1.3 Methodology for Stabilizing Peptide Secondary Structures
11
in the peptide, a covalent bridge linker is formed by either the i, i + 3 amino acids
or i, i + 4 amino acids to stabilize a single helix, or a covalent bridge is formed
in the i, i + 7 amino acids to stabilize two helical turns. Early cyclization methods
included the formation of amide bonds between glutamic acid and lysine residues
[51], or the formation of disulfide bonds [74]. Then the combination of the side
chain coupling strategy and the helix promotion effect of α carbon methylation [75]
led to the production of all-hydrocarbon stapled peptide methodology [76]. These
peptides contain all-hydrocarbon crosslinkers formed by the ruthenium-catalyzed
metathesis (RCM) reaction of two olefin-containing amino acids. More recently, a
protein stapling technology called genetically encoding was reported [77]. In this
method, an unnatural amino acid containing an electrophilic side chain is introduced
into a specific position in a protein by genetic engineering technology. The amino
acid can cross-coupling with adjacent nucleophilic amino acids (lysine, histidine,
cysteine) on the protein. This method is designed to stabilize α-helix sequences
in the protein. When designing stapled peptides with crosslinked side chains, it is
necessary to pay special attention to the position of the bridges and the length of the
side rings. Moreover, it should be noticed that a careful selection of a combination
of helix promoting factors and avoiding unfavorable side chain interference with
the target protein are two basic aspects that should be considered in the design
of stapled peptides. The present methods used to construct PPIs inhibitors mainly
include coupling methods based on thiol-ether, amide, triazole, and all-hydrocarbon
crosslinkers (Fig. 1.6).
1. Crosslinking based on sulfhydryl groups
One of the earliest strategies used to construct structurally constrained helical
peptides was the formation of disulfide bonds by two Cysteine amino acids at positions i and i + 7. To ensure the correct orientation of the side ring, one D-type
Cysteine was used. Compared with unconstrained peptides, cross-coupled disulfide
cyclic peptides show higher helicity [74]. The cross-coupling between the D-cysteine
at position i and the L-cysteine at position i + 3 has also been shown to stabilize
the α-helical peptide [78]. The length of the side chain was shown to affect the
helical content and target recognition. Replacing cysteine with homocysteine can
also form a stable helical peptide [79, 80]. However, disulfide bonds are easily
reduced by the reductive species in eukaryotic cells. Therefore, more chemically
stable thioether bonds are used to replace disulfide bonds. Cysteine is an amino acid
with specific nucleophilic ability among natural amino acids, so the corresponding
electrophilic group can be designed to selectively react with sulfhydryl moieties. A
series of amphiphilic linkers are designed to couple with two cysteines in a peptide
to form side chains to stabilize the peptide structure. Among different electrophilic
molecules, m-xylene reacting with two L-cysteines at positions i, i + 4 was demonstrated the highest helical content proved by circular dichroism and nuclear magnetic
resonance [53]. For longer peptides, the helical content of the constrained peptides
that are formed by brominated diaryl xylenes reacting with L-cysteine at position i
and D-cysteine at position i + 7 is the highest. These side chain-coupled PPI inhibitors
showed higher penetration ability compared to none constrained peptides [54–56].
11
in the peptide, a covalent bridge linker is formed by either the i, i + 3 amino acids
or i, i + 4 amino acids to stabilize a single helix, or a covalent bridge is formed
in the i, i + 7 amino acids to stabilize two helical turns. Early cyclization methods
included the formation of amide bonds between glutamic acid and lysine residues
[51], or the formation of disulfide bonds [74]. Then the combination of the side
chain coupling strategy and the helix promotion effect of α carbon methylation [75]
led to the production of all-hydrocarbon stapled peptide methodology [76]. These
peptides contain all-hydrocarbon crosslinkers formed by the ruthenium-catalyzed
metathesis (RCM) reaction of two olefin-containing amino acids. More recently, a
protein stapling technology called genetically encoding was reported [77]. In this
method, an unnatural amino acid containing an electrophilic side chain is introduced
into a specific position in a protein by genetic engineering technology. The amino
acid can cross-coupling with adjacent nucleophilic amino acids (lysine, histidine,
cysteine) on the protein. This method is designed to stabilize α-helix sequences
in the protein. When designing stapled peptides with crosslinked side chains, it is
necessary to pay special attention to the position of the bridges and the length of the
side rings. Moreover, it should be noticed that a careful selection of a combination
of helix promoting factors and avoiding unfavorable side chain interference with
the target protein are two basic aspects that should be considered in the design
of stapled peptides. The present methods used to construct PPIs inhibitors mainly
include coupling methods based on thiol-ether, amide, triazole, and all-hydrocarbon
crosslinkers (Fig. 1.6).
1. Crosslinking based on sulfhydryl groups
One of the earliest strategies used to construct structurally constrained helical
peptides was the formation of disulfide bonds by two Cysteine amino acids at positions i and i + 7. To ensure the correct orientation of the side ring, one D-type
Cysteine was used. Compared with unconstrained peptides, cross-coupled disulfide
cyclic peptides show higher helicity [74]. The cross-coupling between the D-cysteine
at position i and the L-cysteine at position i + 3 has also been shown to stabilize
the α-helical peptide [78]. The length of the side chain was shown to affect the
helical content and target recognition. Replacing cysteine with homocysteine can
also form a stable helical peptide [79, 80]. However, disulfide bonds are easily
reduced by the reductive species in eukaryotic cells. Therefore, more chemically
stable thioether bonds are used to replace disulfide bonds. Cysteine is an amino acid
with specific nucleophilic ability among natural amino acids, so the corresponding
electrophilic group can be designed to selectively react with sulfhydryl moieties. A
series of amphiphilic linkers are designed to couple with two cysteines in a peptide
to form side chains to stabilize the peptide structure. Among different electrophilic
molecules, m-xylene reacting with two L-cysteines at positions i, i + 4 was demonstrated the highest helical content proved by circular dichroism and nuclear magnetic
resonance [53]. For longer peptides, the helical content of the constrained peptides
that are formed by brominated diaryl xylenes reacting with L-cysteine at position i
and D-cysteine at position i + 7 is the highest. These side chain-coupled PPI inhibitors
showed higher penetration ability compared to none constrained peptides [54–56].