10
1 Introduction
cell penetration ability, and the intracellular p53 reporter gene experiment shows that
the peptide is almost ineffective in vivo. The possible reason for this phenomenon
is that negatively charged amino acids in the peptide interfere with the binding to
the phospholipid bilayer membrane, which prevents the peptide from penetrating the
cell membrane [61, 62]. In our study, we developed peptide constraining methods
based on the thiol-ene photoreaction. The thiol-ther crosslinker generated by this
method can be further modified at the sulfur atom, such as sulfonium modification.
As a result of this modification, the cell-penetrating ability of the peptide is increased
because an excess positive charge was added to the peptide. We also demonstrated
the sulfonium modification can modulate the binding affinity of the peptide [63–66].
Among other modifiable crosslinking methods, Vasco et al. proved that the Ugi reaction can be used to construct α-helix peptides, and after ring formation, exocyclic
functionalities as N-substituents were incorporated [67].
In another example, Assem et al. reported a macrocyclization strategy using
dichloroacetone as the linking group. When this group reacts with the nucleophilic
thiol group on the peptide, the helical conformation of the formed cyclic peptide is
remarkably stabilized. Aside from stabilizing helical structures, the ketone moiety
embedded in the linker can be modified with diverse molecular tags by oxime ligation. The reversible properties of the oximes make it useful for the dynamic covalent
chemical modification of peptides, which will help improve the selectivity of the
peptide and optimize the interaction with the target [68].
A switchable linking group achieves the effect of controlling the structure of
peptides by using light or other conditions to change the conformation of the linking
group. The use of cysteine to couple with azobenzene-containing linking groups has
been demonstrated to be an effective photoswitchable strategy for light-regulating
the physiological activity of peptides [69]. Martin-Quiros et al. used this method to
construct a photoswitchable peptide inhibitor of β-arrestin/β-adaptin protein-protein
interactions, which has a 12.6-fold difference in binding constant in the presence or
absence of light [70]. More interestingly, Belotto et al. proved that peptides containing
azobenzene linker can be selectively bound to protein targets of choices in the phage
display screening, and the affinity of the peptide could be modulated by UV light.
This method is robust and can be applied for the in vitro evolution of photoswitchable
ligands to any targets [71]. In another reversible α-helix formation method, Miller
et al. developed a reversible hydrogen bond surrogate method that utilizes an internal
disulfide linkage. Structural analysis indicates that the dynamic nature of the disulfide bridge allows for the reversible formation of an α-helix through oxidation and
reduction reactions [72]. The most commonly used methods for stabilizing peptide
α-helix are introduced below.
1. Side chain-side chain coupling for helix stabilization
Peptide helix forms intramolecular hydrogen bonds from the carbonyl oxygen of the
i-th amino acid and the protons of the amino group of the i + 4th amino acid. Other
stabilizing effects can be produced by forming a salt bridge between two amino acids
(such as glutamic acid and lysine) on the same face. This type of stabilization was
used to stabilize the helical structure of peptides in early research [73]. Thereafter,
1 Introduction
cell penetration ability, and the intracellular p53 reporter gene experiment shows that
the peptide is almost ineffective in vivo. The possible reason for this phenomenon
is that negatively charged amino acids in the peptide interfere with the binding to
the phospholipid bilayer membrane, which prevents the peptide from penetrating the
cell membrane [61, 62]. In our study, we developed peptide constraining methods
based on the thiol-ene photoreaction. The thiol-ther crosslinker generated by this
method can be further modified at the sulfur atom, such as sulfonium modification.
As a result of this modification, the cell-penetrating ability of the peptide is increased
because an excess positive charge was added to the peptide. We also demonstrated
the sulfonium modification can modulate the binding affinity of the peptide [63–66].
Among other modifiable crosslinking methods, Vasco et al. proved that the Ugi reaction can be used to construct α-helix peptides, and after ring formation, exocyclic
functionalities as N-substituents were incorporated [67].
In another example, Assem et al. reported a macrocyclization strategy using
dichloroacetone as the linking group. When this group reacts with the nucleophilic
thiol group on the peptide, the helical conformation of the formed cyclic peptide is
remarkably stabilized. Aside from stabilizing helical structures, the ketone moiety
embedded in the linker can be modified with diverse molecular tags by oxime ligation. The reversible properties of the oximes make it useful for the dynamic covalent
chemical modification of peptides, which will help improve the selectivity of the
peptide and optimize the interaction with the target [68].
A switchable linking group achieves the effect of controlling the structure of
peptides by using light or other conditions to change the conformation of the linking
group. The use of cysteine to couple with azobenzene-containing linking groups has
been demonstrated to be an effective photoswitchable strategy for light-regulating
the physiological activity of peptides [69]. Martin-Quiros et al. used this method to
construct a photoswitchable peptide inhibitor of β-arrestin/β-adaptin protein-protein
interactions, which has a 12.6-fold difference in binding constant in the presence or
absence of light [70]. More interestingly, Belotto et al. proved that peptides containing
azobenzene linker can be selectively bound to protein targets of choices in the phage
display screening, and the affinity of the peptide could be modulated by UV light.
This method is robust and can be applied for the in vitro evolution of photoswitchable
ligands to any targets [71]. In another reversible α-helix formation method, Miller
et al. developed a reversible hydrogen bond surrogate method that utilizes an internal
disulfide linkage. Structural analysis indicates that the dynamic nature of the disulfide bridge allows for the reversible formation of an α-helix through oxidation and
reduction reactions [72]. The most commonly used methods for stabilizing peptide
α-helix are introduced below.
1. Side chain-side chain coupling for helix stabilization
Peptide helix forms intramolecular hydrogen bonds from the carbonyl oxygen of the
i-th amino acid and the protons of the amino group of the i + 4th amino acid. Other
stabilizing effects can be produced by forming a salt bridge between two amino acids
(such as glutamic acid and lysine) on the same face. This type of stabilization was
used to stabilize the helical structure of peptides in early research [73]. Thereafter,