clark, J. Cottell, B. Crowley,
J. DeMartino, R. Dominique, W. Du, G. Elliott, J. Fuchs,
J. Garfunkle, Y. Ham, A. Hamasaki, W. Han, N. Haq,
C. Hardouin, S. Hong, D. Horne, I. Hwang, H. Ishikawa,
W. Jin, D. Kastrinsky, M. Kelso, G. Kim, B. Lawhorn, S. Lee,
Y. Li, K. MacMillan, J. Nam, S. Pfeiffer, Y. Rew, A. Romero,
M. Schnermann, D. Shin, C. Slown, L. Takaoka, H. Tao,
M. Tichenor, J. Trzupek, J. Velcicky
T
he research interests of our group include the
total synthesis of natural products, development
of new synthetic methods, heterocyclic chemistry,
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
T O TA L S Y N T H E S I S O F N AT U R A L P R O D U C T S
Efforts are under way on the total synthesis of a
number of natural products that constitute agents in
which we have a specific interest. Representative agents
currently under study include (+)-CC-1065 and functional analogs; the duocarmycin class of antitumor
antibiotics, including yatakemycin; tropoloalkaloids;
prodigiosin and roseophilin; the deoxybouvardin and
RA-I class of antitumor agents; vancomycin, teicoplanin,
ristocetin, chloropeptins and related agents; ramoplanin;
the luzopeptins, quinoxapeptins, thiocoraline, BE-22179
and sandramycin; bleomycin A2 and functional analogs;
HUN-7293; chlorofusin; CI-920 (fostriecin); the combretastatins; storniamide A; phomazarin; ningalins;
lamellarin O; lukianol A; piericidins; nothapodytine and
mappicine; rubrolone; and vinblastine (Figs. 2 and 3).
BIOORGANIC CHEMISTRY
The agents listed in the previous paragraph were
selected on the basis of their properties; in many
instances, they are agents related by a projected property. For example, (+)-CC-1065 and the duocarmycins
are antitumor antibiotics and related sequence-selective DNA minor groove alkylating agents. Representa-
CHEMISTRY 2005
71
F i g . 3 . Additional recent total syntheses.
F i g . 2 . Recent total syntheses.
tive of such efforts, studies to determine the structural
features of (+)-CC-1065 and the duocarmycins that
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
contribute to the sequence-selective DNA alkylation
properties of these agents have resulted in the identification of a unique source of catalysis for the DNA alkylation reaction. Efforts are under way to develop DNA
72 CHEMISTRY 2005
cross-linking agents of a predefined cross-link, to further understand the nature of the noncovalent and covalent interactions between agents and DNA, and to apply
this understanding to the de novo design of DNA-binding
and DNA-effector agents. Techniques for the evaluation
of the agent-DNA binding and alkylation properties, collaborative efforts in securing biological data, nuclear
magnetic resonance structures of DNA-agent complexes,
molecular modeling, and studies of DNA-agent interactions are integral parts of the program.
Additional ongoing studies include efforts to define
the fundamental basis of the DNA-binding or cleavage
properties of bleomycin A2, sandramycin, and the luzopeptins; to design inhibitors of the folate-dependent
enzymes glycinamide ribonucleotide transformylase and
aminoimidazole carboxamide ribonucleotide transformylase as potential antineoplastic agents; to establish
the chemical and biological characteristics responsible
for the sleep-inducing properties of the endogenous lipid
oleamide; to inhibit tumor growth through inhibition of
angiogenesis; to inhibit aberrant gene transcription
associated with cancer; and to control intracellular
signal transduction through the discovery of antagonists
or agonists that affect protein-protein interactions, including receptor dimerization.
PUBLICATIONS
Chen, L., Yuan, Y., Helm, J.S., Hu, Y., Rew, Y., Shin, D., Boger, D.L., Walker, S. Dissecting ramoplanin: mechanistic analysis of synthetic ramoplanin analogues as a guide
to the design of improved antibiotics. J. Am. Chem. Soc. 126:7462, 2004.
Crowley, B.M., Mori, Y., McComas, C.C., Tang, D., Boger, D.L. Total synthesis of
the ristocetin aglycon. J. Am. Chem. Soc. 126:4310, 2004.
Ham, Y.W., Boger, D.L. A powerful selection assay for mixture libraries of DNA
alkylating agents. J. Am. Chem. Soc. 126:9194, 2004.
Kastrinsky, D.B., Boger, D.L. Effective asymmetric synthesis of 1,2,9,9a-tetrahydrocyclopropa[c]benzo[e]indol-4-one (CBI). J. Org. Chem. 69:2284, 2004.
Lee, P.S., Du, W., Boger, D.L., Jorgensen, W.L. Energetic preferences for α,β versus β,γ unsaturation. J. Org. Chem. 69:5448, 2004.
Lichtman, A.H., Leung, D., Shelton, C.C., Saghatelian, A., Hardouin, C., Boger,
D.L., Cravatt, B.F. Reversible inhibitors of fatty acid amide hydrolase that promote
analgesia: evidence for an unprecedented combination of potency and selectivity. J.
Pharmacol. Exp. Ther. 311:441, 2004.
Lillo, A.M., Sun, C., Gao, C., Ditzel, H., Parrish, J., Gauss, C.-M., Moss, J., Felding-Habermann, B., Wirsching, P., Boger, D.L., Janda, K.D. A human single-chain
antibody specific for integrin α3β1 capable of cell internalization and delivery of
antitumor agents. Chem. Biol. 11:897, 2004.
Parrish, J.P, Hughes, T.V., Hwang, I., Boger, D.L. Establishing the parabolic relationship between reactivity and activity for derivatives and analogues of the duocarmycin and CC-1065 alkylation subunits. J. Am. Chem. Soc. 126:80, 2004.
Parrish, J.P., Trzupek, J.D., Hughes, T.V., Hwang, I., Boger, D.L. Synthesis and evaluation of N-aryl and N-alkenyl CBI derivatives. Bioorg. Med. Chem. 12:5845, 2004.
Rew, Y., Shin, D., Hwang, I., Boger, D.L. Total synthesis and examination of three
key analogues of ramoplanin: a lipoglycodepsipeptide with potent antibiotic activity.
J. Am. Chem. Soc. 126:1041, 2004.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Shin, D., Rew, Y., Boger, D.L. Total synthesis and structure of the ramoplanin A1
and A3 aglycons: two minor components of the ramoplanin complex. Proc. Natl.
Acad. Sci. U. S. A. 101:11977, 2004.
Tao, H., Hwang, I., Boger, D.L. Multidrug resistance reversal activity of permethyl
ningalin B amide derivatives. Bioorg. Med. Chem. Lett. 14:5979, 2004.
Tichenor, M.S., Kastrinsky, D.B., Boger, D.L. Total synthesis, structure revision, and
absolute configuration of (+)-yatakemycin. J. Am. Chem. Soc. 126:8396, 2004.
Tse, W.C., Boger, D.L. A fluorescent intercalator displacement assay for establishing DNA binding selectivity and affinity. Acc. Chem. Res. 37:61, 2004.
Tse, W.C., Boger, D.L. Sequence-selective DNA recognition: natural products and
nature’s lessons. Chem. Biol. 11:1607, 2004.
Chemical and Functional
Genomic Approaches to
Regenerative Medicine
S. Ding, R. Abu-Jarour, R. Ambasudhan, A. Brunkhorst,
P. Descargues, C. Despon, N. Emre, H.S. Hahm, S. Hilcove,
J. Hsu, S. Takanashi, W. Xiong, S. Yao, D. Yue, Y. Zhao,
X. Zhu
ecent advances in stem cell biology may make
possible new approaches for the treatment of a
number of diseases, including cardiovascular
disease, neurodegenerative disease, musculoskeletal
disease, diabetes, and cancer. These approaches could
involve cell replacement therapy and/or drug treatment
to stimulate the body’s own regenerative capabilities
by promoting survival, migration/homing, proliferation,
and differentiation of endogenous stem/progenitor cells.
However, such approaches will require identification of
renewable cell sources of engraftable functional cells,
an improved ability to manipulate proliferation and
differentiation of stem cells, and a better understanding of the signaling pathways that control the fate of
stem cells.
Equipped with large arrayed molecular libraries—
combinatorial chemical libraries (>100,000 discrete
and diverse small molecules), cDNA overexpression
libraries (>30,000 human and mouse genes), and
small interfering RNA libraries (targeting >20,000
human and mouse genes)—and a high-throughput
screening platform, we are developing and integrating
chemical and functional genomic tools to study stem
cell biology and regeneration. We screen these libraries
to identify small molecules and genes that can control
the fate of stem cells in various systems, including (1)
self-renewal, as well as directed neuronal, cardiac and
R
CHEMISTRY 2005
pancreatic differentiations of pluripotent mouse and
human embryonic stem cells; (2) directed neuronal
differentiation and subtype neuron specification of
human and rodent neural stem cells; (3) directed differentiation of mesenchymal stem cells to osteogenic,
adipogenic, chondrogenic, and myogenic lineages;
(4) functional proliferation of adult cardiomyocytes and
islets/beta cells; (5) cellular plasticity and dedifferentiation of lineage-restricted somatic cells; and (6) developmental signaling pathways.
In addition, we are doing systemic biochemical
and cellular studies, including detailed investigations
of the structure-activity relationship, affinity chromatography for target identification, genome-wide expression
analysis, and cDNA and/or RNA interference complementation screens to map signaling pathways, to characterize the molecular mechanism of these identified
small molecules and genes. The results may ultimately
facilitate the therapeutic application of stem cells and
the development of small-molecule drugs to stimulate
tissue and organ regeneration in vivo.
73
us to consider the triazines (2,4-diamino-triazines and
their oxygen analogs) as alternative nucleobases that
may be able to function as informational base pairs
through a type of hydrogen-bond arrangement that
differs from the canonical Watson-Crick type with its
pairing axis parallel to the nucleosidic bond. Because
carboxyl groups can easily be converted to suitably
functionalized triazine rings, a large variety of oligomer
backbones tagged with informational triazines (instead
of conventional nucleobases) could be envisioned (Fig. 1).
In collaboration with B. Han, Swiss Federal Institute
of Technology, Zürich, Switzerland, we developed the
triazination of the carboxyl group of a variety of α-amino
acids such as glycine, serine, cysteine, aspartic acid,
glutamic acid, β-amino-alanine, and α-carboxy-glycine
to produce correspondingly triazine-tagged building
blocks of potentially informational oligomers.
PUBLICATIONS
Chen, S., Zhang, Q., Wu, X., Schultz, P.G., Ding, S. Dedifferentiation of lineagecommitted cells by a small molecule. J. Am. Chem. Soc. 126:410, 2004.
Ding, S., Schultz, P.G. A role for chemistry in stem cell biology. Nat. Biotechnol.
22:833, 2004.
Liu, J., Wu, X., Mitchell, B., Kintner, C., Ding, S., Schultz, P.G. A small-molecule
agonist of the Wnt signaling pathway. Angew. Chem. Int. Ed. 44:1987, 2005.
Wu, X., Ding, S., Ding, Q., Gray, N.S., Schultz P.G. Small molecules that induce
cardiomyogenesis in embryonic stem cells. J. Am. Chem. Soc. 126:1590, 2004.
Wu, X., Walker, J., Zhang, J., Ding, S., Schultz, P.G. Purmorphamine induces osteogenesis by activation of the hedgehog signaling pathway. Chem. Biol. 11:1229, 2004.
Zheng, L., Liu, J., Batalov, S., Zhou, D., Orth, A., Ding, S., Schultz, P.G. An
approach to genomewide screens of expressed small interfering RNAs in mammalian cells, Proc. Natl. Acad. Sci. U. S. A. 101:135, 2004.
F i g . 1 . 2,4-Diamino-triazine–tagged oligomeric systems.
O L I G O M E R S B A S E D O N T R I A Z I N E - TA G G E D
BACKBONES
Chemical Etiology of the
Structure of Nucleic Acids
A. Eschenmoser, R. Krishnamurthy, O. Munoz, H. Xiong,
G. Kumar, F. De Riccardis, R. Kondreddi, S. Eppacher,
J. Nandy
D
uring the past year, we worked on the following projects.
T R I A Z I N E - TA G G E D A M I N O A C I D D E R I VAT I V E S
Our earlier work on the synthesis of C-nucleosides
with a family of allopurines (formerly isopurines) led
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Of the 2 planned variants (compounds 1 and 2 in
Fig. 1) of ethylenediamine-based oligomer systems containing triazine as recognition elements, we were able
to synthesize and study oligomers (up to dodecamer)
of 1 of them (2 in Fig. 1). As expected, oligomers of
this chemical structure underwent efficient cross-pairing with polyuracil (RNA) and polythymine (DNA)
(Table 1). However, to our surprise, the backbones of
oligomers of this type were unstable because of a triazine-assisted eliminative fragmentation.
A comparative conformational analysis relative to
RNA (tagged with the conventional nucleobases) of
oligomer backbones tagged with triazines predicted
that oligodipeptides of type 2 and 4 (Fig. 1) might be
74 CHEMISTRY 2005
T a b l e 1 . T m values of duplexes formed by the triazine-tagged
oligomers 2–5 with RNA and DNA*
System
Tm (°°C)
2
12-mer
3
6-mer
12-mer
4
6-mer
12-mer
5
8-mer
12-mer
DNA
poly(T)
RNA
poly(U)
DNA
d(T 12)
RNA
r(T 12)
Organic, Materials, and
Analytical Chemistry
M.G. Finn, W.G. Lewis, D. Díaz, S. Punna, V. Rodionov,
S. Sen Gupta
44.1†
29.2†
30.3†
28.5†
<6
26.6
<10
33.1
–
<11.0
–
28.0
41.7
59.4
50.0
65.0
32.2
53.8
42.8
57.2
26.7
35.2
19.7
29.1
n.m.
22.7
n.m.
n.m.
n addition to synthetic chemistry research on viruses,
our program encompasses organic, organometallic,
and materials chemistry. Special emphasis is placed
on methods of chemical synthesis, the discovery of
functional molecules, and catalysis.
I
M E C H A N I S M S A N D A P P L I C AT I O N S O F C L I C K
CHEMISTRY
*Measurements were made at 260 nm, c ≈ 5 µM + 5 µM in 1 M NaCl, 10
mM NaH2PO 4, 0.1 mM Na2EDTA, pH 7.0. T m values are given in degrees
Celsius (°C) and are derived from maxima of the first derivative of the heating curve. – indicates no pairing observed, n.m. = no measurement, † = in
0.15 M NaCl, T = thymine, U = uracil, d = DNA, r = RNA.
oligomer systems that cross-pair with RNA, whereas
oligopeptides of type 3 should not (or less efficiently
so). Experimental results obtained so far are in accord
with the analysis, except that oligopeptides of type 3
also cross-pair with RNA, yet much more weakly than
those of type 4 do (Table 1). The oligopeptides of type
4, composed of a triazine-tagged oligomer consisting
of alternating glutamic and aspartic acid residues,
cross-pairs with RNA (polyuracil) strongly (Table 1).
Studies on the self-pairing and cross-pairing properties
of type 4 oligopeptides are under way.
A variation of oligodipeptide system 3 is the oligodipeptoid of type 5 (Fig. 1), constructed from the
iminodiacetic acid unit, wherein the triazine- and
carboxylate-containing side chains are now appended
to the nitrogen atoms along the backbone, making this
system achiral. We used a solid-support strategy starting from the requisite monomers to synthesize oligomers
(up to dodecamer). The resultant oligodipeptoids crosspaired with RNA (polyuracil) and DNA (polythymine)
(Table 1).
PUBLICATIONS
Ferencic, M., Reddy, G., Wu, X., Guntha, S.G., Nandy, J., Krishnamurthy, R., Eschenmoser, A. Base-pairing systems related to TNA containing phosphoramidate linkages:
synthesis of building blocks and pairing properties. Chem. Biodivers. 1:939, 2004.
Han B., Jaun, B., Krishnamurthy, R., Eschenmoser, A. Mannich type C-nucleosidations in the 5,8-diaza-7,9-dicarba-purine family. Org. Lett. 6:3691, 2004.
Han, B., Rajwanshi, V., Nandy, J., Krishnamurthy, R., Eschenmoser, A. Mannichtype C-nucleosidations with 7-carba-purines and 4-aminopyrimidines. Synlett 744,
2005, Issue 4.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
The copper-catalyzed azide-alkyne cycloaddition
(CuAAC) reaction, discovered in 2002 by V.V. Fokin
and K.B. Sharpless, Department of Chemistry, has been
adopted by chemists all over the world for organic synthesis, drug development, and materials science. In
the past year, we reported the first experimental study
of the mechanism of the reaction (Fig. 1). Two copper
centers are required, and the rate-limiting step of the
catalytic cycle changes under different conditions of
concentration and reactant ratios. Copper acetylide
intermediates are important, and we obtained kinetic
evidence that the existence of a discrete azide-copper
interaction is also important. We observed unusually
large rates for the reactions of 1,3-diazides such as
compound 1 (Fig. 1).
F i g . 1 . Mechanistic outline of the CuAAC reaction as determined
by kinetics measurements.
New ligands have been discovered for copper, which
provide large accelerations in rate in the CuAAC reaction. We used a fluorescence-quenching catalysis screen
in aqueous solutions with low concentrations of reagents
to find systems that operate under conditions optimized
for bioconjugation. We found that a commercially available sulfonated bathophenanthroline ligand imparts
superior performance to the copper catalyst under nitrogen atmosphere. This catalyst has had an extraordinary
CHEMISTRY 2005
impact on our bioconjugation efforts, enabling us to
attach a wide variety of structures to virus particles with
far less material than required by any other method.
We also pioneered the use of the CuAAC reaction
in the synthesis and derivatization of polymeric materials. The deposition of molecules bearing 2, 3, or 4
azide and alkyne groups between metallic copper surfaces makes especially strong adhesive mixtures that
glue the copper pieces to each other. The catalytic copper ions are provided by the surface, and much of the
adhesion power to the metal is provided by the 1,2,3triazole moieties formed in the azide-alkyne cycloaddition process. We can also use the CuAAC process to
connect polymers to each other and to other materials.
Last, the CuAAC reaction mediates an unprecedented
cyclic dimerization of peptides containing azide and
alkyne groups on solid supports (Fig. 2). Rings as large
as 36 amino acids and more than 120 atoms have been
made selectively and in high yield. The bimetallic nature
of the reaction appears to play a crucial role, and we
are exploring its scope with both peptide and nonpeptide structures.
75
Díaz, D.D., Lewis, W.G., Finn, M.G. Acid-mediated amine exchange of N,Ndimethylformamidines: preparation of electron-rich formamidines. Synlett, in press.
Díaz, D.D., Lewis, W.G., Finn, M.G. Activation of urea as a leaving group in substitution reactions of formamidine ureas. Chem. Lett., in press.
Díaz, D.D., Punna, S., Holzer, P., McPherson, A.K., Sharpless, K.B., Fokin, V.V.,
Finn, M.G. Click chemistry in materials synthesis, I: adhesive polymers from coppercatalyzed azide-alkyne cycloaddition. J. Polym. Sci. A Polym. Chem. 42:4392, 2004.
Gissibl, A., Finn, M.G., Reiser, O. Cu(II)-aza(bisoxazoline)-catalyzed asymmetric
benzoylations. Org. Lett. 7:2325, 2005.
Keller, K.A., Guo, J., Punna, S., Finn, M.G. A thermally-cleavable linker for solidphase synthesis. Tetrahedron Lett. 46:1181, 2005.
Lewis, W.G., Magallon, F.G., Fokin, V.V., Finn, M.G. Discovery and characterization of catalysts for azide-alkyne cycloaddition by fluorescence quenching. J. Am.
Chem. Soc. 126:9152, 2004.
Meng, J.-C., Siuzdak, G., Finn, M.G. Affinity mass spectrometry from a tailored
porous silicon surface. Chem. Commun. (Camb.) 2108, 2004, Issue 18.
Narayan, S., Muldoon, J., Finn, M.G., Fokin, V.V., Kolb, H.C., Sharpless, K.B.
“On water”: unique reactivity of organic compounds in aqueous suspension.
Angew. Chem. Int. Ed. 44:3275, 2005.
Punna, S., Díaz, D.D., Finn, M.G. Palladium-catalyzed homocoupling of arylboronic
acids and esters using fluoride in aqueous solvents. Synlett 2351, 2004, Issue 13.
Punna, S., Kuzelka, J., Wang, Q., Finn, M.G. Head-to-tail peptide cyclodimerization by copper-catalyzed azide-alkyne cycloaddition. Angew. Chem. Int. Ed.
44:2215, 2005.
Punna, S., Meunier, S., Finn, M.G. A hierarchy of aryloxide deprotection by boron
tribromide. Org. Lett. 6:2777, 2004.
Rodionov, V.O., Fokin, V.V., Finn, M.G. Mechanism of the ligand-free Cu(I)-catalyzed azide-alkyne cycloaddition reaction. Angew. Chem. Int. Ed. 44:2210, 2005.
F i g . 2 . An example of the cyclodimerization of peptides by the
CuAAC reaction.
SYNTHESIS AND USE OF FORMAMIDINE COMPOUNDS
We have continued to explore the synthesis and
properties of formamidines and formamidine ureas. We
optimized efficient and modular routes to their preparation and found that these compounds are active binders
and inhibitors of several families of enzymes. The properties of greatest importance are the basicity and electrophilicity of the compounds, an unusual combination
that provides multiple ways for them to interact and
react with target proteins. We found that fluorescently
labeled formamidines are strong ligands for acetylcholinebinding proteins and that formamidine urea derivatives
have promise as unique “soft dry” (degradable) binders.
We are exploring these modes of action in collaboration
with P. Taylor, University of California, San Diego.
PUBLICATIONS
Converso, A., Saaidi, P.-L., Sharpless, K.B., Finn, M.G. Nucleophilic substitution
by Grignard reagents on sulfur mustards. J. Org. Chem. 69:7336, 2004.
Díaz, D.D., Finn, M.G. Modular synthesis of formamidines and their formation of
stable organogels. Chem. Commun. (Camb.) 2514, 2004, Issue 21.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Shen, Z., Go, E.P., Gamez, A., Apon, J.V., Fokin, V., Greig, M., Ventura, M.,
Crowell, J.E., Blixt, O., Paulson, J.C., Stevens, R.C., Finn, M.G., Siuzdak, G. A
mass spectrometry plate reader: monitoring enzyme activity and inhibition with a
desorption/ionization on silicon (DIOS) platform. Chembiochem 5:921, 2004.
Stray, S.J., Bourne, C.R., Punna, S., Lewis, W.G., Finn, M.G., Zlotnick, A. A heteroaryldihydropyrimidine activates and can misdirect hepatitis B virus capsid
assembly. Proc. Natl. Acad. Sci. U. S. A. 102:8138, 2005.
Design of Functional
Synthetic Systems
M.R. Ghadiri, G. Ashkenasy, N. Ashkenasy, J. Beierle,
A. Chavochi, N. Gianneschi, W.S. Horne, Z.-Z. Huang,
P. Imming, L. Leman, A. Loutchnikov, A. Montero, L. Motiei,
D. Nicoletti, Y. Norikane, J. Picuri, N. Rahe, D. Radu,
S. Rahimipour, J. Shin, R. Yamasaki, Y.S. Yoo
e are engaged in a multidisciplinary research
effort to uncover new chemical and biochemical approaches for the design of functional
molecular, supramolecular, and complex self-organized
systems. Our endeavors span disciplines ranging from
synthetic organic, bioorganic, and physical organic chemistry to nanotechnology, biophysics, enzymology, and
W
76 CHEMISTRY 2005
molecular biology. Current research includes the design of
synthetic peptide catalysts, antimicrobial self-assembling
peptide nanotubes, semisynthetic allosteric enzymes,
self-replicating molecular systems and emergent networks, single-molecule stochastic DNA sensing, molecular computation, and prebiotic chemistry.
ANTIMICROBIAL PEPTIDE NANOTUBES
F i g . 2 . Schematic representation of an intrasterically inactivated
We showed that appropriately designed cyclic peptide subunits can self-assemble through hydrogen
bond–directed ring stacking into open-ended hollow
tubular structures that have marked antibacterial and
antiviral activities in vitro. The effectiveness of this novel
supramolecular class of bioactive species as selective
antibacterial agents was highlighted by the high efficacy
of one of these antimicrobials against lethal methicillinresistant Staphylococcus aureus infections in mice.
Currently, we are exploring rational design of cyclic
glycopeptides and selections from combinatorial libraries
to discover novel antiviral and anticancer supramolecular compounds (Fig. 1).
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 turnover of substrate.
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.
S T O C H A S T I C A N A LY S I S O F S I N G L E - M O L E C U L E
D N A R O TA X A N E S
We are interested in the study of matter at the level
of single molecules. For these studies we use the transmembrane protein α-hemolysin as a rapid and highly
sensitive sensor element for stochastic analysis of the
molecules lodged or trapped inside the protein pore;
the analysis relies on detecting the perturbations in
the conductance levels produced in the ion channel in
the native protein. Using this technique, we developed
an approach by which a single-stranded DNA molecule can be trapped in a specific configuration inside
an α-hemolysin channel (Fig. 3), manipulated, and
studied with high sensitivity at the single-molecule
level. Moreover, a single adenine nucleotide at a specific location on a strand of polydeoxycytidine can be
detected by its characteristic effect in reducing the ion
conductance in α-hemolysin. We are extending this
approach to the design of rapid single-molecule DNA
sensing and sequencing.
DESIGN OF SIGNAL SELF-AMPLIFYING DNA SENSORS
We constructed a novel sequence-specific DNA
detection system based on rationally designed semisynthetic enzymes. The system is composed of covalently associated inhibitor-DNA-enzyme modules that
function via DNA hybridization–triggered allosteric
enzyme activation and signal amplification through substrate turnover (Fig. 2). The functional capacity of the
system is highlighted by the sequence-specific detection of approximately 10 fmol of DNA in less than 3
minutes under physiologic conditions. Our studies suggest that rationally designed intrasterically regulated
enzymes may be a promising new class of reagents for
highly sensitive, rapid, 1-step detection of label-free
DNA sequences that does not depend on polymerase
chain reactions.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
F i g . 3 . Functional supramolecular chemistry at the single-molecule
level. Single strands of DNA can be captured inside an α-hemolysin
transmembrane pore protein to form single-species pseudorotaxanes
composed of α-hemolysin and DNA. This process can be used to
identify a single adenine nucleotide at a specific location on a
strand of DNA on the basis of the characteristic reductions in the
α-hemolysin ion conductance.
SYNTHETIC NETWORKS
Living cells use complex networks of evolutionary
selected biomolecular interactions and chemical trans-
CHEMISTRY 2005
formations to process multiple extracellular input signals rapidly and simultaneously. We are interested in
understanding and experimentally modeling the organizational and functional properties of biological networks. We have developed a general strategy for the
design and construction of self-organized synthetic peptide networks based on the sequence-selective autocatalytic and cross-catalytic template-directed coiled coil
peptide fragment condensation reactions in aqueous
solutions. The synthetic networks have some of the
basic architectural and dynamic features of the living
networks, reorganize in response to changes in environmental conditions and inputs (Fig. 4), and perform
basic Boolean logic functions such as OR, NOR and
NOTIF logic. We suggest that the ability to rationally
construct predictable chemical circuitry might be useful
in advancing the modeling and in better understanding
some of the basic dynamic information-processing characteristics of the more complex cellular networks.
77
day volcanoes, is a condensing agent that brings about
the formation of peptides from amino acids under mild
conditions in aqueous solution (Fig. 5). We have studied
the carbonyl sulfide–mediated condensations of α-amino
acids under aerobic and anaerobic conditions in the
absence of any added reagents and in the presence of
metal ions, oxidizing agents, or alkylating agents.
Depending on the reaction conditions and additives
used, exposure of α-amino acids to carbonyl sulfide
generates peptides in yields of up to 80% in minutes
to hours at room temperature.
F i g . 5 . Peptide formation under plausibly prebiotic reaction con-
ditions. Carbonyl sulfide, a volcanic gas, is the most simple and
effective amino acid–condensing agent for the formation of peptides
in aqueous solutions.
F i g . 4 . Adaptive reorganization in a synthetic peptide network.
The graph structure or wiring of a synthetic peptide network responds
PUBLICATIONS
Ashkenasy, G., Ghadiri, M.R. Boolean logic functions of a synthetic peptide network. J. Am. Chem. Soc. 126:11140, 2004.
dramatically to changes in the environmental stimuli (pH or salt
content).
Ashkenasy, G., Jagasia, R., Yadav, M., Ghadiri, M.R. Design of a directed molecular network. Proc. Natl. Acad. Sci. U. S. A. 101:10872, 2004.
PREBIOTIC CHEMISTRY
Leman, L., Orgel, L., Ghadiri, M.R. Carbonyl sulfide-mediated prebiotic formation
of peptides. Science 306:283, 2004.
In almost all discussions of prebiotic chemistry, it
is assumed that amino acids, nucleotides, and possibly
other monomers were first formed on Earth or brought
to it in comets and meteorites and that the monomers
subsequently condensed nonenzymatically to form
oligomeric products. Unfortunately, attempts to create
plausibly prebiotic polymerization reactions have met
with limited success. Direct heating of solid mixtures
leads to nonspecific products, and the condensing agents
that have been studied, with the possible exception of
inorganic polyphosphates, are relatively inefficient and/or
marginally prebiotic. We showed that carbonyl sulfide,
a simple gas present in the emissions from presentPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Sánchez-Quesada, J., Saghatelian, A., Cheley, S., Bayley, H., Ghadiri, M.R. Single DNA rotaxanes of a transmembrane pore protein. Angew. Chem. Int. Ed.
43:3063, 2004.
78 CHEMISTRY 2005
A Merging of Chemistry
and Biology
K.D. Janda, J. Ashley, M. Atsumi, C. Berndt, G. Boldt,
A. Brogan, R. Carrera, B. Clapham, T. Dickerson,
L. Eubanks, G. Kaufmann, J. Kennedy, S.-J. Kim, Y.-S. Kim,
B.-S. Lee, M. Lillo, Y. Liu, C. Lowery, H. Ma, S. Mahajan,
H. Matsushita, M. Matsushita, L. McAllister, G. McElhaney,
K. McKenzie, J. Mee, M. Meijler, J. Moss, L. Qi, C. Rogers,
A. Shigenaga, P. Wirsching, Y. Xu, M. Yamashita, B. Zhou
uring the past year, we explored various applications of organic chemistry at the interface of
chemistry and biology. Representative examples
of our results were obtained in 3 research programs:
the immunologic consequences of methamphetaminebased protein glycation, the treatment via viruses of
the effects of exposure to cocaine in the CNS, and the
nonenzymatic formation of a bactericidal product from
a compound known to act as a quorum-sensing agent.
D
IMMUNOLOGIC CONSEQUENCES OF
M E T H A M P H E TA M I N E - B A S E D P R O T E I N G LY C AT I O N
We extended our studies on the aberrant glycation
of proteins by nornicotine to other drugs of abuse that
contain reactive secondary amine groups. In this context, we found that methamphetamine can produce the
corresponding Amadori product in vitro, and we examined the potential roles of this process in vivo. For
example, protein glycation can increase the immunogenicity of the modified protein.
The initial stage of protein glycation is accepted to
proceed via the Amadori rearrangement, although the
specific mechanism varies widely, depending on factors
such as pH, ionic strength, and temperature. We analyzed the reaction of methamphetamine with glucose
in buffer to detect the corresponding Amadori rearrangement product under physiologic conditions. Using commercially available sera containing polyclonal antibodies
to a methamphetamine-protein conjugate, we found
specific covalent modification of bovine serum albumin after extended incubation periods as indicated by
enzyme-linked immunosorbent assays and dot blots
(Fig. 1).
In essence, the biochemical formation of a methamphetamine-derived advanced glycation end product (AGE)
has numerous similarities to traditional hapten preparation, in which a nonimmunogenic molecule is covalently conjugated to a carrier protein via a linker by
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
F i g . 1 . Generation of an aberrant methamphetamine vaccine ini-
tiated by the reaction of methamphetamine and glucose.
using a chemical coupling reagent. Because of this parallel, we postulated that proteins glycated by methamphetamine could evoke an abnormal immune response.
To examine the validity of this hypothesis, we prepared methamphetamine-glycated mouse serum albumin (MSA) and immunized mice with this modified
protein. Serum samples from the mice were analyzed
to determine if an antibody response to methamphetamine-AGE or to MSA had occurred. After a preliminary
series of injections, the mice had appreciable amounts
of antibodies to methamphetamine-AGE but no significant amounts of antibodies to the MSA carrier protein
or to control injections of MSA alone. Of note, no adjuvant was required to achieve significant titers against
methamphetamine-modified MSA, suggesting that the
immune system needs little priming to recognize foreign
glycation motifs.
The discovery that these antibodies bind to methamphetamine is of potential significance in the context
of addiction. Our observations suggest that once a methamphetamine-AGE is administered, an immune response
could develop to the modified protein, and the antibodies produced could bind some proportion of the
serum methamphetamine, thereby reducing the available
concentration of the drug and ensuing high. Furthermore,
autoantibodies against methamphetamine-modified proteins could have undesirable consequences, such as
the misregulated activation of inflammatory pathways,
leading to extensive tissue damage. In total, our results
provide an intriguing possibility for an unrecognized
mechanism underlying methamphetamine addiction
and the associated health consequences.
CHEMISTRY 2005
U S I N G V I R U S E S T O T R E AT C O C A I N E A D D I C T I O N
Cocaine addiction continues to be a major health
and social problem in the United States and other countries. The pharmacologic agents currently used to treat
cocaine abuse are inadequate, and few treatment options
are available. An alternative is to use protein-based
therapeutic agents, such as antibodies, that can eliminate the load of cocaine and thereby attenuate its
effects. This approach is especially attractive because
the therapeutic agents exert no pharmacodynamic action
of their own and therefore have little potential for side
effects. The effectiveness of these agents, however, is
limited by their inability to act directly within the CNS.
Bacteriophages are viruses that infect bacteria, and
unlike animal and plant viruses, they lack intrinsic tropism for eukaryotic cells. The filamentous bacteriophage
fd can be produced at high levels in bacterial culture,
making production simple and economical. However,
perhaps the most important characteristic of this virus
is its genetic flexibility. With phage display technology,
a wide variety of proteins, antibodies, and peptides can
be presented on the phage coat. The recent discovery
that intranasally administered phage can penetrate
the CNS caused us to speculate that by using phage
as a vehicle for CNS entry, a method for treating the
effects of cocaine addiction directly within the brain
could be developed.
To prove this hypothesis, we prepared phage molecules that display the cocaine-binding antibody GNC92H2.
We then administered this modified phage intranasally
to rats over a period of 3 days and assayed the animals’
cocaine-induced locomotor behavior. Gratifyingly, psychomotor responses to cocaine differed significantly
between rats given phage GNC92H2 and rats given
control phage. In rats given phage GNC92H2, movement was almost 50% less than at baseline (before
administration of phage), whereas in the control group,
locomotor activity actually increased compared with
baseline values.
To understand the role of the nasal vaccine, we
investigated potential limitations. The CNS is considered an immune privileged site; however, the possibility
that phage enters the bloodstream cannot be discounted.
Filamentous phage in itself, and with displayed proteins
on its surface, is a foreign entity to the immune system.
However, analysis of serum from vaccinated animals
showed only marginal concentrations of antibodies to
phage and thus provides further evidence that potential toxic side effects are not being manifested in animals administered filamentous phage.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
79
Together these results indicate a novel approach
for treating cocaine addiction directly within the CNS.
We are examining the combination of this phage-based
approach with either passive or active immunization
protocols to determine whether any synergistic benefits
can be obtained. We envision that this new proteinbased treatment for cocaine abuse can be modified to
provide therapeutic agents for treatment of other drug
abuse syndromes in which areas of the CNS are targeted.
N O N E N Z Y M AT I C F O R M AT I O N O F T E T R A M I C A C I D S
FROM QUORUM-SENSING MOLECULES
The term quorum sensing has been coined to
describe the ability of a population of unicellular bacteria to act as a multicellular organism in a cell density–dependent manner, that is, a way to sense “how
many are out there.” Bacteria use small diffusible
molecules to exchange information among themselves.
An important class of “quormones,” or autoinducers,
is the family of N-acylhomoserine lactones used by
gram-negative bacteria. Upon reaching a critical threshold concentration, these compounds bind to their cognate receptor proteins, triggering the expression of
target genes.
Recently, we showed that N-(3-oxo-dodecanoyl)
homoserine lactone (compound 1 in Fig. 2) performs a
previously unrecognized role: the autoinducer itself and
a corresponding degradation product derived from an
unusual Claisen-like condensation reaction function as
innate bactericidal agents. Incubation of compound 1
in water produced an undocumented compound in addition to the expected hydrolysis product (compound 3
in Fig. 2). Structural characterization of this anomalous molecule revealed that it was 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione
(compound 2 in Fig. 2), a molecule that belongs to a
F i g . 2 . Reaction of N-(3-oxo-dodecanoyl) homoserine lactone
(1) to generate lactone hydrolysis product 3 (path a) and 3-(1hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione (compound 2; path b).
80 CHEMISTRY 2005
class of antibacterial and antifungal compounds known
as tetramic acids.
Because Pseudomonas aeruginosa uses compound 1
as the principal autoinducer and because of the known
bactericidal activity of tetramic acids, we hypothesized
that compound 2 could have biological function in the
context of bacterial viability. Indeed, it had significant
antibacterial activity against all tested gram-positive
bacterial strains and no activity against P aeruginosa
or other tested gram-negative bacteria. The effective
concentration of compound 2 is biologically relevant,
because high concentrations of compound 1 have been
detected in P aeruginosa biofilms. Notably, compound 1
was also cytotoxic, suggesting a dual role for N-acylhomoserine lactones in P aeruginosa communities as
both quorum-sensing molecules and as an interference
mechanism against bacterial competitors.
Additionally, compound 2 tightly binds essential metals such as iron, possibly providing a previously unrecognized primordial siderophore. Iron plays an essential
role in physiologic processes and in the pathogenesis
of bacterial infections. Many bacteria produce siderophores to sequester iron, an element that although
essential for their growth has poor solubility under
physiologic conditions. The 3-acetyl-pyrrolidine-2,4dione heterocycle found in compound 2 and in many
other naturally occurring tetramic acids can efficiently
chelate a variety of metal cations, including iron. Our
studies have revealed the ability of compound 2 to
compete for available iron in solution and potentially
provide an additional method for iron solubilization.
We propose that P aeruginosa uses compound 2 as
an interference strategy to preclude encroachment by
competing bacteria. Although the complexation of critical metals such as iron may play a role in this process,
further study is required into the mechanism and scope
of the observed bactericidal activity and the potential of
compound 2 to act as a primordial siderophore.
Dickerson, T.J., Lovell, T., Meijler, M.M., Noodleman, L., Janda, K.D. Nornicotine
aqueous aldol reactions: synthetic and theoretical investigations into the origins of
catalysis. J. Org. Chem. 69:6603, 2004.
Dickerson, T.J., Reed, N.N., La Clair, J.J., Janda, K.D. A precipitator for the
detection of thiophilic metals in aqua. J. Am. Chem. Soc. 126:16582, 2004.
Dickerson, T.J., Yamamoto, N., Janda, K.D. Antibody-catalyzed oxidative degradation of nicotine using riboflavin. Bioorg. Med. Chem. 12:4981, 2004.
Dickerson, T.J., Yamamoto, N., Ruiz, D.I., Janda, K.D. Immunological consequences
of methamphetamine protein glycation. J. Am. Chem. Soc. 126:11446, 2004.
Felding-Habermann, B., Lerner, R.A., Lillo, A., Zhuang, S., Weber, M.R., Arrues, S.,
Gao, C., Mao, S., Saven, A., Janda, K.D. Combinatorial antibody libraries from cancer
patients yield ligand-mimetic Arg-Gly-Asp-containing immunoglobulins that inhibit
breast cancer metastasis. Proc. Natl. Acad. Sci. U. S. A. 101:17210, 2004.
Kaufmann, G.F., Meijler, M.M., Sun, C., Chen, D.W., Kujawa, D.P., Mee, J.M., Hoffman, T.Z., Wirsching, P., Lerner, R.A., Janda, K.D. Enzymatic incorporation of an
antibody-activated blue fluorophore into DNA. Angew. Chem. Int. Ed. 44:2144, 2005.
Kaufmann, G.F., Sartorio, R., Lee, S.H., Rogers, C.J., Meijler, M.M., Moss, J.A.,
Clapham, B., Brogan, A.P., Dickerson, T.J., Janda, K.D. Revisiting quorum sensing: discovery of additional chemical and biological functions for 3-oxo-N-acylhomoserine lactones. Proc. Natl. Acad. Sci. U. S. A. 102:309, 2005.
Kim, Y.S., Moss, J.A., Janda, K.D. Biological tuning of synthetic tactics in solidphase synthesis: application to Aβ(1-42). J. Org. Chem. 69:7776, 2004.
Lee, B.S., Mahajan, S., Janda, K.D. Cross-linked poly(4-vinylpyridine/styrene)
copolymers as a support for immobilization of ytterbium triflate. Tetrahedron
61:3081, 2005.
Lee, B.S., Mahajan, S., Janda, K.D. Molecular iodine-catalyzed imine activation
for three-component nucleophilic addition reactions. Synlett, in press.
Lee, B.S., Mahajan, S., Janda, K.D. Novel method for catalyst immobilization
using an ionic polymer: a case study using recyclable ytterbium triflate. Tetrahedron
Lett. 46:807, 2005.
Lee, S.H., Matsushita, H., Koch, G., Zimmermann, J., Clapham, B., Janda, K.D.
Smart cleavage reactions: the synthesis of an array of ureas from polymer-bound
carbamates. J. Comb. Chem. 6:822, 2004.
Lee, S.H., Yoshida, K., Matsushita, H., Clapham, B., Koch, G., Zimmermann, J.,
Janda, K.D. N-H insertion reactions of primary ureas: the synthesis of highly substituted
imidazolones and imidazoles from diazocarbonyls. J. Org. Chem. 69:8829, 2004.
Lillo, A.M., Sun, C., Gao, C., Ditzel, H., Parrish, J., Gauss, C.-M., Moss, J., Felding-Habermann, B., Wirsching, P., Boger, D.L., Janda, K.D. A human single-chain
antibody specific for integrin α3β1 capable of cell internalization and delivery of
antitumor agents. Chem. Biol. 11:897, 2004.
Lowery, C.A., McKenzie, K.M., Qi, L., Meijler, M.M., Janda, K.D. Quorum sensing
in Vibrio harveyi: probing the specificity of the LuxP binding site. Bioorg. Med.
Chem. Lett. 15:2395, 2005.
Matsushita, H., Lee, S.H., Yoshida, K., Clapham, B., Koch, G., Zimmermann, J.,
Janda, K.D. N-H insertion reactions of Boc-amino acid amides: solution- and solidphase synthesis of pyrazinones and pyrazines. Org. Lett. 6:4627, 2004.
PUBLICATIONS
Carrera, M.R.A., Kaufmann, G.F., Mee, J.M., Meijler, M.M., Koob, G.F., Janda,
K.D. Treating cocaine addiction with viruses. Proc. Natl. Acad. Sci. U. S. A.
101:10416, 2004.
McDunn, J.E., Dickerson, T.J., Janda, K.D. Antibody catalysis of disfavored chemical reactions. In: Catalytic Antibodies. Keinan, E. (Ed.). Wiley & Sons, New York,
2005, p. 184.
Carrera, M.R.A., Meijler, M.M., Janda, K.D. Cocaine pharmacology and current
pharmacotherapies for its abuse. Bioorg. Med. Chem. 12:5019, 2004.
Meijler, M.M, Kaufmann, G.F., Qi, L., Mee, J.M., Coyle, A.R., Moss, J.A., Wirsching,
P., Matsushita, M., Janda, K.D. Fluorescent cocaine probes: a tool for the selection
and engineering of therapeutic antibodies. J. Am. Chem. Soc. 127:2477, 2005.
Carrera, M.R.A., Trigo, J.M., Roberts, A.J., Janda, K.D. Evaluation of the anticocaine monoclonal antibody GNC92H2 as an immunotherapy for cocaine overdose.
Pharmacol. Biochem. Behav., in press.
Moss, J.A., Lillo, A., Kim, Y.S., Gao, C., Ditzel, H., Janda, K.D. A dimerization
“switch” in the internalization mechanism of a cell-penetrating peptide. J. Am.
Chem. Soc. 127:538, 2005.
Dickerson, T.J., Kaufmann, G.F., Janda, K.D. Bacteriophage-mediated protein
delivery into the central nervous system and its application in immunopharmacotherapy. Expert Opin. Biol. Ther. 5:773, 2005.
Reed, N.N., Dickerson, T.J., Boldt, G.E., Janda, K.D. Enantioreversal in the Sharpless
asymmetric epoxidation reaction controlled by the molecular weight of a covalently
appended achiral polymer. J. Org. Chem. 70:1728, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
CHEMISTRY 2005
Rogers, C.J., Dickerson, T.J., Brogan, A.P., Janda, K.D. Hammett correlation of
nornicotine analogues in the aqueous aldol reaction: implications for green organocatalysis. J. Org. Chem. 70:3705, 2005.
Xu, Y., Yamamoto, N., Janda, K.D. Catalytic antibodies: hapten design strategies
and screening methods. Bioorg. Med. Chem. 12:5247, 2004.
Protein Misfolding Diseases:
Cell Biology and Bioorganic and
Biophysical Chemistry
J.W. Kelly, L. Bazhenova, J. Bieschke, D. Bosco, P. Braun,
M.T.A. Dendle, W. D’Haeze, T. Foss, D. Fowler,
K. Frankenfield, Y. Fu, J. Gao, M.-Y. Gao, A. Hurshman,
S. Johnson, E.T. Powers, A. Sawkar, S. Siegel, J.-Y. Suk,
L. Wiseman, I. Yonemoto, Z. Yu, Q. Zhang
ur goal is to contribute to the understanding of
the molecular mechanisms of protein folding and
misfolding; protein misfolding ultimately leads to
neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. We use cell biological
approaches, in collaboration with W.E. Balch, Department of Cell Biology, and spectroscopic and biophysical
approaches in combination with chemical synthesis and
recombinant DNA technology.
O
INHIBITION OF TRANSTHYRETIN AMYLOIDOGENESIS
As a consequence of a mutation or denaturation
stress associated with aging and/or oxidative stress,
the transthyretin tetramer dissociates, and subsequent
changes in the tertiary structure of the monomer make
it competent to misassemble into aggregates, including fibrils. An attractive strategy to slow or prevent the
formation of aggregates is to inhibit the rate-limiting
dissociation of the tetramer and stabilize the native
state. In this respect, we synthesized various structurally distinct small molecules that inhibit transthyretin amyloidogenesis.
For example, bisarylaldoxime ethers substituted with
a carboxylic acid on one aromatic ring and halogens
or a trifluoromethyl group on the other aryl ring substantially inhibit the formation of transthyretin fibrils,
as do oxazoles carrying a carboxyl group at C-4, a 3,5dichlorophenyl group at C-2, and an ethyl, a propyl, or
a trifluoromethyl group at C-5. We showed that hydroxylated polychlorinated biphenyls bind transthyretin in
blood and plasma with a high affinity and specificity
and inhibit the formation of transthyretin fibrils in vitro.
Such small molecules might be suitable drug candiPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
81
dates for treatment of transthyretin amyloidoses, as
illustrated by the drug diflunisal, which is being tested
in humans in a multicenter placebo-controlled clinical
trial because of previous discoveries in our laboratory.
These small-molecule inhibitors typically bind with
negative cooperativity, and thus investigating the ability
of a transthyretin tetramer to undergo amyloidogenesis
when bound to only a single inhibitor is important. We
showed that occupancy of only 1 of the 2 possible sites
was sufficient to stabilize the entire tetramer and prevent amyloidogenesis. In these studies, we tethered a
small-molecule inhibitor to 1 of the 4 transthyretin
subunits, allowing it to bind to 1 of the ligand-binding
sites within the transthyretin tetramer. We also found
that we could stabilize the same interface by linking
the N terminus of one subunit to the C terminus of a
second subunit, a sequence modification that conferred
kinetic stability on the quaternary structure. We developed a new method to demonstrate kinetic stabilization of the tetramer mediated by binding of 1 ligand
under physiologic conditions. In this method, we used
the correlation between the tetramer dissociation rate
and the rate at which unlabeled wild-type transthyretin
homotetramers and wild-type transthyretin homotetramers labeled at the N terminus exchanged subunits.
The latter exchange was dramatically slowed in the
presence of small-molecule inhibitors.
In other research, we found that only the most
destabilized transthyretin variants are degraded by
endoplasmic reticulum–associated degradation, and
then only in certain tissues. We discovered that endoplasmic reticulum–assisted folding determines protein
secretion in a tissue-specific manner, and we propose
that its competition with endoplasmic reticulum–associated degradation may explain the appearance of tissue-selective amyloid diseases, especially for highly
destabilized transthyretin variants that lead to CNSselective disease, because the CNS is the only tissue
that can secrete these highly unstable transthyretin
variants. Surprisingly, we found the brain is a more
permissive transthyretin secretor than is the liver.
O X I D AT I V E M E TA B O L I T E S A N D P R O T E I N
A G G R E G AT I O N
Oxidative stress leads to the generation of oxidative
metabolites that can modify proteins. We discovered
that oxidative metabolites generated upon cholesterol
ozonolysis or lipid peroxidation can covalently modify
amyloid β-peptides (Aβ), dramatically accelerating the
amyloidogenesis of these peptides, which are associ-
82 CHEMISTRY 2005
ated with Alzheimer’s disease. Metabolite modification
of Aβ amyloidogenesis occurs via a 2-step mechanism
involving the nucleation-independent formation of spherical aggregates by Aβ-metabolite adducts before the
generation of fibrillar aggregates. This mechanism may
explain the formation of Aβ aggregates in the brain at
nanomolar concentrations.
α-Synucleinopathies, including Parkinson’s disease
and dementia with Lewy bodies, are characterized by
cytoplasmic α-synuclein–rich aggregates within degenerating dopaminergic neurons in the substantia nigra.
Observations suggest a correlation between oxidative
stress or inflammation and Parkinson’s disease. Feasibly,
reactive oxygen species react with metabolites to generate oxidized metabolites that can interact with α-synuclein, triggering misfolding and subsequent aggregation.
We aim to determine whether such metabolites accelerate the aggregation of α-synuclein in the same way
they accelerate the aggregation of Aβ. Those studies will
provide insight into the correlation between oxidative
stress and α-synucleinopathies.
β-SHEET FOLDING
The native state of a protein is stabilized by hydrophobic interactions between side chains (the hydrophobic effect) and by hydrogen bonding. We studied the
role of backbone hydrogen bonds in the folding kinetics and stability of the PIN WW domain, a 34-residue
β-sheet protein composed of 3 β-strands and 2 intervening loops. For these studies, we used amide-to-ester
backbone mutations, alterations that do not affect backbone conformational preferences or the structure of side
chains. We synthesized 19 variants, allowing the perturbation of 11 backbone hydrogen bonds.
Thermodynamic analysis indicated that the location
of a backbone hydrogen bond determines the extent of
protein destabilization caused by the elimination of a
hydrogen bond by amide-to-ester mutation. Elimination
of buried hydrogen bonds in the hydrophobic core substantially destabilizes the PIN WW domain; in contrast,
elimination of hydrogen bonds present at or near loops
exposed to solvent is only slightly destabilizing. In
addition, we found that the destabilization of the PIN
WW domain was greater when a backbone hydrogenbond donor was eliminated than when a hydrogen-bond
acceptor was weakened. These findings are important
because they suggest that only a subset of hydrogen
bonds is energetically important in protein folding.
Previously, we synthesized a peptidomimetic composed of a dibenzofuran template substituted at C-2
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
and C-8 with propanoic acid linkers to which valinethreonine-valine-threonine peptides were attached via
N-terminal amide bonds. This peptidomimetic forms
protofilaments and filaments via a side-chain hydrophobic collapse model. In this model, the inward
association of the amino acid side chains is such that
intramolecular interactions are mediated by the side
chains and not by hydrogen-bonded intermolecular
formation of β-sheets. Investigation of the means by
which this peptidomimetic self-assembles revealed
that it is strictly analogous to the structure of Aβ fibrils
recently characterized by using solid-state nuclear
magnetic resonance.
FAMILIAL AMYLOIDOSIS OF FINNISH TYPE
Familial amyloidosis of Finnish type is caused by
the D187N/Y mutation in plasma gelsolin. This disease is characterized by amyloid deposits composed
of 5- and 8-kD internal fragments of plasma gelsolin.
The disease is triggered by a loss of a calcium-binding
site in domain 2 that allows aberrant furin cleavage
in the Golgi complex, yielding a 68-kD fragment. This
fragment is then cleaved by a transmembrane matrix
metalloprotease, resulting in 5- and 8-kD fragments
that are deposited as amyloid fibrils in the extracellular matrix.
Currently, we are testing the effect of a variety of
structurally distinct glycosaminoglycans on the rate of
gelsolin amyloidosis. We expect that effective treatment
of gelsolin amyloid disease will involve decreasing the
amount of the amyloidogenic gelsolin fragments. This
decrease can be achieved, for instance, by inhibiting
proteases that cleave mutant gelsolin. Inhibitors of
either or both of these proteases could serve as drugs
for familial amyloidosis of Finnish type.
CHEMICAL CHAPERONES
The N370S mutation in glucocerebrosidase, a lysosomal hydrolase, leads to an accumulation of glucocerebrosidase substrate in lysosomes and consequently to
Gaucher disease, the most common lysosomal storage
disorder. We showed that N-(n-nonyl)deoxynojirimycin
increases the activity of N370S glucocerebrosidase in
a cell line derived from a patient with Gaucher disease.
Most likely, the small molecule acts as a chemical
chaperone for glucocerebrosidase. In other words,
binding of the small molecule stabilizes glucocerebrosidase, allowing the enzyme to be trafficked successfully from the endoplasmic reticulum to the lysosome.
Recently, we examined additional disease-associated
glucocerebrosidase mutants and new classes of small
CHEMISTRY 2005
molecules. We found that some other glucocerebrosidase mutants are also amenable to chemical chaperoning and that several of the compounds tested increased
the activity of multiple mutants.
PUBLICATIONS
Bieschke, J., Zhang, Q., Powers, E.T., Lerner, R.A., Kelly, J.W. Oxidative metabolites accelerate Alzheimer’s amyloidogenesis by a two-step mechanism, eliminating
the requirement for nucleation. Biochemistry 44:4977, 2005.
Deechongkit, S., Dawson, P.E., Kelly, J.W. Toward assessing the position-dependent contributions of backbone hydrogen bonding to β-sheet folding thermodynamics employing amide-to-ester perturbations. J. Am. Chem. Soc. 126:16762, 2004.
83
Total Synthesis,
New Synthetic Technologies,
and Chemical Biology
K.C. Nicolaou, I. Andrews, S. Arseniyadis, W. Brenzovich,
P. Bulgar, G. Carenzi, J. Chen, A. Converso, A. Corbu,
J. Crawford, P. Dagneau, K. Dellios, R. Denton, A. Estrada,
D. Edmonds, R. Faraoni, T. Francis, M. Frederick,
M. Freestone, R. Harbach, D. Harris, S. Harrison, V. Jeso,
Deechongkit, S., Nguyen, H., Powers, E.T., Dawson, P.E., Gruebele, M., Kelly,
J.W. Context-dependent contributions of backbone hydrogen bonding to β-sheet
folding energetics. Nature 430:101, 2004.
Deechongkit, S., Powers, E.T., You, S.-L., Kelly, J.W. Controlling the morphology of
cross β-sheet assemblies by rational design. J. Am. Chem. Soc. 127:8562, 2005.
Foss, T.R., Kelker, M.S., Wiseman, R.L., Wilson, I.A., Kelly, J.W. Kinetic stabilization of the native state by protein engineering: implications for inhibition of transthyretin amyloidogenesis. J. Mol. Biol. 347:841, 2005.
Johnson, S.M., Petrassi, H.M., Palaninathan, S.K., Mohamedmohaideen, N.N.,
Purkey, H.E., Nichols, C., Chiang, K.P., Walkup, T., Sacchettini, J.C., Sharpless,
K.B., Kelly, J.W. Bisaryloxime ethers as potent inhibitors of transthyretin amyloid
fibril formation. J. Med. Chem. 48:1576, 2005.
Petrassi, H.M., Johnson, S.M., Purkey, H.E., Chiang, K.P., Walkup, T., Jiang, X.,
Powers, E.T., Kelly, J.W. Potent and selective structure-based dibenzofuran inhibitors of transthyretin amyloidogenesis: kinetic stabilization of the native state. J.
Am. Chem. Soc. 127:6662, 2005.
Purkey, H.E., Palaninathan, S.K., Kent, K.C., Smith, C., Safe, S.H., Sacchettini,
J.C., Kelly, J.W. Hydroxylated polychlorinated biphenyls selectively bind transthyretin in blood and inhibit amyloidogenesis: rationalizing rodent PCB toxicity. Chem.
Biol. 11:1719, 2004.
Razavi, H., Powers, E.T., Purkey, H.E., Adamski-Werner, S.L., Chiang, K.P., Dendle, M.T.A., Kelly, J.W. Design, synthesis, and evaluation of oxazole transthyretin
amyloidogenesis inhibitors. Bioorg. Med. Chem. Lett. 15:1075, 2005.
Sekijima, Y., Wiseman, R.L., Matteson, J., Hammarström, P., Miller, S.R., Sawkar, A.R.,
Balch, W.E., Kelly, J.W. The biological and chemical basis for tissue-selective amyloid
disease. Cell 121:73, 2005.
Wiseman, R.L., Green, N.S., Kelly, J.W. Kinetic stabilization of an oligomeric protein under physiological conditions demonstrated by a lack of subunit exchange:
implications for transthyretin amyloidosis. Biochemistry 44:9265, 2005.
Wiseman, R.L., Johnson, S.M., Kelker, M.S., Foss, T., Wilson, I.A., Kelly, J.W.
Kinetic stabilization of an oligomeric protein by a single ligand binding event. J.
Am. Chem. Soc. 127:5540, 2005.
You, S.-L., Kelly, J.W. The total synthesis of bistratamides F-I. Tetrahedron
61:241, 2005.
You, S.-L., Kelly, J.W. Total synthesis of didmolamides A and B. Tetrahedron Lett.
46:2567, 2005.
Zhang, Q., Kelly, J.W. Cys-10 mixed disulfide modifications exacerbate transthyretin familial variant amyloidogenicity: a likely explanation for variable clinical
expression of amyloidosis and the lack of pathology in C10S/V30M transgenic
mice? Biochemistry 44:9079, 2005.
F. Kaiser, D. Kim, T. Koftis, S. Lee, T. Ling, D. Lizos,
E. Loizidou, N. Mainolfi, S. Mandal, C. Mathison, R. Milburn,
R. Mogul, A. Nold, R. de Noronha, A. Ortiz, C. Papageorgiou,
L. Pasunoori, G. Petrovic, J. Piper, B. Pratt, A. Roecker,
B. Safina, D. Sarlah, P. Sasmal, C. Schindler, D. Schlawe,
S. Snyder, C. Stathakis, C. Solorio-Alvarado, X. Sun,
W. Tang, C. Turner, H. Xu, M. Zak
e focus on the total synthesis of natural products, the discovery and development of new
synthetic technologies, and chemical biology.
Naturally occurring substances are selected as synthetic
targets because of their novel molecular architectures,
important biological properties, and interesting mechanisms of action. The projects are designed to optimize
the opportunities for discovery and invention in the
areas of chemistry, biology, and medicine. The anticancer
drug paclitaxel (Taxol), the antitumor epothilones, the
neurotoxins brevetoxins A and B, the antibiotic vancomycin, the cholesterol-lowering CP-molecules, the
antibiotic everninomicin, the TNF α–associated trichodimerol, the tetrahydropyran class of natural products, apoptolidin, diazonamide A, thiostrepton, and
azaspiracid-1 exemplify this philosophy. Current projects
include total synthesis of the antibiotic nocathiacin, the
antifeedant azadirachtin, various other azaspiracids,
and the antitumor agents lomaiviticins A and B (Fig. 1).
In addition, we are developing synthetic technologies and strategies for chemical synthesis and chemical biology studies. Our overall aims are to advance
the art and science of chemical synthesis and to develop
enabling technologies for biology and medicine while
maximizing educational opportunities and training of
young men and women in chemistry.
W
PUBLICATIONS
Nicolaou, K.C., Bulger, P.G., Sarlah, D. Metathesis reactions in total synthesis.
Angew. Chem. Int. Ed. 44:4490, 2005.
Nicolaou, K.C., Bulger, P.G., Sarlah, D. Palladium-catalyzed cross-coupling reactions in total synthesis. Angew. Chem. Int. Ed. 44:4442, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
84 CHEMISTRY 2005
Nicolaou, K.C., Sasmal, P.K., Koftis, T.V., Converso, A., Loizidou, E., Kaiser, F.,
Roecker, A.J., Dellios, K., Sun, X.-W., Petrovic, G. Studies toward the synthesis of
azadirachtin, 2: construction of fully functionalized ABCD ring frameworks and
unusual intramolecular reactions induced by close-proximity effects. Angew. Chem.
Int. Ed. 44:3447, 2005.
Nicolaou, K.C., Sasmal, P.K., Roecker, A.J., Sun, X.-W., Mandal, S., Converso, A.
Studies toward the synthesis of azadirachtin, 1: total synthesis of a fully functionalized ABC ring framework and coupling with a norbornene domain. Angew. Chem.
Int. Ed. 44:3443, 2005.
Nicolaou, K.C., Snyder, S.A. Chasing molecules that were never there: misassigned natural products and the role of chemical synthesis in modern structure elucidation. Angew. Chem. Int. Ed. 44:1012, 2005.
Nicolaou, K.C., Snyder, S.A. The essence of total synthesis. Proc. Natl. Acad. Sci.
U. S. A. 101:11929, 2004.
Nicolaou, K.C., Snyder, S.A., Giuseppone, N., Huang, X., Bella, M., Reddy, M.V.,
Rao, P.B., Koumbis, A.E., O’Brate, A., Giannakakou, P. Studies toward diazonamide A: development of a hetero-pinacol macrocyclization cascade for the construction of the bis-macrocyclic framework of the originally proposed structure
[published correction appears in J. Am. Chem. Soc. 126:15316, 2004]. J. Am.
Chem. Soc. 126:10174, 2004.
Nicolaou, K.C., Snyder, S.A., Huang, X., Simonsen, K.B., Koumbis, A.E., Bigot, A.
Studies toward diazonamide A: initial synthetic forays directed toward the originally
proposed structure. J. Am. Chem. Soc. 126:10162, 2004.
Nicolaou, K.C., Snyder, S.A., Longbottom, D.A., Nalbandian, A.Z., Huang, X. New
uses for the Burgess reagent in chemical synthesis: methods for the facile and stereoselective formation of sulfamidates, glycosylamines, and sulfamides. Chemistry
10:5581, 2004.
Nicolaou, K.C., Tang, W., Dagneau, P., Faraoni, R. A catalytic asymmetric threecomponent 1,4-addition/aldol reaction: enantioselective synthesis of the spirocyclic
system of vannusal A. Angew. Chem. Int. Ed. 44:3874, 2005.
Nicolaou, K.C., Vyskocil, S., Koftis, T.V., Yamada, Y.M.A., Ling, T., Chen, D.Y.-K.,
Tang, W., Petrovic, G., Frederick, M.O., Li, Y., Satake, M. Structural revision and
total synthesis of azaspiracid-1, part 1: intelligence gathering and tentative proposal. Angew. Chem. Int. Ed. 43:4312, 2004.
F i g . 1 . Selected target molecules.
Nicolaou, K.C., Carenzi, G.E.A., Jeso, V. Construction of highly functionalized
medium-sized rings: synthesis of hyperforin and perforatumone model systems.
Angew. Chem. Int. Ed. 44:3895, 2005.
Nicolaou, K.C., Chen, D.Y.-K., Huang, X., Ling, T., Bella, M., Snyder, S.A. Chemistry and biology of diazonamide A: first total synthesis and confirmation of the true
structure [published correction appears in J. Am. Chem. Soc. 126:15316, 2004].
J. Am. Chem. Soc. 126:12888, 2004.
Nicolaou, K.C., Xu, H., Wartmann, M. Biomimetic total synthesis of gambogin and
rate acceleration of pericyclic reactions in aqueous media. Angew. Chem. Int. Ed.
44:756, 2005.
Nicolaou, K.C., Zak, M., Safina, B.S., Lee, S.H., Estrada, A.A. Total synthesis of
thiostrepton, 2: construction of the quinaldic acid macrocycle and final stages of
the synthesis. Angew. Chem. Int. Ed. 43:5092, 2004.
Tan, C., de Noronha, R.G., Roecker, A.J., Pyrzynska, B., Khwaja, F., Zhang, Z.,
Zhang, H., Teng, O., Nicholson, A.C., Giannakakou, P., Zhou, W., Olson, J.J., Pereira,
M.M., Nicolaou, K.C., Van Meir, E.G. Identification of a novel small-molecule inhibitor
of the hypoxia-inducible factor 1 pathway. Cancer Res. 65:605, 2005.
Nicolaou, K.C., Estrada, A.A., Zak, M., Lee, S.H., Safina, B.S. A mild and selective method for the hydrolysis of esters employing trimethyltin hydroxide, Angew.
Chem. Int. Ed. 44:1378, 2005.
Nicolaou, K.C., Hao, J., Reddy, M.V., Rao, P.B., Rassias, G., Snyder, S.A., Huang, X.,
Chen, D.Y.-K., Brenzovich, W.E., Giuseppone, N., O’Brate, A., Giannakakou, P.
Chemistry and biology of diazonamide A: second total synthesis and biological investigations [published correction appears in J. Am. Chem. soc. 126:15316]. J. Am. Chem.
Soc. 126:12897, 2004.
Nicolaou, K.C., Koftis, T.V., Vyskocil, S., Petrovic, G., Ling, T., Yamada, Y.M.A., Tang, W.,
Frederick, M.O. Structural revision and total synthesis of azaspiracid-1, part 2: definition
of the ABCD domain and total synthesis. Angew. Chem. Int. Ed. 43:4318, 2004.
Nicolaou, K.C., Lee, S.H., Estrada, A.A., Zak, M. Construction of substituted
N-hydroxyindoles: synthesis of a nocathiacin I model system. Angew. Chem. Int.
Ed. 44:3736, 2005.
Nicolaou, K.C., Montagnon, T., Vassilikogiannakis, G., Mathison, C.J.N. The total
synthesis of coleophomones B, C, and D. J. Am. Chem. Soc. 127:8872, 2005.
Nicolaou, K.C., Safina, B.S., Zak, M., Estrada, A.A., Lee, S.H. Total synthesis of
thiostrepton, 1: construction of the dihydropiperidine/thiazoline-containing macrocycle. Angew. Chem. Int. Ed. 43:5087, 2004.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Chemical, Biological, and
Biophysical Approaches to
Understanding Evolution
F.E. Romesberg, J. Chin, R. Cirz, M. Cremeens, D. Harris, A.
Henry, R. Holmberg, G. Hwang, Y. Kim, A. Leconte, E. Lis, S.
Matsuda, E. Oakman, B. O’Neill, T. Roberts, L. Sagle,
P. Smith, M. Thielges, P. Weinkam, W. Yu, J. Zimmermann
he molecules of biology are unique because they
have been evolved for function. We take a unique
multidisciplinary approach to understanding
these processes.
T
CHEMISTRY 2005
INCREASING THE CHEMICAL AND GENETIC
POTENTIAL OF DNA
Biological information storage is based on the natural
genetic alphabet, composed of the 2 base pairs guaninecytosine and adenine-thymine. We are interested in
increasing the information potential of DNA by expanding the genetic alphabet with a third base pair composed
of unnatural nucleobases. Using hydrophobicity, polarity, shape complementarity, and hydrogen bonding, we
developed several promising unnatural base pairs,
including some that are replicable in vitro. Currently,
we are refining these base pairs and synthesizing and
characterizing additional novel unnatural base pairs.
Nature developed the natural genetic code, not only
by optimizing DNA and RNA but also by evolving the
polymerases that synthesize these nucleic acids. We
developed a selection system that can be used to evolve
polymerases for any desired function. The selection
system is based on the codisplay of DNA polymerases
and their DNA substrates on phage particles (Fig. 1).
Polymerases that can efficiently synthesize DNA containing the unnatural substrates covalently attach a
biotin tag to the corresponding phage particle, allowing selective recovery on a strepavidin solid. Using
this activity-based selection system, we evolved polymerases with a variety of novel functions, including
the synthesis of DNA containing an unnatural base
85
pair. We are optimizing these polymerases and evolving new ones.
DNA DAMAGE RESPONSE
Evolution requires mutation, but mutations also
make cells susceptible to aging and cancer. It is now
understood that at times of sufficient stress, cells
induce error-prone replication to facilitate their own
evolution. We used high-throughput methods to screen
the entire yeast genome for genes involved in both
error-free and error-prone responses to DNA stress. We
identified and characterized a variety of proteins with
functions ranging from cell-cycle control to recombinational repair of stalled replication forks to ubiquitination.
Characterization of the proteins required for mutation
in eukaryotic cells will not only revolutionize our understanding of cancer and aging but also result in identification of targets whose inhibition might actually inhibit
these processes.
Pathogenic bacteria have plagued humanity since
its beginnings. With the advent of antibiotics, many
scientists suggested that this problem had been solved.
However, today, because of their evolution, bacteria
exist that are resistant to all available antibiotics. Consequently, we are also characterizing how prokaryotes
induce mutation, which is required for the evolution of
drug resistance. We have fully characterized the mechanisms in Escherichia coli and are characterizing the
pathways associated with drug resistance in Pseudomonas aeruginosa and Staphylococcus aureus. Perhaps
most exciting, we have initiated efforts to design a drug
that inhibits bacterial mutation and thus evolution.
EVOLUTION OF PROTEIN DYNAMICS
F i g . 1 . Activity-based selection system in which a DNA poly-
merase and its unnatural substrate are codisplayed on the same
phage particle so that activity results in biotinylation and recovery
of active polymerase mutants.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
The products of evolution are molecules with unique
vibrational dynamics. The study of vibrational dynamics
in proteins and nucleic acids has been limited by spectral complexity, but selective deuteration of a protein or
a nucleic acid results in a carbon-deuterium oscillator
that absorbs light in an otherwise transparent region
of the infrared spectrum. The synthesis of selectively
deuterated proteins has provided us with a residue-specific probe of flexibility, function, and folding. We are
also using multidimensional femtosecond spectroscopy
to characterize how protein motion is evolved during the
somatic evolution of antibodies (Fig. 2). We quantitatively showed that somatic evolution systematically
evolves an antibody from a flexible receptor into a more
rigid receptor and that the immune system can manipulate protein dynamics, findings that suggest a role for
these dynamics in molecular recognition.
86 CHEMISTRY 2005
F i g . 2 . Structural and dynamic data are used to understand how
antibodies are evolved for molecular recognition.
PUBLICATIONS
Cirz, R.T., Chin, J.K., Andes, D.R., de Crecy-Lagard, V., Craig, W.A., Romesberg,
F.E. Inhibition of mutation and combating the evolution of antibiotic resistance.
PLoS Biol. 3:e176, 2005.
Henry, A.A., Jimenez, R., Hanway, D., Romesberg, F.E. Preliminary characterization of light harvesting in E. coli DNA photolyase. Chembiochem 5:1088, 2004.
Holmberg, R.C., Henry, A.A., Romesberg, F.E. Directed evolution of novel polymerases. Biomol. Eng. 22:39, 2005.
Matsuda, S., Romesberg, F.E. Optimization of interstrand hydrophobic packing interactions within unnatural DNA base pairs. J. Am. Chem. Soc. 126:14419, 2004.
O’Neill, B.M., Hanway D., Winzeler, E.A., Romesberg, F.E. Coordinated functions
of WSS1, PSY2, and TOF1 in the DNA damage response. Nucleic Acids Res.
32:6519, 2004.
Romesberg, F.E., Schowen, R.L. Isotope effects and quantum tunneling in enzyme-catalyzed hydrogen transfer, I: the experimental basis. Adv. Phys. Org. Chem. 39:27, 2004.
Biological Chemistry
P.G. Schultz, L. Alfonta, E. Brustad, S. Chen, J. Chitturulu,
C. Cho, J. Graziano, J. Grbic, D. Groff, J. Hong, W.Y. Hur,
M. Jahnz, J. Lee, K.-B. Lee, J. Liao, J. Liu, H. Luesch,
F. Marr, S. Matsuda, J. Melnick, J. Mills, K.H. Min,
M. Mukherji, B. Okram, Y. Ryu, S. Schiller, D. Summerer,
L. Supekova, E. Tippmann, M.-L. Tsao, J. Turner, J. Wang,
A. Willingham, J. Xie, H. Zeng, Q. Zhang
A
lthough chemists are remarkably adept at synthesizing molecular structures, they are far less
sophisticated in designing and synthesizing mol-
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
ecules with defined biological or chemical functions.
Nature, on the other hand, has produced an array of
molecules with remarkably complex functions, ranging
from photosynthesis and signal transduction to molecular recognition and catalysis. Our aim is to combine
the synthetic strategies and biological processes of
Nature with the tools and principles of chemistry to
create new molecules with novel chemical and biological functions. By studying the properties of the resulting molecules, we hope to gain new insights into the
molecular mechanisms of complex biological and
chemical systems.
For example, we have shown that the tremendous
combinatorial diversity of the immune response can be
chemically reprogrammed to generate selective enzymelike catalysts. We have developed antibodies that catalyze
a wide array of chemical and biological reactions, from
acyl transfer to redox reactions. Characterization of the
structure and mechanisms of these catalytic antibodies has led to important new insights into the mechanisms of biological catalysis. In addition, the detailed
characterization of the properties and structures of
germ-line and affinity-matured antibodies is revealing
fundamental new aspects of the evolution of binding
and catalytic function, in particular, the role of structural
plasticity in the immune response. Most recently, we
have focused on in vitro evolution methods that involve
the development of novel chemical screens and selections for identifying mutants with enhanced function
and the rational design of proteolytic antibodies.
Our work on catalytic antibodies redirects natural
combinatorial diversity to produce new function. We
are extending this combinatorial approach to many
other problems, including the generation of sequencespecific recombinases, small-molecule regulators of
DNA transcription, and the ab initio evolution of novel
protein domains. We are also generating structure-based
combinatorial libraries of small molecules, including
purine, pyrimidine, and fatty acid derivatives. These
libraries are being used in conjunction with novel cellular and organismal screens to identify important proteins
involved in such cellular processes as differentiation,
proliferation, and signaling. Indeed, we have identified
molecules that control stem cell differentiation and
self-renewal and that dedifferentiate lineage-committed cells. We are using x-ray crystallography and biochemical studies, together with genomics experiments
with gene chip array technology and genetic complementation, to characterize the mode of action of these
CHEMISTRY 2005
compounds and to study their effects on cellular processes. We are also developing modern genomics tools
(cell-based phenotypic screens of arrayed genomic cDNA
and small interfering RNA libraries) and proteomics tools
(mass spectrometric phosphoprotein profiling) and are
applying them to a variety of significant biomedical problems in cancer biology, neurodegenerative disease, aging,
and virology.
We have also developed a general biosynthetic
method that can be used to site specifically incorporate unnatural amino acids into proteins in vitro and
in vivo. Using this method, we have effectively expanded
the genetic codes of bacteria and yeast by adding new
components to the biosynthetic machinery of living cells.
We have added amino acids with novel spectroscopic
and chemical properties (e.g., keto- and heavy atom–
containing amino acids, photocross-linking and photoisomerizable amino acids) to the genetic codes of
Escherichia coli, yeast, and mammalian cells. Our
results have removed a billion-year constraint imposed
by the genetic code on the ability to chemically manipulate the structures of proteins.
PUBLICATIONS
Ding, S., Schultz, P.G. A role for chemistry in stem cell biology. Nat. Biotechnol.
22:833, 2004.
Liu, J., Bang, A., Kintner, C., Orth, A.P., Chanda, S.K., Ding, S., Schultz, P.G.
Identification of the Wnt signaling activator leucine-rich repeat in Flightless interaction
protein 2 by a genome-wide functional analysis. Proc. Natl. Acad. Sci. U. S. A.
102:1927, 2005.
Wang, L., Schultz, P.G. Expanding the genetic code. Angew. Chem. Int. Ed.
44:34, 2004.
Wu, X., Walker, J., Zhang, J., Ding, S., Schultz, P.G. Purmorphamine induces
osteogenesis by activation of the hedgehog signaling pathway. Chem. Biol.
11:1229, 2004.
Xie, J., Wang, L., Wu, N., Brock, A., Spraggon, G., Schultz, P.G. The site-specific
incorporation of p-iodo-L-phenylalanine into proteins for structure determination.
Nat. Biotechnol. 22:1297, 2004.
Catalysis and Click Chemistry
K.B. Sharpless, V.V. Fokin, B. Boren, M. Cassidy,
B. Colasson, T. Chan, A. Feldman, R. Fraser, T.V. Hansen,
B. Hatano, T. Hirose, A. Krasinski, Y. Liu, J. Loren,
R. Manetsch, A. McPherson, S. Narayan, S. Pitram,
L.K. Rasmussen, J. Raushel, S. Röper, W. Sharpless,
S. Silverman, A. Sugawara, X. Wang, J. Wassenaar,
M. Whiting, P. Wu
T
he aim of our research program is the development of reliable chemical transformations that
allow rapid exploration of chemical space. Our
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
87
goal is to find new molecules with desired functions,
whether in medicinal chemistry, materials science, or
organic synthesis.
CLICK CHEMISTRY
The success of any search for new molecules with
desired properties often depends on the degree of diversity of the blocks that are used in synthesis: the greater
the variety of structures and functional groups that can
be used in the construction of candidate compounds,
the more likely it is that useful function will be discovered. However, the number and the sophistication of
methods that allow synthesis of truly diverse collections of compounds still leave much to be desired. The
problems are often due to at least one, and usually a
combination, of the following: limited scope; hard-toobtain starting materials; requirements for inert atmospheres, anhydrous solvents, and protecting groups; and
difficult purifications.
Because organic and medicinal chemistry primarily evolved from the desire to explore and learn from
the chemistry of life, organic and medicinal chemists
naturally favored reactions that are similar to the prototypical biosynthetic pathways. These pathways center on the transformations of the carbonyl group;
Nature’s primary carbon starting material is carbon
dioxide, so exquisite enzymatic pathways evolved to
bring about these essentially thermoneutral transformations. Because we lack these stratagems, we rely
on a more useful and available set of starting materials: a plethora of unsaturated hydrocarbons provided
by the petrochemical industry.
In the past several years, we sought to develop and
use only the best connecting reactions for the synthesis of functional molecules. We coined the term click
chemistry to describe the reactions that fulfill the most
stringent criteria of usefulness and convenience. Most
click reactions form carbon-heteroatom bonds, are tolerant of water, and are often accelerated when water
is used as the sole medium (even if the reagents are
not soluble in water).
C O P P E R - C ATA LY Z E D C Y C L O A D D I T I O N S
Among the best click processes are 1,3-dipolar
cycloadditions, especially formation of 1,2,3-triazoles
from organic azides and alkynes. These modular “fusion”
reactions unite 2 unsaturated reactants and result in
an enormous variety of useful heterocycles. The recently
discovered copper-catalyzed cycloaddition of azides
and terminal alkynes is, arguably, the most convenient
and reliable way to irreversibly fuse a broad variety of
88 CHEMISTRY 2005
blocks by means of the 1,2,3-triazole connection, a
link that is notably stable and inert to severe hydrolytic,
reducing, and oxidizing conditions. Although both alkynes
and azides are highly reactive, their chemoselectivity
profiles are quite narrow, that is, “orthogonal” to an
unusually broad range of reagents, solvents, and other
functional groups. These features allow reliable and
clean sequential transformations of broad scope without the need for any protecting groups.
The uncatalyzed, thermal reaction of azides and
alkynes is often slow, requires elevated temperatures,
and results in mixtures of 2 regioisomeric products.
Therefore, we were pleased to find that copper(I) catalyzes this process, accelerating it by a factor of up to
107 and resulting in regiospecific union of azides and
terminal acetylenes to give only 1,4-disubstituted-[1,2,3]triazoles. The process is experimentally simple and has
enormous scope (Fig. 1).
F i g . 2 . Synthesis of the bis-triazole catalyzed by metallic copper.
The reactants were dissolved in a 1:1 mixture of tert-butanol and
water (left) and were stirred for 24 hours. The reaction was complete then, and the product triazoles were isolated in quantitative
yield by filtration.
F i g . 1 . Copper(I)-catalyzed synthesis of 1,4-disubstituted1,2,3-triazoles.
Although a number of copper(I) sources can be used
directly, we found that the catalyst is often better prepared in situ by reduction of copper(II) salts, which are
less costly and often purer than copper(I) salts. As the
reductant, ascorbic acid (vitamin C) or sodium ascorbate is excellent. Remarkably, even copper metal can
be used as a source of the catalytic species, making
the experimental procedure even simpler; pure triazoles
can be obtained in almost quantitative yield after simply stirring the corresponding azide and alkyne components in water with a small amount of copper turnings
(Fig. 2).
In collaboration with M.G. Finn, Department of
Chemistry, we developed a practical method based on
this high-fidelity transformation to selectively label large
protein structures without affecting their integrity. The
procedure has been used by many researchers around
the world for activity-based profiling of whole proteomes,
selective labeling of bacterial cell walls, and preparation of various bioconjugates. We also discovered that
the ligand tris-(benzyltriazolylmethyl) amine (Fig. 3)
enables efficient coupling of azides and alkynes even
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
F i g . 3 . Tris-(benzyltriazolylmethyl) amine.
in extremely dilute solutions contaminated with myriad
other molecules. The ligand is readily synthesized by
the very same copper-catalyzed transformation and represents a new class of 1,2,3-triazole–derived tripodal
ligands. To date, it has been used in more than 50
laboratories worldwide.
Together with a team of researchers from IBM, San
Jose, California, and the University of California, Santa
Barbara, led by C. Hawker, we devised a high-efficiency
approach to synthesis of dendrimers that is based on
the copper(I)-catalyzed synthesis of triazoles. The
unique properties of these macromolecules, which are
a direct consequence of their regular structure, have
attracted much attention in recent years. However, their
syntheses are often plagued by low yields and tedious
purification. Our approach results in almost quantitative
yields, and, in many instances, simple filtration or solvent extraction is the only method required for purification (Fig. 4). These features represent a significant
CHEMISTRY 2005
89
PUBLICATIONS
Chan, T.R., Hilgraf, R., Sharpless, K.B., Fokin, V.V. Polytriazoles as copper(I)-stabilizing ligands in catalysis. Org. Lett. 6:2853, 2004.
Colasson, B., Feldman, A.K., Sharpless, K.B., Fokin, V.V. The allylic azide
rearrangement: a dynamic [3,3] process. J. Am. Chem. Soc., in press.
Converso, A., Saaidi, P.-L., Sharpless, K.B., Finn, M.G. Nucleophilic substitution
by Grignard reagents on sulfur mustards. J. Org. Chem. 69:7336, 2004.
Díaz, D.D., Punna, S., Holzer, P., McPherson, A.K., Sharpless, K.B., Fokin, V.V.,
Finn, M.G. Click chemistry in materials synthesis, I: adhesive polymers from coppercatalyzed azide-alkyne cycloaddition. J. Polym. Sci. A Polym. Chem. 42:4392, 2004.
Feldman, A.K., Colasson, B., Fokin, V.V. One-pot synthesis of 1,4-disubstituted
1,2,3-triazoles from in situ generated azides. Org. Lett. 6:3897, 2004.
Himo, F., Lovell, T., Hilgraf, R., Rostovtsev, V.V., Noodleman, L., Sharpless, K.B.,
Fokin, V.V. Copper(I)-catalyzed synthesis of azoles: DFT study predicts unprecedented reactivity and intermediates. J. Am. Chem. Soc. 127:210, 2005.
F i g . 4 . Synthesis of 1,2,3-triazole-based dendrimers.
advancement in dendrimer chemistry and illustrate an
evolving synergy between organic chemistry and functional materials.
In collaboration with scientists at Scripps Research
who are involved in finding novel inhibitors of the HIV
type 1 protease, we used the copper-catalyzed cycloaddition to rapidly synthesize a library of potential inhibitors of this crucial enzyme. The absence of byproducts
and almost quantitative yields allowed the direct screening of compounds in a microtiter plate–based assay.
Several compounds with Ki values ranging from 5 to
50 nM, both against the native protease and its mutants,
were identified (Fig. 5).
Johnson, S.M., Petrassi, H.M., Palaninathan, S.K., Mohamedmohaideen, N.N.,
Purkey, H.E., Nichols, C., Chiang, K.P., Walkup, T., Sacchettini, J.C., Sharpless,
K.B., Kelly, J.W. Bisaryloxime ethers as potent inhibitors of transthyretin amyloid
fibril formation. J. Med. Chem. 48:1576, 2005.
Krasinski, A., Radic, Z., Manetsch, R., Raushel, J., Taylor, P., Sharpless, K.B., Kolb,
H.C. In situ selection of lead compounds by click chemistry: target-guided optimization
of aceylcholinesterase inhibitors. J. Am. Chem. Soc. 127:6686, 2005.
Lewis, W.G., Magallon, F., Fokin, V.V., Finn, M.G. Discovery and characterization
of catalysts for azide-alkyne cycloaddition by fluorescence quenching. J. Am. Chem.
Soc. 126:9152, 2004.
Loren, J.C., Sharpless, K.B. The Banert cascade: a synthetic sequence to polyfunctional NH-1,2,3-triazoles. Synthesis, in press.
Manetsch, R., Krasinski, A., Radic, Z., Raushel, J., Taylor, P., Sharpless, K.B.,
Kolb, H.C. In situ click chemistry: enzyme inhibitors made to their own specifications. J. Am. Chem. Soc. 126:12809, 2004.
Mocharla, V.P., Colasson, B., Lee, L.V., Röper, S., Sharpless, K.B., Wong, C.-H.,
Kolb, H.C. In situ click chemistry: enzyme-generated inhibitors of carbonic anhydrase II. Angew. Chem. Int. Ed. 44:116, 2004.
Narayan, S., Muldoon, J., Finn, M.G., Fokin, V.V., Kolb, H.C., Sharpless, K.B.
“On water”: unique reactivity of organic compounds in aqueous suspensions.
Angew. Chem. Int. Ed. 44:3275, 2005.
Wu, P., Feldman, A.K., Nugent, A.K., Hawker, C.J., Scheel, A., Voit, B., Pyun, J.,
Fréchet, J.M.J., Sharpless, K.B., Fokin, V.V. Efficiency and fidelity in a click chemistry route to triazole dendrimers via the copper(I)-catalyzed ligation of azides and
alkynes. Angew. Chem. Int. Ed. 43:3928, 2004.
Chemistry, Biology, and Disease
P. Wentworth, Jr., Y. Chen, L. Eltepu, R. Galvé, R.K. Grover,
F i g . 5 . Synthesis of inhibitors of HIV type 1 protease and its
J. Nieva, M. Puga, A. Shafton, B.D. Song, M.M.R. Peram,
mutants.
C. Takeuchi, S. Tripurenani, R. Troseth, K. Trygvasson,
Studies of other applications, ranging from biology
to materials science, are under way in our laboratories
and in collaboration with M.G. Finn, P.K. Vogt, and
C.-H. Wong, Department of Chemistry; J.H. Elder,
Department of Molecular Biology; and others.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
H. Wang, A.D. Wentworth
ur research is multidisciplinary and involves
bioorganic, biophysical, physical organic, synthetic, and analytical chemistry coupled with
biochemical techniques, cell-based assays, and animal
models. These diverse approaches are combined to facilitate a better understanding of and generate new thera-
O
90 CHEMISTRY 2005
peutic approaches to complex disease states. Ongoing
projects include studies on atherosclerosis, neurodegeneration, ischemia-reperfusion injury, macular degeneration, cancer, inflammation, and infectious diseases.
T H E A N T I B O D Y - C ATA LY Z E D WAT E R O X I D AT I O N
PAT H WAY
Antibodies are the classical adapter molecules of
the immune system, linking recognition and killing of
foreign pathogens. However, we recently discovered
that all antibody molecules, regardless of source or
antigenic specificity, can catalyze the reaction between
singlet oxygen and water to give hydrogen peroxide.
This reaction is being studied as a possibly new effector function of the immune system. Both the chemical
and the biological aspects of this pathway are being
explored intensively, and intriguing new insights into
how it may play a role in immune defense and inflammatory damage are emerging.
In collaboration with I.A. Wilson, Department of
Molecular Biology, we studied, at atomic resolution,
the structural modifications of an antibody Fab fragment that was used as the antibody in the antibodycatalyzed water oxidation pathway. X-ray analysis
revealed surprisingly few but conserved oxidative modifications to certain residues within the Fab structure
(Fig. 1). The most consistent modification is a regioselective hydroxylation of the amino acid tryptophan at
position L163. Such a conserved modification suggests
that the active site of the antibody-catalyzed water oxidation pathway may be close to position L163 and thus
offers an area for site-directed mutagenesis to investigate this observation.
One of the most controversial aspects of this pathway is our assertion that trioxygen species may be generated as intermediates and/or byproducts. To increase our
experimental evidence for such intermediates, we are
generating libraries of molecular probes that are being
tested for both their specificity for ozone in a biological setting and for their usefulness in cell-based assays.
C H E M I S T R Y O F T H E P O LY O X I D E D I H Y D R O G E N
TRIOXIDE
Chemical processes by which molecules containing
polyoxides are generated are of considerable interest.
Although the reaction between hydrogen peroxide and
ozone is a time-honored chemical process, because of
the complexity of the overall mechanism, intermediates
formed when these 2 molecules react are still incompletely characterized. These intermediates recently
became central to biological thinking because of our
discovery that antibodies catalyze the oxidation of
water by singlet oxygen, leading to the formation of
hydrogen peroxide, and it has been proposed that dihydrogen trioxide may be formed by antibodies during the
formation of hydrogen peroxide. In support of this
hypothesis, we recently showed that dihydrogen trioxide is formed in measurable amounts during the thermal
reaction between ozone and hydrogen peroxide (Fig. 2).
Overlaid 1H nuclear magnetic resonance spectra of the
peroxone reaction (A) and the peroxone reaction and authentic dihydrogen trioxide (B). Inset shows the structure of the triplet biradical intermediate theorized as being generated during the reaction
between ozone and hydrogen peroxide.
Fig. 2.
F i g . 1 . Crystal structure of 4C6 Fab prepared after photoirradi-
ation with ultraviolet light shows the Cα trace of the light (L, light
gray) and heavy (H, dark gray) chains. Inset shows Fourier electron density map for tryptophan at position L163 in the 4C6 Fab.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
CHEMISTRY 2005
91
This first experimental report of a link between these 3
oxidants suggests that dihydrogen trioxide may be
involved in oxidation reactions that span biological,
atmospheric, and environmental systems. We are investigating new methods for the formation of dihydrogen trioxide and the chemical properties of this novel oxidant.
inflammatory artery disease. Thus, the atheronal molecules may be a new thread in the already entangled
relationship between cholesterol oxidation, macrophages,
and atherosclerosis.
CHOLESTEROL SECO-STEROLS AND
The ultimate aim of the search for genetic and environmental factors that increase the propensity of a
specific protein to misfold is the understanding and
treatment of disease states as diverse as atherosclerosis, light-chain deposition disease, systemic amyloidosis, Alzheimer’s disease, and Parkinson’s disease. We
recently discovered that the inflammation-derived
atheronal-A and atheronal-B can trigger a deformation
in the secondary structure of the normally folded protein apolipoprotein B-100 to a proamyloidogenic form.
This protein is a component of low-density lipoprotein
particles and is misfolded within atherosclerotic arteries, although the cause had never been known. Therefore, this misfolding of apolipoprotien B-100 induced
by cholesterol seco-sterols is a new link between
inflammation, cholesterol oxidation, protein misfolding,
and atherosclerosis. Our goal is to expand this observation and explore its relevance in many disease states
and to generate molecules that prevent it.
In collaboration with J.W. Kelly, Department of
Chemistry, we showed that these cholesterol seco-sterols
also trigger the misfolding of amyloid β-peptide (1–40),
leading to formation of fibrils similar to those observed
in patients with Alzheimer’s disease. Interestingly,
analysis of the structure-activity relationship revealed
that among a panel of aldehydes, only atheronal-A,
atheronal-B, and 4-hydroxynonenal triggered this misfolding of amyloid β-peptide, suggesting that both electrophilicity and hydrophobicity of the adducting aldehyde
are critical structural aspects.
AT H E R O S C L E R O S I S
We recently discovered that the 5,6-seco-sterols
atheronal-A and atheronal-B of a class of cholesterol
ozonolysis products are present in human atherosclerotic plaques and plasma (Fig. 3). In addition, we
found that the atheronals are present in murine models of atherosclerosis and have a range of biological
properties that, in combination, would increase the
local density of macrophages at sites of vascular inflammation. Atheronal-A and atheronal-B are both chemotactic for cultured macrophages. When in complex with
low-density lipoproteins, atheronal-A induces upregulation of the cell-surface adhesion molecule E-selectin
on vascular endothelial cells.
F i g . 3 . Cholesterol seco-sterols atheronal-A and atheronal-B are
present within the walls of inflamed arteries and in plasma. Bottom
left, Immunohistochemistry of aortic valve atherosclerosis in an aged
mouse that lacked the gene for apolipoprotein E. The primary antibody is a murine monoclonal antibody to atheronal-B, revealing high
localization of atheronal-B in the artery wall. Bottom right, Lipid
accumulation within primary macrophages is triggered by low-density lipoprotein in complex with atheronal-B.
Taken together with our previously shown effects
of atheronals on the formation of foam cells and the
cytotoxic effects of macrophages, these data indicate
that the atheronals have biological effects that would
lead to the recruitment, entrapment, dysfunction, and
ultimate destruction of the major leukocyte player in
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
I N F L A M M AT O R Y A L D E H Y D E - M E D I AT E D P R O T E I N
MISFOLDING
PUBLICATIONS
Ahn, J.-M., Wentworth, P., Jr., Janda, K.D. Probing lipase/esterase libraries for
lipid A hydrolases: discovery of biocatalysts for the detoxification of bacteriallyexpressed recombinant protein. Chem. Commun. (Camb.) 364, 2004, Issue 4.
Chen, Y.P., Eltepu, L., Wentworth, P., Jr. Diastereo- and enantio-selective crotylation of
α-ketoesters using crotyl boronic acid ester complexes. Tetrahedron Lett. 45:8285, 2004.
Granville, D.J., Tashakkor, B., Takeuchi, C., Gustafsson, Å.B., Huang, C., Sayen,
M.R., Wentworth, P., Jr., Yaeger, M., Gottlieb, R.A. Reduction of ischemia and
reperfusion-induced myocardial damage by cytochrome P450 inhibitors. Proc.
Natl. Acad. Sci. U. S. A. 101:1321, 2004.
Nieva, J., Wentworth, P., Jr. The antibody-catalyzed water oxidation pathway: a
new chemical arm to immune defense? Trends Biochem. Sci. 29:274, 2004.
Nyffeler, P., Boyle, N.A., Eltepu, L., Wong, C.-H., Eschenmoser, A., Lerner, R.A.,
Wentworth, P., Jr. Dihydrogen trioxide (HOOOH) is generated during the thermal reaction between hydrogen peroxide and ozone. Angew. Chem. Int. Ed. 43:4656, 2004.
92 CHEMISTRY 2005
Takeuchi, C.T., Wentworth, P., Jr. The antibody-catalyzed water oxidation pathway.
In: Catalytic Antibodies. Keinan, E. (Ed.). Wiley & Sons, New York, 2004, p. 336.
Toker, J.D., Tremblay, M., Wentworth, P., Jr., Janda, K.D. Investigating the scope of the
29G12 antibody-catalyzed 1,3-dipolar cycloaddition reaction. J. Org. Chem., in press.
outside the active site. We are investigating how the
remote changes affect the enantioselectivity in the
active site.
DEVELOPMENT OF INHIBITORS OF ENZYMES AND
Wentworth, A.D., Wentworth, P., Jr., Blackburn, G.M. Transition state analogs
archetype antigens for catalytic antibody generation. In: Catalytic Antibodies.
Keinan, E. (Ed.). Wiley & Sons, New York, 2004, p. 454.
Zhang, Q., Powers, E.T., Nieva, J., Huff, M.E., Dendle, M.A., Bieschke, J., Glabe,
C.G., Eschenmoser, A., Wentworth, P., Jr., Lerner, R.A., Kelly, J.W. Metabolite-initiated protein misfolding may trigger Alzheimer’s disease. Proc. Natl. Acad. Sci.
U. S. A. 101:4752, 2004.
Zhu, X., Wentworth, P., Jr., Wentworth, A.D., Lerner, R.A., Wilson, I.A. Probing
the antibody-catalyzed water-oxidation pathway at atomic resolution. Proc. Natl.
Acad. Sci. U. S. A. 101:2247, 2004.
Bioorganic and Synthetic
Chemistry
C.-H. Wong, C. Behrens, M. Best, A. Brik, M. Fujio, S.
Hanson, Z.-Y. Hong, J. Hsu, C.-Y. Huang, D.-R. Hwang,
A. Krebs, J.-C. Lee, F.-S. Liang, H. Liu, L. Liu, M. Numa,
T. Polat, M. Sawa, P. Schanen, M. Sugiyama, D. Thayer,
S.-K. Wang, L. Whalen, C.-Y. Wu, D. Wu, M. Wuchrer,
Y.-Y. Yang
ur research programs involve development of
new chemical and enzymatic strategies and
methods for the synthesis of biologically active
compounds. We use the synthesized materials as molecular probes to explore carbohydrate-mediated biological recognitions, sequence-specific RNA recognition,
and enzymatic reactions.
O
ORGANIC AND BIOORGANIC SYNTHESIS
Our work in organic and bioorganic synthesis
includes the development of new chemical reactions
and the exploitation of native and engineered enzymes
for organic synthesis. In the past year, we developed
several new synthetic methods. These include development of covalent glycoarrays for high-throughput
analysis of protein-carbohydrate interactions, the use
of aldolases in synthesis of glycosyltransfer enzyme
inhibitors, and enzymatic synthesis of glycoproteins.
Using directed evolution, we developed new aldolase
variants capable of making both enantiomers of sugars.
In collaboration with P.G. Schultz, Department of Chemistry, we evolved a tyrocyl-tRNA synthase to accept
N-acetylgalactosamine α-linked to the side chain of
threonine for incorporation into proteins in vivo in
Escherichia coli. All the mutations found in the new
aldolases and the aminoacyl-tRNA synthase occur
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
RECEPTORS
Our goals in the area of enzyme and receptor inhibitors are to develop new strategies and discover potential new therapeutic agents with high selectivity. Current
strategies involve the design and synthesis of structureand mechanism-based inhibitors of enzymes associated with diseases. Targets for investigation include
bacterial transglycosidase, sulfotransferases, retroviral
proteases, the lethal factor of Bacillus anthracis, and
the enzymes involved in the biosynthesis of carbohydrates essential for biological functions. We developed
new iminocyclitols and derivatives as inhibitors of glycosidases and glycosyltransferases for potential treatment of inflammatory diseases. In addition, we used a
new strategy based on a rapid microscale synthesis
coupled with in situ high-throughput screening to develop
new tight-binding inhibitors of anthrax lethal factor,
a sulfotransferase, and drug-resistant HIV proteases.
We also developed new reactions based on tetrabutylammonium fluoride–mediated N- and O-alkylation in
aqueous solution and used the reactions to identify
potent enzyme inhibitors. Finally, we designed and
synthesized novel aminoglycoside mimetics that target
unique bacterial and oncogenic RNA sequences as
potential new antibiotics and anticancer agents.
C A R B O H Y D R AT E C H E M I S T R Y A N D M O L E C U L A R
G LY C O B I O L O G Y
We continue to improve the programmable 1-pot
oligosaccharide synthesis method for convenient and
rapid preparation of oligosaccharides. So far, we have
designed approximately 600 building blocks and measured the anomeric reactivity of each building block.
Using the computer program OptiMer, developed in
our laboratory, we rapidly assembled a number of
oligosaccharides. We are using this method to define
the specificity of interactions between carbohydrates
and their receptors, with particular focus on optimization of the cancer antigen Globo H and gp120 oligomannose as vaccine candidates and development of
aminoglycosides to target specific RNA sequences.
In collaboration with D.R. Burton, Department of
Immunology, and I.A. Wilson, Department of Molecular
Biology, we are evaluating a designed oligomannoseprotein conjugate as an antigen to elicit antibodies for
neutralizing HIV gp120 and variants. We prepared
CHEMISTRY 2005
some bacterial glycolipids and analogs and found that
they are active ligands for CD cell markers involved in
activation of human natural killer T cells. We also prepared several heparin derivatives and glycoproteins for
investigation of their structures and function. In collaboration with J.C. Paulson, Department of Molecular Biology, we developed new methods for microfabrication
of saccharides on microtiter plates and glass slides for
use in the high-throughput analysis of sugar-protein
interactions. We also developed new methods for the
discovery of enzyme inhibitors.
PUBLICATIONS
Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M.E., Alvarez, R., Bryan,
M.C., Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, D.J., Shekel, J.J.,
van Die, I., Burton, D.R., Wilson, I.A., Cummings, R., Bovin, N., Wong, C.-H.,
Paulson, J.C. Printed covalent glycan array for ligand profiling of diverse glycan
binding proteins. Proc. Natl. Acad. Sci. U. S. A. 101:17033, 2004.
Brik, A., Alexandratos, J., Lin, Y.-C., Elder, J.H., Olson, A.J., Wlodawer, A., Goodsell, D.S., Wong, C.-H. 1,2,3-Triazole as a peptide surrogate in the rapid synthesis
of HIV-1 protease inhibitors. Chembiochem 6:1167, 2005.
93
Hsu, C.-C., Hong, Z., Wada, M., Franke, D., Wong, C.-H. Directed evolution of
D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase. Proc.
Natl. Acad. Sci. U. S. A. 102:9122, 2005.
Hsu, H.-Y., Hua, K.-F., Lin, C.-C., Lin, C.-H., Wong, C.-H. Extract of Reishi polysaccharides induces cytokine expression on TLR4-modulated protein kinase signaling pathways. J. Immunol. 173:5989, 2004.
Kinjo, Y., Wu, D., Kim, G., Xing, G.-W., Poles, M.A., Ho, D.D., Tsuji, M., Kawahara, K., Wong, C.-H., Kronenberg, M. Recognition of bacterial glycosphingolipids
by natural killer T cells. Nature 434:520, 2005.
Klostermeier, D., Sears, P., Wong, C.-H., Millar, D.P., Williamson, J.R. A threefluorophore FRET assay for high-throughput screening of small-molecule inhibitors
of ribosome assembly. Nucleic Acids Res. 32:2707, 2004.
Liang, F.-S., Wang, S.-K., Nakatani, T., Wong, C.-H. Targeting RNAs with tobramycin
analogues. Angew. Chem. Int. Ed. 43:6496, 2004.
Liang, F.-S., Wong C.-H. Surface plasmon resonance study of HCV IRES RNAaminoglycoside interactions. Methods Mol. Biol., in press.
Lin, H., Thayer, D.A., Wong, C.-H., Walsh, C.T. Macrolactamization of glycosylated peptide thioesters by the thioesterase domain of tyrocidine synthetase. Chem.
Biol. 11:1635, 2004.
Liu, J., Numa, M.M.D., Liu, H., Huang, S.-J., Sears, P., Shikhman, A.R., Wong,
C.-H. Novel synthesis and high-throughput screening of N-acetyl-β-hexosaminidase
inhibitor libraries targeting osteoarthritis. J. Org. Chem. 69:6273, 2004.
Brik, A., Wu, C.-Y., Best, M.D., Wong, C.-H. Tetrabutylammonium fluorideassisted rapid N9-alkylation on purine ring: application to combinatorial reactions
in microtiter plates for the discovery of potent sulfotransferase inhibitors in situ.
Bioorg. Med. Chem. 13:4622, 2005.
Mocharla, V.P., Colasson, B., Lee, L.V., Romper, S., Sharpless, K.B., Wong, C.-H.,
Kolb, H.C. In situ click chemistry: enzyme-generated inhibitors of carbonic anhydrase II. Angew. Chem. Int. Ed. 44:116, 2004.
Bryan, M.C., Fazio, F., Lee, H.-K., Huang, C.-Y., Chang, A., Best, M.D., Calarese,
D.A., Blixt, O., Paulson, J.C., Burton, D., Wilson, I.A., Wong, C.-H. Covalent display
of oligosaccharide arrays in microtiter plates. J. Am. Chem. Soc. 126:8640, 2004.
Numa, M.M.D., Lee, L.V., Hsu, C.-C., Bower, K.E., Wong, C.-H. Identification of
novel anthrax lethal factor inhibitors generated by combinatorial Pictet-Spengler
reaction followed by screening in situ. Chembiochem 6:1002, 2005.
Bryan, M.C., Lee, L.V., Wong, C.-H. High-throughput identification of fucosyltransferase
inhibitors using carbohydrate microarrays. Bioorg. Med. Chem. Lett. 14:3185, 2004.
Nyffeler, P.T., Duron, S.G., Burkart, M.D., Vincent, S.P., Wong, C.-H. Selectflour:
mechanistic insight and applications. Angew. Chem. Int. Ed. 44:192, 2005.
Chang, C.-F., Ho, C.-W., Wu, C.-Y., Chao, T.A., Wong, C.-H., Lin, C.-H. Discovery
of picomolar slow tight-binding inhibitors of α-fucosidase [published correction
appears in Chem. Biol. 11:1595, 2004]. Chem. Biol. 11:1301, 2004.
Nyffeler, P.T., Eltepu, L., Boyle, N.A., Wong, C.-H., Eschenmoser, A., Lerner,
R.A., Wentworth, P., Jr. Dihydrogen trioxide (HOOOH) is generated during the thermochemical reaction between hydrogen peroxide and ozone. Angew. Chem. Int.
Ed. 43:4656, 2004.
Chen, H.-S., Tsai, Y.-F., Lin, S., Lin, C.-C., Khoo, K.-H., Lin, C.-H., Wong, C.-H.
Studies on the immuno-modulating and anti-tumor activities of Ganoderma
lucidum (Reishi) polysaccharides. Bioorg. Med. Chem. 12:5595, 2004.
Chen, J.H., Chang, Y.-W., Yao, C.-W., Chiueh, T.-S., Huang, S.-C., Chien, K.-Y.,
Chen, A., Chang, F.-Y., Wong, C.-H., Chen, Y.-J. Plasma proteome of severe acute
respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass
spectrometry. Proc. Natl. Acad. Sci. U. S. A. 101:17039, 2004.
Chien, C.M., Cheng, J.-L., Chang, W.-T., Tien, M.-H., Tsao, C.M., Chang, Y.-H.,
Chang, H.-Y., Hsieh, J.-F., Wong, C.-H., Chen, S.-T. Polysaccharides of Ganoderma
lucidum alter cell immunophenotypic expression and enhance CD56+ NK-cell
cytotoxicity in cord blood. Bioorg. Med. Chem. 12:5603, 2004.
Fan, G.-T., Pan. Y.-S., Lu, K.-C., Cheng, Y.-P., Lin, W.-C., Lin, S., Lin, C.-H.,
Wong, C.-H., Fang, J.-M., Lin, C.-C. Synthesis of α-galactosyl ceramide and the
related glycolipids for evaluation of their activities on mouse splenocytes. Tetrahedron 61:1855, 2005.
Franke, D., Hsu, C.-C., Wong, C.-H. Directed evolution of aldolases. Methods
Enzymol. 388:224, 2004.
Fridman, M., Belakhov, V., Lee, L.V., Liang, F.-S., Wong, C.-H., Baasov, T. Dual
effect of synthetic aminoglycosides: antibacterial activity against Bacillus anthracis
and inhibition of anthrax lethal factor. Angew. Chem. Int. Ed. 44:447, 2005.
Hanson, S.R., Best, M., Bryan, M.C., Wong, C.-H. Chemoenzymatic synthesis of
oligosaccharides and glycoproteins. Trends Biochem. Sci. 29:656, 2004.
Hanson, S.R., Best, M.D., Wong, C.-H. Sulfatases: structure, mechanism, biological
activity, inhibition, and synthetic utility. Angew. Chem. Int. Ed. 43:5736, 2004.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Shie, J.-J., Fang, J.-M., Kuo, C.-J., Kuo, T.H., Liang, P.-H., Huang, H.-J., Yang,
W.-B., Lin, C.-H., Chen, J.-L., Wu, Y.T., Wong, C.-H. Discovery of potent anilide
inhibitors against the severe acute respiratory syndrome 3CL protease. J. Med.
Chem. 48:4469, 2005.
Thayer, D.A., Yu, H.N., Galan, M.C., Wong, C.-H. A general strategy toward S-linked
glycopeptides. Angew. Chem. Int. Ed. 44:4596, 2005.
Tolbert, T.J., Franke, D., Wong, C.-H. A new strategy for glycoprotein synthesis:
ligation of synthetic glycopeptides with truncated proteins expressed in E. coli as
TEV protease cleavable fusion protein. Bioorg. Med. Chem. 13:909, 2005.
Tolbert, T.J., Wong, C.-H. Carbohydrate chains: enzymatic and chemical synthesis.
In: Encyclopedia of Biological Chemistry. Lennarz, W.J., Lane, M.D. (Eds.). Academic Press, San Diego, 2004, Vol. 1, p. 307.
Tolbert, T.J., Wong, C.-H. Conjugation of glycopeptide thioesters to expressed protein fragments: semisynthesis of glycosylated interleukin-2. Methods Mol. Biol.
283:255, 2004.
Tolbert, T.J., Wong, C.-H. Subtilisin-catalyzed glycopetide condensation. Methods
Mol. Biol. 283:267, 2004.
Wong, C.-H. Protein glycosylation: new challenges and opportunities. J. Org.
Chem. 70:4219, 2005.
Wu, C.-Y., Jan, J.-T., Ma, S.-H., Kuo, C.-J., Juan, H.-F., Cheng, Y.-S.E., Hsu, H.-H.,
Huang, H.-C., Wu, D., Brik, A., Liang, F.-S., Liu, R.-S., Fang, J.-M., Chen, S.-T.,
Liang, P.-H., Wong, C.-H. Small molecules targeting severe acute respiratory syndrome human coronavirus. Proc. Natl. Acad. Sci. U. S. A. 101:10012, 2004.
94 CHEMISTRY 2005
Wu, D., Xing, G.-W., Poles, M.A., Horowitz, A., Kinjo, Y., Sullivan, B., BodmerNarkevitch, V., Plettenburg, O., Kronenberg, M., Tsuji, M., Ho, D.D., Wong, C.-H.
Bacterial glycolipids and analogs as antigens for CD1d-restricted NKT cells. Proc.
Natl. Acad. Sci. U. S. A. 102:1351, 2005.
Xing, G.-W., Wu, D., Poles, M.A., Horowitz, A., Tsuji, M., Ho, D.D., Wong, C.-H.
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