40
2 Synthesis of In-Tether Chiral Center Peptides …
clash (3.1 Å distance) with the backbone O atom of the fourth residue, leading to
destabilization and lower population (Cluster 2 in Fig. 2.9d). This steric interference
may also occur for other non-helical conformations (Fig. 2.11). Indeed, this steric
repulsion will limit the rotation of the peptide plane, making the ψ angle of the
4th residue and the φ angle of the 5th residue to favor the α-helical conformation
(Fig. 2.12). On the other hand, the phenyl group is quite comfortable when the
backbone adopts -helix structure (Cluster 1 in Fig. 2.9d). Therefore, peptide 9b has
much stronger preference for -helical conformation, as shown in its , plot.
2.2.8 Cell Permeability Study
The influence of the peptides’ conformations on their biochemical/biophysical properties remains unclear, largely due to the absence of methods for constructing
peptides with minimal differences in chemical composition. Cell permeability is
the major limitation for peptide therapeutics and is influenced by many aspects,
including conformation [44–47]. Scrambling the positions of a few amino acids in a
peptide could dramatically change its permeability and other biophysical properties.
However, our strategy provides an ideal platform for specifically investigating the sole
influence of conformational differences. First, peptide diastereomers 11a/11b FITCβA-[cyclo-CRARS 5 (2-Ph)]-NH 2 and 12a/12b FITC-βA-[cyclo-CRRRS 5 (2-Ph)]NH 2 (βA: beta alanine; FITC: fluorescein isothiocyanate) were synthesized and separated. As shown in Fig. 2.13, the helical diastereomers 11b and 12b could successfully
penetrate HEK293T cells within 2 h while the countpart diastereomers were much
less permeable (Fig. 2.13). This led us to consider if a helical conformation itself
could make peptides permeable. Peptide diastereomers 13a/13b FITC-βA-[cycloCAKAS 5 (2-Ph)]-NH 2 were subsequently tested. Peptide 13b showed enhanced
helicity over peptide 13a (Fig. 2.14a), however; while it outperformed peptide 13a in
penetration of the cell membrane, peptide 13b only showed minimal penetrative efficacy (Fig. 2.14b, c). These results suggested that although helical conformation itself
may not guarantee peptides’ permeability, it is a determining factor for the peptides’
permeability. To date, this method excluded any peptide composition perturbation
and provided an ideal platform to study the solely conformational influence on a
peptide’s permeability and other biophysical properties.
2.2.9 Bioactive Peptide Construction
The structural elucidation of estrogen receptor alpha (ERα) and mammal double
minute 2 (MDM2) with their constrained peptide ligands clearly showed the interaction between the protein targets and the ligand tethers, mostly in the flat hydrophobic
region surrounding the target ligand binding site [31, 32, 41]. Based on these results,
we chose these two model targets to study the influences of peptide helicity and
2 Synthesis of In-Tether Chiral Center Peptides …
clash (3.1 Å distance) with the backbone O atom of the fourth residue, leading to
destabilization and lower population (Cluster 2 in Fig. 2.9d). This steric interference
may also occur for other non-helical conformations (Fig. 2.11). Indeed, this steric
repulsion will limit the rotation of the peptide plane, making the ψ angle of the
4th residue and the φ angle of the 5th residue to favor the α-helical conformation
(Fig. 2.12). On the other hand, the phenyl group is quite comfortable when the
backbone adopts -helix structure (Cluster 1 in Fig. 2.9d). Therefore, peptide 9b has
much stronger preference for -helical conformation, as shown in its , plot.
2.2.8 Cell Permeability Study
The influence of the peptides’ conformations on their biochemical/biophysical properties remains unclear, largely due to the absence of methods for constructing
peptides with minimal differences in chemical composition. Cell permeability is
the major limitation for peptide therapeutics and is influenced by many aspects,
including conformation [44–47]. Scrambling the positions of a few amino acids in a
peptide could dramatically change its permeability and other biophysical properties.
However, our strategy provides an ideal platform for specifically investigating the sole
influence of conformational differences. First, peptide diastereomers 11a/11b FITCβA-[cyclo-CRARS 5 (2-Ph)]-NH 2 and 12a/12b FITC-βA-[cyclo-CRRRS 5 (2-Ph)]NH 2 (βA: beta alanine; FITC: fluorescein isothiocyanate) were synthesized and separated. As shown in Fig. 2.13, the helical diastereomers 11b and 12b could successfully
penetrate HEK293T cells within 2 h while the countpart diastereomers were much
less permeable (Fig. 2.13). This led us to consider if a helical conformation itself
could make peptides permeable. Peptide diastereomers 13a/13b FITC-βA-[cycloCAKAS 5 (2-Ph)]-NH 2 were subsequently tested. Peptide 13b showed enhanced
helicity over peptide 13a (Fig. 2.14a), however; while it outperformed peptide 13a in
penetration of the cell membrane, peptide 13b only showed minimal penetrative efficacy (Fig. 2.14b, c). These results suggested that although helical conformation itself
may not guarantee peptides’ permeability, it is a determining factor for the peptides’
permeability. To date, this method excluded any peptide composition perturbation
and provided an ideal platform to study the solely conformational influence on a
peptide’s permeability and other biophysical properties.
2.2.9 Bioactive Peptide Construction
The structural elucidation of estrogen receptor alpha (ERα) and mammal double
minute 2 (MDM2) with their constrained peptide ligands clearly showed the interaction between the protein targets and the ligand tethers, mostly in the flat hydrophobic
region surrounding the target ligand binding site [31, 32, 41]. Based on these results,
we chose these two model targets to study the influences of peptide helicity and