Cooperative Intramolecular Hydrogen Bonding Strongly Enforces cis-Peptoid Folding

Article abstract of DOI:10.1021/jacs.9b10497

Sequence-defined peptoids, or N-substituted glycines, are an attractive class of bioispired polymer due to their biostability and efficient synthesis. However, the de novo design of folded peptoids with precise three-dimensional structures has been hindered by limited means to deterministically control backbone conformation. Peptoid folds are generally destabilized by the cis/trans backbone-amide isomerization, and few side-chains are capable of enforcing a specific amide conformation. Here, we show that a novel class of cationic alkyl ammonium ethyl side-chains demonstrates significant enforcement of the cis-amide backbone (K_cis/trans up to 70) using an unexpected ensemble of weak intramolecular CH-O and/or NH-O hydrogen bonds between the side-chain and the backbone carbonyl moieties. These interactions are evidenced by X-ray crystallography, variable-temperature NMR spectroscopy, and DFT calculations. Moreover, these side-chains are inexpensive, structurally diverse, hydrophilic, and can be integrated into longer peptoid sequences via solid-phase synthesis. Notably, we extended these concepts to synthesize a water-soluble peptoid 10-mer that adopts one predominant fold in solution, as determined by multidimensional NMR spectroscopy. This decamer, to the best of our knowledge, is the longest linear peptoid sequence atomically characterized to retain a well-folded structure. These findings fill a critical gap in peptoid folding and should propel the development of peptoid applications in a broad range of contexts, from pharmaceutical to material sciences.

Full text of DOI:10.1021/jacs.9b10497

Article
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
pubs.acs.org/JACS
Cooperative Intramolecular Hydrogen Bonding Strongly Enforces
cis-Peptoid Folding
†
,⊥
,†,⊥
,†
†
‡
§
Andrew W. Wijaya, Andy I. Nguyen,*
Leah T. Roe, Glenn L. Butterfoss, Ryan K. Spencer,
†
Nan K. Li, and Ronald N. Zuckermann*
†
The Molecular Foundry, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, United States
Center for Genomics and Systems Biology, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates
Department of Chemistry, Department of Chemical Engineering & Material Science, University of California, Irvine, California
‡
§
9
2697, United States
*
S Supporting Information
ABSTRACT: Sequence-defined peptoids, or N-substituted
glycines, are an attractive class of bioispired polymer due to
their biostability and efficient synthesis. However, the de novo
design of folded peptoids with precise three-dimensional
structures has been hindered by limited means to determin-
istically control backbone conformation. Peptoid folds are
generally destabilized by the cis/trans backbone-amide
isomerization, and few side-chains are capable of enforcing a
specific amide conformation. Here, we show that a novel class
of cationic alkyl ammonium ethyl side-chains demonstrates
significant enforcement of the cis-amide backbone (Kcis/trans up
to 70) using an unexpected ensemble of weak intramolecular
CH−O and/or NH−O hydrogen bonds between the side-
chain and the backbone carbonyl moieties. These interactions are evidenced by X-ray crystallography, variable-temperature
NMR spectroscopy, and DFT calculations. Moreover, these side-chains are inexpensive, structurally diverse, hydrophilic, and
can be integrated into longer peptoid sequences via solid-phase synthesis. Notably, we extended these concepts to synthesize a
water-soluble peptoid 10-mer that adopts one predominant fold in solution, as determined by multidimensional NMR
spectroscopy. This decamer, to the best of our knowledge, is the longest linear peptoid sequence atomically characterized to
retain a well-folded structure. These findings fill a critical gap in peptoid folding and should propel the development of peptoid
applications in a broad range of contexts, from pharmaceutical to material sciences.
INTRODUCTION
backbone, which means that each peptoid residue can adopt
either of the nearly isoenergetic cis or trans conformations
■
Peptoids, or poly N-substituted glycines, are a class of
peptidomimetic polymers privileged with uniquely fast, low-
cost, and modular synthesis that allows for a wide variety of
noncanonical side-chains to be incorporated into an sequence-
defined oligomer with high yield and precision. As peptoids
are resistant to proteolysis, they have been investigated for
(
rotation about ω) (Figure 1a). This isomerization adds to the
flexibility of the peptoid backbone, making it a challenge to
create rigid and stable secondary structures. Despite this,
several peptoid secondary structures have been reported, all
using strategies to control of rotation about ω. These
structures include polyproline-type helices with all cis
1
−3
4
5
,6
numerous biological applications such as drug-delivery,
1
9−22
12
7
8
(PPI)
amide bonds, the all cis sigma strand, the peptoid
antifreeze additives, therapeutics, and antimicrobial
23
24
9
,10
ribbon, and the square helix with an alternating amide
backbone pattern of cis and trans. Thus, in order to promote
the development of novel peptoid functionalities, we must
better understand and extend such strategies to construct new
and diverse monomers that promote predictable peptoid
folding.
agents.
Moreover, amphiphilic peptoid sequences can
form novel nanomaterials via supramolecular self-assembly,
where the morphology is determined by sequence alone,
and the surface of these materials can be precisely tuned to
1
1−16
1
7,18
display useful functionalities such as pathogen binding.
However, despite the demand for biomimetic applications with
peptoids, the development of new functional peptoids has been
hindered by the limited available options to control their
folding into well-defined three-dimensional architectures.
A major challenge in controlling peptoid conformation arises
from the presence of the tertiary amide bond in the peptoid
To date, the most reliable way to control the amide
isomerization in solution is with specialized side-chains (Figure
Received: September 29, 2019
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.9b10497
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Figure 1. Strategies for backbone cis-amide induction. (a) Peptoid backbone dihedral parameter ω controls trans- and cis-amide conformations. A
general approach of tuning ω utilizes sterics and electronic factors by the side-chain. (b) Side-chain examples from the literature include 1-
25
26
27
25
naphthylethyl (1npe), tert-butyl (tBu), (benzyltriazolyl)ethyl (bte), and 4-methylpyridinium (4mpy). (c) Our work features engineered
side-chains that facilitate intramolecular hydrogen bonds as a means to enforce the backbone cis-amide conformation.
1
a). A common approach to favor the cis-amide conformation
catalyzed “click” azide−alkyne cycloaddition (CuAAC) and
electrophilic alkylation, which greatly lengthens the time of
synthesis. Another strategy leverages inductive effects of
fluoroalkyl side-chains to induce moderate cis-amide isomers,
presumably by dipole−dipole interactions between the side-
chain and backbone carbonyl. The ideal structure-inducing
peptoid side-chain should therefore be able to strongly enforce
a specific backbone conformation, and also be inexpensive,
readily available, easily diversifiable, and compatible with a
diverse set of side-chains. Furthermore, it should allow for the
synthesis of long, water-soluble sequences via the solid-phase
submonomer method.
is to use extremely bulky, α-branched side-chains that disfavor
steric interactions present in the trans conformation. A dearth
of these side-chains that are also readily available has led to
repeated use of the same generally hydrophobic peptoid
monomers in generating peptoid structures. For example, the
moderately cis-inducing (Kcis/trans ≈ 6), chiral 1-naphthylethyl
side-chain (N1rnpe or N1snpe) (Figure 1b) is found in nearly
all of the peptoid secondary structures mentioned previ-
30
2
0,23,24
ously.
Additionally, these bulky monomers generally
have low monomer addition yields and create overall structures
that are too hydrophobic for use in aqueous media. The tert-
butyl side-chain and its derivatives are also used to control
amide bond isomerization through steric interactions, showing
Surprisingly, simple intramolecular hydrogen-bonding be-
tween a side-chain and the i − 1 carbonyl oxygen atom has not
been widely recognized in peptoids as a means of stabilizing a
specific amide conformation despite the prevalence and
importance of hydrogen-bonding in natural biopolymers.
Previous studies have experimentally identified instances of
CH−O interactions between side-chains and the backbone in
26,28
preference exclusive to cis-amide isomers in all solvents.
However, these side-chains are extremely nonpolar and are
incompatible with solid-phase peptoid synthesis due to the
instability of the tert-butyl moiety during acid cleavage
conditions, precluding their use in longer peptoid sequences.
A separate class of side-chains leverages attractive intra-
molecular interactions between the amide carbonyl oxygen and
the side-chain moiety to stabilize cis-amide isomers. This
strategy has widely been demonstrated with an n → π*
interaction between the side-chain and the carbonyl oxygen of
the previous residue (i − 1 position). The electronic effect
originates from the lone pair donation (n orbital) of the i − 1
carbonyl oxygen to an empty, low-energy antibonding orbital
26,27,31
peptoids,
but the dominant cis induction mechanism was
attributed to sterics or n → π* stabilization. The free energy
stabilization of a typical hydrogen bond ranges from 1 to 3
32,33
kcal/mol,
and in theory, careful engineering of a hydrogen-
bonding interaction between the carbonyl oxygen of the
previous residue (i − 1) to a polarized hydrogen on the side-
chain should be able to significantly enforce the cis-amide
geometry (a factor of 10 increase in Kcis/trans per ΔG = 1.37
kcal/mol stabilization).
Herein, we describe a series of cationic side-chains that are
both the strongest cis-inducing peptoid side-chains reported to
date and also amenable to solid-phase synthesis, with Kcis/trans
up to 70. Notably, all side-chains herein reported are readily
available commercially or via simple synthesis (with two steps
or fewer from low-cost starting materials), are highly diverse,
and also allow for the synthesis of well-folded, water-soluble
peptoids. Interestingly, this new class of side-chains interacts
with the backbone carbonyl moieties using multiple hydrogen
(
π*) of the side-chain (Figure 1b). A recent report
demonstrated that fluorination of aromatic residues strength-
29
ens this interaction. Potent cis induction (up to Kcis/trans
0) is seen with cationic triazolium and N-alkylpyridinium
side-chains, as well as side-chains with electrophilic carbonyl
≈
2
2
7,25
moieties.
Currently, these side-chains are the strongest
peptoid cis inducers reported in the literature that can be
accessed by solid-phase peptoid synthesis. However, a
potential drawback of triazolium side-chains are the addition
of two extra synthesis steps per monomer incorporation, Cu-
B
DOI: 10.1021/jacs.9b10497
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
a
Chart 1. Peptoid Monomer Structures
a
Cationic peptoids are isolated and characterized in their trifluoroacetate (TFA) salt forms.
bonds, which we characterized with variable-temperature NMR
spectroscopy, X-ray crystallography, and theoretical calcula-
tions. These new monomers allow the synthesis of longer
peptoids (up to a 10-mer), which we also structurally
characterized with atomic precision by solution NMR
spectroscopies. Notably, the peptoid decamer is the longest
linear peptoid known to adopt a predominant well-folded
secondary structure in solution and showcases peptoid side-
chain diversity with 10 individual unique residues. The
development of these readily accessible cationic, hydrogen-
bonding side-chains is an important milestone for advancing
fundamental foldamer chemistry, as well as for the design of
structured, water-soluble peptoids for biological and pharma-
ceutical applications.
Conventional Hydrogen-Bonding Monomers. We com-
menced our study by synthesizing and analyzing peptoids that
feature a potential NH−O hydrogen-bonding interaction.
Previous studies have hinted that side-chains with primary
amine or ammonium functionalities tethered to the backbone
by an alkyl linker, such as N-aminoethyl (Nae), N-aminopropyl
(
Nap), and N-aminobutyl (Nab), may be capable of inducing
the cis-amide geometry in peptoid structures, though these
34,35
studies did not quantify the extent of cis induction.
Moreover, the mentioned side-chains are widely used in
10,12,36
peptoid structures such as in nanosheets and helices.
Peptoid monomer derivatives, 1−3, containing Nae, Nap, and
Nab, respectively, were synthesized to test this hypothesis,
along with other peptoids as controls, N-hydroxyethyl (Nhe)
(
4), N-guanidinylethyl (Ngue) (5), N-2picolyl (N2pic) (6),
RESULTS AND DISCUSSION
and N-3picolyl (N3pic) (7) (Chart 1). Indeed, most of these
■
side-chains favor the cis-amide geometry moderately (Kcis/trans
up to 5.4), with the exception of Nhe (4), which slightly favors
Peptoid Monomer Models. Previous studies by Blackwell
and Taillefumier utilized a peptoid monomer derivative with
an acetyl N-terminus and piperidinyl at the C-terminus to
isolate the side-chain interactions with the backbone and used
that data to predict the cis/trans population (Kcis/trans) in longer
the trans rotamer (Kcis/trans = 0.9 in CDCl , 0.5 in MeOD and
3
D O), and Nab (3), which shows almost no preference for
2
either geometry in all solvents (Kcis/trans ≈ 1) (Table 1).
Comparing the side-chains with N−H moieties, the trend of
monomers that show cis induction follows 2 > 1 > 5 > 3. The
intramolecular hydrogen bond ring size with the backbone
carbonyls leads to a hydrogen bond donor competition
between the i − 1 and i carbonyls, whereby only the
interaction with the i − 1 carbonyl should affect the cis/trans
ratio (Figure 2a). Density-functional theory (DFT) models of
1−3 and 5 reproduce the experimentally observed ordering of
cis favorability (the monomers used for in silico work have an
N,N-dimethyl rather than a 4-methylpiperidinyl C-terminus)
and showcase the expected intramolecular hydrogen-bonding
25,27,28
peptoid oligomers.
system, substituting piperidinyl for 4-methylpiperidinyl, which
still allows for direct comparisons with previously reported
In this study, we utilized a similar
side-chains. The K
ratios were determined by integrating
cis/trans
1
H NMR resonances of the peptoid, and were measured in a
range of solvents (CDCl , MeOD, and D O) to probe the
3
2
effect of solvent polarity and the presence of proton donors.
Peaks for each rotamer were assigned with two-dimensional
NMR techniques, including the rotating-frame nuclear Over-
hauser effect correlation spectroscopy (ROESY) NMR
technique to distinguish between the cis and trans rotamers.
C
DOI: 10.1021/jacs.9b10497
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
a
Table 1. Kcis/trans Values of Peptoid Monomers
monomers
R group
CDCl3
MeOD
D O
2
no.
Kcis/trans ΔG Kcis/trans ΔG
Kcis/trans ΔG
1
Nae
Nae neutral
Nap
Nap neutral
Nab
Nab neutral
Nhe
Ngue
N2pic
N2pic
neutral
1.9
1.0
3.1
1.0
1.0
0.8
0.9
2.0
5.4
0.5
−0.38
0.00
−0.67
0.00
0.00
0.13
2.4
2.2
3.0
1.5
1.0
1.3
0.5
1.5
4.2
1.1
−0.52
−0.47
−0.65
−0.24
0.00
−0.16
0.41
−0.24
−0.85
−0.06
2.2
1.9
2.1
1.0
0.9
1.0
0.5
1.2
3.2
1.0
−0.47
−0.38
−0.44
0.00
0.06
0.00
b
b
b
1
2
2
3
3
4
5
6
6
0.06
0.41
−0.41
−1.00
0.41
−0.11
−0.69
0.00
Figure 3. Energy-minimized structures of peptoid 6 and 7. Lowest
energy conformations (6 pictured left, 7 pictured right) derived at the
b
b
37
B3LYP/6-311++G(2d,2p) level of theory in implicit water feature
an intramolecular hydrogen bond (dotted line with i − 1 carbonyl)
and an n → π* interaction (arrow with i carbonyl) facilitated by the
pyridyl side-chain; the interaction distances are shown in angstroms.
7
7
N3pic
N3pic
neutral
4.3
0.6
−0.86
0.30
3.7
1.6
−0.77
−0.28
2.2
1.3
−0.47
−0.16
8
9
1
1
1
1
1
1
1
Nme ae
22
4.7
0.2
70
70
40
60
13
0.3
−1.83
−0.92
0.95
−2.52
−2.52
−2.32
−2.42
−1.52
0.71
6.1
2.1
0.5
14
−1.07
−0.44
0.41
−1.56
−1.60
−1.29
−1.27
−1.33
0.13
4.1
1.7
−
5.8
6.1
4.4
4.9
5.4
0.8
−0.84
−0.31
−
−1.04
−1.07
−0.88
−0.94
−1.00
0.13
3
Nme ap
3
c
c
0
1
2
3
4
5
5
Nnhx
Net ae
3
Net bnae
15
2
Net pgae
8.8
8.5
9.4
0.8
2
Net tzae
2
Net ae
2
b
b
Net ae
2
Figure 4. Comparison between Lewis structure and a more realistic
neutral
i
charge distribution in an Nme ae side-chain. Formal Lewis structure
3
1
1
6
6
N Pr ae
30
0.3
−2.01
0.71
16
0.7
−1.64
0.21
8.1
0.8
−1.24
0.13
2
fails to depict the distribution of Mulliken charges in the Nme ae side-
i
3
N Pr ae
2
chain. The α-protons of the ammonium core are positively charged
neutral
(blue), while the negative charge (red) is concentrated on the
a
Kcis/trans values in different solvents (CDCl , MeOD, and D O) and
3
2
nitrogen.
their corresponding free energy differences (ΔG in kcal/mol) of
peptoid monomers 1−16. K
values are generally calculated using
cis/trans
1
two sets of H NMR resonances. K
values ≥ 40 are rounded to
cis/trans
b
c
bonding, deprotonation of the amines in peptoids 1 and 2
by addition of 1 equiv of KOH also reduces the cis-population
the nearest ten. Deprotonated to its neutral state. Insoluble in
solvent.
b
b
from 66% and 75%, respectively, to 50% for both 1 and 2 (in
CDCl3).
The difference of Kcis/trans values between Nae (1) and Ngue
(
5) also suggests that the geometry of the N−H proton is a
3
relevant factor for cis induction. Compound 1 has an sp -
hybridized nitrogen with a tetrahedral geometry, whereas the
2
nitrogen of 5 is sp hybridized with a trigonal planar geometry.
Our models suggest that the planarity significantly increases
the ring strain of the seven-membered ring formed between the
guanidine N−H and the i − 1 carbonyl, preventing a proper
hydrogen bond to take place (see Supporting Information). In
the DFT models, the guanidinium side-chain of 5 makes a
hydrogen bond to the i carbonyl in both the most favorable cis
and trans conformations (which are nearly isoenergetic). A cis
conformation with a hydrogen bond to the i − 1 carbonyl is
1
.2 kcal/mol higher in energy, supporting the prediction that
Figure 2. Hydrogen-bonding hypothesis. (a) Hydrogen bond
competition between the backbone carbonyls where interactions
with i − 1 carbonyl O will favor the cis-amide conformation. (b)
Energy-minimized structures of 1−3 (derived at the B3LYP/6-311+
the Ngue side-chain of 5 is too restricted to promote cis.
The N-picolyl monomers, 6 and 7, also show moderate cis
induction (Kcis/trans up to 5.4 and 4.3 for 6 and 7, respectively).
However, unlike the amino alkyl side-chains, these side-chains
are capable of also forming an n → π* interaction with the i −
37
+
G(2d,2p) level of theory in implicit water) showcase NH−O bond
interactions (dotted green line, marked with black arrow) between the
side-chains and the backbone carbonyls with differing ring size.
1
backbone carbonyl, similar to the (benzyltriazolyl)ethyl (bte)
25,27
and 4-methylpyridinium (4mpy) side-chains.
Therefore, cis
interactions (Figure 2b). The predicted relative potential
energies for the best-identified cis versus best-identified trans
conformations give the cis preference rank order as 2 (−1.55
kcal/mol) > 1 (−1.00 kcal/mol) > 5 (−0.44 kcal/mol) > 3
induction to some degree should be present for both 6 and 7,
and the observed Kcis/trans values cannot be attributed to solely
hydrogen bonding. DFT models based on 6 and 7 predict that
both side-chains prefer similar rotameric states. The ring forms
either a pyridyl NH−O hydrogen bond (in 6) or an aromatic
(
0.02 kcal/mol). Moreover, as expected with hydrogen-
D
DOI: 10.1021/jacs.9b10497
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Figure 5. Crystallographic and DFT structures of peptoids 8 and 11. (a) Solid-state structure of peptoid 8 in ball-and-stick representation. Only
one isomer out of two in the asymmetric unit is shown here; BPh anion and toluene molecules are omitted for clarity. Hydrogen bonds are drawn
4
as dotted lines. (b) Electrostatic potential surface (EPS) map of the most favorable DFT structure of peptoid 8. Color scheme: blue indicates
higher positive potential; color intensity is proportional to the magnitude of the charge. (c) Parameters of hydrogen-bonding interactions in
peptoid 8 as observed in the solid state. Distance cutoff for CH−O interactions is 3.7 Å between the donor and acceptor atom. (d) Solid-state
structure of peptoid 11 in ball-and-stick representation. Only one isomer out of four in the asymmetric unit is shown here; BPh anions are omitted
4
for clarity. Hydrogen bonds are drawn as dotted lines. (e) Electrostatic potential surface (EPS) map of the most favorable DFT structure of peptoid
1
1. Color scheme: blue indicates more positively charged; color intensity is proportional to the magnitude of the charge. (f) Parameters of
hydrogen-bonding interactions in peptoid 11 as observed in the solid state. Distance cutoff for CH−O interactions is 3.7 Å between the donor and
acceptor atom.
CH−O hydrogen interaction (7) with the i carbonyl. The cis
state of both appears to be promoted via n → π* interaction
interactions between the i − 1 carbonyl and the pyridyl ring
rather than a hydrogen bond; however, the intramolecular
hydrogen bond interaction with the i carbonyl may assist in cis
induction by forcing the ring to be perpendicular with respect
to the i − 1 carbonyl, thus maximizing the n → π* donation
trimethylammonium-ethyl (Nme ae) and N-trimethylammo-
3
nium-propyl (Nme ap), respectively.
3
Surprisingly, peptoid 8 has an extraordinarily high Kcis/trans of
22 in CDCl
protic solvents, the cis isomer of 8 remained heavily
, about 10 times higher than that of 1. In polar
3
predominant (Kcis/trans = 6.1 in MeOD and 4.1 in D O),
2
which is higher than the popular 1-naphthylethyl side-chain
(
Kcis/trans = 4.6 in MeOD). Similarly, the Kcis/trans values of 9 are
(Figure 3). A similar motif to induce cis has been previously
27
also increased with respect to 2 (Kcis/trans = 4.7 in CDCl3),
albeit much more modestly. This strength of induction
observed in the N(benzyltriazolyl)ethyl (Nbte) monomer.
The existence of attractive electrostatic interactions to
induce cis in 1, 2, 6, and 7 is further supported by the
decrease of Kcis/trans values of these monomers in more polar
between 8 and 9 indicates that the alkyl tether length is a
principal factor for cis induction in this system, similar to the
peptoids that contained N−H donors.
solvents (Kcis/trans up to 5.4 in CD Cl versus K
up to 3.2 in
3
cis/trans
To control for potential steric effects of the larger Nme ae
3
D O). For cases with hydrogen bonding, polar protic solvents
2
side-chain (relative to Nae), we synthesized an isosteric
derivative of 7, peptoid 10, which contains a neutral N-
neohexyl side-chain (Nnhx). The Kcis/trans values of 10 are
significantly less than 1 in all solvents (Kcis/trans = 0.2 and 0.5 in
are able to participate in hydrogen-bonding donation and
acceptance, which competes with the intramolecular peptoid
hydrogen bond. Overall, moderate cis induction has been
observed with four side-chains in the monomeric system: N-
aminoethyl (Nae), N-aminopropyl (Nap), N-2-picolyl
CD Cl and MeOD, respectively), indicating that the trans
3
isomer is favored for this isosteric compound. This observation
(
N2pic), and N-3-picolyl (N3pic). The existence of cis-
dictates that the cis-directing effect of the Nme ae side-chain is
3
inducing electrostatic interactions in these monomers is
consistent with the observed trend in ring size, amine proton
geometry, protonation state, and solvent polarity.
Quaternary Ammonium Side-Chains. To further test our
hypothesis that hydrogen bonding with the backbone
promotes cis-amides, we removed all of the N−H moieties of
not due to sterics, but rather an electronic effect induced by
the quaternary ammonium moiety. In this regard, it is
especially important to recognize a more realistic distribution
of charges in quaternary ammonium moieties. The positive
charge of a quaternary ammonium moiety is known to be
delocalized among the protons surrounding the ammonium
core and not on the nitrogen, as traditionally depicted in
1
and 2 via exhaustive alkylation, forming quaternary
ammonium functionalities. We postulated that the absence of
N−H protons should decrease the K values as the side-
38
formal Lewis structures (Figure 4).
cis/trans
Motivated by the potent cis induction of the Nme ae side-
3
chains can no longer facilitate classical hydrogen bonds to the
backbone carbonyls. Alkylation of peptoids 1 and 2 with excess
iodomethane yielded peptoids 8 and 9 with side-chains N-
chain, we prepared several other peptoids containing novel
quaternary ammonium side-chains, each tethered to the
backbone by an ethylene linker: N-triethylammonium-ethyl
E
DOI: 10.1021/jacs.9b10497
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Scheme 1. Optimized Amine Precursor Synthesis for
Net ae, Net bnae, and Net pgae Side-Chains
3
2
2
Scheme 2. Optimized Solid-Phase Peptoid Synthesis with
a
Quaternary Ammonium Side-Chains
Figure 6. Crystallographic and DFT structures of peptoid 16. (a)
Solid-state structure of peptoid 16 in ball-and-stick representation.
BPh and toluene molecules are omitted for clarity. Hydrogen bonds
4
are drawn as dotted lines. (b) Electrostatic potential surface (EPS)
map of the most favorable DFT structure of peptoid 16. (c)
Parameters of hydrogen-bonding interactions in peptoid 16 as
observed in the solid state. Distance cutoff for CH−O interactions
is 3.7 Å between the donor and acceptor atom. (d) Potential
a
Optimized quaternary ammonium-type side-chains (R) include
Nme ae (17), Net ae (18), Net bnae (19), and Net pgae (20). Full
synthetic details are catalogued in the Supporting Information.
3
3
2
2
Scheme 3. Ketopiperazinium Formation with Net ae
equilibrium process between two intramolecular hydrogen-bonding
2
i
motifs facilitated by a N Pr ae side-chain. The amine hydrogen atom
2
is highlighted with a blue circle and interacts favorably with both the i
carbonyl (left) and the i − 1 carbonyl (right). Barrier of isomerization
is small (0.7 kcal/mol), as estimated by DFT computations.
Table 2. Thermodynamic Parameters of Cis Induction in
Peptoids 8, 11, 12, and 16
a
peptoid
ΔH (kcal/mol)
−TΔS (kcal/mol)
8
+0.50(6)
−0.66(3)
−0.71(3)
−0.52(3)
−1.33(6)
−0.67(3)
−0.70(3)
−0.62(3)
1
1
1
1
2
6
selection alone, providing diverse functionalities for a broad
range of potential applications. Overall, the high Kcis/trans values
demonstrated by peptoids 8 and 11−14 indicate that the
quaternary ammonium cores facilitate a major electronic effect
to enforce the cis-amide backbone geometry.
a
−
TΔS is calculated at 298 K.
Evidence for a cis-stabilizing electronic effect was observed in
(
(
Net ae) (11), N-diethylbenzylammonium-ethyl (Net bnae)
the solid-state structure. X-ray diffraction on single crystals of
3
2
−
12), N-diethyl-propargylammonium-ethyl (Net pgae) (13),
tetraphenylborate (BPh ) salts of 8 and 11 show that the
2
4
and N-diethyl-triazoleammonium-ethyl (Net tzae) (14). Over-
all, these derivatives demonstrate exceptionally high Kcis/trans
peptoids crystallize in their cis rotamer state. The backbone
2
dihedral parameters of 8·BPh fall into the low-energy regions
4
values (Kcis/trans = 40−70 in CD Cl, K
= 8.5−15 in
of the cis peptoid Ramachandran plot: ψ = 180.8 (1)°, φ = 76.6
3
cis/trans
40
MeOD, and Kcis/trans = 4.4−6.1 in D O).
(1)°, ω = 6.0 (1)° (ZRc region). Note that typical peptoid
Ramachandran dihedral angles are ψ (180°), φ (90°), and ω
2
The cis induction of peptoids 11 and 12 is consistently
41
stronger (Kcis/trans ≈ 15 in MeOD and K
≈ 6 in D O)
(0° for cis and 180° for trans). The side-chain ethylene linker
has an anti-conformation, positioning the quaternary ammo-
cis/trans
2
than the best reported in the literature, chiral triazolium-type
side-chains, even in polar protic solvents (K = 11.1 in
nium group of Nme ae onto the backbone carbonyls. Similarly,
cis/trans
3
27
MeOD and 5.5 in D O for Nbte). Notably, 14 was derived
11·BPh also shows backbone dihedral angles that are typical
2
4
39
from 13 via CuAAC “click” reaction. Click chemistry can
of peptoids (ψ (187.5(3)°), φ (84.9(2)°), and ω (0.8(3)°))
greatly increase diversity of the side-chain via azide-partner
and also features an anti-ethylene side-chain for Net ae.
3
F
DOI: 10.1021/jacs.9b10497
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
a
Chart 2. Ribbon Peptoid Chemical Structures
ammonium core than the α protons, allowing them to interact
with the acetyl carbonyl at a better geometry (D(C−O) =
3
.483 (3) Å, θ(C−H ···O) = 164(3)° in 11·BPh compared to
β
4
D(C−O) = 3.344(2) Å, θ(C−H ···O) = 101(2)° and 106(2)°
α
in 8·BPh ). Note that for hydrogen bonds, the optimal X−H−
4
O angle is linear (180°), and deviation from this angle
generally decreases the strength of hydrogen-bonding inter-
52
actions. These interactions can also be observed in the DFT-
computed structures, where the EPS map highlights the
positive nature of the α and β protons on the ethyl groups,
supporting the role of electrostatic stabilization of the cis state
(
Figure 5e). Additionally, DFT models of 12 (Net bnae side-
2
chain) suggest that while the benzyl group may be sterically
hindered from interacting directly with the i − 1 amide, the
observed increases on cis/trans preferences relative to 11 are
likely due to polarization changes introduced by the ring (see
Supporting Information).
Overall, these data suggest that, while an individual CH−O
is weak, the quaternary ammonium group allows for an
ensemble of polarized C−H moieties to interact simulta-
neously with the carbonyl O atoms, which may explain the
remarkably strong cis induction of these side-chains over their
primary ammonium counterparts.
a
Red amide bonds indicate cis-geometry at that position.
a
Table 3. Folded Populations of Designed Ribbon Peptoids
peptoid
population in MeOD (%)
population in D O (%)
2
Tertiary Ammonium Side-Chains. A potential drawback of
quaternary ammonium side-chains is that because an individual
CH−O bond is approximately half as strong as an NH−O
bond, the efficiency of cis induction can be significantly
2
2
5
6
63
52
50
59
a
Population ratios were calculated by peak fitting the formyl NMR
signals.
53,54
reduced in aqueous solution.
Maintaining a high degree of
cis induction in aqueous media remains crucial for foldamer
applications, as the majority of biological applications require
aqueous and buffered media. We envisioned that a synergy
between two types of hydrogen bonds, CH−O and NH−O,
could create side-chains that persist better in the presence of
polar protic solvent. Tertiary amine moieties, which are
generally protonated at pH 7, were hypothesized to facilitate
both an NH−O bond with its amine proton and CH−O bond
with its polarized α or β protons.
The solid-state structures also show several short intra- and
intermolecular CH−O contacts from the both the i and i − 1
carbonyl O atoms to the either α or β protons of the
quaternary ammonium moieties (α and β denote positions
relative to the ammonium N atom). Similar CH−O hydrogen
bonds have already been reported and observed in numerous
42−49
crystal structures of synthetic and biological systems.
For
8
·BPh , the i − 1 (acetyl) carbonyl O has three intramolecular
4
CH−O contacts with the Nme ae side-chain that are
We first synthesized peptoid 15, containing a protonated N-
3
significantly shorter than the sum of their atomic van der
Waals radii (3.7 Å). This observation is consistent with CH−O
hydrogen bonding between the positively polarized hydrogen
atoms α to N and the i − 1 carbonyl O, stabilizing the cis-
diethylamino-ethyl (Net ae) side-chain, as a precursor toward
2
12 and 13. We noticed that 15’s cis induction was somewhat
strong (Kcis/trans up to 13). Motivated by this observation, we
synthesized a bulkier derivative, 16, which features the
5
0,51
i
amide isomer.
Two additional CH−O contacts between
protonated N-diisopropylamino-ethyl (N Pr
2
ae) side-chain.
the quaternary ammonium group of Nme ae with its i carbonyl
We postulated that the polarized C−H, as well as the N−H
groups, will be more protected from solvation and will be able
to interact more strongly with the backbone carbonyls.
Consistent with our hypothesis, the Kcis/trans values of 16
3
are also observed. These interactions likely act cooperatively to
induce the cis isomer by positioning the side-chain to favorably
interact with the i − 1 carbonyl.
The crystal structure conformation of 8 is also favored in
DFT calculations. The two most favorable structures identified
in a monomer model of 8 correspond to the torsional states
observed in the crystal structure, supporting the role of cis
stabilization via polar CH−O interactions. The polarization
can be visualized in the electrostatic potential surface (EPS) of
the molecule (Figure 5b), which is positive over the entirety of
the side-chain electron isodensity surface. The structure-
inducing role of polarization is also supported by the fact that a
model of 10, the neutral isosteric variant of 8, is predicted to
be isoenergic between the cis and trans states (see Supporting
Information), also in line with experimental results.
(Kcis/trans = 30 in CDCl
3
) were increased approximately 3-fold
with respect to 15. Although the cis induction of 16 in
nonpolar media such as CDCl is not as potent as the
3
quaternary-ammonium side-chains (Kcis/trans ≈ 20−70), this
side-chain shows exceptionally large Kcis/trans values in polar
protic solvents (Kcis/trans = 16.0 and 8.1 in MeOD and D O,
2
respectively). To date, these values are the highest Kcis/trans
values of all reported peptoid side-chains achieved in MeOD
and D O. Furthermore, the important role of the N−H amine
2
proton at enforcing the cis-amide geometry was evidenced by
very low Kcis/trans values of the deprotonated, neutral version of
16 (0.3, 0.7, and 0.8 in CDCl , MeOD, and D O, respectively).
3
2
Similar observations are also seen in the X-ray crystal
The solid-state structure of 16·BPh , as determined by X-ray
4
structure of 11·BPh with CH−O interactions from the β
crystallography, gives evidence of synergistic NH−O and CH−
O interactions between the side-chain and backbone (Figure
6). To our surprise, the stronger NH−O bond is formed with
4
protons of the ethyl groups, instead of the α protons, with the
acetyl carbonyl. The β protons extend further away from the
G
DOI: 10.1021/jacs.9b10497
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Figure 7. Structural analyses of peptoid 26 in solution. (a) Inter-residue NOE constraints used for REMD structural solution. Blue lines are side-
chain-to-side-chain correlations, and red lines are backbone-to-backbone correlations. (b) Ensemble of the 10 lowest energy structures; backbone
shown only for clarity. Class 1 structures form the ribbon secondary structure. Class 2 have less regular structure. (c) Space-filling representation of
the lowest energy structure. Magenta residues have N-aryl side-chains, and blue residues have quaternary ammonium side-chains. (d) Cis and trans
Ramachandran plots of the 10 lowest energy structures from residues 1−9. Green points are dihedral combinations of all structures, and red points
are those from the lowest energy structure.
the i carbonyl rather than the i −1 carbonyl, leaving the
polarized methine C−H to interact with the remaining acetyl
carbonyl. Moreover, two protons of the ammonium core (on
C14 and C17) are also within close proximity to the i carbonyl,
suggesting that four hydrogen bonds are working cooperatively
to favor the cis-acetamide conformation. Cooperative inter-
actions also resulted in the side-chain ethylene linker to adopt
a gauche conformation, rather than an anti conformation as
seen in previous structures of 8·BPh and 11·BPh . The
(Table 2). Van’t Hoff plots of these peptoids were constructed
from Kcis/trans values taken in DMSO from 296 K to 353−373 K
(see Supporting Information).
The cis induction by Nme ae in peptoid 8 is actually slightly
3
endothermic, but the large entropy factor makes the folding
spontaneous (ΔH = +0.50 (±0.06) kcal/mol, −TΔS = −1.33
(±0.06) kcal/mol at 298 K). The positive ΔH value dictates
that interactions in peptoid 8 are enthalpically disfavored,
meaning intramolecular interactions are weaker than inter-
4
4
55,56
backbone dihedral parameters also fall within the typical range
molecular interactions with solvent.
However, multiple
for peptoids: ψ = 156.3(1)°, φ = −74.2(2)°, ω = 10.1(2)°
Sc
Likewise, DFT computations on peptoid 16 reveal the
simultaneous intramolecular interactions are able to release
more solvent molecules, which accounts for the significant
entropy gain, a phenomenon analogous to the hydrophobic
4
0
(
Z ).
57
58,59
conformation observed in the crystal structure is among the
three lowest energy structures identified (all three of which are
cis). Surprisingly, the side-chains in the other two low-energy
conformations hydrogen bond with the i − 1 (rather than the
effect and metal chelation.
Although an individual CH−
O bond is relatively weak, an ensemble of such interactions
between the side-chain and backbone are able to release
solvent molecules that were previously interacting with the
quaternary ammonium core and carbonyl O.
i) carbonyl oxygen (Figure 6). This suggests that the NiPr ae
2
side-chain is flexible at room temperature, being able to switch
between different hydrogen-bonding modes, all of which favor
the cis-amide isomer at the i − 1 position. This appears to be
possible because the side-chain can simultaneously form NH−
O and CH−O hydrogen bond interactions with both
surrounding carbonyl oxygens simultaneously.
Thermodynamic Data. The changes in Kcis/trans values with
varying temperature experiments allowed extraction of the
enthalpic (ΔH) and entropic (ΔS) contributions to folding
In contrast to peptoid 8, peptoid 11 and 12’s cis inductions
are exothermic (ΔH = −0.66 (±0.03) kcal/mol and −0.71
(±0.03) kcal/mol, respectively). However, peptoids 11 and 12
still demonstrate entropic gain as a significant factor in their cis
induction (−TΔS = −0.67 (±0.03) kcal/mol and −0.70
(±0.03) kcal/mol at 298 K, respectively). Here, the interaction
between the −CH moiety of the ethyl group with the carbonyl
3
is stronger than in Nme ae, due to a more favorable geometry
3
(vide supra), resulting in a slightly exothermic interaction.
H
DOI: 10.1021/jacs.9b10497
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
However, the entropy gain is smaller than that of 7, possibly
because 11 and 12 must freeze the rotation of an ethyl group
on the side-chain in order to interact with the acetyl carbonyl.
Similar to peptoids 11 and 12, peptoid 16’s cis induction is
also both exothermic and entropy-driven (ΔH = −0.52
methods resulted in cyclized, ketopiperazinium products.
Cyclization yield up to 100% was observed (by HPLC)
following bromoacetylation of Net ae (peptoid 22), while
2
i
N Pr ae (peptoid 23) gave ∼40% cyclized product. We
2
minimized the cyclization by acylating with the less electro-
philic chloroacetic acid and reducing the reaction time from 30
to 2 min. This strategy reduces the cyclization yield to ∼60%
(±0.03) kcal/mol, −TΔS = −0.62 (±0.03) kcal/mol at 298
K). The presence of a classical NH−O interaction, as seen in
the crystal structure, might account for the exothermic cis
induction. Again, entropy is the major driving force for this
process. Altogether, entropic considerations are consistent with
the multiple, intramolecular contacts observed in the crystal
i
with Net ae and ∼0% with N Pr ae. Although this method still
2
2
prevents usage of multiple Net ae residues in longer sequences,
the N Pr ae residue is now readily incorporated. As a proof of
concept, we were able to synthesize pentamer Ts-(N Pr ae) -
2
i
2
i
2
5
structures of 8·BPh , 11·BPh , and 16·BPh and DFT results
4
4
4
NH (peptoid 24) in excellent yield (>95%) (see Supporting
2
on 8, 11, 12, and 16.
Information).
Synthesis of Longer Peptoid Sequences. Amine
Precursor Synthesis. To prepare longer sequences by the
submonomer method, the side-chains are prepared in their
Application toward Water-Soluble Ribbon Structures. To
demonstrate the utility and potent folding properties of these
new monomers, we strived to synthesize long peptoid
sequences that are water-soluble, comprise diverse side-chains,
and possess defined secondary structures. Such properties are
essential for the design of functional peptoid-based materials,
such as therapeutics and nanostructures. We were particularly
interested in the ribbon structure, previously reported by
Blackwell and co-workers, which features alternating cis and
trans backbone−amide bonds. The original ribbon structures
primary amine forms to accommodate the S 2 displacement
N
1
step. For some of these side-chainsNme ae, Net ae, and
3
2
i
N Pr aethe primary amine submonomers are commercially
2
available and can be purchased at reasonable costs. The other
monomers can be easily prepared in gram scale via simple
synthesis (with two steps or fewer from low-cost starting
materials) as demonstrated herein (Scheme 1).
Solid-Phase Peptoid Synthesis Optimization. We then
optimized the conditions for solid-phase synthesis of peptoids
comprising our new monomers by testing the synthesis of 2-
(
up to 6-mer) were made up of two types of alternating
hydrophobic monomers, an Ns1npe side-chain and N-aryl
side-chains, which are strong cis and trans inducers,
mer sequences with the general structure Ts-Sar-X-NH (Ts =
62
2
respectively. This presented an opportunity for us to create
1
p-toluenesulfonyl, Sar = N-methylglycine) (Scheme 2). Our
an amphiphilic ribbon structure by substituting the bulky and
initial attempt at incorporating the quaternary ammonium-type
monomers into the peptoid sequence on Rink-amide MBHA
polystyrene resin using standard conditions (25 °C, acylation
with 1.0 M bromoacetic acid/DIC for 10 min, and displace-
ment with 1.0 M amine for 60 min) was low yielding (<30% by
HPLC), presumably due to the very low basicity and
nonpolar cis inducers (Ns1npe) with our newly described
23
cationic, water-soluble monomers. Moreover, cationic
ammonium moieties have been shown to favorably bind to
electron-rich π systems, thus allowing for additional non-
covalent interactions that promote folding in our ribbon
63
structures.
nucleophilicity of the primary amines (e.g., pK of cholamine
a
6
0
We designed two long sequences: a 6-mer peptoid (25)
is 7.1). This problem was mitigated by the introduction of
.5 M KI to the amine solution (to generate in situ a more
i
where all the Ns1npe are replaced with N Pr ae, and a 10-mer,
2
0
peptoid (26), where we installed five of our monomers,
reactive iodoacetyl electrophile) and extending the displace-
ment step to 24 h at 25 °C. The bromoacylation onto the
quaternary ammonium-containing monomer was also double
coupled (50 min each), again due to the poor basicity of the
N-terminus. Alternatively, synthesis with these amines can be
done at 60 °C to cut the displacement time to 2 h and the
acylation time to 2 min each. With these optimized methods,
the solid-phase coupling efficiency for quaternary-ammonium-
type peptoid monomers exceeds 95%, as demonstrated in the
crude HPLC traces of peptoids 17−20 (see Supporting
Information for all HPLC traces).
i
Net pgae, Net bnae, Net ae, Nme ae, and N Pr ae, at the
2
2
3
3
2
Ns1pe positions (Chart 2). Different N-aryl monomers were
used at the trans positions in peptoids 25 and 26 to facilitate
peak dispersion in the NMR spectra. Both peptoids were also
capped on the N-terminus with formyl, which functions as a
well-separated NMR spectroscopic handle (singlet at ∼8 ppm)
for determining structural homogeneity.
1
H NMR spectroscopy in polar protic solvents (MeOD and
D O) of 25 and 26 reveals only one major conformation
2
(
Table 3), as indicated by the distribution of formyl NMR
signals. In contrast, a structurally undefined hexamer and
Furthermore, the Net pgae side-chain can be easily
2
6
10
decamer would show 2 (64) and 2 (1024) amide isomers,
converted on-resin to a triazole with CuAAC “click” chemistry
in almost quantitative yield allowing for further diversification
of side-chains or postsynthetic conjugation (peptoid 21). As a
proof-of-principle, benzyl azide was conjugated onto Ts-Sar-
1
respectively. The H NMR resonances of 25 and 26 are also
well-dispersed, especially in MeOD, indicative of a well-folded
structure. ROESY NMR spectroscopy confirms a pattern of
alternating cis−trans amides, consistent the ribbon structure (ω
Net pgae in high yield (see Supporting Information for further
2
=
0°, 180°, 0°, ...). The high population of the cis amides at the
details). This reaction should be compatible with most organo-
azide precursors, as the “click” reaction is extraordinarily robust
and can facilitate a wide variety of substituents.
We next optimized solid-phase peptoid synthesis with the
tertiary-amine-containing side-chains. Unlike the quaternary
ammonium side-chains, tertiary-amine side-chains contain
nucleophilic tertiary amino groups capable of intramolecular
cationic residues suggests that CH−O interactions are also
operative in the longer peptoid. Moreover, we observed
ROESY cross-peaks diagnostic of a ribbon fold between the i
side-chain and the i+3 side-chain (Figure 7a). Note that an
alternating cis−trans amide backbone with a different (ψ, φ)
pattern could also produce a “square helix” secondary
61
24
S 2 with the i − 1 haloacetyl residue (Scheme 3). Initial
structure, though this should result in cross-peaks between
N
24
attempts at incorporating these side-chains with normal
the i and i+6 residues, which we do not observe.
I
DOI: 10.1021/jacs.9b10497
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
The NMR distance constraints were used for simulated
annealing via replica exchange molecular dynamics (REMD)
for structural determination of peptoid models corresponding
to 26 (Figure 7a). The 10 lowest energy structures are shown
in Figure 7b. The stereochemistry of the backbone chain is
mostly consistent with that of an extended peptoid ribbon
secondary structure, with backbone dihedrals alternating
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.9b10497.
■
*
S
Full synthetic, physical, theoretical, and crystallographic
details (PDF)
X-ray crystallographic data for 8·BPh (CIF)
between ZSc (ϕ = −90°, ψ = ±180°, ω = 0°) and Z (ϕ =
4
Rt
40
9
0°, ψ = ±180°, ω = 180°). Excluding the termini, the mean
X-ray crystallographic data for 11·BPh (CIF)
4
ϕ and ψ absolute values for the N-aryl residues are |87| ± 18°
and |151| ± 23°, respectively, while the mean ϕ and ψ absolute
values for the ammonium residues were |88| ± 13° and |152| ±
X-ray crystallographic data for 16·BPh (CIF)
NMR structure of 26 (PDB)
4
2
2°, respectively. The standard deviations of the ϕ and ψ
AUTHOR INFORMATION
angles are quite large, indicating that the ensemble, although
retaining the stereochemistry of a ribbon, is still highly flexible,
perhaps due to the absence of chiral side-chains. Our design
hypothesis regarding inter-residual cation → π interactions
between i and i+3 pairs also proved correct. On average, three
pairs of cation → π interactions help to stabilize the fold and
might explain the higher folded population observed for 26
■
Corresponding Authors
*ainguyen@lbl.gov
*rnzuckermann@lbl.gov
ORCID
Andy I. Nguyen: 0000-0003-4137-6453
Ryan K. Spencer: 0000-0002-1043-3913
Ronald N. Zuckermann: 0000-0002-3055-8860
Author Contributions
A.W.W. and A.I.N. contributed equally.
over 25 in D O, as compound 26 can potentially accommodate
2
more cation → π interactions than compound 25. Moreover, a
total of three types of noncovalent interactions (hydrogen
bonds, sterics for N-aryl residues, and cation → π interactions)
work cooperatively to induce folding, and such an ensemble of
interactions is reminiscent of protein folding in nature and
should actively be used in deriving new biomimetic structures.
⊥
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Work at the Molecular Foundry was supported by the Office of
Science, Office of Basic Energy Sciences, of the U.S.
Department of Energy under Contract No. DE-AC02-
SUMMARY AND OUTLOOK
■
In this report, we demonstrate that this new class of
monomers, i.e., cationic alkyl ammonium ethyl side chains,
have the unique ability to control rotation about ω to an
extraordinary degree while being diverse, hydrophilic, and
amenable to solid-phase synthesis, unlike previously reported
steric and electronic methods to cis induction. These peptoid
side-chains leverage an unusual mechanism to enforce the cis-
amide conformation via an ensemble of several weak CH−O
bonds between the side-chains and the backbone carbonyls.
This method is in contrast to the traditional approach of
controlling peptoid cis/trans isomerization, by tuning a single
interaction between the side-chain and the backbone. The
present contribution suggests that a shift in design strategy
toward multiple, cooperative forces may be beneficial in paving
the road toward generating new, diverse monomers that
promote predictable peptoid architectures.
0
5CH11231. Additional support was provided by the
DARPA Fold F(x) program. DFT calculations were carried
out on the High Performance Computing resources at New
York University Abu Dhabi. The funds for the 900 MHz NMR
spectrometer in the QB3 Institute at University of California,
Berkeley, were provided by the NIH Grant GM68933.
Additionally, we would like to thank Dr. Nicholas Settineri
of the UC Berkeley CheXray facility for his assistance in X-ray
diffraction studies, Dr. Jeff G. Pelton for his assistance in NMR
studies, and Victor R. Mann, Michael Connolly, and Zeming
Wang for helpful discussions.
REFERENCES
■
(
1) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H.
Efficient method for the preparation of peptoids [oligo(N-substituted
Given the biological relevance of peptoids, structural
integrity and solubility in water are crucial for application
development. These peptoid submonomers are readily
available and easily diversifiable via simple synthesis and
amenable to solid-phase peptoid synthesis, which allows for
their incorporation into peptoid libraries and longer chains of
sequence-defined polymers. We also reported optimized
synthesis conditions to incorporate these monomers into a
peptoid sequence in over 95% yield along with “click”
glycines)] by submonomer solid-phase synthesis. J. Am. Chem. Soc.
1
(
992, 114, 10646−10647.
2) Yoo, B.; Kirshenbaum, K. Peptoid architectures: elaboration,
actuation, and application. Curr. Opin. Chem. Biol. 2008, 12, 714−721.
(
3) Fowler, S. A.; Blackwell, H. E. Structure−function relationships
in peptoids: Recent advances toward deciphering the structural
requirements for biological function. Org. Biomol. Chem. 2009, 7,
1
508−1524.
(4) Miller, S. M. S.; Reyna, J.; Ng, S.; Zuckermann, R. N.; Kerr, J.
M.; Moos, W. H. Proteolytic studies of homologous peptide and N-
substituted glycine peptoid oligomers. Bioorg. Med. Chem. Lett. 1994,
methodology to convert Net pgae into its triazole form. We
2
4
(
, 2657−262.
5) Vollrath, S. B. L.; Fu
peptoid transporters − synthesis and evaluation. Org. Biomol. Chem.
013, 11, 8197−8201.
6) Kwon, Y.-U.; Kodadek, T. Quantitative Evaluation of the
further demonstrate application of these new monomers to
̈
rniss, D.; Schepers, U.; Brase, S. Amphiphilic
̈
create the longest known example of Blackwell’s ribbon
2
3
peptoid secondary structure. Re-engineering of other
2
(
1
9−21
structures from literature, such as the PPI helix,
peptoid
2
4
square helix, and nanosheets, along with the development of
chiral versions of these monomers are currently underway.
Relative Cell Permeability of Peptoids and Peptides. J. Am. Chem. Soc.
2007, 129, 1508−1509.
J
DOI: 10.1021/jacs.9b10497
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(
7) Huang, M. L.; Ehre, D.; Jiang, Q.; Hu, C.; Kirshenbaum, K.;
Revealed by a Survey of Noncovalent Interactions in Model Systems.
J. Am. Chem. Soc. 2009, 131, 16555−16567.
(26) Roy, O.; Caumes, C.; Esvan, Y.; Didierjean, C.; Faure, S.;
Taillefumier, C. The tert-Butyl Side Chain: A Powerful Means to
Lock Peptoid Amide Bonds in the Cis Conformation. Org. Lett. 2013,
15, 2246−2249.
(27) Caumes, C.; Roy, O.; Faure, S.; Taillefumier, C. The Click
Triazolium Peptoid Side Chain: A Strong cis-Amide Inducer Enabling
Chemical Diversity. J. Am. Chem. Soc. 2012, 134, 9553−9556.
(28) Shyam, R.; Nauton, L.; Angelici, G.; Roy, O.; Taillefumier, C.;
Faure, S. NCα-gem-dimethylated peptoid side chains: A novel
approach for structural control and peptide sequence mimetics.
Biopolymers 2019, 110, e23273.
Ward, M. D. Biomimetic peptoid oligomers as dual-action antifreeze
agents. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 19922.
(
8) Zuckermann, R.; Kodadek, T. Peptoids as Potential Ther-
apeutics. Curr. Op. Mol. Ther. 2009, 11, 299−307.
9) Mojsoska, B.; Carretero, G.; Larsen, S.; Mateiu, R. V.; Jenssen,
(
H. Peptoids successfully inhibit the growth of gram negative E. coli
causing substantial membrane damage. Sci. Rep. 2017, 7, 42332.
(
10) Chongsiriwatana, N. P.; Patch, J. A.; Czyzewski, A. M.; Dohm,
M. T.; Ivankin, A.; Gidalevitz, D.; Zuckermann, R. N.; Barron, A. E.
Peptoids that mimic the structure, function, and mechanism of helical
antimicrobial peptides. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 2794−
2799.
(
11) Jun, J. M. V.; Altoe, M. V. P.; Aloni, S.; Zuckermann, R. N.
(29) Gimenez, D.; Zhou, G.; Hurley, M. F. D.; Aguilar, J. A.; Voelz,
V. A.; Cobb, S. L. Fluorinated Aromatic Monomers as Building Blocks
To Control α-Peptoid Conformation and Structure. J. Am. Chem. Soc.
2019, 141, 3430−3434.
Peptoid nanosheets as soluble, two-dimensional templates for calcium
carbonate mineralization. Chem. Commun. 2015, 51, 10218−10221.
(
Battigelli, A.; Butterfoss, G. L.; Zuckermann, R. N.; Whitelam, S.
Peptoid nanosheets exhibit a new secondary-structure motif. Nature
12) Mannige, R. V.; Haxton, T. K.; Proulx, C.; Robertson, E. J.;
(30) Gimenez, D.; Aguilar, J. A.; Bromley, E. H. C.; Cobb, S. L.
Stabilising Peptoid Helices Using Non-Chiral Fluoroalkyl Monomers.
Angew. Chem., Int. Ed. 2018, 57, 10549−10553.
2
(
015, 526, 415.
13) Mu, P.; Zhou, G.; Chen, C.-L. 2D nanomaterials assembled
from sequence-defined molecules. Nano-Struct. Nano-Obj. 2018, 15,
53−166.
14) Sun, J.; Jiang, X.; Lund, R.; Downing, K. H.; Balsara, N. P.;
(31) Laursen, J. S.; Engel-Andreasen, J.; Fristrup, P.; Harris, P.;
Olsen, C. A. Cis−Trans Amide Bond Rotamers in β-Peptoids and
Peptoids: Evaluation of Stereoelectronic Effects in Backbone and Side
Chains. J. Am. Chem. Soc. 2013, 135, 2835−2844.
1
(
Zuckermann, R. N. Self-assembly of crystalline nanotubes from
monodisperse amphiphilic diblock copolypeptoid tiles. Proc. Natl.
Acad. Sci. U. S. A. 2016, 113, 3954−3959.
(32) Laurence, C.; Berthelot, M. Observations on the strength of
hydrogen bonding. Perspect. Drug Discovery Des. 2000, 18, 39−60.
(33) Petukhov, M.; Rychkov, G.; Firsov, L.; Serrano, L. H-bonding
in protein hydration revisited. Protein Sci. 2004, 13, 2120−2129.
(34) Roe, L. T.; Pelton, J. G.; Edison, J. R.; Butterfoss, G. L.; Tresca,
B. W.; LaFaye, B. A.; Whitelam, S.; Wemmer, D. E.; Zuckermann, R.
N. Unconstrained peptoid tetramer exhibits a predominant
conformation in aqueous solution. Biopolymers 2019, 0, e23267.
(35) Nguyen, A. I.; Spencer, R. K.; Anderson; Christopher, L.;
Zuckermann, R. N. A bio-inspired approach to ligand design: folding
single-chain peptoids to chelate a multimetallic cluster. Chem. Sci.
2018, 9, 8806−8813.
(
15) Vollrath, S. B. L.; Hu, C.; Brase, S.; Kirshenbaum, K. Peptoid
̈
nanotubes: an oligomer macrocycle that reversibly sequesters water
via single-crystal-to-single-crystal transformations. Chem. Commun.
2
(
013, 49, 2317−2319.
16) Yan, F.; Liu, L.; Walsh, T. R.; Gong, Y.; El-Khoury, P. Z.;
Zhang, Y.; Zhu, Z.; De Yoreo, J. J.; Engelhard, M. H.; Zhang, X.;
Chen, C.-L. Controlled synthesis of highly-branched plasmonic gold
nanoparticles through peptoid engineering. Nat. Commun. 2018, 9,
2
(
327.
17) Battigelli, A.; Kim, J. H.; Dehigaspitiya, D. C.; Proulx, C.;
(36) Fuller, A. A.; Yurash, B. A.; Schaumann, E. N.; Seidl, F. J. Self-
Association of Water-Soluble Peptoids Comprising (S)-N-1-
(Naphthylethyl)glycine Residues. Org. Lett. 2013, 15, 5118−5121.
(37) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson,
G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A. V.; Bloino, J.;
Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; V, J.;
Ortiz, A. F. I.; Sonnenberg, J. L.; Williams-Young, D.; Ding, F.;
Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson,
T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.;
Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.;
Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.;
Throssell, K.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.;
Bearpark, M. J.; Heyd, J. J.; Brothers, E. N.; Kudin, K. N.; Staroverov,
V. N.; Keith, T. A.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.;
Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.;
Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J.
Gaussian 16, Revision B.01; Gaussian, Inc.: Wallingford, CT, 2016.
(38) Shirakawa, S.; Liu, S.; Kaneko, S.; Kumatabara, Y.; Fukuda, A.;
Omagari, Y.; Maruoka, K. Tetraalkylammonium Salts as Hydrogen-
Bonding Catalysts. Angew. Chem., Int. Ed. 2015, 54, 15767−15770.
(39) Holub, J. M.; Kirshenbaum, K. Tricks with clicks: modification
of peptidomimetic oligomers via copper-catalyzed azide-alkyne [3 +
2] cycloaddition. Chem. Soc. Rev. 2010, 39, 1325−1337.
Robertson, E. J.; Murray, D. J.; Rad, B.; Kirshenbaum, K.;
Zuckermann, R. N. Glycosylated Peptoid Nanosheets as a Multivalent
Scaffold for Protein Recognition. ACS Nano 2018, 12, 2455−2465.
(18) Jin, H.; Ding, Y.-H.; Wang, M.; Song, Y.; Liao, Z.; Newcomb,
C. J.; Wu, X.; Tang, X.-Q.; Li, Z.; Lin, Y.; Yan, F.; Jian, T.; Mu, P.;
Chen, C.-L. Designable and dynamic single-walled stiff nanotubes
assembled from sequence-defined peptoids. Nat. Commun. 2018, 9,
2
(
70.
19) Armand, P.; Kirshenbaum, K.; Falicov, A.; Dunbrack, R. L.;
Dill, K. A.; Zuckermann, R. N.; Cohen, F. E. Chiral N-substituted
glycines can form stable helical conformations. Folding Des. 1997, 2,
3
(
69−375.
20) Stringer, J. R.; Crapster, J. A.; Guzei, I. A.; Blackwell, H. E.
Extraordinarily Robust Polyproline Type I Peptoid Helices Generated
via the Incorporation of α-Chiral Aromatic N-1-Naphthylethyl Side
Chains. J. Am. Chem. Soc. 2011, 133, 15559−15567.
(21) Roy, O.; Dumonteil, G.; Faure, S.; Jouffret, L.; Kriznik, A.;
Taillefumier, C. Homogeneous and Robust Polyproline Type I
Helices from Peptoids with Nonaromatic α-Chiral Side Chains. J. Am.
Chem. Soc. 2017, 139, 13533−13540.
(22) Wu, C. W.; Kirshenbaum, K.; Sanborn, T. J.; Patch, J. A.;
Huang, K.; Dill, K. A.; Zuckermann, R. N.; Barron, A. E. Structural
and Spectroscopic Studies of Peptoid Oligomers with α-Chiral
Aliphatic Side Chains. J. Am. Chem. Soc. 2003, 125, 13525−13530.
(23) Crapster, J. A.; Guzei, I. A.; Blackwell, H. E. A Peptoid Ribbon
(40) Spencer, R. K.; Butterfoss, G. L.; Edison, J. R.; Eastwood, J. R.;
Whitelam, S.; Kirshenbaum, K.; Zuckermann, R. N. Stereochemistry
of polypeptoid chain configurations. Biopolymers 2019, 110, e23266.
(41) Park, S. H.; Szleifer, I. Structural and Dynamical Characteristics
of Peptoid Oligomers with Achiral Aliphatic Side Chains Studied by
Molecular Dynamics Simulation. J. Phys. Chem. B 2011, 115, 10967−
10975.
Secondary Structure. Angew. Chem., Int. Ed. 2013, 52, 5079−5084.
(24) Gorske, B. C.; Mumford, E. M.; Gerrity, C. G.; Ko, I.; Peptoid
Square, A. Helix via Synergistic Control of Backbone Dihedral Angles.
J. Am. Chem. Soc. 2017, 139, 8070−8073.
(25) Gorske, B. C.; Stringer, J. R.; Bastian, B. L.; Fowler, S. A.;
Blackwell, H. E. New Strategies for the Design of Folded Peptoids
K
DOI: 10.1021/jacs.9b10497
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(
42) Wahl, M. C.; Sundaralingam, M. C-H···O hydrogen bonding in
biology. Trends Biochem. Sci. 1997, 22, 97−102.
43) Yokoyama, T.; Mizuguchi, M.; Nabeshima, Y.; Kusaka, K.;
(
Yamada, T.; Hosoya, T.; Ohhara, T.; Kurihara, K.; Tomoyori, K.;
Tanaka, I.; Niimura, N. Hydrogen-bond network and pH sensitivity in
transthyretin: Neutron crystal structure of human transthyretin. J.
Struct. Biol. 2012, 177, 283−290.
(
44) Ramachandran, G. X.; Chandrasekharan, R. Interchain
hydrogen bonds via bound water molecules in the collagen triple
helix. Biopolymers 1968, 6, 1649−1658.
(45) Scheiner, S.; Kar, T.; Gu, Y. Strength of the CαH··O Hydrogen
Bond of Amino Acid Residues. J. Biol. Chem. 2001, 276, 9832−9837.
46) Steiner, G. D. a. T. The Weak Hydrogen Bond In Structural
Chemistry and Biology; Oxford University Press: Oxford, 2001.
47) Laliberte, D.; Maris, T.; Wuest, J. D. Molecular networks built
(
(
́
from weakly interacting nitro-substituted pentaerythrityl tetraaryl
ethers. CrystEngComm 2005, 7, 158−160.
(48) Iwase, S.; Xiang, B.; Ghosh, S.; Ren, T.; Lewis, P. W.;
Cochrane, J. C.; Allis, C. D.; Picketts, D. J.; Patel, D. J.; Li, H.; Shi, Y.
ATRX ADD domain links an atypical histone methylation recognition
mechanism to human mental-retardation syndrome. Nat. Struct. Mol.
Biol. 2011, 18, 769.
(49) Stamos, J.; Sliwkowski, M. X.; Eigenbrot, C. Structure of the
Epidermal Growth Factor Receptor Kinase Domain Alone and in
Complex with a 4-Anilinoquinazoline Inhibitor. J. Biol. Chem. 2002,
2
77, 46265−46272.
50) Horowitz, S.; Trievel, R. C. Carbon-Oxygen Hydrogen Bonding
in Biological Structure and Function. J. Biol. Chem. 2012, 287,
1576−41582.
51) Jeffrey, G. A. An Introduction to Hydrogen Bonding; Oxford
University Press: New York, 1997.
52) Pimentel, G. C.; McClellan, A. L. The Hydrogen Bond; W.H.
Freeman, 1960.
53) Cannizzaro, C. E.; Houk, K. N. Magnitudes and Chemical
(
4
(
(
(
Consequences of R3N+−C−H···OC Hydrogen Bonding. J. Am.
Chem. Soc. 2002, 124, 7163−7169.
(54) Pike, S. J.; Lavagnini, E.; Varley, L. M.; Cook, J. L.; Hunter, C.
A. H-Bond donor parameters for cations. Chem. Sci. 2019, 10, 5943−
951.
55) Arbely, E.; Arkin, I. T. Experimental Measurement of the
Strength of a Cα−H···O Bond in a Lipid Bilayer. J. Am. Chem. Soc.
004, 126, 5362−5363.
56) Musah, R. A.; Jensen, G. M.; Rosenfeld, R. J.; McRee, D. E.;
5
(
2
(
Goodin, D. B.; Bunte, S. W. Variation in Strength of an
Unconventional C−H to O Hydrogen Bond in an Engineered
Protein Cavity. J. Am. Chem. Soc. 1997, 119, 9083−9084.
(57) Silverstein, T. P. The Real Reason Why Oil and Water Don’t
Mix. J. Chem. Educ. 1998, 75, 116.
58) Frausto da Silva, J. J. R. The chelate effect redefined. J. Chem.
Educ. 1983, 60, 390.
59) Vallet, V.; Wahlgren, U.; Grenthe, I. Chelate Effect and
(
(
Thermodynamics of Metal Complex Formation in Solution: A
Quantum Chemical Study. J. Am. Chem. Soc. 2003, 125, 14941−
1
(
4950.
60) Good, N. E.; Winget, G. D.; Winter, W.; Connolly, T. N.;
Izawa, S.; Singh, R. M. M. Hydrogen Ion Buffers for Biological
Research*. Biochemistry 1966, 5, 467−477.
(
61) Meldal, M.; Tornøe, C. W. Cu-Catalyzed Azide−Alkyne
Cycloaddition. Chem. Rev. 2008, 108, 2952−3015.
(
62) Shah, N. H.; Butterfoss, G. L.; Nguyen, K.; Yoo, B.; Bonneau,
R.; Rabenstein, D. L.; Kirshenbaum, K. Oligo(N-aryl glycines): A
New Twist on Structured Peptoids. J. Am. Chem. Soc. 2008, 130,
1
(
2
6622−16632.
63) Dougherty, D. A. The cation-π interaction. Acc. Chem. Res.
013, 46, 885−893.
L
DOI: 10.1021/jacs.9b10497
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX