Introduction to Peptide Science E-Book

Ian W. Hamley

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

Seitenzahl: 317

Veröffentlichungsjahr: 2020

Beschreibung

Provides an interdisciplinary introduction to peptide science, covering their properties and synthesis, as well as many contemporary applications

Peptides are biomolecules comprised of amino acids which play an important role in modulating many physiological processes in our body. This book presents an interdisciplinary approach and general introduction to peptide science, covering contemporary topics including their applicability in therapeutics, peptide hormones, amyloid structures, self-assembled structures, hydrogels, and peptide conjugates including lipopeptides and polymer-peptide conjugates. In addition, it discusses basic properties and synthesis clearly and concisely.

Taking a logical approach to the subject, Introduction to Peptide Science gives readers the fundamental knowledge that is required to understand the cutting-edge material which comes later in the book. It offers readers in-depth chapter coverage of the basic properties of peptides; synthesis; amyloid and peptide aggregate structures; antimicrobial peptides and cell-penetrating peptides; and peptide therapeutics and peptide hormones.

• Introduces readers to peptide science, including synthesis and properties
• Provides unique content covering properties, synthesis, self-assembly, aggregation, and applications
• Summarizes contemporary topics in an accessible fashion including applications in therapeutics, peptide hormones, amyloid structures, self-assembled structures, hydrogels, and peptide conjugates including lipopeptides
• Presented at an introductory level for the benefit of students and researchers who are new to the subject

Introduction to Peptide Science is an ideal text for undergraduate students of chemistry, biochemistry, and other related biological subjects, and will be a valuable resource for postgraduate students and researchers involved in peptide science and its applications.

Leseprobe

Table of Contents

Cover

Preface

1 Basic Properties

1.1 INTRODUCTION

1.2 PROPERTIES OF AMINO ACIDS

1.3 THE PEPTIDE BOND

1.4 SECONDARY STRUCTURES

1.5 PEPTIDE STRUCTURE AND CONFORMATION CHARACTERIZATION METHODS

1.6 PEPTIDE DATABASES AND WEB SOFTWARE

BIBLIOGRAPHY

2 Synthesis

2.1 INTRODUCTION

2.2 SOLID‐PHASE PEPTIDE SYNTHESIS

2.3 SOLUTION‐PHASE PEPTIDE SYNTHESIS

2.4 METHODS TO PREPARE LONGER PEPTIDES

2.5 PEPTIDE LIBRARY SYNTHESIS

2.6 SYNTHESIS OF CYCLIC PEPTIDES

2.7 PEPTIDOMIMETICS

2.8 POST‐TRANSLATIONAL MODIFICATIONS

2.9 LIPIDATION

2.10 GLYCOSYLATION

2.11 POLYPEPTIDE POLYMERS AND CONJUGATES OF PEPTIDES AND POLYMERS

2.12 NON‐RIBOSOMAL PEPTIDE SYNTHESIS

2.13 PURIFICATION AND ANALYSIS METHODS

BIBLIOGRAPHY

3 Amyloid and Other Peptide Aggregate Structures

3.1 INTRODUCTION

3.2 AMYLOID

3.3 AMYLOID β

3.4 MECHANISMS AND KINETICS OF AMYLOID AGGREGATION

3.5 TOXICITY AND RELEVANCE TO DISEASE

3.6 FIBRILLIZATION OF SMALL PEPTIDES

3.7 BIOLOGICAL FUNCTIONAL AMYLOID AND BIOENGINEERING APPLICATIONS OF AMYLOID MATERIALS

3.8 FIBRILS FROM α‐HELICES

3.9 PEPTIDE HYDROGELS AND TISSUE SCAFFOLDS

3.10 PEPTIDE NANOTUBES

3.11 PEPTIDE AND PEPTIDE CONJUGATE ASSEMBLIES

3.12 CHARACTERIZATION METHODS FOR PEPTIDE ASSEMBLIES

BIBLIOGRAPHY

4 Antimicrobial and Cell‐penetrating Peptides

4.1 INTRODUCTION

4.2 BACTERIAL PATHOGENS, TARGETS OF ANTIBACTERIAL AGENTS, AND ANTIMICROBIAL RESISTANCE PATHWAYS

4.3 TESTING ANTIMICROBIAL ACTIVITY

4.4 BACTERIAL BIOFILMS

4.5 DESIGN OF ANTIMICROBIAL PEPTIDES

4.6 CLASSES OF ANTIBACTERIAL PEPTIDES

4.7 ANTIFUNGAL PEPTIDES

4.8 ANTIVIRAL PEPTIDES

4.9 ANTIPARASITIC PEPTIDES

4.10 MECHANISMS OF ACTIVITY

4.11 CELL‐PENETRATING PEPTIDES

BIBLIOGRAPHY

5 Peptide Hormones and Peptide Therapeutics

5.1 INTRODUCTION

5.2 GENERAL PRINCIPLES OF PEPTIDE THERAPEUTICS

5.3 PEPTIDE HORMONES

5.4 NEUROPEPTIDES AND OTHER PEPTIDES IN VIVO

5.5 VENOM‐DERIVED PEPTIDES

5.6 ANTICANCER PEPTIDES

5.7 MISCELLANEOUS PEPTIDE THERAPEUTICS

5.8 COSMETIC PEPTIDES AND LIPOPEPTIDES

BIBLIOGRAPHY

Index

End User License Agreement

List of Tables

Chapter 1

Table 1.1 Properties of amino acids.

Table 1.2 Side chain protonation depending on pH.

Table 1.3 Some non‐standard amino acids.

Table 1.4 Bond angles and residue spacings for regular peptide conformations.

Table 1.5 Typical FTIR band ranges for peptides.

Table 1.6 Examples of useful peptide software and websites.

Chapter 2

Table 2.1 Common peptide synthesis resins (resin bead shown as filled circle)...

Table 2.2 Typical side chain protecting groups and deprotection conditions.

Chapter 3

Table 3.1 Conditions associated with amyloid‐forming peptides and proteins.

Table 3.2 Effect on Aβ of APP mutations.

Table 3.3 Cell adhesion and related motifs.

Table 3.4 Examples of surfactant‐like peptides, peptide bolaamphiphiles, and ...

Chapter 4

Table 4.1 List of pathogens identified by WHO as priorities due to antimicrob...

Table 4.2 Marketed antimicrobial peptides.

Table 4.3 Examples of anionic antimicrobial peptides.

Table 4.4 Examples of antifungal peptides.

Table 4.5 Examples of antiviral peptides.

Table 4.6 Examples of antiparasitic peptides.

Table 4.7 Lipid composition of the cell membrane of selected bacteria.

Table 4.8 Examples of widely studied cell‐penetrating peptides (CPPs).

Chapter 5

Table 5.1 Examples of established peptide hormone‐based therapeutics on the m...

Table 5.2 Sequences of GnRH agonists with generic sequence (pG)HWSY

x

LRP

y

.

Table 5.3 Sequences of human pancreatic polypeptides with PP‐fold related res...

Table 5.4 Gut‐related peptide families including neurotransmitters (italicize...

Table 5.5 Venom‐derived peptide therapeutics.

Table 5.6 Examples of peptides used in skincare products.

List of Illustrations

Chapter 1

Figure 1.1 Enantiomers of amino acid residues in peptides. Left:

L

‐amino aci...

Figure 1.2 Fischer projections and structures of

L

‐threonine,

D

‐threonine,

L

Figure 1.3 Configurations of a peptide bond.

Figure 1.4 The standard amino acids.

Figure 1.5 Amino acid side chain labelling nomenclature.

Figure 1.6 Resonance structures of (a) arginine and (b) histidine.

Figure 1.7 Hydrophobicity of amino acids, according to the Wimley–White scal...

Figure 1.8 Schematic titration curve for a tetrapeptide NH

2

–EGAK–COOH, plott...

Figure 1.9 Chou–Fasman scale of α‐helix and β‐sheet propensity for protein a...

Figure 1.10 Formation of a peptide bond (amide group highlighted in purple) ...

Figure 1.11 Peptide bond geometry with typical angles shown.

Figure 1.12 A Ramachandran plot. The shaded areas show the ‘normally allowed...

Figure 1.13 Principle regular peptide secondary structures: (a) α‐helix (sho...

Figure 1.14 Example of a helical wheel diagram of a α‐helical peptide sequen...

Figure 1.15 The coil–helix transition is cooperative, leading to a sigmoidal...

Figure 1.16 Example of a β‐hairpin structure.

Figure 1.17 Helical peptide secondary structures. (a) PPII structure, (b) PG...

Figure 1.18 Examples of β‐ and γ‐turn structures. (a) Classical γ‐turn, (b) ...

Figure 1.19 Backbone and side chain interactions that stabilize peptide stru...

Figure 1.20 (a) A dimeric coiled coil structure based on the abcdefg heptad ...

Figure 1.21 Leucine zipper (bZip) structure involved in DNA transcription.

Figure 1.22 Measured CD spectra for peptides. Open circles: Derivative of pe...

Figure 1.23 Chemical shift index plot (for

1

Hα) for 56‐residue sequence from...

Chapter 2

Figure 2.1 Schematic of sequential process in solid‐phase peptide synthesis....

Figure 2.2 Orthogonal protecting groups illustrated for an aspartic acid res...

Figure 2.5 Enolization and oxazolone formation cause peptide racemization. X...

Figure 2.3 Common coupling reagents in peptide synthesis that generate activ...

Figure 2.4 HBTU activation mechanism.

Figure 2.6 Aspartimide formation reaction and subsequent reactions including...

Figure 2.7 Diketopiperazine formation for an on‐resin sequence with C‐termin...

Figure 2.8 Pseudoproline formation strategy: ring‐opening of an oxazolidine ...

Figure 2.9 The Hmb reversible backbone protecting group.

Figure 2.10

O

‐acyl isopeptide conversion into a native peptide.

Figure 2.11 Convergent SPPS, for the case of fragment condensation from the ...

Figure 2.12 Mechanism of native chemical ligation, here the SR group is pres...

Figure 2.13 Recombinant production of peptides/proteins via molecular clonin...

Figure 2.14 Schematic showing the steps of spot peptide synthesis.

Figure 2.15 Split‐mix peptide synthesis, illustrated for a three monomer syn...

Figure 2.16 Schematic of cyclization reactions involving the peptide head (C...

Figure 2.17 Strategy for on‐resin head‐to‐tail peptide cyclization using a r...

Figure 2.18 Representative peptide side chain cyclization reactions. (a) Dis...

Figure 2.19 Molecular structures of two natural bicyclic peptides: (a) phall...

Figure 2.20 Structures of some peptidomimetics.

Figure 2.21 Production of C‐terminal amide group via oxygenation of a termin...

Figure 2.22 Peptide modified at cysteine residue with (a) farnesyl group, (b...

Figure 2.23 Lipopeptides prepared by (a) N‐terminal palmitoylation, (b) C‐te...

Figure 2.24 Synthesis of amidated peptides using amine‐modified linkers.

Figure 2.25 (a)

N

‐glycosylation of an N residue, (b)

O

‐glycosylation of S or...

Figure 2.26 Synthesis of polypeptides via

N

‐carboxyanhydride polymerization:...

Figure 2.27 Representative coupling chemistries. In general R denotes a pept...

Figure 2.28 Side group reactions for peptide functionalization. For lysine a...

Figure 2.29 Peptide ion fragmentation labelling system for tandem MS along w...

Figure 2.30 Reaction of ninhydrin with primary amines.

Figure 2.31 Scheme of the Edman degradation reaction.

Chapter 3

Figure 3.1 Hierarchical structure of amyloid fibrils and ‘cross‐β’ X‐ray dif...

Figure 3.2 TEM images of amyloid fibrils: (a) negative stain TEM image from ...

Figure 3.3 Oligomerization and fibrillization pathways. Species in green are...

Figure 3.4 The amyloid hypothesis for AD.

LTP

(

long‐term potentiation

)...

Figure 3.5 (Box) Amino acid sequences of Aβ peptides Aβ40 and Aβ42, with enz...

Figure 3.6 Structure of Aβ42 amyloid fibrils (pdb file 5KK3).

Figure 3.7 Concentration of species versus time, showing lag and growth phas...

Figure 3.8 Microscopic processes that govern the rate of amyloid fibril form...

Figure 3.9 Schematic fibril growth curves, showing effect of the rate consta...

Figure 3.10 Homologous versus heterologous seeding. Colour scheme as for Fig...

Figure 3.11 Steric zipper structure obtained from single‐crystal X‐ray diffr...

Figure 3.12 Formation of filamentous hyphae by fungi and Streptomyces bacter...

Figure 3.13 Schematic of self‐assembling fibre peptides due to complementary...

Figure 3.14 A peptide hydrogel in a vial with overlaid schematic of microsco...

Figure 3.15 Examples of enzymatic reactions using Fmoc‐ and naphthalene‐modi...

Figure 3.16 There are multiple cues for cell adhesion with the ECM. These in...

Figure 3.17 Three classes of observed peptide nanotube structures: (a) beta‐...

Figure 3.18 Closure of helical ribbons (a) into a peptide nanotube comprisin...

Figure 3.19 Stacking of alternating

D

,

L

‐cyclic peptides to form nanotubes, s...

Figure 3.20 Bottom: AFM height images during the time‐dependent aggregation ...

Figure 3.21 Lipopeptide self‐assembled nanostructures. (a) Fibrils, (b) mice...

Figure 3.22 Schematic phase diagram for lipopeptide assemblies in terms of t...

Figure 3.23 Molecular structure of fluorescent probe dyes mentioned in the t...

Figure 3.24 Congo red staining of β‐sheet ‘amyloid’ fibrils formed by lipope...

Figure 3.25 Representative cryo‐TEM images from different classes of peptide...

Figure 3.26 Dynamic shear moduli measured (at a stress σ = 100 Pa) for hydro...

Chapter 4

Figure 4.1 Targets for antimicrobials, showing examples for each type includ...

Figure 4.2 The distinct cell wall structures of the two different groups of ...

Figure 4.3 Antimicrobial resistance mechanisms.

Figure 4.4 Measurement of inhibition zone of antimicrobial agent on a Petri ...

Figure 4.5 Different classes of antimicrobial agents. (a) Bacteriostatic age...

Figure 4.6 Stages in biofilm formation and strategies to prevent/disrupt or ...

Figure 4.7 Antimicrobial killing kinetics for cultures showing resistance, t...

Figure 4.8 (a) Molecular structure of c‐di‐GMP. (b) Generic molecular struct...

Figure 4.9 Molecular structures of some host defence AMPs: (a) human neutrop...

Figure 4.10 Bidentate binding of guanidinium groups with acidic XOO

−

g...

Figure 4.11 Molecular structures of some arginine‐rich AMPs: (a) gramicidin ...

Figure 4.12 Molecular structures of some AMPs based on cyclic peptides: (a) ...

Figure 4.13 Schematic of the cell membrane of fungi.

Figure 4.14 Molecular structure of several echinocandins: (a) caspofungin, (...

Figure 4.15 Examples of antifungal lipopeptides from among those produced by...

Figure 4.16 Membrane‐disruption modes of antimicrobial activity. The peptide...

Figure 4.17 Structure of heparin.

Figure 4.18 Mechanisms of uptake of CPPs.

Chapter 5

Figure 5.1 Number of approved peptide therapeutics according to treatment fi...

Figure 5.2 SWOT analysis of peptide therapeutics.

Figure 5.3 Delivery routes for marketed peptides.

Figure 5.4 Examples of peptide enzyme inhibitors: (a) amastatin, (b) boroleu...

Figure 5.5 Schematic of a dose–response curve, showing bioactivity versus co...

Figure 5.6 Action of peptide hormones at cell‐surface receptors.

Figure 5.7 The hypothalamic–pituitary system, showing key hormones and their...

Figure 5.8 Molecular structure of (a) GnRH and (b) thyrotropin‐releasing hor...

Figure 5.9 Molecular structure of cetrorelix.

Figure 5.10 Molecular structures of: (a) somatostatin, (b) octreotide and la...

Figure 5.11 Molecular structure of etelcalcetide.

Figure 5.12 Pathways by which insulin and glucagon regulate blood sugar leve...

Figure 5.13 (a) Three‐dimensional crystal structure of insulin, showing inte...

Figure 5.14 Insulin analogue peptides on the market.

Figure 5.15 α‐helical structure of glucagon, obtained from X‐ray diffraction...

Figure 5.16 Vasopressin/oxytocin peptide family members. Mpa = 3‐mercaptopro...

Figure 5.17 Molecular structures of (a) lisinopril and (b) enalapril.

Figure 5.18 Interactions of gut and endocrine hormones with the brain and ho...

Figure 5.19 Molecular structures of: (a) GLP‐1 with residues which are subst...

Figure 5.20 Molecular structure of macimorelin.

Figure 5.21 Venom‐based peptide therapeutics: (a) eptifibatide and (b) bival...

Figure 5.22 Molecular structures of (a) ziconotide, (b) linaclotide, and (c)...

Figure 5.23 Molecular structures of (a) bortezomib, (b) apicidin, (c) romide...

Figure 5.24 Molecular structures of (a) dactinomycin, (b) bleomycin A

2

, and ...

Figure 5.25 Molecular structure of mifamurtide.

Figure 5.26 Molecular structure of cyclosporin.

Figure 5.27 Pam

n

CSK

4

lipopeptides with

n

 = 1, 2, or 3 palmitoyl chains attac...

Guide

Cover

Table of Contents

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Introduction to Peptide Science

IAN W. HAMLEYSchool of Chemistry, University of Reading, UK

This edition first published 2020© 2020 John Wiley & Sons Ltd

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Library of Congress Cataloging‐in‐Publication Data

Names: Hamley, Ian W., author.

Title: Introduction to peptide science / Ian W. Hamley.

Description: First edition. | Hoboken, NJ : Wiley, 2020. | Includes bibliographical references and index.

Identifiers: LCCN 2020013505 (print) | LCCN 2020013506 (ebook) | ISBN 9781119698173 (paperback) | ISBN 9781119698180 (adobe pdf) | ISBN 9781119698197 (epub)

Subjects: MESH: Peptides | Antimicrobial Cationic Peptides | Peptide Hormones | Nanotubes, Peptide

Classification: LCC QP552.P4 (print) | LCC QP552.P4 (ebook) | NLM QU 68 | DDC 612/.015756–dc23

LC record available at https://lccn.loc.gov/2020013505

LC ebook record available at https://lccn.loc.gov/2020013506

Cover Design: WileyCover Image: © MOLEKUUL/Getty Images

Preface

Welcome (or welcome back!) to the wonderful world of peptide science. Peptides are fascinating chain molecules, built from amino acids, that nature has evolved to fulfil an incredible range of structural and functional roles. Of particular importance are peptide hormones, a major class of signalling molecules in the body. But there are many other essential peptides ensuring your body keeps working. Peptides crop up everywhere, along with their larger cousins proteins, as components of biological structures in silk, collagen, amyloid, and many other biomaterials. Amyloid is also now implicated as a ‘pathological’ agent in many diseases that are becoming of increasing concern, including neurodegenerative diseases such as Alzheimer's. Peptide hormones are playing an important role in conditions associated with a modern lifestyle, such as diabetes and the related condition of obesity.

Nature uses an alphabet of 20 natural amino acids to build peptides. Peptides have been synthesized in the laboratory for decades and in the last 50 years automated synthesis methods have been introduced, been rapidly developed, and have become established. Scientists are now designing original sequences and incorporating novel residues, functionalities, and configurations into peptides and are also creating conjugates of peptides with lipids, glyco‐saccharides, and polymers.

This book covers the basic properties of peptides, looking at essential synthesis methods and peptide aggregate structures, including amyloid and other nanostructures such as peptide nanotubes and peptide gels. Medically related applications are considered in the final two chapters, devoted to antimicrobial peptides and peptide therapeutics. These are discussed in the context of peptide hormones, from which many of the most important peptide therapeutics introduced into practice (so far) are derived.

This book is intended to provide a broad coverage of peptide science that is otherwise lacking in existing textbooks. There are several excellent books that cover peptide synthesis in detail and a few books cover basic properties, with the best coverage often being in introductory texts on proteins or general biochemistry. There are also specialist texts on peptide therapeutics and antimicrobial peptides, and a very few on amyloid. However, I felt there was a need for a compact introductory text that covers applications such as therapeutics and biomaterials. This book aims to achieve that. In addition, it covers at an introductory level a number of modern developments in the field that are not included in older texts in the areas of synthesis and aggregation/self‐assembly. The book includes aspects of synthetic chemistry, biochemistry, and biophysics relevant to understanding peptide science. This is an interdisciplinary field, so these are not considered to be exclusive terms, nor is this an exhaustive list of disciplines that feed into or from peptide science.

I have intended this book to be an introduction for senior undergraduates of chemistry, biochemistry, or biology and so should be useful as a supplementary text for junior courses in these and allied subjects. In addition it contains, in a compact and easy to reach form, much material that should be a valuable reference source for researchers in the field. All the texts I consulted in the course of writing the book, including existing books in the field along with key articles (mainly review articles) that I referred to, are cited in the Bibliography sections at the end of each chapter. Of course these do not provide a complete reference list on the subject; it would be impossible to compile such a list, given the huge volume of research in this exciting and fast‐moving field.

I apologize in advance for any errors or omissions and would be grateful to be informed of these. I would also be happy to receive any other feedback on the book as this would be very useful if a future edition emerges. At the moment, I will take a justified break from peptide book writing, although I've enjoyed the process and have learnt many useful new things. I hope you enjoy it in a similar style.

I would like to acknowledge my editor Jenny Cossham for supporting this project and my group of great students and postdocs who have helped immensely as we have learned together over the last couple of decades about peptides and their applications. Also thanks to all my many valued collaborators over the years, from around the world. Finally, I am very grateful to my family for their extracurricular support, and in the case of my wife Valeria for curricular support as well!

Ian W. HamleyUniversity of Reading, UK, 2020

1Basic Properties

1.1 INTRODUCTION

Peptides and proteins are essential biological molecules and assemblies, and many structures and functions of organisms are derived by using them. Peptides and proteins comprise chains of amino acids and are produced from DNA in the ribosome via messenger RNA through three‐letter codons, i.e. each amino acid is represented by a triplet of nucleotides. Peptides are also known as ‘polypeptides’ to signify that they are polymers formed of amino acids. In fact, short peptides as considered in this book have the properties of oligomers rather than polymers.

Although there is no rigorous definition of the difference between peptides and proteins in this book, a typical peptide would have up to 100 residues, and longer chains would be considered proteins. In fact, standard synthesis methods lead to peptides up to 50–60 residues in length, which may also be considered a cut‐off, with somewhat larger chains being described as ‘mini‐proteins’.

This chapter is organized as follows. In Section 1.2, the essential properties of the 20 standard natural amino acids that peptides and proteins are built from are first considered, including an analysis of reactivity, charge, and hydrophobicity. When incorporated in a peptide or protein, the term ‘residue’ is used for the group formed from the corresponding amino acid. Non‐natural residues that are found in a few natural and synthetic peptides are also introduced in Section 1.2, especially those which are mentioned in peptide sequences elsewhere in this book. Then, in Section 1.3, the nature and geometry of the peptide bond are considered. The main secondary structures adopted by peptides are discussed in Section 1.4. There follows (Section 1.5) a description of characterization methods used to determine peptide structure and conformation. The chapter concludes with a list of useful peptide websites and selected software, including databases and property calculators.

1.2 PROPERTIES OF AMINO ACIDS

Amino acids are chiral molecules, meaning that there is a distinct spatial arrangement of substituents around the central backbone carbon atom, which is termed a Cα atom. This is a stereogenic centre. There are two possible arrangements with different ‘handedness’, which are mirror images. These forms are termed enantiomers. There are two forms, termed L‐ and D‐amino acids (note the use of a small capital letter). In the L form of a residue in a peptide, the substituents are arranged as shown in Figure 1.1, which can be remembered via the CORN mnemonic, with the CO, R, and N groups in a clockwise configuration from left, when viewed along the bond to the H atom. In nature, peptide and protein structures are almost entirely built from L‐amino acids. Molecules that contain amino acids may have more than one stereocentre; for example, Figure 1.2 shows the diastereomers of threonine, L‐ and D‐threonine, in the form of Fischer projections. Figure 1.2 also includes the two allo‐ forms, which are rarely found in natural peptides (isoleucine also has four diastereomers). The R,S notation for the stereocentres according to the Cahn–Ingold–Prelog rules are also shown. Fischer projections are planar representations of an enantiomer (named after Emil Fischer) in which bonds denoted as horizontal extend above the plane of the paper and vertical bonds denote those which extend below the plane. In contrast to (mono)stereoisomers, diastereoisomers can have distinct chemical and physical properties (for example melting points).

The peptide bond has partial double bond character, so its length 1.33 Å (0.133 nm) is significantly shorter than a usual C–N bond length of 1.45 Å (0.145 nm). The partial double bond character leads to restricted rotation and a preferred co‐planar arrangement. Two configurations of the planar peptide bond are possible, called trans and cis, shown in Figure 1.3. The trans form is favoured energetically.

Figure 1.1 Enantiomers of amino acid residues in peptides. Left: L‐amino acid, right: D‐amino acid.

Figure 1.2 Fischer projections and structures of L‐threonine, D‐threonine, L‐allothreonine, and D‐allothreonine.

Figure 1.3 Configurations of a peptide bond.

The 20 standard (so‐called canonical) amino acids found in nature with different side chains are shown in Figure 1.4. They are grouped according to amino acid polarity or charge and, for the non‐polar amino acids, according to whether they have an aliphatic or aromatic substituent. All the amino acids except glycine are chiral, being present as either L‐ or D‐enantiomers. In nature, L‐enantiomers are the standard form, although D‐amino acids are present in some natural peptides. Figure 1.4 does not show the enantiomeric forms. Here, and elsewhere, the symbol Me in a chemical formula indicates methyl (CH3).

Figure 1.4 The standard amino acids.

Figure 1.5 Amino acid side chain labelling nomenclature.

The labelling scheme for (non‐hydrogen) atoms in amino acid side chains is shown in Figure 1.5, as exemplified for lysine and tyrosine.

The hydrophilic amino acids comprise two that are normally positively charged (except at low pH, see Table 1.2 and associated discussion) – these are the anionic residues Asp (D) and Glu (E). These differ only by the addition of an extra methylene group in the side chain of E. Although the pKa values of the side chain residues are similar, the length of the side chain (and so the location of the charge) does significantly influence the conformation of the backbone and the reactivity of these groups. Section 2.11.5 discusses some reactions using these residues.

The two basic residues, Lys (K) and Arg (R), are cationic under most conditions, except high pH. Lysine is used for many conjugation reactions, as discussed in Sections 2.11.2 and 2.11.5. Arginine contains a strongly basic guanidino group, which has a resonance structure shown for arginine in the charged state in Figure 1.6, and among its properties it is able to form bidentate hydrogen bonds (see Figure 4.10).

Histidine can also exhibit basic character, although its pKa is within the range of physiological pH values. It is unique among amino acids in having an imidazole group, and in the charged state the positive charge is shared between the nitrogen atoms by resonance. There are also two tautomeric forms in the uncharged state, depending on which nitrogen has an attached hydrogen atom.

Serine and threonine are related polar amino acids containing hydroxyl groups. They differ only by the addition of a methyl group in serine. Both these residues improve the solubility of peptides. Serine is important in the activity of many enzymes, such as serine proteases. There are two diastereomers of threonine, as discussed above (Figure 1.2).

Figure 1.6 Resonance structures of (a) arginine and (b) histidine.

Cysteine is one of the two residues that contain sulfur in the thiol (sulfhydryl) group. This is very commonly used as a tag in bioconjugation reactions, as described further in Sections 2.11.2 and 2.11.5. The thiol group in cysteine can also form disulfide links, which are common stabilizing elements in proteins, and are also used in natural and synthetic cyclic peptides. Disulfide bridges are discussed further in Section 1.4 and the synthesis of cyclic peptides including disulfide bridging is discussed in Section 2.6. The sulfhydryl group ionizes at high pH to give a –CH2–S− side chain.

The two residues Asn (N) and Gln (Q) with side chain amide groups differ only in side chain length. They are the amide forms of the acidic residues Asp (D) and Glu (E) respectively. These residues are polar, but do not ionize. The amide groups act as both hydrogen donor and hydrogen acceptor. The amide groups are labile at very high or low pH values and high temperatures and these residues can undergo deamidation to form Asp and Glu. N‐Terminal glutamine residues can spontaneously cyclize to form pyroglutamic acid, which is shown in Table 1.3.

Glycine is the simplest amino acid, and the only one that is non‐chiral because it only has a second hydrogen atom as its ‘side group’. The amino acids Ala (A), Val (V), Leu (L), and Ile (I) constitute hydrophobic aliphatic residues. The hydrophobicity of these residues increases with the side‐chain size. These residues are inert although they tend to associate through hydrophobic interactions, this being important in the stabilization of β‐sheet structures for example. Ile contains an additional asymmetric centre (Cβ) but only one diastereomer (L‐isoleucine) occurs naturally. Methionine is grouped with the non‐polar aliphatic residues in Figure 1.4, although because it contains a sulfur atom it is related to cysteine. However, the thiol group in cysteine is methylated, leading to a non‐polar character and a lack of ionizability. The sulfur atom in methionine is susceptible to oxidation, forming first a sulfoxide, then a sulfone. Proline is a unique residue in that it does not contain an amide hydrogen able to participate in hydrogen bonds. In addition, the ring in proline leads to a conformational constraint that increases the fraction of cis‐ configured peptide chain before the Pro residue.

The three aromatic amino acids, Phe (F), Tyr (Y), and Trp (W), are hydrophobic. Phe and Trp are not very reactive under most conditions. The –OH group in Tyr is reactive and ionizes at high pH, under which conditions dityrosine formation is possible. The indole group in Trp leads to the intrinsic fluorescence of this residue, which is a useful property in aggregation assays since the fluorescence is sensitive to the local microenvironment. The aromatic groups in Phe, Tyr, and Trp can lead to π‐stacking interactions which can stabilize some secondary and aggregate structures (Section 1.4). In addition, the aromatic groups have characteristic peaks in their UV spectra, which can be used in peptide/protein concentration measurements (Section 1.5.1).

Important properties of the amino acids, including the pKa values of the side chains as well as hydrophobicity, are listed in Table 1.1. Other properties of amino acids are included, such as their typical abundance in proteins (from database analysis), van der Waals volumes, and hydrophobicity. There are a considerable number of published hydrophobicity scales, examples of the most well‐known ones are included in Table 1.1. These are obtained from measurements of free energies of partition. Kyte and Doolittle presented a hydropathy scale based on water‐vapour transfer free energy and the tendency of amino acids to be on the exterior or interior of proteins. This scale correlates to a good degree to the water‐vapour ΔG(transfer). White and Wimley present tables of similarly determined ΔG(partition) values, ΔG(water/lipid interface), and ΔG(water/octanol). The trend in the Wimley–White ΔG values is clear when plotted (Figure 1.7). Other hydrophobicity scales have been proposed.

Since many peptides are charged due to the presence of residues with charged side chains and/or charged termini, analysis of pKa values enables estimation of the overall net charge on the peptides (see Eq. (1.4) below). The isoelectric point (pI) is another useful characteristic of peptides; this corresponds to the pH at which the net charge is zero. Table 1.2 summarizes the state of charged amino acids below and above the respective side chain pKa values. For the termini, typical pKa values are pKa = 9–10 for the N‐terminal NH3 and pKa = 2–3 for the C‐terminal COOH. It can be assumed that when the solution pH = pKa, equal numbers of the charged and uncharged species will be present. It should be noted that pKa values of residues in peptides (such as those quoted in Tables 1.1 and 1.2) are typical values; the pKa of a particular residue will depend on its local environment (for example it will be modified by the presence of other nearby charged residues). Acid or base titrations may be used to experimentally determine pKa values for residues in short charged peptides. Figure 1.8 shows a schematic titration curve.

Table 1.1 Properties of amino acids.

Symbol (one letter code)

Abundance in proteins (%)

Side chain p

K

a

van der Waals volume (Å

3

)

Hydrophobicity: Kyte–Doolittle hydropathy index

Hydrophobicity: Wimley–White interface scale, Δ

G

(kcal mol

−1

)

Hydrophobicity: Wimley–White octanol scale, Δ

G

(kcal mol

−1

)

Gly (G)

7.2

48

−0.4

0.01

1.15

Ala (A)

7.8

67

1.8

0.17

0.50

Val (V)

6.6

105

4.2

0.07

−0.46

Leu (L)

9.1

124

3.8

−0.56

−1.25

Ile (I)

5.3

124

4.5

−0.31

−1.12

Met (M)

2.2

124

1.9

−0.23

−0.67

Pro (P)

5.2

90

−1.6

0.45

0.14

Phe (F)

3.9

135

2.8

−1.13

−1.71

Tyr (Y)

3.2

10.5

141

−1.3

−0.94

−0.71

Trp (W)

1.4

163

−0.9

−1.85

−2.09

Ser (S)

6.8

73

−0.8

0.13

0.46

Thr (T)

5.9

93

−0.7

0.14

0.25

Cys (C)

1.9

8.4

86

2.5

−0.24

−0.02

Asn (N)

4.3

96

−3.5

0.42

0.85

Gln (Q)

4.3

114

−3.5

0.58

0.77

Asp (D)

5.3

3.9

91

−3.5

1.23

3.64

Glu (E)

6.3

4.0

109

−3.5

2.02

3.63

Lys (K)

5.9

10.5

135

−3.9

0.99

2.80

Arg (R)

5.1

12.5

148

−4.5

0.81

1.81

His (H)

2.3

6.0

118

−3.2

0.96

2.33

Figure 1.7 Hydrophobicity of amino acids, according to the Wimley–White scale. The hydrophobicity decreases left‐to‐right.

Close to the pKa, the two forms of the peptide can be represented as an acid–base equilibrium. Using the Henderson–Hasselbalch equation, the charge can be obtained.

The acid dissociation constant is given by

(1.1)

The Henderson–Hasselbalch equation is

(1.2)

Table 1.2 Side chain protonation depending on pH.

Amino acid

pH < p

K

a

p

K

a

pH > p

K

a

Asp (D)

–COOH

3.9

–COO

−

Glu (E)

–COOH

4.0

–COO

−

Lys

–NH

3

+

10.5

–NH

2

Arg (R)

–C(NH

2

)

2

+

12.5

–C(NH

2

)(NH)

His (H)

imH

+

6.0

imH

Tyr (Y)

–PheOH

10.5

–PheO

−

Cys (C)

–SH

8.4

–S

−

im = imidazole.

Figure 1.8 Schematic titration curve for a tetrapeptide NH2–EGAK–COOH, plotting pH versus molar concentration of OH− per mol of tetrapeptide. The peptide net charge is indicated between pKa values (defined as the mid‐point of the pseudo‐plateaus) as shown. Here, pKa(1) is that for the C terminus, pKa(2) is for the E residue, pKa(3) is for the N terminus and pKa(4) is for the K residue. Net charge +2 corresponds to NH3+–(E)GA(K+)–COOH, +1 to NH3+–(E)GA(K+)–COO−, 0 to NH3+–(E−)GA(K+)–COO−, −1 to NH2–(E−)GA(K+)–COO−, and −2 to NH2–(E−)GA(K)–COO−, where E− denotes charged glutamic acid (with COO− side chain terminus) and K+ denotes charged lysine with NH3+ side chain terminus. The pI of this peptide is at pH = 7.

So the fractional charge

(1.3)

For example,