Peptide Chemistry and Drug Design E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 621

Veröffentlichungsjahr: 2015

Beschreibung

This book focuses on peptides as drugs, a growing area of pharmaceutical research and development. It helps readers solve problems of discovering, developing, producing, and delivering peptide-based drugs.

• Identifies promising new areas in peptide drug discovery
• Includes chapters on discovery from natural sources, metabolic modification, and drug delivery
• Overviews separation methods and techniques for analysis, bond formation, and purification
• Offers readers both a professional reference and a text or resource for graduate-level students

Leseprobe

Table of Contents

Cover

Title Page

Copyright

Preface

List of Contributors

Chapter 1: Peptide Therapeutics

1.1 History of Peptides as Drugs

1.2 Factors Limiting The Use of Peptides in The Clinic

1.3 Advances that have Stimulated The Use of Peptides as Drugs

1.4 Development of Peptide Libraries

1.5 Modification of Peptides to Promote Stability and Cell Entry

1.6 Targeting Peptides to Specific Cells

1.7 Formulations to Improve Properties

References

Chapter 2: Methods for The Peptide Synthesis and Analysis

2.1 Introduction

2.2 Solid Supports

2.3 Linkers

2.4 Protecting Groups

2.5 Methods for Peptide Bond Formation

2.6 Solid-Phase Stepwise Synthesis

2.7 Synthesis in Solution

2.8 Hybrid Synthesis–Combination of Solid and Solution Synthesis

2.9 Cyclic Peptides

2.10 Depsipeptides

2.11 Separation and Purification of Peptides

2.12 Characterization of Peptides Through Mass Spectrometry

2.13 Conclusions

Acknowledgments

Abbreviations

References

Chapter 3: Peptide Design Strategies for G-Protein Coupled Receptors (GPCR)

3.1 Introduction

3.2 Classification of GPCR

3.3 Catalog of Peptide-Activated G-Protein Coupled Receptors

3.4 Structure of GPCR: Common Features

3.5 GPCR Activation

3.8 Conclusions

Acknowledgments

References

Chapter 4: Peptide-Based Inhibitors of Enzymes

4.1 Introduction

4.2 Angiotensin-Converting Enzyme and Neprilysin/Neutral Endopeptidase

4.3 Peptide Inhibitors of The HIV-1 Viral Life Cycle

4.4 Matrix Metalloproteinases

4.5 Antrax Lethal Factor Inhibition by Defensins

4.6 Kinases

4.7 Glycosyltransferases (Oligosaccharyltransferases)

4.8 Telomerase Inhibitors

4.9 Tyrosinase

4.10 Peptidyl-Prolyl Isomerase

4.11 Histone Modifying Enzymes

4.12 Putting it all Together: Peptide Inhibitor Applications in Skin Care

4.13 Strategies for The Discovery of Novel Peptide Inhibitors

Acknowledgments

References

Chapter 5: Discovery of Peptide Drugs as Enzyme Inhibitors and Activators

5.1 Introduction

5.2 Enzyme Types That Process Peptides

5.3 Amino Acid Drugs

5.4 Serine Proteases and Blood Clotting

5.5 Diabetes Mellitus

5.6 Renin–Angiotensin–Aldosterone System

5.7 Penicillin and Cephalosporin Antibiotics

5.8 HIV Protease

5.9 Peptide Drugs Under Development

5.10 Discussion

Acknowledgments

References

Chapter 6: Discovery of Peptide Drugs From Natural Sources

6.1 Introduction

6.2 Peptides are Involved in the Host Defense Mechanism of Living Organisms

6.3 Animal Venoms, a Rich Source of Peptides With Therapeutic Potential

6.4 Optimization of Peptides for Drug Development

6.5 Conclusions

Acknowledgments

References

Chapter 7: Modification of Peptides to Limit Metabolism

7.1 Introduction

7.2 Introduction of Unnatural Amino Acids

7.3 Cyclization of Linear Peptides to Improve Stability Toward Blood and Brain Protease Degradation

7.4 Introduction of D-Amino Acids Into Peptides Improves Stability Toward Blood and Brain Protease Degradation

7.5 Introduction of β-Amino Acids Increases the Stability Toward Blood and Brain Protease Degradation

7.6 Introduction of Peptide Bond Isosteres

7.7 Introduction of a N-Methylation of the Amide Bond of Peptides Can Improve the Stability Toward Blood and Brain Protease Degradation

7.8 Use of Unnatural Amino Acids – Use of Topographically Constrained Amino Acid

7.9 Using Glycosylated Amino Acids to Increase the Resistance of the Proteolytic Degradation

7.10 Creation of Peptides as Multiple Antigen Peptide (MAP) Dendrimeric Forms Increases the Stability Toward Blood and Brain Protease Degradation

7.11 Halogenations of Aromatic Residues in Peptides Can Reduce the Enzymatic Recognition Required for Peptide Hydrolysis

7.12 Concluding Discussion

References

Chapter 8: Delivery of Peptide Drugs

8.1 Introduction

8.2 LIpinski'S Rule of Five

8.3 Approaches to Delivering Peptide Drugs

8.4 Parenteral Peptide Drugs

8.5 Topical Peptide Drugs for Local Effects

8.6 Intranasal Peptide Drug Delivery

8.7 Enteral Peptide Drugs

8.8 Different Routes of Administration for Insulin

8.9 Discussion

Acknowledgments

References

Index

End User License Agreement

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Guide

Cover

Table of Contents

Preface

Chapter 1: Peptide Therapeutics

List of Illustrations

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

Figure 2.8

Figure 2.9

Figure 2.10

Figure 2.11

Figure 2.12

Figure 2.13

Figure 2.14

Figure 2.15

Figure 2.16

Figure 2.17

Figure 2.18

Figure 2.19

Figure 2.20

Figure 2.21

Figure 2.22

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

Figure 4.8

Figure 4.9

Figure 4.10

Figure 4.11

Figure 4.12

Figure 4.13

Figure 4.14

Figure 4.15

Figure 4.16

Figure 4.18

Figure 4.19

Figure 4.20

Figure 4.21

Figure 4.22

Figure 5.1

Figure 5.2

Figure 5.3

Figure 5.4

Figure 5.5

Figure 5.6

Figure 5.7

Figure 5.8

Figure 5.9

Figure 5.10

Figure 6.1

Figure 7.1

Figure 7.2

Figure 7.3

Figure 7.4

Figure 7.5

Figure 7.6

Figure 7.7

Figure 7.8

Figure 7.9

Figure 7.10

Figure 7.11

Figure 7.12

Figure 7.13

Figure 7.14

Figure 8.1

Figure 8.2

Figure 8.3

Figure 8.4

Figure 8.5

Figure 8.6

List of Tables

Table 2.1

Table 3.1

Table 3.2

Table 3.3

Table 3.4

Table 4.1

Table 4.2

Table 3

Table 6.1

Table 6.2

Table 6.3

Table 6.4

Table 7.1

Table 7.2

Table 7.3

Table 7.4

Table 7.5

Table 7.6

Peptide Chemistry and Drug Design

Edited by

Ben M. Dunn

Copyright © 2015 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New Jersey

Published simultaneously in Canada

No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, [phone], fax [phone], or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, [phone], fax [phone], or online at http://www.wiley.com/go/permissions.

Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

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Library of Congress Cataloging-in-Publication Data:

Peptide Chemistry and Drug Design / edited by Ben M. Dunn.

p. ; cm.

Includes bibliographical references and index.

Summary: “This book details many of the problems and successes of peptides as potential drugs”–Provided by publisher.

ISBN 978-0-470-31761-7 (hardback)

I. Dunn, Ben M., editor.

[DNLM: 1. Peptides–chemistry. 2. Drug Design. 3. Pharmaceutical Preparations. QU 68]

RM301.25

615.1′9–dc23

2014040280

Preface

This book is result of many conversations with peptide scientists at a variety of meetings, including American Peptide Society Symposia, meetings of the European Peptide Society, the Japanese Peptide Society, and the Australian Peptide Society. Some of these conversations were with the authors of the chapters in this book. One additional influence was a meeting in Dubai, where I had an excellent dinner with Waleed Danho, then with Roche Nutley. Waleed had given an excellent talk about the value of peptide chemistry and peptides as elements in the drug-discovery process. Over a delicious dinner of baked fish and many other courses, we discussed the history of drug discovery and the role that peptides have played in the past. Waleed made the strong point that peptides still have great value in the discovery process and, with appropriate methods to deal with delivery and metabolism issues, can provide excellent drugs for the future.

At around this time, I was contacted by Jonathan Rose of John Wiley & Sons who asked if I would be interested in editing a book on peptides and drug discovery. Sometimes life provides a nice juxtaposition of ideas and I immediately accepted the invitation. Over the following years, I spoke with many scientists, emailed some more, and worked on putting together the chapters for this book. I want to thank Jonathan as well as Kari Capone of John Wiley for their patience and advice over the years it took to bring this together.

The book starts with a chapter provided by Nader Fatouhi, discussing the current state of peptides in drug discovery. I heard Nader speak at the 23rd American Peptide Symposium in the Kona region of the Big Island of Hawaii. As I felt that his presentation provided an update on the thoughts first revealed to me by Waleed Danho, I asked Nader to contribute the opening chapter of the book, as this sets the stage for what follows. In his chapter, Nader discusses the rising importance of peptides as molecules for drug development as well as the issues facing scientists in this field, including cell penetration, stability, and targeting. Tools and techniques are available to address each of these limitations at this time.

Chapter 2 was contributed by Fernando Albericio and colleagues. This presents modern methods of peptide synthesis in a very readable format. Included are sections on solid supports for solid-phase peptide synthesis, which dominates most research level approaches, linkers, protecting groups, methods for peptide-bond formation, and a variety of methods to modify peptides to limit metabolism. In all cases the latest reagents and techniques are featured, thus making this chapter a great starting point for scientists starting out in the peptide field. The authors go on to discuss synthesis of peptides in solution, which still has great value in certain applications, including production of peptides in bulk. In addition, the combination of both solution- and solid-phase methods is discussed for cases where fragment condensation is used to prepare ever larger peptides. This discussion includes native chemical ligation, which permits selectively linking N-termini and C-termini of fragments, and which has several variations with more coming each year. The chapter concludes with a very valuable discussion of separation methods and methods for the analysis of the products of peptide synthesis. Again, this chapter is recommended as a great starting place for new scientists.

Anamika Singh and Carrie Haskell-Luevano have provided Chapter 3 that discusses the important topic of membrane receptors as targets for drug discovery. Due to the vital role of membrane receptors in cell signaling and control of metabolic events, a significant percentage of drugs in current use exert their function by interfering or stimulating binding and signaling events at membrane receptors, also known as G-protein coupled receptors (GPRCs). This chapter provides a catalog of systems where peptides are known to be involved and where it has been shown that synthetic peptides can modulate function. The Haskell-Luevano lab has provided outstanding research on the melanocortin receptors, but this chapter takes a broader approach and discusses a wide variety of these systems, including structural information as known and as modeled by other labs. Anyone involved in aspects of membrane signaling will find this chapter a highly valuable resource for methods, approaches, and strategies for attacking this important area of biology.

Gregg Fields and colleagues present Chapter 4 to introduce the use of peptides as inhibitors of enzymes. In the first part, the authors introduce enzymes and their classification and present several classical examples of the use of peptides to come up with compounds that provide the desired change in enzyme function to overcome a metabolic defect. In a second section, the area of HIV-1 infection and progression to AIDS is described, with emphasis on the value of peptides as modulators of growth and infection. As the human immunodeficiency virus goes through a complicated life cycle, the authors point out that there are multiple targets for approaching therapy and a combination strategy, known as HAART (highly active antiretroviral therapy) has provided the optimal approach to treatment of affected individuals. The Fields lab has made major contributions to discoveries in the area of matrix metalloproteinases and this chapter presents a thorough discussion of this system. The enzymes in this family provide a great example of the development of inhibitors through a process of discovery of aspects of structure and function that can guide the process. The chapter continues with nice discussions of several other systems where peptide chemistry has been key in new discoveries that have driven the drug-development process.

Jeffrey-Tri Nguyen and Yoshiaki Kiso have provided Chapter 5, which continues the discussion of enzyme inhibitors from the aspect of peptides. The highly productive Kiso lab has led the way in creating a very large catalog of peptide derivatives for use in drug discovery in several systems. They begin this chapter by discussing the advantages and disadvantages of peptides as potential drugs and come down on the side of the beneficial role that peptides play. In particular, they make the important point that the use of peptides can frequently define the pharmacophore, or structural model, which can then be transformed into a small molecule of non-peptide nature for further development as a potential drug. This chapter further focuses on the process of the design of potential inhibitors and reviews the history of discovery from natural sources as well as through ab initio design. They discuss the advantages of learning from the natural substrates of an enzyme and introduce the important concept of the transition state analog; the critical role that structural information on the target protein can provide. This chapter provides an excellent discussion of systems where targeting with peptide molecules may provide opportunities for further drug discovery.

Sónia T. Henriques and David J. Craik describe many peptide inhibitors from natural sources in Chapter 6. The introduction to their chapter discusses the value of finding compounds from nature and describes a number of sources, including the antimicrobial peptides from many bacteria. In both bacterial and plant worlds, there is a continual war between competing systems, and this has led to the development through evolution of many natural peptides that serve as defensive molecules. The authors discuss the cyclotides, peptides that are connected end to end and that have multiple disulfide bonds. This arrangement is very stable and the molecules are found in venoms of several species as well as in plants. After this introduction, the authors turn to a discussion of the drug discovery process from their perspective. The chapter continues with an in depth discussion of a variety of systems where many methods are used to modify molecules isolated from nature and where the activity against many targets is tested. The wide diversity of structures and targets is featured in this chapter and the many discoveries have pushed research and drug discovery forward significantly.

Isuru R. Kumarasinghe and Victor J. Hruby have taken on the task of describing methods to limit the metabolism of peptide molecules in humans. This leads to a very detailed discussion of the chemistry of peptide modification. As Victor Hruby is the world leader in this aspect of peptides, the chapter is thoroughly exciting and interesting. A main concern is the digestion of peptides by proteolytic enzymes present in both the digestive tract and the circulation. The first step is to define the pharmacophore residues of a naturally occurring and effective peptide. This will show the absolutely critical functional groups and their stereochemical relationships that must be maintained. Then replacement of some nonessential amino acids by non-natural amino acids, with the d-amino acid isomer, or with peptide-bond isosteres may be sufficient to block degradation by proteases. In addition, cyclization can sometimes provide more stability and also enhance passage of peptides through the blood–brain-barrier. Other strategies include replacement of specific the amino acids with the N-methyl derivatives, with topographically constrained derivatives, or with the halogenated derivatives of aromatic amino acids. Finally, the use of the “multiple-antigenic-peptide” approach where many molecules are attached to a carrier with multiple attachment points can produce molecules that, due to their size, are not recognized by proteases. This chapter emphasizes the role of creative synthetic chemistry is the modification of peptides to achieve stability and bioavailability.

The book concludes with Chapter 8, provided by Jeffrey-Tri Nguyen Yoshiaki Kiso, that discusses the important area of peptide delivery. While progress in the past 50 years has permitted peptide chemists to make almost any sequence of amino acids that is desired in high yield and purity, getting those molecules into humans and into the specific area in the body where they can exert a therapeutic effect is a problem that has not progressed as rapidly. Thus, this chapter is very important for future advances in drug discovery based on peptides. Many of the readers may already be familiar with the Lipinski's Rule of Five that includes recommendations for the size of a molecule, the number of hydrogen bonding atoms, and the lipophilicity. These rules are discussed in this chapter, but much more information is provided regarding solubility, membrane transport, and metabolic stability.

In conclusion, this book provides a primer for anyone in the field of drug discovery and specifically in the area of the use of peptides as molecules for both the discovery phase and, in favorable cases, the final phase of the creation of new molecular entities that can be moved into further studies to evaluate their potential as therapeutic drugs. I want to thank the authors of the chapters for their friendship, for many discussions, and for their excellent writing for this book.

Ben M. Dunn, Ph.D.

September 3, 2014

List of Contributors

Fernando Albericio

, Institute for Research in Biomedicine, Barcelona, Spain; CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Barcelona, Spain; Department of Organic Chemistry, University of Barcelona, Barcelona, Spain; School of Chemistry, University of KwaZulu-Natal, Durban, South Africa; School of Chemistry, Yachay Tech, Yachay City of Knowledge, Urcuqui, Ecuador

Sabrina Amar

, Departments of Chemistry and Biology, Port St. Lucie, FL USA

David J. Craik

, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia

Ayman El-Faham

, Department of Chemistry, Alexandria University, Alexandria, Egypt; Department of Chemistry, King Saud University, Riyadh, Kingdom of Saudi Arabia

Gregg B. Fields

, Departments of Chemistry and Biology, Port St. Lucie, FL USA; Departments of Biochemistry, University of Texas Health Science Center, San Antonio TX, USA

Nader Fotouhi

, Global Alliance for TB Drug Development, Research and Development, New York, NY, USA

Athanassios S. Galanis

, Department of Pharmacy, University of Patras, Patras, Greece

Carrie Haskell-Luevano

, Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, USA

Sónia T. Henriques

, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia

Victor J. Hruby

, Department of Chemistry and Biochemistry, The University of Arizona, Tucson, AZ, USA

Yoshiaki Kiso

, Department of Molecular Pharmacy, Kobe Gakuin University, Kobe, Japan

Anna Knapinska

, Departments of Chemistry and Biology, Port St. Lucie, FL USA

Isuru R. Kumarasinghe

, Department of Chemistry and Biochemistry, The University of Arizona, Tucson, AZ, USA

Jeffrey-Tri Nguyen

, Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto, Japan

Eliandre de Oliveira

, Proteomics Platform Barcelona Science Park, Barcelona, Spain

Trista K. Robichaud

, Departments of Periodontics, University of Texas Health Science Center, San Antonio TX, USA; Departments of Biochemistry, University of Texas Health Science Center, San Antonio TX, USA

Anamika Singh

, Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, USA

Judit Tulla-Puche

, Institute for Research in Biomedicine, Barcelona, Spain; CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Barcelona, Spain

Aikaterini A. Zompra

, Department of Pharmacy, University of Patras, Patras, Greece

1Peptide Therapeutics

Nader Fotouhi

Global Alliance for TB Drug Development, Research and Development, New York, NY, USA

1.1 History of Peptides as Drugs

The advent of molecular biology and our understanding of the physiological and pathological functions of peptides, coupled with advances in synthetic methodologies and peptidomimetics, marked the beginning of a new era in peptide and protein therapeutics, with the vision that there should be no limit to what can be produced as therapeutics. During that period a number of great peptide drugs such as Sandostatin, Lupron, Copaxone, and Zoladex were developed with great therapeutic benefit. The number of approved peptide drugs, however, remains low.

It was not until the last decade that we have seen a significant surge in the number of peptide therapeutics on the market (Figure 1.1). While 10 peptides were approved between 2001 and 2010, the current decade has thus far witnessed the approval of six new peptide therapeutics – a remarkable yearly increase [1, 2]. The number of peptides in development is also steadily growing roughly doubling every decade (Figures 1.2 and 1.3), and there are 400–600 peptides in preclinical studies. This is due to the advances made in our understanding of peptide stability, peptide synthesis, and formulation over the last three decades. Although the market share of peptide drugs is still relatively small (about 2% of the global market for all drugs), the approval rate for peptide drugs is twice as fast as the rate for small molecules, and the market is growing similarly at a rate that is twice the global drug market [3, 4]. While encouraging, the potential for peptide therapeutics is far greater than what it is today.

Figure 1.1 Peptide therapeutics marketed since 2002.

Figure 1.2 Peptides in development over the last three decades.

Figure 1.3 Peptides in clinical trials in 2013.

1.2 Factors Limiting The Use of Peptides in The Clinic

A number of factors have thus far limited the explosion that needs to happen in the peptide field. With the exception of a few peptides, the approved drugs so far target the extracellular compartment, and thus have to compete with biologics. Of the extracellular targets, GPCRs represent the major class, and in most cases, the peptides are agonist. GLP-1 represents one-third of these GPCR targets. We have seen a great advance in extending the circulating half-life of the peptides through the use of unnatural amino acids and formulation technologies, but have not yet reached the half-life achieved by antibodies. The delivery of peptides is still in the great majority of cases limited to i.v. (intravenous), s.c. (subcutaneous), or intranasal. Finally, safety is still a concern as better tissue selectivity is required.

To dramatically heighten their impact, peptides need to access the intracellular space to target protein–protein interactions. These interactions represent a vast source of potential targets with significant biological impact (there are estimated 300,000 such interactions in the cell), and will not in the majority of cases be modulated by small molecules. Peptides and biologics, given their relative size and ability to bind to extended surface areas, are the perfect candidates to inhibit protein–protein interactions. The duration of action of peptides needs to be extended, and while peptides are inherently selective against their targets, they need to more selectively distribute to the desired tissue. Finally, the route of administration needs to be expanded to include oral delivery.

1.3 Advances that have Stimulated The Use of Peptides as Drugs

The many great technological advances that started over a decade ago in drug delivery, peptide design, and synthesis are now maturing, and will undoubtedly address these key challenges and revolutionize the field over the next decades. Many of the technological advances are already proving that it is possible to make peptides permeable to cells, target tissues, have longer half-lives, and be orally bioavailable.

The discovery that certain peptides can penetrate cells and can, therefore, be an effective therapeutic on their own or alternatively bring other drugs into cells allowed for the first time to imagine targeting the intracellular compartment (Figures 1.4 and 1.5) [5]. HIV-enveloped protein tat was one of the first to be recognized for its cell-penetrating ability and, therefore, its potential use to carry bioactive cargo into the cell [6]. Since 2004, more than 200 peptides carried into cells by tat or other naturally occurring cell-penetrating peptides (CPPs) have been in various phases of development [7]. However, the more recent advances in the understanding of how these peptides cross the cell membrane through endocytosis and/or macropinocytosis [8] has allowed the generation of CPPs with intrinsic biological activity [9–12]. It is now possible to take a CPP sequence and synthetically modify it to introduce the key amino acids of an effector peptide into its sequence and create potent peptide antagonists of an intracellular protein–protein interaction with good pharmacokinetic properties [13].

Figure 1.4 HIV Tat.

Figure 1.5 Orally stable and bioavailable peptides (a) Cyclosporin. (b) Destruxin. (c) Kalata B.

1.4 Development of Peptide Libraries

By looking at the list of CPPs in development, one realizes that they are single cases and have to be synthetically prepared and modified to impart some of the desired stability to be a useful therapeutic. It is hard to compete with the screening of the millions of small molecule compounds in various pharmaceutical companies and more recently in many academic centers.

Until now, the available technologies to screen large libraries of peptides of significant length (possessing secondary structure) would only allow us to generate large libraries of natural amino acid sequences through phage display, and if unnatural amino acids were to be introduced, it had to be done with conventional synthetic methodology, and thus be limited to very low numbers of peptides that can be prepared and screened.

Indeed, over the last decade, there has been an explosion of very elegant technologies that now allow the generation of large to extremely large libraries of linear and macrocyclic peptides with unnatural amino acids and unnatural linkers. For the first time, it is possible to engineer stability, cell permeability, and possibly oral bioavailability at once and screen for the desired properties very rapidly. These major advancements have resulted in the generation of a number of companies that are pushing the limits of these technologies to rapidly screen and identify novel peptide therapeutics against protein–protein interaction targets (Figure 1.5).

Ensemble therapeutics utilizing their DNA-programmed chemistry can generate million-member libraries of small macrocycles with MW of 500–1500. On screening these libraries, they have identified potent and orally bioavailable small molecule inhibitors of IL17 [14]. Through medicinal chemistry optimization, they have now identified picomolar inhibitors with good properties [15]. PeptiDream utilizing Professor Suga's mRNA display technology [16] are generating up to trillion-member libraries of larger macrocycles mimicking cyclosporin. These peptides contain a combination of natural, unnatural, and N-methyl amino acids and exhibit good physicochemical properties and membrane permeability [17]. Ra Pharmaceuticals also uses a mRNA display technology developed by Jack Shoztac to generate very large libraries of macrocycles containing unnatural amino acids. They recently presented on their discovery of potent antagonists of mcl-1 and Ras with good cell permeability [18].

1.5 Modification of Peptides to Promote Stability and Cell Entry

The recent focus on another class of macrocycles, containing multiple disulfides, has generated a lot of excitement in maintaining the stability and membrane permeability of the cyclotide kalata B1, or the knottins (the uncyclized version of cyclotides), in order to create potent peptide drugs. David Craik and colleagues at Cyclotide are systematically exchanging the various loops present on cyclotides with sequences that have important biological function [19]. Recently, the introduction of a myelin oligodendrocyte glycoprotein sequence into a cyclotide resulted in a potent peptide in preventing disease progression in a mouse model of MS [20]. Protagonist is taking advantage of the oral stability of the disulfide-rich peptides for local gut delivery of IL6R antagonists for the treatment of irritable bowel disease (IBD). Moreover, novel technologies developed for the rapid generation and screening of extremely large libraries of knottins and cyclotides will undoubtedly have a major impact on this class of peptide therapeutics. Of note is the Intein-based technology from Julio Camarero capable of introducing unnatural amino acids to facilitate screening [21]. Sutro and MitiBio also have very sophisticated and efficient biosynthetic methods to generate very large libraries.

Finally, Verdine and Wollensky and colleagues [22, 23] as well as the investigators at Aileron Therapeutics have developed a novel stapling technology that imparts stability and membrane permeability to alpha helical structure. Using this technology, Aileron Therapeutics were able to discover very potent dual MDM2/MDMx antagonists with low nanomolar activity in cells and excellent pharmacokinetic properties, resulting in excellent antitumor activity in a mouse xenograft model [24]. Even more interesting is the extended efficacy ATSP-7041 exhibits in cells. While the small molecule MDM2 antagonist showed activity over 24 h, ATSP-7041 was still active beyond 48 hours in the same experiment. This is due to the fact that once the peptide enters the cell, the major elimination pathway is through enzymatic catabolism. Not only can stability be tuned for circulating half-life, it can also be tuned to withstand cellular catabolism to lengthen the desired efficacy. This could offer a significant advantage over (small) molecules that passively diffuse through the cell membrane. Additionally, using the same technology, a GHRH antagonist with much extended half-life was discovered and is currently in Phase I clinical trial [25].

1.6 Targeting Peptides to Specific Cells

One of the greatest challenges in drug discovery is the safety of therapeutics. Main reasons for diminished safety are selectivity against the target and tissue/cell specificity. If one could direct a therapeutic to only the site of pathology, then the therapeutic window of the agent increases and correspondingly decreases the side effects. Peptides, due to their specificity against receptors, are perfect candidates to be able to home into one type of cell/tissue versus another. There has been a tremendous amount of progress in identifying homing peptides (cell-penetrating as well as nonpenetrating) that can then be conjugated to a cargo to deliver it to a specific organ [26].

In vivo phage display by Pasqualini and colleagues marked the discovery of the first homing peptide that was able to selectively target the blood vessel of brain and kidney [27]. Since then a number of peptides have been identified that target many other tissues [28]. Arap and colleagues were then the first to perform phage display in humans and discovered a homing peptide to IL11Ra that expresses over 100-fold more on prostate cancer cells versus normal cells [29, 30]. Arrowhead Research is currently in Phase I proof of targeting with a peptide drug conjugate utilizing this homing peptide. Recently, Wen et al., at the Dana Farber, published their first Phase I study result on GRN1005, a peptide drug conjugate that targets the low-density lipoprotein-related protein-1, which mediates blood brain barrier transcytosis. GRN1005 successfully crosses the BBB and delivers its cargo [31].

1.7 Formulations to Improve Properties

While the above advances have and will have significant impact, the ability to administer peptides by the oral route will truly allow them to compete with small molecules and biologics as first line therapies. The majority of advances in this area have been the result of very interesting formulation strategies. A number of companies, including ArisGen, Axcess, Chiasma, Emisphere Tech., Enteris Pharmaceuticals, Lipocine, and Merlion Pharmaceuticals, have had successes in enhancing the oral bioavailability of some peptide therapeutics. They employ a combination of stabilizers, absorption enhancers, and carriers to achieve this. The main mode of absorption is through the paracellular space. However, the bioavailability of the peptides formulated remains relatively low.

While significant, cyclosporin remains the only marketed peptide drug that is administered orally and absorbed into the systemic environment. Learning from nature and systematic studies on macrocyclic peptides will have a tremendous impact in discovering peptide drugs with inherent oral bioavailability that could then be enhanced through formulation to achieve bioavailabilities, which would compete with small molecules. As mentioned earlier, PeptiDream and Ra Pharmaceuticals are generating large libraries of macrocyclic peptides mimicking the core structure of cyclosporin. Ensemble therapeutics are generating small macrocylic structures with molecular weights between 500 and 1500 and have already identified an orally bioavailable IL17 R antagonist. Professors Horst Kessler and Locky are doing the first systematic studies on small cyclic peptides to understand the effect of hydrogen bonding and structure on bioavailability [32, 33]. Their work will undoubtedly form the basis of rational designs of orally active peptide drugs.

In conclusion, the great technological advances over the last two decades are well poised to have a major impact on revolutionizing the field of peptide therapeutics. For the first time, tools are available to create stable, cell permeable, long lasting, and orally bioavailable peptides, allowing them to compete with small molecule drugs and biologics, and thus become first line therapies for many diseases with unmet medical needs.

References

1. Reichert J. (2010). Development Trends for Peptide Therapeutics (2010 Report Summary) 2010

Peptide Therapeutics Foundation

: 1–10.

2. Caspar A, Reichert J. Future directions for peptide therapeutics development.

Drug Discov Today

2013;

18

:807–817.

3. Lax R. The future of peptide development in the pharmaceutical industry.

PharManufacturing Int Pept Rev

2010:10–15.

4. Bellmann-Sickert K, Beck-Sickinger AG. Peptide drugs to target G protein-coupled receptors.

Trends Pharmacol Sci

2010;

31

:434–441.

5. Heitz F, Morris MC, Davita G. Twenty years of cell penetrating peptides: from molecular mechanism to therapeutics.

Br J Pharmacol

2009;

157

:195–206.

6. Franklin AD, Pabo CO. Cellular uptake of the tat protein from the human immunodeficiency virus.

Cell

1988;

55

:1189–1193.

7. Langel U et al. Therapeutic potential of cell-penetrating peptides.

Ther Deliv

2013;

4

:573–591.

8. Duchardt F, Fotin-Mleczek M, Schwarz H, Fischer R, Brock R. A comprehensive model for the cellular uptake of cationic cell-penetrating peptides.

Traffic

2007;

8

:848–866.

9. Verdurmen WPR, Brock R. Biological responses towards cationic peptides as drug carriers.

Cell

2011;

32

:116–127.

10. Low W. Functional cell permeable motifs within medically relevant proteins.

J Biotechnol

2007;

129

:555–564.

11. Jones S, Holm T, Mager I, Langel U, Howl J. Characterization of bioactive cell penetrating peptides from human cytochrome c: protein mimicry and the development of a novel apoptogenic agent.

Chem Biol

2010;

17

:735–744.

12. Milletti F. The Design of Cell-penetrating Peptides with Intrinsic Biological Activity, TIDES: Oligonuleotides and Peptide Therapeutics, from Research through commercialization. Boston, MA, USA 2013.

13. Fotouhi, N. The Evolution of Peptide Therapeutics. The american peptide symposium. 2013.

14. Terret, N. (2013). First in class, orally active small molecule antagonist of IL17. CHI Drug Discovery Chemistry Conference: Constrained Peptides and Macrocylics Drug Discovery Session.

15. Terret N. Producing orally bioavailable biologics.

Drug Discov Dev

2013.

16. Passioura T, Suga H. Flexizymes-mediated genetic reprogramming as a tool for noncanonical peptide synthesis and drug discovery.

Chem-Eur J

2013;

19

:6530–6536.

17. Funamoto S, Sasaki T, Ishihara S, Nobuhara M, Nakano M, Watanabe-Takahashi M, Saito T, Kakuda N, Miyasaka T, Nishikawa K, Saido TC, Ihara Y. Substrate ectodomain is critical for substrate preference and inhibition of g-secretase.

Nat Commun

2013;

4

:3529–3541.

18. Treco D et al. Discovery of high affinity cyclic peptidomimetics binding Mcl-1 and Ras.

Mol Cancer Ther

2013;

12

:C212.

19. Craik D, Swedberg JE, Mylne JS, Cemazar M. Cyclotides as a basis for drug design.

Expert Opin Drug Discov

2012;

7

:179–194.

20. Wang CK, Gruber CW, Cemazar M, Siatskas C, Tagore P, Payne N, Sun GZ, Wang SH, Bernard CC, Craik DJ. Molecular grafting onto a stable framework yields novel cyclic peptides for the treatment of multiple sclerosis.

ACS Chem Biol

2014;

9

:156–163.

21. Jagadish K, Borra R, Lace V, Majumder S, Shekhtman A, Wang L, Camarero JAV. Expression of fluorescent cyclotides using protein trans-splicing for easy monitoring of cyclotides-protein interactions.

Angew Chem

2013;

125

:3208–3213.

22. Walensky LD, Kung AL, Escher I, Malia TJ, Barbuto S, Wright RD, Wagner G, Verdine GL, Korsmeyer SJ. Activation of apoptosis in vivo by a hydrocarbon-stapled BH3 helix.

Science

2004;

305

:1466–1470.

23. Bernal F, Tyler AF, Korsmeyer SJ, Walensky LD, Verdine GL. Reactivation of the p53 Tumor Suppressor Pathway by a Stapled p53 Peptide.

J Am Chem Soc

2007;

129

:2456–2457.

24. Chang YS, Graves B, Guerlavais V, Tovar C, Packman K, To KH, Olson KA, Kesavan K, Gangurde P, Mukherjee A, Baker T, Darlak K, Elkin C, Filipovic Z, Qureshi FZ, Cai HL, Berry P, Feyfant E, Shi XGE, Horstick J, Annis DA, Manning AM, Fotouhi N, Nash H, Vassilev LT, Sawyer TK. Stapled α-helical peptide drug development: A potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy.

Proc Natl Acad Sci U S A

2013;

110

:1–10.

25. Aileron Therapeutics concludes first-in-human study of ALRN-5281 Stapled Peptide drug. Aileron Therapeutics Company announcement May 2013.

26. Laakonen P. Homing peptides as targeted delivery vehicles.

Integr Biol

2010;

2

:326–337.

27. Pasqualini R, Ruoslahti E. Organ targeting in vivo using phage display peptide libraries.

Nature

1996;

380

:364–366.

28. Arap W, Haedicke W, Bernasconi M, Kain R, Rajotte D, Krajewski S, Ellerby HM, Bredesen DE, Pasqualini R, Ruoslahti E. Targeting the prostate for destruction through a vascular address.

Proc Natl Acad Sci U S A

2012;

99

:1527–1531.

29. Arap W, Kolonin MG, Trepel M, Lahdenranta J, Cardo-Vila M, Giordano RJ, Mintz PJ, Ardelt PU, Yao VJ, Vidal CI, Chen L, Flamm A, Valtanen H, Weavind LM, Hicks ME, Pollock RE, Botz GH, Bucana CD, Koivunen E, Cahill D, Troncoso P, Baggerly KA, Pentz RD, Do KA, Logothetis CJ, Pasqualini R. Steps toward mapping the human vasculature by phage display.

Nat Med

2002;

8

:121–127.

30. Zurita AJ, Troncoso P, Cardo-Vila M, Logothetis CJ, Pasqualini R, Arap W. Combinatorial screenings in patients: the interleukin-11 receptor α as a candidate target in the progression of human prostate cancer.

Cancer Res

2004;

64

:435–439.

31. Drappatz J, Brenner A, Wong ET, Eichler A, Schiff D, Groves MD, Mikkelsen T, Rosenfeld S, Sarantopoulos J, Meyers CA, Fielding RM, Elian K, Wang X, Lawrence B, Shing M, Stephen Kelsey S, Castaigne JP, Yen PY. Phase I study of GRN1005 in recurrent malignant glioma.

Clin Cancer Res

2013;

19

:1567–1576.

32. Beck JG, Chatterjee J, Laufer B, Kiran MU, Frank AO, Neubauer S, Ovadia O, Greenberg S, Gilon C, Hoffman A, Kessler H. Intestinal permeability of cyclic peptides: common key backbone motifs identified.

J Am Chem Soc

2012;

134

:12125–12133.

33. White TR, Renzelman CM, Rand AC, Rezai T, McEwen CM, Gelev VM, Turner RA, Linington RG, Leung SSF, Kalgutkar AS, Bauman JN, Zhang YZ, Liras S, Price DA, Mathiowetz AM, Jacobson MP, Lokey RS. On-resin N-methylation of cyclic peptides for discovery of orally bioavailable scaffolds.

Nat Chem Biol

2011;

7

:810–817.

2Methods for The Peptide Synthesis and Analysis

Judit Tulla-Puche

Institute for Research in Biomedicine, Barcelona, Spain

CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Barcelona, Spain

Ayman El-Faham

Department of Chemistry, Alexandria University, Alexandria, Egypt

Department of Chemistry, King Saud University, Riyadh, Kingdom of Saudi Arabia

Athanassios S. Galanis

Department of Pharmacy, University of Patras, Patras, Greece

Eliandre de Oliveira

Proteomics Platform Barcelona Science Park, Barcelona, Spain

Aikaterini A. Zompra

Department of Pharmacy, University of Patras, Patras, Greece

Fernando Albericio

Institute for Research in Biomedicine, Barcelona, Spain

CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Barcelona, Spain

Department of Organic Chemistry, University of Barcelona, Barcelona, Spain

School of Chemistry, University of KwaZulu-Natal, Durban, South Africa

School of Chemistry, Yachay Tech, Yachay City of Knowledge, Urcuqui, Ecuador

2.1 Introduction

Peptides as drugs show unique characteristics (high biological activity, high specificity, and low toxicity) thereby making them particularly attractive therapeutic agents [1]. However, the role of peptides in drug discovery has suffered ups and downs during the last four decades. A first analysis of the new chemical entities (NCEs) accepted by the Food and Drug Administration (FDA) indicated that while 53 NCEs were introduced as drugs in 1996, only 17 were introduced in 2002. This number increased to 31 in 2004, but decreased again in 2005 with just 18 new drugs, 17 in 2007, and a slight increase to 21 in 2008 (Figure 2.1) [2, 3]. An analysis of these 21 drugs approved in 2008 indicated that almost 50% of the new drugs can be considered nonclassical, in the sense that they are nonclassical small molecules.

Figure 2.1 Distribution by chemical structure of the new drugs approved by the FDA in 2008.

Interestingly, peptides represent approximately 20% of the total number of drugs approved by the FDA in 2008 [3]. Thus, Romiplostim from Amgen, which is a thrombopoietin receptor agonist, is a fusion protein conjugated with a 41 amino acid peptide, containing two disulfide bridges. Degarelix from Ferring, which is a gonadotropin-releasing hormone receptor antagonist, is a 10 amino acid peptide. Alvimopan from Adolor, which is a peripherally acting μ-opioid receptor antagonist, is an N-terminal blocked dipeptide. Lacosamide from Schwarz, which selectively enhances slow inactivation of voltage-gated sodium channels and binds to collapsin response mediator protein 2, is a protected O-methylserine [3].

Even more important than the number of peptides accepted by the FDA is the number of peptides that are in clinical phases. In 2008, 39 were in clinical phase I, 77 in phase II, 39 in phase III, and 4 in preregistration [4].

There are several reasons for this renaissance of peptides. The first one is the fact that the number of classical small molecules is not increasing enormously. Furthermore, several comparisons with small molecules are favorable to peptides. Thus, the well-defined peptide chemistry allows an easier way to prepare analogs. Pharmaceutical companies have also detected a better manpower/milestone ratio. Peptides reach clinical phases more easily. In parallel, advances in the fields of formulation and drug delivery technology, and the fact that these technologies are accepted for the introduction of a peptide into the market for the first time, have fueled this field into the drug market. And last but not least, the great developments in peptide synthetic methods over the past few years have improved accessibility of a wider variety of peptides. This translates into the fact that in 2008 more than 90% of peptide production was by chemical synthesis. Another important supporting fact is that while in the 1980s most pharmaceutical peptides contained less than 10 amino acids, nowadays over 50% of peptides in clinical phase have more than 10 amino acids [4].

The purpose of this chapter is to review the latest advances in peptide chemistry that have boosted the peptide field. Even though, and from a synthetic viewpoint, peptides can be prepared in solid phase or in solution; nowadays, it is possible to say that in almost all peptide syntheses a solid-phase step is involved. Thus, the synthesis of small-to-medium-sized peptides is carried out in the solid phase, and the synthesis of large peptides and/or proteins is performed using a convergent approach. In this case, one of the last steps is carried out in solution, but the fragments either protected for a classical strategy or unprotected for a chemical ligation one are prepared in solid phase. Therefore, the solid-phase approach will be covered in detail.

2.2 Solid Supports

In solid-phase peptide synthesis (SPPS), the most important choice becomes the solid support, which needs to accomplish certain features: (i) stability to mechanic stirring, to a range of temperatures, and to different solvents and reagent conditions; (ii) high swelling, so that reagents can access the active sites; (iii) homogeneity: a narrow range of bead sizes, and (iv) biocompatibility: swelling in aqueous buffers if used in biochemical assays. Solid supports for SPPS can be classified into three types [5]: polystyrene (PS), polyethylene glycol–polystyrene (PEG–PS®), and hydrophilic PEG-based resins (Figure 2.2).*

PS resins

. PS is the most widely used solid support for the synthesis of peptides in solid phase because of its good swelling properties and good level of substitution [6, 7]. PS used nowadays contains 1% cross-linked hydrophobic resins obtained by suspension polymerization from styrene and divinylbenzene. PS swells well in nonpolar solvents such as toluene or CH

2

Cl

2

, but can be used in combination with other more polar solvents such as

N

,

N

-dimethylformamide (DMF), dioxane, and tetrahydrofuran. Although it is the polymer of choice for the synthesis of small-to-medium-sized peptides, also from an economic viewpoint, it does present certain limitations in some cases, such as in the synthesis of highly hydrophobic or in the aggregation of peptides. In case of

difficult sequences

, more hydrophilic supports and resins show better performance.

PEG-PS resins

. Due to its amphiphilic properties, which allow solvation in both polar and nonpolar solvents, the addition of PEG was investigated. Thus, based on the early work of Mutter [8], PEG-PS supports, which bear both a hydrophobic PS core and hydrophilic PEG chains, were developed independently by Zalipsky, Albericio, and Barany [9] (PEG-PS) and Bayer and Rapp [10] (Tentagel

®

). The benefits of these resins for the assembly of long peptides prompted the appearance of other supports, such as Champion

®

I and II (NovaGel

®

) [11] and ArgoGel

®

[12]. PEG-PS resins are compatible with both nonpolar and polar solvents.

Hydrophilic PEG-based resins

. Searching to enhance the beneficial swelling properties of PEG's, more hydrophilic PEG resins with a small amount of PS or polyamide (poly(ethylene glycol)-poly(acrylamide) copolymer, PEGA) [13] or acrylate with polymerizable vinyl groups (cross-linked ethoxylate acrylate resin, CLEAR) [14], were developed. While PEGA resin was obtained by inverse suspension radical polymerization of various sizes of linear bis- and branched-tris-2-aminopropyl-PEG samples with acryloyl chloride, CLEAR supports were obtained by copolymerization of branched PEG-containing cross-linkers such as trimethylolpropane ethoxylate triacrylate with amino-functionalized monomers such as allylamine or 2-aminoethylmethacrylate. In the search for more stable and hydrophilic resins, a step forward came with the PEG-based resins developed by Meldal [15], which focused on resins containing only ether bonds, such as polyoxyethylene cross-linked polyoxypropylene (POEPOP), developed from a polymerization of PEG that was partially derivatized with chloromethyloxirane [16]. Although the POEPOP resin is mechanically robust, shows relatively high loading (primary and secondary alcohols), and good performance for organic transformations, the presence of secondary ether bonds implies that this solid support is not totally stable to strong Lewis acids [17]. To overcome this problem, the poly-oxyethylene-poly(3-methylene-3-methyloxethane) copolymer (SPOCC) resin, in which all ether bonds and functional alcohol groups are primary, was developed [18, 19].

Figure 2.2 (a) PS supports; (b) PEG-PS supports; and (c) totally PEG based supports.

At the same time, Côté developed the ChemMatrix (CM) resin [20], a total PEG-based resin comprised of primary ether bonds. Because of its highly cross-linked matrix, CM has surpassed the mechanical stability of other PEG resins. This resin swells well in all of the most common solvents and is, therefore, useful for a broad range of organic chemistries. CM resin performs extremely well compared to PS resins in the solid-phase synthesis of hydrophobic, highly structured peptides such as poly-Arg peptide and β-amyloid (1-42) [21, 22], showing that the presence of PEG chains impairs the aggregation of the growing peptide chain, facilitating the solid-phase synthesis of complex peptides. Furthermore, CM is convenient for the synthesis of oligonucleotides and oligonucleotide peptide conjugates [23].

Compared to earlier PEG-containing resins, these supports are 100% formed by primary ether bonds and thus show improved chemical stability and can reach higher loadings, comparable to those of PS resins. In comparative studies among several resins on the synthesis of human stromal cell-derived factor (SDF)-1α [24] and the acyl carrier protein (ACP 65-74) [25], higher purities were obtained with CM than with PS supports when using similar loadings. Compatibility of all these PEG-based resins with aqueous buffers allows their use for biochemical applications such as on-resin screening of chemical libraries and the development of affinity chromatography [26–29].

2.3 Linkers

A linker is a bifunctional molecule that facilitates the attachment of the growing peptide as well as the final cleavage step. Linkers or handles can be classified into two types: integral and nonintegral [30]. In the first type, the solid support forms part of, or constitutes, the entire linker/handle, as is the case of, for example, 2-chlorotritylchloride resin (6). On the contrary, nonintegral linkers/handles are independent and bifunctional molecules that are attached to the solid support through an ether (e.g., Wang resin, 8) or more commonly, through an amide bond, and they are more recommended because they provide control and flexibility for the synthetic process [31]. Linkage to the solid support should be totally stable to all synthetic processes, including the final treatment that will detach the target compound from the solid support. Sometimes this bond is not totally stable and the carbocation-containing linker is detached from the solid support, causing further heterogeneity of the crude peptide or causing back-alkylation of the target compound [32, 33]. This is the case of linkers attached to p-methylbenzhydrylamine (MBHA) resin when using a tert-butyloxycarbonyl (Boc) (13)/benzyl (Bzl) (20) strategy for preparing peptide amides. To overcome this side reaction, the use of aminobenzyl PS or aminoalkyl resins, which form a more acid-stable bond, is recommended [33]. Similar problems arise with (poly)alkoxybenzyl [34] (Wang (8), backbone amide linker (BAL), Rink (11))-type resins. Incorporation of the p-hydroxybenzyl moiety cleaved from the Wang resin (8) into the N of the C-terminal amide of a peptide during trifluoroacetic acid (TFA) cleavage [35], alkylation of the indol ring of Trp-containing peptides by the p-hydroxybenzyl moiety [36], and formation of O-(4-hydroxy)benzyl derivatives [37], are some of the side reactions encountered. Use of the Wang resin (8) for the solid-phase preparation of small molecules has also led to the introduction of impurities due to the undesired cleavage from the resin (no cleavage at the Bzl position) or from a back-alkylation of the p-hydroxybenzyl cation in the case of furopyridine and furoquinoline target derivatives [38]. To overcome these problems, two resins have been developed based on the activation of the Bzl position by a MeO group, a noncleavable electron-donating group, in either ortho or para position. Thus, Gu and Silverman [39] incorporated the precursor of their backbone linker to the resin through a metal-catalyzed coupling reaction and Colombo et al. [34] the precursor of their Wang-type resin through an amide bond. Linkers generally used in SPPS are those labile to acid, (Figure 2.3) although base and photolabile handles are also used in certain applications. High acid-sensitive linkers (Figure 2.3a) can be used to release side-chain protected fragments that are later used to access cyclic peptides or larger peptides by convergent approaches. On the contrary, C-terminal amide peptides can be constructed using Sieber, which is cleavable with low concentration of TFA (3–5%), 5-(4-aminomethyl-3,4-dimethoxyphenoxy)valeric acid (PAL) (10), and rink linkers (11),† which release the amide function on TFA treatment.

Figure 2.3 (a) Low and (b) high acid-labile linkers and resins.

2.4 Protecting Groups

Temporary Nαprotection. Since in SPPS the peptides are built on the C to N direction, the temporary α-amino protecting group plays a very important role in the overall strategy. The Nα-protected amino acid should be a solid that is easy to handle, soluble in the solvents used in SPPS, to prevent or minimize epimerization during coupling, and the protecting group should allow a fast and clean removal [40]. Two main strategies dominate the synthesis of peptides in solid-phase. The first one relies on using Boc (13) [41–43] as a temporary protecting group for the Nα-amino function and Bzl-type protecting groups as permanent protecting groups for side chains. The main drawback of the Boc (13)/Bzl (20) strategy is the use of HF (hydrogen fluoride) for the final cleavage step, which hampers the application of this methodology to large-scale synthesis. The second strategy and the most employed nowadays uses the 9-fluorenylmethyloxcarbonyl (Fmoc) (14) [44] group as a temporary protecting group and t-butyl (tBu) (19)-type groups for side-chain protection. Fmoc (14)/tBu (19) strategy allows the use of the milder TFA for the final detachment of the peptide from the resin. Several other Nα-amino protecting groups have since appeared, such as the trityl group (Trt) (15), which is removed by very mild acidic treatment (1% TFA), p-nitrobenzyloxycarbonyl (pNZ) (16) [45], also removed by acid (6 M SnCl2, 1 mM HCl), the allyloxycarbonyl (Alloc) (17) group [46, 47], which can be removed under neutral conditions (PhSiH3 (10 equiv), Pd(PPh3)4 (0.1 equiv)), and the photolabile 6-nitroveratryloxycarbonyl (Nvoc) (18) [48], all of which are orthogonal to the Boc (13) and Fmoc (14) groups, and allow for the synthesis of cyclic and branched peptides (Figure 2.4). Trt (15), pNZ (16), and Alloc (17) groups have also found an important application in minimizing diketopiperazine (DKP) in sequences prone to the formation of this side product [49, 45, 50].

Figure 2.4Nα-amino protecting groups in peptide synthesis.

Permanent side-chain protection. These protecting groups need to be stable during the entire elongation of the peptide and are usually removed concomitantly with the cleavage of the peptide from the resin (Figure 2.5). As mentioned earlier, the Fmoc strategy uses mainly tBu (19) and Boc-type protecting groups. For Asp/Glu/Ser/Thr/Tyr, tBu (19) is usually used, whereas the Boc (13) group is applied to Lys. For His/Asn/Gln the Trt (15) group is employed, and for Arg, the bulky pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf) (24) group is used. In the Boc (13) strategy, the Bzl (20) group is usually used for Asp/Glu/Ser/Thr/Tyr, although lately the cyclohexyl (cHx) (21) group is replacing the Bzl (20) group in Asp/Glu, as it better prevents aspartimide formation. Asn and Gln are usually being used without protection. The Lys side-chain is usually protected with the benzyloxycarbonyl (Cbz, Z) (23) or the 2-chlorobenzyloxycarbonyl (2-Cl-Z) group and for His/Arg, the p-toluenesulfonyl (Tos) (22) group is used. As for His, another option is to employ the 2,4-dinitrophenyl group (Dnp) (25), which is removed by thiolysis prior to the HF cleavage step. The Alloc (17) and (1-(4,4-dimethyl-2,6-dioxocylohex-1-ylidene)-3-ethyl) (Dde) (26) group, which is removed by hydrazine, introduce an extra degree of orthogonality, and are used as side-chain protecting groups for Lys to access cyclic and branched peptides.

Figure 2.5 Side-chain protecting groups.

2.4.1 The Special Case of Cysteine

An important number of peptides and proteins possess disulfide bridges, which maintain the structure and biological activity of the molecule [51, 52]. Thus, cysteine residues need special side-chain protecting groups that will allow a postelongation transformation to the corresponding disulfide bridges either in solid phase or in solution. Developing groups for the side-chain of Cys and studying the optimal coupling conditions for these derivatives (Cys residues are very prone to racemization) [53], and deprotection (to prevent back alkylation of the carbocation) has been an active field of research (Figure 2.6) [54, 55]. The most versatile group is maybe S-Trt (27a), which allows both a mild acidic cleavage (∼10% TFA is needed) to later oxidize the thiol moieties, and also the direct formation of disulfide bridges by iodine treatment from the protected Cys. An even milder acid protecting group is the S-methyltrityl (S-Mmt) (27b), which can be removed with less than 1% of a TFA solution. Other groups that form disulfide bridges through iodine-mediated oxidation are S-4-methoxybenzyl (S-Mob) (28), S