GZ-11608, a vesicular monoamine transporter-2 inhibitor, decreases the neurochemical and behavioral effects of methamphetamine

Article abstract of DOI:10.1124/jpet.119.258699

Despite escalating methamphetamine use and high relapse rates, pharmacotherapeutics for methamphetamine use disorders are not available. Our iterative drug discovery program had found that R-N-(1,2-dihydroxypropyl)-2,6-cis-di-(4-methoxyphenethyl)piperidine hydrochloride (GZ-793A), a selective vesicular monoamine transporter-2 (VMAT2) inhibitor, specifically decreased methamphetamine’s behavioral effects. However, GZ-793A inhibited human-ether-a-go-go-related gene (hERG) channels, suggesting cardiotoxicity and prohibiting clinical development. The current study determined if replacement of GZ-793A’s piperidine ring with a phenylalkyl group to yield S-3-(4-methoxyphenyl)-N-(1-phenylpropan-2-yl)propan-1-amine (GZ-11608) diminished hERG interaction while retaining pharmacological efficacy. VMAT2 inhibition, target selectivity, and mechanism of GZ-11608-induced inhibition of methamphetamine-evoked vesicular dopamine release were determined. We used GZ-11608 doses that decreased methamphetamine-sensitized activity to evaluate the potential exacerbation of methamphetamine-induced dopaminergic neurotoxicity. GZ-11608-induced decreases in methamphetamine reinforcement and abuse liability were determined using self-administration, reinstatement, and substitution assays. Results show that GZ-11608 exhibited high affinity (K_i 5 25 nM) and selectivity (92–1180-fold) for VMAT2 over nicotinic receptors, dopamine transporter, and hERG, suggesting low side-effects. GZ-11608 (EC₅₀ 5 620 nM) released vesicular dopamine 25-fold less potently than it inhibited VMAT2 dopamine uptake. GZ-11608 competitively inhibited methamphetamine-evoked vesicular dopamine release (Schild regression slope 5 0.9 6 0.13). GZ-11608 decreased methamphetamine sensitization without altering striatal dopamine content or exacerbating methamphetamine-induced dopamine depletion, revealing efficacy without neurotoxicity. GZ-11608 exhibited linear pharmacokinetics and rapid brain penetration. GZ-11608 decreased methamphetamine self-administration, and this effect was not surmounted by increasing methamphetamine unit doses. GZ-11608 reduced cue- and methamphetamine-induced reinstatement, suggesting potential to prevent relapse. GZ-11608 neither served as a reinforcer nor substituted for methamphetamine, suggesting low abuse liability. Thus, GZ-11608, a potent and selective VMAT2 inhibitor, shows promise as a therapeutic for methamphetamine use disorder. SIGNIFICANCE STATEMENT GZ-11608 is a potent and selective vesicular monoamine transporter-2 inhibitor that decreases methamphetamine-induced dopamine release from isolated synaptic vesicles from brain dopaminergic neurons. Results from behavioral studies show that GZ-11608 specifically decreases methamphetamine-sensitized locomotor activity, methamphetamine self-administration, and reinstatement of methamphetamine-seeking behavior, without exhibiting abuse liability. Tolerance does not develop to the efficacy of GZ-11608 to decrease the behavioral effects of methamphetamine. In conclusion, GZ-11608 is an outstanding lead in our search for a therapeutic to treat methamphetamine use disorder.

Full text of DOI:10.1124/jpet.119.258699

Special Issue: Peptoids – Part 1
Guest Editors: Kent Kirshenbaum (New York University, U.S.A.) and Ronald N. Zuckermann (Lawrence Berkeley National
Laboratory, U.S.A.)
EDITORIAL
Peptoids in Wonderland
Kent Kirshenbaum and Ronald N. Zuckermann, Biopolymers 2019, doi: 10.1002/bip.23279
REVIEW
Design and preparation of organic nanomaterials using self-assembled peptoids
Alessia Battigelli, Biopolymers 2019, doi: 10.1002/bip.23265
FULL PAPERS
Dual-responsive pegylated polypeptoids with tunable cloud point temperatures
Xiaohui Fu, Jiliang Tian, Zheng Li, Jing Sun and Zhibo Li, Biopolymers 2019, doi:
10.1002/bip.23243
Solid-phase synthesis of three-armed star-shaped peptoids and their hierarchical self-assembly
Haibao Jin, Tengyue Jian, Yan-Huai Ding, Yulin Chen, Peng Mu, Lei Wang and Chun-Long Chen, Biopolymers 2019, doi:
10.1002/bip.23258
Polymerization rate difference of N-alkyl glycine NCAs: Steric hindrance or not?
Tianwen Bai and Jun Ling, Biopolymers 2019, doi: 10.1002/bip.23261
Linking two worlds in polymer chemistry: The influence of block uniformity and dispersity in amphiphilic block
copolypeptoids on their self-assembly
Niklas Gangloff, Marcel Höferth, Vladimir Stepanenko, Benedikt Sochor, Bernhard Schummer, Joachim Nickel, Heike Walles,
Randolf Hanke, Frank Würthner, Ronald N. Zuckermann and Robert Luxenhofer, Biopolymers 2019, doi: 10.1002/bip.23259
Peptoids advance multidisciplinary research and undergraduate education in parallel: Sequence effects on conformation and
lipid interactions
Christian J. Jimenez, Jiacheng Tan, Kalli M. Dowell, Gillian E. Gadbois, Cameron A. Read, Nicole Burgess, Jesus E. Cervantes,
Shannon Chan, Anmol Jandaur, Tara Karanik, Jaenic J. Lee, Mikaela C. Ley, Molly McGeehan, Ann McMonigal, Kira L.
Palazzo, Samantha A. Parker, Andre Payman, Maritza Soria, Lauren Verheyden, Vivian T. Vo, Jennifer Yin, Anna L. Calkins,
Amelia A. Fuller and Grace Y. Stokes, Biopolymers 2019, doi: 10.1002/bip.23256
Phosphoramitoids—A submonomer approach to sequence defined N-substituted phosphoramidate polymers
Thomas Horn, Michael D. Connolly and Ronald N. Zuckermann, Biopolymers 2019, doi: 10.1002/bip.23268
Solution effects on the self-association of a water-soluble peptoid
Amelia A. Fuller, Jonathan Huber, Christian J. Jimenez, Kalli M. Dowell, Samuel Hough, Alberto Ortega, Kyra N. McComas,
Jeffrey Kunkel and Prashanth Asuri, Biopolymers 2019, doi: 10.1002/bip.23248

Special Issue: Peptoids – Part 1
Guest Editors: Kent Kirshenbaum (New York University, U.S.A.) and Ronald N. Zuckermann (Lawrence Berkeley National
Laboratory, U.S.A.)
A facile on-bead method for fully symmetric tetra-substituted DOTA derivatizations using
peptoid moieties
Vineeta Rustagi and D. Gomika Udugamasooriya, Biopolymers 2019, doi: 10.1002/bip.23249
Macrocyclization enhances affinity of chemokine-binding peptoids
Kevin Brahm, Julia S. Wack, Stefanie Eckes, Victoria Engemann and Katja Schmitz, Biopolymers
2019, doi: 10.1002/bip.23244

Special Issue: Peptoids – Part 2
Guest Editors: Kent Kirshenbaum (New York University, U.S.A.) and Ronald N. Zuckermann (Lawrence Berkeley National
Laboratory, U.S.A.)
EDITORIAL
Molecular folding science
Kent Kirshenbaum and Ronald N. Zuckermann, Biopolymers 2019, doi: 10.1002/bip.23314
REVIEWS
Peptoids as tools and sensors
Adrian S. Culf, Biopolymers 2019, doi: 10.1002/bip.23285
Peptoid drug discovery and optimization via surface X-ray scattering
Konstantin Andreev, Michael W. Martynowycz and David Gidalevitz, Biopolymers 2019, doi:
10.1002/bip.23274
Polypeptoids synthesis based on Ugi reaction: Advances and perspectives
Yue Tao, Zhen Wang and Youhua Tao, Biopolymers 2019, doi: 10.1002/bip.23288
FULL PAPERS
NCα‐gem‐dimethylated peptoid side chains: A novel approach for structural control and peptide sequence mimetics
Radhe Shyam, Lionel Nauton, Gaetano Angelici, Olivier Roy, Claude Taillefumier and Sophie Faure, Biopolymers 2019, doi:
10.1002/bip.23273
Unconstrained peptoid tetramer exhibits a predominant conformation in aqueous solution
Leah T. Roe, Jeffrey G. Pelton, John R. Edison, Glenn L. Butterfoss, Blakely W. Tresca, Bridgette A. LaFaye, Stephen
Whitelam, David E. Wemmer and Ronald N. Zuckermann, Biopolymers 2019, doi: 10.1002/bip.23267
Oligomers of α‐ABpeptoid/β3‐peptide hybrid
Kang Ju Lee, Ganesh A. Sable, Min-Kyung Shin and Hyun-Suk Lim, Biopolymers 2019, doi: 10.1002/bip.23289
Stereochemistry of polypeptoid chain configurations
Ryan K. Spencer, Glenn L. Butterfoss, John R. Edison, James R. Eastwood, Stephen Whitelam, Kent Kirshenbaum and Ronald
N. Zuckermann, Biopolymers 2019, doi: 10.1002/bip.23266
Peptoid microsphere coatings: The effects of helicity, temperature, pH, and ionic strength
German R. Perez Bakovic, Jesse L. Roberts, Bryce Colford, Myles Joyce and Shannon L. Servoss, Biopolymers 2019, doi:
10.1002/bip.23283
Antibacterial mechanisms of GN-2 derived peptides and peptoids against Escherichia coli
Paola Saporito, Mojsoska Biljana, Anders Løbner Olesen and Håvard Jenssen, Biopolymers 2019, doi: 10.1002/bip.23275
Toward a clinical antifungal peptoid: Investigations into the therapeutic potential of AEC5
Sabrina K. Spicer, Aarthi Subramani, Angelica L. Aguila, R. Madison Green, Erin E. McClelland and Kevin L. Bicker, Biopolymers
2019, doi: 10.1002/bip.23276
Helical side chain chemistry of a peptoid-based SP-C analogue: Balancing structural rigidity and biomimicry
Nathan J. Brown, Jennifer S. Lin and Annelise E. Barron, Biopolymers 2019, doi: 10.1002/bip.23277

Received: 17 December 2018
DOI: 10.1002/bip.23289
Revised: 8 May 2019
Accepted: 14 May 2019
F U L L P A P E R
Oligomers of α-ABpeptoid/β3-peptide hybrid
Kang Ju Lee | Ganesh A. Sable | Min-Kyung Shin | Hyun-Suk Lim
Department of Chemistry and Division of
Abstract
Advanced Material Science, Pohang University
of Science and Technology (POSTECH),
Peptoids, oligomers of N-substituted glycines, have been attracting increasing interest
Pohang, South Korea
due to their advantageous properties as peptidomimetics. However, due to the lack of
chiral centers and amide hydrogen atoms, peptoids, in general, do not form folding
structures except that they have α-chiral side chains. We have recently developed
“peptoids with backbone chirality” as a new class of peptoid foldamers called
α-ABpeptoids and demonstrated that they could have folding conformations owing to
the methyl groups on chiral α-carbons in the backbone structure. Here we report
α-ABpeptoid/β3-peptide oligomers as a unique peptidomimetic structure with a het-
erogeneous backbone. This hybrid structure contains a mixed α-ABpeptoid and
β3-peptide residues arranged in an alternate manner. These α-ABpeptoid/β3-peptide
oligomers could form intramolecular hydrogen bonding and have better cell permeabil-
ity relative to pure peptide sequences. These oligomers were shown to adopt ordered
folding structures based on circular dichroism studies. Overall, α-ABpeptoid/β3-peptide
oligomers may represent a novel class of peptidomimetic foldamers and will find a wide
range of applications in biomedical and material sciences.
Correspondence
Hyun-Suk Lim, Department of Chemistry and
Division of Advanced Material Science,
Pohang University of Science and Technology
(POSTECH), Pohang 37673, South Korea.
Email: hslim@postech.ac.kr
Funding information
National Research Foundation of Korea,
Grant/Award Number: NRF-
2018R1A4A1024713; The National Research
Foundation of Korea, Grant/Award Numbers:
NRF-2017M3A9F6029753, NRF-
2017M3A9E4077447
K E Y W O R D S
foldamer, peptidomimetics, peptoid, α-ABpeptoid, α-ABpeptoid/β3-peptide hybrid
other synthetic peptidomimetics[22,23] and far more cell-permeable com-
pared with native peptides.[24–27] Moreover, a number of primary amines
1
| INTRODUCTION
Artificial oligomers that can emulate three-dimensional folding structures of
natural biopolymers, such as oligonucleotides and proteins, have been
received significant attentions.[1,2] Among them, peptidomimetic foldamers
have promising features as alternatives to native peptides.[3] They often
have better proteolytic stability than peptides. Moreover, they can achieve
larger structural and functional diversities by including various proteogenic
and nonproteogenic side chains. These desirable features may allow them
to serve as an important tool in various biological applications. Indeed, a
myriad of peptidomimetic foldamers have been developed including β- and
γ-peptides,[4–6] oligoureas,[7,8] oligopyrrolidines,[9] γ-AApeptides,[10,11]
α-aminoxy acids,[12] azapeptides,[13,14] oligotriazoles,[15] aromatic
oligoamides,[16,17] and peptoids.[18–20]
as monomer building blocks are readily available, enabling to prepare
structurally diverse peptoid oligomers through efficient solid-phase
submonomer route.[28] In spite of these advantages, peptoids are limited
in using them for biological applications (e.g., as modulators of protein
functions) by their structural flexibility. Because their backbone is com-
posed of tertiary amides devoid of amide protons, peptoids are generally
unable to make intramolecular hydrogen bonds. Moreover, peptoids do
not have main chain chirality. As a result, peptoids are far more flexible
than their peptide counterparts, making it difficult to find peptoids with
high affinity to target proteins. Hence, the development of strategies to
restrict structural flexibility of peptoids has been of significant interest.
For example, macrocyclization has been often used to increase conforma-
tional rigidity of peptoids.[29–37] Notably, it has been proven that peptoids
with α-chiral side chains can form well-defined helical structures, which
resemble polyproline type I helix.[20,38–43] Recently, we have developed a
Peptoids, oligomers of N-substituted glycines, have been attracting
increasing interest due to their advantageous properties as peptido-
mimetics (Figure 1).[21] They are resistant to enzymatic degradation like
Biopolymers. 2019;110:e23289.
wileyonlinelibrary.com/journal/bip
© 2019 Wiley Periodicals, Inc.
1 of 8
https://doi.org/10.1002/bip.23289

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LEE ET AL.
FIGURE 1 General structures of α-peptide,
peptoid, β3-peptide, α-ABpeptoid, and 1:1 β3-
peptide/α-ABpeptoid
new type of peptoids termed α-ABpeptoids (α-alkyl beta-peptoids), oligo-
mers of N-substituted β2-homoalanines (Figure 1).[44] α-ABpeptoids have
methyl groups at chiral α-positions of the β-peptoid-like backbone. The
introduction of these methyl groups into the β-peptoid backbone was
anticipated to induce distinctive folding structures by restricting the con-
figuration of tertiary amide like those in peptoids with α-chiral sides.
Based on the circular dichroism (CD) studies, α-ABpeptoids with (R)-
methyl groups showed characteristic CD spectra with a strong minimum
at 200-210 nm and a weak maximum at 220 to 230 nm, which are remi-
niscent of those of polyproline type II helices. Of note, the enantiomers
harboring (S)-methyl groups displayed mirror images of those of (R)-form
oligomers. These observations indicate that the enantiomeric oligomers
form an identical folding structure with opposite handedness. The result
also suggests that the backbone chirality should contribute to the forma-
tion of the ordered folding structure. In addition, we developed a
submonomer strategy that allows for the rapid and convenient synthesis
of structurally diverse α-ABpeptoid oligomers.[45]
Massachusetts) and used without further purification. Rink amide MBHA
resin (0.45 mmol/g) was purchased from Beadtech, Inc. (Seoul, South
Korea). Analytical high performance liquid chromatography (HPLC) and
liquid chromatography/mass spectrometry were performed on Agilent
systems (Santa Clara, California) with a C18 reversed phase HPLC column
(Eclipse XDB, 3.5 μm, 4.6 mm × 150 mm). A gradient elution of 90% A in
7 minutes followed by 100% B in 13 minutes was used at flow rate of
0.7 mL/min (solvent A: 100% H2O, 0.01% TFA; B: 100% acetonitrile,
0.01% TFA). Preparative HPLC purification was performed on an Agilent
1120 Compact LC system (Agilent Technology) with a C18 reversed-
phase column (Agilent Technology, 5 μM, 21.2 mm × 150 mm) using a lin-
ear gradient from 10% B to 100% B by changing solvent composition over
40 minutes.
2.2 | Hybrid-oligomers of 1:1 β3-phenyl
alanine/benzylated α-ABpeptoid synthesis, (S,R)-4a-f
Rink amide MBHA resin (100 mg, 45 μmol) was swelled with
dimethylformamide (DMF) (1 mL) in a 6-mL fritted syringe for 1 hour.
The Fmoc protecting groups were removed by treating with 20% piper-
idine in DMF (2 × 10 minutes). The resin was treated with (S)-3-(Fmoc-
amino)-4-phenylbutyric acid ((S)-β3, 3.3 equiv), 1-hydroxy-
7-azabenzotriazole (HOAT) (3.3 equiv), 1-[bis(dimethylamino)methy-
lene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
(HATU) (3.3 equiv), and N,N-diisopropylethylamine (DIPEA) (6.6 equiv)
in DMF (1 mL) for 3 hours at room temperature. Then, the reaction
mixture was removed, and the resin was washed with DMF (×7). After
the Fmoc protecting group was removed, the resin was treated with
presynthesized monomer (R)-methyl 3-hydroxy-2-methylpropanoate
((R)-α-AB, 3.3 equiv), HOAT (3.3 equiv), HATU (3.3 equiv), and DIPEA
(6.6 equiv) in DMF (1 mL) for 3 hours at room temperature. After a
thorough washing cycle, the nosyl protecting group was removed by
treating 2-mercaptoethanol (20 equiv) and 1,8-diazabicycloundec-
7-ene (DBU) (10 equiv) in DMF (1 mL) for 3 hours at room temperature.
Then, (S)-β3 and (R)-α-AB were alternatively added to the resin accom-
panying protecting group removal steps to afford hybrid-oligomers
(S,R)-4a-f. The other oligomers (R,S)-4d, (S,S)-4a-d, and (R,R)-4d were
synthesized following the same procedure described above, but enan-
tiomeric monomers, (R)-β3 and (S)-α-AB, were included. After washing
with DMF (3×), CH2Cl2 (2×), MeOH (2×), and CH2Cl2 (5×), the products
were detached from the resin by treating with a cleavage cocktail (95%
trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% water) for 2 hours
In addition to foldamers with homogeneous backbones, peptido-
mimetics possessing heterogeneous backbone structures are of consider-
able interest.[2,3,46,47] The use of heterogeneous backbones could not only
expand the structural complexity and functional repertoire of pep-
tidomimetic foldamers but also exhibit various biological activities. A vari-
ety of heterogeneous backbone foldamers have been developed.
Examples include α/β/γ-peptides,[48,49] urea/amide/carbamate,[50] and α/-
γ-AApeptides.[51] Here we report α-ABpeptoid/β3-peptide oligomers as a
unique peptidomimetic structure with a heterogeneous backbone. This
hybrid structure contains a mixed α-ABpeptoid and β3-peptide residues
arranged in an alternate manner. We envisioned that the α-ABpeptoid/β3-
peptide oligomers could form intramolecular hydrogen bonding, allowing
the hybrid sequences to have more stable and unique folding structures.
Note that peptoids including our α-ABpeptoids are unable to make intra-
molecular hydrogen bonding networks due to the lack of amide protons.
Moreover, owing to the presence of alternating α-ABpeptoid residues,
α-ABpeptoid/β3-peptide oligomers are expected to have better cell perme-
ability relative to pure peptide sequences.
2
| METHODS
2.1 | Materials and general methods
All chemicals and reagents were purchased from commercial suppliers
(Sigma-Aldrich, Saint Louis, Missouri, and Alfa Aesar, Haverhill,

LEE ET AL.
3 of 8
at room temperature. The cleaved products were purified by reverse-
phase HPLC.
delay of 2 seconds and a mixing time of 250 ms with 48 to 56 scans
was used for averaging. All the samples were dissolved in CD3CN and
the spectra were recorded at 300 K. For the H/D exchange experi-
ment, the sample was dissolved in 500 μL of CD3CN and subse-
quently added 50 μL of D2O. Then, 1H NMR spectra was recorded
after 30 minutes and 1 hour at 300 K. Acquisition and processing of
spectra were performed with the Bruker TopSpin 3.1 software.
2.3 | CD spectra measurement
CD spectra were measured on a Jasco J-815 spectropolarimeter
(Jasco Corporation, Tokyo, Japan). The freezing-dried samples were
dissolved in proper solvent to make stock solutions. The CD sample
solutions were prepared in 1.5-mL microtubes by serial dilution of
stock solutions. The spectra were measured in the quartz cuvette with
2 mm path length. Five successive accumulations with 100 nm/min
scan rate was conducted for the measurement. The raw data were
corrected by subtraction of background signal. Data were, then, calcu-
lated in terms of per-residue molar ellipticity (deg cm2/dmol), which
are normalized by the number of amide groups and the molar concen-
tration of the oligomers. Seven points smoothing was performed
afterward.
3
| RESULTS AND DISCUSSION
Initially, we synthesized a series of α-ABpeptoid/β3-peptide oligomers
as a model system, in which both parts of the residues contained phe-
nyl side chains but opposite chiral configurations; (R)-configurations
for α-ABpeptoids and (S)-configurations for β3-peptide parts
(Scheme 1). In order to prepare these heterogeneous oligomers com-
posed of alternating β3-phenyl alanine/benzylated α-ABpeptoids, we
used two building blocks, N-benzyl β2-homoalanine (R)-α-AB, a mono-
mer for α-ABpeptoid part, and (S)-3-(Fmoc-amino)-4-phenylbutyric
acid (S)-β3, a building block for β-peptide part. (R)-α-AB was synthe-
sized by following the previously reported procedure,[44] whereas (S)-
β3 is commercially available. First, (S)-β3 was loaded on Rink amide
MBHA resin using HOAT/HATU coupling conditions to afford 1. After
Fmoc deprotection, (R)-α-AB was coupled to the resulting primary
2.4 | NMR analysis
1H- and 13C-NMR spectra were collected at resonance frequencies of
600 and 150 MHz, respectively, using Bruker AVANCE 600 spectrom-
eter (Bruker, Billerica, Massachusetts). The conventional 1H-COSY
and 1H-13C HSQC 2D experiments were recorded using routine pulse
sequence available in the Bruker TopSpin 3.1 software. 1H-COSY and
1H-13C HSQC spectra were measured with the relaxation delay of
2 and 1.5 seconds, respectively, and 32 to 52 scans were used for
averaging. 2D NOESY experiments were conducted on a Bruker
AVANCE 600 spectrometer equipped with a xyz gradient triple-
resonance probe. The NOESY spectra were recorded with relaxation
amine by the same coupling reaction, providing heterodimer
2
(Figure S1). Nosyl protecting group was then removed by treating
with 2-mercaptoethanol and DBU. Iterative monomer coupling and
deprotection reactions produced oligomers with alternating 1:1 β3-
phenyl alanine/benzylated α-ABpeptoids structures (S,R)-4a-f of dif-
ferent lengths ranging from 4 to 14 mer. The synthesized oligomers
SCHEME 1 Solid-phase
synthesis of oligomers of 1:1 β3-
phenyl alanine/benzylated
α-ABpeptoids (S,R)-4a-f

4 of 8
LEE ET AL.
were cleaved from resin by treating with 95% TFA, and the crude
products were purified by reverse-phase HPLC, providing >95% purity
of final products (Table S1 and Figure S2). Their identity was deter-
mined by MALDI-TOF mass spectrometry as shown in Table 1. It is
noteworthy that our solid-phase synthetic method is robust, affording
heterogeneous backbone oligomers with alternating α-ABpeptoid res-
idues and β3-peptide residues in excellent yields (67%-88% crude
purity as determined by HPLC; Figure 2, Table 1, and Figure S1). In
general, it is very difficult to prepare oligomers of N-alkylated pep-
tides.[52] For example, in the previous study, peptide/peptoid hybrids
were prepared by on-resin coupling reaction of presynthesized
peptide/peptoid heterodimers.[53] In contrast, our method enables to
efficiently prepare peptide/peptoid hybrids without requiring such
cumbersome processes.
~230 nm, which are reminiscent of those of α-ABpeptoids.[44] For
oligomers ranging from 4 to 8 mer (S,R)-4a-c, the intensity of normal-
ized mean residue ellipticity was relatively weak. In contrast, oligo-
mers with 10 to 14 mer (S,R)-4d-f exhibited nearly identical CD
features with saturated signals, suggesting that 10 residues with
5 repeated units are minimally required to form a defined structure.
We then measured CD spectrum of an oligomer with opposite back-
bone chirality (R,S)-4d to examine the influence of backbone chirality
on the three dimensional structures (Figure 3B). A 10 mer composed
of alternating (R)-β3-phenyl alanine and (S)-benzylated α-ABpeptoids
was prepared by the same procedure described above (Scheme 1,
Table 1, and Supporting Information). As expected, the CD spectra of
the oligomer with (S,R)-configurations showed a mirror image to that
of its counterpart with (R,S)-configuration (R,S)-4d. This observation
suggests that the oligomers with opposite backbone chirality have an
identical folding structure with opposite handedness, and chiral
groups on the backbone play a critical role in forming ordered
conformations.
Next, we analyzed the CD spectrum of the synthesized oligomers
to investigate their folding properties. The CD spectra were measured
in 25% solution of 5 mM phosphate buffered saline (PBS) buffer
(pH 7.4) in acetonitrile at 20 ꢀC in the far UV area (190-260 nm)
(Figure 3A). Oligomers with alternating β3-phenyl alanine/benzylated
α-ABpeptoids displayed characteristic CD features with intense mini-
mum at ~205 nm, intense maximum at ~198 nm, and weak maxima at
We next investigated the solvent effect on the conformation of
the oligomers. The CD spectral shape for (S,R)-4a-f did not vary signif-
icantly in trifluoroethanol (a polar protic solvent) and PBS/acetonitrile
(Figure 3C). And the CD spectra of (S,R)-4a-f in polar aprotic solvent
such as acetonitrile were similar (with a slightly reduced CD intensity)
to those measured in PBS buffer (Figure 3D). This result implied that
hydrogen bonding may not be a key driving force for the formation of
folding structures. We also assessed whether the observed CD signals
were resulted from the simple aggregation of the oligomers. To this
end, the CD spectra of a representative oligomer (S,R)-4d was mea-
sured at varying concentrations ranging from 30 to 100 μM. As
depicted in Figure 3E, no significant difference in the intensity and
shape of CD spectra was observed, demonstrating that the heteroge-
neous oligomers may form defined conformations as individual molec-
ular structures rather than forming aggregates.
TABLE 1 Synthesized oligomers of 1:1 β3-phenyl
alanine/benzylated α-ABpeptoids sequence, crude purity, and mass
confirmation
Chain
% Crude
Compd no. length (n) puritya
Calcd mass Obsd massb
(S,R)-4a
(S,R)-4b
(S,R)-4c
(S,R)-4d
(S,R)-4e
(S,R)-4f
(S,S)-4a
(S,S)-4b
(S,S)-4c
(S,S)-4d
(R,S)-4d
(R,R)-4d
2
3
4
5
6
7
2
3
4
5
5
5
76
79
70
76
76
73
77
71
67
81
67
88
689.39
712.35 [M + Na]+
1025.58
1361.76
1697.95
2034.13
2370.31
689.39
1048.5 [M + Na]+
1362.9 [M + H]+
1720.9 [M + Na]+
2057.2 [M + Na]+
2393.4 [M + Na]+
712.41 [M + Na]+
1026.7 [M + H]+
1362.9 [M + H]+
1720.9 [M + Na]+
1721.0 [M + Na]+
1722.0 [M + Na]+
To gain further structural insight into the oligomers, we measured
2D-NMR spectra of (S,R)-4a (4-mer hybrid oligomer) and (S,R)-4d
(10-mer hybrid oligomer) in CD3CN. Since the equilibrium from
dynamic backbone amide rotations mainly determines the conforma-
tion of the oligomers, we evaluated the equilibrium of cis and trans
amide conformers by 2D-NMR analysis. 1H-COSY and 1H-13C HSQC
spectra revealed some segregated cross-peak regions with different
chemical shifts, which correspond to either cis or trans secondary
amide conformers in the β3-peptide parts of the hybrid oligomers
(Figure 4A and Figure S3). Each cross peak was assigned as cis or trans
1025.58
1361.76
1697.95
1697.95
1697.95
aDetermined by analytical reverse-phase HPLC of crude products.
bMass spectrometry data were acquired using MALDI-TOF techniques.
FIGURE 2 HPLC
chromatogram of crude oligomer
(R,R)-4d

LEE ET AL.
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FIGURE 3 CD data. All the data were recorded at 20 ꢀC. A, CD spectra of (S,R)-4a-f in 25% of 5 mM PBS buffer (pH 7.4)/acetonitrile
(60 μM). B, CD spectra of (S,R)-4d and (R,S)-4d in 25% of 5 mM PBS buffer (pH 7.4)/acetonitrile (60 μM). C, CD spectra of (S,R)-4a-f in
trifluoroethanol (60 μM). D, CD spectra of (S,R)-4a-f in acetonitrile (60 μM). E, CD spectra of (S,R)-4d at different concentrations ranging from
30 to 100 μM in 25% of 5 mM PBS buffer (pH 7.4)/acetonitrile. CD, circular dichroism; PBS, phosphate buffered saline
FIGURE 4 1H-COSY spectra of oligomers (A) (S,R)-4a and (B) (S,R)-4d. The panels describe the resonance cross-peaks for the secondary
amide protons
conformers according to NOESY analysis (Figure S4). Although these
specific cross-peak regions indicate that the amount of cis and trans
amides are similar, we could accurately calculate its ratio by the vol-
ume integration of assigned methyl protons on 1H spectrum, which
was ~1:1 as expected (Figure S7). In the case of tertiary amide bonds
in the α-ABpeptoid peptoid parts of the hybrid oligomers, however, it
was hard to differentiate certain segregated regions for comparing cis
and trans ratio due to highly overlapped 1H peaks of methylene

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LEE ET AL.
protons. We thus assumed that the amount of cis or trans tertiary
amides may also be similar according to the comparable intensities of
characteristic nuclear overhauser effect (NOE) signals corresponding
to each conformer (Figure S4).
study of H/D exchange on the oligomer clearly detected the amide
proton signal decay within 30 minutes after the addition of D2O,
suggesting that the amide protons in the oligomer are not involved in
hydrogen bonds (Figure S8). The result was consistent with the CD
results (Figure 3A,C,D). Although intramolecular hydrogen bondings
are not observed in this particular series of oligomers unlike our initial
expectation, the amide protons in α-ABpeptoid/β3-peptide oligomers
could play a critical role when interacting with proteins by making
intermolecular hydrogen bondings, thereby enabling them to bind
more tightly and/or specifically to the target.
In COSY and HSQC spectra of 10-mer oligomer (S,R)-4d, more
degenerated cross-peak signals were observed compared with those
of 4-mer (S,R)-4a (Figure 4B and Figure S5). Based on the assignment,
we found that trans conformers were dominant in the secondary
amides (the calculated cis:trans ratio was 1:3.79; Figure S7). In con-
trast, it was difficult to precisely calculate the cis:trans ratio for tertiary
amides in the α-ABpeptoid peptoid parts. Given that characteristic
NOE signals were observed for trans conformation (Figure S6), while
NOE signals from cis conformation were too weak, it was suggested
that trans conformation would be dominant. These results suggest
that there might be a cooperative effect in forming the folding struc-
ture. Moreover, the results are consistent with CD study on (S,R)-4a
and (S,R)-4d (Figure 3D), suggesting that the higher intensity of (S,R)-
4d is due to cooperatively increased trans amide conformations as the
length of the oligomer is increased.
In addition to (S,R)- and (R,S)-oligomers, we sought to examine the
structural feature of α-ABpeptoid/β3-peptide oligomers with different
stereochemical configurations, such as (S,S)- and (R,R)-oligomers. Het-
erogeneous oligomers composed of (S)-α-ABpeptoid/(S)-β3-peptide
were prepared (Scheme 1, Table 1, and Supporting Information). Their
folding propensities were evaluated by measuring CD spectra in 25%
5 mM PBS/acetonitrile at 20 ꢀC (Figure 5A). Short oligomers ranging
from 4- to 8-mer (S,S)-4a-c did not display apparent CD signals.
Intriguingly, decamer (S,S)-4d showed unique spectral shapes with
maxima at ~186 and ~210 nm and two intense minima at ~198 and
~223 nm. The results suggest that the oligomers longer than 10 mer
may form different folding structures compared with the other stereo-
isomers including (S,R)- and (R,S)-oligomers. We also synthesized a
oligomer with opposite chirality, (R,R)-4d (Scheme 1, Table 1, and
To further investigate whether hydrogen bonds are an important
driving force to induce the ordered secondary structure of (S,R)-4d, a
hydrogen/deuterium (H/D) exchange experiment was conducted. If
the protons participate in hydrogen bonding, they generally show
slower exchange rate than free amide protons.[51] This NMR-based
FIGURE 5 CD data. All the data were recorded at 20 ꢀC. A, CD spectra of (S,S)-4a-d in 25% of 5 mM PBS buffer (pH 7.4)/acetonitrile
(60 μM). B, CD spectra of (S,S)-4d and (R,R)-4d in 25% of 5 mM PBS buffer (pH 7.4)/acetonitrile (60 μM). C, CD spectra of (S,S)-4a-d in
acetonitrile (60 μM). D, CD spectra of (S,S)-4d at different concentrations ranging from 7 to 100 μM in 25% of 5 mM PBS buffer (pH 7.4)/
acetonitrile. E, CD spectra of (S,S)-4d in 40% of water (or PBS buffer)/acetonitrile with different concentrations of urea (60 μM). CD, circular
dichroism; PBS, phosphate buffered saline

LEE ET AL.
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Supporting Information) and compared the CD shape with its stereo-
isomer (S,S)-4d. Not surprisingly, (R,R)-4d gave rise to a clear mirror
image compared with (S,S)-4d. This finding indicates that (S,S)-4d and
(R,R)-4d may adopt identical three-dimensional structures with oppos-
ing handedness (Figure 5B). As observed in (S,R)-4a-f, the spectral
shapes of (S,S)-4d in acetonitrile were similar to those in
PBS/acetonitrile (Figure 5C).
ORCID
Hyun-Suk Lim
https://orcid.org/0000-0003-4083-2998
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ACKNOWLEDGMENTS
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We thank Eun Kyung Jee for the synthesis of intermediate (S)-α-AB.
This work was supported by the National Research Foundation of
Korea (NRF-2017M3A9F6029753, NRF-2017M3A9E4077447, and
NRF-2018R1A4A1024713).

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How to cite this article: Lee KJ, Sable GA, Shin M-K, Lim H-S.
Oligomers of α-ABpeptoid/β3-peptide hybrid. Biopolymers.
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