A peptoid ribbon secondary structure

Article abstract of DOI:10.1002/anie.201208630

Joining the fold: A series of peptoids, or oligomers of N-substituted glycines, adopt a novel secondary structure, designated the peptoid ribbon . This fold was stable at short chain lengths and in a variety of solvents (both organic and aqueous), and arose from a primary sequence of peptoid monomers designed to enforce an alternating pattern of cis and trans main-chain amides. Copyright

Full text of DOI:10.1002/anie.201208630

Angewandte
Chemie
DOI: 10.1002/anie.201208630
Peptoid Foldamers
A Peptoid Ribbon Secondary Structure**
J. Aaron Crapster, Ilia A. Guzei, and Helen E. Blackwell*
Delineating the relationships between sequence, structure,
and function in biopolymers is critical to our understanding of
fundamental biochemical interactions. These relationships
are equally important for the design of functional biomimetic
oligomers (that is, foldamers).[1] Oligomers of N-substituted
glycine, or peptoids (Figure 1a),[2] are an important class of
degradation[5] and their straightforward, modular synthesis[6]
enables the ready incorporation of a range of structurally
diverse amide side chains.[7] However, the design and
prediction of peptoid secondary or higher-order structure
remains a major challenge, and few discrete structures have
been characterized to date for acyclic peptoids.[8] This paucity
of structure–function data limits the potential utility of
peptoids, despite their many advantages.
Structural studies of peptoids have been largely thwarted
by the intrinsic conformational flexibility of the peptoid
backbone itself. This backbone contains C-a methylene units,
lacks hydrogen bond donating atoms, and, perhaps most
notably, is linked by tertiary amides that can be isoenergetic
between cis and trans amide geometries (Figure 1b). We and
others reasoned that the development of peptoid side chains
capable of engendering a high degree of control over
proximate main-chain amide geometries could facilitate the
design of well-defined peptoid structures and expand our
understanding of peptoid folding.[9] We recently designed and
synthesized a range of peptoid model systems to test this
hypothesis, and have identified several classes of amide side
chains that favor cis or trans main-chain amides in peptoid
oligomers, predominantly through steric and stereoelectronic
effects. One such side chain is the a-chiral aromatic (S)-1-(1-
naphthyl)ethyl (s1npe) group (Figure 1), which strongly
favors cis amide bonds (cis/trans > 6.3:1) in peptoid model
systems.[9c] Moreover, Ns1npe homooligomers adopt polypro-
line type I (PPI)-like peptoid helices that contain exclusively
cis amides in the peptoid main chain.[8e] In turn, we found that
the trans amide peptoid rotamer is strongly enforced by N-
aryl side chains (for example, Nph, cis/trans < 0.05:1;
Figure 1), and homooligomers of these residues have been
calculated to give rise to extended, polyproline type II (PPII)-
like peptoid helices with all trans amides.[9b] These peptoid
monomers are thus a set of building blocks with which to
initiate a rational construction of new peptoid secondary
structures. Herein, we report our first test of this modular
design strategy for peptoids and our ensuing discovery of
a new secondary structure containing an alternating sequence
of aromatic residues that we designate as the “peptoid
ribbon”.
Figure 1. a) The primary structure of an a-peptoid oligomer (dihedral
angles omega (w), phi (f), psi (y), and chi (c) labeled) and the
structures of the peptoid side chains discussed in this study. Side-
chain abbreviations: s1npe=(S)-1-(1-naphthyl)ethyl; ph=phenyl;
2,6 mph=2,6-dimethylphenyl; 3,5 mph=3,5-dimethylphenyl; 4fph=4-
fluorophenyl. b) Peptoid cis and trans amide rotamers and the isomeric
preferences engendered by the two amide side chains investigated in
this study in model peptoid monomer systems.
foldamers that have been shown to possess numerous
biological functions[3] and could find use in a range of
fundamental and applied contexts as bio-inspired materials.[4]
Peptoids are highly attractive scaffolds for such purposes, as
their non-native backbones are resistant to proteolytic
[*] Dr. J. A. Crapster,[+] I. A. Guzei, Prof. Dr. H. E. Blackwell
Department of Chemistry, University of Wisconsin-Madison
1101 University Avenue, Madison, WI 53706-1322 (USA)
E-mail: blackwell@chem.wisc.edu
[+] Current address: Department of Chemical and Systems Biology,
Stanford University School of Medicine
269 Campus Drive, Stanford, CA 94305-5174 (USA)
[**] Financial support from the NSF (CHE-0449959), ONR
(N000140710255), Greater Milwaukee Foundation, and Burroughs
Welcome Fund is gratefully acknowledged. Chemistry NMR facilities
at UW-Madison are supported by the NIH (1 S10 RR13866-01) and
the NSF (CHE-0342998 and CHE-9629688). The National Magnetic
Resonance Facility at UW-Madison is supported in part by NIH
grants P41RR02301 (BRTP/NCRR) and P41GM66326 (NIGMS). We
thank Dr. Charles Fry for assistance with NMR spectroscopy, Dr.
Milo Westler for assistance with AMBER calculations, Prof. Samuel
Gellman for thoughtful discussions and the use of his laboratory’s
CD spectrometer, and Dr. Joseph Stringer for experimental guid-
ance.
In view of the strong rotameric preferences enforced in N-
aryl and Ns1npe monomers, we hypothesized that linear
peptoids containing an alternating sequence of these two
residues would adopt a well-defined and novel secondary
structure with a regular, alternating pattern of trans and cis
main-chain amides. A recent X-ray crystallographic report of
an N-aryl/Ns1npe dimer by Kirshenbaum and co-workers
showed that the N-aryl amide bond was trans and that the
Ns1npe amide bond was cis,[10] and served to support our
design hypothesis. The only known a-peptide sequences that
adopt a regular, alternating pattern of trans/cis backbone
Supporting information for this article, including full details of
peptoid syntheses and characterization data (NMR, computations,
X-ray crystallography, CD), is available on the WWW under http://
dx.doi.org/10.1002/anie.201208630.
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amides are alternating l-/d-polyprolines,[11] but no formal
secondary structure designation has been assigned to these
peptides. We reasoned that a systematic study of short,
alternating Nph/Ns1npe peptoid oligomers of increasing size
would allow us to test our design premise.
Ns1npe residues in 2 and 2’ were 8.7 and 10.2, respectively,
while the Kcis/trans values for the Nph residues were 0.1 and
0.05, respectively. We note that the overall amide Kcis/trans for
homo dimers of Ns1npe and Nph in CD3CN were previously
shown to be 10.8[8e] and < 0.1,[9b] respectively, suggesting that
their presence in heterodimers 2 and 2’ did not impact the
rotameric preference of the other residue. The cis or trans
amide preferences of the Ns1npe and Nph residues in 2 and 2’
were also maintained in a variety of solvents (CDCl3
(Table 1); C6D6, CD3OD, [D6]DMSO, and 1:1 CD3CN/D2O;
Supporting Information, Table S1).
We began our investigations by evaluating whether the cis
or trans amide preferences of the Ns1npe and Nph residues in
solution were affected by being adjacent to each other in
a heteropeptoid sequence, or by being placed at either the N-
or C-terminal positions. Two dipeptoids, 2 and 2’ (Table 1),
were synthesized by standard solution-phase methods (see
the Supporting Information for full synthetic details),[9a,12]
purified, and evaluated by NMR spectroscopy. Each peptoid
was capped at the N-terminus with an acetyl group (Ac) and
at the C-terminus as a dimethyl amide (dma) to 1) mimic the
chemical environment of an extended peptoid chain at both
We next designed a trimer through octamer series of
alternating peptoids 3–8 (Table 1) to determine if cis or trans
amide preferences were maintained in longer sequences.
Each was synthesized in solution and purified to homogeneity
by manual column chromatography. As an initial solution-
phase study of these systems, we conducted heteronuclear
single-quantum correlation (HSQC) NMR experiments to
determine the overall Kcis/trans value for the Ns1npe residues of
each heteropeptoid. We anticipated that the N-aryl residues
would adopt nearly exclusively trans-amide conforma-
tions,[9b,d] and thus, the overall Ns1npe-amide Kcis/trans value
would correlate with the degree of overall amide rotamer
homogeneity of a given oligomer. These NMR experiments
were straightforward, as the Ns1npe side-chain a-methine
groups that are oriented in the cis geometry are readily
1
termini; and 2) facilitate conformational analysis by H–1H
nuclear Overhauser effect spectroscopy (NOESY). As
expected, in CD3CN at 248C, the Kcis/trans values for the
Table 1: Sequences, purities, mass spectrometry data, and overall
Ns1npe Kcis/trans values for the peptoids investigated herein.
Peptoid Sequence[a]
Purity Calcd Obs. mass Overall Kcis/
[%][b]
mass (m/z;
trans values for
[M+Na]+)[c] Ns1npe resi-
dues (in
1
distinguishable from those in the trans geometry by H–13C
CDCl3)
HSQC.[8d] In CDCl3 (ca. 5 mm, 248C), we observed an
extremely high preference for cis main-chain Ns1npe
amides in peptoids 3–8 (Kcis/trans = 10–53; Table 1), which, in
general, increased as the peptoid chain length increased. This
trend correlates with that observed for homo-oligomers of
Ns1npe.[8e] Also similar to Ns1npe homo-oligomers, there was
a considerable temperature-dependent decrease in the over-
all Ns1npe amide Kcis/trans values of 8 with increasing temper-
atures, in both CDCl3 and CD3OD, (Supporting Information,
Table S2), suggesting an inherent entropic pressure for multi-
ple rotameric conformers.
Tetramers 4a and 4b (Table 1) were specifically chosen as
models for more detailed conformational analysis by NMR.
Spectra for the tetramers were well-dispersed, with each
residue clearly distinguishable and one conformer predom-
inating. We envisioned that upon adopting a discrete secon-
dary structure with alternating cis and trans amide bonds, the
i and i + 3 side chains of 4a and 4b could be in close proximity,
facilitating the observation of NOEs between substituents on
an N-aryl side chain at the i position and protons of an Ns1npe
side chain at the i + 3 position. Our hypothesis proved correct,
and Figure 2a depicts the NOEs that we detected between the
dimethyl groups on the i (N-aryl) residues and the i + 3
(Ns1npe) methine and methyl protons for 4a and 4b in CDCl3
(10 mm, 248C; see the Supporting Information, Figures S6
and S7 for additional analysis).
2
Ac-Nph-
>98
>98
>98
431.2 454.2
5[d]
Ns1npe-dma
Ac-Ns1npe-
Nph-dma
Ac-Ns1npe-
Nph-Ns1npe-
dma
Ac-N2,6 mph-
Ns1npe-Nph-
Ns1npe-dma
Ac-N3,5 mph-
Ns1npe-Nph-
Ns1npe-dma
Ac-Ns1npe-
(Nph-Ns1npe)2-
dma
2’
3
431.2 454.2
642.3 665.3
5[d]
10[e]
4a
4b
5
>98
>98
>98
803.4 826.4
803.4 826.4
986.5 1009.5
25[e]
19[e]
47[e]
6
Ac-(Nph-
>98
>98
1119.5 1142.5
1195.6 1218.6
30[e]
28[e]
Ns1npe)3-dma
Ac-N3,5 mph-
Ns1npe-
6i
N2,6 mph-ac13C-
Ns1npe-N4fph-
Ns1npe-dma
Ac-Ns1npe-
(Nph-Ns1npe)3-
dma
7
8
>98
>98
1330.6 1353.6
1463.7 1486.7
48[e]
53[e]
Ac-(Nph-
Ns1npe)4-dma
[a] Ac=acetyl, dma=dimethyl amide; see Figure 1a for monomer
abbreviations. [b] Determined by integration of an HPLC trace with UV
detection at 220 nm. [c] Mass spectrometry data were acquired using
high-resolution ESI techniques. [d] Determined by integrating rotamer-
related peaks in 1H NMR spectra (500 MHz, 10 mm). [e] Determined by
integrating rotamer-related peaks of the Ns1npe methine protons in 1H–
13C HSQC spectra (700 MHz, peptoids between 4–10 mm). See the
Supporting Information for full details of methods used.
The NMR data for 4a and 4b inspired our design of
hexamer 6i (Figure 2b), with the goal of determining its
solution-phase structure by NMR. We incorporated the 13C-
labeled main-chain acetyl group into the third residue of 6i
for assignment purposes and the fluoro-substituted N4fph
residue to provide dispersion of resonance peaks. NMR
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repeating turn units that resembled a ribbon-type structure.
Two populations of low energy ribbon conformations were
observed, with variation mainly at the C-terminus. In one
population, the C-termini were extended in a continuation of
the ribbon structure (Figure 2d; ensemble RMSD = 1.0 ꢀ),
while the C-termini of the second group of structures were
curled inwards (ensemble RMSD = 1.1 ꢀ; Supporting Infor-
mation, Figure S10). Not including the C-termini, the mean f
and y values for the Ns1npe residues in 6i were ꢀ558 and
1618, respectively, and the mean f and y values for the Naryl
residues were 648 and ꢀ1628, respectively (see the Supporting
Information, Table S4 for a full listing of ensemble-averaged
main-chain dihedral angles). These values lie closest to the
energetic minima calculated for peptoids in the aD config-
uration (f, y =ꢁ 908, 1808), as defined by Moehle and
Hofmann.[14] We also noted that the structure of 6i clearly
adopts a left-handed helical twist (Figure 2e). This character-
istic has been observed in a-peptide ribbon structures, which
are considered to be subtypes of helical peptide conforma-
tions (b-bend!310-helix).[15] We calculated the helical rota-
tion between two hypothetical planes running through the
two turn units in the hexamer ribbon. These planes were
twisted by 348; therefore, it would take 10.6 turn units (that is,
iꢀi + 3 sets of residues) to complete one full helical turn of the
ribbon. Presumably, the origin of the left-handed spiral in 6i is
the chirality of the s1npe side chains.
Three heteropeptoids were crystalline upon slow evapo-
ration from 1-propanol, and were further analyzed by X-ray
crystallography. Solid-state structures for the synthetic inter-
mediate Br-Nph-Ns1npe-dma (representative of a peptoid
dimer), trimer 3, and tetramer 4a are shown in Figure 3a, and
allow the progressive generation of the ribbon secondary
structure to be viewed as each residue is added. Tetramer 4a is
the shortest peptoid that is capable of adopting one full unit of
the ribbon structure, and the reverse turn of the backbone
approximates the i and i + 2 main-chain methylene carbons of
residues 1 and 3 (C···C distance = 4.97 ꢀ; Figure 3b). Both 3
and 4a adopted nearly identical conformations in the solid
state, despite having different crystal packing interactions
(Supporting Information, Figures S16, S18), which indicates
that an alternating sequence of Ns1npe and N-aryl residues
can adopt a well-defined peptoid ribbon secondary structure
even at short chain lengths. As expected, the amide geo-
metries of all Ns1npe residues were cis and all N-aryl residues
were trans, and the f and y angles of these solid state
structures were in close agreement with the corresponding
values determined for the NMR structure of hexamer 6i (see
the Supporting Information, Table S4 for a full listing of
angles). This agreement is significant, as it constitutes the first
corroboration of a peptoid NMR solution structure with
peptoid solid-state structures containing the same primary
sequences. The similarity is well illustrated by comparison of
the solid state data and NMR data for 4a. The X-ray crystal
structure for 4a shows that the methine proton of the i + 3
Ns1npe side chain is oriented directly towards a methyl group
on the side chain of the N2,6mph (i) residue (C···C distance =
5.05 ꢀ, Figure 3a). Accordingly, an NOE between the
protons of these same groups was observed in our analysis
of 4a in CDCl3 (see above).
Figure 2. a) The structures of peptoid tetramers 4a and 4b; side-
chain–side-chain NOEs are indicated with blue arrows. b) The struc-
ture of hexamer 6i. c) An ensemble of 10 superimposed low-energy
structures of 6i determined by NMR spectroscopy in CDCl3 (hydrogen
atoms are omitted for clarity). d) A length-wise view of the main-chain
atoms of one population of low-energy structures of 6i. e) An axial
view down the N-terminus of 6i showing the left-handed spiral of the
ribbon conformation.
spectra of hexamer 6i were recorded in CDCl3 (10 mm,
158C). One major conformer was observed in all NMR
experiments and the resonances were well-dispersed, ena-
bling the unambiguous assignment of main-chain methylenes,
side-chain s1npe methines, and all methyl groups (Supporting
Information, Figure S9 and Table S3). Rotating frame Over-
hauser effect spectroscopy (ROESY) cross-peaks were
observed between i and i + 3 side chains (residues 1$4 and
2$5), analogous to the NOEs observed in 4a and 4b.
Unfortunately, ROE cross peaks between main-chain meth-
ylenes were not observed. However, the HSQC data
described above provided evidence that each Ns1npe amide
in 6i was primarily in the cis configuration. In total, nine
distance restraints and three main-chain w torsional restraints
from our experimental NMR data were included in simulated
annealing molecular dynamic calculations using AMBER11
software (see the Supporting Information for full details)[13] to
determine an ensemble of calculated solution-phase struc-
tures for hexamer 6i.
The alignment of the 10 lowest energy structures for 6i
(out of 150 calculations) is shown in Figure 2c. These
structures revealed a uniquely folded peptoid backbone of
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Figure 3. a) X-ray crystal structures of Br-Nph-Ns1npe-dma, 3, and 4a. All of the hydrogen atoms except for the s1npe side-chain methines have
been omitted for clarity. The black dashed line indicates a side-chain–side-chain C···C distance. b) The main-chain atoms of the X-ray crystal
structure of 4a illustrate the reverse turn, and the green arrow depicts an n!p*C O interaction. The black dashed line indicates a main chain
=
Ca···Ca distance. c) A displaced aromatic–aromatic stacking interaction was detected between the side chains of residues 1 and 4 in the X-ray
crystal structure of 4a. Only the side-chain and nitrogen atoms are shown. The black dashed line indicates the centroid to centroid distance
between the two aryl rings.
The solution phase and crystal structures were further
analyzed for potential noncovalent interactions that may
stabilize the peptoid ribbon conformation, which lacks
a hydrogen bonding network. Interestingly, in all structures,
every other backbone carbonyl was oriented perpendicular to
one other, but the overall dipole was pointed towards the N-
terminus. At the C-terminus of 4a, we detected an n!p*C
on the ribbon structure. The CD spectra for all of the peptoid
oligomers had nearly identical spectral features in acetoni-
trile, 1:1 acetonitrile/water, and methanol (negative maxima
of ellipticity at 224–226 nm and broad positive peaks of
ellipticity between 197–205 nm; Figure 4), indicating that all
of the peptoids in this series adopted similar secondary
structures in both organic and polar protic solvents (at 30 mm,
208C). Additionally, CD data for peptoid 8 at varying
concentrations (7–80 mm) in acetonitrile suggested that inter-
molecular interactions were not affecting the observed CD
signal (Supporting Information, Figure S19). Interestingly,
the CD spectra of the peptoid ribbon most closely resembled
data reported for poly-ld-Pro sequences,[19] which also adopt
an alternating cis/trans amide backbone pattern. Beyond the
dimer chain length, there were no length-dependent changes
in the CD spectral intensities. Instead, in all solvents, we
observed a difference in signal intensity between even and
odd numbered chain lengths. This effect could potentially be
due to the absorption properties of the s1npe side chains,
which are more solvent-exposed in the ribbon as the C-
terminal residue in odd numbered chain lengths.
=
O
interaction (Figure 3b).[16] Previous X-ray crystallographic
=
studies in our laboratory have revealed that C Oiꢀ1···C’i = O
interactions can exist in peptoid monomers and at the N-
terminus of a peptoid oligomer,[9a,d] yet this is, to our
=
knowledge, the first report of an C Oi + 1···C’i = O interac-
tion in peptoids. We analyzed all sequential backbone
carbonyl groups in the structures of 3, 4a, and 6i for n!
p*C interactions, and although the distances between the
=
O
main-chain carbonyl oxygen atoms and the carbonyl carbon
atoms of the preceding or subsequent residue were under
3.2 ꢀ (as is typical for the n!p* interaction[16]), the angle of
approach was outside of the 109 ꢁ 108 window necessary for
sufficient orbital overlap in all but the C-terminal residue of
4a.[17] We therefore surmise that n!p*C O interactions do not
=
play a major role in enforcing the ribbon conformation in
these peptoids.
The CD spectral shape for peptoids 2–8 varied slightly in
the different solvents. In the acetonitrile/water mixture,
shoulders were apparent in all CD traces from 227–230 nm
(Figure 4b), and the shoulders were more pronounced in
methanol (Figure 4c). These observations could indicate
a slight change in overall conformation that is solvent-
dependent, or a change in the UV adsorption properties or
orientations of the aromatic side chains in these solvents.
Lastly, the thermal stability of 8 (30 mm) was examined in
acetonitrile (15–758C), 1:1 acetonitrile/water (10–758C), and
methanol (10–658C) (Supporting Information, Figures S20–
S22). A linear decrease in signal intensity was observed with
increasing temperatures; however, the spectral shape of 8 in
all solvents was maintained throughout the temperature
ranges investigated. This temperature destabilization can be
attributed, in part, to the increase in amide bond isomer-
We also evaluated the structures of 4a and 6i for
intramolecular aromatic–aromatic interactions between side
chains by measuring the angles between the aryl planes, the
distances between the aryl centroids, and the distances
between nearest interresidue atoms.[18] In the X-ray crystal
structure of 4a, the i and i + 3 side chains are positioned in an
oblique orientation of a displaced stacking interaction (angle
between aromatic planes = 33.58) with a centroid–centroid
ꢀ
distance of 5.4 ꢀ and nearest interresidue C C distance of
4.6 ꢀ (Figure 3c; Supporting Information, Figure S11a). Cor-
respondingly, the ensemble of NMR structures of 6i also
revealed displaced aromatic stacking interactions between
the i and i + 3 residues at the beginning and end of each turn
unit (Supporting Information, Figure S11b–d). Elucidating
the contribution, if any, of these aromatic–aromatic inter-
actions to the thermodynamic stability of the peptoid ribbon
is an important avenue for future study, and is the subject of
ongoing investigations in our laboratory.
1
ization at elevated temperatures that we observed in H–13C
HSQC experiments for 8 (see above).
In summary, we report a new peptoid secondary structure
comprised of a regular alternating sequence of Ns1npe and N-
aryl residues, which we term the “peptoid ribbon”. This
structure is a result of our first rational design of discretely
We next used circular dichroism (CD) spectroscopy to
study the effects of peptoid length, solvent, and temperature
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demands of the bulky, chiral s1npe side chain undoubtedly
play a dominant role in facilitating the ribbon conformation
and enforcing the left-handed spiral of the peptoid ribbon.
Small model systems have proven to be powerful for under-
standing the processes by which foldamers and natural
biopolymers fold.[1a,b,21] The discovery of a peptoid ribbon
secondary structure underscores the value of such model
systems for peptoid design. Looking forward, we note that
several classes of a-peptide ribbons are known to act as
potent and selective antibiotics[22] and cell-membrane-mod-
ifying agents.[23] Helical and cyclic peptoids have previously
been investigated for similar functions,[24] and the peptoid
ribbon could now be considered for such applications, among
others.
Received: October 26, 2012
Revised: January 2, 2013
Published online: April 9, 2013
Keywords: foldamers · peptidomimetics · peptoids · ribbons ·
.
secondary structure
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Angew. Chem. Int. Ed. 2013, 52, 5079 –5084
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