An In-tether Chiral Center Modulates the Helicity, Cell Permeability, and Target Binding Affinity of a Peptide

Article abstract of DOI:10.1002/anie.201602806

The addition of a precisely positioned chiral center in the tether of a constrained peptide is reported, yielding two separable peptide diastereomers with significantly different helicity, as supported by circular dichroism (CD) and NMR spectroscopy. Single crystal X-ray diffraction analysis suggests that the absolute configuration of the in-tether chiral center in helical form is R, which is in agreement with theoretical simulations. The relationship between the secondary structure of the short peptides and their biochemical/biophysical properties remains elusive, largely because of the lack of proper controls. The present strategy provides the only method for investigating the influence of solely conformational differences upon the biochemical/biophysical properties of peptides. The significant differences in permeability and target binding affinity between the peptide diastereomers demonstrate the importance of helical conformation.

Full text of DOI:10.1002/anie.201602806

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International Edition: DOI: 10.1002/anie.201602806
German Edition: DOI: 10.1002/ange.201602806
Peptide Modulators
An In-tether Chiral Center Modulates the Helicity, Cell Permeability,
and Target Binding Affinity of a Peptide
Kuan Hu, Hao Geng, Qingzhou Zhang, Qisong Liu, Mingsheng Xie, Chengjie Sun, Wenjun Li,
Huacan Lin, Fan Jiang, Tao Wang,* Yun-Dong Wu,* and Zigang Li*
Abstract: The addition of a precisely positioned chiral center
in the tether of a constrained peptide is reported, yielding two
separable peptide diastereomers with significantly different
helicity, as supported by circular dichroism (CD) and NMR
spectroscopy. Single crystal X-ray diffraction analysis suggests
that the absolute configuration of the in-tether chiral center in
helical form is R, which is in agreement with theoretical
simulations. The relationship between the secondary structure
of the short peptides and their biochemical/biophysical proper-
ties remains elusive, largely because of the lack of proper
controls. The present strategy provides the only method for
investigating the influence of solely conformational differences
upon the biochemical/biophysical properties of peptides. The
significant differences in permeability and target binding
affinity between the peptide diastereomers demonstrate the
importance of helical conformation.
Figure 1. The a-helical peptide constrained strategies developed by
others and by our group. (HBS (hydrogen bond surrogate), Hcy
(homocysteine), Chiral HS (chiral hydrocarbon staple)).
T
he majority of protein–protein interactions (PPIs) involve
a-helices and are generally considered to be undruggable with
small molecules because of their large interaction areas and
shallow surfaces.[1] Therefore, continuous efforts are invested peptides containing a carbon atom chiral center within the
in developing constrained peptides for modulating PPIs by tether. We have found that a precisely positioned chiral center
enhancing the helicity of short peptides[2–19] and peptidomi- significantly improves the a-helical contents, protease resist-
metics.[20] Numerous strategies have been investigated in this ance, and cell permeability. We also found that the chiral
area, which generally rely on constrained peptides stabilized center modulates target binding affinity. Meanwhile, Moore
by aryl, alkenyl, or amide tethers.[21] The influence of et al. have reported that a chiral center on the tether of
a peptideꢀs conformation on its biochemical/biophysical a stapled peptide has some influences on the peptideꢀs
properties has been studied extensively.[22] However, it is secondary structure.[19]
difficult to examine solely conformational effects on bio-
chemical/biophysical properties when the peptides being a single turn pentapeptide system was employed as a model
compared have different chemical compositions.
system, based on previous literature.[11] The structure–activity
To eliminate possible amino acid residue perturbations,
We wished to examine whether a chiral center in the relationship of the tether ring sizes and chiral center positions
tether of a stapled peptide, as shown in Figure 1, could are summarized in Figure S1 (Supporting Information) and
influence the secondary structure and physical properties of the optimal tether is shown in Figure 2. Cyclic peptides 1a and
the peptide. To this end, we synthesized a series of stabilized 1b were synthesized from the linear peptide 1, cyclo-Ac-
CAAAS5(2-Me)-NH2 (S5 ((S)-pentenylglycine), 2-Me (the
methyl group located at the b-position with respect to the
[*] K. Hu, H. Geng, Q. Zhang, Q. Liu, M. Xie, C. Sun, W. Li, H. Lin,
a-carbon of the amino acid)), by a thiol-ene reaction as shown
in Figure 2A. Peptides 1a and 1b were readily separated by
reverse-phase HPLC, suggesting significant conformational
differences in solution. All peptides categorized in group b
have longer retention times when compared to their dia-
stereomers in group a. Circular dichroism (CD) spectroscopy
measurements clearly show that peptide 1a is a random coil,
while 1b is helical in PBS buffer (pH = 7.0; Figure 2B).
A series of single turn peptides with varying sequences
were tested (peptides 2a/2b to peptides 10a/10b). In all cases,
including glycine residue-containing peptide 8a/8b, the b
Dr. F. Jiang, Prof. Y.-D. Wu, Prof. Z.-G. Li
School of Chemical Biology and Biotechnology
Peking University Shenzhen Graduate School
Shenzhen, 518055 (China)
E-mail: wuyd@pkusz.edu.cn
lizg@pkusz.edu.cn
Prof. T. Wang
Department of Biology
Southern University of Science and Technology
Shenzhen, 518055 (China)
E-mail: wangtao@sustc.edu.cn
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201602806.
diastereomers showed enhanced helicity while the
a
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Figure 3. Conformation analysis of peptide 10a/10b cyclo-Ac-
CAA IS5(2-Me)-NH2. A) NOE summary diagram of 10b (measured in
10% D2O in H2O, 258C). Bar thickness reveals the intensity of the
NOE signals. B) Thermal ellipsoid and backbone H-bonds in a crystal
structures of pentapeptide 10b cyclo-Ac-CAAIS5(2-Me)-NH2. C) Calcu-
lated structure of peptide 10b superimposed with the solved structure.
Each simulation was performed over 200 ns for sufficient sampling.
Snapshots used for analysis were taken at room temperature (300 K)
and conformational clustering conducted using a backbone dihedral-
based method. D) Ramachandran plots of 10a/b from an REMD
simulation; upper 10a, lower 10b.
Figure 2. Helicity enhancements with an in-tether chiral center. A) Con-
strained peptide preparation. Optimization of tether ring size and
chiral center positions are summarized in Figure S1 (Supporting
Information). B) CD spectra of cyclic pentapeptides 1a/1b and
2b–10b in PBS (pH=7.0) at 208C. C) Molar ellipticities and percent-
age of helicity of peptides 1b–10b in PBS (pH=7.0) at 208C. *The
a-helical content of each peptide was calculated as reported previously.
The final helical content presented relative to peptide 2b, where the
helicity of peptide 2b is fixed at 100%.[11]
C5 and A2 (Supporting Information, Figure S4C), consistent
with their involvement in hydrogen bonding typical of an a-
or 310-helix. In summary, the NOE and CD spectra of peptides
1b, 2b, and 10b suggest that they adopt a mixture of 310- and
a-helical conformations in solution.
As shown in Figure 3B, X-ray diffraction analysis of
peptide 10b cyclo-Ac-CAAIS5(2-Me)-NH2 unambiguously
confirms that the absolute configuration of the in-tether chiral
center is R. The backbone dihedral angle set is summarized in
Table SI (Supporting Information), and all dihedral angle
values are close to that of a standard a-helix (except for the C
terminal residue). Additionally, the methyl group at the chiral
center protrudes from the peptide backbone. Therefore,
a chiral center in the tether provides a modifiable site that
can lead to more effective peptide ligands or serve to improve
the drug-like properties of the peptide.
To improve our understanding of the conformational
features of different peptide diastereomers, we performed
replica exchange molecular dynamics (REMD) simulations
with explicit water using the recently developed force field
RSFF2. For peptide 10b, the most distributed structure
derived from the simulation is almost identical to the solved
structure (backbone + Cb rmsd: 0.3 ꢁ) as shown in
Figure 3C. The Ramachandran plots (f,y distribution) of
diastereomers were mainly random coils. These results are
summarized in Figure 2B (see Supporting Information, Fig-
ure S2 for CD spectra of diastereomers 2a–10a). Addition-
ally, peptide 1b remains helical at high temperature and at
high concentrations of guanidinium hydrochloride (Support-
ing Information, Figure S3).
Detailed 1D and 2D 1H-NMR spectroscopic studies of 1b,
2b, and 10b were performed in 10% D2O in H2O at 258C. As
expected, a number of spectral features were observed that
are characteristic of the well-defined structure in the cyclic
pentapeptides, and are specifically characteristic of a-helicity
(except the C terminal residue S5(2-Me/2-Ph)). Firstly, con-
spicuously low coupling constants were observed (3JNH-Cha
<
6 Hz) for all amide resonances except S5(2-Me/2-Ph)
(Figure 3A; Supporting Information, Figure S4A), as nor-
mally observed in a-helical peptides.[22] Secondly, the obser-
vation in NOESY spectra of nonsequential medium range
daN(i, i + 3), dab(i, i + 3), and daN(i, i + 4) NOEs suggest
a helical structure (Figure 3A; Supporting Information, Fig-
ure S4B). Furthermore, the temperature coefficients of the
backbone amide NH chemical shifts of 1b were determined,
with temperature coefficients (Dd/T) less than 4 ppbKÀ for
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the two diastereomers in the simulations, exhibit different
conformational preferences (Figure 3D; Supporting Infor-
mation). For the S-diastereomer 10a, the dominant calculated
structures are shown in Figures S5 and S6 (Supporting
Information), and demonstrate no significant secondary
structures, which is in excellent agreement with the CD
results. Further simulation of a peptide without the in-tether
R-substitution group (Ac-cyclo-CAAAS5(2-H)-NH2) indi-
cates that the polyproline-II (PII) conformation is intrinsi-
cally favored by the residues, and the representative structure
of the most populated cluster is not helical (Supporting
Information, Figure S5A). In this non-helical structure, an
R = CH3/Ph substitution with (S)-chirality can be added
without any steric interference (Supporting Information,
Figure S5B). However, the non-helical structure is signifi-
cantly destabilized when an R = CH3 substitution group is
placed in (R)-chirality, and is very comfortable when the
peptide backbone adopts an a-helical conformation
(Supporting Information, Figure S5C). A larger R = Ph
group in (R)-chirality lead to higher destabilization of non-
helical structures, and a stronger preference for an a-helical
conformation (Supporting Information, Figure S5D). More
detailed information can be found in Figures S6–S8 (Support-
ing Information).
The influence of conformation on the biochemical/bio-
physical properties of peptides remains unclear, largely
because of 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.[23]
Scrambling the positions of a few amino acids in a peptide can
dramatically change permeability and other biophysical
properties. To date, our strategy provides the only method
for specifically investigating the sole influence of conforma-
tional differences. Firstly, peptide diastereomers 11a/
11b FITC-bA-[cyclo-CRARS5(2-Ph)]-NH2 and 12a/12b
FITC-bA-[cyclo-CRRRS5(2-Ph)]-NH2 (bA (beta alanine),
FITC (fluorescein isothiocyanate)) were synthesized and
separated. As shown in Figure 4, the helical diastereomers
11b and 12b could successfully penetrate HEK293T cells
within 2 h while the other diastereomers were much less
permeable (Figure 4A). This led us to consider whether
a helical conformation itself could make peptides permeable.
Figure 4. Cell permeability of pentapeptide diastereomers. A) Fluores-
cent confocal microscopy images of HEK293T cells incubated with
FITC labeled peptides 11a/b and 12a/b (5 mm) at 378C for 2 h (blue
(DAPI), green (FITC)). B) CD spectra of peptides 11a/b and 12a/b at
208C in 50% TFE buffer. C) Flow cytometry measurements of
HEK293T cells with peptide 11a/b, 12a/b, and 13a/b (5 mm) at 378C
for 2 h.
substitution groups at the tether chiral center on the target
binding affinity of the peptide. ER-1 a/b and ER-2 a/b were
synthesized based on their reported sequences (Fig-
ure 5A),[24a] which contain a methyl or phenyl group at the
chiral center, respectively. ER-1b and ER-2b showed a sig-
nificant increase in helicity compared to ER-1a and ER-2a
(Figure 5B). The binding affinity of ER-1b (ca. 1 nm) and
ER-2b (ca. 69 nm) is much better than that for ER-1a (ND)
and ER-2a (> 600 nm; Figure 5C,D). Interestingly, ER-1b
showed a significantly enhanced binding affinity compared to
all previously reported ER-a peptide ligands, which may be
caused by the additional interaction contributed by the
methyl group at the stereocenter in the tether with the ERa
protein. Peptides PDI-1a/b and PDI-2a/b were also synthe-
sized based on their reported sequences.[25] PDI-1b and 2b
showed a remarkable increase in helicity compared to PDI-1a
and 2a (Figure 5E). They also showed significantly better
binding affinities than PDI-1a and 2a (Figure 5F,G). How-
ever, PDI-2b showed unfavorable binding (ca. 504 nm)
compared to PDI-1b (ca. 165 nm), which may be caused by
the steric hindrance imposed by the bulky phenyl group and
MDM2. These results constitute the first direct evidence of
a direct relationship between helical enhancement and
peptide ligand/protein target binding. Moreover, these results
suggest that the substitution group also interacts directly with
the binding groove, providing a valuable modification site for
future applications, such as fragment-based peptide ligand
design.
Peptide
diastereomers
13a/13b
FITC-bA-[cyclo-
CAKAS5(2-Ph)]-NH2 were subsequently tested. Peptide
13b showed enhanced helicity over peptide 13a (Supporting
Information, Figure S9A). However, while peptide 13b out-
performed peptide 13a in terms of penetration of the cell
membrane, it only showed minimal penetrative efficacy
(Supporting Information, Figures S9B and S9C). These
results suggest that, although helical conformation itself
may not guarantee the permeability of peptides, it is
a determining factor. The structural elucidation of estrogen
receptor alpha (ERa) 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.[24] Based on the results, we chose these two
model targets to study the influence of peptide helicity and
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Figure 6. Cell permeability and in vitro serum stability of PDI peptides.
A) Fluorescent confocal microscopy images of MCF-7 cells incubated
with FITC labeled peptides PDI-1a/1b (5 mm) at 378C. B) Fluorescent
confocal microscopy images of MCF-7 cells incubated with FITC
labeled peptides PDI-1b (5 mm) at 48C or treated with NaN3 and
2-deoxyglucose for 1 h (DNA, blue (DAPI) peptides, green (FITC)).
C) FACS measurements of MCF-7 cells treated with ER-1a/1b,
PDI-1a/1b, and PDI-2a/2b (5 mm) at 378C. For separated flow
cytometry figures, see Figure S10 (Supporting Information). D) In vitro
serum digestion assay of PDI-linear, PDI-1b, and PDI-2b peptides.
Figure 5. Target binding affinity of ERa and MDM2 with their peptide
ligand diastereomers. A) Representation of ER-1a/1b, ER-2a/2b,
PDI-1a/1b, and PDI-2a/2b structures. B) CD spectra of ER peptides,
measured in 20% TFE buffer solution at 208C. C and D) Binding of
ER-1a/1b and ER-2a/2b with ERa, respectively. The binding affinities
were measured using fluorescence polarization assays (FP) at 208C.
E) CD spectra of PDI peptides, measured in 20% TFE buffer solution
at 208C. F and G) Binding of PDI-1a/1b and PDI-2a/2b with MDM2,
respectively. The binding affinities were measured using fluorescence
polarization assays (FP) at 208C.
relationship between conformation and biochemical/bio-
physical properties of peptides. We investigated the relation-
ship between the helicities of the peptides and the location of
the chiral center, the stereoconfiguration, the ring size, and
the size of the substitution group. The pentapeptide crystal
structure and computational simulations further validate our
results. Peptide diastereomers were also tested to examine the
sole influence of conformation on cell permeability.
Moreover, this concept was applied to construction of
MDM2 and ERa peptide ligands, which show excellent
a-helicity nucleation properties and dramatically enhanced
binding affinities. More importantly, the significant differ-
ences in the permeability of the long peptide diastereomers
clearly indicate the importance of increasing peptide helicity
in the construction of constrained peptides. Furthermore, the
influence of the substitution groups at the chiral center on the
binding affinity of peptides suggests that this chiral center
could be utilized as an additional modification site away from
the peptide backbone. Consequently, this feature may be
useful for various applications. Studies on the effect of the
chiral center and the implications for biological applications
are currently underway and will be reported in due time.
Notably, cellular uptake experiments using MCF-7 cells
treated with 5 mm peptides (ER-1a/1b, PDI-1a/1b) revealed
that PDI-1b and ER-1b show significantly higher uptakes
than their diastereomers (Figure 6A; Supporting Informa-
tion, Figure S10). These results were further confirmed by
flow cytometry measurement (Figure 6C; Supporting Infor-
mation, Figures S10B–D). The peptide continued to pene-
trate the cell membrane when incubated at 48C or with the
addition of sodium azide (Figure 6B). This observation
suggests that the permeability mechanism partly involves
transduction, which could be explained by the hydrophobic
tether. The latter is produced by the hydrophobic substitution
group as well as cyclization. The in vitro serum stability assay
showed that the PDI-Linear peptide degraded in a few hours,
while more than 70% of peptides PDI-1b and PDI-2b
remained intact after 24 hours (Figure 6D). Notably, the
bulkier substitution group showed better proteolysis resist-
ance. Thus, chiral center-induced helicity enhancement was
successfully translated into longer peptides with good binding
affinity and intriguing cell permeability.
In summary, a precisely positioned in-tether carbon chiral
center was capable of modulating the helicity of a peptide.
This study provides an excellent means for evaluating the
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This work is supported by the National Natural Science
Foundation of China (Grant 21102007 (Z.L.), 31300600
(T.W.), and 21372023 (Z.L.)), MOST 2015DFA31590 (Z.L.),
MOST 2013CB911500 (T.W.). The Shenzhen Science
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Keywords: cell permeability · chirality · helicity · stapled peptide ·
target binding affinity
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Received: March 21, 2016
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Published online: && &&, &&&&
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Peptide Modulators
K. Hu, H. Geng, Q. Zhang, Q. Liu, M. Xie,
C. Sun, W. Li, H. Lin, F. Jiang, T. Wang,*
Y.-D. Wu,* Z.-G. Li*
&&&& — &&&&
An In-tether Chiral Center Modulates the
Helicity, Cell Permeability, and Target
Binding Affinity of a Peptide
Chirality induced helicity: A precisely
positioned in-tether carbon chiral center
was able to modulate the helicity, cell
permeability, and target binding affinity of
a peptide. This study provides an excel-
lent method for studying the relationship
between the conformation and biochem-
ical/biophysical properties of peptides.
6
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!