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Cyclized Helical Peptides
Cyclized Helical Peptides
Synthesis, Properties, and Therapeutic Applications
Zigang Li
Hui Zhao
Chuan Wan
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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
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administer such an oath, but must send for a magistrate competent
for the purpose. Unfortunately for him, the impeachment of Judge
Pickering was a precedent directly opposed to this doctrine. He was
compelled to submit while the Senate unwillingly took the forms of a
court.
Giles’s view of impeachment, which was the same with that of
Randolph, had the advantage of being clear and consistent. The
opposite extreme, afterward pressed by Luther Martin and his
associate counsel for the defence, restricted impeachment to
misdemeanors indictable at law,—a conclusion not to be resisted if
the words of the Constitution were to be understood in a legal sense.
Such a rule would have made impeachment worthless for many
cases where it was likely to be most needed; for comparatively few
violations of official duty, however fatal to the State, could be brought
within this definition. Giles might have quoted Madison in support of
the broader view; and if Madison did not understand the Constitution,
any other Virginian might be excused for error. So far back as the
year 1789, when Congress began to discuss the President’s powers,
Madison said: “I contend that the wanton removal of meritorious
officers would subject him to impeachment and removal from his
own high trust.” Such a misdemeanor was certainly not indictable,
and could not technically be brought within the words of the
Constitution; it was impeachable only on Giles’s theory.
The Senate became confused between these two views, and
never knew on what theory it acted. Giles failed to take from its
proceedings the character of a court of justice; but though calling
itself a court of justice, it would not follow strict rules of law. The
result was a nondescript court, neither legal nor political, making law
and voting misdemeanors for itself as it went, and stumbling from
one inconsistency to another.
The managers added to the confusion. They put forward no
steady theory of their own as to the nature of impeachment; possibly
differing in opinion, they intentionally allotted different lines of
argument to each. In opening the case, Feb. 20, 1805, one of the
managers, George W. Campbell of Tennessee, took the ground that
“misdemeanor” in the Constitution need imply no criminality.
“Impeachment,” said he, “according to the meaning of the
Constitution, may fairly be considered a kind of inquest into the
conduct of an officer merely as it regards his office.... It is more in the
nature of a civil investigation than of a criminal prosecution.” Such
seemed to be the theory of the managers and of the House; for
although the articles of impeachment reported by Randolph in
March, 1804, had in each case alleged acts which were inspired by
an evil intent to oppress the victim or to excite odium against the
Government, and were at least misdemeanors in the sense of
misbehavior, Randolph at the last moment slipped into the indictment
two new articles, one of which alleged no evil intent at all, while both
alleged, at worst, errors in law such as every judge in the United
States had committed. Article V. charged that Chase had issued a
capias against Callender, when the law of Virginia required a
summons to appear at the next court. Article VI. charged that he
had, “with intent to oppress,” held Callender for trial at once, contrary
to the law of Virginia. Every judge on the Supreme Bench had ruled
that United States courts were not bound to follow the processes of
the State courts; Chief-Justice Marshall himself, as Giles threatened,
must be the first victim if such an offence were a misdemeanor in
constitutional law.
That a judge was impeachable for a mistake in declaring the law
seemed therefore to be settled, so far as the House and its
managers could decide the point. Judge Chase’s counsel assumed
that this principle, which had been so publicly proclaimed, was
seriously meant; and one after another dwelt on the extravagance of
the doctrine that a civil officer should be punished for mere error of
judgment. In reply, Joseph H. Nicholson, Randolph’s closest ally,
repudiated the theory on which he had himself acted in Pickering’s
case, and which Giles, Randolph, and Campbell pressed; he even
denied having heard such ground taken as that an impeachment
was a mere inquest of office:—
“For myself, I am free to declare that I heard no such position
taken. If declarations of this kind have been made, in the name of the
managers I here disclaim them. We do contend that this is a criminal
prosecution for offences committed in the discharge of high official
duties, and we now support it,—not merely for the purpose of
removing an individual from office, but in order that the punishment
inflicted on him may deter others from pursuing the baneful example
which has been set them.”
The impeachment, then, was a criminal prosecution, and the
Senate was a criminal court; yet no offence was charged which the
law considered a misdemeanor, while error of judgment, with no
imputed ill-intent, was alleged as a crime.
Staggering under this load of inconsistencies, uncertain what line
of argument to pursue, and ignorant whether the Senate would be
ruled by existing law or invent a system of law of its own, the
managers, Feb. 9, 1805, appeared in the Senate chamber to open
their case and produce their witnesses. Upon the popular
imagination of the day the impeachment of Warren Hastings had
taken deep hold. Barely ten years had passed since the House of
Lords rendered its judgment in that famous case; and men’s minds
were still full of associations with Westminster Hall. The
impeachment of Judge Chase was a cold and colorless performance
beside the melodramatic splendor of Hastings’s trial; but in the
infinite possibilities of American democracy, the questions to be
decided in the Senate chamber had a weight for future ages beyond
any that were then settled in the House of Lords. Whether Judge
Chase should be removed from the bench was a trifling matter;
whether Chief-Justice Marshall and the Supreme Court should hold
their power and principles against this combination of State-rights
conservatives and Pennsylvania democrats was a subject for grave
reflection. Men who did not see that the tide of political innovation
had long since turned, and that the French revolution was no longer
raging, were consumed with anxiety for the fate of Chase, and not
wholly without reason; for had Marshall been a man of less calm and
certain judgment, a single mistake by him might easily have
prostrated the judiciary at the feet of partisans.
By order of the Vice-President the Senate chamber was arranged
in accordance with his ideas of what suited so grave an occasion.
His own chair stood, like that of the chief-justice in the court-room,
against the wall, and on its right and left crimson benches extended
like the seats of associate judges, to accommodate the thirty-four
senators, who were all present. In front of the Vice-President, on the
right, a box was assigned to the managers; on the left, a similar box
was occupied by Justice Chase and his counsel. The rest of the floor
was given to members of the House, foreign ministers, and other
official persons. Behind these a new gallery was erected especially
for ladies, and at each end of this temporary gallery boxes were
reserved for the wives and families of public officers. The upper and
permanent gallery was public. The arrangement was a mimic
reproduction of the famous scene in Westminster Hall; and the little
society of Washington went to the spectacle with the same interest
and passion which had brought the larger society of London to hear
the orations of Sheridan and Burke.
Before this audience Justice Chase at last appeared with his
array of counsel at his side,—Luther Martin, Robert Goodloe Harper,
Charles Lee, Philip Barton Key, and Joseph Hopkinson. In such a
contest weakness of numbers was one element of strength; for the
mere numbers of Congressmen served only to rouse sympathy for
the accused. The contest was unequal in another sense, for the
intellectual power of the House was quite unable on the field of law
to cope with the half-dozen picked and trained champions who stood
at the bar. Justice Chase alone was a better lawyer than any in
Congress; Luther Martin could easily deal with the whole box of
managers; Harper and Lee were not only lawyers, but politicians;
and young Hopkinson’s genius was beyond his years.
In the managers’ box stood no lawyer of corresponding weight.
John Randolph, who looked upon the impeachment as his personal
act, was not only ignorant of law, but could not work by legal
methods. Joseph H. Nicholson and Cæsar A. Rodney were more
formidable; but neither of them would have outweighed any single
member of Chase’s counsel. The four remaining managers, all
Southern men, added little to the strength of their associates. John
Boyle of Kentucky lived to become chief-justice of that State, and
was made district judge of the United States by a President who was
one of the Federalist senators warmly opposed to the impeachment.
George Washington Campbell of Tennessee lived to be a senator,
Secretary of the Treasury, and minister to Russia. Peter Early of
Georgia became a judge on the Supreme Bench of his own State.
Christopher Clark of Virginia was chosen only at the last moment to
take the place of Roger Nelson of Maryland, who retired. None of
them rose much above the average level of Congress; and Chase’s
counsel grappled with them so closely, and shut them within a field
so narrow, that no genius could have found room to move. From the
moment that the legal and criminal character of impeachment was
conceded, Chase’s counsel dragged them hither and thither at will.
Feb. 9, 1805, the case was opened by John Randolph. Randolph
claimed to have drawn all the articles of impeachment with his own
hand. If any one understood their character, it was he; and the
respondent’s counsel naturally listened with interest for Randolph’s
explanation or theory of impeachment, and for the connection he
should establish between his theory and his charges. These charges
were numerous, but fell under few heads. Of the eight articles which
Randolph presented, the first concerned the judge’s conduct at the
trial of John Fries for treason in Philadelphia in 1800; the five
following articles alleged a number of offences committed during the
trial of James Thompson Callender for libel at Richmond in that year;
Article VII. charged as a misdemeanor the judge’s refusal, in the
same year, to dismiss the grand jury in Delaware before indicting a
seditious printer; finally, Article VIII. complained of the judge’s
harangue to the grand jury at Baltimore in May, 1803, which it
characterized as “highly indecent, extrajudicial, and tending to
prostitute the high judicial character with which he was invested to
the low purpose of an electioneering partisan.”
Serious as some of these charges certainly were,—for in the
case of Callender, even more than in that of Fries, Chase’s temper
had led him to strain, if not to violate, the law,—none of the articles
alleged an offence known to the statute-books or the common law;
and Randolph’s first task was to show that they could be made the
subject of impeachment, that they were high crimes and
misdemeanors in the sense of the Constitution, or that in some
sense they were impeachable. Instead of arguing this point, he
contented himself by declaring the theory of the defence to be
monstrous. His speech touched the articles, one by one, adding little
to their force, but piling one mistake on another in its assertions of
fact and assumptions of law.
Ten days passed in taking evidence before the field was cleared
and the discussion began. Then, Feb. 20, 1805, Early and Campbell
led for the managers in arguments which followed more or less
closely in Randolph’s steps, inferring criminality in the accused from
the manifest tenor of his acts. Campbell ventured to add that he was
not obliged to prove the accused to have committed any crime
known to the law,—it was enough that he had transgressed the line
of official duty with corrupt motives; but this timid incursion into the
field of the Constitution was supported by no attempt at argument. “I
lay it down as a settled rule of decision,” said he, “that when a man
violates a law or commits a manifest breach of his duty, an evil intent
or corrupt motive must be presumed to have actuated his conduct.”
Joseph Hopkinson opened for the defence. Friends and enemies
joined in applauding the vigor of this young man’s attack. The whole
effort of Chase’s counsel was to drive the impeachers within the
limits of law, and compel them to submit to the restrictions of legal
methods. Hopkinson struck into the heart of the question. He
maintained that under the Constitution no judge could be lawfully
impeached or removed from office for any act or offence for which he
could not be indicted; “misdemeanor,” he argued, was a technical
term well understood and defined, which meant the violation of a
public law, and which, when occurring in a legal instrument like the
Constitution, must be given its legal meaning. After stating this
proposition with irresistible force, he dealt with Article I. of the
impeachment, which covered the case of Fries, and shook it to
pieces with skill very unlike the treatment of Early and Campbell.
Barton Key next rose, and dealt with Articles II., III., and IV., covering
part of Callender’s case; he was followed by Charles Lee, who
succeeded in breaking down Randolph’s interpolated Articles V. and
VI. Then Luther Martin appeared on the scene, and the audience felt
that the managers were helpless in his hands.
This extraordinary man—“unprincipled and impudent Federalist
bulldog,” as Jefferson called him—revelled in the pleasure of a fight
with democrats. The bar of Maryland felt a curious mixture of pride
and shame in owning that his genius and vices were equally
remarkable. Rough and coarse in manner and expression, verbose,
often ungrammatical, commonly more or less drunk, passionate,
vituperative, gross, he still had a mastery of legal principles and a
memory that overbalanced his faults, an audacity and humor that
conquered ill-will. In the practice of his profession he had learned to
curb his passions until his ample knowledge had time to give the
utmost weight to his assaults. His argument at Chase’s trial was the
climax of his career; but such an argument cannot be condensed in
a paragraph. Its length and variety defied analysis within the limits of
a page, though its force made other efforts seem unsubstantial.
Martin covered the same ground that his associates had taken
before him, dwelling earnestly on the contention that an impeachable
offence must be also indictable. Harper followed, concluding the
argument for the defence, and seeming to go beyond his associates
in narrowing the field of impeachment; for he argued that it was a
criminal prosecution, which must be founded on some wilful violation
of a known law of the land,—a line of reasoning which could end
only in requiring the violation of an Act of Congress. This theory did
not necessarily clash with that of Martin. No hesitation or
inconsistency was shown on the side of the defence; every resource
of the profession was used with energy and skill.
The managers then put forward their best pleaders; for they had
need of all their strength. Nicholson began by disavowing the idea
that impeachment was a mere inquest of office; this impeachment
was, he said, a criminal prosecution intended not merely to remove,
but to punish, the offender. On the other hand, he maintained that
since judges held their commissions during good behavior, and could
be removed only by impeachment, the Constitution must have
intended that any act of misbehavior should be considered a
misdemeanor. He showed the absurdities which would rise from
construing the Constitution in a legal sense. His argument, though
vigorous and earnest, and offering the advantages of a plausible
compromise between two extreme and impracticable doctrines, yet
evidently strained the language of the Constitution and disregarded
law. As Nicholson himself said, he discarded legal usage: “In my
judgment the Constitution of the United States ought to be
expounded upon its own principles, and foreign aid ought never to
be called in. Our Constitution was fashioned after none other in the
known world; and if we understand the language in which it is
written, we require no assistance in giving it a true exposition.” He
wanted a construction “purely and entirely American.” In the mouth
of a strict constructionist this substitution of the will of Congress for
the settled rules of law had as strange a sound as Luther Martin
could have wished, and offered another example of the instinct, so
striking in the Louisiana debate, which not even Nicholson,
Randolph, or Jefferson himself could always resist.
Rodney, the same day, followed Nicholson; and as though not
satisfied with his colleague’s theory, did what Nicholson, in the name
of all the managers, had a few hours before expressly disclaimed,—
he adopted and pressed Giles’s theory of impeachment with all the
precision of language he could command. Nicholson seemed
content to assume impeachment as limited to “treason, bribery, or
other high crimes and misdemeanors;” but in his view misbehavior
might be construed as a misdemeanor in a “purely and entirely
American” sense. Rodney was not satisfied with this argument, and
insisted that the Constitution imposed no limit on impeachment.
“Is there a word in the whole sentence,” he asked, “which
expresses an idea, or from which any fair inference can be drawn, that
no person shall be impeached but for ‘treason, bribery, or other high
crimes and misdemeanors?’... From the most cursory and transient
view of this passage I submit with due deference that it must appear
very manifest that there are other cases than those here specified for
which an impeachment will lie and is the proper remedy.”
The judges held their offices during good behavior; the instant a
judge should behave ill his office became forfeited. To ascertain the
fact “officially, or rather judicially,” impeachment was provided; the
authority of the Senate was therefore coextensive with the complaint.
Rodney stated this principle broadly, but did not rest upon it; on
the contrary, he accepted the respondent’s challenge, and undertook
to show that Chase had been guilty of crimes and misdemeanors in
the technical sense of the term. Probably he was wise in choosing
this alternative; for no one could doubt that his constitutional doctrine
was one into which Chase’s counsel were sedulously trying to drive
him. If Rodney was right, the Senate was not a court of justice, and
should discard judicial forms. Giles had seen this consequence of
the argument, and had acted upon it, until beaten by its inevitable
inconsistencies; at least sixteen senators were willing to accept the
principle, and to make of impeachment an “official, or rather judicial,”
inquest of office. Judge Chase’s counsel knew also that some half-
dozen Republican senators feared to allow a partisan majority in the
Senate to decide, after the fact, that such or such a judicial opinion
had forfeited the judge’s seat on the bench. This practice could end
only in making the Senate, like the House of Lords, a court of last
appeal. Giles threatened to impeach Marshall and the whole
Supreme Court on Rodney’s theory; and such a threat was as
alarming to Dr. Mitchill of New York, or Senator Bradley of Vermont,
as it was to Pickering and Tracy.
When Rodney finished, the theory of impeachment was more
perplexed than ever, and but one chance remained to clear it. All the
respondent’s counsel had spoken in their turn; all the managers had
expounded their theories: John Randolph was to close. Randolph
was an invalid, overwhelmed by work and excitement, nervous,
irritable, and not to be controlled. When he appeared in the box, Feb.
27, 1805, he was unprepared; and as he spoke, he not only made
his usual long pauses for recollection, but continually complained of
having lost his notes, of his weakness, want of ability, and physical
as well as moral incompetence. Such expressions in the mouths of
other men might have passed for rhetoric; but Randolph’s speech
showed that he meant all he said. He too undertook to answer the
argument of Luther Martin, Harper, and Hopkinson on the nature of
impeachment; but he answered without understanding it,—calling it
“almost too absurd for argument,” “a monstrous pretension,” “a
miserable quibble,” but advancing no theory of his own, and
supporting neither Campbell’s, Nicholson’s, nor Rodney’s opinion.
After a number of arguments which were in no sense answers, he
said he would no longer worry the good sense of the Court by
combating such a claim,—a claim which the best lawyers in America
affirmed to be sound, and the two ablest of the managers had
exhausted themselves in refuting.
Randolph’s closing speech was overcharged with vituperation
and with misstatements of fact and law, but was chiefly remarkable
on account of the strange and almost irrational behavior of the
speaker. Randolph’s tall, thin figure, his penetrating eyes and shrill
voice, were familiar to the society of Washington, and his violence of
manner in the House only a short time before, in denouncing
Granger and the Yazoo men, had prepared his audience for some
eccentric outburst; but no one expected to see him, “with much
distortion of face and contortion of body, tears, groans, and sobs,”
break down in the middle of his self-appointed task, and congratulate
the Senate that this was “the last day of my sufferings and of
yours.”[143]
The next day the Senate debated the form of its final judgment.
[144] Bayard moved that the question should be put: “Is Samuel
Chase guilty or not guilty of a high crime or misdemeanor as charged
in the article just read?” The point was vital; for if this form should be
adopted, the Senate returned to the ground it had deserted in the
case of Judge Pickering, and every senator would be obliged to
assert that Chase’s acts were crimes. At this crisis Giles abandoned
the extreme impeachers. He made a speech repeating his old
argument, and insisting that the House might impeach and the
Senate convict not only for other than indictable offences, but for
other than high crimes and misdemeanors; yet since in the present
case the charges were avowedly for high crimes and misdemeanors,
he was willing to take the question as Bayard proposed it, protesting
meanwhile against its establishment as a precedent. Bayard’s
Resolution was adopted March 1, a few moments before the hour of
half-past twelve, which had been appointed for pronouncing
judgment.
The Senate chamber was crowded with spectators when Vice-
President Burr took the chair and directed the secretary to read the
first article of impeachment. Every member of the Senate answered
to his name. Tracy of Connecticut, prostrated by recent illness, was
brought on a couch and supported to his seat, where his pale face
added to the serious effect of the scene. The first article, which
concerned the trial of Fries, was that on which Randolph had
founded the impeachment, and on which the managers had thrown
perhaps the greatest weight. As the roll was called, Senator Bradley
of Vermont, first of the Republican members, startled the audience
by saying “Not Guilty.” Gaillard of South Carolina, and, to the
astonishment of every one, Giles, the most ardent of impeachers,
repeated the same verdict. These three defections decided the
result; but they were only the beginning. Jackson of Georgia, another
hot impeacher, came next; then Dr. Mitchill, Samuel Smith of
Maryland, and in quick succession all the three Smiths of New York,
Ohio, and Vermont. A majority of the Senate declared against the
article, and the overthrow of the impeachers was beyond expectation
complete.
On the second article the acquittal was still more emphatic; but
on the third the impeachers rallied,—Giles, Jackson, and Samuel
Smith returned to their party, and for the first time a majority
appeared for conviction. Yet even with this support, the impeachers
were far from obtaining the required twenty-three votes; the five
recalcitrant Northern democrats stood firm; Gaillard was not to be
moved, and Stone of North Carolina joined him:—the impeachers
could muster but eighteen votes. They did no better on the fourth
article. On the fifth,—Randolph’s interpolated charge, which alleged
no evil intent,—every member of the Senate voted “Not Guilty;” on
the sixth, which was little more than a repetition of the fifth, only four
senators could be found to condemn, and on the seventh, only ten.
One chance of conviction remained, the eighth article, which covered
the judge’s charge to the grand jury at Baltimore in 1803. There lay
the true cause of impeachment; yet this charge had been least
pressed and least defended. The impeachers brought out their whole
strength in its support; Giles, Jackson, Samuel Smith, and Stone
united in pronouncing the judge guilty: but the five Northern
democrats and Gaillard held out to the last, and the managers saw
themselves deserted by nearly one fourth of the Republican
senators. Nineteen voices were the utmost that could be induced to
sustain impeachment.
The sensation was naturally intense; and yet the overwhelming
nature of the defeat would have warranted an excitement still
greater. No one understood better the meaning of Chase’s acquittal
than John Randolph, whose authority it overthrew. His anger showed
itself in an act which at first alarmed and then amused his enemies.
Hurrying from the Senate chamber to the House, he offered a
Resolution for submitting to the States an amendment to the
Constitution: “The judges of the Supreme and all other courts of the
United States shall be removed by the President on the joint address
of both Houses of Congress.” His friend Nicholson, as though still
angrier than Randolph, moved another amendment,—that the
legislature of any State might, whenever it thought proper, recall a
senator and vacate his seat. These resolutions were by a party vote
referred to the next Congress.
Randolph threatened in vain; the rod was no longer in his hands.
His overthrow before the Senate was the smallest of his failures. The
Northern democrats talked of him with disgust; and Senator Cocke of
Tennessee, who had voted “Guilty” on every article of impeachment
except the fifth, told his Federalist colleagues in the Senate that
Randolph’s vanity, ambition, insolence, and dishonesty, not only in
the impeachment but in other matters, were such as to make the
acquittal no subject for regret.[145] Madison did not attempt to hide
his amusement at Randolph’s defeat. Jefferson held himself
studiously aloof. To Jefferson and men of his class Randolph seems
to have alluded, in a letter written a few weeks later, as “whimsicals,”
who “advocated the leading measures of their party until they were
nearly ripe for execution, when they hung back, condemned the step
after it was taken, and on most occasions affected a glorious
neutrality.”[146] Even Giles turned hostile. He not only yielded to the
enemies of Randolph in regard to the form of vote to be taken on the
impeachment, and fairly joined them in the vote on the first article,
but he also aided in offering Randolph a rebuke on another point
connected with the impeachment.
In the middle of the trial, February 15, Randolph reported to the
House, and the House quickly passed, a Bill appropriating five
thousand dollars for the payment of the witnesses summoned by the
managers. When this Bill came before the Senate, Bayard moved to
amend it by extending its provisions to the witnesses summoned by
Judge Chase. The point was delicate; for if the Senate was a court,
and impeachment a criminal procedure, this court should follow the
rules that guided other judicial bodies; and every one knew that no
court in America or in Christendom obliged the State, as a
prosecutor, to pay the witnesses of the accused. After the acquittal,
such a rule was either equivalent to telling the House that its charges
against Chase were frivolous and should never have been
presented, or it suggested that the trial had been an official inquiry
into the conduct of an officer, and not a criminal procedure at law.
The Republicans might properly reject the first assumption, the
Federalists ought to resist the second; yet when Bayard’s
amendment came to a vote, it was unanimously adopted.[147] The
House disagreed; the Senate insisted, and Giles led the Senate,
affirming that he had drawn the form of summons, and that this form
made no distinction between the witnesses for one party and the
other. The argument was not decisive, for the court records showed
at once by whom each witness was called; but Giles’s reasoning
satisfied the Senate, and led to his appointment, March 3, with
Bradley, an enemy of impeachment, as conferrees to meet
Randolph, Nicholson, and Early on the part of the House. They
disagreed; and Randolph, with his friends, felt that Giles and the
Senate had inflicted on them a grievous insult. The Report of the
conference committee was received by the House at about seven
o’clock on the evening of March 3, when the Eighth Congress was
drawing its last breath. Randolph, who reported the disagreement,
moved that the House adhere; and having thus destroyed the Bill, he
next moved that the Clerk of the House should be directed to pay the
witnesses, or any other expense certified by the managers, from the
contingent fund. He would have carried his point, although it violated
every financial profession of the Republican party, but that the House
was thin, and the Federalists, by refusing to vote, prevented a
quorum. At half-past nine o’clock on Sunday night, the 3d of March,
1805, the Eighth Congress came to an end in a scene of total
confusion and factiousness.
The failure of Chase’s impeachment was a blow to the
Republican party from which it never wholly recovered. Chief-Justice
Marshall at length was safe; he might henceforward at his leisure fix
the principles of Constitutional law. Jefferson resigned himself for the
moment to Randolph’s overthrow; but the momentary consolations
passed away, and a life-long disappointment remained. Fifteen years
later his regret was strongly expressed:—
“The Judiciary of the United States,” mourned the old ex-President,
[148] “is the subtle corps of sappers and miners constantly working
underground to undermine the foundations of our confederated fabric.
They are construing our Constitution from a co-ordination of a general
and special government to a general and supreme one alone....
Having found from experience that impeachment is an impracticable
thing, a mere scarecrow, they consider themselves secure for life;
they skulk from responsibility; ... an opinion is huddled up in conclave,
perhaps by a majority of one, delivered as if unanimous, and with the
silent acquiescence of lazy or timid associates, by a crafty chief-judge
who sophisticates the law to his mind by the turn of his own
reasoning.”
The acquittal of Chase proved that impeachment was a
scarecrow; but its effect on impeachment as a principle of law was
less evident. No point was decided. The theory of Giles, Randolph,
and Rodney was still intact, for it was not avowedly applied to the
case. The theory of Judge Chase’s counsel—that an impeachable
offence must be also indictable, or even a violation of some known
statute of the United States—was overthrown neither by the
argument nor by the judgment. So far as Constitutional law was
concerned, President Jefferson himself might still be impeached,
according to the dictum of Madison, for the arbitrary removal of a
useful tide-waiter, and Chief-Justice Marshall might be driven from
the bench, as Giles wished, for declaring the Constitution to be
above the authority of a statute; but although the acquittal of Chase
decided no point of law except his innocence of high crimes or
misdemeanors, as charged in the indictment, it proved impeachment
to be “an impracticable thing” for partisan purposes, and it decided
the permanence of those lines of Constitutional development which
were a reflection of the common law. Henceforward the legal
profession had its own way in expounding the principles and
expanding the powers of the central government through the
Judiciary.
CHAPTER XI.
The Louisiana treaty, signed in May, 1803, was followed by two
years of diplomatic activity. The necessary secrecy of diplomacy
gave to every President the power to involve the country without its
knowledge in dangers which could not be afterward escaped, and
the Republican party neither invented nor suggested means by
which this old evil of irresponsible politics could be cured; but of all
Presidents, none used these arbitrary powers with more freedom
and secrecy than Jefferson. His ideas of Presidential authority in
foreign affairs were little short of royal. He loved the sense of power
and the freedom from oversight which diplomacy gave, and thought
with reason that as his knowledge of Europe was greater than that of
other Americans, so he should be left to carry out his policy
undisturbed.
Jefferson’s overmastering passion was to obtain West Florida. To
this end two paths seemed open. If he chose to conciliate, Yrujo was
still ready to aid; and Spain stood in such danger between England
and France that Godoy could not afford to throw the United States
into the hands of either. If Jefferson wished the friendship of Spain,
he had every reason to feel sure that the Prince of Peace would act
in the same spirit in which he had negotiated the treaty of 1795 and
restored the right of deposit in 1802. In this case Florida must be let
alone until Spain should be willing to cede, or the United States be
ready for war.
On the other hand, the President might alienate Spain and grasp
at Florida. Livingston and Monroe warmly urged this policy, and were
in fact its authors. Livingston’s advice would by itself have had no
great weight with Jefferson or Madison, but they believed strongly in
Monroe; and when he made Livingston’s idea his own, he gave it
weight. Monroe had been sent abroad to buy Florida; he had bought
Louisiana. From the Potomac to the Mississippi, every Southern man
expected and required that by peace or war Florida should be
annexed to the Union; and the annexation of Louisiana made that of
Florida seem easy. Neither Monroe, Madison, nor Jefferson could
resist the impulse to seize it.
Livingston’s plan has been described. He did not assert that
Spain had intended to retrocede Florida to France, or that France
had claimed it as included in the retrocession. He knew the contrary;
and tried in vain to find some one willing to say that the country to
the Perdido ought to be included in the purchase. He made much of
Marbois’s cautious encouragement and Talleyrand’s transparent
manœuvres; but he was forced at last to maintain that Spain had
retroceded West Florida to France without knowing it, that France
had sold it to the United States without suspecting it, that the United
States had bought it without paying for it, and that neither France nor
Spain, although the original contracting parties, were competent to
decide the meaning of their own contract. Believing that Bonaparte
was pledged to support the United States in their effort to obtain
West Florida, Livingston was anxious only to push Spain to the
utmost. Talleyrand allowed him to indulge in these dreams. “I have
obtained from him,” wrote Livingston to Madison,[149] “a positive
promise that this government shall aid any negotiation that shall be
set on foot” for the purchase of East Florida; while as for Florida west
of the Perdido, “the moment is so favorable for taking possession of
that country, that I hope it has not been neglected, even though a
little force should be necessary to effect it. Your minister must find
the means to justify it.”
When the letters written by Livingston and Monroe in May, 1803,
reached Washington, they were carefully studied by the President,
fully understood, and a policy quickly settled. When Jefferson wrote
to Senator Breckenridge his ideas on the unconstitutionality of the
purchase, he spoke with equal clearness on the course he meant to
pursue toward Spain in order to obtain Florida:[150]—
“We have some claims to extend on the sea-coast westwardly to
the Rio Norte or Bravo, and, better, to go eastwardly to the Rio
Perdido, between Mobile and Pensacola, the ancient boundary of
Louisiana. These claims will be a subject of negotiation with Spain;
and if as soon as she is at war we push them strongly with one hand,
holding out a price with the other, we shall certainly obtain the
Floridas, and all in good time.”
This was not Livingston’s plan, but something quite distinct from
it. Livingston and Monroe wanted the President to seize West
Florida, and negotiate for East Florida. Jefferson preferred to
negotiate for West Florida and to leave East Florida alone for the
time.
Madison had already instructed[151] the minister at Madrid that
the Floridas were not included in the treaty, “being, it appears, still
held by Spain,” and that the negotiation for their purchase would be
conducted by Monroe at Madrid. Instructions of the same date were
instantly sent to Monroe,[152] urging him to pursue the negotiation for
Florida, although owing to the large drain made on the Treasury, and
to the “manifest course of events,” the government was not disposed
to make sacrifices for the sake of obtaining that country. “Your
inquiries may also be directed,” wrote Madison, “to the question
whether any, and how much, of what passes for West Florida be
fairly included in the territory ceded to us by France.”
The idea that West Florida could be claimed as a part of the
Louisiana purchase was a turning-point in the second Administration
of Jefferson. Originating in Minister Livingston’s mind, it passed from
him to Monroe; and in a few weeks the President declared the claim
substantial.[153] As the summer of 1803 closed, Jefferson’s plan
became clear. He meant to push this claim, in connection with other
claims, and to wait the moment when Spain should be dragged into
the war between France and England.
These other claims were of various degrees of merit, and
involved France as well as Spain. During the quasi war between the
United States and France, before Jefferson came into power,
American commerce in Spanish waters suffered severely from two
causes. The first consisted in captures made by Spanish cruisers,
and condemnations decided in Spanish courts; the second was due
to captures made by French cruisers, and condemned by French
consuls in Spanish ports, or by courts of appeal in France, without
regard to the rights or dignity of Spain. With much trouble, in August,
1802, at the time when Europe and America were waiting for the end
of Leclerc’s struggle with the negroes and fevers of St. Domingo,
Pinckney succeeded in persuading the Prince of Peace to let the
claims for Spanish depredations go before a commission for
settlement; but Godoy obstinately refused to recognize the claims for
French depredations, taking the ground that Spain was in no way
responsible for them, had never in any way profited by them, and
had no power at the time they occurred to prevent them; that France,
and France alone, had committed the offence, and should pay for it.
Pinckney resisted this reasoning as energetically as possible; but
when Cevallos offered to sign a convention covering the Spanish
depredations, and reserving the Franco-Spanish claims for future
discussion, Pinckney properly decided to accept an offer which
secured for his fellow-citizens five or ten millions of money, and
which left the other claim still open.[154] The convention of Aug. 11,
1802, was sent to the Senate Jan. 11, 1803, in the excitement that
followed Morales’s withdrawal of the entrepôt at New Orleans. The
Senate deferred action until the last moment of the session; and
then, March 3, 1803, after Nicholson and Randolph had appeared at
the bar to impeach Judge Pickering, Pinckney’s claims convention
was taken up, and the nine Federalists were allowed to defeat it by
the absence of Republican senators. The majority reconsidered the
vote and postponed the whole subject till the next session. Thus,
owing to the action of Federalist senators, when Jefferson in the
following summer, after buying Louisiana, looked about for the
means of buying Florida, he found these classes of claims,
aggregating as he supposed between five and ten million dollars,
ready to his hand. Monroe was promptly ordered to insist upon
treating both classes alike, and setting both of them against the
proposed purchase of Florida. “On the subject of these claims you
will hold a strong language,” said Madison.[155]
A third class of claims could be made useful for the same
purpose. Damages had been sustained by individuals in the violation
of their right of deposit at New Orleans in the autumn of 1802.
“A distinction, however, is to be made,” wrote Madison, “between
the positive and specific damages sustained by individuals and the

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

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

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Cyclized Helical Peptides: Synthesis,

Properties, and Therapeutic

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

2.1.2 Amide and Ester

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