1.3 Methodology for Stabilizing Peptide Secondary Structures
13
also be used to stabilize the peptide α helix. Studies have shown that using [1–3]
triazole to replace the amide ring, the helicity of the obtained peptide is similar to that
of the amide ring peptide [85]. In order to obtain a polypeptide with high helicity,
two triazole side loops can be introduced into a peptide, and the obtained peptide is
more stable and binds more tightly to the target [86]. For peptides containing two
azide functional groups located at positions i and i + 7, a linker containing two
terminal alkyne groups with a matching length can be used to react with it to form
a crosslinker that stabilizes two helices [60, 87]. Based on the triazole linker, Lau
et al. developed a modifiable linker that can be conjugated with arginine to improve
the cell permeability, which was proved by a reporter gene experiment [61, 88].
4. All-hydrocarbon crosslinking
The all-hydrocarbon stapling method for peptides was invented by Verdine et al.
[76]. This method has two main characteristics when stabilizing helical peptides.
First, the methylation of the α carbon of the unnatural amino acid of crosslinking.
Second, the utilization of olefin metathesis to generate peptides with all-hydrocarbon
side chain crosslinker. They first systematically studied the length of the crosslinker,
the chirality of stapling residues, and the coupling mode. For crosslinker spanning
one peptide helix, they found that a full hydrocarbon crosslinker (S 5 + S 5 ) of 8 atoms
with the S5 amino acids in the i and i + 4 positions in the peptide has the highest
cyclization efficiency and helix inducing ability. The case for stapling two helices is
a crosslinker of 11 atoms (S5 + R8) with the S5 at the i position and the R8 at i + 7
position. In the comparison of different types of crosslinkers, they found that the i, i
+ 7 side chain system showed the best helix inducing ability. Moreover, the stapling
peptides were revealed greatly improved stability to protease degradation. About ten
years later, this group further published results of how the chirality of amino acids
for stapling affects the membrane permeability of the peptides. They also expanded
the all-hydrocarbon system to i, i + 3 side chain systems. They found that when the
unnatural amino acids are all L-type, the peptide penetration ability is the best, and
the i, i + 3 system is not as good as the i, i + 4 system [89, 90]. However, the expansion of the i, i + 3 system allows them to selectively synthesize bicyclic peptides
(double staple) in one step by collocation of amino acids with different chirality,
using olefin metathesis reaction [91]. In 2013, they explored the influence of chain
length and double bond position on i, i + 3 and i, i + 4 systems, respectively. In
2014, to improve upon the stapling technology for stabilizing a peptide in a bioactive
α-helical conformation, they report the discovery of an efficient and selective bis ringclosing metathesis reaction leading to peptides bearing multiple contiguous staples
connected by a central spiro ring junction [92]. Compared to stapled peptides, the
stitched α-helical peptides showed even increased thermal stabilities, dramatically
enhanced stability against chemical denaturation and proteolytic digestion. Moreover, the stitched peptides show greatly improved cell-penetrating ability compared
to stapled peptides. These features enable stitched peptides potential ligands for
chemical genetics applications, next-generation therapeutic agents, and tools for
macromolecule cargo delivery.
13
also be used to stabilize the peptide α helix. Studies have shown that using [1–3]
triazole to replace the amide ring, the helicity of the obtained peptide is similar to that
of the amide ring peptide [85]. In order to obtain a polypeptide with high helicity,
two triazole side loops can be introduced into a peptide, and the obtained peptide is
more stable and binds more tightly to the target [86]. For peptides containing two
azide functional groups located at positions i and i + 7, a linker containing two
terminal alkyne groups with a matching length can be used to react with it to form
a crosslinker that stabilizes two helices [60, 87]. Based on the triazole linker, Lau
et al. developed a modifiable linker that can be conjugated with arginine to improve
the cell permeability, which was proved by a reporter gene experiment [61, 88].
4. All-hydrocarbon crosslinking
The all-hydrocarbon stapling method for peptides was invented by Verdine et al.
[76]. This method has two main characteristics when stabilizing helical peptides.
First, the methylation of the α carbon of the unnatural amino acid of crosslinking.
Second, the utilization of olefin metathesis to generate peptides with all-hydrocarbon
side chain crosslinker. They first systematically studied the length of the crosslinker,
the chirality of stapling residues, and the coupling mode. For crosslinker spanning
one peptide helix, they found that a full hydrocarbon crosslinker (S 5 + S 5 ) of 8 atoms
with the S5 amino acids in the i and i + 4 positions in the peptide has the highest
cyclization efficiency and helix inducing ability. The case for stapling two helices is
a crosslinker of 11 atoms (S5 + R8) with the S5 at the i position and the R8 at i + 7
position. In the comparison of different types of crosslinkers, they found that the i, i
+ 7 side chain system showed the best helix inducing ability. Moreover, the stapling
peptides were revealed greatly improved stability to protease degradation. About ten
years later, this group further published results of how the chirality of amino acids
for stapling affects the membrane permeability of the peptides. They also expanded
the all-hydrocarbon system to i, i + 3 side chain systems. They found that when the
unnatural amino acids are all L-type, the peptide penetration ability is the best, and
the i, i + 3 system is not as good as the i, i + 4 system [89, 90]. However, the expansion of the i, i + 3 system allows them to selectively synthesize bicyclic peptides
(double staple) in one step by collocation of amino acids with different chirality,
using olefin metathesis reaction [91]. In 2013, they explored the influence of chain
length and double bond position on i, i + 3 and i, i + 4 systems, respectively. In
2014, to improve upon the stapling technology for stabilizing a peptide in a bioactive
α-helical conformation, they report the discovery of an efficient and selective bis ringclosing metathesis reaction leading to peptides bearing multiple contiguous staples
connected by a central spiro ring junction [92]. Compared to stapled peptides, the
stitched α-helical peptides showed even increased thermal stabilities, dramatically
enhanced stability against chemical denaturation and proteolytic digestion. Moreover, the stitched peptides show greatly improved cell-penetrating ability compared
to stapled peptides. These features enable stitched peptides potential ligands for
chemical genetics applications, next-generation therapeutic agents, and tools for
macromolecule cargo delivery.