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Cyclized Helical Peptides
Cyclized Helical Peptides
Synthesis, Properties, and Therapeutic Applications
Zigang Li
Hui Zhao
Chuan Wan
Authors All books published by WILEY-VCH
are carefully produced. Nevertheless,
Prof. Zigang Li authors, editors, and publisher do not
Shenzhen Graduate School of Peking warrant the information contained in
University these books, including this book, to
Chemical Biology and Biotechnology be free of errors. Readers are advised
Lishui Road to keep in mind that statements, data,
Xili Town illustrations, procedural details or other
518055 Shenzhen, Nanshan Disctrict items may inadvertently be inaccurate.
China
Library of Congress Card No.:
Dr. Hui Zhao applied for
Shenzhen Graduate School of Peking
University British Library Cataloguing-in-Publication
Chemical Biology and Biotechnology Data
Lishui Road A catalogue record for this book is
Xili Town available from the British Library.
Nanshan Disctrict
518055 Shenzhen Bibliographic information published by
China the Deutsche Nationalbibliothek
The Deutsche Nationalbibliothek lists
Dr. Chuan Wan this publication in the Deutsche
Shenzhen Graduate School of Peking Nationalbibliografie; detailed
University bibliographic data are available on the
Chemical Biology and Biotechnology Internet at <http://dnb.d-nb.de>.
Lishui Road
Xili Town © 2021 WILEY-VCH GmbH, Boschstr.
Nanshan District 12, 69469 Weinheim, Germany
518055 Shenzhen
China All rights reserved (including those of
translation into other languages). No
Cover Design: Wiley part of this book may be reproduced in
Cover Image: © Molekuul/iStockphoto; any form – by photoprinting,
© ivanastar/iStockphoto microfilm, or any other means – nor
transmitted or translated into a
machine language without written
permission from the publishers.
Registered names, trademarks, etc.
used in this book, even when not
specifically marked as such, are not to
be considered unprotected by law.
Print ISBN: 978-3-527-34342-3

ePDF ISBN: 978-3-527-34347-8
ePub ISBN: 978-3-527-34345-4
oBook ISBN: 978-3-527-34343-0
Typesetting Straive, Chennai, India
Printing and Binding
Printed on acid-free paper
10 9 8 7 6 5 4 3 2 1
v
Contents
1 Introduction 1
1.1 Protein–Protein Interactions and Their Small-Molecule Modulators 1
1.1.1 Characteristics of Protein–Protein Interactions 1
1.1.2 Intervention of Protein–Protein Interactions Using Small Molecules 3
1.1.2.1 Leukocyte Function-Associated Antigen-1 5
1.1.2.2 Inhibitor of Apoptosis Proteins 6
1.1.2.3 Bromodomains 6
1.1.2.4 Human Immunodeficiency Virus Integrase 6
1.1.2.5 B-Cell Lymphoma-2 Family/B-Cell Lymphoma-2 Homology 3 Proteins
Interaction 7
1.1.2.6 Mouse Double Minute 2–p53 Interaction 7
1.2 Features of Peptide as Molecular Tools 8
1.2.1 Advantages of Peptides as Molecular Tools 8
1.2.2 Disadvantages of Peptides as Molecular Tools 11
1.3 Helical Structures and Their Characterization 12
1.3.1 Different Types of Helices 12
1.3.1.1 α-Helix 12
1.3.1.2 310 -Helix 12
1.3.1.3 π-Helix 13
1.3.2 Characterization of Helical Peptides 15
1.3.2.1 Circular Dichroism 15
1.3.2.2 X-ray Crystallography 16
1.3.2.3 Nuclear Magnetic Resonance (NMR) 17
1.4 Stabilization of Peptides 18
1.4.1 Peptide Stabilization via Cyclization 18
1.4.1.1 Monocyclization 18
1.4.1.2 Multicyclization 19
1.4.2 Peptide Stabilization via Backbone Reconstruction 21
1.4.2.1 Methylation 21
1.4.2.2 Foldamers 23
References 27
vi Contents
2 Construction of Constrained Helices 35

2.1 Side-Chain Cross-linking 35
2.1.1 Disulfide Bond 35
2.1.2 Amide and Ester 37
2.1.3 All-Hydrocarbon Stapled Peptide 42
2.1.4 Thioether 48
2.1.5 Azole 56
2.2 End Nucleation 57
2.2.1 Macrocycle-Based N-cap Templates 58
2.2.2 Hydrogen Bond Surrogate Approaches 60
2.2.3 N-Terminal Side Chain Constrains as Helix-Nucleating
Templates 61
References 62
3 Properties of Stabilized Peptides 69

3.1 Helicity 69
3.1.1 Ring Size 69
3.1.2 Rigidity 74
3.1.3 Comparison 79
3.2 Binding Affinity 80
3.2.1 Helicity 80
3.2.2 Cyclization Position 81
3.2.3 Substitution 84
3.3 Cell Permeability 84
3.3.1 Amphiphilicity: Hydrophobicity and Isoelectric Point 84
3.3.2 Helicity 87
3.3.3 Summary 89
3.4 Nonspecific Toxicity 89
3.5 Stability 91
3.5.1 Proteolytic Stability 91
3.5.2 Pharmacokinetic Properties 95
3.6 Additional Features 98
References 103
4 Applications of Constrained Helices 107

4.1 Cancer 107
4.1.1 MDM2/X 107
4.1.2 B-Cell Lymphoma 2
(MCL-1/BCL-2/BCL-XL)-BID/Noxa/BAX/BIM/PUMA 110
4.1.3 NOTCH 114
4.1.4 Insulin Receptor Substrate 1 118
4.1.5 Ras 120
4.1.6 Rab 124
4.1.7 β-Catenin BCL-9/AXIN 126
4.1.8 Epidermal Growth Factor Receptor 130
Contents vii
4.1.9 Estrogen Receptor α 135

4.1.10 Hypoxia-Inducible Factor 138
4.1.11 Embryonic Ectoderm Development – Enhancer of Zeste
Homolog 2 140
4.2 Infectious Disease 143
4.2.1 HIV 143
4.2.2 Respiratory Syncytial Virus F (RSV) 148
4.3 Metabolic Disease 150
4.3.1 Glucokinase-Phospho-BAD 150
References 152
5 Stabilized Peptide Covalent Inhibitors 159

5.1 Methodologies of Peptide Covalent Inhibitor 159
5.1.1 Covalent Warhead 160
5.1.2 Stapling Method 162
5.2 Applications 162
5.2.1 BCL-2 Family Proteins As Target 163
5.2.2 MDM2 and MDM4 As Target 164
5.2.3 Sulfonium Tethered Peptide 168
5.2.4 Others 176
References 177
6 Stabilized Peptide PROTAC 181

6.1 Proteolysis-Targeting Chimera (PROTAC) 181
6.2 Design of Peptide PROTAC 181
6.2.1 Exploitation of E3 Ubiquitin Ligase-Recruiting Ligand 182
6.2.2 Design of Stabilized Peptide Ligand 183
6.2.3 Impact of Linker 184
6.3 Therapeutic Applications of Stabilized Peptide PROTAC 184
6.3.1 Targeting ERα 185
6.3.2 Targeting β-Catenin 188
References 188
7 Stabilized Peptide for Drug Delivery 191

7.1 Cell-Penetraying Peptides (CPPs) 191
7.1.1 Classification of CPPs 192
7.1.2 Mechanism of Cell Penetration of CPPs 193
7.1.3 Applications of CPPs 194
7.2 Cell-Permeable Cyclic Peptides (Cyclic CPPs) 196
7.2.1 Cyclic CPPs-Mediated Drug Delivery 198
7.2.2 Cyclo-RGD 202
7.3 Co-assembly Nanocarrier System 204
7.4 Examples of Stabilized Peptide Drugs 206
References 208
viii Contents
8 Outlook 213
8.1 The Development of Peptide-Stabilizing Methods 213
8.2 Applications of Stabilized Helical Peptides 218
References 220
Index 223
1
Introduction
1.1 Protein–Protein Interactions and Their

Small-Molecule Modulators
1.1.1 Characteristics of Protein–Protein Interactions
Proteins that work and degrade in highly congested and complex environments must
be found by their partners in a large number of non-partners. It is estimated that
human beings have 650 000 different pairs of interactions, which are responsible
for a number of key biomolecular processes [1]. The surface of soluble proteins is
covered by hydrophobic and hydrophilic residues, as well as by hydrophilic back-
bone. The highly specific physical contact between two or more protein molecules
is mainly related to hydrophobic interactions, salt, and hydrogen bonds.
Protein–protein interactions have different affinity and longevity. Some complexes
are weakly and instantaneously clustered; some may continue to form part of a larger
protein complex, stabilized through multiple interactions; some reversible signal
complexes have high pairing affinity, but only limited time; some complexes are sta-
ble, but have built-in timers; the presence of antibodies and antigens and protease
and inhibitor complexes can take up to a day, some of which may be categorized as
irreversible [2].
In addition, protein–protein interactions can be categorized according to the
structural characteristics (Figure 1.1) [3]: the interaction between globular protein
pairs, the interactions between globular proteins and individual peptide chains
with continuous or discontinuous table position, and the interaction between two
segments of peptide chains. Correspondingly, the polypeptide that participates in
protein–protein interactions may adopt a combination of structures: the extension
structure in the groove, β-sheet, α-helix, and even the poly-proline helix.
There is certain regularity in the presence of amino acid residues in proteins [3a].
In the general interface, leucine is the most common residue, followed by arginine.
Furthermore, charged residues are more common than polar residues, and both,
except for arginine and histidine, are generally abundant on the surface. Aromatic
amino acids, except for tryptophan, have a very low abundance on the surface but
have a high abundance at the interface. As is mentioned above, the frequency of
Cyclized Helical Peptides: Synthesis, Properties, and Therapeutic Applications, First Edition.
Zigang Li, Hui Zhao, and Chuan Wan.
© 2021 WILEY-VCH GmbH. Published 2021 by WILEY-VCH GmbH.
2 1 Introduction
PPI class Description Simplified illustration Examples (target- Examples structure

displaced)
Globular protein-helical Helix with a MDM2-p53
peptide, discontinuous discontinuous epitope BCL-XL–BAD and
epitope binding into a groove BCL-XL–BAK*
ZipA–FtsZ
S100B–p53
β–Catenin–TCF3–TCF4
MCL1–BH3 Protein Data Bank (PDB) ID: 2xa0
SUR2–ESX
Globular protein-peptide, Continuous epitope XIAP–SMAC*
continuous epitope on β-sheet or β-strand HIV integrase–LEDGF
and loops binding into Integrins
surface with pockets RAD51–BRCA2
PDZ domains
NRP1–VEGFA
Menin–MLL PDBID: 1g73
Globular protein-peptide, Binding into pocket in KEAP1–NRF2*
continuous epitope a β-propeller WDRS–MLL
PDBID: 2dyh
Globular protein-peptide, Peptide with an Bromodomains*
anchor residue anchor residue owing PDEδ–KRAS
to post-translational SH2 domains
modification binding PLK1 PBD–peptide
into a pocket VHL–HIF1a
PDBID: 3uvvv
Globular protein-globular Two proteins IL-2-IL-2R*
protein, discontinuous both presenting TNF–TNF
epitope discontinuous epitopes E2–E1
PDBID: 1z92
Peptide-peptide A pair of helices with MYC–MAX*
an elongated binding NEMO–IKK
interaction Annexin II–P11 (also
known as S100A10)
PDBID: 1nkp
BAD, BCL-2-associated agonist of cell death: BAK, BCL-2 bomologousantagonist/killer: BCL B cell lymphoma: BH3, BCL-2 bolology domain 3: BRCAZ, breast
Cancer type 2 susceptibility protein: HIF1a, hypoxia-inducible factor 1α: IL-Z, interleukin-2: IL-2R, interleukin-2 receptor: IKK, inhibitor of nuclear factor x8 kinase:
KEAP1, kelch-like ECH- associated protein 1: LEDGF, lens epitheliurn-derived growth factor: MAX MYC-associated factor X: MCL1, myeloid cell leukaemia 1: MLL,
mixed-lineage leukaemia: NEMO, nuclear factor κB essential modulator: NRF2, nuclear factor erythroid 2-related factor 2: NRP1, neuropilin 1: PLK1 PBD, polo-like
kinase 1 polo box domain: S100A10, S100 calcium-binding protein A10: SH2, SRC homology 2: SMAC, second mitochondria- derived activator caspase:SUR2,
mediator of RNA polymerase II transcription subunit 23: TCF3, HMG box trnscription factor 3: TNF, tumor necrosis factor: VEGFA vascular endothelial growth
factor: VHL, Von Hippel-Lindau disease tumor suppressor: WDRS, WD repeat-containing protein 5: XIAP, X-linked inhibitor of apoptosis protein *Example
structure illustrated in the column to the right.
Figure 1.1 Classification of protein–protein interactions and examples [3b]. Source: Scott
et al. [3b]. © 2016, Springer Nature.
occurrence of hydrophobic residues is generally high at the interface and is low on

the surface. Cysteine is particularly rare both on the surface and at the interface.
In addition, based on the results of alanine scanning mutagenesis, the residual
base that has a great influence on the binding affinity is called “hot spot” [4]. Hot
spots are almost always buried in the center of the core, not in contact with solvents.
The hot spot processes the highest sequence conservation [5]. Tryptophan, arginine,
and tyrosine are the most common, accounting for more than half of the total, as hot
spots. These three versatile residues were able to form hydrophobic, aromatic, and
polar interactions, all of which can be wrapped in complementary surfaces to meet
unpaired hydrogen-bonded donors and receptors. In addition, the polar “π-cation”
bond between arginine and tryptophan or tyrosine was found in more than 50% hot
spots [6]. Apart from a “π-cation” bond with arginine, the traditional side chain inter-
action is more common for tyrosine. By contrast, the most common residual at the
interface, leucine, is rarely found in hot spots, while isoleucine is rich.
In the complexes in the protein database, 62% has a helix on the interface [7].
However, the presence of a helix at the interface does not mean that the helix plays a
key role. Analysis shows that in about 60% of the interface, the hot residue is located
on one side of the helix, one-third of the complexes with the hot spots on two faces
of the helix, and about 10% of the complex with all three faces participating in the
interaction with the target protein. In the protein database, the first four major types
of function of protein–protein interactions, where helices are involved, are gene reg-
ulation, enzyme function, cell cycle, and signal transduction.
Analysis of the contribution of each helix residue to the interaction shows that
leucine appears most in the interface area. This is not surprising, because in gen-
eral, leucine is also the most common residue in proteins. After the normalization
of natural abundance, aromatic amino acids, arginine, and leucine are of the highest
frequencies at the helix interface as compared with polar residues [4, 8]. In addition,
polar and charged residues are also important contributors to the interface.
1.1.2 Intervention of Protein–Protein Interactions Using Small

Molecules
Abnormal protein–protein interactions are the basis of multiple diseases, and an
increasing number of researchers are committed to developing molecules to modu-
late protein interactions for therapeutic purposes. Small molecule is a class of entity
with potentially ideal therapeutic potentials. However, the contact surface of some of
the protein interactions is large and shallow (about 1000–6000 Å2 ), especially those
featured by a linear peptide epitope 1–4 amino acids long, compared to the tra-
ditionally small and deep small-molecule binding pockets [9] (Figure 1.2). There-
fore, the interface between proteins is sometimes regarded as a target of “undrug-
gable.” In establishing guidelines for the discovery of protein-protein interaction
(PPI) inhibitors, clinical success cases should be considered in the context of the
type of interface.
Work in recent years has begun to show that some protein–protein interactions
are able to be suppressed by small molecules. Most of the developed inhibitors
target PPIs, where hot spot residues are restricted to small binding pockets
(250–900 Å2 ) [11]. Some small-molecule inhibitors disrupt the interaction between
a globular protein and a single peptide chain with a secondary or tertiary structure,
through binding to the pocket on the globular protein. It is noteworthy that the
secondary structural features processed by the peptide chain, such as α-helices
and β-strands, have important implications for the design of inhibitors that mimic
and replace these peptides. With a better understanding of the structural biology
of the protein–protein interactions, it seems more promising and reasonable to
discover drugs targeting protein–protein interactions with defined structures. In
addition, the hot spots of the interaction interface can be targeted by inhibitors
of protein–protein interactions. The interaction of the rigid globular protein with
a polypeptide may be more suitable for small-molecule interruption because the
polypeptide can contribute more to the binding energy and be replaced by the small
molecule with good design. At present, there are many strategies to discover hits or
leads that interfere with protein–protein interactions, the most notable of which is
high-throughput screening, fragment screening, and optimization.
4 1 Introduction
Complexity of epitope
Primary
Secondary
Tertiary
Figure 1.2 The complexity of the PPI interface affects druggability PPIs can be classified
by whether one side of the interface consists of a primary (linear) protein sequence (green),
a single region of secondary structure (such as an α-helix, yellow), or multiple sequences
requiring tertiary structure (red). There are fewer examples of small-molecule inhibitors of
PPIs as the interface becomes more complex (from primary to secondary to tertiary
epitopes). Structures shown are BRDt/histone (green; Protein Data Bank [PDB]: 2WP1),
MDM2/p53 (yellow; PDB: 1YCR), and IL-2/IL-2Ra (red; PDB: 1Z92) [10]. Source: Arkin et al.
[10]. © 2014, Elsevier.
High-throughput screening is an effective way to find a hit in a traditional drug

target. Most of the high-throughput screening strategies rely on assays such as fluo-
rescence resonance energy transfer, amplified luminescent proximity homogeneous
assay screen, surface plasmon resonance, or fluorescence polarization because they
are highly efficient, sensitive, and reagent-available [12]. However, these methods
can usually disrupt enzyme activity and lead to more false-positive signals. Another
method is based on the label-free strategy, including the refractive index proper-
ties and mass spectrometry [12b]. Their applications may be more extensive, more
quickly developed, and robust because they eliminate the steps associated with intro-
ducing and observing tags. Despite these established methods, it is still difficult to
effectively generate protein–protein interaction inhibitors through high-throughput
screening since the compounds used for screening are mainly targeting traditional
drug targets. Traditional high-throughput screening faces some challenges in deal-
ing with protein–protein interactions – low hit ratio, low activity, and hard to elimi-
nate false positives [12b]. However, high-throughput screening has been successfully
applied in the discovery of the analog of discontinuous epitope on an α-helix.
The fragment-based drug discovery is a strategy to discover molecules from
smaller fragment of drugs or functional groups with low affinity, which can effec-
tively explore the chemical space [13]. These fragments can simplify the calculation
and analysis of ligand binding to improve affinity. The discovery of drug fragments
in the past has become an effective way to target protein–protein interactions. Many
protein interfaces have anchored residues to occupy the pockets of proteins, such
as tyrosine, phenylalanine, tryptophan, or leucine [14]. The pockets of the short
peptide with well-defined structure can effectively become the target of the drug
discovery based on fragments [15]. Fragment drug discovery screening usually
consists of two steps. The first step involves using a surface plasmon resonance
or differential scanning fluorescence for a preliminary rapid screening [16]. The
second step includes more targeted validation of the hit molecule, the use of X-ray
crystallography or protein-based nuclear magnetic resonance (NMR) to define
the spatial aspects of the binding site, the thermodynamic parameters defined
by isothermal calorimetry, and the surface plasmon resonance to define kinetics
[15]. Fragment discovery methods combine fragment space with the enhanced
hit ratio for lower complexity molecule, making itself a powerful lead generation
tool. Compared with high-throughput screening, fragment-based drug discovery
can capture more chemical structures with different hits, providing more hits for a
larger number of protein targets, higher recognition rates and fewer false positives,
and simpler and more reliable detection methods [17]. However, the need for a
large number of proteins is also a problem that fragment-based drug discovery
needs to address. In addition, fragments combine computational analysis aspects,
requiring new hardware design or new concepts and great progress.
Virtual screening based on structure is an important tool to help the discovery and
optimization of potential lead in a fast and cost-effective way based on structural
drug discovery. The virtual screening based on structure is used to select the
large-class drug compound library. Then, the screened out promising compounds
were selected for experimental testing. In the method of de novo design, the
three-dimensional (3D) structure of receptors is used to design novel molecules
that have never been synthesized before using ligand growing programs and the
intuition of medicinal chemists [18]. Compared with high-throughput screening,
the discovery of computer-assisted drugs has the advantage of predicting new
bioactive compounds and their receptor-binding structures, and in some cases
having a greater hit rate.
So far, using the above methods, a number of small-molecule compounds targeting
protein–protein interactions have entered clinical trials. Here are some successful
examples of small-molecule inhibitors that interfere with protein–protein interac-
tions.
1.1.2.1 Leukocyte Function-Associated Antigen-1

Leukocyte function-associated antigen-1 is a β2 integrin that participates in the
activation and adhesion of T cells and is a target in the weakening of inflammatory
immune response [19]. Lymphocyte function-associated antigen 1, the heterodimer
consisting of an α-chain and a β-chain, binds to its ligand intercellular adhesion
molecule-1, which is important for T cell–T cell interactions. The anti-lymphocyte
function-associated antigen 1 antibody efalizumab, an immunosuppressant that
inhibits lymphocyte activation and cell migration by binding to the CD11a subunit
of lymphocyte function-associated antigen 1, had been approved for psoriasis
and then withdrawn for immunosuppression-induced fatal viral infections [20].
Another leukocyte function-associated antigen-1 antagonist lifitegrast, which was
discovered by Sunesis, and then developed clinically by SARcode/Shire, has been
6 1 Introduction
approved as the only drug for the treatment of dry eye disease [21]. The mechanism
of action of the molecule is under debate, which is the inhibition of leukocyte
function-associated antigen-1 from binding to intercellular adhesion molecule 1,
associated with either intercellular adhesion molecule site on the I domain or the
related site on the I-like domain [22]
1.1.2.2 Inhibitor of Apoptosis Proteins

Apoptosis is a programmed cell death mediated by caspases activation. Inhibitor
of apoptosis proteins has been expressed in tumor cells by inhibiting the activity
of apoptosis-inducing protease, which regulates the fate of cells, including death
and immunity of apoptotic cells. X-linked inhibitor of apoptosis proteins is the
most effective inhibitor of apoptosis proteins, which interact with the initiator
caspase-9 through the Baculoviral IAP repeat 3 structure domain and caspase-3/7
through the Baculoviral IAP repeat 1/2 domain [23]. Discovering new compounds
that inhibit the interaction between X-linked inhibitor of apoptosis proteins
and enzymes is thought to be a promising strategy for cancer treatment. Smac
is a natural protein inhibitor of X-linked inhibitor of apoptosis proteins, which
competes with caspase binding to Baculoviral IAP repeat domain through the
alanine–valine–proline–isoleucine tetra-peptide at the nitrogen end [24]. Smac
proteins have attracted the attention of academics and pharmaceutical companies
to the design of small-molecule smac simulators [25]. There are currently two
types of inhibitors, including univalent and bivalent inhibitors. Some of them have
entered phase II clinical stage, such as LCL-161 by Novartis and Debio-1143 by
Debiopharm [12a].
1.1.2.3 Bromodomains
The acetylation of lysine residues or the methylation of lysine and arginine residues
can be “read,” which undertakes central roles in epigenetic regulation. Bromod-
omains and histone interactions are important in controlling gene expression and
DNA repair and in regulating inflammation and cancer. The bromodomains share a
conservative structure consisting of four α-helical bundles, which are connected by
different cyclic regions of variable charge and length. A hydrophobic pocket includes
a conservative aspartic amide and five water molecules that can identify acetylation
of lysine [26]. A quinazoline compound, apabetalone by Resverlogix, which is able
to increase transcription of the ApoA-I gene by inhibiting bromodomain and extra
terminal domain proteins, especially bromodomain-containing protein 4, is in phase
III clinical trials, for the potential treatment of diabetes mellitus, renal impairment,
and cardiovascular diseases [12a].
1.1.2.4 Human Immunodeficiency Virus Integrase

The homotetrameric protein human immunodeficiency virus integrase integrates
the viral genome into human DNA, which is vital for human immunodefi-
ciency virus replication. The protein–protein interactions between the human
immunodeficiency virus 1 integrase and the growth factor/p75 of the host protein
lens epithelium are key to this process, which makes integrase a target for human
immunodeficiency virus. The structure of human immunodeficiency virus integrase
consists of three domains, nitrogen-terminal DNA binding domains, catalytic core

domains, and carbon-terminal DNA binding domains. The catalytic core domain
has several pockets selected as small molecular target for inhibition of enzyme activ-
ity [27]. Now several small molecules targeting the enzyme have been approved for
the treatment of human immunodeficiency virus infections, such as raltegravir by
Merck, dolutegravir by GSK, and elvitegravir by Tobacco, which inhibit the enzyme
activity by binding to the active site. Furthermore, a series of 2-(quinolin-3-yl) acetic
acid derivatives, including clinical compound BI-224436 by Gilead, have been devel-
oped to block the integration step, by inhibiting lens epithelium-derived growth
factor/p75-integrase interaction, which displays a different resistance profile [28].
1.1.2.5 B-Cell Lymphoma-2 Family/B-Cell Lymphoma-2 Homology 3 Proteins

Interaction
B-cell lymphoma family proteins, including members of the family promoting
apoptosis and resistance to family members, are the central effectors of cell
apoptosis. B-cell lymphoma-2 homology 3-containing promotes apoptotic proteins,
such as B-cell lymphoma-2 homologous antagonist killer, through a single helix
binding to hydrophobic pockets that inhibit the apoptosis of B-cell lymphoma-2
proteins. The use of small-molecule compounds to simulate the B-cell lymphoma-2
homology 3 domain has shown significant therapeutic potential [29]. Several
B-cell lymphoma-2 homology 3 simulations were determined by the selection of
NMR-based fragments and the optimization of structures. For example, ABT-737
has an affinity for B-cell lymphoma-2 and with nanometer mole range [30]. This
compound occupies the same hydrophobic bag as a B-cell lymphoma-2 homol-
ogous antagonist killer-derived peptide, which has the same binding position as
B-cell lymphoma-2 homologous antagonist killer’s Leu78 and Ile85 to bind to the
key residues. Another small-molecule therapeutic Venetoclax, which was granted
Breakthrough Therapy Designation by USFDA, has been approved for the treatment
of chronic lymphocytic leukemia.
1.1.2.6 Mouse Double Minute 2–p53 Interaction

The interaction between p53 and its negative regulatory protein mouse double
minute 2 (MDM2) or MDMX is the target of anticancer treatment, and it is also a
common model system to evaluate the new method of protein–protein interaction
inhibition. This interaction is mediated by the short α-helix peptide sequence of p53,
which binds to the globular domain of MDM2 or MDMX. Structurally, N-terminal
domain of MDM2 binds to a short 15-residual α-helix peptide of p53, where three
hydrophobic residues of p53 occupy a well-defined hydrophobic pocket on MDM2
[31]. These structural characteristics make the strategy of targeting MDM2–p53
protein–protein interaction feasible. Various methods had been used to determine
the inhibitors of mdm2–p53 interactions, and a series of cis-imidazoline analogs,
nutlins, were determined by screening the complex library [32]. These molecules are
able to inhibit the interaction between p53 and mdm2 and adopt the same binding
mode as the key residues of p53. Among these analogs, idasanutlin by Roche was in
phase III clinical trial for the potential treatment of acute myelogenous leukemia.
8 1 Introduction
1.2 Features of Peptide as Molecular Tools

1.2.1 Advantages of Peptides as Molecular Tools
Natural or artificial peptides or proteins play a central role in molecular processes,
thanks to their strong molecular recognition capabilities. The strong recognition
ability of peptides can be explained by a large number of different types of func-
tional groups, which are easy to construct. Amino acids are rich in physicochemical
properties. By polarity, they can be classified as basic, acidic, nonpolar, or polar
amino acids, which provide hydrogen-bonded donors, receptors, or hydrophobic
cores. According to rigidity, some amino acids are flexible, such as glycine, while oth-
ers process fixed angles such as proline. This rich building block, combined with an
easy combination of amide keys, makes polypeptides diverse and complex enough to
form macromolecules of a particular nature and mediate important molecular pro-
cesses accordingly. In addition, a large number of posttranslational modifications
and unnatural amino acids greatly enhance their potential function.
In addition, peptide-mediated identification processes are ubiquitous in nature,
including those between proteins/peptides and proteins/peptides, between pro-
teins/peptides and nucleic acids, and between proteins and lipids, all of which
involve all processes of biological systems. These structural information are
known or readily available and can be designed according to complex structures
of polypeptide modulators or further explore the development of small-molecule
inhibitors.
Further, polypeptides and proteins are easy to screen and evolve. Because the
amide bond is easy to construct, the peptide combinatorial library can be easily used
in the screening of active sequences. Protein/polypeptide is located at the end of the
central code, so their molecular evolution can easily be achieved through the appro-
priate size of DNA libraries, biological systems, and Darwinian choices. In contrast,
the direct evolution of small molecules is difficult. An overview of the most com-
mon technologies is presented in Table 1.1. All techniques are somewhat related
and share common steps. The common technical strategies for peptide screening
are described below.
Multi-peptide arrays were synthesized by speckle technique. As a high-throughput
research tool, peptide arrays are a new type of biochip that uses automated instru-
mentation, in situ synthesis, to design hundreds of or even thousands of polypeptides
in very high density. This peptide chip can be incubated directly with a variety of dif-
ferent biological samples. After several washing steps, a secondary antibody, which is
typically labeled by a fluorescent label and can be detected by a fluorescent scanner,
is applied [44].
Protein/peptide evolution techniques are further modified to meet more applica-
tion needs. The use of powerful techniques to generate and screen DNA-encoded
protein libraries helps promote protein development as a drug ligand. However, their
use as drug ligands is limited by their intrinsic characteristics. Two intrinsic limita-
tions include rotational flexibility of the polypeptide backbone and a limited number
Table 1.1 Summary of key features of screening technologies commonly used for cyclic
peptide discovery.
Nonnatural amino
Library size and Screening acids/PTMs/backbone
its restriction host Cyclic modification
One-bead-one- 106, library In vitro Various Yes – all resin

peptide [33] construction chemistries compatible
chemistries
SICLOPPS[34] 109, In Head-to-tail Possible with
Transformation cellulo cyclization via amber codon
efficiency split-intein suppression
chemistry
Peptide on 109, transformation Bacteria Possible
plasmid [35] efficiency
Prokaryotic 109, transformation Bacteria Yes – commonly
[36] efficiency by post
Eukaryotic [37] 107, transformation Yeast translational
efficiency cysteine
alkylation
Phage [38] 109, transformation Phage
efficiency
CIS [39] 1014, translation In vitro Yes – commonly Yes – possible
scale
Ribosome [39]
mRNA [40]
RaPID Yes – commonly Extensive
[41]/TRAP[42] N-acetyl reprogramming
chloride using the FIT
chemistry system
Source: Obexer et al. [43]. © 2017, Elsevier.
(20) of natural amino acids. However, these restrictions can be overcome by using
chemical modifications.
In the one-bead-one-peptide (1B1P) method, pioneered by Lam and Salmon in
1991 [45], split and pool synthesis techniques were developed to generate diverse
libraries of beads (up to 107 compounds currently), each coated with multiple
copies of a unique peptide. Resin-compatible chemistries had been exploited to
make diverse backbones, peptoids, D-amino acids, and peptide cyclizations acces-
sible. Pei Lab presented a method for synthesizing and screening a complex 1B1C
Library of cyclic peptides for biological targets, such as proteins. In the Tentagel
micro-beads, up to 10 million different cyclic peptides were synthesized rapidly
by split-pool synthesis, and followed by multistage screening scheme, including
fluorescent activated cell sorting, magnetic selection, the enzyme-linked reaction
on beads, and the analysis of cyclic peptides in solution by fluorescence anisotropy.
Finally, the most active hits are determined by the partial Edman degradation-mass
spectrometry [33].
10 1 Introduction
Split-intein circular ligation of peptides and proteins (SICLOPPS), an in vivo

method for discovering head-to-tail cyclic peptides, is free from genetic code
reprogramming. The method applies split-intein chemistry to cyclize randomized
peptide sequences. The cyclic peptide library can potentially be of any size, and the
peptide itself may contain unlimited random residues, including unnatural amino
acids [46]. Plasmid propagation within cells bridge genotypes and phenotypes.
Accordingly, transformation efficiency limits the achievable library size to 1079.
Apart from being implemented in Escherichia coli, SICLOPPS has since been
extended to eukaryotic cells [47].
Phage display was for the first time reported by George P. Smith in 1985 [38a].
Phage display technology is considered as a fast and effective method for screen-
ing small peptides. In this technique, a gene encodes an interest protein into the
phage shell protein gene, and the phage displays the protein outside of it for binding
force screening. Phage display is a useful tool for drug discovery, but there are some
deficiencies. First, the library’s capacity can only reach 109, which is limited by trans-
fection efficiency. Second, we need to solve the diversity problem of the polypeptide
library. Third, a small amount of peptide due to its hydrophobicity or because of the
folding of the outer membrane protein cannot be displayed on the phage surface.
During phage display, chemical epoxidation can be incorporated to directly evolve
the cyclic peptide, including the direct evolution of polycyclic polypeptide and heli-
cal peptides. For example, in situ cyclization is easily realized by disulfide bridging
or alkylation via cysteine or enzyme-mediated modifications [48].
Ribosome display techniques are used to perform protein evolution in vitro,
producing proteins that can bind to an ideal ligand [40]. This technique optimizes
the interaction of functional proteins through Plückthun laboratories. The ribo-
some shows the beginning of the polypeptide encoded from the DNA sequence’s
original library. Each sequence is transcribed and then translated into adult foreign
peptides. The DNA library is fused to a lack of a stop codon interval sequence. The
absence of a stop codon prevents the release factor from binding, triggering the
dispersal of the transcription complex. Therefore, this interval sequence remains
connected to the peptide tRNA, occupying the ribosome tunnel, which makes
the protein of interest protruding from the ribosome and folds. The resulting
mRNA, ribosomes, and protein complexes can be screened. The filtered mRNA
was then transcribed to the cDNA and amplified by polymerase chain reaction
(PCR). Since it is carried out completely in vitro, there are two main advantages
over other alternative techniques. First, the diversity of the library is not limited
by the transfection efficiency of bacterial cell but is only affected by the number
of ribosomes and different mRNA molecules in the test tube. Second, random
mutations can be easily introduced after every choice, making proteins evolve over
several generations.
Traditionally, reprogramming was achieved via stop codon suppression or
removal of canonical amino acids. More extensive genetic code reprogramming is
particularly facile when carrying out in vitro display techniques, where enhanced
library diversity can be achieved through reconstituted translation systems giv-
ing compositional freedom [49]. The flexible in vitro translation (FIT) method
remains the most versatile and least labor-intensive procedure for genetic code
reprogramming in vitro. Furthermore, integrating the FIT system with mRNA
display gave rise to the random nonstandard peptide integrated discovery (RaPID)
system [50], the versatility of which is reflected in its broad use in the phar-
maceutical industry. Bashiruddin et al. created tricycles, by combining bicycle
bridging moiety with the classic N-acetyl chloride cyclisation of the RaPID
system [51].
The ligand is an oligonucleotide or peptide molecule that binds to a specific target
molecule. The aptamer is usually created by selecting them from a large random
sequence pool, but the natural ligands also exist in riboswitches. Polypeptide
ligands are artificial protein choices or are designed to bind to specific target
molecules [52]. These proteins are represented by one or more polypeptide loops
by a variable sequence of protein scaffolds. They are usually separated from the
combinatorial library and are often modified by directional mutation or by mutation
and selection of the variable region. In vivo, peptide ligands can bind to cell protein
targets and exert biological effects, including interfering with the normal protein
interactions between their target molecules and other proteins. The library of
polypeptide ligands is used as a “mutation.” In 2013, Shekhtman Laboratories
developed a method to build a combinatorial library of improved peptide adaptation
(clips) of high complexity, containing more than 3 × 1010 independent clones as
molecular tools for the study of biological pathways [53]. The protein skeleton was
modified to improve its solubility, and the aggregation of peptide was eliminated.
Clips is used in yeast two-hybrid screening to determine the peptide adaptation
to the late glycation end product of receptors in different domains. Cell function
detection showed that, in addition to direct interference with the known binding
sites, the combination of polypeptide and distal and ligand sites inhibited the signal
transduction of rage ligand-induced signaling. The findings highlight the potential
of using fragments to select biological targeting inhibitors.
1.2.2 Disadvantages of Peptides as Molecular Tools

Compared with the small molecule, the polypeptide has some disadvantages in
the properties of proprietary medicines, which limited its bioavailability. First,
amide bonds are fragile in vivo, making stability a fatal weakness of natural linear
peptides. There are many ways to improve its stability, such as the insertion of a
loop or unnatural module. In Chapter 2, the stability method of helix peptide is
summarized and discussed. Furthermore, the penetration of the peptides is limited.
The compartments in the organism are mainly composed of lipophilic substances,
which provide the basis for the time and space control of biological processes,
while peptides are generally hydrophilic in nature, which makes it difficult to cross
compartments. Therefore, the penetration of peptides as well as absorption is often
problematic. Regulating the physicochemical properties of peptides, such as substi-
tution, modification, to increase their interaction with biofilms, can improve their
penetrability. In Chapter 3, the factors affecting the permeability of polypeptides
are discussed.
12 1 Introduction
1.3 Helical Structures and Their Characterization

1.3.1 Different Types of Helices
1.3.1.1 𝛂-Helix
A typical α-helix turn is composed of an average of 3.6 amino acid residues with
dihedral angles (𝜑, 𝜓) in backbones close to −55∘ and −45∘ , respectively. As a rise of
1.5 Å/residue or 5.4 Å/turn, only i + 4 and i + 7 positions can make the side chains
of a given residue at the i position and the other residue at the i + n position are on
the same face. α-Helices are stabilized by intramolecular i → i + 4 hydrogen bonds
between a carbonyl group of the residue at position i and an amide proton at position
i + 4 in the main chain, with about 2.72 Å in the distance of nitrogen–oxygen and the
side chains pointing away from the helix axis [54].
Protein–protein interactions are involved in lots of biological processes such as
transcription, signal transduction, exocytosis, and so on [55]. α-Helix is the most
abundant secondary structure motif in proteins, accounting for over 30% in nature.
Meanwhile, α-helix is involved at interfaces of diverse protein–protein interactions,
which was known for α-helix-mediated protein–protein interactions. For its signifi-
cant proportion found in proteins’ structures, it is not surprising to tell that α-helix
is the most fundamental recognition motifs in diverse protein–protein interactions.
According to the study of helical interfaces in protein–protein interactions based on
the Protein Data Bank (PDB), about 13% multi-protein systems contained the helix
interface, ranging from enzymatic activities to protein associations by classification
of their functions, such as energy metabolism, protein synthesis, transcription, DNA
binding, signaling, transport, immune system, and so on [56].
The structural characteristic of α-helix forces residues especially their side chains
to extend out to the surrounding environment for selective and specific recogni-
tion, making it to be a template for designing small-molecule inhibitors or activa-
tors toward protein–protein interactions. The simplest system for α-helix-mediated
protein–protein interactions between two proteins is that one partner binds to its
partner protein by forming a short helical motif.
1.3.1.2 310 -Helix

Besides classical α-helix and β-sheet conformations, the 310 -helix is another
important secondary structural motif occurring in natural proteins, which also
plays significant roles in stabilizing proteins’ conformations and maintaining their
biological functions. Taylor first proposed the 310 -helix structure in 1941. Since then
this structure gained much attention and was studied fully [57]. The short name
of 310 -helix implies that the number of residues per turn is 3 and the number of
atoms contained in each intramolecular hydrogen bond is 10, which indicates that
310 -helix is more tightly packed than α-helix (also called 3.613 -helix). The backbone
torsion angles (𝜑, 𝜓) in 310 -helices are approximately −60∘ and −30∘ , respectively,
which are very close to that in α-helices (𝜑 = −55∘ , 𝜓 = −45∘ ). However, 310 -helices
display significantly distinct hydrogen-bonding pattern of i → i + 3, while α-helices
are stabilized by i → i + 4 intramolecular hydrogen bonds [58]. The 310 -helix is
less stable than the α-helix because of its less favorable van der Waals energy and
nonoptimal hydrogen bond geometry [59]. However, on account of the high struc-
tural similarity between the α-helix and 310 -helix, it is proposed that the α-helix can
be turned into the 310 -helix when side chain interactions happen. Indeed, 310 -helices
are not rare and could be found in globular proteins like aconitase, dienelactone
hydrolase, and phage T4 lysozyme. Barlow and Thornton analyzed globular protein
crystal structures in the database and suggested that at least 3.4% of the residues
are involved in 310 -helices. They also found that the location of 310 -helices is often
close to the N- or C-terminal of an α-helix [60]. Marshall et al. proved that Aib
(α-aminoisobutyric acid or Cα,α -dimethyl-glycine) can promote the formation of
310 -helices by calculations in 1971. Since then, many X-ray diffraction structures
of peptides involving rich Aib indicate their structure preference of 310 -helices
[61]. It is worth noting that an α-helical peptide requests at least seven amino acid
residues, while the formation of 310 -helical peptides has no dependence on main
chain length [62].
1.3.1.3 𝛑-Helix
So far only three helix types α-, 310 -, and π-helix were found in protein structures.
Compared with α-helix (30%) and 310 -helix (4%) in nature, π-helix seems particu-
larly rare, which could be attributed to the instability of corresponding structures.
To be specific, values of dihedral angles in π-helix were very close to the allowed
minimum energy requirements indicated by Ramachandran plot and proven to be
unfavorable [63]. Meanwhile, it was suggested that the required energy cost for sta-
bilizing the intramolecular i → i + 5 hydrogen bond to form a helix was huge [64].
Therefore, many people believe that π-helices are unstable in nature. However, as
researches on π-helix are moving forward, traditional concepts about π-helix are
broke. Researchers found the formation of π-helices in molecular dynamics simu-
lations of peptides [65]. More importantly, π-helices were observed in many protein
structures. Most of naturally occurring π-helices contain at least seven amino acid
residues and minimum two i → i + 5 hydrogen bonds and maximum seven H-bonds.
Along with α-helix and 310 -helix, π-helix can stably exist and may play important
roles in maintaining lots of biological functions.
π-Helices are also called 4.416 -helix where 4.4 is the number of residues in each
turn and 16 is the number of atoms involved in a hydrogen bond [66]. π-Helices are
stabilized by intramolecular i → i + 5 hydrogen bonds between a carbonyl group of
the residue at position i and an amide proton at position i + 4 in the main chain.
α-Helices and 310 -helices are stabilized by repeating i → i + 4 and i → i + 3 hydrogen
bonds, respectively. Therefore, minimal number of residues in a single π-helix is one
more than that in an α-helix and two more than that in a 310 -helix. According to the
structural analysis of π-helices in proteins in PDB, the mean values of dihedral angles
(𝜑, 𝜓) observed in π-helices could be around −76∘ and −41∘ , respectively. However,
it could have slight distinctions according to different models on structure definition
of π-helices. Besides, values of 1.2 Å in an average unit rise, 4.4 residues in each turn,
and 83∘ in an average unit twist were observed in the helical geometry of π-helices.
Like α-helices, π-helices have its featured amino acid preference in sequences. The
14 1 Introduction
distributions of amino acid residues for π-helices showed that aromatic residues like
Tyr, Trp, Phe, and His, as well as bulky aliphatic residues like Ile and Leu have higher
propensities, while small amino acids like Ala, Gly, and Pro are less preferential.
Also, there are amino acid residue preferences in their positions in sequences. For
example, bulky residues such as Phe, Tyr, Trp, Ile, and Leu are more likely to be
located at the beginning and at the end of π-helices. Besides hydrogen bond inter-
actions, other factors facilitated the stabilization of π-helices. Compared with the
α-helix, the π-helix had a lower unit rise (1.2 Å), whose side chains would be closer
to each other in space. Therefore, other interactions between side chains such as the
van der Waals, aromatic ring stacking, and electrostatic interactions became impor-
tant contributors for the stabilization of π-helices. This is why aromatic and large
aliphatic amino acids have higher propensities in π-helices [67].
It is worth noting that some researches revealed that there may be an evolutionary
relationship between α-helices and π-helices. Based on the families of structurally
similar proteins (FSSP) survey on all known π-helix-containing protein structures
in databases [68], in 106 proteins with π-helices, 88 were found to exhibit the α-helix
with one less amino acid residue, accounting for over 80%, which suggested that
nature π-helices may originate from the insertion of one residue into the correspond-
ing α-helices during evolution. Meanwhile, at least three residues could be found in
designated α-helices in over 95% of the analyzed π-helices by FSSP method, which
also suggested that there was a strong association between α-helices and π-helices
[69]. The hypothesis on the originality of π-helices was further confirmed by the phe-
nomenon of α-helix-to-π-helix conversion in some protein families. For example, in
mercuric ion reductases, an α-helix-to-π-helix conversion, which was attributed to
the insertion of a single residue compared to its ancient reductase member, occurred
and put a catalytic Tyr residue into the binding site and triggered the Hg2+ detoxifi-
cation by mercuric reductase [70].
Function of 𝛑-Helix Cellular function depends on highly specific interactions

between biomolecules (proteins, RNA, DNA, and carbohydrates). A basic limitation
of drug development is the inability of traditional “small-molecule” pharmaceuti-
cals to specifically target large protein interfaces, many of which are desirable drug
targets. α-Helices, ubiquitous elements of protein structures, play fundamental
roles in many protein–protein interactions. Stable mimics of α-helices that can
predictably disrupt these interactions would be invaluable as tools in chemical
biology and as leads in drug discovery. There has been exciting progress in the
molecular design of these protein domain mimetics and their remarkable potential
to inhibit challenging interactions in the past decade. Key challenges in the field
including identification of suitable targets and bioavailability of medium-sized
molecules do not conform to empirical rules followed in traditional drug design.
Stabilized α-helices avoid some of the strict limitations that have been placed on
drug discovery. When designing potential drug candidates, medicinal chemists
often adhere to the Lipinski rules, which stipulate that the molecular mass of a drug
should not exceed 500 Da. Recent findings suggest that large synthetic α-helices
can traffic into the cell and efficiently compete with cellular protein–protein
interactions, contrary to predictions based on the Lipinski rules. Although these

molecules have undoubtedly proven their value as probes for decoding biological
complexity, the next big question is whether these molecules can become thera-
peutics. This chapter discusses the applications of constrained helices, based on
properties of protein–protein interactions.
Although π-helices had lower frequency observed in protein structure compared
with other helix types, more and more results revealed that π-helices could exhibit
distinct functional importance, especially in the chelatase family of proteins. For
example, ferrochelatases from Bacillus subtilis, which catalyzes the insertion of Fe2+
into protoporphyrin to generate heme, contained a 10 amino acid residue-length
π-helix.
This π-helix was located near the active site of the enzyme at the distance of 10 Å,
whose residues were proven to have interactions with a hydrated magnesium com-
plex in its bound state [71]. Similar as ferrochelatases from B. subtilis chelatase, the
cobalt chelatase, which was responsible for the insertion of Co2+ into precorin-2 to
generate cobalt-precorin, also possessed a π-helix of seven amino acid residue length
[72]. Dioxygenase is another example, whose three histidine residues H494, H499,
and H504 on the surface of the π-helix were involved in the formation of a metal
binding site, which suggested that π-helices are the unique structural element for
metal binding enzymes.
Besides the chelatase family, π-helices contribute to diverse protein functions.
For example, an π-helix conformation was found in the hydroxylase component of
methane monooxygenase hydroxylase (MMOH), which participates in the binding
of a product analog 6-bromohexan-1-ol at the active site. The long π-helix contains
two π-helices named piB and piD, respectively. In the process of ligand binding,
a peristaltic shift of piD in an esophageal peristalsis-like manner was performed
in the C-terminal direction [73]. Similar phenomenon of π-helical shift could be
observed in the related toluene-4-mono-oxygenase (ToMO) hydroxylase structure
during its binding of the regulatory component (ToMOD) [74], which may provide
a new insight into mechanisms for the activation of bacterial multicomponent
monooxygenases (BMMs) by their regulatory subunits [69].
1.3.2 Characterization of Helical Peptides

The study of the molecular structure can give fine details about the interface that
enables the interaction between proteins. The molecular structures of many protein
complexes have been unlocked by the technique of X-ray crystallography [75]. Later,
NMR also started to be applied with the aim of unraveling the molecular structure
of protein complexes and it is advantageous for characterizing weak protein–protein
interactions [76].
1.3.2.1 Circular Dichroism

Circular dichroism (CD) is a simple and convenient method to analyze secondary
structures of proteins and peptides. Only chiral molecules can exhibit featured
absorption bands in their CD spectra while randomly oriented systems have no
CD intensity. This is why proteins and peptides, which are composed of chiral
16 1 Introduction
amino acid residues, show characteristic CD absorption according to their different

secondary structures. Because of the sensitivity and convenience of CD measure-
ment, CD has become a routine tool in many applications, especially in the study of
determining the helical content of proteins and peptides [77]. Generally speaking,
proteins and peptides in random or denatured structures usually have very weak
signals in their CD spectra, while proteins and peptides with well-defined and stable
conformations such as α-helix can give significant CD intensities and characteristics
in CD spectra. The featured structural characteristics in α-helix, including i/i + 4
hydrogen-bonding pattern, 3.6 amino acid residues per turn, 100∘ turn and 1.5 Å
translation between two peptide units, make α-helix be distinguished from other
structural motifs in proteins and peptides, which exhibits distinct band absorptions
in CD spectra. Absorptions at different wavelengths indicate different kinds of
electron transition such as n → π* , π → π* , n → σ* occurring at 220, 207–190, and
175 nm in CD signals, respectively. The α-helices display two separate negative
maximum signals at both 222 nm (the n → π* transition) and 208 nm (part of the
π → π* transition) as well as another positive signal at 195 nm (part of the π → π*
transition). Compared with α-helices’ featured absorptions, the other common
structural unit β-sheets show a negative band at about 220 nm with another positive
band at about 200 nm in the corresponding CD spectra. It is worth noting that the
prediction of α-helices in proteins and peptides by a CD tool is very sensitive and
has very high accuracy.
1.3.2.2 X-ray Crystallography

X-ray crystallography is a technique used for determining the atomic and molecu-
lar structure of a crystal, in which the crystalline atoms cause a beam of incident
X-rays to diffract into many specific directions. The method has revealed the struc-
ture and function of many biological molecules, including vitamins, drugs, proteins,
and nucleic acids. The crystal structures of proteins were first reported in the late
1950s, beginning with the structure of sperm whale myoglobin. X-ray crystallogra-
phy is now used routinely by scientists to determine how a pharmaceutical drug
interacts with its protein target and to unveil the detailed interface of protein–protein
interactions [78]. Navitoclax is an inhibitor of both BCL-2 and BCL-XL , but the con-
comitant on-target thrombocytopenia caused by BCL-XL inhibition limits the effi-
cacy achievable with this agent. Based on the crystal structure of this compound in
complex with BCL-2 family proteins, Andrew J. Souers et al. re-engineered and opti-
mized navitoclax to create a highly potent, orally bioavailable and BCL-2-selective
inhibitor ABT-199, which showed potent inhibition activity on BCL-2-dependent
tumors in vivo and spares human platelets [79].
The technique of single-crystal X-ray crystallography has three basic steps. The
first step is to obtain an adequate crystal of the molecule under study. The crystal
should be large enough (typically larger than 0.1 mm in all dimensions), regular in
structure, and pure in composition, with no significant internal imperfections such
as cracks or twinning, and this is often the most difficult step. In the second step,
the crystal is illuminated with a finely focused monochromatic beam of X-rays, pro-
ducing a diffraction pattern of regularly spaced spots known as reflections. As the
crystal is gradually rotated, previous reflections disappear, and new ones appear, the
intensity of every spot is recorded at every orientation of the crystal. In the third step,
the 2D images taken at different orientations are converted into a 3D model of the
density of electrons within the crystal using the mathematical method of Fourier
transforms; these data are combined computationally with complementary chemi-
cal information to produce and refine a model of the arrangement of atoms within
the crystal. The crystal structures could give essential details about the structures of
a molecule or macromolecular complex.
1.3.2.3 Nuclear Magnetic Resonance (NMR)

NMR is another fast and powerful method to determine conformations of proteins
and peptides in solution. In 1D and 2D NMR spectroscopies, nuclear overhauser
effects (NOEs), temperature dependence of the NMR spectrum, and coupling con-
stants are three important indicators for the determination of peptides’ secondary
structures [80]. Besides, measurements of the chemical shift index and the rate of
exchange with deuterons are also two regular methods for the conformation analysis
of peptides.
There is a very close correlation between NOEs and the distance between protons.
Generally speaking, NOE signals between two protons can arise when their distance
is under 5 Å in space, indicating strong evidences of secondary structures [81].
The cross-peaks in a 2D scalar-correlated (COSY) spectrum indicate the coupling
patterns for the resonances assigned to specific protons of amino acid residues.
Specifically, sequential connectivities and NH–NH as well as NH–CH cross-peaks
in 2D-ROESY spectra indicate key medium- and long-range ROEs, which strongly
supports the identification of secondary structures. Some representative nonse-
quential medium cross-peaks such as dαN (i, i + 4), dαN (i, i + 3), and dαβ (i, i + 3) in
ROESY spectra support the adoption of helical conformations [82].
The temperature dependence of amide proton resonances is utilized for determin-
ing whether the backbone amino acid residues are involved in the intramolecular
hydrogen-bonding formation. Generally speaking, amide proton resonances exhibit
higher temperature coefficients with increasing temperature, which suggests no
involvement of hydrogen bonds and the complete exposition to solvents. A value of
temperature coefficient (< −4.0 ppb/K) indicates the specific amide proton’s partic-
ipation in the hydrogen-bonding in H2 O, which is so critical for the stabilization of
an α-helix. Values of temperature coefficient for indicating the hydrogen-bonding
are different in various hydrogen-bonding solvents. Compared with H2 O, temper-
ature gradients for the chemical shift of the NH-signal is considered to be under
−3 ppb/K in dimethyl sulfoxide (DMSO).
Vicinal coupling constant 3 J has a close relationship with dihedral angle origi-
nated from the Karplus equations and is very important to analyze conformations
of proteins and peptides. Among these different types of coupling, the homonuclear
3
J HNCαH is easy to be measured and thus particularly important. However, according
to the Karplus curve, there is no one-to-one correspondence between measured cou-
pling constants and bond angles in spite of the dependence of 3 J HNCαH on the angle
𝜑. Indeed, in most cases, one coupling constant can respond to four different bond
18 1 Introduction
angles 𝜑. Therefore, it is ambiguous and hard to achieve compelling conclusions

from 3 J HNCαH measurements, thus viewed as a complement approach for analyzing
conformations of peptides based on the bond angle 𝜑 [83]. It is generally acknowl-
edged that the coupling constant 3 J HNCαH under 6 Hz for amino acid residues indi-
cates the adoption of helical conformations.
Besides, the chemical shift is an equally important index for the determination
of secondary structures of peptides and proteins. It is found that 1 H NMR chemical
shifts have a very close connection with the character of secondary structures. Com-
pared with the random coil value, the 1 H NMR chemical shift of the α-CH proton
of all natural 20 amino acids shows an upfield shift when they are involved in the
helical conformation. Conversely, a downfield shift of the corresponding 1 H NMR
chemical shift is observed when they adopt the β-strand conformations. Therefore,
these observations of 1 H NMR chemical shift characters are very helpful to deter-
mine the location, as well as compare contents of different secondary structural
motifs in proteins and peptides. Compared with traditional NOE-based methods for
secondary structure determination, this method simply requests the measurement
of α-CH 1 H resonance assignments and was proven to be accurate and useful by a
lot of examples [84].
Finally, the rate of exchange with deuterons like added D2 O is a convenient
method for measuring molecular dynamics of peptides. Specifically, the exchange
of amide protons with the deuteron solvent can tell whether these protons are
involved in forming intramolecular hydrogen bonds by measuring the corre-
sponding exchange rate. Generally speaking, the amide proton participating in
intramolecular hydrogen bonds is expected to exhibit lower exchange rate in
deuteron solvents while those protons absent from hydrogen bonds are exchanged
more rapidly [85].
1.4 Stabilization of Peptides

This section describes the methods of stabilizing peptides, including the cyclic and
main alteration.
1.4.1 Peptide Stabilization via Cyclization

1.4.1.1 Monocyclization
Macro cyclization uses additional covalent bonds to limit the distance between two
points. The cyclic increase of peptide resistance to protease reduces the exposure of
hydrogen bond donors or receptors to regulate the physical and chemical properties
of peptides.
Disulfide bonds sensitive to redox reactions are common in nature. The specific
pattern and length of the artificial disulfide bond can be used to stabilize the helix,
hairpin, or loop. Their reversible properties can be used to study the folding of pep-
tides or the uptake of cells [86]. At the same time, the biological system can directly
evolve disulfide bonds. Similarly, the hydrophilic amide or ester bond is widely used
in the construction of peptide analog as a bridge connecting the natural clock. In par-
ticular, the lysine-aspartic acid stable helix shows the highest helicity in the water.
As a kind of high stability bridge connecting mode, the thiol ether bond is often
used as the substitution of disulfide bonds to restrain peptide conformation. The
thiol ether bond can be constructed by using a nucleophilic substitution reaction
of halides or by using a free radical reaction of unsaturated hydrocarbons. In addi-
tion, compared with a component binding strategy, the two-component stabilization
strategies can give the peptide with special characteristics, such as reversibility and
modifying potentials [87]. Like disulfide, the two-ingredient strategy based on cys-
teine can be used in micro-proteins or combined with a screening system.
As a nonpolar connection, the all-hydrocarbon strategy increases the hydropho-
bicity of peptides. The stapled polypeptide, the all-hydrocarbon side chain stabilized
helix, is constructed by ring-closing metathesis. Various types of stapling strategies
have been developed and widely used. In addition, all hydrocarbon chains can be
used in replace of hydrogen bond as an N-terminal template. In addition, azoles, con-
structed using bio-orthogonal reactions, can be used to stabilize the peptide, includ-
ing the helix and hairpin structure [88]. In one-component way, the ring addition
can derive a fluorescent bridge. While for two-component strategy, the functional
stapled polypeptide can be established to further label [89].
1.4.1.2 Multicyclization
Increasing the number of polypeptide rings, such as construction of bicyclic or even
polycyclic peptides, can further improve the therapeutic performance of peptides
and even show the oral dosing potentials. There are many polycyclic peptides in
nature, and in the process of studying these polycyclic peptides, many valuable
techniques and molecules have been obtained. Here we will focus on the polycyclic
polypeptide in nature. There are many bioactive polycyclic peptides in nature,
including bacterial secretions, such as lantibiotics, toxic peptides, such as amatoxin
and phallotoxin, and peptide toxins of animal secretions, such as α-toxins.
Lantibiotics is an important polypeptide antibiotic that is found and produced by a
large number of Gram-positive bacteria (including Streptococcus and Streptomyces)
and attacks other Gram-positive bacteria. The peptide contains the characteristics
of multicyclic lanthionine or methyllanthionine and some unsaturated amino acid
dehydroalanine and 2-aminoisobutyric acid as the basic building blocks. Lanthion-
ine, abbreviation for lanthionine-containing peptide antibiotics, is one of the impor-
tant building blocks of lantibiotics, consisting of two alanine residues, which are
connected by sulfur to their β-carbon atoms [90]. Most of the lantibiotics are consid-
ered to be a class of bacteriocins synthesized by ribosome biosynthesis, with a pilot
polypeptide sequence that is removed when the molecule is transported out of the
synthetic cell. There are four enzymes involved in the construction of lanthionine
rings. This process differs from most of the other natural antibiotics we have pre-
viously known [91]. Lipid II, a glycoprotein of this gram-positive bacterium, is the
target for a large number of lantibiotics. Some lantibiotics causes destructive pores
or inhibits the biosynthesis of peptides. Nisin is a lantibiotics widely used in today’s
commercial preservatives.
20 1 Introduction
Cyclotides are a class of bioactive mini-proteins from plants that have the unique
topological feature of a head-to-tail cyclic backbone combined with intramolecular
disulfide bonds [92]. Because of this structure, they are ultra-stable against degra-
dation at elevated temperatures or in the presence of proteolytic enzymes and have
attracted interest as peptide-based templates for drug design. Their natural function
in plants is acting as insecticidal agents, which provides potential applications in
agriculture. Furthermore, they have a range of pharmaceutically relevant activities,
including anti-HIV, antimicrobial, and uterotonic activities. Their exceptional sta-
bility and facile synthesis make them pharmaceutical templates that can be grafted
into peptides with desired bioactivities.
Several poisonous peptides were found in several genera of poisonous mushrooms,
such as phallotoxin and amatoxin [93]. Unlike many ingested poisons, phallotoxin
and amatoxin are not destroyed by heat, so cooking poisonous mushrooms is no
less lethal. Phallotoxins consists of at least seven amino acids, which are also called
death cap mushrooms from the goose mushrooms. Amatoxins is present in the poi-
sonous death cap and goose mushrooms, consisting of eight amino acid residues.
This compound has a similar structure, with eight amino acid residues arranged
in a conserved double-ring skeleton. Snake venom α-toxin (α-BTX) is a neurotoxin,
which is a multicyclic peptide that is known to be competitive binding to the acetyl-
choline receptor (nachrs), which then causes paralysis of the victim, respiratory
failure, and death. α-Toxin has 74 amino acids with 52 sulfide bridges. Compared
with other snake venom α-toxin, it has a triple-fingered fold structure. Hydrogen
bonds keep the second and third loops roughly parallel to allow parallel β-sheets.
The three-fingered structure is conserved by four of two sulfide bridges, and the
number of these bonds and secondary structures is responsible for the stability of
the neurotoxin, which is not susceptible to degeneration and is proven to be resis-
tant to boiling and low-pH environments [94]. On the basis of detailed studies of the
structure and properties of these toxins, the researchers used these toxins as tem-
plates to embed other functional peptides into the template and to obtain a stable
polypeptide with an interesting function.
In addition to the presence of polycyclic polypeptides in nature, researchers have
developed a number of artificial techniques based on polycyclic peptides. Poly
cyclization can enhance the effects of cyclization on the physical and chemical
properties of peptides, such as the ability of researchers to obtain highly stable
versatile peptides or peptides with better penetration. Existing natural peptides,
mostly knottins and cyclotides, have been modified to derive peptides with new
recognizing abilities (Figure 1.3). Cysteine knot structures, which is a structural
motif comprising three disulfide bridges, with new molecular recognition properties
could be engineered either by molecular grafting of peptide epitopes or by directed
evolution strategies [96]. By replacing existing amino acids, cysteine knot-based
protease inhibitors were converted into inhibitors of homologous proteases [97]. In
addition, polycyclic polypeptide can be directly screened for evolution. In addition
to the stable polycyclic polypeptide of pure disulfide, as mentioned above, on-phage
peptide alkylation is able to realize the screening of a variety of two-ring peptide
libraries, which can generate two-ring peptide ligands with good binding affinity
(a) (b)
Figure 1.3 Epitope grafting in polycyclic peptides. (a) Schematic depiction of the approach
of epitope grafting. A cysteine knot protein is chemically synthesized or recombinantly
expressed with a peptide epitope inserted into one peptide loop. (b) Crystal structure of
porcine pancreatic elastase (PPE) in complex with the engineered trypsin inhibitor EETI-II.
The trypsin binding loop of the 28-amino acid inhibitor was substituted by a peptide
sequence derived from the third domain of turkey ovomucoid inhibitor that was optimized
to inhibit PPE (PDB entry: 1QNJ) [95]. Source: Baeriswyl and Heinis [95]. Reproduced with
permission of Wiley.
[48a]. Finally, poly cyclic peptides could be obtained through rational design. Pei
and coworkers used bicyclic polypeptide as a scaffold to combine cyclic targeting
peptide and cyclic cell penetrating peptides [98]. The derived two-ring peptide
has cellular permeability and retains the ability to identify intracellular targets.
For stapled polypeptides, the addition of a hydrophobic ring can further improve
the physical and chemical properties of peptides. Furthermore, the stability and
permeability of the poly cyclic polypeptides are higher than those of the single-ring
peptides.
1.4.2 Peptide Stabilization via Backbone Reconstruction

1.4.2.1 Methylation
Methylation can affect the physicochemical properties of peptides by dihedral immo-
bilization and hydrogen-bonded donor blockage. The main form of N-methylation
in nature is N-methylation in the polypeptide skeleton or side chain of lysine or argi-
nine with free amino group. For natural N-methylated peptides, N-methyl is mainly
in the trunk, especially the cyclic peptide. Many biological functional peptides are
N-methylated peptides, which are often observed in non-ribosomal peptides. The
N-methylation of lysine and arginine mainly occurs on functional proteins, which
are not discussed here.
First, from a molecular point of view, the N-methylation of NH on the polypeptide
skeleton blocks the potential hydrogen donor of NH, and if NH participates in
the intramolecular hydrogen bond, it will have a great effect on the peptide’s
secondary structure. This effect is particularly important for long peptides, which
introduce H-bonds to build peptide structures such as α-helix and β-slices. For
22 1 Introduction
example, Kapurniotu and coworkers utilized N-methylation into β-sheet peptides

to avoid its aggregation, which is assembled to form a fiber polymer [99]. However,
N-methylation does not destroy the intramolecular hydrogen bond of peptides, so
the additional N-methyl can help to stabilize the peptide from external disturbances
and further optimize the biophysical properties of the polypeptide.
Second, the introduction of N-methylation can increase the resistance and
stiffness of peptides. The addition of N-methylation on the polypeptide skeleton
will strongly reduce the cis–trans equilibrium of the N-methyl amide bond [100].
In addition, as the 3D conformation of the whole peptide, the steric resistance
of N-methyl substituted will further affect the adjacent residues. For example,
natural n-methylated amino acid, proline, the only amino acid where secondary
amine and the ring structure involved the backbone atom, plays an important role
in protein folding. PRO provides CIS amide bonds, which are usually used for
hairpin induction templates. The improvement of steric resistance and stiffness can
further improve the resistance to protease degradation and can maintain biological
function for a long time [101].
In addition, N-methylated peptide has a significant increase in cell permeability
and oral bioavailability. This exciting improvement may come from increasing the
hydrophobicity of the polypeptide skeleton and enhancing the interaction with the
lipid layer. In fact, most natural n-methylated peptides have high oral bioavailability
and are widely used as promising candidates for drug use. A typical n-methylated
cyclic peptide, cyclosporin A, shows 19% oral bioavailability. This improvement
is especially important for multiple N-methylation. Hoffman and coworkers have
established a multiple N-methyl library, with different N-methyl substitutions on
the cyclic hexa peptides. Caco-2 cells and parallel artificial membrane permeability
assay (PAMPA) assay disclosed intestinal permeability of the methylated peptide,
which can be further applied to the design of oral peptide therapy [102].
In addition, the addition of N-methylation can regulate the biological activity of
peptides, such as binding affinity. For peptide inhibitor design, peptides usually
bind to protein surfaces with certain conformation. The target’s functional domain
is typically specific, such as hydrophobicity. Therefore, the interaction of peptide
target binding affinity can be improved by the regulation of the conformation by
N-methylation and enhanced hydrophobicity.
According to the advantages of N-methylation, the researchers have done a great
deal of design and exploration of N-methylation to improve the biophysical function
of peptides.
Further, in addition to N-methylation, there is more general type of n-modified
peptide skeleton that can modulate and optimize the biophysical properties of
peptides called N-alkylation. In particular, the N-alkylation of polypeptide skele-
tons usually results in large changes in peptide structure. Therefore, in general,
N-alkylation can be used as another kind of peptoid. With the use of different func-
tional groups, N-alkylation seems to be more promising than single methylation.
However, due to the difficulty of synthesis, the application of N-alkyl is mainly
restricted. But N-alkylation peptides also show promising biological functions, such
as antibacterial activity.
In the position of methylation, in addition to N-methylation, another methyla-

tion method, α-methylation, can also be considered a design strategy. Similarly,
α-methylation can restrict dihedral angle by steric hindrance, which facilitates the
formation of turns or helices, as is shown by stapled peptides, where the bridging
module is α-methyl. In addition, Aib is often used in the induction of hairpin
structures.
1.4.2.2 Foldamers
For a long time, researchers have wondered whether there are other skeletons,
including modules that have not been chosen by biological evolution, and may have
the ability to support identification, catalysis, or assembly activities while showing
better applicability. The foldamer is based on the skeleton of de novo design, the
conformational order of the epitope simulator. Since many of these activities seem
to require precise spatial positioning of the side chains, foldamer studies tend to
begin with the determination of a class of low polymer with a specific shape.
The foldamers can be divided into two types based on the frame selection of
monomer unit. “Aliphatic” foldamers have a saturated carbon chain separating
amide or urea groups, and the use of aromatic septal skeletons, such as the multiple
pyrrole/imidazole DNA binding oligomer [103]. The foldamers of the aromatic
skeleton is closer to the small molecule and thus has better penetrability. Initial
monomer selection is usually influenced by their synthesis and the ease of struc-
tural characterization. In addition, systems containing identical monomer units are
called “homogeneous” foldamers, while “heterogeneous” foldamers contain more
than one type of subunit [104].
D-type amino acid is a kind of amino acid which is opposite to nature in α-carbon
configuration. Examples of D-type amino acids in nature include opiates and antimi-
crobial peptides from frog skins, snail neuropeptides, shellfish hormones, and spider
venom. These D-amino acids form when L-amino acids change after translation.
Molecular recognition in nature, such as the identification between enzymes and
substrates, antibodies and antigens, is configuration specific. Although the presence
of D-amino acids in nature, most proteins are made of L-amino acids, the rare but
unrecognized D-amino acid insertion makes it difficult for polypeptides to be iden-
tified by shear enzymes or antibodies, which can be used to increase stability or to
reduce the immunogenicity of peptides. On the other hand, it is also important to
ensure the original activity of peptides while improving the stability or immuno-
genicity of polypeptide. Some special strategies have been developed, such as retro
inverso peptide. The direct evolution of the D peptide sequence is also achieved
through the image phage display of D protein, which enables us to obtain D-peptide
with good stability and affinity [105].
Some peptides containing D-amino acids are biologically more powerful. The
ω-agatoxins IVB and IVC peptides that contain D-serine showed higher inhibition
of P-type calcium channel compared with those containing L-serine isomers [106].
As is known to all, β-hairpin is an important secondary conformation, which
can be used to achieve the biological process control. Correspondingly, scientists
have developed a number of ways to stabilize the structure of β-hairpins, mainly
24 1 Introduction
including the nucleation of the turn and the hairpin [107]. As already mentioned
in the preceding article, the induction nucleus requires a special dihedral module.
Dihedral preferences of D-amino acids are different so that they lead to changes
in the conformation of the peptide, which can be used to construct a peptide with
a β-turn or β-hairpin [108]. The scientists found that turn position i + 1 D-amino
acid was used to promote the type II β-turn, thus supporting the formation of
β-hairpins. Studies have shown that D-proline can be used as an auxiliary factor
for β-turn [109]. Templates such as D-pro-l-pro and D-pro-gly dipeptide fragment
are privileged peptides widely used to stabilize parallel β-hairpin, while D-proline
Dadme (1,2-diamino-1,1-dimethylethane) provides parallel β-hairpin [110].
β-Amino acid is another kind of amino acid in nature, which are frequently found
to be important components of bioactive natural products, such as Taxol, bleomycin,
and microcystin [111]. Adding β-amino acids to L-amino acids in natural products
produces unique structures with similar molecular polarity. In addition, similar to
D-amino acids, difficulty to recognize the β-amino acid peptide by protease renders
the peptide greater stability.
It is noteworthy that the use of β-amino acids in order to strengthen the β-sheet
secondary structure has proved to be extremely challenging due to the severe mis-
match between structural characteristics of α- and β-peptide. Because β-amino acids
have no preference for dihedral angle, which leads to a lower conformation change
and a higher flexibility. A single permutation mainly results in the partial stretching
of the structure, and the α,α-di-peptide permutation only provides a modest result
[112].
Recent studies have demonstrated that multiple a → b replacements at carefully
selected positions are able to generate potent hormone analogs with in vivo activ-
ities. Cheloha et al. evaluated α/β-peptide analogs of parathyroid hormone [PTH
(1–34)] that contained exclusively b3 residues in an aaab pattern (Figure 1.4) [114].
Johnson et al. examined α/β-peptide analogs of GLP-1, the activity of which could
be rescued by using ring-constrained b residues [115]. An analog containing five
cyclic b residues along with two Aib residues displayed high resistance to cleavage
by dipeptidyl peptidase-4 or neprilysin, the two in vivo GLP-1 degrading proteases.
As an early example, Gellman and coworkers reported the substitution of β-amino
acids in the phage-display-derived peptide, with hot residues in the last round of
an α-helix and the following turn structures [116]. The final peptide with increased
protease resistance proved the feasibility of the turn mimetic.
β-Amino acid insertion is a relatively less noticeable strategy that can be combined
with more common side chain cross-linking to produce synergistic effects such as
α/β-peptides based on the stapled BH3α-peptide, which contains a hydrocarbon
cross-linking to improve α-helical stability [117]. A fixed α/β-peptide can mimic the
structure and function of the mother’s α-peptide in its ability to enter certain types
of cells and block protein–protein interactions associated with apoptotic signals.
However, the α/β-polypeptide is nearly 100 times more resistant to hydrolysis than
parental stapled peptide. Similarly, β-amino acid insertion can improve the stability
of peptide stability by hydrogen-bonding surrogate strategies [118].
PTH (1–34)
α/β-PTH-1
α/β-PTH-2
GLP-1 (7–37)
α/β-GLP1
(a)
α residues β3 residues Cyclic β residues
O O O O
H H H H H
N N N N N
R O
N
H
(b) Aib (U) ACPC (X) APC (Z)
Figure 1.4 α/β-Peptide mimicry of G protein-coupled receptor agonists. (a) Primary

sequences of PTH (1–34), GLP-1 (7–37), and biologically active α/β-peptide analogs.
Colored circles indicate nonnatural residues, as indicated in (b). (b) Structures of a generic
residue, the Aib residue (green), a generic β3 residue (blue), and cyclic β-residues ACPC and
APC (orange) [113]. Source: Checco and Gellman [113]. © 2016, Elsevier.
The effects of β-residues replacement on the structure and stability of small pro-
teins were studied by Kreitler et al. [119]. First, they evaluated the effect of α → β
modification on the structure and stability of small-study villin-helmet subdomains.
The original state of these 35 residual poly peptides consists of several α-helical seg-
ments wrapped around a small hydrophobic core. After that they examined the α → β
replacement in four solvent exposure positions. In each case, the natural α-residue of
the β3 homologs and the cyclic β-residues were evaluated. According to the variable
temperature CD spectrum, all α → β3 substitutions result in severe instability of the
tertiary structure, although in these locations, the replacement of β3 residues with
cyclic β-residues improves stability. These findings contribute to a basic α-/β-peptide
knowledge base that confirms that β3 amino acid residues can be used as effec-
tive homologous α-amino acid residues in structural simulations in natural tertiary
structures, which support the rational design of the function of natural peptides
α/β-analogs.
Foldamer based on heterogeneous backbone has some benefits relative to only
relying on homogeneous skeleton. Heterogeneous methods allow many different
combinations, which provide a potentially unique way to extend the side chain in
space. By mimicking proteins, valuable foldamer activity is likely to depend on the
placement of a particular 3D functional group; therefore, we can generate a molecule
of clearer shape through foldamers, which is more likely to achieve any particular
activity better. A variety of complementary scaffolds may be required to produce a
wide range of foldamer functions, but the diversity of the skeleton is not sufficient to
achieve this goal; people must also be able to decorate skeletons with different side
chains. The heterogeneous backbone method can greatly promote the generation of
foldamer sets with extensive side chain diversity.
The progress of folding structure synthesis technology, as well as the possi-
ble folding of the backbone in water, have opened up the way for the selective
foldamer–biomolecular interaction and foldamers interfering biological function
26 1 Introduction
[120]. Foldamers set many features in one, so that it can better function with
biomolecules: medium size (MW = 500–5000 g/mol) and large contact areas are
suitable for binding in the extended contact surface of protein, folding predictability,
adjustable type, diversity, and expected resistance to protein hydrolysis. Because
they are structurally well defined, foldamers can be used as scaffolding to accurately
project a combination module in space. Some early work focused on the design of
cationic amphiphilic foldamers, mimicking host–defense peptides and selectively
disrupting bacterial membranes. A new and challenging foldamer application mim-
ics the discovery of folding peptide fragments in proteins, particularly α-helices, to
interfere with protein–protein interactions.
Arora et al. have developed oxopiperazine helix mimetics (OHMs) for modu-
lating α-helical domain of HIF-1a-mediated protein–protein interactions [121]
(Figure 1.5). These oligomers downregulated hypoxia-induced gene expression
and reduced tumor volume in mice bearing MDA-MB-231 xenografts. Wilson and
coworkers have reported significant recent advances in the development of aromatic
oligoamide mimics of short helices containing N-alkylated para-aminobenzoate
units, which was pioneered by Hamilton and coworkers [124]. The three alkyl
groups were intended to reproduce the presentation of three spatially consecutive
side chains along the side of an α-helix. Mimics that engage the p53-recognition cleft
on HDM2 or the BH3-recognition cleft on Mcl-1 showed cell-membrane-penetrating
activities.
Self-assembly has become a very effective method to produce large supramolec-
ular containers with molecular recognition properties. Such containers may be
HN
H
N
N O
O N
O
N O
O N
O O
NH2 NH2
O NH
OH
HIF-1α helix OHM HIF-1α mimic O

(PDB: 1L8C)
p53 helix Aromatic oligoamide
(a) (b) (PDB: 1YCR) p53 mimic
Figure 1.5 α-Helix-mimetic polyamides. (a) Cartoon representation of a portion of the

HIF-1a α-helix (left, PDB: 1L8C) and drawing of an OHM mimic of this helix (right) [122].
Critical protein-contacting side chains are highlighted in red. (b) Cartoon representation of
a portion of the p53 α-helix (left, PDB: 1YCR) and drawing of an aromatic oligoamide mimic
(right) [123]. Critical protein-contacting side chains are highlighted in red. Source: Checco
and Gellman [113]. Reproduced with permission of Wiley.
References 27
based on very simple building blocks and usually have high symmetry. Similarly,
foldamers opens up new avenues for receptor design. The identification may occur
on the foldamer surface or in the cavity of its folding structure. An important class
of foldamer receptors includes a wide helix with a cavity. The groundbreaking
work of Moore and coworkers shows the combination of hydrophobic objects
in oligo-phenylene-ethynylene cavities [125]. Li, and coworkers reported that
saccharides can be bound into aromatic oligomers [126]. These helical receptors are
chiral in nature and can eventually discriminate between different enantiomers.
From the material point of view, foldamers can form materials with morpho-
logical characteristics on nano- or microscale by controlling the self-assembly of
molecules. It is reported that some aliphatic and aromatic foldamers spontaneously
form nano-fibers and nanoparticles [127]. For example, the hydrazide-based
aromatic foldamers containing a long aliphatic side chain exhibits a two-mode
assembly, forming vesicles in polar solvents and forming entangled fibers and gels
in hydrocarbons. Several of the 14-helix β-decapeptides with different combinations
of the lipophilic and hydrophilic side chains are assembled to form a lyotropic
liquid crystal phases in the aqueous solution.
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35
Construction of Constrained Helices
2.1 Side-Chain Cross-linking

Protein–protein interactions (PPIs) play pivotal roles in mediating intracellular bio-
logical processes, and targeting dysfunctional PPIs is broadly utilized for therapeu-
tics development [1]. However, small molecule ligands are less likely to interrupt
PPIs with large, shallow, or discontinued surfaces, which make many PPIs “undrug-
gable” [2]. Over 50% PPIs involve α-helices interactions, however, although numer-
ous peptide-based drugs are commercial, they suffer from poor stability and cell
permeability, which significantly became an obstacle in precise modulation of intra-
cellular targets [3]. Therefore, developing peptide stapling technology which was
demonstrated to be able to stabilize the protein secondary structures is an active
research field to achieve clinically relevant therapeutic agents for decades [4].
One of the most established approaches to mimic these molecular recognition
motifs is to stabilize and recapitulate their functional conformation via the side
chain–side chain or terminus–side chain. Preorganization of peptides into their
functional conformation is reported to increase target binding affinity and protease
resistance, thus leads to a prosperous research area [5].
2.1.1 Disulfide Bond

As a native bond, disulfides in proteins or peptides play a crucial role in the main-
tenance of conformation stability and biological activities. Accordingly, many pio-
neering studies have focused on the development of disulfides as constrains to tune
properties of peptides or proteins.
The series of oxidation, reduction, and disulfide reshuffling reactions lead to the
native set of cysteine residues. Disulfide-containing small molecules or electron
accepting reagents lead to various intermediate species, which contribute to
disulfide bond formation [6] (Figure 2.1). Among the methods of the formation for
disulfide bonds, air oxidation in water is the most commonly used strategy [7]. As
air oxidation usually requires a long duration, the addition of a mixture of reduced
and oxidized glutathione or even stronger reagents such as stronger oxidizing
agents such as I2 and K3 Fe(CN)6 can promote the thiol-disulfide interchange
reactions [8]. And this oxidation strategy is satisfactory within acid conditions.
Cyclized Helical Peptides: Synthesis, Properties, and Therapeutic Applications, First Edition.
Zigang Li, Hui Zhao, and Chuan Wan.
© 2021 WILEY-VCH GmbH. Published 2021 by WILEY-VCH GmbH.
36 2 Construction of Constrained Helices
RS-S
RS-SR S–
S– S
S
S–
RS–
O2
H2O2
HO-S
Unfolded state Native state
–
S
Intermediate species
Figure 2.1 Oxidative folding pathways mediated by disulfide-bond small molecules or

electron-accepting reagents [6]. Source: Gongora-Benitez et al. [6]. © 2014, American
Chemical Society.
Oxidation of cysteine by dimethylsulfoxide (DMSO) had been reported as a novel

efficient method [9]. In general, as the chemical activity of sulfydryl, the cysteine
at appropriate position can be easily oxidative to disulfide in mild conditions with
various reagents to be chosen. Furthermore, as a nature-derived bond that own
DNA encoded building blocks, disulfide bridged peptides can be derived in oxidative
biological systems, which makes them compatible with evolution techniques [10].
There are different modes of disulfide stapling techniques. In general, the forma-
tion of disulfide bond is based on the two active cysteine with dissociative sulfydryl
at position i,i + 3, i,i + 4, or i,i + 7 [11]. Most stabilizing linkers are at the position of
i,i + 4 residues to derive a single turn α-helix, while for relative long peptide or other
applications, the cross-linking position at i,i + 7 is also used as important strategy
for stapled peptide to get a two-turn α-helix. Generally, the i,i + 7 strategy is rela-
tively difficult to synthesis as the increased length of linker. In 1991, Schultz utilized
the disulfide strategy to synthesis a short peptide containing a two-turn α-helix by
a single intramolecular disulfide bond bridging the i,i + 7 residues, which serve as
the presentation of i,i + 7 disulfide-stapled strategy [12]. They use the i,i + 7 cou-
pling to synthesis four short peptides and their helical content showed a dramatic
increases upon formation of disulfide bond by circular dichroism (CD) spectrum.
Finally, many polycyclic peptides in nature, such as cysteine knot protein, are sta-
bilized by disulfides. They provide excellent scaffolds to be engineered to derive
artificial highly stable polycyclic peptides.
The disulfide bond cross-linking showed no perturbation toward the bioactivity
of peptide. An early research by Wemmer using disulfide bond staple strategy to
folding the active bioactive peptides demonstrated that the use of disulfides to stabi-
lize peptide fold did not interfere the activity and function of peptide by bee venom
peptide apamin and S-peptide of ribonuclease A [13]. In some cases, the disulfide
linker of stabled peptide may improve the bioaffinity of peptide for target proteins,
Spatola and coworkers in 2003 utilize the i,i + 3 disulfide-stabled strategy to design
the potential peptide inhibitors for estrogen receptor α (ER-α) with positive results
[11]. So the disulfide linker can improve the helical content of peptide and their
bioactivity such as affinity. Finally, cyclic peptide ligands of targets of choice can be
generated by inserting artificial binding sites into cysteine-containing peptide scaf-
folds, either monocyclic or polycyclic, using rational design or directed evolution
approaches [10].
Thanks to the easy formation and staple-on/off state in oxidation/reduction con-
ditions, disulfide bond can be used as a regulator to control the secondary structure
of peptide in different conditions, or sensor for bioconditions in vivo. For example,
Schneider and coworkers designed a disulfide bond-based stapled peptide, with a
fluorescent group on one side and its quencher on the other. In oxidative environ-
ment, the stapled peptide closer the distance of fluorescence and quencher, while in
reductive environment the reductive disulfide bond makes the fluorescence go free
from its quencher light again [14].
To sum up, the advantages of disulfide bond include the wild reactive condition
and relative high efficiency of formation, convenient exposed sulfydryl from native
cysteine, and the bioactive disulfide bond with high biocompatibility can be fur-
ther designed as regulator for sensing and other applications. However, as the easy
reduction of disulfide bond, the stability of peptide stapled by disulfide bond may be
relatively low, which makes the number of disulfide bond-stapled peptides limited.
Meanwhile, just thanks to the on and off transition in oxidation/reduction condi-
tions, it can be used as a regulator or sensor for different conditions. Finally, build-
ing blocks being encoded by DNA made disulfide-bridged peptides evolutionizable
by screening techniques, which is highly favorable for discovery of molecules with
desired properties. In this sense, cysteine-containing peptides are one of the most
malleable start point for ligands of interest.
2.1.2 Amide and Ester

α-Helical conformation of peptides can be stabilized or induced by introducing cova-
lent links between amino acids side chains through amide bond. Amide bonds are
the key chemical connections of proteins, and the amide bond formation is one of the
most important reactions in peptide synthesis [15]. The current methods for amide
bond formation are remarkably general. In the peptide field, the development of
solid-phase peptide synthesis (SPPS), and the improvements in coupling reagents,
protection groups have made the amide bond formation more easily. Compared to
solid-phase peptide chemistry, the convergent approach of solution-phase methods
allows a greater variety of ways to meet the requirements for selective protection and
de-protection of the backbone and side-chain functional groups that are essential
to the synthesis of lactam-bridged peptides. In addition, solution-phase syntheses
of cyclic peptides allow direct monitoring of the progress of each reaction step, the
purification of key intermediates as necessary, and a high level of control over the
reaction conditions. In particular, the concentration of the peptide during the critical
38 2 Construction of Constrained Helices
cyclization reaction can be adjusted to any level necessary to maximize the yield by
minimizing interchain reactions. However, most peptide chemists have preferred to
adapt and develop the more expedient solid-phase approach to prepare such cyclized
peptides for research applications.
Amide bonds in lactam-bridged peptides are chemically inert to most of the condi-
tions to which peptides are subjected. Second, the availability of a wide variety of pro-
tecting groups for amines and carboxylic acids that are cleavable under orthogonal
or highly selective conditions makes the preparation of complex multicyclic peptides
with overlapping bridge constraints more manageable than would be the synthesis
of peptides incorporating multiple disulfide bridges. Perhaps the simplest and most
common method of enhancing helical structures in long (>15 residues) peptides
has been the use of side chain-to-side-chain amide linkages [16]. This has generally
involved forming a covalent bond between side chains of lysine/ornithine residues
and aspartic/glutamic acid residues, separated by two (i/i + 3) or three (i/i + 4) inter-
vening residues, or through longer linkers between i and i + 7 residues. In 1987,
Baldwin and coworker [17] demonstrated that Glu-Lys salt bridges spaced i,i + 4 in
stabilized helical conformation of short alanine-based peptide. Covalent amide bond
in this circumstance could be a better rigidified bridge.
In 1988, in the pioneering investigation of a helix-stabilizing effect for side-chain
lactam bridges, Asp, Lys side-chain linkages, and the reverse Lys, Asp side-chain
linkages were incorporated to link residues 8 and 12 and residues 21 and 25,
respectively, in human growth hormone releasing factor (GRF) (1–29) analogs
[18]. Demonstrated by CD and nuclear magnetic resonance spectroscopy, peptide
segments in the N-terminal region at residues 7–14 and in the C-terminal region
at residues 21–28 naturally favored the helical conformation. The side-chain
lactam bridged GRF analogs were synthesized through Boc/benzyl chemistry,
where BOP/HOBt coupling reagents, instead of DCC/HOBt, were used for the
cyclization. This lactam bridge was formed on the resin using a combination of
Fmoc deprotection followed by cyclization with BOP/HOBt. This covalent link was
found not only to enhance the bioactive helical conformation of this peptide but
also led to increased biological potency. This strategy also allowed the synthesis of
dicyclic peptides, in which two side-chain lactam bridges were incorporated into
GRF analogs in nonover-lapping positions in the peptide chain [19]. Interestingly,
certain lactam-bridged GRF analogs with larger ring sizes of up to 24 atoms also
appear to have a helix-stabilizing effect, based on their reported CD spectra. And
they found for helix stabilization, the 20-atom ring formed by an (Asp-Lys in
i,i + 4 position) or a (Lys-Asp in i,i + 4 position) bridge is optimal. Smaller ring
sizes (19 atoms or less) are clearly helix destabilizing. Numerous groups have used
the Boc/benzyl/Fmoc protection strategy since the initial report by Felix et al.,
but PyBOP or other modern coupling reagents of the phosphonium ion type,
to avoid generating carcinogenic byproducts, have generally replaced the BOP
reagent. O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluorophosphate
(HBTU) or O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate
(TBTU) have also been used successfully as the coupling reagent for cyclizations.
Similar orientation investigation had been conducted on Lys-Glu combinations. In
1995, Hodges and coworkers [20] made a systemic study of a series of 14 residue
amphipathic α-helical peptides, in which the side chains of Glu and Lys have been
covalently joined, was synthesized in order to determine the effect of spacing,
position, and orientation of these lactam bridges. Adding one methylene group
to the ring by using a Lys-Glu side-chain linkage (21 atoms) has been reported to
give lesser helix-stabilizing effects or no helix stabilization at all, although 21-atom
bridges having the reverse orientation (Glu-Lys) appear to have a strong stabilizing
effect. While peptide sequences longer than 10 residues have been constrained to
varying degrees into α-helical conformations, shorter peptides have rarely been con-
strained into α-helical turns, independent of concentration, 2,2,2-Trifluoroethanol
(TFE), denaturants, and proteases.
Since 1990, Taylor and coworker had reported a series of systematic studies on
different pairs in stabilizing helical peptides, both via single or multiple bridges. In
1990, Taylor and coworker [21] described the synthesis of an amphipathic peptide
containing three Lys-Glu lactam bridges prepared by a combination of cyclization
on a p-nitro benzophenone oxime resin followed by segment condensation. In 1992,
the same group reported that linking the side chains of three Lys-Asp or Lys-Glu
pairs to form multiple lactam bridges stabilizes the α-helical conformation in aque-
ous solution in a 21-residue peptide at neutral pH in the order Lys-Asp ≫ Lys-Glu
bridges > no bridges [22]. Two years later, Taylor and coworkers [23] reported the
Nuclear Magnetic Resonance Spectroscopy (NMR) structure of bicyclic helical
peptide constrained through two i,i + 4 side chain lactam bridge. Similarly in
1996 [24], Taylor group reported a conformationally constrained bicyclic peptide
containing two overlapping i,i + 7 side-chain lactam bridge. The CD spectrum of the
product indicated high helical conformation. In 1999, they [25] reported a similar
stabilizing strategy by using aromatic groups in the diamide linkers between i,i + 7
amino acids, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid receptor
(AMPA) was the best linker among them. The placement of even two lactam
bridges in overlapping positions in a synthetic peptide structure can produce highly
stabilized, unique conformations in intermediate-sized peptide structures. Notably,
the interchain reactions that result in polymeric by-products during solid-phase
cyclization reactions can often result in dramatically low yields when a second
cyclization reaction is performed during a single peptide chain assembly. This
makes the synthesis of bicyclic lactam-bridged peptides by solid-phase methods
significantly less reliable than that of monocyclic peptides. To combine the advan-
tages of rapid peptide chain assembly on solid supports with the advantages of
convergent syntheses by solution-phase methods, Taylor group have proposed the
synthesis of protected, side-chain cyclized peptide building blocks by using the
Kaiser oxime resin [21, 22]. p-Nitrobenzoyl oxime ester and this group is used to
link the side-chain carboxylic acid of one bridging amino acid to the resin. The
peptide chain is built up using Boc/benzyl protection chemistry. Then cyclization
with concomitant cleavage of the protected monocyclic product is achieved by
selective deprotection of the amine component of the bridge, which is then allowed
to react with the oxime ester linkage. Then cyclization with concomitant cleavage
of the protected monocyclic product is achieved by selective deprotection of the
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fell around him. Suddenly he dropped, and hearts sank, thinking his
brief career ended; but he had only tripped over some obstacle. Often
he stumbled, sometimes he fell prostrate, but was quickly up again,
and finally disappeared limping, over the summit, and the Fifty-fifth
saw him no more for several months. As the boy sped away the last
time, the colonel shouted to him: “Bring calibre fifty-four.” General
Sherman’s letter to the War Department will tell the rest of the story.
Headquarters Fifteenth Army Corps,

Camp on Big Black, Aug. 8th, 1863.
Hon. E. M. Stanton, Secretary of War,

Sir:
I take the liberty of asking through you that something be done for a young lad
named Orion P. Howe, of Waukegan, Illinois, who belongs to the 55th Illinois, but
is at present from home, wounded. I think he is too young for West Point, but
would be the very thing for a midshipman.
When the assault at Vicksburg was at its height, on the 19th of May, and I was
in front near the road which formed my line of attack, this young man came up to
me wounded and bleeding, with a good, healthy boy’s cry: “General Sherman,
send some cartridges to Colonel Malmborg, the men are all out.” “What is the
matter, my boy?” “They shot me in the leg, sir, but I can go to the hospital. Send
the cartridges right away.” Even where we stood the shot fell thick, and I told him
to go to the rear at once, I would attend to the cartridges, and off he limped. Just
before he disappeared on the hill, he turned and called to me as loud as he could:
“Calibre 54.”
I have not seen the boy since, and his colonel gave me his address as above, and
says he is a bright, intelligent boy, with a fair preliminary education. What
arrested my attention there, was, and what renews my memory now, is, that one
so young, carrying a musket-ball wound through his leg, should have found his
way to me on that fatal spot, and delivered his message, not forgetting the very
important part even of the calibre of the musket, 54, which you know is an
unusual one. I’ll warrant the boy has in him the elements of a man, and I
commend him to the government as one worthy the fostering care of some of its
national institutions.
I am, with respect, your obedient servant,
W. T. Sherman,
Major-General Commanding.
BE PATRIOTIC.
It may be, my boy, that you will never be able to guide a regiment
of soldiers as did Nathan Beman, or carry cartridges as did young
Howe, but that is no reason why you should not be just as patriotic.
That boy who is law abiding, who opposes everything that tends to
undermine the national fabric, who decries Sabbath desecration, vile
language, bad literature, and all vices, is a patriot in the true sense of
the word, and can be relied upon in times of peace as well as war to
do his best for the country.
Be patriotic. Cultivate the spirit of admiration toward the national
flag. Dowered with priceless traditions its stars and stripes speak of
the sufferings of the past, the prosperity of the present, and the
glories of the future which shall attend the onward march of this
great Republic. It is the hallowed emblem of the world’s greatest
nation, and of its most resplendent civilization. Of Sherman it was
said that he never failed to salute the flag by taking off his hat in its
presence. That flag is the emblem of all we are and all we expect to
be.
“It floats that all the rights of men may every people bless
And God’s own kingdom walk the world in peace and righteousness.”
Be patriotic. Study the questions that have a bearing upon the

well-being of the people. In the past hundred years, more than
twenty-three million foreigners have settled in this land. Many are
God-fearing men, but many more are entirely out of harmony with
our principles and institutions. Truly America is
“The mother with the ever open door,
The feet of many nations on her floor,
And room for all the world about her knees.”
Of the seventy million inhabitants twenty-five per cent. are yet in

gross ignorance, thirteen per cent. cannot read the ballots they cast,
and thousands of such are annually coming to our shores, imbued
with the notions, failings and vices of their native lands. True
patriotism desires and labors not only for a free people, but an
educated one.
To be patriotic requires candor. We must be fair in our judgment
of others who may differ from us concerning methods of dealing with
some vital questions which are always before the nation. We do not
always see and understand alike, but we must strive to promote and
preserve the integrity of the nation. In the opening hours of the
French Revolution Mirabeau roused the rabble of Paris, which
whirled the social order into chaos, provoking Madame Roland’s
dying words, “Oh, liberty, what crimes are done in thy name!” We
have Mirabeaus here, but as educated lovers of our country, we must
antagonize wrong, uphold right, and defend the principles of the
Declaration of Independence.
To be patriotic in the true sense is to permeate every question with
Christianity. It was religious liberty that became the mother of
political liberty in England. De Toqueville said, “America’s liberty
considers Christianity the guardian angel of her struggle and victory,
the cradle of her life, the Divine source of her right.” “God and my
country” is the true patriot’s cry. In the words of the almost forgotten
Oliver Ellsworth to the Grand Jury of Savannah in 1779, “Let us rear
an empire sacred to the rights of men; and commend a government
of reason to the nations of the earth.”
PART III
Relation to God
CHAPTER XXI
Be a Christian
INTRODUCTION TO CHAPTER XXI
By Samuel Fallows
“Early let me seek Thy favor;
Early let me do Thy will;
Blessed Lord, and only Savior,
With Thy love my bosom fill;
Blessed Jesus,
Thou hast loved me, love me still.”
What is it to be a Christian? It is to be born again. What is it to be

born again? The New Testament gives the answer. He that “believeth
that Jesus is the Son of God is born of Him.” (1 John 5:1). He “that
loveth is born of God.” (1 John 4:7). He “that doeth righteousness is
born of Him.” (1 John 2:29).
Faith, love righteousness and trust in Christ, love for Christ, right
deeds through this faith and love in every sphere of life, deeds of
justice, of mercy, of goodness, of purity, of charity for the welfare of
his fellowmen,—these make a Christian.
Be such a Christian, my boy. Be a trusting, brave, noble, strong,
gentle, pure, loving and self-sacrificing follower of Jesus Christ.
CHAPTER XXI
Be a Christian
Having fairly embarked on the voyage which ceases not till the port
of eternity is reached, it is an exhibition of good seamanship to take
one’s bearings. By the log is estimated the progress of the vessel; by
the compass, the direction the ship is pursuing, and by the altitude of
the stars the latitude in which it is. In like manner the Moral
chapters indicate the progress boys should make; the Social, the
course they should take, and the Religious, the latitude in which they
should live. Of these the religious are the most essential, for a boy
cannot be truly religious without being moral and social.
When the Rebellion began a young man went to his mother and
said: “Mother, may I volunteer? I argue the matter in four plain
ways. First, my country needs me. Second, she calls me. Third, I am
able to go. Fourth, I am willing. This makes the duty very clear to me,
unless you interpose a veto, and I think you are too good a patriot to
do that.” She gave her consent, and before he departed, she said:
“You know, my son, how much I have wished to see you a Christian.
Now I want you to look at the claims of Jesus exactly as you have
looked at those of your country, simply and honestly, and see if those
same four plain propositions will not lead you into the service of
heaven.” “I’ll think of it, mother,” was his answer, and they parted.
He did not forget his promise. On his first Sabbath in camp he
resolutely set himself to the fulfilment of his mother’s request.
Remembering how he had argued duty to his country, he brought
before his mind in the same manner the subject of the divine claims
upon his heart and life. “Does Jesus want me? Does He call me? Am I
able to serve Him? Am I willing?” With an open Bible, the first three
questions were quickly answered. At the last one he hesitated, but
duty seemed so clear that he dared not falter, and falling on his knees
he gave himself to Christ. The next letter home announced him to be
a Christian soldier.
A CHRISTIAN.
Many names and titles are significant, but none means so much or
has so much honor attached to it as the word “Christian.” Young
said, “A Christian is the highest style of man.” A Christian is a Christ-
lover and a Christ-worshipper, because he sees God in Christ, and in
the God-man he sees the world’s Redeemer and his own personal
Saviour. He lives in the world, but is not of the world. While in the
world he blesses it by living a godly, upright life. His life work and
influence are a benediction to those among whom he moves. His
purpose is “not to make a living,” as Governor Russell, of
Massachusetts, used to say, “but to make a life.” He is far more
concerned about this than about dying. Death is the least of his
concerns. To live is Christ, and because of this, his life is proof of his
profession.
HOW TO BE A CHRISTIAN.
To become a Christian is not a hard matter, though to live the life

of one is a battle with the world, the flesh and Satan. It is because of
the simple rules laid down whereby one can become a Christian that
many of mature life neglect it. Were it culture, polish, or liberality,
many more would be enrolled as Christians, but because a change of
heart, affections or living is demanded, many cling to their ordinary
life, but at the last deplore it, earnestly pleading for forgiveness and
acceptance by Christ.
Three propositions are given in the New Testament, which,
accepted, will lead any boy to know what it is to be a Christian. First,
repentance: “Jesus came into Galilee, preaching ... repent ye.” (Mark
1:14, 15). Repentance means such sorrow for past conduct as leads to
amendment of life. Second, confession of sin. “If we say we have no
sin, we deceive ourselves, and the truth is not in us. If we confess our
sins, He is faithful and just to forgive us our sins, and to cleanse us
from all unrighteousness.” (1 John 1:8, 9). Third, faith in Christ to
save. Paul said to the jailer, “Believe on the Lord Jesus Christ, and
thou shalt be saved.” (Acts 16:31).
A father and son were once following a perilous path among the
Alps. In passing along they gathered some beautiful flowers, but the
boy, seeing a lovely one waving in the breeze, thoughtlessly hurried
to secure it. His foot slipped and he rolled down an incline until he
was stopped by some tall bushes. With all his strength he seized hold
of the shrubbery and commenced to call for help. The brush grew on
the brink of a yawning abyss. It was impossible for the father to
reach his son with his hands, but he carried a staff on one end of
which was an iron hook. The boy had around him a leathern belt, so
the father reached down and fastened the hook in his girdle. The lad,
however, could not be drawn up without releasing his hold on the
bushes. He could not see his father, nor did he in his fright even feel
that his father held him up; he only heard his voice: “Let go of the
bushes, my son, and I will save you.” To the boy it seemed as though
he would thus hurry himself to destruction, but, relying on his
father’s word, he forsook his hold and was drawn in safety to his
father’s side.
That boy was saved through faith. His firm belief in his father’s
word saved him. Had he persisted in holding on to the bushes
through doubt or hesitation it would have meant his death. To be
saved, every boy must forsake his hold on sin, yield himself to
Christ’s power and mercy, and then will he find to his joy, that Christ
saves to the uttermost. (Heb. 7:25).
THE TIME TO BE A CHRISTIAN.
Solomon said there is “a time to every purpose under the heaven,”

(Eccl. 3:1) and no purpose is greater and no time more important
than when a boy becomes a Christian. Youth is the most important
period of one’s life. It is the time when the faculties are most
susceptible, heart tender and will pliable; the time when tastes and
biases are created, habits acquired and character formed for future
weal or woe. No other period affords greater possibilities of long
usefulness as well as opportunities for peculiar usefulness.
A staff-officer, riding over the field of battle during the Civil War,
was attracted by a body lying under a tree, handsomely dressed, with
a fancy sword. He removed the covering and looked into the sweetest
and handsomest face he had ever seen. It was that of a boy, a
temporary aide to some officer. In his pocket was found a Testament
in which was written “James Simmons, N. Y. My son, ‘Remember
now thy Creator in the days of thy youth.’” (Eccl. 12:1).
That is it, youth. The best and most profitable time for piety.
Jeremiah and John the Baptist loved and worshipped God in their
youth. Josiah knew the Lord at eight years of age. Timothy knew the
Scriptures and loved Christ from a child. Polycarp accepted Christ at
nine, Jonathan Edwards at seven, Isaac Watts at nine, Adam Clarke
at four, William Penn at nine, Matthew Henry at eleven, Robert Hall
at twelve, Augustus Toplady at sixteen, while Joseph Griggs not only
became a Christian very young but wrote the hymn—
“Jesus! and shall it ever be
A mortal man ashamed of Thee!”
when but ten years of age.

Some years ago the “Golden Rule” sent letters of inquiry to
prominent men of the land asking several questions, one of which
was: “At what age did you become a Christian?” It was found on
receiving the answers that out of one hundred and forty-nine less
than one in ten became Christians later than twenty years of age;
twenty-nine were so young that they did not remember; at least
sixty-three professed Christ before they were eighteen. Nine-tenths
of all saved persons are saved before twenty. “Why this?” you ask.
Physiologists say “the cells of the brain change as we grow old until
finally there are ruts in them.” Carlyle explains it thus: “In younger
years the whole mind is, as it were, fluid, and capable of forming
itself into any shape that the owner of the mind pleases. The mind is
in fluid state, but it hardens up gradually to the consistency of rock
or iron, and you cannot alter the habits of the old man, for as he
began he will go on to the last.” To procrastinate in youth is to
jeopardize one’s soul in age.
“REMEMBER.”
“Remember now thy Creator in the days of thy youth,” (Eccl. 12:1)
is the most important exhortation of the Old Testament. Remember
is just the opposite of forget, and the one to remember is the most
exalted and important in the universe, “thy Creator.” Remember His
Word and believe it, for the promise is: “He that heareth My Word
and believeth on Him that sent Me, hath everlasting life.” (John
5:24). Remember His work and accept it, for He was made to “sin for
us, Who knew no sin; that we might be made the righteousness of
God in Him.” (2 Cor. 5:21). Remember His love and return it, for
“herein is love, not that we loved God, but that He loved us,” (1 John
4:10) and “gave His only begotten Son, that whosoever believeth in
Him should not perish but have everlasting life.” (John 3:16).
Remember this Creator now. Only one time is mentioned in the
Scriptures at which eternal life is promised. Cowley sang of an
“everlasting now,” but there is no such time, and no wise boy desires
that there shall be. There is an eternity of the past, an eternity of the
future, but “now” is limited to now. “Behold, now is the accepted
time; behold, now is the day of salvation.” (2 Cor. 6:2). And this—
“Opportunity lost, however deplored
Is eternity gone and is never restored.”
After the overthrow of the French empire by the Germans, Prince

Napoleon joined the English army, and went among the savage tribes
of South Africa. One day while with a squad of soldiers outside the
camp, he was warned by one of the company, who said: “We had
better return. If we don’t hasten we may fall into the hands of the
enemy.” “Oh,” said the Prince, “let us stay here ten minutes and
drink our coffee.” Before the ten minutes had passed a company of
Zulus came upon them and in the skirmish the Prince lost his life.
His mother, when informed of the facts, said, “That was his great
mistake from boyhood. He never wanted to go to bed at night in
time, nor to arise in the morning. He was ever pleading for ten
minutes more. On this account I sometimes called him ‘Mr. Ten
Minutes.’”
The habit of delay was to him what it is to thousands who pass the
tenth, fifteenth and twentieth milestone without accepting Christ, his
ruination. Such delay weakens the force of the will, unfits for action
when opportunity presents, robs the present and blasts the future.
REASONS FOR BEING A CHRISTIAN IN YOUTH.

“If youth,” as Ruskin said, “is essentially one of formation,
edification, instruction,” then is it the proper time to be a Christian,
for “There’s never an hour of it but is trembling with destinies, not a
moment of which, once past, the appointed work can ever be done
again, or the neglected blow struck on cold iron.”
A boy should be a Christian for the sake of safety. As one grows
away from boyhood, he grows away from the opportunities for
salvation. He is liable to drift. There is a point on Niagara River
called “Past Redemption Point,” where the current is too strong for
human power to battle against. Manhood and age have no special
promise like “they that seek Me early shall find Me.” (Prov. 8:17).
A boy should be a Christian that he may be happy. To properly
remember God, to lose oneself in adoration of Him, is to be like Him,
to be “holy as He is holy,” (1 Pet. 1:15, 16) consequently it is to be
happy as He is happy. Holiness and happiness are inseparable. True
love and true joy come together.
A boy should be a Christian to be useful. God’s promise to
Abraham was: “I will bless thee, and thou shalt be a blessing.” (Gen.
12:2). When Joseph dwelt in Potiphar’s house, we read: “The Lord
blessed the Egyptian’s house, for Joseph’s sake.” (Gen. 39:5). And
the boy who loves Christ will be a rich blessing in many ways to
others.
A boy should be a Christian because it is right. Right is better than
might, and worth more than gold. “In the matter of right,” said
Martin Luther, “I will take my stand, I yield to none.” “I’d rather be
right than President,” said Henry Clay. The only proper life to live is
the Christian life. It is sweet on earth, which makes heaven the
sweeter.
My boy, be a Christian. “All men at the head of great movements,”
said Mr. Gladstone, “are Christian men. During the many years I was
cabinet officer, I was brought into association with sixty master
minds, and all but five were Christians.” To be a Christian is the most
satisfactory, honorable, influential course to pursue. It gives
unspeakable joy in life, peace in death, and glory hereafter.
Remember then—
God wants the boys—all kinds of boys—
To love Him, serve Him, do His will;
He wants those boys that make much noise,
And those who keep so very still.
God sent His Son to die for all,

And on the cross His blood was shed.
No boy need spurn His gracious call
Or of the “Bread of Life” be fed.
Then why not to this Christ now flee

And on His mercy cast thyself?
O hear His words: “Come unto Me,”
And answering back, “I yield myself.”
CHAPTER XXII
Be Prayerful
INTRODUCTION TO CHAPTER XXII
By A. C. Lorimer, D. D.
When I was a youth, I loved to climb Arthur’s Seat early in the
morning, for the purpose of breathing the air borne to our inland
home from out the mighty seas; and so it is well for every lad each
day to seek the summit of highest faith, that he may hold
communion with God; that he may inhale something of the
atmosphere of eternal worlds.
It is said that Daniel opened his window when he prayed, toward
Jerusalem. It was doubtless that he might think of the hallowed city.
Better far, however, to open the windows of the soul toward heaven,
not merely that we may think of the hereafter, but that the invisible,
at the present moment, may stream into our being.
Prayer is the soul’s voice. It is the aspiration of the highest part of
man. It is the sublime confidence, that, though foreign, still it is
within the range of possibility to hold communion with the Creator
of us all. Every time we bend the knee before the Throne of Grace, we
declare our belief in our own God-likeness and in our indestructible
affinity for the divine. Therefore, pray, my boy, and keep on praying;
for it is the true Jacob’s ladder that will lead you, round by round, up
to the Everlasting Throne.
CHAPTER XXII
Be Prayerful
A noble characteristic of any boy is love for prayer. Too many

consider common amusements more important than going to some
chamber or church to commune with the loving Saviour. They are
not. The former bring transient happiness and with it a weary frame,
the latter an unexplained peace, rest of body and soul. The former
gratifies for a time without changing selfish desires or promoting
lofty aspirations, the latter moulds into the image of the Christ-
character.
Prayer is not simply a petition or mere forms of a vain repetition.
It is a turning of the life toward God, an opening of the soul toward
heaven, a reaching out of one’s being with desire to appropriate the
Divine. It was a shoemaker’s shop, with bench, half-worn shoes and
not a few boxes. The proprietor was an old friend of the writer, so
deaf that few could converse with him. Visiting the village in which
he lived, I called upon him. After a chat by means of the lips, signs
and paper, he asked if I would like to hear his son play the harp.
Assenting, he called the lad, who brought a beautiful instrument.
Placing his feet on the pedals, he ran his fingers over the wires and
melodious music resounded. When it stopped, I turned to the old
man, and asked by signs: “Did you hear it?” Shaking his head, he
answered, “Not a note.” Then stepping to the stove, he picked up a
long black poker, and putting one end between his teeth and the
other on the harp, he motioned the boy to play. The lad’s fingers
moved as if by magic. The room was flooded with music and passing
pedestrians stopped to listen. Suddenly the musician stopped. I
propounded the same question: “Did you hear anything?” He
laughed and answered: “All that you heard, I heard.” How? That
dirty poker was changed into a conductor of sound. It brought harp
and listener in contact with each other. In like manner prayer brings
God and petitioner into near relation. What one pleads, the other
hears, and answering, God makes music in the soul.
GREAT MEN GREAT IN PRAYING.

Many great men have been great in praying. Men of the Bible, men
of science, history and influence have been firm believers in it.
Charles Simeon and Joseph Alleine spent from four to eight o’clock
in the morning waiting upon God. Wesley gave two hours a day,
Luther the first three hours. Samuel Rutherford was up at three in
the morning to give God praise. Archbishop Leighton was so much
alone with God that he seemed to be in a perpetual meditation.
Bishop Ken was so much alone with God, that his soul was said to be
God-enamored. David Brainerd prayed hour after hour. John
Fletcher spent whole nights in prayer, John Welsh often seven to
eight hours a day. When the hour for devotion arrived, General
Gordon displayed a white handkerchief outside his tent, and as long
as it remained, no one was allowed to disturb him. General Stonewall
Jackson’s servant used to say that when his master got up several
times during the night to pray there was to be a battle next day.
Abraham Lincoln acknowledged that he had been driven to his knees
“by the overwhelming conviction that he had nowhere else to go.”
Gathering his pupils about him at the opening of his school, Agassiz
said, “It is becoming that we first of all bow in the presence of the
Infinite One.” Well might these exclaim with thousands of others:
“For this cause I bow my knees unto the Father of our Lord Jesus
Christ.” (Eph. 3:14).
PRAYER MAKES A BOY BRAVE.
During the Civil War a dozen soldiers were playing cards one night
when one exclaimed: “What on earth was that?” Listening attentively
a moment, he heard a low, solemn voice, coming from the next tent,
occupied by several recruits, who had that day arrived in camp.
Accompanied by the others he approached the tent on tip-toe. “Boys,
he’s praying, or I’m a sinner!” he roared out. “Three cheers for the
parson!” shouted another man of the group as the prayer ended.
“You watch things for three weeks. I’ll show you how to take the
religion out of him,” said the first speaker, laughing. He was a large
burly fellow, prominent in mischief. The recruit was a slight, pale-
faced boy. During the next three weeks the latter was the butt of the
camp. Then several of the boys, conquered by the lad’s gentle
patience and uniform kindness, begged the others to stop annoying
him. “Oh, the little ranter is no better than the rest of us!” answered
the ringleader. “When we get under fire, you’ll see him run. These
pious folk don’t like the smell of gunpowder. I’ve no faith in their
religion.”
In a few weeks, the regiment broke camp, marched toward
Richmond, entered the Wilderness and engaged in that fearful battle.
The company to which the young recruit belonged had a desperate
struggle. The brigade was driven back, and when the line was formed
behind the breastworks they had built in the morning, he was
missing. When last seen, he was surrounded by enemies, fighting
desperately. At his side was the brave fellow who had made the poor
lad a constant object of ridicule. Both were given up as lost. Suddenly
the big man was seen tramping through the underbrush, bearing the
dead body of the boy. Reverently he laid the corpse down, saying as
he wiped the blood from his own face: “Boys, I couldn’t leave him
behind, he fought so. I thought he deserved a decent burial.”
During a lull in the battle the men dug a shallow grave and
tenderly laid him to rest. Then, as one was cutting the name and
regiment upon a board, the big man said, with a husky voice, “I guess
you’d better put the words ‘Praying soldier’ in somewhere. He
deserves the title, and maybe it’ll console him for our abuse.”
There was not a dry eye among those rough men as they stuck the
rudely carved board at the head of the grave. “Well,” said one, “he
was a praying Christian soldier if ever there was one! And,” turning
to the ringleader, “he didn’t run, did he, when he smelt gunpowder?”
“Run!” answered the big man, his voice tender with emotion. “Why,
he didn’t budge an inch! But what’s that to standing for weeks our
fire like a man, and never sending a word back! He just stood by his
flag and let us pepper him, he did; and boys, I have made up my
mind if prayer will make a man as bold, as loving, as forgiving, as
good, as it did that boy, I’m going to resort to it. It did him good and
it’ll do me good,” and as the other fellows bent their heads he prayed
for forgiveness and salvation, at the close of which the others said,
“Amen!”
HOW TO PRAY.
Prayer is a blessed privilege, a vital necessity, an imperative duty,
but many there are who do not know how to pray. A mere repetition
of words or reading prayers is not prayer. Prayer may be a sigh, a
tear, a groan, a bungling utterance, “a true wish” as Phillips Brooks
used to say, “sent God-ward.” It is—
“the soul’s sincere desire
Uttered or unexpressed.
The motive of a hidden fire
That kindles in the breast.”
Prayer should always be accompanied by thanksgiving and

confession. David said, “I will confess my transgressions unto the
Lord,” (Psalm 32:5) and Paul exhorts, “Giving thanks always for all
things unto God and the Father in the name of our Lord Jesus
Christ.” (Eph. 5:20). Prayer should be offered in faith. Faith is taking
one at his word and thus Christ said, “If ye ask anything in My name,
I will do it.” (John 14:14). To pray without faith, the Bible informs us,
is sin, and this is the reason why many of our petitions are not
answered. They are like those blossoms which fall blasted to the
earth. They had a certain beauty and fragrance, but for want of some
conformity to the law of growth, they never developed into fruit.
They are, as Mrs. Stowe says, “drowsy mutterings of unawakened
souls, talking in their sleep.” But real prayer is always answered.
There may be delays as in Daniel’s petition, or tests to strengthen
faith, as when Jesus said to Jairus, “Fear not, only believe,” (Luke
8:50) for what Christ has promised, He will certainly perform.
WHEN AND WHERE TO PRAY.
Prayer should be our vital breath. As with Paul, it should be

“without ceasing,” (Thess. 5:17) our inward desire continually going
up to God. It should be the first exercise of the morning and the last
in the evening. “It is the first hour of the morning,” says a Chinese
proverb, “that gives color to all the others that follow.” Louis XIV.
was awakened every morning with the words: “Arise, Monsieur, you
have great things to do to-day.” But how could they be done properly
without God’s blessing, and how could God’s blessing be secured
without asking? When Arthur P. Stanley the first night went to the
dormitory at Eton School where he with others had to sleep, he knelt
down to say his evening prayers. Instantly a shower of pillows and
shoes flew about him. He prayed on. “Stanley,” said one of the boys
next day, “I ought to have done as you did. I haven’t said my prayers
at night because I was afraid of the ridicule of the boys.” It was not
long before a score of them followed his example. President Garfield
when a boy undertook with a number of students from Williams
College to climb Mount Greylock. Their plan was to spend the night
on the mount. Seated around the campfire they sang college songs
and told stories all the evening. At bedtime Garfield took a
Testament from his pocket and said: “Boys, it is my custom to read a
chapter in the Bible and have prayer before going to bed. Shall we
have it all together?” and though it seemed rather hard to do,
Garfield did it and all were blessed for it.
Two places are mentioned in the Scriptures where a boy should
pray. Those places are the Christian’s arsenal. One is the secret
chamber where communion is sweet because undisturbed, the other
is the church, where in unity believers call upon God. To the devout
boy both are the “Holy of Holies” where God delights to meet him at
the “Mercy Seat.” Blessed is the place of public prayer! Never neglect
it. But the place of secret prayer is still more blessed. Cyprian would
resort to a shady arbor where “no profane listener may hinder my
musings, and no domestic clamor drown them.” Robert Murray
McCheyne declared, “It is my noblest and most fruitful
employment.” Henry Martyn mourned at the close of his saintly life,
that he had devoted “too much time to public works and too little to
private communion with God.” God said, “In quietness and in
confidence shall be your strength.” (Isa. 30:15).
O, the sweetness of one hour at the feet of Jesus. It changes
dispositions, purifies character, overcomes obstacles, imparts
strength to resist temptations, yes, it makes life worth living.
“We kneel, and all around us seems to lower;
We rise, and all, the distant and the near,
Stands forth in sunny outline, brave and clear;
We kneel, how WEAK! we rise, how full of POWER!”
WHAT PRAYER WILL DO.

More things are wrought by prayer than anything else. It opens
heaven’s door, commands God’s ears to hear and hand to bestow,
makes darkened clouds withdraw, climbs—
“the ladder Jacob saw,
Gives exercise to faith and love;
Brings every blessing from above.”
Prayer has brought rain a thousand times since Elijah prayed,

softened kings’ hearts since Nehemiah won the sympathy of
Artaxerxes, shut lions’ mouths since Daniel was cast into their den,
given victory to armies since Amalek was discomfited, liberated
captives since Peter was delivered from prison, abated storms since
Christ said to wind and wave: “Peace! be still,” (Mark 4:39) arrested
hundreds of prodigals since Monica prayed for her wicked son
Augustine, restored health, supplied food, transformed lives and
revolutionized nations.
Prayer is the means that aids to keep in subjection the sinful
tendencies of human nature and though living in the world keeps us
separated from it. It is the means to aid us in winning souls for Jesus.
John Wesley was once riding along when he saw a man kneeling by
the wayside breaking stones. “Ah,” cried he, “I wish I could break the
hearts of some who hear me as easily as you are breaking those
stones.” The man looked up and said, “Did you ever try to break
them on your knees?” Pleading with God should always precede
pleading with souls to come to God, and it is a question whether
anyone has ever come to God who was not earnestly prayed for by
some one.
Prayer will also make a death-bed glorious. “Yea,” saith the
Psalmist, “though I walk through the valley of the shadow of death, I
will fear no evil, for Thou art with me, Thy rod and Thy staff they
comfort me.” (Psalm 23:4). A boy was dying at midnight. He had just
awakened from sleep. “Is it near morning?” he asked his father. “It
soon will be,” replied the parent. “Do you think I will get well?” “I
hope so,” sobbed the father. There was a long silence, then the lad
moved restlessly on the pillow and said, “Hold me up, father, I want
to say my prayers.” Then, clasping his hands together, he repeated:
“Our Father, which art in heaven, hallowed be Thy name, Thy
kingdom come. Thy kingdom come. I can’t remember, father! I can’t
remember!” A short time after the morning light stole into the room.
“Forever and forever,” uttered the boy and he fell asleep in death.
O, my boy, cultivate this glorious habit of praying. To be intimately
acquainted with God cheers, inspires, ennobles. An old man lay
dying. His sons stood around his bed to receive his parting counsel,
and his last blessing. He had fought the battle of life successfully;
and, so far as this world was concerned, had come out crowned with
honors. He had been a pillar in the church; his seat had never been
vacant, his hand always freely opened to every call. For months he
had been laid aside by a lingering and painful illness. “Boys,” he said,
“God has been good to me. He has given me many friends, good
children, a loving wife, and abundant means; but what I thank Him
for now most of all is this long and painful illness. Without it my life
would have been a failure; I should have gone hence without
knowing as I should the only One worth knowing. Boys, whatever
you do or whatever you leave undone, whether you make another
cent of money or not, take time to get acquainted with God.” That’s
it. So acquainted with Him that with simple words you can breathe
your heart’s desire. So acquainted as to talk with Him the first thing
in the morning and the last in the evening. So acquainted as to seek
His favor in everything and to praise Him for anything.
“Implore His aid, in His decisions rest,
Secure, what’er He gives, He gives the best.”

Cyclized Helical Peptides Synthesis Properties and Therapeutic Applications Zigang Li Full Chapter PDF

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Cyclized Helical Peptides: Synthesis,

Properties, and Therapeutic

Silicon Containing Hybrid Copolymers: Synthesis,

Renewable Polymers and Polymer-Metal Oxide Composites:

Polar Organometallic Reagents - Synthesis, Structure,

Conjugated Polymers for Next-Generation Applications,

Therapeutic dressings and wound healing applications

Ternary Quantum Dots: Synthesis, Properties, and

Chiral Building Blocks in Asymmetric Synthesis:

The Mechanics of Hydrogels: Mechanical Properties,

Synthesis, Properties, and Therapeutic Applications

Print ISBN: 978-3-527-34342-3

Typesetting Straive, Chennai, India

Printing and Binding

Printed on acid-free paper

2 Construction of Constrained Helices 35

3 Properties of Stabilized Peptides 69

4 Applications of Constrained Helices 107

4.1.9 Estrogen Receptor α 135

5 Stabilized Peptide Covalent Inhibitors 159

6 Stabilized Peptide PROTAC 181

7 Stabilized Peptide for Drug Delivery 191

1.1 Protein–Protein Interactions and Their

PPI class Description Simplified illustration Examples (target- Examples structure

occurrence of hydrophobic residues is generally high at the interface and is low on

1.1.2 Intervention of Protein–Protein Interactions Using Small

High-throughput screening is an effective way to find a hit in a traditional drug

1.1.2.1 Leukocyte Function-Associated Antigen-1

1.1.2.2 Inhibitor of Apoptosis Proteins

1.1.2.4 Human Immunodeﬁciency Virus Integrase

consists of three domains, nitrogen-terminal DNA binding domains, catalytic core

1.1.2.5 B-Cell Lymphoma-2 Family/B-Cell Lymphoma-2 Homology 3 Proteins

1.1.2.6 Mouse Double Minute 2–p53 Interaction

1.2 Features of Peptide as Molecular Tools

One-bead-one- 106, library In vitro Various Yes – all resin

Source: Obexer et al. [43]. © 2017, Elsevier.

Split-intein circular ligation of peptides and proteins (SICLOPPS), an in vivo

1.2.2 Disadvantages of Peptides as Molecular Tools

1.3 Helical Structures and Their Characterization

1.3.1.2 310 -Helix

Function of 𝛑-Helix Cellular function depends on highly specific interactions

interactions, contrary to predictions based on the Lipinski rules. Although these

1.3.2 Characterization of Helical Peptides

1.3.2.1 Circular Dichroism

amino acid residues, show characteristic CD absorption according to their different

1.3.2.2 X-ray Crystallography

1.3.2.3 Nuclear Magnetic Resonance (NMR)

angles 𝜑. Therefore, it is ambiguous and hard to achieve compelling conclusions

1.4 Stabilization of Peptides

1.4.1 Peptide Stabilization via Cyclization

1.4.2 Peptide Stabilization via Backbone Reconstruction

example, Kapurniotu and coworkers utilized N-methylation into β-sheet peptides

In the position of methylation, in addition to N-methylation, another methyla-

Figure 1.4 α/β-Peptide mimicry of G protein-coupled receptor agonists. (a) Primary

HIF-1α helix OHM HIF-1α mimic O

Figure 1.5 α-Helix-mimetic polyamides. (a) Cartoon representation of a portion of the

34 Lennard, K.R. and Tavassoli, A. (2014). Chemistry (Weinheim an der Bergstrasse,

Schneider-Mergener, J. et al. (2003). The Journal of Biological Chemistry 278:

Construction of Constrained Helices

2.1 Side-Chain Cross-linking

2.1.1 Disulﬁde Bond

Figure 2.1 Oxidative folding pathways mediated by disulﬁde-bond small molecules or

Oxidation of cysteine by dimethylsulfoxide (DMSO) had been reported as a novel