Methods in Enzymology Vol 369: Combinatorial Chemistry, Part B

Methods in Enzymology Volume 369 COMBINATORIAL CHEMISTRY Part B

METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF

John N. Abelson

Melvin I. Simon

DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA

FOUNDING EDITORS

Sidney P. Colowick and Nathan O. Kaplan

Methods in Enzymology Volume 369

Combinatorial Chemistry Part B

EDITED BY

Guillermo A. Morales Barry A. Bunin LIBRARIA, INC. SAN JOSE, CALIFORNIA

This book is printed on acid-free paper. Copyright ß 2003, Elsevier Inc. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2003 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0076-6879/2003 $35.00 Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting ‘‘Customer Support’’ and then ‘‘Obtaining Permissions.’’

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Table of Contents

Contributors to Volume 369

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Volumes in Series . . . . . . . . . . . . . . . . . . . . .

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Preface

Section I. Analytical Techniques 1. High-Throughput Parallel LC/UV/MS Analysis Liling Fang, Jiang Zhao, of Combinatorial Libraries and Bing Yan

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2. Qualitative Colorimetric Tests for Solid Phase Jesu´s Va´zquez, Gregory Synthesis Qushair, and Fernando Albericio

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Section II. Directed Sorting Split-and-Pool Combinatorial Synthesis 3. A Review of Solid-Phase Organic Synthesis on Jack G. Parsons, Craig S. SynPhase‘ Lanterns and SynPhase‘ Crowns Sheehan, Zemin Wu, Ian W. James, and Andrew M. Bray 39 4. Directed Sorting Approach for the Synthesis of Timothy F. Herpin and Large Combinatorial Libraries of Discrete George C. Morton Compounds ´ rpa´d Furka, James W. 5. Split-Mix Synthesis Using Macroscopic Solid A Support Units Christensen, and Eric Healy 6. The Encore Technique: A Novel Approach Viktor Krchnˇa´k and to Directed Split-and-Pool Combinatorial Vı´tecˇek Padeˇra Synthesis

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Section III. Linkers and Their Applications 7. Multifunctional Linkers as an Efficient Tool Stefan Bra¨se, Stefan for the Synthesis of Diverse Small Molecule Dahmen, and Matthias Libraries: The Triazene Anchors E. P. Lormann 8. The Development and Application of Joseph M. Salvino and Tetrafluorophenol-Activated Resins for Roland E. Dolle Rapid Amine Derivatization

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9. The Traceless Solid-Phase Synthesis of Organic David Tumelty, Yijun Pan, Molecules and Christopher P. Holmes 164 10. Unnatural Scaffolds Linkers Chemical

Diamino Acid Derivatives as Robert Pascal, Re´gine Sola, 182 for Creating Diversity and as and Patrick Jouin for Simplifying Screening in Libraries

Section IV. Microwave-Assisted Synthesis 11. Building Dihydropyrimidine Microwave-Assisted Multicomponent Reactions

Libraries via C. Oliver Kappe and Biginelli Alexander Stadler

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12. Microwave-Assisted Solid-Phase Organic Andrew P. Combs, Brian M. Synthesis (MA-SPOS) of Oxazolidinone Glass, and Sharon A. 223 Antimicrobials Jackson

Section V. Oligosaccharide Chemistry 13. Automated Synthesis of Polysaccharides

Obadiah J. Plante, Emma R. 235 Palmacci, and Peter H. Seeberger

14. Solid-Phase Oligosaccharide Chemistry and Its Matthias Grathwohl, Application to Library Synthesis Nicholas Drinnan, Max Broadhurst, Michael L. West, and Wim Meutermans 248

Section VI. Peptide Synthesis and Screening 15. Design, Synthesis, Screening, and Decoding Ruiwu Liu, Jan Marik, and 271 of Encoded One-Bead One-Compound Kit S. Lam Peptidomimetic and Small Molecule Combinatorial Libraries 16. Intelligent Design in Combinatorial Chemistry: Scott M. Cowell, Xuyuan Use of Designed Peptide Libraries to Gu, Josef Vagner, and Explore Secondary and Tertiary Structures Victor J. Hruby in Peptides and Proteins

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17. Synthesis and Screening of ‘‘One-Bead One- Kit S. Lam, Alan L. Lehman, Compound’’ Combinatorial Peptide Libraries Aimin Song, Ninh Doan, Amanda M. Enstrom, and 298 Ruiwu Liu

table of contents 18. Synthetic Combinatorial Libraries as an Sylvie E. Blondelle, Alternative Strategy for the Development Clemencia Pinilla, and of Novel Treatments for Infectious Diseases Ce´sar Boggiano

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Section VII. Polymer-Assisted Approaches 19. Advances in the Applications of Polymer- Alessandra Bartolozzi, Supported Reagents for Organic Synthesis Michael J. Grogan, and Steven A. Kates

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20. Advanced Polymer Reagents Based on Jo¨rg Rademann Activated Reactants and Reactive Intermediates: Powerful Novel Tools in Diversity-Oriented Synthesis

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21. Scavenger Resins CombiChem

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Solution-Phase J. Gabriel Garcia

Section VIII. Small Molecule and Heterocycle Synthesis 22. Cyclative Cleavage Strategies for the Solid- A. Ganesan Phase Synthesis of Heterocycles and Natural Products

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23. Derivatization Reactions of Heterocyclic Eduard R. Felder, Scaffolds on Solid Phase: Tools for the Wolfgang K.-D. Brill, and 435 Synthesis of Drug-Like Molecule Libraries Katia Martina 24. Library Generation via Post condensation Christopher Hulme, Hugues Modifications of Isocyanide-Based Multi- Bienayme´, Thomas Nixey, component Reactions Balan Chenera, Wyeth Jones, Paul Tempest, and 469 Adrian L. Smith 25. Mixture-Based Combinatorial Libraries: From Peptides and Peptidomimetics to Small Molecule Acyclic and Heterocyclic Compounds

Cornelia E. Hoesl, Adel Nefzi, John M. Ostresh, Yongping Yu, and Richard 496 A. Houghten

26. New Strategies for the Solid-Phase Synthesis of Florencio Zaragoza Highly Functionalized, Small Molecules: Sequential Nucleophilic Substitutions on Polymer-Bound Polyelectrophiles

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Author Index . . . . . . . . . . . . . . . . . . . . . .

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Subject Index . . . . . . . . . . . . . . . . . . . . . .

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Contributors to Volume 369 Article numbers are in parentheses and following the names of contributors. Affiliations listed are current.

Fernando Albericio (2), University of Barcelona, Barcelona Biomedical Research Institute, Barcelona Science Park, Josep Samitier 1, Barcelona, 08028, Spain

Balan Chenera (24), Amgen Inc., Department of Small Molecule Drug Discovery, One Amgen Center Drive, Thousand Oaks, California, 91320 James W. Christensen (5), Advanced ChemTech Inc., 5609 Fern Valley Road, Louisville, Kentucky, 40228

Alessandra Bartolozzi (19), Surface Logix, Inc., 50 Soldiers Field Place, Brighton, Massachusetts, 02135

Andrew P. Combs (12), Incyte Corporation, Wilmington, Delaware, 19880-0500

Hugues Bienayme´ (24), Chrysalon Molecular Research, IRC, 11 Albert Einstein Avenue, Villeurbannem, 69100, France

Scott M. Cowell (16), Department of Chemistry, University of Arizona, Tucson, Arizona, 85721

Sylvie E. Blondelle (18), Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California, 92121

Stefan Dahmen (7), Institut fur Organische Chemie, RWTH Aachen, PirletStr. 1, Aachen, 52074, Germany

Ce´sar Boggiano (18), Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California, 92121

Ninh Doan (17), Division of Hematology and Oncology, Department of Internal Medicine, UC Davis Cancer Center, University of California Davis, Sacramento, California, 95817

Stefan Bra¨se (7), Institut fu¨r Organische Chemie, Universita¨t Karlsruhe (TH), Fritz-Haber-Weg 6, Karlsruhe, D-76131, Germany

Roland E. Dolle (8), Senior Director of Chemistry, Department of Chemistry, Adolor Corporation, 700 Pennsylvania Drive, Exton, Pennsylvania, 19345

Andrew M. Bray (3), Mimotopes Pty Ltd., 11 Duerdin Street, Clayton, Victoria, 3168, Australia

Nicholas Drinnan (14), Alchemia Pty Ltd., Eight Mile Plains, Queensland 4113, Australia

Wolfgang K.-D. Brill (23), Discovery Research Oncology, Pharmacia Italy S.p.A, Viale Pasteur 10, Nerviano (MI), I-20014, Italy

Amanda M. Enstrom (17), Division of Hematology and Oncology, Department of Internal Medicine, UC Davis Cancer Center, University of California Davis, Sacramento, California, 95817

Max Broadhurst (14), Alchemia Pty Ltd., Eight Mile Plains, Queensland 4113, Australia

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contributors to volume 369

Liling Fang (1), ChemRx Division, Discovery Partners International, 385 Oyster Point Boulevard, Suite 1, South San Francisco, California, 94080

Richard Houghten (25), Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, Room 2-136, San Diego, California, 92121

Eduard R. Felder (23), Discovery Research Oncology, Pharmacia Italy S.p.A., Viale Pasteur 10, Nerviano (MI), I-20014, Italy

Victor J. Hruby (16), Department of Chemistry, University of Arizona, Tucson, Arizona, 85721

´ rpa´d Furka (5), Eo¨tvo¨s Lora´nd UniverA sity, Department of Organic Chemistry, P.O. Box 32, Budapest 112, H-1518, Hungary A. Ganesan (22), University of Southampton, Department of Chemistry, Highfield, Southampton, SO17 1BJ, United Kingdom J. Gabriel Garcia (20), 4SC AG, Am Klopferspitz 19A, 82152, Martinsried, Germany Brian Glass (13), Incyte Corporation, Wilmington, Delaware, 19880-0500 Matthias Grathwohl (14), Alchemia Pty Ltd., Eight Mile Plains, Queenland 4113, Australia Michael J. Grogan (19), Surface Logix, Inc., 50 Soldiers Field Place, Brighton, Massachusetts, 02135 Xuyuan Gu (16), Department of Chemistry, University of Arizona, Tuscon, Arizona, 85721 Eric Healy (5), Advanced ChemTech Inc., 5609 Fern Valley Road, Louisville, Kentucky, 40228 Timothy F. Herpin (4), Rhoˆne-Poulenec Rorer, 500 Arcola Road, Collegeville, Pennsylvania, 19426 Cornelia E. Hoesl (25), Torrey Pines Institute, Room 2-136, 3550 General Atomics Court, San Diego, California, 92121 Christopher P. Holmes (9), Affymax Inc., 4001 Miranda Avenue, Palo Alto, California, 94304

Christopher Hulme (24), Amgen Inc., Department of Small Molecule Drug Discovery, One Amgen Center Drive, 29-1-B, Thousand Oaks, California, 91320 Sharon A. Jackson (12), Aventis Pharmaceuticals, 202-206, Bridgewater, New Jersey, 08807-0800 Ian W. James (3), Mimotopes Pty Ltd., 11 Duerdin Street, Clayton, Victoria, 3168, Australia Wyeth Jones (24), Amgen Inc., Department of Small Molecule Drug Discovery, One Amgen Center Drive, 29-1-B, Thousand Oaks, California, 91320 Patrick Jouin (10), CNRS UPR 9023, CCIPE, 141, rue de la Cardonille, Montpellier Cedex 05, 34094, France C. Oliver Kappe (11), Institute of Chemistry, Karl-Franzens-University Graz, Heinrichstrasse 28, Graz, A-8010, Austria Steven A. Kates (19), Surface Logix, Inc., 50 Soldiers Field Place, Brighton, Massachusetts, 02135 Viktor Krchnˇa´k (6), Torviq, 3251 West Lambert Lane, Tuscon, Arizona, 85742 Kit S. Lam (15, 17), Division of Hematology and Oncology, Department of Internal Medicine, UC Davis Cancer Center, University of California Davis, Sacramento, California, 95817 Alan L. Lehman (17), Division of Hematology and Oncology, Department of Internal Medicine, UC Davis Cancer Center, University of California Davis, Sacramento, California, 95817

contributors to volume 369 Ruiwu Liu (15, 17), Division of Hematology and Oncology, Department of Internal Medicine, UC Davis Cancer Center, University of California Davis, Sacramento, California, 95817 Matthias Lormann (7), Kekule´-Institut fu¨r Organische Chemie und Biochemie der Rheinischen, Friedrich Wilhelms Universita¨t Bonn, Gerhard-Domagk-Strasse 1, Bonn, D-53121, Germany

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E.R. Palmacci (13), 77 Massachusetts Avenue, T18-209, Cambridge, Massachusetts, 02139 Yijun Pan (9), Affymax Inc., 4001 Miranda Avenue, Palo Alto, California, 94304 Jack G. Parsons (3), Mimotopes Pty Ltd., 11 Duerdin Street, Clayton, Victoria, 3168, Australia

Jan Marik (15), Division of Hematology and Oncology, Department of Internal Medicine, UC Davis Cancer Center, University of California Davis, Sacramento, California, 95817

Robert Pascal (10), UMR 5073, Universite´ de Montpellier 2, CC017, place Euge`ne Bataillon, Montpellier Cedex 05, F-34094, France

Katia Martina (23), Discovery Research Oncology, Pharmacia Italy S.p.A., Viale Pasteur 10, Nerviano (MI), I-20014, Italy

Clemencia Pinilla (18), Torrey Pines Institute for Molecular Studies and Mixture Sciences, Inc., 3550 General Atomics Court, San Diego, California, 92121

Joeseph Maxwell (17), Division of Hematology and Oncology, Department of Internal Medicine, UC Davis Cancer Center, University of California Davis, Sacramento, California, 95817

Obadiah J. Plante (13), Massachusetts Institute of Technology, Department of Chemistry, 77 Massachusetts Avenue, Cambridge, Massachusetts, 02139-4307

Wim Meutermans (14), Alchemia Pty Ltd., 3 Hi-Tech Court, Brisbane Technology Park, Eight Mile Plains, QLD 4113, Australia

Gregory Qushair (2), University of Barcelona, Barcelona Biomedical Research Institute, Barcelona Science Park, Josep Samitier 1, Barcelona, 08028, Spain

George C. Morton (4), Rhoˆne-Poulenc Rorer, 500 Arcola Road, Collegeville, Pennsylvania, 19426 Adel Nefzi (25), Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California, 92121 Thomas Nixey (24), Amgen Inc., Department of Small Molecule Drug Discovery, One Amgen Center Drive, 29-1-B, Thousand Oaks, California, 91320 John M. Ostresh (25), Torrey Pines Institute, Room 2-136, 3550 General Atomics Court, San Diego, California 92121 Vitecek Padeˇra (6), Torvic, 3251 W Lambert Lane, Tucson, Arizona, 84742

Jorg Rademann (21), Eberhard-Karls-University, Tu¨bingen, Institute of Organic Chemistry, Auf der Morgenstelle 18, Tu¨bingen, 72076, Germany Joseph M. Salvino (8), Director of Combinational Chemistry, Adolor Corporation, 700 Pennsylvania Drive, Exton, Pennsylvania, 19345 Peter H. Seeberger (13), Laboratorium fuer Organische Chemie, HCI F 315, Wolfgang-Pauli-Str. 10, ETH-Hoenggerberg, CH-8093 Zu¨rich, Switzerland Craig S. Sheehan (3), Mimotopes Pty Ltd., 11 Duerdin Street, Clayton, Victoria, 3168, Australia

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contributors to volume 369

Adrian L. Smith (24), Amgen Inc., Department of Small Molecule Drug Discovery, One Amgen Center Drive, Thousand Oaks, California, 91320 Re´gine Sola (10), UMR 5076, Ecole Nationale Supe´rieure de Chimie de Montpellier, 8, rue Delaware l’Ecole Normale, Montpellier Cedex 05, F34296, France Aimin Song (17), University of California, UC Davis Cancer Center, Division of Hematology and Oncology, 4501 X Street, Sacramento, California, 95817 Alexander Stadler (11), Institute of Chemistry, Karl-Franzens-University Graz, Heinrichstrasse 28, Graz, A-8010, Austria Paul Tempest (24), Amgen Inc., Department of Small Molecule Drug Discovery, One Amgen Center Drive, 29-1-B, Thousand Oaks, California, 91320 David Tumelty (9), Affymax, Inc., 4001 Miranda Avenue, Palo Alto, California, 94304 Josef Vagner (16), Department of Chemistry, University of Arizona, Tuscon, Arizona, 85741

Jesus Vazquez (2), University of Barcelona, Barcelona Biomedical Research Institute, Barcelona Science Park, Josep Samitier 1, Barcelona, 08028, Spain Michael L. West (14), Alchemia Pty Ltd., Eight Mile Plains, Queensland 4113, Australia Zemin Wu (3), Mimotopes Pty Ltd., 11 Duerdin Street, Clayton, Victoria, 3168, Australia Bing Yan (1), ChemRx Division, Discovery Partners International, 385 Oyster Point, Boulevard, Suite 1, South San Francisco, California, 94080 Yongping Yu (25), Torrey Pines Institute, Room 2-136, 3550 General Atomics Court, San Diego, California, 92121 Florencio Zaragoza (26), Medicinal Chemistry, Novo Nordisk A/S, Novo Nordisk Park, Malov, 2760, Denmark Jiang Zhao (1), ChemRx Division, Discovery Partners International, 385 Oyster Point Boulevard, Suite 1, South San Francisco, California, 94080

Preface

Combinatorial chemistry has matured from a field where efforts initially focused on peptide-based research to become an indispensable research tool for molecular recognition, chemical-property optimization, and drug discovery. Originally used as a method to primarily generate large numbers of molecules, combinatorial chemistry has been significantly influenced and integrated with other important fields such as medicinal chemistry, analytical chemistry, synthetic chemistry, robotics, and computational chemistry. Even though the initial focus of attention was providing larger numbers of molecules with a ‘‘diversity’’ goal in mind, other factors came into play depending upon the problem scientists were trying to solve, such as bioactivity, solubility, permeability properties, PK, ADME, toxicity, and patentability. One can think of combinatorial chemistry and compound screening as an iterative Darwinian process of divergence and selection. Particularly in drug discovery, where time is a critical factor to success, combinatorial chemistry offers the means to test more molecule hypotheses in parallel. We will always be limited to a finite number of molecules that we can economically synthesize and evaluate. Even with all the advances in automation technologies, combinatorial chemistry, and higher-throughput screens that improve our ability to rapidly confirm or disprove hypotheses, the synthesis and screening cycle remains the rate-determining process. Fortunately, we continue to make great strides forward in the quality and refinement of predictive algorithms and in the breadth of the training sets amassed to aid in the drug discovery/compound optimization iterative process. Anyone who has optimized chemical reactions for combinatorial libraries or process chemistry knows first hand how much experimentation is required to identify optimal conditions. Chemical feasibility is at the heart of small molecule discovery and chemotype prioritization since it essentially defines what can and cannot be analoged (i.e., analogability). Although analogability is not the only driving factor, quite often it is overlooked. For example, when commercially-available compounds or complex natural products are screened, the leads generated are often dropped because of the difficulty to rapidly analog them in the lead optimization stage. The desirability of a chemotype is a function of drug-likeness, potency, novelty, and analogability. A particularly attractive feature of combinatorial chemistry is that when desirable properties are identified, they can often be xiii

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optimized through second-generation libraries following optimized synthetic protocols. If this process of exploring truly synthetically accessible chemical spaces could be automated, then it would open up the exciting possibility of modeling the iterative synthesis and screening cycle. Predicting, or even just mapping, synthetic feasibility is a sleeping giant; few people are looking into it, and the ramifications of a breakthrough would be revolutionary for both chemistry and drug discovery. In-roads to predicting (or even just mapping) chemical feasibility have the potential to have as large an impact on drug discovery as computational models of bioavailability and drugability. These are important questions where scientists are now starting to generate a large-enough body of information on high-throughput synthetic chemistry to begin to more globally understand what is cost-effectively possible. Within the biopharmaceutical industry, significant investments in new technologies have been made in molecular biology, genomics, and proteomics. However, with the exception of combinatorial chemistry, relatively little has been done to advance the fundamental nature of chemistry in drug discovery from a conceptual perspective. Now, after having gone through the molecule-generating period where research institutions have a large historical compound collection and the proliferation of combinatorial chemistry services, the trend is now after making more targeted-oriented molecular entities also known as ‘‘focused libraries.’’ An important emerging question is: How can one most effectively make the best possible ‘‘focused libraries’’ to answer very specific research questions, given all the possible molecules one could theoretically synthesize? The first installment in this series (Volume 267, 1996) mostly covered peptide and peptidomimetic based research with just a few examples of small molecule libraries. In this volume we have compiled cutting-edge research in combinatorial chemistry, including divergent areas such as novel analytical techniques, microwave-assisted synthesis, novel linkers, and synthetic approaches in both solid-phase and polymer-assisted synthesis of peptides, small molecules, and heterocyclic systems, as well as the application of these technologies to optimize molecular properties of scientific and commercial interest.

Guillermo A. Morales Barry A. Bunin

METHODS IN ENZYMOLOGY Volume I. Preparation and Assay of Enzymes Edited by Sidney P. Colowick and Nathan O. Kaplan Volume II. Preparation and Assay of Enzymes Edited by Sidney P. Colowick and Nathan O. Kaplan Volume III. Preparation and Assay of Substrates Edited by Sidney P. Colowick and Nathan O. Kaplan Volume IV. Special Techniques for the Enzymologist Edited by Sidney P. Colowick and Nathan O. Kaplan Volume V. Preparation and Assay of Enzymes Edited by Sidney P. Colowick and Nathan O. Kaplan Volume VI. Preparation and Assay of Enzymes (Continued) Preparation and Assay of Substrates Special Techniques Edited by Sidney P. Colowick and Nathan O. Kaplan Volume VII. Cumulative Subject Index Edited by Sidney P. Colowick and Nathan O. Kaplan Volume VIII. Complex Carbohydrates Edited by Elizabeth F. Neufeld and Victor Ginsburg Volume IX. Carbohydrate Metabolism Edited by Willis A. Wood Volume X. Oxidation and Phosphorylation Edited by Ronald W. Estabrook and Maynard E. Pullman Volume XI. Enzyme Structure Edited by C. H. W. Hirs Volume XII. Nucleic Acids (Parts A and B) Edited by Lawrence Grossman and Kivie Moldave Volume XIII. Citric Acid Cycle Edited by J. M. Lowenstein Volume XIV. Lipids Edited by J. M. Lowenstein Volume XV. Steroids and Terpenoids Edited by Raymond B. Clayton

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Volume XVI. Fast Reactions Edited by Kenneth Kustin Volume XVII. Metabolism of Amino Acids and Amines (Parts A and B) Edited by Herbert Tabor and Celia White Tabor Volume XVIII. Vitamins and Coenzymes (Parts A, B, and C) Edited by Donald B. McCormick and Lemuel D. Wright Volume XIX. Proteolytic Enzymes Edited by Gertrude E. Perlmann and Laszlo Lorand Volume XX. Nucleic Acids and Protein Synthesis (Part C) Edited by Kivie Moldave and Lawrence Grossman Volume XXI. Nucleic Acids (Part D) Edited by Lawrence Grossman and Kivie Moldave Volume XXII. Enzyme Purification and Related Techniques Edited by William B. Jakoby Volume XXIII. Photosynthesis (Part A) Edited by Anthony San Pietro Volume XXIV. Photosynthesis and Nitrogen Fixation (Part B) Edited by Anthony San Pietro Volume XXV. Enzyme Structure (Part B) Edited by C. H. W. Hirs and Serge N. Timasheff Volume XXVI. Enzyme Structure (Part C) Edited by C. H. W. Hirs and Serge N. Timasheff Volume XXVII. Enzyme Structure (Part D) Edited by C. H. W. Hirs and Serge N. Timasheff Volume XXVIII. Complex Carbohydrates (Part B) Edited by Victor Ginsburg Volume XXIX. Nucleic Acids and Protein Synthesis (Part E) Edited by Lawrence Grossman and Kivie Moldave Volume XXX. Nucleic Acids and Protein Synthesis (Part F) Edited by Kivie Moldave and Lawrence Grossman Volume XXXI. Biomembranes (Part A) Edited by Sidney Fleischer and Lester Packer Volume XXXII. Biomembranes (Part B) Edited by Sidney Fleischer and Lester Packer Volume XXXIII. Cumulative Subject Index Volumes I-XXX Edited by Martha G. Dennis and Edward A. Dennis Volume XXXIV. Affinity Techniques (Enzyme Purification: Part B) Edited by William B. Jakoby and Meir Wilchek

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Volume XXXV. Lipids (Part B) Edited by John M. Lowenstein Volume XXXVI. Hormone Action (Part A: Steroid Hormones) Edited by Bert W. O’Malley and Joel G. Hardman Volume XXXVII. Hormone Action (Part B: Peptide Hormones) Edited by Bert W. O’Malley and Joel G. Hardman Volume XXXVIII. Hormone Action (Part C: Cyclic Nucleotides) Edited by Joel G. Hardman and Bert W. O’Malley Volume XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems) Edited by Joel G. Hardman and Bert W. O’Malley Volume XL. Hormone Action (Part E: Nuclear Structure and Function) Edited by Bert W. O’Malley and Joel G. Hardman Volume XLI. Carbohydrate Metabolism (Part B) Edited by W. A. Wood Volume XLII. Carbohydrate Metabolism (Part C) Edited by W. A. Wood Volume XLIII. Antibiotics Edited by John H. Hash Volume XLIV. Immobilized Enzymes Edited by Klaus Mosbach Volume XLV. Proteolytic Enzymes (Part B) Edited by Laszlo Lorand Volume XLVI. Affinity Labeling Edited by William B. Jakoby and Meir Wilchek Volume XLVII. Enzyme Structure (Part E) Edited by C. H. W. Hirs and Serge N. Timasheff Volume XLVIII. Enzyme Structure (Part F) Edited by C. H. W. Hirs and Serge N. Timasheff Volume XLIX. Enzyme Structure (Part G) Edited by C. H. W. Hirs and Serge N. Timasheff Volume L. Complex Carbohydrates (Part C) Edited by Victor Ginsburg Volume LI. Purine and Pyrimidine Nucleotide Metabolism Edited by Patricia A. Hoffee and Mary Ellen Jones Volume LII. Biomembranes (Part C: Biological Oxidations) Edited by Sidney Fleischer and Lester Packer Volume LIII. Biomembranes (Part D: Biological Oxidations) Edited by Sidney Fleischer and Lester Packer

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Volume LIV. Biomembranes (Part E: Biological Oxidations) Edited by Sidney Fleischer and Lester Packer Volume LV. Biomembranes (Part F: Bioenergetics) Edited by Sidney Fleischer and Lester Packer Volume LVI. Biomembranes (Part G: Bioenergetics) Edited by Sidney Fleischer and Lester Packer Volume LVII. Bioluminescence and Chemiluminescence Edited by Marlene A. DeLuca Volume LVIII. Cell Culture Edited by William B. Jakoby and Ira Pastan Volume LIX. Nucleic Acids and Protein Synthesis (Part G) Edited by Kivie Moldave and Lawrence Grossman Volume LX. Nucleic Acids and Protein Synthesis (Part H) Edited by Kivie Moldave and Lawrence Grossman Volume 61. Enzyme Structure (Part H) Edited by C. H. W. Hirs and Serge N. Timasheff Volume 62. Vitamins and Coenzymes (Part D) Edited by Donald B. McCormick and Lemuel D. Wright Volume 63. Enzyme Kinetics and Mechanism (Part A: Initial Rate and Inhibitor Methods) Edited by Daniel L. Purich Volume 64. Enzyme Kinetics and Mechanism (Part B: Isotopic Probes and Complex Enzyme Systems) Edited by Daniel L. Purich Volume 65. Nucleic Acids (Part I) Edited by Lawrence Grossman and Kivie Moldave Volume 66. Vitamins and Coenzymes (Part E) Edited by Donald B. McCormick and Lemuel D. Wright Volume 67. Vitamins and Coenzymes (Part F) Edited by Donald B. McCormick and Lemuel D. Wright Volume 68. Recombinant DNA Edited by Ray Wu Volume 69. Photosynthesis and Nitrogen Fixation (Part C) Edited by Anthony San Pietro Volume 70. Immunochemical Techniques (Part A) Edited by Helen Van Vunakis and John J. Langone Volume 71. Lipids (Part C) Edited by John M. Lowenstein

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Volume 72. Lipids (Part D) Edited by John M. Lowenstein Volume 73. Immunochemical Techniques (Part B) Edited by John J. Langone and Helen Van Vunakis Volume 74. Immunochemical Techniques (Part C) Edited by John J. Langone and Helen Van Vunakis Volume 75. Cumulative Subject Index Volumes XXXI, XXXII, XXXIV–LX Edited by Edward A. Dennis and Martha G. Dennis Volume 76. Hemoglobins Edited by Eraldo Antonini, Luigi Rossi-Bernardi, and Emilia Chiancone Volume 77. Detoxication and Drug Metabolism Edited by William B. Jakoby Volume 78. Interferons (Part A) Edited by Sidney Pestka Volume 79. Interferons (Part B) Edited by Sidney Pestka Volume 80. Proteolytic Enzymes (Part C) Edited by Laszlo Lorand Volume 81. Biomembranes (Part H: Visual Pigments and Purple Membranes, I) Edited by Lester Packer Volume 82. Structural and Contractile Proteins (Part A: Extracellular Matrix) Edited by Leon W. Cunningham and Dixie W. Frederiksen Volume 83. Complex Carbohydrates (Part D) Edited by Victor Ginsburg Volume 84. Immunochemical Techniques (Part D: Selected Immunoassays) Edited by John J. Langone and Helen Van Vunakis Volume 85. Structural and Contractile Proteins (Part B: The Contractile Apparatus and the Cytoskeleton) Edited by Dixie W. Frederiksen and Leon W. Cunningham Volume 86. Prostaglandins and Arachidonate Metabolites Edited by William E. M. Lands and William L. Smith Volume 87. Enzyme Kinetics and Mechanism (Part C: Intermediates, Stereo-chemistry, and Rate Studies) Edited by Daniel L. Purich Volume 88. Biomembranes (Part I: Visual Pigments and Purple Membranes, II) Edited by Lester Packer Volume 89. Carbohydrate Metabolism (Part D) Edited by Willis A. Wood

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Volume 90. Carbohydrate Metabolism (Part E) Edited by Willis A. Wood Volume 91. Enzyme Structure (Part I) Edited by C. H. W. Hirs and Serge N. Timasheff Volume 92. Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods) Edited by John J. Langone and Helen Van Vunakis Volume 93. Immunochemical Techniques (Part F: Conventional Antibodies, Fc Receptors, and Cytotoxicity) Edited by John J. Langone and Helen Van Vunakis Volume 94. Polyamines Edited by Herbert Tabor and Celia White Tabor Volume 95. Cumulative Subject Index Volumes 61–74, 76–80 Edited by Edward A. Dennis and Martha G. Dennis Volume 96. Biomembranes [Part J: Membrane Biogenesis: Assembly and Targeting (General Methods; Eukaryotes)] Edited by Sidney Fleischer and Becca Fleischer Volume 97. Biomembranes [Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts)] Edited by Sidney Fleischer and Becca Fleischer Volume 98. Biomembranes (Part L: Membrane Biogenesis: Processing and Recycling) Edited by Sidney Fleischer and Becca Fleischer Volume 99. Hormone Action (Part F: Protein Kinases) Edited by Jackie D. Corbin and Joel G. Hardman Volume 100. Recombinant DNA (Part B) Edited by Ray Wu, Lawrence Grossman, and Kivie Moldave Volume 101. Recombinant DNA (Part C) Edited by Ray Wu, Lawrence Grossman, and Kivie Moldave Volume 102. Hormone Action (Part G: Calmodulin and Calcium-Binding Proteins) Edited by Anthony R. Means and Bert W. O’Malley Volume 103. Hormone Action (Part H: Neuroendocrine Peptides) Edited by P. Michael Conn Volume 104. Enzyme Purification and Related Techniques (Part C) Edited by William B. Jakoby Volume 105. Oxygen Radicals in Biological Systems Edited by Lester Packer Volume 106. Posttranslational Modifications (Part A) Edited by Finn Wold and Kivie Moldave

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Volume 107. Posttranslational Modifications (Part B) Edited by Finn Wold and Kivie Moldave Volume 108. Immunochemical Techniques (Part G: Separation and Characterization of Lymphoid Cells) Edited by Giovanni Di Sabato, John J. Langone, and Helen Van Vunakis Volume 109. Hormone Action (Part I: Peptide Hormones) Edited by Lutz Birnbaumer and Bert W. O’Malley Volume 110. Steroids and Isoprenoids (Part A) Edited by John H. Law and Hans C. Rilling Volume 111. Steroids and Isoprenoids (Part B) Edited by John H. Law and Hans C. Rilling Volume 112. Drug and Enzyme Targeting (Part A) Edited by Kenneth J. Widder and Ralph Green Volume 113. Glutamate, Glutamine, Glutathione, and Related Compounds Edited by Alton Meister Volume 114. Diffraction Methods for Biological Macromolecules (Part A) Edited by Harold W. Wyckoff, C. H. W. Hirs, and Serge N. Timasheff Volume 115. Diffraction Methods for Biological Macromolecules (Part B) Edited by Harold W. Wyckoff, C. H. W. Hirs, and Serge N. Timasheff Volume 116. Immunochemical Techniques (Part H: Effectors and Mediators of Lymphoid Cell Functions) Edited by Giovanni Di Sabato, John J. Langone, and Helen Van Vunakis Volume 117. Enzyme Structure (Part J) Edited by C. H. W. Hirs and Serge N. Timasheff Volume 118. Plant Molecular Biology Edited by Arthur Weissbach and Herbert Weissbach Volume 119. Interferons (Part C) Edited by Sidney Pestka Volume 120. Cumulative Subject Index Volumes 81–94, 96–101 Volume 121. Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) Edited by John J. Langone and Helen Van Vunakis Volume 122. Vitamins and Coenzymes (Part G) Edited by Frank Chytil and Donald B. McCormick Volume 123. Vitamins and Coenzymes (Part H) Edited by Frank Chytil and Donald B. McCormick Volume 124. Hormone Action (Part J: Neuroendocrine Peptides) Edited by P. Michael Conn

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Volume 125. Biomembranes (Part M: Transport in Bacteria, Mitochondria, and Chloroplasts: General Approaches and Transport Systems) Edited by Sidney Fleischer and Becca Fleischer Volume 126. Biomembranes (Part N: Transport in Bacteria, Mitochondria, and Chloroplasts: Protonmotive Force) Edited by Sidney Fleischer and Becca Fleischer Volume 127. Biomembranes (Part O: Protons and Water: Structure and Translocation) Edited by Lester Packer Volume 128. Plasma Lipoproteins (Part A: Preparation, Structure, and Molecular Biology) Edited by Jere P. Segrest and John J. Albers Volume 129. Plasma Lipoproteins (Part B: Characterization, Cell Biology, and Metabolism) Edited by John J. Albers and Jere P. Segrest Volume 130. Enzyme Structure (Part K) Edited by C. H. W. Hirs and Serge N. Timasheff Volume 131. Enzyme Structure (Part L) Edited by C. H. W. Hirs and Serge N. Timasheff Volume 132. Immunochemical Techniques (Part J: Phagocytosis and Cell-Mediated Cytotoxicity) Edited by Giovanni Di Sabato and Johannes Everse Volume 133. Bioluminescence and Chemiluminescence (Part B) Edited by Marlene DeLuca and William D. McElroy Volume 134. Structural and Contractile Proteins (Part C: The Contractile Apparatus and the Cytoskeleton) Edited by Richard B. Vallee Volume 135. Immobilized Enzymes and Cells (Part B) Edited by Klaus Mosbach Volume 136. Immobilized Enzymes and Cells (Part C) Edited by Klaus Mosbach Volume 137. Immobilized Enzymes and Cells (Part D) Edited by Klaus Mosbach Volume 138. Complex Carbohydrates (Part E) Edited by Victor Ginsburg Volume 139. Cellular Regulators (Part A: Calcium- and Calmodulin-Binding Proteins) Edited by Anthony R. Means and P. Michael Conn

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Volume 140. Cumulative Subject Index Volumes 102–119, 121–134 Volume 141. Cellular Regulators (Part B: Calcium and Lipids) Edited by P. Michael Conn and Anthony R. Means Volume 142. Metabolism of Aromatic Amino Acids and Amines Edited by Seymour Kaufman Volume 143. Sulfur and Sulfur Amino Acids Edited by William B. Jakoby and Owen Griffith Volume 144. Structural and Contractile Proteins (Part D: Extracellular Matrix) Edited by Leon W. Cunningham Volume 145. Structural and Contractile Proteins (Part E: Extracellular Matrix) Edited by Leon W. Cunningham Volume 146. Peptide Growth Factors (Part A) Edited by David Barnes and David A. Sirbasku Volume 147. Peptide Growth Factors (Part B) Edited by David Barnes and David A. Sirbasku Volume 148. Plant Cell Membranes Edited by Lester Packer and Roland Douce Volume 149. Drug and Enzyme Targeting (Part B) Edited by Ralph Green and Kenneth J. Widder Volume 150. Immunochemical Techniques (Part K: In Vitro Models of B and T Cell Functions and Lymphoid Cell Receptors) Edited by Giovanni Di Sabato Volume 151. Molecular Genetics of Mammalian Cells Edited by Michael M. Gottesman Volume 152. Guide to Molecular Cloning Techniques Edited by Shelby L. Berger and Alan R. Kimmel Volume 153. Recombinant DNA (Part D) Edited by Ray Wu and Lawrence Grossman Volume 154. Recombinant DNA (Part E) Edited by Ray Wu and Lawrence Grossman Volume 155. Recombinant DNA (Part F) Edited by Ray Wu Volume 156. Biomembranes (Part P: ATP-Driven Pumps and Related Transport: The Na, K-Pump) Edited by Sidney Fleischer and Becca Fleischer Volume 157. Biomembranes (Part Q: ATP-Driven Pumps and Related Transport: Calcium, Proton, and Potassium Pumps) Edited by Sidney Fleischer and Becca Fleischer

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Volume 158. Metalloproteins (Part A) Edited by James F. Riordan and Bert L. Vallee Volume 159. Initiation and Termination of Cyclic Nucleotide Action Edited by Jackie D. Corbin and Roger A. Johnson Volume 160. Biomass (Part A: Cellulose and Hemicellulose) Edited by Willis A. Wood and Scott T. Kellogg Volume 161. Biomass (Part B: Lignin, Pectin, and Chitin) Edited by Willis A. Wood and Scott T. Kellogg Volume 162. Immunochemical Techniques (Part L: Chemotaxis and Inflammation) Edited by Giovanni Di Sabato Volume 163. Immunochemical Techniques (Part M: Chemotaxis and Inflammation) Edited by Giovanni Di Sabato Volume 164. Ribosomes Edited by Harry F. Noller, Jr., and Kivie Moldave Volume 165. Microbial Toxins: Tools for Enzymology Edited by Sidney Harshman Volume 166. Branched-Chain Amino Acids Edited by Robert Harris and John R. Sokatch Volume 167. Cyanobacteria Edited by Lester Packer and Alexander N. Glazer Volume 168. Hormone Action (Part K: Neuroendocrine Peptides) Edited by P. Michael Conn Volume 169. Platelets: Receptors, Adhesion, Secretion (Part A) Edited by Jacek Hawiger Volume 170. Nucleosomes Edited by Paul M. Wassarman and Roger D. Kornberg Volume 171. Biomembranes (Part R: Transport Theory: Cells and Model Membranes) Edited by Sidney Fleischer and Becca Fleischer Volume 172. Biomembranes (Part S: Transport: Membrane Isolation and Characterization) Edited by Sidney Fleischer and Becca Fleischer Volume 173. Biomembranes [Part T: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells] Edited by Sidney Fleischer and Becca Fleischer Volume 174. Biomembranes [Part U: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells] Edited by Sidney Fleischer and Becca Fleischer

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Volume 175. Cumulative Subject Index Volumes 135–139, 141–167 Volume 176. Nuclear Magnetic Resonance (Part A: Spectral Techniques and Dynamics) Edited by Norman J. Oppenheimer and Thomas L. James Volume 177. Nuclear Magnetic Resonance (Part B: Structure and Mechanism) Edited by Norman J. Oppenheimer and Thomas L. James Volume 178. Antibodies, Antigens, and Molecular Mimicry Edited by John J. Langone Volume 179. Complex Carbohydrates (Part F) Edited by Victor Ginsburg Volume 180. RNA Processing (Part A: General Methods) Edited by James E. Dahlberg and John N. Abelson Volume 181. RNA Processing (Part B: Specific Methods) Edited by James E. Dahlberg and John N. Abelson Volume 182. Guide to Protein Purification Edited by Murray P. Deutscher Volume 183. Molecular Evolution: Computer Analysis of Protein and Nucleic Acid Sequences Edited by Russell F. Doolittle Volume 184. Avidin-Biotin Technology Edited by Meir Wilchek and Edward A. Bayer Volume 185. Gene Expression Technology Edited by David V. Goeddel Volume 186. Oxygen Radicals in Biological Systems (Part B: Oxygen Radicals and Antioxidants) Edited by Lester Packer and Alexander N. Glazer Volume 187. Arachidonate Related Lipid Mediators Edited by Robert C. Murphy and Frank A. Fitzpatrick Volume 188. Hydrocarbons and Methylotrophy Edited by Mary E. Lidstrom Volume 189. Retinoids (Part A: Molecular and Metabolic Aspects) Edited by Lester Packer Volume 190. Retinoids (Part B: Cell Differentiation and Clinical Applications) Edited by Lester Packer Volume 191. Biomembranes (Part V: Cellular and Subcellular Transport: Epithelial Cells) Edited by Sidney Fleischer and Becca Fleischer Volume 192. Biomembranes (Part W: Cellular and Subcellular Transport: Epithelial Cells) Edited by Sidney Fleischer and Becca Fleischer

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Volume 193. Mass Spectrometry Edited by James A. McCloskey Volume 194. Guide to Yeast Genetics and Molecular Biology Edited by Christine Guthrie and Gerald R. Fink Volume 195. Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase Edited by Roger A. Johnson and Jackie D. Corbin Volume 196. Molecular Motors and the Cytoskeleton Edited by Richard B. Vallee Volume 197. Phospholipases Edited by Edward A. Dennis Volume 198. Peptide Growth Factors (Part C) Edited by David Barnes, J. P. Mather, and Gordon H. Sato Volume 199. Cumulative Subject Index Volumes 168–174, 176–194 Volume 200. Protein Phosphorylation (Part A: Protein Kinases: Assays, Purification, Antibodies, Functional Analysis, Cloning, and Expression) Edited by Tony Hunter and Bartholomew M. Sefton Volume 201. Protein Phosphorylation (Part B: Analysis of Protein Phosphorylation, Protein Kinase Inhibitors, and Protein Phosphatases) Edited by Tony Hunter and Bartholomew M. Sefton Volume 202. Molecular Design and Modeling: Concepts and Applications (Part A: Proteins, Peptides, and Enzymes) Edited by John J. Langone Volume 203. Molecular Design and Modeling: Concepts and Applications (Part B: Antibodies and Antigens, Nucleic Acids, Polysaccharides, and Drugs) Edited by John J. Langone Volume 204. Bacterial Genetic Systems Edited by Jeffrey H. Miller Volume 205. Metallobiochemistry (Part B: Metallothionein and Related Molecules) Edited by James F. Riordan and Bert L. Vallee Volume 206. Cytochrome P450 Edited by Michael R. Waterman and Eric F. Johnson Volume 207. Ion Channels Edited by Bernardo Rudy and Linda E. Iverson Volume 208. Protein–DNA Interactions Edited by Robert T. Sauer Volume 209. Phospholipid Biosynthesis Edited by Edward A. Dennis and Dennis E. Vance

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Volume 210. Numerical Computer Methods Edited by Ludwig Brand and Michael L. Johnson Volume 211. DNA Structures (Part A: Synthesis and Physical Analysis of DNA) Edited by David M. J. Lilley and James E. Dahlberg Volume 212. DNA Structures (Part B: Chemical and Electrophoretic Analysis of DNA) Edited by David M. J. Lilley and James E. Dahlberg Volume 213. Carotenoids (Part A: Chemistry, Separation, Quantitation, and Antioxidation) Edited by Lester Packer Volume 214. Carotenoids (Part B: Metabolism, Genetics, and Biosynthesis) Edited by Lester Packer Volume 215. Platelets: Receptors, Adhesion, Secretion (Part B) Edited by Jacek J. Hawiger Volume 216. Recombinant DNA (Part G) Edited by Ray Wu Volume 217. Recombinant DNA (Part H) Edited by Ray Wu Volume 218. Recombinant DNA (Part I) Edited by Ray Wu Volume 219. Reconstitution of Intracellular Transport Edited by James E. Rothman Volume 220. Membrane Fusion Techniques (Part A) Edited by Nejat Du¨zgu¨nes, Volume 221. Membrane Fusion Techniques (Part B) Edited by Nejat Du¨zgu¨nes, Volume 222. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part A: Mammalian Blood Coagulation Factors and Inhibitors) Edited by Laszlo Lorand and Kenneth G. Mann Volume 223. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part B: Complement Activation, Fibrinolysis, and Nonmammalian Blood Coagulation Factors) Edited by Laszlo Lorand and Kenneth G. Mann Volume 224. Molecular Evolution: Producing the Biochemical Data Edited by Elizabeth Anne Zimmer, Thomas J. White, Rebecca L. Cann, and Allan C. Wilson Volume 225. Guide to Techniques in Mouse Development Edited by Paul M. Wassarman and Melvin L. DePamphilis

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Volume 226. Metallobiochemistry (Part C: Spectroscopic and Physical Methods for Probing Metal Ion Environments in Metalloenzymes and Metalloproteins) Edited by James F. Riordan and Bert L. Vallee Volume 227. Metallobiochemistry (Part D: Physical and Spectroscopic Methods for Probing Metal Ion Environments in Metalloproteins) Edited by James F. Riordan and Bert L. Vallee Volume 228. Aqueous Two-Phase Systems Edited by Harry Walter and Go¨te Johansson Volume 229. Cumulative Subject Index Volumes 195–198, 200–227 Volume 230. Guide to Techniques in Glycobiology Edited by William J. Lennarz and Gerald W. Hart Volume 231. Hemoglobins (Part B: Biochemical and Analytical Methods) Edited by Johannes Everse, Kim D. Vandegriff, and Robert M. Winslow Volume 232. Hemoglobins (Part C: Biophysical Methods) Edited by Johannes Everse, Kim D. Vandegriff, and Robert M. Winslow Volume 233. Oxygen Radicals in Biological Systems (Part C) Edited by Lester Packer Volume 234. Oxygen Radicals in Biological Systems (Part D) Edited by Lester Packer Volume 235. Bacterial Pathogenesis (Part A: Identification and Regulation of Virulence Factors) Edited by Virginia L. Clark and Patrik M. Bavoil Volume 236. Bacterial Pathogenesis (Part B: Integration of Pathogenic Bacteria with Host Cells) Edited by Virginia L. Clark and Patrik M. Bavoil Volume 237. Heterotrimeric G Proteins Edited by Ravi Iyengar Volume 238. Heterotrimeric G-Protein Effectors Edited by Ravi Iyengar Volume 239. Nuclear Magnetic Resonance (Part C) Edited by Thomas L. James and Norman J. Oppenheimer Volume 240. Numerical Computer Methods (Part B) Edited by Michael L. Johnson and Ludwig Brand Volume 241. Retroviral Proteases Edited by Lawrence C. Kuo and Jules A. Shafer Volume 242. Neoglycoconjugates (Part A) Edited by Y. C. Lee and Reiko T. Lee Volume 243. Inorganic Microbial Sulfur Metabolism Edited by Harry D. Peck, Jr., and Jean LeGall

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Volume 244. Proteolytic Enzymes: Serine and Cysteine Peptidases Edited by Alan J. Barrett Volume 245. Extracellular Matrix Components Edited by E. Ruoslahti and E. Engvall Volume 246. Biochemical Spectroscopy Edited by Kenneth Sauer Volume 247. Neoglycoconjugates (Part B: Biomedical Applications) Edited by Y. C. Lee and Reiko T. Lee Volume 248. Proteolytic Enzymes: Aspartic and Metallo Peptidases Edited by Alan J. Barrett Volume 249. Enzyme Kinetics and Mechanism (Part D: Developments in Enzyme Dynamics) Edited by Daniel L. Purich Volume 250. Lipid Modifications of Proteins Edited by Patrick J. Casey and Janice E. Buss Volume 251. Biothiols (Part A: Monothiols and Dithiols, Protein Thiols, and Thiyl Radicals) Edited by Lester Packer Volume 252. Biothiols (Part B: Glutathione and Thioredoxin; Thiols in Signal Transduction and Gene Regulation) Edited by Lester Packer Volume 253. Adhesion of Microbial Pathogens Edited by Ron J. Doyle and Itzhak Ofek Volume 254. Oncogene Techniques Edited by Peter K. Vogt and Inder M. Verma Volume 255. Small GTPases and Their Regulators (Part A: Ras Family) Edited by W. E. Balch, Channing J. Der, and Alan Hall Volume 256. Small GTPases and Their Regulators (Part B: Rho Family) Edited by W. E. Balch, Channing J. Der, and Alan Hall Volume 257. Small GTPases and Their Regulators (Part C: Proteins Involved in Transport) Edited by W. E. Balch, Channing J. Der, and Alan Hall Volume 258. Redox-Active Amino Acids in Biology Edited by Judith P. Klinman Volume 259. Energetics of Biological Macromolecules Edited by Michael L. Johnson and Gary K. Ackers Volume 260. Mitochondrial Biogenesis and Genetics (Part A) Edited by Giuseppe M. Attardi and Anne Chomyn Volume 261. Nuclear Magnetic Resonance and Nucleic Acids Edited by Thomas L. James

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Volume 262. DNA Replication Edited by Judith L. Campbell Volume 263. Plasma Lipoproteins (Part C: Quantitation) Edited by William A. Bradley, Sandra H. Gianturco, and Jere P. Segrest Volume 264. Mitochondrial Biogenesis and Genetics (Part B) Edited by Giuseppe M. Attardi and Anne Chomyn Volume 265. Cumulative Subject Index Volumes 228, 230–262 Volume 266. Computer Methods for Macromolecular Sequence Analysis Edited by Russell F. Doolittle Volume 267. Combinatorial Chemistry Edited by John N. Abelson Volume 268. Nitric Oxide (Part A: Sources and Detection of NO; NO Synthase) Edited by Lester Packer Volume 269. Nitric Oxide (Part B: Physiological and Pathological Processes) Edited by Lester Packer Volume 270. High Resolution Separation and Analysis of Biological Macromolecules (Part A: Fundamentals) Edited by Barry L. Karger and William S. Hancock Volume 271. High Resolution Separation and Analysis of Biological Macromolecules (Part B: Applications) Edited by Barry L. Karger and William S. Hancock Volume 272. Cytochrome P450 (Part B) Edited by Eric F. Johnson and Michael R. Waterman Volume 273. RNA Polymerase and Associated Factors (Part A) Edited by Sankar Adhya Volume 274. RNA Polymerase and Associated Factors (Part B) Edited by Sankar Adhya Volume 275. Viral Polymerases and Related Proteins Edited by Lawrence C. Kuo, David B. Olsen, and Steven S. Carroll Volume 276. Macromolecular Crystallography (Part A) Edited by Charles W. Carter, Jr., and Robert M. Sweet Volume 277. Macromolecular Crystallography (Part B) Edited by Charles W. Carter, Jr., and Robert M. Sweet Volume 278. Fluorescence Spectroscopy Edited by Ludwig Brand and Michael L. Johnson Volume 279. Vitamins and Coenzymes (Part I) Edited by Donald B. McCormick, John W. Suttie, and Conrad Wagner

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Volume 280. Vitamins and Coenzymes (Part J) Edited by Donald B. McCormick, John W. Suttie, and Conrad Wagner Volume 281. Vitamins and Coenzymes (Part K) Edited by Donald B. McCormick, John W. Suttie, and Conrad Wagner Volume 282. Vitamins and Coenzymes (Part L) Edited by Donald B. McCormick, John W. Suttie, and Conrad Wagner Volume 283. Cell Cycle Control Edited by William G. Dunphy Volume 284. Lipases (Part A: Biotechnology) Edited by Byron Rubin and Edward A. Dennis Volume 285. Cumulative Subject Index Volumes 263, 264, 266–284, 286–289 Volume 286. Lipases (Part B: Enzyme Characterization and Utilization) Edited by Byron Rubin and Edward A. Dennis Volume 287. Chemokines Edited by Richard Horuk Volume 288. Chemokine Receptors Edited by Richard Horuk Volume 289. Solid Phase Peptide Synthesis Edited by Gregg B. Fields Volume 290. Molecular Chaperones Edited by George H. Lorimer and Thomas Baldwin Volume 291. Caged Compounds Edited by Gerard Marriott Volume 292. ABC Transporters: Biochemical, Cellular, and Molecular Aspects Edited by Suresh V. Ambudkar and Michael M. Gottesman Volume 293. Ion Channels (Part B) Edited by P. Michael Conn Volume 294. Ion Channels (Part C) Edited by P. Michael Conn Volume 295. Energetics of Biological Macromolecules (Part B) Edited by Gary K. Ackers and Michael L. Johnson Volume 296. Neurotransmitter Transporters Edited by Susan G. Amara Volume 297. Photosynthesis: Molecular Biology of Energy Capture Edited by Lee McIntosh Volume 298. Molecular Motors and the Cytoskeleton (Part B) Edited by Richard B. Vallee

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Volume 299. Oxidants and Antioxidants (Part A) Edited by Lester Packer Volume 300. Oxidants and Antioxidants (Part B) Edited by Lester Packer Volume 301. Nitric Oxide: Biological and Antioxidant Activities (Part C) Edited by Lester Packer Volume 302. Green Fluorescent Protein Edited by P. Michael Conn Volume 303. cDNA Preparation and Display Edited by Sherman M. Weissman Volume 304. Chromatin Edited by Paul M. Wassarman and Alan P. Wolffe Volume 305. Bioluminescence and Chemiluminescence (Part C) Edited by Thomas O. Baldwin and Miriam M. Ziegler Volume 306. Expression of Recombinant Genes in Eukaryotic Systems Edited by Joseph C. Glorioso and Martin C. Schmidt Volume 307. Confocal Microscopy Edited by P. Michael Conn Volume 308. Enzyme Kinetics and Mechanism (Part E: Energetics of Enzyme Catalysis) Edited by Daniel L. Purich and Vern L. Schramm Volume 309. Amyloid, Prions, and Other Protein Aggregates Edited by Ronald Wetzel Volume 310. Biofilms Edited by Ron J. Doyle Volume 311. Sphingolipid Metabolism and Cell Signaling (Part A) Edited by Alfred H. Merrill, Jr., and Yusuf A. Hannun Volume 312. Sphingolipid Metabolism and Cell Signaling (Part B) Edited by Alfred H. Merrill, Jr., and Yusuf A. Hannun Volume 313. Antisense Technology (Part A: General Methods, Methods of Delivery, and RNA Studies) Edited by M. Ian Phillips Volume 314. Antisense Technology (Part B: Applications) Edited by M. Ian Phillips Volume 315. Vertebrate Phototransduction and the Visual Cycle (Part A) Edited by Krzysztof Palczewski Volume 316. Vertebrate Phototransduction and the Visual Cycle (Part B) Edited by Krzysztof Palczewski

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Volume 317. RNA–Ligand Interactions (Part A: Structural Biology Methods) Edited by Daniel W. Celander and John N. Abelson Volume 318. RNA–Ligand Interactions (Part B: Molecular Biology Methods) Edited by Daniel W. Celander and John N. Abelson Volume 319. Singlet Oxygen, UV-A, and Ozone Edited by Lester Packer and Helmut Sies Volume 320. Cumulative Subject Index Volumes 290–319 Volume 321. Numerical Computer Methods (Part C) Edited by Michael L. Johnson and Ludwig Brand Volume 322. Apoptosis Edited by John C. Reed Volume 323. Energetics of Biological Macromolecules (Part C) Edited by Michael L. Johnson and Gary K. Ackers Volume 324. Branched-Chain Amino Acids (Part B) Edited by Robert A. Harris and John R. Sokatch Volume 325. Regulators and Effectors of Small GTPases (Part D: Rho Family) Edited by W. E. Balch, Channing J. Der, and Alan Hall Volume 326. Applications of Chimeric Genes and Hybrid Proteins (Part A: Gene Expression and Protein Purification) Edited by Jeremy Thorner, Scott D. Emr, and John N. Abelson Volume 327. Applications of Chimeric Genes and Hybrid Proteins (Part B: Cell Biology and Physiology) Edited by Jeremy Thorner, Scott D. Emr, and John N. Abelson Volume 328. Applications of Chimeric Genes and Hybrid Proteins (Part C: Protein–Protein Interactions and Genomics) Edited by Jeremy Thorner, Scott D. Emr, and John N. Abelson Volume 329. Regulators and Effectors of Small GTPases (Part E: GTPases Involved in Vesicular Traffic) Edited by W. E. Balch, Channing J. Der, and Alan Hall Volume 330. Hyperthermophilic Enzymes (Part A) Edited by Michael W. W. Adams and Robert M. Kelly Volume 331. Hyperthermophilic Enzymes (Part B) Edited by Michael W. W. Adams and Robert M. Kelly Volume 332. Regulators and Effectors of Small GTPases (Part F: Ras Family I) Edited by W. E. Balch, Channing J. Der, and Alan Hall Volume 333. Regulators and Effectors of Small GTPases (Part G: Ras Family II) Edited by W. E. Balch, Channing J. Der, and Alan Hall Volume 334. Hyperthermophilic Enzymes (Part C) Edited by Michael W. W. Adams and Robert M. Kelly

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Volume 335. Flavonoids and Other Polyphenols Edited by Lester Packer Volume 336. Microbial Growth in Biofilms (Part A: Developmental and Molecular Biological Aspects) Edited by Ron J. Doyle Volume 337. Microbial Growth in Biofilms (Part B: Special Environments and Physicochemical Aspects) Edited by Ron J. Doyle Volume 338. Nuclear Magnetic Resonance of Biological Macromolecules (Part A) Edited by Thomas L. James, Volker Do¨tsch, and Uli Schmitz Volume 339. Nuclear Magnetic Resonance of Biological Macromolecules (Part B) Edited by Thomas L. James, Volker Do¨tsch, and Uli Schmitz Volume 340. Drug–Nucleic Acid Interactions Edited by Jonathan B. Chaires and Michael J. Waring Volume 341. Ribonucleases (Part A) Edited by Allen W. Nicholson Volume 342. Ribonucleases (Part B) Edited by Allen W. Nicholson Volume 343. G Protein Pathways (Part A: Receptors) Edited by Ravi Iyengar and John D. Hildebrandt Volume 344. G Protein Pathways (Part B: G Proteins and Their Regulators) Edited by Ravi Iyengar and John D. Hildebrandt Volume 345. G Protein Pathways (Part C: Effector Mechanisms) Edited by Ravi Iyengar and John D. Hildebrandt Volume 346. Gene Therapy Methods Edited by M. Ian Phillips Volume 347. Protein Sensors and Reactive Oxygen Species (Part A: Selenoproteins and Thioredoxin) Edited by Helmut Sies and Lester Packer Volume 348. Protein Sensors and Reactive Oxygen Species (Part B: Thiol Enzymes and Proteins) Edited by Helmut Sies and Lester Packer Volume 349. Superoxide Dismutase Edited by Lester Packer Volume 350. Guide to Yeast Genetics and Molecular and Cell Biology (Part B) Edited by Christine Guthrie and Gerald R. Fink Volume 351. Guide to Yeast Genetics and Molecular and Cell Biology (Part C) Edited by Christine Guthrie and Gerald R. Fink

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Volume 352. Redox Cell Biology and Genetics (Part A) Edited by Chandan K. Sen and Lester Packer Volume 353. Redox Cell Biology and Genetics (Part B) Edited by Chandan K. Sen and Lester Packer Volume 354. Enzyme Kinetics and Mechanisms (Part F: Detection and Characterization of Enzyme Reaction Intermediates) Edited by Daniel L. Purich Volume 355. Cumulative Subject Index Volumes 321–354 Volume 356. Laser Capture Microscopy and Microdissection Edited by P. Michael Conn Volume 357. Cytochrome P450, Part C Edited by Eric F. Johnson and Michael R. Waterman Volume 358. Bacterial Pathogenesis (Part C: Identification, Regulation, and Function of Virulence Factors) Edited by Virginia L. Clark and Patrik M. Bavoil Volume 359. Nitric Oxide (Part D) Edited by Enrique Cadenas and Lester Packer Volume 360. Biophotonics (Part A) Edited by Gerard Marriott and Ian Parker Volume 361. Biophotonics (Part B) Edited by Gerard Marriott and Ian Parker Volume 362. Recognition of Carbohydrates in Biological Systems (Part A) Edited by Yuan C. Lee and Reiko T. Lee Volume 363. Recognition of Carbohydrates in Biological Systems (Part B) Edited by Yuan C. Lee and Reiko T. Lee Volume 364. Nuclear Receptors Edited by David W. Russell and David J. Mangelsdorf Volume 365. Differentiation of Embryonic Stem Cells Edited by Paul M. Wassauman and Gordon M. Keller Volume 366. Protein Phosphatases Edited by Susanne Klumpp and Josef Krieglstein Volume 367. Liposomes (Part A) Edited by Nejat Du¨zgu¨nes, Volume 368. Macromolecular Crystallography (Part C) Edited by Charles W. Carter, Jr., and Robert M. Sweet Volume 369. Combinational Chemistry (Part B) Edited by Guillermo A. Morales and Barry A. Bunin Volume 370. RNA Polymerases and Associated Factors (Part C) Edited by Sankar L. Adhya and Susan Garges

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Volume 371. RNA Polymerases and Associated Factors (Part D) Edited by Sankar L. Adhya and Susan Garges Volume 372. Liposomes (Part B) Edited by Negat Du¨zgu¨nes, Volume 373. Liposomes (Part C) (in preparation) Edited by Negat Du¨zgu¨nes, Volume 374. Macromolecular Crystallography (Part D) (in preparation) Edited by Charles W. Carter, Jr. and Robert W. Sweet Volume 375. Chromatin and Chromatin Remodeling Enzymes (Part A) (in preparation) Edited by Carl Wu and C. Davis Allis (in preparation) Volume 376. Chromatin and Chromatin Remodeling Enzymes (Part B) (in preparation) Edited by Carl Wu and C. Davis Allis (in preparation) Volume 377. Chromatin and Chromatin Remodeling Enzymes (Part C) (in preparation) Edited by Carl Wu and C. Davis Allis (in preparation) Volume 378. Quinones and Quinone Enzymes (Part A) (in preparation) Edited by Helmut Sies and Lester Packer (in preparation) Volume 379. Energetics of Biological Macromolecules (Part D) (in preparation) Edited by Jo M. Holt, Michael L. Johnson, and Gary K. Ackers Volume 380. Energetics of Biological Macromolecules (Part E) (in preparation) Edited by Jo M. Holt, Michael L. Johnson, and Gary K. Ackers Volume 381. Oxygen Sensing (in preparation) Edited by Chandan K. Sen and Gregg L. Semenza Volume 382. Quinones and Quinone Enzymes (Part B) (in preparation) Edited by Helmut Sies and Lester Packer Volume 383. Numerical Computer Methods (Part D) (in preparation) Edited by Ludwig Brand and Michael L. Johnson Volume 384. Numerical Computer Methods (Part E) (in preparation) Edited by Ludwig Brand and Michael L. Johnson

Section I Analytical Techniques

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high-throughput LC/UV/MS analysis of libraries

3

[1] High-Throughput Parallel LC/UV/MS Analysis of Combinatorial Libraries By Liling Fang, Jiang Zhao, and Bing Yan Introduction

Combinatorial chemistry and high-throughput organic synthesis allow the preparation of a large number of diverse compounds in a relative short period of time in order to accelerate discovery efforts in the pharmaceutical and other industries. A library can comprise hundreds to thousands of compounds with the need to rapidly analyze those compounds for their identity and purity. Different compound separation and mass spectrometry (MS) techniques have been applied for the characterization of combinatorial libraries. These include separation techniques such as liquid chromatography (LC) and capillary electrophoresis and different ionization methods and mass analyzers.1–3 LC/MS* is the most popular technique used in combinatorial library analysis because it combines separation, molecular weight determination, and relative purity evaluation in a single sample injection. However, the throughput of conventional LC/MS could not meet the need to analyze every member in a large combinatorial library in a timely fashion. Higher-throughput analysis was achieved by utilizing shorter columns at higher flow rates.4 Supercritical fluid chromatography (SFC)/MS has

1

A. Hauser-Fang and P. Vouros, ‘‘Analytical Techniques in Combinatorial Chemistry’’ (M. E. Swartz, ed.). Marcel Dekker, New York, 2000. 2 B. Yan, ‘‘Analytical Methods in Combinatorial Chemistry.’’ Technomic, Lancaster, 2000. 3 D. G. Schmid, P. Grosche, H. Bandel, and G. Jung, Biotechnol. Bioeng. Comb. Chem. 71, 149 (2001). *Abbreviations: CLND, chemiluminescence nitrogen detection; C log P, calculated partition coefficient; ELSD, evaporative light scattering detection; ESI-MS, electrospray ionization mass spectrometry; FWHM, full width at half maximum; i.d., inner diameter; LC, HPLC, liquid chromatography, high-performance liquid chromatography; LC/MS, liquid chromatography – mass spectrometry; LC/MS/MS, liquid chromatography – mass spectrometry – mass spectrometry; LC/UV/MS, liquid chromatography mass spectrometry with a UV detector; LIB, compound library; log P, water/octanol partition coefficient; MUX, multiplexed; RSD, relative standard deviation; SFC, supercritical fluid chromatography; TFA, trifluoroacetic acid; TIC, total ion current; TOF, time of flight; TOFMS, time of flight mass spectrometry. 4 H. Lee, L. Li, and J. Kyranos, Proceedings of the 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, Texas, June 13–17, 1999.

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

4

analytical techniques

[1]

been used to achieve desirable high speed taking advantage of the low viscosity of CO2.5 However, the serial LC/MS approach by its nature does not match the speed of parallel synthesis. Parallel LC/MS is the method of choice to increase throughput while maintaining the separation efficiency. An eight-probe Gilson 215/889 autosampler was incorporated into a quadruple mass spectrometer.6 This arrangement enabled the injection of eight samples (a column from a 96-well microtiter plate) simultaneously for flow-injection analysis/MS (FIA-MS) analysis to achieve a throughput of 8 samples/min. A novel multiplexed electrospray interface (MUX)7 was developed in 1999 and became commercially available for parallel high-throughput LC/UV/MS analysis. The eight-way MUX consists of eight nebulization-assisted electrospray ionization sprayers, a desolvation gas heater probe, and a rotating aperture. It can accommodate all eight high-performance liquid chromatograph (HPLC) streams at a reduced flow rate of <100 l/min per stream and conduct electrospray ionization for all eight streams simultaneously. Ions are continuously formed at the tip of each sprayer and the MUX interface allows sprayers to be sampled sequentially using the rotating aperture driven by a programmable stepper motor. At any given time, only ions from one stream are admitted into the ion sampling cone, while ions from the other seven sprayers are shielded. Each liquid stream is sampled for a preset time with mass spectra acquired in full mass range into eight simultaneously open data files synchronized with the spray being sampled. With a 0.1-s acquisition time per sprayer and 0.05-s intersprayer delay time, the time-of-flight (TOF) mass analyzer can acquire a discrete data file of electrospray ion current sampled from each stream over the entire HPLC separation with a cycle time of 1.2 s. Therefore, this eight-way MUX-LCT was like having eight individual electrospray ionization (ESI)-MS systems working simultaneously. The MUX interface enables the coupling of parallel liquid chromatography to a single mass spectrometer. This technology has had a great impact in high-throughput LC/MS analysis. In drug development, a fourway MUX interface was used on a triple quadrupole mass spectrometer to simultaneously validate LC/MS/MS methods for the quantitation of loratadine and its metabolite in four different biological matrixes8 and of 5

M. C. Ventura, W. P. Farrell, C. M. Aurigemma, and M. J. Greig, Anal. Chem. 71, 2410 (1999). 6 T. Wang, L. Zeng, T. Strader, L. Burton, and D. B. Kassel, Rapid Commun. Mass Spectrom. 12, 1123 (1998). 7 V. De Biasil, N. Haskins, A. Organ, R. Bateman, K. Giles, and S. Jarvis, Rapid Commun. Mass Spectrom. 13, 1165 (1999). 8 M. K. Bayliss, D. Little, D. M. Mallett, and R. S. Plumb, Rapid Commun. Mass Spectrom. 14, 2039 (2000).

[1]

high-throughput LC/UV/MS analysis of libraries

5

diazepam in rat liver microsomes for in vitro metabolic stability.9 The fourchannel LC/MS/MS system was also reported for the quantification of a drug in plasma on both the narrow-bore and capillary scales.10 By incorporating divert valves into this system, aliquots of plasma could be directly analyzed without sample preparation. The four-channel LC/MS/MS has reduced method validation time, increased sample throughput by 4-fold, and afforded adequate sensitivity, precision, and negligible intersprayer crosstalk.8,9 In protein analysis, an eight-way MUX coupled with a TOFMS analyzer has proved to be a powerful tool to monitor the protein purification process by screening fractions from preparative ion-exchange chromatography with a throughput of 50 protein-containing fractions in less than an hour.11 A high-pressure gradient parallel pumping system (JASCO PAR-1500) has been developed to conduct high-throughput parallel liquid chromatography.12 It is a 10-pump system where two pumps are used to generate a binary gradient and eight pumps to deliver the mixed solvent to eight LC columns. Comparing this system to a conventional system with two pumps or a binary pump for LC gradient and a simple splitter to divide the gradient to eight LC columns, this system can ensure uniform flow rates through each LC column. This system has been used for peptides and combinatorial sample,12 protein analysis,13 and bioanalysis.9 We have optimized an eight-way MUX coupled to a TOFMS analyzer to carry out eight-channel parallel LC/UV/MS analysis of combinatorial libraries14 in the past 2 years. This system has not only provided the capacity needed for library analysis, but also enabled simultaneous evaluation of experimental parameters to expedite the method development process. In this chapter, we discuss the optimization of this system and present a high-throughput protocol for combinatorial library analysis. We also compare the eight-channel parallel LC/UV/MS system to a conventional single channel LC/UV/MS system in terms of performance and operation.

9

D. Morrison, A. E. Davis, and A. P. Watt, Anal. Chem. 74, 1896 (2002). L. Yang, T. D. Mann, D. Little, N. Wu, R. P. Clement, and P. J. Rudewicz, Anal. Chem. 73, 1740 (2001). 11 B. Feng, A. Patel, P. M. Keller, and J. R. Slemmon, Rapid Commun. Mass Spectrom. 15, 821 (2001). 12 D. Tolson, A. Organ, and A. Shah, Rapid Commun. Mass Spectrom. 15, 1244 (2001). 13 B. Feng, M. S. McQueney, T. M. Mezzasalma, and J. R. Slemmon, Anal. Chem. 73, 5691 (2001). 14 J. Zhao, D. Liu, J. Wheatley, L. Fang, and B. Yan, Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May, 27–31, 2001. 10

6

[1]

analytical techniques

A

Theophylline O H3C

5-phenyl-1h-tetrazole

Reserpine N

N

N

NH N N N

CH 3 C log P = −0.39 log P = 0.05 D

C

H N

N

O

B

H3CO

C log P = 0.18 log P = 2.41 E

O

N H

O

O

H H3COOC

OCH3

H O O H H OCH3

OCH3 OCH3

Dioctyl phthalate

H3C O

C log P = 3.85 log P = 4.43

H

C log P = 3.32

Fmoc-Asp(OtBu)-OH HO O

N H

H

CH3

O O O

O

H3C

CH3

C log P = 8.39

Fig. 1. The structures of the five commercial compounds (A to E) used to monitor performance.

System Optimization

The high-throughput parallel LC/UV/MS system consists of an autosampler with eight injection probes, two pumps for generating binary gradient, eight UV detectors, and an eight-way MUX with a TOF mass spectrometer. This two-pump arrangement keeps the system simple and cost efficient. However, it does not provide pressure regulation for each LC channel. To ensure flow consistency across each channel, we paid special attention to the selection of tubing, joints, and columns. Columns are from the same manufacturer and the same batch. The tubing is the same length initially for each channel and is further adjusted by checking the flow at the end. With these precautions, the flow from this two-pump system could be split evenly among the eight channels. In addition, a standard mixture is analyzed every 24 injections, and the retention times of these standards are closely monitored to ensure an even flow across the eight channels. Standards and Flow Monitoring Five commercial compounds are chosen as standards in system optimization and quality control (Fig. 1). Theophylline (log P 0.05), 5-phenyl1H-tetrazole (log P 2.41), reserpine (C log P 3.32), Fmoc-Asp(OtBu)-OH (log P 4.43), and dioctyl phthalate (C log P 8.39)15 were selected for our 15

L. Tang, W. Fitch, M. Alexander, and J. Dolan, Anal. Chem. 72, 5211 (2000).

[1] A

B 1.01

1.46

1.03

1.48

1.01

1.46

1.97

2.42

1.45

1.97

2.42

1.03 0.99 1.00

1.0

1.96 2.00

2.42

1d

2.44

7d

1.49 1.44

1.99 1.95 1.95

1.45

1.5

12 d 17 d

2.45

22 d 2.40

26 d

2.5

D

2.0

C

1.5

B

1.0

A

0.5

2.40

31 d 2.0

3.0 Retention time (min)

1.00

0.5

7

high-throughput LC/UV/MS analysis of libraries

2.5

3.0

0.0 0

5

10

15

20

25

30

Time (day)

Retention time (min) Fig. 2. A selection of UV214 chromatograms from channel 5 on different days and the retention times from the eight-channel system on every day for standard compounds (A to D) monitored over a 1-month period. (See color insert.)

standard mixture. Although only three compounds have experimentally determined log P values (see above) their C log P values range from 0.4 to 8.4, which covers most of the elution range for combinatorial library compounds. Without backpressure regulation for each channel, it is necessary to minimize the flow rate fluctuation over time. The relative standard deviation (RSD%) in retention time variation among the eight channels over 1 month for compounds A and B was less than 2% and for C and D it was less than 1%. The RSD% for all channels over a 1-month period for compounds A to D was 3.2, 2.4, 1.6, and 1.5%, respectively. Therefore, this system is well suited for combinatorial library analysis. The UV chromatograms from channel 5 from different days are shown as an example in Fig. 2A. The retention times of the four compounds (compounds A to D) from all eight channels during a 1-month period are shown in Fig. 2B. The throughput of this eight parallel LC/UV/MS system is 3200 compounds per day for a 3.5-min cycle time per injection of eight samples under current optimized conditions. It could be further increased by increasing the gradient slope and flow rate. We have also determined that five compounds in the standard mixture gave a linear response from 0.01 to 0.4 mg/ml.16 16

L. Fang, M. Wang, M. Pennacchio, and J. Pan, J. Comb. Chem. 2, 254 (2000).

8

[1]

analytical techniques

The T-Joint Position A zero dead volume T-joint is used after each UV detector to split the LC eluent to the MS analyzer and the waste to ensure a flow of 100 l/min entering each channel. The position of the T-joint affected the separation in the total ion chromatogram (TIC). When the T-joint was placed close to the UV cell (320 mm from the UV cell), the distance between the T-joint and the eight-way MUX interface is 780 mm. A sample had to travel 12 s to reach the eight-way MUX inlet after the UV detector at a flow rate of 50 l/min. Fmoc-Asp(OtBu)-OH had a full width at half maximum (FWHM) of 0.05 min when detected at the UV detector (Fig. 3C), but the peak width was doubled at the position of the MUX inlet (Fig. 3D). Such a peak broadening in TIC could have jeopardized product identification. To minimize these effects, the T-joint was moved as close as possible to the eight-way MUX inlet. With this modification the sample reached the MUX inlet 2 s after leaving the UV detector. The FWHM in the UV and TIC chromatograms were both 0.05 min (Fig. 3A and B). Thus peak delay and broadening were all eliminated. LC Conditions Unlike a single LC/UV/MS system, reducing solvent consumption is important in this eight-channel LC/UV/MS system. A flow rate of 24 ml/min on eight 4.6  50-mm columns was used initially. This operation resulted in a solvent consumption of 34.5 liters/day. To maintain the same separation

2.31

Configuration A

A

UV214 2.31

Configuration A

B

TIC 2.44

Configuration B

UV214 2.52

C

Configuration B D

TIC 2.2

2.4 2.6 Retention time (min)

2.8

Fig. 3. UV214 and TIC chromatograms of compound D obtained from configurations A (A and B) and B (C and D).

[1]

high-throughput LC/UV/MS analysis of libraries 1.07

0.52

A

2.21

3.40

1.72 0.92

0.36

2.14

B

1.70

0.28

0.72

3.34

1.91

C

1.48

0.24

3.08 1.74

0.60

2.91

1.35

0.0

0.5

1.0

1.5

9

2.0

2.5

3.0

D

3.5

Retention time (min) Fig. 4. UV chromatograms of the standard mixture at one of the eight channels separated using a 4.6-mm-i.d. column at 24 ml/min (A), and a 2.1-mm-i.d. column, at 8 (B), 12 (C), and 16 ml/min (D).

efficiency and minimize solvent consumption, columns with a smaller inner diameter (i.d.), such as 2.1 mm, were evaluated. The standard mixture was analyzed at flow rates of 6, 8, 10, 12, 14, and 16 ml/min. The LC gradient was 10–100% B in 3.0 min and 100% B for 0.5 min. The chromatograms in Fig. 4 show the separation results obtained from the original 4.6–mm-i.d. column at 24 ml/min (A) and 2.1-mm-i.d. column at 8 ml/min (B), 12 ml/ min (C), and 16 ml/min (D). Below 8 ml/min, the very lipophilic dioctyl phthalate did not elute. The separation was good at 12 ml/min and even better at 16 ml/min. A flow rate of 12 ml/min with the 2.1-mm-i.d. column was sufficient to maintain the separation efficiency, and consumed only half the solvent. For a particular library, an optimal LC column needs to be selected. This parallel LC/UV/MS system could evaluate eight different columns in 4 min. Five C18 columns of 2  50 mm packed with 5-m particles made by different manufacturers were evaluated simultaneously based on the separation efficiency of the standard mixture using trifluoroacetic acid (TFA) or acetic acid as modifier. Chromatograms at 12 ml/min using 0.05% TFA from each column are shown in Fig. 5. The Aqua column gave poor peak shape for the early-eluting compound A. The Aqua and Luna columns did not separate compounds B and C (data not shown) using 0.1% acetic acid. The remaining three columns separated the five compounds well.

10

[1]

analytical techniques A B C D E 0.0

0.5

1.0 1.5 2.0 2.5 Retention time (min)

3.0

3.5

Fig. 5. UV chromatograms of the standard mixture separated using 2.1-mm-i.d. columns at 12 ml/min: Polaris (A), Zorbax (B), Omnisphere (C), Luna (D), and Aqua (E).

An Efficient Rerun Protocol

In a high-throughput analysis mode, variable sample concentration sometimes leads to inadequate or saturated signals and at times blocking of the injector ports. Therefore, reanalysis of selected samples became a necessity. However, due to the rigid design of the Gilson 215 Multiprobe liquid handler, MUX-LCT cannot handle reanalysis efficiently. For example, when an injector is blocked during an overnight queue, there may be 24 failed samples on two 96-well plates (Fig. 6). Since the 8-probe injector has to inject an entire column of eight samples for each run, it will take 24 runs or 108 min to complete the analysis. We have developed a new process to improve the efficiency of sample reanalysis. This process includes four steps: data review, replating, reanalysis, and data alignment. We have also generated an Excel template, a Gilson’s Unipoint protocol, and an in-house visual basic program to automate the process. Raw data processed by the OpenLynx program was first reviewed for consistency. Since the full scale of our analog channel is 2.1  106, samples with a UV peak height over that limit saturate the UV detector. Besides the detector saturation, samples can also overload the HPLC column and generate broad peaks (FWHM > 0.1 min) in HPLC chromatogram. Both types of signal saturation were identified, and a dilution factor was estimated for each sample. Low sample concentration was another reason for rerun. Failed external standards and sample carryover are indications of an injector blockage. Samples with hydrophilic diversity eluted with the solvent front using the generic method. These samples needed to be

[1]

11

high-throughput LC/UV/MS analysis of libraries

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 12

A

A

A 1 9 17

B

B

C 1 2 3 4 5 6 7 8 9 10 11 12 D

C 13 14 15 16 17 18 19 20 21 22 23 24

E

E

Reformat

B 2 10 18 C 3 11 19 D 4 12 20 E 5 13 21

D

Alignment

F

F

G

G

F 6 14 22 G 7 15 23

H

H

H 8 16 24

Plate A

Plate B

Target plate

Fig. 6. Reformatting and realigning processes in reanalysis. (See color insert.)

dissolved in water instead of methanol and should be analyzed using a shallower gradient. All the above samples were entered into an Excel template with a plate view. There are three output lists generated automatically by the template: output to liquid handler for plating, output to MassLynx for reanalyzing, and output for realigning the final data. A Gilson liquid handler was programmed with the Unipoint software to dilute and reformat failed samples. A volume of 120 l of solvent (methanol unless specified otherwise) was added to each failed vial. The solution was taken up and released by the sampling needle three times to ensure efficient mixing. A fraction of the liquid was then transferred to the target plate. The fraction volume was determined on the Excel template by the dilution factor, for example 40 l for 2:1 dilution. Solvent was allowed to evaporate at ambient temperature from the new plate, and 200 l of solvent was then added to each well in the plate. The new plate, reformatted and compressed, was analyzed with the MUX-LCT system using the sample list from the template. Using this new format, the 24 samples in the example above (Fig. 6) were reanalyzed in three runs (13.5 min) instead of 24 runs (108 min). This represents an 8-fold improvement in efficiency. We have also created a visual basic program to modify sample location information. Since sample location was hard-coded in the data file, samples on the reformatted plate were of a different location from their original. Once processed by OpenLynx, these samples will cause a ‘‘multiple injection conflict’’ with the samples that were originally in these locations. The visual basic program used Microsoft scripting runtime objects to locate each sample on the reformatted plate. It opened the header file, searched for the sample location, and replaced it with the original location. This program can also append customized information, such as a new identity after ‘‘cherry picking,’’ into the sample file. All added operations, such as plating and alignment, were performed offline of the MUX-LCT. With this new process, sample reanalysis became much more efficient.

12

analytical techniques

[1]

Combinatorial Library Analysis

In LC/MS analysis of combinatorial libraries, the MS determines the product identity and its purity is determined by other on-line detection techniques such as UV, evaporative light scattering detection (ELSD), and chemiluminescent nitrogen detection (CLND).17–20 UV detection is used here to assess product purity based on the assumption of similar absorption coefficients at 214 nm for the desired product and the side-products. To develop a method for combinatorial library analysis, we first analyzed six to eight representative compounds from each library under generic LC/UV/MS conditions. These conditions would be used for library analysis unless adjustments had to be made based on the study of these representative compounds. Evaluation of Representative Library Compounds Five to eight representative compounds were evaluated simultaneously due to the parallel nature of the system. Depending on the structure of a library, this analysis was performed using acetic acid or TFA as modifier. We found that the general LC gradient worked well for most of the library except in a few cases in which very polar compounds eluted early. In these cases, the sample solvent, solvent gradient, or LC column was varied to optimize the retention time. However, we had to adjust ion optics settings for most libraries to ensure that the MHþ ion was the predominant ion to make product identification simple. We found that sample cone voltage was a critical parameter when all other ion optics parameters were kept constant. This was reasonable because the sample cone separates the ionization chamber with a pressure near atmospheric pressure from the vacuum region with a pressure of a few Torr. Ions could be fragmented due to collision with the gas molecules in this region. A higher sample cone voltage would produce more energetic ions to undergo collision-induced dissociation. This eight parallel LC/MS system has dramatically accelerated this process because up to eight compounds can be evaluated simultaneously under the same experimental conditions. Six compounds from library 1 (LIB1) have been analyzed simultaneously at sample cone voltages of 10, 20, 30, and 40 V. The mass spectra of two compounds (LIB1-1 and LIB1-2) are shown in Fig. 7. Only MHþ 17

L. Fang, M. Demee, T. Sierra, J. Zhao, D. Tokushige, and B. Yan, Rapid Commun. Mass Spectrom. 16, 1440 (2002). 18 L. Fang, J. Pan, and B. Yan, Biotechnol. Bioeng. Comb. Chem. 71, 162 (2001). 19 D. A. Yurek, D. L. Branch, and M. Kuo, J. Comb. Chem. 4, 138 (2002). 20 E. W. Taylor, M. G. Qian, and G. D. Dollinger, Anal. Chem. 70, 3339 (1998).

[1]

A

B

PFF115-299-40-3-40V 125 (2.487) Cm (124:126)

TOF MS ES+ 499

423.2

100 316.1 290.1

40V

317.2

773.5

314.1

TOF MS ES+ 480

0 PFF115-299-45-A3-30V 87 (1.737) Cm (86:88) 386.2 265.1 100

30V %

845.4

0 PFF115-299-40-3-20V 122 (2.427) Cm (121:123) 423.2 100

771.4

TOF MS ES+ 438

30V

772.5

%

424.2 316.1

40V

387.2

845.4

0 PFF115-299-40-3-30V 125 (2.487) Cm (124:126) 423.2 100

TOF MS ES+ 368

772.5

313.1

%

424.2

771.4

TOF MS ES+ 383

261.2 287.1

387.2 773.5

0 PFF115-299-45-A3-20V 84 (1.677) Cm (83:85) 386.2 100

TOF MS ES+ 464 771.4

20V %

424.2

0 PFF115-299-40-3-10V 127 (2.527) Cm (126:128) 423.2 100

265.2

TOF MS ES+ 165

387.2 773.5

0 PFF115-299-45-A3-10V 91 (1.817) Cm (91:93) 386.2 100

10V %

845.4 846.4

424.2

20V

772.4

%

845.4

%

TOF MS ES+ 416

10V

771.5 772.5

387.2

high-throughput LC/UV/MS analysis of libraries

%

PFF115-299-45-A3-40V 86 (1.717) Cm (85:87) 265.1 287.1 386.2 100

773.5

0 100

200

300

400

500

600

700

800

900

m/z 1000

0 100

200

300

400

500

600

700

800

900

m/z 1000

13

Fig. 7. Mass spectra of LIB1-1 (A) and LIB1-2 (B) at sample cone voltage of 10, 20, 30, and 40 V. (See color insert.)

14

analytical techniques

[1]

[100% relative abundance (RA)] and 2MHþ (dimer, 50% RA) can be found at 10 V for these compounds. Parent ions have been broken apart as the sample cone voltage increases from 10 to 40 V. A major fragment (m/z ¼ 316.1) with 70% RA could be detected in addition to MHþ (m/z ¼ 423.2, 100% RA) at 40 V for LIB1-1 (Fig. 7A). However, more extensive fragmentation was observed for LIB1-2 (Fig. 7B). Four fragment ions could be encountered along with MHþ (m/z ¼ 386.2, 90% RA) and 2MHþ (m/z ¼ 771.4, 85% RA) at 40 V. In terms of sensitivity, the total ion counts for both of the compounds are lowest at 10 V and highest at 30 V for LIB1-1 and at 20 V for LIB1-2. In general, the higher cone voltage produces the stronger ion intensity. However, higher cone voltage also causes fragmentation, which in turn leads to uncertainty in product identification. As a compromise for six compounds, the sample cone voltage was set to 20 V. The LC/MS chromatogram and mass spectra of all five compounds under optimized conditions are shown in Fig. 8. Six representative compounds from library 2 (LIB2) have also been analyzed to optimize the sample cone voltage. Mass spectra of two compounds (LIB2-1 and LIB2-2) at sample cone voltages of 20, 30, and 40 V are shown in Fig. 9. MHþ ions are shown as the predominant ions only at 40 V. Fragment ions (m/z ¼ 378.3) could be observed with an RA of 100% and 80% for LIB2-1 and LIB2-2 at 30 V. MHþ with 30% RA could be found as a minor ion at 20 V while doubly charged ions with 100% RA were the major ion. With a resolution around 5000, TOFMS made it easy to assign charge states to each ion in the spectrum. Three ions with m/z of 234.6, 378.3, and 468.3 found from LIB2-1 at 30 V are displayed in the 3 amu window in Fig. 10A, B, and C, respectively. Charge states could be easily assigned based on the mass difference between C12 and C13 for each ion observed in the mass spectrum. A mass difference of a half unit indicated that the ion with m/z of 234.6 (Fig. 10A) has a charge state of 2 while ions of 378.3 and 468.3 have a charge state of 1 since a mass difference of one unit was observed. It is concluded that product from LIB2 could be easily identified by a doubly charged ion using a sample cone voltage of 20 V or identified by a singly charged ion at 40 V. Detection sensitivity is higher for the doubly charged ion at 20 V than that of the singly charged ion at 40 V. After method development, a set of optimized ion optics settings was saved and used for future analysis of the library along with the suitable LC conditions. Library Analysis Libraries were analyzed in 10 96-well plate batches. Each QC plate contained 88 sample compounds. The last column of each plate was reserved for sampling blank and standard controls. Standards were analyzed in

[1]

PFF115-63-2-D4-MUX2 100

1: TOF MS ES+ TIC 5.55e3

1.60

PFF115-63-2-D4-MUX2 67 (1.595) Cm (67:69)

%

%

1: TOF MS ES+ TIC 7.41e3

1.39

%

1: TOF MS ES+ TIC 6.06e3

1.09

0 PFF115-45-A3-MUX2 100

1: TOF MS ES+ TIC 1.11e4

1.67

0 PFF115-45-C3-MUX2 46 (1.094) Cm (46:48) 362.1 100

1.12

1.50

0 PFF115-40-3-MUX2 100

723.3

0 PFF115-45-A3-MUX2 70 (1.673) Cm (69:71) 386.1 100

1: TOF MS ES+ 1.17e3 771.3 772.3

387.2 265.1

427.2

0 PFF115-40-3-MUX2 74 (1.761) Cm (73:75) 423.1 TIC 100 7.41e3

1: TOF MS ES+ 917

1: TOF MS ES+ 1.76

0

Time 1.00

1.50

2.00

2.50

3.00

0 100

845.3 846.3

424.2

%

%

0.50

1: TOF MS ES+ 1.11e3

363.1

%

%

1: TOF MS ES+ 988 697.2 698.2

350.1

%

%

855. 2

0 PFF115-63-1-A5-MUX2 58 (1.387) Cm (57:59) 349.1 100

200

300

400

500

600

700

800

900

high-throughput LC/UV/MS analysis of libraries

399.1

%

0 PFF115-45-C3-MUX2 100

913.2 914.2

458.1

1.04

0 PFF115-63-1-A5-MUX2 100

1: TOF MS ES+ 483

457.1

100

m/z 1000

Fig. 8. UV214 chromatogram and mass spectra of LIB1-1 to LIB1-5 under optimized conditions. (See color insert.)

15

16

A

B

PFF107-3-B4-a-1 48 (1.142) Cm (47:49) 468.3

100

1: TOF MS ES+ 450

PFF107-3-D4-a-1 44(1.056) Cm (43:45) 1: TOF MS ES+ 484.3 209 100

40V

40V %

% 333.2 232.1 209.2 290.2 378.3

485.3

469.3

232.1 470.3

0 1: TOF MS ES+ PFF107-3-D4-a 41 (0.984) Cm(40:42) 484.4 968 100

1: TOF MS ES+ 647

378.3 242.7

30V

30V

%

468.4

% 234.7 235.2

379.3

243.2

394.3

485.4

469.4

0 1: TOF MS ES+ PFF107-3-D4-a 40 (0.960) Cm(40:42) 242.6 1.28e3 100

0 PFF107-3-B4-a 45 (1.070) Cm(44:47) 234.6 100

1: TOF MS ES+ 1.17e3

20V 243.2

235.2

484.3 468.3

235.7

0 100

20V %

%

200

300

analytical techniques

0 PFF107-3-B4-a 45 (1.070) Cm(44:47) 378.3 100

333.2 378.3

400

500

243.7

600

700

800

900

m/z 1000

0 100

200

300

485.3

400

500

600

700

800

900

[1]

Fig. 9. Mass spectra of LIB2-1 (A) and LIB2-2 (B) at sample cone voltage of 20, 30, and 40 V. (See color insert.)

m/z 1000

[1]

PFF107-3-B4-a 45 (1.070) Cm (44:48)

100

234.7

1: TOF MS ES+

370

PFF107-3-B4-a 45 (1.070) Cm (44:48) 1: TOF MS ES+

100

378.3

1.09e3

PFF107-3-B4-a 45 (1.070) Cm (44:48)

100

468.4

459

%

%

high-throughput LC/UV/MS analysis of libraries

C

B

A

1: TOF MS ES+

%

235.2 469.4

379.3 235.7 0 234

235

236

m/z 237

0 378

379

380

m/z 381

0 468

469

470

m/z 471

17

Fig. 10. Isotope of three ions found from LIB2-1 at a sample cone voltage of 30 V. (A) Charge state of 2; (B, C) charge state of 1. (See color insert.)

18

[1]

analytical techniques

Number of compounds

2000

Average purity 80.6%

1500

1000

500

0

10

20

30

40

50

60

70

80

90 100

Purity by UV214 (%) Fig. 11. Library LIB2 purity distribution of 5280 compounds measured at UV214. (See color insert.)

every 24 injections during analysis to monitor the performance consistency of all eight channels. The analysis queue was constructed from an Excel spreadsheet and imported into the MassLynx software for execution. After acquisition, the data were processed using MassLynx in batches. Processed data could be reviewed in OpenLynx by selecting a plate and clicking on the desired well. The UV chromatogram and mass spectrum of the desired product in LIB2, plate26, well D1, are shown as an example in Fig. 9. We generated an Excel report that included filename, expected molecular weight, purity of desired products at 214 nm, and a plate view with purity indicated for all compounds in the 10-plate batch. Library LIB2 was composed of 60 plates; it was analyzed in positive ion mode and processed in six batches. The purity distribution of library LIB2 is shown in Fig. 11 with an average of 80.6% for 5280 compounds measured at 214 nm. Figure 11 shows the plate view of all 60 plates. According to this protocol, we have completed more than a half million LC/UV/MS analyses in a period of 15 months with two eight-channel MUX-LCT systems. Comparison of the Eight-Channel LC/UV/MS (MUX-LCT) System with a Conventional Single-Channel LC/UV/MS System

The significant advantage of the parallel LC/MS system is its throughput. Because eight LC/UV/MS analyses can be conducted simultaneously, the total analysis time is decreased by a factor of eight. To analyze every compound in a library of 2500 compounds at 3.5 min cycle time requires 146 h using a single channel LC/UV/MS system. However, it requires only

[1]

high-throughput LC/UV/MS analysis of libraries

19

18.2 h to complete this task using an eight-channel parallel LC/UV/MS system, and this makes it possible to perform LC/UV/MS analysis on every compound for all of our libraries. In addition, this system also speeds up method development because it simultaneously evaluates up to eight parameters or variables such as the performance of eight different columns. UV and TIC Chromatograms An important concern in using an eight-way MUX interface is that the acquisition cycle time (the time required to acquire one data point for each channel) is longer, and the data acquisition time per channel is shorter, than for a single-spray system. Therefore, the sensitivity might be lower and the peak shape could be distorted. In our current system with a timeof-flight mass spectrometer, the minimum time required for each acquisition cycle is 1.2 s with 0.1 s for data acquisition and 0.05 s for intersprayer delay. The chromatographic baseline peak width was between 5 and 6 s in the UV chromatograms and between 6 and 7 s in the TICs under general LC/UV/MS conditions. A maximum of five MS data points could be acquired to define a peak, which resulted in slightly distorted peak shapes in the TICs. On the other hand, peak shapes were much better defined in a single-channel system because more than 10 data points could be easily obtained. For combinatorial library analysis, lower sensitivity is not a problem because the parallel synthesis method always produces enough compound for analysis. The limited number of data points across an LC peak was usually not a problem because the MS data were used only to identify the peak of interest. In theory, one or two data points (TOF mass spectra) should be sufficient to confirm the expected molecular weight. The product purity was obtained from the UV chromatogram, where the number of data points was sufficient to ensure excellent peak shape and precision. Data Acquisition Using Positive and Negative Ionization In a single-spray system, it is common to analyze samples in both positive and negative ion modes by switching polarity during a single data acquisition. This practice makes the best use of precious MS time and identifies products by their presence in both positive and negative ion forms. Both positive and negative ESI modes are available for the eightchannel MUX-LCT system. However, the polarity change within a single data acquisition would make the cycle time much longer. Therefore, we prefer to analyze samples using a single polarity, and conduct a separate experiment with the other polarity if necessary. With this arrangement, high-throughput LC/MS analysis with both positive and negative mode is available.

20

analytical techniques

[1]

Sample Rerun For a conventional single-channel LC/UV/MS system, a single unsatisfied well could be easily reanalyzed. In the eight parallel LC/UV/MS system, the rerun procedure was different from that of the single-spray system. If problems were found in a single channel, such as retention time shift or channel blockage, 12 wells in a row would fail and the whole plate had to be reanalyzed. We have developed a rerun protocol that made the parallel LC/MS analysis as efficient as the single-channel system. Operation and Maintenance In the eight-channel parallel LC/UV/MS system, a standard mixture was analyzed every 24 injections. This was indispensable for the operation. The variation of the retention time across eight channels was monitored closely to ensure consistency for the eight channels. A significant retention time shift indicated problems that usually could be overcome by replacing the frit in the precolumn filter. A diminished peak area or a change in peak shape of standards indicated column deterioration. We started with eight columns from the same batch for sample analysis. Deteriorated columns were replaced individually. This practice gave us satisfactory analysis data for combinatorial library analysis with minimal cost. We anticipated difficulty in maintaining and troubleshooting an eightchannel parallel system because the problems in the autosampler, LC columns, UV detectors, and MS interface would be multiplied by eight. In fact, with the convenience of simultaneous analysis of the other seven channels, the diagnosis and troubleshooting were made easier. The complete system was easily divided into four functions: injection, separation, UV detection, and MS detection. By running the standard mixture on eight channels then switching channels at different function sites and rerunning the standard mixture, problems were easily isolated. Fixing the problems was exactly the same as for the single-spray system. Conclusion

We have optimized an eight-channel parallel LC/UV/MS (MUX-LCT) system for high-throughput LC/UV/MS analysis of large combinatorial libraries. Since the LC gradient is divided into eight LC columns by a simple splitter, the flow fluctuation has been continuously monitored and minimized using a standard mixture during analysis to ensure performance consistency among the eight channels. To preserve the separation integrity in the total ion chromatogram, the zero dead volume T-joint used to split the flow (after UV detection) should be best placed as close to the eight-way

[2]

21

qualitative colorimetric tests for sps

MUX inlet as possible. A flow rate of 12 ml/min on eight 2.1  50 mm Polaris C18 columns was optimal for general purposes in our study. This system could analyze more than 3000 compounds per day for a gradient separation with a cycle time of 3.5 min. We have carried out more than half a million LC/UV/MS analyses in 15 months using two eight-channel parallel LC/UV/MS systems. We found that it was beneficial to evaluate a few representative compounds from each library and optimize ion optics to make product identification simple and reliable. This parallel system has enabled simultaneous evaluation of eight compounds and significantly improved the speed of optimization. The identity and purity of every single product could be obtained from OpenLynx in 10 96-well plate per batch process and transferred into an Excel spreadsheet for the entire library. Compared with a single-channel LC/UV/MS system, the parallel LC/UV/MS system has the advantages of high throughput and simultaneous evaluation of eight parameters. Acknowledgments We thank Jason Cournoyer, Michael Demee, Duayne Tokushige, Melody Wen, and Teresa Sierra for their assistance throughout this work.

[2] Qualitative Colorimetric Tests for Solid Phase Synthesis By Jesu´s Va´zquez, Gregory Qushair, and Fernando Albericio Introduction

Solid-phase synthesis (SPS)* is limited by a shortage of simple and rapid techniques for reaction monitoring, specifically for functional group transformations. The traditional preparation and subsequent analysis (HPLC, *Abbreviations: AliR, alizarin R; BAL, backbone amide linker; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DME, N,N’-dimethylformamide; DTNB, 5,51-dithio(2nitrobenzoic acid) or Ellman’s reagent; Et3N, triethylamine; EtOH, ethanol; HOAc, acetic acid; EtOAc, ethyl acetate; Hex, mixture of hexane isomers plus methylcyclopentane; HPLC, high-pressure liquid chromatography; MeOH, methanol; MG, malachite green; MS, mass spectrometry; NMM, N-methylmorpholine; NMP, N-methylpyrrolidinone; Purpald, 4-amino-3hydrazino-5-mercapto-1,2,4-triazole; SPS, solid-phase synthesis; TCT, trichlorotriazine; THF, tetrahydrofuran; TLC, thin-layer chromatography; TNBSA, trinitrobenzenesulfonic acid; TosCl-PNBP, p-tosylchloride p-nitrobenzylpyridine; TRIS, tris(hydroxymethyl) aminomethane.

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

22

analytical techniques

[2]

MS, etc.) of resin cleavage products are time-consuming processes, hence alternative methods are desirable. The use of colorimetric functional group tests, wherein aliquots of resin are mixed with stock solutions and changes in solution/resin color are used to indicate the presence or absence of functional groups on the resin, was initiated by Kaiser et al.1 in the use of ninhydrin to test for primary amines in the SPS of peptides. Today organic chemists have at their disposal an ever-broadening array of tests, both qualitative and quantitative, for alcohols, aldehydes, amines, carboxylic acids, and thiols. We present a literature overview of the most widely used qualitative tests including instructions on reagent preparation and storage, experimental protocol, and the scope and limitations of each test.2 The majority of these tests can be performed in less than 10 min with simple laboratory equipment and minimal reagent preparation. We also report the results of experiments to determine the functional group response of each test using amino acids as representative organic compounds. It was our intention to provide a central reference for the most common qualitative tests with a special emphasis on substrate compatibility, namely functional group interference. For example, it is known that certain amino acids may give unusual results for a given test (such as cysteine with the ninhydrin test) and some of the original colorimetric test publications include brief reports on the potential for functional group interference (false positives) for a given test. To determine the utility of each test in the presence of multiple functional groups, each of the summarized colorimetric tests was applied against a broad range of amino acids. The aim of this exercise was to determine the universality of each test, and to identify and report those cases with unusual results. The amino acids were tested for the presence of each functional group under all possible levels of amine and lateral chain protection, thus enabling us to determine the extent to which chemical interference by other functional groups could affect each test (see Scheme 1). Table I summarizes qualitative colorimetric tests reported in the literature for various organic functional groups. We also investigated the use of each test at medium (approximately 0.5 mmol/g) and low (approximately 0.025 mmol/g) resin loading. General Experimental Procedures

Resin (polystyrene-based resin, 1% divinylbenzene, 100–200 mesh) substitution and amino acid deprotection were carried out in disposable 1 2

E. Kaiser, R. L. Colescott, C. D. Bossinger, and P. Cook, Anal. Biochem. 34, 595 (1970). To the best of our knowledge, only one other review covering some of the methods described herein exists in the literature: C. Kay, O. E. Lorthioir, N. J. Parr, M. Congreve, S. C. McKeown, J. J. Scicinski, and S. V. Ley, Biotechnol. Bioeng. 71, 110 (2001).

[2]

qualitative colorimetric tests for sps

23

Scheme 1. Amino acids were tested at all possible levels of protection. This enabled us to differentiate between test results caused by a given free functional group and results that may have been caused by other chemical moieties in the molecule.

syringes fitted with polypropylene filter discs using standard solid-phase peptide synthesis procedures. For the majority of the tests described, the experimental protocols were adapted with minor changes from the original publications, none of which we feel jeopardizes the essence of each test (i.e., the underlying chemistry). In the majority of cases, tests were performed immediately after preparation of each resin by aliquoting the resin into equivalent portions using the following technique: to the syringe containing the master quantity of resin is added dichloromethane (DCM, ca. 1 ml/100 mg resin), the resin is agitated with a pipetter by continuously taking up and ejecting a small volume in order to create a uniform suspension, and the desired volume of suspension is quickly removed and transferred to an Eppendorf tube or glass vial and allowed to air dry. This technique is more effective than dispensing dry resin with a spatula since it is faster and more precise for tiny aliquots (1–5 mg) of resin. Eppendorf tubes (2 ml) were used for all tests except the TosCl-PNBS, Kaiser, Va´zquez, and Purpald tests. Disposable glass tubes (800 l) were used for the Kaiser and Va´zquez tests, a disposable syringe (1 ml) fitted with a polypropylene filter disc was used for the TCT, Methyl red, and Purpald

24

analytical techniques

[2]

TABLE I Summary of Qualitative Colorimetric Test Functional group Primary aliphatic amine

Secondary aliphatic amine Primary alcohol Secondary alcohol Tertiary alcohol Phenol Thiol Carboxylic acid Aldehydew a

Tests Kaiser (ninhydrin),a–c trinitrobenzenesulfonic acid (TNBSA),d NF-31,e chloranil,f–h bromophenol blue,i,j nitrophenylisothiocyanate-O-trityl(NPIT),j,k Malachite green isothiocyanate (MGI),j,l Traut’s reagents,j,m and Ellman’s reagents,j,m TNBS,d NF-31,e chloranil,f–h bromophenol blue,i,j MGIi,j TosCl-PNBP,n (1,3,5)-trichlorotriazine (TCT) with fluorescein, Alazarin R, or fuchsin,o,p TosCl-PNBP,n TCT-fluorescein, Alizarin R, or fuchsino,p Diphenyldichlorosilane-methyl redq TosCl-PNBP,n,r TCT-fluorescein, Alizarin R, or fuchsin,o,p diphenyldichlorosilane-methyl redq Ellman’s reagents,t Malachite green,u Cystamine-Ellman’s reagentj,v Va´zquez ( p-anisaldehyde),x Purpaldy

E. Kaiser, R. L. Colescott, C. D. Bossinger, and P. Cook, Anal. Biochem. 34, 595 (1970). V. K. Sarin, S. B. H. Kent, J. P. Tam, and R. B. Merrifield, Anal. Biochem. 117, 147 (1981). c W. Troll and R. K. Cannan, J. Biol. Chem. 200, 803 (1953). d W. S. Hancock and J. E. Battersby, Anal. Biochem. 71, 260 (1976). e A. Madder, N. Farcy, N. G. C. Hosten, H. De Muynck, P. J. De Clercq, J. Barry, and A. P. Davis, Eur. J. Org. Chem. 2787 (1999). f T. Christensen, Acta Chem. Scand. B 33, 763 (1979). g T. Vojkovsky, Peptide Res. 8, 236 (1995). h The chloranil test can also be used to selectively react with primary amines (see experimental section). i V. Krchna´k, J. Va´gner, P. Safar, and M. Lebl, Collect. Czech. Chem. Commun. 53, 2542 (1988). j This test was not reviewed for this publication. k S. S. Chu and S. H. Reich, Bioorg. Med. Chem. Lett. 5, 1053 (1995). l A. Shah, S. S. Rahman, V. de Biasi, and P. Camillero, Anal. Commun. 34, 325 (1997). m T. T. Ngo, Appl. Biochem. Biotechnol. 13, 213 (1986). n O. Kuisle, M. Lolo, E. Quin˜oa´, and R. Riguera, Tetrahedron 55, 14807 (1999). o M. E. Attardi, A. Falchi, and M. Taddei, Tetrahedron Lett. 41, 7395 (2000). p M. E. Attardi, A. Falchi, and M. Taddei, Tetrahedron Lett. 42, 2927 (2001). q B. A. Burkett, R. C. D. Brown, and M. M. Meloni, Tetrahedron Lett. 42, 5773 (2001). r There are conflicting reports in the literature on the utility of this test for phenols.n,q s G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959). t J. P. Baydal, A. M. Cameron, N. R. Cameron, D. M. Coe, R. Cox, B. G. Davis, L. J. Oates, G. Oye, and P. G. Steel, Tetrahedron Lett. 42, 8531 (2001). u M. E. Attardi, G. Porcu, and M. Taddei, Tetrahedron Lett. 41, 7391 (2000). v T. T. Ngo, Appl. Biochem. Biotechnol. 13, 207 (1986). w An aldehyde (BAL) linker was used as a model for this functional group. x J. Va´zquez and F. Albericio, Tetrahedron Lett. 42, 6691 (2001). y J. J. Cournoyer, T. Kshirsagar, P. P. Fantauzzi, G. M. Figliozzi, T. Makdessian, and B. J. Yan, J. Comb. Chem. 4, 120 (2002). b

[2]

qualitative colorimetric tests for sps

25

tests, and the TosCl-PNBP test was performed on TLC plates (silica gel, aluminum backed). NF-31 test sample tubes were heated directly in a preheated, multiwell aluminum block. Kaiser and Va´zquez test sample tubes were heated in a preheated sand bath inside of a laboratory oven. Heating of the silica plates for the TosCl-PNBP test was performed using a laboratory heat gun on high setting. Aliphatic Amines Test: Kaiser (Ninhydrin)1,3,4 Application: detection of primary amines Test time: 4 min Reagent preparation time: 1 day Recommended storage time: 1 month at room temperature in light-proof containers (such as amber bottles) Required Reagents Ninhydrin dissolved in ethanol Phenol dissolved in ethanol aq. KCN dissolved in pyridine Preparation of Reagent Solutions Reagent Solution A. Phenol (40 g) in added to EtOH (10 ml) and the mixture is heated until complete dissolution of the phenol. A solution of KCN (65 mg) in water (100 ml) is added to pyridine (freshly distilled over ninhydrin, 100 ml). Both solutions are stirred for 45 min with Amberlite MB-3 (Merck), filtered, and mixed. Reagent Solution B. A solution of ninhydrin (2.5 g) in absolute EtOH (50 ml) is prepared and maintained in a light-proof container, preferably under inert atmosphere. Experimental Procedure. The resin is washed with appropriate solvents and a small portion (ca. 1–5 mg) is transferred to a small glass tube. To this tube are added three drops of each of the reagent solutions A and B.  The tube is then heated at 100 for 3 min. A negative test, indicating the absence of free primary amines, is communicated by a yellow or orangepink solution and naturally colored beads. A positive test is indicated by a dark blue or purple solution and beads. Variations in the darkness of the solution reflect variations in amine concentration while variations in

3 4

V. K. Sarin, S. B. H. Kent, J. P. Tam, and R. B. Merrifield, Anal. Biochem. 117, 147 (1981). W. Troll and R. K. Cannan, J. Biol. Chem. 200, 803 (1953).

26

analytical techniques

[2]

the color observed (red, green, etc.) are particular to certain substrates and may represent false positives. Notes. The Kaiser test is generally reliable; however, when used to test sterically hindered amines such as aminoisobutyric acid (Aib), results may be difficult to interpret. We found that the color yielded for primary alkyl amine positive tests is strongly influenced by the presence of other functional groups. Specifically we could not establish a clear positive for arginine, asparagine, and cysteine when their lateral chains were deprotected. A similar effect was observed in the case of secondary amines or sterically hindered amines such as Aib. We found that at lower levels of resin functionalization a clear positive was difficult to observe. Test: TNBSA (trinitrobenzenesulfonic acid)5 Application: detection of primary/secondary amines Test time: 5 min Reagent preparation time: minutes Recommended storage time: up to 1 month refrigerated storage Required Reagents A 1% (w/v) solution of TNBSA in DMF A 10% solution of N,N-diisopropylethylamine (DIEA) in DMF Experimental Procedure. The resin is washed with MeOH and a small portion (1–3 mg) is transferred to an Eppendorf tube and suspended in DMF. To this tube is added 1 drop of each of the above solutions. The solution is left for 5 min at room temperature. The resin is washed extensively with DMF. The presence of free amines is indicated by orange or red beads. Notes. The TNBSA test was found to be efficient for primary amines, including sterically hindered amines as seen in our probes with the tertiary amine of Aib. We also found that at lower levels of resin functionalization a clear positive was difficult to observe. Test: NF-316 Application: detection of primary/secondary amines Test time: 10 min Reagent preparation time: 1 week preparation  Recommended storage time: up to 1 month at 4

5 6

W. S. Hancock and J. E. Battersby, Anal. Biochem. 71, 260 (1976). A. Madder, N. Farcy, N. G. C. Hosten, H. De Muynck, P. J. De Clercq, J. Barry, and A. P. Davis, Eur. J. Org. Chem. 2787 (1999).

[2]

qualitative colorimetric tests for sps

27

Required Reagents Disperse Red 1 Ethyl diazoacetate Phosphorous oxychloride p-Nitrophenol Solution Preparation. NF-31 is obtained via a three-step procedure starting from Disperse Red 1. Ether Formation with Ethyldiazoacetate. Disperse Red 1 (2.0 g, 1.0 eq.) and rhodium tetraacetate [Rh2(OAc)4] (47 mg, 0.016 eq.) are dissolved in DCM–toluene (1:1, 50 ml) in a dry two-neck round-bottom flask  and stirred at 40 . A solution of ethyldiazoacetate (2.64 ml, 4.0 eq.) in toluene (13 ml) is added dropwise (Caution) and the solution is left to react overnight. The solution is concentrated to dryness by rotary evaporation. The reaction flask is placed on an ice bath and a solution of 10% aq. HOAc (50 ml) is added to the crude product. The mixture is adsorbed onto silica gel, and the product is purified by column chromatography [silica gel, EtOAc-Hex (1:9)]. Saponification. The purified product (1g, 1.0 eq.) from step 1 and KOH (870 mg, 5 eq.) are dissolved in MeOH–toluene [(5:1), 60 ml]. The  solution is stirred and brought to reflux (ca. 85 ) for 90 min. The reaction is allowed to cool and a red precipitate is observed. The cooled reaction mixture is concentrated by rotary evaporation to a volume of 10 ml. Then 10% aq. HCl (10 ml) is added, followed by water (25 ml). The precipitate is extracted with DCM and the organic phase is washed with water and dried on anhydrous MgSO4. The mixture is filtered, and the filtrate is concentrated by rotary evaporation and the product purified by column chromatography. Condensation with p-Nitrophenol. The product (500 mg, 1.0 eq.) from step 2 and p-nitrophenol (178 mg, 1.0 eq.) are dissolved in DCM (26 ml) and pyridine (22 ml) is then added. The aforementioned solution  is maintained at 15 , a solution of POCl3 (222 l, 1.8 eq.) in DCM (2 ml) is added dropwise, and the solution is then left to react overnight at room temperature. The crude reaction mixture is then washed with aq. saturated NaHCO3 and brine and dried over anhydrous MgSO4. The crude product is concentrated by rotary evaporation and purified by column chromatography [silica gel, EtOAc-Hex (1:9)]. Experimental Procedure. The resin is washed with methanol and a small portion (1–3 mg) is transferred to an Eppendorf tube. To this tube is added NF-31 solution (0.002 M in acetonitrile, 200 l). The tube is heated in an aluminium dry heating block at 70 for 8 min. The resin is washed extensively with MeOH (3), DMF (3), and DCM (3). The presence of free

28

analytical techniques

[2]

amines is indicated by red-colored beads whereas a negative test yields naturally colored beads. Notes. The NF-31 test was found to be highly sensitivite for primary and secondary amines. During our probes of this test we found that false positives are given if the resin is not washed thoroughly with the appropriate solvents. Test: Chloranil7,8 Application: detection of primary/secondary amines Test time: 5 min Reagent preparation time: minutes Recommended storage time: up to 1 month refrigerated storage Required Reagents Acetaldehyde (for detection of primary or secondary amines) or acetone (for detection of secondary amines) Saturated solution of chloranil in toluene Experimental Procedure. The resin is washed with MeOH and a small portion (1–3 mg) is transferred to a small glass tube. To this tube is added acetaldehyde (primary or secondary amines) or acetone (secondary amines) (200 l) followed by the chloranil solution (50 l). The solution is shaken at room temperature for 5 min. The presence of free amines is indicated by a green- or blue-colored solution. Negative samples register as yellow, amber, or brown. Notes. The presence of a secondary amine should be confirmed by a positive result obtained for the secondary test and a simultaneously obtained negative result for the primary test. Likewise the Kaiser test can be used in place of the primary amine version of the chloranil. This test gave excellent results in both of its forms (testing for primary and for secondary amines); a clear positive was observed even at low levels of resin functionalization for the sterically hindered Aib. Alcohols Test: TosCl-PNBP9 Application: detection of alcohols and phenols Test time: ca. 5 min Reagent preparation time: 5 min  Recommended storage time: no more than 2 weeks at 4 7

T. Christensen, Acta Chem. Scand. B 33, 763 (1979). T. Vojkovsky, Peptide Res. 8, 236 (1995). 9 O. Kuisle, M. Lolo, E. Quin˜oa´, and R. Riguera, Tetrahedron 55, 14807 (1999). 8

[2]

qualitative colorimetric tests for sps

29

Required Reagents A solution of p-toluenesulfonyl chloride (0.12 M) in toluene (solution 1) A solution of p-nitrobenzylpyridine (0.30 M) in toluene (solution 2) A 10% (v/v) solution of piperidine in CHCl3 (solution 3) Experimental Procedure. The resin is washed with DCM. A small portion (3–5 mg) of resin is deposited onto a silica plate by pipette as a DCM suspension. The suspension should be pipetted quickly so that it forms a disperse disc (not a mound). Once dry, the resin is treated with one drop of solution 1 and one drop of solution 2. The plate is then heated with a heat gun by swaying the plate in front of the gun from a distance of approximately 5 cm for approximately 1 min. A yellow color should appear and then disappear within the heating time, leaving the resin similar to or slightly darker than its natural color. At this point a drop of solution 3 is added to the resin sample on the plate. Purple coloration of the beads indicates the presence of free hydroxyl groups (light pink or purple at low concentration, dark purple at high concentration). Notes. To get reliable results, concentrations of reagents should be approximately four times higher than that reported in the original paper. To perform several tests on one silica plate, the resin spots should be deposited approximately 1–2 cm from each other in each direction. It is always advisable to carry out control tests, both a positive (a resin bearing either a free alcohol or phenol) and a negative (ideally, an acetylated hydroxy resin). In this case, heating of the plate should be carried out until the positive resin control takes an orange-red color. We found this test to be highly dependent on the quality of the solutions used. Solutions stored at room temperature for prolonged periods of time gave almost 100% false positives. Although there are conflicting reports in the literature on the utility of this test for phenols,9,10 in our hands the tests for the phenol of Tyr gave the expected positive results. Test: TCT-(Fluoresceine, Alizarin R, or Fuchsin)11,12 Application: Detection of alcohols Test time: ca. 30 min Reagent preparation time: minutes  Recommended storage time: No more than 2 weeks at 4

10

B. A. Burkett, R. C. D. Brown, and M. M. Meloni, Tetrahedron Lett. 42, 5773 (2001). M. E. Attardi, A. Falchi, and M. Taddei, Tetrahedron Lett. 41, 7395 (2000). 12 M. E. Attardi, A. Falchi, and M. Taddei, Tetrahedron Lett. 42, 2927 (2001). 11

30

analytical techniques

[2]

Reagents 1,3,5-Trichlorotriazine (TCT) N-Methylmorpholine (NMM) Fluoresceine, Alizarin R, or Fuchsin Experimental Procedure. The procedure is composed of six steps: 1. A few milligrams of resin is placed in a small glass tube and washed with DMF. 2. DMF (3 ml) is added followed by NMM (1 ml) and TCT (5 mg).  3. The tube is heated at 70 for 20 min. 4. The solution is pipetted off and the resin is washed thoroughly with DMF. 5. DMF (3 ml) is then added to the resin followed by AliR (5 mg) (or 3 ml of a 0.025% solution of fuchsin or fluoresceine in NMP) and NMP (1 ml). 6. After 5 min the resin is washed thoroughly with DMF until a clear solution is obtained, then washed with THF and finally with DCM. A positive test is communicated by red beads for the AliR test and green or fluorescent beads in the case of the fluoresceine test. Notes. A drawback found in the use of this test is the formation of a white precipitate during the activation of alcohol with the triazine. If fluoresceine is used, the resin beads must be viewed with ultraviolet light as visible light is not sufficient to determine results. Ambiguous results were often obtained with this test, above all with phenols such as the one of tyrosine. This test also yields positive results in the presence of other nucleophiles such as primary and secondary amines, carboxylic acids, and thiols. Test: Diphenyldichlorosilane-Methyl red10 Application: Detection of alcohols Test time: ca. 25 min Reagent preparation time: minutes Recommended storage time: Up to 1 month at room temperature Required Reagents 10% triethylamine (Et3N) in dry DCM Diphenyldichlorosilane 0.75% (w/v) of methyl red in DMF Experimental Procedure. A few milligrams of resin are moistened with a solution of 10% Et3N in anhydrous DCM (200 l) and treated with diphenyldichlorosilane (100 l) for 10 min. The resin is then filtered, washed

[2]

qualitative colorimetric tests for sps

31

twice with 10% Et3N in anhydrous DCM, at which point a 0.75% (w/w) solution of methyl red in DMF (300 l) is added and the resin allowed to shake for 10 min. The resin is filtered, washed with DMF (5  1 min), and then with DCM (5  1 min). A positive test is indicated by orange beads, which become more reddish with time. In the case of ambiguous results, the beads can be treated with formic acid and will take on a purple color in the case of a positive result. Notes. This test proved to be satisfactory not only for phenols but for primary and secondary alcohols as well. In some cases, light purple beads may be observed even without the addition of formic acid as a true positive. We strongly recommend the use of a blank control run in parallel with the sample to be tested as the colorant used in this test readily stays trapped within resins. This test also yields positive results in the presence of other nucleophiles such as primary and secondary amines, carboxylic acids, and thiols. Thiols Test: Ellman’s reagent13–15 Application: Detection of thiols Test time: 4 min Reagent preaparation time: 5 min  Recommended storage time: up to 1 month at 4 Required Reagent 5,50 -Dithio(2-nitrobenzoic acid), ‘‘DTNB,’’ or ‘‘Ellman’s reagent’’ dissolved in aq. TRIS solution (1 M, pH 8) Experimental Procedure. To a suspension of an aliquot of resin in DMF is added three or four drops of the DTNB solution. The solution is shaken at room temperature for 3 min. The presence of free thiols is indicated by a yellow-orange color. Notes. This test, an adaptation of the original Ellman’s reagent test, worked well for the detection of free thiol groups.

13

G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959). J. P. Baydal, A. M. Cameron, N. R. Cameron, D. M. Coe, R. Cox, B. G. Davis, L. J. Oates, G. Oye, and P. G. Steel, Tetrahedron Lett. 42, 8531 (2001). 15 M. Royo, unpublished results (1991). 14

32

analytical techniques

[2]

Carboxylic Acids Test: Malachite green (MG)16 Application: Detection of carboxylic acids Test time: ca. 5 min Reagent preparation time: minutes  Recommended storage time: up to 1 month at 4 Required Reagents A 0.25% (w/v) solution of malachite green dissolved in ethanol Et3N Experimental Procedure. A small portion (1–3 mg) of resin is transferred to an Eppendorf tube and washed with MeOH. To this tube is added the malachite green solution (1 ml) followed by two drops of Et3N. The solution is left to stand at room temperature for 3 min and the resin is washed extensively with MeOH. The presence of free carboxylic acids is indicated by green beads. Notes. We found this test to be highly reproducible, but the test time is crucial to obtaining accurate results. We observed false positives when resin samples were left in the malachite green solution for more than 15 min, and a loss of color when green beads are left in MeOH for more than 15 min. Aldehydes Test: Va´zquez17 Application: Detection of aldehydes Test time: 5 min Reagent preparation time: ca. 5 min Recommended storage time: up to 1 month at room temperature Required Reagents A solution of EtOH (88 ml), H2SO4 (9 ml), and HOAc (1 ml) p-Anisaldehyde Experimental Procedure. A solution of p-anisaldehyde (26 l) in the first reagent (1 ml) is made. A small portion (1–3 mg) of resin is transferred to a small glass tube and washed with MeOH. To this tube is added the previous EtOH/H2SO4/HOAc solution (500 l). The solution is heated in a  sand bath at 110 for 4 min. The presence of free aldehydes is indicated by orange- to burgundy-colored beads. 16 17

M. E. Attardi, G. Porcu, and M. Taddei, Tetrahedron Lett. 41, 7391 (2000). J. Va´zquez and F. Albericio, Tetrahedron Lett. 42, 6691 (2001).

[2]

qualitative colorimetric tests for sps

33

Notes. This test is very reliable. This test is compatible with acid-labile resins such as the Wang and chlorotrityl resins. While in the case of the Wang resin similar results were obtained, in the case of the chlorotrityl resin solutions also became colored, indicating cleavage of the aldehyde (BAL handle) from the resin. Test: Purpald18 Application: Detection of aldehydes Test time: 25 min Reagent preparation time: minutes Recommended storage time: N/A (see Notes) Required Reagents 4-Amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald) 1 M NaOH Experimental Procedure. The resin is washed with MeOH and a small portion (ca. 5 mg) is transferred to a disposable syringe (1 ml) fitted with a polypropylene filter disc. To the resin is added of DMF (1 ml) and the syringe is capped and shaken for 5 min. The DMF is then drained and a freshly prepared solution of Purpald dissolved in 1 M NaOH (250 l) is added. The syringe is capped and shaken for 5 min. The solution is drained and the resin is washed with DCM (3  1 ml). The resin is then left uncapped for 10 min. The presence of free aldehydes is indicated by brown or purple beads. At lower values of resin loading, a longer air oxidation time may be required for color to develop (up to 20 min). Notes. Due to the instability of Purpald in solution, it is imperative that only freshly prepared reagent solution be used. Conclusions and Summary

Results obtained in the application of these tests are summarized in Table II. We have encountered some variations in the reproducibility and accuracy of some tests. Due to the numerous factors that can influence colorimetric test results (e.g., test reagent stability, resin type, functional group interference, and lability of protecting group) we highly recommend performing a positive and a negative control for any test applied to a new synthesis. We also emphasize the importance of reagent solution purity on the outcome of test results, hence we strongly encourage the use of correctly prepared and carefully stored reactants. To minimize false results 18

J. J. Cournoyer, T. Kshirsagar, P. P. Fantauzzi, G. M. Figliozzi, T. Makdessian, and B. J. Yan, J. Comb. Chem. 4, 120 (2002).

34

TABLE II Summary of Results Obtained in the Application of the Testsa Colorimetric test

Functional group

a

TNBS

NF-31

Chloranil Chloranil TosClfirst amine second amine PNBP

TCT

Methyl red Ellman

MG

Va´zquez Purpald

/ / / / þþ/þ þ/

/ / / / þþ/þ þþ/þ

/ / / / þþ/þþ þþ/þ

/ / / / þþ/þ þ/þ

/ / / / þ/ þ/

þþ/þ þþ/þ þ/ / / /

þþ/þ þþ/þ þþ/þ / þþ/þ þ/þ

þþ/þ þþ/þ þþ/þ / þ/þ þ/

/ / / / / /

/ / / / / /

/ / / þþ/þ / /

/ / / þþ/þ / /

þ/ / / /

þþ/þ / / /

þþ/þþ / / /

þþ/þ / / /

þþ/þ / þ/ /

/ / / /

þ/þ / þ/þ þþ/þ

þ/þ / þ/ þþ/þ

/ / / þ/þ

/ / þþ/þþ /

/ / / /

/ / / /

analytical techniques

Primary alcohol Secondary alcohol Phenol Aldehyde Primary amine Hindered primary amine Secondary amine Guanidine Carboxylic acid Thiol

Kaiser

A/B, A is the result obtained for a 100% loading resin and B is the result for a 5% loading resin; þþ, intense; þ, less intense; þ, not clear; , no difference with a blank control.

[2]

[2]

qualitative colorimetric tests for sps

35

due to lability of the resin–product bond or the product itself (such as Fmoc-protected amino acids), colorimetric tests should be performed with the utmost immediacy in regards to completion of the step to be monitored. Hence the storage of resin over long periods of time (more than 24 h) before testing is not advisable. When the result of a colorimetric test is in doubt we advise repeating the test a few times until a reproducible result is obtained. The use of multiple tests for the same functional group may elucidate ambiguous or otherwise challenging cases. Acknowledgments The authors’ laboratory work was supported by CICYT (BQU2000–0235) and the Generalitat de Catalunya (Grup Consolidat and Centre de Refere`ncia en Biotecnologia). The enthusiastic collaboration of Dr. Miriam Royo, Gloria Sanclimens, Aida Marti´nez, and Meritxell Teixido´ at the Barcelona Biomedical Research Institute, Barcelona Science Park, University of Barcelona in the experimental portion of the work and the preparation of the manuscript is greatly appreciated.

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Section II Directed Sorting Split-and-Pool Combinatorial Synthesis

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[3]

solid-phase synthesis on synphase lanterns

39

[3] A Review of Solid-Phase Organic Synthesis on SynPhase‘ Lanterns and SynPhase‘ Crowns By Jack G. Parsons, Craig S. Sheehan, Zemin Wu, Ian W. James, and Andrew M. Bray Introduction

A decade ago there were few examples of solid-phase organic synthesis outside of the specialized areas of peptide synthesis and oligonucleotide synthesis. It is not hard to understand why solid-phase synthesis is attractive for these target classes. The synthesis comprises a small range of highly optimized repetitive reactions that lend themselves to automation, hence greatly increasing the access to these important biomolecules.1 Early work on small molecule solid-phase synthesis by Leznoff and co-workers2,3 and Frechet and co-workers4,5 illustrated the potential of the approach but did not spark broad interest. Effective solid-phase synthesis required that solution phase chemists develop and become familiar with the new handling methods. It should also be noted that the solid supports used for peptide synthesis were not optimized for nonpeptide synthesis, which requires a far broader range of reaction conditions. Furthermore, unless each reaction step in a solid-phase synthesis is highly optimized, poor quality products are generated.6 Strong interest in the synthesis of small molecule compounds was finally kindled by the benzodiazepine synthesis described by Ellman and Bunin in 1992, which illustrated the potential for the rapid parallel synthesis of compounds of pharmaceutical interest.7 At this time, pharmaceutical and biotechnology companies had an increasing demand for biologically relevant compounds due to the strong growth in highthroughput screening. Through the 1990s, solid-phase synthesis became a well-established tool in the pharmaceutical industry for the generation of large sets of small molecule compounds for lead finding and optimization.8,9 1

W. C. Chan and P. D. White, ‘‘Fmoc Solid-phase Peptide Synthesis: A Practical Approach.’’ Oxford University Press, Oxford, 2000. 2 J. Y. Wong, C. Manning, and C. C. Leznoff, Angew. Chem. Int. Ed. Engl. 13, 666 (1974). 3 C. C. Leznoff, T. M. Fyles, and J. Weatherston, Can. J. Chem. 55, 1143 (1977). 4 M. J. Farrall and J. M. J. Frechet, J. Org. Chem. 41, 3877 (1976). 5 J. M. J. Frechet and E. Seymour, Isr. J. Chem. 17, 253 (1978). 6 A. M. Bray, D. S. Cheifari, R. M. Valerio, and N. J. Maeji, Tetrahedron Lett. 36, 5081 (1995). 7 B. A. Bunin and J. A. Ellman, J. Am. Chem. Soc. 114, 10997 (1992). 8 R. E. Dolle, J. Comb. Chem. 3, 477 (2000). 9 A. Golebiowski, S. R. Klopfenstein, and D. E. Portlock, Curr. Opin. Chem. Biol. 5, 273 (2001).

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

40

combinatorial synthesis

[3]

Fig. 1. Graft polymer devices manufactured and sold by Mimotopes Pty Ltd for solidphase synthesis.

Mimotopes has been involved in the development, use, and commercialization of radiation-grafted polymer surfaces for multiple parallel synthesis since the late 1980s.10–12 Although other workers have reported the use of radiation-graft polymers in solid-phase synthesis,13,14 as far as we are aware, the graft polymer devices manufactured and sold by Mimotopes (SynPhase Crowns, SynPhase Lanterns) are the only current commercial products of this type. These products are presented in Fig. 1. The SynPhase Lanterns are the current design for small molecule synthesis. The initial

10

H. M. Geysen, S. J. Barteling, and R. H. Meloen, Proc. Natl. Acad. Sci. USA 82, 178 (1985). F. Rasoul, F. Ercole, Y. Pham, C. T. Bui, Z. Wu, S. N. James, R. W. Trainor, G. Wickham, and N. J. Maeji, Biopolymers 55, 207 (2000). 12 N. J. Maeji, R. M. Valerio, A. M. Bray, R. A. Campbell, and H. M. Geysen, Reactive Polym. 22, 203 (1994). 13 R. H. Berg, K. Almdal, W. B. Pedersen, A. Holm, J. P. Tam, and R. B. Merrifield, J. Am. Chem. Soc. 111, 8024 (1989). 14 R. Li, X.-Y. Xiao, and A. W. Czarnik, Tetrahedron Lett. 39, 8581 (1998). 11

[3]

solid-phase synthesis on synphase lanterns

41

Fig. 2. The 8  12 Multipin format.

purpose of the technology, which was referred to as Multipin Technology in the late 1980s, was to prepare large sets of very small amounts of support-bound peptide oligomers for use in screening antibodies and other soluble receptors.15 At that time, a suitably functionalized polyacrylic acid graft polymer was used as the support for peptide synthesis. Peptide quantities were small and purities were low, and the peptides were not cleaved from the solid phase. Nevertheless, the support-bound peptide libraries were very effective in rapid epitope determination. To perform multiple peptide synthesis on the micromole scale, it was necessary to develop new graft polymers and new shapes with higher surface area to volume ratios.16 In the mid to late 1990s, Mimotopes was producing rigid injection molded polypropylene devices that were surface grafted with either a hydrophilic copolymer of methacrylic acid/dimethyl acrylamide or the relatively hydrophobic polystyrene.12 The polymer was then suitably derivatized to allow the incorporation of a linker system. In contrast to the various commercial resins available at the time, the Crown was a macroscopic, quantized solid phase. As shown in Fig. 2, the Crowns were typically fitted to a polypropylene stem, which in turn could be fitted into a

15

H. M. Geysen, S. J. Rodda, T. J. Mason, G. Tribbick, and P. G. Schoofs, J. Immunol. Methods 102, 259 (1987). 16 N. J. Maeji, A. M. Bray, and R. M. Valerio, Peptide Res. 8, 33 (1995).

42

combinatorial synthesis

[3]

polypropylene holder that presented the Crowns in an 8  12 matrix that matched the ubiquitous microtiter plate format. Both the Crown and the Lantern have been fitted with individual tags for use in synthesis via the directed ‘‘split and pool’’ approach. Tagging has been achieved using inert colored tags or radiofrequency (RF) tags.17,18 This technology is well described elsewhere.19 A number of successful small molecule library syntheses have been reported on the SynPhase Crown surface. A review summarizes these papers to 1997.19 Nevertheless, we recognized the deficiencies of this design for solid-phase organic synthesis, especially its low loading/volume ratio, and a new high surface area device was developed. The SynPhase Lantern was introduced into the market in 2000.11 The L-series and the larger D-series Lanterns have replaced the earlier designs (see Fig. 1). The Lantern is made up of a series of uniformly spaced flat rings. It resembles a Chinese lantern, hence the name. Although the D-series Lanterns are smaller than the I-series SynPhase Crowns, they have a larger surface area/volume ratio. This is of benefit when using expensive reagents where volumes need to be reduced. The D-series Lantern has a higher loading of 35 mol/unit. A second improvement is that the polystyrene graft surface has been optimized for use in small molecule synthesis.20–22 This was achieved over time by using a comparative combinatorial approach. Method refinement also enabled tight control of loading variation between batches and within batches. In large-scale library synthesis, loading variation results in yield variation, which has been cited as a potential issue with the earlier Crown products.23 Lanterns are now being used primarily by chemists in pharmaceutical companies for the synthesis of libraries of small molecule compounds. Consequently, much of the synthetic work that has been performed has not yet been published, and much of it may never be published. Nevertheless, there is a steady stream of publications on the use of Lanterns in solid-phase synthesis. This review aims to summarize 17

E. J. Moran, S. Sarshar, J. F. Cargill, M. M. Shahbaz, A. Lio, A. M. M. Mjalla, and R. W. Armstrong, J. Am. Chem. Soc. 117, 10787 (1995). 18 R. Giger, Pin sort-and-combine method for HTP synthesis of individual crowns. Paper presented at the Cambridge Healthtec Institute Conference, Barcelona, Spain, 1997. 19 I. W. James, G. Wickham, N. J. Ede, and A. M. Bray, in ‘‘Solid-Phase Organic Synthesis’’ (K. Burgess, ed.), p. 195. Wiley-Interscience, New York, 2000. 20 Z. Wu, F. Ercole, M. FitzGerald, S. Perera, P. Riley, R. Campbell, Y. Pham, P. Rea, S. Sandanayake, M. N. Mathieu, A. M. Bray, and N. J. Ede, J. Comb. Chem. 5, 166 (2003). 21 A. R. Vaino and K. D. Janda, J. Comb. Chem. 2, 579 (2000). 22 S. W. Gerritz, Curr. Drug Discov. 19, (2002). 23 C. G. Boojamra, K. M. Burow, L. A. Thompson, and J. A. Ellman, J. Org. Chem. 62, 1240 (1997).

[3]

solid-phase synthesis on synphase lanterns

SynPhase Crown

43

SynPhase Lantern

Fig. 3. SynPhase Lantern and SynPhase Crown symbols used in this chapter.

work that has been published between 1997 and early 2002, and builds on our earlier review that covered solid-phase organic synthesis on radiationgrafted polymers.19 As summarized in Fig. 3, the Crown is depicted by a star and the Lantern is represented by a rectangular symbol. Heterocycles

Benzodiazepines Benzodiazepines were the first class of heterocyclic compounds to be synthesized on the SynPhase surface. In 1994, Ellman and co-workers24 reported a 192 member library of structurally diverse 1,4-benzodiazepines. These compounds were prepared on Mimotopes pins that were grafted with polyacrylic acid, the surface originally used for antibody epitope elucidation.10 Ellman and co-workers25 subsequently synthesized a 1680member 1,4-benzodiazepine library on SynPhase Crowns that were grafted with a methacrylic acid/dimethylacrylamide copolymer, one of the first SynPhase surfaces designed for solid-phase synthesis. The synthesis was performed on a preformed linker-template system in order to avoid low * aminobenzophenone incorporation; in this case the HMP acid-labile linker 24

B. A. Bunin, M. J. Plunkett, and J. A. Ellman, Proc. Natl. Acad. Sci. USA 91, 4708 (1994). B. A. Bunin, M. J. Plunkett, J. A. Ellman, and A. M. Bray, New J. Chem. 21, 125 (1997). * Abbreviations: AcOH, acetic acid; Ar, general aryl group; BAL, backbone amide linker; BEMP, 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine; Bn, benzyl; Boc, tert-butoxycarbonyl; BOP, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; CDI, carbonyldiimidazole; DEAD, diethyl azodicarboxylate; DIC, N,N-diisopropylcarbodiimide; DIEA, diisopropylethylamine; DMAP, N,N-dimethylaminopyridine; DMF, N,N-dimethylformamide; Fmoc, 9-fluorenylmethoxycarbonyl; HATU, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HBTU, O(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HEMA, 2-hydroxyethyl methacrylamide; HMP, 4-(hydroxymethyl)phenoxyacetyl (linker system); HOAt, 3hydroxy-7-azabenzotriazole; HOBt, 3-hydroxybenzotriazole; HPLC, high-performance liquid chromatography; LCMS, liquid chromatography mass spectrometry; LHP, ‘‘long chain HMP’’: 4-(hydroxymethyl) phenoxypentanoyl (linker system); mCPBA, m-chloroperbenzoic 25

44

[3]

combinatorial synthesis NHfmoc O 2

R

2

R

NH2

R4

R3

R2

NH

R3

O

O

O

HMP

R1 1

N

O

O

HMP

O

N

HMP

R1

R1 3

2

O HMP

OH

N H

O OH

Scheme 1. General solid-phase route to benzodiazepines.

was employed. It should be noted that using the trichloroacetimidate method described by Hanessian and Xie26 phenols can now be readily loaded onto this linker system on the solid-phase.27 Coupling of Fmocprotected amino acids onto 1 was achieved via preformed acid fluorides to afford support-bound amides 2 (Scheme 1). Following Fmoc deprotection, cyclization to the 1,4-benzodiazepine resulted via treatment of the support-bound intermediate with 5% AcOH in NMP. Alkylation was achieved by subsequent treatment with excess lithiated 5-phenylmethyl-2oxazolidinone and the respective alkyl halide to give densely functionalized benzodiazepine compounds 3. Cleavage was achieved with trifluoroacetic acid. A modified version of this synthesis on a polystyrene graft surface has been described (Scheme 2).28 A preformed 2-aminobenzophenone linkertemplate system 5 was coupled to aminomethylated polystyrene Lanterns 4 by the action of DIC and HOBt in DMF. The resulting support-bound aniline 6 was acylated with an in situ generated Fmoc amino acid fluoride to afford the amide 7. Deprotection followed by acid-catalyzed intramolecular cyclization generated the functionalized 1,4-benzodiazepines 8. acid; MD, methacrylic acid/dimethylacrylamide copolymer MA-DMA; Mmt, 4-methoxyltrityl; NMM, N-methylmorpholine; NMP, 1-methyl-2-pyrrolidinone; Py, Pyridine; PyBOP, benzotriazol-1-yloxytris(pyrrolidino) phosphonium hexafluorophosphate; RINK, rink amide forming linker; TFA, trifluoroacetic acid; TFFA, trifluoroacetic anhydride; TFFH, fluoroN,N,N0 ,N0 -tetramethylformadinium hexafluorophosphate; Wang, hydroxymethylphenoxy linker on polystyrene. 26 S. Hanessian and F. Xie, Tetrahedron Lett. 39, 733–736 (1998). 27 Mimotopes SynPhase‘ Application Note SCN005, www.synphase.com 28 Mimotopes SynPhase‘ Application Notes SCN011 and SCN012, www.synphase.com

[3]

45

solid-phase synthesis on synphase lanterns O

HO NH2.TFA DMF/CH Cl 2 2

O

O

O 5

O O

TEA

HMP

NH2

NH2 4 NH2

DIC, HOBt, DMF

TFFH/DIEA/DMF/40 ⬚C I fmoc

6

N H

O

HO fmoc O

H N HN

I O

20% piperidine/DMF 5% AcOH/NMP, 60 ⬚C

HMP

O

7 I

I

O N

HMP

O

O

95% TFA/H2O N

NH

NH

HO

8 O HMP OH

N H

O OH

Scheme 2. Assembly of benzodiazepines using in situ-generated acyl fluorides.

Purines The group of Schultz has synthesized a 406-member purine library on MD grafted SynPhase Crowns with varying substituents at the C2 and C6 positions.29 A common 2-chloropurine derivative was coupled onto

46

[3]

combinatorial synthesis Cl 2

RINK

N 6

H N O

N H

N

Cl 2 N RINK

N

N 6

H N

N O

9

R1

N

N N

O 10

2

R

NH 2

RINK

N 6

H N

N O

R1

N

N N

O 11 O

RINK

NH2

N H

O NH2 MeO

OMe

Scheme 3. Solid-phase synthesis of 2,6-disubstituted purines.

support-bound Rink amide forming linker 9. The first combinatorial step involved acylation of an exocyclic nitrogen attached to C6 to give amides 10 (Scheme 3). An SNAr reaction with diverse amines afforded the final 2,6-substituted purine derivatives 11. Cleavage was achieved with TFA. Compounds from this class were found to inhibit glucose synthase kinase.30 Schultz and co-workers31 also described the preparation of a 2,6,9trisubstituted purine library. A preformed 2-fluoro-6-(4-aminobenzylamino) purine was reductively aminated onto the BAL linker 12. Mitsunobu chemistry was employed to alkylate the C9 position on the support-bound intermediate (Scheme 4). Subsequently, SNAr chemistry was used to incorporate amines at C6. The newly introduced primary and secondary amines bear diverse functional groups and the Mitsunobu reaction allows for incorporation of primary and secondary alcohols lacking acidic hydrogens. The support-bound product 13 was cleaved with 90% TFA/10% H2O to give a library with HPLC purities ranging between 51 and 85%. In a more recent paper, Schultz and co-workers32 extended the technology on SynPhase Crowns to synthesize more diverse 2,6,9-trisubstituted 29

T. C. Norman, N. C. Gray, J. T. Kohand, and P. G. Schultz, J. Am. Chem. Soc. 118, 7430 (1996). S. Rosenberg, K. L. Spear, R. Valerio, and A. Bray, PCT Int. Appl. WO 9705257 (1997). 31 N. S. Gray, S. Kwon, and P. G. Schultz, Tetrahedron Lett. 38, 1161 (1997). 32 N. S. Gray, L. Wodicka, A.-M. Thunnissen, T. C. Norman, S. Kwon, F. H. Espinoza, D. O. Morgan, G. Barnes, S. LeClerc, L. Meijer, S.-H. Kim, D. J. Lockhart, and P. G. Schultz, Science 281, 533 (1998). 30

[3]

47

solid-phase synthesis on synphase lanterns

BAL

BAL

N H

N H

NH 2 N N

SNAr

N9 H

F 6 N 12

R2 Mitsunobu OMe O

BAL

O

O

N

NH 2 N

N6 N H 13

N9 R1

O OMe

Scheme 4. Solid-phase synthesis of 6,9-disubstituted purines.

N H

NH

Cl 2

H N

N O

N H

F

6

N

N

1. Amination at C2 (R1NH2) and/or alkylation at N9 (R2OH)

R1

R3

N 9 and /or amination at C6 (R3NH2) H 2. Acidic cleavage

NH N

N N H

N

N R2

O

Scheme 5. General route to diverse purine libraries.

purine libraries. As summarized in Scheme 5, a number of synthetic routes were used, where the core purine was linked to the solid-phase via the 2, 6, or 9 positions. The synthetic methodologies were essentially as described above. Potent inhibitors of human cyclin-dependent kinase 2 (CDK2) were identified using these trisubstituted purine libraries. Quinazolines Polystyrene grafted SynPhase Lanterns were used by Makino et al.33 at Ajinomoto to synthesize a diverse quinazoline-2-thioxo-4-one library. Lanterns functionalized with long-chain HMP (hydroxymethylphenoxyvaleric amide) linker were coupled with nitrophenols by the action of DIC/ HOAt/DMAP to give nitrobenzenes 14 (Scheme 6). The nitro group was 33

S. Makino, E. Nakanishi, and T. Tsuji, Tetrahedron Lett. 42, 1749 (2001).

48

[3]

combinatorial synthesis NO2-Ar-OH

OH

LHP

LHP

DIC/HOAt/DMAP/NMP

O 14

Ar NO2

N SnCl2/NMP/EtOH

LHP

O 15

Ar NH2

S

CO2Me NMP

O O 16

LHP

1. Br DIEA/NMP HO 2. 95% TFA/H2O

Ar N S

N H

O Ar N S

N H 18

95% TFA/H2O O O

HO Ar N

HO Ar N S

S

+ N H

N 17

HO O LHP

OH

N H

OH O

Scheme 6. Diverse quinazoline-2-thioxo-4-one libraries.

reduced with SnCl2 to efficiently give support-bound amines 15. Subsequent reaction with 2-methoxycarbonylphenyl isothiocyanate gave a transient thiourea that readily cyclized to the required quinazoline-2thioxo-4-ones 16. Products were obtained in high purity, typically greater than 95%. It is interesting to note that Makino observed a number of Salkylated by-products 17 when the synthesis was performed on Wang resin. They stem from reaction with the benzylic linker cations generated by cleavage with 95% TFA/H2O. This side reaction was reported to be far less pronounced in the case of long-chain HMP derivatized Lanterns. Makino investigated the derivatization of quinazoline-2-thioxo-4-ones with alkyl halides. Reaction with DIEA in NMP gave the best results with the addition of allyl bromide giving products 18 in greater than 95% purity. Excellent yields and purities were obtained using a variety of alkyl and aryl halides.

[3]

49

solid-phase synthesis on synphase lanterns

NO2

F

O

O

HMP

NH2

O2N

O

HMP

CO2H

O N H

O

DIC/HOAt/NMP

F

19 NO2

O

R-NH2

HMP

N

O O

N

N

O

N H HN

NMP

N

decalin, 95 ⬚C

R

20 NO2

NO2

O HMP

O

O

HO

95% TFA/H2O

N

N

N

O 21

O

O

N 22

R

O

R

O HMP

OH

N H

O OH

Scheme 7. Synthesis of diverse 1,3-disubstituted quinazoline-2,4-diones.

Makino et al.34 also published a synthesis of a diverse 1,3-disubstituted quinazoline-2,4-dione library on HMP-derived polystyrene SynPhase Lanterns bearing a 4-aminobenzoic ester. As outlined in Scheme 7, 2-fluoro-5nitrobenzoic acid was coupled to the linker-template system to give amide  19. Nucleophilic substitutions with a range of alkyl amines proceeded at 25 to give products 20 with excellent purity. Aryl amines had to be heated to   80 . Next, carbonylation with carbonyl diimidazole in decalin at 95 for 16 h afforded the quinzoline-2,4-diones 21 with 100% conversion. Products were cleaved from the solid support with 95% TFA/H2O to give 22; in most cases purities greater than 95% were obtained. Makino et al. examined several solid supported amines. Quinazoline-2,4-diones were obtained in high purity from 3-aminobenzoic acid ester. Aminocinnamic esters gave significant amounts of a Michael addition product resulting from the addition of imidazole to the cinnamoyl moiety, though -elimination with BEMP catalysis prior to cleavage gave the required products in high purity. Quinazoline-2,4-dione oligomers were synthesized on SynPhase Lanterns by reducing the nitro group to the aniline with SnCl2, and subsequent acylation of the amine followed by SNAr reaction with primary amines to give 23 (Scheme 8). Carbonylation with CDI in decalin at elevated temperature gave the quinazoline-2,4-dione oligomer (4-mer) 24 following cleavage from the solid phase. 34

S. Makino, N. Suzuki, E. Nakanishi, and T. Tsuji, Synlett, 333 (2001).

50

[3]

combinatorial synthesis O O O

N H

19

F

O

1. SnCl2

NO2

N H HN

NO2

2. F

O

F HO2C

NO2

H N

O

O

DIC/HOAt 3. SNAr reaction, propylamine

repeat reduction, substitution, acylation NO2 cycles

1. SnCl2 2. F

NO2

HN H N

O O

HO2C

N H HN

O

DIC/HOAt

O

3. SNAr reaction cyclobutylamine HN H N

O O N H HN

O

HN H N

O N H HN

O

NO2

O

23

1. CDI, decalin 95 ⬚C 2. 95% TFA/H2O

O HO O

N N

N O

O

N

O

O N H HN

O

N N

NO2 O

24

Scheme 8. Stepwise assembly of quinazoline-2,4-dione oligomers.

Makino et al.35 developed a solid-phase synthesis of 1,3-disubstituted 2thioxoquinazoline-4-ones using HMP Lanterns that were derivatized with a 4-aminobenzoate ester. Following the assembly of amide 25 (Scheme 9), SNAr reaction with alkyl amines gave support-bound products 26 with high conversion. The key thiocarbonylation step was achieved with thiocarbo nyldiimidazole in decalin at 95 for 16 h in the presence of DMAP to afford 1,3-disubstituted 2-thioxoquinazoline-4-ones 27. The target compounds 28 35

S. Makino, N. Suzuki, E. Nakanashi, and T. Tsuji, Tetrahedron Lett. 41, 8333 (2000).

[3]

51

solid-phase synthesis on synphase lanterns

HMP

NH2 O2N

O

NO2

O

F

O

HMP

O N H 25

O

CO2H

F

DIC/HOAt/NMP

RNH2, NMP

HMP

N

S

NO2

O

95% TFA/H2O

N O

N

DMAP/decalin 95 ⬚C

NO2

O

N S

N H HN 26 R

O

27

N

O O

HMP

NO2

O

HO N O

N R

28

S

N R

O HMP

OH

N H

O OH

Scheme 9. Synthesis of 2-thioxoquinazoline-4-ones.

were cleaved from the Lanterns using 95% TFA/H2O. The synthesis gave targets with purities generally greater than 95% and yields ranging from 59 to 100%. Arylaminobenzimidazoles Krchnak and co-workers36 at Selectide devised a novel tracking strategy that was demonstrated in the synthesis of a benzimidazole library. Rink amide-derived Crowns were acylated with 4-fluoro-3-nitrobenzoic acid to give 29 and further functionalized via SNAr substitution with eight amines (Scheme 10). The o-nitroaniline-derivatized Crowns 30 were threaded onto a steel string and tied to form a ‘‘necklace.’’ The eight respective R1 groups were defined by the order of placement onto each necklace. Twelve identical necklaces were prepared. The support-bound nitroanilines were reduced with SnCl2 in NMP in bulk and each necklace was treated with a 36

J. M. Smith, J. Gard, W. Cummings, A. Kanizsai, and V. Krchnak, J. Comb. Chem. 1, 368 (1999).

52

[3]

combinatorial synthesis O RINK

O NO2

N H

NH2-R1

F

29

NO2

N H

RINK

NH R1

30 O

SnCl2, NMP

RINK

NH2 R2NCS DIC NH R1

N H

O RINK

N H

O N N 31 R1

N H

R2 H2N HCl (g), 2 h

N N 32 R1

N H

R2

O RINK

NH2

N H

O NH2 MeO OMe

Scheme 10. Synthesis of 2-aminobenzimidazole libraries.

different isothiocyanate and 1 M DIC solution at ambient temperature overnight to form the respective support-bound arylaminobenzimidazoles 31. The 96-member library was cleaved by treatment with gaseous HCl for 2 h and subsequent extraction from the solid phase. Quality of the products 32 ranged from 56 to 99% purity with the average being 83%. Wu et al.37 at Mimotopes developed a one-pot synthesis of benzimidazoles using chloromethylated SynPhase Lanterns functionalized with Wang linker (Scheme 11). The support-bound ester 33 was treated with aliphatic and aromatic amines to afford o-nitroanilines 34 with little, if any, premature cleavage. After some experimentation, one-pot reduction/cyclization to the desired benzimidazole 35 was achieved using two equivalents of aldehyde and 0.75 M SnCl22H2O (10 equivalents) in DMF and heating at  60 for 3 h. The support-bound products were readily cleaved from the Lanterns with 20% TFA/CH2Cl2 to afford the desired disubstituted benzimidazoles 36. A 25-member library was synthesized using five aldehydes and five amines. A benzimidazolium by-product 37 was observed for aliphatic 37

Z. Wu, P. Rea, and G. Wickham, Tetrahedron Lett. 41, 9871 (2000).

[3]

53

solid-phase synthesis on synphase lanterns

Wang

OH

F

DIC/DMAP Wang

CO2H

O

R1NH3

NO2 O

33 NO2 F NHR1 Wang

O

NO2

R1 N

SnCl2.2H2O, R2CHO DMF, 60 ⬚C

Wang

O

R2

N O 35

O 34 R1 N

20% TFA/CH2Cl2

H2N O

Wang

R2

N

OH

36 O

OH

Scheme 11. Synthesis of 5-carboxybenzimidazole libraries.

R1 N R2 HO

N O

37

+ R2

Fig. 4. Benzimidazolium by-product.

aldehydes but not for benzaldehydes (Fig. 4). Target compound purities ranged from 56 to 93% and yields were typically 85%. Pyridin-2-ones SynPhase Crowns with a Rink amide linker were used by Linn et al.38 at Glaxo-Wellcome to synthesize a library of 1,3,5-trisubstituted pyridin2-ones (Scheme 12). The solid bound amide 38 was treated with 3-amino5-methoxycarbonyl-1H-pyridin-2-one with Cs2CO3 in DMF to afford pyridine-2-one 39. Coupling of diphenylacetic acid HATU and DIEA in 38

J. A. Linn, S. W. Gerritz, A. L. Handlon, C. E. Hyman, and D. Heyer, Tetrahedron Lett. 40, 2227 (1999).

54

[3]

combinatorial synthesis CO2Me O O ( ) 4

HO RINK

NH2

Br RINK 38

Ph2CH

O N ( ) 5

N H

NH2 O

39

Br

NH

H2N O

Cs2CO3, DMF 16 h, rt

O

CO2Me

RINK

( )4

N H

CO2Me OH

O

O

HATU, DIEA, DMF, rt, 18 h

RINK

N ( ) 5

N H

CHPh2

N H

O

40 F

CO2H

RINK

N H

1.

O

O

LiOH

F

N ( ) 5 41

OH

F

O

N H

F

CHPh2

F py, TFAA,DMF, 4 h

RINK

N H

1. BnNH2, DMF, 18 h, rt

O

O N ( ) 5 O

NHCH2Ph

O

CO2C6F5

N H

CHPh2 2. 95% TFA(aq), 1 h

O

O H2N

42

N ( ) 5 43

O

N H

CHPh2

O RINK

NH2

N H

O NH2 MeO

OMe

Scheme 12. Synthesis of pyridin-2-one libraries.

DMF gave the required amide 40. Saponification of the ester with 1 M LiOH in 1,4-dioxane gave solid-bound acid 41. The support-bound pentafluorophenol ester 42 was then generated, allowing efficient coupling with a range of benzylamines. Generally, ‘‘reversed’’ couplings are far less efficient than conventional couplings where the carboxilic acid component reacts with a support-bound amine. Cleavage with 95% TFA/H2O afforded a library of trisubstituted pyridin-2-ones 43. Oxazoles Quibell and co-workers39 at Peptide Therapeutics used a 5-(hydroxymethyl)oxazole scaffold 44 to prepare a range of oxazole libraries on HEMA-grafted, glycine-derivatized Gears (a small Crown design). The

[3]

55

solid-phase synthesis on synphase lanterns

O

R

OH O HO O

N H

N 44

Gly-RINK-NH2

1.

R

OH O

fmoc

BOP, HOBt, NMM 2. Piperidine, DMF

Gly RINK

H N

O NH2

N O

O

R

OH O

fmocAA-OC6F5 HOBt, DMF,o/n

Gly RINK

H N

N H

N

O

H N

1. Piperidine, DMF fmoc

2.

O

R

O

O

OC6F5 HOBt, DMF, o/n R

OH O Gly RINK

H N

N O

Gly-RINK

O

O

O

H N

N H

R

O

45 O

H N O

N H

O

, HEMA graft NH2

MeO OMe

Scheme 13. Preparation of libraries based on a 5-(hydroxymethyl)oxazole scaffold.

scaffold was built up on the solid phase as outlined in Scheme 13 on a Rink linker. The scaffold was used to synthesize aryl ethers, thioethers, sulfones, sulfonamides, and carboxamides via the free hydroxymethyl group. Alkyl/aryl ethers 46 were synthesized by a Mitsunobu reaction with DEAD, PPh3, NEt3, and a wide variety of alkyl and aryl alcohols (Scheme 14). Products were obtained in greater than 90% purity. Reaction of 5-(hydroxymethyl)oxazole 45 with CBr4, PPh3 gave bromides 47 that were efficiently converted into thioethers 48 by overnight treatment with thiols in NMP. The synthesis of sulfones 49 was achieved by oxidation of the thioethers precursors with mCPBA in CH2Cl2. Carboxamides 50 were synthesized by converting the free bromides to azides with NaN3, DIEA in NMP/H2O. The azides were reduced to amines with dithiothreitol and DIEA. The amine analogs were acylated with a variety of alkyl, aryl 39

U. Grabowska, A. Rizzo, K. Farnell, and M. Quibell, J. Comb. Chem. 2, 475 (2000).

56

[3]

combinatorial synthesis

O

R

OH O Gly

H N

RINK

O

O

H N

N H

N

R

R 1. PPh3, DEAD, ArOH

O 1. TFA 5% Et3SiH

O

N H

N

H2N

45

O

OH O

R

O

O H N R

46

O

O

CBr4, PPh3 O

R

Br O H N

Gly RINK

O

O

H N

N H

N

R

O

O

R

NH2 O H N

Gly RINK

R

() n SO2

O 1. RSO2Cl DMAP, DMF 2. TFA 5% Et3SiH O

R

OH O

NH O

R

O N

H2N

N H 50

O

R

N H

O H N R

49

O

O

S

H N

N

H2N

R

OH O

OH O

R O

O R

O

H N

1. RCO2H HBTU, HOBt NMM, DMF 2. TFA 5% Et3SiH

N

R

O

O

O

48

O H N

O

O

N H

N

N H

N

H2N

1. NMP/thiol reagent 2. mCPBA 3. TFA/5% Et3SiH

1. NaN3 2. DTT

OH O

R O

2. TFA 5% Et3SiH

47

O

() nS

1.NMP/thiol

N H

N

H2N

51

O H N R

O

O O

O

Scheme 14. An alternative approach to oxazole-based libraries.

carboxylic acids and anhydrides to give high-quality carboxamides 50. The amines were also treated with sulfonyl chlorides to give sulfonamides 51 in greater than 90% purity. Hydantoin/Isoxazolines Kurth and co-workers40 (Novartis) prepared a 990-member compound library of hydantoin and isoxazoline containing heterocycles on aminederived SynPhase Crowns. As shown in Scheme 15, the synthesis was 40

K. Park, J. Ehrler, H. Spoerri, and M. J. Kurth, J. Comb. Chem. 3, 171 (2001).

[3]

57

solid-phase synthesis on synphase lanterns O NHBoc

HO OH

H N

O O

H N

53

O

H N

H N

52

H N

H N

nPrNO

2

PhNCO NEt3 Dioxane

NEt3, CH3CN

N

O

54 O

H N

O

N

NH2

O

O

O

O

O

H N

1. DIC, DMAP DMF/CH2Cl2 2. TFA, NEt3

O

Dioxane PhNCO

O

O NH

O

O

N Major diastereomer

Scheme 15. Synthesis of a hydantoin library via 1,3-dipolar cycloaddition.

performed using simultaneous cleavage and cyclization on a base labile linker. Boc-protected amino acids were coupled onto the free hydroxyl group with DIC/DMAP. Treatment with TFA, followed by neutralization, afforded the amine 52. Reaction with phenylisocyanate gave the solid bound urea 53. Subsequent 1,3-dipolar cycloaddition with a Mukaiyamagenerated nitrile oxide afforded the isoxazoline 54. Treatment with triethylamine produced hydantoins as an 8:1 diastereomeric mixture. An alternative route to the same 990-compound library was developed using 5 amino acids, 9 nitroalkanes, and 22 isocyanates (Scheme 16). The hydroxymethyl function was coupled with the 5 alkene-containing Boc amino acids. Reaction of the resulting esters 55 with 5 nitroalkanes gave the isoxalines 56 after Boc deprotection. Coupling of the amine with the isocyanates afforded ureas 57. Cleavage with triethylamine produced the isoxazolinoimidazolidione final products 58. HPLC purities of greater than 70% were obtained for 90% of the samples in the library. Reaction yields were typically between 40 and 60%. Diversity is further introduced onto the hydantoin and isoxazoline heterocycles with a variety of connecting groups between the two heterocycles.

58

[3]

combinatorial synthesis O NHBoc

HO

O

X OH

H N O

H N

X O

DIC, DMAP DMF/CH2Cl2

NHBoc

O 55

O 1. PhNCO, NEt3

O H N O

57

O

56

N

O

N

R1

O R1

O

R2

H 2 NR

H N X

Dioxane, R3NCO

X O

O

NH2

O

H N

R1CH2NO2 2. TFA, NEt3

NEt3, CH3CN

N NH

O X O

N

R1

58 Scheme 16. Alternative route to hydantoin libraries.

Quinoxalines Wu and Ede41 (Mimotopes) described the first synthesis of quinoxalines on the solid support using polystyrene-grafted SynPhase Lanterns (Scheme 17). Following coupling of 4-fluoro-3-nitrobenzoic acid to SynPhase Rink Lanterns to give 59, amino substitution of the aryl fluoride was achieved by the action of aqueous ammonia solution in 5% DIEA/ DMF at 60 for 5 h. The o-nitroaniline 60 was reduced to the o-phenylenediamine 61 by SnCl22H2O. One-pot N-alkylation with 10 -bromoketones followed by in situ cyclization and oxidation gave regioisomeric quinoxalines 62 and 63. In all cases, good yields were obtained with product purities ranging between 65 and 86% for the sum of quinoxaline isomers. Perhydro-1,4-diazepine-2,5-diones Amblard and co-workers42 from Montpellier University optimized the synthesis of a 3,7-disubstituted perhydro-1,4-diazepine-2,5-dione 64 on polystyrene Crowns (Scheme 18). Seven-membered heterocyclic 41 42

Z. Wu and N. J. Ede, Tetrahedron Lett. 42, 8115 (2001). J. Giovannoni, G. Subra, M. Amblard, and J. Martinez, Tetrahedron Lett. 42, 5389 (2001).

[3]

59

solid-phase synthesis on synphase lanterns O NH2

RINK

1. 20% piperidine, DMF

RINK

2. 4-fluoro-3-nitrobenzoic acid DIC, HOBt, DMF

NO2

N H

F

59

O NH3 (aq), DIEA/DMF

RINK

60 ⬚C, 5 h

NO2

N H

SnCl2.2H2O, NMP.

NH2

60

O

O RINK

N H

NH2 a -bromoketones DMF, 60 ⬚C

61

NH2

RINK

N

N H

N

O

O 20% TFA/CH2Cl2

N

H2N

+

N

H2N

N

N 62

63 O

RINK

NH2

N H

O NH2 MeO

OMe

Scheme 17. Solid-phase quinoxaline synthesis.

compounds lacking a fused aromatic nucleus are difficult to prepare. However, it had been shown that the formation of cyclic compounds such as diketopiperazines was favored in the presence of a secondary amide adopting a cis-configuration. Amblard and co-workers42 used a backbone amide linker (BAL) as both the cis-configuration inductor and to anchor the growing compound to the solid support. The Multipin method was used to rapidly explore a large range of reaction parameters in parallel experiments and determine the optimum reaction conditions. To rapidly optimize the reaction conditions for each step, a Phe-Ala spacer connected to a BAL linker mimic 65 was used to enable evaluation of each step by a single LCMS analysis. This useful technique has been described previously.6 The BAL linker mimic 65, a carboxybenzaldehyde moiety, allowed reductive amination but was stable to acidic treatment. The best conversion to the free diazepin 66 was 94%. The conditions optimized for 65 were directly transferred to

60

[3]

combinatorial synthesis O HN NH O

64 O

O Phe-Ala

Phe-Ala

RINK

RINK

H O BAL linker mimic

N Spacer

O

N O

65

66

O RINK

NH2

N H

O NH2 MeO OMe

Scheme 18. Solid-phase synthesis of a 1,4-diazepine-2,5-dione.

polystyrene Crowns derivatized with the BAL linker (28 mol loading). The benzodiazepine 64 was obtained with 98% purity without further optimization. Although the synthesis of a single diazepinone was described, diversity on the diazepinone core could be introduced by use of various amino acids and their -homologs. Carbohydrates

The synthesis of polymer supported oligosaccharides via n-pentenyl glycosides by Fraser-Reid and co-workers43 was described in a previous review.19 Solid-phase synthesis was used to simplify the synthesis of a library of 14 oligosaccharide-conjugated enediynes 69 (Scheme 19).44 Takahashi and co-workers44 (Tokyo Institute of Technology) synthesized these compounds to study sequence-selective cleavage of DNA. The putative 43 44

R. Rodebaugh, S. Joshi, B. Fraser-Reid, and H. M. Geysen, J. Org. Chem. 62, 5660 (1997). A. Matsuda, T. Doi, H. Tanaka, and T. Takahashi, Synlett, 1101 (2001).

[3]

61

solid-phase synthesis on synphase lanterns O

Et

N H

Si

O

O O (OR)n

Et

SH

Linear or branched Oligosaccharide 67

O

Et

N H

Si

O

Et

O O (OR)n

O

S

O O O

HO 68

HO

O O (OR)n

O

S

O O O

HO A

B

DNA recognition site

DNA cleaving site 69

Scheme 19. Solid-phase synthesis of oligosaccharide-conjugated enediynes as putative DNA-binding and cleaving agents.

DNA-binding systems 69 consist of an oligosaccharide (A) selected to recognize a DNA sequence and a labile nine-membered enediyne moiety (B) that can generate a reactive diradical. These compounds were found to be difficult to handle in a previous solution phase synthesis due to the presence of both the water-soluble sugar (A) and the hydrophobic enediyne moiety (B). The oligosaccharide-trialkylsilyl linker system was prepared by solution phase synthesis and loaded onto SynPhase polystyrene Crowns to give 67 (Scheme 19). As the Crowns were fitted with radiofrequency transponders to allow unambiguous identification, the remaining library synthesis could be performed in a single vial. The enediyne moiety was then conjugated to the support-bound oligosaccharides to give the conjugate 68. The site isolation inherent in solid-phase synthesis prevented the formation of dimeric by-products that are obtained in an analogous solution-phase synthesis. As the assembly proceeded with high efficiency,

62

[3]

combinatorial synthesis O ( )9 O

RINK N H

BnO BnO

SO2 O O BnO

X−

BnO BnO

X O BnO

OMe

OMe

70

71 72 73

X = N3 X=I X = OAc

O RINK

NH2

N H

O NH2 MeO OMe

Scheme 20. Nucleophilic cleavage of monosaccharides from the solid phase to introduce C6 functionality.

high-purity target compounds 69 were obtained following washing of the solid-phase and subsequent cleavage with AcOH/THF/H2O. In contrast, the purification of the target compounds prepared via solution phase conjugation was difficult. Takahashi et al.45 used a 4-hydroxybenzenesulfonate linker on SynPhase polystyrene Crowns to prepare glucose derivatives and macrocycles (Scheme 20). Displacement of the monosaccharide-supported Crowns 70 with nucleophiles such as azide, iodide, and acetate gave the respective 6substituted glucose derivatives 71, 72, and 73 in excellent purities and yields. The authors suggest that this methodology could be utilized for the preparation of oligosaccharide libraries. A similar method was used for the synthesis of a cyclic cyanohydrin ether 75. The cyclization took place by the intramolecular displacement of the polymer-supported cyanohydrin 74 by treatment with lithium hexamethyldisilazide (Scheme 21). Target 75 was obtained in 46% yield. While the yield is moderate, a pure product was obtained after a simple workup procedure. The cyanohydrin ether 75 can be readily converted to a cyclic ketone.46

45 46

T. Takahashi, S. Tomida, H. Inoue, and T. Doi, Synlett, 1261 (1998). T. Takahashi, H. Nemoto, and J. Tsuji, Tetrahedron Lett. 24, 2005 (1983).

[3]

solid-phase synthesis on synphase lanterns

O

TRT

EtO EtO

O2S

LiN(SiMe3)2

O CN

O CN O

74

TRT

63

75

OH

H N

OH

O

Scheme 21. Macrocyclization on the SynPhase surface.

Functionalized Peptide Libraries

Dragovich et al.47 from Agouron Pharmaceuticals synthesized a series of irreversible human rhinovirus 3C protease (HRV 3CP) inhibitors 77. The inhibitors 77 contain a tripeptide-binding domain that provides affinity for the target protein and a Michael acceptor that irreversibly forms a covalent adduct with the active cysteine residue of the 3C enzyme. The N-terminal amides 77 were prepared by coupling of the tripeptide 76 with a variety of carboxylic acids and acid chlorides (Scheme 22). Approximately 500 unique compounds were prepared and their affinity against HRV-14 3CP determined with a high-throughput assay. Eight of the most active compounds were resynthesized by solution-phase techniques for a more accurate determination of their binding constants. The most potent reported compound gave an EC50 value of 250 nM. Liu et al.48 (Academy of Military Medical Science, Beijing) developed a method for the combinatorial synthesis of muramyl dipeptide derivatives 79 (Scheme 23). Two important building blocks, protected muramic acid and Fmoc-d-isoGln, were prepared by solution-phase synthesis. The peptide 78 was then prepared on MD grafted I-series Crowns using standard coupling and Fmoc deprotection methodologies. The peptides were coupled to acids and cleaved from the solid support to give a library of 60 muramyl dipeptide derivatives 79. All compounds in the library were reported to have purities greater than 75%. 47

S. Dragovich, R. Zhou, D. J. Skalitzky, S. A. Fuhrman, A. K. Patick, C. E. Ford, J. W. Meador, III, and S. T. Worland, Bioorg. Med. Chem. 7, 589 (1999). 48 G. Liu, S.-D. Zhang, S.-Q. Xia, and Z.-K. Ding, Bioorg. Med. Chem. Lett. 10, 1361 (2000).

64

[3]

combinatorial synthesis

RINK O

H N

FmocHN

NH

O

O

O N H

O

CO2Et

P

4

N H

NH2

O

H N

N H

O

CO2Et

77

76 O RINK

NH2

N H

O NH2 MeO OMe

Scheme 22. Synthesis of human rhinovirus 3C protease inhibitors.

Cyclic Peptides

Lambert and co-workers49 (University of Melbourne) synthesized a library of cyclic thioether peptides with a pendant 9-aminoacridine moiety as a DNA-binding agent 81. Diversity in the library was achieved by assembling every permutation of four amino acid residues within the cyclic peptide (Scheme 24). The linear peptides 80 were synthesized in parallel with standard Fmoc chemistry on SynPhase Crowns functionalized with a Rink linker. The acridine moiety was incorporated onto the C-terminal lysine side chain using 9-phenoxyacridine. Cysteine deprotection and peptide cyclization also took place under the acidic conditions used for the cleavage of 80 from the solid support. The library of cyclic thioether peptides 81 was obtained in high yields and purity (11 of 12 members had purities >95%). Lambert and co-workers50 also synthesized a pair of cyclic octapeptides 82 and 83 and studied their propensity to form nanotubular aggregates (Fig. 5). A linear peptide was initially synthesized on SynPhase Crowns with a Rink amide handle by standard Fmoc chemistry. The first residue used was aspartic acid protected as the -allyl ester. The support-bound linear peptide was cyclized in a head-to-tail manner by deprotection of 49 50

K. D. Roberts, J. N. Lambert, N. J. Ede, and A. M. Bray, Tetrahedron Lett. 39, 8357 (1998). M. E. Polaskova, N. J. Ede, and J. N. Lambert, Aust. J. Chem. 51, 535 (1998).

[3]

solid-phase synthesis on synphase lanterns OCH2Ph

O

O

65

Ph O

NHAc O

O

H N

O

N H

H N O

O

N H

RINK

NH2 O

NHDde

78

HO

OCH2Ph

O

NHAc

HO O

H N

O

O NH2

H N O

O

N H

NH2 O

NHCOR

79 O RINK

NH2

N H

O NH2 MeO

OMe

Scheme 23. Synthesis of muramyl dipeptide derivatives.

the terminal and aspartic acid residues followed by activation with HATU/ HOAt/DIEA. Acidification of aqueous solutions of 82 initiated formation of needle-like crystals. The morphology and infrared absorption characteristics of these crystals suggested that they were hydrogen-bonded nanotubular aggregates. Ellman and co-workers51,52 synthesized a library of 1152 -turn mimetics 84 (Scheme 25) and screened these compounds for activity against the N-formyl-Mer-Leu-Phe (fMLF) receptor. Antagonists to the fMLF receptor could potentially be used as therapeutic agents for the treatment of inflammatory and infectious diseases. The -turn mimetics 84 consisted of 9- and 10-member rings with an aminoalkylthiol serving as the constraining backbone. A -bromo acid and an Fmoc-protected -amino acid provided 51

A. A. Virgilio, A. M. Bray, W. Zhang, L. Trinh, M. Snyder, M. M. Morrissey, and J. A. Ellman, Tetrahedron 53, 6635 (1997). 52 A. Virgilio and J. A. Ellman, J. Am. Chem. Soc. 116, 11580 (1994).

66

[3]

combinatorial synthesis

N

HN

Mmt-S Br-CH2CO-AA1-AA2-AA3-AA4 N H

CO N H

CO-NH

RINK

80

N

HN

CH2

S

CO-AA1-AA2-AA3-AA4 N H

CO N H

CO- NH

RINK

81 AA1, AA2, AA3 and AA4 = permutations of 2 × Asp, Ile and Lys O RINK

NH2

N H

O NH2 MeO OMe

Scheme 24. Synthesis of acridinyl cyclic peptides as potential DNA-binding agents.

the functionalized i þ 1 and i þ 2 side chains, respectively. The synthesis of 11 of the mimetics 84 was optimized on Rink amide-derivatized Rapp Tentagel. The complete library, comprising all combinations of two backbone components, 32 -amino acids and 18 -bromo acids, was synthesized on MD SynPhase Crowns using the Multipin methodology. The reaction sequence optimized on Tentagel resin performed equally well on the SynPhase Crowns. The mimetics on each Crown were synthesized with a mixture of the two aminoalkylthiol backbone components to give both the 9- and 10-membered rings systems. The library of mimetics 84 was screened in a radioligand-binding assay against a cloned fMLF receptor. Four compounds corresponding to the contents from the two most active wells were prepared on a larger scale and purified. In both instances, the inhibitory activity was due

[3]

67

solid-phase synthesis on synphase lanterns OH

O O

O

H N

H N

O

HN

NH

HN

O NH

H2N O

H N O

Me Me N O

O

O

O

OH

O

N H

O

O NH2

O Me N Me O

H N

NH

H2N O

NH2

O N

HN O Me N Me O

N H

Me Me

O OH

OH O 82

O

O 83

Fig. 5. Cyclic peptides with the potential to form nanotubular aggregates.

predominantly to one of the two compounds from each well. The IC50 values of compounds 85 and 86 were 10 and 13 M, respectively (Fig. 6). Basso and Ernst53 (University of Basel) synthesized a library of cyclic hexapeptides for screening as potential Selectin antagonists. Details of the screening results were not given. The peptides were synthesized on SynPhase Lanterns that were derivatized with a tetrahydropyranyl linker. As outlined in Scheme 26, a series of preformed hydroxyproline dipeptides 87 was coupled onto the solid phase via the hydroproline side chain to give 88. The linear peptide systems 89 were assembled on the solid phase using standard Fmoc chemistry. Head-to-tail cyclization of the peptides was performed on the solid phase using PyBOP/HOAt/DIEA prior to cleavage. The cyclic peptides 90 were obtained in 42–82% purity. The authors reported that the desired cyclic peptide was the major product in all cases. Other Small Molecules

Amines The solution method for the synthesis of polyamines of Bergeron was adapted to the solid-phase by Uriac and co-workers54 as outlined in Scheme 27. Polyamines are of biological interest due to their essential role 53 54

A. Basso and B. Ernst, Tetrahedron Lett. 42, 6687 (2001). S. Tomasi, M. Le Roch, J. Renault, J. C. Corbel, and P. Uriac, Pharm. Pharmacol. Commun. 6, 155 (2000).

68

[3]

combinatorial synthesis H N

Br O

H2N

HN

HN H2N

O

DIC

NH

O

RINK HO

( )n

SS-tBu O NH

RINK

Br

SS-t-Bu

O

O NH

( )n

RINK

O NHFmoc HATU DIEA

HO

i +2

R R

i +2

O i +2

R

O

N

i +1

R

N H

O N

( )n

S

O

1. Bu3P, H2O 2. TMG 3. TFA/Me2S/ H2O (18:1:1)

HN O

N H ( )n

R R

i +1

O

Br SS-t-Bu

NHFmoc N

HN O NH RINK

( )n

SS-t-Bu

O

O

NH2 84

i +2

O

HN

1. 20% piperidine

O

2. DIC (0.5eq) O Br HO i +1 R

NH RINK

n = 1, 2

O RINK

NH2

N H

O NH2 MeO

OMe

Scheme 25. Solid-phase synthesis of macrocyclic -turn mimetics.

in cell growth and the fact that some polyamine analogs are potent anticancer and antiparasitic agents. The classic solution-phase synthesis of Bergeron, which involves the N-alkylation of sulfonamides, is often complicated by the purification of the hydrophilic products. By performing these reactions on the solid phase, Uriac’s group found that purification was simplified and hence the preparation of libraries of these compounds becomes viable.

[3]

69

solid-phase synthesis on synphase lanterns OH

H N

O O

O O

N H

S

N

N H

N

O

S

O

HN

HN

O

O NH2

NH2 86

85

Fig. 6. Macrocyclic -turn mimetics.

O

OH O Fmoc

O

O

N O

O X1

Fmoc N O

N H

O X1

87

88

HO

O

O N X2 X1 NH X3 H N O O

O

Fmoc-Gly-Phe X3 X2

O

O

N H

N O

90

O X1

89 X1 = L-Asp, L-Ser, L-Lys X2 = L-Glu, D-Glu X3 = L-Lys, D-Lys

Scheme 26. Solid-phase synthesis of cyclic peptides using hydroxyproline as a linking site.

70

[3]

combinatorial synthesis

HMP

NO2

O

O O

O HMP

H N

O O

O H2 N

N

N SO2Mes

O

NH2 N SO2Mes · 2CF3CO2H

H2N N

N SO2Mes

O · CF3CO2H

91

92

O HMP

OH

N H

O OH

Scheme 27. Solid-phase synthesis of polyamines.

Synthesis of the orthogonally diprotected homospermidine 91 and the monoprotected homospermidine 92 was performed on SynPhase Crowns with a carbonate handle. The optimization of the reactions was aided by rapid handling of Crowns and performing reactions in parallel. Handlon and Hyman55 from Glaxo Wellcome prepared three libraries totaling 2880 members based around the diphenylmethylamine scaffolds 93, 94, and 95 for use in screening against 7-transmembrane domain receptors. The key step of each of these libraries involved the reductive amination of benzophenones (Scheme 28). The first library (scaffold 93) was prepared on BAL linker Crowns fitted with RF encoding tags.17 The key reductive amination of the solid supported benzophenone intermediates 55

A. L. Handlon and C. E. Hyman, Design and synthesis of a 7TM targeted library: Titanium(IV)-chloride-mediated reductive amination of benzophenones on solid support. Poster presented at 219th ACS National Meeting, San Francisco, CA, March 2000.

[3]

71

solid-phase synthesis on synphase lanterns

O

M1NH2 NaBH(OAc)3

H

DMF/AcOH

BAL

HO

M1 NH

BAL

M1 N

Br O

BAL

DIC/DMF

Br

O X

HO O

Y

Cs2CO3

Ar

M1

H N

X

O

BAL

3

O H 3 N M

1. M NH2, TiCl4 CH2Cl2

Y

M1 N O

O O

X Y

2. NaCNBH3/MeOH 3. 95% TFA/H2O z

z

93 OMe O

O BAL

H

H

O N H

O

OMe

Scheme 28. Synthesis of a diphenylmethylamine library targeted to 7-transmembrane domain receptors.

(40 examples) was performed in two steps by treatment with an amine (40 examples) in the presence of titanium(IV) chloride followed by sodium cyanoborohydride. It was found that this reductive amination step did not take place under standard conditions (amine, catalytic acetic acid, reducing agent) or when boron trifluoride or TiClx(OiPr)y was used in place of titanium(IV) chloride. The use of RF encoding tags allowed the pooling of Crowns into 40 reaction vessels (one vessel for each amine). To perform this reaction in 96-well plates would have required pipetting titanium(IV) chloride into 1600 reaction wells. The two remaining libraries (scaffolds 94 and 95) were prepared on Rink linker-derivatized Crowns. Two libraries were generated after the reductive amination step by either direct cleavage (scaffold 94) or oxidation of the sulfide to a sulfone with hydrogen peroxide followed by cleavage (scaffold 95) (Fig. 7). Ureas and Their Chalogen Analogs Series of ureas 96, thioureas 97, carbamates 98, and semicarbazides 99 were prepared on resin or SynPhase Lanterns by Phoon and Sim56 from Singapore’s Institute of Molecular and Cell Biology. These derivatives 56

C. W. Phoon and M. M. Sim, Synlett, 697 (2001).

72

[3]

combinatorial synthesis O H2 N

O M1

M3

S H N

X

Y

H2N

O M1

M3

S H N

Z 94

O

X

Y

Z 95

Fig. 7. Diphenylmethylamine scaffolds.

were prepared by the attachment of amines or hydrazines onto bromoWang resin followed by treatment with isocyanates, isothiocyanates, or chloroformates (Scheme 29). Ureas 96 were obtained in excellent yields and purity. Thioureas 97 were obtained in lower purity due to the presence of hydroxybenzylated side products, carbamates 98 in good yields and purities, and semicarbazides 99 in good yields but modest purities. A selection of the derivatives was also prepared on SynPhase Lanterns. In comparison to resin, the products from the Lanterns were obtained in higher purity though in reduced overall yields. It should be noted that the reactions on Lanterns employed shorter reaction times than those on resins. Future Perspectives

Solid-phase organic synthesis is now a well-established approach to generating lead finding and lead optimization libraries. Golbiowski et al.9 recently published a review of lead compounds discovered using combinatorial libraries. Many of the examples were generated using solid-phase synthesis. As illustrated throughout this chapter, solid-phase organic synthesis on SynPhase Crowns and SynPhase Lanterns has been adopted by many laboratories for the synthesis of libraries of small molecule compounds. The authors are aware of many other interesting syntheses that may never be published, which are focused on discovering new lead compounds. The main emphasis of this chapter has been the preparation of compounds for screening in the drug discovery process. It should be noted that the applications of combinatorial solid-phase synthesis extend beyond this one field. For example, Gilbertson and co-workers57,58 have made use

[3]

73

solid-phase synthesis on synphase lanterns X S

N R1

S

X N H

R3

R1

Br

S

N

O O

R4

N

NH2

S

N R2

Br

O

R4

98

R2

S

R1 N H

R1 S

R3

X=O X=S

96 97

NHR1 O

S

N H

N H

H N

H N

O R3

O

Bromo-Wang polystyrene resin or Bromo-Wang SynPhase Lantern

R2HN

N H

N H

R3

99

Scheme 29. General solid-phase route to ureas, thioureas, carbamates, and semicarbazides.

of SynPhase Crowns in the development of new support-bound ligands for asymmetric synthesis. The use of the solid phase to ‘‘scavenge’’ reagents and by-products in parallel solution-phase synthesis is a growing field of interest to the practitioner of combinatorial chemistry.59 It should be noted that suitably functionalized SynPhase Lanterns could be used to remove excess reagents from multiple parallel solution-phase reactions. This is a logical extension of the technology, which is now available. Although polystyrene is a useful solid support for a broad range of chemistries, it is not ideally suited to syntheses that require the use of aqueous solvents. To expand the application of SynPhase Lanterns in solidphase synthesis, Mimotopes has developed a new hydrophilic polyamide surface.20 One of the goals of manufacturers of polymeric supports for solid-phase synthesis is to generate surfaces that allow reaction rates and reaction conversions on the solid phase to approach those obtained in solution-phase synthesis. In solid-phase synthesis, reactions are generally driven to completion by using excess reagent, where this is possible. Despite the growing use of solid-phase synthesis in the drug discovery process, there is strong need for more thorough physical chemistry investigations of process on any solid phase, including the SynPhase surfaces, with only limited studies being reported so far. Gerritz22 has undertaken a comparison on the efficiency of small molecule solid-phase synthesis on a range of 57

S. R. Gilbertson and X. Wang, Tetrahedron 55, 11609 (1999). S. R. Gilbertson, S. E. Collibee, and A. Agarkov, J. Am. Chem. Soc. 122, 6522 (2000). 59 S. V. Ley and I. R. Baxendale, Nat. Rev. Drug Discov. 1, 573 (2002). 58

74

[3]

combinatorial synthesis

Si O H N O

H HO HN

O N H

O

O

N H

O NHBoc

O NH

N H 100

Scheme 30. Solid-phase synthesis of 3,9-diazabicyclo[3.3.1]non-6-en-2-one scaffold.

solid phases and found that SynPhase Lanterns gave very favorable results. In a separate study of interest, Gerritz et al.60 reported the high-throughput determination of Hammett relationships for the displacement of a solid supported active ester with a range of anilines on a range of solid supports. It is of interest to note that the observed Hammett reaction constants were dependent on the solid phase. Addendum

Several more publications describing the use of SynPhase Lanterns in solid-phase organic synthesis have recently appeared. Fukase and coworkers61 described the solid-phase synthesis of oligosaccharides using a novel alkyne-based linker. The Sonogashira reaction was used in loading the substrate onto the solid-phase. Workers at Novartis described the solid-phase synthesis of the bicyclic compound 100 via sequential DakinWest and Pectit-Spengler reactions (Scheme 30).62 Ede63 recently published a history of the development of the SynPhase grafted supports with an emphasis on peptide synthesis. Gerritz et al.64 have summarized several years of library production work undertaken at GlaxoSmithKline.

60

S. W. Gerritz, R. P. Trump, and W. J. Zuercher, J. Am. Chem. Soc. 122, 6357 (2000). M. Izumi, K. Fukase, and S. Kusumoto, Synlett, 1409 (2002). 62 D. Orain, R. Canova, M. Dattilo, E. Kloeppner, R. Denay, G. Koch, and R. Giger, Synlett, 1443 (2002). 63 N. J. Ede, J. Immunol. Methods 267, 3 (2002). 64 S. W. Gerritz, M. H. Norman, L. A. Barger, J. Berman, E. C. Bidham, M. J. Bishop, D. H. Drewry, D. T. Garrison, D. Heyer, S. J. Hodson, J. A. Kakel, J. A. Linn, B. E. Marron, S. S. Nanthakumar, and F. J. Navas, III, J. Comb. Chem. 5, 110 (2003). 61

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directed sorting approach for large libraries

75

[4] Directed Sorting Approach for the Synthesis of Large Combinatorial Libraries of Discrete Compounds By Timothy F. Herpin and George C. Morton Introduction

One of the very first steps in the drug discovery process is to find an initial molecule—a hit or a lead—eliciting a desired biological activity. Over the years, many approaches have been used to identify a lead molecule, all of them with some degree of success. The testing of natural product extracts either directly in vivo or in vitro has been a successful source of leads1 in many examples. The rational design of lead compounds based on the knowledge of the natural ligand, the structure of the receptor, or the mechanism of action of the receptor has also been successfully used.2 Recently, methods that screen for low-affinity molecular fragments to be subsequently combined into a lead molecule have been reported to successfully provide lead structures.3 However, the approach that is still considered the most promising is the high-throughput in vitro screening of synthetic compounds and combinatorial libraries.4 It is the most general approach—it can be applied to most types of targets—and when successful, it allows the drug discovery process to start with an easy-to-synthesize small molecule. With the rapid progress of high-throughput screening there has been a growing need for large collections of compounds. In the early 1990s, two main technologies emerged for the rapid synthesis of compound libraries. In 1991, Furka et al.5 described the ‘‘split-and-pool’’ concept for the 1

(a) J. Singh, G. D. Bagchi, A. Singh, and S. Kumar, J. Med. Aromatic Plant Sci. 22/4A-23/ 1A, 554 (2001). (b) J. Josephson, Mod. Drug Discov. 3, 45 (2000). (c) D. G. I. Kingston, Pract. Med. Chem. 101 (1996). 2 (a) D. L. Kirkpatrick, S. Watson, and S. Ulhaq, Comb. Chem. High Throughput Screening 2, 211 (1999). (b) M. Iino, T. Furugori, T. Mori, S. Moriyama, A. Fukuzawa, and T. Shibano, J. Med. Chem. 45, 2150 (2002). (c) S. Flohr, M. Kurz, E. Kostenis, A. Brkovich, A. Fournier, and T. Klabunde, J. Med. Chem. 45, 1799 (2002). (d) G. S. Chen, C. S. Chang, W. M. Kan, C. L. Chang, K. C. Wang, and J. W. Chern, J. Med. Chem. 44, 3759 (2001). 3 (a) J. Fejzo, C. A. Lepre, J. W. Peng, G. W. Bemis, Ajay, and M. A. Murcko, Chem. Biol. 6, 755 (1999). (b) P. J. Hajduk, A. Gomtsyan, S. Didomenico, M. Cowart, E. K. Bayburt, L. Solomon, J. Severin, R. Smith, K. Walter, T. F. Holxman, A. Stewart, S. McGaraughty, M. F. Jarvis, E. A. Kowaluk, and S. W. Fesik, J. Med. Chem. 43, 4781 (2000). (c) D. A. Erlanson, A. C. Braisted, D. R. Raphael, M. Randal, R. M. Stroud, E. M. Gordon, and J. A. Wells, Proc. Natl. Acad. Sci. USA 97, 9367 (2000). 4 A. Golebiowski, S. R. Klopfenstein, and D. E. Portlock, Curr. Opin. Chem. Biol. 5, 273 (2001).

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

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combinatorial synthesis

[4]

generation of large peptide libraries. In 1993, workers at Parke-Davis described the parallel synthesis of small molecules on solid support using a parallel synthesizer.6 For a few years, each technology evolved separately. The split-and-pool approach allowed very large numbers of compounds (100,000s) to be generated but had complications for the screening and the identification of active compounds. Some techniques were invented to improve the identification of active compounds such as tagging methods and fast deconvolution techniques.7 Although solid-phase parallel synthesis offered discrete compounds that were convenient to test, the throughput was limited to a few hundred compounds. When directed sorting was introduced in 1995, it allowed the gap between these two technologies to be bridged.8 The new approach allowed the preparation of combinatorial libraries with the same efficiency as the split-and-pool method, but provided discrete and identified compounds like parallel synthesis. This new method can be used to routinely prepare libraries of several thousand compounds, and it has been quickly adopted by the pharmaceutical industry as one of the techniques of choice. This chapter will review the principle of directed sorting, and it will also present two detailed examples of its application in drug discovery: (1) the synthesis of piperazine-2-carboxamide derivatives, and (2) the synthesis of benzothiazepine derivatives. Directed Sorting Approach

Principle The directed sorting approach (or mix-and-sort approach) uses small synthetic objects, usually called microreactors, which are equipped with a readable encoding system. Typically the synthetic objects can be resin 5

(a) A. Furka, F. Sebestyen, M. Asgedom, and G. Dibo, Int. J. Peptide Protein Res. 37, 487 (1991). (b) A. Furka, L. K. Hamaker, and M. L. Peterson, in ‘‘Combinatorial Chemistry’’ (H. Fenniri, ed.), p. 1. Oxford University Press, Oxford, UK, 2000. (c) A. Furka, Comb. Chem. High Throughput Screening 3, 197 (2000). 6 (a) S. H. DeWitt, J. S. Kiely, C. J. Stankovic, M. C. Schroeder, D. M. R. Cody, and M. R. Pavia, Proc. Natl. Acad. Sci. USA 90, 6909 (1993). (b) S. H. DeWitt, A. W. Czarnik, in ‘‘Combinatorial Chemistry Synthesis and Application’’ (S. R. Wilson and A. W. Czarnik, eds.), p. 25. John Wiley & Sons, New York, 1997. 7 (a) K. S. Lam, M. Lebl, and V. Krchnak, Chem. Rev. 97, 411 (1997). (b) A. W. Czarnik, Curr. Opin. Chem. Biol. 1, 60 (1997). (c) J. J. Baldwin, Mol. Diversity 2, 81 (1996). (d) Z.-J. Ni, D. Maclean, C. P. Holmes, and M. A. Gallop, Methods Enzymol. 267, 261 (1996). 8 (a) K. C. Nicolaou, X.-Y. Xiao, Z. Parandoosh, A. Senyei, and M. P. Nova, Angew. Chem. Int. Ed. Engl. 34, 2289 (1995). (b) X.-Y. Xiao and M. P. Nova, in ‘‘Combinatorial Chemistry Synthesis and Application’’ (S. R. Wilson and A. W. Czarnik, eds.), p. 135. John Wiley & Sons, New York, 1997.

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directed sorting approach for large libraries

77

beads placed in a semi-porous container (MicroKans), a grafted polymer surface, or large plugs of resin beads.9 The coding system can be a radiofrequency tag (RF-Tag), a two-dimensional (2D) optical bar code, a color scheme, or a spatial array. An example of how the directed sorting method works is described in Fig. 1. Imagine a synthesis involving three chemical steps to construct molecules with a common central scaffold incorporating three substituents. At each step three possible reagents can be used. A total of 27 (3  3  3) different compounds can be made through the combination of all of the reagents. At the beginning of the synthesis, the synthetic objects are tagged with unique identifiers for the 27 compounds that can be made. The tags are read in the first sorting step, and the synthetic objects are sorted according to which first reagent they should be reacted with. They are then reacted in batch for the first chemical step. The synthetic objects are then pooled, and sorted again, this time for the second reagent. After a reaction in batch, the process is repeated for the third combinatorial step. At the end of the synthesis, all 27 compounds have been prepared and the tags encode their identity. The compounds can then be released from the synthetic objects and collected in vials or a multiwell plate. The end result of the process affords 27 discrete compounds prepared through 9 chemical reactions and 27 cleavage steps. Equipment Several types of equipment can be used to prepare combinatorial libaries through the directed sorting technique. Irori9b has two types of systems available, one based on the use of RF tags (Accutag9c system) and another one based on the use of 2D optical tags (Nanokan system). The Accutag system is well suited to prepare libraries of 10–10,000 compounds, whereas the Nanokan system is useful for 10,000–100,000 member libraries. Both systems are composed of synthetic objects (MicroKans, Nanokans), tags (RF, 2D optical), an automated sorter, software to track the synthesis (Synthesis Manager), as well as a cleavage system. An alternative cleavage system is available from Bohdan.10 For small libraries, the use of color-coded MicroKans has been reported.11

9

(a) B. Atrash, M. Bradley, R. Kobylecki, D. Cowell, and J. Reader, Angew. Chem. Int. Ed. Engl. 40, 938 (2001). (b) http://www.irori.com. (c) MicroKan, MacroKan, NanoKan, Accutag, Accusort are trademarks of Irori. 10 C. J. Andres, R. T. Swann, K. Grant-Young, S. V. D’Andrea, and M. S. Deshpande, Comb. Chem. High Throughput Screening 2, 29 (1999). 11 J. W. Guiles, C. L. Lanter, and R. A. Rivero, Angew. Chem. Int. Ed. Engl. 37, 926 (1998).

78

[4]

combinatorial synthesis AAA AAB AAC ABA ABB

BAB BAC BBA BBB BBC CAC CBA CBB CCA

ABC ACA ACB ACC BAA BCA BCB BCC CAA CAB CCB CCC CBC

Reagent B

Reagent A

AAA AAC AAB ACB ABA ACA ABB ACC ABC

Reagent C

BBB BBC BAB BAC BBA BCA BCB BCC BAA

Reagent B

Reagent A

CBC CCA CAC CBA CBB CAA CAB CCB CCC

Reagent C

AAA AAC

BBB BBC

CCB CCA

AAB BAB BAC

ABA CBC BBA

ACA ACB ACC

BAA CAC CAA CAB

ABB ABC CBA CBB

BCA BCB BCC CCC

Reagent A

Reagent B

AAA BCA

ACB BBB

BBA CBA CCA

BCB AAB BAB

BAA ACA CAA ABA

ABB CCB CAB CBB

AAA AAB AAC ABA ABB

Reagent C

AAC BBC BAC CBC ACC CAC ABC BCC CCC

BAB BAC BBA BBB BBC CAC CBA CBB CCA

ABC ACA ACB ACC BAA BCA BCB BCC CAA CAB CCB CCC CBC

Fig. 1. Example of directed sorting method. (See color insert.)

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directed sorting approach for large libraries

79

A system based on grafted polymer synthetic objects (lanterns) is available from Mimotopes Pyt. Ltd.12 The system is composed of the synthetic objects (lanterns), RF-Tags (Transtems), software to track the synthesis (TranSort), and a cleavage system. Additional systems that are not commercially available have been reported. Workers at Aventis have developed a highly automated system based on the Mimotope lanterns.13 Recently workers at Millenium Pharmaceuticals reported the StAC system (Stratified Adressable System), a three-dimensional spatial array that allows discrete compounds to be synthesized using the same efficiency as the directed sorting approach.14 Application The directed sorting approach has been widely applied both in industry and in academia. Two examples are detailed in this chapter. Some other recently published examples include a benzopyran library synthesized using the Nanokan system,15 libraries derived from phenolic amino acid scaffolds prepared with the Accutag system,16 and libraries of 1,5-benzodiazepine-2one derivatives.17 Piperazine Carboxamide Libraries The directed sorting method will first be illustrated by the preparation of libraries of piperazine-2-carboxamide derivatives.18 The piperazine2-carboxylic acid scaffold is a pharmacologically important19 center core 12

http://www.mimotopes.com/. J. A. Connelly, New automation technology for the efficient production of large combinatorial libraries of small molecules by ‘‘directed sort and combine’’ methods. Abstracts of Papers, American Chemical Society 221st BTEC-041 (2001). 14 (a) J. C. Reader, C. D. Boden, D. Cowell, C. M. Grant, M. Jones, V. A. Reader, C. C. Renou, and M. Stirling, Multi-dimensional parallel solid-phase synthesis of libraries using StAC technology. Abstracts of Papers, 222nd ACS National Meeting, Chicago, IL, August 26–30, 2001. (b) V. A. Reader, C. J. Boden, D. Cowell, C. M. Grant, M. Jones, J. C. Reader, C. C. Renou, and M. Stirling, Library synthesis using StAC technology: An efficient and inexpensive way of making compounds with 3 points of diversity or more on solid support. Abstracts of Papers, 222nd ACS National Meeting, Chicago, IL, August 26–30, 2001. 15 K. C. Nicolaou, J. A. Pfefferkorn, H. J. Mitchell, A. J. Roecker, S. Barluenga, G.-Q. Cao, R. L. Affleck, and J. E. Lillig, J. Am. Chem. Soc. 122, 9954 (2000). 16 A. D. Morley, Tetrahedron Lett. 41, 7405 (2000). 17 T. F. Herpin, K. G. Van Kirk, J. M. Salvino, S. T. Yu, and R. F. Labaudiniere, J. Comb. Chem. 2, 513 (2000). 18 Work originally published in T. F. Herpin, G. C. Morton, A. K. Dunn, C. Fillon, P. R. Menard, S. Y. Tang, J. M. Salvino, and R. F. Labaudiniere, Mol. Diversity 4, 221 (2000). 19 (a) J. P. Vacca, B. D. Dorsey, W. A. Schleif, R. B. Levin, S. L. McDaniel, P. L. Darke, J. Zugay, J. C. Quintero, and O. M. Blahy, Proc. Natl. Acad. Sci. USA 91, 4096 (1994). 13

80

[4]

combinatorial synthesis

N

N

N O

N

N

N

N

O

O 1

O 2

O

Ph

N H

HO N

N

NH HO

N

O

O

HO N

N

H N

O

O

3

Fig. 2. Examples of piperazine-2-carboxamide derivatives pharmacologically active.

found in angiotensin II antagonist20 1, substance P antagonist21 2, and the aspartyl protease inhibitor indinavir 322 (Fig. 2). Libraries built around this core scaffold are expected to be of general interest for high-throughput screening campaigns. The piperazine-2-carboxylic acid scaffold 4 is well suited for a combinatorial approach as it is a small, constrained structure with three functional groups (one carboxylic acid and two amines) that may be conveniently substituted by solid-phase chemistry. Orthogonal protection of the two amino groups could easily be carried out on a large scale by solution-phase chemistry18 (Scheme 1).

(b) E. Mishani, C. S. Dence, T. J. McCarthy, and M. J. Welch, Tetrahedron Lett. 37, 319 (1996). (c) I. A. Cliffe, C. I. Brightwell, A. Fletcher, E. A. Forster, H. L. Mansell, Y. Reilly, C. Routledge, and A. C. White, J. Med. Chem. 36, 1509 (1993). (d) B. M. Kim, B. E. Evans, K. F. Gilbert, C. M. Hanifin, J. P. Vacca, S. R. Michelson, P. L. Darke, J. A. Zugay, E. A. Emini, W. Schleif, J. H. Lin, I.-W. Chen, K. Vastag, P. S. Anderson, and J. R. Huff, Bioorg. Med. Chem. Lett. 5, 2707 (1995). 20 (a) M. T. Wu, T. J. Ikeler, W. T. Ashton, R. S. L. Chang, V. J. Lotti, and W. J. Greenlee, Bioorg. Med. Chem. Lett. 3, 2023 (1993). (b) W. T. Ashton, W. J. Greenlee, M. T. Wu, C. P. Dorn, M. MacCoss, and S. G. Mills, World Patent WO 9220661 (1992). 21 S. G. Mills, R. J. Budhu, C. P. Dorn, W. J. Greenlee, M. Maccoss, and M. T. Wu, World Patent WO 9413646 (1994). 22 (a) K. Rossen, S. A. Weissman, J. Sager, R. A. Reamer, D. Askin, R. P. Volante, and P. J. Reider, Tetrahedron Lett. 36, 6419 (1995). (b) D. S. Stein, D. G. Fish, J. A. Bilello, S. L. Preston, G. L. Martineau, and G. L. Drusano, AIDS 10, 485 (1996).

[4]

directed sorting approach for large libraries Boc

H N N H 4

a, b CO2H

N

c, d

N CO2H Alloc 5

81

Fmoc N N CO2H Alloc 6

Scheme 1. Synthesis of the protected scaffold. (a) Boc-ON, dioxane:water (1:1), pH 11; (b) Allyl chloroformate, pH 9.5; (c) 50% TFA-DCM; (d) Fmoc-Cl, Na2CO3, dioxane:water.

Two distinct libraries were synthesized and combined to produce 15,000 discrete compounds. Both libraries were prepared with the Irori Accutag system using resin-filled MicroKans as synthetic objects. The first library was prepared according to the chemistry outlined in Scheme 2. Thus the MicroKans were equipped with an RF-tag and resin containing the dimethoxy-benzaldehyde linker 7 (BAL).23* The first combinatorial step was attachment of a primary amine by reductive amination. The MicroKans were then pooled and the resulting secondary amines were acylated with the orthogonally protected piperazine-2-carboxylic acid scaffold 6 in a single batch to yield 9. The fluorenylmethyloxycarbonyl (Fmoc) group was removed with piperidine, and the MicroKans were sorted for the second combinatorial step. The 4-amine was reacted with sulfonyl chlorides, isocyanates, chloroformates, and carboxylic acids. The MicroKans were then pooled, the allyloxycarbonyl (Alloc) group was removed with Pd(PPh3)4, and the MicroKans were sorted for the third combinatorial step. The 1-amine was functionalized similarly to afford sulfonamides, ureas, carbamates, and amides. This chemistry was used to produce a 10,000-member library. See Table I for a representative sample of the reagents used. The second library built around the piperazine-2-carboxylic acid scaffold was designed to fill some of the diversity gaps left by the first library. 23

(a) K. J. Jensen, J. Alsina, M. F. Songster, J. Vagner, F. Albericio, and G. Barany, J. Am. Chem. Soc. 120, 5441 (1998). (b) C. G. Boojamra, K. Burow, L. A. Thompson, and J. A. Ellman, J. Org. Chem. 62, 1240 (1997). * Abbreviations: BAL, backbone amide linker; BSA, bis(trimethylsilyl)acetamide; DBU, 1,8diazabicyclo[5.4.0]undec-7-ene; DCE, dichloroethane; DCM, dichloromethane; DIC, 2diisopropylcarbodiimide; DIEA, diisopropylethyl amine; DMAP, N,N-dimethylaminopyridine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HBTU, [O-(7-azabenzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate; MCPBA, m-chloroperoxybenzoic acid; NMP, Nmethylpyrrolidinone; NMM, N-methylmorpholine; PfP, pentafluorophenol; RT, room temperature; TFA, trifluoroacetic acid; THF, tetrahydrofuran.

82

O

O R1 NH2

O O

O

a

8

7

O

b

NH R1

O

O O

N R1

N Fmoc

9 O c, d

O

O O 10

N R1

Alloc N N R2

Alloc N

O e, d

O

O O

N R1 11

R3 N N R2

O f

R1

N H 12

R3 N N R2

combinatorial synthesis

O

O

6

Scheme 2. A 10,000-member combinatorial library: (a) NaBH(OAc)3, DMF, AcOH; (b) HBTU, DIEA, DMF; (c) piperidine:DMF (1:1); (d) RSO2Cl, NMM, DCM, or RNCO, DCM or ROCOCl, DIEA, DCM, or RCOOH, EDC, NMP; (e) Pd(PPh3)4, morpholine, THF, DMSO, aq. HCl; (f) 50% TFA-DCM.

[4]

[4]

83

directed sorting approach for large libraries TABLE I Representative Sample of the Reagents Used in the 10,000-Member Piperazine-2-carboxamide Library

R1 O H2N

H2N

H2N

N

N

N

O

H2N

H2N

H2N

O

NHBoc

R2 O O

O

O

O Cl

O

HO

Cl

O

HO

H N

O

N

N

F

F

HO

N

HO

O

O O S Cl

O N

O

NHBoc

HO

N

F

O

O

O

HO NHBoc

R3 O O

Cl

O O S Cl

O

HO

HO

O

O

CF3

OH

N

HO

O

N H

O

S

O

N O

O

Cl Cl

O

O

O

O CF3

O

OH O

O

N

HO NHBoc

N H

N

In the first library, all of the compounds contained the 2-secondary amide that was the point of attachment to the resin. Also, none of the compounds contained a basic tertiary amine in the piperazine ring. The synthetic scheme for the second library is outlined in Scheme 3. The scaffold 17 was synthesized on a large scale in solution phase. Bifunctional reagents containing a handle (carboxylic acid or amine) and a halide were loaded onto Wang resin. Bromo- or chlorocarboxylic acids were reacted with Wang resin using either the Yamaguchi method (2,6-dichlorobenzoyl chloride)24 or by conversion to the acid chloride. Alternatively, Fmoc amino acids were loaded onto Wang resin with diisopropylcarbodiimide (DIC) – dimethylaminopyridine (DMAP). The Fmoc group was removed and the free amine was then acylated with bromo- or chlorocarboxylic acids. Symmetric diamines were also loaded on the nitrophenol carbonate derivative of Wang resin and acylated with bromo- or chlorocarboxylic acids. The bromides or chlorides were converted in situ to the corresponding iodide and then reacted with the unprotected amine of 17. Site isolation on the resin ensured clean monoalkylation. The carboxylic acid 24

P. Sieber, Tetrahedron Lett. 28, 6147 (1987).

84

O

O

OH

a

13

HO

N

17 H O

O 14

Alloc N

HO

b R

O

d

O R N

N Alloc 18

16 c O

e, f

NO2

15 R2'

R2' R2' R2 N

R2 N O

i O R N

R N

R2 N O

N Y R3

g, h O R N

O

N Alloc

N Y R3

21 Y= CO, SO2, COO

20

combinatorial synthesis

O

Br O R

O

O

O

NHFmoc

O

19 Y= CO, SO2, COO

Scheme 3. A 5000-member library. (a) Br-R-COOH, dichlorobenzoyl chloride, pyridine, DCM, or Br-R-COCl; (b) (i) piperidine, DMF; (ii) Br-R-COOH, DIC, HOBt, DMF; (c) (i) symmetrical diamine, DMF; (ii) Br-R-COOH, DIC, HOBt; (d) KI, DMF, 80C; (e) DIC, PfP, DMF; (f) R2-NH-R20 , DMF; (g) Pd(PPh3)4, morpholine, THF, DMSO, aq. HCl; (h) RSO2Cl, NMM, DCM, or ROCOCl, DIEA, DCM, or RCOOH, EDC, NMP; (i) 50% TFA-DCM.

[4]

[4]

85

directed sorting approach for large libraries

was then converted to the pentafluorophenyl ester and reacted with amines in a second combinatorial step. After the removal of the alloc group, the third combinatorial step consisted of acylation, sulfonylation, and carbamate formation, all of which were performed under the same conditions as in the first library. Table II lists a representative sample of the reagents used in the 5100-member library. A full matrix of 17  25  10 was constructed for this library. Both libraries were constructed as a combination of a small number of reagents, therefore analysis of a small random portion of the library was expected to be representative of the overall purity. The quality of both libraries was assessed by analyzing 5% of the compounds by high-pressure liquid chromatography (HPLC) with a combination of mass spectrometry (MS) and evaporative light scattering (ELS) detection. The MS detector was used to confirm the identity of the compounds and purity was based on ELS detection. Figure 3 shows the purity distribution for the two libraries. In both cases, the majority of the compounds were present in high purity. For the 10,000-member library, a significant portion of the library was affected by an unexpected side reaction. Compounds containing

TABLE II Representative Sample of the Reagents Used in the 5000-Member 4-Alkyl-piperazine2-carboxamide Combinatorial Librarya R1 H

a

O

d

b

a

HO2C

c

HN

N

Br

HO

Br

O

OH

O

Br

O

O

b

H N

HO

N

Cl

O

Br

R2 O HN

H 2N

N

H 2N

H2N

N

N

N

O

NH2 S O O

H 2N

H 2N

N

O

H2N

O

HN

H2N

N

NHBoc

NH2

R3 O

O

H

Cl

a

O

HO

O HO

Cl

O O

Cl

O S Cl

HO

NHBoc

Notes: (a) Loaded with the Yamaguchi method. (b) Fmoc-amino acid loaded with DIC/ DMAP, deprotected and reacted with bromo-acid. (c) Symmetric diamine loaded on the nitrophenyl carbonate derivative of Wang resin and subsequently reacted with bromoacid. (d) Piperazine carboxamide scaffold reacted with the nitrophenyl carbonate derivative of Wang resin.

[4]

combinatorial synthesis 180 160 140 120 100 80 60 40 20 0

Number of compounds analyzed

Number of compounds analyzed

86

A

10

40

70

100

60

B

50 40 30 20 10 0

5

25

Purity

Purity range

45

65

85

Purity

% of library

Purity range

% of library

76-100%

53%

76-100%

62%

51-75%

12%

51-75%

28%

26-50%

6%

26-50%

5%

0-25%

29%

0-25%

5%

Fig. 3. Purity distribution for (A) the 10,000-member library and (B) the 5000-member library.

aromatic ureas at the R3 position cyclized to form hydantoins in high purity (Scheme 4).25 In the 5000-member library, 95% of the expected compounds were detected and no significant side reaction was observed. Combining Directed Sorting with Parallel Synthesis—Benzothiazepine Library

The directed sorting method has also been used to produce a large amount of compound (up to 100 mg).26 In the following example, the method was used to generate 100 mg of 3-aminobenzothiazepine derivatives. Each derivative was then acylated with 20 different carboxylic acids using the tetrafluorophenol resin (TFP-resin).27 Overall this allowed a large library of 16,000 discrete compounds to be produced. 25

J. Kavalek, V. Machacek, G. Svobodova, and V. Sterba, Collect. Czech. Chem. Commun. 8, 1999 (1987). 26 Work originally published in G. C. Morton, J. M. Salvino, R. F. Labaudiniere, and T. F. Herpin, Tetrahedron Lett. 41, 3029 (2000). 27 (a) J. M. Salvino, N. V. Kumar, E. Orton, J. Airey, T. Kiesow, K. Crawford, R. Mathew, P. Krolikowski, M. Drew, D. Engers, D. Krolinkowski, T. Herpin, M. Gardyan, G. McGeehan, and R. Labaudiniere, J. Comb. Chem. 2, 691 (2000). (b) R. E. Dolle, Methods Enzymol. (2003).

[4]

87

directed sorting approach for large libraries

OMe

O N R1 OMe

O 22

Ar NH

O

Ar N

N

TFA

N R2

DCM

O N

O

N R2 23

Scheme 4. Cyclization of aromatic ureas to hydantoins.

1,5-Benzothiazepine derivatives are of particular interest for lead discovery because they have been shown to have activity against different families of targets. The 1,5-benzothiazepine scaffold has not only been used as a constrained dipeptide mimic in protease inhibitors such as interleukin1-converting enzyme inhibitors,28 elastase,29 and angiotensin-converting enzyme inhibitors,30 but also in antagonists of several G-protein-coupled receptors such as the cholecystokinin31 receptor or the angiotensin II receptor.32 Our synthetic route, depicted in Scheme 5, used the 3-amino group as a point of attachment, and exploited the wide variety of ortho-halonitrobenzene derivatives commercially available and the facile substitution of the benzothiazepine amide nitrogen. The library was made with the Irori Accutag system, using MacroKans containing 400 mg of resin and an RF tag as synthetic object. Cysteine 25 was reacted with the nitrophenyl carbonate derivative of Wang resin 24 by first using bis-(trimethylsilyl)-acetamide33 (BSA) to dissolve the amino acid (Scheme 5). The resin was then loaded into MacroKans, and sorted for the first combinatorial step. The thiol 26 was then reacted with a variety of halonitrobenzene derivatives 27. Formation of up to 25% of cystine by dimerization of the resin-bound cysteine was observed during this step. This could be suppressed by using a strictly inert atmosphere and DBU as base. The MacroKans were combined and reacted in a single batch with tindichloride dihydrate to reduce the nitro group, and then with EDC to cyclize the aniline to the benzothiazepine derivatives 30. The MacroKans were sorted into two batches and further diversity could be obtained by 28

J. M. C. Golec, D. J. Lauffer, D. J. Livingston, M. D. Mullican, P. L. Nyce, A. L. C. Robidoux, and M. W. Wannamaker, World Patent WO 9824804 (1998). 29 J. W. Skiles, R. Sorcek, S. Jacober, C. Miao, P. W. Mui, D. McNeil, and A. S. Rosenthal, Bioorg. Med. Chem. Lett. 3, 773 (1993). 30 J. Slade, J. J. Stanton, D. Ben-David, and G. Mazzenga, J. Med. Chem. 28, 1517 (1985). 31 A. Nagel, World Patent WO 9401421 (1994). 32 P. Buhlmayer and P. Furet, World Patent WO 9413651 (1994). 33 B. A. Dressma, L. A. Spangle, and S. W. Kaldor, Tetrahedron Lett. 37, 937 (1996).

88

X SH O

NO2

O O

+

a

OH

H2N

c O

O

N H

29

H N

30 Z=: 31b Z=O

N H Z

32a Z=: 32b Z=O

R1

S

R1 S Z

R2 N

O

R2 N

N H

Z

N H

30

O

S

O

O

R1 f

H N

O O d

OH

OO O

b

Z

g

combinatorial synthesis

OH

28

e

O

S

O

O O

27

R1

S N H

NO2

OH

H2N

R1

O

R1

26

25 O2N

O

N H

O O

24

SH

O

R1

H2N S Z Z

33a Z=: 33b Z=O

Scheme 5. (a)(1) BSA, DMF, argon, RT; (2) 10% AcOH in DMF; (b) X = Cl, Br, or F, DBU, DMF, argon, RT; (c) SnCl2-2H2O, DMF, RT; (d) EDC, NMP, RT; (e) m-CPBA, DCM, RT; (f) R2-X (X = Br, Cl), KI, DMF, DBU, RT; (g) 50% TFA-DCM, RT, 1 h.

[4]

[4]

89

directed sorting approach for large libraries

oxidation of one batch of the sulfide 30 to the sulfone 31b with m-CPBA. The MacroKans were pooled and sorted for the last combinatorial step. Alkylation of the amide nitrogen could be accomplished with benzamide bases as reported in the solid-phase synthesis of benzodiazepine derivatives,23b but we found that DBU gave cleaner results with a simpler protocol. Cleavage from the resin afforded benzothiazepine derivatives 33a and 33b in high yield and excellent purity (see Table III for a selection). Once released from the resin, the 3-aminobenzothiazepine derivatives could be further reacted with resin-bound tetrafluorophenyl ester of carboxylic acids to form amides (Scheme 6). Carboxylic acids were loaded TABLE III Examples of 1,5-Benzothiazepin-2-one Derivatives Z

Z 8 R1 7

3

NH2

Yieldb

H H CH2-Phc CH2-Phc CH2-Phc CH2-Phc

— O — — — —

82 100 100 95 100 94

78 59 63 68 60 61

CH CH

CH2-Phc CH2-Phc

— O

93 67

65 49

CH N CH CH CH CH CH CH CH CH

CH2-Phc CH2-Phc CH2-Phc (CH2)3-Phd CH2-C(O)-N(Et)2c CH2-4-fluorophenylc CH2-3-chlorothiophenec CH2-3-phenyloxadiazolec (CH2)2 CH3e (CH2)3-O-CH2-Phd

O — — — — — — — — —

94 100 94 100 83 71 70 69 89 84

57 58 62 60 70 53 54 51 60 62

T

1 2 3 4 5 6

H H 7-CF3 7-OMe 7-C(O)-Ph 7-C(O)-(2-methoxymethylpyrrolidine) 7-NH-C(O)-NH-CH2-Ph 7-C(O)-(2-methoxymethylpyrrolidine) 7-NH-C(O)-NH-CH2-Ph 8-OMe 9-Me H H H H H H H

CH CH CH CH CH CH

a

6

N4 O R2

Puritya

R1

9 10 11 12 13 14 15 16 17 18

S

Z

Entry

7 8

T

R2

Purity measured by HPLC/ELSD (evaporative light scattering detector). Yield based on weight of crude extract from the resin. c The alkyl chloride was used for alkylation. d The alkyl bromide and potassium iodide were used for alkylation. e The alkyl iodide was used for alkylation. b

90

[4]

combinatorial synthesis

F

F

F

R3CO2H O

OH F

a

F

F

O

O F

34

O R3

b

R2 N

O O

33 R3

F 35

R1

N H 36

S

Z

Z

Scheme 6. (a) DIC, DMAP, RT; (b) DMF, RT.

onto tetrafluorophenol resin 34 using the standard protocol developed by Salvino et al.27 19F NMR of the resin was used to quantify the loading of each carboxylic acid. These resin bound esters 35 were then reacted with the 3-amino group of the benzothiazepine derivatives in DMF for a few hours. After filtration and evaporation, 3-amido-1,5-benzothiazepine derivatives 36 were obtained in high yields and excellent purity. Table IV illustrates the generality of this reaction. A 16,000-member library was synthesized using this procedure. Twentyone halonitrobenzenes were combined with 20 halides and half were converted to the sulfone, resulting in 840 3-amino-1,5-benzothiazepine derivatives. These were then reacted with 19 carboxylic acids. Table V shows a representative example of the reagents used. A small random sample of 480 compounds from the library was analyzed by HPLC/MS. Figure 4 shows the purity distribution for the library. Most of the compounds were prepared in high purity. Conclusion

The directed sorting approach is a convenient technology to produce large combinatorial libraries of discrete compounds. As can be seen with the two examples presented, it is a very versatile and practical method to produce compounds in a variety of formats. Experimental Section

General Information Chloromethyl polystyrene beads of 150–300 M (loading 2 mmol/g) and Wang resin beads 150–300 M (loading 1.7 mmol/g) were purchased from Polymer Laboratories. 4-Hydroxy-2,6-dimethoxybenzaldehyde was purchased from Perseptive Biosystems. BAL resin was prepared according to the published procedure.23 A loading of 0.8 mmol/g was determined by loading 4-bromobenzylamine to the resin and using elemental analysis. Rink amide resin was purchased from Irori. All other reagents were

[4]

91

directed sorting approach for large libraries

purchased from standard commercial sources and used without further purification. Solvents used were from EM Science of OmniSolv distilled grade unless specified otherwise. 1H NMR and 13C NMR spectra were obtained in 5-mm tubes on a 300-MHz Bruker ARX spectrometer in CDCl3 unless otherwise stated. Mass spectra were recorded on Finnigan 4500 EI and Sciex API 3 IS spectrometers. The libraries were constructed using the Irori system. For the 10,000member library, MicroKans were filled with BAL resin by suspending the resin in an isobuoyant suspension (DMF:DCE 2:1) and dispensing it with a Packard Multiprobe liquid handler. For the 5000-member library, resin bound scaffold 18 was prepared in bulk and loaded into the MicroKans TABLE IV Example of Reaction of 1-Benzyl-3-amino-1,5-benzothiazepine with Resin-Bound Tetrafluorophenyl Esters

F

F

O O

F

O CH 3

N

CH3 CH3

O O

CH3

N

H2N

O O

DMF R3

RT

S

F 35

R

O R3 +

Purity of product (%)b

40

90

90

75

100

79

N S

38

37

Ester resin loading (%)a

N H

R H3C N

O

O

Ester resin loading (%)a

Purity of product (%)b

100

84

100

66

100

78

80

77

60

92

100

78

O

100

80

95

79

N

H N

O CH3

100

71

Cl Cl

a b

Tetrafluorophenol resin loading was measured using 19F NMR. Purity of the crude benzothiazepine derivative measured by HPLC with ELS detection.

92

[4]

combinatorial synthesis

using the method described above. All reactions involving MicroKans were performed in round-bottom flasks equipped with overhead stirrers. The Autosort10K was used to sort the MicroKans between combinatorial steps and cleavage of the library compounds was effected in the Accucleave 96.

TABLE V Representative Sample of the Reagents Used in the 16,000-Member 1,5-Benzothiazepine Combinatorial Library

R1 O + N O−

O

F

O− + N

H N

H N

O + O− N

O

O

F

O−

O

O + N

O O− N+

O

H N

Cl

Cl

Cl

O

R2 N

Cl

Br

Cl

O

N

O

Cl

O

Cl

O

R3

O OH

O

O

Cl

HO

HO

Cl

HO

N

O

O

N

O

HO

O

O

Number of compounds analyzed

70 60 50 40 30 20 10 0 5

15

25

35

45

55

65

75

85

95

Purity Fig. 4. Purity distribution for the 16,000-member 1,5-benzothiazepine library. Purity based on HPLC with ELS detection.

[4]

directed sorting approach for large libraries

93

Resin Bound Amines 8 For each amine, 250 MicroKans (each MicroKan contained 12 mg of 0.8 mmol/g loaded BAL resin 7) were placed into a 1-liter three-necked round-bottom flask fitted with an overhead stirrer. The resin in the MicroKans was swelled in a solution of 1% acetic acid in DMF (300 ml). The air bubbles in the MicroKans were removed by placing the round-bottom flask under house vacuum. The amine (20.0 mmol) and sodium triacetoxyborohydride (4.24 g, 20.0 mmol) were added sequentially. The reaction was stirred at RT for 6.5 h. For workup, each reaction was individually drained and washed with DMF. All of the MicroKans were then combined and washed with 1:1 DMF/MeOH, DMF, DCM, and Et2O. The MicroKans were then dried overnight with a stream of nitrogen gas. Resin Bound Amides 9 The 4500 MicroKans containing resin bound amines 8 were placed into a 12-liter three-necked round-bottom flask fitted with an overhead stirrer. Dimethylformamide (4.5 liters) was added to swell the resin in the MicroKans. 1-Alloc-4-fmoc-piperazine-2-carboxylic acid scaffold 6 (78.6 g, 180.0 mmol) was dissolved into DMF (500 ml) and added to the MicroKans. HBTU (68.3 g, 180.0 mmol) and DIEA (62.7 ml, 360.0 mmol) were then added sequentially. The reaction was stirred at RT for 6.5 h. The solution was drained and the MicroKans were washed with DMF, DCM, and Et2O. The MicroKans were dried overnight with a stream of nitrogen gas. Removal of Fmoc Protecting Group from 9 The MicroKans containing 9 were placed into a 12-liter three-necked round-bottom flask fitted with an overhead stirrer. Dimethylformamide (2.5 liters) and piperidine (2.5 liters) were added to the MicroKans. The reaction was stirred at RT for 3.5 h. The reaction solution was drained and the MicroKans were washed with DMF, DCM, and Et2O. The MicroKans were then dried overnight with a stream of nitrogen gas. Acylation with Carboxylic Acids to Produce 10 or 11 or 20 For each carboxylic acid, 200 MicroKans were placed into a 1-liter three-necked round-bottom flask fitted with an overhead stirrer. The resin in the MicroKans was swelled in NMP (300 ml). The carboxylic acid (20.0 mmol) and EDC (3.83 g, 20.0 mmol) were added sequentially. The reaction was stirred overnight at RT. For workup, each reaction was individually drained and washed once with DMF. All of the MicroKans from

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each acid were then combined and washed with DMF, DCM, and Et2O. The MicroKans were then dried overnight with a stream of nitrogen gas. Acylation with Chloroformates to Produce 10 or 11 or 20 For each chloroformate, 200 MicroKans were placed into a 1-liter three-necked round-bottom flask fitted with an overhead stirrer. The resin in the MicroKans was swelled in anhydrous DCM (300 ml). DIEA (3.5 ml, 20.0 mmol) and the chloroformate (20.0 mmol) were then added sequentially. The reaction was stirred overnight at RT. For workup, each reaction was individually drained and washed once with DMF. All of the MicroKans were then combined and washed with DMF, DCM, and Et2O. The MicroKans were then dried overnight with a stream of nitrogen gas. Urea Formation with Isocyanates to Produce 10 or 11 For each isocyanate, 500 MicroKans were placed into either a 2- or 3liter three-necked round-bottom flask fitted with an overhead stirrer. The resin was swelled in anhydrous DCM (600 ml). The isocyanate (50.0 mmol) was then added neat. The reaction was stirred overnight at RT. For workup, each reaction was individually drained and washed with DMF. All of the MicroKans were then combined and washed with DMF, THF, DCM, and Et2O. The MicroKans were then dried overnight with a stream of nitrogen gas. Sulfonamide Formation to Produce 10 or 11 or 20 For each sulfonyl chloride, 500 MicroKans were placed into either a 2- or 3-liter three-necked round-bottom flask fitted with an overhead stirrer. The resin was swelled in anhydrous DCM (600 ml). NMM (5.5 ml, 50.0 mmol) and the sulfonyl chloride (50.0 mmol) were then added sequentially. The reaction was stirred overnight at RT. For workup, each reaction was individually drained and washed with DMF. All of the MicroKans were then combined and washed with DMF, THF, DCM, and Et2O. The MicroKans were then dried overnight with a stream of nitrogen gas. Removal of Alloc Protecting Group from Resin 10 or 19 The 5000 MicroKans containing resin 10 or 19 were placed into a 12liter three-necked round-bottom flask fitted with an overhead stirrer. THF (2 liters), DMSO (2 liters), and 0.5 N HCl (1 liter) were added to the MicroKans. The reaction flask was then flushed with nitrogen. Pd(Ph3P)4 (8.06 g, 6.98 mmol) and morpholine (218 ml, 2500 mmol) were added sequentially. The reaction was stirred overnight under a flow of

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nitrogen gas. For workup, the reaction was drained and the MicroKans were washed with THF, sodium diethyldithiocarbamate (0.02 M in DMF), DMF, 0.5% DIEA in DCM, DCM, and Et2O. The MicroKans were then dried overnight with a stream of nitrogen gas. Preparation of Resin 16 by the Yamaguchi Method 4-(Bromomethyl)phenylacetic acid (529 mg, 2.34 mmol) was poured into 5 ml of DMF, followed by pyridine (170 l, 2.125 mmol) and 2,6dichlorobenzoylchloride (301 l, 2.125 mmol). The mixture was shaken for 1 h and then the Wang resin (loading 1.7 mmol/g) was added to the mixture. The mixture was shaken overnight, drained, and the resin was washed with DMF, THF, DCM, and ether. The resin was dried overnight under reduced pressure. Preparation of Resin 16 from Acid Chlorides In a flask flushed with nitrogen, the acid (5.5 eq, 2.34 mmol) and oxalyl chloride (3 ml, 6.02 mmol) were mixed in DCM (10 ml). One drop of DMF was added. The mixture was stirred for 1 h. The solvent and oxalyl chloride were then removed by evaporation. The acid choride was dissolved in 9 ml of a mixture of DCM and pyridine (9:1). Wang resin (loading 1.7 mmol/g) was added and the mixture was shaken overnight. The mixture was drained and the resin was washed with DMF, THF, DCM, and ether. The resin was dried overnight under reduced pressure. Preparation of Resin 16 from Amino Acids Wang resin (150 mg, 0.255 mmol) was suspended in DCM (2 ml). The Fmoc amino acid (1.25 mmol) was added, followed by DIC (200 l, 1.25 mmol) and DMAP (6.4 mg, 0.052 mmol). The mixture was shaken overnight. The mixture was drained and the resin was washed with DCM, DMF, THF, DCM, and ether. The resin was then suspended in a mixture of piperidine and DMF (1:1). The mixture was shaken overnight, drained, and the resin was washed with DCM, DMF, THF, DCM, and ether. The resin (100 mg, 0.17 mmol) was suspended in DCM (15 ml). The bromo-acid (2.55 mmol) was added, followed by DIC (400 l, 2.55 mmol). The mixture was shaken overnight, drained, and the resin was washed with DCM, DMF, THF, DCM, and ether. The resin was dried overnight under reduced pressure. Preparation of Resin 16 from Symmetrical Diamines Nitrophenol carbonate resin (100 mg, 0.16 mmol) was suspended in 2 ml of DMF. The diamine (1.6 mmol) was added and the mixture was

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shaken overnight at room temperature. The mixture was drained and the resin was washed with DMF, THF, DCM, and ether. The resin was suspended in DCM (10 ml). The bromo-acid (1.7 mmol) was added, followed by DIC (267 l, 1.7 mmol). The mixture was shaken overnight, drained, and the resin was washed with DCM, DMF, THF, DCM, and ether. The resin was dried overnight under reduced pressure. Alkylation with Piperazine-2-Carboxamide Scaffold to Produce 18 The resin 16 (4.6 g, 7.82 mmol) was suspended in DMF (15 ml). 1-Alloc-2-carboxypiperidine trifluoroacetic acid salt (2.71 g, 23.4 mmol) was added followed by potassium iodide (458 mg, 23.4 mmol) and DIEA  (2.89 ml, 45 mmol). The reaction mixture was heated overnight at 80 , then drained, and the resin was washed with DMF, THF, DCM, and ether. Amide Bond Formation to Produce 19 For each amine, 402 MicroKans (each MicroKan contained 6 mg of 1.7 mmol/g loaded resin 18) were placed into a 1-liter three-necked round-bottom flask fitted with an overhead stirrer. The resin in the MicroKans was swelled in DMF (300 ml). Diisopropylcarbodiimide (4.79 ml) and pentafluorophenol (5.63 g) were added and the resulting mixture was stirred at RT for 2 h. Each reaction was individually drained and washed with DMF. The MicroKans in each round-bottom flask were again suspended in DMF (300 ml) and the corresponding amine (20.4 mmol) was added to each vessel. In the case of hydrochloride salts, DIEA (10 eq) was added. This reaction was stirred at room temperature overnight. Each reaction was individually drained. All of the MicroKans were then combined and washed with DMF, THF, DCM, and ether. The MicroKans were dried overnight with a stream of nitrogen. Cleavage The MicroKans containing BAL resin were cleaved with 50% TFA in DCM for 1 h. Cysteine Carbamate Resin 26 Nitrophenol carbonate resin 24 (320 g, 544.0 mmol) was swelled in anhydrous DMF (5 liters) in a 12-liter three-necked round-bottom flask. Argon gas was bubbled through this slurry for 1 h while stirring with an overhead stirrer. In a second 3-liter three-necked round-bottom flask, anhydrous DMF (500 ml) and BSA (1.5 liter) were added. This solution was degassed for 1 h by bubbling argon gas through the solution.

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dl-Cysteine was then introduced into the BSA solution. After stirring for 30 min, all of the cysteine had dissolved up into solution. This cysteine solution was then canulated into the 12-liter flask containing the resin slurry. The reaction was stirred overnight at room temperature under argon. For the workup, the reaction solution was drained under argon. The resin was then washed with DMF, 10% AcOH in DMF, DMF, THF, DCM, and diethyl ether under argon. The resin was dried overnight with a stream of argon gas. This resin was then dry loaded into Irori MacroKans using a powder dispensing gun (Perry Industries). The MacroKans were manually sorted. Halo-Nitrobenzene Coupling 28 For each halonitrobenzene 27, 42 MacroKans (each containing 300 mg of cysteine resin with a loading of 1.2 mmol/g) were placed into a 1-liter three-necked flask fitted with an overhead stirrer and argon line. The flask was flushed with argon for 30 min. Degassed anhydrous DMF (250 ml) was added to the flask. While stirring the MacroKans under argon, DBU (22.6 ml, 151.2 mmol) was added. After stirring for approximately 5 min, the halonitrobenzene 27 (151.2 mmol) was then added. The reaction was stirred under argon for several hours. The argon lines were then removed, the reaction capped tightly, and stirred overnight at room temperature. For the workup, the reaction solution was drained under argon. The MacroKans were then washed with DMF, 10% HOAc in DMF, 20% aqueous THF, THF, DCM, and diethyl ether. The MacroKans were dried overnight with a stream of nitrogen gas. Reduction of Nitro Group to Prepare 29 A total of 924 MacroKans (each containing 300 mg of resin 28 with a loading of 1.2 mmol/g) was placed into a 12-liter three-necked roundbottom flask fitted with an overhead stirrer and a heating mantle. DMF (6 liters) and tin dichloride dihydrate (750.5 g, 3326.4 mmol) were added  to the flask. The reaction was stirred at 50 overnight. For workup, the reaction solution was drained. The resin was then washed with DMF, aqueous THF, THF, DCM, and diethyl ether. The MacroKans were dried overnight with a stream of nitrogen gas. Cyclization to Benzothiazepine 30 A total of 924 MacroKans (each containing 300 mg of resin 29 with a loading of 1.2 mmol/g) was placed into a 12-liter three-necked roundbottom flask fitted with an overhead stirrer and a heating mantle. The resin

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in the MacroKans was swelled in anhydrous NMP (6 liters). EDC (318.9 g, 1663.2 mmol) was added and the reaction was stirred overnight. For the workup, the MacroKans were washed with DMF, aqueous DMF, DMF, aqueous THF, THF, DCM, and diethyl ether. The MacroKans were dried overnight with a stream of nitrogen gas. Oxidation to Sulfone 31b The resin in 420 MacroKans (each containing 300 mg of resin 30 with a loading of 1.2 mmole/g) was swelled in DCM (4 liters). MCPBA (208.7 g of 50% pure reagent, 604.8 mmol) was added and the reaction was stirred at room temperature for 5.5 h. For the workup, the reaction solution was drained and the resin was washed with DCM, aqueous THF, THF, and diethyl ether. The Microkans were dried overnight with a stream of nitrogen gas. Alkylation Reaction to 32 For each alkyl halide, 44 MacroKans (each containing 300 mg of resin 30 or 31b with a loading of 1.2 mmol/g) were placed into a 1-liter threenecked round-bottom flask fitted with an overhead stirrer. The resin in the MacroKans was swelled with anhydrous DMF (350 ml). The DBU (23.7 ml, 158.4 mmol) was added and the reaction was stirred for 15 min. The alkyl halide (158.4 mmol) was then introduced. KI (158.4 mmol) if needed was added last. The reaction was stirred overnight at room temperature. For the workup, the reaction solution was drained and the MacroKans were washed with DMF, 10% HOAc in DMF, 20% aqueous THF, THF, DCM, and diethyl ether. The MacroKans were dried overnight with a stream of nitrogen gas. Archiving, Cleaving, and Free Basing of Resin 33 For each MacroKan, the cap was removed and the resin and tag were poured into a 16  100-mm glass test tube. Twenty-two racks of 40 tubes each were archived in the Irori system. For each tube, the resin was cleaved with a 50% TFA/DCM solvent mixture for 1 h and then concentrated down. The resulting residue was azeotroped with DCM to remove the remaining traces of TFA. MP-Carbonate resin (411 mg, 1.08 mmol) and DMF (4 ml) were added to the residue (approximately 0.360 mmol of product per MacroKan) in each test tube. After vortexing for several seconds to dissolve the cleaved compounds, the reaction was allowed to sit at room temperature overnight. The liquid above the resin was transferred to another test tube through a filter tube using a Packard

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liquid handler. The resin was then washed twice with DMF (3 ml). Each of these washings was transferred to the collection tube giving approximately 9 ml total of a benzothiazepine free amine stock solution. TFP Resin Loading 35 For each carboxylic acid, TFP resin 34 (20 g with a loading of 1.25 mmol/g) was introduced into a 250-ml glass peptide vessel. The resin was swelled in DMF (120 ml). The carboxylic acid (250 mmol), DMAP (3.05 g, 25 mmol), and DIC (39.14 ml, 250 mmol) were then added sequentially. The reaction was mixed on a wrist shaker overnight. For the workup, the reaction solution was drained and the resin was washed with DMF, THF, DCM, and diethyl ether. The resin was dried in a vacuum oven at room temperature overnight. Reaction with TFP Resin to Prepare 36 Each set of 80 benzothiazepine 33 free amine stock solutions (two racks of 40 tubes each) was reacted with 20 acylated TFP resins 35. Of each stock solution 400 l was added to approximately 15 mg of acylated TFP resin per well. (Note: Each 80-well plate contained the same TFP-activated acid resin in each well but a different amine in each well.) The reactions were mixed on an orbital shaker at room temperature for 3 days. The product suspension was filtered through filter plates into two daughter plates using  a Tomtec. The products were concentrated at 65 .

[5] Split-Mix Synthesis Using Macroscopic Solid Support Units ´ rpa´d Furka, James W. Christensen, and Eric Healy By A Introduction

Split-mix synthesis1–3 made it possible to prepare new compounds in practically unlimited numbers. That procedure was based on the solidphase method4 in which each coupling cycle was replaced by the following operations: 1

´ . Furka, F. Sebestye´n, M. Asgedom, and G. Dibo´, in ‘‘Highlights of Modern A Biochemistry,’’ Proceedings of the 14th International Congress of Biochemistry, Vol. 5, p. 47. VSP, Utrecht, The Netherlands, 1988.

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

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1. dividing the solid support into equal portions; 2. coupling each portion individually with a different building block; and 3. mixing the portions. Repetition of these simple operation steps resulted in an exponential increase in the number of synthesized compounds. The products could be used either as mixtures or as unidentified individual compounds formed on microscopic polymer beads. The parallel synthesis developed by Geysen and his colleagues,5 although less productive than the split-mix method, yielded known products, each in several milligram quantities. To offer the advantages of this parallel synthetic methodology while preserving high productivity, split-mix synthesis was modified by applying macroscopic solid support units instead of the conventional bead form support. The resin was enclosed in permeable capsules. The number of capsules was equal to the number of compounds to be prepared and, after each reaction step, the capsules were redistributed among the reaction vessels. To make this possible, the capsules were encoded by electronic chips also enclosed in the capsules.6,7 The code written into the chip by radiofrequency radiation allows the history of the synthetic transformations to which the capsule was exposed to be tracked. The string synthesis8,9 described below also applies to macroscopic solid support units with the upside that these units are uncoded providing a methodology that is cheaper and faster. Principle

Any combinatorial synthetic method carried out with macroscopic solid support units, even if coding is omitted, has to ensure that the route of every unit in the entire multistep synthetic process can be traced. In string synthesis this can be achieved by ´ . Furka, F. Sebestye´n, M. Asgedom, and G. Dibo´, Abstract of the 10th International A Symposium on Medicinal Chemistry, Budapest, Hungary, p. 288, 1988. 3 ´ A. Furka, F. Sebestye´n, M. Asgedom, and G. Dibo´, Int. J. Peptide Protein Res. 37, 487 (1991). 4 R. B. J. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963). 5 H. M. Geysen, R. H. Meloen, and S. J. Barteling, Proc. Natl. Acad. Sci. USA 81, 3998 (1984). 6 E. J. Moran, S. Sarshar, J. F. Cargill, M. Shahbaz, A. Lio, A. M. M. Mjalli, and R. W. Armstrong, J. Am. Chem. Soc. 117, 10787 (1995). 7 K. C. Nicolaou, X.-Y. Xiao, Z. Parandoosh, A. Senyei, and M. P. Nova, Angew. Chem. Int. Ed. Engl. 36, 2289 (1995). 8 ´ A. Furka, J. W. Christensen, E. Healy, H. R. Tanner, and H. Saneii, J. Comb. Chem. 2, 220 (2000). 9 ´ A. Furka, Comb. Chem. High Throughput Screening 3, 197 (2000). 2

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1. arranging the units into spatially defined groups: one group is formed for each building block of the first reaction step and the relative position of the units need to be maintained during the chemical reactions; 2. redistribution of the units among the groups of the next synthetic step has to be carried out according to a predetermined pattern that can be reflected in a computer; and 3. the redistribution pattern has to ensure formation of all expected members of a combinatorial library. In this case, the products formed on the support units can be identified by computer prediction based on the relative positions of the units occupied in the final groups. There are different possibilities for practical realization of the principles and the string synthesis described below represents only one of them. String Synthesis: Support Units and Strings

The support units used in the string synthesis were Chiron Mimotopes Crowns shown in Fig. 1. They were used together with different color stems, also purchased from Chiron Mimotopes. Spatially ordered groups are formed with the crowns (Fig. 1A) by stringing them together with a thread. The stems serve to string the crowns together and to facilitate redistribution. The stems were modified as shown in Fig. 1. First, a hole was drilled to allow the string to be passed through, and then the stem was carved to keep the holes parallel and facilitate threading them while in the sorting device (see Fig. 4). The modified stems can be A

B

C

D

2 mm

E

F

G

4−5 mm

Fig. 1. Chiron Mimotopes Crowns. (A) Crown; (B) stem; (C) carved stem with hole; (D) support unit; (E) full-length stem labeling the head of the string. The scratches represent the string number; (F) half stem that marks the tail of the string; (G) Mimotopes SynPhase Lantern with stem.

102

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combinatorial synthesis Tail

Head

1

5

10

15

20

25

Fig. 2. Stringed crowns. Full and half-length stems mark the head and the tail end of the string. Positions of the crowns are numbered from the head.

used repeatedly. SynPhase Lanterns (Fig. 1G), available at Mimotopes Pty. Ltd. (http://www.mimotopes.com), may also be used in place of the crowns. The string itself must be resistant to solvents and other reaction conditions involved in the synthesis. In the illustrated example, a polyethylene fishing line was used. The stringed crowns are shown in Fig. 2. To unequivocally define the position of the crowns, the two ends of the string must be distinguishable. The head is marked by a full-length stem (Fig. 1E) and the tail of the string (Fig. 1F) is labeled by a stem cut in half (Figs. 1 and 2). The numbering position of the crowns start at the head. Depending on the number of building blocks used in each consecutive reaction step, a separate string of crowns is incorporated. As a consequence, the strings themselves must be numbered or otherwise labeled. The simplest way to label the strings is by making visible scratches on the stem marking the head. Using colored stems is also a possibility. If lanterns are used as support units, it is worth taking into account that they have a hole in their center, so they can be threaded without the need for stems. The chemical reactions described later in this chapter were carried out on crowns, by coupling amino acids onto the respective strings. The number of crowns used in the synthesis is equal to the number of expected products. Therefor, if five amino acids are used in a coupling step, five different strings are made, each containing the same number of crowns. After threading the crowns, each string was placed into a reaction vessel and coupled with a different amino acid. For the present work, all couplings were carried out in five reaction vessels (Fig. 3). Manual Device for Redistribution

The strings coming from the reaction vessels after completing the first (and any further) coupling reactions are called source strings. Their crowns, which are then rearranged into their strings for the next reaction step, are denoted as destination strings. Redistribution of the crowns is

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1

2

3

4

5

Fig. 3. Strings in reaction vessels and their numbers represented below by the scratches on the stems.

Source tray

Destination tray

Fig. 4. Manual sorting device.

carried out using a very simple device that can be easily made by a machine shop. The device contains two identical pieces shown in Fig. 4. Both pieces are metal plates with several numbered parallel slots and bent at the two edges. The crowns are first positioned in the slots of the source tray (Fig. 5) then sorted by pushing them into the slots of the destination tray (Fig. 6). It is important to place each string into the slot carrying the same number, and position the heads and tails of the source strings into the slots of the source tray as indicated in the figure, otherwise the software used to track the crowns (see Software section) cannot be used. It is also important to number the destination strings according to the numbers of the destination slots and render their heads and tails to the heads and tails of the destination slots. The crowns were loaded into the slots of the source tray while still attached to the string (Fig. 7A), then the string was cut and removed. The crowns were then sorted in string free form (Fig. 7B) and then restrung (Fig. 7C). A device design to sort up to 750 crowns is demonstrated in Fig. 8. The two trays are identical except for the direction of the numbering of the slots. Changing the position of the numbers in any of the trays would make the software inapplicable. If lanterns are used with stems as support units, the width of the slots have to be smaller since the stems of the lanterns

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Fig. 5. Crowns hanging in the slots of the sorting device.

Head 5 4 3 2 1

5 4 3 2 1

Tail Head

Source tray

Tail

Destination tray

Fig. 6. Position of the source and destination trays at the beginning of sorting (top view). Pushing over crowns from source slot No. 5 into destination slot No. 1.

are smaller in size. If the lanterns are used without their stems a different design should be applied. The slots can be replaced by small troughs. Redistribution Pattern

The redistribution sequence made in the course of a synthetic process must ensure formation of all the components expected at the end of a combinatorial synthesis. In the conventional split-mix synthesis this is achieved by pooling the content of all reaction vessels of a given reaction step, thoroughly mixing them, then transferring equal portions of the combined resin into the reaction vessels for the next reaction step. As a result, all products formed in a previous reaction vessel are evenly distributed among the reaction vessels assigned to the next reaction step. This can be considered as the combinatorial redistribution rule. This rule can be translated to string synthesis in the following way: the crowns of a string containing the same product have to be evenly distributed among the strings of the next reaction step. Obeying this rule, there are still many ways to carry out solid support redistributions.9 The semiparallel sorting process described below is designed to ensure a fast redistribution of solid supports. The first column in Fig. 9 depicts redistribution of 125 crowns in a single sorting cycle. In the first redistribution cycle the crowns are delivered from each source slot in groups of five. Relative position 1 (RP 1) illustrates the starting orientation of both source and destination trays. Note that source slot No. 5 is in alignment with destination slot No. 1. From this

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A

B

C

Fig. 7. Crowns in the slot of the sorter. (A) Before cutting the string; (B) string free form before and after sorting; (C) stringing the crowns after sorting.

48 cm 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

40 cm

33 cm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

34 cm Bending

Bending A

B

Fig. 8. Proposed sorting device. (A) Design of the source tray with 15 slots (width 3 mm); (B) top view of the final form of destination tray.

position five crowns are pushed into destination slot No. 1, then the destination tray is repositioned. In RP 2, source slots No. 4 and No 5 are facing destination slots No. 1 and No. 2, respectively. This new position makes it possible to push five crowns into both destination slots No. 1 and No. 2.

106 RP

1

2

3

4

[5]

combinatorial synthesis Sorting step 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5

5 4 3 2 1

6

5 4 3 2 1

7

5 4 3 2 1

8

5 4 3 2 1

9

5 4 3 2 1

Sorting step 2 5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1 5 4 3 2 1 5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

5 4 3 2 1

Fig. 9. Semiparallel sorting of 125 crowns from five source slots into five destination slots. First column: sorting step 1, executed in the nine positions of one distribution cycle; five crowns are delivered from each source slot. Second column: first distribution cycle of sorting step 2: one crown is delivered from each slot. The numbers at the left side of the figure mark the nine different relative positions (RPs) occupied by the source and the destination trays in a redistribution cycle. The figure shows the top views of the two trays. The original and the destination positions of the repositioned crowns are indicated by black circles.

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The destination tray is moved further step by step all the way down to RP 9 pushing groups of five crowns into all destination slots that are in alignment with the slots of the source trays. Thus, in RP 3, RP 4, RP 5, RP 6, RP 7, RP 8, and RP 9, 15, 20, 25, 20, 15, 10, and 5 crowns are pushed into destination slots, respectively. The nine RPs complete a full redistribution cycle since there are no more possible relative positions of the source and destination slots. The semiparallel redistribution follows two simple rules: 1. The destination tray is gradually moved from the start position— when only single slots of both trays are in alignment—to the end position where the other two single slots are facing each other. 2. Delivery of crowns is executed in all slots that are in alignment. Since the delivery of crowns mostly occurs in groups, the redistribution process is fast. Software

The software for the string synthesis technique is written in visual basic and the data appear in Microsoft Excel sheets. This software can be downloaded via the Internet from http://szerves.chem.elte.hu/furka by clicking on the title ExcelBook appearing on the lower part of the main page. This software is compatible with only those PC systems that have Excel installed. The software was created to handle a maximum of 1000 crowns, 20 reagents (building blocks), and 9 reaction steps. The Excel datasheet where the starting data have to be entered is illustrated in Fig. 10. The starting data consist of the number and symbols of the building blocks (monomers) to be used in the coupling steps. The symbols are single-letter abbreviations. In the case of peptide synthesis the symbols correspond to their respective amino acids. When using the software, the areas where data are to be entered appear yellow on the screen. Row 26 (the row following MONOMERS IN COUPLINGS) shows the string numbers. Each of the monomers entered in a column are assigned to successively undergo coupling with the string appearing in the same column. Once all the required data are entered, the software executes the calculation presenting the resulting calculated data in the blue regions of the Excel sheet. The calculated data include the total number of crowns needed in the synthesis as well as the number of coupling steps (Fig. 10, column B). The number of source and destination slots used in the first and subsequent sorting steps (Fig. 10D and E) and the number of crowns occupying these slots (Fig. 10F and G) also appear along with the number of crowns that contain the same product (Fig. 10H). The number of crowns in a group that will have to be moved in every sorting cycle from a source to a destination slot is indicated in Fig. 10, column I.

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Starting Data for Semi-Parallel Sorting SORTING: in every cycle delivery starts from the highest number (rightmost) source slot into the first (leftmost) destination slot. Run: Ctrl + S The number of reagents and the symbols of monomers (A,B,C etc.) have to be entered The number attached to the sequences only show the original position of the units The number of units in a block to be delivered is indicated by red numbers. Do not delete blue cells! Enter data only into yellow cells! Number of building blocks Sort Number of slotes Crowns in tubes Identical number Source crowns (maximum number is 52) Destin. Source Destin. CP 1 5 1 5 5 25 25 25 CP 2 5 2 5 5 25 25 5 0 0 0 CP 3 5 3 0 1 0 0 0 0 CP 4 4 0 0 0 0 0 CP 5 5 0 0 0 0 0 CP 6 6 0 0 0 0 0 CP 7 7 0 0 0 0 0 CP 8 8 0 0 0 0 0 CP 9 9 0 CP 10 Maximum number of reagents 20 Total number of 125 Maximum number of sorter slots 20 crowns Number of coupling 3 Maximum number of crowns 1,000 steps: Pause (in seconds): MONOMERS IN COUPLINGS 1 2 3 4 5 6 7 CP 1 I F L V G CP 2 E F W Y S CP 3 E F W Y S CP 4 CP 5 CP 6 CP 7 CP 8 CP 9 CP 10

A

B

C

D

E

F

G

H

Crowns to move 5 1 0 0 0 0 0 0 0

8

I

Fig. 10. Data sheet of the Excel Book where the starting data can be entered. The symbols of the columns are presented in the last row.

The program can be started by pressing together the ‘‘Ctrl’’ and ‘‘S’’ (‘‘Ctrl S’’) keys of the keyboard. The results of calculations appear in sheets Sort #1 through Sort #9. The sheets show a block of products present in the crowns of the source slots and, below these, a block of products sorted into the destination slots. The positions of the crowns are counted downward from the top. The number of sheets showing the results of couplings and sortings is equal to the number of sortings plus one. The last sheet contains the predicted product distribution on the final strings.

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Coupling 1 Str 1

Str 2

Str 3

Str 4

Str 5

I

F

L

V

G

Str 1

Str 2

Str 3

Str 4

Str 5

E

F

W

Y

S

Str 4 Y

Str 5 S

Sort No. 1

Coupling 2

Sort No. 2

Coupling 3

Products

Str 1 E

Str 2 F

Str 3 W

String 1

String 2

String 3

String 4

String 5

Fig. 11. Flow diagram of the synthesis.

Synthesis of a Library of 125 Tripeptides

The synthesis was carried out using 125 Chiron Mimotopes Crowns (capacity 5.3 mol each) derivatized with an Fmoc*-Rink amide linker. The procedure was started with the formation of five strings by threading 25 crown units on Berkley Fire Line fishing line. Five Fmoc-protected amino acids were used in each coupling position as demonstrated in the flow diagram of the synthesis (Fig. 11).

*

Abbreviations: Fmoc, 9-fluorenylmethoxycarbonyl; DMF, N,N-dimethylformamide; DCM, dichloromethane; HOBt, 1-hydroxybenztriazole; DIC, diisopropylcarbodiimide; NMP, N-methylpyrrolidinone; TFA, trifluoroacetic acid.

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Coupling Couplings were carried out with strings placed in 100-ml flasks. To remove the Fmoc-protecting group 10 ml 1:1 v/v DMF-piperidine was added to each flask and then the flasks were placed on an orbital mixer and shaken for 30 min at room temperature. The solutions were decanted from strings and the strings were then washed with 3  15 ml DMF, 15 ml DCM, 15 ml DMF, 15 ml DCM, and 2  15 ml DMF. The deprotection and washing operation was repeated one more time and finally the strings and the crowns were washed with additional 2  15 ml DCM. After drying all the strings and crowns, individual solutions were prepared with 10 mmol of an Fmoc amino acid, 10 mmol of HOBt, and 15 mmol of DIC in 10 ml NMP. These solutions were added to the flasks placing them on an orbital mixer and shaking them for 2 h at room temperature. The final solutions were decanted and the strings and crowns were washed with 3  40 ml DMF, 40 ml DCM, and 2  40 ml DMF. The above coupling and washing operation was repeated one more time washing at the end the strings and crowns with an additional 2  40 ml DCM. The crowns were dried in an oven and then the strings were removed for sorting. Sorting of the Crowns In the three-step synthesis of the tripeptide library, the crowns are redistributed in two different sorting steps. Sorting step 1 follows the attachment of the amino acids to the solid support at coupling position 1 (CP1). Sorting step 2 is carried out after the second coupling cycle at coupling position 2 (CP2). Before beginning the sorting operations, the starting data were entered into computer (Fig. 10). As showing column I of the datasheet (row CP1), in the first sorting procedure the crowns must be moved from each slot in groups of five. In the second sorting procedure the crowns are moved one at a time (column I, row CP2). Sorting step 1 is demonstrated in the left column of Fig. 9. The sorting of the 125 crowns was finished on the ninth position of a single redistribution cycle. Sorting step 2 is demonstrated in the second column of Fig. 9. In this case only a single crown was moved from slot to slot. On the ninth position of the first distribution cycle shown in Fig. 9, a total of 25 crowns were delivered to the destination tray. The rest of the crowns were redistributed in four additional cycles not shown here. Cleavage To cleave the products from the supports, the crowns were separately placed in test tubes, and the compounds deprotected by adding 1 ml 1:1 v/v piperidine-DMF to each tube. After allowing the mixtures to stand

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for 30 min the crowns were filtered and then washed with 3  2 ml DMF, 2 ml DCM, 2 ml DMF, and 2  2 ml DCM. The crowns were placed back into test tubes, 1 ml 95% TFA/H2O was added to each tube, and the mixtures were allowed to stand for 30 min at room temperature. The final solutions were decanted into vials. The crowns were washed with 1 ml 95% TFA/H2O, the solutions transferred to the same vials, and the solvents were removed using a rotavap. Product Distribution Location of products and intermediates on the strings appear in sheets Sort #1, Sort #2, and Sort #3. Some of the predictions are summarized in Table I. It can be seen that after the first coupling on string 1, as expected, TABLE I Content of No. 1 Strings (Str.) After Couplings (Cpl.) and Sortings (Sort) and Position of Products on the Final Stringsa Cpl. 1 Sort 1 Cpl. 2 Sort 2 Str. 1 Str. 2 Str. 3 Str. 4 Str. 5 Position Str. 1 Str. 1 Str. 1 Str. 1 Products Products Products Products Products 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 a

I I I I I I I I I I I I I I I I I I I I I I I I I

I I I I I F F F F F L L L L L V V V V V G G G G G

EI EI EI EI EI EF EF EF EF EF EL EL EL EL EL EV EV EV EV EV EG EG EG EG EG

EI FI WI YI SI EF FF WF YF SF EL FL WL YL SL EV FV WV YV SV EG FG WG YG SG

EEI EFI EWI EYI ESI EEF EFF EWF EYF ESF EEL EFL EWL EYL ESL EEV EFV EWV EYV ESV EEG EFG EWG EYG ESG

FEI FFI FWI FYI FSI FEF FFF FWF FYF FSF FEL FFL FWL FYL FSL FEV FFV FWV FYV FSV FEG FFG FWG FYG FSG

Amino acids are indicated with one-letter symbols.

WEI WFI WWI WYI WSI WEF WFF WWF WYF WSF WEL WFL WWL WYL WSL WEV WFV WWV WYV WSV WEG WFG WWG WYG WSG

YEI YFI YWI YYI YSI YEF YFF YWF YYF YSF YEL YFL YWL YYL YSL YEV YFV YWV YYV YSV YEG YFG YWG YYG YSG

SEI SFI SWI SYI SSI SEF SFF SWF SYF SSF SEL SFL SWL SYL SSL SEV SFV SWV SYV SSV SEG SFG SWG SYG SSG

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all units contained amino acid I. After the first sorting step, string 1 contained five products in groups of five crowns. The product distribution in the rest of the strings was exactly the same. After the second coupling, string 1 contained five dipeptides in groups of five crowns. After the second sorting step, as Table I shows, all products in string 1 were different. The product distribution in the rest of the strings, not shown in the Table I, was exactly the same. Positions of the formed tripeptides on the five strings after the third coupling are also shown in Table I. Verification of Product Distribution Randomly selected sequences were independently synthesized and compared to those cleaved from the crowns. The data gathered by highperformance liquid chromatography and mass spectrometry unequivocally and positively confirmed the predicted product distribution. Acknowledgments The authors thank the Hungarian government for Grants FKFP/0149/2000, OTKA T34868, and NKFP 1/047.

[6] The Encore Technique: A Novel Approach to Directed Split-and-Pool Combinatorial Synthesis By Viktor Krchnˇa´k and Vi´tecˇek Padeˇra Introduction

The traditional and established way of conducting chemical transformations employs one reaction vessel for each compound synthesized. To prepare 50 different acetamides, 50 reaction vessels are needed, one for each amide. When synthesis of 50 amides is performed at the same time, this approach is referred to as a parallel synthesis. It is, of course, always desirable to simplify the process by reducing the number of vessels used in the synthesis. However, it is not feasible just to mix 50 amines into one reaction vessel and acetylate the mixture of amines because the isolation of 50 amides from the resultant reaction mixture could be complicated and time consuming. Simple separation of individual components was made possible by Merrifield’s solid-phase synthesis.1 Solid support-bound substrates can 1

R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963).

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be pooled into one reaction vessel and the individual substrates are then physically separated by covalent attachment to the carrier. Frank et al.2 recognized the potential of combing a number of different solid-phase bound substrates for a reaction with a single reagent using cellulose-based paper discs as solid-phase support for the synthesis of oligonucleotides. Each disc contained a different substrate (oligonucleotide) and only four reaction vessels were required for the synthesis of any number of oligonucleotides. ‘‘Whenever growing chains on different entities have to be elongated with the same building block these entities are gathered in the same reaction vessel.’’2 Later, Frank and Do¨ring3 applied the pooling strategy to peptide synthesis. Houghten4 expanded this methodology to resin beads, a common support for solid-phase peptide synthesis. He placed resin beads into polypropylene meshed packets similar in appearance to tea (T-)bags. Before the addition of the next amino acid to the resin-bound growing peptide chain, the T-bags were distributed into different reaction vessels, each vessel containing resin-bound intermediates receiving the same amino acid. To carry out reaction transformations common to all peptides (e.g., cleavage of the amino-protecting group) all bags were pooled into one reaction vessel. The concept of reducing the number of reaction vessels and exponentially increasing the number of synthesized compounds was brought to a next level of simplicity by the split-and-pool method of Furka et al.5 The split-and-pool method was independently applied by Lam et al.6 in a onebead–one-compound concept for the combinatorial synthesis of large compound arrays (libraries) and by Houghten et al.7 for the iterative libraries. Now several millions peptides could be synthesized in a few days. In Furka’s method the resin beads receiving the same amino acid were contained in one reaction vessel—identical to Frank’s method—however, the beads were pooled and then split randomly before each combinatorial step. Thus the method is referred to as the random split-and-pool method to differentiate it from Frank’s method in which each solid-phase particle was directed into a particular reaction vessel (the directed split-and-pool method). 2

R. Frank, W. Heikens, G. Heisterberg-Moutsis, and H. Blocker, Nucleic Acids Res. 11, 4365 (1983). 3 R. Frank and R. Do¨ring, Tetrahedron 44, 6031 (1988). 4 R. A. Houghten, Proc. Natl. Acad. Sci. USA 82, 5131 (1985). 5 A. Furka, F. Sebestyen, M. Asgedom, and G. Dibo, Int. J. Peptide Protein Res. 37, 487 (1991). 6 K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, and R. J. Knapp, Nature 354, 82 (1991). 7 R. A. Houghten, C. Pinilla, S. E. Blondelle, J. R. Appel, C. T. Dooley, and J. H. Cuervo, Nature 354, 84 (1991).

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These two methods have two key common features: 1. A limited number of reaction vessels is needed for the synthesis. The number of reaction vessel equals the number of building blocks in any particular reaction step. The number of compounds synthesized exponentially exceeds the number of vessels. 2. The amount of material synthesized depends on the yield from one solid-phase particle (disc) or container (T-bag). The key differences are as follows. In the directed split-and-pool method 1. The chemist controls the distribution of compounds in a library. Any combination of building blocks can be excluded from the synthesis. 2. The chemical history of the particles is recorded (e.g., each T-bag is labeled by an alphanumeric code). In the random split-and-pool method 1. The distribution of compounds in a library is driven by statistical probabilities due to the random split process. Each compound is synthesized numerous times when the number of beads exceeds several times the number of compounds, or only a subset of compounds is produced when the number of beads is lower than the number of possible combinations of building blocks. 2. The chemical history of the beads is lost. After each combinatorial step, the resin beads from all reaction vessels are pooled and randomly split into reaction vessels for the next combinatorial step. The principal differences between those two methods are reflected in their applications. The random split-and-pool method is suited for the synthesis of smaller quantities of large sizable libraries (i.e., million compounds), whereas the directed split-and-pool technique is suited for the synthesis of larger quantities of smaller compound collections (i.e., several hundred to several thousand compounds). In this chapter, a simple technique for directed split-and-pool technique is described. Directed Split-and-Pool Method

Three technical issues have to be solved in order to make the directed split-and-pool method attractive for routine synthesis: 1. One entity of solid-phase support (particle or container) has to provide a sufficient amount of compound. 2. The chemical history of individual entities needs to be recorded.

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3. The process of distribution of entities, particularly for relatively larger libraries, needs to be integrated and/or automated. Solid Support

A single resin bead does not usually provide the required micromolar amount of target material. The sufficient yield from one entity has been solved in two different ways. The first solution is to enclose a sufficient amount of resin beads into a container. Houghten introduced the meshed polypropylene packets (T-bags).4 IRORI changed the shape of a T-bag into a meshed can (MacroKan, MicroKan, and NanoKan for 100, 30, and 8 mg of resin, respectively) in order to allow robot handling.8,9 Recently, very promising resin plugs have been developed10 and commercialized by Polymer Laboratories (StratoSpheres Plugs). Resin beads are mixed with fine powdered high-density polyethylene (HDPE) and heated to melt the HDPE. After cooling, plugs are formed and used as a solid-phase support. An alternative solution is to produce a solid-phase particle that yields micromolar amount of product. Mimotopes Pty. Ltd. has developed and commercialized the SynPhase Crown and later Lanterns, a rigid polypropylene mold with a grafted layer of polystyrene. The solid-phase synthesis takes place on the derivatized graft.11 Encoding

The simplest solution for tracking the chemical history is labeling of individual entities (paper discs, T-bags) by an alphanumeric code that a chemist can read. Alternatively, the reaction containers can also be color coded. Radiofrequency tagging8 and optical encoding12 of containers enabled computer-assisted reading of the tag and automation of the directed split-and-pool process. A radiofrequency tag is inserted into each container with resin beads and, before a combinatorial step, the individual tags are read and the containers distributed into corresponding reaction 8

X. Y. Xiao, R. Li, H. Zhuang, B. Ewing, K. Karunaratne, J. Lillig, R. Brown, and K. C. Nicolaou, Biotechnol. Bioeng. 71, 44 (2000). 9 K. C. Nicolaou, J. A. Pfefferkorn, H. J. Mitchell, A. J. Roecker, S. Barluenga, G. Q. Cao, R. L. Affleck, and J. E. Lillig, J. Am. Chem. Soc. 122, 9954 (2000). 10 B. Atrash, M. Bradley, R. Kobylecki, D. Cowell, and J. Reader, Angew. Chem. Int. Ed. Engl. 40, 938 (2001). 11 F. Rasoul, F. Ercole, Y. Pham, C. T. Bui, Z. Wu, S. N. James, R. W. Trainor, G. Wickham, and N. J. Maeji, Biopolymers 55, 207 (2000). 12 C. Y. Xiao, C. F. Zhao, H. Potash, and M. P. Nova, Angew. Chem. Int. Ed. Engl. 36, 780 (1997).

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Fig. 1. A necklace of SynPhase Lantern. (See color insert.)

vessels. The process is referred to as directed sorting and it has been fully automated and commercialized by IRORI. The Mimotope SynPhase Lanterns have been used in an analogous way. An alternative method for tracking the chemical history is encoding by spatial address. The identity of each entity is defined by its spatial address. A one-dimensional directed split-and-pool procedure, referred to as necklace coding, has been developed for synthesis carried out on SynPhase Crowns and Lanterns.13 Individual supports are strung on a Teflon thread and the position of a particle on the thread (necklace) encoded the previous chemical history (Fig. 1). A similar concept was later reported by Furka and co-workers14,15 Two-and three-dimensional encoding of the directed split-and-pool synthesis platform has been patented by Selectide Corp.16 Process Integration/Automation

The third challenge was to integrate and/or automate the handling of individual particles between combinatorial steps. During the early years of directed split-and-pool methodology the entire process was done 13

J. Smith, J. Gard, W. Cummings, A. Kaniszai, and V. Krchnˇa´k, J. Comb. Chem. 1, 368 (1999). 14 A. Furka, Comb. Chem. High Throughput Screening 3, 197 (2000). 15 A. Furka, J. W. Christensen, E. Healy, H. R. Tanner, and H. Saneii, J. Comb. Chem. 2, 220 (2000). 16 M. Patek, P. Safar, M. Smrcina, E. Wegrzyniak, P. Strop, G. Flynn, and S. A. Baum, World Patent WO 0138268 (2001).

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manually. Manual splitting is still useful for handling small libraries. However, the manual process is tedious and prone to human error. Highly automated and sophisticated robotics systems have been designed to execute unattended directed split-and-pool combinatorial synthesis. IRORI’s fully automated directed sorting robot is based on optical encoding of NanoKans.8,12 An alternative robotic system, based on a sophisticated and complex two- and three-dimensional spatial encoded directed split-and-pool method, has been custom built by Selectide scientists.16 An intermediate level of sophistication was achieved by semiautomated sorting of radiofrequency tagged containers (e.g., MicroKans,8 Lanterns11). A simple instrument that integrates the process of one-dimensional necklace encoding by creating a linear sequences of solid-phase particles is the Encore synthesizer. Description of the Encore Method

The Encore method is based on one-dimensional spatial encoding of the synthesis chemical history, referred to as necklace coding, and applied for the synthesis on SynPhase Crowns and Lanterns.13 One-dimensional spatial encoding eliminates the need to label each solid-phase particle; the chemical history of a particle is encoded by its sequential position on a linear string, a necklace. To preserve the linear sequence during synthesis, Lanterns are strung together using a chemical-resistant Teflon rope. In principle, it is possible to create a single string (necklace) of Lanterns for each reaction vessel. However, this approach would require reshuffling the Lanterns between combinatorial steps. A more effective approach is to create numerous short necklaces after the first combinatorial step, and reshuffle the necklaces after the second combinatorial step (there is no need to distinguish among Lanterns during the first combinatorial step since all Lanterns in one reaction vessel are identical). The current version of the Encore synthesizer has been designed for the synthesis of up to 960 compounds using an algorithm to handle 10, 8, and 12 building blocks in the first, second, and third combinatorial steps, respectively (Fig. 2). The total number of combinatorial compounds produced this way is n ¼ 10  8  12 ¼ 960. For the first combinatorial step, 10 reaction vessels are charged with 96 (8  12) solid-phase supports and the first set of building blocks is chemically attached to solid-phase supports. After this step, all 96 particles per reaction vessel are identical and particles from different reaction vessels differ only by the kind of first building block attached to solid-phase particles. After the first combinatorial step, 96 (8  12) necklaces are formed, each necklace containing 10 particles, one from each of the original 10

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Fig. 2. The principle of the Encore technique. (A) The first combinatorial step is performed in 10 reaction vessels, each charged with 96 scattered Lanterns. (B) Twelve necklaces with 10 stringed Lanterns are placed into each of eight reaction vessels of the second step. (C) The third combinatorial step is performed in 12 reaction vessels with eight necklaces per vessel, each tagged with a different color. (See color insert.)

reaction vessels. The position of a particle on a necklace encodes the first building block. At this stage, all 96 necklaces are identical. The second combinatorial step is performed with eight building blocks for which eight reaction vessels are used with each vessel containing 12 necklaces. To record (encode) the second building block, all necklaces in one reaction vessel are labeled by the same color. Consequently, eight different colors are needed to label necklaces for eight reaction vessels. Obviously, any other suitable ways of labeling could be applicable, including alphanumeric labeling. After finishing the second combinatorial step, one necklace from each of the eight reaction vessels is placed into a reaction vessel for the third combinatorial step. There are 12 new reaction vessels for the third combinatorial step, each vessel containing eight necklaces of different color. After finishing the synthesis, the necklaces are disassembled and the individual particles are placed into a 96-well plate for the cleavage and collection of target compounds. The Encore technique combines three different coding methods: sequential position on a necklace for the first combinatorial step, color coding of individual necklaces for the second combinatorial step, and reaction vessel coding to identify the last building block. Accordingly, we termed this technique Encore for Encoding by a Necklace, Color, and Reaction vessel. The algorithm described above is for a three-step combinatorial synthesis. However, the method is not limited to only three-step combinatorial libraries; the solid-phase support can be derivatized before the directed split-and-pool synthesis on the Encore synthesizer. The necklace coding can also be a very useful tool during the chemistry development process.

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The reactivity of a building block with different polymer-supported substrates can be evaluated in one reaction vessel. Description of the Encore Instrument

The Encore synthesizer has been designed to integrate the process of handling of solid-phase particles in combinatorial synthesis. The current version of the synthesizer enables synthesis in batches of up to 960 compounds on SynPhase Lanterns. The Encore synthesizer facilitates the sorting of Lanterns into sequences (necklace coding) and the plating of the Lanterns after completion of the synthesis for final release of compounds from Lanterns. The Encore synthesizer consists of the following five tools. Arraying Tool The Arraying Tool (Fig. 3) arranges 96 random Lanterns from one reaction vessel after the first combinatorial step (all Lanterns are chemically identical) into a two-dimensional array of 8 rows and 12 columns. The Arraying Tool has a standard 96-well plate footprint. Magazine The Magazine (Fig. 4) is a polyethylene block with 96 shafts. Each shaft accommodates up to 10 Lanterns. The Magazine enables Lanterns to be

Fig. 3. The Arraying Tool. (See color insert.)

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Fig. 4. Magazine (white block at the top) with the dispensing manifold (black block). (See color insert.)

collected into 96 shafts in a predetermined sequence and serves two functions: (1) arrangement of Lanterns for transfer into reaction vessels by the Lapis Tool and (2) arrangement of Lanterns for final distribution into cleavage plates. Lapis Tool The Lantern Picking and Stringing (Lapis) Tool (Fig. 5) is used to secure and pick Lanterns from the Magazine while preserving the sequence of Lanterns in a shaft (necklace coding). Loaded Lapis tools are placed into reaction vessels. The Lapis Tool is made of stainless steel and polyethylene and it is compatible with a wide variety of solvents, reagents, and reaction conditions. Lantern Dispensing Tool After finishing the combinatorial synthesis, Lanterns are distributed into 96-well plates (one Lantern per well) using the Lantern Dispensing Tool (Fig. 6). The Lantern dispensing tool enables the transfer of one ‘‘layer’’ of Lanterns (e.g., 96 Lanterns) from the Magazine into a 96-well plate. Lantern Leveling Tool To ensure a reliable distribution of Lanterns into cleavage plates, Lanterns are stacked tightly in shafts using the Leveling Tool (Fig. 7).

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Fig. 5. The Lapis tools with color tags and 10 Lanterns. (See color insert.)

Fig. 6. Lantern Dispensing Tool. (See color insert.)

Synthetic Protocol

Synthesis of a three-step combinatorial library of 960 compounds using 10, 8, and 12 building blocks in the first, second, and third combinatorial steps, respectively, consists of the following steps: 1. Ten reaction vessels are charged with 96 Lanterns each and the first combinatorial synthetic step is performed. A suitable reaction vessel is a 50-ml syringe and the synthesis can be performed on the Domino Block synthesizer (Torviq, Tucson, AZ). At this point, all the Lanterns in a reaction vessel are identical. There is no need to distinguish among Lanterns in one reaction vessel.

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Fig. 7. Lantern Leveling Tool. (See color insert.)

2. All 96 Lanterns from the first reaction vessel are emptied onto the Arraying Tool and the Arraying Tool is gently shaken in an orbital motion until all Lanterns are sequestered into the openings of the Arraying Tool. This operation typically takes less than 15 sec. 3. The Arraying Tool is placed on top of the Magazine and the stainless-steel plate is removed from the Arraying Tool. This step enables the Lanterns to drop into the shafts of the Magazine. 4. The Leveling Tool is inserted into 96 openings of the Arraying Tool and any stacked Lantern is gently pushed into the shafts of the Magazine. 5. Steps (2) to (4) are repeated nine times to accommodate all the Lanterns from all 10 reaction vessels. When all Lanterns are distributed, the full Magazine contains 960 Lanterns in 96 shafts, each shaft containing 10 Lanterns and all shafts having exactly the same sequence of Lanterns. The position of a Lantern in the sequence codes for the first building block. 6. Ninety-six Lapis Tools are labeled by color tags with eight different colors (or color combinations) to achieve 12 sets of Lapis Tools from each color (or color combination). 7. The Lapis Tool labeled by the first color (or color combination) is pushed through the openings in all 10 Lanterns of a shaft. The sequence of Lanterns from this shaft is stringed on a Lapis Tool. The Lapis Tool carrying a sequence of 10 Lanterns is placed into reaction vessel #1 for the second combinatorial step.

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8. Step (7) is repeated with the remaining Lapis Tools tagged with the first color. As a result, reaction vessel #1 is loaded with 12 Lapis Tools labeled and identified by the same color. 9. The same sequence of steps (7 and 8) is repeated using the next set of 12 Lapis Tools labeled with the second color (or color combination) and reaction vessel #2 is loaded with 12 Lapis Tools. 10. The rest of the Lanterns are strung in an analogous way resulting in eight reaction vessels charged with Lanterns strung on Lapis Tools. Each reaction vessel containing Lapis Tools is labeled with the same color (or color combination) and this color code corresponds to the second building blocks. 11. The second combinatorial step is performed in eight reaction vessels. 12. The Lapis Tools are rearranged before the third combinatorial step in such a way that each of 12 new reaction vessels contains eight necklaces with a different color tag. 13. The third combinatorial step is performed. 14. Lapis Tools with Lanterns are placed into the shafts of the Magazine as described above. Eight Lapis Tools from reaction vessel #1 are moved into the first column of the Magazine in such a way that the sequence of Lapis color tags corresponds to the sequence of second building blocks. 15. Lapis Tools with Lanterns from the remaining reaction vessels are moved into the Magazine in an analogous way. 16. The Lapis Tool from each shaft is removed leaving the sequence of 10 Lanterns in each shaft. 17. Lanterns are distributed for compound release into wells of 96-well plates, one Lantern per well. The Magazine is placed on top of the first 96-well plate. The Magazine is positioned on the right-hand side of the Lantern dispenser and it is secured in this position by two springs. The Lantern Leveling Tool is placed on top of the Lanterns. 18. The Magazine is pushed by hand to the left side. Once the end position is reached, a metal handle located on the side of the Magazine locks it in position. 19. With this motion the bottom layer of Lanterns drops into the Lantern dispensing manifold (a black anodized aluminum piece, see Fig. 4). Completion of the Lantern drop can be visually inspected by observing the Lantern Leveling Tool. The tool has to drop by 5 mm (the height of a Lantern). If a single Lantern does not drop into the Dispensing Tool, the Leveling Tool will not move or will be lopsided. In such a case a gentle push on the Leveling Tool usually moves the Lantern into the Dispensing manifold.

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20. The metal handle is lifted. The Magazine moves back to its original position. At this time, 96 Lanterns are released into the 96-well plate. The Dispensing Tool is lifted and the cleavage plate is visually inspected for the presence of Lanterns. 21. Steps 18 to 21 are repeated. 22. The target compounds are cleaved from the Lanterns in the 96-well plates. Conclusion

Combinatorial chemistry has moved from specially centralized laboratories, often equipped with multimillion-dollar robots, onto the bench of individual medicinal chemists. This change in direction requires the availability of personal chemistry tools that are simple to operate, easy to arrange in the laboratory, and reasonably priced. Such instruments are now available for the effective synthesis of combinatorial libraries. The Encore synthesizer represents a simple and efficient personal chemistry tool that allows the execution of directed split-and-pool combinatorial synthesis. The current version of the Encore synthesizer is designed for solid-phase synthesis on SynPhase Lanterns; however, it can be modified for synthesis on alternative solid supports such as resin plugs from Polymer Laboratories (e.g., StratoSpheres Plugs). The split-and-pool synthesis not only simplifies the complexity of the combinatorial synthetic process, but also offers additional important benefits. To undertake a full range of solid-phase chemical reactions, elaborate reaction conditions are needed for some chemical transformations. These include, but are not limited to, low temperature and inert atmosphere conditions. Parallel synthesis of a thousand compounds requires handling of a thousand reaction vessels. The timely addition of sensitive reagents (e.g.,  butyl lithium) at low temperature (78 ) under inert atmosphere during parallel synthesis is not a trivial task. It can be done if sophisticated automated synthesizer equipment is designed to handle and tolerate such reaction conditions. Such a synthesis can alternatively be performed easily in a manual fashion using a split-and-pool method that requires only a limited number of reaction vessels. Examples from Nicolaou’s17 and Schreiber’s18,19 laboratories have shown that the split-and-pool method is the methodology of choice for the synthesis of complex and diversity-oriented combinatorial libraries. 17

K. C. Nicolaou and J. A. Pfefferkorn, Biopolymers 60, 171 (2001). S. L. Schreiber, Science 287, 1964 (2000). 19 H. Kubota, J. Lim, K. M. Depew, and S. L. Schreiber, Chem. Biol. 9, 265 (2002). 18

Section III Linkers and Their Applications

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[7] Multifunctional Linkers as an Efficient Tool for the Synthesis of Diverse Small Molecule Libraries: The Triazene Anchors By Stefan Bra¨se, Stefan Dahmen, and Matthias E. P. Lormann Introduction

Over the past decade, the drug discovery paradigm has undergone extraordinary changes. With the rapid exploration of potential drug candidates via high-throughput screening now a current challenge is the everincreasing demand for novel small compounds to satisfy and sustain such screening campaigns. As a result, innovative combinatorial approaches toward novel drug-like compounds have become key tools for successful drug discovery programs. One of the major tools in combinatorial chemistry has been rooted in the ingenious solid-phase synthesis of peptides by Merrifield.1 After extending the use of polymer supports into the realm of organic synthesis, the access to small molecule libraries has been accelerated due to the availability of carrying out suitable chemical reactions with the aid of automated synthesis equipment.2,3 While the transformation of chemical functionalities and the assembly of building blocks on solid support are similar to conventional solutionphase chemistry, linkers and their associated strategies play a pivotal role in the successful implementation of solid-phase organic chemistry and their application to combinatorial chemistry.4–6 In general, linkers are bifunctional molecules that act as spacers between the resin and the attached building block. The functional group on the solid support that serves as the point of origin for a synthetic sequence is generally unchanged upon cleavage conditions. However, the bond between the linker and the immobilized compound is sensitive to certain reaction conditions leading to bond breakage freeing the final compound from the immobilized linker. Traditionally, linkers were designed to release one functional group acting more 1

R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963). L. A. Thompson and J. A. Ellman, Chem. Rev. 96, 555 (1996). 3 S. L. Schreiber, Science 287, 1964 (2000). 4 F. Zaragoza Do¨rwald, ‘‘Organic Synthesis on Solid-Phase: Supports, Linkers, Reactions.’’ Wiley-VCH, Weinheim, 2000. 5 S. Dahmen and S. Bra¨se, in ‘‘Handbook of Combinatorial Chemistry’’ (K. C. Nicolaou, R. Hanko, and W. Hartwig, eds.), Chapter 4. VCH, Weinheim, 2002. 6 I. W. James, Tetrahedron 55, 4855 (1999). 2

METHODS IN ENZYMOLOGY, VOL. 369

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Fig. 1. Linker types for solid-phase synthesis.

or less like bulky protecting groups. These types of linkers could be defined as monofunctional linkers (Fig. 1). Although the release of carboxylic acids and amines, which are essential for peptide synthesis, has been extensively studied, the synthesis of small-molecular-weight compound libraries requires more versatile linkers and cleavage strategies.7 One limitation of monofunctional linkers is that they provide only one type of compound in a library. However, the so-called multifunctional linkers offer the important opportunity to incorporate additional diversity upon cleavage. In this case, the number of new functionalities (Fig. 1, type I) can multiply the number of compounds produced (Fig. 2).

7

B. J. Backes and J. Ellman, Curr. Opin. Chem. Biol. 1, 86 (1997).

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Fig. 2. Diversity through multifunctional cleavage. (See color insert.)

If the linker is susceptible to cleavage upon treatment with different building blocks [e.g., nucleophile (A) and electrophiles (B) in Fig. 1, type II], a substantial library of novel molecules could be prepared from one linked compound (Fig. 2). Literature precedence for multifunctional linkers can be found among various types of anchoring groups (Fig. 3). When considering using a multifunctional linker, one must take into account the nature of the cleavage reagent and the cleaving step. For example, cleavage of an immobilized compound anchored via an ester linkage with excess of a Grignard reagent will require an aqueous workup with the potential of losing valuable material, as well as having to develop a tedious workup and product-isolation procedures. Thus, supplementary building blocks need to be easily removed (i.e., must be volatile, soluble in certain solvents, amenable to react with scavenger resins, etc.) and should not interfere with the characteristics of the whole library such as their biological properties. Triazenes as Linkers

The chemistry of diazonium salts provides tremendous opportunities for the construction of a wide range of aromatic compounds. Triazenes not only provide interesting new possibilities for activation of the

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Fig. 3. Examples for multifunctional linkers.4

ortho-position of arenes by coordination of metal ions and/or by lowering the electron density of the arene ring, they are also ideal synthons for diazonium salts. Inspired by the use of triazenes in the total synthesis of vancomycin8 and the pioneering work of Moore and co-workers9 and Tour and co-workers10 in the synthesis of triazenes on a solid support to produce iodoarenes, a whole set of triazene-based linkers has been developed11 (Fig. 4). Triazenes are stable toward daylight, oxygen (air), moisture, reducing agents, oxidizing reagents, and alkyl lithium reagents under certain conditions.12,13 However, triazenes are labile toward Brønsted acids and certain Lewis acids producing diazonium salts and amines. Two linkers based on the triazene chemistry have been developed. While the T1 linker system consists of 3,3-dialkyl-1-aryl triazene bound to

8

K. C. Nicolaou, C. N. C. Boddy, S. Bra¨se, and N. Winssinger, Angew. Chem. 111, 2230 (1999). 9 J. K. Young, J. C. Nelson, and J. S. Moore, J. Am. Chem. Soc. 116, 10841 (1994). 10 L. Jones, J. S. Schumm, and J. M. Tour, J. Org. Chem. 62, 1388 (1997). 11 S. Bra¨se, D. Enders, J. Ko¨bberling, and F. Avemaria, Angew. Chem. Int. Ed. 37, 3413 (1998). 12 M. Lormann, S. Dahmen, and S. Bra¨se, Tetrahedron Lett. 41, 3813 (2000). 13 M. Lormann, S. Dahmen, F. Avemaria, F. Lauterwasser, and S. Bra¨se, Synlett, 917 (2002).

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Fig. 4. The triazene linkers.

the support via the alkyl chain (Scheme 1), the T2 linker family is based on immobilized aryl diazonium salts (compounds 56, 66, and 71).14 The triazene T1 linker has been successfully used as a linker for arenes. Up to now, approximately 100 different anilines 7 have been immobilized.15 In general, the synthesis starts with diazotation of an aniline in an organic solvent using alkyl nitrite reagents. The immobilization on solid support has been successfully carried out via the use of a benzylaminopolystyrene or piperazinylmethylpolystyrene resin, each accessible from Merrifield resin in only one step with loadings generally around 1 mmol/g (1–2% cross-linked with divinylbenzene) (see Experimental section). Although both linkers are equally suitable, the benzylamino resin is more 14

S. Bra¨se, J. Ko¨bberling, D. Enders, M. Wang, R. Lazny, and S. Brandtner, Tetrahedron Lett. 40, 2105 (1999). 15 S. Bra¨se, unpublished results (1998–2003).

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Scheme 1. Concept of the T1 linker.16

sensitive toward strong bases (e.g., BuLi)12 while the piperazine resin is not available with a high loading capacity due to possible cross-linking during its preparation from Merrifield resin. Both resins are now commercially available (Novabiochem). Functionalization on the polymer bead has been demonstrated extensively. Acidic cleavage of the triazene resin yields the amine resin 10, which can be recycled, and the modified aryl diazonium salts 8-R0 which can be further transformed directly at the cleavage step in high yields (>90%) and purities (>90–95% according to GC, NMR, HPLC* analyses) (Scheme 2). Traceless Linkers

One prominent class of monofunctional anchor that provides access to molecules having ‘‘no attachment memory’’ for solid-phase synthesis is called traceless or ‘‘clean break’’ linkers.7,16–18 Although this definition could be used for the classification of linkers, one would advantageously *

Abbreviations: DMF, N,N-dimethylformamide; DMA, N,N-dimethylacetamide; GC, gas chromatography; HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance; RCM, ring closing metathesis; TFA, trifluoroacetic acid; THF, tetrahydrofuran. 16 S. Bra¨se and S. Dahmen, Chem. Eur. J. 6, 1899 (2000). 17 A. B. Reitz, Curr. Opin. Drug. Disc. Dev. 2, 358 (1999). 18 A. C. Comely and S. E. Gibson, Angew. Chem. Int. Ed. Engl. 40, 1012 (2001).

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Scheme 2. Possibilities of the T1 triazene linker.16

define a traceless linker as being able to generate new C—H bonds or to keep all functionalities during cleavage intact/unchanged upon cleavage. Thus, this linker type allows the formation of arenes, alkanes, alkenes, and alkynes bearing no chemical evidence of attachment to a support. For that reason, this anchoring mode has no potentially undesirable constraints on the structure of the products. Therefore, the triazene linkers have been envisaged as traceless linkers. As pointed out above, acidic media (below pH 3) cleave the triazenes to give the diazonium salts. The diazonium salts can be further functionalized as exemplified in the case of the reduction to the hydrocarbon 17-H in THF with the aid of ultrasound11 through a radical pathway. A new reagent

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Scheme 3. The T1 linker for traceless cleavage.

found for this reduction was trichlorosilane (see Experimental section),12 which not only serves as a source of trace-quantity hydrochloric acid to cleave the triazene moiety, but also as a hydride donor reducing the diazonium ions cleanly (Scheme 3). The synthetic utility of the T1 linker has been demonstrated in short reaction sequences. Thus, cinnamic esters were synthesized in a sequence starting from the iodoarene resin 26. A Heck coupling19 with acrylates using palladium catalysis affords immobilized cinnamates, which in turn could be detached either directly with trichlorosilane to afford cinnamate derivative 25, or after further transformations cleaved with an HCl/THF mixture to give 27 in high yields without the need of further purification or workup procedures (Scheme 3). Other traceless linkers have been developed based on silyl linkers,20 acylhydrazines,21 dialkylsulfones,22 olefin metathesis cleavage (RCM), arylsulfonates,23 phosphonium salts,24 or phosphine–chromium complexes.25 In general, the chemistry of traceless linkers is a fast emerging field in the intensive investigated area of solid-phase organic synthesis. Although there was initially some confusion about the definition or classification of the traceless linker, it is now clear that this anchoring mode will play an important role in the design and synthesis of drug-like molecules. With the triazene linker a new chemistry field is now set up for new developments and the investigation of some older ideas that had been overlooked until now. 19

S. Bra¨se, J. Ko¨bberling N. Griebenow, in ‘‘Handbook of Organo-Palladium Chemistry’’ (E˜.-i. Negishi, ed.), Chapter X. 3. Wiley, New York, 2002. 20 M. J. Plunkett and J. A. Ellman, J. Org. Chem. 60, 6006 (1995). 21 F. Stieber, U. Grether, and H. Waldmann, Angew. Chem. Int. Ed. Engl. 38, 1073 (1999). 22 K. W. Jung, X. Y. Zhao, and K. D. Janda, Tetrahedron 53, 6645 (1997). 23 S. J. Jin, D. P. Holub, and D. J. Wustrow, Tetrahedron Lett. 39, 3651 (1998). 24 I. Hughes, Tetrahedron Lett. 37, 7595 (1996). 25 S. E. Gibson, N. J. Hales, and M. A. Peplow, Tetrahedron Lett. 40, 1417 (1999).

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Multifunctional Cleavage

Functionalization during the cleavage process is an attractive objective for the generation of diverse compound libraries. As already shown by Moore and co-workers9 and Tour and coworkers,10 addition of methyl iodide to a triazene resin at elevated tem perature (110 ) gives rise to aryliodides 17-I (Nu ¼ I) in excellent yields. We have shown that aryl halides 17-X (X ¼ Cl, Br, I) are readily available by the action of lithium halides in the presence of an acidic ion-exchange resin or with the corresponding trimethylsilyl halide at room temperature.26 A mixture of acetic anhydride and acetic acid produces phenol acetates 17-OAc.26 Aryl diazonium salts and azide transfer reagents react directly without the need of a catalyst to afford aryl azides. In contrast to the general Sandmeyer reaction, this reaction does not proceed through the cleavage of the carbon–heteroatom bond. In this particular case an open-chain pentazene or a cyclic pentazole is formed, which in turn loses nitrogen to give the desired aryl azide.27 With this information in hand, a solid-phase synthetic protocol was developed for the synthesis of aryl azides. This synthesis was achieved via the cleavage of the triazene resin with 10% TFA in dichloromethane at room temperature in the presence of trimethylsilyl azide, a commercially available azide derivative with no explosive properties.28 After a couple of minutes the mixture was filtered, the solvent was removed, and the aryl azides 17-N3 were isolated in good yields (mostly >90%) and high purity (>95%) without any further purification. The required mild cleavage conditions allow the synthesis of various functionalized arenes. Only small amounts of silyl residues (<5%) were detected by NMR spectroscopy. While the range of electrophiles that could be employed is quite broad, the most versatile benefit was the development of a cleavage cross-coupling strategy.29 Starting from modified triazene resins, a one-pot cleavage crosscoupling reaction was conducted with two equivalents of trifluoroacetic  acid in MeOH at 0 to give a diazonium intermediate 8-R0 . In situ coupling with both electron-deficient or electron-rich alkenes 19 in the presence of catalytic amounts (5% mol) of palladium(II) acetate furnished the corresponding products 20 in excellent yield and purities. This one-pot cleavage cross-coupling reaction affords salt-free products since the resin 10 also 26

S. Bra¨se, M. Lormann, and J. Heuts, Tetrahedron Lett. In preparation (2003). R. N. Butler, A. Fox, S. Collier, and L. A. Burke, J. Chem. Soc. Perkin. Trans. 2, 2243 (1998). 28 W. C. Groutas and D. Felker, Synthesis 861 (1980). 29 S. Bra¨se and M. Schroen, Angew. Chem. Int. Ed. 38, 1071 (1999). 27

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participates as a ‘‘scavenger resin’’ trapping the trifluoroacetic acid in excess. Using palladium on charcoal as the catalyst in the cross-coupling reaction has the advantage of decreasing palladium contamination as well as providing the conditions for a subsequent hydrogenation reaction. This salt-free cleavage cross-coupling strategy allows the clean synthesis of substituted (cyclo)alkenyl- and (cyclo)alkyl (hetero)arene derivatives and it is especially suitable for automated synthesis since there is no need for purification of the final compounds. This modular protocol is amenable to virtually any amino arene as well as alkenes and alkynes tolerating most functional groups providing a synthetic pathway for the formation of highly lipophilic molecules. Multicomponent Heck reactions (e.g., domino Heck Diels–Alder reaction) are possible in this context and lead to further diversification.30 Concept for Heterocycle Synthesis

Heterocycles play a pivotal role in drug discovery.31 It was shown that compounds with biological activity are often derived from heterocyclic structures based on natural and synthetic products possessing various pharmacological properties. Substituted heterocyclic compounds offer a high degree of structural diversity and have proven to be broadly useful as therapeutic agents. With the disclosure of the benzodiazepine libraries by Ellman’s group in 1992,32 the development of strategies for the generation of heterocyclic libraries33 on solid support has become of great interest in combinatorial chemistry.34 Therefore, we envisaged using the triazene T1 linker for the synthesis of diverse heterocycle libraries. While in concept I (Scheme 4) the diazonium ion was directly incorporated into the heterocycle core, the flexibility of the azide functionality has been advantageously used in concept II (Scheme 4). In concept III the heterocyclic core is generated on the bead using (classic) ring-formation reactions followed by a subsequent multifunctional cleavage to give rise to the library members. Concept I in Scheme 4 represents a straightforward synthesis of benzoannelated heterocycles (e.g., cinnolines, benzotriazoles) via a diazonium intermediate and a nucleophilic ortho-substituent. While in the many examples, the diazonium group, upon cleavage from the resin, is lost as 30

A. de Meijere, H. Nu¨ske, M. Es-Sayed, T. Labahn, M. Schroen, and S. Bra¨se, Angew. Chem. Int. Ed. 38, 3669 (1999). 31 S. Bra¨se, C. Gil, and K. Knepper, Bioorg. Med. Chem. 10, 2415 (2002). 32 B. A. Bunin and J. A. Ellman, J. Am. Chem. Soc. 114, 10997 (1992). 33 R. G. Franzen, J. Comb. Chem. 2, 195 (2000). 34 R. E. Dolle, J. Comb. Chem. 2, 383 (2000).

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Scheme 4. Concept for cleavage/heterocyclization with the T1 system.

nitrogen gas, a suitable nucleophilic ortho-substituent favors cyclization to give heterocyclic structures (Scheme 5). Using this approach, various heterocyclic structures are conceivable. Benzotriazoles, for example, are accessible from o-aminoaryl-substituted triazenes after a two-step reaction sequence: a nucleophilic displacement followed by cleavage/heterocyclization.35 The nucleophilic halide displacement of activated haloarenes is an indispensable tool for the synthesis of highly substituted arenes. Fluoronitroarenes in particular have served as excellent precursors in this transformation. Thus, it was appealing to combine this SNAr reaction with the flexibility of diazonium chemistry. In this case, an immobilized fluoronitrophenyl triazene would be the equivalent of the Sanger reagent. Starting from commercially available 2-fluoro-5-nitroaniline, this aniline was diazotized and coupled to benzylaminomethyl polystyrene to give the immobilized triazene 36 (see Experimental section). At this point, it was crucial to avoid using hydroxylic solvents such as methanol in the washing steps since these solvents are known to react with fluorotriazenes. Acetone was found to be a good substitute for methanol. 35

M. E. P. Lormann, C. H. Walker, and S. Bra¨se, Chem. Commun. 1296 (2002).

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Scheme 5. Concept I for cleavage/heterocyclization using the diazonium functionality.

Although 1-alkyl and aryl benzotriazoles have been prepared from symmetrical and unsymmetrical diamines by diazotation in moderate to good yields,36 a synthesis starting from triazenes has not been disclosed. Benzotriazoles are important intermediates, protecting groups, activating groups, and final products in organic synthesis.37 Furthermore, benzotriazoles are indispensable tools for the synthesis of various functional groups. Various 1-alkyl benzotriazoles are biologically active and show nanomolar binding affinities to various proteins despite the fact that this structural unit is not found in nature.38 The antiemetic and neuroleptic compound alizapride (Vergentan) is a 3-unsubstituted 3H-benzotriazole used for the treatment of side effects in chemotherapy where cisplatin (Platinol), an antitumor agent, is used.39 Recently, benzotriazoles have served as linkers and auxiliaries in solid-phase synthesis.40–42 Therefore, the conversion of the polymer-bound fluoronitroarenes to 1-alkyl benzotriazoles was investigated. After nucleophilic displacement with primary amines to furnish the aniline resins 37, the cleavage with trifluoroacetic acid in dichloromethane proceeded smoothly at room temperature within minutes giving the desired 1-alkyl-5-nitro-1H-benzotriazoles 38 in excellent yields and purities (Scheme 6). This route was successfully adopted for the synthesis of a 200-member library by means of automated synthesis.43 36

J. C. Muir, G. Pattenden, and T. Ye, Tetrahedron Lett. 39, 2861 (1998). A. R. Katritzky, X. F. Lan, J. Z. Yang, and O. V. Denisko, Chem. Rev. 98, 409 (1998). 38 C. E. Augelli-Szafran, T. S. Purchase, B. D. Roth, B. Tait, B. K. Trivedi, M. Wilson, N. Suman-Chauhan, and L. Webdale, Bioorg. Med. Chem. Lett. 7, 2009 (1997). 39 K. Munstedt, I. Wunderlich, E. Blauth-Eckmeyer, M. Zygmunt, and H. Vahrson, Oncology 55, 293 (1998). 40 A. R. Katritzky, S. A. Belyakov, and D. O. Tymoshenko, J. Comb. Chem. 1, 173 (1999). 41 A. Paio, A. Zaramella, R. Ferritto, N. Conti, C. Marchioro, and P. Seneci, J. Comb. Chem. 1, 317 (1999). 42 K. Schiemann and H. D. H. Showalter, J. Org. Chem. 64, 4972 (1999). 43 M. E. P. Lormann, M. Es-Sayed, and S. Bra¨se, unpublished results (2002). 37

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Scheme 6. Synthesis of benzotriazoles.

Other heterocyclic systems that can be prepared include cinnolines 41, which are available from o-alkynylaryl triazenes via a von Richter cleavage-cyclization strategy. Cyclizations of diazonium salts having an ortho-positioned electron-rich double or triple bond have been known for over a century through the von Richter (ortho-alkynyl),44 Widman–Stoermer (ortho-alkenyl),45,46 or Borsche–Koelsch (ortho-acetyl)47,48 reactions, all of which yield cinnoline derivatives, an interesting set of building blocks for biologically active compounds. In particular, 4-halocinnolines can serve as valuable starting materials due to the ease of halogen substitution. The first solid-phase von Richter reaction was reported with the triazene T1 linker.49 Starting with the immobilization of diverse ortho-haloaryl diazonium compounds, the palladium-catalyzed cross-couplings were performed  under standard conditions [Pd(OAc)2, NEt3, DMF, 80 ] with different alkynes affording ortho-alkynylarene resins 40. The von Richter cleavage reactions were conducted with aqueous hydrogen chloride or hydrogen bromide in acetone or dioxane for 1 h at room temperature. Filtration and dilution of the filtrates with water afforded the expected cinnoline library format (146 member) in a 47–95% yield range and with 60–95% purity without any further purification (NMR, GC, GC-MS) (Scheme 7). Concept II is based on the flexibility of the azide functionality. Upon thermolysis or photolysis, appropriately substituted aryl azides lose nitrogen gas to give intermediate nitrenes, which in turn cyclize with suitable ortho-substituents to give benzoannelated heterocycles. An unsaturated ortho-substituent consisting of double bonds including carbon, 44

V. von Richter, Ber. Dtsch. Chem. Ges. 16, 677 (1883). O. Widman, Ber. Dtsch. Chem. Ges. 17, 722 (1884). 46 R. Stoermer and H. Fincke, Ber. Dtsch. Chem. Ges. 42, 3115 (1909). 47 W. Borsche and A. Herbert, Liebigs Ann. Chem. 546, 293 (1941). 48 C. F. Koelsch, J. Org. Chem. 8, 295 (1943). 49 S. Bra¨se, J. Heuts, and S. Dahmen, Tetrahedron Lett. 40, 6201 (1999). 45

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Scheme 7. Synthesis of cinnolines.

oxygen, or nitrogen atoms then gives indoles/carbazoles (starting from C — C),50 indazoles (C — N),50,51 benzoisozazoles (C — O), benzooxadiazoles (benzofurazanes) (N — O),52 benzimidazoles (N — C), or benzotriazoles (N — N) (Scheme 8). Alternatively, the intra- or intermolecular aza-Wittig reaction might also be envisaged,53 in which case larger ring sizes (six- and seven-membered rings) are also achievable. One of the first examples of azide thermolysis cyclization reactions was demonstrated in the synthesis of biologically active benzofuroxanes.49 The cyclization of aryl azides 51 based on the ortho-nitro resins 50 was achieved  at ca. 70 to give the benzofuroxanes 52 (Scheme 9). Triazene T2 Linker

Whereas the T1 linker involves an immobilization of a diazonium salt on an amine resin, the T2 linker represents the reversal of this concept. As outlined in Scheme 10, m-aminophenol (54) was attached to Merrifield resin 53 by displacing the chloride group via the phenolic group with the aid of sodium hydride in dimethylformamide (DMF) (see Experimental 50

V. V. Rozhkov, A. M. Kuvshinov, V. I. Gulevskaya, I. I. Chervin, and S. A. Shevelev, Synthesis 2065 (1999). 51 B. J. Clark and R. J. Grayshan, Chem. Res. Miniprint 3786 (1981). 52 M. R. Kamal, M. M. El-Abadelah, and A. A. Mohammad, Heterocycles 50, 819 (1999). 53 S. Bra¨se, C. Gil, K. Kuepper, Bioorg. Med. Chem. 10, 2415–2437 (2002).

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Scheme 8. Concept II for cleavage/heterocyclization using the azide functionality.

Scheme 9. Synthesis of benzofuroxanes.

Scheme 10. Synthesis of the T2 linker.

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section).14,54 m-Aminophenol was used as the linker molecule because it is not as easily oxidized as other aminophenols. Diazotation of the aniline  resin 55 at 10 in THF with tBuONO and boron trifluoride etherate yielded resin 56, which is stable several hours at this temperature. Various diazonium salts have been prepared using this approach55 and thermoanalytically characterized.56 Both the structure of the diazonium moiety and the counterion clearly influence the stability of the diazonium moiety. The thermally stable diazonium ion 71 (Z ¼ Cl, Y ¼ CH2O) [t1/2  (25 ) > 100 days] is also capable of scavenging various nucleophiles (amines, phenols, and anilines).57 This resin with a tetrafluoroborate counterion (resin 66) is called T2* diazonium resin and it is now commercially available from Novabiochem. Coupling of the diazonium resins such as the T2 diazonium resin 56  with various primary or secondary amines at 10 to room temperature led to the formation of a series of new triazene resins (Scheme 10) (see Experimental section). In addition to primary or secondary amines, attachment of hydroxylamine, hydrazines, sulfoximines, or phenols proceeds equally well (Scheme 10). Secondary amines can be cleaved directly from the resin, while primary amines give rise to a different reaction pathway (see below). Primary amines can be derivatized on the free N–H functionality and therefore can be modified to an array of products. Thus, ureas 75,54 thioureas 74,58 guanidines 62,58 and carboxamides 7654 were prepared in excellent yields (see Scheme 14). Solid-phase peptide synthesis (SPPS) has provided various solutions for the linking, chemical transformation, and detachment of amide structures to the chemistry community. In general, these protocols involve the attachment of amine derivatives using a carbon linkage or, in case of amino acids, by their carboxy functionality. Linking by the N–H functionality of the amide bond has been developed through the so-called backbone amide linkers (BAL). Originally designed for the N–H protection of amide bonds to circumvent -turns and other problems during peptide synthesis, these amide-protecting groups can also serve as linkers for SPPS. Barany and co-workers59 described an application of a backbone amide linker for the 54

S. Bra¨se, S. Dahmen, and M. Pfefferkorn, J. Comb. Chem. 2, 710 (2000). S. Bra¨se, S. Dahmen, and M. Schroen, unpublished results (2000). 56 S. Bra¨se, S. Dahmen, C. Popescu, M. Schroen, and F.-J. Wortmann, Polym. Degr. Stab. 75, 329 (2002). 57 S. Dahmen and S. Bra¨se, Angew. Chem. Int. Ed. 39, 3681 (2000). 58 S. Dahmen and S. Bra¨se, Org. Lett. 2, 3563 (2000). 59 K. J. Jensen, J. Alsina, M. F. Songster, J. Vagner, F. Albericio, and G. Barany, J. Am. Chem. Soc. 120, 5441 (1998). 55

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synthesis of oligopeptides based on the peptide amide linker (PAL) concept. Recently, a new backbone amide linker using indole chemistry has also been devised.60 The T2 linker has recently been shown to be a versatile backbone amide anchor. Immobilized disubstituted triazenes were acylated with carboxylic acid anhydrides or chlorides to give amide derivatives. These amides were cleaved under very mild conditions using trimethyl chlorosilane. This sequence thus employs the T2 system as backbone amine linker and was demonstrated in the automated library synthesis of substituted amide derivatives.54 Urea derivatives, which are important biologically active compounds and building blocks for organic syntheses, have been previously synthesized on solid support by various strategies.4,6 In our case, starting with the immobilization of alkene-amines on the T2 linker, the resulting triazenes were treated with isocyanates in THF at room temperature with a catalytic amount of triethylamine. Dihydroxylation of the alkene group was carried out with commercially available AD-mix  (asymmetric dihydroxylation mix) in various solvent mixtures without the use of further additives. The optimum mixture was found to be THF and water (v:v 5:1), which apparently favors the swelling properties as well as the dihydroxylation mechanism. The cleavage was conducted using trimethylsilyl chloride (TMSCI) in dichloromethane (10%) at room temperature. The resulting ureas 58 were isolated in fair to good yields and excellent purities, both of which were consistently greater than 92% (Scheme 11). This synthesis was successfully transferred to a Bohdan Neptune station using a prototype solid-phase unit (see Experimental section) as well as to the Bohdan Miniblock system to yield small libraries of urea derivatives.54 Guanidines are basic molecules with the capacity to form H-bonding interactions. They are a promising class of potentially useful pharmacologically active compounds61 and their liquid phase synthesis has found widespread applications in organic chemistry.62 The solid-phase synthesis of guanidines, however, focuses mainly on three different approaches: the formation of resin bound carbodiimides63 and their reaction with amines, the solid-phase synthesis involving electrophiles in solution,64 and the reaction of supported thioureas with amines.65 60

K. G. Estep, C. E. Neipp, L. M. S. Stramiello, M. D. Adam, M. P. Allen, S. Robinson, and E. J. Roskamp, J. Org. Chem. 63, 5300 (1998). 61 R. G. S. Berlinck, Nat. Prod. Rep. 16, 339 (1999). 62 J. Chen, M. Pattarawarapan, A. J. Zhang, and K. Burgess, J. Comb. Chem. 2, 276 (2000). 63 D. H. Drewry, S. W. Gerritz, and J. A. Linn, Tetrahedron Lett. 38, 3377 (1997). 64 S. Robinson and E. J. Roskamp, Tetrahedron 53, 6697 (1997). 65 P. C. Kearney, M. Fernandez, and J. A. Flygare, Tetrahedron Lett. 39, 2663 (1998).

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Scheme 11. Synthesis of ureas using the T2 linker.

Scheme 12. Synthesis of guanidine libraries.

The triazene T2 linker14 and the improved T2* linker57 systems offer a unique approach to the formation of guanidines in which all three substituents can be varied to a wide extent. Starting from disubstituted triazene on the T2* linker, deprotonation using NaH/DMF and subsequently acylation by the addition of isothiocyanates (Scheme 12) yielded a library of resinbound thioureas. For the reaction of the thioureas with amines, the use

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of mercury(II) oxide (caution: very toxic) proved to be superior over a whole variety of coupling reagents that were envisaged. Traces of the formed insoluble black mercury(II) sulfide could be efficiently removed by simple filtration of the cleavage solution over a short pad of silica. In the final diversification step before cleavage, the reaction of the diazenylthiourea resins 63 with ammonia, primary and secondary amines, was conducted under optimized reaction conditions. Cleavage of polymersupported diazenylguanidines with 10% TFA in dichloromethane yielded guanidines 62 as their trifluoroacetate salts with high purities (>90%). Elution of the TFA salts in methanol over a short column of basic anionexchange resin [strong anion-exchange resin (Lewatit MP 5080, Merck Darmstadt)] efficiently produced the nonprotonated guanidines 62 in high yield (Scheme 12) (see Experimental section). While the cleavage of trisubstituted triazenes gives rise to the formation of secondary amines in excellent yields, the cleavage of disubstituted triazene 69 gives rise to aliphatic diazonium salts. The newly formed diazonium ion undergoes substitution with the nucleophile present in the reaction mixture. Therefore, alkyl halides 79-X,66 alcohols 78-OH,67 ethers 78-OR1,67 as well as alkyl carboxylic 78-OCOR, sulfonic esters 78-OSO2R68 phosphoric esters 78-OPO(OR)2,69 and phosphinic esters 78-OPO(H)(OR)67 can be formed by cleavage with trimethylsilyl halides (X ¼ I, Br, Cl), aqueous trifluoroacetic acid,67 carboxylic acids,70 sulfonic acids, phosphoric acids, and phosphoric acids, respectively. The regioselectivity of the cleavage can be explained by the presence of one tautomer of the triazene in which the hydrogen atom is on the triazene-nitrogen linked to the arene ring. Overall, this reaction sequence provides the means for substituting an amino group for an oxygen or a halogen (Cl, Br, I) atom (Scheme 13). In summary, the triazene T2 linker system displays an original anchoring group with ample possibilities for variations (Scheme 14). Summary and Conclusion

Over the past years, various new types of linkers have emerged. Especially for the synthesis of small molecules on solid support, the design of a new anchoring group might be essential for the success of the synthesis. 66

C. Pilot, S. Dahmen, F. Lauterwasser, and S. Bra¨se, Tetrahedron Lett. 42, 9179 (2001). S. Bra¨se and C. Pilot, unpublished results (1998). 68 N. Vignola, S. Dahmen, D. Enders, and S. Bra¨se, Tetrahedron Lett. 42, 7833 (2001). 69 N. Vignola, S. Dahmen, D. Enders, and S. Bra¨se, J. Comb. Chem. 5, 138 (2003). 70 J. Rademann, J. Smerdka, G. Jung, P. Grosche, and D. Schmid, Angew. Chem. Int. Ed. 40, 381 (2001). 67

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Scheme 13. The multifunctional cleavage of the T2* linker.

Scheme 14. Possibilities with the T2 linker.

Linker, cleavage conditions, and functional groups are appointed to each other. Therefore, the decision to use a specific linker has to be balanced with the nature of the library to be synthesized.

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Experimental Section

Chemicals, Solvents, Reagents Chemicals were purchased from Aldrich, Fluka, Janssen, and Merck. Merrifield resin (1–2% cross-linked, 200–400 mesh) was obtained from Novabiochem or Polymer Laboratories. To obtain the molecular mass of the resin and to calculate the elemental analysis the following calculation has to be performed: molar massnew ¼

1000  ðmolar massSub  molar massAdd Þ Loadingold

(1)

Solvents (benzene, ether, tetrahydrofuran, dichloromethane) for reactions involving organometallic and other sensitive materials were distilled under argon prior to use. All resins were washed sequentially by using a vacuum reservoir connected to a sintered glass frit. Cleavage was conducted using Teflon tubes with a frit connected to a vacuum line or with a glass pipette filled with glass wool. Evaporation of the solvent was achieved using a rota-evaporator and/or high vacuum (ca. 0.1 mbar). T1 Linker: Synthesis of Benzylaminomethyl Polystyrene To a suspension of 10 g of Merrifield resin in 100 ml of dry DMF were added 5 equivalents of benzylamine (20 equivalents for the synthesis of piperazinomethyl polystyrene) and 1 equivalent of potassium iodide. The  reaction mixture was agitated with an overhead stirrer at 80 for 72 h. After cooling the mixture to room temperature, the resin was filtered on a sintered frit and washed with the following solvents (50 ml portions): DMF, methanol, DMF, methanol, water, DMF, methanol, DMF, diethylether, dichloromethane, diethylether, dichloromethane, diethylether, dichloromethane, and diethylether. The resin was then dried under vacuum. Representative Procedure for the Synthesis of Triazene T1 Resins With continuous stirring, 2.5 equivalents of 4-fluoro-3-nitroaniline (4.02 g, 26 mmol) was dissolved in 50 ml of dry dichloromethane and 20 ml of CH3CN. After cooling this mixture to 100, 2.5 equivalents of trifluoroacetic acid were added dropwise followed by the slow addition (two portions with a 30-min interval) of 2.5 equivalents of i-C5H11ONO. The re  action mixture was then stirred for 1 h between 10 and 5 , cooled with  an acetone/dry ice bath to approx. 78 , and then quenched under stirring with ether (50 ml). The solvent was decanted and another 50 ml of ether was added. This washing process was repeated three times. The resulting

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suspension was warmed to 15 and 1 equivalent of benzylaminomethyl polystyrene (10 g) and approximately 20 ml of pyridine were added. The reaction was stirred for a few minutes until all of the resin turned red. The resin was then washed with a 3:1 ratio mixture of absolute DMA and pyridine, then washed once with diethyl ether, and then with distilled pentane to shrink the resin. The resin 36 was then washed with alternate cycles of diethyl ether and dichloromethane until the resin was clean and no colorization could be seen in the solvents after washing. To finish preparing the resin, it was washed with distilled pentane and then freeze-dried overnight. General Procedure for the Traceless Cleavage of the T1 Linker A suspension of the resin (200 mg) prepared as above in dichloromethane was treated with 4 equivalents of trichlorosilane (Caution: corro sive and low boiling material), heated to 40 under stirring for 15 min or at room temperature for 60 min. After cooling the mixture to room temperature, silica gel (100 mg) was added, the mixture was filtered, and the solvent removed. The T2 Linker: 3-Aminophenyl-1-oxymethylpolystyrene (55) A dry 500-ml, three-necked round-bottom flask was fitted with a mechanical stirrer, gas inlet, and addition funnel. The apparatus was purged with argon and charged with 250 ml of dry DMF and 2.52 g (63.0 mmol, 1.51 g, 60% in paraffin) of sodium hydride. After adding 20.0 g (12.8 mmol, loading ¼ 0.64 mmol/g) of Merrifield resin (53), 6.9 g (63 mmol) of maminophenol was added portion wise (caution: H2 evolution). After a reaction time of 20 h, the resin was then washed on an inert gas frit with solvents (three times with each approx. 200 ml): THF, Et2O, and MeOH. Subsequently the resin was dried in vacuo. –IR (KBr): v ¼ 3390 cm1, 3200, 3080, 3060, 3020, 2910, 2840, 2640, 2600, 2310, 2340, 2380, 2260, 2110, 1940, 1870, 1800, 1710, 1670, 1620, 1590, 740, 690. –C125H125ON (1655.0): calcd C 90.63, H 7.55, N 0.85; found C 89.90, H 8.03, N 0.89. Preparation of Diazonium Salt 56 on the Resin The above amine resin 55 (10.0 g, 6.4 mmol) was suspended in dry THF  and cooled by means of a cold bath (EtOH/dry ice) to 20 . After 20 min, BF3Et2O (6.9 ml, 7.7 g, 54 mmol) was added and then after 5 min tertBuONO (5.7 ml, 5.0 g, 49 mmol) was added. After a reaction time of 30 min, the mixture was collected in an inert gas frit, filtered, and washed with chilled THF (4  15 ml/g resin).

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Preparation of Triazene Resin 4 

Resin 56 was swelled in THF (15 ml/g resin) at 10 and treated with an amine (5 equivalents). After a reaction time of 1 h, a solution of MeOH in THF (1:1 v/v) was added to quench the reaction. The resin was then washed on an inert gas frit with the following solvents (three times with each approx. 20 ml for each solvent per 1.00 g resin): THF, Et2O, and MeOH. Subsequently the resin was dried in vacuo. Manual Preparation of Ureas Resin 4 obtained as described above was suspended in dry THF (10 ml/g resin) under argon for 10 min and treated with triethylamine (1 equivalent) and an isocyanate (4 equivalents). After a reaction time of 1 h, a solution of MeOH in THF (50%) was added to quench the reaction. The resin was then washed on an inert gas frit with the following solvents (three times with each approx. 20 ml for each solvent per 1.00 g resin): THF, Et2O, and MeOH. Subsequently the resin was dried in vacuo. A filtration setup, consisting of a glass pipette filled with a plug of glass wool, was filled with 100 mg of the resin and treated three times with 1.5 ml cleavage solution (5% TFA in CH2Cl2) for 5 min at room temperature, by which the resin turns red. The combined filtrates were concentrated in vacuo. Automated Preparation of Amides Resin 4 (1.5 g) was suspended in 7.5 ml CH2Cl2 and 7.5 ml DMF. In each reaction vessel was distributed 2.5 ml (250 mg, 0.1575 mmol, loading 0.64 mmol/g) of this isopycnic resin suspension. After being filtered, the resin was washed twice with 2.0 ml THF and finally suspended in 1 ml THF. The reaction chambers used for the reaction with the acid chlorides were filled with 0.3 ml triethylamine in THF (0.36 mol/liter, 2 ml, 4-fold excess). The reactants were added and the reaction chambers were agitated in a parallel compartment overnight at room temperature. The resin was washed (two cycles with each 4.0 ml CH2Cl2 and MeOH, then 4.0 ml CH2Cl2) and the solvent was removed in vacuo. The cleavage was conducted with 10 ml of a 10% TMSCl solution in CH2Cl2 under mechanical agitation for 1 h. Subsequently, after filtering and washing the resin with 1.0 ml of CH2Cl2, the combined filtrates were concentrated on a parallel evaporator. The yields were measured automatically. Except for the suspending and the weighing process as well as the transport of the shaker unit, all processes have been fully automated.

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Synthesis of Guanidines Using the T2* Linker In a three-necked round-bottom flask equipped with a mechanical stirrer was suspended 10.0 g (5.3 mmol, loading 0.53 mmol/g) of T2* diazonium  resin in THF. The mixture was cooled to 5 to 10 and a primary amine (2.65–5.3 mmol, 5–10 equivalents) was added under stirring. After stirring for 0.5–1 h, the ice bath was removed and the mixture allowed to reach room temperature. The resin was filtered off, washed sequentially with THF and methanol, and dried under high vacuum. The resulting resin (2.0 g, 0.96–0.90 mmol, loading 0.48–0.45 mmol/g) was then suspended in 25 ml of anhydrous DMF under argon. NaH (490 mg, 12 mmol, 60% in mineral oil) was added and the mixture was agitated under a stream of argon. After 2 min, an isothiocyanate (6 mmol, 6.25–6.7 equivalents) was added and the mixture was agitated for another 2 h at room temperature. The reaction was quenched by the addition of methanol, the resin filtered off, sequentially washed with THF and methanol, and then dried under high vacuum. A 15-ml vial was charged with 250 mg (0.11 mmol, loading 0.45 mmol/g) of resin and 100 mg (0.46 mmol, 4.2 equivalents) of orange HgO (caution: very toxic). A magnetic stirring bar was added and the vial was closed. THF (4 ml) and aqueous ammonia solution (2 ml, 25% in water) were added through the septum and the mixture was agitated for  12–24 h at 45 . After being cooled to room temperature, the vials were opened and the resin filtered off and sequentially washed with THF and MeOH. The resin (100–300 mg, 0.14–0.4 mmol) was placed in a 10-ml tube equipped with a frit and 4–6 ml of TFA solution (10% in dichloromethane, v:v) was eluted over the resin. The filtrate was evaporated under reduced pressure. Acknowledgments The chemistry described has been conducted by a young and enthusiastic team, whose names appear in the appropriate references. We thank our academic and industrial partners for fruitful collaborations and comments. This work was supported by the DFG (Deutsche Forschungsgemeinschaft). Bayer, BASF, Novabiochem, and Gru¨nenthal are also gratefully acknowledged.

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[8] The Development and Application of Tetrafluorophenol-Activated Resins for Rapid Amine Derivatization By Joseph M. Salvino and Roland E. Dolle Introduction

Tetrafluorophenol (TFP)*-activated resins1–3 are reactive polymeric acylating and sulfonylating reagents useful for synthesizing pure singlecompound arrays of amides and sulfonamides. TFP-activated resins may be regarded as resin-bound equivalents to acid chlorides. They are easy to prepare in a single step from commercially available polymer-supported TFP, are stable to prolonged storage, and react with a wide range of Nnucleophiles including anilines. This chapter provides detailed protocols for synthesizing the reagents, establishing reagent loading, and reaction with amines. Background

Amines and their amide and sulfonamide congeners are undoubtedly the most prevalent functionality found in drug leads and clinical therapeutics. It is therefore a rather common occurrence that once an amine-bearing lead structure is identified, a derivatization campaign is initiated to establish a structure–activity relationship (SAR). Such a campaign generally makes use of reductive amination, acylation, sulfonylation, and ureido chemistries to create a set or library of amine analogs for biological evaluation. With the advent of combinatorial chemical technology, a number of high-speed synthesis protocols for amine derivatization have been developed, suitable for both parallel (discrete compound) and split- and-pool (mixture synthesis) formats. In cases where the amine species is attached to * Abbreviations: DCM, dichloromethane; DIC, 1,3-diisopropylcarbodiimide; DIEA, diisopropylethylamine; DMAP, 4-dimethylaminopyridine; DMF, N,N-dimethylformamide; ELSD, evaporative light scattering detection; HOBt, hydroxybenzotriazole; IR, infrared; LC/MS, high-pressure liquid chromatography/mass spectrometry; NMM, N-methylmorpholine; NMR, nuclear magnetic resonance; PyBop, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate; SAR, structure–activity relationship; TFP, tetrafluorophenol; THF, tetrahydrofuran. 1 J. S. Salvino, N. V. Kumar, E. Orton, J. Airey, T. Kiesow, K. Crawford, R. Mathew, P. Krolikowski, M. Drew, D. Engers, D. Krolikowski, T. Herpin, M. Gardyan, G. McGeehan, and R. Labaudiniere, J. Comb. Chem. 2, 691 (2000).

METHODS IN ENZYMOLOGY, VOL. 369

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solid support, it may be reacted in a straightforward fashion with a diverse set of derivatizing reagents/conditions and cleaved from resin to provide material for biological testing. However, in cases in which the amine species may not be conveniently attached to a solid support or it is not desired to do so, it is necessary to conduct derivatization directly in solution. One of the more convenient protocols to facilitate the latter process is the reaction of amines with polymeric-activated resins (Table I).4–20 The use of polymeric supported reagents for amine acylation was conceived over 30 years ago primarily for application in peptide synthesis.4,5 When used in excess, activated esters derived from polystyrene-bound nitrophenol6,7 and N-hydroxybenzotriazole (HOBt)8 were shown to effect fast and quantitative acylations with primary amines including amino acids and peptide fragments. Product isolation required simple filtration. Several variations of these reagents have since been reported in the literature,9–11 including HOBt resin (a variant of Patchornik’s original HOBt resin),9 pyrimidine esters,10 dihydropyridino[2,3-d]pyrimidine amides,11 seleno esters,12 phenol esters on macroporous polymer disks,13 activated acyltriazoles,14 hydroxamic esters,15 esters of pyrimidine N-oxide and azoles,16 acyloxyquinolines,17 thiol esters,18 oxime esters,19 and Kenner’s ‘‘safety-catch’’ resin.20 One or more technical deficiencies, however, have prevented their 2

Y. Gong, M. Becker, Y. M. Choi-Sledeski, R. S. Davis, J. M. Salvino, V. Chu, K. D. Brown, and H. W. Pauls, Bioorg. Med. Chem. Lett. 10, 1033 (2000). 3 J. M. Salvino, B. Gerard, H.-F. Ye, B. Sauvagnat, and R. E. Dolle, J. Comb. Chem. 5, 260 (2003). 4 A. Patchornik, M. Fridkin, and E. Katchalsky, German Patent 1913486 (1969). Chem. Abstr. 72, 66932y (1970). 5 D. L. Marshall and I. E. Liener, J. Org. Chem. 35, 867 (1970). 6 R. Kalir, M. Fridkin, and A. Patchornik, Eur. J. Biochem. 42, 151 (1974). 7 B. J. Cohen, H. Karoly-Hafeli, and A. Patchornik, J. Org. Chem. 49, 922 (1982). 8 R. Kalir, A. Warshawsky, M. Fridkin, and A. Patchornik, Eur. J. Biochem. 59, 55 (1975). 9 I. E. Pop, B. P. Deprez, and A. L. Tartar, J. Org. Chem. 62, 2594 (1997). 10 M. Botta, F. Corelli, E. Petricci, and C. Seri, Heterocycles 56, 369 (2002). 11 R. B. Nicewonger, L. Ditto, D. Kerr, and L. Varady, Bioorg. Med. Chem. Lett. 12, 1799 (2002). 12 H. Qian, L.-X. Shao, and X. Huang, Synlett 1571 (2001). 13 J. A. Tripp, F. Svec, and J. M. J. Frechet, J. Comb. Chem. 3, 604 (2001). 14 A. R. Katritzky, A. Pastor, M. Voronkov, and D. Tymoshenko, J. Comb. Chem. 3, 167 (2001). 15 P. N. Sophiamma and K. Sreekumar, Reactive Funct. Polym. 35, 169 (1997). 16 I. Kakobsone, M. Klavins, and A. Zicmanis, Latv. PSR Zinat. Akad. Vestis, Kim. Ser. 483 (1990); 481 (1989). 17 X. Huang, C. C. Chan, and Q. S. Zhou, Makromol. Chem. Rapid Commun. 6, 397 (1985). 18 M. Stern, M. Fridkin, and A. Warshawsky, J. Polym. Chem. Ed. 20, 1569 (1982). 19 W. F. Degrado and T. E. Kaiser, J. Org. Chem. 47, 3258 (1982). 20 G. W. Kenner, J. R. McDermott, and R. C. Sheppard, J. Chem. Soc. Chem. Commun. 636 (1971).

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TABLE I Selected Examples of Known Linkers for Generating Activated Resins for Amine Derivatization Entry

Linkera

Transfer group(s) (reference)

1

–C(O)R7

2

–C(O)R5

3

–C(O)R8

4

–C(O)R20

5

–C(O)R,–SO2R, –CH2CO2R1

6

–C(O)R10

7

–C(O)R11

a

The asterick (*) indicates the atom to which the transfer group is attached.

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widespread use in the production of single compound arrays. These deficiencies include the following: the inherent reactivity of the polymeric activated resin can limit the scope of N-nucleophiles that may react with the reagents; the instability of the activated resin makes resin manipulation and storage inconvenient; the method of activation can be a potential issue, e.g., side products are observed using hydrogen peroxide5 or alkylating agents20 in activation protocols; it may be difficult to establish the absolute loading of the activated resins to ensure that a limiting amount of amine is used during derivatization; and known resin linkers are largely limited to amide bond formation and are not generally suitable for expansion to sulfonylations, carbamoylations, urea synthesis, and carbon–carbon bond formation. Recently, Salvino et al.1 developed TFP-activated resins as novel reagents to specifically address many of the liabilities listed above. Polymeric TFP-activated carboxylate esters react with amines with broad chemical reactivity, analogous to the well-known reactivity of pentafluorophenol carboxylate esters commonly used for solution amide coupling. Due to the electron-withdrawing carbonyl group in the 4-position of the TFP linker, polymeric TFP sulfonate esters are sufficiently activated to react smoothly with amines yielding sulfonamides. By virtue of the fluorine atoms present on the linker, the loading (esterification) to the resin and its subsequent release (amide/sulfonamide formation) can be quantitatively measured using 19 F NMR.1,21,22 Synthesis of Polymeric TFP

The synthesis of polymeric TFP-activated resins is performed with simple equipment and apparatus. Commercially available aminomethyl polystyrene (Polymer Labs) is acylated with 4-hydroxy-2,3,5,6-tetrafluorobenzoic acid hydrate (Aldrich) using HOBt and 1,3-diisopropylcarbodiimide (DIC) as coupling reagents in dimethylformamide (DMF; Fig. 1) to give the TFP-linked resin. During the coupling procedure, ca. 10% TFP tetrafluorobenzoic acid ester is formed by reaction of the unprotected phenol oxygen with the activated fluoroinated benzoic acid. Ester formation is clearly evident by the ester carbonyl stretch at 1765 cm1 in the IR spectra of the resin and by the two extra F signals in the 19F NMR. Treating the resin with a slight excess of piperidine in DMF liberates the phenol. It is 21

M. Drew, E. Orton, P. Krolikowski, J. M. Salvino, and N. V. Kumar, J. Comb. Chem. 2, 8 (2000). 22 A. Svensson, K.-E. Bergquist, T. Fex, and J. Kihlberg, Tetrahedron Lett. 39, 7193 (1998) and references therein.

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Fig. 1. Preparation of TFP resin from aminomethyl polystyrene and 4-hydroxy-2,3,5,6tetrafluorobenzoic acid.

essential that the piperidine treatment be carried out because residual phenol ester generates a corresponding 4-hydroxy-2,3,5,6-tetrafluorobezamide or -sulfonamide contaminant upon amine derivatization. Following acidification of the resin, washing, and drying, the TFP resin is ready for activation with carboxylic and sulfonic acids. The loading of TFP to methylaminopolystyrene resin is determined by elemental analysis by F ion selective chromatography. Typical loading values are within 70% of the starting polystyrene loading. Synthesis of TFP-Activated Resins

Acylation of the TFP resin to furnish carboxylate-activated resins may be carried out by using three complementary protocols (Fig. 2). One optimized protocol is TFP resin esterification with a carboxylic acid and DIC in dichloromethane (DCM).3 TFP resin is first swelled with DCM for 10 min min with mild agitation. The acid (3.5 equiv.) is added and agitated gently until it dissolves. In cases where the acid is poorly soluble in DCM, it may first be dissolved in DMF. Hydroxybenzotriazole (HOBt, 0.1 equiv.) is then added to the reaction mixture followed by the addition of DIC (3.5 equiv.). The acylation reaction is agitated for 12 h at room temperature after which time the resin is collected by filtration and washed with DMF, THF (tetrahydrofuran), and DCM (dichloromethane), and dried in vacuo. In an earlier protocol,1 DMAP [4-(N,N-dimethylamino)pyridine] was used in place of HOBt, but it was found that loadings were irreproducible (unpublished observation). Alternative protocols for phenol acylation employ either acid chlorides [N-methylmorpholine (NMM) as base in DCM] or carboxylic acid sodium salts and PyBop in DMF. The activated TFP reagents are stable when dry and no special handling is required. Although no detailed stability

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Fig. 2. Preparation of polymeric TFP-activated carboxylate and sulfonate ester resins.

studies have been carried out, many TFP reagents in our laboratories have been stored at room temperature for >2 years without loss of activity. The reaction of TFP resin with sulfonyl chlorides (3 equiv.) in the presence of diisopropylamine (DIEA; 3 equiv.) provides the corresponding sulfonate-activated resins (Fig. 2). Alternatively, sulfonic acids may be loaded directly onto TFP using DIC (via the intermediate sulfonic acid anhydride) as the coupling reagent. Analogous to the carboxylate esters, the sulfonate ester resins are stable when dry and may be kept at room temperature for an extended period of time without decomposition. This is one of the clear advantages of the polymeric TFP reagents versus acid chlorides; the latter decompose once exposed to air and moisture. Quality Control of TFP-Activated Resins

Activated TFP resins may be created from a wide selection of structural classes (Table II). The coupling yield or loading of carboxylic or sulfonic acids to TFP resin varies as a function of acid. For carboxylic acids, typical loadings achieved are on the order of 70–80% while sulfonic acid loading approaches 100%. However, loadings from 100% to <10% are observed depending on the reactivity of the acid and the presence of other functional groups and, hence, it must be empirically determined. Examples of acids that do not load well include phenylacetic acids (likely forming ketenes upon addition of amine), acids containing amine functionality, acids containing a pendent functionality that may assist in ester bond cleavage, and certain oxazole carboxylic acids. Determination of loading is critical to library purity and, therefore, it is essential to reliably establish coupling yield. There are two options available to do this: on-resin 19F NMR analysis or by reaction of a standard amine with resin and measuring the conversion

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TABLE II Examples of Diverse Building Blocks Used in the Preparation and Reactions of TFP-Activated Resins

to amide off-resin via 1H NMR. Each custom prepared activated TFP resin is subjected to one of these options as a rigorous quality control step. Both the quality of TFP resin and the resulting polymeric-activated resins may be quantitatively determined by 19F NMR spectroscopy.1 This nondestructive technique takes advantage of the high sensitivity, natural abundance (the 1/2 spin nucleus 19F is 100% abundant), and large chemical shift dispersion (200 ppm) of the 19F nucleus. The 19F NMR spectrum of TFP resin shows two resonances at 148 and 165 ppm (Fig. 3), each corresponding to 2 equiv. fluorine nuclei. The 19F NMR spectrum of TFPactivated carboxylate and sulfonate resins shows two resonances at ca. 145 and 154. Gel phase 19F NMR spectra are easy to interpret. There is only one resonance line per nonequivalent F and nonfluoroinated residuals are transparent to the method. Relative resin loading23 is quantified 23

Absolute loading is established by elemental analysis (F ion chromatography).

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Fig. 3. 19F NMR illustration indicating resonances for the two equivalent F nuclei in nonactivated and activated TFP resins.

by simply integrating the F signals resulting from free and activated forms of polymeric TFP. The analytical method can be done in a high-throughput mode with 100 samples processed overnight using 50 mg of resin suspended in deuterated DMF in a standard 5-mm NMR tube. In lieu of 19F NMR spectroscopy, a second method for determining loading involves the reaction of an accurately weighed sample of resin with a known quantity of a reactive amine, e.g., 2-methylbenzylamine. The reaction product is evaluated by 1H NMR and LC/MS. Loading is determined by integration and comparison of the aromatic methyl protons and the -methylene protons of the product amide relative to the starting amine. Reaction of TFP Reagents with N-Nucleophiles

The reaction of a TFP reagent with an amine is performed by first suspending a 20% excess of activated resin (loading established) in DMF. A limiting amount of amine, 0.8 equiv. based on the resin loading, is added to the resin as a dilute solution in a solvent such as DMF.24 The reaction

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tfp-activated resins for amine derivatization

159

Fig. 4. Reaction of polymeric TFP-activated reagents with amines.

mixture is incubated for a minimum of 3 h, but typically is allowed to run overnight as a matter of convenience. Workup of the reaction requires filtration of the excess TFP-containing resin and collecting the filtrate. A small sample of the filtrate is analyzed by LC/MS and, optionally, evaporative light scattering (ELSD), to assess product purity (generally >95%) and to confirm compound identity. The remaining sample is concentrated to dryness to afford the derivatized amine that may be submitted for biological evaluation (Fig. 4). The process of library synthesis is outlined in Table III. For large library campaigns, 80 wells of a 96-deepwell plate (2 ml/well) are formatted in such a fashion that one-half of the plate (40 wells) is charged with one TFP resin, and the remaining half plate charged with a second TFP resin. Formatting is facilitated via a 96-well pipetting robot, such as the Tomtec Quadra 3, or an eight-tip liquid handling robot, such as the Packard Multiprobe equipped with wide-bore needles. Resin can be accurately dispensed into the plate via a gently agitating resin slurry in THF–DCM (4:1). The solvent is evaporated by allowing the plate to stand in a ventilated fume hood or concentrated using a plate evaporator, such as the Genevac HT-1 centrifuge evaporator. Upon dispensing 2  40 different amines as dilute solutions into the plate, 80 different reaction mixtures are created. After standing for 12 h, the mixtures are aspirated and dispensed into a 96-well filter plate fitted with a 30-m filter. A 96-deepwell plate is placed directly underneath the filter plate and the filtrate is collected. Dry products are obtained upon concentrating the deepwell plate in a Genevac

24

The N-nucleophile may be a primary or secondary amine, or an aniline. N-Nucleophiles that react poorly with TFP reagents include very hindered amines (e.g., o-substituted anilines and 1-aminonaphthalene), deactivated anilines, and aminothiazoles.

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TABLE III Process for Library Synthesis Using Polymeric TFP-Activated Reagents Step

Task

Activity

1

4

Procure building blocks Generate TFP reagents Quality control reagents Distribute resins

5

Add amines

6

Incubate reaction mixtures Filter reaction mixtures Product analysis

Purchase or synthesize amines, acids, acid chlorides, sulfonic acids, and sulfonyl chlorides Load carboxylic acid and sulfonyl chloride building blocks to TFP resin Use 19F NMR spectroscopy or react each resin with 2-methylbenzylamine followed by 1H NMR analysis Measure portions of resins into vials or plates and swell with DMF and calculate reagent equivalents Accurately weigh out amines and prepare dilute (10 M) stock solutions in DMF; dispense 0.8 equivalent of each amine into vials or plates Reaction mixtures agitated at room temperature for 12 h

2 3

7 8

9

Final sample preparation

Collect filtrates and discard spent resin Submit small portions of filtrates for LC/MS analysis; in case of plates, create daughter plate(s) and submit for same; establish purity and confirm identity of new derivatives Remove solvent from filtrates and dry sample in vacuo; register compounds or plates and submit products for biological testing

centrifuge.25 Analytical data are collected on approximately 20% of the compounds in libraries with >1000 members. For focused libraries, where one amine is converted to ca. 80 analogs, 80 polypropylene fritted vessels or wells of a 96-deepwell plate are charged with 80 different TFP reagents. The focused library is obtained following incubation of the dispensed resins with one amine and workup as described above. Analytical data are collected on all the members of the library. Adolor Corporation currently possesses a 300-member custom TFP reagent kit that continues to increase in number. Focused libraries are produced in as little as 2–3 days by derivatizing an amine with a subset of the reagents. Analytical data are collected on all the members of the library. Focused libraries find utility in SAR exploration and patent protection.2 For larger library campaigns, which require 3–4 weeks to complete, 25

It is convenient to produce ca. 10 M of product per well by dispensing ca. 12–15 mg of TFP-activated resin per well, assuming a loading of ca. 0.95 mmol/g and an 80–90% reaction yield. This quantity of product is sufficient for evaluation in multiple biological assays.

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tfp-activated resins for amine derivatization

161

the entire reagent kit is coupled to a set of 40–50 commercially available or custom prepared amines3 to furnish 12,000–15,000 compounds. Large arrays, requiring 3–4 weeks to complete, are screened across a broad range of molecular targets for new lead discovery.26 Experimental Protocols

Preparation of TFP Resin (Scheme 1) Reagents DMF (6 liter total) Aminomethyl polystyrene (100 g; 1.6 mmol/g) TFP in DMF (57 g TFP dissolved in 240 ml DMF) HOBt hydrate in DMF (32.3 g dissolved in 60 ml DMF) DIC (37.5 ml) Piperidine (17.3 ml) 2 M aqueous HCl–DMF (150 ml of 2 M HCl added portion-wise to 1.3 liters of DMF with stirring) THF (2 liter total) DCM (2 liter total) Procedure. A 2-liter three-necked flask fitted with an overhead stirrer and thermometer was charged with DMF (1 liter) and aminomethyl polystyrene resin (Polymer Labs). Freshly prepared solutions of TFP in DMF and HOBt in DMF were sequentially added followed by DIC. The reaction mixture was stirred at room temperature for 16 h and then filtered and washed well with DMF, THF, and DCM to give the TFP resin containing ca. 10% of the undesired TFP-tetrafluorobenzoic acid ester. Hydrolysis of the undesired ester was carried out as follows. The washed resin cake obtained above was placed into a 2-liter three-necked round-bottom flask, again fitted with an overhead stirrer and thermometer. DMF was added (1 liter) and gentle stirring was initiated. Piperidine was added and the reaction mixture was stirred for 2 h, after which time the reaction mixture was filtered and washed thoroughly with DMF (1 liter). The resin cake of TFP piperidine salt was transferred into a 2liter three-necked round-bottom flask, again fitted with an overhead stirrer and thermometer. The resin was resuspended (gentle stirring) in a solution of 2 M aqueous HCl–DMF. The reaction mixture was stirred for 26

Screening hits from lead finding campaigns are resynthesized in 10 mg amounts using either TFP technology or solution-phase chemistry to confirm their biological activity and to provide accurate primary and functional in vitro data (Ki, IC50, EC50, etc.)

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linkers and their applications

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1.5 h and then filtered using a Buchner funnel. The resin cake was washed with DMF (0.6 liter), THF (1 liter), and DCM (0.6 liter). The resin was  dried in vacuo at 45 to give a beige solid (140 g). FT-IR 1650 cm1 (ab1 sence of the 1765 cm absorption indicating complete hydrolysis of the unwanted ester). 19F NMR  148 and 165 ppm. Loading of resin was determined by elemental analysis by F ion selective chromatography. Found %F ¼ 7.2 corresponding to 0.95 mmol/g. Note that several vendors sell TFP resin including Argonaut Technologies and Polymer Laboratories. Preparation of TFP-Activated Carboxylic Acid Esters (Scheme 2) Reagents DCM (150 ml total) TFP resin (2 g; 1.9 mmol, 0.95 mmol/g) Carboxylic acid building block (3.8 mmol) HOBt hydrate (51.3 mg) DIC (0.6 ml) DMF (150 ml total) Procedure. TFP resin was added to a 100-ml polypropylene reaction vial. The resin was swelled in DCM (30 ml) for 15 min followed by the addition of a carboxylic acid building block. Upon dissolution of the acid, HOBt, DIC, and DCM (20 ml) were added. The reaction mixture was gently agitated for 3 h and filtered, and the resin was washed with DMF (3  30 ml) and DCM (3  30 ml), and dried in vacuo for 12 h  at 25 . Preparation of TFP-Activated Sulfonic Acid Esters (Scheme 2) Reagents DMF (120 ml total) TFP resin (2 g; 1.9 mmol, 0.95 mmol/g) Sulfonyl chloride building block in DMF (2.85 mmol dissolved in 10 ml) DIEA (1 ml) DCM (90 ml) Procedure. TFP resin was added to a 100-ml polypropylene reaction vial. The resin was swelled with DMF (20 ml) for 15 min. Diisopropylamine (DIEA) was added followed by a solution of a sulfonyl chloride building block previously dissolved in DMF (10 ml). The reaction mixture was gently agitated for 3 h and filtered, and the resin was washed with DMF  (3  30 ml) and DCM (3  30 ml), and dried in vacuo for 12 h at 25 .

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Loading Determination (Chemical Type) Reagents TFP-activated carboxylic or sulfonate ester (50 mg; ca. 0.05 mmol, ca. 0.95 mmol/g) 2-Methylbenzylamine in DMF (5.74 mg, 0.05 mmol dissolved in 2.5 ml DMF) Procedure. A TFP-activated resin was placed in a small reaction vessel to which was added a DMF solution of 2-methylbenzylamine. The reaction mixture was gently agitated for 12 h and filtered. The filtrate was evaporated to dryness and the residue dissolved in CDCl3 or other deuterated solvent. The sample was analyzed by 1H NMR. Loading was determined by integration and comparison of the aromatic methyl protons and the -methylene protons of the product amide relative to the starting amine. Amine Derivatization with TFP-Activated Esters Reagents (Reaction in vials) DMF (150 ml total) TFP resin (90 mg; 0.08 mmol, 0.95 mmol/g) Amine (0.065 mmol) DCM (6 ml total) Procedure. A 3-ml polypropylene reaction vial was charged with a TFPactivated resin, either the carboxylate or sulfonate ester type, and 1 ml of DMF. The reaction mixture was gentle agitated for 10 min and an amine (0.065 mmol) was added. The reaction mixture was further agitated for 16 h and filtered. The resin was washed with DCM (3  2 ml) and the filtrate and washings were combined. The solvent was removed in vacuo and the amide or sulfonamide (>85% yield) so obtained was analyzed by LC/MS for purity and identity.

164

[9]

linkers and their applications

[9] The Traceless Solid-Phase Synthesis of Organic Molecules By David Tumelty, Yijun Pan, and Christopher P. Holmes Introduction

As the field of solid-phase organic synthesis continues to progress and expand, one aim of practitioners is to synthesize molecules that do not signal their solid-phase ‘‘origins’’ by the presence of extraneous remnants left after cleavage from the resin. These often appeared in earlier work as primary or secondary carboxamides or carboxyl groups appended to the target molecules and conveniently overlooked. These additional functionalities could hinder or obscure biological activity in an otherwise promising target compound or scaffold, as well as being chemically rather unaesthetic. As solid-phase routes and linkers have become increasingly sophisticated in recent years, many workers in the field have forwarded novel chemical methods to attempt to overcome some of these previously mentioned limitations.1 This chapter describes two such approaches, which illustrate different tactics used in the goal of synthesizing organic compounds in a traceless manner. In the first strategy, a linker and scaffold combine synergistically to achieve a traceless synthesis of diverse substituted benzimidazole compounds and libraries.2 Second, a novel linker is used in a more global fashion to synthesize target compounds by activation of chemically diverse phenols.3,4 Traceless Solid-Phase Synthesis of Benzimidazoles Background

Several solid-phase syntheses of benzimidazoles have been reported in recent years.5,6,7 Recently some have been described in which the final products could be regarded as traceless.2,8–10 Our initial goal was to design 1

V. Krchnak and M. W. Holladay, Chem. Rev. 102, 61 (2002). D. Tumelty, K. Cao, and C. P. Holmes, Org. Lett. 3, 83 (2001). 3 Y. Pan and C. P. Holmes, Org. Lett. 3, 2769 (2001). 4 Y. Pan, B. Ruhland, and C. P. Holmes, Angew. Chem. Int. Ed. Engl. 40, 4488 (2001). 5 D. Tumelty, M. K. Schwarz, K. Cao, and M. C. Needels, Tetrahedron Lett. 40, 6185 (1999). 6 D. Tumelty, M. K. Schwarz, and M. C. Needels, Tetrahedron Lett. 39, 7467 (1998). 7 J. P. Mayer, G. S. Lewis, C. McGee, and D. Bankaitis-Davis, Tetrahedron Lett. 39, 6655 (1998). 2

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

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a synthetic route that would permit the traceless release of benzimidazoles from single beads to support Affymax’s encoded combinatorial library screening technologies.11 We required that the final compounds would be released without the benefit of further solution-phase reactions to complete the synthesis and that purification steps would not be carried out (in this particular format). This necessitated the development of a novel synthetic strategy in which the required products were synthesized on the solid support in a quaternary salt form, and treatment with base released the products in a Hofmann elimination reaction. The basic concept has been previously reported for the synthesis of simple amines on the REM linker.12 Our modified plan to synthesize and release the benzimidazole compounds is shown in Fig. 1. Here, we envisage building the benzimidazole scaffold directly onto the linker and, by analogy with a regular tertiary amine synthesis on the REM linker, we can quaternize the resin-bound benzimidazole compounds by reaction with reactive bromides. The quaternary salt can then be liberated by a Hofmann elimination reaction upon treatment with base. Development of the Traceless Route

The uncertain part of the synthesis scheme (prior to testing it experimentally) was whether quaternization on the ring nitrogen that was not directly attached to the resin would provide a sufficiently strong electronwithdrawing force to permit the Hofmann elimination under mild conditions. This idea was initially tested in a double-linker scheme as shown in Fig. 2. This construct, although not the final chemical route, was extremely valuable in proving that the concept was valid and enabling various portions of the scheme to be optimized more effectively. This construct has also proven useful in the development of related chemistries using a similar Hofmann elimination strategy. In this scheme, Fmoc*--alanine is coupled to ArgoGel-Wang resin using standard methodology to give resin 1, and then the Fmoc group is 8

V. Krchnak, J. Smith, and J. Vagner, Tetrahedron Lett. 42, 1627 (2001). A. Mazurov, Bioorg. Med. Chem. Lett. 10, 67 (2000). 10 W. Huang and R. M. Scarborough, Tetrahedron Lett. 40, 2665 (1999). 11 Z. J. Ni, D. Maclean, C. P. Holmes, and M. A. Gallop, Methods Enzymol. 267, 261 (1996). 12 J. R. Morphy, Z. Rankovic, and D. C. Rees, Tetrahedron Lett. 37, 3209 (1996). * Abbreviations: AcOH, acetic acid; Alloc, allyloxycarbonyl; ArB(OH)2, a generalized aromatic boronic acid; BINAP, (R)-(þ)-2,20 -Bis(diphenylphosphino)-1,10 -binaphthyl, a chiral chelating ligand useful in palladium-mediated reactions; DCM, 1,2-dichloromethane, a common organic solvent (caution: suspected carcinogen); DIEA, N,N0 -diisopropylethylamine, a hindered organic base; DMF, N,N0 -dimethylformamide, a polar organic solvent; 9

166

[9]

linkers and their applications O

R2 + HN R1

O

R2 N R1

O

O

NH2 O

R2 O

O

N N

O

R1

R3 N

R2 R2 O

+

N

R3

R1

O

O

R2

N

+

O

R3 N R1

R1

R3 N

R2 N

R1

Fig. 1. Comparison of a regular tertiary amine synthesis on REM resin (left) with the planned traceless benzimidazole route.

DMSO, dimethylsulfoxide, a polar organic solvent; dppp, 1,3-bis(diphenyl-phosphino)propane; Fmoc, 9-fluorenylmethyloxycarbonyl, a protecting group of amines; GC-MS, combined gas chromatography and mass spectrometry instrumentation; HPLC, highpressure (performance) liquid chromatography; LC-MS, combined liquid chromatography and mass spectrometry instrumentation; MeOH, methanol; Na(CN)BH3, sodium cyanoborohydride, a reducing agent often used for the reduction of imines to amines; NMP, Nmethylpyrrolidinone, an organic solvent; Na2S2O4, sodium hyposulfite (sodium dithionite), a mild, water-soluble reducing agent; Oxone, potassium peroxymonosulfate, an oxidizing agent (Dupont); Pd(dppf)Cl2, [1,10 -bis(diphenylphosphino)ferrocene]dichloropalladium(II); Pd(OAc)2, palladium(II) acetate; PEG-PS, a resin composed of low-cross-linked polystyrene linked with polyethylene glycol of various lengths; PFS linker/resin, perfluoroalkylsulfonyl linker attached to resin; SnCl22H2O, tin(II) dichloride dihydrate, often used for reduction of aromatic nitro groups to corresponding anilines; tBoc, tertbutyloxycarbonyl, a common, acid-labile protecting group for amines; TEA, triethylamine, an organic base; TFA, 1,1,1-trifluoroacetic acid, a strong organic acid; THF, tetrahydrofuran, an organic solvent; TLC, thin-layer chromatography.

[9]

167

traceless solid-phase organic synthesis O H N

O O

O

O

a,b

O

H N

O

O

+

N

O

R1

O

1

2 c R2

O O

N

R1

e R2 HO

3

f

R2

O

4a

O

R1

O

N N

O

R1

N

g

R3 N

R2 O

+ O

R3 N Br +

5

O

O

NH2

H N

O

O

4

O

d

N

R1

e

R2 HO

N

R3 N CF3CO2+

N O

5a

R1

R1 + TEA.HBr 6

Fig. 2. Double-linker route used in development: (a) 20% piperidine/NMP; (b) ortho nitrofluoro/-chloro-R1-arene, DIEA, NMP, 12 h, 60 ; (c) SnCl22H2O, NMP, 12 h; (d) R2  CHO, NMP, 12 h, 50 ; (e) TFA, DCM, 30 min; (f) R3-bromide, NMP, 18 h, 60 ; (g) TEA,  DCM, 18 h, 25 .

removed with piperidine. The exposed resin-bound amine acts as the starting anchor for building diverse benzimidazole compounds by the sequential reaction of three components, namely ortho-fluoro- or ortho-chloronitroarenes, aldehydes, and alkyl- or benzylbromides. The fluoronitroarenes are added to give resin 2, with the expected steric and electronic considerations dictating the kinetics of the nucleophilic substitution reaction. We compensate for the differing kinetics to a large extent by using as high a concentration of the amine as possible in a polar organic solvent, such as NMP or DMSO. Several chloroarenes are useful in the scheme, although they tend to require heating and the reactions are often difficult to force to completion. The extent of the reaction is monitored by the ninhydrin test and in cases where an incomplete reaction occurs, the unreacted resin-bound

168

linkers and their applications

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amine is capped by reaction with acetic anhydride/pyridine/DMF for 20 min. Fortunately, this capping step does not acetylate the resin-bound aniline under these conditions, presumably due to the low nucleophilicity of the resin-bound ortho-nitroaniline. The resin-bound phenylene diamine intermediates 3 are then generated by nitro group reduction with tin(II) chloride in NMP and cyclization/ aromatization with a wide variety of aldehydes gave the resin-bound benzimidazole intermediates 4. The treatment of this intermediate with 50% TFA/DCM liberates the substituted 3-(benzoimidazol-1-yl)-propionic acid derivative 4a. Analysis of this intermediate by HPLC and LC-MS gave a measure of the purity of the resin-bound product and enabled the optimization of conditions for the incorporation of the R1-nitroarenes and R2-aldehydes by an iterative process. Incorporation of the third and final diversity element to give resin 5 is achieved via quaternization of the resin-bound benzimidazole intermediates using a large excess of a primary alkyl or benzyl bromide. A high concentration solution of the desired bromide in NMP or DMSO (at least 1.5 M, with at least 30 equivalents over the estimated resin loading) proved effective in most cases, and heating this reaction to around a maximum of  55 helps to achieve a higher final yield in this model system. We observed some premature release of the product from the resin when heating at 60 and above. For the final scheme (see below), modifications to the linker enabled a higher temperature to be used for the reaction with bromides, which increases the yields of the final products by an average of about 20%. Despite the slight temperature sensitivity of this two-linker model system, it was valuable in determining the optimal conditions for quaternization using different classes of alkylating agent. Treatment of 5 with TFA releases the quaternary salt derivative 5a, which enables assessment of the success of the alkylation conditions. Again, evaluating different conditions enabled us to optimize this reaction for a range of alkylating agents. Despite the fact that the quaternization event occurs on a nitrogen atom that is not directly attached to the resin, we were gratified to observe that the products 6 are indeed released by base treatment. Release of the desired compounds from the resin was carried out in the development stages with various proportions of TEA or DIEA in DCM. We later observed that release could be carried out in several different solvents using one of a variety of organic and inorganic bases.13 For single compound 13

For example, ammonia in dioxane or methanol and inorganic bases, such as sodium hydroxide or ammonium hydroxide solutions. Such cleavages gave satisfactory results in a variety of solvents, such as N-methylpyrrolidinone, dimethyl sulfoxide, acetonitrile, methanol, dioxane, and acetone.

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traceless solid-phase organic synthesis

169

work at the development stage, a single treatment with 5% TEA in DCM for 16 h gives the highest yields of compound recovery. The excess TEA salts can be removed by extraction, prior to purification of the desired compound (if necessary), to recover the product 6 as a white solid in most cases. The double-linker construct again proved valuable in determining the end point and yield of the cleavage reaction under different reaction conditions. After treatment with base to release the product, the resin is thoroughly washed with DCM then subjected to TFA treatment. This allows us to examine the products remaining on the resin. If there was incomplete Hofmann elimination of the desired product, compound 5a is observed. By optimization of the basic conditions, the presence of 5a can be reduced to low levels or often completely eliminated. This procedure is allied with standard gravimetric analysis of the expected compound to determine the cleavage yields for a range of compounds. In our hands, the cleavage yields (i.e., target compound removed from the resin) is typically >80%, with generally less than 10% of the desired material remaining bound to the resin.14 Library Rehearsal

Despite the variable yields of the products, we observe that they are very pure, almost without exception, directly after cleavage from the resin (usually >90% of target compound by HPLC trace integral). Interestingly, combinations of the R1, R2, and R3 monomers that interact unfavorably to produce a low yield of target compound (as judged by both the resin and gravimetric analyses as outlined above) always give pure products upon cleavage. It became obvious that optimization of the R1 and R2 monomer combinations (that lead to high-purity resin-bound intermediate 4) would give the best chance for eventual product formation. Most commercially available aromatic and some aliphatic aldehydes work well in the synthetic scheme. Some examples of the R1 monomers used for subsequent library formation are shown in Fig. 3. Having determined the most successful R1/R2 combinations, we screened a wide variety of alkylating agents for their ability to quaternize resin intermediate 4. For several types of resin intermediate 4 (chosen 14

To further elucidate this point, we were able to remove from the resin about four-fifths of the quaternized target compound that had been synthesized (this was judged using the double-linker approach mentioned above). The variability in the actual amount of target material synthesized on (and competent to be released from) the resin was dictated by the reaction (in)compatibility of the three monomers comprising each individual benzimidazole compound.

170

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linkers and their applications

F O2N

O2 N

O2N

F

F

Cl

O2N

S

O O

F

O

F

Cl

O

O

O2 N F

F

F

Cl O

O2 N F

F Br

O2 N

F

O2N

F

Cl

O O2 N

F

O2N

O2N

O2N O

F

F

O O2N Cl

F

O2 N

O

O2N

Br N

Cl

O

Fig. 3. A selection of nitroarenes used in library production.

to have differing steric and electronic properties), a certain number of alkylating species give acceptable formation of resin 5 under common condi tions (2 M in NMP, 18 h, 60 ). The yield of this reaction largely determines the final yield of the reaction, almost independently of the conditions used for the final base-promoted elimination. Several of the alkylating species used in subsequent library production are shown in Fig. 4. Final Improved Reaction Route

Several modifications were made to the scheme to improve the stability of some of the resin intermediates and provide improved overall yields of the cleaved products. The final scheme used to synthesize tagged libraries is shown in Fig. 5. Two major changes to the double-linker resin used in development were introduced: a modification to the REM-type linker itself and the inclusion of tags for chemical encoding. The new resin/linker is easily made from commercially available reagents. In our hands, the use of PEG-PSbased resins works best for the scheme, which does call for both organic and aqueous reaction conditions. We have previously reported the use of an unencoded version of this route.2 A halogenated PEG-PS resin (TentaGel-Br or ArgoGel-Cl) is reacted with tert-butyl-N-(2-mercaptoethyl)carbamate to give resin 7. Coupling of this reagent to the resin introduces both the amine function that serves as an anchor for benzimidazole synthesis, as well as a sulfur group. The sulfur is later oxidized to a sulfoxide providing the driving force for the elimination reaction that ultimately releases the final products. For library synthesis,

[9]

171

traceless solid-phase organic synthesis Br

Br

Br

Br

Br

Br

F

Br

F

F

F

F Br

Br

NC

Br O2 N

Cl

F Br

Br

CF3 O

Br

Br

CF3

F CF3 Br

Br

Cl

Br

O O2 N

N

S

CF3

O

N

Br

CF3

Br

O

Br

O

Br

Br

Br

NH2 Br

O

Fig. 4. A selection of alkyl and benzyl bromides used in library production.

H N

S HN

Alloc

h, i, b

R2 O S O HN

Tag1 Tag2

S

tBoc

7

HN

R1

R1

10

O S O

f HN

S

R1 HN

R2

N

Tag 1 Tag 2

N

g

9

Tag1 Tag2

R3 N Br + R1

11

N N

j, d, h

8

Tag1 Alloc

N

R2

NO2

H N

O S O HN

Tag1 Tag2

12

R3 N

R2 +

k

N R1 + TEA.HBr

6

Fig. 5. Final library scheme. Conditions as in Fig. 2 and (h) Alloc removal and tag coupling;  (i) TFA, dimethylsulfide, DCM, 2  30 min, then DIEA, DCM, 2  30 min, 25 ; (j) Na2S2O4,   water, MeOH, 16 h, 25 ; (k) aqueous Oxone, 12 h, 25 .

we have devised a method for differentiating the resin, such that approximately one-tenth of the available functionality was reserved for encoding procedures. Prior to acidolysis to remove the tBoc protecting group, the resin is divided and each resin pool is encoded using the tagging strategy. Each tagged resin pool is then treated with the nitroarenes as before to give resin 8 and the resins are pooled prior to nitro group reduction.

172

linkers and their applications

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The nitro group reduction is carried out on the pooled resin using sodium hydrosulfite in water to form the resin-bound substituted phenylene diamine precursor.15 The resin is again split into smaller pools and the second diversity elements (various aliphatic and aromatic aldehydes) are added. As previously observed, no exogenous oxidants are necessary to form the required resin-bound benzimidazoles. After the final round of tagging, the resin 9 is once again pooled and treated with a chemical oxidant to convert the sulfur group to a sulfoxide. Aqueous Oxone proved most effective, with complete conversion observed after overnight treatment. Prewashing of the resin with methanol aids in its subsequent solvation by the Oxone solution. The resin is then split into pools for addition of the final diversity elements, the alkyl and benzyl bromides. This proved to be the step in the synthesis scheme that largely determines the final yield of product, as noted before. Quaternization of the resin-bound intermediates 10 is carried out with a high concentration solution of the desired alkylating agent in NMP or DMSO to give resin 11. Heating this reaction to around a maximum of  70 helps to achieve higher final yield. We had previously determined that this sulfur-based linker has increased temperature stability compared to that used in the development stage. In model studies, we observe some premature release of the product from this resin only when heating at 90 and above. These final diversity elements were ‘‘spatially encoded,’’ i.e., the resin pools are kept separated and assayed separately, to allow for identification of the final monomers without the need for a further chemical encoding step. As a result no further pooling was necessary after the quaternization reaction and we were able to tailor the most favorable reaction conditions for incorporation of the required alkylating agents in each reaction. The release of the final products from the beads can be achieved using several different procedures, as previously determined at the development stage. For library production, we are able to release sufficient compound by solvating the beads with DMSO and subjecting them to treatment with ammonia gas.16 After release of the desired compound 6, we recover the encoding bead 12 and carry out the decoding procedures to determine the identity of the R1 and R2 monomers from beads of interest (after various assays have been carried out on the released compounds).

15 16

R. A. Scheuerman and D. Tumelty, Tetrahedron Lett. 41, 6531 (2000). R. Brown, J. Comb. Chem. 1, 283 (1999).

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traceless solid-phase organic synthesis

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Conclusion

This section described the successful development and implementation of a traceless synthetic route to create libraries of chemically diverse benzimidazole compounds. The chemical route delivers compounds in moderate yields but in high purity directly after cleavage from the solid support. The basic concept of this traceless approach has been applied to several other related heterocyclic systems that will be reported in due course.17 Experimental

Reagents and General Methods We have previously described the basic resin handling and washing procedures, as well as nitro group reduction, cyclization with aldehydes to form the benzimidazole ring, and chemical encoding procedures for a related benzimidazole system.18 Reagents and solvents used are available from Aldrich (Milwaukee, WI) and Calbiochem-Novabiochem (San Diego, CA). General Procedure for Coupling of o-Fluoro/Chloro-Nitroarenes The same procedure is used for the formation of resins 2 and 8 by reaction of the nitroarenes with the resin-bound -alanine or mercaptan/ amine linker, respectively. The o-fluoronitroarenes (Fig. 3) are dissolved in DMSO or NMP (at a concentration between 1.5 and 2 M) and added to the resin, followed by diisopropylethylamine (10 equivalents) and additional DMSO or NMP (if required) to ensure resin solvation. Although many nitroarenes react rapidly at room temperature, in a library format  the resin/nitroarene mixture is heated overnight at 50 to help achieve equivalent reaction kinetics between different monomers. Under the same conditions, some o-chloronitroarenes are synthetically useful. The extent of the reaction can be assessed qualitatively (or quantitatively, if desired) by carrying out a ninhydrin test to check for the presence of free amine. In any case, the resins are acetylated with five equivalents of acetic anhydride/pyridine/DMF (1:1:10) for 20 min to cap any unreacted amine.

17 18

D. Tumelty, unpublished results (2000). D. Tumelty, L.-C. Dong, K. Cao, L. Le, and M. C. Needels, in ‘‘High Throughput Synthesis’’ (I. Sucholeiki, ed.), p. 93. Marcel Dekker, New York, 2001.

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[9]

Procedures for Reduction of the Aromatic Nitro Group For formation of resin 3, the resin is washed in NMP (20 ml/g of resin), filtered, and left solvated. Separately, tin(II) chloride dihydrate (approximately 40 equivalents with respect to resin-bound nitro groups) is dissolved in NMP with vigorous stirring, then the solution is added to the resin and mixed by nitrogen bubbling for 12 h at room temperature. The resin is filtered, washed, and left solvated prior to the next synthetic step. For reduction of resin 8, concerns about the possibility of traces of tin by-products contaminating subsequent assays led to the adoption of a different reduction procedure for library production.15 The tagged resins 8 are combined into one pool in a large peptide synthesis vessel, washed with methanol, filtered, and the resin left solvated. Separately, an aqueous solution of 0.5 M aqueous sodium hydrosulfite/0.5 M potassium carbonate is prepared and added to the resin (40 ml/g of resin) and then the resin/solution is bubbled with nitrogen at room temperature for 16 h. The resulting resin is washed with water, water/MeOH (1:1), MeOH, MeOH/NMP (1:1), NMP, DCM, MeOH, and ether, then filtered and dried overnight in vacuo prior to the next step. General Procedure for Quaternization with Alkyl/benzyl Bromides Resin 4 or 10 is solvated by washing in NMP. For quaternization, a benzyl or alkyl bromide (50 equivalents) is dissolved in NMP to give a final solution with a concentration of 2 M. This solution is added to the  resin in a glass vial and stirred at 50–70 for 18 h. After this time, the dark brown resin is transferred to a polypropylene tube and washed with NMP, DCM, MeOH, then finally diethyl ether and dried overnight in vacuo. Preparation of Resin 7 A PEG-PS resin is subjected to a proprietary procedure where about 90% of the initial amine functionality is loaded with a moiety bearing a reactive bromide, while the remainder has an amine function protected by an allyloxycarbonyl group. The resin (40 g, approximately 15 mmol with respect to the bromine group) is solvated with NMP (200 ml) in a 1-liter pear-shaped flask, fitted with a nitrogen-bubbler. t-Butyl-N-(2-mercaptoethyl)carbamate (Aldrich, 20 ml, 7 equivalents; caution: Stench!) is added, followed by solid potassium carbonate (9 g, 4 equivalents), and the  resulting resin/solution is stirred with an overhead paddle-stirrer at 60 for 12 h in a thermostatically controlled oil bath. After this time, the slurry is transferred to a 2-liter peptide synthesis vessel and the resin is subsequently washed under vigorous nitrogen bubbling using NMP, NMP/water, MeOH/ water, water, MeOH/water, NMP/water, NMP, DCM, MeOH, and ether

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(3  250 ml each), and finally dried overnight in vacuo. A pale yellow resin is obtained (43 g). A small resin sample is taken and, after removal of the tBoc group, the loading of the resin is assessed using two complementary methods: either a quantitative ninhydrin test19 or coupling an Fmoc group to the exposed amine, deprotecting with piperidine in DMF (1:4), and quantitatively assessing the concentration of the dibenzofulvene adduct formed at 302 nm.20 Either method usually gives loading values between 0.30 and 0.33 mmol/g for the amine linker. Preparation of Resin 10 The tagged resins are pooled into one large batch in a 2-liter peptide vessel, washed with methanol, and then left solvated. Separately, solid Oxone is dissolved in water (to a final concentration of 0.4 M), sonicating for 5 min to aid in solvation. The aqueous Oxone solution (10 equivalents with respect to the nitro group loading of the resin) is added to the methanolsolvated resin and stirred/bubbled for 16 h at room temperature. The resulting resin 10 was then washed with water, MeOH/water (1:1), MeOH, MeOH/NMP, NMP, DCM, and ether, then dried in vacuo overnight prior to the next step. Traceless Syntheses Using a Novel Triflate-Type Linker Background

Our goal for this work was somewhat different from the preceding traceless benzimidazole syntheses. Here we aimed to develop a novel solid-phase linker that would serve as an activating group for a wide variety of phenols, permitting subsequent transformations to occur between the resin-bound phenol and a variety of different classes of input molecules. The strategy is based on the well-known activation properties of triflates, which are widely used as precursors for aryl and vinyl cations due to their excellent leaving group properties.21 Once an oxygen atom on the phenol moiety is activated by the triflate (trifluoromethanesulfonyl) group, it becomes possible to carry out a reductive cleavage (essentially deoxygenating the phenol) or cross-coupling reactions, e.g., through palladium-catalyzed Suzuki, Stille, and Heck reactions.22 This gives rise to a variety of 19

V. K. Sarin, S. B. H. Kent, J. P. Tam, and R. B. Merrifield, Anal. Biochem. 117, 147 (1981). M. K. Schwarz, D. Tumelty, and M. A. Gallop, J. Org. Chem. 64, 2219 (1999). 21 J. F. Hartwig, Angew. Chem. Int. Ed. Engl. 37, 2046 (1998). 22 B. A. Lorsbach and M. J. Kurth, Chem. Rev. 99, 1549 (1999). 20

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substituted aromatics or olefins at the ‘‘inert’’ phenolic or vinyl oxygen position. Our goal therefore was to design a triflate-like linker upon which we can conduct such triflate-directed transformations on solid-phase resin and this section describes the successful implementation of this strategy. Perfluoroalkylsulfonyl (PFS) Linker/Resin

We recently reported the synthesis of a perfluoroalkylsulfonyl linker attached to TentaGel resin 13 (Fig. 6), which proves to act in a fashion similar to triflates as we had hoped, and demonstrated its application for the traceless cleavage of phenols using palladium-catalyzed reduction and Suzuki cross-coupling reactions.3,4 The new polymer-supported linker allows the attachment of phenolic rings to the solid phase through the formation of aryl nonflates and subsequent traceless cleavage of the hydroxyl groups on aryl rings. A variety of phenols can be attached to this resin through the formation of the polymersupported perfluoroalkylsulfonates 14 in DMF at room temperature using potassium carbonate as base (Fig. 7). The attachment of phenols to this linker is especially attractive since the mild reaction conditions allow many useful functional groups (such as aldehydes, nitro, carboxylic acids, ketones, and alcohols) to be incorporated without additional protection, and these

H N O

O O S F F F

F F FF O O S O F F F FF

13 Fig. 6. Structure of the perfluoroalkylsulfonyl (PFS) linker/resin.

O O S F F F 13

l R1 HO

O O S O F F 14

m

R1

R1

o

R1 R2 R2

n

R1 N R3

Fig. 7. Divergent syntheses from a PFS-derivatized linker. (l) Substituted phenol, potassium carbonate, DMF; (m) Suzuki reaction; (n) reduction; (o) amine displacement.

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177

groups can themselves serve as combinatorial sites for the synthesis of large libraries. We have found that the resin-bound perfluoroalkylsulfonate species have similar reactivities to aryl triflates, such that most of the known palladium-catalyzed reactions involving aryl triflates were possible on the support (Fig. 7). Thus, a cleavage/cross-coupling strategy of simultaneously introducing diversity while liberating the desired molecule from the support provides a powerful technique for the traceless synthesis of molecules. We have initially targeted the Suzuki and Buchwald amination reactions as methods for generating biaryls and anilines, respectively. Cleavage of the Resin-Bound Phenols Using the Suzuki Coupling Reaction

The Suzuki coupling reaction is a powerful tool for carbon–carbon bond formation in combinatorial library production.23 Many different reaction conditions and catalyst systems have been reported for the cross-coupling of aryl triflates and aromatic halides with boronic acids in solution. After some experimentation, we found that the ‘‘Suzuki cleavage’’ of the resinbound perfluoroalkylsulfonates proceeded smoothly by using [1,10 -bis (diphenylphosphino)ferrocene]dichloropalladium(II), triethylamine, and boronic acids in dimethylformamide at 80 within 8 h afforded the desired biaryl compounds in good yields.24 The desired products are easily isolated by a simple two-phase extraction process and purified by preparative TLC to give the biaryl compounds in high purity, as determined by HPLC, GC-MS, and LC-MS analysis. A small library of biaryl compounds was synthesized in order to examine the scope and generality of the resin-bound PFS linker and the traceless ‘‘Suzuki cleavage’’ strategy, as shown in Fig. 8. The aryl perfluoroalkylsulfonate resin 15 is prepared by attaching 4-hydroxybenzaldehyde to resin. Resin 16 is prepared by a reductive amination of 15 with primary amines using sodium cyanoborohydride as the reducing agent. The presence of some acetic acid is also important in this step to promote the reductive amination reaction. The secondary amines generated in this step are used as another diversity site through functionalization of this amine. Biaryls 18 are produced in a traceless fashion from resin 17 in yields ranging from 65 to 90% upon exposure to the Suzuki conditions. We have observed that most boronic acids are suitable for this cleavage/cross-coupling procedure to generate a wide variety of molecules.

23 24

J. Hassan, M. Sevignon, C. Gozzi, E. Schulz, and M. Lemaire, Chem. Rev. 102, 1359 (2002). B. Ruhland, A. Bombrun, and M. A. Gallop, J. Org. Chem. 62, 7820 (1997).

178

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linkers and their applications O O S O F F 15

O

O O S O F F 17

N R1

NH R1

O O S O F F 16

p

O

q

O R2

r

N R1

Ar

R2

18

Fig. 8. A four-step synthesis on the PFS linker. (p) R1-NH2, Na(CN)BH3, AcOH, THF; (q) R2-NH2, TEA, CH2Cl2; (r) ArB(OH)2, Pd(dppf)Cl2, TEA, DMF.

Cleavage of the Resin-Bound Phenols Using Catalytic Reductive Elimination

The deoxygenation of phenols by a palladium-mediated reduction in solution phase is well established.25 Reductive cleavage of the polymersupported aryl triflate-type species allows phenols to be cleaved from the resin without any trace of the phenolic hydroxyl group. This strategy is valuable for combinatorial syntheses since the structure of the final product cleaved from the resin is independent of the position of the hydroxyl group on the starting phenol and enables greater flexibility in choosing the building blocks for library syntheses. The deoxygenation of the polymersupported aryl nonaflate species was studied through a palladium-mediated reduction reaction. We discovered that the polymer-bound aryl nonaflates were efficiently cleaved with a mixture of triethylamine and formic acid in the presence of a catalytic amount of palladium(II) acetate and 1,3-bis(diphenylphosphino)propane to afford high yields of the reduced arenes under mild conditions. The desired products are again isolated by a twophase extraction and the trace metal catalyst is removed by eluting the organic solution through a thin pad of silica gel. The resulting products are obtained in good yields as determined by HPLC, GC-MS, and LC-MS. Figure 9 shows two examples of the traceless cleavage of the resin-bound phenols using this palladium-catalyzed reductive elimination. It is notable that the cleaved product 20 does not have any remnant of the anchoring hydroxyl group and also that the polymer-bound perfluoroalkylsulfonate served here as a linker, a protecting group, and an activating group for the phenols. The aryl perfluoroalkylsulfonyl resin also permits monoattachment of a symmetric bisphenol to form resin 21 and only the attached

25

S. Cacchi, P. G. Ciattini, E. Morera, and G. Ortar, Tetrahedron Lett. 27, 5541 (1986).

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traceless solid-phase organic synthesis O O S O F F 19

O O S O F F 21

N

s N 20

s OH

OH 22

Fig. 9. Reductive cleavage from the PFS linker. (s) Pd(OAc)2, dppp, TEA-HCO2H,

phenol group is deoxygenated, leading to a nonsymmetrical phenol 22 upon reductive cleavage. The application of resin 13 to the solid-phase synthesis of other useful target compounds was also explored and an example of this is the multistep synthesis of Meclizine (Fig. 10).26 The starting material, 3-methyl-4-hydroxybenzaldehyde, is attached to the PFS linker, and a polymer-bound amine intermediate is prepared by a reductive amination of resin 23 with amine 24. The resulting resin 25 is subjected to a palladium-mediated reductive cleavage to give Meclizine 26 in 80% yield, based on the original resin loading. Cleavage of the Resin-Bound Phenols Using Catalytic Amination

Substituted anilines often appear as a key element in biologically active compounds. The palladium-catalyzed amination of aryl triflates has drawn increasing interest as a synthetic route to a wide variety of aryl amines.27 The diversity of phenols and amines that is available, along with the simple attachment of phenols to the PFS linker, suggested to us that the catalytic amination of resin-bound aryl triflate species would provide another useful synthetic route to aryl amines. Figure 11 shows a general solid-phase protocol for the traceless cleavage of phenols from the PFS linker using catalytic amination. The reaction is carried out with phenolloaded resins 19 or 28, palladium(II) acetate, BINAP, cesium carbonate,  and the corresponding amines in THF at 80 for 16 h. The desired products 27 and 29 are obtained in 70–80% yields based on the actual loading of the 26

Meclizine is an oral antiemetic used to treat nausea, vomiting, and dizziness associated with motion sickness. Compound 26, synthesized by our route, was spectroscopically identical with a commercially obtained sample (Sigma). 27 ˚ hman and S. L. Buchwald, Tetrahedron Lett. 38, 6363 (1997). J. A

180 O O S O F F 23

[9]

linkers and their applications

O

Cl

HN N

Cl

N

O O S O F F 25

p

N

24 Cl

N

s

N

26 Meclizine

Fig. 10. Synthesis of Meclizine on the PFS linker.

O O S O F F 19

N

t HN

O O S O F F 28

N

N N 27

N Et

N

t

N

O

N HN

O

29

Fig. 11. Cleavage from the PFS linker with amines. (t) Pd(OAc)2, BINAP, Cs2CO3, THF.

phenols. The traceless ‘‘amination cleavage’’ approach permits the introduction of a new aromatic amine functionality at the phenolic oxygen position during cleavage and provides a powerful method to synthesize libraries with rich synthetic diversity. Conclusion

The resin-bound perfluoroalkylsulfonyl linker is compatible with many common solid-phase reactions, such as tin dichloride-mediated aromatic nitro group reduction, trifluoroacetic acid-mediated tBoc deprotection, reductive amination reactions, acylation, and sulfonation. It is possible to perform several sequential synthetic reactions on the nonflate resin so that multistep syntheses can be carried out. The solid-phase approach provides an operationally simple, inexpensive, and general protocol for the cleavage

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181

of the aryl-oxygen bond. We anticipate that the ease of preparation, excellent stability, and synthetic versatility of the polymer-supported linker will prove useful in solid-phase and combinatorial chemistry. Since a large number of phenols with a variety of functional groups are commercially available, and a variety of palladium-mediated reactions can be used for the resin-bound aryl triflates, our novel resin-bound perfluoroalkylsulfonyl linker will provide a powerful method to synthesize structurally diverse libraries. Experimental

Reagents and General Methods All starting materials were obtained from Aldrich (Milwaukee, WI). TentaGel resin was obtained from Rapp Polymere (Tubingen, Germany). Procedures for the synthesis of the PFS linker and its attachment to resin to form 13 have been previous described.3 General Procedure for Attachment of Phenols to Resin 13 (to form 14, 15, 19, 21, 23, or 28) The phenol (20 equivalents), potassium carbonate (22 equivalents), and resin 13 (1 equivalent with respect to the sulfonyl fluoride group) are mixed with DMF (10 ml/g of resin) and shaken overnight at room temperature. The resin is filtered and washed with water, DMF, and DCM, and then is dried under vacuum overnight to give the required resin-bound phenol. General Procedure for Cleavage of Phenols Using the Suzuki Coupling Reaction: Preparation of Resins 15–17 and Compounds 18 A mixture of 4-hydroxybenzadehyde (5.0 mmol), potassium carbonate (6.6 mmol), and resin-bound linker 13 (3.0 g, 1.0 mmol) is added to DMF (8.0 ml) and the mixture was shaken at room temperature overnight. The resin was filtered and washed with water, DMF, and DCM, and then dried under vacuum overnight to give resin 15. A portion of the dried resin (0.50 g, 0.16 mmol) is then mixed with a primary amine (R1-NH2, 2.0 mmol), THF (2.0 ml), Na(CN)BH3 (1 N solution in THF, 2.0 ml, 2.0 mmol), mmol), and acetic acid (0.11 ml, 1.95 mmol) and the mixture is shaken overnight at room temperature. The beads are filtered and washed with water, DMF, and DCM, and dried under vacuum overnight to give resin 16. To a portion of the dried amine resin 15 (0.20 g, 0.066 mmol) is added TEA (2.3 mmol), DCM (4.0 ml), and an acid chloride (R2-COCl, 1.6 mmol)  mmol) at 0 . The mixture was allowed to warm-up and then shaken at room

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temperature overnight. The resin is filtered, washed with DMF and DCM, and dried under vacuum overnight, as before, to give resin 17. For the Suzuki cleavage reaction, a portion of the dry resin 17 (0.20 g, 0.07 mmol) is mixed with Pd(dppf)Cl2 (7.2 mg), an arylboronic acid (0.25 mmol), TEA (0.1 ml, 0.60 mmol), and DMF (2.0 ml) in a glass vial  under nitrogen. The mixture is then shaken at 90 overnight. The polymer beads are next filtered and washed several times with Et2O and the combined organic phase is washed with aqueous 2% sodium carbonate and water and then evaporated to dryness. The crude products are purified by preparative TLC (or other suitable methods) to give the desired products 18 in 65–90% yields, with >98% purity as determined by HPLC. General Procedure for Cleavage of Phenols by a Reductive Elimination Reaction: Preparation of Compounds 20 and 22 To dried resins 19 and 21 (0.1 g resin, approximately 0.04 mmol with respect to the loading of the phenol) are added Pd(OAc)2 (8.0 mg), 1,3-bis (diphenyl-phosphino)propane (dppp, 17.0 mg), DMF (1.4 ml), and a mix ture of HCO2H (0.2 ml) and TEA (0.8 ml). The mixture is shaken at 85 for 2 h, and then the resin is filtered and washed several times with diethyl ether. The combined organic phase is washed with aqueous sodium carbonate solution then water and evaporated to dryness. The residue obtained is dissolved in diethyl ether and eluted through a short column of alumina to remove any remaining inorganic residues. The crude products are purified by preparative TLC (or other suitable methods) to give the desired products 20 and 22 in >95% purity.

[10] Unnatural Diamino Acid Derivatives as Scaffolds for Creating Diversity and as Linkers for Simplifying Screening in Chemical Libraries By Robert Pascal, Re´gine Sola, and Patrick Jouin Introduction

The introduction of conformational restrictions into flexible active molecules is a well-known strategy for trying to increase their potency and/or selectivity toward their biological targets.1 Several methods have been used for constraining flexible molecules. Cyclic derivatives of linear peptides or peptidomimetics can thus be prepared by reactions involving side-chain

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

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NH2 H2N

R

H2N

COOH 1

H2N

COOH 2

H2N (CH2)n HN

COOH

3 (n = 2,3)

Fig. 1. Unnatural aliphatic diamino acids.

functional groups and/or C- or N-termini.2 For this purpose, lactam bridges linking Lys and Asp residues have been introduced in peptides.3 However, selecting linear oligomers prior to introducing conformational restrictions is not essential and combinatorial chemistry has indeed been successful in the direct production of lead compounds from chemical libraries.4 A powerful method is to build libraries starting from suitable scaffolds that display several pendant functional groups to introduce diversity.1,5–7 In this context, amino acids bearing an extra amino functionality are potentially very attractive either for peptide cyclization or as central scaffold structures displaying three points of diversity. A major advantage of carboxyl and amino groups is their compatibility with standard protocols of peptide synthesis. Moreover, hydrophilic amide linkages present in the products are likely to increase their bioavailability as potential drugs. Finally, constructions based on amide bonds are usually chemically stable and their stability toward proteases is likely to be increased if unnatural diamino acids (Fig. 1) are involved. In spite of these useful features, few unnatural diamino acids with appropriate protecting groups have been reported and still fewer are commercially available. A survey of such structures is presented here and some of their possible applications in combinatorial chemistry are mentioned or illustrated by methodological developments carried out in our research group: the use of derivatives of benzoic acid as scaffolds for creating diversity and a procedure for handling a linker for solid-phase synthesis derived from l-2,3-diaminopropionic acid 1

E. M. Gordon, R. W. Barrett, W. J. Dower, S. P. A. Fodor, and M. A. Gallop, J. Med. Chem. 37, 1385 (1994). 2 J. N. Lambert, J. P. Mitchell, and K. D. Roberts, J. Chem. Soc. Perkin Trans. 1, 471 (2001). 3 W. Zhang and J. W. Taylor, Tetrahedron Lett. 37, 2173 (1996) and references cited therein. 4 A. Golebiowski, S. R. Klopfenstein, and D. E. Portlock, Curr. Opin. Chem. Biol. 5, 273 (2001). 5 A. J. Souers and J. A. Ellman, Tetrahedron 57, 7431 (2001). 6 J. A. Ellman, Acc. Chem. Res. 29, 132 (1996). 7 M. Royo, M. del Fresno, A. Frieden, W. Van Den Nest, M. Sanseverino, J. Alsina, S. A. Kates, G. Barany, and F. Albericio, React. Funct. Polym. 41, 103 (1999).

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4 H2 N

NH2

H2N

NH2

COOH

COOH

COOH

H2N

H2N

*

6

*

* COOH N H 7

NH2

H2N

5

COOH * COOH

HN NH2

N H 8

9

H N

HN

COOH

COOH NH 10

N H 11

Fig. 2. Unnatural cyclic diamino acids.

(1) (Dpr) and allowing the mild release of molecules in media that can be readily made compatible with biological assays. Two lower homologues of lysine, Dpr3,8–10 and l-2,4-diaminobutyric acid (2) (Dab), have often been used to introduce conformational restrictions on peptides by lactam bridges. The Dpr to Asp linkage can also be used as a stable surrogate of disulfide bonds for stabilizing loops.11,12 Except for small cyclic systems, the residual conformational flexibility is not likely to provide an appropriate rigidity to structures based on Dpr or Dab residues. A similar finding can be applied to the backbone-to-backbone cyclization strategy based on the use of N-aminoalkyl amino acid residues 3.13 In our opinion, better rigidity may be expected from cyclic building blocks (Fig. 2). Compounds 4–6 containing three- or four-membered rings have been prepared under conveniently protected forms.14,15 They have 8

J. Rizo, S. C. Koerber, R. J. Bienstock, J. Rivier, A. T. Hagler, and L. M. Gierasch, J. Am. Chem. Soc. 114, 2852 (1992). 9 C. H. Hassall, R. G. Tyson, and K. K. Chexal, J. Chem. Soc. Perkin Trans. 1, 2010 (1976). 10 P. Wipf and H.-Y. Kim, Tetrahedron Lett. 33, 4275 (1992). 11 D. Limal, J.-P. Briand, P. Dalbon, and M. Jolivet, J. Peptide Res. 52, 121 (1998). 12 C. Mendre, R. Pascal, and B. Calas, Tetrahedron Lett. 35, 5429 (1994). 13 B. Mu¨ller, D. Besser, P. Kleinwa¨chter, O. Arad, and S. Reissmann, J. Peptide Res. 54, 383 (1999). 14 T. Wakamiya, Y. Oda, H. Fujita, and T. Shiba, Tetrahedron Lett. 27, 2143 (1986). 15 E. Gershonov, R. Granoth, E. Tzehoval, Y. Gaoni, and M. Fridkin, J. Med. Chem. 39, 4833 (1996).

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185

been mainly used as constrained analogues of lysine or ornithine or as intermediates in the preparation of analogues of arginine. Synthetic routes giving access to building blocks derived from all the stereoisomers of 4-aminoproline (7) and its homologue 8 have also been devised.16–18 Residue 9 has been used to promote helix formation in peptides.19 Carboxylic acids 10 and 11, derived from piperazine and imidazolidine, respectively, have been synthesized with orthogonal amino-protecting groups.20–22 The structure of residue 10 is also found in several -turn mimetics.5 Although many residues in Fig. 2 might be used as scaffolds for combinatorial synthesis, it is interesting to point out that only cis-aminoprolines 7 have been considered for such applications.7 Several other protected diamino acids involve aromatic rings (Fig. 3).23–27 Their structures may also be suitable as scaffolds for building synthetic libraries, but this application has been proposed only for compounds 12, 15, and 16.24,25 Whereas most of the structures displayed in Fig. 2 require stereoselective syntheses, the preparation of diamino acids 12–17 is facilitated by the absence of an asymmetric center. One of the most important limitations in the use of these diamino acids is the need of an additional orthogonal protection for the amino groups. Using the t-butoxycarbonyl/benzyl (Boc/Bzl) strategy of solid-phase peptide synthesis, this additional orthogonality can be easily provided by a base-labile protecting group such as the 9-fluorenylmethyloxycarbonyl 16

T. R. Webb and C. Eigenbrot, J. Org. Chem. 56, 3009 (1991). Z. Zhang, A. Van Aerschot, C. Hendrix, R. Busson, F. David, P. Sandra, and P. Herdewijn, Tetrahedron 56, 2513 (2000). 18 M. Tamaki, G. Han, and V. J. Hruby, J. Org. Chem. 66, 1038 (2001). 19 C. L. Wysong, T. S. Yokum, G. A. Morales, R. L. Gundry, M. L. McLaughlin, and R. P. Hammer, J. Org. Chem. 61, 7650 (1996). 20 B. D. Dorsey, R. B. Levin, S. L. McDaniel, J. P. Vacca, J. P. Guare, P. L. Darke, J. A. Zugay, E. A. Emini, W. A. Schleif, J. C. Quintero, J. H. Lin, I.-W. Chen, M. K. Holloway, P. M. D. Fitzgerald, M. G. Axel, D. Ostovic, P. S. Anderson, and J. R. Huff, J. Med. Chem. 37, 3443 (1994). 21 A. M. Warshawsky, M. V. Patel, and T.-M. Chen, J. Org. Chem. 62, 6439 (1997). 22 L. Rene´, L. Yaouancq, and B. Badet, Tetrahedron Lett. 39, 2569 (1998). 23 R. M. Keenan, J. F. Callahan, J. M. Samanen, W. E. Bondinell, R. R. Calvo, L. Chen, C. DeBrosse, D. S. Eggleston, R. C. Haltiwanger, S. M. Hwang, D. R. Jakas, T. W. Ku, W. H. Miller, K. A. Newlander, A. Nichols, M. F. Parker, L. S. Southhall, I. Uzinskas, J. A. Vasko-Moser, J. W. Venslavsky, A. S. Wong, and W. F. Huffman, J. Med. Chem. 42, 545 (1999). 24 B. R. Neustadt, E. M. Smith, T. Nechuta, and Y. Zhang, Tetrahedron Lett. 39, 5317 (1998). 25 R. Pascal, R. Sola, F. Labe´gue`re, and P. Jouin, Eur. J. Org. Chem. 3755 (2000). 26 M. H. Gelb and R. H. Abeles, J. Med. Chem. 29, 585 (1986). 27 V. Santagada, F. Fiorino, B. Severino, S. Salvadori, L. H. Lazarus, S. D. Bryant, and G. Caliendo, Tetrahedron Lett. 42, 3507 (2001). 17

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Fmoc NH

NH Boc

NH2 COOH

Boc NH COOH

Z N H

COOH

H2N 12

13

Boc N H H2N

COOH 15

Fmoc N H H2N

14

COOH

Boc N H Fmoc HN

16

COOH 17

COOH N

H 2N

Fmoc

18

Fig. 3. Unnatural arylamino amino acid building blocks.

(Fmoc) group. Using the Fmoc/t-butyl strategy, three main orthogonal classes of protecting groups are available and have been recently reviewed.28 The first class consists of highly acid-labile groups such as 2-(4-biphenyl)isopropoxycarbonyl (Bpoc), trityl (Trt), or derivatives, and , -dimethyl -3,5-dimethoxybenzyloxycarbonyl (Ddz), which can be removed in the presence of the t-butyl group with practically complete selectivity. The second one involves groups removed by hydrazine such as the 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) group or preferentially its isovaleryl analogue, Ddiv, which may avoid the intramolecular migration observed with the Dde group.29 The third one involves the palladium-labile allyloxycarbonyl group (Alloc). However, in the case of the aromatic protected scaffolds 12, 15, and 16, the protection of the arylamino group was shown to be useless because it can be replaced by the choice of selective coupling conditions.24,25 Our interest in diamino acid building blocks or scaffolds is connected with the studies of a new type of safety-catch linkers based on a diamino acid residue that we have carried out.30–32 Safety-catch linkers33 for 28

F. Albericio, Biopolymers 55, 123 (2000). S. R. Chhabra, B. Hothi, D. J. Evans, P. D. White, B. W. Bycroft, and W. C. Chan, Tetrahedron Lett. 39, 1603 (1998). 30 R. Sola, P. Saguer, M.-L. David, and R. Pascal, J. Chem. Soc. Chem. Commun. 1786 (1993). 29

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H2N H2N

COOH

i, ii, iii

H2N

COOH

iv

15 or 16

AmAbz

Fig. 4. Preparation of the AmAbz scaffold and of its protected derivatives 15 and 16. (i) MeOCOCl, Na2SO4, dioxane; (ii) N-hydroxymethylphthalimide, 96% H2SO4–H2O (9:1, v/v); (iii) NaOH, H2O; (iv) Boc2O or Fmoc–OSu, NaOH, dioxane-H2O.

solid-phase synthesis are designed to be cleaved by performing two different reactions with the advantage of providing an increased stability during the synthesis. Indeed, the Dpr(Phoc) linker30–32 (Phoc ¼ phenyloxycarbonyl) is incorporated by formation of amide bonds as stable as any peptide bond, but it can be activated as a cyclic N-acylurea and, thereby, made sensitive to nucleophiles at the end of the synthesis. It is well suited for the synthesis of peptides or peptidomimetics on hydrophilic supports and it is compatible with Boc and Fmoc strategies. Moreover, due to the stability of this linker in strongly acidic media, side-chain-protecting groups can be removed in an independent step preceding the cleavage in weakly alkaline aqueous solution. This linker could then become a powerful tool for the preparation of libraries of peptides or peptidomimetics free of deprotection contaminants and suitable for direct biological assays after addition of an appropriate buffer. The Dpr(Phoc) linker is therefore fully compatible with the high-throughput screening of libraries synthesized via solid phase, which requires ready purification procedures. Indications that related aromatic structures might improve the cleavage rate have also been reported.34 Methodology for the Use of the Protected Aromatic Scaffold 16

4-Amino-3-(aminomethyl)benzoic acid (AmAbz) can be easily prepared in three steps by amidomethylation of aminobenzoic acid (Fig. 4).25 Then the benzylamino group can be selectively protected by reaction with mild reagents such as Boc2O or Fmoc–OSu capable of discriminating between the two amino groups to give the building blocks AmAbz(Boc) (15) and AmAbz(Fmoc) (16), respectively (by convention, the 4-amino group is defined here as the main chain and the 3-aminomethyl group as 31

R. Sola, J. Me´ry, and R. Pascal, Tetrahedron Lett. 37, 9195 (1996). R. Pascal and R. Sola, Tetrahedron Lett. 38, 4549 (1997). 33 M. Patek and M. Lebl, Biopolymers 47, 353 (1998). 34 R. Pascal, D. Chauvey, and R. Sola, Tetrahedron Lett. 35, 6291 (1994). 32

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Fig. 5. Solid-phase synthesis of the AmAbz scaffold-containing heptapeptide 19. (i) Piperidine–DMF (1:4);(ii) amino acid building block, BOP/HOBt/DIEA, DMF;(iii) FmocPhe, DIC, CH2Cl2; (iv) TFA, H2O, TIS (95:2.5:2.5, v/v/v).

the side chain for building abbreviations). The potential application of this scaffold to the preparation of a library containing the 6.4  107 heptapeptides obtained by combination of the 20 natural amino acids is illustrated by the solid-phase synthesis of the branched heptapeptide 19 (Fig. 5). Aminomethylpolystyrene resin (0.56 mmol/g) is derivatized with the Rink amide linker and the two residues (Gly and Val) at the C-terminus can be introduced by standard Fmoc-based solid-phase methods of peptide synthesis. At that time, AmAbz(Fmoc), which is now commercially available, can be introduced using BOP* activation in the presence of HOBt. With *

Abbreviations: BOP, benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate; DCC, N,N0 -dicyclohexylcarbodiimide; DIC, N,N0 -diisopropylcarbodiimide; DME, dimethoxyethane; DMF, N,N-dimethylformamide; DIEA, N-ethyldiisopropylamine; Et2O, diethyl ether; HOBt, 1-hydroxybenzotriazole; HOSu, N-hydroxysuccinimide; t-BuOMe, tert-butylmethylether; TFA, trifluoroacetic acid; TIS, triisopropylsilane.

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189

this acylation method, involving hydroxybenzotriazole (HOBt) active esters, the free arylamino group is unaffected.25 The next residues can be selectively attached to the benzylamino group by using the same method. Then, more powerful conditions are required to acylate the arylamino group. Although PyBroP with diisopropylethylamine (DIEA) in CH2Cl2 has been reported to be efficient,24 we have preferred a coupling procedure involving diisopropylcarbodiimide (DIC) activation in CH2Cl2 because basic conditions are likely to promote the racemization of the activated amino acid. As a matter of fact, the arylamino group is not protonated in the medium owing to its low basicity and can thus be acylated even in the absence of base. The next residues can be introduced using standard coupling conditions. The side-chain-protecting groups can be removed and the product released by acidolysis. Methodology for the Use of Dpr(Phoc) Linker

The attachment of the Dpr(Phoc) residue to the support can be made, as previously reported,30–32 by coupling the Boc-Dpr(Phoc) building block. It is worth mentioning that this building block could not be obtained as a crystalline solid, a practical consideration to keep in mind when using this linker. An improvement based on the preparation of the crystalline activated ester Boc-Dpr(Phoc)–OSu is presented here as a convenient alternative route as well as the procedures for applying it to Boc or Fmoc methods of solid-phase synthesis (Fig. 6). Before the cleavage by mild alkaline hydrolysis into carboxylic acid, the stable C-terminal amide linkage must be converted into the labile acylurea via an intramolecular reaction induced by the breakdown of the phenyl carbamate moiety under mild alkaline conditions. At this activation stage, high selectivity is needed. To prevent any side reaction of the preceding amide group at this stage, the linker must be attached to solid supports bearing secondary amino groups. Thus Tentagel S-NH2 resin can be modified with Boc-Sar (Sar ¼ N-methylglycine). After the deprotection step, Boc-Dpr(Phoc)–OSu is reacted with the resin in the presence of HOBt and DIEA. The reaction generally proceeds to completion within 15–20 h as indicated by the chloranil test.35 The resin can then be used for solid-phase synthesis. Using Fmoc strategy, the linker must be cyclized into the Imc (Imc ¼ 2-oxoimidazoline-5-carboxylic acid residue) form, which is resistant to piperidine treatment.31 This operation can be better carried out after the attachment of the C-terminal residue except for sequences that are prone to diketopiperazine formation, which then

35

T. Vojkovsky, Peptide Res. 8, 236 (1995).

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linkers and their applications

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Fig. 6. Use of Dpr(Phoc) linker in Boc (left) or Fmoc (right) chemistries. PG, Boc or Fmoc; scPGs, side-chain-protecting groups.

would require special treatment.31 After peptide chain elongation, the sidechain-protecting groups can be removed with the usual reagents for Boc or Fmoc strategies prior to the alkaline cleavage of the linker; purification steps are not essential when using this procedure since the deprotection

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contaminants are removed by simply washing the resin. Finally, crude products of acceptable purity can generally be released in solution from the support by a 1–3 h treatment with 0.01 N NaOH in water or 2-propanol–water (7:3, v/v) at room temperature. Experimental Protocols

General A reaction vessel equipped with a fritted disc, a solvent inlet, and a device to transfer in and out liquids under low nitrogen pressure was used to carry out solid-phase syntheses. This manual apparatus enables suspensions of resin to be stirred either by gently rocking the vessel on a shaker or by bubbling nitrogen through the fritted plate. Chemicals Aminomethylpolystyrene resin (0.56 mmol/g, NovaBiochem, La¨ufelfingen, Switzerland) TentaGel S-NH2 resin (0.28 mmol/g, RappPolymere, Tu¨bingen, Germany) Fmoc-Rink amide linker (NovaBiochem, La¨ufelfingen, Switzerland) AmAbz(Fmoc) (16) (prepared according to Pascal et al.25 or commercially available from Senn Chemical AG, Dielsdorf, Switzerland) Use of AmAbz Building Blocks in the Solid-Phase Synthesis of Peptidomimetics: The Typical Example of Heptapeptide 19 25 Elongation. Aminomethylpolystyrene resin (1 g, 0.56 mmol) is first washed with DMF, CH2Cl2, TFA/CH2Cl2 (1:1, v/v), CH2Cl2, DIEA/ CH2Cl2 (1:20, v/v), CH2Cl2, and DMF four times each. Then, Fmoc-Rink linker (0.45 g, 0.84 mmol) is added to the resin with DMF (3 ml), BOP (0.37 g, 0.84 mmol), and DIEA (0.22 ml, 1.26 mmol) and the mixture is shaken for 90 min. The resin is filtered and washed with DMF, and then DIEA (0.29 ml, 1.68 mmol) and acetic anhydride (0.53 ml, 5.6 mmol) are added to cap unreacted amino groups. Except for Fmoc-Phe, the incorporation of the next amino acids is carried out as follows: (1) the Fmoc group is removed with piperidine/DMF (1:4, v/v) (1  1 min þ 3  3 min) and the resin is filtered and washed with DMF (4  1 min); (2) the N-protected amino acid building block (1.85 mmol) and HOBtH2O (0.26 g, 1.68 mmol) mmol) are then added to the resin using a minimum volume of DMF, then DIEA (0.44 ml, 2.52 mmol) and BOP (0.743 g, 1.68 mmol) are added and

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linkers and their applications

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the mixture is shaken for 60 min. The resin is filtered and washed with DMF (4  1 min) and subjected to the next deprotection step or, at the end of the synthesis, it is filtered, washed with DMF, CH2Cl2, MeOH, 100% EtOH, and Et2O, and then dried. Acylation of the Arylamino Group. The resin is washed with DMF (4  1 min) and CH2Cl2 (4  1 min). A solution of Fmoc-Phe (0.716 g, 1.85 mmol) in CH2Cl2 (10 ml) is added to the resin then DIC (0.263 ml, 1.68 mmol) is added to the mixture. CH2Cl2 (10 ml) is added 5 min later because of the precipitation of a Fmoc-Phe-activated species and the reaction mixture is shaken for 3 h. Fmoc-Phe coupling is then repeated using a similar procedure except that DMF (6 ml) is added after 5 min and CH2Cl2 is evaporated off by nitrogen bubbling during the reaction. Release and Recovery of Peptide 19. The dried resin (0.21 g) is suspended in TFA/H2O/TIS (95:2.5:2.5, v/v/v, 8.4 ml) and the mixture is shaken for 3 h. The suspension is filtered and the resin is washed with TFA (2  2 ml). The filtrate is concentrated, diluted with Et2O (100 ml), and then extracted with water (2  20 ml). The combined aqueous layers are concentrated and freeze-dried to give peptide 19 as a white solid (20 mg). Preparation and Use of Dpr(Phoc) Linker Preparation of Boc-Dpr.30,36 A mixture of diacetoxyiodobenzene (24.16 g, 75 mmol), acetonitrile (100 ml), and water (100 ml) is stirred until almost complete dissolution. Acetic acid (8.6 ml, 150 mmol) is then added and solid Boc-Asn (11.61 g, 50 mmol) is introduced into the flask with acetonitrile (25 ml) and water (25 ml). The mixture is stirred at room temperature for 24 h. The phenyl iodide by-product is removed by extraction with t-BuOMe (2  100 ml). The aqueous layer is concentrated under reduced pressure and the solid residue is suspended in cold EtOH (100 ml), collected by filtration, and washed with cold EtOH then with Et2O, and dried under vacuum to give crude Boc-Dpr as a white crystalline solid (7.62 g, 75%). Preparation of Boc-Dpr(Phoc).30 Crude Boc-Dpr (4.52 g, 22.1 mmol) is dissolved in water (110 ml) with sodium bicarbonate (4.65 g, 55.3 mmol). The mixture is stirred vigorously at room temperature, while phenyl chloroformate (3.35 ml, 26.7 mmol) is added in five portions over 30 min. Stirring is continued for 4 h then the mixture is transferred into a separating 36

L.-h. Zhang, G. S. Kauffman, J. A. Pesti, and J. Yin, J. Org. Chem. 62, 6918 (1997); 63, 10085 (1998).

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funnel and washed with t-BuOMe (2  100 ml). The t-BuOMe layer is separated and the aqueous layer is acidified with 1 M NaHSO4 (33 ml) and extracted with ethyl acetate (2  75 ml). The combined extracts are washed with water and brine, then dried (Na2SO4) and concentrated under reduced pressure. The oily residue is diluted with CH2Cl2 (5 ml), then the solvent is evaporated under reduced pressure to give Boc-Dpr(Phoc) as a white solid foam (5.53 g, 77%). Preparation of Boc-Dpr(Phoc)–OSu. A solution of Boc-Dpr(Phoc) (11.06 g, 34.1 mmol) and HOSu (4.12 g, 35.8 mmol) in DME (20 ml) is  cooled to 0 then a solution of DCC in DME (15 ml) is added. The mixture   is stirred at 0 for 60 min and allowed to stand overnight at 4 . The precipitate is filtered off, washed with cold DME, and the filtrate is concentrated under reduced pressure. Toluene (100 ml) is added to the residue and crystallization is initiated by ultrasonic irradiation (15 s) and continued over night at 4 . The solid is collected by filtration and washed with pentane. Recrystallization from toluene gives Boc-Dpr(Phoc)–OSu as a white solid (11.2 g, 78%). Preparation of the Resin Carrying Boc-Dpr(Phoc). The Tentagel resin (1 g, 0.28 mmol) is swollen in DMF (10 ml) for 30 min and then filtered and washed with DMF four times. A mixture of Boc-Sar (0.175 g, 0.93 mmol) HOBtH2O (0.129 g, 0.84 mmol) and DIC (0.132 ml, 0.84 mmol) is stirred for 5 min in CH2Cl2 (1 ml) then added to the resin with a minimum volume of DMF to allow stirring. DIEA (0.146 ml, 0.84 mmol) is added and the suspension is shaken for 60 min. The resin is filtered and washed with DMF and CH2Cl2 four times each and Boc-protecting groups are removed with TFA/CH2Cl2 (1:1, v/v) (5 ml, 1  1 min þ 1  30 min). Then the resin is filtered and washed with CH2Cl2 (4  1 min), neutralized with DIEA/ CH2Cl2 (1:20, v/v) (5 ml, 3  2 min), and washed with CH2Cl2 and DMF four times each. Boc-Dpr(Phoc)–OSu, 0.177 g (0.42 mmol) and HOBtH2O (0.064 g, 0.42 mmol) are added with DMF (2 ml). The suspension is shaken, then DIEA (0.073 ml, 0.42 mmol) is added and shaking is continued for 24 h at room temperature, then the resin is filtered and washed with DMF four times. Acetic anhydride (0.26 ml, 2.8 mmol) and pyridine/ CH2Cl2 (1:19, v/v) (5 ml) are added and allowed to react for 15 min to cap unreacted amino groups. The resin is filtered and washed with DMF, CH2Cl2, MeOH, EtOH, and Et2O four times each and dried under vacuum. Boc-protecting groups are removed with TFA/CH2Cl2 and the C-terminal residue of the target peptide is coupled to the resin (as an NBoc- or N-Fmoc-protected building block) using the Boc protocol described below. Then, elongation can be continued using either the Boc or the Fmoc protocols as follows.

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Boc Protocol Deprotection and Coupling. The resin (0.28 mmol) is treated with TFA/CH2Cl2 (1:1, v/v) (5 ml, 1  1 min þ 1  30 min), then filtered and washed (1 min) with CH2Cl2, MeOH, and DMF four times each. The amino acid building block (0.84 mmol) is activated with HOBtH2O (0.129 g, 0.84 mmol) and DIC (0.132 ml, 0.84 mmol) by stirring for 5 min in CH2Cl2 (1 ml). Then the mixture is added to the resin with a minimum volume of DMF to allow stirring. DIEA (0.122 ml, 0.70 mmol) is added and the suspension is shaken for 30 min. Further DIEA is added (0.073 ml, 0.42 mmol) and shaking is continued for 30 min. The resin is filtered and washed with DMF then CH2Cl2 four times each. Fmoc Protocol Cyclization of the Linker. After the N-Fmoc-protected C-terminal residue has been coupled, the resin (0.28 mmol) is filtered, washed with DMF, and then repeatedly treated with a solution of PhONa-PhOH in DMF (7 ml, 10  20 min) prepared as follows: a mixture of phenol (0.264 g, 2.80 mmol) and 1 N NaOH (1.4 ml, 1.4 mmol) is concentrated under reduced pressure without heating; DMF (10 ml) is added to the residue then the solvent is evaporated under reduced pressure; phenol (0.033 g, 0.35 mmol) is added to the residue and the final volume of the solution is adjusted to 70 ml with DMF. The resin is filtered and washed with DMF four times. Deprotection and Coupling. The resin (0.28 mmol) is treated with piperidine/DMF (1:4, v/v) (5 ml, 1  1 min þ 3  3 min), then washed with DMF (4  1 min). A mixture of the N-Fmoc amino acid building block (0.84 mmol), HOBtH2O (0.129 g, 0.84 mmol), and DIC (0.132 ml, 0.84 mmol) is stirred for 5 min in CH2Cl2 (1 ml) then added to the resin with a minimum volume of DMF to allow stirring. DIEA (0.073 ml, 0.42 mmol) is added and the suspension is shaken for 30 min. Further DIEA is added (0.073 ml, 0.42 mmol) and shaking is continued for 30 min. The resin is filtered and washed with DMF four times.

Section IV Microwave-Assisted Synthesis

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[11] Building Dihydropyrimidine Libraries via Microwave-Assisted Biginelli Multicomponent Reactions By C. Oliver Kappe and Alexander Stadler Introduction

Multicomponent reactions (MCRs) are of increasing importance in organic and medicinal chemistry.1–5 In times where a premium is put on speed, diversity, and efficiency in the drug discovery process,6 MCR strategies offer significant advantages over conventional linear-type syntheses.1–5 In such MCR reactions three or more reactants come together in a single reaction vessel to form novel products that contain portions of all the components.1–5 In an ideal case, the individual building blocks are commercially available or easily prepared covering a broad range of structural variations. MCRs can rapidly provide products with the diversity needed for the discovery of new lead compounds or lead optimization employing combinatorial chemistry techniques.2–6 The search and discovery for new MCRs on one hand,7 and the full exploitation of already known multicomponent reactions on the other, are of considerable current interest. One MCR that belongs in the latter category is the venerable Biginelli dihydropyrimidine synthesis. In 1893 Italian chemist P. Biginelli reported the acidcatalyzed cyclocondensation reaction of ethyl acetoacetate, benzaldehyde, and urea, as shown in Eq. (1).8 Ph EtO2C

Ph +

Me

O

H

NH2 O

+

H2N

O

EtOH/HCl

EtO2C Me

NH N H

O

Surprisingly, the synthetic potential of this heterocycle synthesis remained unexplored for quite some time. In the 1970s and 1980s interest for the original Biginelli cyclocondensation reaction slowly increased and 1

I. Ugi, A. Do¨mling, and W. Ho¨rl, Endeavour 18, 115 (1994). R. W. Armstrong, A. P. Combs, P. A. Tempest, S. D. Brown, and T. A. Keating, Acc. Chem. Res. 29, 123 (1996). 3 L. F. Tietze and M. E. Lieb, Curr. Opin. Chem. Biol. 2, 363 (1998). 4 S. L. Dax, J. J. McNally, and M. A. Youngman, Curr. Med. Chem. 6, 255 (1999). 5 A. Do¨mling, Comb. Chem. High Throughput Screen. 1, 1 (1998). 6 M. Plunkett and J. A. Ellman, Sci. Am. 276, 68 (1997). 7 L. Weber, K. Illgen, and M. Almstetter, Synlett 366 (1999). 8 P. Biginelli, Gazz. Chim. Ital. 23, 360 (1893). 2

METHODS IN ENZYMOLOGY, VOL. 369

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microwave-assisted synthesis R2 E

O

O

+

R2 E

NH

NH2

+ R1

H

H

HN R3

Z

[11]

R1

N

Z

E = ester, amide, acyl, nitro Z = O, S, NR R1-R3 = H, alkyl, (het)aryl

R3 1, DHPM

Fig. 1. Building blocks and points of diversity in the Biginelli three-component dihydropyrimidine synthesis.

its scope was gradually extended by variation of all three building blocks allowing access to a large number of multifunctionalized dihydropyrimidines (DHMP) of type 1.9 Today all three-component condensations involving suitable CH-acidic-containing carbonyl compounds, aldehydes, and urea-type building blocks are considered Biginelli condensations as indicated in Fig. 1.10 A high degree of diversity can therefore be introduced by variation of all three components.9 Of the three building blocks in the Biginelli reaction, it is the aldehyde component that can be varied to the largest extent introducing a point of diversity at the C4 position of the DHPM scaffold 1 (R2 in Fig. 1). In general, it has been found that the Biginelli reaction works best with aromatic aldehydes carrying either electron-withdrawing or electron-donating groups at the o, m, or p positions. Interestingly, good yields are usually obtained with m- or p-electron-withdrawing substituted aromatic aldehydes. Heterocyclic aldehydes derived from furan, thiophene, and pyridine rings also generally furnish acceptable DHPM yields. Traditionally, simple alkyl acetoacetates are employed as CH-acidic-containing carbonyl building block (E, R1 in Fig. 1), but other types such as 3-oxoalkanoic esters or thioesters can also be used successfully in the Biginelli reaction. Benzoylacetic esters can also be used, but yields are usually significantly lower and the overall condensation process is more sluggish. Primary, secondary, and tertiary acetoacetamides can be used in place of esters to produce pyrimidine-5-carboxamides. In addition, -diketones serve as viable substrates in Biginelli reactions and the condensation can also be applied to cyclic -diketones. Furthermore, nitroacetone is a good building block leading to 5-nitro-substituted DHPM derivatives generally with very high yields. The urea analog is the component in the Biginelli reaction that faces the most restrictions in terms of allowed structural diversity (Z, R3 in Fig. 1). Therefore, most of the published Biginelli examples involve urea itself as building block. However, 9 10

C. O. Kappe, Tetrahedron 49, 6937 (1993). C. O. Kappe, Acc. Chem. Res. 33, 879 (2000).

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simple monosubstituted alkyl ureas in general react equally well in a regiospecific manner and provide good yields of exclusively N1-substituted DHPMs. Although thiourea and substituted thioureas follow the same general reactivity pattern as in ureas, they require longer reaction times to achieve good conversions. Yields are typically lower as compared to the corresponding urea derivatives. This flexibility in building block selection allows for considerable diversity to be introduced in the DHPM products.10 In the context of library production and screening, the Biginelli multicomponent protocol is particularly attractive since the resulting DHPM scaffold covers a wide range of biological targets. In the past decades, a broad range of biological effects, such as antiviral, antitumor, antibacterial, and antiinflammatory activities, has been ascribed to these partly reduced pyrimidine derivatives.11 More recently, appropriately functionalized DHPMs have emerged as orally active antihypertensive agents12–14 or 1a-adrenoceptor-selective antagonists.15 Furthermore, the structurally rather simple DHPM derivative monastrol has been identified as a novel cell-permeable molecule that blocks normal bipolar spindle assembly in mammalian cells causing cellcycle arrest.16 Monastrol specifically inhibits the mitotic kinesin Eg5 motor protein and can be considered as a new lead for the development of anticancer drugs.16 Apart from synthetic DHPM derivatives, several marine natural products containing the dihydropyrimidine-5-carboxylate core have recently been isolated exhibiting interesting biological activities.17 Most notably among these natural products are the batzelladine alkaloids A and B, which inhibit the binding of HIV envelope protein gp120 to human CD4 cells serving as potential new leads for AIDS therapy.18 11

C. O. Kappe, Eur. J. Med. Chem. 35, 1043 (2000). K. S. Atwal, B. N. Swanson, S. E. Unger, D. M. Floyd, S. Moreland, A. Hedberg, and B. C. O’Reilly, J. Med. Chem. 34, 806 (1991). 13 G. C. Rovnyak, K. S. Atwal, A. Hedberg, S. D. Kimball, S. Moreland, J. Z. Gougoutas, B. C. O’Reilly, J. Schwartz, and M. F. Malley, J. Med. Chem. 35, 3254 (1992). 14 G. J. Grover, S. Dzwonczyk, D. M. McMullen, D. E. Normandin, C. S. Parham, P. G. Sleph, and S. Moreland, J. Cardiovasc. Pharm. 26, 289 (1995). 15 J. C. Barrow, P. G. Nantermet, H. G. Selnick, K. L. Glass, K. E. Rittle, K. F. Gilbert, T. G. Steele, C. F. Homnick, R. M. Freidinger, R. W. Ransom, P. Kling, D. Reiss, T. P. Broten, T. W. Schorn, R. S. L. Chang, S. S. O’Malley, T. V. Olah, J. D. Ellis, A. Barrish, K. Kassahun, P. Leppert, D. Nagarathnam, and C. Forray, J. Med. Chem. 43, 2703 (2000). 16 T. U. Mayer, T. M. Kapoor, S. J. Haggarty, R. W. King, S. L. Schreiber, and T. J. Mitchison, Science 286, 971 (1999). 17 L. Heys, C. G. Moore, and P. J. Murphy, Chem. Soc. Rev. 29, 57 (2000). 18 A. D. Patil, N. V. Kumar, W. Kokke, M. F. Bean, A. J. Freyer, C. De Brosse, S. Mai, A. Truneh, D. J. Faulkner, B. Carte, A. L. Breen, R. P. Hertzberg, R. K. Johnson, J. W. Westley, and B. C. M. Potts, J. Org. Chem. 60, 1182 (1995). 12

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Fig. 2. Solid-phase Biginelli condensation using -aminobutyric acid -derived urea on Wang resin.

Previous Dihydropyrimidine Libraries

Since the experimental conditions for the traditional Biginelli reaction are rather straightforward, small libraries of DHPMs are readily accessible by parallel synthesis. Along these lines the generation of a 140-member single-compound DHPM library by combination of 25 aldehydes, 6 ureas/ thioureas, and 7 acetoacetates or acetoamides under standard reaction conditions has been reported.19,20 Apart from these conventional solutionphase methods to prepare DHPM libraries, it is also possible to employ polymer-supported reagents to aid in the purification and workup protocol. Polymer-assisted solution-phase chemistry using polymer-supported Lewis acid [Yb-(III)-reagent supported on Amberlyst 15] in combination with polymer-supported urea scavenging resins (Amberlyst 15 and Ambersep 900 OH) permits a rapid parallel Biginelli synthesis with a simple and efficient purification strategy.21 Solid-phase protocols allow an even higher degree of throughput and automation as shown in the example in Fig. 2.22 In this example, -aminobutyric acid-derived urea was attached to Wang resin using standard procedures. The resulting polymer-bound urea was then condensed with an excess of a -ketoester and aromatic aldehydes (such as benzaldehyde, Fig. 2) in the presence of a catalytic amount of hydrochloric acid to afford the corresponding immobilized DHPMs. Subsequent cleavage of the product from the polystyrene resin with trifluoroacetic acid provided DHPMs in high yields and excellent purities. In a variation of the above protocol, the Biginelli synthesis was easily adapted to fluorous-phase conditions.23,24 Here a fluorous urea derivative 19

K. Lewandowski, P. Murer, F. Svec, and J. M. J. Fre´chet, Chem. Commun. 2237 (1998). K. Lewandowski, P. Murer, F. Svec, and J. M. J. Fre´chet, J. Comb. Chem. 1, 105 (1999). 21 A. Dondoni and A. Massi, Tetrahedron Lett. 42, 7975 (2001). 22 P. Wipf and A. Cunningham, Tetrahedron Lett. 36, 7819 (1995). 20

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Fig. 3. Solid-phase Biginelli condensations using Wang resin-bound acetoacetates.

was prepared by attaching a suitable fluorous tag to hydroxyethylurea. The fluorous urea was then condensed with excess of acetoacetates and aldehydes in a suitable solvent containing hydrochloric acid. After extraction of the fluorous DHPMs with fluorous solvent, desilylation with tetrabutylammonium fluoride followed by extractive purification provided the ‘‘organic’’ Biginelli products in good overall yields. Considering the simple experimental techniques used in this fluorous chemistry, automation should be feasible, thus allowing the preparation of DHPM libraries. In addition to the methods described above where the urea component is linked to a solid (or fluorous) support, it is also possible to link instead the acetoacetate building block to the solid support as shown in the example in Fig. 3.25 Thus, Biginelli condensation of Wang-bound acetoacetates with excess aldehydes (such as 2-trifluoromethylbenzaldehyde, Fig. 3) and urea/thiourea provides the desired DHPMs on solid support. Subsequent cleavage with trifluoroacetic acid furnishes the free carboxylic acids in high overall yields. There are alternative solid-phase protocols described in the literature for the generation of DHPMs, not via the classic three-component Biginelli approach but through related modifications.26,27 By employing any of the solid-phase synthesis methods described above, large libraries of DHPMs can potentially be generated in a relatively straightforward fashion. Because of the inherent benefits of solution-phase protocols over solid-phase strategies, we herein describe an automated solution-phase method using 23

A. Studer, S. Hadida, R. Ferritto, S.-Y. Kim, P. Jeger, P. Wipf, and D. P. Curran, Science 275, 823 (1997). 24 A. Studer, P. Jeger, P. Wipf, and D. P. Curran, J. Org. Chem. 62, 2917 (1997). 25 M. G. Valverde, D. Dallinger, and C. O. Kappe, Synlett 741 (2001). 26 L. D. Robinett, K. M. Yager, and J. C. Phelan, 211th National Meeting of the American Chemical Society, New Orleans, 1996, ORGN 122. American Chemical Society, Washington, DC, 1996. 27 C. O. Kappe, Bioorg. Med. Chem. Lett. 10, 49 (2000).

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microwave irradiation to prepare small focused DHPM libraries. The key element in our strategy relies on reaction enhancement by microwave irradiation. Microwave-Assisted Organic Synthesis

Parallel to the recent developments in combinatorial chemistry, microwave-enhanced synthesis has attracted a substantial amount of attention in recent years. During the past decade an ever-increasing number of reports have been published that advocate the advantages and the use of microwave irradiation to carry out organic synthesis.28 Significant increases in rates, yields, and purities of final products have frequently been observed when employing this nonconventional and energy-efficient heating method. Using microwave dielectric heating, the microwave radiation passes through the walls of the reaction vessel heating only the reactants and solvent, but not the reaction vessel. The energy transfer is not produced by conduction or convection, but by dielectric loss. Thus, the degree to which a sample could undergo microwave heating depends on the dielectric properties that are represented by the so-called loss tangent (tan ).29 Materials dissipate microwave energy by two main mechanisms: dipole rotation and ionic conduction. When molecules with a permanent dipole are submitted to an electric field, they become aligned. If this field oscillates, the orientation changes with each alternation. The strong agitation, provided by the reorientation of molecules, in phase with the electrical field excitation, causes an intense internal heating. During ionic conduction, as the dissolved charged particles in a sample (usually ions) oscillate back and forth under the influence of the microwave field, they collide with their neighboring molecules or atoms. This collision causes agitation or motion, creating heat. The main benefits of performing reactions under microwave irradiation conditions are the significant rate enhancements and the higher product yields that can frequently be observed. This method has been successfully applied in various fields of synthetic organic chemistry28–41 such as 28

P. Lidstro¨m, J. Tierney, B. Wathey, and J. Westman, Tetrahedron 57, 9225 (2001). C. Gabriel, S. Gabriel, E. H. Grant, B. S. J. Halstead, and D. M. P. Mingos, Chem. Soc. Rev. 27, 213 (1998). 30 A. de la Hoz, A. Di´az-Ortis, A. Moreno, and F. Langa, Eur. J. Org. Chem. 3659 (2000). 31 R. S. Varma, J. Heterocycl. Chem. 36, 1565 (1999). 32 N. Elander, J. R. Jones, S.-Y. Lu, and S. Stone-Elander, Chem. Soc. Rev. 29, 239 (2000). 33 M. Larhed, C. Moberg, and A. Hallberg, Acc. Chem. Res. 35, 717 (2002). 34 A. Loupy, A. Petit, J. Hamelin, F. Texier-Boullet, P. Jacquault, and D. Mathe´, Synthesis 1213 (1998). 29

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cycloaddition reactions,30 heterocycle synthesis,31 radiolabeled materials,32 transition metal-catalyzed processes,33 solvent-free reactions,34 and phasetransfer catalysis.35 In fact, it is becoming evident that microwave-assisted approaches could be developed for many chemical transformations requiring heat. Not surprisingly, these features have recently started to attract the interest of the drug discovery/medicinal chemistry communities where reaction speed is of great importance.36–39 The combination of microwave heating technology and combinatorial chemistry applications therefore seems a logical consequence of the increased speed and effectiveness offered by using microwave irradiation instead of conventional heating methods. While different hypotheses have been proposed to account for the observed rate enhancements under microwave irradiation, a generally accepted rationalization remains elusive.40 Regardless of the origin/existence of a special microwave effect, this novel heating source is extremely efficient and applicable to a broad range of practical synthesis. Although many of the early experiments in microwave-assisted organic synthesis have been carried out in domestic microwave ovens, the current trend clearly is to use specialized instruments for this type of chemical synthesis. Experiments carried out in domestic ovens have been found to be difficult to reproduce, owing to the lack of temperature and pressure control, pulsed irradiation, uneven electromagnetic field distributions, and the unpredictable formation of hotspots. For several years a number of commercial microwave systems have been available offering either batch or continuous flow-type reactors for chemical synthesis. Most microwave reactors commercially available today feature built-in magnetic stirrers, direct temperature control of the reaction mixture with the aid of fiberoptic probes or infrared (IR) sensors, and software that enables on-line temperature/pressure control by regulation of microwave power output. In recent years all suppliers of microwave instrumentation for organic synthesis have moved toward combinatorial/highthroughput platforms.41 Currently two different philosophies with respect to microwave reactor design are emerging: multimode and monomode (single-mode) reactors. In the so-called multimode instruments (conceptually similar to a domestic oven), the microwaves that enter the cavity are being reflected by the walls and the load over the typically large cavity. 35

S. Deshayes, M. Liagre, A. Loupy, J.-L. Luche, and A. Petit, Tetrahedron 55, 10851 (1999). M. Larhed and A. Hallberg, Drug Discov. Today 6, 406 (2001). 37 J. L. Krstenansky and I. Cotterill, Curr. Opin. Drug Discov. Dev. 4, 454 (2000). 38 A. Lew, P. O. Krutzik, M. E. Hart, and A. R. Chamberlin, J. Comb. Chem. 4, 95 (2002). 39 C. O. Kappe, Curr. Opin. Chem. Biol. 6, 314 (2002). 40 L. Perreux and A. Loupy, Tetrahedron 57, 9199 (2001). 41 A. Loupy, ‘‘Microwaves in Organic Synthesis.’’ Wiley-VCH, Weinheim, 2002. 36

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A mode stirrer ensures that the electromagnetic field distribution is as homogeneous as possible. In the much smaller mono- or single-mode cavities, only one mode is present and the electromagnetic irradiation, through an accurately designed wave guide, is focused directly onto the reaction vessel mounted at a fixed distance from the radiation source. For applications of combinatorial chemistry the key difference between these two types of reactor systems is that whereas in multimode cavities several reaction vessels can be irradiated simultaneously in multivessel rotors (parallel synthesis), in monomode systems only one vessel can be irradiated at the time. In the latter case high throughput can be achieved by integrated robotics that move individual reaction vessels in and out of the microwave cavity (automated sequential synthesis). For the current preparation of a DHPM library, the latter concept has been employed. Microwave-Assisted Biginelli Reactions

The concept of speeding up the Biginelli dihydropyrimidine synthesis by microwave irradiation has already been reported several times in the literature. Those procedures involve microwave heating open vessels containing a solution of the building blocks in a suitable solvent (i.e., ethanol, acetic acid) with an acidic catalyst (HCl or montmorillonite clay).42–46 Alternatively, several authors have exploited the inherent benefits of carrying out microwave-heated Biginelli reactions in the absence of solvent (‘‘dry-media synthesis’’).34 In this case the building blocks are either adsorbed on an inorganic support or admixed with a suitable nonvolatile catalyst.46–50 In most cases, significant rate enhancements have been reported in those studies where reaction times are reduced from several hours when using conventional reflux conditions to a few minutes. Moreover, the final yields were also improved as compared to the outcome of standard thermal protocols. Both procedures, however, have employed domestic microwave ovens that have severe limitations when used to carry out chemical transformations. In fact, in some instances it was proven that 42

R. Gupta, A. K. Gupta, S. Paul, and P. L. Kachroo, Ind. J. Chem. 34B, 151 (1995). A. Dandia, M. Saha, and H. Taneja, J. Fluorine Chem. 90, 17 (1998). 44 J. S. Yaday, B. V. Subba Reddy, E. Jagan Reddy, and T. Ramalingam, J. Chem. Res. 354 (2000). 45 S. J. Tu, J. F. Zhou, P. J. Cai, H. Wang, and J. C. Feng, Synth. Commun. 32, 147 (2002). 46 A. Stadler and C. O. Kappe, J. Chem. Soc. Perkin Trans. 1, 1363 (2000). 47 R. Gupta, S. Paul, and A. K. Gupta, Ind. J. Chem. Technol. 5, 340 (1998). 48 J. L. Krstenansky and Y. Khmelnitsky, Bioorg. Med. Chem. 7, 2157 (1999). 49 H. A. Stefani and P. M. Gatti, Synth. Commun. 30, 2165 (2000). 50 C. O. Kappe, D. Kumar, and R. S. Varma, Synthesis 1799 (1999). 43

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experimental artifacts were responsible for the observed enhancements.46 In contrast, very few publications have reported Biginelli condensations using dedicated microwave reactors for chemical syntheses. In those cases ethanol was used as solvent and the reactions were carried out in sealed vessels under fully controlled conditions (temperature/pressure) showing the expected rate enhancements.46,51,52 However, so far what has been somewhat neglected in microwave-assisted processes is throughput and automation. In one case a dedicated parallel reactor with 36 compartments was used for the preparation of multiple DHPM derivatives in one single irradiation experiment in a dedicated multimode microwave cavity.51 One limitation of this approach, however, is that all reaction vessels during library production are exposed to the same irradiation conditions in terms of reaction time and microwave power. An alternative way to achieve high throughput in microwave-assisted synthesis would be to perform reactions sequentially in an automated fashion. The benefit of this approach is that apart from the achievable throughput in the library production, fast iterations in protocol development and in optimization of reaction conditions can be realized. Here we report the generation of a small library of 48 dihydropyrimidine derivatives via the three-component Biginelli condensation via automated sequential microwave-assisted synthesis using a commercially available microwave synthesizer. Microwave Chemistry Utilizing the Emrys Synthesizer

The microwave synthesizer used in this work (Fig. 4) is composed of a monomode (also referred to as single-mode) microwave cavity that operates at a frequency of 2.45 GHz with continuous microwave irradiation power from 0 to 300 W. The reaction vials are glass-made 10-ml closed tubes, sealed with Teflon septa and an aluminum crimp top. The vials are available in two different designs to carry out reactions in 0.5–2.0 or 2.0– 5.0 ml scale. Magnetic stirring bars are available for both types of vials. The vials are moved in and out of the microwave cavity in an automated fashion by a gripper incorporated into the platform. Inside the microwave  cavity these vessels can be exposed to up to 20 bars of pressure and 250 . The reaction temperature is measured with an IR sensor (infrared thermometry) on the outer surface of the reaction vial. Specialized software regulates the microwave output power so that the preselected maximum temperature is maintained for the desired reaction/irradiation time. Reagents can be added manually into the vials before capping or through 51 52

M. Nu¨chter, W. Lautenschla¨ger, B. Ondruschka, and A. Tied, LaborPraxis 25, 28 (2001). A. Stadler and C. O. Kappe, J. Comb. Chem. 3, 624 (2001).

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Fig. 4. Monomode microwave reactor with integrated robotic platform for automated use (left). A liquid handler allows dispensing of reagents into Teflon-sealed reaction vials, while a gripper moves each vial in and out of the microwave cavity after irradiation. The instrument processes up to 120 reactions per run with a maximum throughput of 12–15 reactions/h. The temperature is measured by an IR sensor on the outside of the reaction vessel. Details of the cavity/gripper (top right) and reaction vials (bottom right) are also displayed (Emrys Synthesizer, Personal Chemistry AB). Reprinted with permission from Wiley-VCH.41 (See color insert.)

the Teflon septum via the platform’s liquid handler. When using the liquid handler, the system can be programmed to deliver a sequence of reagents from different stock solutions into different process vials. Upon completion of the irradiation period process, the reaction vessels are cooled down rapidly (20–80 s) to ambient temperature by using a stream of compressed air (gas jet cooling). Synthesis Criteria for Dihydropyrimidine Library

To construct a small library of DHPMs via microwave-enhanced Biginelli condensation, we felt that several criteria should be met. 1. The DHPMs should be synthesized in solution using standard Biginelli three-component condensation reaction mixtures. Although microwave-assisted Biginelli reactions have been published under solvent-free conditions,46–50 we felt that such methods would

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2.

3.

4.

5.

6.

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not allow the production of high-quality libraries in an automated fashion. The solvent system employed should be able to dissolve most of the building blocks at room temperature so the liquid handler could be used to dispense all building blocks into the appropriate reaction vials. The resulting DHPM products should be only sparingly soluble in the solvent system used at room temperature to facilitate their isolation. All building blocks used for the construction of the DHPM library should be commercially available covering a reasonable diversity range. Since the reaction conditions will be run in a sequential format, individual reaction times need to be kept at a minimum (10–20 min) to achieve a reasonable throughput. Reaction scale must deliver multi-100 mg quantities of material for further scaffold decoration work.

Reaction Optimization in the Emrys Synthesizer

Today there is a great variety of suitable reaction conditions for carrying out Biginelli condensations. For the originally published condensation of ethyl acetoacetate with benzaldehyde and urea more than 40 different experimental protocols are known.53 Traditionally, Biginelli condensations are carried out in a solvent such as ethanol or methanol, but more recently aprotic solvents such as tetrahydrofuran, dioxane, or acetonitrile have also been used successfully. In some cases it is necessary to use acetic acid as solvent. This is particularly important in those cases in which condensation of an aromatic aldehyde and urea will lead to precipitation of an insoluble bisurea derivative, i.e., ArCH(NHCONH2)2,54 which may not react any further along the desired pathway when ethanol alone is used as a solvent. Biginelli reactions in water and ionic liquids are also reported. Although these methods are for solution-phase synthesis, a recent trend involves the condensation without any solvent using the components either adsorbed on an inorganic support or in the presence of a suitable catalyst. The Biginelli condensation strongly depends on the amount of acidic catalyst present in the reaction medium. Traditionally, strong Brønsted acids such as hydrochloric or sulfuric acid have been employed, but today the use Lewis acids such as BF3OEt2, LaCl3, FeCl3, Yb(OTf)3, InCl3, 53 54

C. O. Kappe and A. Stadler, Org. React. 63, (2003). C. O. Kappe, J. Org. Chem. 62, 7201 (1997).

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BiCl3, LiClO4, Mn(OAc)3, or ZrCl4 is prefered.53 It is worth noting that it is also possible to use a solid acid catalyst such as an acidic clay, zeolite, or Amberlyst material. As far as the molar ratio of building blocks is concerned, Biginelli reactions generally employ an excess of the CH-acidic carbonyl or urea components, and not of the aldehyde. Since the resulting DHPM products of a Biginelli-type condensation are usually only sparingly soluble in solvents such as methanol or ethanol at room temperature, in many cases workup involves only isolation of the formed product by a simple filtration. Alternatively, it is also possible to precipitate the products by addition of water. For our microwave-promoted high-throughput library synthesis we decided to use a protocol that utilizes a combination of Lewis acids and Brønsted acids as catalysts. Step 1: Choice of Solvent To make full use of the liquid handler incorporated in the microwave reactor platform (see Fig. 4) we attempted to dissolve all three types of building blocks (Fig. 1) in solvents that are compatible with the reaction conditions. Disappointingly, most urea components, in particular urea itself, were not soluble in sufficient amounts at room temperature in any of the following standard solvent systems: ethanol, acetic acid, tetrahydrofuran, dioxane, acetonitrile, N-methylpyrrolidone, and N,N-dimethylformamide. Water could not be used as a solvent due to its incompatibility with the high-temperature microwave conditions. Since many of the published protocols employ either ethanol or acetic acid as solvents in Biginelli-type condensations, we decided to explore the use of a 3:1 mixture of acetic acid (AcOH) and ethanol (EtOH) to develop a microwave-assisted solution phase protocol. Both solvents effectively couple with microwave  irradiation (tan  at 2.45 GHz/25 : EtOH ¼ 0.941, AcOH ¼ 0.174).29 This allows the reaction mixture to heat up very rapidly under microwave irradiation conditions leading to so-called microwave flash heating conditions. Attempts using other solvents, such as dioxane or THF, proved far less effective both in terms of heating rates and product yields. Not only did the AcOH/EtOH solvent combination have the advantage that all building blocks are soluble under the reaction conditions at elevated temperatures, but the resulting DHPM products were also comparatively insoluble at room temperature facilitating product isolation. Thus, for the automated library synthesis protocols, the CH-acidic carbonyl components were dissolved in AcOH and the aldehyde building blocks in EtOH, whereas the urea components and the catalyst had to be dispensed as solids into the process vials.

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Fig. 5. The influence of the catalyst on the Biginelli condensation involving ethyl acetoacetate, benzaldehyde, and urea [see Eq. (1)] in a 3:1 AcOH/EtOH solvent mixture  under microwave irradiation (120 , 10 min).

Step 2: Selection of Catalyst The next parameter involved the selection of a suitable catalyst. From our previous experience with microwave-assisted Biginelli condensations at high temperature it became evident that hydrochloric acid was not the most suitable reaction promoter due to its role in the then explored decomposition of the urea components to ammonia, leading to unwanted byproducts.46 We then explored the use of more tolerable Lewis acids such as Yb(OTf)3,55 InCl3,56 FeCl3,57 and LaCl3,58 all recently reported to be very effective catalysts for Biginelli condensations, presumably by stabilizing the key N-acyliminium ion intermediates.54 An initial screen of all these catalysts for the model system ethyl acetoacetate, benzaldehyde, and urea revealed that 10 mol% Yb(OTf)3 was the most effective catalyst with the AcOH/EtOH 3:1 solvent system providing the desired DHPM in 92% isolated yield (Fig. 5). The use of only 5 mol% of the same catalyst furnished a significantly lower yield (68%). In the conventionally heated Biginelli reactions a period of 2–3 h of refluxing conditions (in ethanol, concentrated hydrochloric acid as catalyst) is typically required to achieve a 60–70% yield. For all experiments displayed in Fig. 5 an initial set of  conditions involving 10-min microwave flash heating at 120 was chosen.

55

Y. Ma, C. Qian, L. Wang, and M. Yang, J. Org. Chem. 65, 3864 (2000). B. C. Ranu, A. Hajra, and U. Jana, J. Org. Chem. 65, 6270 (2000). 57 J. Lu, Y. Bai, Z. Wang, B. Yang, and H. Ma, Tetrahedron Lett. 41, 9075 (2000). 58 J. Lu and H. Ma, Synlett 63 (2000). 56

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Step 3: Optimization of Time and Temperature Having identified an efficient solvent/catalyst combination [AcOH/ EtOH 3:1, 10 mol% Yb(OTf)3] we next dealt with the issue of reaction time and temperature. One of the benefits of using microwave flash heating in sealed vessels is the fact that one is not limited by the boiling point of the solvents or reagents as in conventional synthesis using refluxing conditions.  After a few optimization cycles we discovered that 120 proved to be an optimum reaction temperature (Fig. 6). While higher temperatures would lead to decreased yields due to the formation of undesired by-products, lower reaction temperatures on the other hand required longer reaction times for complete conversion. For the model system displayed in Eq. (1)  a total irradiation time of 10 min at 120 resulted in 92% isolated yield of pure product. The final DHPM product precipitated directly after the active cooling period (Fig. 7) and showed no traces of impurities as determined by 1H NMR analysis. Although an increase in reaction time to 15 min would further increase the yield for this example to 94%, we have decided generally not to use longer reaction times unless warranted by the specific building block combinations (see Step 4). All reactions were run in the larger 2.0–5.0 ml process vials using 4.0 mmol of each building block in  a total of 1.6–2.0 ml of solvent mixture. At a temperature of 120 this led to a pressure of ca. 3–4 bar in the vial, well below the accessible limit of 20 bar. Although it has been shown that higher yields of DHPMs can be

Fig. 6. Optimization of reaction time and temperature for the Biginelli condensation involving ethyl acetoacetate, benzaldehyde, and urea [see Eq. (1)] in a 3:1 AcOH/EtOH solvent mixture with 10 mol% Yb(OTf)3 as a catalyst.

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6

120

5

100

4

80 3 60 2

40

Pressure (bar)

Temperature (8C)

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1

20 0

0 0

60

120

180

240

300

360

420

480

540

600

660

Time (sec) Fig. 7. Temperature (bold) and pressure profiles for the Biginelli condensation involving ethyl acetoacetate, benzaldehyde, and urea [Eq. (1)] in AcOH/EtOH (3:1) under sealed vessel/microwave irradiation conditions. Microwave flash heating (300 W, 0–40 s),  temperature control using the feedback from IR thermography (constant 120 , 40–600 s), and active cooling (600–660 s).

obtained by employing either the carbonyl or urea building blocks in excess, we have not optimized for molar ratios of reagents using equimolar amounts of building blocks in all experiments. Step 4: Optimization for Troublesome Building Blocks Having identified an optimized set of reaction conditions (conditions A, Fig. 8) for the model substrate of the planned DHPM library, we next looked at potentially troublesome reagents and reagent combinations in our selection of building blocks. Thioureas, for example, are known to give significantly lower yields when employed in the Biginelli condensation.9 We have discovered that for thioureas LaCl3 is usually the preferred catalyst in a microwave-assisted protocol (conditions B, Fig. 8). In the case of acid-sensitive aldehydes such as furane-2-carbaldehyde, we devised a  modified protocol in which neat ethanol was used as a solvent at 100 (conditions C, Fig. 8). Other examples of reaction conditions fine-tuned to specific building block combinations are also presented in Fig. 8. The yields for the optimized microwave-assisted Biginelli condensations are in general comparable or higher than the yields obtained using the standard reflux conditions. More importantly, however, reaction times have been brought

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O

O EtO

EtO

NH N H

Me

Me

O

NH N H

S

B: AcOH/EtOH 3:1, LaCl3 (10 mol%) 120 ⬚C, 10 min, 56% yield

A: AcOH/EtOH 3:1, Yb(OTf )3 (10 mol%) 120 ⬚C, 10 min, 56% yield

NO2 O

O

O EtO Me

NH N H

O

C: EtOH, Yb(OTf )3 (10 mol%) 120⬚C, 20 min, 50% yield

EtO Me

O NH N Me

S

D: EtOH, LaCl3 (10 mol%) 120 ⬚C, 10 min, 41% yield

H2N Me

NH N H

O

E: EtOH, HCl (10 mol%) 120 ⬚C, 15 min, 59% yield

Fig. 8. Optimized reaction conditions A–E for selected examples of DHPM products.

down from several hours (4–12 h) under reflux conditions to 10–20 min using superheated solvents and direct in-core microwave-flash heating. The optimization cycles described above can be carried out within a few hours, providing optimized sets of conditions (conditions A–E, Fig. 8) useful for synthesizing a larger library. Automated Sequential Library Production

With a set of five optimized reaction conditions (solvent, catalyst, time) in hand for a variety of representative Biginelli condensations (Fig. 8), we next turned our attention toward the production of a small library of DHPMs. A set of structurally diverse representative building blocks was chosen: 17 CH-acidic carbonyl compounds, 25 aldehydes, and 8 urea/ thioureas (the structures of the building blocks can be determined from the structures of the final DHPM products displayed in Fig. 9). A combination of all these building blocks in a Biginelli-type fashion would lead to a library of 3400 unique DHPMs. To demonstrate the practicality of our synthetic protocol we decided to generate a representative subset library of 48 DHPM analogs involving all building blocks mentioned above. While the optimization experiments described previously were carried out manually (i.e., adding individual reagents, catalysts, and solvents into the process vials before capping of the vial) we now attempted to automate this

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O

O

EtO

NH N H

EtO

EtO

NH

O

N Et

O

EtO

NH

O

N H

Ph A/120/10/43

EtO

NH

O

N

A/120/10/18

A/120/10/92

O

O

NH

S

N Me

B/120/10/56

D/120/10/41 F

OMe OH O

NO2

EtO

NH N H

OMe

O NH N H

O

EtO

NH

S

N H

C/120/20/45

A/120/10/54

F

O

EtO

O

EtO

NH

O

HN

EtO

NH N H

C/120/10/61

N

EtO

NH N H

EtO N

O

EtO

NH

O

C/120/10/78

S

O

O

O

NH

O

EtO N H

OMe OMe

O EtO

EtO

NH

EtO

NH

NH

MeO

N O H C/100/15/46

F

OMe

O

MeO

O N H C/120/20/67

O N H A/120/10/35

N O H C/100/20/50

MeO

O

O

O NH

O

A/120/10/89

A/120/10/30

C/120/10/41

NH

O

N H

OH O

O

OEt

O

A/120/20/49

NH N H

A/120/10/68

O CF3

EtO

O

N H

C/120/10/52

O

Cl

H N

O

O

S

NH O N H A/120/10/62

O2N

NO2

O O

O MeO

NH N H

O

A/120/10/81

O

O

MeO

NH N H

S

D/120/20/58

MeO

NH N H

O

C/120/10/73

Fig. 9. (continued)

O

MeO

NH N H

O

C/100/10/34

O

NH N O Me A/120/10/51

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O

O O

O

O

NH

NH

O

NH

O N H

O

OMe MeO

NH

NH

MeO

O N H C/120/10/68

O N H C/120/10/53

F OMe O

MeO

NH

NH N H

O

N H

O

Cl

O

O NH O

N H

O

O

NH

MeO

N O Me C/120/10/26

A/120/10/35

NO2

F

Cl

MeO

NH

N H

A/120/10/64

N Ph

E/120/15/21

Me

NH N H

S

EtO

O

NH

Ph

C/120/10/41

N H

O

A/120/20/40

Br

O

H2N

NH

O

N H

O

O

O

EtO

NH

NO2

H2N

NH O N H C/120/20/35

NO2

F

MeO MeO

MeO

E/120/20/31

C/120/20/31

NO2

F

O

O

O

O

NH O N Me A/120/10/25

O

C/120/10/49

A/120/10/34

Cl

O

NH

O

N

C/120/15/73

C/120/10/50

Ph

O

O

N H

O

N H

Cl

NO2

NO2

NH

E/120/15/59

O

Et2N

O

N H

O

O

NO2

N H

NH N H

A/120/10/66

O

A/120/10/61

Ph

N H

NH N H

O

A/120/10/55

OH OMe O Ph

O

N H

NH N H

O

E/100/15/28

Ph

Cl

N H

NH N H B/120/10/89

S

O2N

NH N H

O

B/100/15/83

Fig. 9. Structural representation of a 48-member DHPM library. Given are the reaction  parameters in the format: conditions/temperature ( )/reaction time (min)/yield (%). Conditions refers to the solvent/catalyst systems A–E specified in Fig. 8.

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process as far as possible using of the liquid handler/gripper devices of the microwave synthesizer. For that purpose, stock solutions of all CH-acidic carbonyl components in acetic acid were prepared and stored in designated rack positions [for experiments employing methods C–E (Fig. 8) EtOH was used as solvent]. Similarly, all solid aldehydes were dissolved in absolute ethanol, the only exception being aldehydes that would not be soluble in the required concentration in ethanol. These were weighed directly in the reaction vials. Liquid CH-acidic carbonyl compounds and aldehydes were not used as stock solutions, they were dispensed neat. All urea/thiourea derivatives and the catalysts Yb(OTf)3 and LaCl3 were weighed directly into the process vials before capping. After all building blocks and reaction conditions were entered into the software, the sequential irradiation of all 48 process vials was programmed identifying the rack position of the corresponding stock solutions. The appropriate building blocks were first dispensed into the corresponding vials through the Teflon septa, then each vial was sequentially moved in and out of the microwave cavity by the gripper. Irradiation using the conditions specified in Fig. 9 produced the desired DHPMs in 18–92% isolated yield. It can be determined from the data presented in Fig. 9 that an ample degree of diversity in all the three building blocks is well tolerated. Thus all five variable substituents (R1– R3, E, Z, see Fig. 1) around the DHPM scaffold can be modified, increasing the structural diversity of DHPM analogs that can be synthesized. In the majority of cases, the solid DHPM derivatives would precipitate directly from the reaction mixture after active cooling. The examples that would not crystallize directly upon cooling were crystallized after the crude reaction mixture was poured onto ice water. 1H NMR spectra were obtained from all samples to determine their chemical identity and purity. All products had at least >90% purity; in most cases no foreigner signals other than the ones for the products could be identified from their 1H NMR spectrum. While the 48 examples of DHPM analogs presented in Fig. 9 are a representative subset of the full 3400-member library, it should be stressed that not all combinations of building blocks may lead to DHPMs in such a high state of purity. For some of the examples given in Fig. 9 the yields were not as high as previously reported using different (e.g., solvent-free) Biginelli protocols. Undoubtedly, these yields could be further optimized by finetuning the microwave-assisted reaction conditions, i.e., varying the molar ratios of reagents or using different solvent/catalyst combinations. No attempts were made along these lines since the protocols described above deliver quantities of several 100 mg of pure DHPM products. Recent evidence from this laboratory also indicates that these reactions could be carried out to a significantly smaller or larger scale without reoptimization of reaction conditions.59 The high reaction speed and ease of product

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generation/isolation far outweigh the higher yields that may be obtainable by other protocols, such as solvent-free procedures, where the protocols cannot be easily automated. With an average irradiation time of ca. 15 min (including the time needed for dispensing reagents and moving vials in and out of the cavity) the generation of the 48-member library could be achieved within 12 h. Since the instrument is designed for fully automated unattended operation, a library of this size can conveniently be prepared overnight. Concluding Remarks

Very recently, a report was published51 covering high-speed parallel Biginelli reactions carried out in specifically designed multimode microwave reactors. While this method allows for a considerable throughput due to the relatively short timeframe of a microwave-enhanced chemical reaction, the individual control over each reaction vessel in terms of reaction temperature/pressure is limited. As an alternative to parallel synthesis we have reported the automated sequential synthesis of DHPM libraries. Contrary to the parallel mode where all reaction vessels are exposed to the same irradiation conditions, irradiating each individual reaction vessel separately not only gives better control over the reaction parameters, but also allows for the rapid optimization of reaction conditions. To ensure similar temperatures in a parallel set-up, the same amount of the identical solvent has to be used in each reaction vessel due to the dielectric properties involved. For the preparation of relatively small libraries where delicate chemistries are to be performed, a sequential synthetic format is the mode of choice. Microwave-assisted synthesis in general is likely to have a tremendous impact in the medicinal/combinatorial chemistry communities because it shortens reaction times, improves final yields and purities, and can carry out reactions that previously were thought impossible to do. It should be stressed that in general the rate enhancements seen in microwave-assisted synthesis can be attributed to the very rapid heating of the reaction mixture (flash heating) and the high temperatures that can be reached, rather than to any other specific or nonthermal microwave effect.40 Moreover, the short reaction times open up new approaches for rapid testing of ideas and fast iterations in protocol development as demonstrated here successfully for the Biginelli reaction. While microwave heating is today still considered by some as a laboratory curiosity, we believe that this technology will be used extensively in the future for many 59

A. Stadler, S. Pichler, G. Horeis, and C. O. Kappe, Tetrahedron 58, 3177 (2002).

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chemical processes requiring heat. For this, new developments in microwave reactor and vessel design are still warranted to address issues such as scale-up, higher throughput in monomode cavities, more reliable parallel reactors with improved temperature control, and specialized vessel design for solid-phase organic synthesis. Despite these current limitations it is clear that the use of microwave heating in combinatorial chemistry will continue to grow and is likely to become a standard procedure in the next few years. Experimental Section

Reagents and General Methods All building blocks were purchased from commercial sources and used without further purification. Lewis acid catalysts Yb(OTf)3H2O (Aldrich 40,532-9), LaCl37 H2O (Aldrich 26,207-2), InCl3 (Aldrich 42,941-4), and FeCl36 H2O (Aldrich 20,792-6) were purchased from Aldrich Chemical Co. in the specifications given. 1H NMR spectra were recorded on a Bruker AMX360 or AMX500 instrument in CDCl3 or DMSO-d6, operating at 360 or 500 MHz, respectively. Microwave Irradiation Experiments The Emrys synthesizer (PersonalChemistry AB)60 was used in the standard configuration as delivered including proprietary Workflow Manager software (version 1.1). Optimization experiments were performed in ‘‘single-run’’ mode (i.e., by manual filling of reaction vials) and by specifying the irradiation time and maximum temperature. The DHPM library generation (Fig. 9) was done in an automated fashion using the liquid handler capabilities of the instrument/software. For that purpose, each of the required 48 2.0–5.0 ml process vials was filled with 4.0 mmol of the corresponding urea/thiourea building blocks and 10 mol% (0.4 mmol) of Lewis acid catalyst [for method E 100 l of 4 M HCl in dioxane (Aldrich 34,554-7) was employed]. The vials were sealed with the Teflon septum and aluminum crimp using an appropriate crimping tool. Each vial was then placed in its correct position in the rack of the Smith synthesizer as specified by the software. Then 3.3 M stock solutions of CH-acidic carbonyl compounds in AcOH and 10.0 M stock solutions of aldehydes were prepared and similarly placed in designated rack positions. If solutions could not be prepared because of insufficient solubility, the insoluble 60

PersonalChemistry AB, Kungsgatan 76, SE-753 18 Uppsala, Sweden; phone: (internat.) þ46-18-4899000; fax: (internat.) 46-18-4899100; http://www.personalchemistry.com.

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building block was added manually to the urea/thiourea and catalyst components directly into the vial. In cases where the building block was a liquid, dispensing was done using the reagent neat in which case additional solvent mixture was added to reach the minimum filling volume of 2.0 ml (ca. 1.5 ml). For conditions C–E stock solutions of the corresponding CH-acidic carbonyl compounds in EtOH were additionally prepared. Using the liquid handler, 4.0-mmol aliquots of the corresponding aldehydes (400 l) and carbonyl compounds (1200 l) were dispensed to the process vials containing the appropriate urea/thiourea and catalyst (an additional 400 l of solvent was added to reach the minimum volume of 2.0 ml). After charging all the reagents in an individual vessel, the vial was moved in and  out of the microwave cavity where irradiation for 10–20 min at 100–120 (Figs. 8 and 9) was performed. After the full irradiation sequence was com pleted, racks containing the processed vials were stored at 4 for 8 h. In case of precipitation, the solid products were filtered, washed with cold  (4 ) EtOH, and dried. Where no precipitation was experienced, the crude reaction mixture was treated with 10 ml of ice water and allowed to stand  for 12 h at 4 . The solid DHPM products were filtered and treated as above. The purity of all DHPM products was >90% according to 1H NMR measurements (360 MHz). 1H NMR spectral data for all 48 DHPMs, including melting points (mp), are given below. Spectral Data for DHPM Library Ethyl 6-methyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 978 mg (92%); mp 204 ; 1H NMR (DMSO-d6)  1.12 (t, J ¼ 7.5 Hz, 3H), 2.28 (s, 3H), 4.03 (q, J ¼ 7.5 Hz, 2H), 5.17 (d, J ¼ 3 Hz, 1H), 7.22–7.41 (m, 5H), 7.78 (br s, 1H), 9.22 (br s, 1H). Ethyl 6-methyl-4-phenyl-1-ethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate: yield 208 mg (18%); mp 124–125 ; 1H NMR (DMSO-d6)  1.08 (m, 6H), 2.31 (s, 3H), 3.83 (m, 2H) 4.03 (q, J ¼ 7.5 Hz, 2H), 5.14 (d, J ¼ 3 Hz, 1H), 7.20–7.34 (m, 5H), 7.87 (br s, 1H). Ethyl 6-methyl-4-phenyl-1-benzyl-2-oxo-1,2,3,4-tetrahydropyrimidine5-carboxylate: yield 608 mg (43%); mp 155–56 ; 1H NMR (DMSO-d6)  1.11 (t, J ¼ 7.5 Hz, 3H), 2.39 (s, 3H), 4.03 (q, J ¼ 7.5 Hz, 2H), 4.88, 5.11 (2d, J ¼ 17.5 Hz, 2H), 5.25 (d, J ¼ 3.0 Hz, 1H), 7.05–7.39 (m, 10H), 8.13 (d, J ¼ 3.0 Hz, 1H). Ethyl 6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carb oxylate: yield 620 mg (56%); mp 205 ; 1H NMR (DMSO-d6)  1.12 (t, J ¼ 7.5 Hz, 3H), 2.31 (s, 3H), 4.02 (q, J ¼ 7.5 Hz, 2H), 5.20 (d, J ¼ 3.0 Hz, 1H), 7.20–7.41 (m, 5H), 9.68 (br s, 1H), 10.31 (br s, 1H).

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Ethyl 1,6-dimethyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate: yield 480 mg (41%); mp 144–147 ; 1H NMR (DMSO-d6)  1.18 (t, J ¼ 7.5 Hz, 3H), 2.55 (s, 3H), 3.51 (s, 3H), 4.12 (q, J ¼ 7.5 Hz, 2H), 5.22 (d, J ¼ 3.5 Hz, 1H), 7.18–7.41 (m, 5H), 9.88 (br s, 1H). Ethyl 6-methyl-4-(2-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine 5-carboxylate: yield 662 mg (54%); mp 215–216 ; 1H NMR (DMSO-d6)  0.94 (t, J ¼ 7.5 Hz, 3H), 2.30 (s, 3H), 3.88 (q, J ¼ 7.5 Hz, 2H), 5.81 (d, J ¼ 3.0 Hz, 1H), 7.49–7.98 (m, 5H), 9.39 (br s, 1H). Ethyl 6-methyl-4-(3-hydroxyphenyl)-2-thioxo-1,2,3,4-tetrahydropyrimi dine-5-carboxylate: yield 531 mg (45%); mp 179 ; 1H NMR (DMSO-d6)  1.07 (t, J ¼ 7.5 Hz, 3H), 2.28 (s, 3H), 4.03 (q, J ¼ 7.5 Hz, 2H), 5.09 (d, J ¼ 4 Hz, 1H), 6.63 (m, 3H), 7.11 (m, 1H), 9.43 (br s, 1H), 9.58 (s, 1H), 10.27 (br s, 1H). Ethyl 6-methyl-4-(3,4-dimethoxyphenyl)-2-oxo-1,2,3,4-tetrahydropyri midine-5-carboxylate: yield 670 mg (52%); mp 175 ; 1H NMR (DMSO-d6)  1.15 (t, J ¼ 7.5 Hz, 3H), 2.28 (s, 3H), 3.73 (s, 6H), 4.12 (q, J ¼ 7.5 Hz, 2H), 5.11 (d, J ¼ 3 Hz, 1H), 6.70–7.95 (m, 3H), 7.69 (br s, 1H), 9.17 (br s, 1H). Ethyl 6-methyl-4-(2-chlorophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine 5-carboxylate: yield 805 mg (68%); mp 210–212 ; 1H NMR (DMSO-d6)  1.08 (t, J ¼ 7.5 Hz, 3H), 2.32 (s, 3H), 3.91 (q, J ¼ 7.5 Hz, 2H), 5.67 (d, J ¼ 2.5 Hz, 1H), 7.22–7.46 (m, 4H), 7.72 (br s, 1H), 9.30 (br s, 1H). Ethyl 6-methyl-4-(3,4-difluorophenyl)-2-oxo-1,2,3,4-tetrahydropyrimi dine-5-carboxylate: yield 723 mg (61%); mp 188 ; 1H NMR (DMSO-d6)  1.11 (t, J ¼ 7.5 Hz, 3H), 2.29 (s, 3H), 4.02 (q, J ¼ 7.5 Hz, 2H), 5.17 (d, J ¼ 3.0 Hz, 1H), 7.05–7.50 (m, 3H), 7.80 (br s, 1H), 9.28 (br s, 1H). Ethyl 6-methyl-4-(2-trifluoromethylphenyl)-2-oxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylate: yield 644 mg (49%); mp 202–204 ; 1H NMR (DMSO-d6)  0.97 (t, J ¼ 7.5 Hz, 3H), 2.45 (s, 3H), 3.97 (q, J ¼ 7.5 Hz, 2H), 5.37 (br s, 1H), 5.82 (br s, 1H), 7.32–7.70 (m, 4H), 8.46 (br s, 1H). Diethyl 1,4-phenylen-di-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine 5-carboxylate: yield 692 mg (78%); mp >330 (dec.); 1H NMR (DMSOd6)  1.09 (t, J ¼ 7.5 Hz, 6H), 2.19 (s, 6H), 3.96 (q, J ¼ 7.5 Hz, 4H), 5.11 (d, J ¼ 3 Hz, 2H), 7.17 (s, 4H), 7.67 (br s, 2H), 9.14 (br s, 2H). Ethyl 6-methyl-4-(1-naphthyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate: yield 575 mg (41%); mp 104–106 ; 1H NMR (DMSO-d6)  0.79 (t, J ¼ 2.5 Hz, 3H), 2.56 (s, 3H), 3.80 (q, J ¼ 3.0 Hz, 2H), 4.32 and 4.42 (2d, J ¼ 4 Hz, 2H), 5.13 (d, J ¼ 3.0 Hz, 1H), 5.17 (m, 1H), 5.89 (m, 1H), 6.07 (s, 1H), 7.40–7.97 (m, 7H), 8.30 (s, 1H). Ethyl 6-methyl-4-(3-pyridinyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate: yield 325 mg (31%); mp 209–211 ; 1H NMR (DMSO-d6)  1.08 (t, J ¼ 7.5 Hz), 2.01 (s, 3H), 3.97 (q, J ¼ 7.0 Hz), 5.19 (d, J ¼ 3.0 Hz, 1H), 7.35–7.62 (m, 2H), 7.78 (br s, 1H), 8.45 (s, 2H), 9.28 (br s, 1H).

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Ethyl 6-methyl-4-(2-thienyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate: yield 951 mg (89%); mp 214–217 ; 1H NMR (DMSO-d6)  1.19 (t, J ¼ 7.5 Hz, 3H), 2.28 (s, 3H), 4.10 (q, J ¼ 7.5 Hz, 2H), 5.43 (d, J ¼ 3.0 Hz, 1H), 6.89–7.11 (m, 2H), 7.33–7.42 (d, J ¼ 6.0 Hz, 1H), 7.92 (br s, 1H), 9.33 (br s, 1H). Ethyl 6-methyl-4-(2-furanyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate: yield 503 mg (50%); mp 193–194 ; 1H NMR (DMSO-d6)  1.21 (t, J ¼ 7.5 Hz, 3H), 2.36 (s, 3H), 4.13 (q, J ¼ 7.5 Hz, 2H), 5.48 (d, J ¼ 3.0 Hz, 1H), 5.83 (br s, 1H), 6.12 (d, J ¼ 3 Hz, 1H), 6.27 (s, 1H), 7.32 (s, 1H), 7.79 (br s, 1H). Ethyl 6-methyl-4-(5-pentyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carb oxylate: yield 357 mg (35%); mp 148–150 ; 1H NMR (DMSO-d6)  0.86 (t, J ¼ 7.5 Hz, 3H), 1.18 (t, J ¼ 7.5 Hz, 3H), 1.20–1.37 (m, 6H), 2.15 (s, 3H), 3.99–4.13 (m, 4H), 5.39 (m, 1H), 7.30 (br s, 1H), 8.90 (br s, 1H). Ethyl 4,6-dimethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate:  yield 534 mg (67%); mp 194 ; 1H NMR (DMSO-d6)  1.08 (d, J ¼ 5.5 Hz, 3H), 1.20 (t, J ¼ 7.5 Hz, 3H), 2.15 (s, 3H), 4.00–4.14 (m, 3H), 7.19 (br s, 1H), 8.96 (br s, 1H). Methyl 6-methyl-4-(3-methoxy-4-hydroxyphenyl)-2-oxo-1,2,3,4-tetrahy dropyrimidine-5-carboxylate: yield 539 mg (46%); mp 245–247 ; 1H NMR (DMSO-d6)  2.24 (s, 3H), 3.54 (s, 3H), 3.72 (s, 3H), 5.06 (d, J ¼ 3.0 Hz, 1H), 6.58–6.80 (m, 3H), 7.62 (br s, 1H), 8.89 (s, 1H), 9.10 (br s, 1H). Methyl 6-methyl-4-(3,4,5-trimethoxyphenyl)-2-oxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylate: yield 834 mg (62%); mp 203–204 ; 1H NMR (DMSO-d6)  2.25 (s, 3H), 3.57 (s, 3H), 3.63 (s, 3H), 3.73 (s, 6H), 5.11 (d, J ¼ 3.0 Hz, 1H), 6.65 (d, J ¼ 3.0 Hz, 2H), 7.70 (br s, 1H), 9.19 (br s, 1H). Methyl 6-methyl-4-(4-fluorophenyl)-2-oxo-1,2,3,4-tetrahydropyrimi dine-5-carboxylate: yield 860 mg (81%); mp 188–190 ; 1H NMR (DMSOd6)  2.25 (s, 3H), 3.97 (s, 3H), 5.38 (d, J ¼ 3.0 Hz, 1H), 7.12–7.27 (m, 4H), 7.75 (br s, 1H), 9.22 (br s, 1H). Methyl 6-methyl-4-(4-methylphenyl)-2-thioxo-1,2,3,4-tetrahydropyri midine-5-carboxylate: yield 643 mg (58%); mp 153–155 ; 1H NMR (DMSO-d6)  2.26 (s, 3H), 2.28 (s, 3H), 3.63 (s, 3H), 5.13 (d, J ¼ 3.5 Hz, 1H), 7.08–7.15 (m, 4H), 9.61 (br s, 1H), 10.26 (br s, 1H). Methyl 6-methyl-4-(2-methylphenyl)-2-oxo-1,2,3,4-tetrahydropyrimi dine-5-carboxylate: yield 760 mg (73%); mp 235–237 ; 1H NMR (DMSOd6)  2.31 (s, 3H), 2.43 (s, 3H), 3.48 (s, 3H), 5.41 (d, J ¼ 4.0 Hz, 1H), 7.11–7.22 (m, 4H), 7.62 (br s, 1H), 9.19 (br s, 1H). Methyl 6-methyl-4-(5-nitro-2-furyl)-2-oxo-1,2,3,4-tetrahydropyrimi dine-5-carboxylate: yield 385 mg (34%); mp 237–239 ; 1H NMR (DMSOd6)  2.26 (s, 3H), 3.60 (s, 3H), 5.31 (d, J ¼ 3.0 Hz, 1H), 6.59, 7.60 (2d, J ¼ 4.5 Hz, 2H), 8.000 (br s, 1H), 9.48 (br s, 1H).

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Allyl 1,6-dimethyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimi dine-5-carboxylate: yield 677 mg (51%); mp 122–123 ; 1H NMR (DMSOd6)  2.51 (s, 3H), 3.20 (s, 3H), 4.52 (m, 2H), 5.10–5.20 (m, 2H), 5.45 (d, J ¼ 3.5 Hz, 1H), 5.70–5.90 (m, 1H), 6.82 (d, J ¼ 3.5 Hz, 1H), 7.30–7.60 (m, 2H), 8.00–8.10 (m, 2H). Isopropyl 6-methyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate: yield 550 mg (50%); mp 192–193 ; 1H NMR (DMSO-d6)  1.00, 1.19 (2d, J ¼ 7.5 Hz, 6H), 2.28 (s, 3H), 4.82 (m, 1H), 5.13 (d, J ¼ 3.0 Hz, 1H), 7.20–7.31 (m, 5H), 7.71 (br s, 1H), 9.17 (br s, 1H). Isopropyl 6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimi dine-5-carboxylate: yield 654 mg (73%); mp 199–201 ; 1H NMR (DMSOd6)  1.00, 1.18 (2d, J ¼ 6.0 Hz, 6H), 2.28 (s, 3H), 4.84 (m, 1H), 5.31 (d, J ¼ 3.0 Hz, 1H), 7.65–7.77 (m, 2H), 7.91 (br s, 1H), 8.09–8.19 (m, 2H), 9.38 (br s, 1H). Isopropyl 6-methyl-4-(3-nitrophenyl)-1-allyl-2-oxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylate: yield 490 mg (34%); mp 115–118 ; 1H NMR (DMSO-d6)  1.00, 1.18 (2d, J ¼ 7.0 Hz, 6H), 2.47 (s, 3H), 4.03, 4.46 (2d, J ¼ 7.5 Hz, 2H), 4.86 (m, 1H), 4.95 (d, J ¼ 7.5 Hz, 1H), 5.10 (d, J ¼ 5.5 Hz, 1H), 5.32 (d, J ¼ 3.0 Hz, 1H), 5.83 (m, 1H), 7.64–7.69 (m, 2H), 8.10 (br s, 1H), 8.14 (d, 2H). tert-Butyl 6-methyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate: yield 566 mg (49%); mp 200–202 ; 1H NMR (DMSO-d6)  1.30 (s, 9H), 2.24 (s, 3H), 5.12 (d, J ¼ 3.0 Hz, 1H), 7.20–7.39 (m, 5H), 7.69 (br s, 1H), 9.09 (br s, 1H). Benzyl 1,6-dimethyl-4-(2,3-dichlorophenyl)-2-oxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylate: yield 412 mg (25%); mp 125–126 ; 1H NMR (DMSO-d6)  2.57 (s, 3H), 3.14 (s, 3H), 4.52 (m, 2H), 4.96–5.16 (m, 2H), 5.74 (d, J ¼ 3.5 Hz, 1H), 6.90–7.60 (m, 8H), 8.10 (d, J ¼ 3.5 Hz, 1H). 6-Methyl-5-acetyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine: yield  490 mg (53%); mp 240–242 ; 1H NMR (DMSO-d6)  2.01 (s, 3H), 2.29 (s, 3H), 5.25 (d, J ¼ 3 Hz, 1H), 7.26–7.32 (m, 5H), 7.80 (br s, 1H), 9.16 (br s, 1H). 6-Methyl-5-acetyl-4-(3-methylphenyl)-2-oxo-1,2,3,4-tetrahydropyri midine: yield 666 mg (68%); mp 250–252 ; 1H NMR (DMSO-d6)  2.09 (s, 3H), 2.28 (s, 6H), 5.21 (d, J ¼ 3 Hz, 1H), 7.05–7.20 (m, 4H), 7.75 (br s, 1H), 9.12 (br s, 1H). Methyl 4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 290 mg (31%); mp 226–227 ; 1H NMR (DMSO-d6)  3.55 (s, 3H), 5.11 (d, J ¼ 3.0 Hz, 1H), 7.25–7.35 (m, 6H), 7.69 (s, 1H), 9.20 (br s, 1H). Methyl 4-(3,4,5-trimethoxyphenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine 5-carboxylate: yield 401 mg (31%); mp 216–219 ; 1H NMR (DMSO-d6)  3.56 (s, 3H), 3.63 (s, 3H), 3.73 (s, 6H), 5.10 (d, J ¼ 3.0 Hz, 1H), 7.32 (d, J ¼ 7 Hz, 1H), 6.55 (s, 2H), 7.64 (s, 1H), 9.18 (d, J ¼ 3.0 Hz, 1H).

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Methyl 4-(3,4-difluorophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate: yield 377 mg (35%); mp 229–231 ; 1H NMR (DMSO-d6)  3.56 (s, 3H), 5.15 (d, J ¼ 3.0 Hz, 1H), 7.11 (br s, 1H), 7.22–7.45 (m, 3H), 7.75 (s, 1H), 9.28 (br s, 1H). Methyl 6-methoxymethyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylate: yield 450 mg (35%); mp 182–184 ; 1H NMR (CDCl3)  3.48 (s, 3H), 3.65 (s, 3H), 4.66 (m, 2H), 5.50 (d, J ¼ 3.0 Hz, 1H), 6.33 (br s, 1H), 7.49–7.68 (m, 3H), 8.14 (d, J ¼ 7 Hz, 1H), 8.17 (br s, 1H). Methyl 6-ethyl-4-(2,3-dichlorophenyl)-1-methyl-2-oxo-1,2,3,4-tetrahy dropyrimidine-5-carboxylate: yield 360 mg (26%); mp 163 ; 1H NMR (DMSO-d6)  1.17 (t, J ¼ 7.5 Hz, 3H), 2.98 (m, 2H), 3.17 (s, 3H), 3.49 (s, 3H), 5.60 (d, J ¼ 3.0 Hz, 1H), 7.25–7.55 (m, 3H), 8.02 (br s, 1H). Methyl 6-ethyl-4-(3,4-difluorophenyl)-2-oxo-1,2,3,4-tetrahydropyrimi dine-5-carboxylate: yield 762 mg (64%); mp 186 ; 1H NMR (DMSO-d6)  1.13 (t, J ¼ 7.5 Hz, 3H), 2.69 (m, 2H), 3.57 (s, 3H), 5.18 (d, J ¼ 3.0 Hz, 1H), 7.03–7.49 (m, 3H), 7.82 (br s, 1H), 9.32 (br s, 1H). Ethyl 6-propyl-4-(4-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate: yield 549 mg (41%); mp 173–175 ; 1H NMR (DMSO-d6)  0.90 (t, J ¼ 7.5 Hz, 3H), 1.10 (t, J ¼ 7.5 Hz, 3H), 1.57 (q, J ¼ 7.5 Hz, 2H), 2.60 (m, 2H), 4.00 (q, J ¼ 7.5 Hz, 3H), 5.27 (d, J ¼ 3.5 Hz, 1H), 7.49 (d, J ¼ 8.5 Hz, 2H), 7.86 (br s, 1H), 8.21 (d, J ¼ 8.5 Hz, 2H), 9.30 (br s, 1H). Ethyl 6-phenyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine 5-carboxylate: yield 591 mg (40%); mp 233–235 ; 1H NMR (DMSO-d6) 0.74 (t, J ¼ 7.5 Hz, 3H), 3.74 (q, J ¼ 7.5 Hz, 2H), 5.39 (d, J ¼ 3.0 Hz, 1H), 7.32–8.35 (m, 9H), 8.02 (br s, 1H), 9.48 (br s, 1H). 6-Methyl-1,4-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbox amide: yield 277 mg (21%); mp 198–200 ; 1H NMR (DMSO-d6)  1.73 (s, 3H), 5.28 (d, J ¼ 3 Hz, 1H), 7.18, 7.21 (2 br s, 2H), 7.33–7.45 (m, 10H), 9.62 (br s, 1H). 6-Methyl-4-(4-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-car boxamide: yield 652 mg (59%); mp 281 (dec.); 1H NMR (DMSO-d6)  2.07 (s, 3H), 5.36 (d, J ¼ 3 Hz, 1H), 6.97 (br m, 2H), 7.49 (d, J ¼ 8.0 Hz, 2H), 7.66 (br s, 1H), 8.21 (d, J ¼ 8.0 Hz, 2H), 8.72 (br s, 1H). Methyl 6-methyl-4-(2-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine 5-carboxamide: yield 766 mg (66%); mp 142–144 ; 1H NMR (DMSO-d6)  1.76 (s, 3H), 2.45 (s, 3H), 5.37 (d, J ¼ 3 Hz, 1H), 7.24 (br s, 1H), 7.47–7.90 (m, 4H), 8.72 (br s, 1H). Diethyl 6-methyl-4-(4-bromophenyl)-2-oxo-1,2,3,4-tetrahydropyrimi dine-5-carboxamide: yield 895 mg (61%); mp 227–230 ; 1H NMR (DMSO-d6)  1.05 (t, J ¼ 7.0 Hz, 6H), 1.64 (s, 3H), 3.05 (q, J ¼ 7.0 Hz, 4H), 4.99 (d, J ¼ 3 Hz, 1H), 7.15 (d, J ¼ 8.0 Hz, 2H), 7.32 (br s, 1H), 7.51 (d, J ¼ 8.0 Hz, 2H), 8.48 (br s, 1H).

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6-Methyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxanil lide: yield 680 mg (55%); mp 245–247 ; 1H NMR (DMSO-d6)  2.04 (s, 3H), 5.40 (d, J ¼ 3 Hz, 1H), 6.99 (br s, 1H), 7.24–7.71 (m, 10H), 8.69 (s, 1H), 9.54 (s, 1H). 6-Methyl-4-(3-methoxy-4-hydroxyphenyl)-2-oxo-1,2,3,4-tetrahydropyr imidine-5-carboxanillide: yield 400 mg (28%); mp 238–241 ; 1H NMR (DMSO-d6)  2.03 (s, 3H), 3.66 (s, 3H), 5.32 (d, J ¼ 3 Hz, 1H), 6.69–7.55 (m, 9H), 8.62 (s, 1H), 8.89 (s, 1H), 9.49 (s, 1H). 6-Methyl-4-(2-chlorophenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine 5-carboxylanillide: yield 1278 mg (89%); mp 195–197 ; 1H NMR (DMSOd6)  2.02 (s, 3H), 5.74 (d, J ¼ 3.0 Hz, 1H), 6.99–7.87 (m, 9H), 9.49 (br s, 1H), 9.63 (br s, 1H), 10.04 (br s, 1H). 6-Methyl-5-nitro-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine: yield  775 mg (83%); mp 188–190 ; 1H NMR (DMSO-d6)  1.58 (s, 3H), 5.65 (d, J ¼ 3.0 Hz, 1H), 7.26–7.47 (m, 6H), 8.07 (br s, 1H).

[12] Microwave-Assisted Solid-Phase Organic Synthesis (MA-SPOS) of Oxazolidinone Antimicrobials By Andrew P. Combs, Brian M. Glass, and Sharon A. Jackson Introduction

Microwave-assisted organic synthesis (MAOS)* has dramatically evolved since its first use in the mid-1980s. Recent advances in microwave equipment have provided homogeneous microwave heating in safe and reliable instruments for use in the chemistry laboratory. Microwave-assisted heating has been utilized with many solution-phase reactions, typically reducing reaction times from days or hours to minutes or even seconds. The observed rapid microwave dielectric heating is primarily due to the continuous realignment of polar molecules with the paramagnetic oscillating field. The microwave radiation is increasingly more absorbed by molecules with larger dipole moments and thus reactions are often performed in *

Abbreviations: Bal, 4-formyl-3,5-dimethoxyphenoxy linker; DCM, dichloromethane; DIEA, diisopropylethylamine; DMF, dimethylformamide; ELSD, evaporative light scattering detector; ESI MS, electrospray ionization mass spectroscopy; EtOAc, ethylacetate; HPLC, high-performance liquid chromatography; LC/MS, liquid chromatography/mass spectroscopy; MA, microwave assisted; MAOS, microwave-assisted organic synthesis; MeOH, methanol; PEG-PS, polyethyleneglycol grafted on 1% cross-linked polystyrene; SPOS, solid-phase organic synthesis; TFA, trifluoroacetic acid; THF, tetrahydrofuran.

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solvents with high dielectric constants. Several in-depth reviews are available on the general principles and recent advances in the microwave-assisted organic synthesis field.1 The successful application of microwave dielectric heating to solidphase organic reactions has also been reported recently.2 Microwaveassisted solid-phase organic synthesis (MA-SPOS) provides an additional means by which solid-phase reactions can be driven to completion in much shorter reaction times. MA-SPOS has proven to be an invaluable optimization method, since optimization of solution-phase or solid-phase chemistries is by far the most time-consuming portion of developing reliable and general chemical library syntheses. Many reaction parameters can be tested in just a few hours or days due to the shortened reaction times and thus solid-phase chemistries can be optimized in dramatically reduced times. Once a solid-phase synthesis is derived, a library of compounds can be efficiently prepared. Several commercially available microwave instruments are currently available for performing up to 50 reactions in parallel or robotic sequential microwave heating of hundreds of individual reactions at specified temperatures, pressures, and times. Instrumentation

In 1996, we initiated our microwave-assisted organic synthesis research using a domestic Sharp carousel microwave oven. We soon demonstrated the effectiveness of this synthetic tool for solution-phase and solid-phase synthesis of libraries of compounds directed toward our drug discovery efforts.3 Though we were excited by the potential of this technology, there remained many limitations and safety concerns associated with the use of a domestic microwave oven. The most prominent limitations were the lack of temperature and pressure control. Nonhomogeneous heating of laboratory samples within these microwave sources can result in an explosion. While several multimode laboratory safe microwave instruments were commercially available at the time we opted for the relatively new single-mode Smith synthesizer from Personal Chemistry, shown in Fig. 1. The Smith synthesizer features an enclosed reaction chamber with temperature, 1

Several reviews are available: (a) B. Wathey, J. Teirney, P. Lidstrom, and J. Westman, Drug Discov. Today 7, 373 (2002). (b) P. Lidstrom, J. Tierney, B. Wathey, and B. Westman, Tetrahedron 57, 9225 (2001). (c) M. Larhed and A. Hallberg, Drug Discov. Today 6, 406 (2001). (d) S. Caddick, Tetrahedron 51, 10403 (1995). 2 A. Lew, P. O. Krutzik, M. E. Hart, and A. R. Chamberlin, J. Comb. Chem. 4, 95 (2002). 3 (a) A. P. Combs, S. Saubern, M. Rafalski, and P. Y. S. Lam, Tetrahedron Lett. 40, 1623 (1999). (b) B. M. Glass and A. P. Combs, ‘‘High-Throughput Synthesis,’’ p. 123. Marcel Dekker Inc., New York, 2001.

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225

Fig. 1. Personal Chemistry Smith synthesizer. (Photograph compliments of Personal Chemistry Inc.) (See color insert.)

pressure, and time control for microwave-heating sealed-glass vessels. Reaction pressures up to 300 psi are tolerated by this system. The integrated Gilson robotic platform has proven robust for automated sequential reaction optimization and/or library production. The system is also easy to use and has superb safety features. Solid-Phase Biaryloxazolidinones

A microwave-assisted solid-phase synthesis of the antimicrobial oxazolidinone pharmacophore is described herein as a demonstration of the utility of this emerging technology toward drug discovery.4 The optimization process and full experimental details for the synthesis of a small library of oxazolidinones are exemplified. The oxazolidinones comprise a unique class of potent antibacterial agents.5 This antibacterial pharmacophore structure (1) was discovered nearly two decades ago by scientists at E. I. Du Pont De Nemours and Company.6 Several compounds advanced to preclinical safety studies, including Dup 721 and E3656, are shown in Fig. 2. Unfortunately, these compounds were dropped from development due to dose-limiting toxicities. In the late 1990s, scientists at the Upjohn Company reported the discovery and phase I clinical trials of two oxazolidinone antibacterial agents, linezolid and eperezolid,7 displaying diminished toxicities. Pharmacia-Upjohn’s persistence resulted in the recent approval of linezolid as the first new class of antimicrobials in over a decade. 4

C. D. Dzierba and A. P. Combs, Annu. Rep. Med. Chem. 37, 247, (2002). S. J. Brickner, Curr. Pharm. Design 2, 175 (1996). 6 A. M. Slee, M. A. Wuonola, and R. J. McRipley, Antimicrob. Agents Chemother. 31, 1791 (1987). 5

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microwave-assisted synthesis R2

O

O

O N

O N NH R1

Oxazolidinone Pharmacophore 1

O NHAc

Dup 721

O N N

O

O F N

N

O

O

Linezolid

E3656

NHAc

NHAc

Fig. 2. Oxazolidinone antimicrobial pharmacophore.

Renewed interest at DuPont-Merck Pharmaceuticals Company in this class of antibacterial compounds led to a second discovery chemistry effort. Hundreds of compounds had been previously synthesized over the decade long program at DuPont, but a systematic variation of many functional groups had not been explored thoroughly. Oxazolidinones, such as E3656, containing the customary acetamide at the R1 position and the biaryl functionality at the R2 position were targeted for extensive analoging, due to the excellent potency for the series and the incomplete SAR available. A method for the rapid parallel synthesis of this class of compounds was thus desired to enable thorough analoging around this core in hopes of identifying new potent clinical candidates lacking the toxicological liabilities of the previous candidates. Retrosynthetic analysis of the oxazolidinone pharmacophore 1 shown in Fig. 3 revealed that the oxazolidinone aminomethyl group could serve as an attachment site to the solid support during the elaboration of a suitably substituted scaffold. We envisioned amine scaffold 2 could be readily coupled to a resin-bound aldehyde. Attachment of two points of diversity via derivatization of the solid-supported secondary amine 3 with a variety 7

(a) G. E. Zurenko, C. W. Ford, D. K. Hutchinson, S. J. Brickner, and M. R. Barbachyn, Exp. Opin. Invest. Drugs 6, 151 (1997). (b) S. J. Brickner, Curr. Pharm. Design 2, 175 (1996). (c) S. J. Brickner, D. K. Hutchinson, M. R. Barbachyn, P. R. Manninen, D. A. Ulanowicz, S. A. Garmon, K. C. Grega, S. K. Hendges, D. S. Toops, C. W. Ford, and G. E. Zurenko, J. Med. Chem. 39, 673 (1996).

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227

Fig. 3. Retrosynthetic analysis of the oxazolidinone pharmacophore.

of acylating agents and palladium-mediated couplings of arylboronic acids to the solid-supported aryl-iodide 4 would provide arrays of the desired fully functionalized oxazolidinones 5. Oxazolidinone scaffold 2 was synthesized in six steps by literature methods.7c Coupling of only 1.5 equivalents of the valuable scaffold amine 2 to Bal-resin under reductive amination conditions afforded the resinbound secondary amine 3, as shown in Fig. 4. The resulting resin-bound secondary amine 3 was acylated with several acylating reagents, including acetic anhydride, to afford the N-acyl-aryliodide 4 with a resin loading of 0.27 mmol/g. The Suzuki coupling of arylboronic acids to the solid-supported arylhalide 4 was initially performed by conventional heating methods, DMF at  85 in an oil bath for 16 h, to provide the desired products 6 after TFA cleavage. Due to the extended heating times necessary for the Suzuki reaction, full optimization of the reaction conditions were not investigated prior to compound library synthesis. Reaction conditions sufficient for the library synthesis were determined within a couple of weeks affording moderate yields of the desired oxazolidinones. Libraries of hundreds of

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Fig. 4. Solid-phase synthesis of oxazolidinones.

oxazolidinones were synthesized using a variety of acylating reagents and arylboronic acids using this methodology. HPLC purification of the desired products was typically necessary due to the modest yields and purities of the various library members. Attempts to perform these same Suzuki couplings in a domestic microwave afforded the desired product, but yields and purities were inconsistent, presumably due to the nonhomogeneity of the heating and lack of sufficient temperature and pressure controls. We have subsequently revisited this reaction and successfully optimized the Suzuki microwave-assisted coupling conditions using the Smith synthesizer. Several parameters were investigated, including the palladium catalysts, the reaction temperatures, and the reaction times (Table I). Optimization reactions were run in the Smith synthesizer using 50 mg of resin 7 and 6 equivalents of 4-methoxyphenylboronic acid to afford oxazolidinone 8. In just a few days, optimized conditions were identified that afforded the desired product in excellent yields and purities with reactions times of only 5–10 min.8 A small library of oxazolidinones was then synthesized using the robotics of the Smith synthesizer to run sequentially each new boronic acid in the Suzuki reaction. Cleavage of the products and filtration through a small plug of silica provided excellent yields and purities of the desired oxazolidinones, including compound 12, the previous clinical candidate E3656, in 96% yield and 96% purity (Table II). This MA-SPOS of the oxazolidinone class of antimicrobial/antibiotics allows for the rapid synthesis of libraries of compounds that simultaneously 8

M. Larhed, G. Lindeberg, and A. Hallberg, Tetrahedron Lett. 37, 8219 (1996).

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MA-SPOS of oxazolidinones TABLE I Optimization of Suzuki Reaction Conditions

Entry

b

Catalyst

% conversiona

Pd(PPh3)4 Pd(PPh3)4 Pd(PPh3)4 PdCl2(PPh3)2 PdCl2(PPh3)2

74 83 66 66 88

Time (min)

140 160 180 140 180

1 2 3 4 5 a



Temp ( C)

5 5b 10 5 5

Conversion based on HPLC UV analysis of cleaved product vs. unreacted aryl iodide. Resin was subjected to Suzuki coupling conditions with 4-methylarylboronic acid twice. TABLE II MA-SPOS of Oxazolidinones O

R N

O NH O

Compound

R-Group

Purity (%)a

Yield (%)b

9 10 11 12 13 14

3-MeO 4-MeO 3-F 4-F 3-pyridyl 4-Me

89 95 95 95 96 85

72 78 91 92 96 94

a b

Purity assessed by LC/MS with quantitation by ELSD. Crude yields based on gravimetric analysis.

vary both the N-acyl functionality and the biarylsubstituent of the oxazolidinone pharmacophore. Many potent antibacterial compounds were identified from the compound libraries produced using variations of this rapid parallel synthesis strategy. Compound structures and associated biological

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activities of these antimicrobial compound libraries will be reported in due course. Conclusion

MA-SPOS is a valuable methodology for the rapid synthesis of novel and diverse chemical entities. While solid-phase technologies have previously allowed for the rapid purification of reaction products by simply washing excess reagents from the resin-bound products, recent advances in microwave technologies now enable the chemist to drive chemical reactions to completion via microwave dielectric heating in unprecedented timeframes. The combinatorial chemist is now able to break through the solid-phase synthetic chemistry optimization bottleneck and develop new reaction conditions in hours or days rather than weeks or months. The rapid production of libraries of novel compounds has thus been enabled by synergizing the best aspects of MA chemistry and SPOS. Reagents and General Methods

Bal-PEG-PS (HL) resin (loading of 0.45 mmol/g) was purchased from Perceptive Biosystems. Commercially available starting materials and reagents were purchased from Aldrich and used without further purification. Microwave-assisted chemistry was performed in a Personal Chemistry Smith synthesizer. All other reactions were performed in capped polypropylene-fritted tubes manufactured by Jones Chromatography. The polypropylene tubes were mixed using a Labquake tube rotor/rocker manufactured by Thermolyne. LC/MS analyses were performed on a Micromass ZMD Electrospray spectrometer equipped with a Gilson 215 liquid handler, a Sedere Sedex 75 ELS detector, and a Waters Symmetry chromatography column (C18, 5 M, 2.1  50 mm). The HPLC gradient ran from 0% acetonitrile/water containing 0.1% TFA to 100% over 8 min at 1.5 ml/min flow rate. Peaks areas were integrated by evaporative light scattering. Experimental Section

Coupling of Iodoaryloxazolidinone to BAL Resin Bal resin (0.4 g, 0.45 mmol/g, 0.18 mmol, Perseptive Biosystems) was weighed into a fritted polypropylene tube and swelled with 4 ml of DCM. To the suspension was added oxazolidinone HCl salt 2 (96 mg, 0.27 mmol), sodium triacetoxyborohydride (190 mg, 0.90 mmol), and acetic acid (80 l,

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2% final concentration). The resin was mixed for 16 h, and then washed with DCM, THF, and MeOH (3 each). Acylation of Solid-Supported Iodoaryloxazolidinone Resin 3 was swelled with 4 ml of DCM followed by treatment with acetic anhydride (85 l, 0.90 mmol) and DIEA (160 l, 0.90 mmol). The resin was mixed for 30 min, then washed with DCM, THF, and MeOH (3 each). A portion of the resin (50 mg) was cleaved with 50% TFA in DCM and dried in vacuo. The loading of resin 4 (R1 ¼ CH3) was determined to be 0.27 mmol/g by gravimetric analysis of the cleaved product. Microwave-Assisted Solid-Phase Suzuki Coupling Resin 4 (R1 ¼ CH3) (50 mg, 0.014 mmol) was weighed into a conical Personal Chemistry Smith process vial equipped with a conical-shaped stir bar. To the resin was added 4-fluorophenylboronic acid (11 mg, 0.08 mmol), DMF (1 ml), 2 M aqueous sodium carbonate (80 l), and dichlorobis(triphenylphosphine)palladium(II) (1–2 mg). The heavy walled glass vial was crimp sealed and placed on the Gilson platform. The micro wave program was set to a 5 min duration at 180 on the normal absorption setting and the vial was then processed. After completion, the vial was decrimped and resin 5 (R1 ¼ CH3, R2 ¼ 4-F-phenyl) was transferred to a fritted tube and washed with DMF, H2O, MeOH, and DCM (3 each). Cleavage of Biaryloxazolidinone from the Solid Support Resin 5 (R1 ¼ CH3, R2 ¼ 4-F-phenyl) was mixed with 2 ml of 50% TFA in DCM for 30 min. The resin was washed with DCM and the filtrates were collected and concentrated in vacuo. The residue was dissolved in EtOAc and filtered through a short plug of silica (75 mg) in a thin fritted polypropylene tube to remove residual palladium. The product was dried under vacuum to give the desired product 12 in good yield (4.1 mg, 92%) and purity (95%). ESI MS: Theor: 329.1 (M þ H)þ. Found: 329.2 (M þ H)þ.

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Section V Oligosaccharide Chemistry

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[13] Automated Synthesis of Polysaccharides By Obadiah J. Plante, Emma R. Palmacci, and Peter H. Seeberger Introduction

Carbohydrates are the most structurally diverse class of biopolymers. In addition to branching and anomeric configuration, structural diversity is further complicated when considering that natural structures are often found attached to proteins, lipids, or both. The properties attributed to carbohydrates and glycoconjugates are as varied as their structure, ranging from sources of bioenergetics to critical markers for cancer metastasis.1,2 A better understanding of the biological roles of oligosaccharides and glycoconjugates is needed to advance the field of glycobiology. To meet this challenge and create new approaches to carbohydrate production, an automated synthesis method was developed recently and is the focus of this chapter. Background

The major challenges in carbohydrate synthesis are two-fold: (1) how to control the stereochemistry of each newly formed glycosidic linkage and (2) how to incorporate various degrees of branching (Fig. 1). Unlike peptide and nucleic acid synthesis, carbohydrate synthesis requires the installation of a new stereocenter during each elongation event. Furthermore, many degrees of branching are common in carbohydrates whereas the other biopolymers are strictly linear in sequence. The current state of the art in peptide and DNA synthesis is a line of fully automated synthesizers that allows for the production of oligomers by nonspecialists.3,4 The same is not true for carbohydrate synthesis and no general method has emerged despite years of research.5 The most widely adopted methods for carbohydrate synthesis utilize either enzymatic or chemical approaches. Enzymatic carbohydrate synthesis is a vibrant area of research that has been reviewed thoroughly and will 1

C.-H. Wong, S. L. Haynie, and G. M. Whitesides, J. Org. Chem. 47, 5418 (1983). S. Hakomori and Y. Zhang, Chem. Biol. 4, 97104 (1997). 3 M. H. Caruthers, G. Beaton, J. V. Wu, and W. Wiesler, Methods Enzymol. 211, 3 (1992). 4 G. B. Fields, Z. Tian, and G. Barany, in ‘‘Synthetic Peptides: A User’s Guide’’ (H. Grant, ed.). Freeman, New York, 1992. 5 K. Toshima and K. Tatsuta, Chem. Rev. 93, 1503 (1983). 2

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Branching can occur at any position

HO OH O HO HO

OH O O

HO OH

O O

OR

AcHN OH

New stereocenter at each glycosidic linkage

Fig. 1. Stereochemistry and regiochemistry in carbohydrates.

not be discussed in detail here.6 Enzymatic methods offer exquisite stereochemical and regiochemical control although the limitations with regards to substrate specificity preclude their widespread use. Chemical methods, on the other hand, are used frequently to produce highly branched complex carbohydrates.7 By incorporating unique protecting groups within a carbohydrate monomer, many patterns of complex structures can be accessed. Furthermore, several strategies are available for ensuring the appropriate stereochemical outcome of the coupling reaction. The requirement of multiple synthetic steps limits the scope of this method and necessitates an expertise in carbohydrate synthesis in order to be successful. Despite the almost limitless diversity that chemical synthesis offers, no general method for the synthesis of carbohydrates exists. Carbohydrate synthesis has been considered an art form rather than a science based on the finding that subtle changes in solvent, reagent, temperature, concentration, and/or substrate may alter the stereo- and regiochemical outcome of an elongation event. The lack of predictive tools for carbohydrate synthesis has precluded all previous efforts to develop a general method. Compounding the intricacies of carbohydrate synthesis further is the tedious nature of the synthetic process. A typical heptasaccharide synthesis requires at least 14 distinct synthetic steps and purification events with overall yields commonly in the 0.1–5% range after months of manual labor. To address all of the variables in carbohydrate synthesis, researchers have attempted to generalize the glycosylation conditions for a particular set of building blocks.8 This technique has proven useful for small libraries of carbohydrates but has yet to offer a universal solution for complex carbohydrate synthesis. To address the need for new synthetic methods we developed a procedure that utilizes solid-phase methods to screen for appropriate building block reactivity and that assembles complex carbohydrates with minimal manual labor.9 Rather than confine a group of building blocks to a given 6

K. Koeller and C.-H. Wong, Chem. Rev. 100, 4465 (2000). K. C. Nicolaou and H. J. Mitchell, Angew. Chem. Int. Ed. Engl. 40, 1576 (2001). 8 L. Yan, C. M. Taylor, R. Goodnow, and D. Kahne, J. Am. Chem. Soc. 116, 6953 (1994). 7

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set of reaction conditions, the automated method described below provides a framework for exploring potential reaction conditions while at the same time minimizing the amount of effort required to create complex carbohydrates. It is anticipated that further advances in automated carbohydrate synthesis will shape the field of glycobiology in much the same manner that gene and peptide synthesis machines have shaped the nature of biotechnology over the past two decades. Overview

The general strategy for automated carbohydrate synthesis is based on solid-phase techniques.10 For carbohydrate synthesis, the acceptor-bound method involves the sequential addition of carbohydrate building blocks (glycosyl donors) to an insoluble polystyrene support (Scheme 1).11 Side products, reagents, and unreacted starting material are washed away and the carbohydrate chain is covalently attached to the polymer support. A unique protecting group is removed to provide another nucleophilic position for subsequent elongation. At the conclusion of the synthesis, the carbohydrate is liberated from the support, deprotected, and purified. The solid-phase platform is ideal for automation due to the repetitive nature of the process. The successful application of automated methods for carbohydrate synthesis requires the appropriate choice of polymer support, linker, building blocks, reagents, temperature control, and cleavage conditions. We investigated each of the aforementioned variables and selected the most promising set of conditions compatible with automation.12

Scheme 1. Solid-phase synthesis using the acceptor-bound method.

9

O. J. Plante, E. R. Palmacci, and P. H. Seeberger, Science 291, 1523 (2001). W.-C. Haase and P. H. Seeberger, Chem. Rev. 100, 4349 (2000). 11 P. H. Seeberger and S. J. Danishefsky, Acc. Chem. Res. 31, 685 (1998). 12 O. J. Plante, E. R. Palmacci, and P. H. Seeberger, Adv. Carbohydr. Chem. Biochem. 58, 35 (2003). 10

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Fig. 2. Schematic description of the automated carbohydrate synthesis machine. (See color insert.)

The instrument chosen for the evaluation of carbohydrate synthesis was an ABI-433 peptide synthesizer (Fig. 2). The instrument was adapted for carbohydrate synthesis and customized coupling cycles were developed. A specially designed low-temperature reaction vessel was installed and interfaced with a commercially available cooling device.13 The necessary reagents were loaded onto the instrument ports and reaction conditions were programmed on the computer, in a fashion similar to the automated synthesis of peptides. Detailed Description

An automated solid-phase method necessitates a polymer support and a linker that are compatible with the reagents used in carbohydrate synthesis. Several strategies have been developed that address functional group compatibility and swelling of the polymer support. Of these strategies, a family of olefinic linkers has proven to be readily cleaved under neutral 13

A Julabo circulating cooler was used with an ethanol/ethylene glycol mixture.

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239

Scheme 2. Automated carbohydrate synthesis using glycosyl trichloroacetimidates.

conditions at the end of a synthesis.14,15 Olefin-based linkers are also stable to a variety of acidic glycosylation conditions when attached to polystyrene resins. Both lightly cross-linked, swellable resins such as Merrifield’s (1% cross-linked) resin and rigid macroreticular resins (Argopore) are amenable to functionalization with olefinic linkers. Initial studies determined that the polystyrene resins were compatible with common glycosylation conditions and our olefinic linker.14 When initiating a synthesis, the choice of glycosylating agent and protecting groups governs the selection of activating and deblocking reagents. The automated method described here has proven useful with glycosyl trichloroacetimidate and glycosyl phosphate building blocks. Temporary protecting groups such as levulinate esters, silyl ethers, and acetate esters also are compatible with automation. Using this set of reagents we anticipate that the majority of natural carbohydrate linkages can be accessed. A typical coupling cycle (Scheme 2) for glycosyl trichloroacetimidates is outlined in Table I. A polystyrene support, functionalized with an olefinic linker, is loaded into a reaction vessel in the instrument.16,17 The activating reagent (trimethylsilyl trifluoromethanesulfonate (TMSOTf*/ 14

R. B. Andrade, O. J. Plante, L. G. Melean, and P. H. Seeberger, Org. Lett. 1, 1811 (1999). L. Knerr and R. R. Schmidt, Eur. J. Org. Chem. 2803 (2000). 16 One percent cross-linked polystyrene and Argopore resin performed equally well. The compatibility with polar solvents makes Argopore the most versatile resin when investigating new reaction conditions. 17 An Applied Biosystems 433A was adapted for carbohydrate synthesis. * Abbreviations: DMF, N,N-dimethylformamide; DMT, 4,40 -dimethoxytrityl; HPLC, highpressure liquid chromatography; NPG, n-pentenyl glycoside; TEA, triethylamine; TCA, trichloroacetic acid; THF, tetrahydrofuran; TMSOTf, trimethysilyl trifluoromethanesulfonate. 15

240

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oligosaccharide chemistry TABLE I Automated Coupling Cycle Using Glycosyl Trichloroacetimidatesa

Step

Function

Reagent

Time (min)

1 2 3 4 5 6

Couple Wash Couple Wash Wash Deprotection

10 equiv. donor and 0.5 equiv. TMSOTf Dichloromethane 10 equiv. donor and 0.5 equiv. TMSOTf Dichloromethane Methanol:dichloromethane (1:9) 2  10 equiv. NaOMe (methanol: dichloromethane) (1:9) Methanol:dichloromethane (1:9) 0.2 M acetic acid in tetrahydrofuran Tetrahydrofuran Dichloromethane

30 6 30 6 6 80

7 8 9 10

Wash Wash Wash Wash a

4 4 6 6

Scale: 25 mol.

CH2Cl2) and deblocking reagent (e.g., NaOMe) are inserted into the instrument along with the glycosyl imidate building blocks. The synthesis is performed in an iterative manner according to the programmed coupling cycle. In general, double glycosylations (95–98%) result in (5%) greater yield per coupling when compared to single glycosylations (90–95%). In addition to glycosyl trichloroacetimidate building blocks, we developed a similar coupling cycle for glycosyl phosphate building blocks (Table II). Glycosyl phosphates are versatile building blocks that are activated under mild conditions to form glycosidic linkages in high yield.18 The overall synthesizer configuration is consistent with the imidate method, however, the reagent solutions are modified to accommodate phosphate reactivity and protecting group removal. A reaction vessel designed for low temperature and a cooling apparatus are required to enable the 15 temperature necessary for productive phosphate couplings. In this cycle, the deprotection events [N2H4/Pyr:AcOH (3:2)] were quantitative and all coupling steps were >90% yield. An example of automated carbohydrate synthesis using glycosyl phosphates is shown in Scheme 3. The coupling protocols described in the two previous examples can be combined to allow for the synthesis of branched carbohydrates.19 The automated solid-phase synthesis of a tetrasaccharide is illustrated in Scheme 4. Both glycosyl phosphate and glycosyl imidate building blocks were used along with acetate and levulinate esters as temporary protecting groups. 18 19

O. J. Plante, R. B. Andrade, and P. H. Seeberger, Org. Lett. 1, 211 (1999). M. C. Hewitt and P. H. Seeberger, Org. Lett. 3, 3699 (2001).

[13]

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automated synthesis of polysaccharides TABLE II Automated Coupling Cycle Used with Glycosyl Phosphatesa

Step

Function

Reagent

Time (min)

1 2 3 4 5 6 7

Couple Wash Couple Wash Wash Wash Deprotection

5 equiv. donor and 5 equiv. TMSOTf Dichloromethane 5 equiv. donor and 5 equiv. TMSOTf Methanol:dichloromethane (1:9) Tetrahydrofuran Pyridine:acetic acid (3:2) 2  20 equiv. hydrazine (pyridine: acetic acid) (3:2) Pyridine:acetic acid (3:2) Methanol:dichloromethane (1:9) 0.2 M acetic acid in tetrahydrofuran Tetrahydrofuran Dichloromethane

15 6 15 4 4 3 30

8 9 10 11 12

Wash Wash Wash Wash Wash a

3 4 4 4 6

Scale: 25 mol.

Scheme 3. Automated carbohydrate synthesis using glycosyl phosphates.

After a successful synthesis, the resin is transferred to a round-bottom flask for cleavage. The most convenient method of cleavage involves reaction of the resin with Grubbs’ catalyst in CH2Cl2 under an atmosphere of ethylene. The product is liberated to afford an n-pentenyl glycoside (NPG). NPGs serve as versatile intermediates in carbohydrate synthesis and are readily converted into various functionalities useful for immobilization to a surface, conjugation to proteins, or fluorescent labeling.20 20

T. Buskas, E. Soderberg, P. Konradsson, and B. Fraser-Reid, J. Org. Chem. 65, 958 (2000).

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oligosaccharide chemistry

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Scheme 4. Synthesis of a tetrasaccharide using a variety of reaction conditions.

Purification of the synthetic carbohydrate is accomplished using either flash silica gel chromatography or HPLC depending on the purity of the sample. The most common side-products in automated solid-phase synthesis are deletion sequences (n1, n2, etc.). The prevalence of deletion sequences complicates purification of the final product. To aid in the purification process, a capping procedure was developed that allows for the facile removal of deletion sequences.21 Following each coupling event, unreacted hydroxyl groups that may give rise to deletion sequences are subjected to a capping reagent that renders these sites silent in subsequent couplings (Fig. 3). The caps also function as a handle to readily separate all unwanted capped sequences from the desired uncapped products. For example, installation of a polyfluorinated silyl ether F-tag onto unreacted hydroxyl groups after glycosylation precludes further elongation of the deletion sequence. Following cleavage from the resin all of the fluorinated intermediates are easily removed by passing through a pad of fluorinated silica gel. This modification of the automated coupling cycles greatly facilitates the purification of synthetic carbohydrates.

21

E. R. Palmacci, M. C. Hewitt, and P. H. Seeberger, Angew. Chem. Int. Ed. Engl. 40, 4433 (2001).

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automated synthesis of polysaccharides

243

Fig. 3. Schematic description of the use of an F-tag reagent for the purification of compounds synthesized on a solid support.

State of the Art

Automated solid-phase carbohydrate synthesis utilizes instrumentation and reaction design in order to remove the most time-consuming aspect of carbohydrate production. This process reduces the challenge of carbohydrate synthesis to the production of simple building blocks, for which there is ample synthetic precedence. Although a full set of building blocks has yet to be established, the variety of possible building blocks will enable the production of diverse libraries of complex carbohydrates. Carbohydrate libraries have been sought for many years and technologies for their production will find widespread application in academic and industrial laboratories. A representative sample of the carbohydrate sequences prepared using automated solid-phase synthesis is shown in Fig. 4. Automated carbohydrate synthesis allows for the production of complex carbohydrates orders of magnitude faster than other approaches. This advance has the potential to parallel the breakthroughs achieved by researchers in the peptide and DNA fields that opened up the proteomic and genomic eras in biotechnology. By increasing the scope of the carbohydrate building block library and streamlining the reaction conditions further we anticipate that automated solid-phase carbohydrate synthesis will become the method of choice for carbohydrate production.

244

[13]

oligosaccharide chemistry O BnO BnO BnO

OAc O

BnO BnO

BnO O O BnO BnO OBn BnO BnO O O O O BnO BnO OPiv O

BnO BnO BnO BnO

7

BnO BnO BnO

O O O

OAc O

BnO BnO BnO

O

n=3

BnO BnO BnO

O

O O

O

O OAc 8

n = 3, 5, 8 O

9 n =3 10 n = 5 11 n = 8

BnO OBn O BnO

PivO

OBn O BnO

O O NPhth BnO O BnO BnO O

12

BnO BnO

LevO OBn3 n=3 O O BnO O O PivO BnO O BnO O BnO PivO OBn O BnO O BnO O O BnO PivO BnO BnO 13 BnO

n = 1, 3 O O PivO

Fig. 4. Representative sequences prepared by automation.

Experimental Procedures

Materials and Methods All reactions were performed in oven-dried glassware under an atmosphere of argon unless noted otherwise. Reagent grade chemicals were used as supplied except where noted. TMSOTf was purchased from Acros Chemicals. N,N-Dimethylformamide (DMF) was obtained from Aldrich Chemical Co. (Sure-Seal Grade) and used without further purification. Merrifield’s resin (1% cross-linked) was obtained from Novabiochem. Argopore resin was purchased from Argonaut Technologies. Dichloromethane (CH2Cl2) and tetrahydrofuraran (THF) were purchased from J. T. Baker (Cycletainer) and passed through neutral alumina columns prior to use. Toluene was purchased from J. T. Baker (Cycletainer) and passed through a neutral alumina column and a copper(II) oxide column prior to use. Pyridine, triethylamine, and acetonitrile were refluxed over calcium hydride and distilled prior to use. Analytical thin-layer chromatography was performed on E. Merck silica column 60 F254 plates (0.25 mm). Compounds were visualized by dipping the plates in a cerium sulfate– ammonium molybdate solution followed by heating. Liquid column chromatography was performed using forced flow of the indicated solvent ˚ pore diameter) silica gel. on Silicycle 230–400 mesh (60 A

[13]

automated synthesis of polysaccharides

245

HPLC analysis was performed on a Waters Model 600E Multisolvent ˚ , 4-m, 3.6  150-mm) delivery system using analytical (Nova-Pak 60-A ˚ and preparative (Nova-Pak 60-A, 6-m, 7.8  300-mm) silica columns. Synthesis of Linker-Functionalized Resin 1. 8-(4,40 -Dimethoxytrityl)-4(Z)-octenol (4.6 g, 11 mmol, 4.0 equiv.) was dissolved in DMF (20 ml) and  cooled to 0 . NaH (60% dispersion in mineral oil, 0.53 g, 11 mmol, 4.0 equiv.) was added and the solution was stirred for 1 h. Merrifield’s resin [1% cross-linked (1.2 mmol/g): 2.21 g, 2.65 mmol, 1.0 equiv.] was loaded into a solid-phase flask and swollen with DMF (20 ml). The linker solution was transferred to the resin suspension via cannula and tetrabutylammonium iodide (98 mg, 0.27 mmol, 0.1 equiv.) was added. After shaking for  1 h at 0 in the dark, the reaction mixture was warmed to room temperature and shaken for 12 h. Capping of unreacted sites was accomplished by reaction with methanol (1.0 ml) and NaH (60% dispersion in mineral oil, 0.10 g) for 16 h. Methanol (5 ml) was added and the resin was transferred to a fritted funnel. The resin was washed with 3  50 ml of each of the following: MeOH:DMF (1:1), DMF, MeOH:THF (1:9), THF, MeOH:CH2Cl2 (1:9), and CH2Cl2. Drying under vacuum over P2O5 afforded 3.38 g resin. Analysis of a small sample of resin (10 mg) via a dimethoxytrityl cation assay revealed the loading to be 0.57 mmol/g (see below). Deprotection of the DMT functionalized resin was accomplished by washing the resin with 3  50 ml of 3% dichloroacetic acid/CH2Cl2. Further washing with 3  50 ml of CH2Cl2, 1% TEA/CH2Cl2, THF, and CH2Cl2 and drying under vacuum afforded 2.39 g linker-functionalized resin (0.78 mmol/g). Trityl Cation Assay.22 DMT-functionalized resin (10–20 mg) was weighed into a 10-ml volumetric flask. Trichloroacetic acid (3% solution ¼ 1.50 g TCA in 50 ml dichloromethane) in CH2Cl2 was added and a 200- to 300-l aliquot was transferred to another 10-ml volumetric flask and diluted with 3% TCA/ CH2Cl2. Analysis was done by UV-VIS absorption at 504 nm (A504). Loading calculation: Calculation: [(A504)(10 ml)]/76 ¼ X micromole ¼ 0.00 X millimole in final solution [0.00X mmol/(vol. aliquot in ml)](10 ml) ¼ number of millimoles in initial solution Loading: Number of millimoles in initial solution/(mass resin in g) ¼ number of millimoles/g 22

R. T. Pon, in, ‘‘Methods in Molecular Biology 20: Protocols for Oligonucleotides and Analogs’’ (S. Agrawal, ed.), p. 467. Humana Press, Totowa, New Jersey, 1993.

246

oligosaccharide chemistry

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Example: 12.9 mg resin was loaded into a 10-ml volumetric flask. Trichloroacetic acid (3%) in CH2Cl2 was added and a 200-l aliquot was transferred and diluted to 10 ml. Analysis by UV-VIS gave A504 ¼ 1.05. Calculation: [(1.05)(10 ml)]/(76 ml/mol) ¼ 0.138 mol ¼ 0.000138 mmol (0.000138 mmol/0.20 ml)(10 ml) ¼ 0.00692 mmol 0.00692 mmol/0.0129 g ¼ 0.54 mmol/g Synthesizer Configuration. An Applied Biosystems peptide synthesizer model 433A (ABI-433A) was adapted for carbohydrate synthesis. The necessary reagents were attached to reagent ports 1–7 and CH2Cl2 and THF were installed as the bulk solvents on ports 9 and 10. Glycosyl donor building blocks were weighed and loaded into cartridges as were the capping reagents. The coupling cycles were programmed and all synthesis steps were performed by the instrument. Low-temperature applications were achieved by attachment of a Julabo circulating bath to a customized reaction vessel. The temperature of the reaction vessel was manually controlled on the Julabo. Automated Synthesis Using Glycosyl Trichloroacetimidates. Octenediol functionalized resin (25 mol, 83 mg, 0.30 mmol/g loading) was loaded into a reaction vessel and inserted into a modified ABI-433A peptide synthesizer. The resin was glycosylated with donor 2 (10 equiv., 0.25 mmol, 160 mg) delivered in CH2Cl2 (4 ml) and TMSOTf (0.5 equiv., 1 ml, 0.0125 M TMSOTf in CH2Cl2). Mixing of the suspension was performed (10 s vortex, 50 s rest) for 30 min. The resin was washed with CH2Cl2 (6  4 ml each) and glycosylated a second time. Upon completion of the double glycosylation the resin was washed with CH2Cl2 (6  4 ml each) and MeOH:CH2Cl2 (1:9) (4  4 ml each). The glycosylated resin was subjected to the deprotection protocol for acetyl esters (see below) or to the standard cleavage conditions. General Deprotection Conditions (Acetate Ester/NaOMe). Deprotection of the acetyl ester was carried out by treatment of the glycosylated resin with sodium methoxide (10 equiv., 0.5 ml, 0.5 M NaOMe in MeOH) in CH2Cl2 (5 ml) for 30 min. The resin was then washed with MeOH:CH2Cl2 (1:9) (1  4 ml) and subjected to the deprotection conditions a second time for 30 min. Removal of any soluble impurities was accomplished by washing the resin with MeOH:CH2Cl2 (1:9) (4  4 ml each), 0.2 M AcOH in THF (4  4 ml each), THF (4  4 ml each), and CH2Cl2 (6  4 ml each). The deprotected polymer-bound glycoside was then elongated by reiteration of the above glycosylation/deprotection protocol or subjected to the standard cleavage protocol.

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automated synthesis of polysaccharides

247

Automated Synthesis Using Glycosyl Phosphates. Octenediol functionalized resin (25 mol, 83 mg, 0.30 mmol/g loading) was loaded into a reaction vessel equipped with a cooling jacket and inserted into a modified ABI-433A peptide synthesizer. The resin was glycosylated using donor 4 (5 equiv., 0.125 mmol, 90 mg) delivered in CH2Cl2 (4 ml) and TMSOTf  (5 equiv., 1 ml, 0.125 M TMSOTf in CH2Cl2) at 15 . Mixing of the suspension was performed (10 s vortex, 50 s rest) for 15 min. The resin was then washed with CH2Cl2 (6  4 ml each) and glycosylated a second time. Upon completion of the double glycosylation the resin was washed with MeOH:CH2Cl2 (1:9) (4  4 ml each), THF (4  4 ml), and pyridine:acetic  acid (3:2) (3  4 ml) and warmed to 15 . The glycosylated resin was subjected to the deprotection protocol for levulinate esters (see below) or to the standard cleavage conditions. General Deprotection Conditions (Levulinate:N2H4). Deprotection of the levulinate ester was carried out by treating the glycosylated resin with hydrazine acetate [40 equiv., 4 ml, 0.25 M N2H4-HOAc in pyridine:acetic acid (3:2)] for 15 min. The resin was subjected to the deprotection conditions a second time for 15 min. Removal of any soluble impurities was accomplished by washing the resin with pyridine:acetic acid (3:2) (3  4 ml), 0.2 M AcOH in THF (4  4 ml each), THF (4  4 ml each), and CH2Cl2 (6  4 ml each). The deprotected polymer-bound glycoside was then elongated by reiteration of the above glycosylation/deprotection protocol or subjected to the standard cleavage protocol. Oligosaccharide Cleavage from the Polymer Support. The glycosylated resin (25 mol) was dried in vacuo over phosphorous pentoxide for 12 h and transferred to a 10-ml flask. The flask was purged with ethylene and Grubb’s catalyst [bis(tricyclohexylphosphine)benzylidine ruthenium(IV) dichloride, 4.1 mg, 0.005 mmol, 20 mol%] was added. The reaction mixture was diluted with CH2Cl2 (3 ml) and stirred under 1 atm ethylene for 36 h. Triethylamine (111 ml, 0.80 mmol, 160 equiv.) and tris hydroxymethylphosphine (50 mg, 0.40 mmol, 80 equiv.) were added and the resulting solution was stirred at room temperature for 1 h.23 The pale yellow reaction mixture was diluted with CH2Cl2 (25 ml) and washed with water (3  25 ml), saturated aqueous NaHCO3 (3  25 ml), and brine (3  25 ml). The aqueous phase was extracted with CH2Cl2 (3  25 ml) and the combined organics were dried over Na2SO4, filtered, and concentrated. The resulting oligosaccharides were purified either by flash column chromatography on silica gel or high-pressure liquid chromatography (HPLC).

23

H. D. Maynard and R. H. Grubbs, Tetrahedron Lett. 40, 4137 (1999).

248

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oligosaccharide chemistry

F-Tag Protocols Incorporation of the F-Tag Cap into the Automated Solid-Phase Synthesis Cycle. The resin (50 mol) was swelled in a 0.1 M solution of 2,6-lutidine in CH2Cl2 (4 ml, 8.0 equiv.). After vortexing for 5 s, a 0.1 M solution of the F-Tag triflate in CH2Cl2 (2.5 ml, 5.0 equiv., loaded into cartridges) was delivered to the reaction vessel. Mixing of the suspension was performed (10 s vortex, 50 s rest) for 15 min. Oligosaccharide Cleavage from the Polymer Support and Purification Using the F-Tag Method. The glycosylated resin was cleaved as described above. The crude mixture was dissolved in CH2Cl2/MeOH (1:1) (1 ml) and added to a column of tridecafluoro [Si-(CH2)2-(CF2)5-CF3)3] functionalized silica gel (Silicycle) equilibrated in 80% MeOH/20% H2O. One column length of 80% MeOH/20% H2O was eluted, and the solvent was changed to 100% MeOH. Nonfluorinated material typically eluted in fractions 1–4, while fluorinated material remained on the column until the gradient was increased to 100% MeOH. The desired nonfluorinated fractions were concentrated and analyzed by HPLC. Recycling of the fluorous silica gel was possible after washing with three column lengths MeOH, four column lengths CH2Cl2, and drying with nitrogen.

[14] Solid-Phase Oligosaccharide Chemistry and Its Application to Library Synthesis By Matthias Grathwohl, Nicholas Drinnan, Max Broadhurst, Michael L. West, and Wim Meutermans Introduction

Biological processes are controlled at the molecular level through networks and cascades of molecular interactions, primarily involving three classes of biomolecules: peptides, oligonucleotides, and oligosaccharides. Oligosaccharides play a major role in cell recognition and cell-signaling events through the involvement of carbohydrate-recognizing proteins such as lectins and selectins.1,2 The information of affinity and selectivity of a carbohydrate substrate to its target is stored in the nature of its constituents and the three-dimensional structure, or connectivity profile. A simple comparison illustrates the virtually endless size of structural diversity that 1 2

A. Varki, Glycobiology 3, 97 (1993). R. A. Dwek, Chem. Rev. 96, 683 (1996).

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

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solid-phase oligosaccharide chemistry B O

O

O O

P

B

O

O

O

N H

H N

O

O

O O

O

O O

O O

O O

O

O

O O

P

O

64 trinucleotides

64 tripeptides

? (>10,000 trisaccharides)

Fig. 1. Number of trimers that can be generated from four monomer building blocks for nucleotides, peptides, and carbohydrates. The number of trisaccharides that can be generated in theory using four monosaccharide building blocks depends on what one wants to include. If ) symbolizes the direction of the donor to the acceptor then two types of accessible trisaccharides can be envisaged: (1) A)B)C and (2) A)B(C. The number of possible combinations, in the absence of trehaloses, but including the possibility of  and  anomers is 83  (4  4) for option 1 and 83  (4  3) for option 2. Total number of possibilities is 14,336.

can be theoretically accessed through linking carbohydrate monomers, especially when compared to peptides and oligonucleotides (Fig. 1). Most of the carbohydrate-based processes, however, are poorly understood largely due to the fact that determining the structures and connectivities of natural substrates remains difficult and identification of novel carbohydrate-based substrates is impeded by a general lack of access to carbohydrates. Combinatorial peptide and oligonucleotide chemistry is now commonplace and enables large libraries of diverse sequences to be readily generated in a short time frame. This has not only drastically accelerated the process of hit discovery in many drug discovery projects, but has also provided essential tools to validate targets and determine protein function in the world of peptides and oligonucleotides. Thus, there is a clear need for combinatorial oligosaccharide approaches to enable access to libraries of structurally diverse oligosaccharides.3 With such diverse libraries at hand, the chances of identifying biologically active oligosaccharides should become far greater, which should help unravel key carbohydrate entities involving biological processes, and therefore assist the development of new therapeutic drugs. In the following sections we will first address some recent developments at Alchemia in solid-phase

3

L. A. Marcaurelle and P. H. Seeberger, Curr. Opin. Chem. Biol. 6, 289 (2002).

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oligosaccharide chemistry

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carbohydrate chemistry, then we will cover suggested methods to develop structurally diverse oligosaccharide libraries. Solid-Phase Oligosaccharide Synthesis (SPOS)

The synthesis of carbohydrates on solid support has recently received renewed interest with some concomitant successes.4,5 Although not yet commonplace, the solid-phase synthesis of carbohydrates is becoming more prevalent and indeed more accepted as an alternative to the established solution-phase methods of oligosaccharide synthesis. A wide variety of solid supports, including controlled pore glass,6 soluble mpeg/dox-based resins,7,8 and more classic polystyrene and grafted polystyrene-based resins9 have been employed for the synthesis of carbohydrates with various degrees of success. Critical to a successful solid-phase synthesis methodology is the linker that connects the growing oligosaccharide to a solid support. The linker is generally recognized as a modified protecting group and, as such, it must display complete orthogonality to any other protecting groups employed during the synthesis. This is particularly pertinent in oligosaccharide synthesis, which typically requires a number of orthogonal protecting groups to distinguish between different hydroxyl functions and different saccharide linkages on the carbohydrate rings. A novel N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde)* linker resin 1 has recently demonstrated potential for solid-phase synthesis

4

M. I. Osborn and T. H. Khan, Tetrahedron 55, 1807 (1999). P. H. Seeberger and W.-C. Haase, Chem. Rev. 100, 4349 (2000). 6 A. Heckel, E. Mross, K. H. Jung, J. Rademann, and R. R. Schmidt, Synlett 171 (1998). 7 G. Hodosi and J. J. Krepinsky, Synlett 159 (1996). 8 S. P. Douglas, D. M. Whitfield, and J. J. Krepinsky, J. Am. Chem. Soc. 117, 2116 (1995). 9 L. G. Melean, W.-C. Haase, and P. H. Seeberger, Tetrahedron Lett. 41, 4329 (2000). * Abbreviations: Ac, acetyl; Ac2O, acetic anhydride; AcOH, acetic acid; Bz, benzoyl, C6H5C — O; ClAc, monochloroacetyl, ClCH2C( — O); ClBz, p-chlorobenzoyl, ClC6H4C( — O); CSPOS, combinatorial solid-phase oligosaccharide synthesis; 1,2-DCE, 1,2-dichloroethane; DCM, dichloromethane; Dde, 1-(4,4-dimethyl-2,6-dioxocyclohexylidene) ethyl; DMAP, 4-dimethylaminopyridine, p-(CH3)2N-C5H4N; DMF, N,N-dimethylformamide; ,-DMT, ,-dimethoxytoluene; DMTST, dimethyl(methylthio)sulfonium triflate; DVB, divinylbenzene; ELSD, evaporative light scattering detection; Fmoc-Cl, 9-fluorenylmethyloxycarbonyl chloride; Lev, levulinoyl, CH3C( — O)CH2 CH2C( — O); MBHA, methylbenzhydrylamine; MeCN, acetonitrile; MeOH, methanol; min, minute(s); MS, molecular sieves; NaOMe, sodium methoxide; NEt3, triethylamine; Piv, pivaloyl, (CH3)3CC — O; Pn, protecting group P in hydroxyl position n; PMB, p-methoxybenzyl, MeOC6H4CH2; PS, polystyrene; Py, pyridine; SPOS, solid-phase oligosaccharide synthesis; Rf, retention factor; Rt, retention time; TBDPS, tert-butyldiphenylsilyl; TBDMS, tert-butyldimethylsilyl; THF, tetrahydrofurane; TsOH, p-toluenesulfonic acid. 5

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251

Fig. 2. Different attaching strategies for the solid-phase synthesis with the Dde linker system 1 leading to sugar loaded resins 2 and 3.

(Fig. 2).10,11 Dde-based linkers have proved stable to most of the chemical conditions commonly employed in carbohydrate synthesis and are readily cleaved by treatment with ammonia, hydrazine, or primary aliphatic amines. Solid-phase synthesis with the Dde linker can be undertaken either by directly attaching a carbohydrate amino group to the linker resin 1 as in construct 2, or alternatively by immobilizing a primary sugar residue via a spacer to the linker resin 1 to form construct 3. This chapter report is concerned with the former method, including potential applications this solid-phase methodology might have for the creation of a combinatorial oligosaccharide system (Fig. 2). The initial experiments were designed with the intent to pursue a high loading and high yielding solid-phase synthesis of a biologically active carbohydrate. To this end MBHA resin (0.7 mmol/g) was employed with the aim of preparing tens to hundreds of milligrams of desired product. Trisaccharide 4, a mammalian cell surface epitope known to elicit a high antibody response in humans as a result of the high titer of the anti-Gal antibody in human sera,12,13 was chosen as the target oligosaccharide (Fig. 3). 10

I. Toth, G. Dekany, and B. Kellam, PCT/AU98/00808 (1996). N. Drinnan, M. West, M. Broadhurst, B. Kellam, and I. Toth, Tetrahedron Lett. 42, 1159 (2001). 12 U. Galili, B. A. Macher, J. Buehler, and S. B. Shohet, J. Exp. Med. 165, 573 (1985). 13 U. Galili, S. B. Shohet, E. Kobrin, C. L. Stults, and B. A. Macher, J. Biol. Chem. 263, 17755 (1988). 11

252

oligosaccharide chemistry

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Fig. 3. Target -Gal trisaccharide 4 and resin-bound glucosamine 5.

This decision was based on the critical roles 4 plays in both the binding of Clostridium difficile associated toxin A, an etiological agent of antibioticassociated diarrhea and Pseudomembranus colitis, and the role that trisaccharide 4 plays in xenograft rejection. The resin bound glucosamine 5 has been previously described and is the ideal construct from which to commence the present synthesis.12 A sugar linker conjugate was initially synthesized in solution phase and then coupled to a relatively high loading MBHA resin. The cleavage of the carbohydrate products from the resin is effected with a 5% hydrazine hydrate/DMF solution. The critical building block in the synthetic sequence to trisaccharide 4 is intermediary galactose block 6, which required differential protection (Fig. 4). The 3-hydroxyl group of the galactosyl ring (protected by temporary group P1) needed to be accessed to provide for further chain elongation, and the 2-hydroxyl group needed to be protected with a participating group P2 so as to provide 1,2-trans stereochemistry. The terminal galactosyl building block 7 should enable generation of a highly stereoselective  linkage leading to the target trisaccharide 4. Accordingly, a set of galactose building blocks 6 containing different acyl substituents in the 2-position was synthesized (Fig. 5). There was a spectrum of reactivities anticipated with the chosen range of acyl subsituents but, as expected,14,15 the donor sugar 6a protected by the 2-O-pivaloyl group provided the best stereochemical outcome (/; 1:16). It is pertinent to note at this stage that the 2-Obenzoyl block 6b provided a very serviceable outcome (/; 1:10). The temporary protecting group (P1) chosen was 9-fluorenylmethoxycarbonyl (Fmoc) due to its high base lability but excellent acid stability; it resulted in a group that could be cleaved easily but would demonstrate excellent stability to glycosylation conditions (Fig. 5). 14 15

T. Nukada, A. Berces, and D. M. Whitfield, J. Org. Chem. 64, 9030 (1999). T. Nishimura, T. Takano, F. Nakatubo, and K. Murakami, Mokuzai Gakkaishi 31, 40 (1993).

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253

solid-phase oligosaccharide chemistry

Fig. 4. Synthetic strategy with resin-bound glucosamine acceptor 5 and galactosyl donors 6 and 7 for the assembly of human antigen trisaccharide 4. 7a, X ¼ Cl; 7b, X ¼ H; 6a P2 ¼ Piv; 6b P2 ¼ Bz.

O O

O

OTBDMS a) O SMe OH

O

OTBDMS b) O SMe OR

Ph

HO

OH OH O SMe OR

Ph O

O

d) O

FmocO

6a R = Piv OR 6b R = Bz 6c R = 4-ClBz 6d R = Ac

c)

O O O

SMe

HO

SMe OR

Fig. 5. Sequence to core galactosyl donors 6a–6d, bearing different stereodirecting groups in position 2. Reagents and conditions: (a) DMAP, 1,2-DCE, R-Cl; (b) MeCN/MeOH, TsOH; (c) ,-DMT, MeCN, TsOH; (d) Fmoc-Cl, DMAP, 1,2-DCE.

Resin bound monosaccharide 5 was glycosylated with pivaloate donor 6a in the presence of dimethyl(methylthio)trifluoromethanesulfonate (DMTST, Fig. 6). Due to the effectiveness of DMTST in solution-phase chemistry, it was used as the sole promoter or leaving group activator in all glycosylations during our studies. The resin was then subjected to 20% NEt3/DMF to effectively cleave the Fmoc group yielding disaccharide acceptor resin 8. The final glycosylation was effected with donor sugar 7a and the

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Fig. 6. Solid-phase synthesis sequence of human antigen trisaccharide 4. Reagents and ˚ , (ii) 20% NEt3/DMF; (b) 7a, DMTST, DCM, MS 4 conditions: (a) (i) DMTST, DCM, MS 4 A ˚ ; (c) 5% hydrazine hydrate/DMF; (d) (i) Ac2O, Py, (ii) NaOMe/MeOH, reflux, (iii) H2, Pd/ A C, THF, AcOH, MeOH/THF.

resin-bound trisaccharide 9 was subjected to cleavage conditions using 5% hydrazine hydrate/DMF. Trisaccharide 10 was treated with acetic anhydride/pyridine to acetylate the amino function generated from the cleavage, followed by treatment with NaOMe/methanol under reflux to cleave the pivaloate ester. All benzyl groups were then removed by hydrogenolysis. The overall yield for the synthesis of trisaccharide 4 after purification by column chromatography was 76%. The fully deprotected trisaccharide was compared with a sample prepared by solution-phase methods and showed matching physical properties.16 After the success of this initial synthesis it was noted that by introducing minor modifications to the synthesis, an extended human blood group determinant could be easily synthesized and that in fact with these modifications there would be quite a high degree of structural diversity accessible. To demonstrate the growing versatility of the system, tetrasaccharide 11, which is considered as either an extended H or B type blood group determinant, was chosen as the next target (Fig. 7). To achieve the synthesis of the branched tetrasaccharide 11, a further building block fucosyl donor 12 was chosen, which promised a highly stereoselective glycosylation outcome. By using the benzoyl galactosyl 16

G. Dekany, L. Bornaghi, J. Pappageorgiou, M. West, and N. Drinnan, PCT AU01/00028 (2000).

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255

solid-phase oligosaccharide chemistry OH OH O OH OH HO O HO HO O O O CH3

O

OH HO

NHAc O OH

OH

BzO

OH

O

CH3

OBz

11

SMe OBn

12

Fig. 7. Blood group determinant tetrasaccharide 11 and fucosyl donor 12.

building block 6b instead of the pivaloyl-protected building block 6a, deprotection of the 2-hydroxyl group could be effected due to the far greater lability of the benzoate ester to base cleavage (Fig. 5). The next critical modification required was the substitution of the chlorobenzylated galactose donor 7a for a benzylated donor 7b (Fig. 4). Terminal donor 7a was originally chosen for its highly crystalline characteristics, but under the conditions of hydrogenolysis the chlorobenzyl groups liberate HCl, in this case four molar equivalents of it. Considering the known sensitivity of fucosyl glycosidic linkages it was deemed expedient to go with simple benzyl groups. Resin sugar conjugate 5 was glycosylated with donor sugar 6b in a fashion similar to that described for the synthesis of 4 (Fig. 6) to form resin-bound disaccharide 13 (Fig. 8). Similarly, Fmoc deprotection followed by glycosylation with donor sugar 7b produced resin-bound trisaccharide 14. This resin was then subjected to a mixture of NaOMe in MeOH/THF to provide resin-bound trisaccharide 15. After the resin was washed and dried it was glycosylated with the fucose building block 12 to furnish resin-bound tetrasaccharide 16. The resin was cleaved with a 5% hydrazine hydrate/DMF solution, followed by the addition of an Ac2O/ Py mixture. After workup the residue was treated with a solution of NaOMe/MeOH and then chromatographed to give the anomerically pure tetrasaccharide 17 in 46% yield over nine steps (Fig. 8). The synthesis of the tetrasaccharide 17 proved to be a high yielding and facile synthesis. Analyses of cleavage solutions indicate that the ‘‘on resin’’ chemistry proceeds extremely clean. Accumulation of alternative anomers appears to account for the majority of reaction by-products. Similarly, the results for the solid-phase synthesis of 4 compare very favorably with those achieved via comparable solution-phase synthesis.17 Overall, 50 mg of the free trisaccharide 4 was prepared. While this system demonstrates a certain level of versatility, there are several criteria to be met to achieve fully generalized combinatorial methodology based on 17

P. J. Garegg and S. Oscarson, Carbohydr. Res. 136, 207 (1985).

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Fig. 8. Solid-phase synthesis sequence to fully protected blood group determinant tetrasaccharide 17 starting from disaccharide acceptor resin 13. Reagents and conditions: ˚ (b) NaOMe, MeOH, THF; (c) 12, DMTST, DCM, MS 4 A ˚ (d) (a) 7b, DMTST, DCM, MS 4 A (i) 5% hydrazine hydrate, DMF, (ii) Ac2O, Py; (e) NaOMe/MeOH.

this kind of technology. The primary issues to address involve selection of appropriate protecting groups (Fig. 9). In this system, although it is possible to synthesize a range of lactosamine-based tri- and tetrasaccharides employing a variety of different monosaccharide donors (Fig. 9), the protecting groups presently used are not fully orthogonal and therefore not amenable to a generalized combinatorial methodology. In this system, the Fmoc group would be cleaved under the same conditions as the benzoyl-protecting group and so is invalidated due to lack of orthogonality. A silyl-protecting group instead of Fmoc would provide the appropriate stability and orthogonality and, therefore, advances itself as a good alternative. The benzylidene ring opening

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257

Fig. 9. General methodology for the preparation of lactosamine-based libraries.

reaction is well established as a typical solution-phase carbohydrate synthetic technique, but its use in solid-phase techniques is rarely mentioned. This reaction could allow discriminate access to either the 4- and 6hydroxyl groups of a pyranose ring while at the same time successfully capping the other hydroxyl group. It is conceivable that a fully orthogonal oligosaccharide synthetic strategy could be accessed by the development of such building blocks. In the following section we will describe the use of a silyl-protected building block in the synthesis of structurally diverse galactosyl oligosaccharides. Combinatorial Solid-Phase Oligosaccharide Synthesis (CSPOS)

A combinatorial approach should provide the ability to generate arrays of compounds instead of single molecules by linear syntheses, and provide the chemist with the opportunity to fully exploit different aspects of diversity, whether it be structural, functional, or otherwise.18,19 This is particularly relevant when considering monosaccharides and carbohydrates due to the high functional density and stereoisomeric forms found with these molecules. Consequently, a juxtaposition of traditional solidphase synthetic carbohydrate techniques with combinatorial synthesis techniques to provide a combinatorial solid-phase oligosaccharide synthesis (CSPOS) platform is an ideal approach for the production of carbohydrate structures to enable the investigation of the biological roles of carbohydrates and to further drug discovery. However, compared to 18

N. K. Terrett, M. Gardner, D. Gordon, R. J. Kobylecki, and J. Steele, Tetrahedron 51, 8135 (1995). 19 F. Balkenhohl, C. von dem Bussche-Huennefeld, A. Lansky, and C. Zechel, Angew. Chem. 108, 2436 (1996); Angew. Chem. Int. Ed. Engl. 35, 2288 (1996).

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Fig. 10. Issues and criteria in SPOS and CSPOS.

conventional solid-phase oligosaccharide synthesis,4,5 the CSPOS methodology is still in its infancy.20–23 One of the major problems to overcome in order to fully develop an efficient system is the requirement to generate suitably protected building blocks for each monomer type (e.g., glucose, galactose, mannose) as well as for each linkage position per monomer type (i.e., for the 1!2, 1!3, 1!4, and 1!6 connections), orthogonal protected to each other.24,25 Further basic considerations and additional requirements of both, SPOS and CSPOS, include (Fig. 10) the following: Support, linker, and cleavage compatible with target structure? Orthogonal protecting groups and elongation method? Common, flexible, and automation-friendly protocol? Hydroxyl and donor reactivity and regiochemistry? Defined stereochemistry at the anomeric carbon? In the synthesis of diverse oligosaccharide libraries, a conventional SPOS methodology would involve laborious solution-phase preparation of many building blocks and that number would increase if increased diversity of the library is to be obtained. To develop a versatile and practical approach, it is necessary to reduce the number of building blocks ideally 20

R. Liang, L. Yan, J. Loebacg, Y. Uozumi, K. Sekanina, N. Horan, J. Gildersleeve, C. Thompson, A. Smith, K. Biswas, W. C. Still, and D. Kahne, Science 274, 1520, (1996). 21 T. Zhu and G.-J. Boons, Angew. Chem. 110, 2000 (1998); Angew. Chem. Int. Ed. Engl. 37, 1898 (1998). 22 D. J. Silva, H. Wang, N. M. Allanson, R. K. Jain, and M. J. Sofia, J. Org. Chem. 64, 5926 (1999). 23 T. Takahashi, H. Inoue, Y. Yamamura, and T. Doi, Angew. Chem. 113, 3330 (2001); Angew. Chem. Int. Ed. Engl. 40, 3230 (2001). 24 G. Baranay and R. B. Merrifield, J. Am. Chem. Soc. 116, 7363 (1977). 25 M. Schelhaas and H. Waldmann, Angew. Chem. 108, 2192 (1996); Angew. Chem. Int. Ed. Engl. 35, 2056 (1996).

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259

n. Selective deprotection LevO

OTBDPS O

PMBO

O

OClAc

O OMe

Combinatorial approach in solution

Disaccharides Trisaccharides Tetrasaccharides Pentasaccharides

n + 1. Glycosylation

Fig. 11. Combinatorial oligosaccharide library approach in solution with an orthogonal protecting group strategy. [See C.-H. Wong, X.-S. Ye, Z. Zhang, J. Am. Chem. Soc. 120, 7137 (1998).]

employing only one single, orthogonal protected universal building block per monomer type.26 This block should allow us to access each linkage position on the carbohydrate residue independently and with no preference in order of the removal of protecting groups (i.e., a completely permutable order of all protecting group manipulations). Wong et al.27 described a similar method and the first combinatorial application thereof in solution phase in 1998. As part of this methodology, the use of a methyl 6-hydroxyhexanate galactoside building block is presented (Fig. 11). This strategy employs individual deprotection steps for every hydroxyl group prior to the sequential glycosylation with different glycosyl donors, linking the new residue to each of the four galactose hydroxyl groups. By this means a library of di-, tri-, tetra-, and pentasaccharides was prepared. The limitation of this method is that the building block cannot be used iteratively to form linear or branched linear oligosaccharides. After each deprotection there is essentially a capping step with the next glycosylation. The incumbent building block is not differentially protected and therefore does not allow for any further chain elongation. Essentially, substitutions may occur only around one monosaccharide scaffold. In addition, the high variation in donor/acceptor reactivities leads to a high level of uncertainty in predicting both the extent and the stereochemical outcome of each reaction.28,29 In our search for more versatile approaches toward linear oligosaccharides we developed a new solid-phase method combining the use of an orthogonal25,26 protected universal building block with powerful perbenzylated acceptors bound on the resin. After initial attachment of the first building 26

G. Dekany, L. Bornaghi, and J. Pappageorgiou, US PCT 09/889687 (2000). C.-H. Wong, X.-S. Ye, and Z. Zhang, J. Am. Chem. Soc. 120, 7137 (1998). 28 Z. Zhang, I. R. Ollmann, X.-S. Ye, R. Wischnat, T. Baasov, and C.-H. Wong, J. Am. Chem. Soc. 121, 6527 (1999). 29 X.-S. Ye and C.-H. Wong, J. Org. Chem. 65, 2410 (2000). 27

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Fig. 12. Iterative CSPOS sequence.

block to the resin, the synthesis follows an iterative, combinatorial process as stepwise outlined below (Fig. 12): 1. Removal of protecting groups at nonlinking positions [step (1), P2, P4, P6)]. These positions will be free hydroxyl functionalities in the final product. 2. Temporarily cap these hydroxyl positions (e.g., benzylation) generating a highly electron-rich carbohydrate residue [step (2), capping]. 3. Removal of the temporary protecting group at the selected linking position [step (3), P3 deprotection]. 4. Stereoselective solid-phase glycosylation of the highly reactive, fully benzylated acceptor using the orthogonal protected building block bearing a neighbor participating group (P2) [step(4), glycosylation]. Branched oligosaccharides might be approached by this method in a similar fashion using benzyl-protected building blocks in the capping step. This new method offers rapid access to a whole variety of glycostructures in a simple manner. A maximum in structural diversity can be gained by using only a minimum number of building blocks. Compared to SPOS approaches, this CSPOS method should be able to generate more value through a much higher efficiency in terms of building blocks. In our first study on the combinatorial assembly of galactose oligomers, we designed thiomethyl glycoside 1830 as universal building block (Fig. 13).27 Thiomethyl galactoside 18 is readily available via a high yielding six-step synthesis starting from methyl 1-thio--d-galactoside.17,31 Thiodonors have already been applied successfully in the combinatorial synthesis of 30

M. Grathwohl, M. Broadhurst, and W. Meutermans, Oral and Poster Presentation, 21th International Carbohydrate Symposium, Cairns, Australia, 2002, OP 051 and PP 204. 31 P. Fu¨gedi, P. J. Garegg, H. Lo¨nn, and T. Norberg, Glycoconjugate J. 4, 97 (1987).

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261

Fig. 13. Universal building block 18, photo linker resin 19, and CSPOS sequence to the fully benzylated, resin-bound monosaccharide acceptors 21, 22, 23, and 24. Reactions: (a) loading, (b) transesterification, (c) TBDPS cleavage, (d) alkylation, (e) ring opening [! 6OH], (f) ring opening [! 4-OH], (g) acidic TBDPS cleavage, (h) neutral/acidic benzylation.

oligosaccharides22,24 and offer the additional advantage of an easy to handle donor type. In these types of donors, the leaving group at the anomeric center may be introduced stereoselectively at an early stage and serves throughout the whole synthesis as a protecting group at C1, being stable toward most conditions used in oligosaccharide construction.31 On position C2 of our proposed universal building block 18, we have chosen the ClBz group (p-chlorobenzoyl) as a hydroxyl protection with stereodirecting properties, on C3 the O-TBDPS group, and in positions C4 and C6 a cyclic benzylidene acetal protection (Fig. 13). All protecting groups have proven to fulfill the given requirements of orthogonality for a permutable, combinatorial access to each linkage position of the core residue (Fig. 12). In our initial experiments on Wang

262

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resin and on Rink resins with a carbamate type linkage, the regioselective ring opening of the 4,6-O-benzylidene acetal to the corresponding benzyl ethers catalyzed by Lewis acids, was accompanied by the loss of product from the resin. To avoid these problems and to increase the stability of the linkage against acids, we choose the 6-nitroveratryl-based photo linker support 19 with an amino methyl-PS-core resin.32,33 Photo-labile resin approaches were already successful applied in SPOS,34–38 as well as in combinatorial approaches.39 Loading of the universal building block 18 onto resin 19 was performed via treatment with DMTST [dimethyl(methylthio)˚ ) yielding polysulfonium triflate] under anhydrous conditions (MS 4 A meric support 20 in a quantitative manner. Loading in this sequence was determined by mass gain as well as by preparative cleavage of the corresponding hemiacetals (h, 365 nm, THF) from the resin. The sequences to fully benzylated acceptor resins 21 and 22, bearing unprotected hydroxyl groups at C4 and C6, start both with cleavage of the acyl group in position 2 via transesterification under Zemple´n conditions40 [reaction (b), NaOMe, THF/MeOH (5:1)], followed by removal of the 3-O-TBDPS group [reac tion (c), TBAF, DMF/THF (2:1), 65 ]. Independent results have shown that this sequence may be shortened by one step through a simultaneous cleavage of both groups under conditions c). Alternatively, selective access to both groups proved to be possible using milder and slightly acidic conditions as described for the synthesis of acceptor 24. Finally, double alkylation led to the common intermediate for both sequences, e.g., 2,3di-O-benzyl-4,6-O-benzylidene galactosyl resin [reaction(d), KOtBu, BnBr, DMF].41 Initial experiments to generate the 4-hydroxy derivative 32

C. P. Holmes and D. G. Jones, J. Org. Chem. 60, 2318 (1995). C. P. Holmes, J. Org. Chem. 62, 2370 (1997). 34 N. Winssinger, J. Pastor, F. DeRoose, and K. C. Nicolaou, J. Am. Chem. Soc. 119, 449 (1997). 35 R. Rodebaugh, S. Joshi, B. Fraser-Reid, M. H. Geysen, and G. M. Paul, J. Org. Chem. 62, 5660 (1997). 36 R. Rodebaugh, S. Joshi, B. Fraser-Reid, and M. H. Geysen, Tetrahedron Lett. 38, 7653 (1997). 37 K. C. Nicolaou, N. Watanabe, J. Li, J. Pastor, and N. Winssinger, Angew. Chem. 110, 1636 (1998); Angew. Chem. Int. Ed. Engl. 37, 1559 (1998). 38 A. B. Kantchev and J. R. Parquette, Tetrahedron Lett. 40, 8049 (1999). 39 M. J. Sofia, N. Allanson, N. T. Hatzenbuhler, R. Jain, R. Kakarla, N. Kogan, R. Liang, D. Lui, D. J. Silva, H. Wang, D. Gange, J. Anderson, A. Chen, F. Chi, R. Dulina, B. Huang, M. Kamau, C. Wang, E. Baizman, A. Branstrom, N. Bristol, R. Goldman, K. Han, C. Longley, S. Midha, and H. R. Axelrod, J. Med. Chem. 42, 3194 (1999). 40 G. Zemple´n, Ber. Dtsch. Chem. Ges. 60, 1555 (1927). 41 T. Wunberg, C. Kallus, T. Opatz, S. Henke, W. Schmidt, and H. Kunz, Angew. Chem. 110, 2620 (1998); Angew. Chem. Int. Ed. Engl. 37, 2503 (1998). 33

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22 via the benzylidene ring opening system Et3SiH/TFA did not lead to the desired product. Finally, both regioselective ring opening reactions were successfully achieved and furnished 6-hydroxy derivative 21 [reaction (e), BH3THF, Bu2BOTf, CH2Cl2]42–44 and the corresponding 4-hydroxy resin ˚ , CH2Cl2].45 It is worth mention22 [reaction (f), NaCNBH3, HCl, MS 4 A ing that both regioisomers showed remarkable differences in their physical properties [6-hydroxy derivative 21: Rf (Tol/Ac, 2:1) ¼ 0.34, Rt (LCMS)46 ¼ 5.25; 4-hydroxy derivative 22: Rf (Tol/Ac, 2:1) ¼ 0.54, Rt (LCMS)46 ¼ 5.31]. Permutation of the protecting group manipulation steps, i.e., ring opening [reaction (e)], transesterification [reaction (b)], followed by a double benzylation of positions 2 and 6 and cleavage of the TBDPSprotecting group at the desired linking position furnished the perbenzylated regioisomer 23, ready for linkage through its 3 position. The sequence to fully benzylated, 2-OH acceptor resin 24 starts with a slightly acidic cleavage of the TBDPS group in position 3 [reaction (g), HFPy, Py]47 leaving the ester protection in position 2 unaffected.48 Reductive ring opening of the 4,6-O-benzylidene acetal to the corresponding 4-O-Bn derivative [reaction (e), BH3THF, Bu2BOTf] furnished the 3,6-diol resin in a clean and regioselective manner. To generate the 2-hydroxy acceptor resin 24, the nonlinking sites have to be capped via benzylation prior to deprotection of the 2-O-C1Bz group. Preferentially, this alkylation step is to be performed under acidic or neutral conditions in order to avoid acyl group migration. Unfortunately, the conditions for the acidic double benzylation in positions 3 and 6 [reaction (h), BnOC(NH)CCl3, BF3OEt2], though successfully employed in solution-phase synthesis,49,50 have so far not been transferable to resin chemistries. We are currently investigating alternative alkylation reagents and conditions. Having access to acceptor resins 21, 22, and 23, solid-phase glycosylation to the corresponding, -linked disaccharides 25, 26, and 27 was performed using again donor 18 (Fig. 14).

42

L. Liang and T.-H. Chan, Tetrahedron Lett. 39, 355 (1998). X. Wu, M. Grathwohl, and R. R. Schmidt, Org. Lett. 3, 747 (2001). 44 X. Wu, M. Grathwohl, and R. R. Schmidt, Angew. Chem. 114, 4664 (2002); Angew. Chem. Int. Ed. Engl. 41, 4489 (2002). 45 P. J. Garegg, Pure Appl. Chem. 56, 845 (1984). 46 System parameters: All LCMS spectra were recorded on a Micromass LCZ/LCT system Mux 4 [gradient, MeCN/H2O (5:95) to (100:0); time, 12 min; flow rate, 2 ml/min; column, Zorbax, SB-C18 (4.6  50 mm); pore size, 5 m] after analytical cleavage from the corresponding resin. 47 B. M. Trost, C. G. Caldwell, E. Murayama, and D. Heissler, J. Org. Chem. 48, 3252 (1983). 48 M. Grathwohl and R. R. Schmidt, Synthesis 2263 (2001). 49 H.-P. Wessel, T. Iversen, and D. R. Bundle, J. Chem. Soc. Perkin Trans. I 2247 (1985). 50 H.-P. Wessel and D. R. Bundle, J. Chem. Soc. Perkin Trans. I 2251 (1985). 43

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Fig. 14. CSPOS sequence to disaccharides 25, 26, and 27 via the fully benzylated, resinbound monosaccharide acceptors 21, 22, and 23. Reagents and conditions: (a) DMTST, 18 (2.0 ˚ , CH2Cl2, two runs. equiv.), MS 4 A

In this elongation cycle, using 2.0 equivalents of donor 18 and repeating the glycosylation reaction once to ensure complete conversion, achieved best results. The use of molecular sieves proved to be crucial; removal of the finely grounded powder after reaction was done via simple washings through a polyethylene porous disc. To prove the method and to extend it to the next generation of linkages, the trisaccharides, we decided to synthesize an example of the Gal(1!6)Gal(1!3)Gal trisaccharide resin 29 following the sequence outlined below (Fig. 15). Starting from fully protected disaccharide resin 27, the primary resinbound acceptor 28 was synthesized according to the conditions described above for the monosaccharide cases. All conditions could be successfully transferred to the disaccharide stage and the final elongation to resinbound trisaccharide 29 was performed under standard conditions [condi˚ , CH2Cl2, two runs] to generate tions (e), DMTST, 18 (2.0 equiv.), MS 4 A an anomeric mixture of hemiacetals 30 after preparative cleavage from the resin (Fig. 16). Performing the elongation via repeated glycosylation cycles led to a virtually complete consumption of disaccharide acceptor 28 (starting material <2%) and afforded the target trisaccharide

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265

Fig. 15. CSPOS sequence to the final Gal(1!6)Gal(1!3)Gal trisaccharide resin 29, starting from fully protected disaccharide resin 27. Reagents and conditions: (a) HFPy, Py; (b) NaOMe, THF/MeOH (5:1); (c) KOtBu, BnBr, DMF; (d) BH3THF, Bu2BOTf, CH2Cl2; ˚ , CH2Cl2, two runs. (e) DMTST, 18 (2.0 equiv.), MS 4 A

O-(4,6-O-benzylidene-3-O-tert-butyldiphenylsilyl-2-O-para-chlorobenzoyl-d-galactopyranosyl)-(1!6)-(2,3,4-tri-O-benzyl--d-galactopyranosyl)(1!3)-2,4,6-tri-O-benzyl-/-d-galactopyranose 30 in over 88% purity (LCMS trace of the crude cleavage mixture, Rt (LCMS)46 ¼ 10.42, Rf (Tol/Ac, 2:1) ¼ 0.86; C90H93ClO7Si, calcd: 1526.59 [MNH4þ], found: 1526.69 [MNH4þ]) after a 12-step solid-phase synthesis (Fig. 16). Conclusion

The overwhelming potential for creating diversity in oligosaccharide libraries, at least from a theoretical point of view, is inevitably linked with growing complexity in the required synthetic routes. A truly versatile method would provide parallel access to structurally diverse oligosaccharide libraries but is currently not at hand. The solid-phase concept that we proposed here of selective deprotection and capping nonlinking positions prior to glycosylating perbenzylated acceptors enables access to combinatorial libraries of structurally different oligosaccharides with a minimal number of building blocks. Our initial results indicate that this is a realistic approach, though much more development work needs to be done before its full potential can be explored. Here we have demonstrated that by using one universal building block we can access variously linked di- and trisaccharides in a regio- and stereoselective manner on solid-phase, thereby opening the possibilities of generating combinatorial libraries of structurally diverse oligosaccharides.

266

Ph O O O TBDPSO

Mw=1508

BnO O

ClBzO BnO

AL098-67_AN#2609 100 Mw=1508

AL098-67_AN#2609127 (10.361) Cm (127:130)

OH OBn

O

C90H93ClO17Si

PR

O

O BnO

30

100

OBn BnO

1528.6877

ELSD An1 2.36e5

10.42 1: Scan ES+ 4.07e3

1526.6973

Time 7.42 8.11 10.42

Height 23279 6057 218744

Area Area% 3283.00 9.48 623.34 1.80 30717.55 88.72

Exact Mass: 1508.59

oligosaccharide chemistry

1581.6956

%

1582.7329

%

1492.7949

1583.6792

1059.3802 1061.6107

0 800

1584.8055 1649.7124

1831.6660

m/z 900

1000

1100

1200

1300

1400

1500

1700

1600

7.42

1800

1900

SM 8.11

Time 3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

Fig. 16. ELSD chromatogram and mass spectrum from CSPOS sequence of the crude trisaccharide product 30 after cleavage from resin 29 (12 steps). Glycosylation to 29 was performed in two runs using 2.0 equivalents of donor 18 per run. (See color insert.)

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Acknowledgments This work was supported in part by the Australian Research Grants Council and the Government of Queensland. The authors thank all Alchemia core facilities that participated in these study, especially Mr. Hoan The Vu for his support in analytical tasks and the entire VAST Ô team for their support of this research.

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Section VI Peptide Synthesis and Screening

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encoded peptidomimetic and small molecule libraries

271

[15] Design, Synthesis, Screening, and Decoding of Encoded One-Bead One-Compound Peptidomimetic and Small Molecule Combinatorial Libraries By Ruiwu Liu, Jan Marik, and Kit S. Lam Introduction

In 1991, we first introduced the ‘‘one-bead one-compound’’ (OBOC*) combinatorial library method.1 Since then, it has been successfully applied to the identification of ligands for a large number of biological targets.2,3 Using well-established on-bead binding or functional assays, the OBOC method is highly efficient and practical. A random library of millions of beads can be rapidly screened in parallel for a specific acceptor molecule (receptor, antibody, enzyme, virus, etc.). The amount of acceptor needed is minute compared to solution phase assay in microtiter plates. The positive beads with active compounds are easily isolated and subjected to structural determination. For peptides that contain natural amino acids and have a free N-terminus, we routinely use an automatic protein sequencer with Edman chemistry, which converts each -amino acid sequentially to its phenylthiohydantoin (PTH) derivatives, to determine the structure of peptide on the positive beads. We have recently reported methods for sequencing peptides containing many unnatural -amino acids.4,5 However, there is no simple way to *

Abbreviations: AllocOSu, allyloxycarbonyl-N-hydroxysuccinimide; BCIP, 5-bromo-4chloro-3-indolyl; Boc, tert-butyloxycarbonyl; tBu, tert-butyl; DCM, dichloromethane; Dde, 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl; Ddz, ; -dimethyl-3,5-dimethoxybenzyloxycarbonyl; DIC, N,N0 -diisopropylcarbodiimide; DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; FmocOSu, 9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide; OBOC, one-bead one-compound; PBS, phosphate-buffered saline; Pmc, 2,2,5,7,8pentamethylchroman-6-sulfonyl; PTH, phenylthiohydantoin; RR, relative reactivities; Teoc, 2-(trimethylsilyl)ethoxycarbonyl; TFA, trifluoroacetic acid; Trt, trityl. 1 K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, and R. J. Knapp, Nature 354, 82 (1991). 2 K. S. Lam, T. Sroka, M. L. Chen, Y. Zhao, Q. Lou, J. Wu, and Z. G. Zhao, Life Sci. 62, 1577 (1998). 3 K. S. Lam, M. Lebl, and V. Krchnak, Chem. Rev. 97, 411 (1997). 4 R. Liu and K. S. Lam, Anal. Biochem. 295, 9 (2001). 5 R. Liu and K. S. Lam, in ‘‘Peptides: The Wave of the Future. Proceedings of the Second International and the Seventeen American Peptide Symposium’’ (M. Lebl and R. A. Houghten, eds.), p. 299. American Peptide Society and Kluwer Academic Publishers, San Diego, 2001.

METHODS IN ENZYMOLOGY, VOL. 369

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directly determine the structure of peptidomimetic and small molecule compounds on a single bead. Typical spectroscopic methods such as mass spectrometry (MS), nuclear magnetic resonance (NMR), and infrared (IR) will not provide sufficient information for complete structure elucidation of compounds contained in a single bead. Another approach is to use an encoding strategy. Various physical and chemical encoding methods have been reported by several groups and the subject has been recently reviewed.3,6–9 These encoding methods are successful to a varying degree, but are far from ideal. Physical encoding methods are unable to encode a large number of compounds, such as more than 100,000. Furthermore, some of these methods are not applicable to OBOC bead library technology. While currently available chemical encoding methods are useful for OBOC bead library, they have some limitations. First, the coding molecule is randomly present at the surface, which may interfere with the binding of the target protein to the testing compound resulting in a false-positive result. A spatial segregation of the ligand and the encoding tag is necessary to minimize the unwanted interaction. Second, the current chemical encoding method requires that the synthetic chemistry of the coding tag and the testing compound be orthogonal, which doubles the number of required synthetic steps. To overcome these limitations, we have recently reported a novel, highly efficient, and robust encoding system for OBOC peptidomimetic and small molecule combinatorial libraries.10 In this method, only the testing compounds are present on the exterior of the bead (i.e., exposed to the target proteins), and the coding peptide molecules are placed in the core of the bead. A novel strategy has also been employed to eliminate the extra synthetic steps and minimize the amount of undesirable side products by combining the synthetic steps of both coding molecules and testing compounds. The use of polyglutamic acid11 or proteases11,12 to prepare topologically segregated bifunctional beads is successful to a certain degree, but far from ideal since they are technically difficult to control, laborious, and not reproducible from batch to batch. Recently we have developed a highly efficient 6

A. W. Czarnik, Curr. Opin. Chem. Biol. 1, 60 (1997). X. Y. Xiao, Front. Biotechnol. Pharm. 1, 114 (2000). 8 C. Barnes and S. Balasubramanian, Curr. Opin. Chem. Biol. 4, 346 (2000). 9 R. L. Affleck, Curr. Opin. Chem. Biol. 5, 257 (2001). 10 R. Liu, J. Marik, and K. S. Lam, J. Am. Chem. Soc. 124, 7678 (2002). 11 M. Lebl, K. S. Lam, S. E. Salmon, V. Krchnak, N. Sepetov, and P. Kocis, U. S. Patent 5 840 485 (1998). 12 J. Vagner, G. Barany, K. S. Lam, V. Krchnak, N. F. Sepetov, J. A. Ostrem, P. Strop, and M. Lebl, Proc. Natl. Acad. Sci. USA 93, 8194 (1996). 7

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method termed the ‘‘biphasic solvent approach’’ for spatial segregation on individual TentaGel beads (Rapp Polymere). This approach makes use of the two immiscible solvents system in which TentaGel resin has approximately equal swelling volumes in both phases. The crucial requirement is to keep the first solvent inside the bead and the second solvent outside the bead while the reaction on the outer layer is occurring. The derivatizing reagents that modify the surface of the bead should be soluble in the outer solvent only. Water and a mixture of dichloromethane (DCM)/diethyl ether do meet these requirement. The ratio of the DCM/diethyl ether may be calculated from the swelling volumes and then adjusted experimentally for the given reaction. For TentaGel beads, the resin is swollen in the first solvent (water) and then the coupling reaction to the surface of the wet bead is carried out in the second solvent (DCM/diethyl ether). We have discovered that the optimized ratio of DCM/diethyl ether is 55/45. The derivatizing reagent, e.g., 9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide (FmocOSu) or allyloxycarbonyl-N-hydroxysuccinimide (AllocOSu), in the solvent mixture reacts with the amino group on the surface of the TentaGel bead rapidly (normally less than 30 min), whereas the amino group in the interior of the bead in water remains intact. Thus, only the outer layer of the bead is derivatized. By adjusting the amount of derivatizing reagent used, the thickness of the outer layer can be easily controlled. Encoded libraries are synthesized on topologically segregated bifunctional beads described above. To efficiently generate an encoded chemical library, the appropriate orthogonal protecting groups and synthetic schemes must be chosen so that the number of necessary chemical reactions can be minimized by coupling the building blocks to the testing and coding arms simultaneously. For example, the orthogonal protecting groups for amine can be Fmoc, Alloc, tert-butyloxycarbonyl (Boc), 1(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), ,-dimethyl-3,5dimethoxybenzyloxycarbonyl (Ddz), and 2-(trimethylsilyl)ethoxycarbonyl (Teoc) groups. In our encoding system, the testing compound is a peptidomimetic or small molecule, and the coding molecule is a peptide containing a large number of unnatural -amino acids derived from different building blocks that are used for generating the peptidomimetic and small molecules. The retention time of these unnatural -amino acids must be predetermined by a protein sequencer. The general synthetic and decoding scheme of an encoded library is shown in Scheme 1. The amino group on the outer layer of the bead is first topologically protected with a protecting group P, such as Fmoc, Alloc, and a free amino group in the interior. A coding arm precursor, (P3)A0 -(P2)Y (P1)X, is then constructed in the interior of the bead. (P1)X and (P2)Y represent trifunctional amino acids, which are preprotected by orthogonal

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P2 H2N

H2N

H 2N

_ Y _ X P3 _ A9 _ P NH

_ P NH P2 P3

F

E

_ A9 _ Y _ X

H2N

P3 _ A9

P2

P1

_ Y _ X

P1 P

S _ D

_ D P2

E

_ A9 _ Y _ X

S _ D

S F

_ D

P1

P3 _ A9 _ Y _ X

Edman degradation

E

E

E P2

P2

P1

F

Identify D, E, F

S

E

_ _ PTH A9 PTH _ Y PTH X

D

F

Chemical structure

Scheme 1. General synthetic and encoding/decoding scheme of encoded library.

groups, P1 and P2, respectively. D represents a bifunctional building block that links the scaffold to the bead, and it can be coded by amino acid A0 , which represents a single amino acid or a combination of two amino acids. S stands for the scaffold of the testing arm with preprotected multifunctional groups onto which building blocks can be covalently attached. To minimize the synthetic steps, the protecting groups on the scaffold are carefully chosen so that they are identical or very similar to the types (e.g., Fmoc and Dde both can be removed by 2% hydrazine) used on the coding arm. During the peptidomimetic and small molecule library synthesis, both the deprotection of protecting groups on both the coding arm and testing arm, and the coupling of each building block (E or F) to both the testing arm and coding arm are achieved simultaneously. Therefore, each step of the peptidomimetic or small molecule synthesis can proceed as if there is no coding arm. The decoding process is straightforward since there is no need of coding tag cleavage and retrieval prior to Edman microsequencing. The testing molecule is stable to the Edman condition, and the coding peptide consists of -amino acids that can be readily sequenced (Scheme1). The signal of the amino acid (A0 ) in the first cycle corresponds to D of the testing compound. The signal of PTH-(F)Y in the second cycle corresponds to the building block F. The signal of PTH-(E)X in the third cycle corresponds to the last coupling moiety (E). By combining all the decoded structural information, the complete chemical structure of the testing molecule is easily determined. Alternatively, the coding tag can be released (if a cleavable linker is used) and decoded unambiguously by mass spectrometry, if each of the coding subunit(s) has a unique molecular mass.

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TABLE I Coding Units and Corresponding Reactions Coding Units Lysine, 4-amino phenylalanine Lysine, 4-amino phenylalanine Lysine, 4-amino phenylalanine Lysine, 4-amino phenylalanine 4-Bromophenylalanine 4-Bromophenylalanine Glutamic acid Serine Tyrosine Cysteine 1-Amino-4-ketocyclohexyl carboxylic acid 2-Aminopent-4-enoic acid 2-Amino-3-acryloyloxypropionic acid 2-Amino-3-(4-formylbenzoylamino)propionic acid

Building Blocks

Chemical Reactions

Carboxylic acids, acyl halides, sulfonyl halides, acid anhydride Isocyanates, isothiocyanates

Amide formation

Aldehydes (aliphatic, aromatic, heterocyclic) Alkyl halides

Reductive alkylation

Aryl boronic acids Amines Amines Carboxylic acids, acyl halides, sulfonyl halides, acid anhydride Alcohols Alkyl halides Ph3P ¼ CHR RCH2NO2

Urea, thiourea formation

Nucleophilic substitution (N-alkylation) Suzuki coupling Nucleophilic substitution Amide formation Acylation Mitsunobu reaction S-Alkylation Wittig reaction

Secondary amines

1,3-Dipolar cycloaddition reaction Michael addition

Ketones

Aldol condensation

In principle, various types of encoded peptidomimetic and small molecule libraries can be generated using this method via different chemical reactions (Table I). The trifunctional amino acids listed in Table I are either commercially available or can be easily synthesized. These amino acids with orthogonal protecting groups are used as encoding units. The side chains of these -amino acids are sites to which building blocks are attached via a large number of organic reactions, including C–C bond-forming reactions (e.g., Suzuki coupling and Wittig reaction). In summary, the biphasic approach to derivatize a resin bead topologically and bifunctionally is highly efficient, inexpensive, and easy to control. The advantages of this encoding method include (1) the chemical stability of the peptide tag under many organic reaction conditions, (2) the ease of simultaneous coupling of common building blocks to the scaffold of the testing compounds and the side chains of the -amino acids on the coding peptide, and (3) straightforward and unambiguous decoding with automatic microsequencing or mass spectrometry. One added advantage is

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the potential reduced usage of expensive scaffold if the majority of substitution of beads is coding tag, which is relatively cheap in general. Besides eliminating a number of synthetic steps by combining the synthesis on the testing and encoding arms in many of the coupling steps, this method also enables the coding arm precursor to be prepared in bulk prior to the synthesis of the libraries. Consequently, encoded peptidomimetic and small molecule libraries may be prepared as if there is no coding arm. General Synthetic Procedures for Encoded Peptidomimetic and Small Molecule Libraries

In general, an Fmoc/Boc orthogonal protecting strategy is most often used in library synthesis. Therefore, the protecting groups P and P3 in Scheme 1 are Fmoc and Boc, respectively. 1. The coding-precursor strand is first constructed in the interior of the topologically segregated bifunctional TentaGel beads described above. a. Couple the first coding unit, Boc-protected amino acid Boc-(P1)X, to the beads. Boc-(P1)X (3 equiv. to the free NH2 of the total resin) and 1-hydroxybenzotriazole (HOBt) (3 equiv.) are dissolved in N,N-dimethylformamide (DMF), then added to the resin followed by addition of N,N0 -diisopropylcarbodiimide (DIC) (3 equiv.). The beads are shaken at room temperature for 2 h. b. Wash the beads with DMF, MeOH, and DCM, three times each, respectively. c. Remove Boc with 50% trifluoroacetic acid (TFA) in DCM at room temperature for 30 min; after filtration, the bead is neutralized with 2% N,N-diisopropylethylamine (DIEA) in DMF. d. Couple the second coding unit, Boc-protected amino acid Boc-(P2)Y, in the same manner as in step (a). e. Repeat steps (b) and (c). f. Split the peptide-resin into n equal portions (n equal to the number of the first testing blocks). g. Couple the third coding units, n different Boc-A0 (single Bocamino acids or a combination of two Boc-amino acids, one in each well, 3 equiv.), in the same manner as in step (1). Usually, the third coding unit can be a large number of amino acids (up to 253). After coupling, keep it in a spatially separated format. h. Remove Fmoc with 20% piperidine in DMF (twice, 5 min, 15 min). i. Wash the beads with DMF, MeOH, and DMF, three times each, respectively.

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2. Couple the first testing building block, e.g., n different Fmoc-amino acids in the same manner as above. Dissolve all Fmoc-amino acids in equal volumes of HOBt solution in DMF and add the solutions to the corresponding wells (i.e., one amino acid in each well), followed by the addition of DIC. The coupling proceeds at room temperature for 2 h. 3. Combine and mix all the beads, and then deprotect the ‘‘Fmoc’’ as step (1 h). 4. Couple the scaffold (P1)S(P2) using compatible chemistry. 5. Remove the protecting group P1 of both the coding arm and scaffold by means of a compatible reaction. 6. The beads are split into m equal portions (m is equal to the number of the second testing building blocks). 7. Couple common building block E to both the coding and testing arms of individual beads by means of compatible chemistry. 8. Combine and mix all the beads and wash with different solvents. 9. Remove the protecting group P2 of both the coding arm and scaffold by means of a compatible reaction. 10. Split the beads into I equal portions (I is equal to the number of the third testing building blocks). 11. Couple common building block F to both the coding and testing arms of individual beads by means of compatible chemistry. 12. Combine and mix all the beads, and wash with DMF, MeOH, and DCM, three times each, respectively; dry the beads under vacuum for 1–3 h. 13. Remove the Boc-protecting group of the coding molecule and all other protecting groups of any testing and coding molecule with TFA-containing reagent, e.g., reagent K (TFA:phenol:thioanisole:water:1,2-ethanedithiol/82.5:5:5:5:2.5, v/v/v/v/v); the deprotection proceeds at room temperature for 2 h. 14. Neutralize the beads with 2% DIEA in DMF twice, then wash sequentially with DMF, MeOH, DCM, DMF, DMF/water, water, and finally PBS buffer. The library is ready for screening.

On-Bead Screening of Encoded Peptidomimetic and Small Molecule Libraries

Various on-bead binding or functional screening methods for OBOC combinatorial libraries are described in Chapter 17 in this volume and will not be repeated here. The positive beads are physically isolated under a microscope for structure determination.

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Decoding of Peptidomimetic and Small Molecule Libraries

Decoding of peptide-encoded peptidomimetic and small molecule library can be accomplished by two methods: (1) direct microsequencing of a bead-bound peptide tag on an automatic protein sequencer,10 and (2) mass spectroscopic analysis of a released peptide tag retrieved from a bead.13 The latter approach requires a cleavable linker. Sample processing for decoding by the first method is easy and straightforward as there is no need for cleavage and retrieval of the coding tag. Since the testing molecule is unsequencable, only the signals from the coding peptide are observed. Comparing the coding signals (retention time) with the external reference (predetermined retention time of PTH amino acid derivatives), the structure of the building blocks is determined. By combining the structure information of scaffold (known) and building blocks, the entire chemical structure of the peptidomimetic or small molecule is easily determined. Peptide sequencing is performed on a protein sequencer following the manufacturer’s instructions but with a slightly modified gradient program and method as described in chapter 17 in this volume. Materials and Methods

All solvents and chemicals were purchased from Aldrich and were analytical grade if not given otherwise. FmocOSu and protected l-amino acids were purchased from ChemImpex Inc, Wood Dale, IL. Perkin-Elmer/ Applied Biosystems protein sequencer (model 494) was used for the sequencing of the peptides. Preparation of Topologically Segregated Bifunctional Beads with 70% Fmoc Outside and 30% Free NH2 Inside TentaGel S NH2 beads (2.0 g, loading 0.52 mmol) were swollen in water for 24 h. Water was removed by filtration, and the solution of FmocOSu (122.8 mg, 0.364 mmol) in DCM/diethyl ether (100 ml, 55/45) mixture was added to the wet beads, followed by addition of DIEA (126 l, 0.728 mmol). The mixture was shaken vigorously at room temperature for 30 min. The beads were washed three times with DCM/diethyl ether mixture and six times with DMF to remove water from the beads. Synthesis of an Encoded Peptidomimetic Library, an Example The synthesis of a 158,400-membered encoded peptidomimetic library (96  50  33) is given in Scheme 2, using -Phe(4-NH2) as a trifunctional scaffold for the testing compound and Phe(4-NH2) as a peptide backbone 13

A. H. Franz, R. Liu, A. Song, K. S. Lam, and C. B. Lebrilla, J. Comb. Chem. 5, 125 (2003).

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Fmoc-OSu

H2 N

H2N Fmoc-NH

Boc-Aa1(P)-Phe(4-NO2)-Lys(Dde) FmocNH CONH

Aa 2 NH P

Couple coding Deprotect Boc Couple coding Deprotect Boc

block Boc-Lys(Dde) with 50% TFA/DCM block Boc-Phe(NO2) with 50% TFA/DCM

5) Couple 96 coding blocks Boc-Aa1(P) 6) Remove Fmoc with 20% piperidine 7) Couple 96 testing blocks Fmoc-Aa2(P)

Boc-Aa1(P)-Phe(4-NO2)-Lys(Dde) 1) Remove Fmoc with 20% piperidine 2) Couple Fmoc-beta-Phe(4-NO2) Fmoc-Aa2-NH

1) Reduce NO2 with SnCl2 2) Couple 33 R1COOH, R1COCl or R1SO2Cl 3) Remove Dde and Fmoc with 20% piperidine 4) Couple 50 R2COOH or (R2CO)2O 5) Remove Boc and side chain protecting groups of 96 amino acids

P

Coding peptide NH2-Aa1-Phe(4-NHCOR1)-Lys(COR2) R2CONH

CONH

Aa 2 NH

NHCOR1

NO2

Testing molecule Scheme 2. Synthesis of encoded model library.

encoded peptidomimetic and small molecule libraries

H2N

1) 2) 3) 4)

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for the coding tag. Their corresponding precursors -Phe(4-NO2) and Phe(4-NO2) have been selected for the attachment of the building blocks because the NO2 group can be treated like a protected amino group, and it can be easily converted to amino groups by reduction with SnCl2. The side chain amino groups of these -amino acids are acylated by a large number of carboxylic acids, acid anhydrides, acyl chlorides, or sulfonyl chlorides. The elution profiles of many PTH derivatives of lysine and Phe(4-NH2) have been predetermined using an improved sequencing gradient program.10 Forty-eight carboxylic acids and two acid anhydrides have been selected as building blocks to couple to the side chain of lysine. Twenty-seven carboxylic acids, three acyl chlorides, and three sulfonyl chlorides have been chosen to acylate the amino group of aniline. To obtain an unambiguous sequence, the principle of choosing a building block is that two amino acid derivatives must have a retention time difference greater than 0.10 min. In the encoded library shown in Scheme 2, the first testing building block is 96 amino acids. They are encoded with 22 single amino acids (19 natural amino acids plus norvaline, norleucine, and cyclohexylglycine) and 74 combinations of two amino acids from the 22 amino acids. To ensure that a signal from each of the doublet amino acids is detectable, preferably in ‘‘equipeak height’’ ratio, the ratio of the two encoding Boc-amino acids has to be adjusted according to their relative reactivities (RR) (see Table II).10 In the encoding strategy, the reduction of the NO2 group, the removal of protecting groups Fmoc and Dde of NH2 (using 2% hydrazine in DMF), and the acylation of both coding and testing arms are achieved simultaneously. This greatly reduces the number of synthetic steps. The experiment TABLE II Relative Reactivities (RR) of 22 Boc-amino Acidsa Amino Acids

RR

Amino Acids

RR

Amino Acids

RR

Boc-Asp(OtBu)-OH Boc-Asn(Trt)-OH Boc-Ser(tBu)-OH Boc-Gln(Trt)-OH Boc-Thr(tBu)-OH Boc-Gly-OH Boc-Met-OH Boc-Val-OH

8.38 6.40 1.47 2.44 2.29 3.50 4.77 2.98

Boc-Nva-OH Boc-Trp(Boc)-OH Boc-Phe-OH Boc-Ile-OH Boc-Glu(OtBu)-OH Boc-His(Trt)-OH Boc-Ala-OH

5.18 3.08 5.44 1.28 7.28 1.00 6.08

Boc-Arg(Pmc)-OH Boc-Tyr(tBu)-OH Boc-Pro-OH Boc-Lys(Boc)-OH Boc-Leu-OH Boc-Nle-OH Boc-Chg-OH

1.73 5.19 3.98 3.00 4.09 4.98 2.56

a

Natural amino acids are designated by the standard three-letter code. Nva, norvaline; Nle, norleucine; Chg, cyclohexylglycine; tBu, tert-butyl; Trt, trityl; Pmc, 2,2,5,7,8pentamethylchroman-6-sulfonyl.

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detail of the synthesis of an encoded model peptidomimetic library is as follows. Topologically segregated bifunctional TentaGel resin (2.0 g, 0.52 mmol) (30% substitution of the beads for the coding tag) as prepared above is used for the library synthesis. The coding-precursor strand is first built in the interior of the bead: a solution of Boc-Lys(Dde) (250 mg, 0.468 mmol), HOBt (71.6 mg, 0.468 mmol), and DIC (73.2 l, 0.468 mmol) in DMF (20 ml) is added to the beads. After coupling at room temperature for 2 h, the ninhydrin test is negative, i.e., the coupling reaction is finished. The beads are then washed with DMF, MeOH, and DCM, three times each. Then 50% TFA in DCM (20 ml) is added to the beads. After 30 min, the beads are washed with DCM, neutralized with 2% DIEA in DMF twice, and washed with DMF three times. A solution of Boc-Phe(4-NO2) (145.2 mg, 0.468 mmol) and HOBt (71.6 mg, 0.468 mmol) in DMF (20 ml) is added to the beads followed by addition of DIC (73.2 l, 0.468 mmol). The amino acid coupling and the subsequent Boc group deprotection are achieved in the same manner as above. The peptide-resin is split into 96 equal portions in a 96well plate. Ninety-six different ‘‘coding molecules’’ (22 single Boc-amino acids and 74 combinations of two Boc-amino acids, one in each well, 3 equiv.) are dissolved in a solution of HOBt (3 equiv.) and DIC (3 equiv.) in DMF, and are added into 96 wells (each well receives only one amino acid). The coupling is carried out at room temperature for 2 h. After filtration, the beads are washed with DMF, MeOH, and DMF, respectively, three times each. Twenty percent piperidine in DMF (20 ml, twice, 5 min, 15 min) is used to remove the Fmoc group in a 96-well plate. The beads are washed with DMF, MeOH, and DMF, respectively, three times each. Ninety-six Fmoc-amino acids (0.0114 mmol) are dissolved in 96 equal volumes of a solution of HOBt (total 167.2 mg HOBt, 1.092 mmol) and DIC (total 171 l, 1.092 mmol) in DMF (57.6 ml). The solutions are added to the corresponding wells (i.e., one amino acid in each well). The coupling reaction proceeds at room temperature for 2 h. After filtration, all resin beads in 96 wells are combined, mixed, and washed with DMF, MeOH, and DMF, respectively, three times each. The ‘‘Fmoc’’ deprotection is achieved with 20% piperidine in the same manner. A solution of Fmoc--Phe(4-NO2) (472 mg, 1.092 mmol), HOBt (167.2 mg, 1.092 mmol), and DIC (171 l, 1.092 mmol) in DMF is added to the resin. The coupling is conducted at room temperature for 2 h. The beads are washed with DMF, MeOH, and DMF, respectively. NO2 groups in both coding and testing arms are reduced with 2 M SnCl22H2O in DMF (30 ml) at room temperature for 2 h, twice. The resin is split into 33 equal portions and transferred to 33 disposable polypropylene columns with a polyethylene frit. Acylation of the

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amino group in aniline is as follows: 27 individual carboxylic acids (20 equiv. each) in a solution of DIC and DIEA in DCM are added to the beads of 27 columns (each column receives one carboxylic acid). Three acyl chlorides and three sulfonyl chlorides in a solution of DIEA in DCM are added to the beads of six columns separately. The coupling reaction is carried out at room temperature overnight. After filtration, all 33 portions of beads are combined and randomized, and then washed with DMF, MeOH, and DMF again. Two percent hydrazine in DMF (30 ml) is added to the beads. The reaction proceeds at room temperature for 5 min, and repeats for another 10 min. Fmoc in the testing arm and Dde in the coding arm are removed simultaneously. The resin is then distributed into 50 portions to 50 columns, and 48 different carboxylic acids (20 equiv. each) are preactivated by BOP in DMF/DIEA and added to the corresponding columns. Two acid anhydrides, i.e., diglycolic anhydride and glutaric anhydride, are dissolved in a solution of DIEA in DMF (0.5 ml) and added to two separate columns. The acylation is conducted at room temperature for 2 h and repeated once. After filtration, all 50 portions of resin are combined and washed with DMF, methanol, and DCM. The beads are then dried under vacuum for 1 h. Side chain deprotection is carried out with reagent K (40 ml) at room temperature for 2 h. After neutralization with 2% DIEA/DMF twice, the resin is washed sequentially with DMF, MeOH, DCM, DMF, DMF/water, water, and finally PBS. The library is now ready for screening. Fmoc-amino acids used as building blocks of testing compounds are as follows: Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Ile-OH, Fmoc-His(Trt)-OH, Fmoc-Tyr(tBu)-OH, FmocArg(Pmc)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-Trp(Boc)-OH, Fmoc-d-Ala-OH, Fmoc-d-Arg(Pmc)-OH, Fmoc-d-Trp(Boc)-OH, Fmoc-d-Cys(Trt)-OH, Fmoc-d-Asp(OtBu)-OH, Fmoc-d-Glu(OtBu)-OH, Fmoc-d-His(Trt)-OH, Fmoc-d-Gln(Trt)-OH, Fmoc-d-Leu-OH, Fmoc-d-Met-OH, Fmoc-d-ProOH, Fmoc-d-Ser(tBu)-OH, Fmoc-d-Lys(Boc)-OH, Fmoc-d-Tyr(tBu)OH, Fmoc-d-Thr(tBu)-OH, Fmoc-d-Phe-OH, Fmoc-d-Asn(Trt)-OH, Fmoc-3-(4-pyridyl)alanine, Fmoc-d-3-(3-pyridyl)alanine, Fmoc-4-tertbutoxyproline, Fmoc-3-chlorophenylalanine, Fmoc-norleucine, Fmoc-2cyclohexylglycine, Fmoc-2-aminoisobutyric acid, Fmoc-tranexamic acid, Fmoc-(R,S)-3-amino-3-(2-furyl)propionic acid, Fmoc-(R,S)-(6,7-dimethoxy)-1,2,3,4-tetrahydroquinoline-3-carboxylic acid, Fmoc-(R,S)-3amino-3-(4-hydroxyphenyl)propionic acid, Fmoc-(R,S)-3-aminovaleric acid, Fmoc-(R,S)-3-amino-3-(3,4-dichlorophenyl)propionic acid, Fmocisonipecotic acid, Fmoc-(R,S)-3-amino-3-(3,4-methylenedioxyphenyl)

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propionic acid, Fmoc-(R,S)-3-amino-3-(4-tert-butylphenyl)propionic acid, Fmoc-d-3-(2-thienyl)-l-alanine, Fmoc-(R,S)-3-amino-3-(4-methoxyphenyl)propionic acid, Fmoc-3,5-diiodotyrosine, Fmoc-(R,S)-3-amino-3-(4N,N-dimethylaminophenyl)propionic acid, Fmoc-cis-3-amino-2-cyclohexane carboxylic acid, Fmoc-(R,S)-3-amino-3-(4-bromophenyl)propionic acid, Fmoc-(R,S)-3-amino-3-(2-thienyl)propionic acid, Fmoc-(R,S)-3amino-3-(1-naphthyl)propionic acid, Fmoc-(R,S)-3-amino-3-(3-pyridyl)propionic acid, Fmoc-trans-3-amino-2-cyclohexane carboxylic acid, 4-NFmoc-amino-4-carboxytetrahydropyran, 2-N-Fmoc-amino-3-(4-N-Boc-piperidinyl)propionic acid, 4-N-Fmoc-amino-4-carboxy-1,1-dioxo-tetrahydrothiopyran, N-Fmoc-(R,S)-amino-(3-N-Boc-piperidinyl)carboxylic acid, N-Fmoc-amino-(4-tert-butoxycyclohexyl)carboxylic acid, N-Fmoc-(R,S)amino-2-naphthylacetic acid, N-Fmoc-amino-(4-N-Boc-piperidinyl)carboxylic acid, N-Fmoc-amino-4-ketocyclohexyl carboxylic acid, Fmoc(2S,5R)-5-phenylpyrrolidine-2-carboxylic acid, Fmoc-4-(3-carboxymethyl2-keto-one-benzimidazolyl)piperidine, Fmoc-cis-3-amino-1-cyclohexane carboxylic acid, Fmoc-4-phenylpiperidine-4-carboxylic acid, Fmoc-d-thiazolidine-4-carboxylic acid, Fmoc-(R)-(þ)-2-piperidinecarboxylic acid, Fmoc-5H-thiazolo[3,2-a]pyridine-3-carboxylic acid, Fmoc-3-Boc-2, 3-diaminopropionic acid, Fmoc-3-carboxymethyl-1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one, Fmoc-2-aminoindane-2-carboxylic acid, Fmoc-3benzothienylalanine, Fmoc-3,3-diphenylalanine, Fmoc-2-amino-5-ureidovaleric acid, Fmoc-d-3,4-dichlorophenylalanine, Fmoc-(3-aminomethyl)benzoic acid, Fmoc-2-amino-4-tert-butoxybutanoic acid, Fmoc-O-benzylhydroxyproline, Fmoc-O-methyltyrosine, Fmoc-3,5-dibromotyrosine, Fmoc-4-tert-butyloxycarbonylaminophenylalanine, Fmoc-4-benzoylphenylalanine, Fmoc-d-tetrahydroisoquinoline-3-COOH, Fmoc-thiazolidine-4carboxylic acid, Fmoc-d-propargylglycine, Fmoc-3-(2-naphthyl)alanine, Fmoc-d-3-(2-naphthyl)alanine, Fmoc-3-aminobenzoic acid, (2S,4S) Fmoc4-amino-1-Boc-pyrrolidine-2-carboxylic acid. (d stands for d configuration.) Carboxylic acids and anhydrides used for the derivatization of Lys are as follows: diglycolic anhydride, 2,5-thiophenedicarboxylic acid, (S)-()-2pyrrolidone-5-carboxylic acid, glutaric anhydride, malonic acid, trans-4-cotininecarboxylic acid, levulinic acid, propionic acid, 3,5-dihydroxybenzoic acid, Boc-1-aminocyclopropane-1-carboxlic acid, 2-pyrazinecarboxylic acid, 4-acetamidobenzoic acid, 3-pyridinepropionic acid, hydroxyphenylacetic acid, trans-3-(3-pyridyl)acrylic acid, 3-hydroxy-2-quinoxaline carboxylic acid, isobutyric acid, butyric acid, 4-bromo-3,5-dihydroxybenzoic acid, 3-oxo-1-indancarboxylic acid, 3-thiophenecarboxylic acid, 2-thiophenecarboxylic acid, (S)-(þ)-oxo-4-phenyl-3-oxazolidineacetic acid, 2,6dichloronicotic acid, 3-nitrophenylacetic acid, 4-(dimethylamino) phenylacetic acid, 4-(dimethylamino)benzoic acid, 2,5-dimethoxybenzoic acid,

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phenoxyacetic acid, 3-(dimethylamino)benzoic acid, 3-benzoyl-2-pyridine carboxylic acid, hexanoic acid, 3,4-difluorobenzoic acid, indole-2-carboxylic acid, phenylpropionic acid, 4-chlorophenylacetic acid, 4-bromobenzoic acid, bromophenylacetic acid, 3,5-dimethylbenzoic acid, 1-naphthylacetic acid, 2-biphenylcarboxylic acid, 2-phenoxybutyric acid, 2,4-dichlorophenylacetic acid, 3,4-dichlorophenylacetic acid, (2-naphthoxy)acetic acid, (1naphthoxy)acetic acid, 4-biphenylcarboxylic acid, 4-phenoxybenzoic acid, 2-phenyl-4-quinoline carboxylic acid, 5-(4-chlorophenyl)-2-furoic acid. Carboxylic acids, acyl chlorides, and sulfonyl chlorides used for derivatization of 4-aminophenylalanine and -4-aminophenylalanine are as follows: 5-hydantoinacetic acid, trans-4-cotininecarboxylic acid, isonicotinic acid, 3-pyridinepropionic acid, 4-hydroxyphenylacetic acid, 2-butynoic acid, 2-pyrazinecarboxylic acid, cyclopropanecarboxylic acid, 3-hydroxy-2-quinoxaline carboxylic acid, 5-bromovaleric acid, propargyl chloroformate, 3,4-dimethoxybenzoyl chloride, 2-thiophenesulfonyl chloride, 3-thiophenecarboxylic acid, 2-thiophenecarboxylic acid, 2-methylbutyric acid, 2-thiopheneacetyl chloride, benzoic acid, furylacrylic acid, 4-nitrophenyl acetic acid, 2,5-dimethoxyphenylacetic acid, p-toluenesulfonyl chloride, 4-(dimethylamino)phenylacetic acid, 3-indolepropionic acid, phenoxyacetic acid, 3-(dimethylamino)benzoic acid, cyclohexanecarboxylic acid, naphthalenesulfonyl chloride, 4-bromophenylacetic acid, 4-bromobenzoic acid, 2phenoxybutyric acid, 3,4-dichlorophenylacetic acid, (1-naphthoxy)acetic acid. Screening of an Encoded Peptidomimetic Library with Streptavidin-Alkaline Phosphatase Conjugate An enzyme-linked colorimetric assay14,15 is used to screen for ligands that bind to streptavidin (also see Chapter 17 in this volume). To find the strongest ligands, an extremely dilute streptavidin-alkaline phosphatase conjugate concentration (1:100,000) is used. Library beads (0.1 ml) are transferred into a disposable polypropylene column with a polyethylene frit. The beads are washed with phosphate-buffered saline (PBS) (10  4 ml), and then with 0.1% Tween/0.1% gelatin/0.05% sodium azide/PBS (PBSTG-azide, 3  4 ml). The bead-supported library is filtered and blocked with PBSTG-azide (4 ml) at room temperature for 1 h. The beads are filtered and washed with 0.1% Tween-20/0.1% gelatin/0.05% sodium azide/PBS (3  4 ml). Streptavidin-alkaline phosphatase conjugate 14

G. Liu and K. S. Lam, in ‘‘Combinatorial Chemistry, A Practical Approach Series’’ (H. Fenniri, ed.), p. 33. Oxford University Press, New York, 2000. 15 K. S. Lam and M. Lebl, ImmunoMethods 1, 11 (1992).

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TABLE III Peptidomimetic Ligands for Streptavidin

Aa2

O

O

HN

R1 O

Entry 01

Aa2 d-Arg

Structure H2N

Isonicotinic acid

COOH H N

R1COOH

Structure N

NH2

COOH

R2COOH 3-Thiophene carboxylic acid

02

COOH H N

d-Arg

COOH S

NH H2N

Structure

NH2

Isonicotinic acid

N COOH

2-Thiophene carboxylic acid

S

COOH

NH

03

Ckbpa

HN

N N COOH

Isonicotinic acid

COOH

N COOH

3-Thiophene carboxylic acid S

O H2N

04

COOH H N

d-Arg

NH2

Isonicotinic acid

O

N COOH

3-Benzoyl-2-pyridine carboxylic acid

COOH

4-(Dimethylamino)phenylacetic acid

NH H2N

05

COOH

Isonicotinic acid

d-Thi S

N

N

N

COOH

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H N

H N

R2

COOH

285

(continues)

286

TABLE III (Continued) Entry

Aa2

N

N

HN

COOH

R1COOH Isonicotinic acid

Structure N

O

07

Cptd

08

Cptd -Phe(4-tBu)

09

-Phe(4-tBu)

10

a b

N HN

N HN

N

COOH

Isonicotinic acid

N

COOH

2-Pyrazine carboxylic acid

COOH NH2

2-Pyrazine carboxylic acid

COOH NH2

2-Pyrazine carboxylic acid

4-(Dimethylamino)phenylacetic acid

COOH

4-(Dimethylamino)phenylacetic acid

N N

O

COOH

N

O

4-(Dimethylamino)phenylacetic acid

COOH N

COOH N

COOH N

COOH

(S)-(+)-2-Oxo-4-phenyl-3oxazolidineacetic acid

O

COOH

(S)-(+)-2-Oxo-4-phenyl-3oxazolidineacetic acid

O

N N

Structure

COOH

N N

R2COOH

COOH

N O

COOH

N

peptide synthesis and screening

Cptdb

06

Structure

O

Ckbp, 4-(3-carboxymethyl-2-keto-one-benzimidazolyl)-piperidine. Cptd, 3-Carboxymethyl-1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one.

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solution is added at a dilution of 1:100,000 (original concentration is 1 mg/ ml) in 0.1% Tween-20/0.1% gelatin/0.05% sodium azide/PBS (4.0 ml). The bead library is incubated at room temperature for 1 h. The beads are filtered and washed with 0.1% Tween-20/PBS (3  4 ml), followed by washing with Tris buffer saline (TBS, 4 ml). The beads are placed into two polystyrene Petri dishes (15 cm diameter). Each dish then receives the alkaline phosphatase substrate 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt (BCIP) in BCIP buffer (0.5 ml) to develop color at room temperature for 1 h. The enzymatic reaction is stopped with 1.0 M HCl (1.0 ml). The dark turquoise colored positive beads are retrieved and treated with 6.0 M guanidine–HCl pH 1.0 solution to strip the protein off the beads. The positive beads are then washed with water and sequenced. The following chemical composition of buffers was used. PBS: 8.0 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4; TBS: 2.5 mM Tris–HCl, 13.7 mM NaCl, 0.27 mM KCl, pH 8.0; BCIP substrate in BCIP buffer: 1.65 mg BCIP in 10 ml of 0.1 M Tris–HCl, 0.1 M NaCl, and 2.34 mM MgCl2, pH 8.8. Ten dark beads have been picked up and sequenced. The result is shown in Table III. Ligands with high consensus have been identified. All the ligands have an aromatic N-heterocyclic structure (R1) attached to the amino group of aniline. Although there is some variation in the first building units (Aa2) and the last building blocks (R2), they can be cataloged into several different groups, respectively. Some ligands even have an identical structure, e.g., compounds 6 and 7 and compounds 9 and 10. The screening result is very similar to our previous data with the library using Phe(4-NH2) as a scaffold.6 It validates the fact that the encoding system for the OBOC peptidomimetic and small molecule libraries is rapid, direct, and reliable. Very recently, we reported on a novel chemical encoding method that uses separate coding tags with a cleavable linker in the bead interior. These coding tags can be released for decoding with mass spectrometry.16 Acknowledgments The author would like to thank Ms. Amanda M. Enstrom for editorial support. This work was supported by NIH R33CA86364. Ruiwu Liu is supported in part by the University of California Systemwide Biotechnology Research Program, Grant 2001-07.

16

A. Song, J. Zhang, C. B. Lebrilla, K. S. Lam, J. Am. Chem. Soc. 125, 6180 (2003).

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[16] Intelligent Design in Combinatorial Chemistry: Use of Designed Peptide Libraries to Explore Secondary and Tertiary Structures in Peptides and Proteins By Scott M. Cowell, Xuyuan Gu, Josef Vagner, and Victor J. Hruby Introduction

It is hard to imagine all the possible roles of combinatorial chemistry in modern science. Combinatorial chemistry became a major tool in medicinal chemistry by solving two major problems simultaneously. A need existed to dramatically increase the number of new chemical entities and, at the same time, reduce the assay time of these entities to find novel ligands. Because of its ability to very quickly (a few days) synthesize millions of compounds that can be tested for possible lead compounds in drug discovery, combinatorial chemistry has become a critical component of drug design in modern pharmaceutical science. It subsequently spurred research to reduce assay times to screen these millions of compounds. Combinatorial chemistry could be seen as the ‘‘engineering’’ of the science of discovery of novel ligands. The future of combinatorial science seems endless as other areas of science adapt the methodology. Although it has developed rapidly in other areas of chemistry, the origins of combinatorial chemistry were in the area of peptide design. While the methodology has embraced many different facets of synthetic organic chemistry, the role of combinatorial chemistry in peptide science is far from over, mainly because it still is one of the few areas of organic chemistry in which true combinatorial science can be pursued. Peptide synthesis in combinatorial chemistry is a viable field in finding lead compounds and in the evaluation of other aspects of protein research. These cannot be fully duplicated by most other areas of synthetic organic chemistry, because of the very high fidelity of organic peptide synthesis, and because of the central significance of the proteins and peptides in living systems.1 Peptide libraries can be used to better understand everything from protein folding and evaluation of secondary structures of proteins, to the ability to fully examine which key amino acid residues are needed to impart biological functions of these molecules.

1

V. J. Hruby, J.-M. Ahn, and S. Liao, Curr. Opin. Chem. Bio. 1, 114 (1997).

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

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With today’s technology, one can theoretically prepare virtually limitless numbers of peptide compounds. While this can be accomplished using automated methods, it still requires intelligent design by the scientist. Intelligent design formulates specific questions to be answered without the need to synthesize libraries with millions of different compounds. Intelligent design also endeavors to evaluate the library for a particular question quickly. This chapter will discuss the evolution of intelligent design in combinatorial peptide synthesis and its use in the evaluation of secondary and tertiary structures in proteins. Background

The roots of combinatorial chemistry are in Merrifield’s2 work on solidphase peptide synthesis, a technique that has revolutionized peptide chemistry. However, the limiting factor of traditional solid-phase peptide synthesis is the linearity of the method. Perhaps the first example of a peptide library (not combinatorial) was accomplished by Geysen et al.3 This involved the synthesis of a library of peptides on pins to identify an immunologically important coat protein of foot-and-mouth disease virus. The study centered on a particular 7-mer sequence of the coat protein, and scanning the amino acids on this peptide to determine the significance of each amino acid. Similar peptide libraries have been produced using phage display.4,5 Another very early example of a solid-phase peptide library is the early studies by Houghten,6 who made peptides using ‘‘teabags’’ to synthesize 248 different 13-mer peptides in less than 4 weeks, and evaluated their binding using a particular antibody–antigen reaction. A significant advance in the field came with the design of a solid-phase peptide synthesis for the evaluation of ligand-binding activity using a true combinatorial approach.7 Here literally millions of individual peptides were produced using standard peptide synthesis on ‘‘resin’’ beads, which were then evaluated directly using a marker attached to a monoclonal antibody against -endorphin. This method succeeded in pinpointing single beads that contained the proper sequence to bind to the antibody. The particular bead was then isolated and microsequenced to find the lead peptide for further evaluation. The concept of ‘‘one bead-one peptide’’ 2

R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963). H. M. Geysen, R. H. Meloen, and S. J. Barteling, Proc. Natl. Acad. Sci. USA 81, 3998 (1984). 4 T. Clackson, H. R. Hoogenboom, A. D. Griffiths, and G. Winter, Nature 352, 624 (1991). 5 J. K. Scott and G. P. Smith, Science 249, 386 (1990). 6 R. A. Houghten, Proc. Nat. Acad. Sci. USA 82, 5131 (1985). 7 K. S. Lam. S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, and R. J. Knap, Nature 354, 82 (1991). 3

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was coined.8 A very large library (tens of millions or more) could be produced in much less time than previously reported. One of the advantages of this process is that a variety of unnatural amino acids could now be incorporated into the libraries. For example, an all d-amino acid-containing ligand was discovered for the -endorphin receptor.8 This could not be accomplished using phage display libraries. Furthermore, since the synthesis of the peptides was direct and did not contain the natural bias of phage libraries, one had direct control of the combinatorial conditions in which the libraries were synthesized. More recently, a variation of this ‘‘one bead-one peptide’’ method was used to determine the molecular recognition of a synthetic receptor to a peptide library designed to be similar to a vancomycin-resistant peptide.9 A question of how to properly screen peptide mixtures that are released from solid support was being asked. This involved the idea that the fast release of all peptides into a mixture might cause interference with the test. For example, in screening for novel ligands for G-protein-coupled receptors, done by releasing literally thousands of peptides into solution, there is a chance that multiple peptides will interact with the receptor. Some of these peptides might act as agonists while others act as antagonists. This can cause problems in finding which peptides are responsible for an observed receptor interaction. One group designed a system that allows for the slow release of peptides utilizing gaseous trifluoroacetic acid (TFA) to the TFA-resistant linker methylbenzhydrylamine (MBHA).10 This controlled release approach allows for the screening of one library using the same bioassay multiple times and, under the proper circumstances, allows for the isolation of the one bead that is responsible for an agonist response that was coupled to a color change. At the same time that combinatorial libraries were examined on solid support, scientists were able to evaluate combinatorial libraries in solution with success.11–14 It was suggested that the attached peptides might give 8

K. S. Lam, M. Lebl, V. Krchnak, S. Wade, F. Abdul-Latif, R. Ferguson, C. Cuzzocrea, and K. Wertman, Gene 137, 13 (1993). 9 R. Xu, G. Greiveldinger, L E. Marenus, A. Cooper, and J. A. Ellman, J. Am. Chem. Soc. 121, 4898 (1999). 10 C. K. Jayawickreme, G. F. Graminski, J. M. Quillan, and M. R. Lerner, Proc. Natl. Acad. Sci. USA 91, 1614 (1994). 11 R. A. Houghten, C. Pinilla, S. E. Blondelle, J. R. Appel, C. T. Dooley, and J. H. Cuervo, Nature 354, 84 (1991). 12 H. Han, M. M. Wolfe, S. Brenner, and K. D. Janda, Proc. Natl. Acad. Sci. USA 92, 6419 (1995). 13 R. N. Zuckermann, J. M. Kerr, M. A. Siani, S. C. Banville, and D. V. Santi, Proc. Natl. Acad. Sci. USA 89, 4505 (1992). 14 C. Pinilla, J. R. Appel, and R. A. Houghton, Gene 128, 71 (1993).

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different data as compared with peptides in solution. However, similarity between the binding of ligands on solid supports and in solution are observed. For example, a  opioid receptor antagonist with nanomolar binding was found by screening a library of 52,128,400 hexapeptides.15 This was followed up in the same group by doing positional scanning on each residue of the hexapeptide16 as well as a tetrapeptide17 library to further examine the effects of individual amino acids. Positional scanning with phage libraries were used to discover the amino acid residues in peptides responsible for binding to the calciumbinding protein calmodulin.18 In this case, the binding polypeptide needed Trp as a key residue located in the fourteenth position from the N-terminus for strong binding. Positional scanning on peptides is one of the strengths of combinatorial chemistry and it was used successfully to find antithrombic activity in a tetrapeptide series.19 Another form of positional scanning was used to determine the optimal hexapeptide for prostate-specific antigen. This was accomplished by varying residues 1 through 4. This has led to novel compounds that are being further defined.20 An additional example of positional scanning was the isolation of pentapeptides responsible for erythromycin resistance in Escherichia coli.21 In this case, the pentapeptides interfere with the binding of the erythromycin to the ribosome. This interaction was also studied using a pentapeptide library in vivo to determine the properties of the pentapeptide that cause resistivity to occur. It was shown that hydrophobic residues on the peptide in the 1, 3, and 5 positions were responsible for the major resistance of the drugs. It should be noted that it is not always libraries with large peptides that give lead compounds. In one case, a dipeptide library used for conformational studies led to peptoid ligands. In this case, the conformation of the dipeptides held the key to binding activity. Therefore, it is the conformation of the peptides that will give lead compounds to peptidomimetics. A good antagonist for the human tachykinin NK3 was found using this approach.22 15

C. T. Dooley, N. N. Chung, P. W. Schiller, and R. A. Houghten, Proc. Natl. Acad. Sci. USA 90, 10811 (1993). 16 C. T. Dooley and R. A. Houghten, Life Sci. 52, 1509 (1993). 17 C. T. Dooley, P. Ny, J. M. Bidlack, and R. A. Houghten, J. Biol. Chem. 273, 18848 (1998). 18 B. K. Kay and N. B. Adey, Gene 169, 133 (1996). 19 B. Kundu, M. Bauser, J. Betschinger, W. Kraas, and G. Jung, Bioorg. Med. Chem. Lett. 8, 1669 (1998). 20 C. F. Yang, E. S. Porter, J. Boths, D. Kanyi, M. Hsieh, and B. S. Cooperman, J. Peptide Res. 54, 444 (1999). 21 T. Tenson, L. Xiong, P. Kloss, and A. S. Mankin, J. Biol. Chem. 272, 17425 (1997).

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Exploration of Antimicrobial Peptides Using Combinatorial Chemistry

Another use for combinatorial libraries has been the screening of peptides for antimicrobrial properties. In this case, the design of the library is based on antimicrobial peptides found in nature. A combinatorial synthesis is used to find alternative unnatural amino acids expected to mimic the antimicrobial properties.23 Peptide libraries also have been used to find compounds that could bind the lytic peptide mellitin.24 The library was synthesized in solution phase, purified, and evaluated using time-of-flight mass spectrometry (TOF-MS). The sequences determined to bind to mellitin contained hydrophobic pairs. By binding to mellitin, they were able to prevent the cell-surface mellitin interaction. This is an example of a peptide library able to afford compounds that interact with other small peptides without having to find an interacting protein first. The exploration of antimicrobial peptides was taken a step further when the major consideration of the target molecule is the secondary structure it can adopt. Blondelle et al. designed a library in which the goal was to increase the amphipathic characteristic of the peptide. However, it soon became clear from early results that the presence of the helix-breaking Pro in the molecule causes a large increase in the lytic properties of the molecule.25 This demonstrates the power of libraries for determining pathways that otherwise would not have been explored. Moreover, the secondary structures of the amphipathic peptides have been studied using peptide libraries in which it was observed that the smaller the amphipathicity, the greater the antimicrobial activity.26 The use of these libraries for the study of secondary structures in the peptide has been of great value to derive better antimicrobial drugs. The assay method relies on the deconvolution of the mixture of peptides in order to elucidate the structure of biologically active compounds. Amphipathic peptides have been used as a starting point for the intelligent design of tertiary structures of proteins.27 In this case, the residues responsible for the amphipathicity were held constant while the rest were varied to try to overcome the limitations necessary to stabilize the 22

P. Boden, J. M. Eden, J. Hodgson, D. C. Horwell, J. Hughes, A. T. McKnight, R. A. Lewthwaite, M. C. Pritchard, J. Raphy, K. Meecham, G. S. Ratcliffe, N. Suman-Chauhan, and G. N. Woodruff, J. Med. Chem. 39, 1664 (1996). 23 S. E. Blondelle, E. Takhashi, P. A. Weber, and R. A. Houghten, Antimicrob. Agents Chemother. 38, 2280 (1994). 24 S. E. Blondelle, R. A. Houghten, and E. Perez-Paya, J. Biol. Chem. 271, 4093 (1996). 25 S. E. Blondelle, E. Takahashi, R. A. Houghten, and E. Perez-Paya, Biochem. J. 313, 141 (1996). 26 S. E. Blondelle and K. Lohner, Biopolymers 55, 74 (2000). 27 E. Perez-Paya, R. A. Houghton, and S. E. Blondelle, J. Biol. Chem. 271, 4120 (1996).

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formation of the tertiary structures. These factors included van der Waals interactions, electrostatic interactions, etc. This study determined the optimal amphipathic peptides for aggregation to occur and proved that simple protein design can be achieved using combinatorial peptide synthesis. Design Strategies in Combinatorial Libraries of Peptides

A paper published in the 1990s represents a breakthrough in the design of peptide libraries. Sepetov et al.28 approached the design of the library from the standpoint of the functionality of the final peptide. The premise from which they started was that not all residues in a peptide are needed for biological activity. These ‘‘noncritical’’ residues are needed merely scaffolding and contribute to the proper orientation for the other residues needed for interaction. Therefore, the proper design of peptide libraries should be the identification and isolation of critical residues with the focus of design being only to modify these residues necessary for optimal interactions (the ‘‘pharmacophores’’ elements). With this approach, the size of the library could be greatly reduced. Pharmacophores are identified usually through the literature for a particular ligand. The pharmacophore moieties are strategically placed at certain positions in the peptide chain varying the ‘‘scaffolding’’ residues. The pharmacophores are then varied in location in the peptide chain iteratively. One can view this as a modified alanine scan where certain residues are held constant and other residues are varied. This strategy greatly streamlines the process of building libraries with the knowledge as to which residues are key to desired interactions with target receptors. An example of this methodology is reported by de Koster et al.29 This research group used a 15-mer template generated from a phage library as a starting point for further optimization of the peptide. Once they determined that two of the residues were inconsequential to the antibody interactions, they generated a limited 12-residue library via amino acid scanning to optimize antibody interactions. This study led to the identification of novel compounds with high binding affinity for which it is speculated that a second-generation library could provide greater success with a smaller library. Combinatorial libraries have been used to find the binding affinities to a transporter associated with antigen processing (TAP).30 Uebel et al.30 28

N. F. Sepetov, V. Krchnak, M. Stankova, S. Wade, K. S. Lam, and M. Lebl, Proc. Natl. Acad. Sci. USA 92, 5436 (1995). 29 H. S. de Koster, R. Amons, W. E. Benckhuijsen, M. Feijlbrief, G. A. Schellekens, and J. W. Drijfhout, J. Immunol. Methods 187, 179 (1995). 30 S. Uebel, W. Kraas, S. Kienle, K. H. Wiesmuller, G. Jung, and R. Tampe, Proc. Natl. Acad. Sci. USA 94, 8976 (1997).

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found that the residues at the termini of the peptides are key for binding to TAP, while the other portions were needed only as a backbone for the molecule. A variation of the pharmacophore library was seen in an approach used to determine CD4þ T cell epitopes.31 In a 14-mer peptide, the researchers keep residues in positions 1, 4, and 6 constant since they functioned as ‘‘anchor positions’’ for binding receptor, and proceeded to vary the other residues in the peptide. This allowed the use of a shorter synthetic route compared to one that would be needed if all positions were to be varied. Limiting the size of the library allowed the authors to obtain better assays. By doing a partial release of the peptides, the authors were able to find the final peptide that represented the epitope of the T cell receptor. In another example, the epitope to inhibit stimulation of the thyrotropin receptor also was found via combinatorial libraries.32 Since the synthesis of a totally random hexapeptide library was deemed impractical, the authors opted to hold one position constant while the other five residues were randomized. This method was repeated for each residue in the peptide. The residues that were determined to be the most active were used as a basis for a second-generation library. The only limitation of the library was not the quantity of product synthesized, but to properly pinpoint the peptides in an assay. Another method for designing libraries is to formulate target molecules based on physicochemical properties such as the ability to cross the blood– brain barrier. Based on literature or other sources, data are obtained that show certain residues or peptide sequences could be developed to study and predict a particular task in question. With this information, an algorithm, commonly known as a ‘‘filter,’’ is created to screen a virtual library. Using quantitative structure–activity relationships (QSAR), the computer is essentially predicting which moiety in the virtual library may lead to a novel ligand. The practicality of this method lies in the time savings for library design, thus decreasing the size of the library to a more manageable number.33 In a similar method, computer models can also be developed to screen virtual libraries based on the binding pockets of receptors.34 In this case, the computational models directly probe the binding sites in receptors 31

H. S. Hiemstra, G. Duinkerken, W. E. Benckhuijesen, R. Amons, R. R. P. de Vries, B. O. Roep, and J. W. Drijfhout, Proc. Natl. Acad. Sci. USA 94, 10313 (1997). 32 J. Y. Park, I. J. Kim, M. H. Lee, J. K. Seo, P. G. Suh, B. Y. Cho, S. H. Ryu, and C. B. Chae, Endocrinology 138, 617 (1997). 33 Ajay, G. W. Bemis, and M. A. Murcko, J. Med. Chem. 42, 4942 (1999). 34 C. M. Murray and S. J. Cato, J. Chem. Inf. Comput. Sci. 39, 46 (1999).

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and screening libraries to find peptides with a high probability of binding to the pocket. This approach, now referred to as ‘‘combinatorial docking,’’ gives researchers a more educated starting point to design compound libraries.35 Protein Design

Combinatorial libraries have been used to design a four-helix bundle.36 The libraries were designed to cover the packing of the ligand in the libraries around a heme group after being bound to a template attached to a cellulose backbone. The library was varied to determine the affects of different core amino acids on the packing of the heme in the bundle. This work demonstrates the use of peptide libraries to design tertiary structures such as a heme bundle. How are stable proteins separated from those that are forming mutant globular proteins? One research group was able to separate de novo proteins using a histidine tagging method on a phage-generated combinatorial library and then using proteases to cut the proteins that were not packed in a hydrophobic core.37 The experiments were monitored using surface plasmon resonance, which demonstrated the presence of protein after the exposure to the protease. The design of tertiary structures using combinatorial libraries led one group to propose that the evolution of tertiary structures occurs similarly in vivo. Riechmann and Winter were able to show that comparable tertiary structures could occur by combinatorial shuffling of polypeptide fragments. This group was able to elicit the formation of a folding similar to the -barrel domain of a CspA fragment using a phage display library with a polypeptide not possessing the same sequence homology as the in vivo CspA molecule. It is possible that Nature might have used combinatory libraries in evolution but this has not been proved at this time.38 Cyclization

Tertiary structures also have been explored by modifying the flexibility of peptides in libraries. One of the first papers that introduced the head-totail cyclization of a library was by Spatola et al.39 The researchers relied on 35

H.-J. Bohm and M. Stahl, Curr. Opin. Chem. Biol. 2, 282 (2000). H. K. Rau, N. DeIong, and W. Haehnel, Angew. Chem. Int. Ed. Engl. 39, 250 (2000). 37 M. D. Finucane, M. Tuna, J. H. Lees, and D. N. Woolfson, Biochemistry 38, 11604 (1999). 38 L. Riechmann and G. Winter, Proc Natl. Acad. Sci. USA 97, 10068 (2000). 39 A. F. Spatola, Y. Crozet, D. de Wit, and M. Yanagisawa, J. Med. Chem. 39, 3842 (1996). 36

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a side chain attachment of the first amino acid residue to the resin. Although no novel hits were obtained for the antagonist in question, the paper validated the method and acted as a proof of concept. Positional scanning of a cyclic peptide was used to identify chymotrypsin inhibitors.40 The libraries were cyclized on the resin via oxidation of the cysteine residues. Residues at positions 1, 2, and 4 were scanned. In other studies, a method for ring closing metathesis of a portion of a growing peptide was developed.41 Though not attempted in an actual library, this method does offer some unique advantages in the design of tertiary structures in a library. The cyclization of peptides on the resin has always been important since the decrease in the degrees of freedom of a peptide increases the chance to induce selectivity. Therefore, there is a better chance of having selectivity of the molecules. One example of this relies on the cyclization on resin using an Asp resin.42 One of the many examples of how a library can be used to explore the rational for a particular binding is the use of a cyclic minilibrary where an NK-1 peptide was converted into a somatostatin receptor ligand.43 While the cyclization did not occur on the resin, the authors used the library to give them a lead compound that they might not have found without the use of the small library. Hinges in Peptide Libraries

A drawback of combinatorial libraries is the large number of compounds needed to explore all structural possibilities as the length of peptide chains grows. Intelligent design is needed in order to decrease the size of the libraries and obtain relevant information. This concept was shown in the incorporation of various structural elements to enhance turn parameters in a library.44 Novel -turns play an important role in nature, a fact that has prompted some investigators to build libraries to find novel -turns.45 To potentially have greater ligand affinities and explore secondary structures of peptides, a -turn mimetic has been incorporated into 40

J. D. McBride, H. N. Freeman, and R. J. Letherbarrow, J. Peptide Sci. 6, 446 (2000). J. F. Reichwein, B. Wels, J. A. W. Kruijtzer, C. Versluis, and R. M. J. Liskamp, Angew. Chem. Int. Ed. Engl. 38, 3684 (1999). 42 A. F. Spatola, K. Darlak, and P. Romanovskis, Tetrahedron Lett. 37, 591 (1996). 43 R. Hirshmann, W. Yao, M. A. Cascieri, C. D. Strader, L. Meaechler, M. A. Cichy-Knight, J. Hynes, Jr., R. D. van Rihn, P. A. Sprengeler, and A. B. Smith III, J. Med. Chem. 39, 2441 (1996). 44 G. J. Moore, Drug Dev. Res. 42, 157 (1997). 45 A. J. Souers, A. A. Virgilio, A. Ro`senquist, W. Fenuik, and J. A. Ellman, J. Am. Chem. Soc. 121, 1817 (1999). 41

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peptide libraries. This has proven to be very effective in providing leads for novel ligands.46 Becker et al.,46 coining the term ‘‘turn-scan,’’ discovered a possible inverse agonist for the  opioid receptor and an agonist for the orphan receptor ORL1 using this method. The use of the -turn in libraries provides an approach to determine the impact on conformation individual residues have on ligands. The incorporation of mimetic units into a peptide to change the properties of the peptide also has been used to obtain better protease inhibitors.47 A cyclohexanone was incorporated in the middle of a tetramer in order to generate strong binding interaction with chosen protease inhibitors. By conformationally constraining the tetramer using cyclohexanone, they were able to gain insight into the nature of the binding pocket of the proteases. -Amino acids have been incorporated into peptidomimetics to facilitate cyclization of the libraries.48 Similarly, peptidomimetics have been synthesized using -methylated amino acids. The difference between peptide and peptidomimetics is further blurred as different compounds are substituted in peptides in place of amino acid residues to influence the structures of the resultant libraries.49 Conclusion

Although combinatorial chemistry has shown much promise for the evaluation of molecules, combinatorial chemistry is a tool, and success greatly depends on the ingenuity and intuition of the scientists. Traditionally, insight into the natural world came slowly with each molecule synthesized. This approach allows us to obtain systematic chemical insight into bioactivity. Combinatorial chemistry, with its ability to synthesize many compounds with fewer synthetic steps, has caused this level of insight to be sacrificed for the ability to produce thousands of compounds at one time. Now thousands of chemical entities are designed to answer one particular question. Combinatorial chemistry gives rise to a higher level of insight with proper library design. Whether a shift from traditional solution-phase chemistry to solid-phase combinatorial chemistry is causing a loss of insight at a more fundamental level is a debate for the future. At this point, combinatorial chemistry is a part of chemistry and its proper use and the application of intelligent design from the chemists and engineers who accomplish this are providing us with new insights into the physical world. 46

J. A. J. Becker, A. Wallace, A. Garzon, P. Ingallinella, E. Bianchi, R. Cortese, F. Simonin, B. L. Kieffer, and A. Pessi, J. Biol. Chem. 274, 27513 (1999). 47 P. A. Abato, J. L. Conroy, and C. T. Seto, J. Med. Chem. 42, 4001 (1999). 48 E. A. Jefferson and E. E. Swayze, Tetrahedron Lett. 40, 7757 (1999). 49 S. W. Kim, Y. S. Shimn, and S. Ro, Bioorg. Med. Chem. Lett. 8, 1665 (1998).

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[17] Synthesis and Screening of ‘‘One-Bead One-Compound’’ Combinatorial Peptide Libraries By Kit S. Lam, Alan L. Lehman, Aimin Song, Ninh Doan, Amanda M. Enstrom, Joeseph Maxwell, and Ruiwu Liu Introduction

We first reported the ‘‘one-bead one-compound’’ (OBOC)* combinatorial library method in 1991.1 In this method, compound beads are prepared by a ‘‘split-mix’’ synthesis approach1–3 (Fig. 1) that results in the display of many copies of the same compound on one single bead.1,4 Tens of thousands to millions of these compound beads can easily be prepared. The bead library is then mixed with a target molecule, such as a protein, an *Abbreviations: Acm, acetamidomethyl; All, allyl; ATP, adenosine triphosphate; BCIP, 5-bromo-4-chloro-3-indolyl-phosphate; Bn, benzyl; t-Boc, tert-butyloxycarbonyl; BOP, benzotriazol-1-yl-oxy-tris(dimethylamine)-phosphonium hexafluorophosphate; t-But, tert-butyl; coupling DMF, HPLC grade DMF; DABCYL, 4-[ [40 -(dimethylamino)phenyl]azo]-benzoic acid; DCC, N,N0 -dicyclohexylcarbodiimide; DCM, dichloromethane; DCU, dicyclohexylurea; Dde, 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl; DIC, N,N0 -diisopropylcarbodiimide; DIEA, N,N-diisopropylethylamine; Dmab, 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)3-methylbutyl]-amino}benzyl; DMEM, Dulbecco’s modified Eagle’s medium; DMF, N, N-dimethylformamide; DMSO, Dimethyl sulfoxide; Dnp, 2,4-dinitrophenyl; Dns, dansyl; DPTU, diphenylthiourea; EDANS, 5-[(2-aminoethyl)-amino]naphthelensulfonic acid; EDT, 1,2-ethanedithiol; FCS, fetal calf serum; Fm, 9-fluorenylmethyl; Fmoc, 9-fluorenylmethoxycarbonyl; N-Fmoc-AAs, N-fluorenylmethoxycarbonyl -amino acids; HBTU, 2-(1Hbenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HF, hydrofluoric acid; HOAt, 1-hydroxy-7-azabenzotriazole; HOBt, N-hydroxybenzotriazole; HOPfp, pentafluorophenol; HPLC, high-performance liquid chromatography; NMM, N-methylmorpholine; OBOC, one-bead one-compound; PBSTG-azide, phosphate-buffered saline/0.1% Tween-20/ 0.1% gelatin/0.05% sodium azide; PEG, polyethyleneglycol; PEGA beads, [bis(2-acrylamidoprop-1-yl) poly(ethylene glycol) cross-linked dimethyl acrylamide and mono-2-acrylamidoprop1-yl [2-aminoprop-1-yl] poly(ethylene glycol)]; PITC, phenylisothiocyanate; Pmc, 2,2,5,7,8pentamethylchroman-6-sulfonyl; PMTC, phenylmethylthiocarbamate; PTH, phenylthiohydantoin; PyBOP, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate; PyBroP, bromo-tris-pyrrolidino-phosphonium hexafluorophosphate; RT, retention time; TBS, Tris buffer saline; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TEA, triethylamine; TFA, trifluoroacetic acid; Tis, tiisopropylsilane; Trt, trityl. 1 K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, and R. J. Knapp, Nature 354, 82 (1991). 2 R. A. Houghten, C. Pinilla, S. E. Blondelle, J. R. Appel, C. T. Dooley, and J. H. Cuervo, Nature 354, 84 (1991). 3 A. Furka, F. Sebestyen, M. Asgedom, and G. Dibo, Int. J. Peptide Protein Res. 37, 487 (1991). 4 K. S. Lam, M. Lebl, and V. Krchnak, Chem. Rev. 97, 449 (1997).

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

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Split

I (coupling step)

L

N

M

L

N

M Mix/Split

II (9 dipeptides)

L

N

M ML MM MN

LL LM LN

NL NM NN Mix/Split

III (27 tripeptides)

L L L L L L L L L L

LL LM LN ML MM MN NL NM NN

N

M M M M M M M M M M

LL LM LN ML MM MN NL NM NN

N N N N N N N N N

LL LM LN ML MM MN NL NM NN

Fig. 1. Split/mix synthesis approach to generate an OBOC combinatorial library.

enzyme, or other biomolecule, or whole cells and intact microorganisms such as viruses, bacteria, and yeasts. Compound beads that interact with these target molecules are identified by an appropriate reporter system. These positive beads are then physically isolated for structural determination by instruments such as a protein sequencer. In our initial work, peptide libraries and enzyme-linked colorimetric assays were used.1 Later other screening methods were developed to identify peptide ligands and enzyme substrates against a number of target molecules.4 Small molecules and peptidomimetic libraries that require special encoding systems to decode these unsequencable compounds were also developed.4–9 In this chapter, we shall focus on methods for preparing peptide libraries, and the various screening methods that can be applied to both peptide and 5

A. Borchardt and W. C. Still, J. Am. Chem. Soc. 116, 373 (1994). R. Liu, J. Marik, and K. S. Lam, J. Am. Chem. Soc. 124, 7678 (2002). 7 A. W. Czarnik, Curr. Opin. Chem. Biol. 1, 60 (1997). 8 C. Barnes and S. Balasubramanian, Curr. Opin. Chem. Biol. 4, 346 (2000). 9 R. L. Affleck, Curr. Opin. Chem. Biol. 5, 257 (2001). 6

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small molecule libraries. In Chapter 15, this volume, we describe a novel encoding method for OBOC small molecule and peptidomimetic libraries. Synthesis of Linear and Cyclic Peptide Libraries

Lam et al.4 reviewed the choice of solid supports for the OBOC combinatorial chemistry methodology. TentaGel resin (Rapp Polymere, Tubingen, Germany) is a good choice for OBOC peptide libraries due to its uniformity in size as well as its nonstickiness. It consists of hydroxyethylpolystyrene beads onto which have been grafted 3000- to 4000-Da polyethyleneglycol (PEG) linkers. A functional group, e.g., hydroxyl or amino, is located at the end of the linkers. This resin can be swollen in a wide range of solvents from water to toluene. The PEG linker is advantageous for many biological assays. For protease substrate screening, porous beads such as PEGA beads {bis(2-acrylamidoprop-1-yl) poly(ethylene glycol) cross-linked dimethyl acrylamide and mono-2-acrylamidoprop-1-yl [2aminoprop-1-yl] poly(ethylene glycol)} (Calbiochem-Novabiochem, San Diego) are preferable. In principle, either the Fmoc/t-But (9-fluorenylmethoxycarbonyl/tertbutyl) or t-Boc/Bn (tert-butyloxycarbonyl/benzyl) amino acid protection strategies could be used for library synthesis on TentaGel. However, hydrofluoric acid (HF), which is highly toxic and requires a special apparatus, is needed to cleave off the side-chain-protecting groups for the t-Boc/ Bn chemistry. Since it partially degrades the PEG chain on the resin, the Fmoc/t-But strategy is preferable for the synthesis of peptide libraries on TentaGel. Efficient and unambiguous peptide-bond formation requires chemical activation of the carboxyl group of the N-protected amino acid. Many of the commercially available activating reagents can be used for solidphase peptide library synthesis, including DIC (N,N0 -diisopropylcarbodiimide), BOP (benzotriazol-1-yl-oxy-tris(dimethylamine)-phosphonium hexafluorophosphate), HBTU [2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate], TBTU (2-(1H-benzotriazole-1-yl)-1, 1,3,3-tetramethyluronium tetrafluoroborate], PyBOP (benzotriazole1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate), and PyBroP (bromo-tris-pyrrolidino-phosphonium hexafluorophosphate). The classic condensation reagent, DCC (N,N0 -dicyclohexylcarbodiimide), is not suitable for peptide library synthesis because it will produce an insoluble DCU (dicyclohexylurea) byproduct. An additive reagent to generate an active ester can significantly increase the rate of the coupling reaction and reduce the racemization of N-protected amino acids. HOBt (1-hydroxybenzotriazole) and HOPfp (pentafluorophenol) are usually

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used. When BOP, HBTU, TBTU, PyBOP, or PyBroP is used as the activating reagent, a base is needed to neutralize the acidic side-product, which may induce undesired side-reactions. DIEA (N,N-diisopropylethylamine), NMM (N-methylmorpholine), and TEA (triethylamine) are recommended for this purpose. In this chapter, we describe a procedure that uses DIC as an activating reagent and HOBt as an additive to synthesize peptide libraries. Preparation of the Amino Acid Solutions Freshly made amino acid solutions are recommended for peptide synthesis, although stock solutions of N-Fmoc-AAs (N-fluorenylmethoxycarbonyl -amino acids) with HOBt in DMF (N,N-dimethylformamide) can  be stored at 4 for a week. Since the synthesis of a hexapeptide or octapeptide library usually takes only 2–3 days, all the amino acid solutions may be prepared before the synthesis. A 3-fold excess of N-Fmoc-AAs is needed in each coupling step to ensure completion of the coupling reaction. We have been using the following equations to calculate the amount of necessary reagents. Waa ¼ ðMWaa Þð3LÞðgÞðN Þ=X

(1)

WHOBt ¼ ð153:2Þð3LÞðgÞðN Þ

(2)

VHOBt ¼ ð0:7ÞðV 0 ÞðX ÞðN Þ

(3)

VDIC ¼ ð126:2Þð3LÞðgÞ=ð0:806ÞðX Þ

(4)

Waa ¼ mass of each protected amino acid required for library synthesis WHOBt ¼ total amount of HOBt necessary for the whole library VHOBt ¼ total volume of HOBt solution needed VDIC ¼ volume of DIC required for each amino acid coupling reaction MWaa ¼ molecular weight of the protected amino acid L ¼ loading of the resin in mmol/g g ¼ mass of the resin N ¼ total number of amino acid residues in the peptide sequence X ¼ total number of amino acids used in each coupling step V0 ¼ volume of amino acid/HOBt solution to be added to each reaction vessel (for a reaction vial containing 100 mg TentaGel resin, we generally use 1.0 ml amino acid/HOBt solution) 153.2 ¼ molecular weight of HOBtH2O (HOBt monohydrate) 126.2 ¼ molecular weight of DIC 0.806 ¼ density of DIC

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Reagents. N-Fmoc-AAs with the appropriate side-chain-protecting groups are used in our library synthesis, e.g., N-Fmoc-Arg(Pmc)-OH, NFmoc-Asn(Trt)-OH, N-Fmoc-Asp(Ot-Bu)-OH, N-Fmoc-Cys(Trt)-OH, N-Fmoc-Gln(Trt)-OH, N-Fmoc-Glu(Ot-Bu)-OH, N-Fmoc-His(Trt)OH, N-Fmoc-Lys(Boc)-OH, N-Fmoc-Ser(t-Bu)-OH, N-Fmoc-Thr(tBu)-OH, N-Fmoc-Trp(Boc)-OH, and N-Fmoc-Tyr(t-Bu)-OH. Other amino acids without side-chain-protecting groups are N-Fmoc-Ala-OH, N-Fmoc-Gly-OH, N-Fmoc-Ile-OH, N-Fmoc-Leu-OH, N-Fmoc-MetOH, N-Fmoc-Phe-OH, N-Fmoc-Pro-OH, and N-Fmoc-Val-OH. Many other Fmoc unnatural amino acids are commercially available. HPLC (highperformance liquid chromatography) grade DMF is used for coupling and analytical reagent grade DMF is used for washing of the resins. TentaGel S NH2 resin (e.g., 0.27 mmol/g) is used for library preparation. (Natural amino acids are designated by the standard three-letter code.) Procedure. To prepare a linear heptapeptide library of 20 amino acids and 2 g of resins, we use Eqs. (1)–(4) to calculate the amount of reagents: Waa ¼ 3  0.27  2.0  7  MWaa/20 ¼ 0.567  MWaa mg; WHOBt ¼ 153.2  3  0.27  2.0  7 ¼ 1737.3 mg; VHOBt ¼ 0.7  1.0  20  7 ¼ 98 ml; VDIC ¼ 126.2  3  0.27  2.0/(0.806  20) ¼ 12.7 l. Then 1737.3 mg HOBtH2O in DMF is dissolved until the final volume is 98 ml and 4.9 ml of HOBt solution is added to each of the 20 amino acid containers and then coupling DMF is added until the final volume is (V0 )  (N) ¼ 1.0  7 ¼ 7.0 ml. The amino acid should be dissolved by vortexing; however, sonication can be used if dissolution is incomplete. One milliliter of amino acid and HOBt solution (V0 ) is added into each reaction vessel at each coupling step. Prior to transfer into the reaction vessel the suspension must be completely dissolved. Monitor Coupling Reaction The most popular test for the presence or absence of free amino groups is the Kaiser test.10 The test is simple and quick; however it should be noted that some deprotected amino acids do not show the expected dark blue color typical of free primary amino groups (e.g., serine, asparagine, aspartic acid).11 Furthermore, for secondary amines such as proline, the resin will turn brown instead of blue. For secondary amines and aromatic amines, the chloranil test is recommended.12 In this volume, Albericio’s research

10

E. Kaiser, R. L. Colescott, C. D. Bossinger, and P. I. Cook, Anal. Biochem. 34, 595 (1970). J. D. Fontenot, J. M. Ball, M. A. Miller, C. M. David, and R. C. Montelaro, Peptide Res. 4, 19 (1991). 12 T. Vojkovsky, Peptide Res. 8, 236 (1995). 11

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group has included a comprehensive study on colorimetric tests for solid-phase synthesis. The Kaiser test uses three solutions: ninhydrin (5 g in 100 ml of ethanol), phenol (80 g crystalline phenol in 20 ml ethanol), and pyridine/KCN [dilute 2 ml of 0.001 mol/liter aqueous solution of KCN (potassium cyanide) with 98 ml of pyridine]. The test is performed by transferring a few resin beads to a 6  50-mm glass test tube. The beads are washed with ethanol and a drop of each of the three testing solutions is added. The tube  is then heated to 100 for 5 min in a heating block. Blue (brown for proline)-colored beads and solution (positive test) indicate the presence of free amino groups on the resin. Colorless beads (negative test) indicate complete coupling. The quality of phenol is important. Impure phenol may result in a false-positive Kaiser test. Although the order in which the three reagents are added does not affect the test result, we recommend adding the KCN/pyridine solution first, because ethanol makes the beads adhere to the wall of tube. The chloranil test uses two reagents: 2% acetaldehyde in DMF (v/v) and 2% p-chloranil in DMF (w/v). A few resin beads are transferred to a small glass test tube, washed with coupling DMF, and one drop of each of the two reagents is added. After standing at room temperature for 5 min, min, the beads will turn blue or dark green indicating the presence of free amino groups on the resin. Synthesis of a Linear Heptapeptide Library with 19 Eukaryotic Amino Acids (Cysteine Excluded) For linear peptide library synthesis, cysteine is usually excluded to avoid undesired formation of disulfide bonds. Reagents. Reagents are TentaGel S NH2 resin (80–90 m), loading: 0.27 mmol/g, protected amino acid stock solutions, 25% piperidine/DMF (v/v), Kaiser test reagents, DIC, analytical reagent grade DMF (washing DMF), methanol, HPLC grade DMF (coupling DMF), dichloromethane (DCM), cleavage solution: phenol/thioanisole/water/1,2-ethanedithiol (EDT)/trifluoroacetic acid (TFA) (7.5:5:5:2.5:82.5, w/v/v/v/v), and PBS (phosphate-buffered saline): 137 mmol/liter NaCl, 2.68 mmol/liter KCl, 8.0 mmol/liter Na2HPO4, and 1.47 mmol/liter KH2PO4, pH 7.4. Procedure. TentaGel S NH2 resin (1.9 g, 0.27 mmol/g) is swollen in DMF overnight. The resin is then equally distributed into 19 polypropylene columns each with a tight-sealing cap, a frit, and a stopper. The solvent is drained under reduced pressure. One milliliter of amino acid/HOBt solution and 12.7 l of DIC is added to each column. The reaction mixtures are agitated for 1 h at room temperature. Complete coupling is confirmed

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with the Kaiser test. For those columns with incomplete coupling reactions, prolonged reaction time or a ‘‘repeat coupling’’ by adding fresh amino acid/ HOBt solution and DIC is applied until the Kaiser test is negative. After all the coupling reactions are completed, the resin is combined and transferred to a siliconized glass column with a frit at the bottom. The resin is washed with washing DMF twice (15 ml each wash) and coupling DMF (15 ml  3). The N-Fmoc-protecting group is removed by incubation twice in 10 ml of 25% piperidine/DMF for 15 min each. The resin is then washed with washing DMF (15 ml  3), methanol (15 ml  3), and coupling DMF (15 ml  4). The Kaiser test is performed to confirm the Fmoc deprotection. The cycle of resin distribution, amino acid coupling, Kaiser test, resin mixing, resin washing, Fmoc deprotection, resin washing, and Kaiser test again is repeated until amino acid assembly is completed. The NFmoc-protecting group on the last amino acid should be removed prior to side-chain deprotection. The resin is washed with DCM (15 ml  3) and is dried under vacuum for 2 h. The side-chain-protecting groups are removed by agitating the resin with 30 ml cleavage solution for 4 h at room temperature. The bead-supported library is then washed with washing DMF (15 ml  5), methanol (15 ml  3), DCM (15 ml  3), coupling DMF (15 ml  3), 30% water/DMF (15 ml  1), 60% water/ DMF (15 ml  1), water (15 ml  1), and PBS buffer (15 ml  10), and  is stored in 0.05% sodium azide/PBS at 4 . Synthesis of a Disulfide Cyclic Peptide Library Efficient disulfide formation requires a careful choice of oxidation methods and protecting groups for cysteine. There are several methods to cyclize a peptide via disulfide bond formation. We recommend the DMSO oxidation method13,14 for free thiol oxidation and the iodine oxidation method15 for the protected Cys(S-Trt)/Cys(S-Acm) oxidation. A procedure using the dimethyl sulfoxide (DMSO) oxidation method for the synthesis of a disulfide cyclic peptide library is described in this section. Cys(STrt) is placed at the first and last residue position of the library during library synthesis. After library synthesis is finished, all the side-chainprotecting groups including the Trt group on cysteines are removed with TFA. DMSO is used to oxidize the two cysteines to form a disulfide bond. The Ellman test is carried out to monitor the completion of disulfide bond 13

A. Otaka, T. Koide, A. Shide, and N. Fujii, Tetrahedron Lett. 32, 1223 (1991). J. P. Tam, C. R. Wu, W. Liu, and J. W. Zhang, J. Am. Chem. Soc. 113, 6657 (1991). 15 M. Pohl, D. Ambrosius, J. Groetzinger, T. Kretzschmar, D. Saunders, A. Wollmer, D. Brandenburg, D. Bitter-Suermann, and H. Hoecker, Int. J. Peptide Protein Res. 41, 362 (1993). 14

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formation.16,17 Triisopropylsilane (TIS) is used as the scavenger in the cleavage solution instead of EDT, since the latter can reduce disulfide bonds during the oxidation step. Reagents. N-Fmoc-Cys(S-Trt)-OH, N-Boc-Cys(S-Trt)-OH, reagents for peptide synthesis, Ellman’s reagent: 4.0 mg 5,50 -dithiobis(2-nitrobenzoic acid) in 1.0 ml of 20 mmol/liter sodium phosphate buffer pH 8.0; oxidation solution: water/acetic acid/DMSO (75:5:20, v/v/v), the pH is adjusted with ammonium hydroxide to 6.0 before DMSO is added; cleavage solution: phenol/thioanisole/water/TIS/TFA (7.5:5:5:2.5:82.5, w/v/v/v/v). Procedure. A solution of N-Fmoc-Cys(S-Trt)-OH (949 mg, 3.0 equiv.) and HOBtH2O (248 mg, 3.0 equiv.) in 15 ml DMF is added to 2.0 g TentaGel S NH2 resin (0.27 mmol/g) preswollen in DMF. DIC (255 l, 3.0 equiv.) is added. The reaction mixture is shaken overnight at room temperature. After the coupling is completed, the resin is washed with washing DMF (15 ml  2) and coupling DMF (15 ml  3). The N-Fmoc-protecting group is removed by incubation twice in 10 ml of 25% piperidine/DMF for 15 min each. The resin is then washed with washing DMF (15 ml  3), methanol (15 ml  3), and coupling DMF (15 ml  4). The Kaiser test is performed to confirm the Fmoc deprotection. The peptide library is then assembled following the method described above. After the peptide library assembly is completed, N-Boc-Cys(S-Trt)-OH is coupled to the last amino acid using the same method as for N-Fmoc-Cys(S-Trt)-OH. The resin is washed with DCM (15 ml  3) and is dried under vacuum for 2 h. The side-chain-protecting groups are removed by adding 30 ml of the cleavage solution to the resin for 4 h at room temperature with gentle shaking. The cleavage solution is drained, and the bead-supported library is washed with DMF (15 ml  5), methanol (15 ml  3), DCM (15 ml  3), DMF (15 ml  3), 30% water/DMF (15 ml  1), 60% water/DMF (15 ml  1), and water (15 ml  5). A few resin beads are transferred to a glass test tube and a few drops of Ellman’s reagent are added. In this step, the Ellman test must be positive. The yellow color of the solution indicates the presence of free thiol group. The bead-supported library is then oxidized in 1 liter of the oxidation solution with gentle shaking in a 2-liter bottle for 48 h until the Ellman test is negative (colorless solution). Magnetic stirring is not recommend in this step as the stirring bar may break the beads. The resin is then thoroughly washed with water, followed by PBS. The bead-supported cyclic library is stored in 0.05% sodium azide/  PBS at 4 . 16 17

G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959). G. L. Ellman, K. D. Courtney, V. Andres, Jr., and R. M. Featherstone, Biochem. Pharmacol. 7, 88 (1961).

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On-Resin Synthesis of a Cyclic Peptide Library Using Lys and Glu Side-Chains The amide bond formation between the side-chain carboxyl and amino groups of glutamic acid (or aspartic acid) and lysine, respectively, provides an excellent alternative approach to cyclize peptides on solid support.18,19 Various protection strategies can be used for this purpose. Many sidechain-protected derivatives of Glu, Asp, and Lys, e.g., N-Boc-Asp (OFm)-OH, N-Boc-Glu(OFm)-OH, N-Fmoc-Asp(OAll), N-FmocGlu(OAll)-OH, N-Boc-Asp(ODmab)-OH, N-Boc-Glu(ODmab)-OH, N-Fmoc-Lys(Dde)-OH, and N-Boc-Lys(Fmoc)-OH, are commercially available. Similar to the N-Fmoc-protecting group, the Fm group is labile to 20% piperidine/DMF, while the allyl group can be selectively removed by Pd(PPh3)4 in CHCl3:AcOH:N-methylmorpholine (37:2:1) without affecting the other protecting groups.20 Both the Dmab and Dde groups are removed by treating the resin with 2% hydrazine/DMF. In this section, we describe a procedure for the synthesis of a cyclic peptide library using N-Fmoc-Lys(Dde)-OH and N-Boc-Glu(ODmab)-OH as building blocks for the cyclization. The peptides are cyclized with an amine bond between the -carboxyl group of Glu and the e-amino group of Lys. Reagents. Reagents for peptide synthesis are N-Fmoc-Lys(Dde)OH and N-Boc-Glu(ODmab)-OH, acetic anhydride, PyBOP, DIEA, 1-hydroxy-7-azabenzotriazole (HOAt), and 2% hydrazine/DMF. Procedure. Two grams TentaGel S NH2 resin (0.27 mmol/g) is swollen in DMF overnight. The loading of the resin is decreased using the following steps: (1) mix 0.30 mmol of N-Fmoc-Gly-OH (89 mg), 0.31 mmol of HOBtH2O (47 mg), and 0.31 mmol of DIC (48.5 l) in 15 ml of coupling DMF; (2) add the mixture to the TentaGel S NH2 resin and shake gently overnight at room temperature; (3) after washing the resin with DMF and DCM, block the exposed amino group with 15% acetic anhydride in DCM for 30 min. In this case, the final loading of the down-substituted resin will be 0.15 mmol/g. After Fmoc deprotection and resin washing as above, a solution of N-Fmoc-Lys(Dde)-OH (320 mg, 2.0 equiv.), HOBtH2O (92 mg, 2.0 equiv.), and DIC (94 l, 2.0 equiv.) in 15 ml of coupling DMF is added to the resin. The mixture is shaken overnight at room temperature. The Kaiser test is performed to confirm complete coupling reaction. After Fmoc deprotection and resin washing, the peptide library is assembled as described above, until the desired cyclization 18

A. F. Spatola and P. Romanovskis, ‘‘Combinatorial Peptide and Nonpeptide Libraries: A Handbook,’’ p. 327. VCH Verlagsgesellschaft mbH, Weinheim, 1996. 19 J. S. McMurray, Peptide Res. 7, 195 (1994). 20 ‘‘Novabiochem Catalog and Peptide Synthesis Handbook (1997/1998),’’ p. S33.

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position (Glu) is reached. The N-Boc-Glu(ODmab)-OH is coupled to the last amino acid by adding a solution of N-Boc-Glu(ODmab)-OH (735 mg, 2.0 equiv.), HOBtH2O (92 mg), and DIC (94 l) in 15 ml DMF to the resin. The reaction mixture is shaken overnight or until the Kaiser test is negative. The resin is then washed with DMF (15 ml  5), and incubated twice in 80 ml of 2% hydrazine/DMF for 3 min each. After washing with DMF (15 ml  6), the cyclization step is performed by adding 5.0 equiv. of PyBOP and HOAt and 10 equiv. of DIEA in DMF. The reaction mixture is shaken at room temperature overnight, until the Kaiser test is negative. Finally, the side-chain-protecting groups are removed using the same method as for the linear peptide libraries. The bead-supported library is then washed with DMF (15 ml  5), methanol (15 ml  3), DCM (15 ml  3), DMF (15 ml  3), 30% water/DMF (15 ml  1), 60% water/DMF (15 ml  1), water (15 ml  1), and PBS buffer (15 ml  10),  and is stored in 0.05% sodium azide/PBS at 4 . The Synthesis of ‘‘Fluorescence-Quench’’ Combinatorial Library for Protease Substrate Determination We have found that porous PEGA bead resin is more suited for protease substrate screening than TentaGel resin. The porosity of the PEGA beads allows more protease to reach the interior of the beads, which produces a favorable fluorescent signal-to-noise ratio. The peptide synthesis follows the Fmoc chemistry scheme and mix-split synthesis method except that the first amino acid in this library is lysine or any other amino acid with a free nitrogen on the side chain that can be coupled to a fluorescent (donor) molecule. The last amino acid in this library should be a quencher (acceptor). The following possible fluorescence-quencher pairs can be used: (1) ortho-aminobenzoic acid as the donor with Phe(4-NO2), nitrobenzylamide, 2,4-dinitrophenyl (Dnp), Tyr(3-NO2), or 4-nitroaniline as the receptor; (2) aminocoumarin as the donor and aminoquinolinone type flourophores and nitroaromatic derivatives as the acceptor; (3) 5-[(2aminoethyl)-amino]naphthelensulfonic acid (EDANS) as the donor and 4-[[40 -(dimethylamino)phenyl]azo]-benzoic acid (DABCYL) as the quencher; and (4) tryptophan as the donor and the dansyl (Dns) as the acceptor. If a significant number of false-positive fluorescent beads remain after library synthesis, the library may be capped with a different quencher that utilizes a different coupling chemistry. This capping step should be performed prior to side-chain deprotection. Therefore, before the final deprotection step, we routinely sample the library for inspection under the fluorescent microscope. We describe below a procedure for the synthesis of a fluorescence quench combinatorial library using ortho-aminobenzoic acid as the fluorophore and 3-nitrotyrosine as the quencher.

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Reagents. Reagents include amino PEGA resin (0.30 mmol/g), NFmoc-Lys(Boc)-OH, N-Boc-Abz-OH (N-Boc-2-aminobenzoic acid), NFmoc-Tyr(3-NO2)-OH, 50% TFA/DCM, 2.5% DIEA/DCM, and reagents for peptide synthesis. Procedure. A solution of N-Fmoc-Lys(Boc)-OH (422 mg, 3.0 equiv.) and HOBtH2O (138 mg, 3.0 equiv.) in 10 ml DMF is added to 1.0 g amino PEGA resin (0.3 mmol/g) preswollen in DMF. DIC (141 l, 3.0 equiv.) is added. The reaction mixture is shaken at room temperature until the Kaiser test is negative (about 2 h). The resin is washed with washing DMF (10 ml  3) and coupling DCM (10 ml  5). The Ne-Boc-protecting group is removed by incubation in 15 ml of 50% TFA/DCM for 20 min at room temperature. The resin is immediately washed with DCM (10 ml  2), 2.5% DIEA/DCM (10 ml  3), DCM (10 ml  2), methanol (10 ml  3), and DMF (10 ml  4). The Kaiser test is performed to confirm Boc deprotection. A solution of N-Boc-Abz-OH (356 mg, 5.0 equiv.), HOBtH2O (230 mg, 5.0 equiv.), and DIC (235 l, 3.0 equiv.) in 10 ml DMF is added to the resin. The mixture is shaken at room temperature overnight, until the Kaiser test is negative. The peptide library is then assembled on the -amino group of lysine after Fmoc deprotection and resin washing using the method described above. At the end of the peptide library construction, N-Fmoc-Tyr(3-NO2)-OH is coupled to the N-terminus by adding a solution of N-Fmoc-Tyr(3-NO2)-OH (673 mg, 5.0 equiv.), HOBtH2O (230 mg, 5.0 equiv.), and DIC (235 l, 3.0 equiv.) in 10 ml of DMF to the resin. The reaction mixture is shaken overnight, until the Kaiser test is negative. After Fmoc deprotection and resin washing, the side-chainprotecting groups are removed using the same method as for normal linear peptide libraries. The ‘‘fluorescence-quench’’ bead library is then washed with washing DMF (10 ml  5), methanol (10 ml  3), DCM (10 ml  3), DMF (10 ml  3), and methanol (10 ml  5), and is stored in methanol in  the dark at 4 . Library Screening

To screen OBOC combinatorial libraries, tens of thousands to millions of compound beads are first mixed with a target molecule. The beads that interact with the target molecule will be identified and then isolated for structure determination. For target molecules that cannot be visualized directly through a microscope, a reporter group such as an enzyme, fluorescent probe, or radionuclide is conjugated to the target molecule. If an antibody to the target molecule is readily available, an alternative method uses the antibody with a reporter group. Otherwise, the target molecule can be biotinylated and probed with streptavidin with the reporter group. Protein

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kinase, protease substrates determination, and other functional assays use [-32P]ATP (adenosine triphosphate) and fluorescence-quench peptides, respectively, to identify the positive beads. For identification of ligands that bind to the surface of whole microorganisms or eukaryotic cells, the easiest approach is to directly inspect the library under a microscope for organism or cell-bound beads. The screening method described below can be used to screen both peptide and small molecule OBOC libraries.4 Enzyme-Linked Colorimetric Assay This method is suitable for a broad range of experimental sizes. Between 2  103 and 2  106 beads can easily and rapidly be screened with this method.1,21 Colorimetric screening relies on tight binding between the target molecule and specific beads in the combinatorial library, which must be maintained throughout subsequent washing and color development steps. The enzyme-linked colorimetric screening method is divided into four basic steps: (1) preblocking, (2) marker molecule prescreening and removal, (3) target molecule incubation, and (4) color development. The preblocking step utilizes blocking protein, typically gelatin or bovine serum albumin, to coat surfaces on the combinatorial bead that bind protein nonspecifically. Subsequent bead incubation steps are also done in the presence of blocking protein to minimize nonspecific binding of the marker and target molecules. Marker molecule prescreening is done to identify and remove compound beads that interact directly with the marker molecules. The target molecule incubation step incubates the bead library and the target molecule. Color development methods vary with the type of marker molecules used in previous steps. The protocol presented herein will use a biotin label on the target molecule and a streptavidin-alkaline phosphatase conjugate (Zymed, South San Francisco, CA) to mark the positive beads. Reagents. Unless noted, all reagents are commonly available from a number of sources. The following buffers and solutions are needed for the colorimetric screening: phosphate-buffered saline/0.1% Tween-20/0.1% gelatin/0.05% sodium azide (PBSTG-azide): 8 mM, Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.2, 0.1% Tween-20 (v/v, Sigma), 0.1% Gelatin (Sigma), and 0.05% sodium azide (Sigma); 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) buffer: 0.1 M Tris–HCl, pH 8.8, 0.1 M NaCl, and 2.34 mM MgCl2; BCIP stock: 50 mg/ml BCIP (Amresco, Solon, OH or Biosynth AG, Staad, Switzerland) in DMF; Tris buffer saline (TBS): 2.5 mM Tris–HCl, 13.7 mM NaCl, and 2.7 mM KCl, pH 8.0; guanidine: 8 M guanidine–HCl in H2O. 21

K. S. Lam, Methods Mol. Biol. 87, 7 (1998).

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Methods. A number of suitable labeling strategies are available for colorimetric screening of OBOC combinatorial libraries. In general these methods utilize alkaline phosphatase to dephosphorylate BCIP. The resulting dephosphorylated BCIP molecules dimerize to form indigo, which is turquoise colored and precipitates on the surface of the TentaGel bead, thereby marking the bead containing the interacting peptide (Fig. 2A). Other marker strategies include target molecule-directed antibodies with alkaline phosphatase-conjugated secondary antibodies, as well as tagging strategies such as penta-His tags and nickel-alkaline phosphatase. Biotinylation of Target Protein. Protein samples are biotinylated essentially as described by the manufacturer (Pierce, Milwaukee, WI). Briefly, EZ-Link Sulfo-NHS-LC-Biotin is employed to attach a biotin molecule via a linker to primary amines on the surface of the target protein. The molar ratio of biotin linker to protein is adjusted so that each target protein molecule carries approximately one biotin. Overbiotinylation of the target protein is discouraged as this may alter the structure or function of the protein. Colorimetric Screening. All incubations are performed at room tem perature or at 4 on a bench top rotator (Barnstead Thermolyne— Labquake) with incubation times as noted. After incubation, the solution is removed either by gravity or by vacuum applied to the bottom of the column and followed by addition and removal of the wash solution two more times. The level of the wash solution is never allowed to fall below the level of settled beads in the column during solution removal. There are approximately 750,000 TentaGel beads (80–90 m in diameter) per ml of settled beads in solution. Between 5000 and 50,000 beads are transferred to a 1-ml microcolumn (Perkin Elmer Life Sciences, Boston, MA). Larger columns or containers can be used when larger numbers of beads are to be screened. The column is drained and the beads are washed three times with PBSTG-azide. PBSTG-azide (1 ml) is added and the column is capped with a top closure. The beads are incubated for 1 h on a rotator at  room temperature or at 4 . The gelatin in the buffer will coat nonspecific protein-binding sites on the TentaGel beads. Following the gelatin preblock, the column is washed three times with PBSTG-azide and incubated with streptavidin-alkaline phosphatase conjugate (Zymed, 1.5 mg/ml) diluted in PBSTG-azide for 1 h. The streptavidin-alkaline phosphatase prescreen always employs higher concentrations of streptavidin-alkaline phosphatase than the subsequent marking steps. Dilutions typically range between 1:3000 and 1:10,000. The streptavidin-alkaline phosphatase concentration can be adjusted up or down if the color intensity of the prescreen needs to be adjusted. In preparation for color development, the column is washed once with TBS and twice with BCIP buffer. Special care is taken to

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Fig. 2. Screening methods: (A) enzyme-linked colorimetric method for ligands that bind a to soluble protein target; photomicrograph of a BCIP-stained bead in a background of many negative beads; (B) whole-cell binding assay for cell surface ligands; photomicrograph of a peptide bead surrounded by a monolayer of Jurkat T-lymphoma cells; (C) protease substrate determination; ‘‘fluorescence-quench’’ library cleaved by a protease and the brightly blue fluorescent beads are positives; (D) protein kinase substrate determination; autoradiograph showing positive beads labeled with [-32P]ATP in the presence of a purified protein kinase. (See color insert.)

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wash away all traces of streptavidin-alkaline phosphatase from the previous step. Unbound streptavidin-alkaline phosphatase in the colorimetric reaction will dephosphorylate BCIP nonspecifically, resulting in high background coloration. After washing with BCIP buffer, a solution of BCIP in BCIP buffer is prepared by the addition of 3.3 l of BCIP stock solution per ml of BCIP buffer. The BCIP solution is added to the column, which is then incubated for 1–2 h. Columns that have retained a large amount of streptavidin-alkaline phosphatase conjugate will begin to show a faint blue color after approximately 15 min. The column is washed three times with PBSTG-azide to stop the reaction and remove residual BCIP. Bluecolored beads are removed manually with the aid of a gel loading pipette tip and a dissecting stereomicroscope. Depending on the combinatorial library being screened, upward of 1 in 1000 beads will be stained darkly. All beads showing color should be removed from the library at this point. Following the prescreening and washing, the beads are incubated with biotinylated target protein in PBSTG-azide for 1–2 h. Different target protein samples will require titration of amounts and incubation times to show adequate signal in subsequent color development steps. Typically, incubation of 1 g of protein per 1 ml column volume for 1 h is an adequate titration starting point. To mark bound target proteins, the column is washed three times with PBSTG-azide and is incubated with a reduced concentration of streptavidin-alkaline phosphatase a second time. Typical dilutions are 2- to 3-fold lower than the first streptavidin-alkaline phosphatase incubation, e.g., 1:3000 followed by 1:10,000 or 1:10,000 followed by 1:20,000. Incubation times can be adjusted to alter sensitivity. Typical incubation times are 1–2 h. For resolution of color on beads containing bound target and marker molecules, the column is washed as above, first with TBS and then twice with BCIP buffer solutions. The color is developed as above and the reaction is stopped after 1 h. The newly colored beads are isolated and their ligands characterized. In some experiments, one may want to decolorize these positive beads with 100% DMF, and restain them with the molecular target using an orthogonal reporting system, or in the presence and absence of a known competing ligand. These latter steps could greatly increase the chance that the selected compound beads are true positives. Whole Cell Binding Assay Screening combinatorial peptide libraries with intact cells is a rapid way of discovering cell surface-binding ligands.22–25 The procedure is divided into three sections: (1) preparation and sterilization of the bead library,

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(2) incubation with the target cells, and (3) recovery and characterization of the positive beads. Reagents. The following buffers and solutions are needed for whole cell screening: growth media: Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal calf serum (FCS), 100 units/ml penicillin, and 100 g/ml streptomycin, PBS: 8 mM Na2HPO4, 1.4 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.2. Methods. Whenever possible, all work is done under sterile conditions in a laminar flow hood. Whole-cell binding assays can incubate for as short as 5 min or as long as days. To ensure that the reaction is not contaminated, the washing column and the combinatorial library are sterilized as follows: 200 l of settled beads (150,000 beads) are placed in a 1-ml disposable column (Perkin Elmer Life Sciences). The column and beads are washed five times with PBS and five times with H2O. The final wash step is with 70% ethanol, which is added to the column but not removed until the library is needed for incubation with the target cells. When the cells are ready, the ethanol is removed from the column and the beads are rinsed once with culture media and resuspended in 0.6 ml of culture media. Using a sterile sixwell tissue culture plate, three wells are prepared with 3  105 target suspension cells, such as lymphoma cells, in 3 ml of culture media. Then 0.2 ml of beads (containing 50 K beads) is added to each of the three wells. The beads and cells are incubated on a platform shaker at 70–110 rpm for 15 min to  48 h at 37 with 5% CO2. Some cells may bind beads rapidly (i.e., several minutes) and other cells may take longer. After gentle mixing and settling of the beads, free cells are removed with a sterile pipette. If the target cell type is adherent, this protocol can be modified. For loosely adherent cells it may be adequate to manually remove the cells using a cold PBS wash and centrifugation followed by removal of PBS and resuspension in culture media. This allows for preservation of the cell surface adhesion molecules, which may result in more rapid binding. However, care should be taken to avoid unnecessary cell aggregation. For these cells, the shaking step may be omitted and the cells incubated with  beads for 30 min to 1 day at 37 with 5% CO2. For more strongly bound adherent cells, trypsinizing the cells will allow the cells to revert to suspension cells rapidly with minimal mechanical damage to the cells. The trypsin reaction is stopped with an equal volume of media containing FBS as soon as 22

M. E. Pennington, K. S. Lam, and A. E. Cress, Mol. Diversity 2, 19 (1996). I. B. DeRoock, M. E. Pennington, T. C. Sroka, K. S. Lam, T. Bowden, E. L. Bair, and A. E. Cress, Cancer Res. 61, 3308 (2001). 24 S. Park, M. Renil, B. Vikstrom, N. Amro, L.-W. Song, B. Xu, and K. S. Lam, Lett. Peptide Sci. 8, 171 (2002). 25 D. H. Lau, L. Guo, R. Liu, A. Song, C. Shao, and K. S. Lam, Biotechnol. Lett. 24, 497 (2002). 23

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the cells release from the tissue culture plastic. Following centrifugation, the trypsin is removed and the cells are resuspended in culture media and counted. The cells can be plated into six-well plates as above or in various tissue culture dishes at a final concentration of about 70% confluency as suspension cells. Beads are then added as above, and for the case of trypsinized cells the plates can either be placed on a shaker at 70 rpm to facilitate cell–bead contact or incubated without shaking. In the case of trypsinized cells, bead binding may be delayed due to the action of the trypsin on the surface receptors. The dishes should be examined under a microscope at various time points to identify any beads that are covered in bound cells. Under an inverted microscope, beads with bound cells are retrieved. The estimated number of bound cells on each bead is recorded and each retrieved bead is placed in an individual well of a 96- or 24-well plate or groups of similarly binding beads from the same library and cell type can be placed into a small Petri dish. Strongly bound beads should be covered with cells (Fig. 2B). Subsequent wash steps may be performed on the bench top under nonsterile conditions. The retrieved beads in the 96-well plate are washed by carefully removing each bead in series and placing it in a Petri dish filled with 8 M guanidine–HCl. Incubate until no cells are visible on the surface of the bead (approximately 15 min). At this time, the bead is removed and placed in a second Petri dish containing H2O. Each bead is washed no fewer than three times in H2O to ensure protein removal from the bead surface. In some experiments, the cleaned positive beads can be retested for binding against the same or a different cell line in the same fashion or in the presence of a blocking antibody to the suspected target. If bead sequencing is desired, each individual bead is carefully placed on glass filter paper and submitted for sequencing. Screening for Protease Substrates Meldal and co-workers26,27 first described the fluorescence-quenching approach to screen the OBOC combinatorial libraries for protease substrates. A ‘‘fluorescence-quench’’ combinatorial library prepared from the porous PEGA bead resin (see above) is first inspected under a fluorescent microscope and the fluorescent beads are removed and discarded. After all of the false-positive beads have been removed, the peptide beads are transferred into an Eppendorf tube and washed 5 with water, followed by a 5 wash with the appropriate protease buffer. Protease is then added 26

M. Meldal, I. Svendsen, K. Breddam, and F. I. Auzanneau, Proc. Natl. Acad. Sci. USA 91, 3314 (1994). 27 M. Meldal and I. Svendsen, J. Chem. Soc. Perkin Trans. I 1591 (1995).

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to the Eppendorf tube and incubated at room temperature or 37 for 1–3 h. The beads are then washed with PBSTG, transferred to a Petri dish, and examined under a fluorescent microscope (excitation: 254–310 nm, emission: 420 nm). The positive beads appear bright blue (Fig. 2C). The fluorescent beads are manually isolated, washed with 8 M guanidine–HCl and water, and then submitted for sequence analysis. We expect that some of the peptides on the positive beads will be cleaved at the proteolytic site. As a result, the PTH-amino acids detected during each cycle of Edman degradation will have been generated from both of these peptides. This complicates the sequence analysis somewhat. However, it also allows us to determine the proteolytic cleavage site of the peptide, in addition to its uncleaved sequence. COPAS Screening Recently we have employed the COPAS Biobead bead sorter (Union Biometrica, Somerville, MA) to isolate positive beads from combinatorial libraries. Combinatorial libraries are incubated as described above with a fluorescently tagged protein. The beads are washed to remove unbound proteins and then sorted based on the fluorescence intensity of the protein bound to individual beads. Combinatorial beads can be sorted at a rate of 50–100 thousand beads per hour and the positive beads dispensed into 96well plates. If necessary, the combinatorial beads can be prescreened to remove beads with excessive fluorescence background. Protein Kinase Substrate Assay Radionuclide-based screening for protein kinase substrates is a quick and effective method for determining peptide substrates for protein kinases.28–31 The method presented here is based on the screening of random synthetic combinatorial peptide libraries with a protein kinase and is divided into two main parts: (1) the phosphorylation of the peptide bead library and (2) the recovery and characterization of positive beads. Reagents. Unless noted, all reagents are commonly available from a number of sources. The following buffers and solutions are needed for kinase screening: MES buffer: 30 mM 2-(N-morpholino)ethanesulfonic acid (MES), 10 mM MgCl2, and 0.4 mg/ml bovine serum albumin (BSA) pH 28

J. Wu, Q. N. Ma, and K. S. Lam, Biochemistry 49, 14825 (1994). K. S. Lam, J. J. Wu, and Q. Lou, Intl. J. Protein Peptide Res. 45, 587 (1995). 30 Q. Lou, M. E. Leftwich, and K. S. Lam, Bioorg. Med. Chem. 5, 677 (1996). 31 J. J. Wu, D. E. H. Afar, H. Phan, O. N. Witte, and K. S. Lam, Comb. Chem. High Throughput Screen. 5, 83 (2002). 29

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6.8; HCl: 1 N HCl; 5 PBS: 40 mM Na2HPO4, 7 mM KH2PO4, 680 mM NaCl, 13 mM KCl, with the addition of 0.05% Tween-20, pH 7.2. Procedure. The kinase screening reaction is performed in a 1-ml polypropylene column (Perkin Elmer Life Sciences, Boston, MA). [-32P]ATP (25 Ci/mmol) is used as substrate for the kinase labeling reaction. The type of protein kinase being screened determines the choice of combinatorial library. Typically, the combinatorial libraries will consist of a serine, threonine, or tyrosine residue flanked by three to five additional random amino acids on both the N- and C-terminal sides and can be constructed to satisfy the needs of individual screening experiments. We have found that background phosphorylation can be greatly reduced by using PEGA beads instead of TenteGel beads. Approximately 0.5 ml settled volumes of beads are placed in the column and washed five times with MES. The level of buffer is allowed to drop to the level of the combinatorial beads before additional washings are performed. At no time is the level of the buffer allowed to fall below the level of the beads. The reaction is assembled inside the column in a volume equal to twice the volume of the settled beads. A typical reaction contains 500 l of settled beads, 0.1 M [ 32P]ATP, 0.4 mg/ml BSA, 10 mM MgCl2, and 30 mM MES buffer pH 6.8 in a final volume of 1 ml. The kinase reaction is initiated by the addition of 1–2 g/ml of purified protein kinase and is incubated at room temperature for 1 h on a bench top rotator (Barnstead Thermolyne—Labquake). The reaction is stopped by washing the column 5 times with 5 PBS containing 0.05% Tween-20. Additional wash steps can be performed as necessary if unbound ATP is present in the eluted PBS wash. To further reduce nonspecific binding of ATP to the  bead library, 1 ml of 1 N HCl is added to the column, which is heated to 95 for 10 min to hydrolyze the ATP, and then washed 10 times with 5 PBS. Recovery of Positive Beads. After washing, the combinatorial beads are suspended in 1% agarose (SeaPlaque, FMC BioProducts) heated to 70–75 and poured onto a 8.5  11-in. sheet of transparency film on a flat and level surface. Two to three milliliters of agarose should be thinly spread over an area of 16 in2 (100 cm2). The beads and the agarose are evenly spread across the surface, allowed to cool, and air dried overnight. A rectangular box marked by a wax pencil on the transparency helps to keep the agarose solution on the transparency prior to gelling. Thickly spread agarose will crack as it dries. Autoradiogram markers or other forms of marking material are placed on three locations on the transparency film, which is then covered with Saran wrap and then exposed to X-ray film (Kodak, double sided) for 24–48 h at room temperature. The film is developed and the markers are used to precisely align the radiolabeled bead image map on the developed film with the beads on the transparency (Fig. 2D). Retrieval of beads carrying a radiolabel from the agarose on the transparency is

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accomplished using a hollow punch to precisely excise the region corresponding to the positive signal on the aligned film from the agarose on the transparency. The excised beads and agarose are swollen in 200 l of H2O  and heated to 70–75 to dissolve the agarose. After dissolving the agarose, the beads are washed five times in 5 PBS with 0.05% Tween-20. One percent agarose is used to resuspend the beads and they are then exposed to film as above for secondary screening. The bead density on the second transparency film should be low enough that individual beads can be aligned with positive signals on the film. If necessary, tertiary screening can be performed. Individual beads are excised by adding 20 l of H2O to the region corresponding to the positive signal to swell and soften the agarose. The positive bead can then be retrieved from the agarose with a 25-gauge needle or gel loading pipette tip. Individual recovered beads are washed twice with 8 M guanidine–HCl and twice with H2O prior to microsequencing. Microsequencing of Peptide Beads

The structure of peptides containing 20 eukaryotic natural amino acids is now routinely determined by the use of automatic protein microsequencer, which uses Edman chemistry to convert each -amino acid sequentially to its phenylthiohydantoin (PTH) derivative. The formed PTH-amino acids can be identified by their retention times on HPLC systems by comparison with reference standards derived from the 20 natural amino acids. For an OBOC peptide library composed of natural amino acids, the sequencing protocols of the automatic sequencer are well developed and standardized. However, structure determination of peptides composed of unnatural -amino acids requires modification of the standard sequencing program.32 For peptides composed of non--amino acids, one can use an encoding strategy or mass spectrometry if a cleavable linker is employed. In this chapter, we shall focus on the new sequencing method we have developed for unnatural -amino acids. A large number of unnatural -amino acids are commercially available. Many of them are more hydrophobic than natural amino acids. To detect and clearly separate hydrophobic PTH-amino acids using the sequencer, we have extended the elution time from a total of 22 min to 28 min prior to washing the column with 90% solvent B (88% acetonitrile/12% isopropanol, v/v) (Table I). To use the existing software in the protein sequencer to read all 20 PTH-natural amino acids automatically, the first 18 min of the gradient profile is identical to that recommended by the manufacturer. This ensures that the elution profile of natural amino acids remains unchanged. 32

R. Liu and K. S. Lam, Anal. Biochem. 295, 9 (2001).

318

[17]

peptide synthesis and screening TABLE I Normal and Modified Gradient Program for PTH-Amino Acid Elutiona

Normal Time (min) % B Modified Time (min) % B a

0.0 6 0.0 6

0.3 6 0.3 6

0.4 16 0.4 16

18.0 46 18.0 46

18.5 90 24.5 90

21.5 90 27.5 90

22.0 50 28.0 50

Solution B: 88% acetonitrile/12% isopropanol v/v (solvent B2). TABLE II Cycles and Gradients of Normal and Modified Methods

Normal (PL PVDF peptide)

Cartridge cycle Flask cycle Gradient

Modified

Cartridge cycle Flask cycle Gradient

Default Cart PL PVDF peptide Flask Normal Fast Normal I Default Cart PL PVDF peptide Flask Normal Modified

None

None

Cart begin

Prep Pump Prep Pump

Flask Blank Fast Normal I None

Flask Standard Fast Normal I Cart Begin

Flask Blank Fast Normal I

Flask Standard Fast Normal I

None

Prep Pump Prep Pump

To reduce the sequencing time for each peptide containing unnatural amino acids, the normal gradient (Fast Normal I) is used in Flask Blank and Flask Standard cycles, except during Flask Normal when the modified gradient is used (Table II). This results in 12 min in total being saved in each peptide sequence (6 min in Flask Blank and 6 min in Flask Standard). By increasing the buffer concentration from 20 ml to 25 ml of Premix buffer concentrate per liter of solvent A3 (3.5% tetrahydrofuran/water), a better separation of PTH-amino acids near the region of PTH-His and PTH-Arg has been achieved, which increases the ability to use more unnatural amino acids. To eliminate the phenylmethylthiocarbamate (PMTC) peak [an adduct of the original coupling reagent phenylisothiocyanate (PITC) and methanol], a new coupling base R2C (methyl piperidine/water/butanol/ isopropanol) is used to replace R2B (methyl piperidine/water/methanol). PMTC usually is the largest peak in the chromatogram, and it elutes right after the Edman chemistry by-product diphenylthiourea (DPTU), which may interfere with integration of hydrophobic PTH-amino acids. In

[17]

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319

addition, it mislabels the reference peak causing the misidentification of the PTH-amino acids, resulting in incorrect sequence calling. DPTU is an excellent internal reference peak because DPTU appears on each amino acid residue cycle during the process of sequencing. Under the same sequencing conditions (i.e., the same column, gradient, column temperature, and mobile phase), the shift of a certain PTH-amino acid peak itself and the relative retention time (RT) difference to DPTU is less than 0.05 min. Therefore, it is not difficult to distinguish two amino acids with an RT difference greater than 0.10 min. It is not necessary to run a blank and a standard cycle prior to sequencing each peptide if the DPTU peak is eluted at the same position as that of the standard. In this case, a previously established standard can be used as a reference standard for amino acid assignment. By setting the cycle number to the number of peptide residues plus 1 and using a further-modified method (the same as Table II except no Flask Blank and Flask Standard cycles), 44 min (22 min min for the blank cycle and 22 min for the standard cycle) is saved for each peptide. However, when the column is changed or a new bottle of mobile phase is used, larger shifts of the PTH-amino acid elution positions may occur. It is then necessary to run an external reference standard containing both natural and unnatural amino acids. While the existing program in the sequencer can read the natural amino acids, unnatural amino acids have to be manually identified by comparing their RT with the external reference. We generate the external reference standard of PTH-amino acids in situ by placing amino acids mixtures that are covalently coupled to beads in the sequencing cartridge and letting the sequencer run through one sequencing cycle. Amino acid beads can be easily prepared by coupling a mixture of equal-molar Fmoc-unnatural and Fmoc-natural amino acids to the TentaGel NH2 beads in a one-pot reaction using standard HOBt/DIC coupling (see synthesis of peptide in this chapter), with subsequent Fmoc and side-chain deprotection, resulting in all amino acids covalently attached to each bead. We have selected many unnatural -amino acids to study their sequencing profiles. Table III summarizes the RT of 74 PTH derivatives of both natural and unnatural amino acids. Through Table III it is easy to select 40–45 amino acids with RT greater than 0.10 min as building blocks to construct peptide libraries. This greatly increases the diversity of the peptide libraries that can be generated. For OBOC small peptide libraries or libraries with a fixed position, it is not always necessary to sequence individual beads. In cases in which structural consensus can be expected, it is sometimes much faster to sequence a multiplicity of beads. From the sequencing data, the information about the structure–activity relationships can be obtained. In the cycles with a requirement for high specificity only one particular amino acid can be found, while

320

TABLE III RT of PTH-Amino Acids (Natural and Unnatural) on an ABI Protein Sequencera RT (min)

Aspartic acid

4.17

1-Aminocyclopropane1-carboxylic acid Asparagine Serine Glutamine

4.59

Threonine

5.85

Homoserine

5.95

Citrulline Glycine Glutamic acid Homocitrulline

6.07 6.11 6.49 6.98 7.57,8.75 7.80

RT (min)

Amino Acid

RT (min)

-Aminoisobutyric acid -Aminobutyric acid Proline Methionine Valine

10.90

Leucine

18.36

10.99

3,5-Dibromotyrosine

18.43

13.02 13.70 14.03

18.88 18.97 19.19

1-Amino-1-cyclopentane carboxylic acid 4-Cyanophenylalanine

14.78

Norvaline 3-(2-Thienyl)alanine 3-Nitrotyrosine ,-Diaminopropionic acid "-Benzoyllysine , -Diaminopropionic acid

15.32 15.61 15.62 15.75

Norleucine 4-Aminophenylalanine 1-Amino-1-(3-piperidinyl)carboxylic acid 1-Amino-1-(4-piperidinyl)carboxylic acid 2-Amino-3-(4-piperidinyl)propionic acid 4-Methylphenylalanine 3,5-Diiodotyrosine 4-Azidophenylalanine 2-Aminoindane-2carboxylic acid 3-Chlorophenylalanine Homophenylalanine

14.81

15.79 16.32

19.22 20.05 20.38 20.67 20.83 20.89 21.13 21.66

[17]

Hydroxyproline -Aminohexanedioic acid

4.70 5.34 5.61

Amino Acid

peptide synthesis and screening

Amino Acid

21.86

16.48 16.66 16.83 17.09 17.24

22.01 22.11 22.41 22.65 22.72

9.23

4-Nitrophenylalanine

17.27

9.95

-tert-Butylglycine

17.65

3-Benzothienylalanine 4-Benzoylphenylalanine 3-(2-Naphthyl)alanine 3-(1-Naphthyl)alanine 2-Amino-2-naphthylacetic acid 3,4-Dichlorophenylalanine O-Benzyl-hydroxyproline

10.01 10.03

-Benzyloxycarbonyl Isoleucine

17.72 17.76

Cyclohexylalanine 3,3-Diphenylalanine

23.98 24.02

10.13 10.50

Lysine 1-Amino-1-cyclohexane carboxylic acid

18.05 18.13

Di-n-propylglycine

27.17

"-Acetyl lysine Alanine 3-(4-Pyridyl)alanine 3-(3-Pyridyl)alanine 4-Amino-4-carboxy-1, 1-dioxotetrahydrothiopyran 1-Amino-1-(4-hydroxycyclohexyl)carboxylic acid 1-Amino-1-(4-ketocyclohexyl)carboxylic acid Propargylglycine 4-Amino-4-carboxytetrahydropyran Arginine Tyrosine

a

Internal reference, DPTU, 14.82 min.

23.23 23.31

oboc combinatorial peptide libraries

-Cyclohexylglycine

8.08

[17]

16.46

8.13 8.37 8.59 8.71 8.81

Thiazolidine-4carboxylic acid Tryptophan O-Methyltyrosine Ornithine Phenylglycine Phenylalanine

Histidine

321

322

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peptide synthesis and screening

in positions not essential for the activity numerous amino acids can be found. To prevent the loss of sequencing information of a unique bead (e.g., where only one positive bead was found in the screening and verified to be specific) caused by unforeseeable problems such as malfunctioning instruments or a power failure, the bead can be cut in half under a microscope with an injection needle and half of the bead can be submitted for sequencing. Edman Sequencing Peptide sequencing is performed on a Perkin-Elmer/Applied Biosystems Protein Sequencer (Procise 494). The equipment parameters are as follows: column, Spheri-5 C18 PTH column, 5 m, 2.1  220 mm; column    temperature, 55 ; cartridge temperature, 48 ; flask temperature, 64 . Premix buffer concentrate (concentration: 17.5 mM sodium acetate, 33 mM acetic acid, 10 mM hexanesulfonic acid sodium salt) is purchased from the manufacturer. Reservoir A: 25 ml premix buffer added to 1 liter solvent A3 (3.5% v/v tetrahydrofuran in water). Reservoir B: 88% acetonitrile/12% isopropanol, v/v (solvent B2). For sequencing peptides containing natural amino acids, the manufacturers instructions are followed. For sequencing peptides consisting of both natural and unnatural amino acids, a slightly modified gradient program and modified method are employed (refer to Tables I and II). Acknowledgments This work was supported by NIH R01-CA78868, R33-CA86364, R33-CA89706, the California Cacner Research Program, Contract No. 00-00764V-20133. Ruiwu Liu is supported in part by the University of California Systemwide Biotechnology Research Program, Grant 2001–07.

[18] Synthetic Combinatorial Libraries as an Alternative Strategy for the Development of Novel Treatments for Infectious Diseases By Sylvie E. Blondelle, Clemencia Pinilla, and Ce´sar Boggiano Background

Infectious diseases are now the leading cause of death worldwide (25% of all world death is caused by infectious diseases and 45% in low-income countries), the main cause of death among children (65%), and the third leading cause of death in the United States. In particular, diarrhea, human

METHODS IN ENZYMOLOGY, VOL. 369

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immunodeficiency virus/acquired immunodeficiency syndrome (HIV/ AIDS), malaria, measles, pneumonia, and tuberculosis cause 90% of infectious disease-related deaths. Antimicrobial drug resistance is a major concern in infectious diseases such as acute respiratory infections, tuberculosis, and diarrheal diseases, while the arsenal of drugs available to treat infectious diseases is being progressively depleted. For example, in the area of hospital infection control, there is an increasing concern regarding the rapid emergence of drug-resistant strains with high epidemic potential.1,2 Of even greater concern is the fact that a number of bacteria, such as Staphylococcus aureus, the most common cause of nosocomial wound infections, are becoming resistant to vancomycin, a drug of last resort.3 Although there are reports of about a dozen new drugs that show promising antibacterial activity by interfering with protein synthesis, cell wall formation, or DNA replication, a new generation of antibiotics remains urgently needed to combat the ever increasing spread of antibiotic-resistant bacterial strains. Life-threatening infections caused by pathogenic fungi are also becoming increasingly common, especially in individuals with suppressed immune systems such as a number of cancer patients and individuals with AIDS.4 Unfortunately, there are only a limited number of antifungal compounds available to counter such infections, which implies a strong need for development of new compounds with antifungal activities. The main groups of antifungals currently available are polyenes (e.g., amphotericin B, nystatin, and pimaricin), imidazoles (e.g., ketoconazole, fluconazole, miconazole), antimetabolites (e.g., 5-fluorocytosine), echinocandins, and pneumocandins. While over 33 million people are living with HIV/AIDS, the impact of HIV therapies has been shown by a nationwide decline in AIDS-related deaths. The vast majority of anti-HIV therapeutic agents have been limited to reverse transcriptase and protease inhibitors.5,6 However, the development of resistance to these inhibitors7 and the toxic side effects associated with these compounds8 indicate a major need for further discovery and development of alternative strategies such as those targeting other stages 1

J. T. Cross, Jr. and G. D. Campbell, Jr., Clin. Chest Med. 20, 499 (1999). C. O. Solberg, Scand. J. Infect. Dis. 32, 587 (2000). 3 K. Hiramatsu, Lancet Infect. Dis. 1, 147 (2001). 4 J. Ponton, R. Ruchel, K. V. Clemons, D. C. Coleman, R. Grillot, J. Guarro, D. Aldebert, P. Ambroise-Thomas, J. Cano, A. J. Carrillo-Munoz, J. Gene, C. Pinel, D. A. Stevens, and D. J. Sullivan, Med. Mycol. 38, 225 (2000). 5 Z. Temesgen, Expert Opin. Pharmacother. 2, 1239 (2001). 6 R. P. van Heeswijk, A. Vedkamp, J. W. Mulder, P. L. Meenhorst, J. M. Lange, J. H. Beijnen, and R. M. Hoetelmans, Antiviral Ther. 6, 201 (2001). 7 D. Pillay, Antiviral Ther. 6, 15 (2001). 8 C. Vigouroux, S. Gharakhanian, Y. Salhi, T. H. Nguyen, N. Adda, W. Rozenbaum, and J. Capeau, Diabetes Metab. 25, 383 (1999). 2

324

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of the HIV life cycle to prevent transmission and spread of HIV as well as to improve the quality of life of HIV-infected patients. Synthetic combinatorial chemistry approaches have created a vast new source of molecular diversity for the potential identification of lead compounds. This revolutionary field enables hundreds to thousands of times more compounds to be synthesized and screened in shorter periods of time relative to traditional approaches. In particular, novel antibacterials and/or antifungals9,10 as well as inhibitory peptides of either HIV proteases11,12 or HIV integrase proteins13 were identified following the screening of synthetic combinatorial libraries (SCLs). As described below, SCLs have been used by this laboratory to develop antimicrobial and antifungal compounds, as well as novel treatments for HIV infection. Mixture-Based Combinatorial Libraries

Mixture-based SCLs are built around a given pharmacophore by generating all possible combinations of selected sets of functional groups at a defined number of positions, which are readily screened as mixtures of soluble compounds.9,14 Each SCL differs from the others by its chemical class (i.e., from peptides to organic molecules), size (i.e., total number of individual compounds present in the library), number and location of the diversity positions, and format driving the deconvolution process toward the identification of the active components. Although the majority of combinatorial chemistry programs prefer the preparation of large numbers of individual compounds by parallel synthesis methods, more than 100 separate studies in which active compounds have been identified from mixturebased libraries of various classes have now been reported. These studies have been carried out by nearly 50 separate research groups in assays ranging from binding assays to cell-based assays.9 The number of compounds present within different mixture-based libraries tested ranged from less than 10 to greater than 1014, and the number of individual compounds 9

R. A. Houghten, C. Pinilla, J. R. Appel, S. E. Blondelle, C. T. Dooley, J. Eichler, A. Nefzi, and J. M. Ostresh, J. Med. Chem. 42, 3743 (1999). 10 S. E. Blondelle and K. Lohner, Biopolymers (Peptide Sci.) 55, 74 (2000). 11 R. A. Owens, P. D. Gesellchen, B. J. Houchins, and R. D. DiMarchi, Biochem. Biophys. Res. Commun. 181, 402 (1991). 12 T. A. Rano, Y. Cheng, T. T. Huening, F. Zhang, W. A. Schleif, L. Gabryelski, D. B. Olsen, L. C. Kuo, J. H. Lin, X. Xu, T. V. Olah, D. A. McLoughlin, R. King, K. T. Chapman, and J. R. Tata, Bioorg. Med. Chem. Lett. 10, 1527 (2000). 13 R. A. P. Lutzke, N. A. Eppens, P. A. Weber, R. A. Houghten, and R. H. Plasterk, Proc. Natl. Acad. Sci. USA 92, 11456 (1995). 14 R. A. Houghten, C. Pinilla, S. E. Blondelle, J. R. Appel, C. T. Dooley, and J. H. Cuervo, Nature 354, 84 (1991).

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325

per mixture ranged from less than 10 to more than 2  1010. It should be noted, however, that the levels in activity (micromolar to fentomolar) of the individual compounds identified from these libraries are independent of the complexity and chemical nature of the library tested. Indeed, the most active compounds derived from a given library may have micromolar range activity against a specific target system, while other individual compounds derived from the same library may have nanomolar or subnanomolar activity in a different bioassay system. Deconvolution procedures are an essential element in the identification of active individual compounds from mixture-based libraries. In particular, the positional scanning approach15 uses a positional scanning SCL (PSSCL), in which the key functionality(ies) at each position of the active compound(s) can be determined directly from the library screening data. Thus, a PS-SCL is composed of sublibraries, each addressing a single position of the sequence while representing the same collection of individual compounds (Table I). When used in concert, the data derived from each sublibrary yield information about the key functionalities for each position. If the key functionalities at each position are found to be connected to each other (i.e., represent the same active compound(s) present in the corresponding mixtures), then combinations of these functionalities will lead to active individual compounds. Individual compounds that represent all possible combinations of the selected functionalities at each position are then synthesized in order to confirm the screening data and determine their relative activities. Mixture-based PS-SCLs therefore have the advantage of greatly decreasing the economics and time constraints of single compound array systems. Screening of millions of compounds can be accomplished even in assays that are not formatted for conventional high throughput. Development of Antifungal Agents Using the PS-SCL Approach

Candida albicans and Cryptococcus neoformans are two of the most common fungi responsible for opportunistic infections. For instance, oropharyngeal candidiasis is the fungal infection most frequently associated with HIV-positive patients.16 Cr. neoformans is the causative agent of cryptococcosis, which is the leading cause of morbidity and mortality due to fungi in patients with AIDS.17 In a continuing effort to generate and screen 15

C. Pinilla, J. R. Appel, P. Blanc, and R. A. Houghten, Biotechniques 13, 901 (1992). W. G. Powderly, K. H. Mayer, and J. R. Perfect, AIDS Res. Hum. Retroviruses 15, 1405 (1999). 17 K. J. Kwon-Chung, T. C. Sorrell, F. Dromer, E. Fung, and S. M. Levitz, Med. Mycol. 38, 205 (2000). 16

326

TABLE I Illustration of a Tripeptide PS-SCL Structurea Sublibrary 1: OXXb

AAA AAC ... AKA AKC ... AYW AYY a

...

LXX LAA LAC ... LKA LKC ... LYW LYY

...

YXX

XAX

YAA YAC ... YKA YKC ... YYW YYY

AAA AAC ... KAA KAC ... YAW YAY

...

XLX ALA ALC ... KLA KLC ... YLW YLY

...

Sublibrary 3: XXO XYX

XXA

AYA AYC ... KYA KYC ... YYW YYY

AAA ACA ... KAA KCA ... YWA YYA

...

XXL AAL ACL ... KAL KCL ... YWL YYL

...

XXY AAY ACY ... KAY KCY ... YWY YYY

The tripeptide PS-SCL has three diversity positions and thus consists of three sublibraries, each of which has a single defined amino acid at one position and an equimolar mixture of amino acids at each of the other two positions. Twenty l-amino acids are used at each defined position, and there are 20 mixtures per sublibrary, as well as at each mixture position. Thus there are 202 ¼ 400 peptides per mixture and 203 ¼ 8,000 peptides in the entire library. The same 8,000 peptides are present in each sublibrary, but are arranged differently within the mixtures of each sublibrary. b ‘‘O’’ (referred as ‘‘the defined position’’) represents the l-amino acid common to every peptide present at this position in a given mixture; ‘‘X’’ (referred as ‘‘the mixture position’’) represents all 20 l-amino acids that can be present at this position in a peptide contained in a given mixture. All peptides are present in a close to equimolar concentration.

peptide synthesis and screening

AXX

Sublibrary 2: XOX

[18]

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327

various classes of antifungal compounds against C. albicans and Cr. neoformans, a number of mixture-based PS-SCLs made up of heterocyclic compounds were evaluated for the identification of novel antifungal candidate drugs.18,19 Heterocyclic compounds offer a high degree of structural diversity and have proven to be broadly and economically useful as therapeutic agents, particularly nitro-carbon heterocyclic structures.20 To illustrate the PS-SCL approach, we report the generation and identification of N-benzyl aminocyclic ureas having anticandidal activities.18 Screening of a PS-SCL for Antifungal Activity An N-benzyl aminocyclic urea PS-SCL was prepared as described elsewhere.21,22 This library was built with a diversity of 40, 37, and 80 separate functionalities at position 1, 2, and 3, respectively. As shown in Table II, the complete PS-SCL contains 118,400 compounds (40  37  80) and consists of three sublibraries, each of which has a single defined functionality at one position and a mixture of functionalities at each of the other two positions. The number of individual compounds per mixture varied from one sublibrary to the others since this is based on the diversity at the defined position of that sublibrary. Thus, each sublibrary contains the same 118,400 compounds and differs from the others by how the compounds are grouped within each mixture. Each mixture was screened at 250 g/ml against 1–5  105 colonyforming unit (CFU)/ml blastoconidial C. albicans prepared by inoculating two colonies of a newly grown C. albicans culture in yeast media (YM) broth (Difco Laboratories, Detroit, MI), vortexed, and diluted 10-fold in YM. The assay was carried out in 96-well flat-bottom tissue culture plates (Costar, Pleasanton, CA). An equal volume (100 l) of yeast suspension in 2 YM broth was added to the mixtures and the plates incubated for 48 h  at 30 . The relative percent growth of C. albicans found in the presence of each mixture was determined by the optical density at 620 nm (OD620) using a Spectra Max 250 microplate spectrophotometer. Duplicate samples of each mixture were used in each assay, and the assays were repeated at least twice. As shown in Fig. 1, a small number of mixtures exhibited 18

S. E. Blondelle, A. Nefzi, J. M. Ostresh, and R. A. Houghten, Pure Appl. Chem. 70, 2141 (1998). 19 S. E. Blondelle, E. Crooks, J. M. Ostresh, and R. A. Houghten, Antimicrob. Agents Chemother. 43, 106 (1999). 20 H. J. Roth and A. Kleeman, ‘‘Pharmaceutical Chemistry.’’ John Wiley & Sons, New York, 1988. 21 A. Nefzi, J. M. Ostresh, J.-P. Meyer, and R. A. Houghten, Tetrahedron Lett. 38, 931 (1997). 22 C. E. Hoesl, A. Nefzi, J. M. Ostresh, Y. Yu, and R. A. Houghten, Chapter 25, this volume.

328

TABLE II N-benzyl Aminocyclic Urea PS-SCL Descriptiona

O

RX

Number of mixtures Number of compounds per mixture Total number of compounds in sublibrary a

N

RO N

Sublibrary 2 O

RX

BZ NH

RX

N

N

Sublibrary 3 O

RX

BZ NH

RO

N

N

RX

RO

RX

40 37  80 ¼ 2960

37 40  80 ¼ 3200

80 40  37 ¼ 1480

40  2960 ¼ 118; 400

37  3200 ¼ 118; 400

80  1480 ¼ 118; 400

BZ NH

Ro represents a defined functionality, common to all compounds present in a given mixture; Rx represents a close to equimolar mixture of all functionalities used to build the library at a given position.

peptide synthesis and screening

Sublibrary 1

[18]

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combinatorial libraries in infectious diseases

100%

329

Sublibrary 1

80% 60% 40% 20% 0%

Sublibrary 2 100% 80% 60% 40% 20% 0% 100%

Sublibrary 3

80% 60% 40% 20% 0% Fig. 1. Anticandidal activity of N-benzyl aminocyclic urea PS-SCL. Each graph represents the activity of a sublibrary, and each bar within a graph represents the percent inhibition of the growth of C. albicans ATCC 10231 by a separate mixture. The line represents the average activity for all of the mixtures making up the corresponding sublibrary.

330

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peptide synthesis and screening TABLE III Anticandidal Activity of Most Active N-benzyl Aminocyclic Urea Mixtures RO1a

RO2

RO3

(S)-Cyclohexylmethyl (R)-Cyclohexylmethyl (R)-Isobutyl (S)-Dibenzylaminoethyl X

X X X X (S)-N-Methylindole-3methyl (S)-n-Butyl (S)-Cyclohexylmethyl X X X

X X X X X

173 182 188 219 93

X X 4-Dimethylaminobenzyl 3,3-Dicyclohexylpropyl 4-Cyclohexylbutyl

140 151 149 180 210

X X X X X a b

IC50 (g/ml)b

Functionality defining the mixture. The IC50 values are determined against C. albicans ATCC 10231.

significant antifungal activity. To better differentiate the most active mixtures, the concentration necessary to inhibit 50% growth (IC50) was determined for those mixtures that exhibited more than 50% inhibition at 250 g/ml by testing them at concentrations varying from 250 to 2 g/ml in H2O derived from serial 2-fold dilutions (Table III). These IC50 values were calculated using a sigmoidal curve fitting software (Graphpad—ISI, San Diego, CA). Deconvolution Process Based on the screening results of the PS-SCL, a limited number of mixtures were selected to carry out a first deconvolution process according to criteria of high antifungal activity and significant differences in the chemical nature of the defined functionalities within each sublibrary. Thus, 12 N-benzyl aminocyclic ureas, which corresponded to all possible combinations of the functionalities defining selected mixtures at each position (two mixtures from sublibraries 1 and 3, and three mixtures from sublibrary 2, 2  3  2 ¼ 12), were generated in order to confirm the screening data and determine their relative activities. The IC50 values of these compounds varied from 10 M to greater than 500 M. Similar activities were observed when tested against Cr. neoformans. The structures of the two most active N-benzyl aminocyclic ureas are shown in Table IV.

[18]

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combinatorial libraries in infectious diseases TABLE IV Antifungal Activity of the Two Most Active N-benzyl Aminocyclic Ureasa

O N N

N O

NH

N N

N

IC50 (M) C. albicans Cr. neoformans

48 16

MIC (M)b 60–120 60–120

NH

IC50 (M)

MIC (M)b

36 9

40–60 30–60

a

The fungi strains used in these studies were C. albicans ATCC 10231 and Cr. neoformans   ATCC 32045. Each compound was incubated for 48 h at 30 or 72 h at 26 for C. albicans or Cr. neoformans, respectively. b The minimum inhibitory concentrations (MICs) were defined as the lowest concentration of compound at which less than 2% growth was detected.

Development of Antibacterial Agents Using the PS-SCL Approach

Continued development of novel therapeutics for the treatment of bacterial infection has become an important clinical imperative. This is especially true due to the emergence and dissemination of new opportunistic pathogens in a growing immune system-debilitated host population, as well as to the emerging resistance of common or resurgent pathogens to standard antibiotics in community-acquired infection.2,4,23 In particular, colonization with methicillin-resistant S. aureus (MRSA) is a growing clinical challenge in hospitals and community care centers and has been recognized as a major nosocomial pathogen in hospitals.2,24 Vancomycin and a number of fluoroquinolones are currently the agents of choice against MRSA.25 However, fluoroquinolone-resistant MRSA strains26 as well as vancomycin-resistant MRSA strains3 have now been clinically isolated. Pseudomonas aeruginosa is another major human pathogen that causes disease in patients with impaired host defenses.27 Ps. aeruginosa is primarily associated with ultimately fatal chronic respiratory infection in cystic fibrosis and other forms of bronchiectasis.28 Despite strong inflammatory 23

J. P. Metlay, Curr. Opin. Infect. Dis. 15, 163 (2002). F. M. Hussain, S. Boyle-Vavra, and R. S. Daum, Pediatr. Infect. Dis. J. 20, 763 (2001). 25 J. M. Boyce, J. Hosp. Infect. 48, S9 (2001). 26 J. F. Acar and F. W. Goldstein, Clin. Infect. Dis. 24, S67 (1997). 27 R. Wilson and R. B. Dowling, Thorax 53, 213 (1998). 28 A. L. Brennan and D. M. Geddes, Curr. Opin. Infect. Dis. 15, 175 (2002). 24

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antibody response and intensive antibiotic therapies, Ps. aeruginosa is rarely eliminated once colonization of the airways is established. To reach the sites of infection, antibiotics must be able to penetrate into bronchial mucosa, which renders Ps. aeruginosa resistant to a large number of antibiotics.27 Among existing antibiotics, quinolone derivatives such as ciprofloxacin have the greatest ability to penetrate the mucosal barrier. However, resistance to quinolone derivatives has also emerged.29 The SCL approach was then undertaken to identify novel structures having activity against MRSA and/or Ps. aeruginosa using standard microdilution susceptibility assays as described above for antifungal activity screening.9,10 Examples of the ranges of activities and chemical structures of antimicrobial compounds identified from SCLs are shown in Table V. The activities were determined following overnight incubation at 37 in Mueller Hinton (MH) broth against MRSA and Ps. aeruginosa culture prepared by inoculating 100 l of an overnight grown bacterial cultures in 10 ml of 2 MH broth,  incubated for 2 h at 37 , and diluted 40-fold in 2 MH broth, for an approximate final assay concentration of 1–5  105 CFU/ml. Development of HIV-1 Antagonists Using the SCL Approach

Although the introduction of highly active antiretroviral therapy (HAART) significantly improved the quality of life of HIV-1-infected individuals, the reverse transcriptase and protease inhibitors used in HAART cannot completely eradicate HIV-1 from infected people,30 and the emerging transmission of resistant variants and long-term side effects of these inhibitors31,32 raise the need for novel strategies to fight HIV-1 infection. The discovery in the early 1990s that peptides corresponding to segments of HIV-1 gp41 env potently inhibit virus entry into target cells,33,34 combined by the recent emergence of structural and functional information on HIV-1 envs and membrane fusion process,35–38 opened a new strategy 29

T. Ko¨hler, M. Michea-Hamzehpour, P. Plesiat, A.-L. Kahr, and J.-C. Pechere, Antimicrob. Agents Chemother. 41, 2540 (1997). 30 D. Finzi, J. Blankson, J. D. Siliciano, J. B. Margolick, K. Chadwick, T. Pierson, K. Smith, J. Lisziewicz, F. Lori, C. Flexner, T. C. Quinn, R. E. Chaisson, E. Rosenberg, B. D. Walker, S. Gange, J. Gallant, and R. F. Siliciano, Nat. Med. 5, 512 (1999). 31 N. L. Michael and J. P. Moore, Nat. Med. 5, 740 (1999). 32 J. Martinez-Picado, M. P. DePasquale, N. Kartsonis, G. J. Hanna, J. Wong, D. Finzi, E. Rosenberg, H. F. Gunthard, L. Sutton, A. Savara, C. J. Petropoulos, N. Hellmann, B. D. Walker, D. D. Richman, R. Siliciano, and R. T. D’Aquila, Proc. Natl. Acad. Sci. USA 97, 10948 (2000). 33 C. Wild, T. Oas, V. McDanal, D. Bolognesi, and T. Matthews, Proc. Natl. Acad. Sci. USA 89, 10537 (1992). 34 S. Jiang, K. Lin, N. Strick, and A. R. Neurath, Nature 365, 113 (1993).

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combinatorial libraries in infectious diseases TABLE V Example of Acyclic and Heterocyclic Antimicrobial Structures Identified from PS-SCLs

MIC (g/ml) of Most Active Compoundsa Structure R

R H N

H2N

4

N H

Number of compounds in SCL

MRSA

Ps. aeruginosa

49,521,980

2–4

16–32

125,934

2–4

nd

32,400

8–16

nd

6,525

2–4

nd

102,459

4–8

62–125

NH2

R O H N

H N

R

R O

R

O

H

O

R

C

N

CH

N N

R R

O

R

O

HN

R

N

CONH2

S

R R

N N N R a

The bacterial strains used in these studies are MRSA ATCC 33591 and Ps. aeruginosa ATCC 27853.

for the development of potentially useful therapeutics. Thus, therapeutic approaches involving the inhibition of the membrane fusion are gaining importance. The major issues when considering peptides as potential drugs 35

W. A. Weissenhorn, A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley, Nature 387, 426 (1997). 36 D. C. Chan and P. S. Kim, Cell 93, 681 (1998). 37 P. D. Kwong, R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson, Nature 393, 648 (1998). 38 E. A. Berger, P. M. Murphy, and J. M. Farber, Annu. Rev. Immunol. 17, 657 (1999).

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are the peptides’ susceptibility to proteolysis, immune clearance, and lack of bioavailability. Compounds less than 1000 Da, which represent the vast majority of clinically useful drugs, are indeed more desirable for mass treatment. Furthermore, the crystal structure of the gp41 core39 showed the presence of three prominent pockets that could accommodate molecules with a molecular weight of approximately 500 Da. The SCL approach was then used to develop small molecules having inhibitory activity for HIV-1-mediated membrane fusion as novel lead anti-HIV compounds. Determination of Inhibition of HIV-1 Mediated Fusion The intrinsic fusion selectivity of the viral envelope glycoprotein plays a major role for infection of CD4þ T cell lines versus primary macrophages.40 This selectivity is determined by ‘‘fusion cofactors’’ (CXCR4 for T cell lines and CCR5 for macrophages) that belong to the chemokine receptor family.38 To address the two processes and enable a rapid screening of various PS-SCls, a 96-well plate-based assay was adapted from the envmediated membrane fusion assay described by Berger’s group.41 This system uses two different cell populations, effector cells (expressing HIV1 env and T7 RNA pol using vaccinia virus) and target cells (expressing constitutively CD4 and the required coreceptor, and -galactosidase gene downstream of a T7 promotor also using a vaccinia construct). Thus, effector cells (HeLa) and target cells (HeLa CD4þ for the X4 system and HOS CD4þ CCR5þ for the R5 system) were diluted to a final density of 2  106 cells/ml for the R5 system and 6  105 cells/ml for the X4 system. Effector and target cells (25 l each) were dispensed into individual wells of a 96-well, polystyrene, half-area flat-bottom, high-binding plate containing 50 l of test samples at varying concentrations derived from serial 2-fold dilutions or media in the control wells. The positive controls contained effector and target cells and the negative control contained uncleaved control cells and target cells. Plates were centrifuged at 400 g for 2 min and then incubated for 3 h to allow fusion. To determine the level of fusion between effector and target cells, 25 l of 2% (v/v) Tween-20 was added to each well, mixed by pipetting, frozen, and thawed twice to facilitate cell lysis. Then 25 l of lysate was transferred to a 96-well half-area plate and 25 l of substrate solution--gal (8 mM chlorophenol red--d-galactopiranoside, 60 mM Na2HPO4H2O, 40 mM NaH2PO4H2O, 10 mM KCl, 1 mM MgSO4H2O, 50 mM 39

D. C. Chan, C. T. Chutkowski, and P. S. Kim, Proc. Natl. Acad. Sci. USA 95, 15613 (1998). C. C. Broder and E. A. Berger, Proc. Natl. Acad. Sci. USA 92, 9004 (1995). 41 O. Nussbaum, C. C. Broder, and E. A. Berger, J. Virol. 68, 5411 (1994). 40

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2-mercaptoethanol) was added. The OD570 was measured and the percent inhibition was calculated relative to the positive controls. Identification of Inhibitors of HIV-1-Mediated Fusion from PS-SCLs An example of PS-SCL screening for anti-HIV activity is shown in Fig. 2 with the screening of a PS-SCL of diethylenetriamines and a PS-SCL of Nmethyl-1,4,5-trisubstituted 2,3-diketopiperazines in the X4 system. The two libraries were generated using the same building blocks and the diethylenetriamine PS-SCL served as starting material for the synthesis of the N-methyl-1,4,5-trisubstituted 2,3-diketopiperazine library following the ‘‘library from library’’ concept.42–44 Thus, N-methyl-1,4,5-trisubstituted 2,3-diketopiperazines were obtained by overnight treatment with oxalyldiimidazole of resin-bound reduced acylated dipeptides. These reduced acylated dipeptides led to diethylenetriamines following cleavage from the resin with anhydrous HF, extraction, and lyophilization. Interestingly, a significantly different profile of activity was observed between the two libraries (i.e., mixtures defined with the same functionality showed a different relative activity in the two libraries). These results indicate that the presence of the cyclic structure plays a role in the activity of the individual compounds. Based on the screening results, 27 diethylenetriamines and 24 Nmethyl-1,4,5-trisubstituted 2,3-diketopiperazines were generated and assayed in the X4 and R5 systems. The most active diethylenetriamines had IC50 values varying from 30 to 500 nM and 840 nM to 1.1 M in the R5 and X4 systems, respectively, while the most active diketopiperazines had IC50 values varying from 5 to 36 M in both systems. Inhibition of HIV-1 Replication The fusion assays described above combined with the SCL approaches represent a straightforward and rapid means for the development of novel compounds that inhibit HIV-1 env-mediated membrane fusion. Although based on recombinant glycoproteins, the process of cell-to-cell fusion is generally thought to be analogous to the processes involved in fusion of viral membrane during virus penetration.40 To further investigate the inhibitory activity of the newly identified antagonists, their ability to inhibit HIV-1 infectivity was evaluated by carrying out replication assays. Thus, two well-characterized HIV-1 isolates were selected for this assay: HIV-1 IIIb 42

J. M. Ostresh, G. M. Husar, S. E. Blondelle, B. Do¨rner, P. A. Weber, and R. A. Houghten, Proc. Natl. Acad. Sci. USA 91, 11138 (1994). 43 A. Nefzi, J. M. Ostresh, and R. A. Houghten, Tetrahedron 55, 335 (1999). 44 A. Nefzi, M. A. Giulianotti, and R. A. Houghten, Tetrahedron 56, 3319 (2000).

336

Diethylenetriamine PS-SCL Sublibrary 2

Sublibrary 1

Sublibrary 3

80

80

60

60

60

40

40

40

20

20

20

0

0

0

N-methyl-1,4,5-trisubstituted-2,3-diketopiperazine PS-SCL Sublibrary 2

Sublibray 1

Sublibrary 3

80

80

80

60

60

60

40

40

40

20

20

20

0

0

0

peptide synthesis and screening

80

Fig. 2. Inhibitory activity of HIV-1-mediated fusion by diethylenetriamine and N-methyl-1,4,5-trisubstituted 2,3-diketopiperazine PS-SCLs. Each bar within a graph represents the percent inhibition as measured in the X4 fusion system by a separate mixture at 2.5 g/ml for the diethylenetriamines and at 25 g/ml for the diketopiperazines. The line represents the average activity for all of the mixtures making up the corresponding sublibrary.

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N H

H N

337

N H

Fig. 3. Chemical structure of the diethylenetriamine identified from the PS-SCL showing the greatest inhibition of HIV-1 replication.

(X4 virus) and HIV-1 SF162 (R5 virus). These viruses were propagated and titrated in peripheral blood monocellular cells (PBMCs) from HIV-1negative donors. Preactivated PBMCs with phytohemagglutinin (PHA) and interleukin (IL)-2 were infected in a 15-ml tube with 0.03–0.01 tissue cul ture infectious dose (TCID) virus and incubated overnight at 37 under a 5% CO2 atmosphere. Following three washes with RPMI containing 10% fetal calf serum, the cells were resuspended in 96-well plates at 30,000 cells/well in the presence or absence of different dilutions of test compounds, and the  plates were incubated for 7 days at 37 under 5% CO2 atmosphere. The presence of HIV-1 p24 was tested using commercial ELISA kits (Perkin Elmer, Life Sciences). For the most active compounds (structure shown in Fig. 3) 44% and 62% inhibition of the replication of the X4 and R5 viruses, respectively, were observed at 3 M. Although the mechanism of inhibition remains to be determined, due to the small size of the identified inhibitors, they might interact with the gp41 N36 binding pocket.39 Novel Strategies toward the Development of Vaccines against Infectious Diseases

The worldwide significance of infectious diseases has led to great interest in generating vaccines to cure or prevent such diseases. Potent and universally applicable vaccines must be recognized as nonself by the immune systems of many individuals of a wide patient population. T lymphocytes with the help of activated B lymphocytes regulate both the cellular and humoral responses of the immune system. By their T cell receptor (TCR) T cells identify antigens (short peptides) bound to major histocompatibility complex (MHC) molecules on the antigen-presenting cells (APC), and bind, recognize, and are activated by these antigens. The two major T cell subpopulations, CD8þ cytolytic T lymphocytes (CTL) and CD4þ helper T lymphocytes, which act as effectors during infection, or as inducers of T and B cell functions, are involved in both physiological and pathological immune responses. CD8þ T cells typically recognize 9 amino acid-long

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peptides in the context of MHC class I molecules, whereas CD4þ T cells recognize 11–15 amino acid-long peptides in the context of MHC class II molecules. An approach directed toward the identification of antigens that stimulate T cell responses important to clear infection, i.e., through the identification of immunodominant epitopes and epitope mimics, is anticipated to lead to a successful development of prophylactic and therapeutic vaccines to combat infectious diseases. Identification of Epitope Mimics The use of peptide libraries to study T cell specificity was first demonstrated by Gundlach et al.45 Since then studies have focused on T cell specificity with emphasis on the identification of immunodominant epitopes in infectious diseases and autoimmune disorders, and on the determination of tumor antigens. The PS-SCL approach, as described above, was successfully used to study T cell recognition and to identify and optimize peptides having a range of activities in stimulating proliferative responses, cytokine production, and/or lysis by CD4þ and CD8þ murine and human T cell clones and T cell lines of known and unknown specificity.46–53 In the cases of clones of known specificity, approximately 25% of the identified epitope mimics were found to be superagonists, having EC50 values (concentration of peptide that causes a half-maximal response by a clone or a line) that are 1 to 3 orders of magnitude more effective than the native ligand.48,49,51 Importantly, most of these agonists are effective immunogens capable of inducing T cell-mediated potent immune responses 45

B. R. Gundlach, K.-H. Wiesmu¨ller, T. Junt, S. Kienle, G. Jung, and P. Walden, J. Immunol. Methods 192, 149 (1996). 46 C. Pinilla, R. Martin, B. Gran, J. R. Appel, C. Boggiano, D. B. Wilson, and R. A. Houghten, Curr. Opin. Immunol. 11, 193 (1999). 47 B. Hemmer, B. Gran, Y. Zhao, A. Marques, J. Pascal, A. Tzou, T. Kondo, I. Cortese, B. Bielekova, S. Straus, H. F. McFarland, R. A. Houghten, R. Simon, C. Pinilla, and R. Martin, Nat. Med. 5, 1375 (1999). 48 D. B. Wilson, C. Pinilla, D. H. Wilson, K. Schroder, C. Boggiano, V. Judkowski, J. Kaye, B. Hemmer, R. Martin, and R. A. Houghten, J. Immunol. 163, 6424 (1999). 49 B. Hemmer, C. Pinilla, B. Gran, M. Vergelli, N. Ling, P. Conlon, H. F. McFarland, R. A. Houghten, and R. Martin, J. Immunol. 164, 861 (2000). 50 R. Martin, B. Gran, Y. Zhao, S. Markovic-Plese, B. Bielekova, A. Marques, M. H. Sung, B. Hemmer, R. Simon, H. F. McFarland, and C. Pinilla, J. Autoimmun. 16, 187 (2001). 51 C. Pinilla, V. Rubio-Godoy, V. Dutoit, P. Guillaume, R. Simon, Y. Zhao, R. A. Houghten, J.-C. Cerottini, P. Romero, and D. Valmori, Cancer Res. 61, 5153 (2001). 52 C. La Rosa, R. Krishnan, S. Markel, J. P. Schneck, R. Houghten, C. Pinilla, and D. J. Diamond, Blood 97, 1776 (2001). 53 V. Rubio-Godoy, C. Pinilla, V. Dutoit, E. Borras, R. Simon, Y. Zhao, J.-C. Cerottini, P. Romero, R. A. Houghten, and D. Valmori, Cancer Res. 62, 2058 (2002).

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to the native peptide ligand in vivo. An additional 25% are effective antagonists, and are able to inhibit responses to prepulsed agonists. It is noteworthy that most of these identified epitope mimics have multiple nonconservative changes of sequence that would have been impossible to predict by traditional and restricted analoging methods. An example of the identification of epitope mimics for CD8þ T cell clones in infectious diseases is the generation of nonapeptide analogs of the pp65495–503 CTL epitope for cytomegalovirus (CMV).52 CMV is an important human pathogen affecting immunosuppressed patients.54 The pp65495–503 CTL epitope is universally recognized among CMV-seropositive individuals who express HLA-A*0201.55,56 However, the relative binding affinity of this epitope to HLA-A*0201 is moderate, and an increased immunogenicity might prove beneficial in its use as a CTL epitope vaccine. The different steps involved in the identification of pp65495–503 epitope mimics from the screening of the library are described in Fig. 4, pathway A. Thus, each mixture of a C-terminal amidated nonapeptide PS-SCL was incubated at 50 g/ml with cloned T cells (3-3F4—a CMV-specific CD8þ HLA-A*0201-restricted T cell clone) and 51Cr-labeled T2 cells (HLA-A2þ, TAP-deficient human lymphoblastoid hybridoma often used for peptide presentation protocols)57 for 4 h and the levels of chromium release were determined using a Cobra II gamma counter. Upon screening the nonapeptide PS-SCL against the 3-3F4 CMV-specific T cell clone, one to three mixtures exhibited significant activities in each sublibrary, and five of nine amino acids defining the most active mixtures corresponded to the residues of the native peptide epitope. In particular, the mixtures defined by the known anchor residues at positions 2 and 9 (Leu and Val, respectively) were among the highest responders at those positions.58 Based on the screening results, a series of 16 individual nonapeptides was generated. The two most active peptides, despite having four amino acid substitutions, were 1000- and 10,000-fold more active than the native epitope. To verify whether the increases seen in recognition by the HLA-A*0201-restricted T cell clone 3-3F4 would be common to all T cell clones that recognize the native epitope, a chromium release assay was also performed using a cohort of four additional T cell clones derived from four different CMV-seropositive volunteers. The two most active peptides 54

D. Salmon-Ceron, HIV Med. 2, 255 (2001). M. P. Weekes, M. R. Wills, K. Mynard, A. J. Carmichael, and J. G. Sissons, J. Virol. 73, 2099 (1999). 56 A. Solache, C. L. Morgan, A. I. Dodi, C. Morte, I. Scott, C. Baboonian, B. Zal, J. Goldman, J. E. Grundy, and J. A. Madrigal, J. Immunol. 163, 5512 (1999). 57 R. D. Salter, D. N. Howell, and P. Cresswell, Immunogenetics 21, 235 (1985). 58 H.-G. Rammensee, T. Friede, and S. Stevanovic, Immunogenetics 41, 178 (1995). 55

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Screen PS-SCL

A: Identification of epitope mimics that do not necessarily correspond to natural epitopes

Few mixtures are clearly active

Synthesize all combinations of amino acids defining the most active mixtures

Many mixtures show similar activity

Generate and screen a biased library with reduced complexity based on screening results

B: Biometrical analysis: Identification of natural epitopes in protein databases

Generate a matrix based on the activity of each mixture of the PS-SCL

Score and rank all peptides from proteins contained in database(s)

Synthesize top ranked peptides

Test individual peptides for T cell stimulatory activity

Fig. 4. Flowchart of the successive steps involved in the optimization and identification of T cell epitopes using (A) PS-SCLs and (B) PS-SCL-based biometrical analysis.

were not recognized by any of these clones. The disparity in recognition between native pp65495–503 and these peptides may result from the peptides’ inability of interact with many different TCRs, which might be due to the high amino acid substitutions relative to the native sequence. These results therefore suggest that the native sequence is recognized by a diverse group of TCRs, and that its moderate immunogenicity is likely a compromise to maintain broad recognition and protective immunity against CMV. In contrast, the two epitope mimics identified from the library are highly antigenic but recognized by a highly restricted number of TCRs, thus at the expense of losing universal recognition. To maintain ‘‘broad recognition’’ while preserving improved activity, a series of p65405–503 peptide analogs were designed by substituting native amino acids with library-directed changes. Using this approach three peptides were identified that were 100-fold more active than the native ligand and, at the same time, conserved ‘‘broad recognition.’’ Furthermore, all three analogs expanded peripheral blood CTLs from CMV-seropositive individuals, which not only recognized peptide-coated targets, but were able to lyse CMV-infected targets. The extent of CD8þ T cell activation can alternatively be assessed by quantitative evaluation of cytokine (e.g., -interferon) production and release measured by standard sandwich ELISA. The assay to be used for evaluating the extent of T cell activation needs to be selected and

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optimized for each T cell clone and T cell line prior to testing a PS-SCL. It is indeed essential to optimize the T cell assay conditions in order to achieve the best level of sensitivity and reproducibility and to successfully select the most active mixtures. The basic protocols as well as optimization strategies have been described in detail.59 If a limited number of mixtures show significant activity in each sublibrary, then a series of individual peptides representing all combinations of amino acids defining those mixtures are generated as described in the examples above. Alternatively, in those cases in which too many mixtures show similar high activity in several sublibraries, and in turn, the number of individual peptides to be synthesized is too high, a biased PS-SCL is generated by fixing the positions where amino acids having high specificity could be identified following the original library screening, and including a limited number of amino acids at the other positions selected based on the results of the primary screening. This strategy was successfully applied for the identification of peptide ligands specific for a diabetogenic T cell clone.60 Identification of Natural Epitopes in Protein Databases With the inclusion of computational methods in T cell immunology, a number of strategies have emerged that use computer algorithms to predict which peptide fragments from an untested protein will fit into an MHC molecule and trigger an immunological response.61 Although there is an increasing number of different computer algorithms, they all are based on the available databases for the MHC anchors, together with the assumption of an independent contribution of each amino acid in the peptide sequence to MHC binding.62 The recent development of a large database of the sequences of natural epitopes eluted from both MHC class I and class II molecules has provided a new basis for computer models to predict epitopes.63 PS-SCL-based biometrical analysis is a novel strategy that uses the power of computers and the information available in the databases to systematically compare the data derived from a PS-SCL screening with all peptide fragments present in protein databases.64 Unlike other strategies using 59

C. Pinilla, J. R. Appel, and R. A. Houghten, in ‘‘Current Protocols in Immunology’’ (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds.), p. 9.5.1. John Wiley & Sons, New York, 2001. 60 V. Judkowski, C. Pinilla, K. Schroder, L. Tucker, N. Sarvetnick, and D. B. Wilson, J. Immunol. 166, 908 (2001). 61 M. Hagmann, Science 290, 80 (2000). 62 T. Sturniolo, E. Bono, J. Ding, L. Raddrizzani, O. Tuereci, U. Sahin, M. Braxenthaler, F. Gallazzi, M. P. Protti, F. Sinigaglia, and J. Hammer, Nat. Biotechnol. 17, 555 (1999). 63 V. Brusic, J. Zeleznikow, and N. Petrovsky, J. Immunol. Methods 238, 17 (2000). 64 Y. Zhao, B. Gran, C. Pinilla, S. Markovic-Plese, B. Hemmer, A. Tzou, L. W. Whitney, W. E. Biddison, R. Martin, and R. Simon, J. Immunol. 167, 2130 (2001).

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mathematical algorithms, the biometrical analysis is not based on the described MHC motifs nor is any previous knowledge of the restriction element for a given T cell clone required. The different steps of the biometrical analyses are described in Fig. 4, pathway B. The first example of the PS-SCL-based biometrical approach was the study of the specificity of GP5F11,64 a CD4þ T cell clone that was established from peripheral blood of a patient with multiple sclerosis using influenza virus hemagglutinin (HA) peptide 309–318 as an antigen. The biometrical analysis based on the screening of a decapeptide PS-SCL predicted the native peptide (HA 309–318) as the sixth peptide out of over 20 million decapeptides analyzed having the highest stimulatory index for this clone, and two analogs of the same HA sequence ranked third and eighth. Synthesis of these peptides confirmed their activity for GP5F11. Similarly, the native ligand was predicted among peptides having high stimulatory activity from the biometrical analysis based on the screening of a nonapeptide PS-SCL using a CD8þ T cell clone that recognized HIV-1 Gag p17-derived epitope SL9.65 Thus, the SL9 epitope was ranked 116 out of over 22 million nonapeptides with high predicted stimulatory activity and the nonapeptides that ranked first and second were naturally occurring double substitutions of this epitope. Other examples with T cell clones of known specificity have been reported for a Melan-A (a melanoma associated antigen)-specific CD8þ T cell clone, with a well known epitope (Melan-A 26–35)51 and for a tyrosinase (another melanoma-associated antigen)-specific CD8þ T cell clone, with a known epitope (tyrosinase peptide 368–376).53 Melan-A 26–35 was predicted as the fourth decapeptide with high stimulatory activity, while tyrosinase peptide 368–376 ranked tenth following the biometrical analyses based on the screening of a nonapeptide PS-SCL and twenty-ninth when the analysis was based on the screening of a decapeptide PS-SCL. These examples using T cell clones of known specificity clearly demonstrate the strength of the PS-SCL-based biometrical analysis approach for the identification and optimization of native ligands from T cell clones of both known and unknown specificity. This is further supported in the following example in which the clone specificity was unknown. Thus, the PS-SCL-based biometrical analysis was applied for the identification of T cell ligands for CSF-3, a CD4þ clone of unknown specificity that was isolated from the cerebrospinal fluid of a patient with chronic Lyme disease of the central nervous system.47 Each mixture of a decapeptide PS-SCL 65

C. Boggiano, C. Pinilla, B. D. Walker, and S. E. Blondelle, in ‘‘Peptides: The Wave of the Future. Proceedings of the 2nd International/17th American Peptide Symposium’’ (R. A. Houghten and M. Lebl, eds.), p. 1023. Kluwer Academic Publishers, Norwell and Dordrecht, 2001.

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was assayed for activation of CSF-3 in a standard proliferation assay that measure the incorporation of [3H]thymidine into newly synthesized DNA (using irradiated MHC matching PBMCs as APCs). A matrix was then generated based on the screening results. The entry for a given amino acid in a specific position was based on the stimulation index obtained for the mixture from the PS-SCL corresponding to that amino acid defined in that position. The biometrical analysis then used this matrix to score all decapeptides in the entire Borrelia burgdorferi (the agent causing Lyme disease) genome as well as in databases compiling all known human or viral proteins by moving a scoring window across the known protein sequences in increments of one amino acid and adding the individual stimulatory values relevant to each amino acid and its position of each decamer sequence. This way, peptides present in databases can be identified in a complete unbiased fashion and with unprecedented efficiency, and ranked according to a score that is predictive of their stimulatory potency. Each peptide was then ranked based on their predicted stimulatory score. The percentage of sequences with high scores was substantially greater in the B. burgdorferi genome than in the human and viral databases, suggesting a higher probability of the T cell clone recognizing B. burgdorferi antigens. The best ranking sequences derived from B. burgdorferi, human, or viral proteins were synthesized and tested to confirm their stimulatory capacity. As anticipated, the most active peptides derived from B. burgdorferi. The primary amino acid sequences of these peptides differed substantially, indicating that little or no sequence homology was required for cross-recognition. These studies demonstrate the effectiveness of the PS-SCL-based biometrical approach for the identification of specific target epitopes for an organ-infiltrating T cell population that had been stimulated with a crude lysate from an infectious organism. Conclusions

One of the fundamental objectives in organic and medicinal chemistry is the design, synthesis, and production of molecules having value as human therapeutic agents. The successes of organic and medicinal chemistry combined with the rapid expansion of combinatorial chemistry have fundamentally changed human therapeutics over the past 30–40 years. It can be expected from the current successes that the ability to synthesize and densely search the molecular space of therapeutically important receptors will permit highly active analogs of existing pharmacophores to be identified. Furthermore, the use of combinatorial variance of synthetic reaction conditions will allow the identification of entirely new synthetic approaches to novel pharmacophores. Combinatorial chemistry and synthetic approaches can then be expected to rapidly lead to more active, more specific,

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safer, and less expensive therapeutics. Mixture-based SCL approaches as described may be considered an ‘‘optimized approach’’ of the identification of novel pharmacophores from natural sources. Indeed, both SCLs and natural product extracts are composed of mixtures of compounds that are directly screened without intensive initial purification. In both cases, a large number of active individual compounds were identified from such mixtures. Although not thoroughly studied in the area of infectious disease, the reported successes over the past 12 years of combinatorial library approaches show the ability to synthesize and densely search the molecular space against therapeutically important targets such as drug-resistant microorganisms and HIV, permitting highly active antimicrobial, antifungal, and antiviral compounds to be identified. Compounds such as those described here can serve as starting points for the development of therapeutic agents that inhibit or treat bacterial, fungal, or viral infection. In particular, the identified small molecules have the desired physicochemical properties for oral therapeutic application. Thus, they are small in size (molecular weight less than 1000 Da) and are unlikely to be subject to proteolysis. The efficiency of PS-SCLs combined with biometrical analyses to identify highly active B cell and T cell ligands has now been clearly demonstrated. These studies have also led to the identification of immunologically relevant peptides for use as potential preventive and therapeutic vaccines, provided insight into the requirements for TCR interactions with peptide/MHC complexes in immunogenicity, and established new principles regarding the level of cross-reactivity in immunological recognition. As shown in the examples described above, the use of PS-SCLs in studies of T cell specificity can be directed toward the identification of two types of T cell ligands: (1) those that do not necessarily correspond to sequences in reported proteins (epitope mimics), and (2) when combined with biometrical analyses, those corresponding to proteins present in a database (optimal native epitopes). Such strategies are then expected to greatly increase the potential to develop sequences with multiple clonal recognition and cross-reactivity ability and to be of great value for the development of vaccines against infectious diseases. Acknowledgments The authors thank Dr. Houghten, Dr. Nefzi, and John Ostresh at Torrey Pines Institute for Molecular Studies for their scientific discussion and Marc Giulianotti, Ed Brehm, and Ema Crooks for their technical assistance. The authors also thank Dr. Berger at the National Institute of Allergy and Infectious Diseases for providing the vaccinia virus constructs, and Dr. Mosier at the Scripps Research Institute for providing the HIV-1 isolates. This research was supported in part by Universitywide AIDS Research Program Grant R-98-TPI-064, National Institute of Dental and Craniofacial Research Grant RO1-DE12923, and Mixture Sciences, Inc., San Diego, California.

Section VII Polymer-Assisted Approaches

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[19]

applications of polymer-supported reagents

347

[19] Advances in the Applications of Polymer-Supported Reagents for Organic Synthesis By Alessandra Bartolozzi, Michael J. Grogan, and Steven A. Kates Introduction

In the last half of the twentieth century organic chemistry evolved to a level of extreme sophistication in which complex molecules could be prepared from basic building blocks. The process typically involves performing a reaction in an organic solvent followed by isolation of a desired product from excess reagents and reaction by-products. Subject to chemical properties unique to the individual components, these reactions and purifications are tedious and time consuming, and require considerable expertise. Bruce Merrifield was the first to apply an alternative approach for the iterative synthesis of polypeptides, for which he was awarded the Nobel Prize in 1984.1 By covalently anchoring the starting amino acid to a solid support, he was able to perform synthetic steps with known solution-phase chemistry, and purification of bound intermediates could be effected with quick solvent washes. Following chain elongation, the bound polypeptide was cleaved from the solid support with concomitant release of the sidechain-protecting groups. The physical differences between compounds that are so pronounced for solution-phase synthesis are leveled for solid-phase synthesis by the common denominator for each reaction, the solid support. Key advantages to the solid-phase technique include simple filtration and washing without manipulative losses as well as lending itself easily to automation. Subsequent to Merrifield’s discovery, other repetitive processes such as oligonucleotide synthesis2 and protein sequencing3 have been adapted to automated solid-phase methods. Beginning in the early 1990s, solid-phase techniques were combined with modern synthetic methods to construct small molecules for drug discovery. With a starting material on solid support, sets of reactants are coupled in a combinatorial fashion. Various strategies of solid-phase organic synthesis, with or without automation, are used to prepare libraries of low-molecular-weight structures. Common to all approaches is the simple ability to wash the solid support in order to add reagents or remove

1

R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963). R. L. Letsinger and M. J. Kornet, J. Am. Chem. Soc. 85, 3045 (1963). 3 R. A. Laursen, Eur. J. Chem. 20, 89 (1971). 2

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

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impurities, enabling the rapid execution of parallel reactions to generate sets of compounds. As an alternative to building compound libraries on resin, solid-supported synthetic reagents have been developed. This has the advantage that two additional steps, compound attachment and cleavage associated with solid-phase synthesis, are not required, and yet the key advantage of purification by filtration remains possible. Supported reagents are therefore particularly useful for quick reaction execution and compound optimization necessary for drug discovery. Functionalized polymers have also been used as scavengers to purify solution-phase reactions, whereby one reactant may be used in large excess and the polymer is added to capture either the product or impurities. Supported scavengers and reagents for a wide range of synthetic conversions have been developed and are becoming commercially available. Compared to the linkers incorporated in solid-phase synthesis, these reagents are more generally accessible. This chapter discusses new developments in polymer-supported reagents for 2001–2002, with reagents grouped by different classes of chemical transformations. The features and characteristics of the common solid supports, polystyrene (PS),* polyethylene glycol (PEG), and silica, have been extensively reviewed and will not be discussed in this work.4 For other discussions of solid-supported reagents and solid-phase synthesis, a number of excellent reviews have been written.5,6

Abbreviations: AIBN, 2,20 -azobisisobutyronitrile; Alk, alkyl; Ar, aryl; Bn, benzyl; Boc, tertbutyloxycarbonyl; t-But, tert-butyl; Cbz, benzyloxycarbonyl; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DMAP, 4-dimethylaminopyridine; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; Fmoc, 9-fluorenylmethoxycarbonyl; GC, gas chromatography; Hal, halogen; HOSu, N-hydroxysuccinimide; Me, methyl; PEG, polyethylene glycol; PS, polystyrene; PS-TEMPO, 4-(polystyrylmethyloxy)-2,2,6,6-tetramethylpiperidin-1-yloxy; RCM, ring-closing metathesis; ROMP, ring-opening metathesis polymerization; RP-HPLC, reverse-phase high-performance liquid chromatography; Sar, sarcosine; Suc, succinoyl; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin-layer chromatography; UV, ultraviolet. 4 S. A. Kates and F. Albericio, Eds., ‘‘Solid-Phase Synthesis: A Practical Guide.’’ Marcel Dekker, New York, 2000. 5 For previous reviews, see (a) S. V. Ley, I. R. Baxendale, R. N. Bream, P. S. Jackson, A. G. Leach, D. A. Longbottom, M. Nesi, J. S. Scott, R. L. Storer, and S. J. Taylor, J. Chem. Soc. Perkin Trans I 3815 (2000). (b) A. Kirschning, H. Monenschein, and R. Wittenberg, Angew. Chem. Int. Ed. Engl. 40, 650 (2001). (c) Y. R. de Miguel, E. Brule, and R. G. Margue, J. Chem. Soc. Perkin Trans I 3085 (2001). (d) J. Eames and M. Watkinson, Eur. J. Org. Chem. 1213 (2001). (e) B. Clapham, T. S. Reger, and K. D. Janda, Tetrahedron 57, 4637 (2001). 6 (a) C. Blackburn, F. Albericio, and S. A. Kates, Recent Res. Dev. Org. Chem. 1, 477 (1997). (b) C. Blackburn, F. Albericio, and S. A. Kates, Drugs Future 22, 1007 (1997). (c) S. A. Kates, Biopolymers 47, 309 (1998). *

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applications of polymer-supported reagents

349

Acids and Bases

Polymer-supported acids and bases have been used extensively as ionexchange resins for purifications and reagents for synthetic transformations. Their handling properties guarantee easy manipulations allowing rapid reaction optimization and parallel synthesis development. Using various polystyrene-amine resins in three different synthetic steps, YadavBhatnagar et al.7 performed the parallel synthesis of cysteine trap protease inhibitors. A set of benzyloxycarbonyl (Cbz)-protected amino acids is converted with diazomethane to -diazoketones by activation with a polystyrene-supported amine (Table I, entry 1). The -diazoketones are converted to the corresponding -bromoketones by treatment with polystyrene-based ammonium bromide (entry 2). Nucleophilic displacements of these bromides by carboxylic acids, thiols, and amines are promoted with polystyrene-supported dimethylamine affording a library of 80 highly pure peptide-based compounds (entry 3). Kirschning et al.8 succeeded in promoting glycosylations of alcohols with a supported silylsulfonate as a Lewis acid (entry 4). Treatment of a glycosyl acetate with the polymer-bound silyl triflate efficiently transfers the carbohydrate to a range of alcohols, affording the corresponding 2-iodo--glycosides in high yields. Subsequent reductions provide 2-deoxy sugars. Dondoni and Massi9 executed a Bignelli synthesis to prepare dihydropyrimidinones starting from aldehydes, 1,3-dicarbonyl compounds, and urea with ytterbium(III) supported on Amberlyst 15 resin as a Lewis acid (entry 5). After condensation, the reaction mixture is treated with both strongly basic and acid resins to sequester reaction by-products. For more than 30 reported examples, dihydropyrimidones obtained directly from concentration of the filtrates are produced in high yields and in excellent purities. To recycle a valuable amine acylation catalyst, Janda and co-workers10 attached a proline-based catalyst to a polymeric support for the enantioselective kinetic resolution of alcohols (entry 6). The resin-bound catalyst behaves similarly to the soluble catalyst, providing good yields of secondary alcohols and their corresponding esters with good to excellent enantioselectivities for various substrates.

7

N. Yadav-Bhatnagar, N. Desjonque`res, and J. Mauger, J. Comb. Chem. 4, 49 (2002). A. Kirschning, M. Jesberger, and A. Scho¨nberger, Org. Lett. 3, 3623 (2001). 9 A. Dondoni and A. Massi, Tetrahedron Lett. 42, 7975 (2001). 10 B. Clapham, C.-W. Cho, and K. D. Janda, J. Org. Chem. 66, 868 (2001). 8

350

[19]

polymer-assisted approaches TABLE I Acids and Bases

Entry number

Supported reagent

Transformation R Cbz

1

Reference

O OH

N H

R

1)

Cbz

O Cl PS-amine, DMF

O

2) CH2N2, CH2Cl2

Cbz

7

N2

O

R = Bn, Alk, Indolyl

R

2

N H

R CH2Cl2

N H

N2

Cbz

N H

PS-HBr

O

7

Br O

R Cbz

N H

R

3

Cbz

RCOOH or RSH N H

Cbz

Br

N H

or R1R2NH

O

OCOR O R

7

SR O R

Cbz

I

4

BzO TBSO

N H

NR1R2 O

I

O

BzO TBSO

R OH OAc

O

8

OR

CH2Cl2 or Et2O

R = Alk, Bn, sugar, steroid

O O

5 R1

6

R2

R3

Toluene

+

+ R2

OH R1

O

O

O

H

H2N

NH2

OBz

BzCl, NEt3

R2

NH R1

N H

9

O

OH +

R1

R3

R2

R1

R2

10

R1, R2 = Alk, Bn

Synthesis of Dihydropyrimidones (Entry 5) A screw-capped vial containing a magnetic stir bar was charged with Yb(III) resin (160 mg), urea (1.50 mmol), aldehyde (0.50 mmol), and  dicarbonyl (0.50 mmol) and heated at 120 for 5 min. Yb(III) resin  (170 mg) was added and the mixture was heated at 120 for 48 h with  gentle stirring. After cooling to 60 , methanol (1 ml) was added. The suspension was stirred for an additional 30 min then the resin was filtered and washed thoroughly with ethyl acetate. Amberlyst 15 (400 mg) and

[19]

applications of polymer-supported reagents

351

TABLE II Oxidations–Reductions Entry number

Supported reagent

Transformation OH

7

R

Reference O

KBr, NaOCl

R1

R

CH2Cl2

R1

11

R1 = H, Alk

KBr, NaOCl

8

R

OH

CH3CN

O R

12

OH R = Alk, Bn

OH

O

9

10

H

R

R1

CH2Cl2

RhCl(PPh3)3

H

R

13

R1

DMSO R = Et, Ph

14

R1 = COOH, COOMe, CN, COMe, CHO

Ambersep 900 OH (400 mg) were added to the combined filtrates. The suspension was shaken for 2 h and then the resins were filtered and washed thoroughly with methanol. The combined filtrates were concentrated to give dihydropyrimidinones. Oxidations and Reductions

The Anelli oxidation of alcohols to aldehydes and ketones has been accomplished using polymer-supported nitroxyl radical catalysts. The practicality of removing polymer-supported reagents by filtration to simplify product purification is highlighted by these examples. Bolm and coworkers11 demonstrated that a silica-supported nitroxyl catalyst is easily filtrated after use from the reaction solution, recovered and recycled, and the residual inorganic salts present in the reaction mixture are separated from the organic product by aqueous extraction (Table II, entry 7). 11

T. Fey, H. Fischer, S. Bachmann, K. Albert, and C. Bolm, J. Org. Chem. 66, 8154 (2001).

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polymer-assisted approaches

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As an extension to this synthetic method, Yasuda and Ley12 performed the Anelli oxidation with a polystyrene-supported nitroxyl radical and a polymer-supported chlorite (entry 8). The latter is incorporated in the reaction to oxidize the intermediate aldehydes to carboxylic acids. The choice of reagents and reaction design affords pure carboxylic acids in high yields. Various polymer-supported hydrides have been applied successfully to reductions of both carbonyl and olefin groups. Rajasree and Devaky13 describe a cross-linked polystyrene-supported ethylenediamine borane reagent for the selective reduction of aldehydes in the presence of ketones (entry 9). This borane reagent is easily prepared and can be recycled after completion of the reaction. This is a practical alternative to standard borane reagents such as diborane, borane-amine, or borane-sulfide complexes. Desai and Danks14 reported the use of a polymer-supported formate for transfer hydrogenation to alkenes with Wilkinson’s rhodium catalyst (entry 10). A range of unsaturated carbonyl compounds is reduced to the corresponding alcohols by the formate resin under microwave irradiation. Reactions occur rapidly and afford the desired product in high yields. Typical reagents for solution-phase transfer hydrogenations such as ammonium formate salts have the propensity to sublime and be hygroscopic. The polymer-supported formate circumvents these drawbacks providing greater reproducibility, particularly when optimizing and scaling reaction conditions. Synthesis of Carboxylic Acids (Entry 8) Prior to use, 4-(polystyrylmethyloxy)-2,2,6,6-tetramethylpiperidin1-yloxy (PS-TEMPO) (18.8 mg, 0.03 mmol) was washed with acetonitrile (2  2 ml). A solution of NaOCl (8.5 mg, 0.01 mmol) in water (0.5 ml) was added to a suspension of washed PS-TEMPO, PS-chlorite (0.70 g, 1.50 mmol), PS-dihydrogen phosphate (154 mg, 0.30 mmol), and KBr (3.6 mg, 0.03 mmol) in acetonitrile (1 ml). After 10 min at room temperature, a solution of the alcohol (0.30 mmol) in acetonitrile (1 ml) was added. The mixture was stirred at room temperature for 24–48 h, then passed through a short plug of Celite (1 g). After removal of the solvent under reduced pressure, the residue was dissolved in acetonitrile (0.5 ml) and passed through a short plug of silica (0.3 g) eluting with 25% methanol in chloroform (10 ml). The filtrate was concentrated to give the desired carboxylic acid derivative. 12

K. Yasuda and S. V. Ley, J. Chem. Soc. Perkin Trans. I 1024 (2002). K. Rajasree and K. S. Devaky, J. Appl. Polym. Sci. 82, 593 (2001). 14 B. Desai and T. N. Danks, Tetrahedron Lett. 42, 5963 (2001). 13

[19]

applications of polymer-supported reagents

353

Hydrogenation of Alkenes (Entry 10) E-3-Phenylpropenoic acid (0.025 g, 0.16 mmol), Amberlite IRA 938supported formate (0.2 g) and Wilkinson’s catalyst (0.04 g, 0.0043 mmol) were suspended in dimethylsulfoxide (0.5 ml) in a reaction tube. The mixture was irradiated with microwaves at 100 W for 30 s. On cooling, the mixture was diluted with dichloromethane (5 ml) and filtered. The filtrate was washed with water (2  5 ml). The organic layer was dried over anhydrous magnesium sulfate, the solvent removed under reduced pressure, and the residue was purified by filtration through a plug of alumina to give 3-phenylpropanoic acid (0.024 g, 95% yield). Sulfur and Phosphorous Transfer Reagents

New polymer-supported reagents for sulfide transfer have been developed to avoid exposure to malodorous and toxic sulfur reagents. Ley et al.15 prepared a stable aminothiophosphate polystyrene resin for the conversion of secondary and tertiary amides to thioamides in high conversion and purity (Table III, entry 11). This procedure is extremely clean and affords the desired product with short reaction times in comparison to Lawesson’s reagent. In addition, the aminothiophosphate resin dehydrates primary amides to nitriles. A supported sulfide source has been demonstrated by Zhang et al.16 to synthesize oligonucleotide phosphorothioates (entry 12). Aminodithiazolethione attached to a methacrylate-ethyleneglycol copolymer is used to efficiently convert a nucleotide phosphite to a phosphorothioate. The product nucleotide possesses protecting groups suitable for solid-phase oligonucleotide synthesis and thus is a valuable building block for nucleic acid therapeutics. Ley and Taylor17 reported a polymer-supported oxazaphospholidine to convert isothiocyanides to isonitriles for subsequent application in Ugi three-component coupling reactions (entry 13). This method affords clean isonitriles and facilitates the handling of a toxic and unstable reactant. Glycosyl phosphates are important building blocks in carbohydrate synthesis. Hindsgaul and co-workers18 applied a capture-phosphorylation method with a polystyrene-supported phosphoramidite to simplify their synthesis (entry 14). Unreacted alcohols are removed by washing the resin and the desired glcyophosphates are released in high purity by a standard 15

S. V. Ley, A. G. Leach, and R. I. Storer, J. Chem. Soc. Perkin Trans I 358 (2001). Z. Zhang, Y. Han, J. X. Tang, and J.-Y. Tang, Tetrahedron Lett. 43, 4347 (2002). 17 S. V. Ley and S. J. Taylor, Bioorg. Med. Chem. Lett. 12, 1813 (2002). 18 K. Parang, E. J.-L. Fournier, and O. Hindsgaul, Org. Lett. 3, 307 (2001). 16

354

[19]

polymer-assisted approaches TABLE III Sulfur -Phosphorous Transfer Reagents

Entry number

Supported reagent

Transformation Ionic solvent:

O

11

Me N R

N

Reference S

Me PF6

NMe2 200⬚ C, microwave

R

NMe2

15

R = Alk, Ar

S

12

R1O

P OR2 OR3

13

R N C S

CH2Cl2

R1O

Toluene Microwave

16

P OR2 OR3

1) THF, 1H -tetrazole, PS-phosphoamidite 2) t -BuOOH, THF

14

17

R N C

−

O

O P O R

R OH 3) DBU, THF 4) NaOMe, MeOH, dioxane

O−

18

R = sugar, nucleoside

three-step procedure. This method avoids using excess phosphoramidites and reduces polyphosphorylation of diols and thus is a flexible tool for the synthesis of carbohydrate and nucleotide monophosphates. Carbon Transfers

To provide a source of allyl nucleophile for additions to aldehydes, Barrett and co-workers19 prepared an allylboronate nucleophile attached to a norbornene diol gel generated by ring-opening metathesis polymerization (ROMP) (Table IV, entry 15). This ROMP gel reagent has physical properties more desirable than the commonly used low-weight boranes and boronic acid reagents. The homoallylic alcohols are produced in high yield starting from a wide range of aldehydes. To avoid the introduction of soluble phosphines into a reaction mixture, Westman20 performed a one-pot phosphorous ylide formation and Wittig 19 20

T. Arnauld, A. G. M. Barrett, and R. Seifried, Tetrahedron Lett. 42, 7899 (2001). J. Westman, Org. Lett. 3, 3745 (2001).

[19]

355

applications of polymer-supported reagents TABLE IV Carbon Nucleophiles

Entry number

Supported reagent

Transformation

O

n

15

O

O

OH H

R1

Reference

R

R1

B

19 R1 = Alk, Ar

R = H, Me

R

O

16

R1

H

+

Br

R2

K2CO3 MeOH

R1

R2

20 R1 = Ar, Heterocycle R2 = COOMe, Ph, CN, COPh

17

R1

O O S OH

O

R1

O O R S O

O

21

R = Alk, Bn R1 = Alk, Ar, Bn

reaction on solid support (entry 16). Microwave irradiation of various aldehydes and alkyl halides in the presence of a polymer-supported triphenylphosphine produced the desired olefins in high purity with reaction times <5 min. To prepare alkyl sulfonate esters, sulfonic acids were treated with supported alkyl triazenes (entry 17).21 The acids decompose the triazenes to diazoalkanes, which subsequently alkylate the sulfonates in situ. These supported triazenes avoid the dangerous handling of pure diazoalkanes. Synthesis of Olefins via Wittig Reaction (Entry 17) An aldehyde was mixed with solid supported triphenylphosphine oxide (3 equiv.), alkyl halide (4 equiv.), and potassium carbonate (4 equiv.) in  methanol (2 ml). The mixture was heated at 150 for 5 min. The residue was filtered through a short plug of silica gel and washed. The solution was concentrated and purified by reverse-phase high-performance liquid chromatography (RP-HPLC).

21

N. Vignola, S. Dahmen, D. Enders, and S. Bra¨se, Tetrahedron Lett. 42, 7833 (2001).

356

[19]

polymer-assisted approaches

Electrophilic Reagents and Radical Reaction Reagents

Polystyrene-bound selenium bromide has made organic transformations involving electrophilic selenium readily accessible.22 Compared to solution phase, the supported reagent simplifies the purification step reducing the exposure to toxic materials. Huang and Sheng23 have used this versatile reagent to capture phosphorous carbon ylides on the support (Table V, entry 18). These selenoalkylidenes react with aldehydes to form vinylic selenides that, upon acidic hydrolysis, are cleaved to release aldehydes or ketones from the resin. With a polymer-bound chiral selenium bromide reagent, the stereoselective activation of olefins has been accomplished (entry 19).24 Uehlin and Wirth24 found that both mesoporous silica and polystyrene-supported

TABLE V Electrophilic and Radical Reactions Entry number

Supported reagent

Transformation O R2

18

Reference

1) Supported ylide

R1

R2

H

2) H+

23

O

R2 = Alk, Ar

R

Ph

1) PS-SeBr R1

19

Ph

OH

R OR1

2) Bu3SnH

24

R = H, Alk, OAlk R1 = Alk, Bn

SnMe2 H

20

Activated carbon

R

1) Heat

I +

R H

25

2) Activated carbon R = Alk, Ar

21

22

26

K. C. Nicolaou, J. Pastor, S. Barluenga, and N. Winssinger, Chem. Commun. 1947 (1998). X. Huang and S.-R. Sheng, Tetrahedron Lett. 42, 9035 (2001). 24 L. Uehlin and T. Wirth, Org. Lett. 3, 2931 (2001). 23

[19]

applications of polymer-supported reagents

357

reagents gave clean products with moderate to good enantiopurities after reductive, nucleophilic, or oxidative cleavage of the organoselenium intermediates. For all these examples, the recovered selenium bromide reagent can be regenerated for repeated use. Trialkyltin hydrides are common reagents in organic radical chemistry, but the toxic by-products are extremely difficult to remove from reaction products. To facilitate their removal, a pyrene-functionalized tin hydride has been prepared (entry 20).25 After standard solution-phase radical reactions with the stannane, filtration through activated carbon traps the tin species to afford pure products. As a general platform for converting carboxylic acids or alcohols into reactive radicals, a supported N-hydroxythiazole-thione has been developed (entry 21).26 The intermediate heterocyclic carboxylate esters are transformed into pure alkyl bromides by photolysis in the presence of BrCCl3. In a second application, the support contained an alkyl ether as opposed to a hydroxyl group. Cleavage under similar conditions provided a furan by the cyclization of a released oxygen radical. The heterocycle necessary for this radical process remains attached to the Wang resin, presenting an alternative to commonly used thiohydroxamates.27 Reaction of Pyrene-Functionalized Tin Hydride with an Alkyl Halide (Entry 20) A solution of substrate (0.20 mmol), pyrene-dimethyltinhydride (1.2 equiv.), and 2,20 -azobisisobutyronitrile (AIBN, 0.1 equiv.) in dry degassed benzene (1–2 ml) was stirred under reflux for 1 h [thin-layer chromatography (TLC) monitoring] under inert atmosphere. The mixture was cooled to room temperature and evaporated. Methanol/dichloromethane (3:2, 4 ml) was added followed by the addition of activated carbon (800 mg). The suspension was stirred for 30 min [the adsorption of the pyrene core was monitored by ultraviolet (UV)]. After filtration, the activated carbon was washed with methanol and the combined filtrates were concentrated to yield the product in pure form. Supported Transition Metal Catalysts

Organometallic complex catalysts have been removed from reaction mixtures with solid-supported phosphines as well as other ligands to facilitate reagent handling and product purification. Alternatively, Leadbeater28 25

S. Gastaldi and D. Stien, Tetrahedron Lett. 43, 4309 (2002). L. De Luca, G. Giacomelli, G. Porcu, and M. Taddei, Org. Lett. 3, 855 (2001). 27 D. H. R. Barton, D. Crich, and W. B. Motherwell, Tetrahedron 41, 3901 (1985). 28 N. E. Leadbeater, J. Org. Chem. 66, 2168 (2001). 26

358

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polymer-assisted approaches

used polystyrene-supported triphenylphosphine as a ligand for an oxidation ruthenium(II) complex (Table VI, entry 22). The utility of this complex for the hydrogenation of ketones to alcohols as well as for the oxidation of methylenes or alcohols to ketones is demonstrated. For the examples described, the yields of the reactions are similar or higher than those reported for the soluble ruthenium complex. TABLE VI Transition Metal Catalysts Entry number

Supported reagent

Transformation

Reference O

1,2-dichloroethane R

22

R1

R

Peracetic acid

R1

28

R,R1 = Alk, Ar EtOOC

EtOOC

COOEt

COOEt

CH2Cl2

23

29

R

R Ar

X + HN R1

24

Pd(OAc)2

25

N R1

30

R, R1 = H, Ar, Alk

X = Hal

Ar

Ar

X + Ar1

OH B Pd2(dba)3 OH

Ar

Ar1

30

X = Hal

I SPh

50

26

O O

DMF, NEt3

+

R O

R

T = 1208C

31

Pd Cl

R = COOMe, Ph, COO-t-Bu, CN

11

SPh

COOBu

X O

27

OBu

Pd(OAc)2 Bu4NOAc scCO2

+

32

R X = Br, I

R

[19]

applications of polymer-supported reagents

359

A variant of the widely used Grubbs ruthenium alkylidene catalyst for the ring-closing metathesis (RCM) and ROMP of olefins was constructed on a polystyrene resin by Mol and co-workers29 (entry 23). The phosphine ligands in the ruthenium complex are potential points of attachment to solid phase; however, they must disassociate for olefin metathesis to occur. Thus, a supported carboxylate ligand is chosen to replace one of the anionic chloride ligands of the Grubbs complex to anchor to the resin. This strategy ensures catalyst activity without loss of metal complexes in solution phase. This supported catalyst is highly effective for both RCM and self-methathesis of olefins, and can be regenerated and stored without significant loss of activity. Parrish and Buchwald30 performed couplings with a polystyrenesupported biphenyl-phosphine palladium complex between aryl halides and either amines (entry 24) or boronic acids (entry 25). The resin-bound complex is analogous to the corresponding homogeneous compound and is effective for couplings to unactivated aryl halides, including aryl chlorides. The complex is air-stable and retains activity after recovery without apparent loss of palladium. Pollino and Weck31 executed Heck olefinations with a palladium complex containing an unusual sulfur–carbon–sulfur trivalent ligand supported on a ROMP polymer backbone (entry 26). This is a very efficient catalyst and can be recycled with good retention of activity. The controlled spacing of the metal complexes along the ROMP polymer backbone suggests the potential of supported complexes with designed intermetal distances. Gordon and Holmes32 used a supported triphenylphosphine–Pd(II) complex as an effective catalyst for Heck and Suzuki couplings in supercritical carbon dioxide (entry 27). After optimization of the amine base for the reaction, the final products have been isolated in good yields and high purity with no traces of metal. Synthesis of Arylamines (Entry 24) A solution of Pd(OAc)2 in toluene (1.00 ml of a 7.50 mM solution, 7.50 mol) was added to an oven-dried test tube (16  100 mm) containing dialkylarylphosphine resin (9.8 mol) and sealed with a septum. The mix ture was stirred at 2 for 30–75 min. Sodium tert-butoxide (108 mg, 29

P. Nieczypor, W. Buchowicz, W. J. N. Meester, F. P. J. T. Rutjes, and J. C. Mol, Tetrahedron Lett. 42, 7103 (2001). 30 C. A. Parrish and S. L. Buchwald, J. Org. Chem. 66, 3820 (2001). 31 J. M. Pollino and M. Weck, Org. Lett. 4, 753 (2002). 32 R. S. Gordon and A. B. Holmes, Chem. Commun. 640 (2002).

360

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[19]

1.12 mmol) was added to the flask, and the system was purged with Argon. An aryl halide (0.75 mmol), an amine (0.975 mmol), and toluene (0.50 ml)  were added sequentially via syringe. The flask was placed into an 80 oil bath, and the reaction mixture was stirred for 15–20 h. After cooling, the mixture was diluted with ether (5 ml), filtered through Celite, and rinsed with ether (2  5 ml). The combined organic layers were diluted with ether (50 ml) and washed with a saturated aqueous bicarbonate solution (3  50 ml). The organic phase was dried over anhydrous sodium sulfate and removal of the solvents under reduced pressure provided pure final products. Synthesis of Biphenyls Via Suzuki Coupling (Entry 25) Tetrahydrofuran (1.00 ml) was added to an oven-dried test tube (16  100 mm) sealed with a septum containing dialkylarylphosphine resin (5.2 mol) and Pd2(dba)3 (1.7 mg, 1.9 mol). The mixture was stirred at room temperature for 30–75 min. An aryl halide (0.75 mmol) was added via syringe. Cesium carbonate (489 mg, 1.50 mmol) and a boronic acid (0.90 mmol) were added simultaneously to the flask, and the system was purged with Argon Tetrahydrofuran (0.50 ml) was added via syringe. The  flask was placed into a 65 oil bath, and the reaction mixture was stirred for 17–21 h. After cooling, the mixture was diluted with ether (5 ml), filtered through Celite, and rinsed with ether (2  5 ml). The combined organic layers were transferred to a separatory funnel and diluted with ether (50 ml). The organic layer was washed with 1 N aqueous NaOH solution (3  50 ml), dried over sodium sulfate, and concentrated in vacuo to provide a pure product. Esterification and Amide-Coupling Reagents

Carboxylic acids are activated by a wide variety of reagents for condensations with amines, alcohols, and other nucleophiles. Many condensation reactions generate by-products that are difficult to remove, thus supported forms of the reagents can greatly improve the purification step. As an example, Zander and Frank33 used a polystyrylsulfonyl chloride resin to activate carboxylic acids for esterification with a range of alcohols, including phenols and tert-butanol (Table VII, entry 28). Also, Keck et al.34 improved a macrolactonization with a polymer-bound carbodiimide (entry 29). Compared to the solution-phase reagent, fewer equivalents of the supported 33 34

N. Zander and R. Frank, Tetrahedron Lett. 42, 7783 (2001). G. E. Keck, C. Sanchez, and C. A. Wager, Tetrahedron Lett. 41, 8673 (2000).

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applications of polymer-supported reagents TABLE VII Activation and Condensation of Carbonyl Compounds

Entry number

Supported reagent

Transformation O

HO

NHFmoc

O

OH

R1

28

O

NHFmoc

O

R1

FmocNH

Reference

33

O

R2OH

FmocNH

OH

OR2

R1, R2 = Alk, Ar

O

29

HO

N OH

n

n = 8, 9, 11, 12

Ph

HCl, CHCl3

O

N O

O

Ph

N O

R1 R1O

O

K2CO3

NH2 O

O O

33

34

N H

F3C

35

OMe

R = Alk, Bn

R n

O

R

R NH2

OMe CF3

32

n

O

n

31

O

O

O

30

O

N

R R = H, t-But

R2 R1O O

N H

R

O O

36,37 R

R1

NH R2

1) Supported phosgene O equivalent R1 R N N 4 R 2) HN 4 R2 R3 R3

38

39

carbodiimide are necessary to give the macrolactones in equivalent or higher yields. The isolation of reactive sites on the solid support suppresses undesired oligomerizations of the hydroxy acids. Depending on their stability, activated esters can be generated and stored as pure reagents on solid supports. The by-products of carbodiimides

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and similar reagents for carboxylic acid activation can be removed before couplings with amines or other nucleophiles. This greatly simplifies purifications and reduces reagent consumption. As an example, the widely used Mosher’s acid has been activated and incorporated as a polymersupported N-hydroxysuccinimide ester (entry 30).35 Treatment of amines with this reagent produces Mosher amides with high purities allowing easy derivatization of chiral amines for stereochemical determinations. Group Transfer Reagents

For the 9-fluorenylmethoxycarbonyl (Fmoc) protection of amino acids, Chinchilla et al.36,37 prepared a similar ROMP-polymer that supports an activated N-hydroxysuccinimide Fmoc-carbonate (Table VII, entry 31). Various Fmoc-amino acids are prepared in pure form after removal of the polymer reagent by filtration and aqueous phase separation. Analogous to these amide-forming reactions, polymer-supported benzotriazole has been used to facilitate urea syntheses by capturing and activating phosgene for sequential additions of amines (entry 32).38 The first amine is reacted with the resin at room temperature to form an acylamine intermediate to the triazole support. The second amine is reacted with the supported intermediate at higher temperature. The discrete temperature difference between the first and second amine additions ensure high purity of the asymmetric ureas. These coupling conditions have been optimized for automated parallel synthesis, and over 150 ureas have been rapidly produced and with good purities. To synthesize guanidines, a carbamate-linked electrophilic reagent was prepared on polystyrene (entry 33).39 Displacement of the reagent’s sulfonamide leaving group with different amines generates resin-bound intermediates that yield pure guanidines after treatment with trifluoroacetic acid. The reagent is effective even for less reactive secondary amines and simplifies the purification and handling of the highly polar guanidine products.

35

T. Arnauld, A. G. M. Barrett, B. T. Hopkins, and F. J. Ze´cri, Tetrahedron Lett. 42, 8215 (2001). 36 R. Chinchilla, D. J. Dodsworth, C. Na´jera, and J. M. Soriano, Tetrahedron Lett. 42, 7579 (2001). 37 R. Chinchilla, D. J. Dodsworth, C. Na´jera, and J. M. Soriano, Bioorg. Med. Chem. Lett. 12, 1817 (2002). 38 A. Paio, R. F. Crespo, P. Seneci, and M. Ciraco, J. Comb. Chem. 3, 354 (2001). 39 C. W. Zapf, C. J. Creighton, M. Tomioka, and M. Goodman, Org. Lett. 3, 1133 (2001).

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Synthesis of Esters (Entry 28) N-Methylimidazole (22.5 l) was added to a mixture of Fmoc-glycinol (20.0 mg, 0.07 mmol), Fmoc-glycine (27.3 mg, 1.3 equiv.), and polystyrylsulfonyl chloride resin (61.6 mg, 1.3 equiv., 1.49 mmol/g) in dichloromethane (1 ml). After 2 h, aminomethylated PS-resin (138 mg, 2 equiv.) was added and the suspension shaken for 30 min. After filtration through a fritted filter, the resin was rinsed thoroughly with dichloromethane, and the combined solvents were concentrated to 3 ml. Amberlyte 15 (176 mg) was added and the suspension was shaken again for 30 min and filtered as described above. After evaporation of the solvent, the pure product was obtained as a colorless foam 93% yield. Synthesis of Fmoc-Amino Acids (Entry 31) A solution of the amino acid (0.40 mmol) and potassium carbonate (39 mg, 0.40 mmol) in water (15 ml) was added to a suspension of FmocP-OSu (270 mg, 0.40 mmol) in acetone (20 ml). The suspension was stirred at room temperature for 24 h and the solvents were removed under reduced pressure. Toluene was added (20 ml) and the resulting solid was filtered. The solid was suspended in water (20 ml) and the P-HOSu (Nhydroxysuccinimide) was removed by filtration. The filtrate was acidified with concentrated HCl (2 ml) and extracted with ethyl acetate (3  20 ml). The combined organic layers were dried over sodium sulfate and evaporated to give the pure Fmoc-protected amino acids. Synthesis of Guanidines (Entry 33) Guanidine hydrochloride (7.5 g, 78.5 mmol) and di-tert-butyldicarbonate (Boc2O, 13.71 g, 62.9 mmol) were stirred for 15 h at room temperature in a mixture of 4 N aqueous sodium hydroxide and dioxane (1:2, 120 ml). This intermediate N-Boc (tert-butyloxycarbonyl) guanidine (2.39 g, 15 mmol) was treated with p-nitrophenyl carbonate Wang resin (5.0 g, 3 mmol) in N,N-dimethylformamide (DMF) for 15 h in the presence of triethylamine (2.1 ml, 5 equiv.) and a catalytic amount of 4-dimethylaminopyridine (DMAP). Trifluoromethanesulfonic anhydride  (2.52 ml, 5 equiv.) was added at 78 to thoroughly rinsed and dried resin.  The reaction mixture was allowed to warm to 0 , filtered, and thoroughly rinsed with methanol and dichloromethane. The resin (87 mg, 42 mol) was treated with H-Sar-OMe (sarcosine methyl ester, 43 mg, 10 equiv.) for 20 h and subsequently washed with methanol and dichloromethane. Treatment with dichloromethane-trifluoroacetic acid (1:1, 2 ml) for 3 h liberated the pure guanidine from the solid support.

364

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Scavengers

In addition to supported reagents, extensive use of polymers for purification of solution-phase reactions has been reported. For example, triarylphosphines are common reagents for organic and organometallic reactions, but the chromatographic separation of phosphines and phosphine oxides from the desired products can be difficult. To simplify this step, Lipshutz and Blomgren40 applied a Merrifield chloromethyl polystyrene support for the electrophilic capture of triphenylphosphine and the corresponding oxide (Table VIII, entry 34). Following Pd(PPh3)4-mediated crosscouplings or after reduction of an azide with PPh3, the concentrated reaction mixtures were heated in acetone with sodium iodide and the capture resin. This method is highly effective for a variety of compounds, including amines and alcohols, and does not impair product yields. Combination of amines and dendrimers functionalized proton scavenger resins efficiently removes excess reagents from amide-formation reactions. Marsh et al.41 used a supported branched polyamine and TABLE VIII Scavengers Entry number

Supported reagent

Transformation

Reference

40

34

O

35

O Ph

NH2

Cl Cl

−HCl

Ph

36

40 41

41

N H

B. H. Lipshutz and P. A. Blomgren, Org. Lett. 3, 1869 (2001). A. Marsh, S. J. Carlisle, and S. C. Smith, Tetrahedron Lett. 42, 493 (2001).

Cl

42

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dichlorotriazene as acid and amine scavengers, respectively, for amide formation reactions (entry 35). The highly branched structures have high loading capacities for efficient purification of amides and sulfonamides. Similarly, a monolithic polymer of PolyHIPE functionalized with tris(aminoethyl)amine captures acid chlorides in solution with high efficiency (entry 36).42 Contrary to suspensions of polymer beads, the porous polymer monolith is used in a flow-through reaction format. Scavenging of Phosphines and Phosphinoxides (Entry 34) The crude reaction was filtered, and the filtrate [containing triphenylphosphine (75 mg, 0.28 mmol)] was concentrated in vacuo. Acetone (1.5 ml), sodium iodide (84 mg, 0.56 mmol), and high loading Merrifield resin (140 mg, 4.38 mmol of Cl/g) were added, and the slurry was allowed to stir at room temperature. After 18 h, the mixture was filtered and washed with tetrahydrofuran (3  3 ml), water (3  3 ml), acetone (3  3 ml), and methanol. Gas chromatography (GC) analysis of combined filtrates and weight gain of the resin indicated complete removal of triphenylphosine from the reaction mixture.

Fig. 1. Multistep synthesis of nicotines. 42

P. Krajnc, J. F. Brown, and N. R. Cameron, Org. Lett. 4, 2497 (2002).

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Multistep Synthesis Using Polymer-Supported Reagents

With the many types of supported reagents available, efficient multistep syntheses of complex organic structures are being performed in solution phase. A recent synthesis of nicotine alkaloids employs several classes of the reagents reviewed above (Fig. 1). Starting from pyridine-3-aldehyde (1), a Grignard addition was quenched with Amberlite carboxylic acid resin. The product alcohol 2 was cleanly transformed with a supported triarylphosphine to alkyl bromide 3. Nucleophilic displacement with a supported azide to prepare 4 and subsequent reductive amination/cyclization produced, following ion-exchange purification, the nornicotine 5 in >90% purity. From this route, the advantages of supported reagents for simplifying purifications are obvious and also enable rapid diversity-oriented parallel syntheses for preparation of compound libraries. Using different supported reagents, the amine 5 was converted in high purity to a set of compounds including alkyl amines, amides, ureas, and sulfonamides.

[20] Advanced Polymer Reagents Based on Activated Reactants and Reactive Intermediates: Powerful Novel Tools in Diversity-Oriented Synthesis By Jo¨rg Rademann Introduction: Current Challenges in Combinatorial Chemistry Research

Combinatorial chemistry provides an array of concepts and methods to solve molecular optimization problems—in drug research and beyond— more rapidly and efficiently than classic synthetic approaches.1 Since specific molecular interactions between proteins and their ligands have been recognized as the molecular basis of most biological processes including disease, it became possible to study and optimize the interactions between drugs and their target proteins on a molecular level. Thus, drug development has been turned into a rational and systematic process of optimization. The establishment of combinatorial chemistry has created novel demands for basic as well as for applied research. Among the prominent challenges is the development of an efficient synthetic methodology for diversity-oriented synthesis2 as well as the creation of powerful interfaces 1

J. Rademann, in ‘‘Molecular Pharmacology—An Encyclopedic Reference’’ (S. Offermanns and W. Rosenthal, eds.). Springer, Heidelberg, in press, 2003.

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

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between synthesis and screening steps.3 For diversity-oriented synthesis, reagents and reactions have to be devised allowing for reliable and robust transformations of simple, chemically diverse starting materials to diverse, complex products. These efforts have to be supplemented by the further refinement of the phase systems devised for synthetic purposes as well as for bioassays. One of the major innovations in combinatorial and medicinal chemistry in recent years aiming at efficient diversity-oriented synthesis has been the implementation of polymer reagents in polymer-assisted solution phase (PASP)* synthesis. This contribution will present—following an introduction to the field—a concept of advanced polymer reagents based on reactive intermediates and active reactants that should extend the scope PASP synthesis significantly. Experimental procedures describing preparation and use of the novel polymer reagents are included. Polymer-Assisted Solution Phase (PASP) Synthesis

Complex organic molecules can be constructed in a homogeneous solution. However, for diversity-oriented purposes it is often advantageous to employ a multiple phase system to greatly facilitate isolation and separation procedures as well as the removal of excess reagents and the completion of reactions. Solid-phase synthesis is the most widely applied example for multiple phase systems in combinatorial chemistry, possessing significant advantages in comparison to homogeneous single phases. Surfaces, soluble polymers, fluorous biphasic systems, or supercritical carbon dioxide are alternative examples of multiple phase systems employed in synthesis, compound purification, or compound screening. Synthesis in solution, however, continues to possess indisputable advantages in respect to the versatility and reliability of applicable reactions, the ease of analytical monitoring, and the accumulated knowledge of synthetic 2

S. L. Schreiber, Science 287, 1964 (2000). J. Rademann and G. Jung, Science 287, 1946 (2000). * Abbreviations: ATR IR, Fourier transform attenuated reflection infrared spectroscopy; DIPEA, diisopropylethylamine; DCM, dichloromethane; DMF, dimethylformamide; DMSO, dimethylsulfoxide; ENDOR, electron-nuclear double resonance spectroscopy; ESI, electrospray ionization; ESR, electron spin resonance; Et2O, diethylether; FT, Fourier transform; GC, gas chromatography; HPLC, high-performance liquid chromatography; HR MAS, high-resolution magic angle spinning; IBX, 1-hydroxy-(1H)-benzo-1,2-iodoxol-3-one1-oxide, 2-iodoxybenzoic acid; ICR, ion cyclotron resonance; MS, mass spectrometry; NBS, N-bromosuccinimide; NMO, N-methylmorpholino-N-oxide; NMR, nuclear magnetic resonance; PASP, polymer-assisted solution phase; PEI, polyethylene imine; RT, room temperature; TEMPO, 2,2,6,6-tetramethylpiperidinoxyl radical; THF, tetrahydrofuran; TPAP, tetrapropylammonium perruthenate. 3

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Fig. 1. Polymer-assisted solution phase (PASP) synthesis combines the merits of solutionphase chemistry with the advantages of facilitated phase separation by using polymer reagents (a) or scavenger resins (b).

protocols. Thus, an ideal synthetic strategy would be to combine the merits of solution-phase with the advantages of solid-phase synthesis protocols, specifically the ability to use reagents in high excess, to remove them by filtration, and to employ automated multiple synthesizers. This combination is realized by PASP synthesis using functional polymers either as scavengers for purification or as reactants involved directly in a chemical transformation (Fig. 1).4–6 Polymer reagents can be used in high excess and then removed by filtration, facilitating product purification for analysis and for further chemical transformations. They are especially suitable for parallel and split-and-pool combinatorial synthesis. They allow the preparation of complex libraries by multistep syntheses in solution, they can be utilized in automated and in flow-through systems, and they can be employed to transform single compounds as well as complex mixtures. The first polymer-supported reagents were derived from ion-exchange resins immobilizing ionic reagents on macroporous polystyrene resins.7 This approach grants easy access to many reagents. For preparation, a solution of a salt is added in excess to the resin, the mixture is allowed to equilibrate, and then the resin is washed with nonionic solvents. Leaching of the reactive ions, however, is a general problem of this type of support. In principle, the immobilized ions can be exchanged by other competing ions available in solution. Several important polymer reagents are 4

A. Akelah and D. C. Sherrington, Chem. Rev. 81, 557 (1981). A. Kirschning, H. Monenschein, and R. Wittenberg, Angew. Chem. 113, 670 (2001); Angew. Chem. Int. Ed. Engl. 40, 650 (2001). 6 S. V. Ley, I. R. Baxendale, R. M. Bream, P. S. Jackson, A. G. Leach, D. A. Longbottom, M. Nesi, J. S. Scott, R. I. Storer, and S. J. Taylor, J. Chem. Soc. Perkin Trans. I, 3815 (2000). 7 F. Helfferich, ‘‘Ion Exchange.’’ McGraw-Hill, New York, 1962. 5

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Fig. 2. First polymer reagents, either in the form of ion-exchange resins or covalently attached, were employed for various simple transformations in the 1960s and 1970s.

still based on ion-exchange resins, such as the borohydride resins8,9 for reductive amination and the perruthenate resin10 for oxidation. The next generation of polymer-supported reagents was based on covalently linked reactants. The concept was initiated in the 1960s and 1970s with the introduction of peptide coupling reagents such as supported carbodiimides,5,11 active esters,12 phosphines,13 and bases (Fig. 2). A major advantage of covalent linking is avoiding leaching of the immobilized reagents from the resin. At that time polymer-supported chemistry was still limited to a few fundamental chemical transformations and was not widely accepted as a useful synthetic method beyond the areas of peptide and oligonucleotide chemistry. It was only when solid-phase chemistry became a powerful tool for combinatorial chemistry in the 1990s that polymersupported reagents gained acceptance for the generation of compound libraries and multistep syntheses of natural products. 8

B. Sansoni and O. Sigmund, Naturwissenschaften 48, 598 (1961). H. W. Gibson and F. C. Bailey, J. Chem. Soc. Chem. Commun. 815 (1977). 10 B. Hinzen and S. V. Ley, J. Chem. Soc. Perkin Trans. I 1907 (1997). 11 Y. Wolman, S. Kivity, and M. Frankel, J. Chem. Soc. Chem. Commun. 629 (1967). 12 R. Kalir, A. Warshawsky, M. Fridkin, and A. Patchornik, Eur. J. Biochem. 59, 55 (1975). 13 W. Heitz and R. Michels, Angew. Chem. 84, 296 (1972); Angew. Chem. Int. Ed. Engl. 12, 298 (1972). 9

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A Concept for Advanced Polymer Reagents

Despite the current success and popularity of polymer reagents, the availability of functional resins has been a severe limitation in recent years. For many synthetically important transformations, reliable reagents were not available. Moreover, polymer-assisted synthesis was usually restricted to small scale applications, and also suffered from the inherent limitations of the standard support material (e.g., cross-linked polystyrene) such as solvent incompatibility, adsorption of reagents,14 or the chemical reactivity of the resin backbone. Better designed polymer reagents should extend the scope of polymerassisted transformations to more challenging chemistries. Novel reagents should be able to replace hazardous, toxic, and other undesirable reactants with clean and reliable polymer-supported alternatives. Ideally, these polymer reagents ought to be recyclable and efficient in catalytic amounts. Novel, high-loading polymer supports should enable applications of advanced polymer reagents on a large scale as well. The generation and investigation of activated reactants and intermediates in polymer gels have extended the field of polymer-assisted conversions considerably. In this chapter we will discuss four selected examples of advanced polymer-supported reagents including reactive oxidants employed on alcohols and in single electron oxidations involving radical ion intermediates (example 1), generation and release of reactive intermediates and activated reactants on polymer supports including the release of carbenium ions (example 2, alkylating resins), the synthesis and applications of supported carbanion equivalents (example 3), and release of radicals (example 4). It will be demonstrated as well that generation or release of reactive intermediates from polymer gels allows for the exploitation of solid-phase specific reactivities. Investigation of these specific solid-phase effects will contribute to improved understanding and advancement in polymer-supported organic chemistry.15 As a result, in example 5 the development of high-loading ULTRA resins that were especially designed for polymer reagents will be demonstrated. Example 1: Oxidizing Polymers

The oxidation of alcohols to carbonyl compounds is one of the most relevant transformations in organic synthesis, due to the large diversity of products that can be obtained from aldehyde and ketone precursors. 14

J. Rademann, M. Barth, R. Brock, H.-J, Egelhaaf, and G. Jung, Chem. Eur. J. 7, 3884 (2001). 15 J. Rademann, Angew. Chem., submitted (2003).

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A variety of methods have been described to solve the task in solution.16 Common oxidative agents for this transformation include various heavy-metal reagents such as chromium-or ruthenium-based oxides, pyridine-SO3, and dimethylsulfoxide (DMSO) in combination with acetic anhydride, carbodiimide, or oxalyl chloride for activation. One of the most prominent methods for the reliable conversion of sensitive compounds is the Dess–Martin reagent or its nonacetylated equivalent, 1-hydroxy(1H)-benzo-1,2-iodoxol-3-one-1-oxide (2-iodoxybenzoic acid, IBX). Polymer-Supported Heavy-Metal Oxides There are several examples of polymer-assisted oxidation reagents, including heavy-metal oxides bound to ion-exchange resins. Perruthenate resin,10 the immobilized analog of tetrapropylammonium perruthenate (TPAP), can be employed stochiometrically as well as catalytically. In the latter case, additional cooxidants [e.g., N-methylmorpholino-N-oxide (NMO)] are required. The use of elemental oxygen has been described as well. Perruthenate resin has been recently employed in a reaction sequence leading to heterocycles (Fig. 3),17 although its usage is limited to benzylic alcohols. In general, ion-exchange resins suffer from the potential leaching of heavy metals into the solution mixture, as heavy metals can be exchanged for other anions present in the solution. Oxidations with Immobilized Oxoammonium Salts The 2,2,6,6-tetramethylpiperidinoxyl radical (TEMPO) was first prepared in 1960 by Lebedev and Kazarnovskii by oxidation of its piperidine precursor.18 The steric hindrance of the NO bond in TEMPO makes it a highly stable radical species, resistant to air and moisture. Paramagnetic TEMPO radicals can be employed as powerful spin probes for elucidating the structure and dynamics of both synthetic and biopolymers (e.g., proteins and DNA) by ESR spectroscopy.19 Unlike solid-phase 1H-NMR where magic angle spinning is required in order to reduce the anisotropic effects in the solid-phase environment, solid-phase ESR spectroscopy can be conducted without specialized equipment. Thus, we conducted comparative ESR studies of various polymers with persistent radical labels, and we also determined rotational correlation times as a function of 16

L. A. Paquette, ed. ‘‘Encyclopedia of Reagents for Organic Synthesis.’’ Wiley, Chichester, 1995. 17 F. Haunert, M. H. Bolli, B. Hinzen, and S. V. Ley, J. Chem. Soc. Perkin Trans. I 2235 (1998). 18 O. L. Lebedev and S. N. Kazarnovskii, Zhur. Obshch. Khim. 30, 1631 (1960). 19 S. L. Regen, J. Am. Chem. Soc. 96, 5175 (1974).

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Fig. 3. Perruthenate resin, an oxidizing resin based on ion exchange of heavy metal oxides, has been successfully employed in the preparation of heterocycle libraries. In this example, benzaldehydes were generated and reacted in aldol reactions with Nafion-TMS as Lewis acid.

solvent, temperature, and resin type. For TEMPO radicals a versatile redox chemistry was reported in which radical species can be transformed by a two-electron reduction to the respective hydroxylamine or by a twoelectron oxidation to the oxoammonium salt.20 One-electron oxidations involving oxoammonium salts have been postulated as well.21 Commonly, TEMPO is employed under phase-transfer conditions with, for example, sodium hypochlorite as the activating oxidant in the aqueous phase. In oxidations of primary alcohols, carboxylic acids are often formed by overoxidation in addition to the desired aldehydes. For catalytic oxidations the oxoammonium salt was postulated as the active intermediate.22 Thus, isolation of oxoammonium salts on insoluble, cross-linked polymer supports was investigated along with their implementation in polymer-assisted solution-phase synthesis.23 These isolated oxoammonium salts could be employed in a water-free system to generate highly reactive oxidation agents without the overoxidation problems normally seen in the presence of water. 20

A. E. J. de Nooy, A. C. Besemer, and H. van Bekkum, Synthesis 1153 (1996). M. F. Semmelhack, C. R. Schmid, and D. A. Cortes, Tetrahedron Lett. 27, 1119 (1986). 22 P. L. Anelli, C. Biffi, F. Montanari, and S. Quici, J. Org. Chem. 52, 2559 (1987). 23 S. Weik, G. Nicholson, G. Jung, and J. Rademann, Angew. Chem. 113, 1489 (2001); Angew. Chem. Int. Ed. Engl. 40, 1436 (2001). 21

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The 4-hydroxy-TEMPO radical was coupled to Merrifield resin (chloromethyl polystyrene resin cross-linked with 1% divinylbenzene) employing sodium hydride as base yielding resin 1 with a loading of 0.93 mmol/g (Fig. 4). ESR spectroscopy detected the presence of the free radical electron displaying the characteristic triplet signal coming from the coupling of the 14N nucleus. Likewise, the HR-MAS NMR spectrum displayed significant line broadening that can be attributed to the enhanced relaxation of the nuclei due to interaction with the persistent electron spins. Oxidation of the radical resin 1 to the oxoammonium resin 2 was best performed with N-chlorosuccinimide. Following oxidation, resin 2 displays strong absorption at 1700 cm1 in the FT-ATR-IR spectrum, a characteristic of the N — O double bond of the reactive species. This oxidation is accompanied by a distinct color change from colorless to either a bright orange-red when chloride or brown-red when bromide is the counterion. Chloride proved to be superior as a counterion being more reactive and leading to fewer by-products. Protocol for Oxidations Employing Oxoammonium Resins 22 Preparation of Oxoammonium Resin. 2. N-Chlorosuccinimide (6 equiv.) was dissolved in DCM and then 4 M HCl in dioxane was added (5 equiv.). After 5 min the solution was added to resin 1 (1 equiv.) swollen in dry DCM. Agitation for 15 min was followed by filtration of the resin and washing with dry DCM. The Half-life (t1/2) of the activated form was  ca. 1 week when stored in vacuo at 4 . Oxidation of Alcohols. Alcohols (1 equiv.) were dissolved in dry DCM. Freshly prepared oxoammonium resin 2 (5 equiv. as calculated from the loading of resin 1) was added and agitated at RT for 1 h for primary alcohols and 2 h for secondary alcohols. The resin was filtered and washed with DCM, and the combined filtrates were analyzed by GC and GC-MS. Yields

Fig. 4. Polymer-supported oxoammonium salts; resins 2 are highly reactive oxidants generated in situ by oxidation of TEMPO radical resin 1 with N-chlorosuccinimide.

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were determined for 10 mg of starting alcohol. After a 1-h reaction, the resin was washed four times with 3 ml DCM and the solvent was evaporated at RT. Representative examples are the oxidation of piperinyl alcohol to piperonal (8.9 mg, 90% yield), cinnamic alcohol to cinnamic aldehyde (8.7 mg, 88% yield), and (þ)-borneol to (þ)-campher (9.2 mg, 91% yield). The identity of the isolated products was also confirmed by NMR analysis (250 MHz, CDCl3). The versatility of the novel reagent was investigated with a diverse selection of alcohols at RT with 3 equiv. of the reagent for 1 h. Results of the oxidations can be summarized as follows. Clean, fast, and quantitative conversion to the respective aldehyde or ketone product was observed for all benzylic, allylic, and primary aliphatic as well as for most secondary aliphatic alcohols in yields around 90%. As expected, diols yielded lactones in the secondary oxidation step. Easily enolizable primary ketones obtained from cyclohexanol, 1-phenylpropan-2-ol, and cholesterol could be further converted to the respective 1,2-diones. In this reaction the primary oxidation product (e.g., cyclohexanone) is transformed to the final diketone product via an enolized intermediate. Interesting cascade-like reactions were observed with (-e)-unsaturated terpene alcohols such as geraniol and citronellol. Monitoring by GC-MS revealed that with an excess of resin 2, the primary oxidation products, the open-chain terpene aldehydes were cyclized in an acid-catalyzed ene reaction (Prins reaction), to furnish (secondary) cyclohexyl alcohols. For example, starting from citronellol, the intermediary citronellal is cyclized yielding the secondary alcohol isopulegol. In the case of geraniol, the secondary alcohol obtained was even further oxidized to yield the respective cyclohexanone in good purity. In addition, oxidating resin 2 was effective in the conversion of a compound collection of 15 chemically diverse alcohols. Under the described nonaqueous conditions the oxoammonium resin, however, failed with nitrogen-containing substrates such as protected amino alcohols, presumably due to a single electron oxidation reported earlier.20 In summary, the polymer-bound oxoammonium reagent was highly efficient in polymer-supported oxidations of various alcohols and was capable of cleanly converting chemically diverse compound collections. No overoxidation to carboxylic acids was observed. It is obvious that this reagent shall be of great value in polymer-supported transformations in solution, in automated parallel synthesis operations, and in flow-through reactors in up-scaled production processes. Catalytic applications employing TEMPO resin 1 are particularly desirable for preparations on larger scale, increasing the efficiency of the polymer-supported reagent. As additional work-up is required for the removal of cooxidants or mediators, catalytic applications do not fit well into

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Fig. 5. Reactive oxoammonium resins 2 can be regenerated catalytically in a three-phase system. With only 6% of TEMPO resin 1, gram amounts of alcohols can be converted efficiently.

parallel and high-throughput synthetic formats. Polymer-supported TEMPO can be employed with sodium hypochlorite in stochiometric amounts for the oxidation of alcohols. An interesting alternative is the use of oxone as an insoluble cooxidant together with tetrabutylammonium bromide as a transfer reagent.24 This reagent system can be easily extended to a three-phase system, employing immobilized TEMPO, dissolved transfer reagent, and insoluble oxone as cooxidant (Fig. 5).25 Elemental oxygen can also be employed as cooxidant for oxidations mediated by TEMPO resin 1. Soluble copper(II) salts can be employed as mediators. However, the rate of oxidation is considerably slower than with oxone as cooxidant. Immobilized TEMPO has been used for the one-pot oxidation of alcohols to carboxylic acids as well.26 For this purpose TEMPO resin 1 was combined with two ion-exchange resins loaded with chlorite anions and hydrogen phosphate in the presence of catalytic amounts of potassium bromide and sodium hypochlorite in solution. The reaction required work-up for the removal of salts, but tolerated several protecting schemes and afforded pure products in good to excellent yields. The reaction is initiated by catalytic TEMPO oxidation of alcohols to aldehydes driven by dissolved hypochlorite followed by oxidation to the carboxylic acids effected by chlorite. 24

C. Bolm, A. S. Magnus, and J. P. Hildebrand, Org. Lett. 2, 1173 (2000). S. Barthelemy, S. Weik, and J. Rademann, unpublished results (2001). 26 K. Yasuda and S. V. Ley, J. Chem. Soc. Perkin Trans. I 1024 (2002). 25

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Oxidations with Immobilized Periodinanes 27 In recent years hypervalent iodine compounds have been extensively investigated yielding many results of practical synthetic importance. Iodinane reagents [e.g., iodoso or iodine(III)] have been prepared in a supported fashion by several groups, mainly as the bis-acetoxy-iodoso derivative28–30 or as the respective dihalogeno compounds.31 Iodoso reagents are employed in the oxidation of hydroquinones as well as phenols, and have been exploited in the formation of spiroketals from various tyrosines. On the contrary, periodinanes [e.g., iodoxo or iodine(V) reagents] are widely employed in the oxidation of sensitive and complex alcohols, preferably as IBX32,33 or its acetylation product, the Dess–Martin reagent.34 Periodinanes have not been prepared on a polymer support so far, although a silica-supported IBX has been reported recently.35 The limitations discovered for oxoammonium resins in respect to the oxidation of nitrogen-containing moieties prompted the investigation of polymer-supported periodinanes as potential alternatives.26 To obtain a functional iodine(V) reagent, a derivative of 2-iodobenzoic acid was required suitable for immobilization and still retaining oxidation properties similar to the parent compound. 4-Hydroxy-2-iodo-benzoic acid esters permit efficient immobilization to chloromethyl polystyrene via the phenoxide. Methyl 5-hydroxy-2-iodobenzoate was obtained in two steps from 3-hydroxyanthranilic acid by a Sandmeyer reaction followed by esterification with thionylchloride in methanol. It was coupled to chloromethyl polystyrene cross-linked with 1% divinylbenzene (1.20 mmol/g) by using cesium carbonate as base (Fig. 6). Loading of the resin was determined by elemental analysis finding it close to the theoretical loading (98%). Saponification was effected by treatment with potassium trimethylsilanoxide in THF yielding resin 4.

27

G. Sorg, A. Mengel, G. Jung, and J. Rademann, Angew. Chem. 113, 4532 (2001); Angew. Chem. Int. Ed. Engl. 40, 4395 (2001). 28 M. L. Hallensleben, Angew. Makromol. Chem. 27, 223 (1972). 29 S. V. Ley, A. W. Thomas, and H. Finch, J. Chem. Soc. Perkin Trans. I 669 (1999). 30 G.-P. Wang and Z.-C. Chen, Synthetic Commun. 29, 2859 (1999). 31 M. Zupan and A. Pollak, J. Chem. Soc. Chem. Commun. 715 (1975). 32 M. Frigerio and M. Santagostino, Tetrahedron Lett. 35, 8019 (1994). 33 C. Hartmann and V. Meyer, Chem. Ber. 26, 1727 (1893). 34 D. B. Dess and C. Martin, J. Org. Chem. 48, 4156 (1983). 35 M. Mu¨lbaier and Giannis, Angew. Chem. 113, 4530 (2001); Angew. Chem. Int. Ed. Engl. 40, 4393 (2001).

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Fig. 6. Polymer-supported IBX (resin 5) can be activated and recycled with monoperoxy sulfonic acid (Caro’s acid). The polymer reagent is capable of alcohol oxidations, dehydrogenations, and radical cyclization reactions.

Protocol for Oxidation with and Reactivation of Polymer-Supported Periodinane 26 (Re)activation of Resin 4. Resin 4 (100 mg, 0.092 mmol) was treated with a solution of tetrabutylammonium oxone (460 mg, 0.46 mmol, active oxygen 1.6%) and methylsulfonic acid (30 l, 0.46 mmol) in dry DCM (1.2 ml) and agitated for 3 h at RT. The product was washed thoroughly with DCM, Et2O, DCM, Et2O, DCM, Et2O (seven times each) and dried yielding resin 4. IR: = 1578, 1602, 1655 cm1. Iodine content: 10.8%. Loading: 0.85 mmol/g. Taking into account the mass increase, this corresponds to 93% conversion of the chloromethyl groups over three steps. The oxidative activity of resin 5 (0.8 mmol/g) was determined by converting an excess of piperonyl alcohol as test substrate. Oxidation of Alcohols. Alcohols (1 equiv.) were dissolved in dry DCM (15 mmol/liter) and treated with resin 5 (1.75 equiv.) for 3 h at RT. The resin was filtered off and washed with dry DCM. From the filtrate the volatile compounds were analyzed by GC-MS. The nonvolatile compounds were analyzed by HPLC-MS. For product isolation the collected filtrates from several washings (DCM, 3  2 ml) were evaporated yielding 5 mg of starting alcohols: piperonal 4.2 mg (84% yield) and Fmoc-l-phenylalaninal 4.1 mg (82% yield). Purity and identity of the products were determined by GC or HPLC and by NMR spectroscopy.

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Oxidation of resin 4 to resin 5 was investigated under various conditions. Initial screening for oxidizing activity was conducted by HPLC analysis of the reaction with piperonylalcohol as test substrate. Potassium bromate and potassium hydromonoperoxosulfate triple salt (Caroate, Oxone) failed in aqueous solvent mixtures. By employing the phase transfer catalyst 18crown-6 together with the caroate in a triphase system, traces of piperonal were detected. To avoid the presence of water and to ensure proper swelling of the resin, tetrabutylammonium oxone in dichloromethane (DCM) was selected, yielding a low resin activity of 0.1–0.2 mmol/g. Monoperoxysulfonic acid (Caro’s acid) is a stronger oxidant than its anion, which is present in Oxone and was therefore examined in further oxidation experiments. An equimolar mixture of tetrabutylammonium oxone with methyl sulfonic acid (DCM, RT, 3 h) furnished resin 5 with a high activity of 0.8 mmol/g. Resin 5 was characterized by IR spectroscopy, elemental analysis, and MASNMR. Elemental analysis indicated a loading of 0.84 mmol/g, corresponding to a yield of 94% in respect to the initial loading, taking into account the mass increase of the resin. No loss of iodine was observed under the strongly acidic reaction conditions. The oxidizing polymer 5 was stable toward air and moisture and it could be stored without loss of activity. The oxidation properties of periodinane resin 5 (1.75 equiv., DCM, RT, 3 h) were investigated by reaction with a collection of diverse alcohols including benzylic, allylic, primary aliphatic alcohols including the unsaturated terpene alcohols citronellol and geraniol, secondary aliphatic alcohols, and the carbamate-protected aminoalcohols Fmoc-Phe-ol and Fmoc-Ile-ol. All reactions were followed by GC-MS or by HPLC (UV at 215 and 280 nm). Products were identified by NMR spectroscopy and by mass spectrometry (EI, 70 eV); isolated yields were determined by weight. Most alcohols were converted to the respective aldehyde or ketone products in good to excellent yields and purities. Following extensive washings, resin 5, which had not been exposed to temperatures higher than RT, could be recycled by repeated oxidation. In addition to the oxidation of alcohols, further important transformations effected by IBX were investigated with resin 5. Cyclohexanol reacted with resin 5 in a closed vessel (2.3 equiv.,  DCM, 2 h, 65 ) yielding ,-unsaturated cyclohexenone via cyclohexanone and a postulated iodine-enol ether intermediate.36 The unsaturated carba mate 6 was treated with resin 5 (4 equiv., THF/DMSO 10:1, 90 , 16 h) in order to undergo radical cyclization affording product 7 with 30% yield.37 It should be noted that IBX at elevated temperatures can oxidize benzylic 36 37

K. C. Nicolaou, Y.-L. Zhong, and P. S. Baran, J. Am. Chem. Soc. 122, 7596 (2000). K. C. Nicolaou, Y.-L. Zhong, and P. S. Baran, Angew. Chem. 112, 639 (2000); Angew. Chem. Int. Ed. Engl. 39, 625 (2000).

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positions, which are abundant in the polystyrene backbone of resin 4 and might account for a competing reaction pathway. Resin 5 was prepared as the first polymer-supported periodinane reagent. This resin was obtained with high loading (0.8 mmol/g) and was capable of converting a collection of diverse alcohols, including complex and sensitive structures, efficiently in good to excellent yields to the respective carbonyl compounds. In addition, the ,-desaturation of carbonyl compounds and the radical cylization of an unsaturated carbamate were demonstrated. This novel reagent is likely to find broad application in polymer-assisted solution-phase synthesis. Furthermore, this new oxidizing resin should be well suited for integration into parallel polymer-supported reaction sequences for the production of novel compound libraries. This reagent has also been efficient in the oxidation of a selection of medium to large-sized peptide alcohols. With 5 equiv. of resin 5 the respective peptide aldehydes were obtained in good to excellent purity. For example, the natural peptide alcohol alamethicin F-30 containing as many as 19 amino acids was efficiently oxidized to its aldehyde derivative as verified by ESI-FT-ICR MS. Example 2. Alkylating Polymers38

Many alkylating agents, such as diazoalkanes, sulfates, sulfonates esters, and alkyl halogens, are highly toxic, mutagenic, or explosive compounds making a safer resin-bound alternative an attractive substitution. Solid-supported sulfonate esters have been employed in alkylations of amines and thiols at elevated temperatures. Sulfonate alkylations of carboxylic acids were reported following our work, requiring the addition of a base that can be removed with a scavenger resin.39 Because elemental nitrogen is an excellent leaving group in alkylations, alkylating polymers ideally work best when releasing carbenium ions and nitrogen from precursors bound to insoluble polystyrene gels.38 The alkylating species were generated from solid-phase bound 1-aryl-3-alkyltriazenes under acidic conditions and were demonstrated as very reactive, mild, and versatile alkylation reagents. As higher reactivity of electron-rich triazenes was observed, p-alkoxysubstituted anilines were selected as an efficient starting material. Solidsupported triazenes were prepared via a solid-phase bound nitroaryl, which was reduced to the polymeric ammonium hydrochloride resin 8 (Fig. 7). 38

J. Rademann, J. Smerdka, G. Jung, P. Grosche, and D. Schmid, Angew. Chem. 113, 390 (2001); Angew. Chem. Int. Ed. Engl. 40, 381 (2001). 39 N. Zander and R. Frank, Tetrahedron Lett. 42, 7783 (2001).

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Fig. 7. Triazenes as versatile polymer-supported diazoalkane analogues (resins 10) were obtained from polymeric diazonium salts (resins 9) and releasing carbenium ions upon acidic activation. The reaction can be employed for the alkylation of carboxylic acids with a reaction half life of ca. 5 min.

The latter resin was treated with tert-butyl nitrite in DCM at 18 resulting in diazotation. The best results in the diazotation reaction were obtained by directly employing the ammonium hydrochloride salt resin 8. The diazonium salt resin 9 was reacted with various primary amines including methylamine, n-butylamine, n-dodecylamine, several allyl-and benzylamines, 20 -amino-2-ethylpyridine, and diamines including 2-morpholinoethyl amine and 1,13-diamino-4,7,10-tridecane (diamino-PEG-200), to furnish the supported triazene resins 10. Starting from polystyrene-containing chloromethyl groups (2.0 mmol/g) methyl resin 10 was obtained with a loading of 1.54 mmol/g of reactive sites. Taking into account the mass increase during the reaction sequence, this loading corresponds to an excellent conversion (94%) of chloromethyl groups into triazenes. Completion of aryl ether formation, reduction, and diazotation as well as triazene formation could be monitored by following characteristic vibrational bands in the attenuated total reflection IR spectrum (FT-ATR-IR) obtained directly from washed and dried resin samples. The methyl triazene resin 10 was stable at room temperature for at least several months when kept in the dark. Various reactions were investigated using the novel polymer reagent resin 10. First, esterifications were studied with acids representing a broad

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pKa range, molecular weight, steric constraints, as well as diverse functionalities. As the reaction requires acidic conditions to activate the triazene moiety by protonation, the pKa of the substrate plays a crucial role in this reaction. Furthermore, there is evidence pointing at carbenium ion intermediates either as ion pairs in the case of benzyl ions or via a concerted mechanism in case of primary alkyl ions. Protocol for the Alkylation of Carboxylic Acids by Use of Alkylating Resins37 Representative Example. A carboxylic acid (5 mg) was dissolved in DCM (5 ml) (or DCM/MeOH 9:1, or THF, or dioxane) and treated with n-butyl triazene resin 10 (R1 = n-butyl, 5 equiv.) for 6 h. The resin was washed with DCM, MeOH, and DCM (2  2.5 ml) and the solvent was removed by evaporation furnishing 5.3 mg of n-butyl product in the case of benzilic acid (79.5% yield). HPLC analysis was conducted to determine the purity of the products. Sterically demanding benzilic acid was used to optimize the reaction conditions in respect to reagent excess and reaction time. Complete conversion (98%) of benzilic acid to the corresponding methyl ester was obtained with two equivalents of the methyl triazene resin 10 after 6 h; 96% conversion was obtained when using the n-butyl triazene resin 10. The reaction between p-nitrophenylacetic acid (1 equiv., 2 mg/ml) and the polymer-supported triazene (2 equiv.) in DCM was monitored by HPLC. A 53% conversion from the acid to the ester product was observed after 5 min; data analysis indicated a second-order reaction as observed in homogeneous solution. A diverse selection of acids was converted to highly pure ester products when treated with 5 equiv. of the alkylating polymers 10 for 6 h. Representative yields were in the range of 80%. In case of the esterification of benzilic acid with n-butyl triazene resin, NMR analysis was employed to validate the structure of the expected n-butyl ester. The NMR spectrum did not display any signals corresponding to an isobutyl ester by-product formed by rearrangement. In contrast, alkylation to the tert-butyl esters failed, though the generation of gaseous products indicated decomposition of the triazene moiety. Likely, the intermediary tert-butyl cation undergoes proton abstraction yielding isobutene. Functional groups that can be tolerated by resin 10 include aliphatic hydroxy groups, enolizable carbonyl functions, and nitrogen heterocycles with limited basicity as in pyridine (pKa 5.25) and pyrazoles. The conversion of acid-sensitive structures was exemplified with penicillin V. Esterification of this especially labile structure failed under acidic and basic conditions as well as with diazomethane.

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The efficiency of this polymer-supported alkylation was investigated using various protected amino acids and peptides including a decapeptide bearing various acid-labile protecting groups and a mass of 2924.5 Da. The latter was reacted with methyltriazene resin 10 (R1 ¼ CH3, 5 equiv.). After 6 h the starting peptide was consumed and the product was confirmed by HPLC and ESI-MS. Uncatalyzed etherification was successful only with strongly acidic phenols such as pentafluorophenol (70% purity). Triazene resins based on diamines were also found to be efficient in the alkylation reaction. In addition, the conversion of a compound collection of drug-like heterocycles as might be used in a medicinal chemistry program was investigated. An equimolar mixture of 20 pyrazole acids, synthesized by a splitand-mix approach, was treated with methyl resin 10 (R1 ¼ CH3, 5 equiv.) for 6 h to yield the respective pyrazole esters. All 20 pyrazole acids in the starting reaction mixture and all their corresponding 20 pyrazole methyl esters in the product mixture could be identified by FT-ICR MS coupled to micro-HPLC with a relative mass error <2.2 ppm. In summary, alkylating resins 10 have been demonstrated to be very efficient and versatile esterification reagents that allow the conversion of diverse, highly functionalized substrates under extremely mild reaction conditions. These novel reagents might be valuable especially in the parallel conversion of mixtures of highly functionalized compound collections such as those used in lead optimization efforts. Alkylating polymers should also be useful in flow-through applications allowing in situ conversions (e.g., in gas chromatography). Further potential of the presented alkylation reagents can be expected for other nucleophiles such as alcohols, phenols, amines, thiols, and various C-nucleophiles including cyanide ion. Example 3. Polymer-Supported Carbanion Equivalents

To establish more complex reaction sequences with polymer reagents, generation of reactive electrophiles either by oxidizing polymers or by the release of carbenium ions is supplemented well by polymer-supported carbanion equivalents. For example, the combination of an oxidizing resin with a support carrying carbanion equivalents will be especially rewarding, allowing reaction sequences with C–C coupling steps and therefore access to a wealth of potentially relevant products. Classically, polymer-supported carbanion equivalents are obtained from polymeric phosphines,13 which are used for the generation of polymeric Wittig salts, ylides, and finally olefines. The -hydroxy carbonyl moiety 11 (Fig. 8 is an important structural motif found in many natural products including carbohydrates, fatty acids, and secondary metabolites. Potent protease inhibitors and antitumor

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Fig. 8. Resins 12 and 13 as examples of polymeric acyl anion equivalents.

agents contain this motif as well. A general polymer-supported access to these compounds has not been established so far. In nature, this motif is provided via acyl anion equivalents (‘‘active aldehydes’’) derived from thiamine pyrophosphate (TPP, vitamin B1) as enzymatic cofactor. To introduce diversity-oriented access to this important structural motif, polymersupported acyl anion equivalents were selected as starting points. Various thiamine derivatives were prepared on polymer support and were tested in C–C coupling reactions.40 The most straightforward approach was the alkylation of substituted 1,3-thiazoles with chloromethyl polystyrene resin yielding supported thiazolium chloride salts. Alternatively, resin-supported thiamine derivatives were constructed in a two-step process by alkylation of the primary alcohol with chloromethyl polystyrene followed by alkylation of the ring nitrogen yielding resin 12 (Fig. 8). Not only does this pathway introduce a spacer between the active thiamine 40

S. Weik and J. Rademann, Angew. Chem. 115, 2595 (2003); Angew. Chem. Int. Ed. Engl. 42, 2491 (2003).

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and the polymer backbone, it allows for the diverse substitution of the ring nitrogen for variation of steric demand at the nucleophilic 2-position or for the introduction of a chiral thiamine as the C–C bond-forming catalyst. Resin 12 can be efficiently employed for benzoin condensations with catalytic amounts of the supported thiamine (10 mol%). The reaction has to be performed under exclusion of air to avoid formation of benzylic or  benzoic acids. Ethanol equilibrium is reached after 6 h (60 ) with a maximum of 40% conversion. Thiamine-catalyzed transformations are reversible, thus N,N-dialkyl hydrazones were selected as alternative acyl anion equivalents that were reported to react with electrophiles without acidic activation.41 One especially reactive example, formaldehyde hydrazone resin 13, was constructed from polymer-supported hydrazines and was employed in the first polymersupported, uncatalyzed acyl anion additions (Fig. 8).38 As test substrates, nitroalkenes (as Michael acceptors) and activated aldehydes were selected. Reactivity of these acyl anion equivalents depended critically not only on the nature of the starting hydrazine, but also on the protocol for hydrazine formation. Example 4. Radical Release from Polymer Gels42

The generation of radical intermediates inside of polymer gels is appealing not only for synthetic purposes but as well for understanding polymer-supported reactivity. The relative isolation or hindered diffusion of radicals inside of polymer gels might influence the lifetime of radicals so that reactivities are specific for solid-supported reaction systems (specific solid-phase effects). For example, the recombination of radicals could be disfavored in the solid-supported reaction. In addition, reactivity of radicals with the polymer backbone also has to be considered. To investigate solid-supported radical chemistry, analytical techniques for the observation of reactive intermediates in polymer gels and product distribution are required. Until now, the chemistry of radicals on solid supports has been investigated mostly in respect to intramolecular radical cyclizations and radical chain reactions. One reason for refraining from free radical transformations is the chemical nature of the polystyrene with its abundance of benzylic positions that are prone to H-radical abstraction and oxidation. Symmetric azo compounds are useful initiators of radical chain reactions. However, in many instances, the radicals are released from the initial R. Ferna´ndez, E. Marti´n-Zamora, C. Pareja, and J. M. Lassaletta, J. Org. Chem. 66, 5201 (2001). 42 J. Rademann, P. Schuler, and G. Nicholson, unpublished results (2002). 41

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radical pair only to a small extent and react preferably by recombination or disproportion. In contrast, two radicals of very different reactivity generated from an asymmetric precursor molecule can be released from the initial radical pair to a high percentage, if alternative reaction pathways are accessible.43 Trityl-azo-aryl resins 14 were obtained from trityl chloride resin (0.96 mmol/g) in two steps. Aryl hydrazines (3 equiv.) were coupled in the presence of diisopropylethyl amine (DIPEA) at RT, followed by oxidation with N-bromosuccinimide (NBS) in DCM/pyridine (9:1). The reaction was analyzed by elemental analysis and FT-ATR IR, respectively. Radical thermolysis was monitored by ESR spectroscopy in order to determine the onset of radical generation, to characterize and study the persistence of the formed radicals. A sample of resin 14 swollen in CHCl3 at room tempera ture did not contain any detectable radicals, but heating to 40 generated a distinct ESR signal. Signal intensity was further increased by warming to  60 , resulting in vigorous gas evolution. ENDOR (electron-nuclear double  resonance) spectroscopy of the sample at 60 was used to characterize the radical structure yielding coupling constants that could be fully assigned the protons of the trityl-type radical. These radicals are highly persistent, being detected for hours at room temperature. To investigate putative matrix effects, thermolytic radical fragmentation of 4-bromophenyl-azo-triphenylmethyl was conducted at various concentrations with a suspension of resin 14 in an H-donating solvent  (CHCl3, 60 , 2 h). The product distribution for solution and solid-phase experiments was analyzed by GC. In solution experiments even at high concentrations (0.1 M) only a few recombined products between aryl and trityl radicals were detected (2.7%). Low recombination, however, is essential for good recovery of fragmentation products from the solid support. In solution and on solid support, the main product formed from the aryl radicals was bromobenzene, while 4,40 -dibromo biphenyl was formed as a by-product from the recombination of two bromophenyl radicals. Formation of the recombined product was concentration dependent. A minor by-product was bromo-4-chlorobenzene. Benzophenone was detected to a significant extent in the presence of oxygen, probably formed via disproportion of the intermediate trityl peroxide. Comparing the results from radical release in solution and on solid support, it was determined that the amounts of recombined product were reduced significantly in the matrix-supported reaction even at higher concentrations (0.1 M solution: 2.6%, 0.16 M polymer support: 0.4%). On the other hand, in both solution and polymer gel experiments, the 43

T. B. Patrick, R. P. Willaredt, and D. J. DeGonia, J. Org. Chem. 50, 2232 (1985).

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amount of bromo-4-chlorobenzene was not concentration dependent. These findings clearly indicate a matrix effect that can be attributed to the relative isolation and/or hindered diffusion of released radicals inside the polymer. This matrix effect was found in both released aryl radicals and polymer-attached trityl radicals (Fig. 9). The high purity (>95%) of the released product from resin 14 prompted us to investigate the potential of radical release as a traceless linker concept. To our knowledge, thermolytic radical fragmentation of covalent bonds is a mechanism of bond dissociation that has not been exploited so far for linker chemistry and for solid-phase transformations. Cleavage yields, determined by releasing nonvolatile products, were found to be higher than 90%. Radical fragmentation of 2-nitrophenyl-azo-trityl resin was studied in the presence of various radical acceptor solvents to elucidate possible radical reaction pathways. When using benzene as solvent, only 2-nitro-biphenyl was formed as the product of radical substitution reaction (SNR) in 67% yield. Hydrogen-radical abstraction from the polymer backbone (e.g., from the benzylic units of polystyrene) was completely suppressed. When toluene was used as solvent, a mixture of the following products was obtained: nitrobenzene, 4-methyl-20 -nitrobisphenyl, 2-methyl-20 -nitrobisphenyl, and 3-methyl-20 -nitrobisphenyl (9:9:1:1). In the case of toluene, the nitro-aryl radicals undergo H-abstraction with radical substitution as a competing reaction pathway. These results indicate that H-abstraction

Fig. 9. Thermolytic radical release from polymer-supported trityl-azo-arenes resins 14.

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occurs on the toluene side chain rather from the benzylic positions of polystyrene. With pyridine as solvent, no product was detected in solution, despite the fact that gas evolution indicated radical formation. Residual nitro groups detected in the IR spectrum suggest that radical scavenging by the resin backbone was the predominant reaction pathway. Aryl radicals can be generated inside of polystyrene gels by thermolysis of trityl-azo-aryl compounds. Radical formation and the long lifetime of the trityl radicals can be monitored by ESR spectroscopy. Matrix effect studies of radicals inside of polymer gels indicated a significant reduction of radical recombination as well as a prolonged lifetime of radicals inside of polymer gels. In addition, we have shown that radical quenching by the polystyrene backbone is strongly disfavored in comparison to H-abstraction from the solvent. In the absence of efficient radical reaction pathways involving solvent interactions, radical quenching by the trityl polystyrene resin became the major reaction pathway. These findings will be helpful for the further exploitation of matrix-supported or matrix-released radicals and could be of particular interest for the investigation of radicals of biological importance studied using the presented methodology. Example 5. Optimization of the Polymer Support: Highly Loading Resins for Polymer-Supported Reagents44

To date the majority of polymer-supported chemistry is conducted only on a few solid support materials. Recently, it has been documented that specific solid-phase effects have significant impact on the success or failure of polymer-supported reactions.15 Considering the limitations of polystyrene, which is the standard material for most applications today, it becomes even more evident that innovations in the area of support materials will open the door to novel opportunities for polymer-supported chemistries. One major drawback of the current methods is the low atom economy45 of solid-supported chemistry with conventional resins in comparison to solution-phase synthesis. The low loadings are one important reason for excluding solid-supported methods from many resource-and cost-sensitive applications such as scale-up projects. Furthermore, polystyrene-based resins are restricted by solvent compatibility, thermal and chemical stability, and extensive adsorption of reagents. Large-scale polymer-supported chemistries could significantly accelerate the drug discovery process, particularly for the generation of lead 44

J. Rademann and M. Barth, Angew. Chem. 114, 3087 (2002); Angew. Chem. Int. Ed. Engl. 41, 2975 (2002). 45 B. Trost, Angew. Chem. 107, 285 (1991); Angew. Chem. Int. Ed. Engl. 30, 214 (1991).

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compounds in larger quantities. Moreover, matrix-supported synthesis is of strong interest for ‘‘green chemistry’’ providing environmentally friendly production processes. Efficient scale-up requires higher yields per reaction volume and has to be more time efficient than can be provided with the current generation of support materials. Increasing the loading of polymer supports, i.e., the millimoles of reactive sites per gram of polymer, is one important prerequisite for a scale-up resin. High-loading resins have been prepared using several approaches. Dendrimer synthesis on polystyrene resins is one possibility.46 However, the synthesis is relatively tedious and only the loading per bead (not per gram) is increased significantly due to the large weight increase. Unfortunately, loading per gram is not much increased by dendrimer synthesis. An alternative approach is grafting on polystyrene by living radical polymerization.47 The O-TEMPO group is attached to the benzylic units in  polystyrene resin. Heating to 140 generates free benzyl radicals that initiate the radical polymerization of olefines in the solution (e.g., acrylamides). High loadings of grafted functional styrene monomers are obtained by this route leading to so-called RASTA resins. Resins constructed from low-molecular-weight monomers with efficient functionalization sites at each monomer would possess greatly enhanced resin capacity. The maximum theoretical loading of poly-(4-chloromethyl) styrene is 6 mmol/g. In commerically available polystyrene supports, however, only a small fraction of this can be obtained due to methylene cross-linking under Friedel–Crafts conditions. Polyvinyl alcohol as well as polyethylene imines (PEI) possess significantly higher loadings up to 23 mmol/g functional groups. Since polyvinyl alcohols or esters are prone to elimination or acid hydrolysis, polyethylene imine was preferred as the lead polymer for resins with significantly higher loading and atom economy than conventional polymers. To control the degree of cross-linking and the degree of amine substitution in the resin product, linear polyethylene imine was selected as the polymeric starting material.42 Cross-linking of poly (ethylene imine) was investigated with polyaldehydes, polyacids, and multivalent alkylating agents. Dialdehydes furnishing the resin structure via a thermodynamically controlled equilibrium reaction were found to be superior allowing the preparation of resins with robust protocols from inexpensive and easily available precursors. The dialdehyde was crucial for the success of the reaction and for the mechanical properties of the polymer formed. The rigid terephthalic dialdehyde afforded the chemically, mechanically, and thermally robust ULTRA resin 15. Whereas 46 47

V. Swali, N. J. Wells, G. J. Langley, and M. Bradley, J. Org. Chem. 66, 4902 (1997). L. C. Hodges, L. S. Harikrishnan, and S. Ault-Justus, J. Comb. Chem. 2, 80 (2000).

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glutaric dialdehyde yielded a softer resin, glyoxal delivered no resin at all. Presumably, under the described conditions only a rigid dialdehyde cross-linking between two polymer chains was favored relative to the intramolecular reaction within one chain. ULTRA resins were prepared from varying molar ratios between dialdehydes and the linear PEI and with PEI of varying length (Fig. 10). In all cases resin micropellets of a defined size range were generated by polymer extrusion in the swollen state, followed by sieving. ULTRA resins were employed for the preparation of polymer reagents as well as for solid-phase synthesis. ULTRA resin 15 can be directly employed as a polymeric base with a loading of 15.2 mmol/g. Following reductive amination with formaldehyde, a resin containing 13.2 mmol/g of tertiary amines was obtained. An ULTRA resin for ion exchange was prepared by alkylation of the tertiary amine resin with methyl iodide; the maximum loading of the resin with chloride was 8 mmol/g corresponding to a chlorine content of 28%. Starting from ULTRA resin 15, 4-chloropyridinium hydrochloride and triethylamine under microwave-assisted condi tions at 220 afforded a resin analogous to the acylation catalyst dimethylamino pyridine. For solid-phase synthesis various linker molecules were constructed on the ULTRA resin 15. To determine the optimal spacer length, ULTRA resins were coupled with spacers of variable length. With 4-(40 -acetoxymethyl-30 -methoxy-phenoxy) butyrate and a loading of 2.5 mmol/g the synthesis of heterocycles and peptides was investigated. A pyrazole carboxylic acid was prepared using a procedure established for Wang polystyrene

Fig. 10. High-loading ULTRA resins 15 based on the reductive cross-linking of linear polyethylene imines.

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without modification. Starting with 5 mg of the ULTRA resin 15, this three-step process produced 8.8 mg of the heterocycle (80% purity in the raw product, 65% yield following chromatography). Limitations in product size were investigated by preparing several peptides of various lengths. Peptides containing 7, 9, 13, and 19 amino acid residues were prepared on a synthesis robot employing Fmoc-protected amino acids and carbodiimide-hydroxybenzotriazol activation (0.25 M, 1 h) without detection of deletion products. To illustrate the remarkable economy of the ULTRA resin, 3.4 mg of the starting resin 15 sufficed for the synthesis of 42 mg resin with fully protected tridecapeptide. The raw product was obtained by ethereal trituration in excellent purity and yield (90% purity in the raw product, 78% yield, 13.1 mg after preparative HPLC). In summary, ULTRA resins can be prepared with extremely high loading compared to standard resins in use today. Secondary amine groups of the resin were very accessible to various derivatizations and even larger product molecules could be assembled successfully in the resin interior. Thus, these resins allowed solid-supported chemistry that was greatly improved in atom economy and provide a significant contribution to the efficient scale-up of polymer-supported syntheses. Conclusions

The generation and release of polymer-supported reactive intermediates or activated reactants have been demonstrated to be a powerful concept leading to novel insights of matrix-assisted reactivity. Following this concept, several novel advanced polymer reagents for important transformations have been introduced. The reagents are capable of cleanly converting sensitive single compounds as well as complex mixtures in solution, and additionally, they are recyclable and/or can be employed catalytically. Despite the significant advances reached in recent years, further research and innovations are required to overcome the most significant limitations in polymer-supported chemistry. Still too little is understood about the influence of the physical and chemical properties of support materials on solid-supported reactivity. The impact of polymer reagents will rise considerably, if innovative support materials become available that allow the scale-up of polymer-supported reaction sequences. In addition, biocompatible supports will extend the opportunities of solid-supported chemistry toward biochemical transformations and screening applications. ULTRA resins, as a novel resin concept with extreme high loading and enormous swelling in water, will contribute significantly to these future developments.

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[21] Scavenger Resins in Solution-Phase CombiChem By J. Gabriel Garcia Introduction

Combinational chemistry jointly with high-throughput biological screening has dramatically increased the number of potential drug candidates that need to be synthesized.1 As a result, they are being widely used in medicinal chemistry as a tool for accelerating synthesis and drug discovery. Combinatorial chemistry is a highly effective method for the generation of multiple small molecule libraries of drug-like compounds,2–6 and has the ability to generate a large set of structurally related analogues. Therefore, it has become a legitimate tool for increasing productivity in the functional assessment of compound libraries and the rapid development of structure–activity relationships.7 To keep up with the fast-paced progress of both solution- and solid-phase combinatorial chemistry, an increased number of commercially available materials with a multitude of applications has been generated.3,4,8–10 The most time-consuming factor in organic synthesis is the purification of the desired product, thus the bottleneck in combinatorial chemistry is the rapid purification of library compounds. Traditional purification methods such as aqueous extraction and chromatography become time consuming as the performed number of simultaneous (parallel) reaction increases. Novel methodologies are thus being developed to overcome this problem with the aim of implementing automated work-up procedures for crude solution-phase reactions. The most popular methodology gaining 1

G. M. Coppola, Tetrahedron Lett. 39, 8233 (1998). S. W. Kaldor, M. G. Siegel, J. E. Fritz, B. A. Dressman, and P. J. Hahn, Tetrahedron Lett. 37, 7193 (1996). 3 L. A. Thompson and J. A. Ellman, Chem. Rev. 96, 555 (1996). 4 E. M. Gordon, M. A. Gallop, and D. V. Patel, Acc. Chem. Res. 29, 144 (1996). 5 S. H. DeWitt and A. W. Czarnik, Acc. Chem. Res. 29, 114 (1996). 6 L. M. Gayo and M. J. Suto, Tetrahedron Lett. 38, 513 (1997). 7 R. E. Dolle and K. H. Nelson, Jr., J. Comb. Chem. 1, 235 (1999). 8 R. Storer, Drug Discov. Today 1, 248 (1996). A. Chuckolowski, T. Masquelin, D. Obrecht, J. Atdlweiser, and J. M. Villagordo, Chimica 50, 525, (1996). D. M. Coe and R. Storer, Annu. Rep. Comb. Chem. Mol. Divers. 1, 50 (1997). A. T. Merritt, Comb. Chem. High Throughput Screen. 1, 57 (1998). 9 A. Chesney, P. Barnwell, D. F. Stonehouse, and P. G. Steel, Green Chem. 57 (2000). 10 F. Balkenhohl, C. v.d. Bussche-Huennefeld, A. Lansky, and C. Zechel, Angew. Chem. Int. Ed. Engl. 35, 2288 (1996). J. C. Hogan, Jr., Nat. Biotechnol. 15, 328 (1997). 2

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

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widespread acceptance for solution-phase combinatorial chemistry11 is the use of scavenger resins, which are also known as polymer-supported quenching/scavenging reagents.2,12 Additional methodologies applied for the same purpose of rapid purification include chemical tagging of reagents, solid-phase extraction, and fluorous-phase extraction.13,14 Recently, a number of methods have been developed for the aqueous extraction of a large number of organic reactions in parallel. However, most of these techniques involve expensive robotic systems and are unable to deal with emulsions, and thus these techniques are restricted to using solvents denser than water.14 Solution-phase combinatorial synthesis provides a homogeneous reaction medium and overcomes the drawbacks of a solid-phase strategy. An easy and reliable purification method is required in solution-phase combinatorial (parallel) synthesis to facilitate automation. The throughput in solution-phase automated synthesis is directly related to the facility of performing a purification process (work-up), compound separation, etc.15 This chapter will cover an overview of recent advances in solid-phaseassisted solution-phase combinatorial synthesis, specifically the use of scavenger resins in assisting in the isolation of pure product without the need for chromatography. In addition, an experimental section has been included. During the past few years, polymer-assisted solution-phase synthesis has become the prevalent method for the parallel synthesis of chemical libraries as confirmed by the increasing number of publications and reviews on the subject.16–29 A key step in the parallel solution-phase combinatorial 11

R. J. Booth and J. C. Hodges, Acc. Chem. Res. 32, 18 (1999). R. J. Booth and J. C. Hodges, J. Am. Chem. Soc. 119, 4882 (1997). 13 D. L. Flynn, R. V. Devraj, and J. J. Parlow, Curr. Opin. Drug Discov. Dev. 1, 41 (1998). J. J. Parlow, R. V. Devraj, and M. S. South, Curr. Opin. Chem. Biol. 3, 320 (1999). 14 E. Maslana, R. Schmitt, and J. Pan, J. Autom. Methods Manage. Chem. 22, 187 (2000). N. Bailey, W. J. Cooper, M. J. Deal, A. W. Dean, A. L. Gore, M. C. Hawes, D. B. Judd, A. T. Merritt, R. Storer, S. Travers, and S. P. Watson, Chimica 51, 832 (1997). M. Rabinowitz, P. Seneci, T. Rossi, M. DalCin, M. Deal, and G. Terstappen, Bioorg. Med. Chem. Lett. 10, 1007 (2000). 15 R. Ferritto and P. Seneci, Drugs Future 23, 643 (1998). 16 A comprehensive review article: S. V. Ley, I. R. Baxendale, R. N. Bream, P. S. Jackson, A. G. Leach, D. A. Longbottom, M. Nesi, J. S. Scott, R. I. Storer, and S. J. Taylor, J. Chem. Soc. Perkin Trans. I 3815 (2000). 17 H. N. Weller, Mol. Divers. 4, 47 (1999). 18 R. Ferritto and P. Sensci, Drugs Future 23, 643 (1998). 19 H. Y. An and P. D. Cook, Chem. Rev. 100, 3311 (2000). 20 J. G. Breitenbucher, K. L. Arienti, and K. J. McClure, J. Comb. Chem. 3, 528 (2001). 21 B. A. Bunin, ‘‘The Combinatorial Index.’’ Academic Press, New York, 1998. G. Yung, ‘‘Combinatorial Chemistry: Synthesis, Analysis, Screening.’’ Wiley-VCH, Weinheim, 1999. 12

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synthesis of compound collections involves the purification of each member of the library. The possibility of avoiding aqueous work-up, crystallization, and chromatographic procedures makes the whole process more suitable for automation and enhances its efficiency from economic and ecological viewpoints. Alternatively, this rationale can be used in solution-phase synthesis where reactive scavenger resins are added to the completed reaction to remove any excess reagents. The advantage of this technique is that the reaction can be monitored, the product remains in solution, and product purity can be determined by standard methods. Combinatorial chemistry, now a widely practiced technique in the pharmaceutical and biotechnology industry, is regarded as an important component of the drug discovery process.30 While traditional combinatorial chemistry is carried out on solid supports, the use of solution-phase techniques for library generation has gained momentum since the late 1990s.31 The introduction of solid-supported scavengers facilitated this trend12,32 and a diverse set of scavengers is now commercially available. Reactive groups (e.g., amines, aldehydes, thiols, hydrazines, isocyanates) linked to poly(styrene-divinylbenzene) beads (typically 1 or 3% crosslinked) are used to quench excess or to selectively react with starting materials of complementary reactivity in solution. These quenching reagents are particularly useful in solution-phase combinatorial chemistry, where the purification of large numbers of compounds is difficult to achieve using traditional methods such as crystallization or flash chromatography. However, the use of quenching reagents does have some drawbacks. First, they must be used in solvents with good polymer swelling properties such as

22

J. A. Ellman and L. A. Thompson, Chem. Rev. 96, 555 (1996). J. J. Parlow and J. E. Normansell, Mol. Divers. 1, 266 (1995). D. C. Sherrington and P. Hodge, ‘‘Syntheses and Separations Using Functional Polymers.’’ Wiley, Chichester, UK, 1988. A. Akelah and D. C. Sherrington, Chem. Rev. 81, 557 (1981). 24 A. Studer, S. Hadida, R. Ferritto, S. Kim, P. Jeger, P. Wipf, and D. P. Curran, Science 275, 823 (1997). 25 J. S. Fru¨chtel and G. Jung, Angew. Chem. Int. Ed. Engl. 35, 17 (1996). 26 A. Kirschning, H. Monenschein, and R. Wittenberg, Angew. Chem. Int. Ed. Engl. 40, 650 (2001). 27 A. Dondoni and A. Massi, Tetrahedron Lett. 42, 7975 (2001). 28 G. L. Bolton, R. J. Booth, M. W. Creswell, J. C. Hodges, J. S. Warmus, M. W. Wilson, and R. M. Kennedy, U. S. Patent 9742230 (1997). 29 J. Eames and M. Watkinson, Eur. J. Org. Chem. 1213 (2001). 30 R. B. Nicewonger, L. Ditto, and L. Varady, Tetrahedron Lett. 41, 2323 (2000). 31 R. E. Dolle and K. H. Nelson, Jr., J. Comb. Chem. 1, 235 (1999). 32 D. L. Flynn, J. Z. Crich, R. V. Devraj, S. L. Hockerman, J. J. Parlow, M. S. South, and S. Woodard, J. Am. Chem. Soc. 119, 4874 (1997). 23

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DMF,* methylene chloride, or THF. Second, these solvents are undesirable when running thousands of reactions (DMF is difficult to remove from the final product, methylene chloride is toxic, and THF may contain peroxides). Third, according to most manufacturer’s instructions,16 an excess of quenching reagent is required for a few hours to overnight to completely remove impurities, which translates into longer synthesis times when running large number of reactions. A goal to overcoming the latter is to find alternative base matrices for solid-phase quenching reagents that would rapidly remove impurities (15 min or less) in a broader range of solvents. Macroporous support, a highly cross-linked polystyrene material class, is the support of choice for solid-phase organic synthesis in acetonitrile because it swells equally in polar and nonpolar solvents as long as the solvent wets the surface.33 In addition, macroporous resins are widely used as chromatography matrices where effective mass transfer between the pores and the bulk solvents can overcome the slow diffusion kinetics experienced when 1–2% cross-linked gels are used.34 The combination of solution-phase with solid-phase reagents allows for the selective removal of excess reagents and/or by-products within a certain reaction containing a wide variety of functional groups.2,12,32,35–44 These *

Abbreviations: Amberlite IRA 400 borohydride resin, commercially available resin for selective reduction of ,-unsaturated aldehydes and ketones; DBU, 1,8-diazabicyclo [5.4.0]undec-7-en; DCM, dichloromethane; DMF, dimethylformamide; EtOAc, ethyl acetate; Et2NH, diethylamine; Et3N, triethylamine; HBr, hydrobromic acid; HCl, hydrochloric acid; MS, mass spectrometry; MeOH, methyl alcohol; NH4OH, ammonium hydroxide; NaBH4, sodium borohydride; NaOH, sodium hydroxide; NaOMe, sodium methanolate; PAMAM, polyaminoamide resin (commercially available); RT, room temperature; THF, tetrahydrofuran. 33 A. K. Ghosh, P. Mathivanan, and J. Cappiello, Tetrahedron Lett. 38, 2427 (1997). 34 C. A. Doyle and J. G. Dorsey, in ‘‘Handbook of HPLC’’ (E. Katz, R. Eksteen, P. Schoenmakers, and N. Miller, eds.), p. 293. Marcel Dekker, New York, 1999. 35 S. W. Kaldor, J. E. Fritz, J. Tang, and E. R. McKinney, Bioorg. Med. Chem. Lett. 6, 3041 (1996). 36 S. D. Brown and R. W. Armstrong, J. Am. Chem. Soc. 118, 6331 (1996). 37 T. A. Keating and R. W. Armstrong, J. Am. Chem. Soc. 118, 2574 (1996). 38 S. D. Brown and R. W. Armstrong, J. Org. Chem. 62, 7076 (1997). 39 A. Cheminat, C. Benezra, M. J. Farral, and J. M. J. Frechet, Tetrahedron Lett. 21, 617 (1980). 40 A. Cheminat, C. Benezra, M. J. Farral, and J. M. J. Frechet, Can. J. Chem. 59, 1405 (1981). 41 J. M. J. Frechet, M. J. Farral, C. Benezra, and A. Cheminat, Polym. Prep. Am. Chem. Soc. Div. Polym. Chem. 21, 101 (1980). 42 J. J. Parlow, D. A. Mischke, and S. S. Woodard, J. Org. Chem. 62, 5908 (1997). 43 J. J. Parlow, W. Naing, M. S. South, and D. L. Flynn, Tetrahedron Lett. 38, 7959 (1997). 44 A. J. Shuker, M. G. Siegel, D. P. Matthews, and L. O. Weigel, Tetrahedron Lett. 38, 6149 (1997).

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Fig. 1. General approaches to the removal of excess reagents using nucleophilic and electrophilic scavenger resins.

compounds are anchored onto the resin leaving the desired material in solution so a simple filtration renders pure product without the need for further chromatographic purification as seen in Fig. 1. This principle makes use of innovative resins with a variety of functional groups backed by more than 20 years of research on the subject.11,12,32,38–47 A recent descriptive review summarizes a great amount of data.16 A considerable amount of scavenger resins is commercially available for these specific work-up purposes depending upon their applicability.48 In addition, specific scavenger resins can easily be custom prepared by simple chemical transformations on the available resins.37 When reacting two substrates in solution (solution phase) to form a desired product (R1-El-Nu-R2 in Fig. 1), a resin with the desired characteristics (solid phase) is utilized to trap undesired material. A scavenging resin, usually added upon reacting of the substrates, interacts with the undesired reagent, thus forming a chemically modified new resin. Upon simple filtration, this resin is separated from the reaction mixture providing (in some cases clean) product without further purification being necessary. For a solution-phase parallel synthesis to be efficient, a complete conversion of reactants to product with little or no formation of by-products or impurities is required. Here the concept of solid-phase-assisted solution-phase organic synthesis comes into play. The important characteristic to have in mind about polymer-assisted solution-phase organic synthesis 45

D. C. Sherrington and P. Hodge, ‘‘Synthesis and Separations Using Functional Polymers.’’ John Wiley & Sons, New York, 1988. 46 P. Lazlo, ‘‘Preparative Chemistry Using Supported Reagent.’’ Academic Press, New York, 1987. 47 D. L. Flynn, Med. Res. Rev. 19, 408 (1999). 48 Some representative examples can be found at www.sigma-aldrich.com, www.argotech.com, www.albmolecular.com, www.polymerlabs.com, www.glycopep.com, www.huric.com, www.novabiochem.com, and www.silicycle.com.

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should be the added simplicity in manipulating both reaction work-ups and purifications. This characteristic centers on simple resin filtration to separate pure product from starting material, thus allowing the chemist to use excess reagents to drive reactions to completion. Additional characteristics should be the cost efficiency factor and the ability to obtain larger amounts of final products.49 The use of scavenger resins in manual or automated parallel synthesis work-ups is intended to reduce purification times. Due to the commercial availability of a great number of scavenger resins, large amounts of synthetic organic reactions are benefited. One of the potential drawbacks these resins may encounter in organic synthesis is that in some instances a large quantity of resin is necessary to clean up a typical product, thus presenting physical difficulties since the beads themselves swell in solvent,49 a particularly difficult task when the reaction is performed on a small scale. To broaden the scope of applications for scavenger resins, the use of a combination of different resins50 is advised when the extraction of a variety of undesired products and/or excess starting materials is desired. Several polymer-supported quenching reagents have been designed to remove a wide variety of functionalities. These resins vary in applications depending upon their inherent chemical nature. Some of the most widely used scavenger supports include resins specific for trapping HCl,51 acid halides,28 alcohols,28 aldehydes,52,53 alkyl halides,28 amine scavengers,2,12,26,28,29 boronic acids,54,55 carboxylic acids,56,57 isocyanates,12,28,58 isothiocyanates,12,28,58 and sulfonyl chloride.2,12,28,30,32,51,55,59 In addition, some of these resins have been expanded into dendrimer-type resins with applications in combinatorial chemistry45,60 as high loading proton, nucleophile, or electrophile scavengers. Two creative examples are shown in 49

R. Santini, M. C. Griffith, and M. Qi, Tetrahedron Lett. 39, 8951 (1998). D. Cork and N. Hird, Drug Discov. Today 7, 56 (2002). 51 C. Gennari, S. Ceccarelli, U. Piarulli, C. Montalbetti, and R. F. W. Jackson, J. Org. Chem. 63, 5312 (1998). 52 N. Bic¸ak and B. F. s,enkal, J. Polym. Sci. 35, 2857 (1997). 53 M. Panunzio, M. Villa, A. Missio, T. Rossi, and P. Seneci, Tetrahedron Lett. 39, 6585 (1998). 54 P. Hodge and J. Waterhouse, J. Chem. Soc. Perkin Trans. I 2319 (1983). 55 A. G. M. Barrett, M. L. Smith, and F. J. Zecri, J. Chem. Soc. Chem. Commun. 2317 (1998). 56 S. Affrossman and J. P. Murray, J. Chem. Soc. B 1015 (1966). 57 I. C. Chisem, J. Rafelt, M. T. Shieh, J. Chisem, J. H. Clark, R. Jachuck, D. Macquarrie, C. Ramshaw, and K. Scott, J. Chem. Soc. Chem. Commun. 1949 (1998). 58 J. Habermann, S. V. Ley, and J. S. Scott, J. Chem. Soc. Perkin Trans. I 3127 (1998). 59 M. Caldarelli, J. Habermann, and S. V. Ley, Bioorg. Med. Chem. Lett. 9, 2049 (1999). 60 L. Williams and S. M. Neset, 4th Int. Elect. Conf. Synth. Org. Chem. (ECSOC-4), B0011 (2000). R. M. Kim, M. Manna, S. M. Hutchins, P. R. Griffin, N. A. Yates, A. M. Bernick, and K. T. Chapman, Proc. Natl. Acad. Sci. USA 93, 10012 (1996). A. B. Kantchev and J. R. Parquette, Tetrahedron Lett. 40, 8049 (1999). 50

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Fig. 2. Resin 1 is a [1,3,5]triazin-2-oxy-based resin with morpholine end tips. Resin 2 is a simple starbust polyaminoamide (PAMAM) commercially available resin with 64 surface primary amino groups.54 Commercially available basic resins that are available employed for neutralizing HCl51 contain a tertiary amine or pyridinyl functionality, which readily traps proton species. Some examples are amine-based morpholinomethyl (3) and piperidinemethyl (4) resins (Fig. 3). Commercially available acidic resins, as well as ion exchangers, that are used for scavenging amines or other basic compounds contain an acidic functionality, which interacts with the counter basic moiety, thus forming an easily removable salt. Some representative examples are those of benzoic acid (5) and sulfonic acid (6) resins (Fig. 4). Carbonate resins are generally used to neutralize strong mineral acids (e.g., HBr) when generated in situ as a by-product on certain reactions. These resins normally contain a quaternary ammonium carbonate salt functionality. Specific examples are triethylammonium carbonate (7) and trimethylammonium bicarbonate (8) resins (Fig. 5) ion exchanging the anionic halogen for carbonate. To capture electrophilic substrates such as acid halides, aldehydes, alkyl halides, isocyanates, and isothiocyanates, a variety of nucleophilic resins are commonly used. Some commercially available and representative examples are tris-(2-aminoethyl)amine (9), thiophenol (10), sulfonylhydrazide (11), triphenylphosphine (12), and methylthiourea (13) polymer resins (Fig. 6). To capture nucleophilic substrates such as alcohols, amines, triphenylphosphine, carboxylic acids, and the like, a variety of electrophilic resins have been developed. Some interesting examples are benzaldehyde (14), methylisocyanate (15), methylisothiocyanate (16), chloromethystyrene (17, Merrifield resin),61 benzenesulfonyl chloride (18), and N-methylisatoic anhydride (19) resins (Fig. 7). Among the custom made resins that have been developed recently for scavenging electrophilic substrates, a few examples are worth mentioning: oligo(ethyleneimine) (20), morpholinodiethanolamine (21), amine/aminoalcohol (22), guanidine (23), and 4-phenol-substituted (24) (Fig. 8). Some rather interesting custom made electrophilic resins that have been developed lately as nucleophile scavenging materials are the acid chloride (25) and arenesulfonyl chloride (26) resin shown in Fig. 9. Resin 26 is stable to various reaction conditions over resin 18 and can be used while conducting subsequent chemical steps.62 61 62

R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963). J. K. Rueter, S. O. Nortey, E. W. Baxter, G. C. Leo, and A. B. Reitz, Tetrahedron Lett. 39, 975 (1998). H. M. Zhong, M. N. Greco, and B. E. Maryanoff, J. Org Chem. 62, 9326 (1997).

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Fig. 2. [1,3,5]Triazin-2-oxy-based resin with morpholine end tips (1) and starbust polyaminoamide (2) commercially available resins.

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Fig. 3. Morpholino and piperidinemethyl-based commercially available basic resins.

Fig. 4. Benzoic and sulfonic acid-based commercially available acidic resins.

Fig. 5. Triethylammonium carbonate (7) and trimethylammonium bicarbonate (8)-based commercially available neutralizing resins.

Fig. 6. Tris-(2-aminoethyl)amine (9), thiophenol (10), sulfonylhydrazide (11), triphenylphosphine (12), and methylthiourea (13)-based commercially available nucleophilic resins.

Scavenger Resins Synthetic Applications

The synthesis of compound 2712,63 is of special interest since it shows the application of different types of resins (basic and electrophilic) being used sequentially at different stages during the synthesis for the removal 63

W. Murray, M. Wachter, D. Barton, and Y. Forero-Kelly, Synthesis 18 (1991).

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Fig. 7. Benzaldehyde (14), methylisocyanate (15), methylisothiocyanate (16) chloromethystyrene (17, Merrifield resin), benzenesulfonyl chloride (18), and N-methylisatoic anhydride (19)-based commercially available electrophilic resins.

Fig. 8. Oligo(ethyleneimine) (20), morpholinodiethanolamine (21), amine/aminoalcohol (22), guanidine (23), and 4-phenolsubstituted (24)-based custom made electrophilic scavenging resins.

Fig. 9. Acid chloride (25) and arenesulfonyl chloride (26)-based custom made nucleophilic scavenging resins.

of hydrochloric acid and excess of 4-hydrazinobenzoic acid, respectively. In the first step of the synthesis the reaction mixture is treated with commercially available morpholine resin 3 to trap hydrochloric acid,

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then after filtration of protonatated resin 3 the isocyanate resin 15 is added. Thus, an aminourea resin is being generated that can also be extracted from the reaction mixture by simple filtration affording clean 4-(3-methyl-5-phenyl-pyrazol-1-yl)-benzoic acid 27 (Fig. 10). Another interesting example encountering numerous applications was reported by Bolton et al.28 It comprises the implementation of a commercially available high-loading nucleophilic resin 9 for the removal of excess starting material 2-bromobenzoyl chloride (28) during the synthesis of a simple amide such as that of N-benzyl-2-bromo-N-methylbenzamide (29), as indicated in Fig. 11.

Fig. 10. Synthesis of 4-(3-methyl-5-phenyl-pyrazol-1-yl)-benzoic acid (27).

Fig. 11. Synthesis of N-benzyl-2-bromo-N-methylbenzamide (29).

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Rueter et al.62 described an efficient and clean synthesis of diethyl-(2-ptolyl-ethyl)amine (30) from 2-p-tolylethanol and diethylamine by making use of the benzenesulfonyl chloride resin (26) to ‘‘catch’’ the intermediate O-alkylated substrate (31) followed by the ‘‘release’’ (from the intermediate resin) of the final product (30) upon treatment with diethylamine (Fig. 12). When benzylbromide is allowed to react with 4-(3-mercapto-5-phenyl[1,2,4]triazol-4-ylmethyl)-benzamide (32) in order to synthesize the corresponding thiobenzyl adduct 4-(3-benzylsulfanyl-5-phenyl-[1,2,4]triazol-4-ylmethyl)-benzamide (33), a nucleophilic sulfur-based resin (34) is employed to trap excess benzylbromide from the reaction mixture affording a final clean product (33).28 In addition, a basic Amberlite (OH) is added to assist in the efficient deprotonation of the mercapto functionality in (34) as depicted in Fig. 13. A very interesting reported application of a resin combination (specifically for the synthesis of a wide variety of amides)12,63 is depicted by the synthesis of N-benzyl-2-bromo-N-methy-benzamide (35), shown in Fig. 14, from simple starting materials. At first, benzylmethylamine is reacted with

Fig. 12. Synthesis of diethyl-(2-p-tolyl-ethyl)amine (30).

Fig. 13. Synthesis of 4-(3-benzylsulfanyl-5-phenyl-[1,2,4]triazol-4-ylmethyl)-benzamide (33).

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Fig. 14. Synthesis of N-benzyl-2-bromo-N-methyl-benzamide (35).

Fig. 15. Synthesis of 1-benzyl-3-phenyl-thiourea (36).

2-bromobenzoyl chloride in the presence of the basic morpholine resin (3), which acts as a proton scavenger for hydrochloric acid generated in situ. Second, addition of the electrophilic (isocyanate) resin (15) yields a new chemically modified resin in the form of a urea when reacting with excess benzylmethylamine. Finally, upon filtration of the final urea resin, pure N-benzyl-2-bromo-N-methyl-benzamide (35) is isolated. The synthesis 1-benzyl-3-phenyl-thiourea (36) requires the application of a recently described and commercialized isatoic resin (19).1 Benzylamine readily reacts with isothiocyanato-benzene to afford 1-benzyl-3phenyl-thiourea (36). However, excess benzylamine needs to be removed by adding resin (19), thus providing pure product (36) upon simple filtration of the amide resin formed as depicted in Fig. 15. Recently, a novel commercially available acetoacetoxyethyl metacrylate resin (37) is finding wide applications as a selective electrophilic scavenger resin. This resin has the ability to differentiate primary amines from a mixture where secondary amines are present.64,65 An illustrative example is depicted in the synthesis of dibenzylamine (38, Fig. 16) from benzaldehyde and benzylamine. Unreacted benzylamine is selectively removed from the reaction mixture upon treatment with the ketoacetate resin (37). 64

Z. Yu, S. Alesso, D. Pears, P. A. Worthington, R. W. A. Luke, and M. Bradley, Tetrahedron Lett. 41, 8963 (2000). 65 Z. Yu, S. Alesso, D. Pears, P. A. Worthington, R. W. A. Luke, and M. Bradley, J. Chem. Soc. Perkin Trans. I 1947 (2001).

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Fig. 16. Synthesis of dibenzylamine (38).

Fig. 17. Synthesis of a boronic acid-based resin.

The newly developed boronic acid scavenger diethanolamine resin (39)66 (Fig. 17) has found applications in a wide variety of important coupling reactions, such as the Suzuki couplings.67 This resin is largely used in the pharmaceutical industry for drug development. A representative example published by Hall et al.66 shows the relatively fast immobilization of 4-carboxyboronic acid at room temperature. This resin undoubtedly will find extensive applications in modern synthetic organic chemistry as well, as demonstrated by the increased number of carbon–carbon,68 carbon– nitrogen,69,70 and carbon–oxygen71 bond formation, in addition to urea72

66

D. G. Hall, J. Tailor, and M. Gravel, Angew. Chem. Int. Ed. Engl. 38, 3064 (1999). N. Miyaura and A. Suzuki, Chem. Rev. 95, 2457 (1995). 68 A. Suzuki, J. Organomet. Chem. 576, 147 (1999). 69 J. F. Hartwig, Angew. Chem. Int. Ed. Engl. 37, 2046 (1998). J. P. Wolfe, S. Wagaw, J.-F. Marcoux, and S. L. Buchwald, Acc. Chem. Res. 31, 805 (1998). B. H. Yang and S. L. Buchwald, J. Organomet. Chem. 576, 125 (1999). 70 G. Mann, J. F. Hartwig, M. S. Driver, and C. Fernandez-Rivas, J. Am. Chem. Soc. 120, 827 (1998). B. C. Hamann and J. F. Hartwig, J. Am. Chem. Soc. 120, 7369 (1998). D. W. Old, J. P. Wolfe, and S. L. Buchwald, J. Am. Chem. Soc. 120, 9722 (1998). J. F. Hartwig, M. Kawatsure, S. I. Hauck, K. H. Shaughnessy, and L. M. Alcazar-Roman, J. Org. Chem. 67

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Fig. 18. Synthesis of tetra-substituted pyrazoles (40).

and ketone73 formation reactions in which boronic acids are being employed. Some useful resins that have found their way into quite substantial applications are the carbonate resins. For instance, trimethylammonium bicarbonate resin (8) is used to neutralize carboxylic acids or strong mineral acids formed in situ in specific reactions. An interesting example is the quenching/scavenging of acid bromide reported by Staufer and Katzenellenbogen74 during the synthesis of tetra-substituted pyrazoles (40) from immobilized starting materials as seen in Fig. 18. A nucleophilic resin with the potential for being transformed into a high-loading or dendrimer-type of resin is the triethylenetetraamine resin (9) employed in the scavenging of excess electrophiles. Representative and useful reactions for this resin are in the preparations of ureas and thioureas. For example, when 2-(2-isocyanato-ethyl)thiophene is reacted with butylamine during the preparation of 1-butyl-3-(2-thiophen-2-yl-ethyl) urea (41), triethylenetetraamine resin (9) readily scavenges any excess of 2-(2-isocyanato-ethyl)thiophene. This excess material is easily removed from the reaction mixture by simple filtration as the urea adduct resin (42).12 Similarly, in the synthesis of 1-(3-isopropoxy-propyl)-3-phenethylthiourea (43), triethylenetetraamine resin (9) readily scavenges excess (2-isothiocyanato-ethyl)benzene by forming a bis-isothiourea resin (44) easily removable by filtration from the reaction mixture,12 as indicated in Fig. 19. A quite useful resin in nucleophilic carbon–carbon bond formation reactions is the carboxylic resin (5). The soft metal from the nucleophile is 64, 5575 (1999). J. P. Wolfe and S. L. Buchwald, J. Org. Chem. 65, 1144 (2000). J. P. Wolfe, H. Tomori, J. P. Sadighi, J. Yin, and S. L. Buchwald, J. Org. Chem. 64, 1158 (2000). 71 D. A. Evans, J. L. Katz, and T. R. West, Tetrahedron Lett. 39, 2937 (1998). 72 G. A. Artamkina, A. G. Sergeev, and I. P. Beletskaya, Tetrahedron Lett. 42, 4381 (2001). 73 M. Haddach and J. R. McCarthy, Tetrahedron Lett. 40, 3109 (1999). 74 S. R. Stauffer and J. A. Katzenellenbogen, J. Comb. Chem. 2, 318 (2000).

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trapped in the form of a metal carboxylic salt resin (45) as depicted in Fig. 20. Upon completion of the carbon-carbon bond formation reaction, the resin is then added to the mixture. The resin addition insures both carboxylic salt formation as well as protonation of the hydroxy anion formed during the reaction. An illustrative example is the alkylation of benzaldehyde with buthyllithium.32 The reaction between these two reactants takes  place from 78 C to room temperature over a 2.5 h time period. The reaction mixture is then treated with carboxylic acid resin (5). Upon simple filtration, clean 1-phenylpentan-1-ol (46) is obtained. The synthesis of 3-benzyl-2-phenylthiazolidin-4-one (47, Fig. 21) demonstrates the mercaptane scavenging applicability of the aminoethanethiol resin (48). Benzaldehyde is allowed to react with benzylamine in the presence of mercapto acetic acid (49) in toluene under refluxing conditions.

Fig. 19. Synthesis of 1-butyl-3-(2-thiophen-2-yl-ethyl)urea (41) and 1-(3-isopropoxypropyl)-3-phenethylthiourea (43).

Fig. 20. Synthesis of 1-phenylpentan-1-ol (46).

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Fig. 21. Synthesis of 3-benzyl-2-phenylthiazolidin-4-one (47).

Fig. 22. Synthesis of benzylamine.

The addition of the aminoethanethiol resin (48) follows, and an aminoethanesulfanyl acetic acid resin (50) is being formed alongside the desired product. Upon filtration of the resin, clean 3-benzyl-2-phenylthiazolidin-4-one (47) is obtained.75 One of the most used resins in solid-phase combinatorial organic synthesis, which has found a myriad of applications, is the Merrifield resin (17).61 This resin is also the building block for a tremendous amount of novel resins being developed in combinatorial chemistry with applications in both solid-phase as well as solid-phase-assisted solution-phase combinatorial chemistry. A recent, useful, and novel example is the report of its being employed as a triphenylphosphine scavenging resin.76 During the conversion of azidomethylbenzene (51) into benzylamine, excess triphenylphosphine is allowed to react with Merrifield resin (17) in the presence of sodium iodide in acetone. A phosphonium-substituted resin (52) is thus formed. Upon simple filtration, pure benzylamine is isolated as shown in Fig. 22.

75 76

S. E. Ault-Justus, J. C. Hodges, and M. W. Wilson, Biotechnol. Bioeng. 61, 17 (1998). B. H. Lipshutz and P. A. Blomgren, Org. Lett. 3, 1869 (2001).

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Concluding Remarks

Described are varied resins available for wide application in solidphase-assisted solution-phase combinatorial chemistry during the process of the purification of reaction mixtures. Therefore, what was once a complex work-up procedure was transformed into a simple reaction mixture filtration with the possibility of avoiding liquid-phase extraction protocols for reaction quenching and work-ups. Hence, this process has some remarkable advantages. Primarily, there is minimization or avoidance of chromatographic purification of the desired product. This results in the feasibility of being implemented in high-throughput parallel automated synthesis. As a consequence, the reaction protocols allow for excess reagents to be added to drive solution-phase reactions to completion. In conclusion, solid-phaseassisted solution-phase combinatorial chemistry processes result in reduced time and costs in overall synthetic development. Experimental Section

Reagents and General Methods Unless otherwise indicated, all reactions are run in capped glass vials, without the use of an inert atmosphere, and were shaken on an orbital shaker. THF was purchased distilled in a sure-seal bottle. Other reagents and anhydrous solvents were commercially available and used without further purification. Scavenger resins are available from a wide variety of suppliers48 and used without further purification. Resin Preparation Oligo(ethyleneimine) Resin (20). A cold (0 ) solution of the chlorosulfonylated resin (18)77 (15 g, 0.12 mol) in dimethoxyethane (40 ml) was treated with a dimethoxyethane solution (20 ml) of triethylenetetraamine (0.596 mol) dropwise. The resulting reaction mixture was allowed to stir for 24 h at RT. Upon filtration, the hydrochloride salt form of the resin formed was washed with ethanol (20 ml) followed by water in excess amounts. The resin was further washed with a 5% NaOH solution (100 ml) under stirring for 30 min. Upon decantation, water (100 ml) was added and the resulting mixture was boiled for 30 min. The reaction mixture was filtered and washed successively with water in excess followed  by ethanol (20 ml). The resin was then dried under vacuum at 40 for 24 h.

77

N. Bic¸ak and B. F. s,enkal, React. Funct. Polym. 29, 123 (1996).

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Aminodiol/morpholine Resin (21). A suspension of Merrifield resin (2 g, 4.3 mmol Cl/g resin, 8.6 mmol) in DMF (20 ml) was treated with diethanolamine (1.5 g, 14.3 mmol) and morpholine (1.2 g, 14.3 mmol) by  shaking the resulting reaction mixture at 65 for 6 h under a nitrogen atmosphere. Upon cooling to RT, the resin was filtered and washed with MeOH, DMF, Et3N, MeOH, DCM, Et3N, MeOH, DCM, MeOH, DCM,  and MeOH. The resulting resin was then dried at 45 under vacuum. Amine/aminoalcohol Resin (22). A suspension of Merifield resin (5 g, 4.3 mmol Cl/g resin, 21.5 mmol) and morpholine (5.2 ml, 60 mmol) in DMF (35 ml) was treated with piperidin-3-yl-methanol (2.3 g, 20 mmol) at  65 for 6 h under a nitrogen atmosphere. Upon cooling to RT, the resin was filtered and washed with DMF, MeOH, Et3N, DMF, MeOH, Et3N, MeOH, DCM, MeOH, DCM, and EtOAc. The resulting amine/aminoalcohol resin  was dried at 45 under vacuum overnight. Guanidine Resin (23). A suspension of Merrifield resin (5 g, 1.7 mmol Cl/g resin) in DMF (100 ml) was treated with guanidine hydrochloride (5 g) and a solution (1 M) of potassium tert-butoxide in THF (50 ml).  The resulting reaction mixture was heated at 90 for 24 h. Upon cooling, the resulting resin was filtered and washed with DMF/DBU (7:3), DMF, dioxane, water, THF, and ethyl ether. The resin thus produced was dried under vacuum at RT overnight. Phenol Resin (24). A solution of 3-(4-hydroxyphenyl)propionic acid (2.2 g, 13.3 mmole), N-ethyl-N0 -dimethylaminopropylcarbodiimide (2.5 g, 13 mmol) and 1-hydroxybenzotriazole monohydrate (1.8 g, 12 mmol) in DMF (20 ml) was treated with aminomethylpolystyrene resin (1.0 g, 4.5 mmol N/g resin). The resulting reaction mixture was stirred for 36 h at room temperature. The product resin was obtained upon filtration and washings with DCM, MeOH, a solution of NH4OH:MeOH (1:1), DMF, DCM, MeOH, DCM, and hexanes (2) followed by drying under vacuum  at 40 . Acid Chloride Resin (25). A suspension of 1-substituted benzylamineHCl resin (2 g, 0.83 mmol N/g resin, 1.66 mmol) in DCM (20 ml) and N-methylmorpholine (1.2 ml, 10.9 mmol) was treated with benzene-1,3,5tricarboxylic acid chloride (0.93 g, 3.5 mmol) by shaking the reaction mixture for a few minutes. The resulting suspension was stirred at RT for 1 h, diluted with DCM (200 ml), and filtered. The resin was then washed with DCM (2) followed by EtOAc (2) and dried at 35 under vacuum. Arenesulfonyl Chloride Resin (26). To a suspension of chlorobenzyl Merrifield resin (5.0 g, 4.9 mmole) and anhydrous 4-hydroxybenzenesulfonic acid sodium salt (2.94 g, 15 mmole; commercial material was dehy drated at 110 in vacuo for 8 h) in N,N-dimethylacetamide (50 ml) was  added NaOMe (0.81 g, 15 mmol), and the mixture was stirred at 90 for 2

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days. The resin product (phenylsulfonic acid) was collected by filtration, washed (sequentially with DMF, 1.0 N HCl, MeOH, and DCM), and dried  under vacuum at 50 for 24 h. The resin was treated with 1:1 Et2NH–DMF (50 ml) for 1 h and washed with DMF. The reaction was suspended in DMF (50 ml) followed by the addition of PCl5 (5.2 g, 25 mmol) in several  portions, and the resulting suspension was stirred at 23 for 4 h. After washing the mixture with DMF and DCM, the resin was dried in a vacuum  oven at 50 overnight to afford the arenesulfonyl chloride resin suitable for further applications. Solid-Phase Assisted Solution-Phase Synthesis 4-(3-Methyl-5-phenyl-pyrazol-1-yl)-benzoic Acid (27). A mixture of 4-hydrazino-benzoic acid hydrochloride (113 mg, 0.6 mmol) and morpholine-based resin (3) in MeOH (2 ml) was treated with 1-phenyl-butane1,3-dione (81.5 mg, 0.5 mmol) and shaken for 2.5 h. The MeOH was allowed to evaporate under a stream of N2. DCM (4 ml) was then added to the reaction mixture followed by the addition of isocyanate resin (15) (350 mg). The resulting reaction mixture was shaken for 16 h at which time additional amounts of isocyanate resin (15) (120 mg) were added. The mixture was shaken for 4 h followed by filtration. The filtered resin was washed with DCM (2  1.5 ml). Upon concentration of the organic filtrate, clean product was obtained. MS: 278.11 (M + 1). N-Benzyl-2-bromo-N-methyl-benzamide (29). A solution of benzyl-Nmethylamine (0.4 mmol), triethylamine (3 mmol), and 2-bromobenzoylchloride (0.6 mmol) in DCM (1 ml) was shaken for 4 h. Amine-based resin (9) (100 mg) was added and the reaction mixture was shaken overnight. Upon filtration and concentration, the residue was partitioned between aqueous NaOH and EtOAc. Concentration of the organic layer afforded the purified product. MS: 304, 306 (M + 1). Diethyl-(2-p-tolyl-ethyl)-amine (30). A solution of 2-(p-tolyl)-ethanol (3–5 mole-equiv.), triethylamine (3–5 mole-equiv.), and arenesulfonyl chloride resin (26) in DCM (10 ml/mmol) was allowed to react at RT for 48 h. The resulting resin (31) was then filtered and washed with DCM (2), MeOH (2), and DCM (2). Upon treatment of resin (31) with  excess diethylamine at 60 for 6 h, diethyl-(2-p-tolyl-ethyl)-amine (30) was obtained. 4-(3-Benzylsulfanyl-5-phenyl-[1,2,4]triazol-4-ylmethyl)-benzamide (33). A solution of 4-(3-mercapto-5-phenyl-[1,2,4]triazol-4-ylmethyl)-benzamide (32, 0.1 mmol) in THF (6 ml) was treated with Amberlite resin (OH form, 0.1 mmol OH) and benzyl bromide (0.15 mmol). The resulting mixture was shaken at room temperature until complete consumption

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of the starting thiol. Aminothiol resin (34, 100 mg) was added and the mixture was shaken at RT for 1 h. TLC showed that the excess benzyl bromide was consumed. The resulting resins were removed by filtration and washed with DCM. Upon concentration of the combined filtrates, 4-(3-benzylsulfanyl-5-phenyl-[1,2,4]triazol-4-ylmethyl)-benzamide (33) was obtained. N-Benzyl-2-bromo-N-methyl-benzamide (35). A suspension of morpholine resin (3, 0.63 mmol) in DCM (2 ml) was treated with N-methylbenzylamine (0.23 mmol) and 2-bromobenzoyl chloride (0.146 mmol). The reaction mixture was shaken for 5 h. Isocyanate resin (15, 0.2 g) was added followed by DCM (1 ml). The reaction mixture was then shaken for 16 h. Filtration of the resin followed by concentration of the filtrate gave the purified product. 1-Benzyl-3-phenylthiourea (36). A solution of isothiocyanatobenzene (1 mmol) in DCM (7 ml) was treated with benzylamine (1.2 mmol). The resulting reaction mixture was allowed to stir at room temperature overnight. Then isatoic anhydride resin (19, 100 mg, 3.2 mmol anhydride/g) was introduced into the reaction vessel. The contents were shaken at RT for 1.5 h followed by resin filtration. Upon evaporation of the filtrate solvent, pure benzylamine-free 1-benzyl-3-phenyl-thiourea (36) was obtained. Dibenzylamine (38). A solution of benzaldehyde (0.5 mmol) in isopropanol (1.5 ml) was treated with benzylamine (0.8 mmol). The resulting reaction mixture was shaken at RT for 2 h. Then Amberlite IRA 400 borohydride resin (2.5 mmol NaBH4/g resin) was added and the mixture was shaken at RT for 24 h. DCM (1.5 ml) was introduced followed by the addition of the acetoacetoxyethyl metacrylate resin (37). The contents were shaken at RT for 36 h. Upon filtration of the resin followed by solvent evaporation of the filtrate, pure benzylamine-free dibenzylamine (38) was obtained. 1-Butyl-3-(2-thiophen-2-yl-ethyl)urea (41). A solution of 2-(thienyl-2yl)ethyl isocyanate (47 mg, 0.3 mmol) in DCM (2 ml) was treated with nbutylamine (25 l, 0.25 mmol) at RT. The reaction mixture was shaken for 1 h followed by addition of resin (9) (50 mg, 4.3 mmol N/g). After 2 h the resin was filtered and washed with DCM (2  1.5 ml). The combined organic filtrates, when concentrated to dryness, gave the desired urea (44 mg, 99% yield) as an oil that crystallizes upon prolonged standing. 1-(3-Isopropoxy-propyl)-3-phenethyl-thiourea (43). A solution of 3isopropoxypropyl amine (25 mg, 0.25 mmol) and 2-phenylethylisothiocyanate (44 l, 0.3 mmol) in DCM (2 ml) was shaken for 1.5 h at RT. The triethylenetetraamine resin (9, 50 mg) was then added and the resulting reaction mixture was shaken for 2 h. Upon filtration, the resin was washed with DCM (2  1.5 ml). Evaporation of the filtrates gave the starting-material-free thiourea.

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1-Phenylpentan-1-ol (46). A solution of benzaldehyde (0.5 mmol) in  THF (2 ml) was treated at 78 with a solution of n-buthyllithium (0.36 ml, 1.6 M solution in hexanes, 0.57 mmol). The resulting reaction mixture was allowed to slowly reach RT and stirred for 2.5 h. Then carboxylic acid resin (5, 0.80 g, 8 mmol, 10.0 mequiv./g) was added and the suspension was stirred for 4 h. The resin was filtered and rinsed with THF a few times until complete washing of the product. Upon evaporation of the filtrate, lithium-free product was obtained, which was then dissolved in DCM followed by the addition of polyamine resin (0.50 g, 1.49 mmol). This suspension was stirred at room temperature for 5 h. The reaction mixture was then filtered and the resin rinsed with DCM until complete washing of the product. Upon evaporation of the solvent, pure (benzaldehyde-free) 1-phenylpentan-1-ol was obtained. 3-Benzyl-2-phenylthiazolidin-4-one (47). A solution of benzylamine (0.2 ml, 0.2 mmol) in toluene (0.2 ml) was treated with a benzaldehyde (1.2 ml, 0.30 mmol) solution in toluene (1.2 ml), followed by the addition of a mecaptoacetic acid (49, 1.2 ml, 0.6 mmol) solution in toluene ˚ ). The reaction mixture was heated to (1.2 ml) and molecular sieves (3 A  80 for 1.5 h. Then 2-aminoethanethiol resin (48, 0.3 g, 1.0 mmol) was added and the mixture was allowed to cool to RT overnight. The mixture was then treated with basic alumina (0.5 g) and shaken for 1 h. Additional toluene (5 ml) was added. Upon filtration and evaporation of the solvent, the product (47) was thus obtained. Benzylamine. A solution of azidomethylbenzene (51) in THF was treated with water followed by excess addition of triphenylphosphine (75 mg, 0.28 mmol). The reaction mixture was allowed to react at RT. Upon concentration under vacuum, acetone (1.5 ml) was added followed by the addition of sodium iodide (84 mg, 0.56 mmol) and high loading Merrifield resin (17, 140 mg, 4.38 mmol of Cl/g). The resulting mixture was allowed to stir at RT overnight. The resin was filtered and washed with THF (3  3 ml), water (3  3 ml), acetone (3  3 ml), and finally methanol affording triphenylphosphine-free benzylamine.

Section VIII Small Molecule and Heterocycle Synthesis

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[22] Cyclative Cleavage Strategies for the Solid-Phase Synthesis of Heterocycles and Natural Products By A. Ganesan Introduction

For many years, solid-phase organic synthesis was predominantly employed in the stepwise assembly of peptides and nucleotides, using the C-terminal carboxylic acid and the 30 -terminal alcohol, respectively, as attachment points to the resin. Because these functional groups are part of the final oligomer, their unmasking by resin cleavage at the end of the synthesis is not an issue. More recently, solid-phase techniques have become tremendously popular for combinatorial chemistry, particularly in the areas of drug and catalyst discovery. In these applications, the functional group used for immobilization may serve no other purpose. Consequently, the release of this dangling functionality (colloquially referred to as a ‘‘navel’’) upon cleavage can be an undesirable ‘‘memory’’ or ‘‘trace’’ of the point of attachment. Since resin attachment usually involves nucleophilic and often ionizable heteroatom functional groups such as carboxylic acids, amines, alcohols, and thiols, this can seriously perturb the structural properties of the target molecule. One solution that circumvents the above problem is to design a solid-phase synthesis in which the last step is an intramolecular cyclization reaction involving the point of attachment (Fig. 1). This strategy was first termed ‘‘cyclative cleavage’’ in a review by DeWitt and Czarnik1 describing the Diversomer effort at Parke-Davis. Others have used ‘‘cyclitive cleavage,’’ ‘‘cyclization cleavage,’’ ‘‘cyclorelease,’’ ‘‘cycle-elimination,’’ or ‘‘traceless cleavage’’ interchangeably, although the original definition2 reserves ‘‘traceless’’ synthesis strictly for the formation of a C–H bond upon cleavage. Compared to conventional resin cleavage methods, the cyclative strategy has two principal advantages. First, the resulting cyclic molecule does not have a free vestigial functional group at the point of resin attachment. Second, only the final intermediate on solid phase has the necessary functionality for the cyclization reaction. This is of great value when carrying out multiple reaction steps on solid phase, as any by-products or failed intermediates of earlier reactions are unable to undergo the final cyclization. Thus, the overall yield of a cyclative cleavage sequence may be low or high depending on the efficiency of each step. However, even in those 1 2

S. H. DeWitt and A. W. Czarnik, Acc. Chem. Res. 29, 114 (1996). M. J. Plunkett and J. A. Ellman, J. Org. Chem. 60, 6006 (1995).

METHODS IN ENZYMOLOGY, VOL. 369

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Fig. 1. Conventional versus cyclative solid-phase cleavage.

cases in which the yield is low, the purity of cyclatively cleaved material should still be high. Often, compounds can be submitted to biological assays without further purification. In the following reaction schemes, the yields quoted are usually overall for the total sequence on solid phase. For simplicity, the exact nature of the solidphase linker is not detailed. While some of these cyclizations are likely to be efficient regardless of the linker used, others can be highly susceptible to steric and electronic effects. When in doubt, the original literature should be consulted, bearing in mind that it is the exception rather than the rule for authors to explain why a particular resin or linker was chosen. To facilitate an appreciation of the underlying chemistry, the examples are classified according to cyclization reaction rather than type of molecule produced. The literature coverage3 extends to mid-2002. The focus is on small molecules, and the synthesis of cyclic peptides4 is deliberately omitted. Nitrogen Nucleophile Attacking sp2 or sp3 Carbon: Five-Membered Ring Formation

This category represents the most popular subclass of cyclative cleavage reactions. There are many solid-phase sequences with resin attachment via carboxylic acids, followed by elaboration to a free amine five centers away 3

For other recent reviews on solid-phase cleavage strategies, see (a) A. C. Comely and S. E. Gibson, Angew. Chem. Int. Ed. Engl. 40, 1012 (2001). (b) V. Krchnˇa´k and M. W. Holladay, Chem. Rev. 102, 61 (2002). (c) P. Blaney, R. Grigg, and V. Sridharan, Chem. Rev. 102, 2607 (2002). 4 For a review, see J. N. Lambert, J. P. Mitchell, and K. D. Roberts, J. Chem. Soc., Perkin Trans. I 471 (2001).

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Fig. 2. Examples of hydantoin synthesis by cyclative cleavage.

and cleavage by intramolecular cyclization. A pioneering example5 is the acid-catalyzed cyclization reported by the Parke-Davis group (Fig. 2). Using the Diversomer apparatus for parallel synthesis, 39/40 hydantoins were successfully prepared. Later, the hydantoin cyclization was accomplished6 by a Lilly group under basic conditions by heating with excess triethylamine. An array of 800 hydantoins was prepared, and a random sampling of 15% of the library showed product formation in 90% of the cases. This was soon followed by a report by Kim et al.7 of similar cleavage at room temperature using neat diisopropylamine. In the hydantoin synthesis by Hanessian and Yang,8 the initial cyclization product undergoes solution-phase loss of benzyl alcohol and nucleophilic attack to give 5-alkoxyhydantoins, while that by Wilson et al.9 provides access to 1-aminohydantoins (Fig. 3). Cyclization in a more complex setting is illustrated by the Affymax10 transformation of a bicyclic scaffold resulting from 1,3-dipolar cycloaddition to a tricyclic hydantoin. Due to its importance in drug discovery, there are numerous other examples11 of hydantoin and thiohydantoin synthesis via cyclative cleavage. 5

S. H. DeWitt, J. S. Kiely, C. J. Stankovic, M. C. Schroeder, D. M. Reynolds Cody, and M. R. Pavia, Proc. Natl. Acad. Sci. USA 90, 6909 (1993). 6 B. A. Dressman, L. A. Spangle, and S. W. Kaldor, Tetrahedron Lett. 37, 937 (1996). 7 S. W. Kim, S. Y. Anh, J. S. Koh, J. H. Lee, S. Ro, and H. Y. Cho, Tetrahedron Lett. 38, 4603 (1997). 8 S. Hanessian and R.-Y. Yang, Tetrahedron Lett. 37, 5835 (1996). 9 L. Wilson, M. Li, and D. E. Portlock, Tetrahedron Lett. 39, 5135 (1998). 10 G. Peng, A. Sohn, and M. A. Gallop, J. Org. Chem. 64, 8342 (1999). 11 (a) J. Matthews and R. A. Rivero, J. Org. Chem. 62, 6090 (1997). (b) S. W. Kim, J. S. Koh, S. Ro, and E. J. Lee, Mol. Divers. 3, 129 (1997). (c) A. Boeijen, J. A. W. Kruitzer, and

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Fig. 3. Further examples of hydantoin synthesis by cyclative cleavage.

R. M. J. Liskamp, Bioorg. Med. Chem. Lett. 8, 2375 (1998). (d) J. Stadlwieser, E. P. EllmererMu¨ller, A. Tako´, N. Maslouh, and W. Bannwarth, Angew. Chem. Int. Ed. Engl. 37, 1402 (1998). (e) W. Karnbrock, M. Deeg, J. Gerhardt, and W. Rapp, Mol. Divers. 4, 165 (1998). (f) Y.-D. Gong, S. Najdi, M. M. Olmstead, and M. J. Kurth, J. Org. Chem. 63, 3081 (1998). (g) K.-H. Park, M. M. Olmstead, and M. J. Kurth, J. Org. Chem. 63, 6579 (1998). (h) S.-H. Lee, S.-H. Chung, and Y.-S. Lee, Tetrahedron Lett. 39, 9469 (1998). (i) Y. Hamuro, W. J. Marshall, and M. A. Scialdone, J. Comb. Chem. 1, 163 (1999). (j) K.-H. Park and M. J. Kurth, Tetrahedron Lett. 40, 5841 (1999). (k) K.-H. Park and M. J. Kurth, J. Org. Chem. 64, 9297 (1999). (l) K.-H. Park and M. J. Kurth, Tetrahedron Lett. 41, 7409 (2000). (m) F. Albericio, J. Garcia, E. L. Michelotti, E. Nicolas, and C. M. Tice, Tetrahedron Lett. 41, 3161 (2000). (n) M. Lamothe, M. Lannuzel, and M. Perez, J. Comb. Chem. 4, 73 (2002).

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A number of other five-membered ring nitrogen heterocycles have been prepared by cyclative cleavage. The illustrative examples (Fig. 4) depict the synthesis of pyrazolones,12 succinimides and phthalimides,13 pyrrolo[3,4-b] pyridines,14 2-aminoimidazolones,15 imidazo[4,5-b]pyridin-2-ones,16 and 1,2,4-triazoline-3,5-diones.17 The final example in this section features a rare instance where the electrophilic center is sp3-hybridized carbon, as most cyclative cleavages involve the attack of carbonyl derivatives. Oxazolidinones are formed cyclatively18 by the displacement of a sulfonate ester by an acylsulfonamide (Fig. 5). In a variant19 of this cyclization, a quasi-meso bis-sulfonate partitions into a pair of quasi-enantiomeric sulfonates, one resin bound and the other cleaved, depending on the direction of intramolecular cyclization. The resin-bound enantiomer can then be displaced by an external nucleophile. Nitrogen Nucleophile Attacking sp2 Carbonyl: Six-Membered Ring Formation

Among the six-membered ring heterocycles, diketopiperazines are most commonly prepared by cyclative cleavage. Indeed, diketopiperazine formation is often observed as an undesirable by-product during peptide synthesis20 and the facile nature of this cyclization makes it an obvious choice for library generation. In an early example from Pfizer,21 a set of 10 immobilized -amino acids was reductively alkylated with 10 aldehydes, followed by acylation with 10 -amino acids and cyclization (Fig. 6). By 12

(a) L. Tietze and A. Steinmetz, Synlett 667 (1996). (b) L. Tietze, A. Steinmetz, and F. Balkenhohl, Bioorg. Med. Chem. Lett. 7, 1303 (1997). (c) O. Attanasi, P. Filippone, B. Guidi, T. Hippe, F. Mantellini, and L. F. Tietze, Tetrahedron Lett. 40, 9277 (1999). (d) O. A. Attanasi, L. De Crescentini, P. Filippone, F. Mantellini, and L. F. Tietze, Tetrahedron 57, 5855 (2001). 13 (a) D. R. Barn and J. R. Morphy, J. Comb. Chem. 1, 151 (1999). (b) Z. Xiao, K. Schaefer, S. Firestone, and P.-K. Li, J. Comb. Chem. 4, 149 (2002). 14 A. Bhandari, B. Li, and M. A. Gallop, Synthesis 1951 (1999). 15 (a) D. H. Drewry and C. Ghiron, Tetrahedron Lett. 41, 6989 (2000). (b) M. Li and L. J. Wilson, Tetrahedron Lett. 42, 1455 (2001). 16 M. Ermann, N. M. Simkovsky, S. M. Roberts, D. M. Parry, and A. D. Baxter, J. Comb. Chem. 4, 352 (2002). 17 K.-H. Park and L. J. Cox, Tetrahedron Lett. 43, 3899 (2002). 18 (a) P. ten Holte, L. Thijs, and B. Zwanenburg, Tetrahedron Lett. 39, 7407 (1998). (b) P. ten Holte, B. C. J. van Esseveldt, L. Thijs, and B. Zwanenburg, Eur. J. Org. Chem. 2965 (2001). 19 P. ten Holte, L. Thijs, and B. Zwanenburg, Org. Lett. 3, 1093 (2001). 20 E. Pedoroso, A. Grandas, X. de las Heras, R. Eritja, and E. Giralt, Tetrahedron Lett. 27, 743 (1986). 21 D. W. Gordon and J. Steele, Bioorg. Med. Chem. Lett. 5, 47 (1995).

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Fig. 4. Five-membered azole heterocycles prepared by cyclative cleavage.

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Fig. 5. Cyclative cleavage at sp3 hybridized carbon.

Fig. 6. An early example of diketopiperazine library synthesis by Pfizer.

mix-and-split synthesis, a library of 1000 diketopiperazines was prepared as 10 mixtures, each potentially containing 100 components. Many groups have subsequently reported11i,22 syntheses of diketopiperazines and related scaffolds such as monoketopiperazines and diketomorpholines by cyclative cleavage. Among examples that are more complex 22

(a) J. Kowalski and M. A. Lipton, Tetrahedron Lett. 37, 5839 (1996). (b) A. Chucholowski, T. Masquelin, D. Obrecht, J. Stadlwieser, and J. M. Villalgordo, Chimia 50, 525 (1996). (c) A. K. Szardenings, T. S. Burkoth, H. H. Lu, D. W. Tien, and D. A. Campbell, Tetrahedron 53, 6573 (1997). (d) B. O. Scott, A. C. Siegmund, C. K. Marlowe, Y. Pei, and K. L. Spear, Mol. Divers. 1, 125 (1996). (e) A. K. Szardenings, D. Harris, S. Lam, L. Shi, D. Tien, Y. Wang, D. V. Patel, M. Navre, and D. A. Campbell, J. Med. Chem. 41, 2194 (1998). (f) R. A. Smith,

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Fig. 7. Examples of bi- and polycyclic diketopiperazine synthesis.

are the -turn mimetics prepared by Golebiowski et al.,23 and the solidphase total synthesis of demethoxyfumitremorgin C reported by Wang and Ganesan24 (Fig. 7). The research groups of Ganesan24 and Koomen25 have both utilized this procedure for the synthesis of analogues of fumitremorgin alkaloids, which have attracted attention as neuroactive agents, mammalian cell cycle inhibitors, and antagonists of the breast cancer resistance protein. M. A. Bobko, and W. Lee, Bioorg. Med. Chem. Lett. 8, 2369 (1998). (g) P. P. Fantauzzi and K. M. Yager, Tetrahedron Lett. 39, 1291 (1998). (h) M. del Fresno, J. Alsina, M. Royo, G. Barany, and F. Albericio, Tetrahedron Lett. 39, 2639 (1998). (i) W.-R. Li and S.-Z. Peng, Tetrahedron Lett. 39, 7373 (1998). (j) K. Shreder, L. Zhang, J.-P. Gleeson, J. A. Ericsson, V. V. Yalamouri, and M. Goodman, J. Comb. Chem. 1, 383 (1999). (k) F. Berst, A. B. Holmes, M. Ladlow, and P. J. Murray, Tetrahedron Lett. 41, 6649 (2000). (l) J. C. Gonzalez-Gomez, E. Uriarte-Villares, and S. Figueroa-Perez, Synlett 1085 (2002). 23 A. Golebiowski, S. R. Klopfenstein, J. J. Chen, and X. Shao, Tetrahedron Lett. 41, 4841 (2000). 24 H. Wang and A. Ganesan, Org. Lett. 1, 1647 (1999). 25 (a) A. van Loevezijn, J. H. van Maarseveen, K. Stegman, G. M. Visser, and G. J. Koomen, Tetrahedron Lett. 39, 4737 (1998). (b) A. van Loevezijn, J. D. Allen, A. H. Schinkel, and G. J. Koomen, Bioorg. Med. Chem. Lett. 11, 29 (2001).

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Fig. 8. Cyclative cleavage to give dihydropyrimidinediones, quinazolinediones, and quinolinones.

The reaction of immobilized -amino acids with isocyanates provides ureas that can be cyclatively cleaved26 to dihydropyrimidinediones, while the analogous reaction27 with anthranilic acids affords 2,4-quinazolinediones (Fig. 8). A similar process is the cyclization28 of -keto esters derived from substituted anthranilates to furnish 4-hydroxyquinolinones, while the hydrazones of immobilized -keto esters are cyclatively cleaved29 to dihydropyridazinones. 26

S. A. Kolodziej and B. C. Hamper, Tetrahedron Lett. 37, 5277 (1996). (a) L. Gouilleux, J.-A. Fehrentz, F. Winternitz, and J. Martinez, Tetrahedron Lett. 37, 7031 (1996). (b) A. L. Smith, C. G. Thomson, and P. D. Leeson, Bioorg. Med. Chem. Lett. 6, 1483 (1996). (c) H. Shao, M. Colucci, S. Tong, H. Zhang, and A. L. Castelhano, Tetrahedron Lett. 39, 7235 (1998). (d) H.-Y. P. Choo, M. Kim, S. K. Lee, S. W. Kim, and I. K. Chung, Bioorg. Med. Chem. 10, 517 (2002). 28 M. M. Sim, C. L. Lee, and A. Ganesan, Tetrahedron Lett. 39, 6399 (1998). 29 N. Gouault, J.-F. Cupif, S. Picard, A. Lecat, and M. David, J. Pharm. Pharmacol. 53, 981 (2001). 27

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Fig. 9. Cyclative cleavage syntheses of quinazolinones.

A Roche synthesis22b,30 (Fig. 9) illustrates the basic principle behind the cyclization of substituted anthranilates to quinazolinones, a route similar to other31 examples. The concise solid-phase total synthesis32 of the fumiquinazoline alkaloid glyantrypine features a different approach whereby a piperidine amidine undergoes thermal reorganization to the natural product via two six-membered ring closures. The paper also describes the preparation of unnatural analogues. Nitrogen Nucleophile Attacking sp2 Carbonyl: Seven-Membered and Larger Ring Formation

Cyclative cleavage to five- and six-membered ring heterocycles is dominated by the synthesis of hydantoins and diketopiperazines, respectively. In the seven-membered ring case, it is the benzodiazepine nucleus that has attracted attention, due to its status as a ‘‘privileged structure’’ in drug discovery. In the precombinatorial days, Camps et al.33 reported a 30

J. M. Villalgordo, D. Obrecht, and A. Chucholowsky, Synlett 1405 (1998). (a) R.-Y. Yang and A. Kaplan, Tetrahedron Lett. 41, 7005 (2000). (b) A. P. Kesarwani, G. K. Srivastava, S. K. Rastogi, and B. Kundu, Tetrahedron Lett. 43, 5579 (2002). (c) Y. Yu, J. M. Ostresh, and R. A. Houghten, J. Org. Chem. 67, 5831 (2002). 32 H. Wang and A. Ganesan, J. Comb. Chem. 2, 186 (2000). 33 F. Camps, J. Cartells, and J. Pi, An. Quim. 70, 848 (1974). 31

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Fig. 10. Cyclative cleavage syntheses of benzodiazepines.

solid-phase route to benzodiazepinones that was later adapted to parallel synthesis5 by the Parke-Davis group (Fig. 10). A related approach34 provides access to 1,4-benzodiazepine-2,5-diones. Due to the extra degrees of freedom and entropic cost, cyclization reactions that are suitable for five- and six-membered ring formation can be a lot slower and proceed in lower yields for the seven-membered ring case. While the cyclization22g of a tetrahydro--carboline proceeded reasonably (Fig. 11), the cleavage35 of an acyclic sulfonamide needed extended reaction times and a variation of the fumiquinazoline total synthesis32 for seven-membered ring formation proceeded in poor yield. A cyclization of !-amino acids is the sole report36 of even larger ring sizes being formed. In this case, the acid was activated as an HOBt ester on solid phase. Oxygen Nucleophiles

Although cyclization using an amine is more common, there are examples with oxygen nucleophiles. An alcohol was employed37 to give both five- and six-membered lactones (Fig. 12). In a similar vein are the 34

J. P. Mayer, Z. Jingwen, K. Bjergarde, D. M. Lenz, and J. J. Gaudino, Tetrahedron Lett. 37, 8081 (1996). 35 D. B. A. de Bont, W. J. Moree, and R. M. J. Liskamp, Bioorg. Med. Chem. 4, 667 (1996). 36 W. Huang and A. G. Kalivretenos, Tetrahedron Lett. 36, 9113 (1995).

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Fig. 11. Other cyclative cleavages forming seven-membered or larger sized rings.

syntheses of oxazolidinones38 and phthalides39 by cyclative cleavage. A coumarin synthesis40 relied on photochemical isomerization as the trigger for cleavage, and the method was demonstrated for quinolone synthesis when the phenol was replaced with an aniline.

37

(a) C. Le Hetet, M. David, F. Carreaux, B. Carboni, and A. Sauleau, Tetrahedron Lett. 38, 5153 (1997). (b) S. Kobayashi, T. Wakabayashi, and M. Yasuda, J. Org. Chem. 63, 4868 (1998). (c) N. Gouault, J.-F. Cupif, A. Sauleau, and M. David, Tetrahedron Lett. 41, 7293 (2000). 38 H.-P. Buchstaller, Tetrahedron 54, 3465 (1998). 39 (a) P. Garibay, P. H. Toy, T. Hoeg-Jensen, and K. D. Janda, Synlett 1438 (1999). (b) P. Garibay, P. Vedsø, M. Begtrup, and T. Hoeg-Jensen, J. Comb. Chem. 3, 332 (2001). 40 Y. Kondo, K. Inamoto, and T. Sakamoto, J. Comb. Chem. 2, 232 (2000).

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Fig. 12. Examples of cyclative cleavage by oxygen nucleophiles.

Carbon Nucleophiles

The first cyclative cleavage of a small molecule was Crowley and Rapoport’s study41 of intramolecular Dieckmann condensations on solid phase (Fig. 13), which was complicated by the reversible nature of the cyclization. Despite the unique opportunities afforded by C–C rather than C–X bond formation during cleavage, there are few examples from the combinatorial age. A modern version of the Claisen-type condensation by Kulkarni and Ganesan42 uses a strongly acidic active methylene group to ensure unidirectional cyclization, and furnishes tetramic acids with three points of diversity.

41

(a) H. Rapoport and J. I. Crowley, J. Am. Chem. Soc. 92, 6363 (1970). (b) J. I. Crowley and H. Rapoport, J. Org. Chem. 45, 3215 (1980). 42 B. A. Kulkarni and A. Ganesan, Tetrahedron Lett. 39, 4369 (1998).

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Fig. 13. Cyclative cleavages based on Claisen-type condensations.

Soon thereafter, the same route was published43 by three industrial groups with slight variations in substrate and cleavage conditions. Organometallic Reactions

Many transition metal-catalyzed cross-coupling reactions have potential for effecting cyclative cleavage, and exhibit a wide tolerance of functional groups. The Nicolaou group demonstrated44 the feasibility of macrocyclizations via Stille coupling in a total synthesis of zearalenone (Fig. 14). The cyclative cleavage yielded a bis-MEM ether, which upon deprotection afforded the natural product. The application of ring closing metathesis for the synthesis of Friedinger lactams was studied by Piscopio et al.45 (Fig. 15), as well as other groups,46 while the formation of macrocycles was accomplished by Blechert’s group.47 43

(a) J. Mathews and R. A. Rivero, J. Org. Chem. 63, 4808 (1998). (b) L. Weber, P. Iaiza, G. Biringer, and P. T. Barbier, Synlett 1156 (1998). (c) T. Romoff, L. Ma, Y. Wang, and D. A. Campbell, Synlett 1341 (1998). 44 K. C. Nicolaou, N. Winssinger, J. Pastor, and F. Murphy, Angew. Chem. Int. Ed. Engl. 37, 2534 (1998). 45 (a) A. D. Piscopio, J. F. Miller, and K. Koch, Tetrahedron Lett. 38, 7143 (1997). (b) A. D. Piscopio, J. F. Miller, and K. Koch, Tetrahedron Lett. 39, 2667 (1998). (c) A. D. Piscopio, J. F. Miller, and K. Koch, Tetrahedron 55, 8189 (1999). 46 (a) J. H. van Maarseveen, J. A. J. den Hartog, V. Engelen, E. Finner, G. Visser, and C. G. Kruse, Tetrahedron Lett. 37, 8249 (1996). (b) J. J. N. Veerman, J. H. van Maarseveen, G. M. Visser, C. G. Kruse, H. E. Schoemaker, H. Hiemstra, and F. P. J. T. Rutjes, Eur. J. Org. Chem. 2583 (1998). (c) R. C. D. Brown, J. L. Castro, and J.-D. Moriggi, Tetrahedron Lett. 41, 3681 (2000). 47 J. Pernerfoster, M. Schuster, and S. Blechert, Chem. Commun. 1949 (1997).

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Fig. 14. Cyclative cleavage via Stille macrocyclization.

Fig. 15. Cyclative cleavage via ring closing metathesis.

The most impressive solid-phase sequence leading to a nonoligomeric molecule is the Nicolaou group’s total synthesis48 of the epothilone mitotic spindle poisons. Here, ring closing metathesis resulted in a mixture of four separable diastereomeric macrocylic olefins (Fig. 16). These were deprotected and epoxidized in solution phase, and one of the diastereomers thus converted to synthetic epothilone A. The method was also applied to the synthesis of diverse analogues for biological screening.

48

(a) K. C. Nicolaou, N. Winssinger, J. Pastor, S. Ninkovic, F. Sarabia, Y. He, D. Vourloumis, Z. Yang, T. Li, P. Giannakakou, and E. Hamel, Nature 387, 268 (1997). (b) K. C. Nicolaou, D. Vourloumis, T. H. Li, J. Pastor, N. Winssinger, Y. He, S. Ninkovic, F. Sarabia, H. Vallberg, F. Roschangar, N. P. King, M. R. V. Finlay, P. Giannakakou, P. VerdierPinard, and E. Hamel, Angew. Chem. Int. Ed. Engl. 36, 2097 (1997).

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Fig. 16. Cyclative cleavage as part of an epothilone A total synthesis.

‘‘Reverse’’ Cyclative Cleavage In polar cyclative cleavages, it is invariably the terminal functional group that is a nucleophile in attacking the point of resin attachment. However, the reverse disconnection whereby the functional group at the point of immobilization serves as the nucleophile is possible. This can also result in cyclization and cleavage from the resin, although it strictly falls outside the original definition of cyclative cleavage. Haloetherification49 and halolactonization50 reactions, for example, were studied by Kurth’s group, while cyanogen bromide-mediated cleavage51 of methionine residues was another route to lactones (Fig. 17). The intramolecular ring opening of epoxides,37a trapping of N-acyliminium ions to give bicyclic -lactams,52 and a synthesis of benzisoxazoles53 are other examples of heterocycles prepared in this manner. To date, ‘‘reverse’’ cyclative cleavage with C–C bond formation has relied on ylid chemistry. A single example was reported54 of the intramolecular Wittig condensation of an immobilized amide to give a twosubstituted indole (Fig. 18). Macrocyclization through an immobilized

49

(a) X. Beebe, N. E. Schore, and M. J. Kurth, J. Am. Chem. Soc. 114, 10061 (1992). (b) X. Beebe, N. E. Schore, and M. J. Kurth, J. Org. Chem. 60, 4196 (1995). 50 (a) H.-S. Moon, N. E. Schore, and M. J. Kurth, J. Org. Chem. 57, 6088 (1992). (b) H.-S. Moon, N. E. Schore, and M. J. Kurth, Tetrahedron Lett. 35, 8915 (1994). (c) D. A. Ockey, D. R. Lane, J. A. Seeley, and N. E. Schore, Tetrahedron 56, 711 (2000). 51 D.-H. Ko, D. J. Kim, C. S. Lyu, I. K. Min, and H.-S. Moon, Tetrahedron Lett. 39, 297 (1998). 52 (a) B. Furman, R. Thurmer, Z. Kałuza, W. Voelter, and M. Chmielewski, Tetrahedron Lett. 40, 5909 (1999). (b) B. Furman, R. Thu¨rmer, Z. Kałuza, R. Lysek, W. Voelter, and M. Chmielewski, Angew. Chem. Int. Ed. Engl. 38, 1121 (1999). 53 (a) S. D. Lepore and M. R. Wiley, J. Org. Chem. 64, 4547 (1999). (b) S. D. Lepore and M. R. Wiley, J. Org. Chem. 65, 2924 (2000). 54 I. Hughes, Tetrahedron Lett. 37, 7595 (1996).

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Fig. 17. ‘‘Reverse’’ cyclative cleavage by oxygen and nitrogen nucleophiles.

-ketophosphonate was the pivotal step in Nicolaou’s approach55 to muscone. Conjugate addition of dimethyl lithiocuprate completed the total synthesis. Another recent example56 involved macrocyclization through a sulfur ylid and ejection by cyclopropane formation. 55

K. C. Nicolaou, J. Pastor, N. Winssinger, and F. Murphy, J. Am. Chem. Soc. 120, 5132 (1998). 56 E. La Porta, U. Piarulli, F. Cardullo, A. Paio, S. Provera, P. Seneci, and C. Gennari, Tetrahedron Lett. 43, 761 (2002).

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Fig. 18. ‘‘Reverse’’ cyclative cleavage involving phosphorus and sulfur chemistry.

Summary

In solid-phase synthesis, cyclative cleavage can be achieved by any of a broad range of reaction chemistries. It is a powerful strategy for effecting resin cleavage that often proceeds under mild reaction conditions, sometimes requiring only heating in the absence of acids or bases. Further advances can be anticipated in both the types of reactions used for cyclative cleavage as well as the classes of target molecules released. Experimental

Reagents and General Methods Fmoc*-amino acids loaded on Wang resin are purchased from CN Biosciences (San Diego, CA). All other reagents and solvents are from Sigma-Aldrich (Milwaukee, WI). *

Abbreviations: DMF, N,N-dimethylformamide; Fmoc, (9H-fluoren-9-ylmethoxy)carbonyl; Phe, phenylalanine; THF, tetrahydrofuran; Trp, tryptophan; Val, valine.

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Solid-Phase Synthesis Terminated by Diketopiperazine Cyclative Cleavage (Scheme 1). The Fmoc-l-Trp resin (loading 0.5 mmol/g) is first deprotected with 20% piperidine in DMF for 20 min, and the resin washed and dried. The resin (1 g, 0.62 mmol/g) is shaken with benzaldehyde (0.63 ml, 10 molar equiv.) and trimethyl orthoformate (1.36 ml, 20 molar equiv.) in 7 ml CH2Cl2 overnight, followed by washing and drying to give resin 1. The resin is swollen in CH2Cl2, and solutions of Fmoc-l-Phe-Cl (0.5 M in CH2Cl2, 10 molar equiv.) and pyridine (4 M in CH2Cl2, 15 molar equiv.) were added. After agitation for 40 h, the resin 2 is filtered, washed, and dried. Cyclative cleavage is achieved by the addition of 20% piperidine in CH2Cl2 for 20 min. The supernatant is filtered off, and the resin washed. The combined filtrates are concentrated and triturated with hexanes to remove the nonpolar dibenzofulvene-piperidine adduct. The residue is purified by preparative thin-layer chromatography on silica. Two major bands, corresponding to the cis and trans diastereomers of the tetrahydro--carboline 3, are collected to furnish the desired material in a total of 85% yield (based on the loading of the Trp-Wang resin), cis–trans ratio 57/43. Solid-Phase Synthesis Terminated by Tetramic Acid Cyclative Cleavage (Scheme 2). The Fmoc-l-Val resin (loading 0.7 mmol/g) is first deprotected with 20% piperidine in DMF for 20 min, and the resin washed and dried. The resin (0.3 g, 0.67 mmol/g) is shaken with p-anisaldehyde (0.25 ml, 10 molar equiv.) and sodium triacetoxyborohydride (0.65 g, 15 molar equiv.) in 10 ml CH2Cl2 for 8 h, followed by washing and drying to give resin 4.

Scheme 1. Synthesis of a tetrahydro--carboline-diketopiperazine.

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Scheme 2. Synthesis of a tetramic acid.

The resin is swollen in CH2Cl2, and hydroxybenzotriazole hydrate (0.41 g, 15 molar equiv.) and cyanoacetic acid (0.26 g, 15 molar equiv.)  were added. After cooling to 0 , diisopropylcarbodiimide (0.64 ml, 20 molar equiv.) is added. The reaction mixture is warmed to room temperature and agitated for 18 h, after which the resin 5 is filtered, washed, and dried. Cyclative cleavage is achieved by the addition of 1 M tetrabutylammonium hydroxide in MeOH (0.82 ml) and THF (10 ml) for 6 h. The supernatant is filtered off, the resin washed, and the filtrates combined to afford the tetramic acid 6 as its tetrabutylammonium salt. The filtrate is acidified with Amberlyst A-15 sulfonic acid ion-exchange resin (0.71 g) and the resin washed. The ion-exchange resin treatment is repeated, and the combined filtrates concentrated. After trituration with hexanes, the free tetramic acid is obtained in 91% yield (based on the loading of the Val-Wang resin) as a colorless solid. Acknowledgments The work described in the experimental section was carried out at the Institute of Molecular and Cell Biology, National University of Singapore, and funded by the National Science and Technology Board of Singapore.

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[23] Derivatization Reactions of Heterocyclic Scaffolds on Solid Phase: Tools for the Synthesis of Drug-Like Molecule Libraries By Eduard R. Felder, Wolfgang K.-D. Brill, and Katia Martina Introduction

The quest to identify new molecular entities with useful, novel, or enhanced property profiles has intensified over the past years, among other reasons because of the incessant rise of new assays having high-throughput screening capacity. The pharmaceutical industry is particularly committed to the generation of large numbers of patentable compounds to choose from, in view of the high attrition rate experienced in selecting most effective and well-tolerated new drugs. In the earliest research phases the compounds must match a precisely defined property profile in order to confirm their potential to progress rapidly toward market application. Combinatorial chemistry is a rich source for the rapid generation of new compounds, but its value as a drug discovery tool goes beyond the prolific delivery of molecules and resides also in its inherent flexibility to adapt to specific problem solving. The first applications underscored the powerful numeric productivity resulting from the basic principles of combinatorial chemistry formulated by Furka et al.1 For years the chemical nature of combinatorial libraries was centered around oligomeric compounds such as peptides and analogs thereof, i.e., chemical classes with serious limits in the role of drug candidates or lead structures. The potentially much broader scope of the technology became evident once the combinatorial principle was shown to be applicable not only by linking monomeric building blocks to chain-like molecules, but also by combining chemical transformations, cyclizations, or, for that matter, any type of derivatization.2 Combinatorial chemistry is now used broadly throughout the drug discovery process,3 not only for the production of large libraries for lead finding, but also for the optimization of leads with focused libraries, designed with maximum input from existing structural information. The drug industry pays great attention to ensuring that newly generated molecules are as ‘‘drug-like’’ as possible, thus possessing the kind of 1

A. Furka, F. Sebestyen, M. Asgedom, and G. Dibo, Abstr. 14th Int. Cong. Biochem. Prague 5 (Abstr. FR:013), 47 (1988). 2 B. A. Bunin and J. A. Ellman, J. Am. Chem. Soc. 114, 10997 (1992). 3 R. E. Dolle, J. Comb. Chem. 3, 1 (2001).

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

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physicochemical properties compatible with bioavailability and favorable pharmacokinetics. A seminal paper by Lipinski et al.4 has contributed to raising consciousness about applying simple, but statistically validated, relevant predictor rules as filters on large compound lists when planning syntheses or compound acquisitions. The aim is to limit the appearance of compound-associated problems (e.g., lack of oral absorption) in drug development. Lipinski’s caution concerning poor absorption or permeation is commonly referred to as the ‘‘rule-of-five,’’ because the cutoffs for each of its four parameters are all close to five or a multiple of five. Unless specific biological transport mechanisms exist, compounds matching more than one of the following descriptions are likely to be poorly absorbed: (1) there are more than five H-bond donors, (2) the molecular weight is over 500, (3) the log P is over 5, and (4) there are more than 10 H-bond acceptors. Computational methods have been designed to estimate these and other predictive properties in silico prior to the production of compound libraries.5 Heterocycles turn out to be an excellent chemical platform for the generation of drug-like molecules, taking into account the criteria just described. In retrospect, this is confirmed by the observation that heterocycles are very common among drugs. According to the CMC2001.1 database, 56.8% of the current drugs contain heterocyclic entities.6 A few simple additional considerations further substantiate the excellent prerequisites of heterocyclic compounds in terms of ‘‘drug-likeness.’’ Lowmolecular-weight compounds predominantly bind to hydrophobic pockets on proteins. Although hydrogen bonds or electrostatic interactions within a hydrophobic environment enhance binding dramatically, the hydrophobic contacts between drug and receptor have to be maximized as a consequence of the Lennard–Jones potential. This can be the case only if the drug molecule has a shape complementing that of its binding site on a protein. Polar drugs often have liabilities, which ultimately originate from the energy barrier associated with dismantling the solvation shield (dehydration) in processes critical for bioavailability (transport across membranes). A tightly bound drug molecule is likely to be buried deeply in a hole or a fold of its receptor. The geometries of optimal hydrogen bonding between polar residues have to be fulfilled. The interacting functionalities have to be preoriented so that binding results in minimal conformational strain on drug 4

C. A. Lipinski, F. Lombardo, B. W. Dominy, and P. J. Feeney, Adv. Drug. Delivery Rev. 23, 3 (1997). 5 H. Matter, K.-H. Baringhaus, T. Naumann, T. Klabunde, and B. Pirard, Comb. Chem. High Throughput Screen. 4, 453 (2001). 6 The CMC 2001.1 database is a product of MDL Information Systems Inc. (San Leandro, CA, USA).

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437

and target. Electric fields within the binding pocket should be compensated. The conformational flexibility should be as low as possible. In narrow protein folds the optimal orientation of clustered functional groups may be achieved upon fixation onto or integration into cyclic structures.7 Aromatic heterocycles allow hydrophobic interactions and dipole interactions to be fine tuned by electronic alterations of their -systems. The chemistry of many types of heterocycles is well known and versatile methodologies exist for attaching a wide variety of functional groups. These features are very favorable when large numbers of analogs of a certain scaffold type have to be synthesized using multiparallel synthesis. Solid-phase synthesis being a key preparative methodology in the industrial context of high-throughput processes and laboratory automation, heterocyclic chemistry has been adapted to this format over recent years. A detailed protocol about the solid supported synthesis of 1,4-benzodiazepine libraries has been published.8 A vast number of publications related to solid-phase heterocyclic chemistry have appeared and reviews on this subject reflect this intense activity.9,10 We noticed, however, that this literature mostly focuses on the formation of heterocycles from noncyclic precursor building blocks. Derivatizations of already preformed heterocyclic scaffolds with straightforward standard reactions are covered only occasionally. Here we illustrate selected examples of common derivatization reactions on heterocycles grafted on the solid phase. The aim is to provide a sense of the relevant factors and experimental conditions that allow application of known chemical reactions on solid supported heterocyclic substrates for the preparation of novel compounds. In industry, it is not uncommon to seek and secure novelty in a whole chemical class rather than in single derivatives. A direct route to broad coverage aims at the development of a novel heterocyclic scaffold, which in turn is derivatized with standard reactions in order to create a thematic library. The novel scaffold material may be prepared in bulk, typically in solution. Subsequently it is loaded onto a solid support, which is then appropriately portioned for multiple derivatizations in parallel. The latter may involve common reactions, but the resulting products are novel. Examples of such reactions, ordinary in nature, but powerful when applied to innovative heterocyclic scaffolds, will be discussed. Attention is focused on specific steps of interest, which stand out as representatives 7

O. Brummer, B. Clapham, and K. D. Janda, Curr. Opin. Drug Discov. Dev. 3, 462 (2000). B. A. Bunin, M. J. Plunkett, and J. A. Ellman, Methods Enzymol. 267, 448 (1996). 9 P. M. S. Chauhan and S. K. Srivastava, Comb. Chem. High Throughput Screen. 4, 35 (2001). 10 E. R. Felder and A. L. Marzinzik, in ‘‘Combinatorial Chemistry—A Practical Approach’’ (W. Bannwarth and E. R. Felder, eds.), p. 157. Wiley, Weinheim, 2000. 8

438

small molecule and heterocycle synthesis

[23]

of broader significance. Lengthy descriptions of complete pathways (and the related information overlap on repetitive steps) are beyond the scope of this chapter. Particular emphasis is put on nucleophilic aromatic displacements (SNAr), a type of reaction that is potentially useful on a large variety of appropriate precursors, for example, on halogenated cores. In solid-phase synthesis intermediates and products are bound to a solid support via a covalent linker. The linker must allow selective removal of the final product from the support, but must be stable under the reaction conditions throughout the synthesis. The advantage of a solid-phase approach is that reagents can be used in large excess to drive reactions to completion and most side products are just washed off from the solid support. However, the solid-phase implies steric constraints onto the reactions performed. The choice of method depends on the synthetic problem; it is often not obvious and usually results from a reaction optimization study. Nucleophilic Aromatic Displacements

SNAr reactions can be used to bind scaffolds onto linkers, to perform ring closure reactions to afford a scaffold, or to diversify the scaffold with various groups or functionalities. Grafting a scaffold to a polymeric support sometimes involves heterocycles having more than one atom susceptible to SNAr reactions. Characteristic examples for syntheses involving SNAr resin capture reactions are derivatizations of pyrazines,11 pyrimidines,12 and triazines.13,14 Ding et al.15 captured a number of dichloroheterocyclic scaffolds, where one chloro atom is prone to nucleophilic aromatic substitution onto resinbound amine nucleophiles, as shown in Fig. 1. They found the PAL* linker 11

I. Parrot, C. G. Wermuth, and M. Hilbert, Tetrahedron Lett. 40, 7975 (1999). F. Guillier, P. Roussel, H. Moser, P. Kane, and M. Bradley, Chem. Eur. J. 5, 3450 (1999). 13 M. Stankova and M. Lebl, Mol. Divers. 2, 75 (1996). 14 T. Masquelin, N. Meunier, F. Gerber, and G. Rosse, Heterocycles 48, 2489 (1998). 15 S. Ding, N. S. Gray, X. Wu, Q. Ding, and P. G. Schultz, J. Am. Chem. Soc. 124, 1594 (2002). * Abbreviations: BuOH, n-butanol; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane; DEAD, diethylazodicarboxylate; DiAD, diisopropylazodicarboxylate; DIC, diisopropylcarbodiimide; DIPEA, diisopropylethylamine; DMA, N,N-dimethylacetamide; DME, dimethoxyethane; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; Dppp, 1,3-bis-(diphenylphosphino)-propane; EtOAc, ethylacetate; EtOH, ethanol; HATU, (N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide; HOAc, acetic acid; HOAt, 1-hydroxy-7-azabenzotriazole; KOtBu, potassium tert-butoxide; mCPBA, meta-chloroperbenzoic acid; MeOH, methanol; MMMP, magnesium monoperoxyphthalate; NMP, N-methylpyrrolidone; NaBH(OAc)3, sodium triacetoxyborohydride; NaOMe, sodium methylate; nBu4NBr, tetrabutylammoniumbromide; PAL, peptide amide linker; P(Ph)3, triphenylphosphine; P(tBu)3, tri-tert-butylphosphine; Pd(OAc)2, palladiumacetate; Pd2(dba)3, tris(dibenzylideneacetone) 12

[23]

derivatization reactions of heterocyclic scaffolds

439

advantageous to immobilize various amines onto the solid support using reductive amination. The polymer-bound amines were then acting as nucleophiles in the reaction with various electron-deficient dichloro heterocycles. The heterocycles in Fig. 1 participated in nucleophilic displacement in n-butanol-containing diisopropylethylamine (Hu¨nig’s base, DIPEA). Choosing the most appropriate solvent is beneficial for the reaction rate and the swelling of the solid support, which renders the polymer-bound functionalities accessible. Electron-deficient dichloro heterocycles (e.g., pyrimidines) can be captured at room temperature, while electron-richer heterocycles, such as pyrazines, phthalazines, and pyridazines, require elevated temperatures. In turn, indole and pyridine derivatives failed to react with PAL-resin-bound primary bound amines. Nucleophilic displacement or the Pd-mediated coupling reaction on the remaining chloro substituent has been investigated. It turns out that only a small subset of the polymer-bound heterocycles could be modified by SNAr reactions under standardized conditions. The less reactive C2 chloro group of pyrimidine and quinazoline (see Fig. 2) was found to react with various amines  quantitatively at high concentration (>2 M for 12 h at 100 ). 16 Brill et al. confirmed earlier observations that for the substitution on the C2 of pyrimidines the less nucleophilic anilines are actually more reactive than aliphatic amines, presumably because of an auto-acid-catalyzed mechanism. In turn, less reactive centers like the C6 chloro group of pyrimidines could be displaced with anilines in the presence of tert-butoxide as a base (see the experimental section). Instead, palladium-catalyzed reactions offer the most versatility in terms of substrate structure. Since they usually require inert conditions, their use in a parallel synthesis setting is more demanding with regards to laboratory equipment and logistics. To expand the diversity of their libraries Brill et al.16 also modified various heterocycles by alkylation, acylation, or metal-mediated coupling reaction prior to resin capture. A remaining chloro substituent was still available for nucleophilic displacement or a palladium-mediated coupling reaction with anilines, phenols, and boronic acids on solid phase [see Fig. 10 for the preparation of purine derivative (62)]. Guillier et al.12 avoided the problem of regioselectivity in the substitution of chloro atoms in polyhalogenated pyrimidines by capturing a symmetrical dipalladium(0); PrOH, n-propanol; pTSOH, p-toluenesulfonic acid; Py, pyridine; RT, room temperature; TBAN, tetrabutylammoniumnitrate; TEAA, triethylammoniumacetate; TFA, trifluoroacetic acid; TFAA, trifluoroacetic acid anhydride; THF, tetrahydrofuran; TMSCl, trimethylsilyl chloride. 16 W. K.-D. Brill, C. Riva-Toniolo, and S. Mueller, Synlett 1097 (2001).

i

O

P

N H

P

440

Cl

Cl R1

3 ii

N

P

2

1

Cl Pyrimidines:

R1

Cl

4

OMe

3

PAL-resin

O

Cl

1

N 4 1 2 N 6

OMe

N Cl

N

3a

3b

Cl

N 6

8 N H 9

Cl Cl

N

Cl N

N1 N

N 2 Cl 3

Cl

N

8 7

N 2 Cl 1

6

Phthalazine: Cl 8

6

4 N 3 Cl

5

5

1

N2

7

4

N3

6

1 4 5

Cl

Cl 3l

3m

N 3d

Quinazolines:

3n

Cl

2 N 1 3f

Cl

5 6

3N 4

Cl

H2N

3c

Cl

Pyridazine:

Cl

3k

3j

3e

Quinoxaline:

N

N

Cl 7 N

Cl

Pyrazine

Purine:

Cl

N

7

N

8

3g

N2 Cl

Cl

N3

Cl

small molecule and heterocycle synthesis

O O

P

Cl 5

N

N N

Cl

N 3i

Cl

Fig. 1. Capture of diverse dichloroheterocycles on solid phase by nucleophilic aromatic substitution.

[23]

3h

[23]

441

derivatization reactions of heterocyclic scaffolds O

OMe HN

R1

R -NH2

N N

OMe

R1

2

N

c (if R2 = aliphatic) d (if R2 = aromatic)

Cl N

e

N

5a

N R2 H

6a Cl

b

N OMe O

N

R1-NH2 a

O

Cl

OMe H 1 N R

O

OMe

Cl

OMe

N N

Cl

b O

OMe

R1 R1

N OMe 5b

N N

HN 2 R -NH2

Cl c (if R2 = aliphatic) d (if R2 = aromatic)

N e

N 6b

N R2 H

Fig. 2. Derivatizing the C2 chloro group of resin-bound pyrimidines and quinazolines. (a)  NaBH(OAc)3, 1% HOAc, THF; (b) DIPEA (3 equiv.), heterocycle (2 equiv.), BuOH, 90 ,   24 h; (c) amine (2 M), 100 , 12 h; (d) arylamine (0.2 M), 80 , 12 h; (e) 45% TFA/DCM, 2 h.

4,6-dichloro-2-(methylthio)pyrimidine (8) (Fig. 3). They generally focused on heterocycles bearing unsubstituted amino functions and thus used bulky Rink-amine resin 7 as the solid-phase capture component, as shown in Fig. 3. The latter offers on the one hand the advantage of mild cleavage conditions, but on the other hand it also limits the choice of amines that can be immobilized onto the support and still remain nucleophilic enough for subsequent resin capture. The capture of 4,6-dichloro-2-(methylthio)pyrimidine (8) was performed in DMF with diisopropylethylamine (DIPEA, Huenig’s base) as a  base and tetrabutylammonium bromide as a catalyst at 90 . The substitution of the remaining chlorine atom on the polymer-bound scaffold requires harsher conditions. Thus the immobilized 6-chlorothiomethylpyrimidine  (9) could be substituted with aliphatic amines in neat amine at 140 . The coupling with anilines could be afforded consistently only by using KOtBu as base and [18]crown-6. Also, the use of Pd catalysts gave positive results, but failures were observed occasionally. Finally, the substitution of the thiomethyl group in resin-bound 2-(methylthio)pyrimidine-4,6-diamines

P

442

NH2

Rink-resin: NH2 :

S(On)Me O

O

7

O

N R1

N

N SMe N

iii

P

10

N H

P N

R1

n = 1: 11b n = 2: 11c

11b

+

N

Cl 11a

ii Cl

8 i

N

P

N H

R1

SMe

SMe

P 7

N

Cl

N

Cl NH2

P

N H

N

R 1⬘

N H

N

N

iv Cl

9

P

small molecule and heterocycle synthesis

SO2Me

R1⬘

N

N

R2⬘

v

N

N H

16

N

vi

11b,c

P

R2

N

N H

R1⬘ N N

12

R2⬘

R2

vii SMe N

P

N H

N

ii

N

SMe 13

R1

SO2Me

P

N H

N

14

N

vi SO2Me

N

P

N H

R1⬘ N

iv

12

SO2Me 15 

[23]

Fig. 3. Capturing symmetrically functionalized pyrimidine on Rink 0 resin for subsequent derivatization. (i) DIPEA, nBu4NBr, DMF, 90 ; 0 0   (ii) mCPBA, dioxane, 1 M NaOH, RT; (iii) R1R1 NH, 15 h, RT; (iv) R2R2 NH, 15 h, 140 ; (v) MMPP, EtOH/DMF 1:4, 2 h, 0 ; (vi) R1R1 NH, 15 h,   140 ; (vii)NaSMe (15)crown-5, EtOH/DMF (1/4), 15 h, 130 .

[23]

derivatization reactions of heterocyclic scaffolds

443

(16) was investigated. For the oxidation of the thiomethyl group it was found that magnesium monoperoxyphthalate (MMPP) gives the best results. Although the sulfoxide form (11b) was predominant, contamination with sulfone 11c was unavoidable. Nonetheless, these two functional groups would be displaced during the next reaction. Interestingly, there was no report of any formation of N-oxidized products. The final displacement  was then achieved again with a set of neat amines including aniline at 140 . In a separate report, the regioselectivity and reactivity problems in the substitution of pyrimidines were avoided using 4,6-dichloro-5-nitropyrimidine as starting material,17 a very electron-poor heterocycle, which is highly reactive in nucleophilic aromatic substitutions. It reacts readily with the free amino group of the (trialkoxybenzhydrylamine) Rink linker on solid phase. This heterocycle could serve as a scaffold by itself and could also be used as a building block (precursor) to make other heterocycles such as purines. Triazine is another scaffold of interest not only for herbicides and pharmaceuticals, but also for the development of novel catalysts18 and the construction of affinity chromatography matrices. As a result, several triazine libraries have been synthesized on various matrices such as traditional polystyrene Wang-type resins, polypropylene membranes, agarose, and glass surfaces, including the microscale. A general approach to synthesize substituted 2,4,6-triamino-s-triazines begins with a polymer-bound amine, which is used to capture symmetrical trichlorotriazine. Further selective sequential substitution of the two remaining chloro atoms can be performed using amines incrementing the reaction temperature.19 However, the selectivity of the monosubstitution is often variable,20 and partial solution-phase approaches are being reported. Thus, Masquelin et al.14 began by reacting cyanuric chloride (17) with an amine in solution (Fig. 4), a very convenient approach since many products can be isolated by crystallization or precipitation without difficult chromatographic procedures. The resulting dichloro-s-triazines (18) are then captured by a thiol resin 19. Finally, the remaining chloro function is substituted with various amines, typically using elevated temperatures. The resin attachment can be cleaved after first oxidizing the thioether function with N-phenylsulfonyl-3phenoxaziridine21 followed by treatment with very nucleophilic and preferably volatile amines. Although this method provides clean final products, 17

R. Di Lucrezia, I. H. Gilbert, and C. D. Floyd, J. Comb. Chem. 2, 249 (2000). S. Masala and M. Taddei, Org. Lett. 9, 1355 (1999). 19 M. Stankova and M. Lebl, Mol. Divers. 2, 75 (1996). 20 D. Scharn, H. Wenschuh, U. Reinecke, J. Schneider-Mergener, and L. Germeroth, J. Comb. Chem. 2, 361 (2000). 21 F. A. Davis and O. D. Stringer, J. Org. Chem. 47, 1774 (1982). 18

444

Cl N Cl

[23]

small molecule and heterocycle synthesis

HN i

N N 17

Cl

N Cl

R1

SH 19 ii

N N

HN N S

Cl

18

R1 iii

N N

R1

HN N S

Cl

20

N N

N

21

R2'

R2

O N

S O O

iv

22

HN N N

R1

HN

H N N

N 24

N R2 N R2´

v

S

R1 N

N

O 23

R2 N R2⬘

Fig. 4. Triazine derivatization in a combined solution/solid-phase approach. (i) For R1¼H:      1 aq. NH3, Et 2O, 20 –0 ; for R NH2: acetone, 2 N NaOH, 20 –0 ; (ii) dioxane, DIPEA, 40 ;   2 20 (iii) R R NH, DMA, 45 ; (iv) CH2Cl2; (v) pyrrolidine, dioxane, 60 .

there is a limit to how nucleophilic and volatile amines can be for use in the final step. Alternative activation of chloro atoms in triazines is the substitution of the chloro atom by a ‘‘transient nucleophile’’ such as N-methylmorpholine.22,23 Several solid-phase approaches to afford purines were performed. Some may involve selective substitution of the purine directly; others begin by construction of a pyrimidine scaffold followed by closure of the imidazole ring. One type of synthesis was described by Ding et al.15 (Fig. 1). A drawback to this approach is the low reactivity of a polymer-bound amine. This problem may be alleviated to some extent by activating the heterocycle with ammonium salts16 and by the choice of linker. Mainly acid-labile indole24 (28) and PAL linkers25 (26) have been employed. The possibility of activating C2 by acylation of N626 of the purine scaffold was not 22

Z. J. Kaminiski, P. Paneth, and J. Rudzinski, J. Org. Chem. 63, 4248 (1998). S. Masala and M. Taddei, Org. Lett. 9, 1355 (1999). 24 K. G. Estep, C. E. Neipp, L. M. S. Stramiello, M. D. Adam, M. P. Allen, S. Robinson, and E. J. Roskamp, J. Org. Chem. 63, 5300 (1998). 25 F. Albericio, N. Kneib-Cordonier, S. Biancolana, L. Gera, R. I. Masada, D. Hudson, and G. Barany, J. Org. Chem. 55, 3730 (1990). 26 Y.-T. Chang, N. S. Gray, G. R. Rosania, D. P. Sutherlin, S. Kwon, T. C. Norman, R. Sarohia, M. Leost, L. Meijer, and P. G. Schultz, Chem. Biol. 6, 361 (1999). 23

[23]

445

derivatization reactions of heterocyclic scaffolds 1

i

O

P

No

N H

P

R

OMe O

25

26

OMe

O Crowns or PS-resin O

27

O

28

N

N H AMS resin

R1

Cl N

N

N H

N

ii X

N

iii

N

N

N

N R

R1

X

R3

N

3

32

HN v

Y

N N R3

N

R1 N

N

30

P

N

P N

N H

29

iv

N

P N

N

X

31

R1 N 2 2 2⬘ Y=OAr, NHR , NR R , Ar

N

Y

33

Fig. 5. Derivatization of resin-bound purines. (i) R1NH2, [Me4N]þ [HB(OAc)3], then  NaBH3CN; (ii) 26 or 28, DIPEA, THF, 60 , 16 h; (iii) R2OH (10 equiv.) P(Ph)3 (10 equiv.),  DEAD (10 equiv.), THF; (iv) for X ¼ F: R2NH2, n-BuOH/DMSO, 120 ; for X ¼ Cl: ArOH, 2 2 20 ArB(OH)2, R NH2, or R NR H Pd cat.; (v) 5% TFA/DCM.

exploited in the example described here (Fig. 5). However, the use of Pd-catalyzed reactions, as described later, allowed the substitution of the C2–C1 atom by amino, aryloxy, and aryl groups. In fact, a way to overcome the lack of reactivity of chlorine at the purine C2 position and poorly reactive halides on other heterocycles is the use of Pd-catalyzed C–N and C–C formations, as illustrated in more detail in Fig. 10 and related procedures in the experimental section. An interesting synthetic approach has been used in the synthesis of adenosine analogs. Rodenko et al.27 generated a leaving group at C2 of 6-chloropurinylribofuranoside (34) via electrophilic nitration (Fig. 6). The replacement 6-chloro function was very facile in the electron-poor purine (35). In turn, the 2-nitro group of the 2-nitro-9-ribosylpurin-6- amines  (36) could be displaced by aliphatic amines at 80–90 . 27

B. Rodenko, M. J. Wanner, and G.-J. Koomen, J. Chem. Soc. Perkin Trans I 1247 (2002).

446

[23]

small molecule and heterocycle synthesis

N

O O

N O

N

N

O

N

O

N

O

i

O

N

N

NO2

N O

O

O 34

O

H2N

N

O

R

O

1

N

N

ii 1

O

R

HN

Cl

Cl

NO2

O 36

35

2

H2N

iii

HN N

R

HN

1

N

N

O

N

O N 2

HO

N H

R

R

iv, v

O

N O O

R

1

N 2

N

N H

R

O

OH OH 38

37

Fig. 6. Adenosine analogs via electrophilic nitration for the activation of the purine  scaffold. (i) Bu4Nþ NO3 (TBAN), DCM, TFAA, 0 , 2.5 h; (ii) amine, DCM, DIPEA, 4 h,  RT; (iii) amine, NMP, DIPEA, 80–90 , 24 h; (iv) pTsOHH2O, DCM/MeOH 97:3; (v) NaOMe, THF, MeOH, 1 h, RT.

Quinazolines are of great interest in the pharmaceutical industry as protein tyrosine kinase inhibitors. Dener et al.28 described a synthesis starting from 2-methoxybenzaldehyde, Wang, or Rink resins. With the aldehyde resin reductive aminations were undertaken to yield polymerbound secondary amines (Fig. 7). The latter were subjected to 2,4-dichloro6,7-dimethoxyquinazoline to give the 4-amino-substituted derivatives. These were then allowed to react with primary or secondary amines at  135–140 in the presence of DBU in DMA. As a result of a detailed scope and limitation study, Dener et al.28 note that some bifunctional amines, such as piperazine, give to some extent dimeric derivatives. Dener et al.28 also indicated a list of amine functionalities that fail to displace the 2-chloro group. In turn, Wu et al.29 performed similar chemistry displacing the chloro group at the C2 position under milder conditions (lower temperature), but this methodology is restricted to more electrondeficient and more reactive quinazolines bearing further chloro groups at C6, C7, or C8. 28

J. M. Dener, T. G. Lease, A. R. Novack, M. J. Plunkett, M. D. Hocker, and P. P. Fantauzzi, J. Comb. Chem. 3, 590 (2001). 29 Z. Wu, J. Kim, R. M. Soll, and D. S. Dhamoa, Biotechnol. Bioeng. Comb. Chem. 71, 88 (2000/2001).

[23]

447

derivatization reactions of heterocyclic scaffolds Cl N Cl 1

P

N H

R2 N

R1 40

R

N

P

i

N Cl

R2

R1

N

ii

R2

R3 N

N 41

O

N

P

R3'

N 42

R1 N H

39: O

Fig. 7. Immobilization and subsequent derivatization of quinazolines. (i) DIPEA, THF, 0   60 ; (ii) R3R3 NH, DMA, 135–140 .

Cobb et al.30 constructed 4-amino-2-carboxy-6-chloroquinazolin-4-one on a solid support using the 2-carboxylic ester linkage as the resin point of attachment. The quinazolinone (43) was converted to the resin-bound 4-chloroquinazoline (44) with SOCl2. The chloro group was then displaced even with anilines under acid-catalyzed conditions at room temperature. Finally, the resin-bound quinazoline-2-carboxylic ester (45) was cleaved from the resin and decarboxylated with TMSCl/NaI (Fig. 8) (yield 69%, purity 95%). There are a few examples for arylations of aliphatic amines, which are SNAr reactions. However, most of these examples are restricted to a highly nucleophilic piperazine scaffold. A broader approach, which may have more general value since it does not require the presence of an activating additional nitro or chloro substituent, has been developed by Ruhland et al.31 (Fig. 9). The authors formed an iron- -complex upon reaction of a dichloroarene (52) with ferrocene. The resulting ferrocene-hybrid complex (53) is capable of undergoing SNAr reactions much more readily than the ‘‘electron-richer’’ parent chloroarene (52). Using this methodology polymer-bound piperazine and 1,4-diazepane resins 51a and 51b (Fig. 9) could be readily arylated. Ruhland et al.31 showed that in the case of dihaloarenes, the second halogen group on the iron-complexed aromatic could undergo further aminations, etherifications, thiolations, and phosphine and seleno-ether formations. Unfortunately, the scission of iron-aryl 30

J. M. Cobb, M. T. Fiorini, M.-E. T. Goddard, and C. Abell, Tetrahedron Lett. 40, 1045 (1999). 31 T. Ruhland, K. S. Bang, and K. Andersen, J. Org. Chem. 67, 5257 (2002).

448

[23]

small molecule and heterocycle synthesis NH2 Cl

O Cl

HN

i

O

O

N O

N

ii O

N O

43

Br Cl

HN Cl Br

N

N O

44

45

iii Mechanism of cleavage I

N O

HN

Br Cl

HN

N

Si O

N

O

Si

Br Cl

N N

N

O 47

Br Cl

HN

HN

48

N

I H

O

Br Cl

46 CO2

N O

49

Fig. 8. Quinazolines via decarboxylation of resin-bound carboxylic esters. (i) SOCl2, DMF,  reflux; (ii) HCl/PrOH/DMF, RT, 18 h; (iii) TMSCl, NaI, CH3CN/dioxane, 75 , 72 h.

Cl

N

i

NH n n=1 or 2 51a or 51b

50 Merrifield-resin

n

Fe Cl Cl Cl

+

ii Al, AlCl3

52

PF−6

53

R

O O

X N

O

N n 57a,b

R

O vi

Fe+

54

Cl

Fe+

Cl

N

N

iii

X Na+ iv

N N hn (visible light)

Cl N

N n 56

X

R

v

X

N

N

n

R

Fe+

55

Fig. 9. Nucleophilic aromatic substitution promoted by iron- -complex formation on solid   phase. Piperazine or diazepane, THF/DMF 1: (i) 50 , 18 h; (ii) (1) 95 , 4 h; (2) NH4PF6; (iii)   K2CO3, THF, 60 , 16 h; (iv) various nucleophiles, 45–90 ; (v) CH3CN/H2O, visible light; (vi)  1,2-dichloroethane, 0 , RT, 16 h.

[23]

derivatization reactions of heterocyclic scaffolds

449

-bonds is not very facile. A photochemical reaction was used to liberate the polymer-bound arene (56) after the SNAr substitution. The latter was then cleaved from the resin with methyl chloroformate to give 57a and 57b (Fig. 9). This approach provides a robust alternative to Pd-, Ni-, or Cu-catalyzed aminations and etherifications, which often require rigorous exclusion of oxygen. In conclusion, SNAr reactions are often facilitated by polar protic solvents such as butanol or by polar solvents in the presence of Brønsted or Lewis acids. When displacements of halides with amines are used, both polar and protic solvents can solubilize the ammonium halides resulting from the reactions, and lead to the formation of Brønsted acids. Fluoride or chloride ions are preferred leaving groups for this reaction. Sulfinyl and sulfenyl groups require somewhat harsher conditions. If SNAr reactions are to be performed, it is advisable to render the scaffold as electron deficient as possible. Thus, thioethers may be oxidized to sulfoxide or sulfonyl groups, and nitrogen-containing functionalities should be amides or nitro groups prior to a substitution. Of course, these derivatizations are limited by the reactivity of the corresponding heterocycle, since more than one functionality may be displaced, if multiple leaving groups are present. The following sections highlight some additional reaction types, which can be used proficiently for the derivatization of heterocyclic scaffolds. Palladium-Catalyzed Reactions

As mentioned earlier, Ding et al.15 captured a number of dichloroheterocyclic scaffolds where one chloro atom is prone to nucleophilic aromatic substitution onto resin-bound amine nucleophiles (Fig. 1). Even though it was demonstrated that in many cases the second chlorine may be substituted with SNAr reactions, it was pointed out that palladium-catalyzed reactions offer the most versatility in terms of substrate structure. When introducing amino, aryloxy, and aryl groups, Ding et al.15 reported Pdcatalyzed reactions as a way to overcome the lack of reactivity of chlorine at the purine C2 position and poorly reactive halides on other heterocycles (Fig. 10). The reaction sequence starts by anchoring 2,6-dichloropurine onto the solid-phase PAL-amine at the more reactive C6 position with exclusive regioselectivity. A multitude of PAL-amine resins 59 can be prepared ahead by reductive amination of commercial (4-formyl-3,5-dimethoxyphenoxy)-methylpolystyrene. The N9 position of the purine (60) may be modified by Mitsunobu alkylation. The final derivatization step involves a palladium-catalyzed cross-coupling reaction in position 2. This reaction

450

[23]

small molecule and heterocycle synthesis OMe O O

OMe 58

1 N R

a

1

N R H

R1-NH2

b

PAL

PAL

N

N Cl

59

N

N H

60 1 N R

c

N

60 Mitsunobu

Cl

N 61

N

choice of nucleophile

N

Pd-chemistry

R2

R3 N R4

1 N R

N

N X

N 62

N R2

X=

R3 O R3

R3

N H

d, e, f, or g

Fig. 10. Multiple derivatization of purines including palladium-catalyzed reaction at the poorly reactive C2 position. (a) NaBH(OAc)3, 1% HOAc, THF; (b) 59 (0.5 equiv.),  2,6-dichloropurine (1 equiv.), DIEA (1.5 equiv.), BuOH 80 ; (c) R2OH, PPh3, DiAD (1.5:2:1.3) in excess, THF, RT; (d) boronic acids (5 equiv.), 7% Pd2(dba)3, 14% carbene  ligand, Cs2CO3 (6 equiv.), 1,4-dioxane, 90 , 12 h; (e) anilines (5 equiv.), 7% Pd2(dba)3, 14%  carbene ligand, KOtBu (6 equiv.), 1,4-dioxane, 90 , 12 h; (f) phenols (5 equiv.), 7% Pd2(dba)3,  28% phosphine ligand, K3PO4 (7 equiv.), toluene, 90 , 12 h; (g) primary or secondary amines  (5 equiv.), 90 , 12 h.

requires 5 equivalents of the coupling partner (arylboronic acids, anilines, or phenols), 7 mol% Pd2(dba)3, 14 mol% of a suitable ligand, and 6  equivalents of the base at 80 in dioxane (for C–C and C–N formation) or toluene (for C–O formation). Over the past 5 years the number of reports on the use of palladiumcatalyzed reactions for solid-phase derivatizations has greatly increased. In this section (and throughout this chapter), we limit our scope to representative applications for the modification of solid-supported heterocyclic scaffolds. A more general overview of the versatility of Suzuki, Heck, and Stille reactions on solid supports was recently provided by Franzen.32 Extensive use of Pd-catalyzed reactions was included in the synthesis of 2,6,8-trisubstituted purines (Fig. 11).33 The synthesis started by anchoring dichloropurine to Rink resin via N9 linkage. Polymer-bound 2,6-dichloropurine (63) was selectively substituted at C6 via acid-catalyzed SNAr substitutions. In the absence of Pd catalysis, the substitution on C2 could be performed only with strongly nucleophilic amines. To expand the scope of C2 substitution, catalytic amounts of Pd were used. Under these reaction conditions arylboronic acids and amines successfully substituted the chloro atom on C2 to afford C2–C and C2–N bonds. Subsequently, the C8 position was brominated with a bromine–lutidine complex33 (66) to give resin 67. 32 33

R. Franzen, Can. J. Chem. 78, 957 (2000). W. K.-D. Brill and C. Riva-Toniolo, Tetrahedron Lett. 42, 6515 (2001).

[23]

451

derivatization reactions of heterocyclic scaffolds R1 Cl OH

N

i

N N

N

N

P

N

ii

P

63

R1 N

N

N

iii

N

N

Cl

R2

N

N

Cl P

64

R2

R3

N 65

Rink resin: Br :

N

iv P

R2 = Aryl or prim./sec. alkyl- or (aryl-)amino

polystyrene/DVB

R1 N R4

N N H

R1

R2

N vi

N N 69

Br

O

O

O

N

R4

R

P

R1

R2

N

N

N

3

N

66

v 3

R

68

N

N

Br N P

R2

N

R3

67

Fig. 11. Bromination and subsequent Stille coupling at the C8 position of purines on solid phase. (i) TFAA, 2,6-lutidine, then 2,6-dichloropurine, NMP, 2,6-lutidine; (ii) primary or  secondary alkylamine (4 equiv.), TFA (0.4 equiv.) in NMP, 55 , 90 h; (iii) ArB(OH)2 or RNH2 or RNHR0 (8.3 equiv.), K3PO4 (31.4 equiv.), Pd2(dba)3, P(tBu)3, NMP; (iv) 66 (5)  NMP, 3 h, RT; (v) Stille coupling: Pd(OAc)2 dppp, stannane (8.3 equiv.), NMP, 100 , 20 h; (vi) 20% TFA in CH2Cl2.

The latter intermediate could undergo Stille couplings to afford trisubstituted purine (68). In many cases, high yields (>75% based on polymer loading) of the final product were obtained. Some drawbacks to this approach include partial dehalogenation of (67) back to (65) during the Stille coupling, the presence of Pd impurities in some products, and structural and chemical constraints imposed by the bromination reaction. Acylations, Alkylations, Reductive Alkylations (Aminations, Alkaminations)

Following Ellman’s pioneering work on benzodiazepines,2 another study from the same laboratory illustrated the vast potential of combining a variety of chemical reactions on drug-like heterocycles. In this case a preformed heterocycle was grafted as a whole onto the solid phase to be subsequently derivatized via a three-component palladium-mediated coupling reaction followed by a reductive alkylation or an acylation (Fig. 12).34 34

J. S. Koh and J. A. Ellman, J. Org. Chem. 61, 4494 (1996).

452

[23]

small molecule and heterocycle synthesis Teoc O O

O +

Teoc

N

N

a

b

Pd

N SiMe3

O

O

70

Br

PPh3 71

THP OH

THP

Teoc N c, or d, or e

R1 R2

H N f

O

R3 R1 R2

N

g or h

R1 R2

O 73

72 THP

THP R1: aryl R2: aryl, H, alkinyl

X

X

O

74

THP

N i

OH

R3 R1 R2

75

1

X: CO, CH2

R : aryl 2 R : aryl, H, alkinyl X: CO, CH2

Fig. 12. Versatile derivatization scheme of a tropane template, including palladium mediated coupling. (a) TsOH, CH2Cl2; (b) aryl bromide, Pd(PPh3)4, THF, 66 ; (c) arylboronic   acid, 2 N Na2CO3, PPh3, THF, or anisole, 66 ; (d) formic acid, Et3N, PPh3, DMF, 66 ; (e)   phenylacetylene, CuI, Bu4NCl, DMF, 66 ; (f) Bu4NF, THF, 50 ; (g) aldehyde, NaBH(OAc)3, DMF; (h) HATU, Et3N, benzoic acid, HOAt, DMF; (i) TFA/H2O (20:1).

The derivatization of the secondary amine functionality of a tropane template is described in more detail in the experimental section. The acylation is carried out with a classic protocol employing a modern coupling reagent of the uronium type, namely the highly effective HATU,35,36 which contains an azabenzotriazolyl moiety, having demonstrated advantages over the formerly used benzotriazolyl derivatives. Acylations on solid phase have been studied intensely for decades in the context of peptide chemistry. Experience from innumerable optimization studies reported in this field benefits the combinatorial chemist by providing a rich choice of protocols with well-documented scope and limitations. Optimal results cannot be obtained with any single protocol in all cases. The observation is that the relative performances of distinct combinations of activating agents and reaction conditions often vary depending on the substrates. Informative overviews on coupling methodologies for amide and ester bond formation are available.37,38 Much of the effort in these 35

L. A. Carpino, J. Am. Chem. Soc. 115, 4397 (1993). L. A. Carpino and A. El-Faham, J. Org. Chem. 60, 3561 (1995). 37 F. Albericio and L. Carpino, Methods Enzymol. 289, 104 (1997). 38 F. Albericio and S. A. Kates, in ‘‘Solid-Phase Synthesis’’ (S. A. Kates and F. Albericio, eds.), p. 275. Marcel Dekker Inc., New York, 2000. 36

[23]

derivatization reactions of heterocyclic scaffolds

453

studies has been devoted to preventing racemization during the coupling of chiral amino acids. When working with nonchiral building blocks, this aspect can be neglected and more aggressive approaches can be applied as well. Such methods may involve acyl halogenides or, for the acylation of unreactive alcohols, the use of n-butyllithium in the presence of an acid chloride39 or superbase-promoted acylations, for example, with the commercially available nonionic superbase P(MeNCH2CH2)3N.40 Acylations on amines usually do not need the presence of excess base, unless the amino group must be liberated from a protonated salt form or if the coupling agents or adjuvants are of an acidic nature (e.g., hydroxybenzotriazol derivatives). If carbodiimide-based coupling reagents are used in relatively polar solvents such as DMF, a preactivation of the carboxylic acid to form an activated ester [which can be done in situ, e.g., with (aza)benzotriazolyl derivatives] prevents formation of acylurea side products. The solid-phase reaction format has the advantage that these side products can be easily washed away, if the activated ester is in solution. Any partial depletion of the acyl component has negligible effects on yields if a sufficient molar excess is applied. A caveat for long preactivation times is the tendency toward racemization of the activated species, although this is irrelevant when nonchiral building blocks are used. With CH2Cl2 as a solvent, the straight use of carbodiimides without active ester preformation is possible and the acylurea side reaction is not observed. On the other hand, if the amine component is exposed to a coupling reagent of the uronium type before the carboxylic acid is added, there is risk of guanidino side product formation. Particularly in parallel synthesis the optimal timing of reagent addition cannot always be maintained for logistical reasons and the consequences of a prolonged temporary absence of one of the reaction components must be assessed in advance. Where possible, it is usually preferable to have the activated species (e.g., the active ester) in solution and the substrate (e.g., the amine component) on the solid phase. If the case requires an inverted situation, as for the synthesis of a thiazolidinone library (see Fig. 13),41 identification of the best reaction conditions may be more challenging, considering the relative scarcity of literature examples. The example previously described in Fig. 12 involves a reductive alkylation, a widely used derivatization reaction in combinatorial chemistry. The use of sodium triacetoxyborohydride has been thoroughly validated for the solid-phase reaction format. With this reagent the pH is maintained 39

E. Mc Kaiser and R. A. Woodruff, J. Org. Chem. 35, 1198 (1970). B. A. D’Sa and J. G. Verkade, J. Org. Chem. 61, 2963 (1996). 41 M. C. Munson, A. W. Cook, J. A. Josey, and C. Rao, Tetrahedron Lett. 39, 7223 (1998). 40

454

[23]

small molecule and heterocycle synthesis

R1

O O

R

a then b N H

N

S H2N

Wang

R2

R1

2

c

H2N

N

S

R3

O O

O

N R3 H

77

COOH

76

Fig. 13. Acylation via activation of a resin-bound carboxylic acid. (a) Pentafluorophenyl trifluoroacetate (6 equiv.) in Py/DMF (1:10), 1 h; (b) amine (6 equiv.) CH2Cl2/DMF; (c) TFA/ DCM (1:1) 1 h. O

O

O

R2

HN

HN

b

a

c

HN

HN

R1

R1 O 78

N

N

NH ArgoGel Rink

R2

H2N

O 79

N R3

R1 O 80 

Fig. 14. Stepwise selective amine and amide alkylation. (a) R2-CH2-X (2 M), DMF, 50 , 24–48 h; (b) lithiated 4-Bz-2-oxazolidinone, R3-CH2-X (15 equiv.), THF/DMF, 1 h; (c) 90% TFA/DCM, 0.5 h.

in the desired, slightly acidic range. Secondary amine functions (like the described tropane scaffold) are unaffected by the potential bisalkylation side-reactions, which may occur between aliphatic aldehydes and primary amines. One way of minimizing the risk of bis-alkylations is the preformation of the imine intermediate in the absence of the reducing agent42 and, subsequent, time-delayed (>20 min) addition of the borohydride. Straight N-alkylations with alkyl halogenides were commonly used at the inception of combinatorial chemistry, when large oligo-N-substituted glycine libraries were prepared for lead finding.43 The same reaction type can be conveniently used on heterocyclic scaffolds, since selectivity is the most critical aspect if multiple alkylation sites are present. The example illustrated in Fig. 1444 indicates that stepwise selective alkylation without differential protection is also feasible on solid phase, if the nucleophilicity difference of the targeted nitrogen atoms is sufficiently high. A discussion on protecting group chemistry and the related strategies to mask the reactivity of one functional group while allowing the modification of another group in the same molecule is beyond the scope of this chapter. 42

D. W. Gordon and J. Steele, Bioorg. Med. Chem. Lett. 5, 47 (1995). R. N. Zuckermann, J. M. Kerr, S. B. H. Kent, and W. H. Moos, J. Am. Chem. Soc. 114, 10646 (1992). 44 M. K. Schwarz, D. Tumelty, and M. A. Gallop, Tetrahedron Lett. 39, 8397 (1998). 43

[23]

OH

OH H N

455

derivatization reactions of heterocyclic scaffolds

N H

fmoc

O c

b

a

N

N O2N

NO2 O

O

F O d

N NH2 O

e, f

81 O

H N

H N O

O O

82

Fig. 15. Reduction of an aromatic nitro group on solid phase. (a) Twenty percent piperidine, DMF, 20 min; salicylaldehyde, HC(OMe)3 then NaBH(OAc)3, DMF, 12 h, RT; (b) 2-fluoro-5-nitrobenzoic acid, HOAt, DIC, DMF, 24 h, RT; (c) 5% DBU, DMF, 24 h, RT; (d) SnCl22H2O, DMF, 12 h, RT; (e) furfuryl chloride, Hu¨nig’s base, DMF, 8 h, RT; (f) 20% TFA, CH2Cl2, 40 min.

Protocols of peptide chemistry and, to some extent, biooligomer synthesis (e.g., nucleotides, saccharides) are valuable sources of information on this topic with regards to solid-phase synthesis peculiarities. Here we focus on a particular functional group transformation, which takes the role of ‘‘deprotecting’’ a masked functionality, namely the nitro-to-amine reduction. This approach provides a versatile tool for planning multistep derivatizations of heterocycles, as exemplified in Fig. 15.45 A key step in tin-based reductions on the solid phase is the washing procedure, which is used for the removal of residual tin salts. To be on the safe side, numerous washes with a very broad spectrum of solvents are recommended. In our experience good results are obtained with polystyrene-based resins when washes with warm DMF/water (1:1) are included. Name Reactions

As of today, the variety of chemical reactions already applied in the solid-phase synthesis format is very rich and comprises the majority of the organic synthesis reactions with significant preparative scope. However, the number of examples describing the use of classic reactions for the ‘‘decoration’’ of solid supported scaffolds, i.e., aiming at standardized derivatization of already formed heterocycles, is still surprisingly low. The reaction types previously mentioned in this chapter seem to be the most frequently applied for this specific purpose. In this section we cover 45

X. Ouyang and A. S. Kiselyov, Tetrahedron 55, 8295 (1999).

456

small molecule and heterocycle synthesis

[23]

some name reactions, which were used in this context. Again, we realized that the overall pool of examples to choose from is rather limited. The Mitsunobu reaction is a very powerful and versatile tool in solidphase chemistry. This was also recognized early for the preparation of combinatorial libraries.46 It effectively leads to the nucleophilic substitution of an alcohol by the conjugate base of an acidic reactant with sterical inversion at the alcohol carbon. This reaction is mediated by the redox combination of a phosphine with a dialkyl azodicarboxylate. The sequence of reagent addition has little effect on the yield of the reaction if the time delays are kept minimal (the adduct forming the redox system has limited stability). In general, the adduct is prepared first in order to reduce potential side reactions related to the triphenylphosphine nucleophilicity. In combinatorial chemistry this reaction is often used for N-alkylations and ether formations. Esterifications are an attractive option as well since the activation process is reversed (compared to the classic activation of the acyl component). This provides more flexibility in avoiding activation of the immobilized species on the solid phase. Due to the instability of the redox system it makes little sense to prolong reaction times beyond 2 h. Repeating the reaction with fresh reagents is more effective and easy to do in the solid-phase format. As washes with methanol or isopropanol are not uncommon in working with polystyrene-based supports, such solvents should be avoided previous to a Mitsunobu reaction or utmost care has to be applied in removing residual alcohol traces prior to the reaction. Arylsulfonamides are acidic enough to be substrates in Mitsunobu reactions. Electron-withdrawing substituents on the arylsulfonyl group facilitate the reaction, but they also render the moiety more sensitive to cleavage with nucleophiles. As illustrated in Fig. 16 (step b)47 this fact can be exploited for the monoalkylation of an amino group, where trifluoroacetylation activates the group making it accessible for Mitsunobu reaction. The trifluoroacetyl residue can be removed subsequently by treatment with a nucleophile. Purines were also derivatized by Mitsunobu reaction at the endocyclic N9 as already illustrated in Fig. 1015 and reported by Dorff and Garigipati.48 The base facilitated Knoevenagel reaction has been used on an oxindole scaffold as indicated in Fig. 17.49 Position 3 of this heterocycle consists of an activated methylene function, which lends itself to classic 46

V. Krchnak, J. Vagner, Z. Flegelova, A. S. Weichsel, G. Barany, and M. Lebl, in ‘‘Peptides: Chemistry, Structure and Biology, Proceedings of the 14th American Peptide Symposium,’’ p. 307, Columbus, OH, 1996. 47 T. C. Norman, N. S. Gray, J. T. Koh, and P. G. Schultz, J. Am. Chem. Soc. 118, 7430 (1996). 48 P. H. Dorff and R. S. Garigipati, Tetrahedron Lett. 42, 2771 (2001). 49 Y.-L. Chou, M. M. Morrissey, and R. Mohan, Tetrahedron Lett. 39, 757 (1998).

[23]

457

derivatization reactions of heterocyclic scaffolds Cl

Cl N

N

a N

N

H2N

O

O

H

O

O

H

F3C

N H

N H

83

N

N N

N Linker

O 84 R2 HN

Cl O

b F3C

N

c, d HN

N

N

R1

N

N

N

N

O

N

N

OH

R1

Linker

85

86

Fig. 16. Mitsunobu reaction for exocyclic N2 alkylation on a purine scaffold. (a)  Trifluoroacetic anhydride (0.2 M), 0.3 M 4-methyl-2,6-di-tert-butylpyridine, CH2Cl2, 37 , 4 h;  1 (b) 0.2 M R -OH, 0.4 M PPh3, 0.2 M DEAD, THF, 10 to RT, 6 h; (c) 0.25 M amine,  DMSO, 70 , 12 h; (d) TFA/H2O, 90:10 (v/v), RT, 1 h. tBu O N H TIPSO

OH H N

a

O O

O

N

87

HO

tBu O

b N

88

Fig. 17. Knoevenagel reaction on an immobilized oxindole scaffold. (a) 4-Hydroxy-3,5-ditert-butylbenzaldehyde (3 equiv.), pyrrolidine (3 equiv.), CH2Cl2/MeOH (4:1), 24 h; (b) 1 M Bu4NF/THF, 1 h.

formation of C–C bonds with carbonyl components. The analogous position of the fully aromatized indole scaffold can be addressed with a Mannich reaction as described by Zhang et al.50 (see Fig. 18). These two methods are quite valuable for the generation of biologically interesting indole-based chemical libraries and for the solid-phase synthesis of indole alkaloids. Conclusion

Substituted heterocyclic compounds offer a high degree of structural diversity and have proven to be broadly useful as therapeutic agents. The 50

H.-C. Zhang, K. K. Brumfield, L. Jaroskova, and B. E. Maryanoff, Tetrahedron Lett. 39, 4449 (1998).

458

[23]

small molecule and heterocycle synthesis

R1

R1 N a

O N H

N H 89

O

N R2 b

N H

N H

R2

O H2N

N H

90

Rink amide resin

Fig. 18. Mannich reaction on a resin-bound indole scaffold. (a) Amine (1.5 equiv.), HCHO (1.5 equiv.), HOAc/1,4-dioxane (1:4), 1.5 h RT; (b) 30% TFA, CH2Cl2, 1 h, RT.

derivatization on the solid phase of preassembled heterocyclic core scaffolds (which may be previously prepared in large scale in solution) is an attractive approach for the high-throughput synthesis of new chemical entities for activity screens. Validated protocols of standard reactions applicable to a variety of solid-supported substrates are useful tools, which can be used repeatedly as long as the supply of novel scaffolds is maintained. In this chapter we have exemplified this type of derivatization strategy on a number of cases, all of which were reported in the literature with a well-documented experimental protocol. We expect that a growing number of examples will be published in the coming years, both in the patent literature (because of the industrial focus of the field) and in scientific journals. The added value of parallel and combinatorial chemistry will be further substantiated by these examples. Experimental

Substitution of Remaining Chloro Group with Amines via Non-Palladium-Catalyzed Amination Reaction without Base (Fig. 2)15 A resin of type 5 (0.05 mmol) is suspended in the solution of an amine  (2 M in n-butanol, 0.2 ml). After 12 h of heating at 80 in a sealed reaction vessel under argon, the resin is washed with methanol (4  1 ml) and dichloromethane (4  1 ml) and dried under vacuum. The product (6) is obtained by cleavage in DCM:TFA:Me2S:H2O 45:45:5:5 (0.3 ml) for 2 h. Substitution of Remaining Chloro Group with Amines via Non-PalladiumCatalyzed Amination Reaction with KOtBu as Base (Fig. 2)15 To a suspension of a resin of type 5 (0.05 mmol) in THF (anhydrous, 0.25 mmol) an amine is added (0.25 mmol), followed by addition of KOtBu  solution (in THF, 1.0 M, 0.25 ml, 0.25 mmol). After 12 h of heating at 70

[23]

derivatization reactions of heterocyclic scaffolds

459

in a sealed reaction vessel under argon, the resin is washed with methanol (4  1 ml) and CH2Cl2 (4  1 ml) and dried under vacuum. The product is subsequently cleaved using CH2Cl2:TFA:Me2S: H2O 45:45:5:5 (0.3 ml) for 2 h to afford the desired final product of type 6. General Reaction Conditions for the Conversions in Fig. 312 Small scale reactions (<250 mg of resin) and resin washing and drying procedures are performed at room temperature in polypropylene filtration tubes (1,3, or 6 ml) with polyethylene frits and a Visprep solid-phase extraction vacuum manifold supplied by Supelco. Cleavage reactions for high-performance liquid chromatography (HPLC) analysis are performed using approximately 5 mg of resin in 1.9-ml polypropylene microcentrifuge tubes (Eppendorf) while agitating on a tube rotator. Otherwise the reactions are performed in a round-bottom flask with magnetic stirring set at the lowest possible speed to avoid grinding of the resin. General Procedure for the Synthesis of Rink Resin-Bound 6-Chloro-2-methane sulfenyl pyrimidine (9) (Fig. 3)12 Rink amide resin 7 (4.01 g, loading: 0.73 mmol/ g, 2.93 mmol) is swollen in DMF (75 ml) for 30 min. Then 4,6-dichloro-2-methylthiopyrimidine (8) (2.84 g, 14.6 mmol), tetrabutylammonium bromide (1.84 g, 5.7 mmol), and diisopropylethylamine (5.1 ml, 29.3 mmol) are added and the mixture  gently stirred and heated overnight at 90 . The resin is filtered and washed thoroughly with DMF (2  40 ml), CH2Cl2 (2  40 ml), MeOH (2  40 ml), and Et2O (2  40 ml). The resin is first predried by passing air through the filter tube, and then placed under high vacuum. A negative ninhydrine test of the resin indicates full substitution. Of the monochlorinated pyrimidine resin 4.33 g is obtained (9). Cleavage of 4-Amino-6-chloro-2-methanesulfenyl Pyridine from (9) (Fig. 3)12 An aliquot of resin 9 (50 mg) is swollen in CH2Cl2 (250 l). A solution of trifluoroacetic acid in water 95:5 (4.75 ml) is added and the mixture is stirred for 3 h at room temperature. The suspension is filtered and the resin is washed with CH2Cl2(2  5 ml), CH3CN (2  5 ml), and CH2Cl2 (2  5 ml). The filtrates are combined and the solvents evaporated under reduced pressure. The residue is then taken up in a CH3CN/ water solution (2 ml, 1:1) and freeze-dried overnight. The resulting residue is purified by silica gel chromatography or semipreparative reverse phase (RP)-HPLC.

460

[23]

small molecule and heterocycle synthesis

Resin-Bound 2-Methylthio-6-piperidinopyrimidine (16) 0 (R2NR2 ¼ Piperidine) (Fig. 3)12 Resin 9 (130 mg, 0.51 mmol/g, loading 0.066 mmol) is swollen in the minimal amount of N-methylpyrrolidone (1.5 ml) for 30 min. Piperidine (4 ml) is added and the mixture is stirred gently (or better shaken) and refluxed overnight. The resin is then filtered, washed thoroughly with DMF (2  5 ml), CH2Cl2 (2  5 ml), MeOH (2  5 ml), and DMF (2  5 ml). The resin is predried by passing air through the filter tube, which is then 0 placed under high vacuum. Resin 16 (R2NR2 ¼ piperidine) is obtained with a theoretical loading of 0.50 mmol/g. Cleavage of 2-Methylthio-6-piperidinopyrimidine from Resin 16 (Fig. 3)12 The cleavage is performed according to the previously described method. The crude product is further subjected to flash column chromatography on silica gel using ethyl acetate/petroleum ether giving 4-amino-2methansulfenyl-6-piperidinopyrimidine. Oxidation of the Methylthio Group (Fig. 3)12 0

Resin-Bound 2-Methanesulfinyl-6-piperidinopyrimidine (9b) (R2NR2 ¼ Piperidine) (11b). Resin 16 (500 mg, 0.50 mmol/ g loading, 0.25 mmol) is  swollen in DMF (20 ml) for 30 min. The mixture is cooled to 0 and a solution of magnesium monoperoxyphthalate (MMPP, 212 mg, 0.34 mmol) in  DMF (5 ml) is added dropwise and shaking continues for 2 h at 0 . After the resin is washed and dried in the usual manner, an aliquot of the resin (5 mg) is taken and cleaved off. RP-HPLC analysis indicates that approximately 6% starting material remained unreacted and a new oxidation cycle is performed with MMPP (0.14 mmol) for 1 h at 0 . Analysis confirmed the total conversion of resin 16 into a mixture of resin-bound sulfoxide (11b) (79%) and sulfone (11c) (15%). A theoretical loading of 0.50 mmol/g is assumed for subsequent work. Displacement of Methanesulfinyl (11b) or Methanesulfonyl (11c) Groups (Fig. 3)12 0

4-Amino-2-benzylamino-6-piperidin-1-yl Pyrimidine (12) (R1NR1 H ¼ 0 Benzylamine, R2NR2 H ¼ Piperidine). Resin mixture 11b, 11c (130 mg, 0.50 mmol/g loading, 0.065 mmol) is swollen in the minimal amount of N-methylpyrrolidone (1.5 ml) for 30 min. Benzylamine (4 ml) is added  and the mixture is shaken overnight at 140 . The resin is filtered and washed thoroughly with DMF (2  5 ml), MeOH (2  5 ml), then Et2O (2  5 ml). The resin is predried by passing air through the filter tube, which is then subjected to high vacuum.

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Cleavage of the Resin 12 (Fig. 3)12 Resin 12 (110 mg, 0.054 mmol) is treated with TFA as previously described for resin 9. The crude product (purity 88%) is subjected to chromatography on silica using EtOAc/ petroleum ether as the mobile phase. The purified product 4-amino-2-benzylamino-6-piperidin-1-yl pyrimidine is obtained in >70% yield. Synthesis of 2,4,6-Amino-Substituted s-Triazines Using a Partial Solution Phase Approach (Fig. 4)14 Example: 4,6-Dichloro-N-(3, 5-dichlorophenyl)-s-triazin-2-amine (R1 ¼ 3,5-Dichlorophenyl) (18) (Fig. 4)14. A finely dispersed suspension of (17) is prepared by adding water (175 ml) into a stirred solution of 2,4,6-trichloro1,3,5-triazine (17) (15 g, 79.7 mmol) in acetone (150 ml). After cooling to  20 a solution of 3,5-dichloroaniline (13.3 g, 79.7 mmol) in acetone is added, followed by the dropwise addition of NaOH (2 N, 40 ml). The mix ture is stirred for 2 h at 0 . The acetone is evaporated. The precipitate is filtered off, washed with water, and dried over MgSO4. Approximately 23 g of product (18) (R1 ¼ 3,5-dichlorophenyl) is obtained in >95% yield. Polymer-Bound Thiol (19) (Fig. 4)14 A mixture of Merrifield resin (chloromethylpolystyrene 0.5–2% crosslinked with DVB) (100 g, 1.8 mmol/g), thiourea (68.5 g, 900 mmol), and  DMA (1 liter) is shaken at 85 for 20 h. Washes with isopropyl alcohol (1  5 min), dioxane (2  4 min), dioxane/H2O (1:1) (6  4 min), DMA (3  4 min), and isopropyl alcohol (5  4 min) at RT are performed on an automated washing station using about 1 liter of the appropriate solvent per wash. Drying under high vacuum for 20 h affords polymer-bound thiuronium salt with >90% yield based on elemental analysis. Of this resin 91 g  is suspended in dioxane/pyrrolidine (4:1, 900 ml) and stirred gently at 110 for 2 h, preferably using an overhead stirrer. Resin 19 is washed and dried as described for the thiuronium salt. It can be used for the resin capture strategy. The thiol content is 88% of the initial loading. Polymer-Bound Triazine Attached via a Thiol Linker (20) (Fig. 4)14 The resin bearing a thiol function (19) (4 g, 11.2 mmol) is washed with  DMA (3  15 ml) at 40 . 6-(3,5-Dichloroanilino)-2,4-dichloro-s-triazine (5.49 g, 14.0 mmol), DIPEA (2.45 ml, 14 mmol), and DMA (15 ml) are  added and the mixture is shaken at 40 for 18 h. The polymer-bound tria zine is washed successively at 40 with DMA (15 ml), isopropyl alcohol  (15 ml), and hexane (15 ml). Resin 20 is dried at 50 under high vacuum

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and is analyzed by elemental analysis. The same general procedure can be applied for 2-amino-4,6-dichloro-1,3,5-triazine (R1 ¼ NH2). Displacement of the Remaining ChloroAtom to (21) (Fig. 4)14 To dry resin 20 (0.92 g, 0.25 mmol) are added an amine (such as 3,5dichloroaniline) (0.625 mmol), DIPEA (0.215 ml, 1.25 mmol), and DMA  (1.5 ml). After shaking the mixture at 45 for 17 h, the resin is washed successively with DMA (2  5 ml), isopropyl alcohol (5 ml), and DCM (3  5 ml). Oxidation of the Thio Group (Fig. 4)14,51 The resin is treated with N-phenyloxaziridine (0.075 g, 0.288 mmol) and DCM (2.5 ml) for 14 h at RT. The oxidized resin is washed with DCM (5 ml), isopropyl alcohol (5 ml), and dioxane (2  5 ml). Cleavage and Release of the 2,4-Bis-(3,5-dichloroanilinyl)-6-pyrrolidinyl1,3,5-triazine (24) (R1, R2 ¼ 3,5-Dichlorophenyl, R2 ¼ H) from Resin 23 upon Substitution of the Oxidized Thioether Link (Fig. 4)14 A solution of pyrrolidine (0.041 ml, 0.50 mmol) in dioxane (2.5 ml) is  added to resin 23 and the mixture is shaken for 6 h at 60 . The filtrate is lyophilized to yield the substituted 2,4,6-triamino-s-triazine (24), such as 2benzylamino-4-(3,5-dichloroanilinyl)-6-pyrrolidinyl-1,3,5-triazine (R1 ¼ 0 benzyl; R2 ¼ 3,5-dichlorophenyl, R2 ¼ H) in high purity and 31% yield. Conversion of a Resin-Bound Quinazolin-4-one (43) into a 4-Chloroquinazoline (44) (Fig. 8)30 To a resin bearing 2-carboxyquinazolin-4-one (43) (200 mg; 60 mol) swollen in anhydrous DMF (5 ml) is added thionyl chloride (5 ml). The suspension is refluxed for 3 h, then the resin is filtered and reswollen in DMF for the subsequent substitution of the 4-chloro group. Preparation of Amino-Substituted Quinazoline (45) and Cleavage from the Resin to Obtain the 2-Unsubstituted 4-Arylaminoquinazoline (46) (Fig. 8)30 Resin 44 (preswollen in 5 ml DMF) is exposed to 3-bromoaniline (150 l, 5 equiv.) in propan-2-ol (5 ml) containing four drops of conc. HCl. The reaction is shaken at room temperature overnight. The resin 45 is filtered, washed with DMF (5  20 ml), CH2Cl2 (5  20 ml), and diethyl 51

F. A. Davis and O. D. Stringer, J. Org. Chem. 47, 1774 (1982).

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ether (5  20 ml) and dried in vacuo. Resin 45 (73 mg) is swollen in dioxane (4 ml) and CH3CN (4 ml). NaI (100 mg) and trimethylsilylchloride  (200 l) are added. The suspension is heated at 75 for 72 h and then filtered. The filtrate is concentrated in vacuo and taken up in water. The product is extracted with DCM (3  10 ml), washed with aq. Na2S2O4, dried over MgSO4, and concentrated in vacuo to give 4-(3-bromoanilino)6-chloroquinazoline (46). Facilitated Arylations via Iron- Complex 6-1,2-Dichlorobenzyne-5-cyclopentadienyliron(II) Hexafluorophosphate (53) (Fig. 9)31. 1,2-Dichlorobenzene (500 g, 3.4 mol), anhydrous aluminum chloride (91.2 g, 68 mol), aluminum powder (9.2 g, 0.34 mol), and  ferrocene (63.2 g, 0.34 mol) are heated to 95 for 4 h under vigorous stir ring. The reaction is cooled to 0 and H2O (500 ml) is added slowly, while  keeping the temperature below 50 . (Caution: Very exothermic!) Et2O (500 ml) is added and the reaction mixture is stirred for 30 min. The aqueous phase is isolated, filtered, and extracted with Et2O (2  200 ml). The dark-green aqueous phase is filtered and saturated aqueous ammonium hexafluorophosphate solution is added in small portions until precipitation of the desired product is completed (38% yield). This procedure can also be applied to 1,3-dichlorobenzene, 1,4-dichlorobenzene, and 1,3dichloro-2-methylbenzene. Representative Reaction with Piperazine Bound to Polymeric Supports (Fig. 9)31. Piperazin-1-yl methyl polystyrene resin 51a (24 g, 24.2 mmol), 6-1,2-dichlorobenzene-5-cyclopentadienyl iron(II) hexafluorophosphate (53) (25 g, 69.6 mmol), and potassium carbonate (13.4 g, 97 mmol) are suspended in dry THF (400 ml) at RT. (Caution: Exothermic reaction!) The reaction mixture is agitated overnight. Resin 54 is washed with dry THF (2  100 ml), CH2Cl2 (3  100 ml), MeOH (2  100 ml), and CH2Cl2  (3  100 ml), and then dried in vacuo at 40 to afford a red resin. Reaction of Resin-Bound Iron Complex (54) with Aliphatic Alkoxides (Fig. 9)31. A solution of 3-methoxyphenylmethoxide (0.50 M) is prepared by slowly adding 3-methoxyphenylmethanol to sodium hydride (60% suspension in mineral oil) in dry THF at room temperature. (Caution: Generation of heat and hydrogen.) The mixture is stirred for an additional 30 min after gas generation ceased. The alcoholate (11 mmol) is transferred to the resins bearing the aryl-iron complex (54) (3 g, 1.9–2.2 mmol) and THF (45 ml) is added. The reaction is stirred using an overhead  stirrer at 60 for 48 h. The resin 55 is filtered and washed with THF (2  100 ml), H2O (2  100 ml), THF (2  100 ml), MeOH (2  100 ml), and CH2Cl2 (3  100 ml) and dried in vacuo at 40 to give a red resin.

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Reaction of Resin-Bound Iron Complex (54) with Alkyl Mercaptans, Thiophenols, and Phenols (Fig. 9)31. Sodium thiolates are prepared analogously to the alkoxides from thiol and sodium hydride, except that dry DMF is used as a solvent. The substitution on the polymer-bound arene (54) is performed at 70 in DMF within 16 h. The resin is filtered and washed with DMF (2  50 ml), MeOH (2  50 ml), H2O (2  50 ml), MeOH (2  50 ml), and CH2Cl2 (3  50 ml) and then dried in vacuo  at 40 to yield a red resin. Representative Reaction of Resin Bound Iron Complex (54) with Alkylamines (Fig. 9)31. A resin bearing the aryl-iron complex (3 g, 1.9– 2.2 mmol), 4,4-(10,10-dimethyl-9,10-dihydroanthracene-9-yl)-piperidine (3.2 g, 11 mmol), and diisopropylethylamine are suspended in dry DMF (40 ml) and stirred at 90 for 96 h. Resin 55 (XR ¼ NHR) is filtered and washed with DMF (2  50 ml), MeOH (2  50 ml), H2O (2  50 ml), MeOH  (2  50 ml), and CH2Cl2 (3  50 ml), and then dried in vacuo at 40 . 31 Decomplexation on Solid Phase (Fig. 9) . To prepare a 0.5 M 1,10phenanthroline solution, to 1,10-phenanthroline (180 g, 1 mol) CH3CN (600 ml) and H2O (200 ml) are added in that order under stirring. More water is added (approximately 100 ml) until the solution becomes clear. Resins bearing the aryl-iron complexes (55) are suspended in a lighttransparent reactor tube with the phenanthroline solution (10 ml/g of resin). The suspensions are agitated under irradiation with visible light for 12 h. The appearance of an intensively red color of the liquid phase indicates the progress of the decomplexation. The resulting resins 56 are filtered and washed with MeOH (3  50 ml), THF (3  50 ml), and MeOH (3  50 ml) until the washing solutions are colorless (approximately five cycles). The irradiation and washing procedure is repeated until the decomplexation is complete. The resins are washed with MeOH (3  50 ml), THF (3  50 ml), a 1 M solution of triethylamine in THF (3  50 ml), MeOH (2  50 ml), CH2Cl2 (3  50 ml), MeOH (2  50 ml), and CH2Cl2 (3  50 ml)  and dried in vacuo at 50 . Standard Cleavage from the Polymeric Support (Fig. 9)31. To a suspension of the decomplexed resin 56 (2.41 g, 2 mmol) in 1,2-dichloroethane  (30 ml) at 0 is added slowly methylchloroformate (25 mmol). After 1 h  of agitation at 0 , the suspension is allowed to come to RT and the reaction to continue overnight. The resin is filtered and washed with H2O (1  30 ml), CH2Cl2 (3  30 ml), and H2O (1  30 ml). The liquid phase is rendered alkaline using NaOH (1 M, 30 ml) and then separated. The aqueous layer is extracted with CH2Cl2 (3  30 ml). The combined organic phases are washed with brine and dried over MgSO4. Flash column chromatography (silica, heptane/EtOAc) is used for the purification of (57) (up to 72% isolated yield).

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Derivatization of Dichloroheterocyclic Scaffolds (Fig. 10)15 Reductive Amination for Synthesis of PAL-Resin-Bound Amines (59) (Fig. 10)15. To a suspension of 4-formyl-3,5-dimethoxyphennoxymethyl functionalized polystyrene resin (PAL)25 (10.0 g, 11.3 mmol) in DMF (350 ml) a primary amine (56.5 mmol) is added followed by addition of sodium triacetoxyborohydride (7.18 g, 33.9 mmol) and acetic acid (6.52 ml, 113 mmol). The suspension is shaken gently at RT overnight. Resin 59 is washed with methanol (4  300 ml) and CH2Cl2 (4  300 ml) and dried under vacuum. Subsequent derivatization products can be cleaved from the resin by a 2 h treatment with CH2Cl2/TFA/Me2S/H2O 45:45:5:5. Mitsunobu Reaction for Endocyclic N9-Alkylation on Purine Scaffold (60) (Fig. 10).15 This is described later in the section dedicated to Mitsunobu reactions. Suzuki Coupling to Displace the Remaining Chloro Atom on C2 with Aryl Groups (Fig. 10).15 To a 10-ml flame-dried Schlenk flask containing the solid supported intermediate 61 (0.10 mmol, 1.0 equiv.) are added an arylboronic acid (0.50 mmol, 5.0 equiv.), Pd2(dba)3 (0.007 mmol, 0.07 equiv.), 1,3-bis(2,6-diisopropylphenyl)-1H-imidazol-3-ium chloride (carbine ligand, 0.014 mmol, 0.14 equiv.), and Cs2CO3 (0.60 mmol, 6.0 equiv.). The Schlenk flask is purged with Ar and charged with anhyd rous 1,4-dioxane (1.0 ml). The reaction is heated to 80 under Ar. After 12 h the resin is filtered, washed (4  1 ml) with a sodium diethyldithiocarbamate solution (0.05 M in DMF), CH2Cl2 (4  1 ml), and MeOH (4  1 ml). The resin is dried in vacuo. Pd-Catalyzed Displacement of the Remaining Chloro Atom on C2 with Amines (Fig. 10).15 To a 10-ml flame-dried Schlenk flask with resin 61 (0.10 mmol, 1.0 equiv.) are added an amine or aniline (0.50 mmol, 5.0 equiv.), Pd2(dba)3 (0.007 mmol, 0.07 equiv.), 1,3-bis-(2,6-diisopropylphenyl)-1H-imidazol-3-ium chloride (carbine ligand, 0.014 mmol, 0.14 equiv.), and KOtBu (0.60 mmol, 6.0 equiv.). The Schlenk flask is purged with Ar and charged with anhydrous 1,4-dioxane (1.0 ml). The  reaction is heated to 80 under Ar. After 12 h the resin is washed with a sodium dithiocarbamate solution (0.05 M in DMF, 4  1 ml), CH2Cl2 (4  1 ml), and MeOH (4  1 ml) and is subsequently dried in vacuo. Pd-Catalyzed Displacement of the Remaining Chloro Atom on C2 by Phenols (Fig. 10).15 To a 10-ml flame-dried Schlenk flask with resin 61 (0.10 mmol, 1.0 equiv.) are added a phenol (0.50 mmol, 5.0 equiv.), Pd2(dba)3 (0.007 mmol, 0.07 equiv.), 1,10 -biphenyl-2-yl-[di-(tert-butyl)]phosphine (phosphine ligand) (0.028 mmol, 0.28 equiv.), and K3PO4 (0.70 mmol, 7.0 equiv.). The Schlenk flask is purged with Ar and charged with anhydrous toluene (1.0 ml). The reaction is heated to 80 under Ar.

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After 12 h the resin is washed with a sodium diethyldithiocarbamate solution (0.05 M in DMF, 4  1 ml), CH2Cl2 (4  1 ml) and MeOH (4  1 ml), and is subsequently dried in vacuo. Preparation of 2,6-Disubstituted Purines (65) Using Pd-Mediated Reactions Involving Amines and Boronic Acids (Fig. 11)33 The following reactions are performed in a 96-well polypropylene reaction block with 1.8-ml wells. To a well with resin 64 (30 mg, 0.015 mmol) and an amine or boronic acid (R2 ¼ primary or secondary aliphatic or aromatic amine, or aryl boronic acid) (0.125 mmol, 8.3 equiv.) the inorganic salt K3PO4 [100 ( 10) mg, 0.471 mmol, 31.4 equiv.] is added as a dry solid. The reactor block is sealed and the wells purged with Ar by applying vacuum and Ar in an alternating fashion three times successively. Subsequently 1 ml of a freshly prepared solution of Pd2dba3/CHCl3 (0.00298 M, 0.2 equiv.) and P(tBu)3 (0.04 M, 2.7 equiv.) in previously degassed Ar-saturated NMP is added. The reaction wells are purged with Ar another three times. The reaction block is then kept at 100 for 40 h. After draining all the wells, the resin is washed with 0.5 ml of aq. 0.25 M TEAA, in DMA/H2O 4:1 (10), 5% sodium N,N-diethyl dithiocarbonate in DMA (10), DMA (10), DCM (5), MeOH (5), DCM (5), and then n-pentane (5). Compounds of type (65) with C2–C and C2–N bonds can be obtained after cleavage. The main impurity was found to be unreacted starting material (64). Stille Coupling on Support-Bound Compounds 67 (Fig. 11)33 NMP is distilled over CaH2 under Ar at 20 Torr. Resins containing brominated intermediates of type 67 (60 mg, 0.030 mmol) and Cu2O (36.0 mg, 0.25 mmol, 8.3 equiv.) are placed in the reaction wells as solids. The reaction block is sealed and purged with Ar. Through the septa in the reaction block are added 0.5 ml of a freshly prepared stock solution of Pd(OAc)2 (1.38 mg, 0.006125 mmol, 0.2 equiv.) and 1,3-bis-(diphenylphosphino)propane (dppp) (5.15 mg, 0.01249 mmol, 0.41 equiv.) in previously degassed Ar-saturated NMP. Subsequently, 0.25 mmol (8.3 equiv.) of the appropriate stannanes is added as a 0.25 M solution in NMP. The reaction  block is kept at 100 for 20 h. The reaction wells are washed with the following solvents (10  0.5 ml):(1) aq. 0.25 M TEAA, in DMA/H2O (4:1), (2) acetonitrile/HOAc/DMA 2:1:2 (this wash solubilizes the excess Cu2O, which may clot frits), and (3) 5% sodium N,N-diethyl dithiocarbonate in DMA. Alternating washes follow with MeOH (5), CH2Cl2 (5), again CH2Cl2 (5), and n-pentane successively. The resin 68 is dried in vacuo for 2 h and product 69 is obtained by appropriate cleavage.

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Acylations, Alkylations, Reductive Alkylations (Aminations, Alkaminations) Reductive Alkylation of Resin-Bound Tropane 73 (Fig. 12).34 A solution of an aldehyde (0.6 mmol) in 1% HOAc in DMF (9 ml) is added to 0.4 g of dry resin 73 (0.3 mmol/g, 0.12 mmol). The suspension is gently stirred for 1 h and then 127 mg of sodium triacetoxyborohydride is added (0.6 mmol). Stirring is maintained for 35 h at which point methanol is added to quench the excess of the reducing agent and to dissolve the borate salts. The solution is then removed from the resin via filtration cannula and rinsed with DMF (3  10 ml), DMF/H2O (1:1; 3  10 ml), THF (3  10 ml), and DCM (3  10 ml). The resin 74 (X ¼ CH2) is dried under vacuum to constant weight. Acylation of Resin-Bound Tropane (73) (Fig. 12).34 Intermediate 73 (0.12 mmol) is added to the following solution: HATU (228 mg, 0.6 mmol, 5 equiv.), triethylamine (120 mg, 1.2 mmol, 10 equiv.), benzoic acid (73 mg, 0.6 mmol, 5 equiv.), and HOAt (16 mg, 0.12 mmol, 1 equiv.). The suspension is gently stirred at RT overnight. The solution is removed via filtration cannula and the resin (74, X ¼ CO) is washed with DMF (3  10 ml), CH2Cl2 (3  10 ml) and dried in vacuo. Acylation via Activation of Resin-Bound Carboxylic Acid (Fig. 13)41. Resin 76 is swelled in pyridine/DMF (1:10) and reacted with pentafluorophenyl trifluoroacetate (6 equiv.) in pyridine/DMF (1:10) for 1 h. Once filtered, the resin is washed with DMF, then subjected to an 18 h treatment with an appropriate amine building block (6 equiv.). Once filtered, the resin is washed with DMF (5), CH2Cl2 (2), and MeOH (3) and dried in vacuo to provide the product grafted on solid support. Final products of type (77) are obtained with an average purity of 65% by cleavage with TFA/DCM (1:1) for 1 h and subsequent rinsing with appropriate solvents. The cleavage solution and all organic washes are combined and evaporated (yields up to 85%). Stepwise Selective Amine and Amide Alkylation (Fig. 14).44 A first alkylation step is performed by suspending (78) in a 2 M solution of a suitable alkyl halide in DMF at 50 for 24–48 h. After thorough washing with DMF (3), CH2Cl2 (3), and THF (3) intermediate (79) (usually formed with >85% purity) is subjected to the final alkylation. The reaction flask is sealed with a fresh rubber septum and flushed with nitrogen  followed by cooling to 0 . In a separate flame-dried 25-ml round-bottom flask 12 equiv. (with respect to 79) of 5-phenylmethyl-2-oxazolidinone is added. To the reaction flask freshly distilled THF is added (the appropriate volume to provide a 0.2 M solution of the 5-phenylmethyl-2-oxazolidinone). The resulting clear solution is then cooled to 78 and 1.6 M n-butyl

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lithium in hexanes is added dropwise by syringe under stirring (10 equiv.  with respect to 79). The solution is stirred at 78 for 15 min and trans ferred by cannula to the solid support (79) with stirring at 0 . The resulting  slurry is stirred at 0 for 1.5 h, at which point 15 equiv. of the appropriate alkyl halide is added by syringe followed by addition of anhydrous DMF to reach a final solvent ratio of approx. 70:30 THF/DMF. The slurry is allowed to warm to ambient temperature under stirring. After 3–12 h at RT the solvent is removed by filtration cannula. The support is then washed with THF (1), THF/H2O (1:1; 2), THF (2), and CH2 Cl2 (2). To the fully derivatized product on solid support an excess of TFA/CH2Cl2 (90:10) is added and the cleavage reaction is allowed to continue for 0.5 h at RT. The cleavage solution is removed by filtration cannula, and the resin is rinsed with an appropriate solvent (e.g., MeOH/CH2Cl2). Concentration of the combined filtrates provides the crude product (80) obtained in >80% yield and >80% purity. Purification by RP-HPLC leads to >95% purity and yields in the range of 56–69%. Reduction of an Aromatic Nitro Group on Solid Phase (Fig. 15).45 Resin 81 (1 g, 0.47 mmol/g) is suspended in 20 ml of a 1.5 M solution of SnCl22H2O in DMF. The mixture is shaken for 12–24 h and filtered. The resin is washed with MeOH, CH2Cl2, DMF, dioxane, and Et2O and dried in vacuo to obtain (82). Mitsunobu Reaction for Exocyclic N2-Alkylation on a Purine Scaffold (Fig. 16).47 2,6-Di-tert-butyl-4-methylpyridine (1.54 g, 7.5 mmol, 0.3 M) is  added to freshly distilled CH2Cl2 (25 ml) at 0 under N2. To this solution is added trifluoroacetic anhydride (706 l, 5.0 mmol, 0.2 M) and the mixture is stirred for 5–10 min. The solution is then transferred to the resin 83 and the flask is vortexed and vented several times to relieve pressure. The flask is shaken for 6–12 h after which the solvent is removed and the resin washed with dry CH2Cl2 (5  25 ml) with vortexing between each rinse. The resulting resin 84 is dried. Diethylazodicarboxylate (394 l, 2.5 mmol) is added dropwise to triphenylphosphine (1.31 g, 5.0 mmol) dissolved in dry 1:1 THF/CH2Cl2   (5 ml) at 0 . After stirring at 0 for 1 h, the solution is transferred by Teflon cannula to the trifluoroacetylated resin 84 (180 mg) simultaneously with the dropwise addition of the appropriate alcohol (2.0 mmol) in THF (100 l). The flask is vortexed and vented several times and shaken for 12 h. The solvent is removed and resin 85 washed with DMF (6  10 ml) followed by CH2Cl2 (6  10 ml). The rinsed resin 85 is dried. Knoevenagel Reaction on an Oxindole Scaffold (87) (Fig. 17)49. To a suspension of resin 87 (1 g, 0.32 mmol/g, 0.32 mmol) in 10 ml of a mixture CH2Cl2/MeOH (4:1), 4-hydroxy-di-tert-butylbenzaldehyde (243 mg, 1 mmol) and pyrrolidine (83 l, 1 mmol) are added. The suspension is

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stirred for 24 h, and the resin is filtered, washed with appropriated solvents, and dried under vacuum. The resin is subsequently subjected to cleavage conditions (1 h treatment with 1 M tetrabutylammonium fluoride) and the product 88 is obtained by liquid–liquid extraction into organic solvent. Mannich Reaction on a Resin-Bound Indole Scaffold (89) (Fig. 18).50 Commercially available indole 5- or 6-carboxylic acid supported on Rink amide resin is suspended in HOAc/1,4-dioxane (1:4). A secondary amine (1.5 equiv.) and formaldehyde (1.5 equiv.) are added. The suspension is shaken for 1.5 h at RT. The resin is then filtered, washed with appropriate solvents, and dried under vacuum to obtain (90).

[24] Library Generation via Postcondensation Modifications of Isocyanide-Based Multicomponent Reactions By Christopher Hulme, Hugues Bienayme´, Thomas Nixey, Balan Chenera, Wyeth Jones, Paul Tempest, and Adrian L. Smith Introduction

New developments in the search for novel pharmacological agents over the past decade have focused on the preparation of chemical libraries as sources for new leads for drug discovery. To aid this search a plethora of personal synthesizers and new automation technologies have emerged to help fuel the lead discovery engines of drug discovery organizations. In fact, multistep solid-phase syntheses of diverse libraries in excess of 10,000 products are now feasible via split and mix techniques. At the same time, a multitude of more efficient, diverse, or target-oriented solutionphase chemical methodologies have appeared in the chemical literature, which have enabled the relatively facile construction of successful lead generation libraries with low FTE* input and little capital expenditure. *

Abbreviations: AcCl, acetyl chloride; BDP, benzodiazepine; Boc, tert-butyloxycarbonyl; DCE, 1,2-dichloroethane; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; EtOAc, ethyl acetate; FTE, full-time employee; HPLC, high-pressure liquid chromatography; HRMS, high-resolution mass spectroscopy; IMCR, isocyanide multicomponent condensation reaction; LC/MS, liquid chromatography/mass spectrometry; MCR, multicomponent condensation reaction; MeOH, methanol; MP-carbonate, macroporous carbonate resin (Argonaut Technologies); NMR, nuclear magnetic resonance; PS, polystyrene; PS-DIEA, polystyrene-bound diisopropylethylamine;

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

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small molecule and heterocycle synthesis

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Several groups have pioneered the use of such multicomponent condensation reaction (MCR) technologies, including those led by Ugi, Bienayme, Domling, and Weber, spawning new chemically driven companies that have rapidly built up their own internal corporate collections. One particular branch of MCR methodologies, namely postcondensation modifications (or secondary reactions) of IMCRs (isocyanide – based multicomponent reactions), forms the basis of this chapter. UDC (Ugi/De-Boc/Cyclize) Methodology

Benzodiazepines have been of pharmacological interest for decades and represent one of the most intensely studied heterocyclic templates known in drug discovery circles. Reports of the biological utility of 1,4-benzodiazepine-2,5-diones (BDPs), in particular, have appeared in many areas, including applications as antagonists of the platelet glycoprotein IIb-IIIa,1 anticonvulsant agents,2 antihypnotic agents,3 reverse transcriptase inhibitors,4 and selective cholecystokinin (CCK) receptor subtype A or B antagonists.5 Many routes to libraries of this class of molecule have been developed, including one of the early pioneering solidphase methods reported by Bunin and Ellman.6 A shorter and more efficient route to this class of molecule was initially proposed by Keating and Armstrong7 employing the Ugi MCR8 with anthranilic acids and 1-isocyanocyclohexene, 2 (Armstrong’s convertible isocyanide)9 as the acid PS-TsNHNH2, polystyrene-bound tosylhydrazine; PS-NCO, polystyrene-bound isocyanate; QC, quality control; RPR, Rhoˆne-Poulenc Rorer; RT, room temperature; TBAF, tetrabutylammonium fluoride; TFA, trifluoroacetic acid; TFP, tetrafluorophenol; THF, tetrahydrofuran; TMSN3, trimethylsilyl azide; TOF, time of flight mass spectrometry; TsNHNH2, p-toluenesulfonylhydrazine; UDC, Ugi/DeBoc/Cyclize; Wang, 4-(hydroxymethyl)phenoxy polystyrene. 1 R. S. McDowell, B. K. Blackburn, T. R. Gadek, L. R. McGee, T. Rawson, M. E. Reynolds, K. D. Robarge, T. C. Somers, E. D. Thorsett, M. Tischler, R. R. Webb, and M. C. Venuti, J. Am. Chem. Soc. 116, 5077 (1994). 2 N. S. Cho, K. Y. Song, and C. Parkanyi, J. Heterocycl. Chem. 26, 1807 (1989). 3 L. H. Sternbach, J. Med. Chem. 22, 1 (1979). 4 R. Pauwels, K. Andries, J. Desmyler, D. Schols, M. J. Kukla, H. J. Breslin, A. Raeymaeckers, J. Van Gelder, R. Woostenborgles, J. Heykants, K. Schellekens, M. A. C. Jansen, E. D. Clercq, and P. A. J. Janssen, Nature 343, 470 (1990). 5 M. G. Bock, R. M. Dipardo, B. E. Evans, K. E. Rittle, W. L. Whitter, D. F. Veber, P. S. Anderson, and R. M. Freidinger, J. Med. Chem. 32, 13 (1989). 6 B. A. Bunin and J. A. Ellman, J. Am. Chem. Soc. 114, 10997 (1992). 7 (a) T. A. Keating and R. W. Armstrong, J. Am. Chem. Soc. 118, 2574 (1996). For earlier syntheses of 1,4-benzodiazepines see (b) M. Gates, J. Org. Chem. 45, 1675 (1980). (c) M. Uskokovic, J. Iacobelli, and W. Wenner, J. Org. Chem. 27, 3606 (1962). 8 (a) I. Ugi, Angew. Chem. Int. Ed. Engl. 1, 8 (1962). (b) I. Ugi and C. Steinbruckner, Chem Ber. 94, 734 (1961). (c) I. Ugi, A. Domling, and W. Horl, Endeavor 18, 115 (1994).

[24]

471

isocyanide-based multicomponent reactions

O R4

R1 CHO OH R NH 2 Boc 2

N R3 1

NC 2

R4

O R1 H N N R2 O N R3 Boc

O O R N+ 1 R2 NH2 4

H+

O R1 NH+ N R2 O NH2 5

3

O AcCl/MeOH or 10% TFA/DCE

N

O R1

O N R2 O NH2 6

R2

R1 N O R3 7

Scheme 1. Synthetic route for the preparation of 1,4-benzodiazepine libraries.

and isocyanide inputs, respectively. Subsequent postcondensation acidcatalyzed cyclization of the anthranilic amine (the so-called ‘‘internal nucleophile’’ proceeding via an intermediate munchnone) produces the desired BDP, with reported isolated yields for the two-step procedure ranging from 15 to 50%.10 Intrigued with this report and attracted by the broad biological activity and desirable pharmacokinetics of 1,4benzodiazepine derivatives, workers at Rhoˆne-Poulenc Rorer (RPR; Aventis) developed an alternative protocol that employed N-Boc-protected anthranilic acids, 1 (Scheme 1).11 It was thought that protection of the anthranilic nitrogen could prevent competing and undesired participation in the Ugi reaction, a potential explanation for the moderate yields reported for the parent synthetic route.7 Boc removal and cyclization to BDP could be sequentially achieved on treatment with acid in ‘‘one pot.’’ Cyclization may potentially occur upon acid treatment via any of the three intermediates 4, 5, and 6. Syntheses of five BDPs, designated 8, 9, 10, 11, and 12 (Fig. 1) were evaluated with isolated yields from 70 to 95%. The chemistry was then successfully advanced to production stage and the generation of two 96-well plates of 1,4-benzodiazepine-2,5-diones was reported. The LC/MS A% (area%) yields taken from two 96-well plates are presented in Table I, along with the participating reagents (Fig. 2). 9

(a) T. A. Keating and R. W. Armstrong, J. Am. Chem. Soc. 117, 7842 (1995). (b) F. K. Rosendahl and I. Ugi, Ann. Chem. 666, 65 (1963). 10 T. A. Keating and R. W. Armstrong, J. Org. Chem. 61, 8935 (1996). 11 C. Hulme, S.-Y. Tang, C. J. Burns, I. Morize, and R. Labaudiniere, J. Org. Chem. 63, 8021 (1998).

472

[24]

small molecule and heterocycle synthesis O

O

O

N H 8

N H

O N

N

N

N O

O

O

N

N H

N H O 10

O 9

N N H

O 11

O 12

Fig. 1. Five fully characterized examples of 1,4-benzodiazepines.

NH2

NH2 15 NH2 O

21

H2 N

S

O

20 27

NH2 O

N

O

19 NH2

N

14

NH2

NH2

18

NBoc

NHBoc 13

17

16

CO2H

CO2H

NH2

29

28

NH2

O

O O

22

O

30

NH2 24

O NH2

N

O

O O

33

26

25

32

31

O

N

O

O

23

NH2

O

34

Fig. 2. Diversity reagents employed in the preparation of a 96-member array of BDPs.

TABLE I LC/MS Area% Purities of a 96-Member Array of BDPs

27 28 29 30 31 32 33 34

15

16

17

18

19

20

21

22

23

24

25

26

40/16 85/87 88/84 87/80 45/10 79/49 89/87 85/64

40/29 82/72 85/73 72/52 37/24 74/61 86/69 86/63

40/27 77/64 89/68 69/43 39/22 63/51 88/66 80/67

54/15 79/72 92/81 79/70 36/12 75/12 85/82 85/82

40/25 82/69 92/78 70/41 34/20 66/53 89/63 84/75

39/40 84/67 88/68 80/51 33/12 69/54 85/70 85/64

26/16 81/73 90/75 87/63 41/7 59/10 90/74 82/69

18/21 78/67 82/73 81/64 28/26 74/61 83/69 84/67

41/15 82/74 84/79 81/70 44/10 83/67 86/84 86/75

0/0 43/10 39/8 51/25 9/0 6/0 38/8 27/11

39/31 88/74 76/77 75/60 37/16 71/49 84/74 84/69

47/30 84/73 85/72 80/62 39/20 67/64 88/78 83/61

[24]

isocyanide-based multicomponent reactions

473

The first percentage corresponds to A% yields with N-Boc anthranilic acid 13 and the second percentage is for the N-methylated analogue 14. Note: For A% yields x/y: The first yield x represents that for reactions with N-Boc anthranilic acid, 13. The second yield y represents that for N-Me-Boc anthranilic acid, 14. Row 27 represents yields of reactions with aldehyde 27. Column 15 represents yields of reactions with amine 15. The reaction appeared general for both a range of commercially available aldehydes (e.g., aldehydes with attached ester, heteroaryl, aryl, amido, thioalkyl, alkyl, and cycloalkyl functionality, such as aldehydes 27 through 34) and primary amines (e.g., with attached alkyl, aryl, heteroaryl, acidic, and basic functionality such as amines 15 through 26). The process is also viable, albeit lower yielding, for N-methylated-Boc anthranilic acids. As such this synthetic route, coupled with a facile production protocol, has now been adopted throughout the pharmaceutical industry as a preferred methodology to access arrays of 1,4-benzodiazepines-2,5-diones. Noteworthy is the fact that since the first report of this route, the number of commercially available N-Boc anthranilic acids in the ACD (available chemicals directory) has increased from 2 to 35.12 The abbreviation UDC (Ugi/DeBoc/Cyclize) was thus introduced to describe the one-pot sequence of events, which was subsequently extended to several other pharmacologically relevant templates utilizing the ‘‘universal isontrile’’ concept. For example, application of N-Boc-protected -amino acids allows conversion to diketopiperazines13 35 with four potential diversity points. Similarly, Boc-protected ethylene diamines and mono-N-Boc-protected phenylene diamines afford ketopiperazines14 37 and dihydroquinoxalinones,15 12

N-Boc anthranilic acids are readily accessible in multigram quantities via the synthetic route shown in the scheme below from the corresponding anthranilic acid or isatin. N-Boc diamines are readily available as described in A. P. Krapcho, M. J. Maresh, and J. Lunn, Synthetic Commun. 23, 2443 (1993). The majority of available N-Boc anthranilic acids may be purchased from Anaspec AA. CO2H NH2

HCl(g) MeOH

O NaOMe, MeOH N H 13

O

H2O2

CO2Me NH2

(Boc)2O

CO2Me

K2CO3, MeOH

THF, reflux

N(Boc)2

THF

CO2H NHBoc

C. Hulme, M. Morrissette, F. Volz, and C. Burns, Tetrahedron Lett. 39, 1113 (1998). (a) C. Hulme, J. Peng, B. Louridas, P. Menard, P. Krolikowski, and N. V. Kumar, Tetrahedron Lett. 39, 8047 (1998). (b) Note that MP-carbonate (Argonaut Technologies) was used to facilitate cyclization in the ketopiperazine series. (c) I. Ugi and A. V. Zychlinski, Heterocycles 49, 29 (1998). 15 T. Nixey, P. Tempest, and C. Hulme, Tetrahedron Lett. 43, 1637 (2002). 14

474

[24]

small molecule and heterocycle synthesis O N

R2 R1

R5 R1 40

R3

R4 N R2

N R4

O

O

R4

7

R3

R3 UDC

35

R1

N N

R2

O

UDC R2

N

N

O

NC

R1

UDC

O

O

UDC UDC

39

R3

36

UDC R2 38 R4

O

N R1 O N R3

37

O

R3

R2 N

R1

N H

O

R2 N

R1

N

O

R3 R4

Scheme 2. Examples of templates available from cyclohexenyl isonitrile and UDC methodology.

respectively 36. Both monocyclic lactams 38 (from N-Boc--amino aldehydes) and bicyclic-lactams 39, where postcondensation modification occurs after a tethered input has been used for the Ugi reaction, are readily accessible.16 Additionally, if no internal nucleophile is present, the munchnone may still be trapped by an external dipolarophile to yield polysubstituted pyrroles, 4017 (Scheme 2). The convertible isocyanide also enables transformation of the secondary amide in the Ugi product to a carboxylic acid, ester, or thioester, which is thus amenable to further functionalization. The aforementioned templates are all readily accessible via manufacture in 96-well plates using 96-well plate liquid handlers. The initial condensations are optimal with excess aldehyde (2 equiv.), which can be subsequently removed via a simple scavenging and filtration step with PS-TsNHNH2.18 Several ‘‘universal’’ resin-bound isocyanides have also been developed to exploit UDC methodology for the generation of the above heterocyclic products. 16

C. Hulme, L. Ma, J. Romano, M. P. Cherrier, J. Salvino, and R. Labaudiniere, Tetrahedron Lett. 41, 1889 (2000). 17 (a) T. A. Keating and R. W. Armstrong, J. Am. Chem. Soc. 118, 2574 (1996). (b) A. M. M. Mjalli, S. Sarshar, and T. J. Baiga, Tetrahedron Lett. 37, 2943 (1996). 18 PS-TsNHNH2 was purchased from Argonaut Technologies, Foster City, CA.

[24]

isocyanide-based multicomponent reactions

475

Fig. 3. Examples of immobilized convertible isonitriles.

Piscopio and co-workers19 originally produced an immobilized version of cyclohexenyl isocyanide, 41, and Ugi and co-workers20 utilized a solutionphase carbonate convertible isocyanide, recently extended to solid phase by Kennedy et al. 43.21 A universal rink isocyanide resin has also been reported by workers at Proctor and Gamble.22 Of particular note is the safety-catch linker isocyanide resin 44, developed at Rhoˆne-Poulenc Rorer (now Aventis), which releases the multicomponent adduct through N-Boc activation (i.e., the safety catch) and subsequent hydrolysis, or esterification, of the amide carbonyl (Fig. 3).23 This allows the generation of a methyl ester, which can be further manipulated in solution to give the range of heterocycles accessible via cyclohexenyl isocyanide (Scheme 3). The methoxide safety-catch clipping strategy and subsequent solution-phase cyclization offer similar advantages to a traceless linker as no functionality derived from clipping remains at the end of the synthetic protocol.24 The UDC concept can be further extended by application of ethyl glyoxylate (a ‘‘convertible aldehyde’’). Simple reaction of 48 in the Ugi MCR with N-Boc anthranilic acids, N-Boc--amino acids, mono-N-Boc diamines, and mono-N-Boc phenylenediamines, followed by acid treatment and in some cases proton scavenging, affords 1,4-benzodiazepines 49, diketopiperazines 50, ketopiperazines 51, and dihydroquinoxalinones 52, respectively.25 Note that products differ from those obtained from convertible isocyanides in that they contain an additional exocyclic amide 19

J. F. Miller, K. Koch, and A. D. Piscopio, 214th ACS National Meeting, Las Vegas, Nevada, ORGN-232 (1997). 20 T. Lindhorst, H. Bock, and I. Ugi, Tetrahedron 55, 7411 (1999). 21 A. L. Kennedy, A. M. Fryer, and J. A. Josey, Org. Lett. 4, 1167 (2002). 22 J. J. Chen, A. Golebiowski, J. McClenaghan, S. R. Klopfenstein, and L. West, Tetrahedron Lett. 42, 2269 (2001). 23 (a) C. Hulme, J. Peng, G. Morton, J. M. Salvino, T. Herpin, and R. Labaudiniere, Tetrahedron Lett. 39, 7227 (1998). (b) D. L. Flynn, R. E. Zelle, and P. A. Grieco, J. Org. Chem. 48, 2424 (1983). 24 M. J. Plunkett and J. A. Ellman, J. Org. Chem. 60, 6006 (1995). 25 C. Hulme and M. P. Cherrier, Tetrahedron Lett. 40, 5295 (1999).

476

small molecule and heterocycle synthesis

[24]

Scheme 3. Applications of a safety-catch isonitrile resin in UDC methodology.

O N R3 49

N R4

R2

O HN R1 O

R4

HN R1 O

R3

UDC

UDC

O N O

O O

UDC O

R2

O

N

N R1 H

R3 N 51

N

R4

O

R2

50

UDC

O 48

O

R2 O N

R3

N H

N R1 H O 52

Scheme 4. Accessible templates via use of ethyl glyoxalate.

group. Representative examples, 53 through 60, with A% purities as judged by ELS (evaporative light scattering detection) are shown in Fig. 4. Workers at RPR put several of these chemistries into full production. Of particular note was the preparation of a 12,480-member diketopiperazine library via the aforementioned solution phase approach and produced as a 30 (RNH2)  16 (RNC)  26 (N-Boc--amino acid) full array. Details report a 2-week production time (1 FTE) with four wells/plate analyzed (624 samples) using a selection algorithm that repeated every six plates to ensure a set of representative compounds from the full library. The overall purity distribution is excellent (Fig. 5). Reported physicochemical properties of the collection were also favorable, showing a 66% pass rate for Lipinski’s ‘‘rule of five’’ and 80% of the library possessed a PSA (polar ˚ 2 suggesting good permeability surface area) value between 50 and 140 A 26 properties. Also of note from reagent selection is that 60% of the library members contained at least one or more acid or basic functional groups through R1–R4. These are of particular value for the discovery of 26

J. Kelder, P. D. J. Grootenhuis, D. M. Bayada, L. P. C. Delbressine, and J.-P. Ploemen, Pharm. Res. 16, 1514 (1999).

[24]

477

isocyanide-based multicomponent reactions

53

O N

O

82%

57 N

N

58

O HN

N HN

N

HN N H O 60%

Cl

O O

O

N

N N H

O 70%

O

N H

O

O

HN O 63%

O

N

O

N H

56

O N O

100%

60 O

O N

N O H 20%

100%

100%

HN

59

O

N H

O HN

55

O

O

N

HN O

N H

54

O

N H

Fig. 4. Representative examples of BDPs and diketopiperazines from ethylglyoxalate employing UDC methodology.

% of samples

80 60 40 20 0

0-25%

26-50%

51-75%

76-100%

Purity ranges (ELS) Fig. 5. Purity distribution for 12,480 diketopiperazines.

high-affinity ligands where charge-reinforced H-bonds often play pivotal roles.27 The ability to introduce acids and bases is compatible with the actual cyclization step, where simple selection of reagents, unable to compete with the Boc-protected internal nucleophile for cyclization, yet containing tethered Boc-protected amines or t-butyl esters, are unmasked during the synthetic protocol. Twelve of the amino acids used were revealed and included tyrosine, histidine, proline, aspartic acid (t-Bu protected), asparagine, lysine (Boc protected), methionine, phenylalanine, valine, glycine, and alanine. Repositioning the electrophilic carbonyl tethered to the primary amine 61 to use in the initial condensation also adds to the number of potential 27

A. M. Davis and S. J. Teague, Angew. Chem. Int. Ed. Engl. 38, 736 (1999).

478

small molecule and heterocycle synthesis

[24]

Scheme 5. BDPs and DKPs from immobilized -amino acids. Reagents and conditions: (i) R1CHO (3 equiv.), 61 (3 equiv., Wang resin), R3NC (3 equiv.), R4CO2H ¼ N-Boc anthranilic acid (3 equiv.), all 0.5 M solutions (MeOH/CH2Cl2, 1:1), RT 24 h. Wash resin CH2Cl2 (3), MeOH (3). (ii) R1CHO ¼ N-Boc--amino-aldehyde (3 equiv.), R3NC (3 equiv.), R4CO2H (3 equiv.), 61 (3 equiv., hydroxymethyl resin) all 0.5 M solutions (MeOH/CH2Cl2, 1:1). Wash resin CH2Cl2 (3), MeOH (3). (iii) Ten percent TFA in CH2Cl2, wash resin CH2Cl2 (2).

Scheme 6. Preparation of diketomorpholines.

sites for an appropriately positioned amino internal nucleophile to attack and has been reported by Hulme and co-workers (Scheme 5).28 Note that the benzodiazepines 62, are accessible via Wang resin, either by direct cyclocleavage or cyclization onto the clipped carboxylic acid. However, access to the ketopiperazines 63 is higher yielding utilizing hydroxymethyl resin as opposed to Wang, and mechanistically results only from direct cyclocleavage. Interestingly, Szardenings et al.29 have also reported this methodology and successfully extended it to the synthesis of diketomorpholines 64 (Scheme 6). As such these heterocycles are versatile synthetic precursors to optically active -hydroxy acids,30 however, only limited reports of biological utility exist.31 28

C. Hulme, L. Ma, and R. Labaudiniere, Tetrahedron Lett. 41, 1509 (2000). A. K. Szardenings, T. S. Burkoth, H. H. Lu, D. W. Tien, and D. A. Campbell, Tetrahedron 53, 6573 (1997). 30 G. Frater, U. Muller, and W. Gunther, Tetrahedron Lett. 22, 4221 (1981). 31 M. S. Iyer, K. M. Gigstad, N. D. Namdev, and M. Lipton, J. Am. Chem. Soc. 118, 4910 (1996). 29

[24]

O

R1

O N R4 R5

H N

R2 = R3

O

R4

R N 5 Boc

R1 = O O

R1 N R2 O

NH

O R5

479

isocyanide-based multicomponent reactions

N

R1 H N N R3 O

R4

O R3 66

N

H N

Boc N R5

R5 HN R4 O H N O N R3 R O

R4 R3

2

65

R5

R2 N R1 O

N O 68

67

N R2

R4 H NR O

3

Scheme 7. Ureas and hydantoins from postcondensation modifications of the Ugi-5CR. Reagents and conditions: (i) Ten percent trifluoroacetic acid in dichloroethane. (ii) Na2CO3 (sat.). (iii) 1 N KOH in MeOH/THF/H2O, 3 days; then conc. HCl.

Alcohols are in equilibrium with their carbamic acid in the presence of CO2 and thus employing the CO2/MeOH reagent combination (MeOCO2H), coupled with a UDC strategy, affords access to a variety of biologically important heterocycles.32 Named the Ugi-5CR, this modification of the Ugi reaction was originally reported in 1961 and only two further reports of this reaction have appeared since that date.33 Recent investigations by Keating and Armstrong34 have, however, extended the scope of this condensation via the use of CS2 and COS as oxidized carbon sources. Thus, the Ugi-5CR condensation product 65, derived from N-Boc--amino aldehydes and N-Boc diamines, followed by acid deprotection and base treatment, affords the two cyclic ureas, 66 and 67, respectively (Scheme 7). During the course of this work it was also found that simple treatment of the carbamate condensation product 65 with 1 M KOH gave the corresponding hydantoin, 68. Reports of the biological utility of cyclic ureas and hydantoins have appeared in several areas, including applications as inhibitors of integrins and kinases.35 Representative examples of each core, 69 through 76, with isolated yields are shown in Fig. 6. N-Boc--aminoaldehyde condensation products 78 are also precursors for nucleophilic attack via an internal nucleophile onto the carbonyl derived from the carboxylic acid of the classical Ugi adduct in an acidcatalyzed process. Thus, reaction in the Ugi followed by TFA treatment and prolonged evaporation in a Savant or GeneVac evaporator (8 h) affords imidazolines 79 containing four potential points of diversity in good 32

C. Hulme, L. Ma, J. Romano, G. Morton, S.-Y. Tang, M. P. Cherrier, S. Choi, and R. Labaudiniere, Tetrahedron Lett. 41, 1883 (2000). 33 I. Ugi and C. Steinbruckner, Chem. Ber. 94, 2802 (1961). 34 T. A. Keating and R. W. Armstrong, J. Org. Chem. 63, 867 (1998).

480

[24]

small molecule and heterocycle synthesis

O

O

O

N

N

N

N

70

75 %

H N

N

N O

71

60 %

H N

O

N

N O

O 69

O

N

N

O N 72

74 %

38 %

S H N

H N

H N

O N N

H N

O N O

O N 73

38 %

74

H N

50 %

O N

H N

O N N

O

H N

N O

O

N 75

25%

76

< 10%

Fig. 6. Representative examples of ureas and integrins.

yield (Scheme 8).36 Any uncyclized material may be removed by one-pot scavenging with PS-trisamine and PS-NCO.37 This may be further extended by utilizing N-Boc-protected phenylene diamines 80 to give arrays of benzimidazoles 82 via a solution phase procedure in excellent overall yield (Scheme 9).38 Both represent examples of UDC methodology and have been extended to scale production runs (>10 K) by workers at both Amgen and RPR. Imidazolines have been shown to have biological utility as antidepressants, and imidazoline ligands are known for a number of receptors widely distributed in both the peripheral and central nervous system.39 The imidazoline moiety has also been extensively studied as an amide bond 35

(a) K. Karabelas, M. Lepisto, and P. Sjo, World Patent WO9932483 (1999). (b) K. E. Miller, J. F. Carpenter, and R. R. Brooks, Cardiovasc. Drugs Ther. 12, 83 (1998). (c) A. E. Busch, B. Eigenberger, N. K. Jurkiewicz, J. J. Salata, A. Pica, H. Suessbrich, and F. B. Lang, Br. J. Pharmacol. 123, 23 (1998). (d) J. J. Edmunds, S. Klutchko, J. M. Hamby, A. M. Bunker, C. J. C. Connolly, R. T. Winters, J. Quin, I. Sircar, and J. C. Hodges, J. Med. Chem. 38, 3759 (1995). For previous syntheses of hydantoins see (e) S. Hanessian and R.-Y. Yang, Tetrahedron Lett. 37, 5835 (1996). (f) K. M. Short, B. W. Ching, and A. M. M. Mjalli, Tetrahedron Lett. 37, 7489 (1996). (g) B. A. Dressman, L. A. Spangle, and S. W. Kaldor, Tetrahedron Lett. 37, 937 (1996). (h) S. W. Kim, S. Y. Ahn, J. S. Koh, J. H. Lee, S. Ro, and H. Y. Cho, Tetrahedron Lett. 38, 4603 (1997). 36 C. Hulme, M. Morrissette, and L. Ma, Tetrahedron Lett. 40, 7925 (1999). 37 PS-NCO was purchased from Argonaut Technologies, Foster City, California. 38 P. Tempest, M. Kelly, and C. Hulme, Tetrahedron Lett. 42, 4959 (2001). 39 (a) M. Pigini, P. Bousquet, A. Carotti, M. Dontenwill, M. Gianella, R. Moriconi, A. Piergentili, W. Quaglia, S. K. Tayebati, and L. Brasili, Bioorg. Med. Chem. 5, 833 (1997). (b) M. Harfenist, D. J. Heuser, C. T. Joyner, J. F. Batchelor, and H. L. White, J. Med. Chem. 39, 1857 (1996). (c) H. C. Jackson, I. J. Griffin, and D. J. Nutt, Br. J. Pharmacol. 104, 258 (1991). (d) E. Tibirica, J. Feldman, C. Mermet, F. Gonon, and P. Bousquet, J. Pharmacol. 134, 1 (1987).

[24]

R1 CO2H

481

isocyanide-based multicomponent reactions

NH2 R2

MeOH

CHO

BocHN

R3

NC R4

Boc HN O R1

R3

N

U

R2

77

R3 H N

N

TFA/DCM R4

R1

DC

O

H N N R2

78

R4

O

79

Scheme 8. Formation of imidazolines via UDC methodology. Reagents and conditions: (i) 77 (2 equiv.), R1CO2H, R2NH2, R4NC, RT, 48 h. (ii) PS-tosylhydrazine (3 equiv.), PS-Nmethylmorpholine (3 equiv.). THF: CH2Cl2, 24 h. (iii) Thirty percent TFA/CH2Cl2, 12 h.

R3

CO2H NH2 H N

CHO NC

R2

80

Boc

MeOH R4

U

R1 H Boc N 81

R2

R3

O

R1

H N

N O

N

TFA/DCM R4

R1 N

DC

H N R 4

R3 R2

O 82

Scheme 9. Formation of benzimidazoles via UDC methodology. Reagents and conditions: (i) R3CHO (2 equiv.), R1CO2H, 80, R4NC, RT, 48 h. (ii) PS-tosylhydrazine (3 equiv.), PS-Nmethylmorpholine (3 equiv.). THF: CH2Cl2, 24 h. (iii) Thirty percent TFA/CH2Cl2, 12 h.

replacement in biologically active peptides.40 Benzimidazoles have been shown to exhibit a wide range of biological function, including utility as Factor Xa inhibitors, NPY 1 antagonists, and proton pump inhibitors.41 Clearly rapid access to large numbers of these classes of molecule is of major significance for new lead generation in the pharmaceutical sector. In a recent report, Nixey et al.42 combined glyoxylic acids with N-Boc phenylenediamines 80 and supporting reagents to give the condensation product 83. TFA treatment gave the quinoxalinone, 84, containing four diversity inputs generally in good to excellent yield (Scheme 10). Quinoxalinones have been shown to exhibit a wide range of biological functions, including utility as kinase inhibitors and GABAA receptor agonists acting through the BDP-binding site.43 Four representative examples are shown in Fig. 7. The methodology was reported to be compatible with 40

(a) I. Gilbert, D. C. Rees, and R. S. Richardson, Tetrahedron Lett. 32, 2277 (1991). (b) R. C. F. Jones and G. J. Ward, Tetrahedron Lett. 29, 3853 (1988). 41 (a) Z. Zhao, D. Arnaiz, B. Griedel, S. Sakata, J. Dallas, M. Whitlow, L. Trinh, J. Post, A. Liang, M. Morrissey, and K. Shaw, Bioorg. Med. Chem. Lett. 10, 963 (2000). (b) H. Zarrinmayeh, A. Nunes, P. Ornstein, D. Zimmerman, B. Arnold, D. Schober, S. Gackenheimer, R. Bruns, P. Hipskind, T. Britton, B. Cantrell, and D. Gehlert, J. Med. Chem. 41, 2709 (1998). (c) J. Horn, Clin. Ther. 22, 266 (2000). 42 T. Nixey, P. Tempest, and C. Hulme, Tetrahedron Lett. 43, 1637 (2002). 43 K. Masumoto, 118th Annual Meeting of the Pharmacological Society of Japan, March 31–April 12, Kyoto, 1998, Abstr. 01(XD) 10–1.

482 O

[24]

small molecule and heterocycle synthesis R1

CO2H

R3 NH2

CHO

H N Boc

O NC MeOH R4 (i) U

R2

R1

H Boc N

80

83

R2

R3

O

H N

N

N

R1

N

O

TFA/DCM R4

O

(ii)

O

R3

DC R2

HN

84

R4

Scheme 10. Formation of quinoxalinones via UDC methodology.

O H N

N

85

N HO

O H N

O H N

N

O 86

O

N

N

N

N

N

N

O H N

N O

O

O

O 88

87

Fig. 7. Representative examples of quinoxalinones.

% of samples

80 60 40 20 0 0-25%

26-50%

51-75%

76-100%

Purity ranges (ELS) Fig. 8. Purity distribution after production of 96 quinoxalinones.

96-well production and the purity distribution for a plate of compounds is shown in Fig. 8.44 Miscellaneous Postcondensation Modifications

TMSN3 Modified Ugi Reactions Originally reported in 1961,8 the TMSN3-modified Ugi reaction involves condensation of an appropriately substituted aldehyde or ketone with a primary or secondary amine. Reaction of the Schiff base with an 44

LC/MS analysis was performed using a C18 Hypersil BDS 3-m 2.1  50 mm column (UV 220 nm) with a mobile phase of 0.1% TFA in CH3CN/H2O, gradient from 10% CH3CN to 100% over 5 min.

[24]

N

CHO R2

NH2

CN

R2

R2

O R1

483

isocyanide-based multicomponent reactions

O

NH

R3

TMSN3

N

R1

N

N 89

90

CO2R3

N

CO2R3

N

R1 HCl

N N

N

N

N

91

Scheme 11. Bicyclic tetrazoles via the TMSN3-modified Ugi reaction.

Boc

R6 R2 = H

R1= HN Boc

R3 HN

O O

N N N N

(i) R3 O

NC O 92

O

TMSN3 R4R5NH

R1

U R2

O O

R3

R1

N +

R6 (ii), (iii), (iv) R5 N DC R4

H N

O R3

R5 N N R4 N N N

93

R2

R6

94

R5 N R4

N3−

(i) R5 = H

O O

R3

R1

N N N N

R2 NH R4

(v) C

R1 R4

R2 N N N N N O

R3

95

Scheme 12. Synthetic routes to fused 6,5-ketopiperazine-tetrazoles and 7,5-azepinetetrazoles. Reagents and conditions: (i) R1R2C ¼ O (1.5 equiv., 0.1 M in MeOH), TMSN3 (1 equiv., 0.1 M in MeOH), R3NH2 (1 equiv., 0.1 M in MeOH), 92 (1 equiv., 0.1 M in MeOH), 24 h, RT. (ii) 10% TFA in CH2Cl2. (iii) PS-DIEA, DMF/dioxane, 1:1, reflux. (iv) PS-NCO, PS-TsNHNH2 THF/DCE, 1:1. (v) Reflux, MeOH.

isocyanide and trapping of the intermediate nitrilium ion with azide, affords monocyclic tetrazoles 90 in good overall yield. The initial tetrazole-forming reaction is particularly well suited for the solution-phase preparation of monocyclic tetrazoles, and efficient enough to generate libraries with three points of diversity in the 10,000-member range. Bienayme was the original pioneer in utilizing this version of the Ugi for combinatorial and lead generation purposes. As such, a combination of the TMSN3modified Ugi reaction with a subsequent Michael 1,4-addition elimination process yields bicyclic tetrazoles in a two-step one-pot protocol using an isocyanide that contains a -dimethylaminoacrylate moiety 89.45 Thus, highly elaborate and rigid molecules can be prepared directly from simple precursors (Scheme 11) using this methodology. Workers at Amgen further exploited this reaction in an attempt to access 6,5-fused ketopiperazine-tetrazoles 96 and 7,5-fused azepine-tetrazoles 94, respectively (Scheme 12). Thus, reaction of methyl-isocyanoacetates 92, 45

For an early application of the TMSN3-modified Ugi reaction producing fused tetrazoles see H. Bienayme, Tetrahedron Lett. 39, 2735 (1998).

484

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small molecule and heterocycle synthesis

H N N

O

N N NN

H N

N N

N

N

N 97

84%

98

N N N N 80%

N

N

O

N N N N 91% 100

N

O

N

O

N N N N 101 89%

N

N

O

N N N N

N 99

75%

N

N

O

N N N N 102 72%

Fig. 9. Representative examples of 7,5-azepine tetrazoles and 6,5-ketopiperazinetetrazoles.

N-Boc--aminoaldehydes, TMSN3, and secondary amines was found to proceed with high yield and final product purity (in most cases >70% A% as judged by LC/MS at UV 215 nm), giving the Ugi adduct 93. Acid treatment, proton scavenging, and PS-NCO clean-up afforded arrays of azepine-tetrazoles 94 and, as such, represent an example of UDC methodology.46 Switching to a primary amine resulted in an appropriately positioned internal nucleophile 95 for subsequent cyclization to fused ketopiperazine-tetrazoles 96.47 Representative examples (A% purities as judged by LC/MS UV 220 nm) for both cores are shown in Fig. 9, and isolated yields are reported to be similar. Postcondensation Passerini Reactions Utilizing a postcondensation modification of the Passerini reaction,48 both Banfi and workers at Amgen49 recognized the potential for a onepot, two-step transformation to produce nor-statines with the general structure 104 containing three points of potential diversity. Recognizing their 46

T. Nixey, M. Kelly, D. Semin, and C. Hulme, Tetrahedron Lett. 43, 3681 (2002). T. Nixey, M. Kelly, and C. Hulme, Tetrahedron Lett. 41, 8729 (2000). 48 (a) M. Passerini, Gazz. Chim. Ital. 51, 126 (1921). (b) M. Passerini, Gazz. Chim. Ital. 51, 181 (1921). 49 (a) L. Banfi, G. Guanti, and R. Riva, Chem. Commun. 985 (2000). (b) W. Jones, S. Tadesse, B. Chenera, V. Viswanadhan, and C. Hulme, A.C.S, 223nd American Chemical Society Meeting & Exposition, April 2002, New Orleans. 47

[24]

485

isocyanide-based multicomponent reactions R1 Boc

NH R2 R3

R1 CHO

NC CO2H

i)

Boc

HN O

O

O R3

R1

O N H

R2

ii) iii)

103

R3

N H

O N OH H

R2

104

Scheme 13. Application of the Passerini reaction for the preparation of nor-statines. Reagents and conditions: (i) 0.1 M solutions in MeOH, 18 h, RT, then PS-TsNHNH2 in CH2Cl2. (ii) Ten percent trifluoroacetic acid (TFA) in CH2Cl2. (iii) PS-N-methylmorpholine in CH2Cl2.

utility as known transition state mimetics for the inhibitors of aspartyl proteases,50 the Amgen group advanced the methodology to full production readiness and has reported the preparation of a 9600-member hit generation library. A full array was produced with N-Boc--aminoaldehydes (8), isocyanides (20), and carboxylic acids (60) being employed in conjunction with a variety of immobilized scavenger resins. The key step was simple TFA treatment of the Passerini product 103 followed by proton scavenging to promote full acyl transfer to 104 (Scheme 13). Primary screening interest for the set was the aspartyl protease -secretase,51 one of two proteases that cleave the -amyloid precursor protein (APP) to produce -amyloid peptide (A) in the human brain, a key event in the pathogenesis of Alzheimer’s disease.52 No biological data have yet been reported. However, representative examples and a full QC distribution taken from a 10% random selection of the final library are shown in Figs. 10 and 11. The methodology appears amenable to a range of functionality and amply demonstrates the efficiency of Passerini MCR methodology when compared to analogous linear syntheses. Also note that the overall purity of the library (judged by LC/MS at UV 215 nm) is excellent, a reflection of the short route and compatibility with solution-phase protocols enabling resin-bound removal of excess reagents.

50

(a) B. M. Dunn, ‘‘Structure and Function of the Aspartic Proteases: Genetics, Structures, and Mechanisms,’’ Vol. 306, p. xviii. Plenum Press, New York, 1991. (b) K. Takahashi, ‘‘Aspartic Proteinases: Structure, Function, Biology, and Biomedical Implications.’’ Plenum Press, New York, 1995. (c) C. E. Lee, E. K. Kick, and J. A. Ellman, J. Am. Chem. Soc. 120, 9735 (1998) and references therein. 51 R. Vassar, B. Bennett, and S. Babu-Khan, Science 286, 735 (1999). 52 (a) D. Selkoe, Trends Cell Biol. 8, 447 (1998). (b) D. Selkoe, Nature 399A, 23 (1999). (c) S. Sinha and I. Lieberburg, Proc. Natl. Acad. Sci. USA 96, 11049 (1999).

486

[24]

small molecule and heterocycle synthesis

O

O

O N H

OH

N H

O

O

O N H

105

OH

O N H

O

O

106

O N H

OH

N H

107

Fig. 10. Representative examples of nor-statines.

% of samples

80 60 40 20 0

0-25%

25-50%

50-75%

75-100%

Purity ranges (UV 215) Fig. 11. Purity distribution after production of 9600 nor-statines.

TMSN3-Modified Passerini Reaction Continuing the role of MCRs in the generation of transition state mimetics and potential aspartyl protease inhibitors, the underused TMSN3-modified version of the Passerini condensation was applied to produce tetrazole-based cis-constrained nor-statine isosteres, 110 (mimetics of a mimetic).53 Thus, replacing the standard carboxylic acid input of the Passerini with TMSN3 and combining with N-Boc--aminoaldehydes and isocyanides, tetrazoles 108 and 109 were produced. Treatment of the silylated product, 108, with TBAF afforded 109 and acid deprotection followed by N-capping gave the desired cis-constrained nor-statinetetrazole mimetic 110 (Scheme 14). Earlier work by Abell and Foulds54 clearly demonstrated the importance of tetrazole-based cis-constrained hydroxyethylamine isosteres as a new class of HIV-1 protease inhibitor, although the linear synthesis was lengthy and not well suited to the production of arrays. Several representative examples of the initial condensation together with isolated yields were reported (111 through 115; Fig. 12) and the protocol was advanced to production readiness employing 80 resin-bound 53 54

T. Nixey and C. Hulme, Tetrahedron Lett. 43, 6833 (2002). A. D. Abell and G. J. Foulds, J. Chem. Soc. Perkin Trans. I 2475 (1997).

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487

isocyanide-based multicomponent reactions

Scheme 14. Synthetic route to tetrazole analogs of nor-statines using the TMSN3-modified Passerini reaction.

H N

OH N

N N N

O 111

83%

NH H N

CN OH

H N

N

N N N

O 112

76%

O 113

S

OH N

N N N 70%

H N

OH N

N N N

O 114

H N

N

OH N N N N

O

80%

115

80%

Fig. 12. Representative examples of tetrazole-based nor-statine isosteres.

TFP (tetrafluorophenol) esters55 in a 96-well filter plate, encapsulated in a Calypso frame assembly. Final compound purities were improved (13% on average) by the addition of PS-NCO. Automation

One attractive feature of the multicomponent reaction is the relative ease of its automation. Discrete reactions may be run in parallel by either solution- or solid-phase protocols in a standard 96-well format. Reagent dispensing is facile and rapid and may be performed with a range of commercially available automated 96-well of X, Y dispensers (Quadra 96, Rapid Plate, Hydra 96, Tecan Genesis, Gilson 215 etc.). Figure 13 shows the Quadra 96 with six plates on deck, consisting of one plate with 8 rows, one with 10 columns, two open plates, the wash station (in black), and the final 96-well plate. The typical cycle time for one plate with 20 mol of product targeted is a mere 6 min. Also noteworthy are the commercially available Calypso reaction frame assemblies (Charybdis technologies) (Fig. 14) that, combined with 96-well plate polyfiltronic filter plates, allow automation of a range of solid-phase syntheses and parallel purification 55

(a) J. Salvino, V. N. Kumar, E. Orton, J. Airey, T. Kiesow, K. Crawford, M. Rose, P. Krolikowski, M. Drew, D. Engers, D. Krolinkowski, T. Herpin, M. Gardyan, G. McGeehan, and R. Labaudiniere, J. Comb. Chem. 2, 691 (2000). (b) M. Drew, E. Orton, P. Krolikowski, J. Salvino, and N. V. Kumar, J. Comb. Chem. 2, 8 (2000). (c) W. Jones, D. Overland, L. Poppe, J. Cardenas, M. Pate, and C. Hulme, LabAutomation 2002, T002.

488

small molecule and heterocycle synthesis

[24]

Fig. 13. Production ready Quadra96 with six modified plates on deck. (See color insert.)

Fig. 14. Zymark rapid plate fitted with Calpyso frame assembley. (See color insert.)

with immobilized scavenger resins. The blocks are ideally suited for use with the Zymark Rapid Plate. Filtration of the reaction mix from such scavenger resins is straightforward, as shown in Fig. 15. The operation may be performed in one step via use of the polyfiltronic filter plate loaded

[24]

isocyanide-based multicomponent reactions

489

Fig. 15. Production ready Quadra96 with scavenger resin filtration setup. (See color insert.)

on top of a new collection plate. Note that the majority of these automated tools are conceptually simple and the range of operational conditions they tolerate can be restricting. When optimizing the chemistry of a specific compound class, these constraints must be kept in mind. Hence, powerful MCR transformations, which are often operationally friendly, offer a special bonus and should be considered with extra attention. Conclusion

The chemistry of isocyanides began in the 1850s and was largely ignored until the discovery of the classical Passerini (1921)47 and Ugi reactions (1959).8 Forty years have elapsed since those early days of peptide-like Ugi products to the more recent highly elaborate heterocycles, exemplified by Bienayme, Domling, Weber, Schreiber, Armstrong, Bossio, and others.56 IMCR methodologies have in fact now touched most stages 56

(a) R. W. Armstrong, S. D. Brown, T. A. Keating, and P. A. Tempest, in ‘‘Combinatorial Chemistry. Synthesis and Application’’ (S. R. Wilson and A. W. Czarnik, eds.), p. 153. John Wiley, New York, 1997. (b) C. Blackburn, B. Guan, P. Fleming, K. Shiosaki, and S. Tsai, Tetrahedron Lett. 39, 3635 (1998). (c) H. Bienayme and K. Bouzid, Angew, Chem. Int. Ed. Engl. 37, 2234 (1998). (c) R. Bossio, S. Marcaccini, P. Paoli, and R. Pepino, Synthesis 672 (1994). (d) S.-J. Park, G. Keum, S.-B. Kang, H.-Y. Koh, and Y. Kim, Tetrahedron Lett. 39, 7109 (1998). (e) A. Domling, Comb. Chem. High Throughput Screen. 1, 1 (1998). (f) S. L. Schreiber, Science 287, 1964 (2000). (g) L. Weber, S. Wallbaum, C. Broger, and K. Gubernator, Angew. Chem. Int. Ed. Engl. 34, 2280 (1995).

490

small molecule and heterocycle synthesis

[24]

of the drug discovery process spanning lead discovery, lead optimization, and final drug manufacture. Examples of the latter include the preparation Xylocain and, more recently, the HIV protease inhibitor Crixivan.57 Additional utility is now emerging for the preparation of natural product-like diversity libraries targeting protein–protein interactions encompassed by the emerging field of chemical genomics.58 What does the future hold for isocyanide-based multicomponent reactions? With the advent of functional proteomics delivering hundreds of new targets to drug discovery, ultra-high-throughput screening, and a premium on novel biologically active entities, it seems reasonable to speculate that the discovery of new IMCRs will continue, spawning multiple postcondensation possibilities via secondary reactions. Unions of IMCRs may also continue to receive attention56e and more elaborate preparations of heterocycles amenable for further diversification will no doubt be found. In summary, isocyanide-based multicomponent reactions and subsequent secondary reactions have experienced a resurgence of interest in the past decade and appear well positioned for growth as we enter the new millenium. Experimental Section

Reagents were obtained from commercial sources and used as received. N-Boc anthranilic acid 13 was purchased from Advanced ChemTech and N-Boc-methyl anthranilic acid 14 was purchased from Aldrich. Proton nuclear magnetic resonance (1H NMR) spectra were run at 500 MHz. LC/MS analysis was performed using a C18 Hypersil BDS 3-m 2.1  50-mm column (UV 220 nm) with a mobile phase of 0.1% TFA in CH3CN/H2O, gradient from 10% CH3CN to 100% over 5 or 15 min and APcI ionization. Typical Experimental Procedure (Plate Production) Stoichiometric amounts (0.1 mL) of 0.1 M solutions of the four Ugi components in methanol were combined in order of their participation in the Ugi reaction (aldehyde first, amine second, isocyanide third, and the carboxylic acid fourth) and shaken at room temperature for 20 h. The reagents were dispensed into the 96-well plate using a Quadra 96 (Tomtec) multidispensing system. The solvent was then evaporated in vacuo in a 57

(a) K. Rossen, P. J. Pye, L. M. DiMichele, R. P. Volante, and P. J. Reider, Tetrahedron Lett. 39, 6823 (1998). (b) K. Rossen, J. Sager, and L. M. DiMichele, Tetrahedron Lett. 38, 3183 (1997). 58 S. L. Schreiber, Bioorg. Med. Chem. 6, 1127 (1998).

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isocyanide-based multicomponent reactions

491



Savant evaporator at 65 for 2 h. The Ugi products were then treated with a 10% AcCl/MeOH solution (400 l/well) and shaken overnight at room  temperature. The solvent was then removed in vacuo at 65 with a Savant evaporator for 2 h. LC/MS analyses were performed on every well and A% yields of the desired product are reported in Fig. 5. Typical Experimental Procedure (Scale Up) Stoichiometric amounts (1.75 mL) of 0.1 M solutions of the four Ugi components were combined in order of their participation in the Ugi reaction and stirred at room temperature for 20 h. The solvent was evap orated in vacuo at 40 and dried under high vacuum. A 10% solution of AcCl in MeOH (7 mL) was added to the crude material and stirred at room temperature for 15 h. The solvent was then evaporated in vacuo  at 40 . The crude material was preabsorbed onto flash silica and purified by flash column chromatography (EtOAc:hexane) to yield the desired product. The following benzodiazepines were prepared via this procedure. (R,S)-3-Isopropyl-4-(4-methoxybenzyl)-1,4-benzodiazepine-2,5-dione (8). See scale-up general procedure, isolated yield 81%. For major diastereomer only: 1H NMR (500 MHz, CDCl3, ppm): 8.9 (s, 1H), 7.99– 8.01 (m, 1H), 7.41–7.45 (m, 1H), 7.31–7.33 (m, 2H), 7.21–7.24 (m, 1H), 6.85–6.89 (m, 1H), 6.82–6.84 (m, 2H), 5.14–5.17 (m, 1H), 4.44–4.47 (m, 1H), 3.74 (s, 3H), 3.62–3.65 (m, 1H), 1.65–1.70 (m, 1H), 0.82–0.83 (m, 3H), 0.66–0.67 (m, 3H). For both diastereomers: 13C (125 MHz, CDCl3, ppm): 172.2, 166.3, 159.5, 134.9, 132.8, 131.7, 130.4, 128.8, 127.0, 125.0, 120.0, 114.2, 71.3, 55.4, 54.9, 27.8, 19.8, 19.6. (R,S)-4-Benzyl-3-(2-pyridyl)-1,4-benzodiazepine-2,5-dione (9). See scale-up general procedure, isolated yield 32% of a 10:1 mixture of conformers. For major diastereomer only: 1H NMR (500 MHz, CDCl3, ppm): 8.68 (1H, br s), 8.19–8.20 (1H, m), 7.68–7.70 (1H, m), 7.53–7.55 (2H, m), 7.26–7.36 (4H, m), 7.14–7.17 (1H, m), 6.93–6.96 (1H, m), 6.86– 6.89 (1H, m), 6.73–6.77 (1H, m). For major diastereomer only: 13C (125 MHz, CDCl3, ppm): 171.5, 167.2, 154.0, 148.7, 136.4, 136.2, 134.7, 132.1, 131.0, 129.2, 128.9, 128.2, 127.2, 124.5, 122.4, 120.2, 119.6. (R,S)-3-Ethyl-4-hexyl-1,4-benzodiazepine-2,5-dione (10). See scale-up general procedure, isolated yield 42% of a 2:1 mixture of conformers. For major diastereomer only: 1H NMR (500 MHz, CDCl3, ppm): 0.80–0.98 (6H, m), 1.20–1.30 (6H, m), 1.50–1.70 (4H, m), 3.25–3.35 (1H, m), 3.90– 3.94 (1H, m), 4.00–4.10 (1H, m), 6.90–6.95 (1H, m), 7.2–7.25 (1H, m), 7.40–7.5 (1H, m), 7.90–7.96 (1H, m), 8.60 (1H, br s). For both diastereomers: 13C (125 MHz, CDCl3, ppm): 172.4, 172, 171.7, 168.2, 165.6, 135.6,

492

small molecule and heterocycle synthesis

[24]

134.49, 132.3, 132.1, 131.7, 131.4, 128.0, 127.1, 125.2, 124.8, 120.3, 119.7, 66.5, 57.0, 52.3, 51.7, 42.5, 31.5, 28.6, 28.0, 26.6, 26.4, 22.7, 22.5, 19.6, 14.0, 11.2, 10.8. (R,S)-4-Isobutyl-3-(2-phenylethyl)-1,4-benzodiazepine-2,5-dione (11). See scale-up general procedure, isolated yield 79% of a 2:1 mixture of conformers. 1H NMR (500 MHz, CDCl3, ppm): 0.88–0.89 (3H, m), 0.93–0.94 (3H, m), 1.70–1.78 (1H, m), 1.85–1.90 (1H, m), 1.91–2.00 (1H, m), 2.50– 2.62 (2H, m), 2.90–2.93 (1H, m), 3.96–4.04(2H, m), 6.98–7.01 (2H, m), 7.1–7.3 (5H, m), 7.4–7.45 (1H, m), 7.95–8.0 (1H, m), 8.78(1H, s). For both diastereomers: 13C (125 MHz, CDCl3, ppm): 172.3, 171.8, 168.5, 166.0, 140.4, 139.7, 135.6, 134.5, 132.5, 132.2, 131.7, 131.6, 128.5, 128.5, 128.4, 128.3, 128.3, 127.7, 127.0, 126.4, 126.4, 126.2, 125.2, 125.0, 120.3, 119.8, 117.3. (R,S)-3-Hexyl-4-decyl-1,4-benzodiazepine-2,5-dione (12). See scale-up general procedure, isolated yield 65% of a 2:1 mixture of conformers. For major diastereomer only: 1H NMR (500 MHz, CDCl3, ppm): 8.84 (1H, br s), 7.93–7.96 (1H, m), 7.41–7.45 (1H, m), 7.21–7.27 (1H, m), 6.95–6.99 (1H, m), 3.98–4.04 (2H, 2  m), 3.29–3.33 (1H, m), 1.09–1.32 (26H, m), 0.87–0.89 (6H, m). For both diastereomers: 13C (125 MHz, CDCl3, ppm): 172.7, 172.2, 171.9, 168.2, 165.7, 135.6, 134.6, 132.3, 132.1, 131.6, 131.4, 130.3, 127.9, 127.1, 125.1, 124.8, 120.3, 119.7, 117.4, 116.3, 65.1, 55.6, 52.3, 51.7, 42.6, 31.9, 31.6, 31.5, 31.3, 29.6, 29.5, 29.3, 29.2, 29.1, 29.0, 28.6, 28.5, 28.1, 27.0, 26.8, 26.6, 26.3, 26.1, 22.7, 22.6, 22.5, 22.4, 14.1, 14.0, 14.0, 13.9. Diketopiperazines (50) Typical Experimental Procedure (Scale Up). Stoichiometric amounts (6.2 mL) of 0.1 M methanolic solutions of the three supporting Ugi components and ethyl glyoxalate (7.75 ml) were combined and stirred at reflux overnight. The solvent was evaporated in vacuo and crude Ugi product dried under high vacuum. A 10% solution of AcCl in MeOH (25 mL) or a 10% solution of TFA (trifluoroacetic acid) in dichloroethane (25 mL) was added to the crude material and stirred at room temperature overnight. The solvent was evaporated in vacuo. The crude material was preadsorbed onto flash silica and purified by flash column chromatography (EtOAc:hexane, 1:4) to yield the desired product 57 (192 mg, 71%) as a white solid. Typical Experimental Procedure (Plate Production). Equal amounts (0.1 mL) of 0.1 M solutions in methanol of the four components are employed generating a theoretical 10 mol of final product. Reagents were dispensed into a 96-well plate using either a Quadra 96 or Rapid Plate (Zymark) 96-well dispenser. The deprotection/cyclization steps were

[24]

isocyanide-based multicomponent reactions

493

performed using either a 10% solution of acetyl chloride in methanol or a 10% solution of TFA in dichloroethane. Evaporations were performed at  65 in a Savant evaporator. 1-Benzyl-5-(1-benzyl-1H-imidazol-4-ylmethyl)-3,6-dioxo-piperazine2-carboxylic Acid Cyclohexylamide (109). For major diastereomer: 1H NMR (500 MHz, CD3OD, ppm): 8.91 (s, 1H), 7.32–7.44, 7.23–7.32, 7.11–7.24 (3  m, 12H), 5.36–5.39 (m, 2H), 4.77–4.93 (m, 2H), 4.68–4.70, 4.36–4.38 (m, 1H), 4.03–4.13 (m, 1H), 3.11–3.55 (m, 2H), 3.15–3.21 (m, 1H), 1.60–1.79, 1.10–1.29 (2  m, 10H). 13C (125 MHz, CDCl3, ppm): 168.68, 167.61, 166.65, 166.32, 166.28, 164.93, 136.88, 136.49, 136.38, 136.08, 135.50, 135.37, 131.53, 131.53, 130.58, 130.55, 130.52, 130.43, 130.42, 129.98, 129.95, 129.84, 129.78, 129.69, 129.59, 129.51, 129.26. M. spec. (TOF MS ESþ) 500 (MHþ). For minor diastereomer. 1H NMR (500 MHz, CD3OD, ppm): 8.92 (s, 1H), 7.39–7.44, 7.23–7.34, 7.11–7.13 (3  m, 12H), 5.36–5.39 (m, 2H), 4.77–4.93 (m, 2H), 4.68–4.70, 4.35–4.38 (m, 1H), 4.03–4.13 (m, 1H), 3.37–3.55 (m, 2H), 3.14–3.18 (m, 1H), 1.58–1.93, 1.11–1.29 (2  m, 10H). 13C (125 MHz, CDCl3, ppm): 168.49, 167.45, 166.52, 166.14, 164.77 136.73, 136.34, 136.25, 135.38, 135.25, 131.35, 130.44, 130.37, 130.29, 129.84, 129.81, 129.70, 129.55, 129.45, 129.36, 129.12, 121.92, 121.65, 56.09, 54.84, 53.90, 53.85, 50.66, 50.59, 50.06, 50.00, 33.47, 33.40, 33.12, 32.15, 28.28, 26.50, 25.89, 25.82. M. spec. (TOF MS ESþ) 500 (MHþ). 5-Benzo[b]thiophen-3-ylmethyl-3,6-dioxo-1-propyl-piperazine-2-carboxylic Acid Benzylamide (1). For major diastereomer: 1H NMR (500 MHz, CDCl3, ppm): 7.65–7.80 (s, 2H), 7.10–7.40 (3  m, 7H), 6.95 (s, 1H), 6.5 (s, 1H), 4.35–4.55 (m, 2H), 4.48 (s, 2H), 4.05–4.2 (m, 2H), 3.7–3.9(m, 2H), 3.2– 3.6 (m, 2H), 2.8–2.9 (m, 2H), 1.4–1.6 (m, 2H), 1.2–1.3 (m, 2H), 0.8–0.9 (m, 3H). 13C (125 MHz, CDCl3, ppm): 165.8, 164.8, 163.9, 140.5, 138.2, 137.5, 130.7, 128.7, 127.6, 124.7, 124.4, 124.3, 122.8, 121.6, 64.1, 55.9, 48.2, 44.1, 34.4, 20.1, 11.2. For minor diastereomer: 1H NMR (500 MHz, CDCl3, ppm): 7.75–7.9 (m, 2H), 7.35–7.45 (m, 2H), 7.1–7.3 (m, 5H), 6.78 (s, 1H), 6.05 (s, 1H), 4.4–4.5 (2  m, 3H), 3.8–4.0, 4.2–4.3 (2  m, 3H), 2.85–2.95, 3.05–3.15 (2  m, 2H), 1.4–1.6 (m, 2H), 0.85–0.95 (m, 3H) 13C (125 MHz, CDCl3, ppm): 166.5, 166.5, 164.4, 140.8, 138.1, 137.1, 130.5, 128.7, 127.6, 124.6, 123.1, 121.7, 65.2, 53.0, 48.2, 44.2, 31.3, 20.4, 11.2. Azepine-tetrazoles (94) Typical Experimental Procedure (Scale Up). The following procedure was followed for the large-scale preparation of 99: Solutions of N-(tertbutoxycarbonyl)-d-prolinal (0.1 M, 10 ml in MeOH), 1-(2-pyrimidyl) piperazine (0.1 M, 10 ml in MeOH), methyl isocyanoacetate (0.1 M,

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10 mL in MeOH), and TMSN3 (0.1 M, 10 mL in MeOH) were added to a round-bottom flask and stirred at room temperature for 18 h. The solution was concentrated and the resulting oil was redissolved in 10% TFA/DCM. After an additional 18 h the solution was concentrated and PS-DIEA (3.54 mmol/g, 0.85 g, 3 mmol) was added to the oil followed by a solution of DMF/dioxane (50%, 60 ml). The slurry was heated in a shaker-oven  at 80 for 96 h, followed by filtration and evaporation of the solvent. The oil was purified by flash column chromatography (2% MeOH/chloroform) to yield an off-white solid, 99 (267 mg, 75%). 1H (400 MHz, CDCl3): 8.28 (2H, d, J ¼ 4.5 Hz), 6.50 (1H, dd, J ¼ 4.5, 4.5 Hz), 5.41 (1H, d, J ¼ 15.5 Hz), 5.13 (1H, d, J ¼ 15.5 Hz), 4.37 (1H, m), 4.01 (1H, d, J ¼ 11.5 Hz), 3.85 (4H, m), 3.64 (1H, m), 3.52 (1H, m), 2.99 (2H, m), 2.56 (1H, m), 2.41 (3H, m), 2.05 (2H, m). 13C (100 MHz, CDCl3): 163.9, 161.3, 157.7, 151.5, 110.1, 63.0, 56.9, 52.5, 50.0, 47.2, 43.8, 30.7, 22.3. FTIR: 3272, 1633, 1150, 636 cm1. HRMS: MHþ theoretical value 356.1947: Actual value 356.1952. dM/M ¼ 1.4 ppm. Typical Experimental Procedure (Plate Production). Production of an 80-member array was successfully completed using a Charybdis 96-well Teflon block, encapsulated in a Calypso reaction frame assembly. Reagents were transferred into the 96-well plate using either a Quadra 96 or Rapid Plate 96. The blocks were then heated at 65 for 3 days and the solvent  evaporated in vacuo at 65 . Scavenging with PS-TsNHNH2 (6 equiv.) and PS-NCO (1 equiv.) was performed at the plate level and the resins were added using a Millipore column loader. Evaporation was performed in a Savant evaporator for 2 h. Ketopiperazine Tetrazoles (96) Typical Experimental Procedure (Scale Up). A mixture of phenpropionaldehyde (0.1 M, 15 mL in MeOH), phenpropylamine (0.1 M, 15 mL in MeOH), methyl isocyanoacetate (0.1 M, 15 mL in MeOH), and TMSN3 (0.1 M, 15 mL in MeOH) was stirred at reflux for 48 h. LC/MS analysis after 2 days revealed 72% (product at UV 220 nm, 9% acyclic) and 30% (product at UV 254 nm, 18% acyclic). The solvent was evaporated in vacuo and material dried under high vacuum for 1 h. The crude material was redissolved in THF:DCM (1:1, 20 ml) and PS-NCO (300 mg) was added. The suspension was shaken for 15 h at room temperature. The resin was filtered, crude product preabsorbed onto flash silica, and purified by column chromatography (EtOAc:hexane, 1:2) to yield 102 (321 mg, 60%) as an oil. 1H (400 MHz, CDCl3): 7.07–7.11, 7.17–7.20, 7.23–7.27 (10H, 3  m, 2  C6H5), 4.71–4.97 (2H, m, CH2CO), 4.85–4.86 (1H, m, CH), 4.07– 4.14, 2.99–3.03 (2H, 2  m, NCH2), 2.60–2.65 (3H, m, CH2CH2), 2.36–2.41

[24]

isocyanide-based multicomponent reactions

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(2H, m, CH2), 1.89–2.02, 2.10–2.12 (3H, 2  m, CH2CH2); 13C(100 MHz, CDCl3): 161.37, 150.31, 140.88, 139.13, 129.14, 128.82, 128.68, 128.37, 127.11, 126.59, 53.92, 52.26, 48.12, 45.28, 35.20, 33.52, 30.19, 28.08. HMBC (heteronuclear multibond correlation) revealed connectivities between the two ring methine protons and one methide proton, confirming the cyclic structure. HRMS: theoretical value 362.1981; actual value 362.2000. dM/ M ¼ 5.24 ppm. Nor-statines (104) Typical Experimental Procedure (Plate Production). Production of a 9600-member array was successfully completed in a 96-well format. N-Boc--amino aldehydes (0.2 M, 200 L in MeOH), isonitrile (0.1 M, 200 L in MeOH), and carboxylic acids (0.1 M, 200 L in MeOH) were dispensed using a Quadra 96 liquid dispenser. Each plate represented a 1 (acid)  8 (aldehyde)  10 (isonitrile) array with one unique acid per plate. Plates were capped and agitated gently for 36 h. Solvent was evapor ated in a Savant evaporator for 3 h on the high setting (60 ). PS-TsNHNH2 (>3 equiv., Argonaut) was added with a top resin loader. Crude mixtures were then dissolved in 1:1 THF:DCE via use of a Rapid Plate 96 (600 L) and plates agitated for 12 h. Filtration was performed by transfer of solutions to 96-well filter plates sat above new collection vessels using the Quadra 96. Solvent was then evaporated on high for 2 h. Then 600 L of 10% TFA:DCM was added to each well via the Quadra 96 and plates agitated for a further 12 h. Solvent was evaporated in a Savant evaporator on high setting for 2 h. PS-N-methylmorpholine (>3 equiv.) was added to each well with a top resin loader and the crude material dissolved in dichloroethane (600 L) and plates agitated for 12 h. Filtration was performed by transfer of solutions to 96-well filter plates sat above new collection vessels using the Quadra 96. Solvent was then evaporated on high for 2 h. LC/MS analysis was performed using a C18 Hypersil BDS 3-m 2.1  50-mm column with a mobile phase of 0.1% TFA in CH3CN/H2O, gradient from 10% CH3CN to 100% over 15 min using APcI ionization. QC of four samples from each plate (480 compounds) was performed using a repeating algorithmn. A% purities are reported in Fig. 8. Tetrazole-nor-statine Mimetics (110) Typical Experimental Procedure (Scale Up). Compound 109 (12 mg, 0.036 mmol) was treated with TFA (50% in DCM) for 5 min, evaporated, and the oil was dissolved in DCM. MP-carbonate (3.15 mmol/g, 50 mg) was added and the mixture was shaken overnight, filtered, and evaporated.

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The resulting amine was dissolved in DMF (1.2 mL) and was added to polymer bound 3-cyanobenzoate TFP ester (101 mg, 85 mmol/g, 0.085 mmol).  The slurry was heated at 60 for 16 h, filtered, and the DMF was removed in vacuo. The resulting oil was purified by preparative HPLC to give 113 as an off-white solid (12 mg, 86% yield). 1H (400 MHz, CDCl3): 9.85 (1H, s), 9.35 (1H, s), 9.12 (1H, m), 8.40 (1H, d, J ¼ 8.5 Hz), 8.18 (1H, d, J ¼ 8.5 Hz), 8.08 (1H, dd, J ¼ 8.5, 8.5 Hz), 7.89 (1H, dd, J ¼ 8.5, 8.5 Hz), 7.35 (1H, dd, J ¼ 7.5, 7.5 Hz), 7.21 (1H, d, J ¼ 7.5 Hz), 7.19 (1H, d, J ¼ 7.5 Hz), 4.98 (1H, m), 4.00 (2H, m), 1.99 (3H, s), 1.90 (3H, s). 13C (100 MHz, CDCl3): 157.5, 155.5, 136.4, 135.1, 132.2, 130.9, 128.8, 128.6, 80.6, 64.4, 44.6, 28.2(3C), 17.5, 17.3. FTIR: 3272, 1633, 1150, 636 cm1. HRMS: MHþ theoretical value 363.1565; actual value 363.1569. dM/M ¼ 1.1 ppm. Typical Experimental Procedure (Plate Production). Production of an 80-member array was successfully completed using a 96-well filter plate encapsulated in a Calypso reaction frame assembly. The assembly was  heated at 60 for 18 h, cooled, and the slurry was then filtered into a  collection plate and the solvent was evaporated in vacuo at 65 . Scavenging with PS-NCO (1 equiv.) was performed at the plate level and the resins were added using a Millipore column loader. LC/MS analysis was performed using a C18 Hypersil BDS 3-m 2.1  50-mm column with a mobile phase of 0.1% TFA in CH3CN/H2O, gradient from 10% CH3CN to 100% over 15 min.

[25] Mixture-Based Combinatorial Libraries: From Peptides and Peptidomimetics to Small Molecule Acyclic and Heterocyclic Compounds By Cornelia E. Hoesl, Adel Nefzi, John M. Ostresh, Yongping Yu, and Richard A. Houghten Introduction

The recent emergence of combinatorial libraries made up of millions of chemically diverse compounds has revolutionized the drug discovery process. In contrast to the expectations of ‘‘rational drug design,’’ which enables compounds to be designed based on a detailed understanding of molecular interactions, chemical library diversity allows both direct de novo discovery of lead compounds, as well as enhancement of the activity of existing compounds. Combinatorial chemistry has proven to be a

METHODS IN ENZYMOLOGY, VOL. 369

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powerful tool for providing important information for the fundamental understanding of molecular recognition.1 In the postgenomic era, the library approach can be extended to identify a small molecule partner for every gene product and furthermore to determine the function and biological role of the gene.2 The key feature of combinatorial chemistry is the simultaneous synthesis of a large number of analogs under similar reaction conditions. Historically, the concept was first developed for the synthesis of peptide libraries due primarily to the fact that robust solid-phase chemistry3 was available for peptides. In the 1980s, various multiple synthesis techniques for the rapid parallel synthesis of large numbers of peptides emerged including the synthesis on plastic pins,4 on glass chips,5 on paper,6 and in ‘‘tea-bags.’’7 Inspired by the multiple-synthesis techniques, Lam et al.8 and Houghten et al.9 published simultaneously in 1991 the first practical and broadly applicable validations of the combinatorial library approach. Since then, a wide range of bioactive peptides10 has been identified by employing combinatorial chemistry methods, including novel antibacterials,11 potent agonists and antagonists of opioid receptors,12 inhibitors of melittin’s

1

(a) J. Eichler and R. A. Houghten, Biochemistry 32, 11035 (1993). (b) C. Pinilla, J. R. Appel, and R. A. Houghten, Biochem. J. 301, 847 (1994). (c) J. R. Appel, J. Buencamino, R. A. Houghten, and C. Pinilla, Mol. Divers. 2, 29 (1996). (d) B. Hemmer, M. Bergelli, C. Pinilla, R. A. Houghten, and R. Martin, Immunol. Today 19, 163 (1998). 2 G. Dorma´n, P. Krajcsi, and F. Darvas, Curr. Drug Discov. 21 (October 2001). http:// www.currentdrugdiscovery.com. 3 (a) R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963). (b) R. B. Merrifield, Science 232, 341 (1986). 4 H. M. Geysen, R. H. Meloen, and S. J. Barteling, Proc. Natl. Acad. Sci. USA 81, 3998 (1984). 5 S. P. A. Fodor, R. J. Leighton, M. C. Pirrung, L. Stryer, A. T. Lu, and D. Solas, Science 251, 767 (1991). 6 R. Frank and R. Do¨ring, Tetrahedron 44, 6031 (1988). 7 R. A. Houghten, Proc. Natl. Acad. Sci. USA 82, 5131 (1985). 8 K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, and R. J. Knapp, Nature 354, 82 (1991). 9 R. A. Houghten, C. Pinilla, S. E. Blondelle, J. R. Appel, C. T. Dooley, and J. H. Cuervo, Nature 354, 84 (1991). 10 (a) R. A. Houghten, C. Pinilla, J. R. Appel, S. E. Blondelle, C. T. Dooley, J. Eichler, A. Nefzi, and J. M. Ostresh, J. Med. Chem. 42, 3743 (1999). (b) R. E. Dolle, Mol. Divers. 3, 199 (1998). 11 (a) S. E. Blondelle, E. Pe´rez-Paya´, and R. A. Houghten, Antimicrob. Agents Chemother. 40, 1067 (1996). (b) S. E. Blondelle, E. Takahashi, R. A. Houghten, and E. Pe´rez-Paya´, Biochem. J. 313, 141 (1996). 12 C. T. Dooley, N. N. Chung, P. W. Schiller, and R. A. Houghten, Proc. Natl. Acad. Sci. USA 90, 10811 (1993).

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hemolytic activity, antigenic peptides recognized by monoclonal antibodies,13 and potent endothelin antagonists.10 Highly active chymotrypsin inhibitors have also been found upon screening of a cyclic peptide template combinatorial library.14 In general, the peptide library approaches presented to date fall into three broad categories. The first category represents synthetic approaches, in which peptide mixtures are synthesized, cleaved from the solid support, and the non-support-bound synthetic combinatorial library is assayed in solution, which allows each compound within the mixture to freely interact with a given receptor.15 The focus of the research in our laboratory is directed to the preparation, screening, and deconvolution of non-supportbound combinatorial libraries. The second category involves the chemical synthesis of peptide libraries, which are still attached to the solid support during the screening.8 Alternatively to chemical techniques, recombinant DNA methods, as the third category of peptide library preparation, have been successfully used to generate millions of peptides expressed randomly in a fusion phage or other vector system.16 This method, however, remains restricted to the 20 proteinogenic amino acids, whereas chemical approaches allow the incorporation of nonproteinogenic and nonnatural amino acids as well as chemically modified amino acids and their carboxylic acids. Two different approaches, the ‘‘divide, couple and recombine’’ (DCR) method9 (also known as ‘‘split-resin’’ method8) and the reagent mixture method17 are widely used for the chemical generation of immense mixtures and will be explained here in detail. The low oral bioavailability, the susceptibility to proteolytic breakdown, and the inability to pass through the blood–brain barrier make peptides of less general utility as pharmaceutical leads as compared to nonpeptidic compounds. Substituted heterocyclic compounds offer a high degree of structural diversity and have proven to be broadly useful as therapeutic agents. In the last decade, the design and development of 13

(a) D. R. Burton, C. F. Barbas III, M. A. A. Persson, S. Koenig, R. M. Chanock, and R. A. Lerner, Proc. Natl. Acad. Sci. USA 88, 10134 (1991). (b) C. Motti, M. Nuzzo, A. Meola, G. Galfre´, F. Felici, R. Cortese, A. Nicosia, and P. Monaci, Gene 146, 191 (1994). 14 J. Eichler, A. W. Lucka, and R. A. Houghten, Peptide Res. 7, 300 (1994). 15 C. Pinilla, J. R. Appel, P. Blanc, and R. A. Houghten, BioTechniques 13, 901 (1992). 16 (a) S. E. Cwirla, W. A. Peters, R. W. Barrett, and W. J. Dower, Proc. Natl. Acad. Sci. USA 87, 6378 (1990). (b) J. K. Scott and G. P. Smith, Science 249, 386 (1990). (c) J. J. Devlin, L. C. Panganiban, and P. E. Devlin, Science 249, 404 (1990). (d) M. B. Zwick, J. Q. Shen, and J. Scott, Curr. Opin. Biotechnol. 9, 427 (1998). 17 J. M. Ostresh, J. H. Winkle, V. T. Hamashin, and R. A. Houghten, Biopolymers 34, 1681 (1994).

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strategies for the synthesis of individual and combinatorial libraries of small molecules have become an area of intense research. Many wellknown reactions of organic chemistry have been successfully accomplished on solid supports, including nucleophilic aromatic substitution, condensation reactions, cycloaddition reactions, reduction and oxidation reactions, organometallic reactions, olefin formation reactions, and multicomponent reactions.18 The solid-phase reaction repertoire is continuously growing. Taking advantage of the large diversity afforded by peptide libraries and the well-understood chemistry that provides peptides and peptidomimetics in excellent synthetic purity, one research focus in our group has been on the development of methods to chemically transform existing resin-bound peptide libraries to libraries of peptidomimetics and acyclic and heterocyclic compounds. Termed the ‘‘libraries from libraries’’ concept,19 the postsynthetic chemical modification of peptide libraries leads to combinatorial peptidomimetic libraries as well as acyclic and heterocyclic low-molecular-weight organic compound libraries having different physical, chemical, and biological properties compared to the peptide libraries used as starting materials. Initial examples of this approach include the peralkylation and/or exhaustive reduction of the amide bonds in peptides yielding peptidomimetics and polyamines.20 Case studies illustrating the synthesis of a range of heterocyclic compounds from resin-bound amino acids, peptides, and modified resin-bound peptides are presented here. Preparation of Mixture-Based Synthetic Combinatorial Libraries

Two major techniques are generally employed in solid-phase chemistry to generate mixture-based synthetic combinatorial libraries (SCL) of millions of compounds. Multiple functionalities at diverse positions within the library are incorporated either by mixing multiple resins or by using mixtures of incoming reagents. Resin Mixtures The ‘‘divide-couple-recombine’’ (DCR) method,9 also known as ‘‘splitresin’’ synthesis,8 involves the coupling of reactants to individual aliquots of resin followed by thorough mixing of the resin portions. The resin is then 18

W. D. Bennett, in ‘‘Combinatorial Chemistry’’ (H. Fenniri, ed.), p. 139. Oxford University Press, Oxford and New York, 2000. 19 J. M. Ostresh, G. M. Husar, S. E. Blondelle, B. Do¨rner, P. A. Weber, and R. A. Houghten, Proc. Natl. Acad. Sci. USA 91, 11138 (1994). 20 J. M. Ostresh, C. C. Schoner, V. T. Hamashin, A. Nefzi, J.-P. Meyer, and R. A. Houghten, J. Org. Chem. 63, 8622 (1998).

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divided again for the next building block incorporation. Thereby, a onebead one-compound library is generated. The procedure ensures that an approximately equimolar representation of all individual compounds within the library is obtained. Due to the statistical distribution of beads at each step, a crucial step is the determination of the appropriate amount of resin to be used in order to achieve a complete inclusion of all compounds in the library. The number of beads should be at least 10 times the theoretical number of compounds in the library to ensure that all library components are present with statistical probability. In contrast to the ‘‘reagent-mixture’’ method, described below, the DCR method is more labor and cost intensive, because the amount of resin and work increases proportionally with the number of building blocks incorporated at a particular position. A more detailed presentation of the DCR method, including the experimental procedure, can be found in Ostresh et al.21 Reagent Mixtures The ‘‘reagent mixture method’’ is an alternative way for the introduction of mixture positions using a mixture of incoming building blocks that are reacted with the resin-bound compounds. To ensure an equal distribution of individual compounds within the library, the proportion of each building block in the reaction mixture is varied inversely to its reaction rate, i.e., the higher the reaction rate of a particular building block, the lower is the concentration of this building block in the reaction mixture. To determine the reaction rates and thereby the ratio of every building block in the reagents mixture, a competition experiment is performed involving the reaction of an equimolar reagents mixture with the resin-bound material and determination of the relative composition of the products by HPLC.* Since in solid-phase synthesis typically a large excess of incoming reagents is used, the reaction can be considered a pseudo-first-order reaction (i.e., the reaction rates of the incoming building blocks are independent of the resin-bound compound with which they react). This was shown 21

J. M. Ostresh, S. E. Blondelle, B. Do¨rner, and R. A. Houghten, Methods Enzymol. 267, 220 (1996). * Abbreviations: Boc, t-butyloxycarbonyl; (COIm)2, 1,10 -oxalyldiimidazole; DBU, 1,8diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane; DIC, N,N0 -diisopropylcarbodiimide; DIEA, N,N0 -diisopropylethylamine; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; Fmoc, 9-fluorenylmethoxycarbonyl; HBTU, N-[(1H-benzotriazol-1yl)dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; HOBt, 1-hydroxybenzotriazole; HPLC, high-performance liquid chromatography; RP-HPLC, reverse-phase high-performance liquid chromatography; IPA, isopropyl alcohol; MBHA, p-methylbenzhydrylamine; SPPS, solid-phase peptide synthesis; TFA, trifluoroacetic acid; THF, tetrahydrofuran.

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for mixtures of incoming amino acids,17 aldehydes,22 and carboxylic acids23 reacting with resin-bound amino groups. It is important that the relative ratios of the incoming reagents are similar equal and show similar reactivity in the reaction with the resin-bound reagent (e.g., similar nucleophilicity, no significant steric hindrance). The use of reagent mixtures requires a thorough knowledge of the mechanism and kinetics involved in the specific reactions carried out. In the synthesis of peptides with great length, conformational effects that can alter the relative rates must be taken into account. However, once the isokinetic ratios are determined under controlled reaction conditions, the reagent mixture concept offers the advantage that both defined and mixture position can be incorporated into a molecule at any given position. This is a requirement for the synthesis of positional scanning combinatorial libraries. By simply establishing the isokinetic ratio of amino acids in the coupling reaction with a resin-bound amino function and using the ‘‘reagent mixture’’ method, we have made a wide range of peptide libraries accessible. Furthermore, those libraries were used as starting materials in the synthesis of a variety of peptidomimetic and small molecule libraries. Libraries from Libraries: Generation of Peptidomimetic Libraries Using the ‘‘libraries from libraries’’ concept,19 the diversity of existing combinatorial libraries is leveraged through successive transformations of these libraries. Entirely new chemical diversities are generated by the chemical modification of existing libraries providing different compounds with physical, chemical, and biological properties. Two requirements have to be fulfilled for the chemical modification of an existing library: (1) one must begin with a well-defined library, and (ii) one must use a chemical reagent that can effectively alter chemical moieties, while leaving either all of the compound mixture on the resin or alternatively removing all of the mixture from the resin (in the latter case, the employed reagents must be selectively removed from the cleaved mixture). The ‘‘libraries from libraries’’ approach was first demonstrated by the peralkylation of an existing hexapeptide library,19 since the integrity of the peptide library had been well established in earlier work. Modification of peptide libraries is highly desirable due to the limitations of peptides as potential drugs (lack of oral activity, rapid breakdown by proteolytic enzymes, rapid clearance 22

J. M. Ostresh, C. C. Schoner, M. A. Giulianotti, M. J. Kurth, and R. A. Houghten, in ‘‘Peptides, Frontiers of Peptide Science, Proceedings of the 15th American Peptide Symposium’’ (J. Tam and P. T. P. Kaumaya, eds.), p. 57. Kluwer, Dordrecht, The Netherlands, 1999. 23 J. M. Ostresh and R. A. Houghten, U. S. Patent US5856107 (1999).

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Fig. 1. Libraries from libraries. Generation of peptidomimetic libraries by chemical modification of an existing dipeptide library. (The same transformations were applied to longer peptides.)

from circulation, and typical inability to pass through the blood–brain barrier to effect central nervous system activity). Indeed, the enzymatic susceptibility of the prepared permethylated peptide library was very low.19 Figure 1 summarizes the chemical transformations that have been successfully performed in the past 10 years to yield linear peptidomimetic and small molecule libraries. A vital modification is the exhaustive reduction of the peptide backbone leading to polyamines.24 Polyamines have been shown to be pharmacologically interesting compounds. They are ideally suited to bind to and then condense DNA,25 and multiple amine functionalities are common in drugs active within the central nervous system. In addition, the polyamine libraries were further transformed yielding peptidomimetic libraries such as N-terminally acylated polyamine libraries, peracylated polyamine libraries, and polyurea libraries. Furthermore, the resin-bound polyamines served as templates for the generation of different classes of azaheterocyclic compounds. Chemistry Optimization Generally, when developing a mixture-based synthetic combinatorial library, reaction conditions for all the steps in the reaction scheme need to be optimized to achieve the widest possible breadth of diversity and 24 25

A. Nefzi, J. M. Ostresh, and R. A. Houghten, Tetrahedron 55, 335 (1999). D. R. Morris and L. J. Marton, in ‘‘Polyamines in Biology and Medicine,’’ p. 183. Marcel Dekker, Inc., New York, 1981.

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reproducibility. Variation of the reaction conditions using control compounds derived from the most reactive and least reactive building blocks is required. We usually determine the purity of the product by RP-HPLC. Within a single class of compounds, physical properties like absorbance or ionization can vary widely. Thus, the detection method used should be based on the particular structures being synthesized. After establishing the best reaction conditions, every proposed building block should be tested before inclusion in the library and only those yielding acceptable products under the criteria of >80% yield and purity are considered for inclusion in the library synthesis. The scale-up of the reaction needs to be investigated, since the library synthesis involves a much larger amount of resin and reagents compared to the initial experiments. To determine the breadth of the synthetic approach, we vary the building blocks for the first position of diversity, while keeping the other positions fixed, followed by the variation of the second position, and so on. If adverse interactions between certain building blocks are expected, additional controls must be included. Concurrently with the mixture-based library synthesis, the selected control compounds incorporating the different building blocks have to be prepared to verify the completeness and the reproducibility of all individual reaction steps. Finally, mass spectral analysis of mixtures within the library is used to confirm that the expected range of masses is present. Solid-Phase Synthesis of Heterocyclic Compounds from Resin-Bound Amino Acids, Short Peptides, and Polyamines A large number of drugs feature a heterocyclic component. Thus, the design, synthesis, and evaluation of heterocyclic libraries have rapidly become a major field of organic chemistry. Over the past decade, we have developed synthetic routes to a wide range of different heterocycles starting from resin-bound amino acids, short peptides, and polyamines. As an example of libraries made starting from resin-bound amino acids, a mixture-based combinatorial library containing 16,000 different 2,3,5-trisubstituted 4H-imidazolones 1 was prepared using 40 different amino acids, 20 different isothiocyanates, and 20 different amines (Fig. 2).26 The synthetic strategy involves the conversion of resin-bound amino acids to resin-bound thioureas using isothiocyanates. Reaction of the thioureas with HgCl2 and a wide range of primary and secondary amines gave resin-bound guanidine intermediates. Under acidic cleavage conditions, cyclization yielded the imidazolones 1. Using the DCR method, a mixture-based library of 95 mixtures of 48 benzothiazepine-5-ones 2 (Fig. 2) was produced in eight synthetic 26

Y. Yu, J. M. Ostresh, and R. A. Houghten, J. Comb. Chem. 3, 521 (2001).

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Fig. 2. Solid-phase synthesis of heterocyclic compounds from resin-bound amino acids.

steps.27 2-Fluoro-5-nitrobenzoic acid was coupled to the resin-bound amino acid N--Fmoc-l-cysteine by nucleophilic substitution of the fluoro group forming a thioether functionality. The Fmoc group was cleaved followed by the reductive alkylation of the amine. Cyclization to nitrobenzothiazepine-5-ones was achieved by intramolecular amide bond formation of the resulting secondary amine function and the carboxylic acid moiety of the aromatic ring. Reduction with SnCl2, N-acylation of the resulting amino group and cleavage from the resin using HF/anisole yielded compounds 2. Starting from N--Fmoc-aspartic acid -t-butylester, a wide range of 1,3,4,7-tetrasubstituted perhydro-1,4-diazepine-2,5-diones 3 were synthesized (Fig. 2).28 After deprotection of the aspartic acid amino function and reductive alkylation, a second amino acid was coupled and a second reductive alkylation was carried out. Following tBu cleavage, the thermodynamically favorable coupling of the resulting secondary amine to the side chain of aspartic acid was readily accomplished. A wide range of 1,2,5-trisubstituted 4-imidazolidinones 4 (Fig. 2)29 was prepared by a synthetic approach based on the formation of a reactive adduct of benzotriazole and aldehyde.30 The N-[1-(benzotriazol-1-yl)alkyl] derivative from the -amino group of a resin-bound amino acid underwent spontaneous intermolecular nucleophilic substitution with the

27

A. Nefzi, N. A. Ong, M. A. Giulianotti, J. M. Ostresh, and R. A. Houghten, Tetrahedron Lett. 40, 4939 (1999). 28 A. Nefzi, J. M. Ostresh, and R. A. Houghten, Tetrahedron Lett. 38, 4943 (1997). 29 M. Rinnova´, A. Vidal, A. Nefzi, and R. A. Houghten, J. Comb. Chem. 4, 209 (2002). 30 A. R. Katrizky, S. Rachwal, and B. Rachwal, J. Chem. Soc. Perkin Trans. I 799 (1987).

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Fig. 3. Solid-phase synthesis of hydantoin and thiohydantoin libraries from resin-bound dipeptides.

nearest amide to form the five-membered 4-imidazolidinone ring in a nonstereospecific manner. A variety of heterocyclic compounds was generated starting from resin-bound dipeptides. The reaction of the N-terminal amino group of resin-bound dipeptides with carbonyldiimidazole or thiocarbonyldiimidazole gave intermediate isocyanates or thioisocyanates that further reacted intramolecularly to form the hydantoins 5 or thiohydantoins 6 having two positions of diversity (Fig. 3). We have also synthesized branched hydantoins 7 (or thiohydantoins 8) starting from resin-bound orthogonally protected diamino acids (amino acids having amino group in the side chain) (Fig. 3). Following coupling of a second amino acid and hydantoin (or thiohydantoin) formation, the side chain of the diamino acid was deprotected and the free amino group was then either N-acylated with various carboxylic acids or reacted with isocyanates to urea moieties. Cleavage from the solid support by HF/anisole yielded the corresponding hydantoins 7 or thiohydantoins 8.31 To increase the number of diversities, the hydantoin (or thiohydantoin) formation reaction was performed starting from N-alkylated dipeptides (Fig. 3). In the last synthesis step, the hydantoin (or thiohydantoin) ring was alkylated followed by the cleavage from the resin. Using 54 different amino acids for the first position of diversity (R1), 60 different amino acids for the second position of diversity (R2), and four different alkylating 31

A. Nefzi, J. M. Ostresh, M. Giulianotti, and R. A. Houghten, Tetrahedron Lett. 39, 8199 (1998).

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Fig. 4. Solid-phase synthesis of an indolepyridoimidazole library and an imidazoimidazolone library from resin-bound acylated dipeptides.

reagents (R3), two libraries (38,880 hydantoins 9 and 38,880 thiohydantoins 10) were prepared.32 Starting from resin-bound N-acylated dipeptides 11 having tryptophan or a tryptophan analog as the C-terminal amino acid, a mixture-based combinatorial library of 46,750 indolepyridoimidazoles 12 was synthesized by double cyclodehydration under Bischler–Napieralski conditions (POCl3,  dioxane, 100 ) (Fig. 4).23 Twenty-two tryptophan analogs, 25 amino acids, and 85 carboxylic acids were used for the library synthesis. The synthetic strategy was extended to the parallel synthesis of [3,5,7]1H-imidazo[1,5-a]imidazol-2(3H)-ones 13 starting from resin-bound N-acylated dipeptide 11 having various nonaromatic amino acids as the C-terminal amino acid (Fig. 4).33 The chloriminium ion, which is generated as an intermediate from the peptidic amide bond using POCl3, is reacted with the second amide group instead of an aromatic ring to form the amidine structure. A second generated chloriminium ion induced the formation of the second heterocycle and, thereby, led to compounds 13. A variety of reaction conditions were tried and the best results were obtained  using 15 equiv. of freshly distilled POCl3 at 100 in dioxane for 18 h. Dehydration of monoacylated diamines using POCl3 yielded substituted imidazolines 16 and 19 (Fig. 5). Selective acylation of the primary amino group of the diamines 14 and 17 was successfully achieved using a 32

A. Nefzi, C. Dooley, J. M. Ostresh, and R. A. Houghten, Bioorg. Med. Chem. Lett. 8, 2273 (1998). 33 Y. Yu, H. M. El Abdellaoui, J. M. Ostresh, and R. A. Houghten, Tetrahedron Lett. 42, 623 (2001).

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Fig. 5. Solid-phase synthesis of imidazolines from resin-bound acylated diamines.

moderate excess of a wide range of carboxylic acids (5 equiv, 0.1 M in DMF) in the presence of HBTU and DIEA. Treatment of the amides 15 and 18 with POCl3 in dioxane led to cyclodehydration of the resulting in situ-formed chloriminium intermediates to generate the resin-bound imidazolines.34 When the diamines 14 were acylated by 4-fluoro-3-nitrobenzoic acid, following dihydroimidazole formation, the fluoro and nitro group could be successfully utilized to construct a 2-alkylthiobenzimidazole, dihydroquinoxalin-2,3-dione, and 2-iminobenzimidazole ring (Fig. 6).35 The fluoro group of 21 was displaced by nucleophilic substitution with a primary amine. The nitro group was reduced and the resulting o-dianilino compounds were treated with three different reagents to yield the biheterocyclic dihydroimidazole analogs 22, 23, and 24. Cyclization with cyanogen bromide (CNBr) generated the disubstituted dihydroimidazolyl 2-iminodihydrobenzimidazoles 22. Treatment with 1,10 -oxalyldiimidazole (COIm)2 followed by N-alkylation using DBU and alkyl halides led to the trisubstituted dihydroimidazolyl dihydroquinoxalin-2,3-diones 23. The reaction with thiocarbonyldiimidazole gave dihydroimidazolyl dihydrobenzimidazol-2-thiones, which were subsequently S-alkylated with alkyl halides in the presence of a weak base (1-methylimidazole) yielding, after cleavage from the resin, the dihydroimidazolyl 2-alkylthiobenzimidazoles 24. Compounds 24 were easily oxidized to trisubstituted dihydroimidazolyl 2-alkylsulfonylbenzimidazoles using hydrogen peroxide under weakly basic conditions [1 M (NH4)2CO3 in 50% acetonitrile in water]. 34 35

A. N. Acharya, J. M. Ostresh, and R. A. Houghten, J. Org. Chem. 66, 8673 (2001). A. N. Acharya, J. M. Ostresh, and R. A. Houghten, J. Comb. Chem. 4, 214 (2002).

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Fig. 6. Solid-phase synthesis of biheterocyclic dihydroimidazole derivatives from resinbound diamines.

Fig. 7. Solid-phase synthesis of 1,7-disubstituted-1,3,5-triazepane-2,4-diones and a 2-aryliminoimidazolidine library from resin-bound diamines.

Starting from resin-bound diamines 25, the parallel synthesis of 1,7disubstituted 1,3,5-triazepane-2,4-diones 26 was carried out using phenyl isocyanatoformate (Fig. 7).36 To avoid regioselectivity problems in the cyclization step, amino acids that generate reactive functionalities after reduction (asparagine, glutamine, lysine) were not included in the R1 position. In an efficient one-pot reaction, diamines 25 were converted to 1,5disubstituted 2-aryliminoimidazolidines 27 via mercury(II)-activated trisubstituted thioureas using arylisothiocyanates, HgCl2, and triethylamine in DMF at room temperature (Fig. 7).37 A mixture-based combinatorial 36

Y. Yu, J. M. Ostresh, and R. A. Houghten, Org. Lett. 3, 2797 (2001).

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Fig. 8. Solid-phase synthesis of a trisubstituted bicyclic guanidine library.

Fig. 9. Solid-phase synthesis of an urea-tethered bicyclic guanidine library from resinbound polyamines.

library containing 16,000 (40 R1  20 R2  20 R3) 1,5-disubstituted 2-aryliminoimidazolidines 27 was synthesized. Starting from resin-bound reduced acylated dipeptides, a mixture-based combinatorial library of 102,459 protonated bicyclic guanidines 30 was synthesized in the positional scanning format. Following exhaustive reduction of the resin-bound N-acylated dipeptides 28, the resin-bound triamines 29 were treated with thiocarbonyldiimidazole to generate after HF cleavage the trisubstituted bicyclic guanidines (Fig. 8).20 Amino acids, such as arginine or lysine, which yield reactive funtionalities following amide reduction, were not included in the library. Forty-nine amino acids for the first variable position of diversity, 51 amino acids for the second position, and 41 carboxylic acids for the third position were found to yield the product in a purity greater than 80% after cyclization, thereby meeting the library criteria imposed. To increase the amount of diversity, the solid-phase synthesis of an urea-linked library containing 47,600 bicyclic guanidines 33 was achieved starting from a glutamine-containing resin-bound N-acylated dipeptide library (Fig. 9).38 Following exhaustive reduction, the selective protection of the primary amine with trityl yielded the resin-bound polyamines 31. Treatment of the three secondary amines with thiocarbonyldiimidazole generated the resin-bound bicyclic guanidines 32. After trityl deprotection, coupling of an amino acid, N-deprotection of the just-coupled amino acid, isocyanate treatment, and final HF cleavage, the urea-linked bicyclic guanidines 33 were obtained. 37 38

Y. Yu, J. M. Ostresh, and R. A. Houghten, J. Org. Chem. 67, 3138 (2002). A. N. Acharya, A. Nefzi, J. M. Ostresh, and R. A. Houghten, J. Comb. Chem. 3, 189 (2001).

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Solid-Phase Synthesis of Bis-Heterocyclic Compounds from Resin-Bound Acylated and Nonacylated Polyamines An efficient, practical solid-phase synthesis of a variety of bis-heterocyclic compounds was developed starting from resin-bound orthogonally protected lysine (Fig. 10). Tetraamines 36 were synthesized by exhaustive reduction of resin-bound tetraamides 35. Cyclization with different commercially available bifunctional reagents such as cyanogen bromide, thiocarbonyldiimidazole, carbonyldiimidazole, and oxalyldiimidazole yielded the corresponding bis-heterocyclic compounds bis-cyclic guanidines 37,39 bis-cyclic thioureas 38, bis-cyclic ureas 39, and bis-diketopiperazines 40, respectively.40 Reduction of compounds 40 led to bis-piperazines 41.

Fig. 10. Solid-phase synthesis of bis-heterocyclic libraries from resin-bound orthogonally protected lysine.

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Fig. 11. Solid-phase synthesis of bis-cyclic thioureas and bis-cyclic guanidines from resinbound reduced tripeptides.

Extending the above-mentioned cyclization reactions to other polyamines, resin-bound tetraamines 42 from resin-bound tripeptides containing three secondary amines and one terminal primary amine were treated with thiocarbonyldiimidazole41 or cyanogen bromide42 (Fig. 11). Kinetically, the primary amine reacts first with thiocarbonyldiimidazole (or cyanogen bromide), followed immediately by cyclization to form the energetically favorable five-membered ring. The two remaining secondary amines then further react to form the second cyclic thiourea (or cyclic guanidine). It is important to work at lower concentrations with small excesses of the reagents to minimize the formation of undesired impurities most likely due to cyclization between the two internal secondary amines. In support of the kinetic hypothesis, it was found that the reaction of resin-bound polyamines containing four secondary amines with thiocarbonyldiimidazole led to the formation of multiple products. Parallel Synthesis of Combinatorial Libraries As continuation and extension of the parallel synthesis and ‘‘libraries from libraries’’ concept, there is the simultaneous synthesis of various libraries (i.e., a resin-bound library is converted in parallel into several new libraries employing different reagents). Figure 12 illustrates the strategy. The initial step is the preparation of the dipeptide library 45. Introduction of the benzyl group was achieved by selective N-alkylation of N-terminally trityl-protected resin-bound amino acids in the presence of lithium t-butoxide and benzyl bromide. As expected, the alkylation of the amide nitrogen 39

A. A. 41 A. 42 A. 40

N. Acharya, J. M. Ostresh, and R. A. Houghten, J. Comb. Chem. 3, 578 (2001). Nefzi, M. A. Giulianotti, and R. A. Houghten, J. Comb. Chem. 3, 68 (2001). Nefzi, M. A. Giulianotti, N. A. Ong, and R. A. Houghten, Org. Lett. 2, 3349 (2000). N. Acharya, J. M. Ostresh, and R. A. Houghten, Tetrahedron 57, 9911 (2001).

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Fig. 12. Parallel synthesis of combinatorial libraries of heterocycles via resin-bound triamines.

next to the resin dramatically increased the acid sensitivity of the MBHA resin-bound amino acids, excluding the use of Boc-amino acids in any further coupling step. Therefore, Fmoc-amino acids were used to obtain the second position of diversity. Following Fmoc removal and N-acylation, exhaustive reduction of the amide bonds yielded the triamine library 46, which served as starting material for the parallel synthesis of five different heterocyclic libraries employing various cyclization reagents. The reaction of 46 with carbonyldiimidazole or (thiocarbonyldiimidazole) led to cyclic ureas 47 or (thioureas 48).43 Diazepinediones 49 were formed by cyclization using malonyl chloride. Treatment of library 46 with oxalyldiimidazole afforded the 2,3-diketopiperazines 50.44 Following cyclization with oxalyldiimidazole, piperazines 51 were obtained by reduction of the oxamide moiety on the solid support. Ninety-six different building

43 44

A. Nefzi, J. M. Ostresh, J. -P. Meyer, and R. A. Houghten, Tetrahedron Lett. 38, 931 (1997). A. Nefzi, M. A. Giulianotti, and R. A. Houghten, Tetrahedron 56, 3319 (2000).

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blocks (29 Boc-protected amino acids for the first position of diversity, 27 Fmoc-protected amino acids for the second position of diversity, and 40 carboxylic acids for the third position of diversity) were included for the parallel synthesis of libraries 47–51. For the parallel cyclization using five different cyclization reagents, 480 individual control compounds (96  5) needed to be prepared concurrently to the synthesis of the 480 mixtures (96  5) to ensure the completeness and reproducibility of the performed cyclization reactions. Mixtures, containing in total 156,600 structurally different heterocycles, were prepared in an efficient, reliable, and fast manner using 960 ‘‘teabags.’’ Along the same lines, in addition to the N-benzylated compounds, the corresponding N-methylated compounds were synthesized employing the parallel synthesis of libraries. Experimental Procedure for the Parallel Synthesis of Heterocyclic Positional Scanning Libraries 47 to 51

General Requirements for the Synthesis BOC-amino acid derivatives, Fmoc-amino acid derivatives, HOBt, and HBTU were purchased from Calbiochem-Novabiochem Corp. (San Diego, CA), Bachem Bioscience Inc. (Philadelphia, PA), and Bachem California (Torrance, CA). MBHA resin, 1% divinylbenzene, 100–200 mesh, 0.81 mmol/g substitution, was purchased from Chem Impex Intl. (Wood Dale, IL). DIC was purchased from Chem Impex Intl., trifluoroacetic acid (TFA) from Halocarbon (River Edge, NJ), and hydrogen fluoride from Air Products (San Marcos, CA). All other reagents and anhydrous solvents (DMSO and THF) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Resin packets (teabags) are made with polypropylene mesh (74 m) using an impulse sealer. Teabags are numbered, filled with 100 mg of MBHA resin (1% divinylbenzene, 100–200 mesh, 1 mmol/g substitution) to each bag and sealed. For standard reactions, the teabags were put into a polypropylene bottle, containing the reagents solution, and the bottle was shaken using a reciprocating shaker. Air-sensitive reactions were performed either within a glovebox or, when heating was required, in a Pyrex resin kettle. Completeness of the coupling reactions was verified by the ninhydrin test.45

45

E. T. Kaiser, R. L. Colescott, C. D. Blossinger, and P. I. Cook, Anal. Biochem. 34, 595 (1970).

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Coupling of Boc Amino Acids to MBHA Resin to Introduce the First Position of Diversity Standard SPPS (solid-phase peptide synthesis) methodology was used (6 equiv. of Boc amino acid or Boc amino acid mixture, 6 equiv. DIC/ HOBt, 1 h in DMF). Twenty-nine Boc amino acids were coupled individually to 145 resin-filled teabags (numbered 471–29, 481–29, 491–29, 501–29, 511–29). A mixture, containing 29 Boc amino acids in a predetermined isokinetic ratio,17 was coupled to 335 resin-filled teabags (numbered 4730–96, 4830–96, 4930–96, 5030–96, 5130–96). The teabags were pooled together, the Boc group was removed (55% TFA in DCM, 30 min), and the resin was neutralized (5% DIEA in DCM). Trityl Protection of the Resin-Bound Amino Acids The resin-bound amino acids (480 teabags) were treated with 15 equiv. of trityl chloride (0.1 M in 90% DMF/10% DCM) containing 20 equiv. of DIEA for 3 h. Following washes with DMF, the resin-filled teabags were dried overnight under high vacuum. Alkylation of Trityl-Protected Resin-Bound Amino Acids All manipulations were carried out under nitrogen atmosphere in a glovebox. Lithium t-butoxide (0.5 M, 20 equiv.) in THF was added to the 480 teabags. After shaking for 15 min, the base solution was removed. Sixty equivalents of benzyl bromide in DMSO was added and the teabags were shaken for 2 h. The alkylation solution was removed. Deprotonation and alkylation were repeated three times. Following washes with DMF, IPA, and DCM, the resin packets were dried under high vacuum. Removal of the Trityl Group The trityl-protecting group was removed by treatment of the 480 teabags with 2% TFA in DCM (twice for 10 min). Coupling of Fmoc Amino Acids to Introduce the Second Position of Diversity Standard SPPS methodology was used (6 equiv. of Fmoc amino acid or Fmoc amino acid mixture, 6 equiv. DIC/HOBt, 1 h in DMF). Twentyseven Fmoc amino acids were coupled individually to 135 resin-filled teabags (numbered 4730–56, 4830–56, 4930–56, 5030–56, 5130–56). A mixture, containing 27 Fmoc amino acids in a predetermined isokinetic ratio,46 was coupled to 345 resin-filled teabags (numbered 471–29, 4757–96, 481–29,

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4857–96, 491–29, 4957–96, 501–29, 5057–96, 511–29, 5157–96). The teabags were pooled together and the Fmoc group was removed (20% piperidine in DMF, 2  10 min), followed by washes with DMF and DCM. N-Acylation to Introduce the Third Position of Diversity Standard SPPS methodology was used (10 equiv. of carboxylic acid or carboxylic acid mixture, 10 equiv. DIC/HOBt, 12 h in DMF). Forty carboxylic acids were coupled individually to 200 resin-filled teabags (numbered 4757–96, 4857–96, 4957–96, 5057–96, 5157–96). A mixture, containing 40 carboxylic acids in a predetermined isokinetic ratio,23 was coupled to 280 resin-filled teabags (numbered 471–56, 481–56, 491–56, 501–56, 511–56). The resin-filled teabags were dried overnight under high vacuum. Exhaustive Reduction of the Amide Bond All the teabags were put into a resin kettle under nitrogen. Fifteen equivalents of boric acid and 15 equiv. of trimethylborate were added followed by the slow addition of 45 equiv. of borane (1 M in THF). After  hydrogen production ceased, the reaction was heated at 65 for 72 h. The reaction solution was decanted and quenched by the slow addition of methanol. The resin was washed with methanol, THF, and piperidine. The polyamine–borane complex was disproportionated by overnight treat ment (16 h) with piperidine at 65 followed by washes with DMF, DCM, and methanol. Parallel Synthesis of Heterocyclic Libraries 47 to 51 The cyclization reactions were performed under nitrogen atmosphere in a glovebox. All teabags were prewashed with anhydrous DCM prior to cyclization reactions. Five equivalents of carbonyldiimidazole (0.1 M in anhydrous DCM) was added to 96 teabags (471–96) and the reaction was shaken 16 h to afford, following washes with DCM and IPA and HF cleavage, the resin-bound cyclic urea library 47. Synthesis of the resin-bound cyclic thiourea library 48 was achieved by treatment of 96 teabags (numbered 481–96) with 5 equiv. of thiocarbonyldiimidazole (0.1 M in anhydrous DCM) for 16 h followed by washes with DCM and IPA and HF cleavage. Cyclization of 96 teabags (numbered 491–96) using 5 equiv. of malonyl chloride in anhydrous DCM for 16 h led, following HF cleavage, 46

J. Eichler, C. Pinilla, S. Chendra, J. R. Appel, and R. A. Houghten, in ‘‘Innovation and Perspectives in Solid Phase Synthesis’’ (R. Epton, ed.), p. 33. Mayflower Worldwide Limited, Birmingham, 1994.

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to the resin-bound diazepinedione library 49. Teabags (192) (numbered 501–96, 511–96) were treated with 5 equiv. of oxalyldiimidazole (0.1 M in anhydrous DCM) for 16 h. Following washes with DCM and IPA, two sets of the resin-bound 2,3-diketopiperazine library 50 were obtained. One set was cleaved to generate the 2,3-diketopiperazine library 50. The second set (teabags, numbered 511–96) was reduced in the presence of BH3-THF as described for the amide reduction. Following HF cleavage the resin-bound piperazine library 51 was obtained. Cleavage of Resin-Bound Heterocyclic Libraries from the MBHA Resin To obtain the non-support-bound libraries 47 to 51, the heterocyclic compounds, still contained within their teabags, were cleaved for 7 h with hydrogen fluoride/anisole using a multivessel apparatus47 for simultaneous cleavage. Simultaneous Synthesis of Control Teabags To ensure the completeness and reproducibility of all reaction steps, concurrently to the library synthesis, 480 control teabags (numbered 47C1–C96, 48C1–C96, 49C1–C96, 50C1–C96, 51C1–C96) were treated under the same reaction conditions described above. To 145 teabags (47C1–C29, 48C1–C29, 49C1–C29, 50C1–C29, 51C1–C29), the 29 Boc-amino acids were individually coupled as the first amino acid. Following trityl protection, alkylation was carried out as described above. Fmoc-phenylalanine was coupled followed by N-acylation with phenylacetic acid. Teabags (135) (47C30–C56, 48C30–C56, 49C30–C56, 50C30–C56, 51C30–C56) were treated with Boc-phenylalanine, trityl protected, and alkylated, followed by the second coupling of 27 individual Fmoc-amino acids and N-acylation with phenylacetic acid. N-Acylation of 200 teabags (47C57–C96, 48C57–C96, 49C57–C96, 50C57–C96, 51C57–C96) was performed with 40 individual carboxylic acids after coupling of Boc-phenylalanine, trityl protection, alkylation, and coupling with Fmoc-phenylalanine. Conclusion

Employing the teabag approach for parallel synthesis, the reagent mixture method, and the ‘‘libraries from libraries’’ concept, a wide range of different small molecule compounds was successfully prepared in our laboratory during the past decade. Starting from amino acids and short 47

R. A. Houghten, M. K. Bray, S. T. DeGraw, and C. J. Kirby, Int. J. Peptide Protein Res. 27, 673 (1986).

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peptides, new reactions for the solid-phase synthesis of heterocycles were developed and optimized for library synthesis. Using the positional scanning technique, screening and deconvolution of the libraries led to the identification of various biologically active compounds. The synthesis of combinatorial libraries has found broad acceptance in the field of medicinal chemistry since it greatly increases the likelihood of finding useful therapeutic and diagnostic agents. In the emerging field of proteomics, combinatorial library synthesis has reached new importance as a tool for the identification of new targets for drug development. The scope and versatility of synthetic combinatorial libraries can be expected to further expand their significance for basic research and drug discovery.

[26] New Strategies for the Solid-Phase Synthesis of Highly Functionalized, Small Molecules: Sequential Nucleophilic Substitutions on Polymer-Bound Polyelectrophiles By Florencio Zaragoza Introduction

For the fast identification of lead compounds for novel, small molecule enzyme inhibitors or other ligands for proteins, the screening of large and diverse arrays of compounds prepared on insoluble supports is one of the most efficient approaches.1–8 Parallel solid-phase synthesis has been found to be particularly well suited for the preparation of such arrays of diverse compounds since multistep synthetic sequences on insoluble supports can be conducted on fully automated synthesizers. 1

R. E. Dolle, Mol. Divers. 3, 199 (1998). J. L. Conroy, P. Abato, M. Ghosh, M. I. Austermuhle, M. R. Kiefer, and C. T. Seto, Tetrahedron Lett. 39, 8253 (1998). 3 M. Renil, M. Ferreras, J. M. Delaisse, N. T. Foged, and M. Meldal, J. Peptide Sci. 4, 195 (1998). 4 J. C. Spetzler, V. Westphal, J. R. Winther, and M. Meldal, J. Peptide Sci. 4, 128 (1998). 5 T. S. Haque, A. G. Skillman, C. E. Lee, H. Habashita, I. Y. Gluzman, T. J. A. Ewing, D. E. Goldberg, I. D. Kuntz, and J. A. Ellman, J. Med. Chem. 42, 1428 (1999). 6 M. Steger and D. W. Young, Tetrahedron 55, 7935 (1999). 7 A. K. Szardenings, V. Antonenko, D. A. Campbell, N. DeFrancisco, S. Ida, L. Shi, N. Sharkov, D. Tien, Y. Wang, and M. Navre, J. Med. Chem. 42, 1348 (1999). 8 D. S. Yamashita, X. Dong, H. J. Oh, C. S. Brook, T. A. Tomaszek, L. Szewczuk, D. G. Tew, and D. F. Veber, J. Comb. Chem. 1, 207 (1999). 2

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

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Each synthetic step of syntheses performed on insoluble supports must generally be optimized to such an extent to yield intermediates of high purity, because these polymer-bound intermediates cannot be purified. Once such an optimized solid-phase synthesis has been developed, final products of high purity can often be obtained directly after cleavage from the insoluble support and evaporation of the cleavage reagent (e.g., trifluoroacetic acid). Such products may be screened directly without any further purification. Because no purification on the final products is required in such instances, parallel solid-phase synthesis can be used to prepare arrays of compounds with a broad range of lipophilicity, charge, and molecular weight.9 Such arrays would be difficult to prepare by parallel solutionphase chemistry not only because each compound would require a different work-up strategy, but also because the purification of small, hydrophilic molecules is often a difficult task. Large arrays of compounds are mostly used at the beginning of medicinal chemistry projects for the fast identification of new lead structures. To increase the odds of finding innovative ligands, such arrays should not contain large numbers of closely related compounds (which are likely to have similar biological properties), but highly diverse compounds with widely different properties. Moreover, because new leads and not finished drugs are initially sought, such arrays should consist of lead-like, not drug-like compounds. Lead compounds tend to be smaller and more hydrophilic than drugs, and consequently compound arrays for the discovery of new leads should mainly contain low-molecular-weight (e.g., MW < 350 g mol1) and hydrophilic (e.g., log P < 3) molecules.10,11 Because an excessive increase in the complexity of a molecule severely reduces its chances of binding to a given protein,11 the total number of functional groups capable of strongly interacting with proteins (pharmacophores) per molecule should also have an upper limit. Finally, a high-quality lead structure should also enable the quick preparation of a large number of closely related analogs. It is crucial that synthetic chemistry procedures do not become the bottleneck in the optimization process since this process can move forward swiftly only when a large set of compounds is available for the determination of their pharmacological properties. Large compound arrays, however, will be meaningful only for the fast optimization of those parameters that can 9

F. Zaragoza, ‘‘Organic Synthesis on Solid Phase.’’ Wiley-VCH, Weinheim, New York, 2000. S. J. Teague, A. M. Davis, P. D. Leeson, and T. Oprea, Angew. Chem. Int. Ed. Engl. 38, 3743 (1999). 11 M. M. Hann, A. R. Leach, and G. Harper, J. Chem. Inf. Comput. Sci. 41, 856 (2001). 10

[26]

substitutions on polymer-bound polyelectrophiles

519

also be quickly determined, for instance, by in vitro assays obtaining the binding affinity to an enzyme or receptor and selectivity. If the time required for testing one compound in a biological assay becomes significantly longer than the time required for synthesizing the compound, large arrays of compounds generally do not accelerate the optimization process. The reason for this is that a large array of compounds is designed without any knowledge about the biological properties of each individual compound. This results in the generation of redundant compounds that would not have been prepared if the test result of a selection of compounds of the array had been known beforehand. In the case of slow and labor-intensive biological assays it is therefore more efficient to prepare and test small arrays of compounds. This is, for instance, the case when properties such as clearance, half-life, oral availability, or brain–plasma ratio are to be optimized. The determination of these parameters requires in vivo pharmacology, which has a rather low throughput when compared to in vitro assays. Compound arrays are usually prepared by performing the same reaction sequence in each reactor, but using different reagents. For this reason all the compounds of such an array will generally contain a repetitive structural element. Diverse libraries will be obtained only if this repetitive element is both small and pharmacophore poor, and if the synthesis enables the incorporation of highly variable, pharmacophore-rich side chains. Synthetic routes for the preparation of lead-like arrays should therefore be based on readily available, pharmacophore-rich reagents such as amines, alcohols, carboxylic acids, and thiols. Less suitable chemicals would include hard-to-find reagents (e.g., difunctional reagents, such as haloketones, diols, diamines, dicarboxylic acids, and amino acids) or reagents that are inherently pharmacophore poor (e.g., acyl halides, isocyanates, and strong alkylating agents). Chemistry leading to repetitive structural elements displaying important pharmacophores (e.g., diketopiperazines, benzodiazepines, and peptides) will lead to sparsely diverse compound arrays because the repetitive structural element will be the dominating feature of all the members of the array. Such chemistries are therefore less suitable for the preparation of diverse compound libraries and should be avoided. Sequential Nucleophilic Substitutions on Insoluble Supports

What kind of chemistry and reagents will then be capable of delivering arrays of compounds suitable for the identification of high-quality leads as outlined above? As discussed in the introduction, for the preparation of highly diverse, lead-like libraries the incorporation of pharmacophore-rich reagents into the final products is required. The majority of important

520

[26]

small molecule and heterocycle synthesis

pharmacophores, such as the residues of proteinogenic amino acids (hydroxyl, amino, guanidino, mercapto, carboxyl, 4-imidazolyl, and 3-indolyl groups), are nucleophilic functional groups, and electrophilic reagents containing such pharmacophores will generally require protection of the latter to avoid polymerization (first equation, Fig. 1). Partially protected electrophilic reagents, however, are not available in large number and high diversity, and are often expensive. The use of protective groups also increases the number of synthetic operations and thereby the costs of library production. Because synthetic intermediates cannot be purified during solid-phase synthesis, an increased number of synthetic operations will likely lead to less pure final products. For these reasons, syntheses based on the reaction of electrophilic reagents with polymer-bound nucleophiles are of limited utility for the preparation of lead-like compound arrays. Nucleophilic reagents containing additional nucleophilic functional groups, such as diamines or aminoalcohols, on the other hand, are often cheap, easily available, and can be used without any protective groups. With this type of reagents the most nucleophilic group will react first usually with no interference of any other nucleophilic group present (second equation, Fig. 1). The reaction of nucleophilic reagent with polymer-bound electrophiles is therefore a valuable strategy for the preparation of lead-like compound arrays since it allows the chemist to introduce highly variable pharmacophores into the final product. Electrophilic reagents + resin-bound nucleophile: FG

+

COX

H2N

Pol

FG

H N

Pol

S

Pol

O

FG = NR-PG, O-PG, S-PG; protective groups are required Nucleophilic reagents + resin-bound electrophile: FG

SH

+

Cl

Pol

FG

FG = NR2, OH, SH; protective groups are not required Sequential nucleophilic substitutions: 1

X 2

X

Po l

E 3

X

1. Nu1H 2. Nu2H 3. Nu3H

Nu1 Nu

2

Pol

E Nu

3

Fig. 1. Nucleophilic substitutions as strategy for the use of unprotected, pharmacophorerich reagents. FG, functional group; Nu, nucleophile; PG, protective group; Pol, polymeric support; X, leaving group for nucleophilic displacement.

[26]

substitutions on polymer-bound polyelectrophiles

521

The advantages of using nucleophilic reagents for library production can be exploited even further by performing sequential nucleophilic substitutions on a polymer-bound polyelectrophile. A suitable polyelectrophile would be a polymer-bound compound with two or more leaving groups that could be orthogonally displaced by different nucleophiles. Because many pharmacophore-rich nucleophilic reagents are commercially available, this strategy represents a simple method for the preparation of highly diverse, pharmacophore-rich compounds. Various examples of 2-fold sequential nucleophilic substitutions on insoluble supports have been reported in the literature. Suitable polyelectrophiles are polyhalo triazines,12,13 pyrimidines,12,14 and purines,12,15–17 and the most common nucleophiles are amines, thiols, and phenols. The use of 2,3-dichloropropionic acid18 and 4,5-difluoro-2-nitrobenzoic acid19 is discussed below to illustrate the scope and limitations of this strategy for the preparation of lead-like compound arrays. 2,3-Dichloropropionic Acid Derivatives as Polyelectrophile

2,3-Dichloropropionic acid is a cheap reagent that can be linked to insoluble polymers either as an ester or an amide (1, Fig. 2). Although both chlorine atoms of this substrate were expected to show a slightly different reactivity toward nucleophiles, all attempts to displace these two leaving groups by two different nucleophiles failed. Regardless of the reaction conditions and the type of nucleophile used, both chlorine atoms were displaced by the first nucleophile. On the other hand, further experiments revealed that treatment of immobilized 2,3-dichloropropionic acid derivatives 1 with thiols afforded dithioethers 2, which were found to be unstable in the presence of bases, undergoing clean -elimination when treated with 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU)* to yield acrylic acid derivatives 3. When this 12

D. Scharn, L. Germeroth, J. Schneider-Mergener, and H. Wenschuh, J. Org. Chem. 66, 507 (2001). 13 D. Scharn, H. Wenschuh, U. Reineke, J. Schneider-Mergener, and L. Germeroth, J. Comb. Chem. 2, 361 (2000). 14 F. Guillier, P. Roussel, H. Moser, P. Kane, and M. Bradley, Chem. Eur. J. 5, 3450 (1999). 15 W. K.-D. Brill and C. Riva-Toniolo, Tetrahedron Lett. 42, 6515 (2001). 16 W. K.-D. Brill, C. Riva-Toniolo, and S. Mu¨ller, Synlett 1097 (2001). 17 K. Kim and B. Wang, Chem. Commun. 2268 (2001). 18 F. Zaragoza and H. Stephensen, Angew. Chem. Int. Ed. Engl. 39, 554 (2000). 19 M. Grimstrup and F. Zaragoza, Eur. J. Org. Chem. 3233 (2001). * Abbreviations: DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DMF, N,N-dimethylformamide; MCPBA, 3-chloroperbenzoic acid; NMP, N-methyl-2-pyrrolidinone; Pol, undefined polymeric support; PS, cross-linked polystyrene; Wang resin, 1–2% cross-linked polystyrene with p-benzyloxybenzyl alcohol linker.

522

small molecule and heterocycle synthesis O Cl

X

Pol

O

? 2

X

Cl Cl

Pol

X

Nu

Cl O

Nu

R1SH, DIPEA NMP, 20 ⬚C, 22 h

R1S

X

R2NH, DBU NMP, 20−80 ⬚C, 22 h

Pol

1

2 O

O X SR

Pol

1

O

SR

1

[26]

1

3

Pol

R2 N

X SR

Pol

1

4

Fig. 2. Two-fold sequential nucleophilic substitution at support-bound 2,3-dichloropropionic acid for the synthesis of 3-amino-2-thiopropionic acid derivatives. X ¼ O, NR.

elimination was performed in the presence of an aliphatic or aromatic amine, addition of the latter to the electrophilic double bond of intermediates 3 took place to yield 3-amino-2-thiopropionic acid derivatives 4.18 The elimination/addition reaction already proceeded at room temperature when the dichloropropionic acid had been linked as an ester to the support, but required heating when an amide linkage had been chosen. When amines with low nucleophilicity were used, such as aniline or amino acid esters, higher reaction temperatures were also beneficial. Occasional by-products for this reaction sequence were acrylic acid derivatives or the corresponding hydrogenated products (2-thiopropionic acid derivatives). These by-products were usually formed when a very small excess of amine was used in the elimination/addition step. Both the thiols and the amines used in this reaction sequence could be polyfunctional, as illustrated by the examples sketched in Fig. 3. 4,5-Difluoro-2-nitrobenzamides as Polyelectrophile

With the aim of identifying a suitable polyelectrophile to enable three sequential nucleophilic substitutions, we investigated the reactivity of support-bound 4,5-difluoro-2-nitrobenzoic acid toward nucleophilic reagents.19 To minimize the risk of premature cleavage from the support during the substitution reactions it was decided to immobilize this acid via an amide linkage with Wang-resin-bound piperazine. We anticipated that the optimized reaction conditions using this linker would be directly transferable to other linkers for amides, such as backbone amide linkers.9

[26]

523

substitutions on polymer-bound polyelectrophiles O

CO2H

N

N H

SPh Cl 90% pure 75% yield

72% pure 31% yield

Ph

NH

S

SPh

73% pure 49% yield

N H Cl 82% pure 79% yield

NH2

N SPh

NH

61% pure 90% yield O

CO2H

N H

HO

CO2Me N H

O Ph

N

Ph N

S

O N

NH

HO

N SPh

NH

91% pure 94% yield

Fig. 3. Examples of products prepared by sequential nucleophilic substitution at supportbound 2,3-dichloropropionic acid derivatives. Purities were determined by HPLC monitoring at 214 nm.

Sequential aromatic nucleophilic substitutions on resin-bound 4,5difluoro-2-nitrobenzamides required a careful selection of the nucleophiles and optimization of each step (Fig. 4). We found that the use of amines as first nucleophile proceeded cleanly, but the resulting 5-amino-4-fluoro-2nitrobenzamides were too unreactive toward the nucleophilic displacement of the nitro group. Because the reactivity of arenes toward nucleophiles is strongly influenced by the electronic properties of its substituents, we found that for our purposes the first substitution had to be done with thiols and not with a more electron-donating nucleophile. Treatment of the intermediate thioethers 7 with amines led to a clean substitution of the second fluoride, but again, the resulting 4-amino-5-thio-2-nitrobenzamides were too unreactive toward nucleophiles to undergo the desired substitution of the nitro group. To facilitate the nitro group displacement reaction, the thioethers 7, obtained by treatment of difluoride 6 with thiols, were oxidized with 3-chloroperbenzoic acid to afford the corresponding sulfones 8 (Fig. 4). The resulting sulfones 8 showed the expected high reactivity toward nucleophiles, as demonstrated by the efficient displacement of both the second fluoride and the nitro group with two different aliphatic amines to yield the highly substituted benzamides 10. As illustrated by the examples sketched in Fig. 5, pharmacophore-rich thiols and amines could be used in all three displacement reactions to yield substituted benzamides displaying highly variable arrangements of different pharmacophoric groups.

524

[26]

small molecule and heterocycle synthesis O F

O X

Pol

Nu1

?

X

NO2

F

Pol

Nu3

Nu2

5 NO2 O N

BnSH (1 mole/liter, 13 eq) AcOH (13 eq) PS NMP, 22 ⬚C, 3 h

NO2 O N PS

R

F

R

F F

S

6

Ethylenediamine (0.5 mole/liter, 14 eq) NMP, 22 ⬚C, 2 ⫻ 2 h H2N

NO2 O

N H

N R O2S

MCPBA (0.5 mole/liter, 9 eq) DCM, 22 ⬚C, 2 ⫻ 2 h

Ph

NO2 O N

F Ph

O2S

7

Piperidine (2 mole/liter, 100 eq) NMP, 100 ⬚C H2N PS

N

N H

Ph

9

8

O N

2⫻3h

PS

R

PS

R Ph

O2S

10

Fig. 4. Three-fold sequential nucleophilic substitution at support-bound 4,5-difluoro-2nitrobenzoic acid.

O H2N

NH O

HO

N H

NH O2S

N

N

N H

NH O2S

OH

NH

N O2S

N N H

N H 81% pure (214 nm) 46% yield

N

N

N Ph

O

N

NH O

57% pure (214 nm) 68% yield

84% pure (214 nm) 61% yield

Fig. 5. Examples of substituted benzamides prepared by 3-fold sequential nucleophilic substitution on an insoluble support.

Conclusion

As illustrated by the examples given in Figs. 3 and 5, sequential nucleophilic substitutions on support-bound polyelectrophiles give quick access to highly functionalized, pharmacophore-rich small molecules. Because no protective groups are required, the scope of these syntheses is broad, and a multitude of different pharmacophoric patterns can be generated by using exclusively simple, commercially available reagents. The broad scope

[26]

substitutions on polymer-bound polyelectrophiles

525

of this new synthetic methodology should enable the quick optimization of hits by preparing further, more focused compound arrays using the same chemistry. Experimental Section

Reagents and General Methods All reagents were used as supplied. p-Benzyloxybenzyl alcohol resin (Wang resin) with a loading of approx. 1 mmol g1 was purchased from Bachem (Bubendorf, Switzerland). All reactions were performed in fritted Teflon reactors, using a setup as described in Zaragoza.9 Further experimental details are given in Grimstrup and Zaragoza.19 2-Phenylsulfanyl-3-(piperidin-1-yl)propionic Acid Trifluoroacetate CO2H

N S

CF3CO2H

To Wang resin (0.60 g, approx. 0.60 mmol) was added a solution of 2,3-dichloropropionic acid (0.87 g, 6.09 mmol) in dichloromethane (12 ml) followed by the addition of N,N0 -diisopropylcarbodiimide (0.48 ml, 3.07 mmol) and 4-dimethylaminopyridine (0.06 ml, 1 M in DMF, 0.06 mmol). The mixture was shaken at room temperature for 20 h, filtered, and the resin was washed with dichloromethane (3  6 ml). This resin was suspended in NMP (9.0 ml), treated with diisopropylethylamine (0.90 ml, 5.17 mmol) and thiophenol (0.60 ml, 5.84 mmol), and the resulting mixture was shaken at room temperature for 22 h. After filtration and washing with NMP (3  6 ml), the resin was suspended in NMP (9.0 ml), and piperidine (0.60 ml, 6.07 mmol) and DBU (0.90 ml, 6.02 mmol) were added. The mixture was shaken at room temperature for 22 h, filtered, and the resin was extensively washed with NMP, dichloromethane, and methanol, and resuspended in 1,2-dichloropropane. After shaking overnight at room temperature to remove traces of NMP the mixture was filtered, and the resin was suspended in a mixture of dichloromethane (4.5 ml) and trifluoroacetic acid (4.5 ml). After shaking at room temperature for 0.5 h the mixture was filtered, the resin was washed twice with dichloromethane, and the combined filtrates were concentrated. Crystallization of the residue from a mix ture of ethyl acetate and heptane at 20 yielded the title compound

526

small molecule and heterocycle synthesis

[26]



(144 mg, 63% yield) as a colorless solid, mp 81–83 . LCMS m/z 266 (MHþ); 1 H NMR (400 MHz, DMSO-d6)  1.51 (s, br, 2H), 1.72 (s, br, 4H), 2.98– 3.29 (m, 4H), 3.34 (dd, J ¼ 13 Hz, 5 Hz, 1H), 3.53 (dd, J ¼ 13 Hz, 8 Hz, 1H), 4.29 (dd, J ¼ 5 Hz, 8 Hz, 1H), 7.38–7.43 (m, 3H), 7.50–7.55 (m, 2H). Anal. Calcd. for C14H19NO2SC2HF3O2 (379.40): C, 50.65; H, 5.31; N, 3.69. Found: C, 50.94; H, 5.38; N, 3.63. 1-[5-Benzenesulfonyl-2-(piperidin-1-yl)-4-(pyridin-4-ylmethylamino) benzoyl]piperazine Trifluoroacetate

N

N

O N

N H

NH

O

2 CF3CO2H

S O

Wang-resin-bound piperazine20 (5.9 g, approx. 6.4 mmol) was treated with a mixture of 4,5-difluoro-2-nitrobenzoic acid (3.3 g, 16 mmol), dichloromethane (25 ml), NMP (25 ml), 1-hydroxybenzotriazole (2.5 g, 19 mmol), N,N0 -diisopropylcarbodiimide (2.5 ml, 16 mmol), and diisopropylethylamine (1.1 ml, 6.3 mmol) at room temperature for 2.5 h. After washing and drying, part of the resulting resin-bound difluoroarene (2.0 g, approx. 1.6 mmol) was treated with a mixture of NMP (20 ml), thiophenol (2.0 ml, 20 mmol), and acetic acid (1.1 ml, 19 mmol) at room temperature for 3 h. After washing and drying, part of the resulting resinbound thioether (1.5 g, approx. 1.1 mmol) was treated twice with a solution of MCPBA (2.5 g, 70%, 10 mmol) in dichloromethane (20 ml) at room temperature for 2 h. After washing and drying, part of the resulting resin-bound 2-fluoroarylsulfone (1.0 g, approx. 0.74 mmol) was treated twice with a solution of 4-(aminomethyl)pyridine (1.0 ml, 10 mmol) in NMP (20 ml) at room temperature for 2 h. After washing and drying, part of the resulting resinbound nitroaniline (0.5 g, approx. 0.35 mmol) was treated twice with a  solution of piperidine (4.0 ml, 40 mmol) in NMP (20 ml) at 100 for 3 h. After extensive washing with dichloromethane and methanol, part of this 20

F. Zaragoza and S. V. Petersen, Tetrahedron 52, 10823 (1996).

[26]

substitutions on polymer-bound polyelectrophiles

527

resin (164 mg, approx. 0.111 mmol) was treated with a mixture of trifluoroacetic acid and dichloromethane (1:1) at room temperature for 0.5 h. Filtration, washing with dichloromethane, and concentration of the combined filtrates yielded 0.081 mmol (determined by 1H NMR with internal standard; 73% yield) of the title compound. LCMS m/z 520 (MHþ); 1H NMR (400 MHz, DMSO-d6) 1.45 (br s, 6H), 2.74 (br s, 2H), 2.87 (br s, 2H), 3.02 (br s, 2H), 3.18 (br s, 3H), 3.46 (br s, 2H), 4.10 (br s, 1H), 4.74 (d, J ¼ 6 Hz, 2H), 5.84 (s, 1H), 7.19 (t, J ¼ 6 Hz, 1H), 7.54 (d, J ¼ 6 Hz, 2H), 7.58–7.76 (m, 4H), 8.02 (d, J ¼ 7 Hz, 2H), 8.70 (d, J ¼ 6 Hz, 2H), 9.21 (br s, 2H). Acknowledgments The technical assistance of Henrik Stephensen during the development of sequential nucleophilic substitutions at support-bound 2,3-dichloropropionic acid is gratefully acknowledged. Thanks are also extended to Marie Grimstrup, who optimized the synthesis of substituted benzamides.

This Page Intentionally Left Blank

Author Index

Numbers in parentheses are footnote reference numbers and indicate that an author’s work is referred to although the name is not cited in the text.

A

Amons, R., 293, 294 Amro, N., 313 An, H. Y., 392 Andersen, K., 447, 463(31), 464(31) Anderson, J., 262 Anderson, P. S., 80, 185, 470 Andrade, R. B., 239, 240(18), 241 Andres, C. J., 77 Andres, V., Jr., 305 Andries, K., 470 Anelli, P. L., 372, 373(22) Anh, S. Y., 417 Antonenko, V., 517 Appel, J. R., 113, 290, 298, 324, 325, 338, 341, 497, 498, 498(9; 10), 499(9), 514(46), 515 Arad, O., 184 Arienti, K. L., 392 Armstrong, R. W., 42, 70(17), 100, 197, 394, 395(37; 38), 470, 470(9), 471, 471(7), 474, 479, 489 Arnaiz, D., 481 Arnauld, T., 354, 355(19), 361(35), 362 Arnold, B., 481 Artamkina, G. A., 404(72), 405 Asgedom, M., 75(5), 76, 99(2; 3), 100, 113, 298, 435 Ashton, W. T., 80 Askin, D., 80 Atdlweiser, J., 391 Atrash, B., 77, 115 Attanasi, O., 419 Attardi, M. E., 24, 29, 32 Atwal, K. S., 199 Augelli-Szafran, C. E., 138 Ault-Justus, S., 388, 407 Aurigemma, C. M., 4 Austermuhle, M. I., 517 Auzanneau, F. I., 314 Avemaria, F., 130, 133(11)

Abato, P. A., 297, 517 Abdul-Latif, F., 290 Abeles, R. H., 185 Abell, A. D., 486 Abell, C., 447, 462(30) Acar, J. F., 331 Acharya, A. N., 507, 509, 510(39), 511 Adam, M. D., 143, 444 Adda, N., 323 Adey, N. B., 291 Afar, D. E. H., 315 Affleck, R. L., 79, 115, 272, 299 Affrossman, S., 396 Agarkov, A., 72(58), 73 ˚ hman, J., 179 A Ahn, J.-M., 288 Ahn, S. Y., 480 Airey, J., 86, 90(27), 151, 154(1), 487 Ajay, 75, 294 Akelah, A., 368 Albericio, F., 21, 24, 32, 81, 90(23), 142, 183, 185(7), 186, 348, 418, 422, 444, 452, 465(25) Albert, K., 351 Alcazar-Roman, L. M., 404 Aldebert, D., 323, 331(4) Alesso, S., 403 Alexander, M., 6 Allanson, N. M., 258, 261(22), 262 Allen, J. D., 422 Allen, M. P., 143, 444 Almdal, K., 40 Almstetter, M., 197 Alsina, J., 81, 90(23), 142, 183, 185(7), 422 Amblard, M., 58, 59(42) Ambroise-Thomas, P., 323, 331(4) Ambrosius, D., 304

529

530

author index

Axel, M. G., 185 Axelrod, H. R., 262

B Baasov, T., 259 Baboonian, C., 339 Babu-Khan, S., 485 Bachmann, S., 351 Backes, B. J., 128, 132(7) Badet, B., 185 Bagchi, G. D., 75 Bai, Y., 209 Bailey, F. C., 369 Bailey, N., 392 Bair, E. L., 313 Baizman, E., 262 Balasubramanian, S., 272, 299 Balkenhohl, F., 257, 391, 419 Ball, J. M., 302 Bandel, H., 3 Banfi, L., 484 Bang, K. S., 447, 463(31), 464(31) Bankaitis-Davis, D., 164 Bannwarth, W., 418 Banville, S. C., 290 Baran, P. S., 378 Barany, G., 81, 90(23), 142, 183, 185(7), 235, 258, 261(24), 272, 422, 444, 456, 465(25) Barbachyn, M. R., 225(7), 226 Barbas, C. F. III, 498 Barbier, P. T., 428 Barger, L. A., 74 Baringhaus, K.-H., 436 Barluenga, S., 79, 115, 356 Barn, D. R., 419 Barnes, C., 272, 299 Barnes, G., 46 Barnwell, P., 391 Barrett, A. G. M., 354, 355(19), 361(35), 362, 396 Barrett, R. W., 183, 498 Barrish, A., 199 Barrow, J. C., 199 Barry, J., 24, 26 Barteling, S. J., 40, 43(10), 100, 289, 497 Barth, M., 370, 387 Barthelemy, S., 375 Bartolozzi, A., 347 Barton, D., 357, 399, 402(63)

Basso, A., 67 Batchelor, J. F., 480 Bateman, R., 4 Battersby, J. E., 24, 26 Baum, S. A., 116, 117(16) Bauser, M., 291 Baxendale, I. R., 73, 348, 368, 392, 394(16) Baxter, A. D., 419 Baxter, E. W., 397, 402(62) Bayada, D. M., 476 Bayburt, E. K., 75 Baydal, J. P., 24, 31 Bayliss, M. K., 4, 5(8) Bean, M. F., 199 Beaton, G., 235 Becker, J. A. J., 297 Becker, M., 151(2), 152, 160(2) Beebe, X., 430 Befzi, A., 335 Begtrup, M., 426 Beijnen, J. H., 323 Beletskaya, I. P., 404(72), 405 Belyakov, S. A., 138 Bemis, G. W., 75, 294 Benckhuijsen, W. E., 293, 294 Ben-David, D., 87 Benezra, C., 394, 395(39–41) Bennett, B., 485 Bennett, W. D., 499 Berces, A., 252 Berg, R. H., 40 Bergelli, M., 497 Berger, E. A., 332(38), 333, 334, 334(38), 335(40) Bergquist, K.-E., 154 Berlinck, R. G. S., 143 Berman, J., 74 Bernick, A. M., 396 Berst, F., 422 Besemer, A. C., 372, 374(20) Besser, D., 184 Betschinger, J., 291 Bhandari, A., 419 Biaga, T. J., 474 Bianchi, E., 297 Biancolana, S., 444, 465(25) Bic¸ak, N., 396, 408 Biddison, W. E., 341, 342(64) Bidham, E. C., 74 Bidlack, J. M., 291

author index Bielekova, B., 338, 342(47) Bienayme, H., 469, 483, 489 Bienstock, R. J., 184 Biffi, C., 372, 373(22) Biginelli, P., 197 Bilello, J. A., 80 Biringer, G., 428 Bishop, M. J., 74 Biswas, K., 258 Bitter-Suermann, D., 304 Bjergarde, K., 425 Blackburn, B. K., 470 Blackburn, C., 348, 489 Blahy, O. M., 79 Blanc, P., 325, 498 Blaney, P., 416 Blankson, J., 332 Blauth-Eckmeyer, E., 138 Blechert, S., 428 Blocker, H., 113 Blomgren, P. A., 364, 407 Blondelle, S. E., 113, 290, 292, 298, 322, 324, 327, 335, 342, 497, 498(9; 10), 499, 499(9), 500, 501(19), 502(19) Blossinger, C. D., 513 Bobko, M. A., 422 Bock, H., 475 Bock, M. G., 470 Boddy, C. N. C., 130 Boden, C. D., 79 Boden, C. J., 79 Boden, P., 291(22), 292 Boeijen, A., 417 Boggiano, C., 322, 338, 342 Bohm, H.-J., 295 Bolli, M. H., 371 Bolm, C., 351, 375 Bolognesi, D., 332 Bolton, G. L., 392(28), 393, 396(28), 401(28), 402(28) Bombrun, A., 177 Bondinell, W. E., 185 Bono, E., 341 Boojamra, C. G., 42, 81, 89(23b), 90(23) Boons, G.-J., 258 Booth, R. J., 392, 392(28), 393, 393(12), 394(12), 395(11; 12), 396(12; 28), 399(12), 401(28), 402(12; 28), 405(12) Borchardt, A., 299 Bornaghi, L., 254, 259

531

Borras, E., 338, 342(53) Borsche, W., 139 Bossinger, C. D., 22, 24, 25(1), 302 Bossio, R., 489 Boths, J., 291 Botta, M., 152 Boule-Vavra, S., 331 Bousquet, P., 480 Bouzid, K., 489 Boyce, J. M., 331 Bradley, M., 77, 115, 388, 403, 438, 459(12), 460(12), 461(12), 521 Braisted, A. C., 75 Branch, D. L., 12 Brandenburg, D., 304 Brandtner, S., 131, 142(14), 144(14) Branstrom, A., 262 Bra¨se, S., 127, 130, 131, 132, 132(12), 133(11; 16), 134, 134(12), 135, 136, 137, 138, 139, 140, 140(49), 142, 142(14), 143(54), 144(14; 57), 145, 355 Brasili, L., 480 Braxenthaler, M., 341 Bray, A. M., 39, 40, 41, 42, 43, 43(19), 46, 59(6), 60(19), 64, 65, 73(20) Bray, M. K., 516 Bream, R. M., 348, 368, 392, 394(16) Breddam, K., 314 Breen, A. L., 199 Breitenbucher, J. G., 392 Brennan, A. L., 331 Brenner, S., 290 Breslin, H. J., 470 Briand, J.-P., 184 Brickner, S. J., 225, 225(7), 226 Brightwell, C. I., 80 Brill, W. K.-D., 435, 439, 444(16), 450, 466(33), 521 Bristol, N., 262 Britton, T., 481 Broadhurst, M., 248, 251, 260 Brock, R., 370 Broder, C. C., 334, 335(40) Broger, C., 489 Brook, C. S., 517 Brooks, R. R., 480 Broten, T. P., 199 Brown, J. F., 364(42), 365 Brown, K. D., 151(2), 152, 160(2) Brown, R., 172

532

author index

Brown, R. C. D., 24, 29, 30(10), 115, 117(8), 428 Brown, S. D., 197, 394, 395(38), 489 Brule, E., 348 Brumfield, K. K., 457 Brummer, O., 437 Bruns, R., 481 Brusic, V., 341 Bryant, S. D., 185 Buchowicz, W., 358(29), 359 Buchstaller, H.-P., 426 Buchwald, S. L., 179, 358(30), 359, 404, 405 Budhu, R. J., 80 Buehler, J., 251, 252(12) Buencamino, J., 497 Buhlmayer, P., 87 Bui, C. T., 40, 115, 117(11) Bundle, D. R., 263 Bunin, B. A., 39, 43, 136, 392, 435, 437, 451(2), 470 Bunker, A. M., 480 Burgess, K., 143 Burke, L. A., 135 Burkett, B. A., 24, 29, 30(10) Burkoth, T. S., 421, 478 Burns, C. J., 471, 473 Burow, K. M., 42, 81, 89(23b), 90(23) Burton, D. R., 498 Burton, L., 4 Busch, A. E., 480 Buskas, T., 241(20), 242 Bussche-Huennefeld, C. v. d., 391 Busson, R., 185 Butler, R. N., 135 Bycroft, B. W., 186

C Cacchi, S., 178 Caddick, S., 224 Cai, P. J., 204 Calas, B., 184 Caldarelli, M., 396 Caldwell, C. G., 263 Caliendo, G., 185 Callahan, J. F., 185 Calvo, R. R., 185 Cameron, A. M., 24, 31 Cameron, N. R., 24, 31, 364(42), 365 Camillero, P., 24

Campbell, D. A., 421, 425(22g), 428, 478, 517 Campbell, G. D., Jr., 323 Campbell, R. A., 40, 42, 73(20) Camps, F., 424 Cannan, R. K., 24, 25 Cano, J., 323, 331(4) Canova, R., 74 Cantrell, B., 481 Cao, G.-Q., 79, 115 Cao, K., 164, 170(2), 173 Capeau, J., 323 Cappiello, J., 394 Carboni, B., 425(37), 426 Cardenas, J., 487 Cardullo, F., 431 Cargill, J. F., 42, 70(17), 100 Carlisle, S. J., 364 Carmichael, A. J., 339 Carotti, A., 480 Carpenter, J. F., 480 Carpino, L., 452 Carreaux, F., 425(37), 426 Carrillo-Munoz, A. J., 323, 331(4) Carte, B., 199 Cartells, J., 424 Caruthers, M. H., 235 Cascieri, M. A., 296 Castelhano, A. L., 423 Castro, J. L., 428 Cato, S. J., 294 Ceccarelli, S., 396 Cerottini, J.-C., 338, 342(51; 53) Chadwick, K., 332 Chae, C. B., 294 Chaisson, R. E., 332 Chamberlin, A. R., 202(38), 203, 224 Chan, C. C., 152 Chan, D. C., 332(36), 333, 334, 337(39) Chan, T.-H., 263 Chan, W. C., 39, 186 Chang, C. L., 75 Chang, C. S., 75 Chang, R. S. L., 80, 199 Chang, Y.-T., 444 Chanock, R. M., 498 Chapman, K. T., 324, 396 Chauhan, P. M. S., 437 Chauvey, D., 187 Cheifari, D. S., 39, 59(6) Cheminat, A., 394, 395(39–41)

author index Chen, A., 262 Chen, G. S., 75 Chen, I.-W., 80, 185 Chen, J., 143 Chen, J. J., 422, 475 Chen, L., 185 Chen, M. L., 271 Chen, T.-M., 185 Chen, Z.-C., 376 Chendra, S., 514(46), 515 Chenera, B., 469, 484 Cheng, Y., 324 Chern, J. W., 75 Cherrier, M. P., 474, 475, 479 Chervin, I. I., 140 Chesney, A., 391 Chexal, K. K., 184 Chhabra, S. R., 186 Chi, A., 262 Chinchilla, R., 361(36; 37), 362 Ching, B. W., 480 Chisem, I. C., 396 Chisem, J., 396 Chmielewski, M., 430 Cho, B. Y., 294 Cho, C.-W., 349, 350(10) Cho, H. Y., 417, 480 Cho, N. S., 470 Choi, S., 479 Choi-Sledeski, Y. M., 151(2), 152, 160(2) Choo, H.-Y. P., 423 Chou, Y.-L., 456 Christensen, J. W., 99, 100, 116 Christensen, T., 24, 28 Chu, S. S., 24 Chu, V., 151(2), 152, 160(2) Chucholowski, A., 391, 421, 424, 424(22b) Chung, I. K., 423 Chung, N. N., 291, 497 Chung, S.-H., 418 Chutkowski, C. T., 334, 337(39) Ciattini, P. G., 178 Cichy-Knight, M. A., 296 Ciraco, M., 361(38), 362 Clackson, T., 289 Clapham, B., 348, 349, 350(10), 437 Clark, B. J., 140 Clark, J. H., 396 Clement, R. P., 5 Clemons, K. V., 323, 331(4)

Clercq, E. D., 470 Cliffe, I. A., 80 Cobb, J. M., 447, 462(30) Cody, D. M. R., 76 Coe, D. M., 24, 31, 391 Cohen, B. J., 152 Coleman, D. C., 323, 331(4) Colescott, R. L., 22, 24, 25(1), 302, 513 Collibee, S. E., 72(58), 73 Collier, S., 135 Colucci, M., 423 Combs, A. P., 197, 223, 224, 225 Comely, A. C., 131, 416 Congreve, M., 22 Conlon, P., 338 Connelly, J. A., 79 Connolly, C. J. C., 480 Conroy, J. L., 297, 517 Conti, N., 138 Cook, A. W., 453 Cook, P., 22, 24, 25(1) Cook, P. D., 392 Cook, P. I., 302, 513 Cooper, A., 290 Cooper, W. J., 392 Cooperman, B. S., 291 Coppola, G. M., 391 Corbel, J. C., 67 Corelli, F., 152 Cork, D., 396 Cortes, D. A., 372 Cortese, I., 338, 342(47) Cortese, R., 297, 498 Cotterill, I., 202(37), 203 Cournoyer, J. J., 24, 33 Courtney, K. D., 305 Cowart, M., 75 Cowell, D., 77, 79, 115 Cowell, S. M., 288 Cox, L. J., 419 Cox, R., 24, 31 Crawford, K., 86, 90(27), 151, 154(1), 487 Creighton, C. J., 361(39), 362 Crespo, R. F., 361(38), 362 Cress, A. E., 313 Cresswell, P., 339 Creswell, M. W., 392(28), 393, 396(28), 401(28), 402(28) Crich, D., 357

533

534

author index

Crich, J. Z., 393, 394(32), 395(32), 396(32), 406(32) Crooks, E., 327 Cross, J. T., Jr., 323 Crowley, J. I., 427 Crozet, Y., 295 Cuervo, J. H., 113, 290, 298, 324, 497, 498(9), 499(9) Cummings, W., 51, 116, 117(13) Cunningham, A., 200 Cupif, J.-F., 423, 425(37), 426 Curran, D. P., 200(23; 24), 201, 392(24), 393 Cuzzocrea, C., 290 Cwirla, S. E., 498 Czarnik, A. W., 40, 76, 272, 299, 391, 415

D Dahmen, S., 127, 130, 131, 132, 132(12), 133(16), 134(12), 139, 140(49), 142, 143(54), 144(57), 145, 355 Dalbon, P., 184 DalCin, M., 392 Dallas, J., 481 Dallinger, D., 201 Dandia, A., 204 D’Andrea, S. V., 77 Danishefsky, S. J., 237 Danks, T. N., 351(14), 352 D’Aquila, R. T., 332 Darke, P. L., 79, 80, 185 Darlak, K., 296 Darvas, F., 497 Dattilo, M., 74 Daum, R. S., 331 David, C. M., 302 David, F., 185 David, M., 423, 425(37), 426 David, M.-L., 186, 189(30), 192(30) Davis, A. E., 5 Davis, A. M., 477, 518 Davis, A. P., 24, 26 Davis, B. G., 24, 31 Davis, F. A., 443, 462 Davis, R. S., 151(2), 152, 160(2) Dax, S. L., 197 Deal, M. J., 392 Dean, A. W., 392 de Biasi, V., 4, 24 de Bont, D. B. A., 425

De Brosse, C., 185, 199 De Clercq, P. J., 24, 26 De Crescentini, L., 419 Deeg, M., 418 DeFrancisco, N., 517 DeGonia, D. J., 385 Degrado, W. F., 152 DeGraw, S. T., 516 DeIong, N., 295 Dekany, G., 251, 254, 259 de Koster, H. S., 293 de la Hoz, A., 202, 203(30) Delaisse, J. M., 517 de las Heras, X., 419 Delbressine, L. P. C., 476 del Fresno, M., 183, 185(7), 422 De Luca, L., 356(26), 357 Demee, M., 12 de Meijere, A., 136 de Miguel, Y. R., 348 De Muynck, H., 24, 26 Denay, R., 74 Dence, C. S., 80 Dener, J. M., 446 den Hartog, J. A. J., 428 Denisko, O. V., 138 de Nooy, A. E. J., 372, 374(20) DePasquale, M. P., 332 Depew, K. M., 124 Deprez, B. P., 152 DeRoock, I. B., 313 DeRoose, F., 262 Desai, B., 351(14), 352 Deshayes, S., 202(35), 203 Deshpande, M. S., 77 Desjonqueres, N., 349, 350(7) Desmyler, J., 470 Dess, D. B., 376 Dessen, A., 332(35), 333 Devaky, K. S., 351(13), 352 Devlin, J. J., 498 Devlin, P. E., 498 Devraj, R. V., 392, 393, 394(32), 395(32), 396(32), 406(32) de Vries, R. R. P., 294 de Wit, D., 295 DeWitt, S. H., 76, 391, 415, 417, 425(5) Dhamoa, D. S., 446 Diamond, D. J., 338, 339(52) Dı´az-Ortis, A., 202, 203(30)

535

author index Dibo, G., 75(5), 76, 99(2; 3), 100, 113, 298, 435 Didomenico, S., 75 Di Lucrezia, R., 443 DiMarchi, R. D., 324 DiMichele, L. M., 490 Ding, J., 341 Ding, Q., 438, 444(15), 449(15), 458(15), 465(15) Ding, S., 438, 444(15), 449(15), 458(15), 465(15) Ding, Z.-K., 63 Dipardo, R. M., 470 Ditto, L., 152, 393, 396(30) Doan, N., 298 Dodi, A. I., 339 Dodsworth, D. J., 361(36; 37), 362 Doi, T., 60, 62, 258 Dolan, J., 6 Dolle, R. E., 39, 136, 151, 151(3), 152, 391, 393, 435, 517 Dollinger, G. D., 12 Dominy, B. W., 436 Domling, A., 470, 482(8), 489, 489(8), 490(56e) Do¨mling, S., 197 Dondoni, A., 200, 349, 350(9), 392(27), 393 Dong, L.-C., 173 Dong, X., 517 Dontenwill, M., 480 Dooley, C. T., 113, 290, 291, 298, 324, 497, 498(9; 10), 499(9), 506 Dorff, P. H., 456 Do¨ring, R., 113, 497 Dorma´n, G., 497 Dorn, C. P., 80 Do¨rner, B., 335, 499, 500, 501(19), 502(19) Dorsey, B. D., 79, 185 Dorsey, J. G., 394 Douglas, S. P., 250 Dower, W. J., 183, 498 Dowling, R. B., 331 Doyle, C. A., 394 Dragovich, S., 63 Dressman, B. A., 87, 391, 392(2), 394(2), 396(2), 417, 480 Drew, M., 86, 90(27), 151, 154, 154(1), 487 Drewry, D. H., 74, 143, 419 Drijfhout, J. W., 293, 294 Drinnan, N., 248, 251, 254

Driver, M. S., 404 Dromer, F., 325 Drusano, G. L., 80 D’Sa, B. A., 453 Duinkerken, G., 294 Dulina, R., 262 Dunn, A. K., 18(80), 79 Dunn, B. M., 485 Dutoit, V., 338, 342(51; 53) Dwek, R. A., 248 Dzierba, C. D., 225 Dzwonczyk, S., 199

E Eames, J., 348, 392(29), 393, 396(29) Ede, N. J., 42, 43(19), 58, 60(19), 64, 73(20), 74 Eden, J. M., 291(22), 292 Edmunds, J. J., 480 Egelhaaf, H.-J., 370 Eggleston, D. S., 185 Ehrler, J., 56 Eichler, J., 324, 497, 498, 498(10), 514(46), 515 Eigenberger, B., 480 Eigenbrot, C., 185 El-Abadelah, M. M., 140 El Abdellaoui, H. M., 506 Elander, N., 202, 203(32) El-Faham, A., 452 Ellis, J. D., 199 Ellman, G. L., 24, 31, 305, 392(22), 393 Ellman, J. A., 39, 42, 43, 65, 81, 89(23b), 90(23), 128, 132(7), 134, 136, 183, 185(5), 197, 290, 296, 391, 415, 435, 437, 451, 451(2), 467(34), 470, 475, 485, 517 Ellmerer-Mu¨ller, E. P., 418 Emini, E. A., 80, 185 Enders, D., 130, 131, 133(11), 142(14), 144(14), 145, 355 Engelsen, V., 428 Engers, D., 86, 90(27), 151, 154(1), 487 Enstrom, A., 298 Eppens, N. A., 324 Ercole, F., 40, 42, 73(20), 115, 117(11) Ericsson, J. A., 422 Eritja, R., 419 Erlanson, D. A., 75 Ermann, M., 419

536

author index

Ernst, B., 67 Espinoza, F. H., 46 Es-Sayed, M., 136, 138 Estep, K. G., 143, 444 Evans, B. E., 80, 470 Evans, D. A., 404(71), 405 Evans, D. J., 186 Ewing, B., 115, 117(8) Ewing, T. J. A., 517

F Falchi, A., 24, 29 Fang, L., 3, 5, 7, 12 Fantauzzi, P. P., 24, 33, 422, 446 Farber, J. M., 332(38), 333, 334(38) Farcy, N., 24, 26 Farnell, K., 54(39), 55 Farrall, M. J., 39, 394, 395(39–41) Farrell, W. P., 4 Faulkner, D. J., 199 Featherstone, R. M., 305 Feeney, P. J., 436 Fehrentz, J.-A., 423 Feijlbrief, M., 293 Fejzo, J., 75 Felder, E. R., 435, 437 Feldman, J., 480 Felici, F., 498 Felker, D., 135 Feng, B., 5 Feng, J. C., 204 Fenuik, W., 296 Ferguson, R., 290 Fernandez, M., 143 Ferna´ndez, R., 384 Fernandez-Rivas, C., 404 Ferreras, M., 517 Ferritto, R., 138, 200(23), 201, 392, 392(24), 393 Fesik, S. W., 75 Fex, T., 154 Fey, T., 351 Fields, G. B., 235 Figliozzi, G. M., 24, 33 Figueroa-Perez, S., 422 Filippone, P., 419 Fillon, C., 18(80), 79 Finch, H., 376 Fincke, H., 139

Finlay, M. R. V., 429 Finner, E., 428 Finucane, M. D., 295 Finzi, D., 332 Fiorini, M. T., 447, 462(30) Fiorino, F., 185 Firestone, S., 419 Fischer, H., 351 Fish, D. G., 80 Fitch, W., 6 FitzGerald, M., 42, 73(20) Fitzgerald, P. M. D., 185 Flegelova, Z., 456 Fleming, P., 489 Fletcher, A., 80 Flexner, C., 332 Flohr, S., 75 Floyd, C. D., 443 Floyd, D. M., 199 Flygare, J. A., 143 Flynn, D. L., 392, 393, 394, 394(32), 395, 395(32; 43), 396(32), 406(32), 475 Flynn, G., 116, 117(16) Fodor, S. P. A., 183, 497 Foged, N. T., 517 Fonon, F., 480 Fontenot, J. D., 302 Ford, C. E., 63 Ford, C. W., 225(7), 226 Forero-Kelly, Y., 399, 402(63) Forray, C., 199 Forster, E. A., 80 Foulds, G. J., 486 Fournier, A., 75 Fournier, E. J.-L., 353, 354(18) Fox, A., 135 Frank, R., 113, 360, 361(33), 379, 497 Frankel, M., 369 Franz, A. H., 278 Franzen, R. G., 136, 450 Fraser-Reid, B., 60, 241(20), 242, 262 Frater, G., 478 Frechet, J. M. J., 39, 152, 200, 394, 395(39–41) Freeman, H. N., 296 Freidinger, R. M., 199, 470 Freyer, A. J., 199 Fridkin, M., 152, 184, 369 Friede, T., 339 Frieden, A., 183, 185(7) Frigerio, M., 376

author index Fritz, J. E., 391, 392(2), 394, 394(2), 396(2) Fru¨chtel, J. S., 392(25), 393 Fryer, A. M., 475 Fu¨gedi, P., 260, 261(31) Fuhrman, S. A., 63 Fujii, N., 304 Fujita, H., 184 Fukase, K., 74 Fukuzawa, A., 75 Fung, E., 325 Furet, P., 87 Furka, A., 75(5), 76, 99, 99(2; 3), 100, 104(9), 113, 116, 298, 435 Furman, B., 430 Furugori, T., 75 Fyles, T. M., 39

G Gabriel, C., 202, 208(29) Gabriel, S., 202, 208(29) Gabryelski, L., 324 Gackenheimer, S., 481 Gadek, T. R., 470 Galfre´, G., 498 Galili, U., 251, 252(12) Gallant, J., 332 Gallazzi, F., 341 Gallop, M. A., 76, 165, 175, 177, 183, 391, 417, 419, 454, 467(44) Ganesan, A., 415, 422, 423, 424, 425(32), 427 Gange, D., 262 Gange, S., 332 Gaoni, Y., 184 Garcia, J. G., 391, 418 Gard, J., 51, 116, 117(13) Gardner, M., 257 Gardyan, M., 86, 90(27), 151, 154(1), 487 Garegg, P. J., 255, 260, 260(17), 261(31), 263 Garibay, P., 426 Garigipati, R. S., 456 Garmon, S. A., 225(7), 226 Garrison, D. T., 74 Garzon, A., 297 Gastaldi, S., 356(25), 357 Gates, M., 470, 471(7) Gatti, P. M., 204, 206(49) Gaudino, J. J., 425 Gayo, L. M., 391 Geddes, D. M., 331

537

Gehlert, D., 481 Gelb, M. H., 185 Gene, J., 323, 331(4) Gennari, C., 396, 431 Gera, L., 444, 465(25) Gerard, B., 151(3), 152 Gerber, F., 438, 443(14), 461(14), 462(14) Gerhardt, J., 418 Germeroth, L., 443, 521 Gerritz, S. W., 42, 53, 73(22), 74, 143 Gershonov, E., 184 Gesellchen, P. D., 324 Geysen, H. M., 40, 41, 43(10), 60, 100, 289, 497 Geysen, M. H., 262 Gharakhanian, S., 323 Ghiron, C., 419 Ghosh, A. K., 394 Ghosh, M., 517 Giacomelli, G., 356(26), 357 Gianella, M., 480 Giannakakou, P., 429 Giannis, 376 Gibson, H. W., 369 Gibson, S. E., 131, 134, 416 Gierasch, L. M., 184 Giger, R., 42, 74 Gigstad, K. M., 478 Gil, C., 136, 140 Gilbert, I. H., 443, 481 Gilbert, K. F., 80, 199 Gilbertson, S. R., 72(57; 58), 73 Gildersleeve, J., 258 Giles, K., 4 Giovannoni, J., 58, 59(42) Giralt, E., 419 Giulianotti, M. A., 335, 501, 504, 505, 510(40), 511, 512 Glass, B. M., 223, 224 Glass, K. L., 199 Gleeson, J.-P., 422 Gluzman, I. Y., 517 Goddard, M.-E. T., 447, 462(30) Goldberg, D. E., 517 Goldman, J., 339 Goldman, R., 262 Goldstein, F. W., 331 Golebiowski, A., 39(9), 40, 72(9), 75, 183, 422, 475 Golec, J. M. C., 87

538

author index

Gomtsyan, A., 75 Gong, Y., 151(2), 152, 160(2) Gong, Y.-D., 418 Gonzalez-Gomez, J. C., 422 Goodman, M., 361(39), 362, 422 Goodnow, R., 236 Gordon, D. W., 257, 419, 454 Gordon, E. M., 75, 183, 391 Gordon, R. S., 358(32), 359 Gore, A. L., 392 Gouault, N., 423, 425(37), 426 Gougoutas, J. Z., 199 Gouilleux, L., 423 Gozzi, C., 177 Grabowska, U., 54(39), 55 Graminski, G. F., 290 Gran, B., 338, 341, 342(47; 64) Grandas, A., 419 Granoth, R., 184 Grant, C. M., 79 Grant, E. H., 202, 208(29) Grant-Young, K., 77 Grathwohl, M., 248, 260, 263 Gravel, M., 404 Gray, N. C., 46, 46(29) Gray, N. S., 438, 444, 444(15), 449(15), 456, 458(15), 465(15) Grayshan, R. J., 140 Greco, M. N., 397, 402(62) Greenlee, W. J., 80 Grega, K. C., 225(7), 226 Greig, M. J., 4 Greivedinger, G., 290 Grether, U., 134 Griebenow, N., 134 Grieco, P. A., 475 Griedel, B., 481 Griffin, I. J., 480 Griffin, P. R., 396 Griffith, M. C., 396 Griffiths, A. D., 289 Grigg, R., 416 Grillot, R., 323, 331(4) Grimstrup, M., 521, 522(19), 525(19) Groetzinger, J., 304 Grogan, M. J., 347 Grootenhuis, P. D. J., 476 Grosche, P., 3, 145, 379 Groutas, W. C., 135 Grover, G. J., 199

Grundy, J. E., 339 Gu, X., 288 Guan, B., 489 Guanti, G., 484 Guare, J. P., 185 Guarro, J., 323, 331(4) Gubernator, K., 489 Guidi, B., 419 Guiles, J. W., 77 Guillaume, P., 338, 342(51) Guillier, F., 438, 459(12), 460(12), 461(12), 521 Gulevskaya, V. I., 140 Gundlach, B. R., 338 Gundry, R. L., 185 Gunthard, H. F., 332 Gunther, W., 478 Guo, L., 313 Gupta, A. K., 204, 206(47) Gupta, R., 204, 206(47)

H Haase, W.-C., 237, 250, 258(5) Habashita, H., 517 Habermann, J., 396 Haddach, M., 405 Hadida, S., 200(23), 201, 392(24), 393 Haehnel, W., 295 Haggarty, S. J., 199 Hagler, A. T., 184 Hagmann, M., 341 Hahn, P. J., 391, 392(2), 394(2), 396(2) Hajduk, P. J., 75 Hajra, A., 209 Hakomori, S., 235 Hales, N. J., 134 Hall, D. G., 404 Hallberg, A., 202, 202(36), 203, 203(33), 224, 228 Hallensleben, M. L., 376 Halstead, B. S. J., 202, 208(29) Haltiwanger, R. C., 185 Hamaker, L. K., 75(5), 76 Hamann, B. C., 404 Hamashin, V. T., 499, 509(20) Hamby, J. M., 480 Hamel, E., 429 Hamelin, J., 202, 203(34), 204(34) Hammer, J., 341

author index Hammer, R. P., 185 Hamper, B. C., 422 Hamuro, Y., 418, 421(11i) Han, G., 185 Han, H., 290 Han, K., 262 Han, Y., 353, 354(16) Hanashin, V. T., 498, 501(17) Hancock, W. S., 24, 26 Handlon, A. L., 53, 70 Hanessian, S., 44, 417, 480 Hanifin, C. M., 80 Hann, M. M., 518 Hanna, G. J., 332 Haque, T. S., 517 Harfenist, M., 480 Harikrishnan, L. S., 388 Harper, G., 518 Harris, D., 421, 425(22g) Harrison, S. C., 332(35), 333 Hart, M. E., 202(38), 203, 224 Hartmann, C., 376 Hartwig, J. F., 175, 404 Haskins, N., 4 Hassall, C. H., 184 Hassan, J., 177 Hatzenbuhler, N. T., 262 Hauck, S. I., 404 Haunert, F., 371 Hauser-Fang, A., 3 Hawes, M. C., 392 Haynie, S. L., 235 He, Y., 429 Healy, E., 99, 100, 116 Heckel, A., 250 Hedberg, A., 199 Heikens, W., 113 Heissler, D., 263 Heisterberg-Moutsis, G., 113 Heitz, W., 369, 382(13) Hellmann, N., 332 Hemmer, B., 338, 341, 342(47; 67), 497 Hendges, S. K., 225(7), 226 Hendrickson, W. A., 332(37), 333 Hendrix, C., 185 Henke, S., 262 Herbert, A., 139 Herdewijn, P., 185 Herpin, T. F., 18(80), 75, 79, 86, 90(27), 151, 154(1), 475, 487

539

Hersh, E. M., 113, 271, 289, 298, 299(1), 309(1), 497, 498(8), 499(8) Hertzberg, R. P., 199 Heuser, D. J., 480 Heuts, J., 135, 139, 140(49) Hewitt, M. C., 240(19), 242, 242(21), 245 Heyer, D., 53, 74 Heykants, J., 470 Heys, L., 199 Hiemstra, H. S., 294, 428 Hilbert, M., 438 Hildebrand, J. P., 375 Hindsgaul, O., 353, 354(18) Hinzen, B., 369, 371, 371(10) Hippe, T., 419 Hipskind, P., 481 Hiramatsu, K., 323, 331(3) Hird, N., 396 Hirshmann, R., 296 Hocker, M. D., 446 Hockerman, S. L., 393, 394(32), 395(32), 396(32), 406(32) Hodge, P., 392(23), 393, 395, 396, 397(54) Hodges, J. C., 392, 392(28), 393, 393(12), 394(12), 395(11; 12), 396(12; 28), 399(12), 401(28), 402(12; 28), 405(12), 407, 480 Hodges, L. C., 388 Hodgson, J., 291(22), 292 Hodosi, G., 250 Hodson, S. J., 74 Hoecker, H., 304 Hoeg-Jensen, T., 426 Hoesl, C. E., 327, 496 Hoetelmans, R. M., 323 Hogan, J. C., Jr., 391 Holladay, M. W., 164, 416 Holloway, M. K., 185 Holm, A., 40 Holmes, A. B., 358(32), 359, 422 Holmes, C. P., 76, 164, 165, 170(2), 176(3; 4), 181(3), 262 Holub, D. P., 134 Holxman, T. F., 75 Homnick, C. F., 199 Hoogenboom, H. R., 289 Hopkins, B. T., 361(35), 362 Horan, N., 258 Horeis, G., 215(59), 216 Ho¨rl, W., 197, 470, 482(8), 489(8)

540

author index

Horn, J., 481 Horwell, D. C., 291(22), 292 Hosten, N. G. C., 24, 26 Hothi, B., 186 Houchins, B. J., 324 Houghten, R. A., 113, 115(4), 289, 290, 291, 292, 298, 324, 325, 327, 335, 338, 339(52), 341, 342(47; 51; 53), 424, 496, 497, 498, 498(9; 10), 499, 499(9), 500, 501, 501(17; 19), 502, 502(19), 503, 504, 505, 506, 506(23), 508, 508(37), 509, 509(20), 510(39; 40), 511, 512, 514(46), 515, 515(23) Howell, D. N., 339 Hruby, V. J., 113, 185, 271, 288, 289, 298, 299(1), 309(1), 497, 498(8), 499(8) Hsieh, M., 291 Huang, B., 262 Huang, W., 164(10), 165, 425 Huang, X., 152, 356 Hudson, D., 444, 465(25) Huening, T. T., 324 Huff, J. R., 80, 185 Huffman, W. F., 185 Hughes, I., 134, 430 Hughes, J., 291(22), 292 Hulme, C., 469, 471, 473, 473(15), 474, 475, 478, 479, 480, 481, 484, 486, 487, 489(47) Husar, G. M., 335, 499, 501(19), 502(19) Hussain, F. M., 331 Hussain, J. P., 331 Hutchins, S. M., 396 Hutchinson, D. K., 225(7), 226 Hwang, S. M., 185 Hyman, C. E., 53, 70 Hynes, J., Jr., 296

I Iacobelli, J., 470, 471(7) Iaiza, P., 428 Ida, S., 517 Iino, M., 75 Ikeler, T. J., 80 Illgen, K., 197 Inamoto, K., 426 Ingallinella, P., 297 Inoue, H., 60, 62, 258 Iversen, T., 263

Iyer, M. S., 478 Izumi, M., 74

J Jachuck, R., 396 Jackson, H. C., 480 Jackson, P. S., 348, 368, 392, 394(16) Jackson, R. F. W., 396 Jackson, S. A., 223 Jacober, S., 87 Jacquault, P., 202, 203(34), 204(34) Jagan Reddy, E., 204 Jain, R. K., 258, 261(22), 262 Jakas, D. R., 185 James, I. W., 39, 42, 43(19), 60(19), 127, 143(6) James, S. N., 40, 115, 117(11) Jana, U., 209 Janda, K. D., 42, 134, 290, 348, 349, 350(10), 426, 437 Jansen, M. A., 470 Janssen, P. A. J., 470 Jaroskova, L., 457 Jarvis, M. F., 75 Jarvis, S., 4 Jayawickreme, C. K., 290 Jefferson, E. A., 297 Jeger, P., 200(23; 24), 201, 392(24), 393 Jensen, K. J., 81, 90(23), 142 Jesberger, M., 349, 350(8) Jiang, S., 332 Jin, S. J., 134 Jingwen, Z., 425 Johnson, R. K., 199 Jolivet, M., 184 Jones, D. G., 262 Jones, J. R., 202, 203(32) Jones, L., 130 Jones, M., 79 Jones, R. C. F., 481 Jones, W., 469, 484, 487 Josephson, J., 75 Josey, J. A., 453, 475 Joshi, S., 60, 262 Jouin, P., 182, 185, 186(25), 187(25), 189(21), 191(25) Joyner, C. T., 480 Judd, D. B., 392 Judkowski, V., 338, 341

author index Jung, G., 3, 145, 291, 293, 338, 367, 370, 372, 376, 379, 392(25), 393 Jung, K. H., 250 Jung, K. W., 134 Junt, T., 338 Jurkiewicz, N. K., 480

K Kachroo, P. L., 204 Kahne, D., 236, 258 Kahr, A.-L., 332 Kaiser, E., 22, 24, 25(1), 302, 513 Kaiser, T. E., 152 Kakarla, R., 262 Kakel, J. A., 74 Kakobsone, I., 152 Kaldor, S. W., 87, 391, 392(2), 394, 394(2), 396(2), 417, 480 Kalir, R., 152, 369 Kalivretenos, A. G., 425 Kallus, C., 262 Kaluza, Z., 430 Kamal, M. R., 140 Kamau, M., 262 Kaminiski, Z. J., 444 Kan, W. M., 75 Kane, P., 438, 459(12), 460(12), 461(12), 521 Kang, S.-B., 489 Kaniszai, A., 51, 116, 117(13) Kantchev, A. B., 262, 396 Kanyi, D., 291 Kaplan, A., 424 Kapoor, T. M., 199 Kappe, C. O., 197, 198, 199, 199(10), 201, 202(39), 203, 204, 205, 205(46), 206(46; 50), 207, 208(53), 209(46; 54), 215(59), 216 Karabelas, K., 480 Karnbrock, W., 418 Karoly-Hafeli, H., 152 Kartsonis, N., 332 Karunaratne, K., 115, 117(8) Kassahun, K., 199 Kassel, D. B., 4 Katchalsky, E., 152 Kates, S. A., 183, 185(7), 347, 348, 452 Katritzky, A. R., 138, 152, 504 Katz, J. L., 404(71), 405 Katzenellenbogen, J. A., 405

541

Kauffman, G. S., 192 Kavalek, J., 86 Kawatsure, M., 404 Kay, B. K., 291 Kay, C., 22 Kaye, J., 338 Kazarnovskii, S. N., 371 Kazmierski, W. M., 113, 271, 289, 298, 299(1), 309(1), 497, 498(8), 499(8) Kearney, P. C., 143 Keating, T. A., 197, 394, 395(37), 470, 470(9), 471, 471(7), 474, 479, 489 Keck, G. E., 360, 361(34) Keenan, R. M., 185 Kelder, J., 476 Kellam, B., 251 Keller, P. M., 5 Kelly, M., 480, 484, 489(47) Kennedy, A. L., 475 Kennedy, R. M., 392(28), 393, 396(28), 401(28), 402(28) Kenner, G. W., 152, 154(20) Kent, S. B. H., 24, 25, 175, 454 Kerr, D., 152 Kerr, J. M., 290, 454 Kesarwani, A. P., 424 Keum, G., 489 Khan, T. H., 250, 258(4) Khmelnitsky, Y., 204, 206(48) Kick, E. K., 485 Kiefer, M. R., 517 Kieffer, B. L., 297 Kiely, J. S., 76, 417, 425(5) Kienle, S., 293, 338 Kiesow, T., 86, 90(27), 151, 154(1), 487 Kihlberg, J., 154 Kim, B. M., 80 Kim, D. J., 430 Kim, H.-Y., 184 Kim, I. J., 294 Kim, J., 446 Kim, K., 521 Kim, M., 423 Kim, P. S., 332(36), 333, 334, 337(39) Kim, R. M., 396 Kim, S., 392(24), 393 Kim, S.-H., 46 Kim, S. W., 297, 417, 423, 480 Kim, S.-Y., 200(23), 201 Kim, Y., 489

542

author index

Kimball, S. D., 199 King, N. P., 429 King, R. W., 199, 324 Kingston, D. G. I., 75 Kirby, C. J., 516 Kirkpatrick, D. L., 75 Kirschning, A., 348, 349, 350(8), 368, 392(25), 393, 396(25) Kiselyov, A. S., 455 Kivity, S., 369 Klabunde, T., 75, 436 Klavins, M., 152 Kleeman, A., 327 Kleinwa¨chter, P., 184 Kling, P., 199 Kloeppner, E., 74 Klopfenstein, S. R., 39(9), 40, 72(9), 75, 183, 422, 475 Kloss, P., 291 Klutchko, S., 480 Knapp, R. J., 113, 271, 289, 298, 299(1), 309(1), 497, 498(8), 499(8) Kneib-Cordonier, N., 444, 465(25) Knepper, K., 136 Knerr, L., 239 Ko, D.-H., 430 Kobayashi, S., 425(37), 426 Ko¨bberling, J., 130, 131, 133(11), 134, 142(14), 144(14) Kobrin, E., 251 Kobylecki, R. J., 77, 115, 257 Koch, G., 74 Koch, K., 428, 475 Kocis, P., 272 Koeller, K., 236 Koelsch, C. F., 139 Koenig, S., 498 Koerber, S. C., 184 Kogan, N., 262 Koh, H.-Y., 489 Koh, J. S., 417, 451, 467(34), 480 Koh, J. T., 456 Kohand, J. T., 46, 46(29) Ko¨hler, T., 332 Koide, T., 304 Kokke, W., 199 Kolodziej, S. A., 422 Kondo, T., 338, 342(47) Kondo, Y., 426

Konradsson, P., 241(20), 242 Koomen, G.-J., 422, 445 Kornet, M. J., 347 Kostenis, E., 75 Kowalski, J., 421 Kowaluk, E. A., 75 Kraas, W., 291, 293 Krajcsi, P., 497 Krajnc, P., 364(42), 365 Krapcho, A. P., 473 Krchnak, V., 24, 51, 76, 112, 116, 117(13), 164, 164(8), 165, 271, 272, 272(3), 290, 293, 298, 299(4), 300(4), 309(4), 416, 456 Krepinsky, J. J., 250 Kretzschmar, T., 304 Krishnan, R., 338, 339(52) Krolikowski, D., 86, 90(27), 487 Krolikowski, P., 86, 90(27), 151, 154, 154(1), 473, 487 Krstenansky, J. L., 202(37), 203, 204, 206(48) Kruijtzer, J. A. W., 296, 417 Kruse, C. G., 428 Krutzik, P. O., 202(38), 203, 224 Kshirsagar, T., 24, 33 Ku, T. W., 185 Kubota, H., 124 Kuisle, O., 24, 28 Kukla, M. J., 470 Kulkarni, B. A., 427 Kumar, D., 204, 206(50) Kumar, N. V., 86, 90(27), 151, 154, 154(1), 199, 473, 487 Kumar, S., 75 Kumar, V. N., 487 Kundu, B., 291, 424 Kuntz, I. D., 517 Kunz, H., 262 Kuo, L. C., 324 Kuo, M., 12 Kurth, M. J., 56, 175, 418, 430, 501 Kurz, M., 75 Kusumoto, S., 74 Kuvshinov, A. M., 140 Kwon, S., 46, 444 Kwon-Chung, K. J., 325 Kwong, P. D., 332(37), 333 Kyranos, J., 3

author index

L Labahn, T., 136 Labaudiniere, R. F., 18(80), 79, 86, 90(27), 151, 154(1), 471, 474, 475, 478, 479, 487 Labe´gue`re, F., 185, 186(25), 187(25), 189(21), 191(25) Ladlow, M., 422 Lam, K. S., 76, 113, 271, 272, 272(3), 278, 278(10), 280(10), 284, 289, 290, 293, 298, 299, 299(1; 4), 300(4), 307, 309(1; 4), 313, 315, 317, 497, 498(8), 499(8) Lam, P. Y. S., 224 Lam, S., 421, 425(22 g) Lambert, J. N., 64, 183, 416 Lamothe, M., 418 Lan, X. F., 138 Lane, D. R., 430 Lang, F. B., 480 Langa, F., 202, 203(30) Lange, J. M., 323 Langley, G. J., 388 Lannuzel, M., 418 Lansky, A., 257, 391 Lanter, C. L., 77 La Porta, E., 431 Larhed, M., 202, 202(36), 203, 203(33), 224, 228 La Rosa, C., 338, 339(52) Lassalette, J. M., 384 Lau, D. H., 313 Lauffer, D. J., 87 Laursen, R. A., 347 Lautenschla¨ger, W., 205, 216(51) Lauterwasser, F., 130, 145 Lazarus, L. H., 185 Lazlo, P., 395 Lazny, R., 131, 142(14), 144(14) Le, L., 173 Leach, A. G., 348, 353, 354(15), 368, 392, 394(16) Leach, A. R., 518 Leadbeater, N. E., 357, 358(28) Lease, T. G., 446 Lebedev, O. L., 371 Lebl, M., 24, 76, 186(32), 187, 271, 272, 272(3), 284, 290, 293, 298, 299(4), 300(4), 309(4), 438, 443, 456 Lebrilla, C. B., 278 Lecat, A., 423

543

LeClerc, S., 46 Lee, C. E., 485, 517 Lee, C. L., 423 Lee, E. J., 417 Lee, H., 3 Lee, J. H., 417, 480 Lee, M. H., 294 Lee, S.-H., 418 Lee, S. K., 423 Lee, W., 422 Lee, Y.-S., 418 Lees, J. H., 295 Leeson, P. D., 423, 518 Leftwich, M. E., 315 LeHetet, C., 425(37), 426 Lehman, A. L., 298 Leighton, R. J., 497 Lemaire, M., 177 Lenz, D. M., 425 Leo, G. C., 397, 402(62) Leost, M., 444 Lepisto, m., 480 Lepore, S. D., 430 Leppert, P., 199 Lepre, C. A., 75 Lerner, M. R., 290, 498 Lerner, R. A., 498 Le Roch, M., 67 Letherbarrow, R. J., 296 Letsinger, R. L., 347 Levin, R. B., 79, 185 Levitz, S. M., 325 Lew, A., 202(38), 203, 224 Lewandowski, K., 200 Lewis, G. S., 164 Lewthwaite, R. A., 291(22), 292 Ley, S. V., 22, 73, 348, 351(12), 352, 353, 354(15; 17), 368, 369, 371, 371(10), 375, 376, 376(26), 392, 394(16), 396 Leznoff, C. C., 39 Li, B., 419 Li, J., 262 Li, L., 3 Li, M., 417, 419 Li, P.-K., 419 Li, R., 40, 115, 117(8) Li, T., 429 Li, T. H., 429 Li, W.-R., 422 Liagre, M., 202(35), 203

544

author index

Liang, A., 481 Liang, L., 263 Liang, R., 258, 262 Liao, S., 288 Lidstrom, P., 202, 224 Lieb, M. E., 197 Lieberburg, I., 485 Liener, I. E., 152, 154(5) Lillig, J. E., 79, 115, 117(8) Lim, J., 124 Limal, D., 184 Lin, J. H., 80, 185, 324 Lin, K., 332 Lindeberg, G., 228 Lindhorst, T., 475 Ling, N., 338 Linn, J. A., 53, 74, 143 Lio, A., 42, 70(17), 100 Lipinski, C. A., 436 Lipshutz, B. H., 364, 407 Lipton, M. A., 421, 478 Liskamp, R. M. J., 296, 418, 425 Lisziewicz, J., 332 Little, D., 4, 5, 5(8) Liu, D., 5 Liu, G., 63, 284 Liu, R., 271, 272, 278, 278(10), 280(10), 298, 299, 313, 317 Liu, W., 304 Livingston, D. J., 87 Lockhart, D. J., 46 Loebacg, J., 258 Lohner, K., 292, 324 Lolo, M., 24, 28 Lombardo, F., 436 Longbottom, D. A., 348, 368, 392, 394(16) Longley, C., 262 Lo¨nn, H., 260, 261(31) Lori, F., 332 Lormann, M., 127, 130, 132(12), 134(12), 135, 137, 138 Lorsbach, B. A., 175 Lorthioir, O. E., 22 Lotti, V. J., 80 Lou, Q., 271, 315 Loupy, A., 202, 202(35; 40; 41), 203, 203(34), 204(34), 216(40) Louridas, B., 473 Lu, A. T., 497 Lu, H. H., 421, 478

Lu, J., 209 Lu, S.-Y., 202, 203(32) Luche, J.-L., 202(35), 203 Lucka, A. W., 498 Lui, D., 262 Luke, R. W. A., 403 Lunn, J., 473 Lutzke, R. A. P., 324 Lysek, R., 430 Lyu, C. S., 430

M Ma, H., 209 Ma, L., 428, 474, 478, 479, 480 Ma, Q. N., 315 Ma, Y., 209 MacCoss, M., 80 Machacek, V., 86 Macher, B. A., 251, 252(12) Maclean, D., 76, 165 Macquarrie, D., 396 Madder, A., 24, 26 Madrigal, J. A., 339 Maeji, N. J., 39, 40, 41, 59(6), 115, 117(11) Magnus, A. S., 375 Mai, S., 199 Makdessian, T., 24, 33 Makino, S., 47, 49, 50 Mallett, D. M., 4, 5(8) Malley, M. F., 199 Mankin, A. S., 291 Mann, G., 404 Mann, M., 396 Mann, T. D., 5 Manninen, P. R., 225(7), 226 Manning, C., 39 Mansell, H. L., 80 Mantellini, F., 419 Marcaccini, S., 489 Marcaurelle, L. A., 249 Marchioro, C., 138 Marcoux, J.-F., 404 Marenus, L. E., 290 Maresh, M. J., 473 Margolick, J. B., 332 Margue, R. G., 348 Marik, J., 271, 272, 278(10), 280(10), 299 Markel, S., 338, 339(52) Markovic-Plese, S., 338, 341, 342(64)

author index Marlowe, C. K., 421 Marques, A., 338, 342(47) Marron, B. E., 74 Marsh, A., 364 Marshall, D. L., 152, 154(5) Marshall, W. J., 418, 421(11i) Martin, C., 376 Martin, R., 338, 341, 342(47; 64), 497 Martina, K., 435 Martineau, G. L., 80 Martinez, J., 58, 59(42), 423 Martinez-Picado, J., 332 Martı´n-Zamora, E., 384 Marton, L. J., 502 Maryanoff, B. E., 397, 402(62), 457 Marzinzik, A. L., 437 Masada, R. I., 444, 465(25) Masala, S., 443, 444 Masko-Moser, J. A., 185 Maslana, E., 392 Maslouh, N., 418 Mason, T. J., 41 Masquelin, T., 391, 421, 424(22b), 438, 443(14), 461(14), 462(14) Massi, A., 200, 349, 350(9), 392(27), 393 Masumoto, K., 481 Mathe´, D., 202, 203(34), 204(34) Mathew, R., 86, 90(27), 151, 154(1) Mathews, J., 428 Mathieu, M. N., 42, 73(20) Mathivanan, P., 394 Matsuda, A., 60 Matter, H., 436 Matthews, D. P., 394, 395(44) Matthews, J., 417 Matthews, T., 332 Mauger, J., 349, 350(7) Mayer, J. P., 164, 425 Mayer, K. H., 325 Mayer, T. U., 199 Mazurov, A., 164(9), 165 Mazzenga, G., 87 McBride, J. D., 296 McCarthy, J. R., 405 McCarthy, T. J., 80 McClenaghan, J., 475 McClure, K. J., 392 McDanal, V., 332 McDaniel, S. L., 79, 185 McDermott, J. R., 152, 154(20)

545

McDowell, R. S., 470 McFarland, H. F., 338, 342(47) McGaraughty, S., 75 McGee, C., 164 McGee, L. R., 470 McGeehan, G., 86, 90(27), 151, 154(1), 487 McKaiser, E., 453 McKeown, S. C., 22 McKinney, E. R., 394 McKnight, A. T., 291(22), 292 McLaughlin, M. L., 185 McLoughlin, D. A., 324 McMullen, D. M., 199 McMurray, J. S., 306 McNally, J. J., 197 McNeil, D., 87 McQueney, M. S., 5 McRipley, R. J., 225 Meador, J. W. III, 63 Meaechler, L., 296 Medal, M., 517 Meecham, K., 291(22), 292 Meenhorst, P. L., 323 Meester, W. J. N., 358(29), 359 Meijer, L., 46, 444 Meldal, M., 314, 314(27), 315, 517 Melean, L. G., 239, 250 Meloen, R. H., 40, 43(10), 100, 289, 497 Meloni, M. M., 24, 29, 30(10) Menard, P. R., 18(80), 79, 473 Mendre, C., 184 Mengel, A., 376 Meola, A., 498 Mermet, C., 480 Merrifield, R. B., 24, 25, 40, 99(4), 100, 112, 127, 175, 258, 261(24), 289, 347, 397, 407(61), 497 Merritt, A. T., 391, 392 Metlay, J. P., 331 Meunier, N., 438, 443(14), 461(14), 462(14) Meutermans, W., 248, 260 Meyer, J.-P., 327, 499, 509(20), 512 Meyer, V., 376 Mezzasalma, T. M., 5 Miao, C., 87 Michael, N. L., 332 Michea-Hamzehpour, M., 332 Michelotti, E. L., 418 Michels, R., 369, 382(13) Michelson, S. R., 80

546

author index

Midha, S., 262 Miller, J. F., 428, 475 Miller, K. E., 480 Miller, M. A., 302 Miller, W. H., 185 Mills, S. G., 80 Min, I. K., 430 Mingos, D. M. P., 202, 208(29) Mischke, D. A., 394, 395(42) Mishani, E., 80 Missio, A., 396 Mitchell, H. J., 79, 115, 236 Mitchell, J. P., 183, 416 Mitchison, T. J., 199 Miyaura, N., 404 Mjalli, A. M. M., 42, 70(17), 100, 474, 480 Moberg, C., 202, 203(33) Mohammad, A. A., 140 Mohan, R., 456 Mol, J. C., 358(29), 359 Monaci, P., 498 Monenschein, H., 348, 368, 392(25), 393, 396(25) Montalbetti, C., 396 Montanari, F., 372, 373(22) Montelaro, R. C., 302 Moon, H.-S., 430 Moore, C. G., 199 Moore, G. J., 296 Moore, J. P., 332 Moore, J. S., 130, 135(9) Moos, W. H., 454 Morales, G. A., 185 Moran, E. J., 42, 70(17), 100 Moree, W. J., 425 Moreland, S., 199 Moreno, A., 202, 203(30) Morera, E., 178 Morgan, C. L., 339 Morgan, D. O., 46 Mori, T., 75 Moriconi, R., 480 Moriggi, J.-D., 428 Moriyama, S., 75 Morize, I., 471 Morley, A. D., 79 Morphy, J. R., 165, 419 Morris, D. R., 502 Morrison, D., 5 Morrissette, M., 473, 480

Morrissey, M. M., 65, 456, 481 Morte, C., 339 Morton, G. C., 18(80), 75, 79, 86, 475, 479 Moser, H., 438, 459(12), 460(12), 461(12), 521 Motherwell, W. B., 357 Motti, C., 498 Mross, E., 250 Mueller, S., 439, 444(16) Mui, P. W., 87 Muir, J. C., 138 Mu¨lbaier, M., 376 Mulder, J. W., 323 Mu¨ller, B., 184 Mu¨ller, S., 521 Muller, U., 478 Mullican, M. D., 87 Munson, M. C., 453 Munstedt, K., 138 Murakami, K., 252 Murayama, E., 263 Murcko, M. A., 75, 294 Murer, P., 200 Murphy, F., 428, 431 Murphy, P. J., 199 Murphy, P. M., 332(38), 333, 334(38) Murray, C. M., 294 Murray, J. P., 396 Murray, P. J., 422 Murray, W., 399, 402(63) Mynard, K., 339

N Nadji, S., 418 Nagarathnam, D., 199 Nagel, A., 87 Naing, W., 394, 395(43) Najdi, S., 418 Na´jera, C., 361(36; 37), 362 Nakanishi, E., 47, 49, 50 Nakatubo, F., 252 Namdev, N. D., 478 Nantermet, P. G., 199 Nanthakumar, S. S., 74 Naumann, T., 436 Navas, F. J. III, 74 Navre, M., 421, 425(22 g), 517 Nechuta, T., 185, 186(24), 189(24) Needels, M. C., 164, 173

547

author index Nefzi, A., 324, 327, 496, 497, 498(10), 499, 502, 504, 505, 506, 509, 509(20), 510(40), 511, 512 Neipp, C. E., 143, 444 Nelson, J. C., 130, 135(9) Nelson, K. H., Jr., 391, 393 Nemoto, H., 60, 62 Neset, S. M., 396 Nesi, M., 348, 368, 392, 394(16) Neurath, A. R., 332 Neustadt, B. R., 185, 186(24), 189(24) Newlander, K. A., 185 Ngo, T. T., 24 Nguyen, T. H., 323 Ni, Z.-J., 76, 165 Nicewonger, R. B., 152, 393, 396(30) Nichols, A., 185 Nicholson, G., 372, 384, 388(42) Nicolaou, K. C., 76, 79, 100, 115, 117(8), 124, 130, 236, 262, 356, 378, 428, 429, 431 Nicolas, E., 418 Nicosia, A., 498 Nieczypor, P., 358(29), 359 Ninkovic, S., 429 Nishimura, T., 252 Nixey, T., 469, 473(15), 474, 481, 484, 486, 489(47) Norberg, T., 260, 261(31) Norman, M. H., 74 Norman, T. C., 46, 46(29), 444, 456 Normandin, D. E., 199 Normansell, J. E., 392(23), 393 Nortey, S. O., 397, 402(62) Nova, M. P., 76, 100, 115, 117(12) Novack, A. R., 446 Nu¨chter, M., 205, 216(51) Nukada, T., 252 Nunes, A., 481 Nu¨ske, H., 136 Nussbaum, O., 334 Nutt, D. J., 480 Nuzzo, M., 498 Ny, P., 291 Nyce, P. L., 87

O Oas, T., 332 Oates, L. J., 24, 31 Obrecht, D., 391, 421, 424, 424(22b)

Ockey, D. A., 430 Oda, Y., 184 Oh, H. J., 517 Olah, T. V., 199, 324 Old, D. W., 404 Ollmann, I. R., 259 Olmstead, M. M., 418 Olsen, D. B., 324 O’Malley, S. S., 199 Ondruschka, B., 205, 216(51) Ong, N. A., 504, 511 Opatz, T., 262 Oprea, T., 518 Orain, D., 74 O’Reilly, B. C., 199 Organ, A., 4, 5 Ornstein, P., 481 Ortar, G., 178 Orton, E., 86, 90(27), 151, 154, 154(1), 487 Osborn, M. I., 250, 258(4) Oscarson, S., 255, 260(17) Ostovic, D., 185 Ostrem, J. A., 272 Ostresh, J. M., 324, 327, 335, 424, 496, 497, 498, 498(10), 499, 500, 501, 501(17; 19), 502, 502(19), 503, 504, 505, 506, 506(23), 508, 508(37), 509, 509(20), 510(39), 511, 512, 515(23) Otaka, A., 304 Ouyang, X., 455 Overland, D., 487 Owens, R. A., 324 Oye, G., 24, 31

P Padera, V., 112 Paio, A., 138, 361(38), 362, 431 Palmacci, E. R., 235, 236(9), 237, 242(21), 245 Pan, J., 7, 12, 392 Pan, Y., 164, 176(3; 4), 181(3) Paneth, P., 444 Panganiban, L. C., 498 Panunzio, M., 396 Paoli, P., 489 Pappageorgiou, J., 254, 259 Paquette, L. A., 371 Parandoosh, Z., 76, 100 Parang, K., 353, 354(18) Pareja, C., 384

548

author index

Parham, C. S., 199 Park, J. Y., 294 Park, K., 56 Park, K.-H., 418, 419 Park, S., 313 Park, S.-J., 489 Parkanyi, C., 470 Parker, M. F., 185 Parlow, J. J., 392, 392(23), 393, 394, 394(32), 395(32; 42; 43), 396(32), 406(32) Parquette, J. R., 262, 396 Parr, N. J., 22 Parrish, C. A., 358(30), 359 Parrot, I., 438 Parry, D. M., 419 Parsons, J. G., 39 Pascal, J., 338, 342(47) Pascal, R., 182, 184, 185, 186, 186(25; 31; 32), 187, 187(25), 189(21; 30–32), 190(31), 191(25), 192(30) Passerini, M., 484 Pastor, A., 152 Pastor, J., 262, 356, 428, 429, 431 Patchornik, A., 152, 369 Pate, M., 487 Patek, M., 116, 117(16), 186(32), 187 Patel, A., 5 Patel, D. V., 391, 421, 425(22 g) Patel, M. V., 185 Patick, A. K., 63 Patil, A. D., 199 Patrick, T. B., 385 Pattarawarapan, M., 143 Pattenden, G., 138 Paul, G. M., 262 Paul, S., 204, 206(47) Pauls, H. W., 151(2), 152, 160(2) Pauwels, R., 470 Pavia, M. R., 76, 417, 425(5) Pears, D., 403 Pechere, J.-C., 332 Pedersen, W. B., 40 Pedoroso, E., 419 Pei, Y., 421 Peng, G., 417 Peng, J. W., 75, 473, 475 Peng, S.-Z., 422 Pennacchio, M., 7 Pennington, M. E., 313 Pepino, R., 489

Peplow, M. A., 134 Perera, S., 42, 73(20) Perez, M., 418 Perez-Paya, E., 292, 497 Perfect, J. R., 325 Pernerfoster, J., 428 Perreux, L., 202(40), 203, 216(40) Persson, M. A. A., 498 Pessi, A., 297 Pesti, J. A., 192 Peters, W. A., 498 Petersen, S. V., 526 Peterson, M. L., 75(5), 76 Petit, A., 202, 203, 203(34), 204(34; 35) Petricci, E., 152 Petropoulos, C. J., 332 Petrovsky, N., 341 Pfefferkorn, J. A., 79, 115, 124 Pfefferkorn, M., 142, 143(54) Pham, Y., 40, 42, 73(20), 115, 117(11) Phan, H., 315 Phelan, J. C., 201 Phoon, C. W., 71 Pi, J., 424 Piarulli, U., 396, 431 Pica, A., 480 Picard, S., 423 Pichler, S., 215(59), 216 Piergentili, A., 480 Pierson, T., 332 Pigini, M., 480 Pillay, D., 323 Pilot, C., 145 Pinel, C., 323, 331(4) Pinilla, C., 113, 290, 298, 322, 324, 325, 338, 339(52), 341, 342, 342(47; 51; 53; 64), 497, 498, 498(9; 10), 499(9), 514(46), 515 Pirard, B., 436 Pirrung, M. C., 497 Piscopio, A. D., 428, 475 Plante, O. J., 235, 236(9), 237, 239, 240(18), 241 Plasterk, R. H., 324 Plesiat, P., 332 Ploemen, J.-P., 476 Plumb, R. S., 4, 5(8) Plunkett, M. J., 43, 134, 197, 415, 437, 446, 475 Pohl, M., 304 Polaskova, M. E., 64

author index Pollak, A., 376 Pollino, J. M., 358(31), 359 Ponton, J., 323, 331(4) Pop, I. E., 152 Popescu, C., 142 Poppe, L., 487 Porcu, G., 24, 32, 356(26), 357 Porter, E. S., 291 Portlock, D. E., 39(9), 40, 72(9), 75, 183, 417 Post, J., 481 Potash, H., 115, 117(12) Potts, B. C. M., 199 Powderly, W. G., 325 Preston, S. L., 80 Pritchard, M. C., 291(22), 292 Protti, M. P., 341 Provera, S., 431 Purchase, T. S., 138 Pye, P. J., 490

Q Qian, C., 209 Qian, H., 152 Qian, M. G., 12 Quaglia, W., 480 Quibell, M., 54(39), 55 Quici, S., 372, 373(22) Quillan, J. M., 290 Quinn, T. C., 332 Quin˜oa´, E., 24, 28 Quintero, J. C., 79, 185 Qushair, G., 21

R Rabinowitz, M., 392 Rachwal, B., 504 Rachwal, S., 504 Raddrizzani, L., 341 Rademann, J., 145, 250, 366, 367, 370, 372, 375, 376, 379, 383, 384, 387, 388(42) Raeymaeckers, A., 470 Rafalski, M., 224 Rafelt, J., 396 Rahman, S. S., 24 Rajasree, K., 351(13), 352 Ramalingam, T., 204 Rammensee, H.-G., 339

Ramshaw, C., 396 Randal, M., 75 Rankovic, Z., 165 Rano, T. A., 324 Ransom, R. W., 199 Ranu, B. C., 209 Rao, C., 453 Raphael, D. R., 75 Raphy, J., 291(22), 292 Rapoport, H., 427 Rapp, W., 418 Rasoul, F., 40, 115, 117(11) Rastogi, S. K., 424 Ratcliffe, G. S., 291(22), 292 Rau, H. K., 295 Rawson, T., 470 Rea, P., 42, 52, 73(20) Reader, J. C., 77, 79, 115 Reader, V. A., 79 Reamer, R. A., 80 Rees, D. C., 165, 481 Regen, S. L., 371 Reger, T. S., 348 Reich, S. H., 24 Reichwein, J. F., 296 Reider, P. J., 80, 490 Reilly, Y., 80 Reinecke, U., 443, 521 Reiss, D., 199 Reissmann, S., 184 Reitz, A. B., 131, 397, 402(62) Renault, J., 67 Rene´, L., 185 Renil, M., 313, 517 Renou, C. C., 79 Reynolds, M. E., 470 Reynolds Cody, D. M., 417, 425(5) Richardson, R. S., 481 Richman, D. D., 332 Riechmann, L., 295 Riguera, R., 24, 28 Riley, P., 42, 73(20) Rinnova´, M., 504 Rittle, K. E., 199, 470 Riva, R., 484 Riva-Toniolo, C., 439, 444(16), 450, 466(33), 521 Rivero, R. A., 77, 417, 428 Rivier, J., 184 Rizo, J., 184

549

550 Rizzo, A., 54(39), 55 Ro, S., 297, 417, 480 Robarge, K. D., 470 Roberts, K. D., 64, 183, 416 Roberts, S. M., 419 Robidoux, A. L. C., 87 Robinett, L. D., 201 Robinson, J., 332(37), 333 Robinson, S., 143, 444 Rodda, S. J., 41 Rodebaugh, R., 60, 262 Rodenko, B., 445 Roecker, A. J., 79, 115 Roep, B. O., 294 Romano, J., 474, 479 Romanovskis, P., 296, 306 Romero, P., 338, 342(51; 53) Romoff, T., 428 Rosania, G. R., 444 Roschangar, F., 429 Rose, M., 487 Rosenberg, E., 332 Rosenberg, S., 46 Ro`senquist, A., 296 Rosenthal, A. S., 87 Roskamp, E. J., 143, 444 Rosse, G., 438, 443(14), 461(14), 462(14) Rossen, K., 80, 490 Rossi, T., 392, 396 Roth, B. D., 138 Roth, H. J., 327 Roussel, P., 438, 459(12), 460(12), 461(12), 521 Routledge, C., 80 Rovnyak, G. C., 199 Royo, M., 31, 183, 185(7), 422 Rozenbaum, W., 323 Rozhkov, V. V., 140 Rubio-Godoy, V., 338, 342(51; 53) Ruchel, R., 323, 331(4) Rudewicz, P. J., 5 Rudzinski, J., 444 Rueter, J. K., 397, 402(62) Ruhland, B., 164, 176(4), 177 Ruhland, R., 447, 463(31), 464(31) Rutjes, F. P. J. T., 358(29), 359, 428 Ryu, S. H., 294

author index

S Sadighi, J. P., 405 Safar, P., 24, 116, 117(16) Sager, J., 80, 490 Saguer, P., 186, 189(30), 192(30) Saha, M., 204 Sahin, U., 341 Sakamoto, T., 426 Sakata, S., 481 Salata, J. J., 480 Salhi, Y., 323 Salmon, S. E., 113, 271, 272, 289, 298, 299(1), 309(1), 497, 498(8), 499(8) Salmon-Ceron, D., 339 Salter, R. D., 339 Salvadori, S., 185 Salvino, J. M., 18(80), 79, 86, 90(27), 151, 151(2; 3), 152, 154, 160(2), 475, 487 Salvino, J. S., 151, 154(1) Samanen, J. M., 185 Sanchez, C., 360, 361(34) Sandanayake, S., 42, 73(20) Sandra, P., 185 Saneii, H., 100, 116 Sanseverino, M., 183, 185(7) Sansoni, B., 369 Santagada, V., 185 Santagostino, M., 376 Santi, D. V., 290 Santini, R., 396 Sarabia, F., 429 Sarin, V. K., 24, 25, 175 Sarohia, R., 444 Sarshar, S., 42, 70(17), 100, 474 Sarvetnick, N., 341 Saubern, S., 224 Sauleau, A., 425(37), 426 Saunders, D., 304 Sauvagnat, B., 151(3), 152 Savara, A., 332 Scarborough, R. M., 164(10), 165 Schaefer, K., 419 Scharn, D., 443, 521 Schelhaas, M., 258, 259(25) Schellekens, G. A., 293 Schellekens, K., 470 Scheuerman, R. A., 172, 174(15) Schiemann, K., 138 Schiller, P. W., 291, 497

author index Schinkel, A. H., 422 Schleif, W. A., 79, 80, 185, 324 Schmid, C. R., 372 Schmid, D., 379 Schmid, D. G., 3, 145 Schmidt, R. R., 239, 250, 263 Schmidt, W., 262 Schmitt, R., 392 Schneck, J. P., 338, 339(52) Schneider-Mergener, J., 443, 521 Schober, D., 481 Schoemaker, H. E., 428 Schols, D., 470 Scho¨nberger, A., 349, 350(8) Schoner, C. C., 499, 501, 509(20) Schoofs, P. G., 41 Schore, N. E., 430 Schorn, T. W., 199 Schreiber, S. L., 124, 127, 199, 366(2), 367, 489, 490 Schroder, K., 338, 341 Schroeder, M. C., 76, 417, 425(5) Schroen, M., 135, 136, 142 Schuler, P., 384, 388(42) Schultz, P. G., 46, 46(29), 438, 444, 444(15), 449(15), 456, 458(15), 465(15) Schulz, E., 177 Schumm, J. S., 130 Schuster, M., 428 Schwartz, J., 199 Schwarz, M. K., 164, 175, 454, 467(44) Scialdone, M. A., 418, 421(11i) Scicinski, J. J., 22 Scott, B. O., 421 Scott, I., 339 Scott, J. K., 289, 498 Scott, J. S., 348, 368, 392, 394(16), 396 Scott, K., 396 Sebestyen, F., 75(5), 76, 99(2; 3), 100, 113, 298, 435 Seeberger, P. H., 235, 236(9), 237, 239, 240(18; 19), 241, 242, 242(21), 245, 249, 250, 258(5) Seeley, J. A., 430 Seitfried, R., 354, 355(19) Sekanina, K., 258 Selkoe, D., 485 Selnick, H. G., 199 Semin, D., 484 Semmelhack, M. F., 372

551

Seneci, P., 138, 361(38), 362, 392, 396, 431 Senkal, B. F., 396, 408 Sensci, P., 392 Senyei, A., 76, 100 Seo, J. K., 294 Sepetov, N., 272, 293 Sergeev, A. G., 404(72), 405 Seri, C., 152 Seto, C. T., 297, 517 Severin, J., 75 Severino, B., 185 Sevignon, M., 177 Seymour, E., 39 Shah, A., 5, 24 Shahbaz, M. M., 42, 70(17), 100 Shao, C., 313 Shao, H., 423 Shao, L.-X., 152 Shao, X., 422 Sharkov, N., 517 Shaughnessy, K. H., 404 Shaw, K., 481 Sheehan, C. S., 39 Shen, J. Q., 498 Sheng, S.-R., 356 Sheppard, R. C., 152, 154(20) Sherrington, D. C., 368, 392(23), 393, 395 Shevelev, S. A., 140 Shi, L., 421, 425(22g), 517 Shiba, T., 184 Shibano, T., 75 Shide, A., 304 Shieh, M. T., 396 Shimn, Y. S., 297 Shiosaki, K., 489 Shohet, S. B., 251, 252(12) Short, K. M., 480 Showalter, H. D. H., 138 Shreder, K., 422 Siani, M. A., 290 Sieber, P., 83 Siegel, M. G., 391, 392(2), 394, 394(2), 395(44), 396(2) Siegmund, A. C., 421 Sierra, T., 12 Sigmund, O., 369 Siliciano, J. D., 332 Siliciano, R., 332 Sillivan, D. J., 323, 331(4) Silva, D. J., 258, 261(22), 262

552

author index

Sim, M. M., 71, 423 Simkovsky, N. M., 419 Simon, R., 338, 341, 342(47; 51; 53; 64) Simonin, F., 297 Singh, A., 75 Singh, J., 75 Sinha, S., 485 Sinigaglia, F., 341 Sircar, I., 480 Sissons, J. G., 339 Sjo, P., 480 Skalitzky, D. J., 63 Skehel, J. J., 332(35), 333 Skiles, J. W., 87 Skillman, A. G., 517 Slade, J., 87 Slee, A. M., 225 Slemmon, J. R., 5 Sleph, P. G., 199 Smerdka, J., 145, 379 Smith, A., 258 Smith, A. B. III, 296 Smith, A. L., 423, 469 Smith, E. M., 185, 186(24), 189(24) Smith, G. P., 289, 498 Smith, J. M., 51, 116, 117(13), 164(8), 165 Smith, K., 332 Smith, M. L., 396 Smith, R., 75 Smith, R. A., 421 Smith, S. C., 364 Smrcina, M., 116, 117(16) Snyder, M., 65 Soderberg, E., 241(20), 242 Sodroski, J., 332(37), 333 Sofia, M. J., 258, 261(22), 262 Sohn, A., 417 Sola, R., 182, 185, 186, 186(25; 31; 32), 187, 187(25), 189(21; 30–32), 190(31), 191(25), 192(30) Solache, A., 339 Solas, D., 497 Solberg, C. O., 323, 331(2) Soll, R. M., 446 Solomon, L., 75 Somers, T. C., 470 Song, A., 278, 298, 313 Song, K. Y., 470 Song, L.-W., 313 Songster, M. F., 81, 90(23), 142

Sophiamma, P. N., 152 Sorcek, R., 87 Sorg, G., 376 Soriano, J. M., 361(36; 37), 362 Sorrell, T. C., 325 Souers, A. J., 183, 185(5), 296 South, M. S., 392, 393, 394, 394(32), 395(32; 43), 396(32), 406(32) Southhall, L. S., 185 Spangle, L. A., 87, 417, 480 Spatola, A. F., 295, 296, 306 Spear, K. L., 46, 421 Spetzler, J. C., 517 Spoerri, H., 56 Sprengeler, P. A., 296 Sreekumar, K., 152 Sridharan, V., 416 Srivastava, G. K., 424 Srivastava, S. K., 437 Sroka, T., 271, 313 Stadler, A., 197, 204, 205, 205(46), 206(46), 207, 208(53), 209(46), 215(59), 216 Stadlwiesser, J., 418, 421, 424(22b) Stahl, M., 295 Stankova, M., 438, 443 Stankove, M., 293 Stankovic, C. J., 76, 417, 425(5) Stanton, J. J., 87 Stauffer, S. R., 405 Steel, P. G., 24, 31, 391 Steele, J., 257, 419, 454 Steele, T. G., 199 Stefani, H. A., 204, 206(49) Steger, M., 517 Stegman, K., 422 Stein, D. S., 80 Steinbruckner, C., 470, 479, 482(8), 489(8) Steinmetz, A., 419 Stephensen, H., 521, 522(18) Sterba, V., 86 Stern, M., 152 Sternbach, L. H., 470 Stevanovic, S., 339 Stevens, D. A., 323, 331(4) Stewart, A., 75 Stieber, F., 134 Stien, D., 356(25), 357 Still, W. C., 258, 299 Stirling, M., 79 Stoermer, R., 139

author index Stone-Elander, S., 202, 203(32) Stonehouse, D. F., 391 Storer, R. I., 348, 353, 354(15), 368, 391, 392, 394(16) Strader, C. D., 296 Strader, T., 4 Stramiello, L. M. S., 143, 444 Straus, S., 338, 342(47) Strick, N., 332 Stringer, O. D., 443, 462 Strop, P., 116, 117(16), 272 Stroud, R. M., 75 Stryer, L., 497 Studer, A., 200(23; 24), 201, 392(24), 393 Stults, C. L., 251 Sturniolo, T., 341 Subba Reddy, B. V., 204 Subra, G., 58, 59(42) Suessbrich, H., 480 Suh, P. G., 294 Suman-Chauhan, N., 138, 291(22), 292 Sung, M. H., 338 Sutherlin, D. P., 444 Suto, M. J., 391 Sutton, L., 332 Suzuki, A., 404 Suzuki, N., 49, 50 Svec, F., 152, 200 Svendsen, I., 314, 314(27), 315 Svensson, A., 154 Svobodova, G., 86 Swali, V., 388 Swann, R. T., 77 Swanson, B. N., 199 Swayze, E. E., 297 Sweet, R. W., 332(37), 333 Szardenings, A. K., 421, 425(22g), 478, 517 Szewczuk, L., 517

T Taddei, M., 24, 29, 32, 356(26), 357, 443, 444 Tadesse, S., 484 Tailor, J., 404 Tait, B., 138 Takahashi, E., 292, 497 Takahashi, K., 485 Takahashi, T., 60, 62, 258

553

Takano, T., 252 Tako´, A., 418 Tam, J. P., 24, 25, 40, 175, 304 Tamaki, M., 185 Tampe, R., 293 Tanaka, H., 60 Taneja, H., 204 Tang, J. X., 353, 354(16), 394 Tang, J.-Y., 353, 354(16) Tang, L., 6 Tang, S.-Y., 18(80), 79, 471, 479 Tanner, H. R., 100, 116 Tartar, A. L., 152 Tata, J. R., 324 Tatsuta, K., 235 Tayebati, S. K., 480 Taylor, C. M., 236 Taylor, E. W., 12 Taylor, J. W., 183, 184(3) Taylor, S. J., 348, 353, 354(17), 368, 392, 394(16) Teague, S. J., 477, 518 Teirney, J., 224 Teitze, L. F., 419 Temesgen, Z., 323 Tempest, P. A., 197, 469, 473(15), 474, 480, 481, 489 ten Holte, P., 419 Tenson, T., 291 Terrett, N. K., 257 Terstappen, G., 392 Tew, D. G., 517 Texier-Boullet, F., 202, 203(34), 204(34) Thijs, L., 419 Thomas, A. W., 376 Thompson, C., 258 Thompson, L. A., 42, 81, 89(23b), 90(23), 127, 391, 392(22), 393 Thomson, C. G., 423 Thorsett, E. D., 470 Thunnissen, A.-M., 46 Thurmer, R., 430 Tian, Z., 235 Tibirica, E., 480 Tice, C. M., 418 Tied, A., 205, 216(51) Tien, D. W., 421, 425(22g), 478, 517 Tierney, J., 202 Tietze, L. F., 197, 419 Tischler, M., 470

554

author index

Tokushige, D., 12 Tolson, D., 5 Tomasi, S., 67 Tomaszek, T. A., 517 Tomida, S., 60, 62 Tomioka, M., 361(39), 362 Tomori, H., 405 Tong, S., 423 Toops, D. S., 225(7), 226 Toshima, K., 235 Toth, I., 251 Toy, P. H., 426 Trainor, R. W., 40, 115, 117(11) Travers, S., 392 Tribbick, G., 41 Trinh, L., 65, 481 Tripp, J. A., 152 Trivedi, B. K., 138 Troll, W., 24, 25 Trost, B. M., 263, 387 Trump, R. P., 74 Truneh, A., 199 Tsai, S., 489 Tsuji, J., 60, 62 Tsuji, T., 47, 49, 50 Tu, S. J., 204 Tucker, L., 341 Tuereci, O., 341 Tumelty, D., 164, 170(2), 172, 173, 174(15), 175, 454, 467(44) Tuna, M., 295 Tymoshenko, D. O., 138, 152 Tyson, R. G., 184 Tzehoval, E., 184 Tzou, A., 338, 341, 342(47; 64)

U Uebel, S., 293 Uehlin, L., 356 Ugi, I., 197, 470, 473, 475, 479, 482(8), 489(8) Ulanowicz, D. A., 225(7), 226 Ulhaq, S., 75 Unger, S. E., 199 Uozumi, Y., 258 Uriac, P., 67 Uriarte-Villares, E., 422 Uskokovic, M., 470, 471(7) Uzinskas, I., 185

V Vacca, J. P., 79, 80, 185 Vagner, J., 24, 81, 90(23), 142, 164(8), 165, 272, 288, 456 Vahrson, H., 138 Vaino, A. R., 42 Valerio, R. M., 39, 40, 41, 46, 59(6) Vallberg, H., 429 Valmori, D., 338, 342(51; 53) Valverde, M. G., 201 Van Aerschot, A., 185 van Bekkum, H., 372, 374(20) Van Den Nest, W., 183, 185(7) van Esseveldt, B. C. J., 419 Van Gelder, J., 470 van Heeswijk, R. P., 323 Van Kirk, K. G., 79 van Loevezijn, A., 422 van Maarseveen, J. H., 422, 428 van Rihn, R. D., 296 Varady, L., 152, 393, 396(30) Varki, A., 248 Varma, R. S., 202, 203(31), 204, 206(50) Vassar, R., 485 Vastag, K., 80 Va´zquez, J., 21, 24, 32 Veber, D. F., 470, 517 Vedkamp, A., 323 Vedsø, P., 426 Veerman, J. J. N., 428 Venslavsky, J. W., 185 Ventura, M. C., 4 Venuti, M. C., 470 VerdierPinard, P., 429 Vergelli, M., 338 Verkade, J. G., 453 Versluis, C., 296 Vidal, A., 504 Vignola, N., 145, 355 Vigouroux, C., 323 Vikstrom, B., 313 Villa, M., 396 Villagordo, J. M., 391, 421, 424, 424(22b) Virgilio, A. A., 65, 296 Visser, G. M., 422, 428 Viswanadhan, V., 484 Voelter, W., 430 Vojkovsky, T., 24, 28, 189, 303 Volante, R. P., 80, 490

author index Volz, F., 473 von dem Bussche-Huennefeld, C., 257 von Richter, V., 139 Voronkov, M., 152 Vourloumis, D., 429 Vouros, P., 3

W Wachter, M., 399, 402(63) Wade, S., 290, 293 Wagaw, S., 404 Wager, C. A., 360, 361(34) Wakabayashi, T., 425(37), 426 Wakamiya, T., 184 Walden, P., 338 Waldmann, H., 134, 258, 259(25) Walker, B. D., 332, 342 Walker, C. H., 137 Wallace, A., 297 Wallbaum, S., 489 Walter, K., 75 Wang, B., 521 Wang, C., 262 Wang, G.-P., 376 Wang, H., 204, 258, 261(22), 262, 422, 424, 425(32) Wang, K. C., 75 Wang, L., 209 Wang, M., 7, 131, 142(14), 144(14) Wang, T., 4 Wang, X., 72(57), 73 Wang, Y., 421, 425(22g), 428 Wang, Z., 209 Wannamaker, M. W., 87 Wanner, M. J., 445 Ward, G. J., 481 Warmus, J. S., 392(28), 393, 396(28), 401(28), 402(28) Warshawsky, A., 152, 185, 369 Watanabe, N., 262 Waterhouse, J., 396, 397(54) Wathey, B., 202, 224 Watkinson, M., 348, 392(29), 393, 396(29) Watson, S., 75 Watson, S. P., 392 Watt, A. P., 5 Weatherston, J., 39 Webb, R. R., 470 Webb, T. R., 185

Webdale, L., 138 Weber, L., 197, 428, 489 Weber, P. A., 292, 324, 335, 499, 501(19), 502(19) Weck, M., 358(31), 359 Weekes, M. P., 339 Wegrzyniak, E., 116, 117(16) Weichsel, A. S., 456 Weigel, L. O., 394, 395(44) Weik, S., 372, 375, 383 Weissenhorn, W. A., 332(35), 333 Weissman, S. A., 80 Welch, M. J., 80 Weller, H. N., 392 Wells, J. A., 75 Wells, N. J., 388 Wels, B., 296 Wenner, W., 470, 471(7) Wenschuh, H., 443, 521 Wermuth, C. G., 438 Wertman, K., 290 Wessel, H.-P., 263 West, L., 475 West, M. L., 248, 251, 254 West, T. R., 404(71), 405 Westley, J. W., 199 Westman, J., 202, 224, 354, 355(20) Westphal, V., 517 Wheatley, J., 5 White, A. C., 80 White, H. L., 480 White, P. D., 39, 186 Whitesides, G. M., 235 Whitfield, D. M., 250, 252 Whitlow, M., 481 Whitney, L. W., 341, 342(64) Whitter, W. L., 470 Wickham, G., 40, 42, 43(19), 52, 60(19), 115, 117(11) Widman, O., 139 Wiesler, W., 235 Wiesmu¨ller, K.-H., 293, 338 Wild, C., 332 Wiley, D. C., 332(35), 333 Wiley, M. R., 430 Willaredt, R. P., 385 Williams, L., 396 Wills, M. R., 339 Wilson, D. B., 338, 341 Wilson, D. H., 338

555

556

author index

Wilson, L. J., 417, 419 Wilson, M., 138 Wilson, M. W., 392(28), 393, 396(28), 401(28), 402(28), 407 Wilson, R., 331 Winkle, J. H., 498, 501(17) Winssinger, N., 130, 262, 356, 428, 429, 431 Winter, G., 289, 295 Winternitz, F., 423 Winters, R. T., 480 Winther, J. R., 517 Wipf, P., 184, 200, 200(23; 24), 201, 392(24), 393 Wirth, T., 356 Wischnat, R., 259 Witte, O. N., 315 Wittenberg, R., 348, 368, 392(25), 393, 396(25) Wodicka, L., 46 Wolfe, J. P., 404, 405 Wolfe, M. M., 290 Wollmer, A., 304 Wolman, Y., 369 Wong, A. S., 185 Wong, C.-H., 235, 236, 259, 260(27) Wong, J., 332 Wong, J. Y., 39 Woodard, S., 393, 394, 394(32), 395(32; 42), 396(32), 406(32) Woodruff, G. N., 291(22), 292 Woodruff, R. A., 453 Woolfson, D. N., 295 Woostenborgles, R., 470 Worland, S. T., 63 Worthington, P. A., 403 Wortmann, F.-J., 142 Wu, C. R., 304 Wu, J., 315 Wu, J. J., 315 Wu, J. V., 235, 271 Wu, M. T., 80 Wu, N., 5 Wu, X., 263, 438, 444(15), 449(15), 458(15), 465(15) Wu, Z., 39, 40, 42, 52, 58, 73(20), 115, 117(11), 446 Wuin, J., 480 Wunberg, T., 262 Wunderlich, I., 138 Wuonola, M. A., 225

Wustrow, D. J., 134 Wyatt, R., 332(37), 333 Wysong, C. L., 185

X Xia, S.-Q., 63 Xiao, C. Y., 115, 117(12) Xiao, X.-Y., 40, 76, 100, 115, 117(8), 272 Xiao, Z., 419 Xie, F., 44 Xiong, L., 291 Xu, B., 313 Xu, R., 290 Xu, X., 324

Y Yadav-Bhatnagar, N., 349, 350(7) Yaday, J. S., 204 Yager, K. M., 201, 422 Yalamouri, V. V., 422 Yamamura, Y., 258 Yamashita, D. S., 517 Yan, B., 3, 5, 12 Yan, B. J., 24, 33 Yan, L., 236, 258 Yanagisawa, M., 295 Yang, B., 209 Yang, B. H., 404 Yang, C. F., 291 Yang, J. Z., 138 Yang, L., 5 Yang, M., 209 Yang, R.-Y., 417, 424, 480 Yang, Y., 517 Yang, Z., 429 Yao, W., 296 Yaouancq, L., 185 Yasuda, K., 351(12), 352, 375, 376(26) Yasuda, M., 425(37), 426 Yates, N. A., 396 Ye, H.-F., 151(3), 152 Ye, T., 138 Ye, X.-S., 259, 260(27) Yin, J., 192, 405 Yokum, T. S., 185 Young, D. W., 517 Young, J. K., 130, 135(9)

author index Youngman, M. A., 197 Yu, S. T., 79 Yu, Y., 327, 424, 496, 503, 506, 508, 508(37), 509 Yu, Z., 403 Yung, G., 392 Yurek, D. A., 12

Z Zal, B., 339 Zander, N., 360, 361(33), 379 Zapf, C. W., 361(39), 362 Zaragoza, F., 517, 518, 521, 522(9; 18; 19), 525(9; 19), 526 Zaragoza Do¨rwald, F., 127, 130(4), 143(4) Zaramella, A., 138 Zarrinmayeh, H., 481 Zechel, C., 257, 391 Ze´cri, F. J., 361(35), 362, 396 Zeleznikow, J., 341 Zelle, R. E., 475 Zemple´n, G., 262 Zeng, L., 4 Zhang, A. J., 143 Zhang, F., 324 Zhang, H., 423 Zhang, H.-C., 457 Zhang, J. W., 304

557

Zhang, L.-h., 192 Zhang, S.-D., 63 Zhang, W., 65, 183, 184(3) Zhang, Y., 185, 186(24), 189(24), 235 Zhang, Z., 185, 259, 260(27), 353, 354(16) Zhao, C. F., 115, 117(12) Zhao, J., 3, 5, 12 Zhao, X. Y., 134 Zhao, Y., 271, 338, 341, 342(47; 51; 53; 64) Zhao, Z. G., 271, 481 Zhong, H. M., 397, 402(62) Zhong, Y.-L., 378 Zhou, J. F., 204 Zhou, Q. S., 152 Zhou, R., 63 Zhu, T., 258 Zhuang, H., 115, 117(8) Zicmanis, A., 152 Zimmerman, D., 481 Zuckermann, R. N., 290, 454 Zuercher, W. J., 74 Zugay, J. A., 79, 80, 185 Zupan, M., 376 Zurenko, G. E., 225(7), 226 Zwanenburg, B., 419 Zwick, M. B., 498 Zychlinski, A. V., 473 Zygmunt, M., 138

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Subject Index

A

Benzotriazole library, synthesis using T1 triazene linkers, 136–138, 147–148 Benzylamine, polymer-assisted solution phase synthesis using scavenger resins, 407, 412 N-Benzyl-2-bromo-N-methylbenzamide, polymer-assisted solution phase synthesis using scavenger resins, 403, 403, 410–411 3-Benzyl-2-phenylthiazolidin-4-one, polymer-assisted solution phase synthesis using scavenger resins, 406–407, 412 1-Benzyl-3-phenyl-thiourea, polymer-assisted solution phase synthesis using scavenger resins, 403, 411 4-(3-Benzylsulfanyl-5-phenyl-[1,2,4]triazol-4ylmethyl)-benzamide, polymer-assisted solution phase synthesis using scavenger resins, 402, 410–411 1-Butyl-3-(2-thiophen-2-yl-ethyl)urea, polymer-assisted solution phase synthesis using scavenger resins, 405–406, 411

4-Amino-3-hydrazino-5-mercapto-1,2,4triazole, aldehyde assay in solid-phase synthesis, 33 p-Anisaldehyde, aldehyde assay in solid-phase synthesis, 32–33

B Benzimidazole library isocyanide-based multicomponent reactions, 481 synthesis using SynPhase Crowns and Lanterns, 51–53 traceless synthesis o-floro/chloro-nitroarene coupling, 173 overview of development and optimization, 164–173 quaternization with alkyl/benzyl bromides, 174 reagents, 173 reduction of aromatic nitro group, 174 resin preparation, 174–175 Benzodiazepine library applications, 470 cyclative cleavage in solid-phase synthesis, 424–425 isocyanide-based multicomponent reactions nuclear magnetic resonance characterization, 491–492 overview, 470–471, 473, 478 plate production, 490–491 scale-up, 491–492 synthesis using SynPhase Crowns and Lanterns, 43–45 Benzofuroxane library, synthesis using T1 triazene linkers, 140–141, 147–148 Benzothiazepine library, synthesis with directed sorting and parallel synthesis, 86–87, 89–90

C Carbohydrate library oligosaccharide synthesis, see Oligosaccharide solid-phase synthesis synthesis using SynPhase Crowns, 60–63 Chloranil, amine assay in solid-phase synthesis, 28, 303 Cinnoline library, synthesis using T1 triazene linkers, 136–137, 139–140 Claisen-type condensation, cyclative cleavage in solid-phase synthesis, 427–428 Cyclative cleavage, solid-phase synthesis advantages in heterocycle synthesis, 415–416 carbon nucleophiles, 427–428 559

560

subject index

Cyclative cleavage, solid-phase synthesis (cont.) diketopiperazine cyclative cleavage, 433 five-membered ring formation by nitrogen nucleophile attack of sp2 or sp3 carbon, 416–419 organometallic reactions, 428–429 overview, 415 oxygen nucleophiles, 425–427 prospects, 432 reverse cyclative cleavage, 430–431 seven-membered ring formation by nitrogen nucleophile attack of sp2 carbon, 424–425 six-membered ring formation by nitrogen nucleophile attack of sp2 carbon, 419, 421–424 tetramic acid cyclative cleavage, 433–434

D Diamino acid linkers 4-amino-3-(aminomethyl)benzoic acid scaffold preparation and peptide synthesis, 187–189, 191–192 constructs unnatural aliphatic amino acids, 183–184 unnatural arylamino amino acids, 185–186 unnatural cyclic amino acids, 184–185 2,4-diaminobutyric acid, 184 2,3-diaminoproprionic acid(Phoc) linker attachment to support, 189–191, 193 deprotection and coupling in peptide synthesis, 194 stability, 187 structure, 183–184 synthesis, 192–193 safety-catch linkers, 186–187 2,3-Diaminoproprionic acid(Phoc) linker, see Diamino acid linkers 1,4-Diazepine-2,5-dione library, synthesis using SynPhase Crowns, 58–60 Dibenzylamine, polymer-assisted solution phase synthesis using scavenger resins, 404, 411 2,3-Dichloroproprionic acid, derivatives as polyelectrophiles for sequential nucleophilic substitutions, 521–522

Diethyl-(2-p-tolyl-ethyl)amine, polymer-assisted solution phase synthesis using scavenger resins, 402, 410 4,5-Difluoro-2-nitrobenzamine, derivatives as polyelectrophiles for sequential nucleophilic substitutions, 522–523 Dihydroimidazole library, mixture-based synthetic combinatorial libraries, 507–508 Dihydropyrimidine library Biginelli synthesis, see Microwave-assisted Biginelli multicomponent reactions clinical applications, 199 parallel synthesis, 200 solid-phase synthesis approaches, 200–202 Diketomorpholine library, isocyanide-based multicomponent reactions, 478 Diketopiperazine library cyclative cleavage in solid-phase synthesis, 421–422, 433 isocyanide-based multicomponent reactions nuclear magnetic resonance characterization, 493 overview, 476–478 plate production, 492 scale-up, 492 Diphenyldichlorosilane-methyl red, alcohol assay in solid-phase synthesis, 30–31 Diphenylmethylamine library, synthesis using SynPhase‘ Crowns, 70–71 Directed sorting, see Solid-phase synthesis 5,50 -Dithio(2-nitrobenzoic acid), thiol assay in solid-phase synthesis, 31

E Ellman’s reagent, see 5,50 -Dithio(2nitrobenzoic acid) Emrys synthesizer, microwave-assisted organic synthesis, 205–206 Encore synthesis advantages of split-and-pool synthesis, 124 instrumentation Arraying Tool, 119 Lantern Dispensing Tool, 120 Lantern Leveling Tool, 120 Lapis Tool, 120 Magazine, 119–120 overview, 117–119

561

subject index steps in combinatorial library synthesis, 121–124

G Guanidine library mixture-based synthetic combinatorial libraries, 509 polymer-assisted solution phase synthesis, 363 synthesis using T2 triazene linkers, 143–145, 149–150

H Heterocycle library synthesis, see also specific compounds mixture-based synthetic combinatorial libraries, see Mixture-based synthetic combinatorial libraries cyclative cleavage, see Cyclative cleavage, solid-phase synthesis derivatization reactions on solid-phase acylation, 451–453, 467 alkylation, 451, 453–454, 467–468 aromatic nitro group reduction, 455, 468 derivatization of C2 chloro group of resin-bound heterocycles, 439, 441, 458–459 facilitated arylations via iron-

complex, 447–449, 463 Knoevenagel reaction, 456–457, 468–469 Mannich reaction, 457, 469 Mitsunobu reaction, 456, 468 nucleophilic aromatic displacements, 438–449 overview, 437–438 palladium-catalyzed reactions, 449–451, 465–466 prospects, 457–458 purines and adenosine analog synthesis, 445 quinazolines, 446–448, 462–463 Rink resin compound capture and derivatization, 441–443, 459–461 Stille coupling, 450–451, 466 triazine derivatization, 443–444, 461–462 drug development applications, 435–437, 469

multicomponent condensation reactions, see Isocyanide-based multicomponent reactions triazene T1 linkers, 136–140, 147–148 High-performance liquid chromatography, combinatorial library analysis, see Liquid chromatography/ultraviolet/mass spectrometry, combinatorial library analysis HIV, see Human immunodeficiency virus HPLC, see High-performance liquid chromatography Human immunodeficiency virus synthetic combinatorial libraries for drug development membrane fusion inhibition assay, 334–335 peptide considerations as drugs, 334 replication inhibition assay, 335, 337 therapeutic targets, 323–324, 332 Hydantoin library cyclative cleavage in solid-phase synthesis, 417–418 isocyanide-based multicomponent reactions, 479 mixture-based synthetic combinatorial libraries, 505–506 synthesis using SynPhase‘ Crowns, 56–58

I Imidazoline library isocyanide-based multicomponent reactions, 480–481 mixture-based synthetic combinatorial libraries, 506–507 Imidazolone library, mixture-based synthetic combinatorial libraries, 503–505 Indolepyridoimidazole library, mixturebased synthetic combinatorial libraries, 506 Isocyanide-based multicomponent reactions automation, 487–489 azepine-tetrazole preparation, 493–494 benzodiazepine library synthesis nuclear magnetic resonance characterization, 491–492 overview, 470–471, 473, 478 plate production, 490–491

562

subject index

Isocyanide-based multicomponent reactions (cont.) scale-up, 491–492 diketopiperazine library synthesis nuclear magnetic resonance characterization, 493 overview, 476–478 plate production, 492 scale-up, 492 ketopiperazine tetrazole preparation, 494–495 nor-statine preparation, 495 postcondensation Passerini reactions, 484–485 prospects, 489–490 tetrazole-nor-statine preparation, 495–496 trimethylsilyl azide-modified Passerini reaction, 486–487 trimethylsilyl azide-modified Ugi reactions, 482–484 Ugi multicomponent condensation reactions benzimidazole library synthesis, 481 deprotection, 473–474 diketomorpholine library synthesis, 478 ethyl glyoxylate templates, 475–477 hydantoin library synthesis, 479 imidazoline library synthesis, 480–481 immobilized convertible isonitriles, 475 quinoxalinone library synthesis, 481–482 UDC concept, 473–475 urea library synthesis, 479–480 1-(3-Isopropoxypropyl)-3-phenylthiourea, polymer-assisted solution phase synthesis using scavenger resins, 405–406, 411

K Knoevenagel reaction, derivatization reactions on solid-phase, 456–457, 468–469

L Liquid chromatography/ultraviolet/mass spectrometry, combinatorial library analysis comparison of eight-channel system with single-channel system

data acquisition in positive and negative ion modes, 19 operation and maintenance, 20 reanalysis of samples, 20 sensitivity, 19 time of analysis, 18–19 eight-way multiplexed electrospray interface, 4–5 high-throughput modifications, 3–4 instrumentation, 6 library evaluation conditions, 14, 18 optimization column selection, 9 flow monitoring, 7 flow rates, 8–9, 21 standards, 6–7 T-joint positioning, 8, 20–21 pumping system, 5 reanalysis of samples, 10–11 representative library compound evaluation, 12, 14, 21

M Malachite green, carboxylic acid assay in solid-phase synthesis, 32 Mannich reaction, derivatization reactions on solid-phase, 457, 469 Mass spectrometry, combinatorial library analysis, see Liquid chromatography/ ultraviolet/mass spectrometry, combinatorial library analysis 4-(3-Methyl-5-phenyl-pyrazol-1-yl)-benzoic acid, polymer-assisted solution phase synthesis using scavenger resins, 399–401, 410 Microwave-assisted Biginelli multicomponent reactions advantages, 216–217 Biginelli dihydropyrimidine synthesis, 197–198 dihydropyrimidine library synthesis automated sequential library production, 212, 215–216 criteria, 206–207 microwave irradiation, 217–218 nuclear magnetic resonance characterization, 218–223 reaction optimization catalyst selection, 209

563

subject index overview, 207–208 solvent selection, 208 time and temperature, 210–211 troublesome building blocks, 211–212 reagents, 217 Emrys synthesizer, 205–206 multicomponent reaction advantages, 197 overview, 204–205 Microwave-assisted organic synthesis dihydropyrimidines, see Microwave-assisted Biginelli multicomponent reactions instrumentation, 203–206, 224–225 oxazolinidones, see Oxazolinidone library principles, 202–203, 223–224 solid-phase synthesis, 224 Mitsunobu reaction, derivatization reactions on solid-phase, 456, 468 Mixture-based synthetic combinatorial libraries chemistry optimization, 502–503 heterocyclic compound synthesis from resin-bound compounds bis-heterocyclic compound synthesis from resin-bound polyamines, 510–511 dihydroimidazoles, 507–508 guanidines, 509 hydantoins, 505–506 imidazolines, 506–507 imidazolones, 503–505 indolepyridoimidazoles, 506 overview, 496–499 parallel synthesis of heterocyclic positional scanning libraries alkylation of trityl-protected amino acids, 514 amide bond reduction, 515 Boc amino acid coupling to resin, 514 cleavage conditions, 516 control teabag synthesis, 516 Fmoc amino acid coupling, 514–515 N-acylation, 515 overview, 511–513 parallel synthesis, 515–516 requirements, 513 trityl group removal, 514 trityl protection of resin-bound amino acids, 514

peptide library generation, 324–325 peptidomimetic library generation, 501–502 prospects, 516–517 reagent mixtures, 500–501 resin mixtures, 499–500 MS, see Mass spectrometry Multicomponent reaction advantages, 197 Biginelli dihydropyrimidine synthesis, see Microwave-assisted Biginelli multicomponent reactions multicomponent condensation reactions, see Isocyanide-based multicomponent reactions

N NF-31, amine assay in solid-phase synthesis, 26–28 Ninhydrin, amine assay in solid-phase synthesis, 25–26, 303 NMR, see Nuclear magnetic resonance Nuclear magnetic resonance benzodiazepine library characterization, 491–492 dihydropyrimidine library characterization, 218–223 diketopiperazine library characterization, 493 tetrafluorophenol-activated resin quality control, 156–158

O Oligosaccharide solid-phase synthesis applications, 243, 248–249 automated synthesis branching, 240 cleavage conditions, 241, 247 deprotection, 246–247 F-tag capping and purification of products, 242, 248 glycosyl phosphate coupling, 240–241, 247 glycosyl trichlooacetimidate coupling, 239–240, 246 instrumentation, 238, 243, 246 linkers, 238–239, 250–251

564

subject index

Oligosaccharide solid-phase synthesis (cont.) materials, 244–245 overview, 236–238 resin synthesis, 245 trityl cation assay, 245–246 blood group determinant tetrasaccharide preparation, 255–257 branching problem, 235–236 combinatorial oligosaccharide library synthesis, 257–265 human antigen trisaccharide preparation, 251–255 prospects, 265 stereochemistry problem, 235–236 One-bead one-compound library Boc-amino acid relative reactivity, 280 decoding, 273–274, 278 encoding advantages, 275–276 biphasic solvent approach for bead segregation, 273, 275, 278, 281 coding units and reactions, 275 overview, 272 peptidomimetic and small molecule library synthesis, 273–274, 276–278, 280–284 historical perspective, 289–290 peptide library synthesis activating agents, 300–301 amino acid solution preparation, 301–302 coupling reaction monitoring, 302–303 cyclic peptide library synthesis using lysine and glutamate residues, 306–307 disulfide cyclic peptide library synthesis, 304–306 fluorescence quench library synthesis for protease substrate determination, 307–308 linear hexapeptide synthesis, 303–304 solid supports, 300 screening encoded libraries, 277, 284, 287, 299–300 enzyme-linked colorimetric assay, 309–310, 312 protease substrates, 314–315 protein kinase substrate assay, 315–317 whole cell binding assay, 313–314

sequencing Edman sequencing, 322 microsequencing, 317–319, 322 retention time of derivatized amino acids on protein sequencer, 320–321 split-mix synthesis, 298–299 structural characterization of compounds, 271–272 Oxazole library, synthesis using SynPhase‘ Crowns, 54–56 Oxazolinidone library antimicrobial activity, 225–226 microwave-assisted synthesis acylation of attached iodoaryloxazolidinone, 231 cleavage of biaryloxazolidinone from solid support, 231 iodoaryloxazolidinone coupling to resin, 230–231 optimization of Suzuki reaction, 228–229 overview, 228–230 reagents, 230 Smith synthesizer, 228 solid-phase Suzuki coupling, 231 retrosynthetic analysis of oxazolinidone pharmacophore, 226–228

P Parallel synthesis, see Solid-phase synthesis PASP synthesis, see Polymer-assisted solution phase synthesis Passerini reaction, see Isocyanide-based multicomponent reactions Peptide combinatorial library antibacterial agent development using positional scanning synthetic combinatorial libraries pathogens, 331–332 screening, 332 antifungal agent development using positional scanning synthetic combinatorial libraries deconvolution, 330 pathogens, 326–327 screening, 327, 330 antimicrobial peptide library design, 292–293 applications, 288–289 combinatorial docking, 295

subject index cyclization, 295–296 design strategies, 293–295, 297 diamino acid linkers in synthesis, see Diamino acid linkers hinges in libraries, 297–297 historical perspective, 289–290 human immunodeficiency virus antagonist development membrane fusion inhibition assay, 334–335 peptide considerations as drugs, 334 replication inhibition assay, 335, 337 mixture-based synthetic combinatorial libraries, see Mixture-based synthetic combinatorial libraries one-bead one-compound libraries, see One-bead one-compound library positional scanning, 291, 325 screening approaches, 290–291 split-and-pool approach in synthesis, 75–76 synthesis using SynPhase‘ Crowns cyclic peptides, 64–67, 69 muramyl peptide derivatives, 63, 65 rhinovirus protease inhibitors, 63–64 tripeptide library string synthesis cleavage, 110–111 coupling, 110 overview, 109 product distribution and verification, 111–112 sorting of crowns, 110 synthesis using T2 triazene linkers, 142–143 tertiary structure design, 295 vaccine development epitope mimic identification, 338–341 natural epitope identification in protein databases, 341–343 overview, 337–338 prospects, 344 Perfluoroalkylsulfonyl linker cleavage of resin-bound phenols catalytic amination, 179–180 catalytic reductive elimination, 178–179, 182 Suzuki coupling reaction, 177, 181–182 phenol attachment, 175–176, 181 prospects for traceless library synthesis, 180–181 synthesis, 176–177

565

1-Phenylpentan-1-ol, polymer-assisted solution phase synthesis using scavenger resins, 406, 412 Piperazine-2-carboxamide library, synthesis with directed sorting, 79–81, 83, 85–86 Polyamine library, synthesis using SynPhase Crowns, 67–68, 70 Polymer-assisted solution phase synthesis, see also Polymer-supported reagents advantages over solid-phase synthesis, 392 alkylating polymers carboxylic acid alkylation, 381–382 overview, 379–381 carbanion equivalent reagents, 382–384 highly-loading resins for polymer-supported reagents, 387–390 oxidizing polymers heavy-metal oxide reagents, 371 overview, 370–371 oxoammonium salt resins alcohol oxidation, 373–375 overview, 371–373 preparation, 373 periodinane resins alcohol oxidation, 377–379 overview, 376 reactivation, 377 principles, 367–369 prospects, 370, 390 radical release from polymer gels, 385–387 scavenger resins benzylamine synthesis, 407, 412 N-benzyl-2-bromo-N-methylbenzamide synthesis, 401, 403, 410–411 3-benzyl-2-phenylthiazolidin-4-one synthesis, 406–407, 412 1-benzyl-3-phenyl-thiourea synthesis, 403, 411 4-(3-benzylsulfanyl-5-phenyl[1,2,4]triazol-4-ylmethyl)benzamide synthesis, 402, 410–411, 399–401, 410 1-butyl-3-(2-thiophen-2-yl-ethyl)urea synthesis, 405–406, 411 dibenzylamine synthesis, 404, 411 diethyl-(2-p-tolyl-ethyl)amine synthesis, 402, 410 historial perspective, 393

566

subject index

Polymer-assisted solution phase synthesis (cont.) 1-(3-isopropoxypropyl)-3phenylthiourea synthesis, 405–406, 411 4-(3-methyl-5-phenyl-pyrazol-1-yl)benzoic acid synthesis overview of types, 396–397 1-phenylpentan-1-ol synthesis, 406, 412 principles, 394–396 quenching reagent criteria, 393–394 rationale, 391–393, 396 synthesis of resins acid chloride resin, 409 amine/aminoalcohol resin, 409 aminodiol/morpholine resin, 409 arenesulfonyl chloride resin, 409–410 boronic acid diethanolamine resin, 404–405 guanidine resin, 409 oligo(ethyleneimine) resin, 408 phenol resin, 409 Polymer-supported reagents acids/bases and dihydropyrimidone synthesis, 349–351 amide-coupling reagents, 360–362 carbon transfer reagents and olefin synthesis via Wittig reaction, 354–355 electrophilic reagents, 356–357 esterification reagents, 360–362 group transfer reagents ester synthesis, 363 Fmoc-amino acid synthesis, 363 guanidine synthesis, 363 overview, 362 historical perspective, 368–369 multistep synthesis applications, 366 oxidations and reductions alkene hydrogenation, 353 carboxylic acid synthesis, 352 overview, 351–352 polymer-assisted solution phase synthesis, see Polymer-assisted solution phase synthesis radical reaction reagents, 356–357 scavengers overview, 364–365 phosphine and phosphinoxide scavenging, 365

sulfur and phosphorous transfer reagents, 353–354 transition metal catalysts arylamine synthesis, 359–360 biphenyl synthesis via Suzuki coupling, 360 overview, 357–359 Purine library cyclative cleavage, solid-phase synthesis, 445 synthesis using SynPhase‘ Crowns, 45–47 Purpald, see 4-Amino-3-hydrazino-5mercapto-1,2,4-triazole Pyrin-2-one library, synthesis using SynPhase‘ Crowns, 53–54

Q Quinazoline library derivatization reactions on solid-phase, 446–448, 462–463 synthesis using SynPhase‘ Lanterns, 47–51 Quinazolinone library, cyclative cleavage in solid-phase synthesis, 424 Quinoxaline library, synthesis using SynPhase‘ Lanterns, 58–59 Quinoxalinone library, isocyanide-based multicomponent reactions, 481–482

R Radiation-graft polymers, see SynPhase‘ Crown; SynPhase‘ Lantern Rink resin, compound capture and derivatization, 441–443, 459–461

S Scavenger resins, see Polymer-assisted solution phase synthesis Solid-phase-assisted-solution-phase combinatorial synthesis, see Polymer-assisted solution phase synthesis; Polymer-supported reagents Solid-phase synthesis, see also specific compounds colorimetric tests

subject index alcohols diphenyldichlorosilane-methyl red test, 30–31 p-toluenesulfonyl chloride/ p-nitrobenzylpyridine test, 28–29 1,3,5-trichlorotriazine-(fluorescein, alizarin R, or fuchsin) test, 29–30 aldehydes 4-amino-3-hydrazino-5-mercapto1,2,4-triazole test, 33 p-anisaldehyde test, 32–33 aliphatic amines chloranil test, 28 NF-31 test, 26–28 ninhydrin test, 25–26 trinitrobenzenesulfonic acid test, 26 carboxylic acids, malachite green test, 32 overview, 22–25 reproducibility, 35 specificity of tests, 33–34 thiols, 5,5’-dithio(2-nitrobenzoic acid) test, 31 cyclative cleavage, see Cyclative cleavage, solid-phase synthesis directed sorting approach acylation with carboxylic acids, 93–94 acylation with chloroformates, 94 alkylation reaction, 98 amide bond formation, 96 beads and resins, 90–91 benzothiazepine library synthesis with directed sorting and parallel synthesis, 86–87, 89–90 cleavage conditions, 96, 98–99 cyclization to benzothiazepine, 97–98 equipment, 77, 79 halo-nitrobenzene coupling, 97 nitro group reduction, 97 piperazine-2-carboxamide library synthesis, 79–81, 83, 85–86 principles, 76–77 protecting group removal, 93–95 resin preparation, 95–97, 99 sulfonamide formation, 94 sulfone oxidation, 98 urea formation with isocyanates, 94 washing of beads, 93 historical perspective, 39, 112–113, 347

567

linkers, see also Diamino acid linkers; Tetrafluorophenol-activated resins; Triazene anchors amine derivatization linkers, 153 monofunctional linker limitations, 128 multifunctional linker advantages, 129 oligosaccharide solid-phase synthesis, 238–239, 250–251 traceless linkers, see also Perfluoroalkylsulfonyl linker; Triazene anchors definition, 132–133, 164 types, 134 types, 127–130 microwave-assisted synthesis, see Microwave-assisted organic synthesis oligosaccharides, see Oligosaccharide solid-phase synthesis parallel synthesis, 76 polymer-bound polyelectrophiles and sequential nucleophilic substitutions 1-[5-benzenesulfonyl-2-(piperidin-1-yl)4-(pyridin-4ylmethylamino)benzoyl]piperazine trifluoroacetate synthesis, 526–527 2,3-dichloroproprionic acid derivatives as polyelectrophiles, 521–522 4,5-difluoro-2-nitrobenzamines as polyelectrophiles, 522–523 2-phenylsulfanyl-3-(piperidin-1yl)propionic acid trifluoroacetate synthesis, 525–526 principles, 517–521 radiation-graft polymers from Chiron Mimitopes, see SynPhase‘ Crown; SynPhase‘ Lantern split-mix synthesis, see Split-mix synthesis tea bag synthesis, 113 traceless synthesis of benzimidazole library o-floro/chloro-nitroarene coupling, 173 overview of development and optimization, 164–173 quaternization with alkyl/benzyl bromides, 174 reagents, 173 reduction of aromatic nitro group, 174 resin preparation, 174–175

568

subject index

Split-and-pool synthesis advantages, 124 directed synthesis, see also Encore synthesis encoding, 115–116 process integration and automation, 116–117 random synthesis comparison, 113–114 solid supports, 115 peptide libraries, 75–76 Split-mix synthesis one-bead one-compound library, 298–299 principles, 99–100 string synthesis manual redistribution of crowns, 102–104 overview, 100–101 redistribution pattern, 104–105, 107 software, 107–108 stringing of support units, 101–102 SynPhase‘ Crowns as support units, 101–102 tripeptide library synthesis cleavage, 110–111 coupling, 110 overview, 109 product distribution and verification, 111–112 sorting of crowns, 110 SPS, see Solid-phase synthesis String synthesis, see Split-mix synthesis Suzuki reaction biphenyl synthesis with transition metal catalysts, 360 microwave-assisted synthesis of oxazolinidones optimization, 228–229 solid-phase coupling, 231 perfluoroalkylsulfonyl linker, cleavage of resin-bound phenols, 177, 181–182 SynPhase‘ Crown benzimidazole synthesis, 51–53 benzodiazepine synthesis, 43–44 carbohydrate synthesis, 60–63 1,4-diazepine-2,5-dione synthesis, 58–60 diphenylmethylamine library, 70–71 directed synthesis, see Encore synthesis hydantoin synthesis, 56–58 overview, 41–43

oxazole synthesis, 54–56 peptide synthesis cyclic peptides, 64–67, 69 muramyl peptide derivatives, 63, 65 rhinovirus protease inhibitors, 63–64 tripeptide library string synthesis cleavage, 110–111 coupling, 110 overview, 109 product distribution and verification, 111–112 sorting of crowns, 110 polyamine synthesis, 67–68, 70 prospects, 72–73 purine synthesis, 45–47 pyrin-2-one synthesis, 53–54 string synthesis, see Split-mix synthesis urea synthesis, 72 SynPhase‘ Lantern benzimidazole synthesis, 52–53 benzodiazepine synthesis, 44–45 directed synthesis, see Encore synthesis overview, 40–44 prospects, 73–74 quinazoline synthesis, 47–51 quinoxaline synthesis, 58–59 urea synthesis, 71–72

T Tetrafluorophenol-activated resins amine derivatization applications, 151–152, 154 library synthesis, 158–161, 163 carboxylic acid ester preparation, 162 loading determination, 163 polymeric tetrafluorophenol synthesis, 154–155 quality control, 156–158 sulfonic acid ester preparation, 162 synthesis of resins, 155–156, 161–162 TNBS, see Trinitrobenzenesulfonic acid p-Toluenesulfonyl chloride/ p-nitrobenzylpyridine test, alcohol assay in solid-phase synthesis, 28–29 Triazene anchors acidic cleavage, 132–133 applications in solid-phase synthesis, 129–130

569

subject index heterocycle derivatization, 443–444, 461–462 heterocycle library synthesis with T1 linkers, 136–140, 147–148 multifunctional cleavage, 135–136, 145 T2 linkers guanidine library synthesis, 143–145, 149–150 peptide library synthesis, 142–143 synthesis, 140–142, 148–149 urea library synthesis, 143, 149 traceless cleavage, 132–134, 148 types, 130–132 1,3,5-Trichlorotriazine-(fluorescein, alizarin R, or fuchsin) test, alcohol assay in solid-phase synthesis, 29–30 Trinitrobenzenesulfonic acid, amine assay in solid-phase synthesis, 26

U Ugi multicomponent condensation reactions, see Isocyanide-based multicomponent reactions Urea library isocyanide-based multicomponent reactions, 479–480 synthesis using SynPhase‘ Crowns and Lanterns, 71–72 synthesis using T2 triazene linkers, 143, 149

W Wittig reaction, carbon transfer reagents and olefin synthesis, 354–355

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