A precisely positioned chiral center in an: I, i + 7 tether modulates the helicity of the backbone peptide

Article abstract of DOI:10.1039/c7cc03799f

In some cases, helical peptides stabilized by an i, i + 7 tether exhibit better target binding and cellular functions compared to their i, i + 4 analogues. Herein, we carried out a systematic study of the effects of an in-tether chiral center on the i, i + 7 system. We screened the optimal cross linking mode, tether length, in-tether chiral center positions, and absolute configurations. From these studies, we determined that a chiral center of R absolute configuration at the γ-position to the C-terminal of a 10-membered tether could function to efficiently induce helicity of the backbone peptides. This is an important addition to the current i, i + 4 in-tether chiral center induced helicity strategy (CIH strategy), and could have broad biological applications.

Full text of DOI:10.1039/c7cc03799f

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DOI: 10.1039/C7CC03799F
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A precisely positioned chiral center in an i, i+7 tether modulates
the helicity of the backbone peptide
Kuan Hu,a,† Chengjie Sun,a,† Dan Yang,a,† Yujie Wu,b Chuan Shi,a Longjian Chen,a Tao Liao,a Jialin
Received 00th January 20xx,
Accepted 00th January 20xx
Guo,c Yinghuan Liu,a Zigang Lia,*
DOI: 10.1039/x0xx00000x
www.rsc.org/
In some cases, helical peptides stabilized by an i, i+7 tether exhibit indicate that the CIH strategy could be applicable for the
better target binding and cellular functions compared to their i, i+4 stabilization of multiple secondary structures.
analogues. Herein, we carried out a systematic study of the effects
The development of constraining methods between different
of an in-tether chiral center on the i, i+7 system. We screened the positions is desirable. In order to choose a proper tethering
optimal cross linking mode, tether length, in-tether chiral center position is largely dependent on both the sequence and the
positions, and absolute configurations. From these studies, we target. In some cases, constraint peptides that are stabilized
determined that a chiral center of R absolute configuration at the with a two helical turn tether, namely an i, i+7 tether, could
γ-position to the C-terminal of a 10-membered tether could provide better helix stabilization, target binding and cellular
function to efficiently induce helicity of the backbone peptides. This functions than a single turn i, i+4 tether.5, 31-33Compared to the
is an important addition to the current i, i+4 in-tether chiral center single turn tether, the two turn tether generally requires more
induced helicity strategy (CIH strategy), and could have broad atoms (i.e. an 8 atom tether for an i, i+4 all hydrocarbon stapled
biological applications.
peptide and an 11 atom tether for an i, i+7 stapled peptide).
When taking the tether length and the size of the peptides into
consideration, finding a properly positioned chiral center in an
i, i+7 tether is actually more challenging, which is due to the fact
that this chiral center should exhibit a nucleating effect on a
more flexible tether and a larger size peptide. (Scheme. 1)
Constraint peptides with preferable biophysical properties
have been developed to target various protein–protein
interactions (PPIs).1-10 The side chain cross-linking strategy is a
critical strategy in the development of stabilized peptides, and
numerous chemical methods have been applied to generate
peptides with fixed secondary structures (mostly helical) for
various purposes.11-23 We recently reported that a precisely
positioned chiral centre in an i, i+4 tether could dominate the
backbone peptide’s helicity, target binding affinity, and cellular
uptake (chirality induced helicity strategy, CIH).24,25 The validity
of this CIH strategy was verified using both an in-tether carbon
chiral center and a sulfur chiral center.26-29 The synergetic effect
Scheme 1. Schematic representation of an in-tether chiral
center induced helical peptide via an i, i+4 and i, i+7 cross linking
system. Notably, the L-amino acids were used for tethering the
i, i+4 system, while in i, i+7 system, a D-amino acid was placed
at the N-terminus and a L-amino acid was placed at the C-
terminus.
was also reported in the dual in-tether chiral centers (DCIH
)
system to constrain a peptide.30 With the exception of helicity
stabilization, a sulfilimine chiral center in an i, i+3 tether was
found to induce a type-III β turn structure26. These results
Herein, we systematically investigated the effects of the
chiral centre on an i, i+7 tether as shown in Scheme 1. We chose
an octapeptide as a model to study the in-tether chiral center
effects on the i, i+7 tether, as this could provide minimum strain
and steric hindrance, and could avoid possible sequence
perturbations. As demonstrated in our previous reports, a
hydrocarbon chiral center could be translated into sulfoxide or
sulfilimine chiral centers as shown in Scheme 1. Therefore, we
began with sulfoxide chiral centers in order to screen for the
a.School of Chemical Biology and Biotechnology, Shenzhen Graduate
School of Peking University, Shenzhen, 518055, E-mail: lizg@pkusz.edu.cn
b Department of Biology, Southern University of Science and Technology, Shenzhen,
Guangdong, 518055, P. R. China
c Shenzhen Middle School, Shenzhen, China, 518001
† These authors contributed equally to this work.
Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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proper chiral center position, for relatively simplified synthesis. other six residues had low amide coupling constaVnitesw A(3rtJicNlHe-OCHnαline
≤
DOI: 10.1039/C7CC03799F
As shown in Fig. 1A, a panel of macrocyclic peptides Ac-cyclo- 6), suggesting that they were
X(n)AAAAAAC-NH2(n=7-10) tethered by thioether linkages
consisting of different numbers of atoms (a 9 to 13 membered
tether with sulphur atoms at different positions) were prepared
via a thiol-ene reaction3. We found that the cyclization
efficiency of the radical reactions was influenced by the side
chain length of amino acid Xn. An Xn with a shorter side chain, as
in entry
entry
1
,
8
2
,
5
exhibited higher cyclization conversion than
4
,
7
,
with a longer side chain (Fig. 1B). Circular dichroism
(CD) spectroscopy studies clearly indicated that a simple
thioether tether was not able to induce α-helicity (Fig. S1),
which is in agreement with our previous reports.24,27,28,34,35
Next, all peptides were subjected to 5% H2O2 oxidation in
order to generate peptide sulfoxide epimers. These peptide
sulfoxide epimers were then subsequently separated by reverse
phase HPLC (rpHPLC). The epimers were designated as peptides
A and B, referring to their respective HPLC retention time (tA<tB).
Figure 1. (A) The chemical structure of the amino acids used for
constructing CIH peptides at i and i+7 positions. The synthetic
route of sulfoxide CIH peptides are shown. Abbreviation: hCys,
homocysteine; Xn, (R)-alkenylglycine. (B) A panel of peptides
For peptide entries
retention time between the
1
and
2
S
, the significant difference in
and epimers indicated the
used for optimizing the cross-linker length and chiral center
R
position. Notes: aThe conversion of the thiol-ene reaction was
presence of structural variations within the solution. In the case
of the other peptide entries, we found that the retention time
difference was decreased, indicating little structural variation
within the solution (see selected HPLC curve for 2-A/B and 7-
A/B in SI-Fig. S2). The CD spectroscopy study of peptides 1–
8(A/B) with different tether lengths and chiral center positions
(γ and δ position) were performed in H2O and were summarized
in Fig. 1C-D. All peptides exhibited random coil structures, while
peptides 1-B and 2-B, both with a γ positioned chiral center
b
determined by LC-MS integration. The percentage of helicity
for the B epimer was calculated based on the Luo and Baldwin
rule.13 “-” indicates the helical content is unavailable. (C, D) CD
spectra of the sulfoxide epimer A or B (~20
H2O, 20°C , respectively.
M) of peptides 1-8,
part of helical structures (Fig.2A). The observation in NOESY
spectra of non-sequential medium-range dαN(i, i + 3) and dαβ(i, i
+ 3) NOEs in 9-B further suggests helical structures (Fig. S5). In
addition, the low temperature coefficient (∆δ/T < 4.5 ppb K-1) of
Arg5, Ala6, Leu7 and Cys8 in 9-B were also indicative of
(tether length of
9 or 10 atoms), exhibited significantly
enhanced α-helical contents (Fig. 1D). Notably, we found that
either increasing the tether length (peptide 3/4) or switching
the chiral center positions (peptide 5-8) resulted in a largely
diminished helical structure. We found that peptide 5-B
possessed comparable ellipticity at 222 nm with peptide 1-B or
2-B. However, the negative maximum ellipticity of 5-B shifted
to 202 nm (208 nm for a regular α-helix), along with a low
θ222/θ202 ratio, which suggests the existence of a potential 310
helicity/ random coil structure.14 Based on our previous study,
we know that the N-γ position is non-essential in the i, i+4 CIH
system24-28 (Scheme 1). Here, we aimed to understand whether
this phenomenon holds true in the i, i+7 system. We prepared
the control peptides S-I and S-2 with the chiral centre at the N
terminus. Both of the peptides were separated as one peak in
the HPLC chromatogram (Fig. S3), which is the mixture of the
R/S isomers. The CD spectra demonstrated that both of the
peptides exist as random coils in H2O (Fig. S4). We found that
the preparation of X8 was more efficient than that of X7, and the
8+2 tether was also found to be slightly better than the 7+2
tether in helix stabilization. Thus, we used the 8+2 tether as the
standard linking pattern in further studies.
hydrogen bonding patterns typical of
Additionally, the CD spectra of 9-A/B were showed in Fig. 2B,
which suggested 9-B is a more helical structure than 9-A
 helical structure (Fig. S6).
.
Therefore, the NOE and CD spectra of peptides 9-B clearly
showed that a precisely positioned chiral centre in an i, i+7
peptide could induce helicity of the backbone peptide in
aqueous solution.
Figure 2. (A) NOE summary diagram of 9-B (measured in 10%
D2O in H2O, 20℃, 2mM). Bar thickness reveals the intensity of
the NOE signals. (B) CD spectra of peptides 9-A/B (~20 M) was
measured in water at 20°C.
To better illustrate the secondary structure of the i, i+7
peptide’s conformation in aqueous solution, a detailed 1D and
2D 1H-NMR spectroscopy study of peptide 9-B (Ac-cyclo-
X8ELARALC(O)-NH2) was performed in 90% H2O: 10% D2O (V:V).
Except for the residues Cys and a Leu (closed to N-termini), the
We further examined whether the helical inducing effect of
an in-tether sulfoxide chiral center in an i, i+7 system could be
transferred into a carbon chiral center as shown in Fig. 3A. The
enantiomerically-pure unnatural amino acids S8(Ph-R) and
S8(Ph-S) were prepared according to reported procedures.36
Peptides 11-S/R Ac-cyclo-(d)CAAAAAAS8(2-Ph)-NH2-S/R were
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synthesized. We found that the epimers exhibited both a
distinguished retention time (Fig. S7) and CD spectra, as shown
in Fig. 3B. The peptide 11-R exhibited an-helical structure
while the 11-S epimer exhibited a random coil structure. This
confirmed the modulating effect of an in-tether hydrocarbon
chiral center in the i, i+7 system. The i, i+4 tethered version of
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DOI: 10.1039/C7CC03799F
peptide 11 was also synthesized, namely peptide 10-S/R
.
Peptide 11-R displayed obvious better helical conformation
than 10-R. We also synthesized peptide S-3 to demonstrate that
the N-γ position is nonessential (Fig. S8). Finally, we investigated
the thermal and chemical stability of peptide 11-R (Fig. S9). The
slight decrease of ellipticity observed at 222 nm by CD
measurements suggests the high stability of peptide 11-R
.
Figure 4. (A) Representation of 12
,
13 (PDI-i, i+4) and 14 (PDI-i,
i+7 CIH) peptide chemical structures. (B) The comparison of
mean residue helicity of 12 13 (i, i+4) and 14 (i, i+7) CIH peptides.
CD spectral measurements were performed in H2O, pH 7, 20°C .
(C) Binding affinity of 12 13 (PDI-i, i+4) and 14 (PDI-i, i+7) with
MDM2, respectively. The binding affinities were measured
using fluorescence polarization assays (FPA) at 20°C .
,
Figure 3. (A) The chemical structures and (B) CD spectra of 10-
S/R, 11-S/R. The CD measurements were performed in H2O,
20°C at ~20 M. (d)C indicates the D-cysteine.
,
Based on these results, we enriched the chemical toolbox of
constructing i, i+7 CIH peptides. Several co-crystal structures of
peptide modulators-MDM2/MDMX revealed that the tether is
involved in interactions with the flat hydrophobic region
surrounding the target ligand binding site. In addition, it was
shown that a larger hydrophobic tether of the i, i+7 stapled
peptide exhibited enhanced performance over their i, i+4
analogues.31-33 Thus, the development of a CIH peptide
modulator of p53-MDM2/MDMX interactions (PDI36,37) would
provide a suitable model for the study of performance
differences between i, i+4 and i, i+7 CIH peptides. Peptides 13
-
FITC- A-LTF[cyclo-CEYWS5(Ph-R)]QLTSAA-NH2, 14 FITC- A-
β
-
β
LTF[cyclo-(d)CEYWAQLS8(Ph-R)]SAA-NH2, and their linear
peptide 12 were prepared as shown in Fig. 4A. We showed that
the i, i+7 tethered peptide exhibited enhanced helical content
compared to the i, i+4 tether (19.6% and 15.7%, respectively,
Fig. 4B), and both of them were more helical than peptide 12
.
As shown in Fig. 4C, fluorescence polarization assay
demonstrates that the i, i+7 CIH peptide exhibited enhanced
binding affinity with MDM2 protein (374 nm). This
phenomenon could be attributed to the higher helical content
of i, i+7 peptide, as well as its enlarged tether interaction
interface, observations that are in agreement with previous
reports.38,39
In addition, in vitro serum stability assays showed that the i,
i+7 peptide exhibited increased resistance to protease
degradation and remained approximately 85% intact after 24
hours (Fig. 5A). We also evaluated cellular uptakes of the i, i+7
Figure 5. (A) In vitro serum stability of CIH peptides. Peptides
12-14 were incubated with human serum at 37°C . (B)
peptide or i, i+4 peptide by fluorescence-activated cell sorting Comparison of cellular uptakes of peptides 12
,
13 and 14. Hela
cells were incubated with peptides (5
M) for 2 hours and the
(FACS). These studies showed that the i, i+7 peptide possessed
higher cellular uptake compared to the i, i+4 peptide (Fig. 5B&
fluorescence intensity was measured by flow cytometry. Error
bars represent the standard error of mean (SEM) from more
than two experiments. (C) Live cell Confocal images of peptides
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the Shenzhen Science and Technology Innovation Committee
JSGG20140519105550503,
JCYJ20150403101146313,
JCYJ20150331100849958,
JCYJ20160301111338144,
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