A Peptoid with Extended Shape in Water

Article abstract of DOI:10.1021/jacs.9b04371

The term "peptoids" was introduced decades ago to describe peptide analogues that exhibit better physicochemical and pharmacokinetic properties than peptides. Oligo(N-substituted glycine) (oligo-NSG) was previously proposed as a peptoid due to its high proteolytic resistance and membrane permeability. However, oligo-NSG is conformationally flexible, and ensuring a defined shape in water is difficult. This conformational flexibility severely limits the biological application of oligo-NSG. Here, we propose oligo(N-substituted alanine) (oligo-NSA) as a peptoid that forms a defined shape in water. The synthetic method established in this study enabled the first isolation and conformational study of optically pure oligo-NSA. Computational simulations, crystallographic studies, and spectroscopic analysis demonstrated the well-defined extended shape of oligo-NSA realized by backbone steric effects. This new class of peptoid achieves the constrained conformation without any assistance of N-substituents and serves as a scaffold for displaying functional groups in well-defined three-dimensional space in water, which leads to effective biomolecular recognition.

Full text of DOI:10.1021/jacs.9b04371

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Article
A Peptoid with Extended Shape in Water
Jumpei Morimoto, Yasuhiro Fukuda, Daisuke Kuroda, Takumu Watanabe, Fumihiko Yoshida, Mizue
Asada, Toshikazu Nakamura, Akinobu Senoo, Satoru Nagatoishi, Kouhei Tsumoto, and Shinsuke Sando
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b04371 • Publication Date (Web): 12 Aug 2019
Downloaded from pubs.acs.org on August 13, 2019
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A Peptoid with Extended Shape in Water
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Jumpei Morimoto,
Yasuhiro Fukuda, Daisuke Kuroda, Takumu Watanabe, Fumihiko
†
¶
¶
†
†,§,‖
Yoshida, Mizue Asada, Toshikazu Nakamura, Akinobu Senoo, Satoru Nagatoishi, Kouhei
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Tsumoto,
and Shinsuke Sando
†Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-8656, Japan.
§Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
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¶Department of Materials Molecular Science, Institute for Molecular Science, 38 Nishigo-naka, Myodaiji, Okazaki 444-8585,
Japan.
‖Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108–8639, Japan.
ABSTRACT: The term “peptoids” was introduced decades ago to describe peptide analogs that exhibit better physicochemical and
pharmacokinetic properties than peptides. Oligo(N-substituted glycine) (oligo-NSG) was previously proposed as a peptoid due to its
high proteolytic resistance and membrane permeability. However, oligo-NSG is conformationally flexible and ensuring a defined
shape in water is difficult. This conformational flexibility severely limits the biological application of oligo-NSG. Here, we propose
oligo(N-substituted alanine) (oligo-NSA) as a peptoid that forms a defined shape in water. The synthetic method established in this
study enabled the first isolation and conformational study of optically pure oligo-NSA. Computational simulations, crystallographic
studies and spectroscopic analysis demonstrated the well-defined extended shape of oligo-NSA realized by backbone steric effects.
This new class of peptoid achieves the constrained conformation without any assistance of N-substituents and serves as a scaffold
for displaying functional groups in well-defined three-dimensional space in water, which leads to effective biomolecular
recognition.
compromise water solubility and/or restrict the choice of N-
substituents. The introduction of charged groups is effective to
compensate the water solubility of these peptoids,
these restrictions reduce the design flexibility of oligo-NSGs.
After the pioneering works by Zuckermann and coworkers on
oligo-NSGs, “peptoids” has been recognized as the term
describing oligo-(N-substituted amides) and many other
peptoids have been developed, e.g. β-peptoids,
hydrazinoazapeptoids,
INTRODUCTION
2
3–26
Peptidomimetic synthetic oligoamides with better
physicochemical and pharmacokinetic properties than peptides
have been long sought and many examples of such oligomers
but
1,2
have been reported, e.g. β-peptides,
oligomers of α-
3
,4
5,6
aminoisobutyric acids, and oligoureas. Oligo(N-substituted
2
7–30
glycine) (oligo-NSG) was proposed as one such oligoamide
and called “peptoid” in 1992. Oligo-NSG is a rare example
of synthetic oligomers that combines high proteolytic
resistance and high membrane permeability derived from
its N-substituted amide backbone. In addition, submonomer
synthetic methods of oligo-NSG allow the introduction of a
diverse set of proteinogenic and nonproteinogenic functional
7
,8
9
31
32
aminooxy
peptoids,
and
3
3,34
arylopeptoids.
However, for these oligomers also, it is
1
0
11
difficult to achieve conformationally constrained oligomers
with good water solubility.
The introduction of methyl substituents on the backbone α-
carbon of oligo-NSG to make oligo(N-substituted alanine)
oligo-NSA) (Figure 1a) was previously proposed by Kodadek
and coworkers to constrain the conformation of peptoids via
the local steric effects on the backbone.
12
groups as N-substituents. These important unique features of
(
oligo-NSG are advantageous over other peptidomimetic
oligoamides for generating drug candidates. However, the
backbone of the NSG-type peptoid is intrinsically flexible;
therefore, a peptoid that exhibits high affinity to biomolecules
has not been reported except for rare exceptions. To
overcome this limitation, extensive research studies have
constrained the conformation of oligo-NSG using elaborate N-
substituents. These efforts successfully realized oligo-NSGs
with defined shapes in organic solvents, such as helices similar
35,36
The constrained
conformation was reasoned from an analogy of N-methylated
peptides. More specifically, the introduced methyl substituent
1
3
would produce steric repulsion, known as pseudo-1,3-allylic
37,38
strain,
with the carbonyl oxygen of the preceding residue
and the N-substituent of the following residue and restrict
rotation about the φ and ψ angles (Figure 1b). Moreover, steric
repulsion between two methyl substituents on neighboring
residues would destabilize the cis conformation of the
intervening amide bond and bias the amide bond to trans, i.e.
1
4–22
to polyproline type I and type II helices.
conformational regulation generally requires introduction of
multiple bulky hydrophobic N-substituents that severely
However, such
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dimethylamide, were subjected to quantum mechanical (QM)
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calculations to generate Ramachandran-type energy diagrams
over φ and ψ angles. The diagram of oligo-NSG had a
relatively large region within 10 kcal/mol from the lowest
energy point (Figure 2a), which is consistent with the previous
3
9,40
reports of similar calculations.
In contrast, the diagram of
oligo-NSA had a narrow region with the lowest energy at
around (φ, ψ) = (−120°, 90°) and the surrounding region was
predominantly favored over other regions (Figure 2b). To
understand how the N-substituents will be oriented on oligo-
NSA, we have also examined the preferred χ angles of acetyl-
N-ethylalanine dimethylamide with fixed φ and ψ angles of the
lowest energy point; (φ, ψ) = (−120°, 90°). As a result, 100°
and −100° were determined to be the χ angles of the lowest
energy (Figure S1). Based on this χ scan, energy diagrams of
NSA over φ and ψ angles were re-examined using the χ angles
of 100° and −100° for acetyl-N-ethylalanine dimethylamide as
a minimal model of oligo-NSA with N-substituents larger than
methyl. For the χ angle of 100°, the lowest energy angles were
slightly shifted and became (φ, ψ) = (−105°, 105°), but, overall
for both χ angles, the allowed region was similar to the above-
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mentioned
landscape
of
acetyl-N-methylalanine
dimethylamide (Figure 2c and d), which suggests that the
structure of N-substituent does not largely affect the backbone
conformation. These results support the hypothesis that
introduction of a methyl substituent on the α-carbon of NSG
would locally restrict the bond rotations about all the backbone
dihedral angles. (For the definition of each dihedral angle, see
Figure 2g and h.)
Figure 1. Chemical structure and proposed conformational
regulation by steric effects of oligo-NSA. (a) Chemical structures
of peptide and peptoid. (b) The bond rotations about φ and ψ
angles of oligo-NSA are restricted due to the pseudo-1,3-allylic
strains. (c) ω angle of oligo-NSA is biased to ~180° due to the
steric repulsion between backbone structures.
Based on the QM calculation of the monomeric NSA
structure, we generated model structures of oligo-NSA. The
models were built using dihedral angles (χ, φ, ψ, ω) of either
(100°, −105°, 105°, 180°) or (−100°, −120°, 90°, 180°) and
optimized with QM calculations using an implicit water model
(Figure 2e and f). With either set of dihedral angles, the
optimized structure of the oligo-NSA resulted in an extended
shape. The conformation with χ angle of −100° was 1.4
kcal/mol more stable than the conformation with χ angle of
100°, indicating that the χ angle forms −100° a little more
preferably in an oligomer. The extended shape of oligo-NSA is
realized by the iterative regulation of backbone dihedral angles
driven by the steric effects. This backbone-dictated shape
would be useful as a scaffold for biomolecular recognition
because the backbone forms a constrained conformation N-
substituent-independently and functional groups introduced as
N-substituents would be displayed in well-dispersed space
without causing intramolecular association of hydrophobic
residues.
the ω angles are restricted to the value around 180° (Figure
1
c). Because it does not require introduction of bulky N-
substituents, but requires the introduction of only a small
methyl substituent, this strategy is suitable for realizing a
peptoid that forms a defined shape in biological environment
and displays various functional groups as N-substituents into
well-defined three-dimensional spaces in water. Despite the
potential utility of oligo-NSA, optically pure oligo-NSAs
possessing N-substituents other than methyl have not been
reported because of the synthetic difficulty of such oligomers.
Thus, the validity of the constrained conformation of oligo-
NSA remains to be examined and establishment of synthetic
scheme for such a useful oligomer has been awaited.
Here, we established a synthetic method for oligo-NSA
and isolated optically pure oligo-NSAs bearing N-substituents
larger than methyl for the first time. Using the synthesized
oligo-NSAs, conformational studies of oligo-NSAs were
conducted and the studies revealed that oligo-NSA achieves a
defined shape in water and serves as a scaffold of an extended
shape displaying functional groups in a well-defined three-
dimensional space in water. The well-defined extended shape
of oligo-NSA was utilized to rationally generate a protein
ligand, which demonstrated the utility of this new peptoid for
biological applications.
Synthesis. To experimentally validate the extended
conformation of oligo-NSA, optically pure oligomers must be
isolated in multimilligram quantities. Although peptides or
oligo-NSGs with nonconsecutive NSA residues have been
3
5,41–44
reported,
there have been no reports for oligomers with
consecutive NSA residues with N-substituents other than
methyl. The extended shape of oligo-NSA would be realized
only by such oligomers with consecutive NSA residues.
Therefore, we first established a synthetic method for optically
pure oligo-NSAs. The most promising synthetic method was
the submonomer synthetic method using chiral 2-
RESULTS AND DISCUSSION
Quantum mechanical studies. First, we performed
computational simulations to predict how the pseudo-1,3-
allylic strain would constrain the conformation of peptoid and
what the conformation of oligo-NSA would look like. Minimal
components of oligo-NSG, i.e. acetyl-N-methylglycine
dimethylamide, and oligo-NSA, i.e. acetyl-N-methylalanine
bromopropionic acid and primary amine (Figure S2a)
35,45
proposed by Zuckerman and Kodadek
because this method
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Figure 2. (a) An energy diagram of acetyl-N-methylglycine dimethylamide as a minimal model of oligo-NSG. (b) An energy
diagram of acetyl-N-methylalanine dimethylamide as a minimal model of oligo-NSA. (c, d) An energy diagram of acetyl-N-
ethylalanine dimethylamide as a minimal model of oligo-NSA with N-substituents larger than methyl. The χ angle was fixed to
1
00° for c and –100° for d based on the results of χ scan (Figure S1). For these diagrams, regions that have energies above 10
kcal/mol from the lowest energy point were colored in white. (e) A model structure of acetyl-N-ethylalanine pentamer that was
optimized from an initial conformation of (χ, φ, ψ, ω) = (100°, −105°, 105°, 180°) with a QM calculation. (f) A model structure of
acetyl-N-ethylalanine pentamer that was optimized from an initial conformation of (χ, φ, ψ, ω) = (−100°, −120°, 90°, 180°) with a
QM calculation. (g) Definitions of φ angle (Ci-1, N
i
i i i i i
, Cα , C ) and ψ angle (N , Cα , C , Ni+1) of oligo-NSA. (h) Definitions of ω angle
(Cαi-1, Ci-1, N
i
, Cα ) and χ angle (Cα , N , CNα , CNβ
i
i
i
i
i
) of oligo-NSA.
does not require prior preparation of individual N-substituted
alanines, but only requires commercially available simple
submonomers. However, due to the racemization prone nature
due to the highly acid labile nature of NSAs47,48 and a
piperazine spacer was inserted between the trityl linker and
NSA oligomers to prevent diketopiperazine formation during
synthesis. All 1–5 mer of NSAs were obtained with decent
yields (Figures 3b and S5–8). There have been reports about
4
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of 2-bromopropionic acid, efficient synthesis of optically
pure oligo-NSAs using the method was unsuccessful (Figure
S2b–d). Therefore, we turned to another synthetic method for
oligo-NSAs reported by Pels and Kodadek that utilizes Fmoc-
3
5,42–44,49–51
oligo-NSGs containing skipped NSA residues,
but
this is the first report of the isolation of optically pure oligo-
NSAs containing consecutive NSA residues with N-
substituents other than methyl.
42
alanine and aldehydes as submonomers (Figure S3a). This
method also only requires commercially available
submonomers, which is equally attractive to the
abovementioned submonomer synthetic method. However, in
their report, consecutive introduction of NSA residues was
avoided due to the inefficient coupling reaction of Fmoc-
alanine on N-substituted alanine terminus. Therefore, we
investigated various coupling conditions for Fmoc-alanine on
N-substituted alanine terminus to achieve introduction of
consecutive NSA residues (Figure S3b). Among the tested
conditions, coupling reaction using 1-ethoxycarbonyl-2-
ethoxy-1,2-dihydroquinoline (EEDQ) in dioxane gave the
highest coupling yield (Figure S3c and Figure S4). Double
coupling at 60 °C for 3 h each or at 100 °C by microwave
heating for 1 h each using EEDQ gave a quantitative
conversion with no detectable amount of racemization (Figure
S3c and d). Encouraged by the quantitative conversion for the
challenging acylation reaction, 1–5 mer of NSAs were
synthesized on resin using the optimized conditions (Figure
To demonstrate that polar residues can be also introduced
on oligo-NSA, we synthesized heterooligomers containing
amine and carboxylic acid on N-substituents. We synthesized
pentamers with either Boc/tBu-protected amine/carboxylic
acid (S6) or Alloc/Allyl-protected amine/carboxylic acid (S7)
(Figure S9a and b). Although the yields were low (1% and 3%,
respectively), both the pentamers were successfully
synthesized and isolated. Deprotection of Alloc/Allyl groups
of S7 on resin successfully led to isolation of a pentamer with
free amine and carboxylic acid on N-substituents (S8). (Figure
S9c)
It should be noted that even the longest oligo-NSA 5 with
no polar N-substituents exhibited good water solubility. Oligo-
NSA 5 was soluble in water up to 270 mM (Figure S10). As a
control, an oligo-NSG with the same chain length and the
same N-substituents (5-NSG) was synthesized and its water
solubility was evaluated. The oligomer was soluble up to 320
mM, which is not significantly different from that of oligo-
NSA. This validates our strategy that the introduction of
methyl substituents on backbone carbons constrains the
3
a). Note that a trityl linker was recruited to allow cleavage of
the synthesized oligomers from resin with a mild acidic
condition (30% hexafluoroisopropanol in dichloromethane)
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Figure 3. Submonomeric synthesis of oligo-NSA using Fmoc-Ala-OH and aldehyde. (a) Synthetic scheme of oligo-NSA. Resin with a
trityl linker was utilized to allow cleavage of the synthesized compounds from resin using mild acidic conditions. The piperazine spacer
was introduced between the trityl linker and oligo-NSAs to prevent diketopiperazine formation during synthesis. (b) Yields of N-
isobutylalanine 1–5 mer (1–5). HPLC chromatograms of crude and purified products are listed on Figures S5 and S6. For monomer (1), 1H
and 13C NMR spectra are shown on Figures S7 and S8.
conformation of peptoids without reducing their water
solubiliy. As a piperazine group was previously shown to
were well separated on the oligomer and no obvious contacts
with backbone or other N-substituents were observed,
suggesting that the extended shape of oligo-NSA is dictated
solely by backbone steric effects and independently from N-
substituents. This makes the oligo-NSA a favorable scaffold
for displaying functional groups into well-dispersed three-
dimensional space without causing intramolecular association
of hydrophobic residues. N-substituents on every other residue
are displayed in the same directions. The distance from one
residue to the one after the next is 6.5–7.0 Å in the crystal. N-
methylated peptides were also previously reported to exhibit
similar side chain orientations with similar intervals in
2
6
improve the water solubility of oligo-NSG, the oligo-NSA
without the terminal piperazine group (5-amide) was also
synthesized to investigate the influence of the terminal group
on water solubility. As expected, this oligomer exhibited lower
water solubility than the oligomer with a piperazine group, but
the oligomer with a C-terminal amide was still soluble up to 87
mM. (Figure S11 shows the HPLC chromatograms of 5-NSG
and 5-amide after purification.) These results demonstrated the
utility of the oligo-NSA for biological applications.
X-ray crystallographic study and molecular dynamics
simulations. With the optimized synthetic method, several
oligo-NSAs were synthesized and X-ray crystallographic
analysis was conducted to experimentally reveal a preferred
conformation of oligo-NSAs. Among the various conditions
tested for the synthesized oligomers, crystals of N-
benzylalanine pentamer (Figure 4a, compound 6) were grown
via slow evaporation from a cosolvent of hexane and
dichloromethane. The structure was solved by X-ray
crystallographic analysis (Figures 4b and c, and S12). In the
crystal, the oligomer exhibited an extended shape that is
consistent with the predicted steric effects and QM
calculations (Figure 4c). First, all the amide bonds were trans
as expected from the steric repulsion of backbone structures.
Second, pseudo-1,3-allylic strains were seen at all the φ and ψ
angles and the dihedral angles of all the residues were
consequently restricted to the region within 2 kcal/mol from
the lowest energy point on the energy diagram generated by
the QM calculations (Figure S13). To assess how the crystal
structure relates to the above-described model structure, the
crystal structure was overlaid with one of the model structures
shown in Figure 2f that gave a lower energy than the other
model structure shown in Figure 2e. Overall, the backbone
conformation of the crystal structure and the model structure
well overlapped with each other except for the N-terminal
residue, which is inevitably different because the N-terminal
amine of the crystalized oligomer is not acylated and does not
experience pseudo-1,3-allylic strain (Figure 4d). The root-
5
2
crystals. This is reasonable considering the same pseudo-1,3-
allylic strains are the determining factors of the constrained
conformations of these two oligomers. It is notable that the
relative arrangements of side chains from backbones differ for
these two oligomers, which may lead to different types of
applications for each oligomer. Furthermore, a combination of
these two types of oligomers may expand their future utility.
To obtain insights into how much the conformation of
oligo-NSA observed in the crystal is stable in aqueous
solution, molecular dynamics (MD) calculations of the N-
benzylalanine pentamer were conducted using the crystal
structure as the initial conformation with explicit solvent. The
accuracy of computational simulations depends on the physical
models of interactions. MD simulations of peptoids have been
conducted based on traditional protein force fields, which
successfully reproduced some experimental observations, such
53–
as major conformations of NMR or X-ray crystal structures.
5
5
Therefore, we exploited the CHARMM36m/CGenFF force
field to assess the stability and dynamic behavior of oligo-
NSAs in solution. The calculation was performed for 500 ns at
298 K with five trials using the TIP3P water model. As a
result, the φ and ψ angles were in the region within 4 kcal/mol
from the lowest energy point on the energy diagram generated
from the QM calculations for 95% of the simulated time
(Figure S14). The ω angles were also kept at around 180° for
all the simulated time (Figure S15), although this does not
necessarily support the stability of trans amide bond because
the kinetics of trans/cis isomerization N-substituted amide is
α
mean-square deviation (RMSD) of backbone atoms (N, C ,
5
6
and carbonyl C) other than the N-terminal residues for the
model and the crystal structures was 0.289 Å. As seen in the
model structure and also in the crystal, all the N-substituents
much slower than the simulated time. Therefore, the ω angles
must be also evaluated spectroscopically (see the NMR
analysis
below).
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Figure 4. (a) Chemical structure of oligo-NSA 6 subjected to X-ray crystallographic analysis. (b) Dihedral angles of 6 observed in the
crystal. (c) Crystal structure of 6. (d) An overlay of the crystal structure and the model structure shown in Figure 2f (cyan). (e) RMSD
value of the oligo-NSA 6 during each MD run. The average and standard deviations are shown above each plot. (f) RMSD value of the
oligo-NSG during each MD runs. (g) Distribution of end-to-end distance of oligo-NSA (7) and (h) oligo-NSG (7-NSG) measured by
DEER experiment. The distributions of distance between the two spins were calculated with Tikhonov regularization (solid black line) and
Gaussian model fitting (dashed red line). The estimated length of oligo-NSA when it forms the predicted extended shape is labeled above
the structure. The average length of the oligomer in the Gaussian model fitting is described in gray in the graph.
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Because of the restricted rotations about these backbone
label is small and rigid in structure, the end-to-end distance
was not largely affected by the labels. The oligomer in 1:1
O/DMSO mixture was rapidly frozen at 20 K and subjected
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dihedral angles, the oligomer well sustained an extended shape
with a RMSD value of the α-carbons of the oligomer of only
0.5–0.9 Å on average, when compared to the initial crystal
structure, for all the five runs (Figure 4e). As an exception, the
RMSD value of the third run increased to ~2 Å at around 400
ns. The conformations observed during the last 100 ns of the
third run were little bent from the initial extended
conformation due to transitions of φ angles from around at
H
2
to the DEER measurement (Figures S21 and 22). This flush-
freezing process is supposed to preserve the conformational
6
4
distribution of peptides in solution at ambient temperature.
As a result, the mean distance of oligo-NSA determined from
Gaussian fitting was 27 Å and the distance distribution was 5
Å (Figure 4g). In the model extended shape of oligo-NSA
(Figure 2f), the distance between the two amide nitrogens of
neighboring residues was 3.3 Å on average. Therefore, the
NSA hexamer with an N-terminal alanine residue is expected
to be ~23 Å in length when it forms the predicted extended
structure. As a C-terminal piperazine spacer and two spin
labels are appended to the oligomer, it is reasonable to
consider that the observed 27 Å indicates that the oligo-NSA
forms the predicted extended shape in solution. As a control
oligomer, oligo-NSG (7-NSG) with the same chain length, the
same N-substituents, and the same terminal structures were
synthesized and measured. This oligomer exhibited a much
shorter mean length than oligo-NSA, 21 Å (Figure 4h) and a
larger distribution, 8 Å. The average distance is too short to
consider that the oligomer forms an extended shape. This
result suggests that oligo-NSG is more flexible than oligo-
NSA and does not persistently form an extended shape in
solution. Recently, Hawker and coworkers reported similar
DEER measurements of oligo-NSG and an oligomer with
alternate NSA/NSG residues and showed that both the
−
120° to around at 70°. However, the dihedral angles seen in
the bent conformations correspond to a high-energy region on
the Ramachandran-type plot (the upper-right blue region of
Figure 2b); therefore, the conformations are presumably a
minor population. In addition, the χ angles of the oligomers
were also restricted to around −100° and 100°, which were
calculated to be low in energy in the QM calculations (Figure
S16). In contrast, when the same MD simulations were run for
the oligomer of NSG backbone using the same backbone
conformation as the initial structure of oligo-NSA, the φ and ψ
angles of all the residues were distributed in much larger
ranges (Figure S17) and the RMSD value was also much larger
at 1.7 Å on average (Figure 4f). Interestingly, when an
oligomer with alternate NSA and NSG residues were subjected
to the same calculation, the dihedral angles of NSA residues
were constrained but those of NSG residues were not
constrained (Figure S18a), which led to distortion of the initial
extended shape and a large conformational fluctuation (Figure
S18b). This result agrees with the assumption that the extended
shape is only realized by oligomers with consecutive NSA
residues with no intervening NSG residues. This is consistent
with the previous report that oligo-NSGs with alternate NSA
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oligomers are intrinsically disordered. Therefore, we also
measured the oligomer with alternate NSA/NSG residues and
confirmed that the mean distance (20 Å) and distance
distribution (8 Å) is more similar to those of oligo-NSG rather
than those of oligo-NSA (Figure S23). These results support
the validity of the conclusion from the MD simulations, i.e.
oligomers with consecutive NSA residues form an extended
shape in water and the shape persistence is higher than
oligomers with NSG residues or alternating NSA/NSG
residues.
4
3
residues are intrinsically disordered.
To confirm the validity of our MD simulations, we also
performed both QM and molecular mechanics (MM) dihedral
(φ, ψ) scanning, and described one-dimensional energy
profiles as a function of each dihedral (Figure S19). The
energy minima of the MM potential were in the close vicinity
to those of the QM results, also corresponding to the dihedral
angles observed in the crystal structure. These results imply
that our simulations were able to capture the microscopic
detail of the peptoid dynamics.
Spectroscopic studies. NMR studies were conducted to
evaluate the solution structure of oligo-NSA. Several
heteropentamers that are expected to exhibit dispersed proton
peaks on NMR spectrum were designed and synthesized to
obtain information about spatial proximity of protons from 2D-
NMR spectrum. Among those, NSA heteropentamer 8
Although our computational results were in good
agreement with the experimental results as described later,
there is still room for improvement in describing molecular
interactions. In addition to the continuing developments of
protein force fields,57 a new force field has been parameterized
for an NSG peptoid based on experimental data (NMR and X-
α
satisfied the requirement and all the α-protons and N protons
of the oligomer on 1H NMR spectrum (Figure S24) were
successfully assigned unambiguously for this oligomer using
COSY (Figure S25), 13C NMR (Figure S26) and HMBC
spectra (Figure S27). Although the N-substituents differ
between oligomer 6 used for crystallographic analysis and
5
8,59
ray crystallography) and QM calculations.
Likewise, our
success of experimentally synthesizing oligo-NSAs and
solving the crystal structure will also enable us to develop a
new force field for oligo-NSA peptoids in future.
To complement our computational simulations and to
experimentally validate the stability of the extended shape of
oligo-NSA in water, we measured the end-to-end distance of
oligo-NSA using electron paramagnetic resonance (EPR)
spectroscopy. More specifically, we used the EPR-based
oligomer
8 used for NMR analysis, their backbone
conformation must closely resemble each other, not to say be
identical, because the extended backbone conformation is
dictated without the aid of N-substituents as described above.
The NOESY spectrum of the pentamer 8 was measured to
obtain information about spatial proximities among backbone
atoms in an oligomer (Figures 5a, S28, and S29). To evaluate
backbone conformation, interresidue NOEs among protons on
method, i.e.
(DEER/PELDOR) experiment.
a
double electron–electron resonance
6
0–63
We synthesized an NSA
hexamer (7) composed of ethyl and isobutyl N-substituents and
labeled a nitroxide radical on both termini of the oligomer
α, β, and N
amide bonds were confirmed to be in trans from interresidue
NOEs between protons on an N -carbon and the backbone α-
proton of the preceding alanine residue (Figure 5a left).
Restriction of bond
α
positions were analyzed. As a result, first, all
(Figure S20). A non-N-substituted alanine residue was inserted
α
between the N-terminal label and the N-terminus of oligo-NSA
to enhance the labeling reaction. Because the nitroxide spin
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Figure 5. Conformational studies of oligo-NSAs in aqueous solution. (a) A summary of spatial proximity information obtained from a
NOESY measurement. Protons with strong NOEs that suggest rotational restrictions about ω (left), φ (middle), and ψ (right) are indicated
with red solid arrows. Protons with a weak NOE related to ψ (right) are indicated with a red dashed arrow. 1H NMR spectrum, COSY
spectrum, 13C NMR spectrum, HMBC spectrum and NOESY spectrum are listed in Figures S24–S29. For the middle and right figures, a
part of the crystal structure is shown to visualize the proximities of N
α α
protons and β protons (middle) or N protons and α proton (right).
(b) Chemical structures and CD spectra of NSA pentamers 8, 9, and 5. (c) Chemical structures and CD spectra of oligo-NSA 2–5. The CD
spectra were recorded at 25 °C with 100 µM solution of each oligomer in phosphate buffer (pH 7.2). The Y-axis was normalized to molar
ellipticity per residue.
rotations about φ angles by pseudo-1,3-allylic strain is
expected to result in the intraresidue β and N protons being
close to each other while intraresidue α and N protons are
away from each other. The existence of strong NOEs between
3 4
same oligomer in acetonitrile-d and methanol-d to check the
α
solvent dependency of the conformation. In both solvents,
almost the same NOEs were observed, indicating that the
oligomer forms the same conformation in organic solvents
(Figure S31–42). This supports our hypothesis that the
conformation of oligo-NSA is dictated by local steric
interactions and not dictated by other remote interactions such
as hydrophobic interactions or electronic interactions among
N-substituents.
α
intraresidue β and N
intraresidue α and N
α
protons and absence of NOEs between
protons supports the restricted rotation
α
about φ angles around the value observed at the crystal
structure (Figure 5a, middle). Similarly, the restriction of bond
rotations about ψ angles by pseudo-1,3-allylic strain is
expected to result in the N
preceding alanine residue to be close to each other, while N
protons and β protons of preceding alanine are away from each
other. The existence of strong interresidue NOEs between N
protons and α proton of the preceding alanine residue supports
the restricted rotation about ψ angles around the value
observed at the crystal structure (Figure 5a, right). The validity
of the interpretation of the observed NOEs was ensured with
QM calculations of a model NSA dimer where a φ or ψ angle
α
protons and α proton of the
We also conducted a CD spectroscopic analysis of oligo-
NSA to evaluate the effects of the N-substituents,
concentration, solvent and length of the oligomer on
conformation of oligo-NSA. First, CD spectra of oligomer 8,
another heteropentamer 9, and a homopentamer 5 were
measured to check the sequence dependency of the backbone
conformation. All three oligomers exhibited strong signals and
a similar spectral shape with a maximum around at 195 nm
and a minimum around at 225 nm (Figure 5b). This result
indicates a specific set of backbone dihedral angles is repeated
in an oligomer that is independent from N-substituent
structures. This is consistent with the highly restricted
backbone dihedral angles in the QM calculation. The spectrum
of homopentamer 5 did not show concentration dependence of
the oligomer, which eliminates the possibility that the ordered
structure is realized by aggregation of the oligomer (Figure
S43a). The spectral shape did not change in acetonitrile or
methanol, which are less polar solvent than water (Figure
S43b). This confirms that the conformation is realized by local
steric effects on backbone. The spectral shape in aqueous
α
α
was systematically rotated and distances between N
α
protons
and α or β protons on each optimized structure were measured
th
(
Figure S30). Because the β protons of 4 residue exhibited a
th
α
weak NOE with the N protons of 5 residue, despite the fact
that those protons are more than 3 Å away from each other in
the crystal structure (Figure 5a, right, red dashed lines), some
degree of rotation may be allowed for the ψ angle. Together
with the absence of NOEs among nonneighboring remote
residues, the overall NMR results indicate that the extended
conformation of oligo-NSAs seen in the crystal is persistent in
aqueous solution. We also measured the NMR spectrum of the
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Figure 6. Design and binding assays of MDM2-binding oligo-NSA. (a) (Left) The crystal structure of p53-TAD binding to MDM2 from
PDB 1YCR. (Right, top) An enlarged view of p53-TAD in the left figure. The bonds connecting the α carbon (C ) and β carbon (C ) of
hot-spot residues (Phe19, Trp23 and Leu26) are shown with bold sticks. (Right, middle) The model structure of oligo-NSA pentamer from
Figure 2f. For oligo-NSA, the bonds connecting amide nitrogen (N) and N carbon (N ) of the 1st, 3rd, and 5th residues are shown with
bold sticks colored in magenta. (Right, bottom) The overlay of the above two structures. The RMSD value for C and C of hot-spot
residues in p53-TAD and N and N of 1st, 3rd, and 5th residues of oligo-NSA is described at the bottom. (b) Structures of oligo-NSA (10),
oligo-NSG (10-NSG) and oligomer with alternating NSA/NSG residues (10-NSA/NSG) that bears functional N-substituents (magenta)
corresponding to hot-spot residues of p53-TAD. (c) ITC profiles of interaction between the oligo-NSA (10) and MDM2. ITC data with full
parameters obtained from the fitting are shown in Figure S43a. (d) Inhibitory curves of 10 (red), 10-NSG (blue) and 10-NSA/NSG (green)
against the interaction between fluorescently labeled p53-TAD peptide and MDM2 generated from a competitive fluorescence polarization
assay. Error bars represent standard deviations of triplicates. The competitive binding data with full parameters obtained from the fitting
are shown in Figure S45.
α
β
α
α
α
β
α
buffer was also insensitive to temperature from 5 °C to 85 °C,
which indicates the extended structure is thermally stable
rotations about backbone dihedral angles and oligomers with
consecutive NSA residues uniformly exhibit a constrained
conformation composed of repetition of a similar set of
backbone dihedral angles, most probably around (φ, ψ) = (–
120°, 90°), which was found to be the lowest energy point in
the QM calculation.
(Figure S43c and d). Shorter oligomers, tetramer and trimer,
exhibited similar spectra to the pentamers and even dimer
exhibited a similar spectral shape, although the intensity was
little weaker than in longer oligomers (Figure 5c). This result
suggests that an NSA residue is intrinsically restricted in
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Application of oligo-NSA to biomolecular recognition.
the binding is much weaker than the corresponding oligo-NSA
(10) (Figure S44b). In addition, the oligo-NSG did not
compete with p53-TAD peptide for MDM2 binding up to 300
µM (Figure 6d), which further supports the much lower
affinity of the oligo-NSG compared to the corresponding
oligo-NSA. An oligomer with alternating NSA and NSG
residues bearing the same N-substituents with the oligo-NSA
10 (10-NSA/NSG) also did not generate as much heat as oligo-
NSA 10 did as seen in the ITC profile (Figure S44c) and the
competitive fluorescence polarization assay showed that the
oligomer (10-NSA/NSG) binds to MDM2 much less strongly
than oligo-NSA 10 (Figure 6d).
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Thus far, we have shown that the rotations about all the three
backbone dihedral angles, φ, ψ and ω, of oligo-NSA are
restricted per-residue basis, which leads to the persistent
formation of the extended shape of the oligomer in solution.
This rigid structure together with its good water solubility and
facile introduction of various N-substituents makes oligo-NSA
a promising scaffold for displaying multiple functional groups
in a well-defined three-dimensional space. Therefore, we
hypothesized that oligo-NSA displaying hot spot residues
involved in protein-protein interactions in an appropriate order
would effectively bind to proteins. To examine this hypothesis,
we designed an oligo-NSA that binds to MDM2. MDM2 is
recognized by three hot spot residues, Phe19, Trp23 and
Leu26, displayed on the transactivation domain of p53 (p53-
TAD). When a previously reported crystal structure of p53-
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These results demonstrated the utility of oligo-NSA as a
scaffold for protein binders. Besides, the importance of
conformational constraints on peptoid for achieving the
efficient protein binding was also demonstrated, at least for the
current example. The α-helix mimetic based on the β-strand-
like scaffold is reminiscent of the β-hairpin scaffold reported
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5
TAD binding to MDM2 was overlaid with the model
structure of oligo-NSA generated from the QM calculations
6
9
(Figure 2f), N-substituents of every other residue of oligo-NSA
by Robinson and co-workers. The per-residue conformational
control of oligo-NSA realized a smaller α-helix mimic than the
β-hairpin-based mimic.
were approximately overlapped with the three hot spot
residues of p53-TAD (Figure 6a). More specifically, C
α
and C
β
atoms of the three hot-spot residues of p53-TAD and N and N
α
Many preceding reports described the mimicry of α-helical
protein–protein interactions using small molecules, e.g.
terphenyl and oligobenzamides, and some of these molecules
exhibited comparable or even better MDM2-binding affinities
than the oligo-NSA 10. However, the synthetic flexibility of
oligo-NSA is its advantageous feature as a molecular scaffold
over other molecules. In future, this unique feature will
facilitate (1) medicinal chemistry on the lead compound 10 to
produce more potent MDM2 inhibitors and (2) rational design
of inhibitors of other protein–protein interactions, especially
those mediated by α-helices and β-sheets.
atoms of the 1st, 3rd, and 5th residues of the model oligo-NSA
matched with RMSD value of 0.88 Å. Based on this result, we
designed and synthesized oligo-NSA 10 that bears benzyl,
indolylmethyl and isopentyl substituents at the 1st, 3rd, and
5
th residues (Figure 6b) assuming that the oligo-NSA mimics
p53-TAD and binds to MDM2. During the synthesis of the
designed oligomer, it was found that the indolylmethyl group
was difficult to install using the reductive amination protocol
described above. This is probably because an aldehyde with an
electron-rich aromatic group like indole directly connected to
the carbonyl carbon poorly reacts with the terminal amine of
oligo-NSA. Therefore, we recruited reductive amination
conditions reported previously where acidic conditions and a
dehydrating solvent, trimethyl orthoformate, are used for imine
CONCLUSIONS
In this study, we achieved efficient synthesis of optically
pure oligo-NSA and demonstrated the following three notable
features of oligo-NSA: 1) all three backbone dihedral angles,
φ, ψ and ω, of oligo-NSA are controlled per residue basis
irrespective of the structure of N-substituents, which leads to
the well-defined extended shape of the oligomer, 2) the
oligomer exhibits good water solubility, and 3) the efficient
submonomer synthetic method allows facile introduction of
various functional groups as N-substituents. These features
make the extended shape of oligo-NSA an attractive scaffold
for rational design of protein ligands. The utility of oligo-NSA
as a scaffold was clearly demonstrated by the generation of a
MDM2 ligand. The backbone mutational study conducted on
the MDM2 ligand successfully demonstrated that the
introduction of conformational restriction on peptoid is
important to realize efficient protein binding. The side-chain
independent formation of the well-defined backbone shape
allowed the facile generation of protein ligands via the rational
approach.
As stated in the introduction, many oligo(N-substituted
amides) including oligo-NSG have been reported as peptoids.
Among the large collection of such peptoids, oligo-NSA is
unique for the per-residue program of backbone
conformations. This allows oligo-NSA to form a well-defined
shape that is well-predictable from the monomer structure and
liberate N-substituents, or side chains for biomolecular
recognitions or other functions.
6
6,67
formation.
With this reductive amination conditions,
indolylmethyl group was successfully installed as N-
substituents and the designed oligo-NSA was successfully
synthesized.
The synthesized ligand was confirmed to bind to MDM2
by isothermal titration calorimetry (ITC). The dissociation
D
constant (K ) was determined to be 1.1 µM (Figures 6c and
S44a). The binding was enthalpy-driven (ΔH = −10.8
kcal/mol, −TΔS = 2.7 kcal/mol, and ΔG = –8.1 kcal/mol),
suggesting that the interaction is not caused by nonspecific
hydrophobic contacts, but realized by specific interactions.
The designed oligo-NSA was confirmed to bind to MDM2
at the same binding site with p53-TAD by a competitive
fluorescent polarization assay using a fluorescently labeled
p53-TAD peptide. The oligo-NSA 10 competed with the p53-
i
TAD peptide with K value of 0.93 µM (Figures 6d and S45).
(As a reference, the binding isotherm of the fluorescently
labeled p53-TAD and MDM2 is shown in Figure S46.)
Finally, we examined the importance of the
conformationally constrained oligo-NSA backbone for the
MDM2 binding because protein ligands based on a rigid
scaffold are not necessarily always optimal ligands.68 The
same set of binding assays and competitive binding assays of
control oligomers that completely or partially lose NSA
backbones was conducted. Oligo-NSG bearing the same N-
substituents with the oligo-NSA 10 (10-NSG) did not generate
substantial amount of heat upon titration on MDM2 suggesting
There are also limitations in the current state of oligo-NSA
chemistry. First, although we successfully synthesized oligo-
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NSA up to pentamer, the yields were modest or low.
Page 10 of 14
The computations were performed using Research Center for
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Especially, for heteropentamers with polar residues, the yields
were very low. In addition, the synthesis involves long
coupling reactions. To accelerate further research on oligo-
NSA, more efficient and rapid synthetic methods should be
developed in future. Second, the shape of oligo-NSA is limited
to the extended shape when only L-alanine is used as
submonomer. The extended shape can be utilized as a helix
mimetic as demonstrated in this study and possibly also as a β-
strand mimetic. As protein-protein interaction interfaces
consist of a diverse tertiary structure, expansion of the shape
diversity is desirable to utilize oligo-NSA as more widely
applicable protein capture agents. Introduction of amino acids
that prefer different area of the Ramachandran plot is a
promising strategy to achieve the goal. Plausible candidates
are β-branched amino acids, e.g. valine, and D-alanine. Also,
insertion of other conformationally preorganized amino acids,
such as L/D-proline, would realize different folded shapes.
Third, the oligo-NSA lacks backbone amide hydrogens, which
prohibit the backbone to make hydrogen bonds with target
proteins. This limitation needs to be backed up by introducing
hydrogen bonding donors on N-substituents in the current
design of oligo-NSA or by introducing other peptoid-type
backbone units possessing hydrogen bonding donors, such as
Computational Science, Okazaki, Japan. We thank Dr. H. Sato
Mr. S. Suginome, Dr. S. Kusumoto, Dr. M. Akiyama, and Dr.
Okitsu at the University of Tokyo and Dr. H. Sato at Rigaku for
the support on X-ray crystallographic analysis. We thank Drs. Y.
Goto and H. Suga at the University of Tokyo for the use of a CD
spectrometer. The X-ray diffraction data of the crystal was
collected using an instrument at Advanced Characterization
Nanotechnology Platform of the University of Tokyo, supported
by “Nanotechnology Platform” of the Ministry of Education,
Culture, Sports, Science and Technology (MEXT), Japan. This
work was partly supported by Platform Project for Supporting
Drug Discovery and Life Science Research (Basis for Supporting
Innovative Drug Discovery and Life Science Research (BINDS))
from AMED (JP18am0101094). This work was partly supported
by Nanotechnology Platform Program (Molecule and Material
Synthesis) of MEXT, Japan. This work was partly supported by
the World-leading Innovative Graduate Study Program for Life
Science and Technology, The University of Tokyo, as part of the
WISE Program (Doctoral Program for World-leading Innovative
& Smart Education), MEXT, Japan.
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AUTHOR INFORMATION
Corresponding Author
Non-Peptide
Helical
Foldamers
by
Sequence
J.M.: jmorimoto@chembio.t.u-tokyo.ac.jp
S.S.: ssando@chembio.t.u-tokyo.ac.jp
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‡
These authors contributed equally.
Funding Sources
S.S. acknowledges
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acknowledges financial support from JSPS (JP17K13265 and
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and
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(JP18fm0208022h).
K.T.
acknowledges financial support from JSPS (JP16H02420 and
JM17H06109) and AMED (JP19am0101094).
Notes
The authors (J. M., Y. F. and S. S.) have filed a patent application
(PCT/JP2019/124016 ).
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