Extraordinarily robust polyproline type i peptoid helices generated via the incorporation of α-chiral aromatic N -1-naphthylethyl side chains

Article abstract of DOI:10.1021/ja204755p

Peptoids, or oligomers of N-substituted glycines, are a class of foldamers that have shown extraordinary functional potential since their inception nearly two decades ago. However, the generation of well-defined peptoid secondary structures remains a diff

Full text of DOI:10.1021/ja204755p

ARTICLE
pubs.acs.org/JACS
Extraordinarily Robust Polyproline Type I Peptoid Helices Generated
via the Incorporation of α-Chiral Aromatic N-1-Naphthylethyl Side
Chains
Joseph R. Stringer, J. Aaron Crapster, Ilia A. Guzei, and Helen E. Blackwell*
Department of Chemistry, University of Wisconsin—Madison, 1101 University Avenue, Madison, Wisconsin 53706-1322, United States
S Supporting Information
b
ABSTRACT:
Peptoids, or oligomers of N-substituted glycines, are a class of foldamers that have shown extraordinary functional potential since
their inception nearly two decades ago. However, the generation of well-defined peptoid secondary structures remains a difficult
task. This challenge is due, in part, to the lack of a thorough understanding of peptoid sequenceꢀstructure relationships and,
consequently, an incomplete understanding of the peptoid folding process. We seek to delineate sequenceꢀstructure relationships
through the systematic study of noncovalent interactions in peptoids and the design of novel amide side chains capable of such
interactions. Herein, we report the synthesis and detailed structural analysis of a series of (S)-N-(1-naphthylethyl)glycine (Ns1npe)
peptoid homo-oligomers by X-ray crystallography, NMR spectroscopy, and circular dichroism (CD) spectroscopy. Four of these
peptoids were found to adopt well-defined structures in the solid state, with dihedral angles similar to those observed in polyproline
type I (PPI) peptide helices and in peptoids with α-chiral side chains. The X-ray crystal structure of a representative Ns1npe tetramer
revealed an all cis-amide helix, with approximately three residues per turn, and a helical pitch of approximately 6.0 Å. 2D-NMR
analysis of the length-dependent Ns1npe series showed that these peptoids have very high overall backbone amide Kcis/trans values in
acetonitrile, indicative of conformationally homogeneous structures in solution. Additionally, CD spectroscopy studies of the
Ns1npe homo-oligomers in acetonitrile and methanol revealed a striking length-dependent increase in ellipticity per amide. These
Ns1npe helices represent the most robust peptoid helices to be reported, and the incorporation of (S)-N-(1-naphthylethyl)glycines
provides a new approach for the generation of stable helical structure in this important class of foldamers.
’ INTRODUCTION
The breadth of peptoid function continues to expand,5 highlighted by
recent investigations that have yielded potent antimicrobial agents,6,7
modulators of proteinꢀprotein interactions,8ꢀ11 RNA-binding
agents,12ꢀ15 lung surfactants,16,17 antibiofouling agents,18 asymmet-
ric catalysts,19 and self-assembled nanostructures.20,21 Peptoids
can be synthesized in a straightforward modular fashion22 and
possess desirable characteristics such as proteolytic stability23
and increased cellular permeability relative to their α-peptide
equivalents.24,25 These attributes make them highly attractive
scaffolds for multivalent ligand display13,26,27 and as potential
therapeutic agents.28 While certain recent studies have revealed
The design and construction of synthetic foldamers capable of
mimicking the structure and function of natural biopolymers is
an active area of research.1ꢀ3 The multitudinous functions
performed by biological polymers such as proteins and RNA
are made possible by the precise, three-dimensional arrange-
ments of chemical functionality arising from distinct folds within
their polymeric backbones. Thus, the ability to accurately and
predictably assemble well-folded, higher order structures is
paramount to understanding structureꢀfunction relationships
within a particular foldameric class.
Peptoids, or N-substituted glycines, are a class of foldamers
that have shown extraordinary promise since their inception
nearly two decades ago as part of drug discovery efforts (Figure 1A).4
Received: May 24, 2011
Published: August 24, 2011
r
2011 American Chemical Society
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Figure 1. (A) Comparison of the primary structures of an α-peptoid to an α-peptide. (B) N-Substituted glycine side chains discussed in this study.
that a well-defined structure is not an absolute requirement for
biological activity in peptoids,11 there remains a persistent
need to delineate peptoid sequenceꢀstructure relationships
in order to create well-folded, three-dimensional biomimetic
structures.
residues were needed to promote a stable and predictable
secondary structure. Additional structural diversity could then
be installed onto the peptoid backbone relative to Nspe helices,
and this enhancement in structural tunability would be a
significant advance for the design of biologically active peptoid
helices.
Initial research on peptoid structure focused primarily on pep-
toids containing sterically bulky α-chiral side chains (Figure 1B).
In these early studies, molecular modeling experiments predicted
that peptoid oligomers composed of α-chiral aromatic (S)-N-
(1-phenylethyl)glycine (Nspe) residues would adopt an all cis-
amide helix with approximately three residues per turn and
dihedral angles similar to that of a polyproline type I (PPI)
peptide helix.29 The peptoid helical sense was hypothesized to
be dictated by the stereochemistry of the α-chiral side chain,
with R or S stereocenters producing a left- or right-handed helix,
respectively. These modeling studies were later supported by
2D-NMR and X-ray crystallographic experiments. In a 2D-
NMR study in methanol, the major conformer of a peptoid
pentamer containing five para-substituted Nspe residues was
found to adopt an all cis-amide, right-handed helix with three
residues per turn and a pitch of ∼6 Å, analogous to a PPI helix.30
Interestingly, the CD spectrum of this peptoid pentamer in
methanol resembled that of an α-helix, as opposed to a PPI
helix, with minima near 220 nm and near 205 nm. It is important
to note, however, that the major NMR conformer of the Nspe
pentamer only represented 50ꢀ60% of the total molecules in
solution due to cis/trans isomerization of the backbone tertiary
amides, and the Nspe pentamer CD spectrum was weak relative
to longer Nspe peptoids.30 This suggested that while the Nspe
pentamer can sample a helical conformation, it was conforma-
tionally heterogeneous.
In addition to Nspe-type oligomers, peptoids with α-chiral
aliphatic side chains can also adopt helical structures. X-ray
crystallographic analysis of an (R)-N-(1-cyclohexylethyl)glycine
(Nrch, Figure 1B) pentamer revealed an all cis-amide left-handed
helix with approximately three residues per turn and a helical
pitch of approximately 6.7 Å.33 The dihedral angles observed in
the solid-state structure of this peptoid pentamer were similar to
those observed in PPI peptide helices, and were comparable to
those observed for the solution structure of the para-substituted
Nspe pentamer introduced above. The CD spectra of the Nrch
pentamer in acetonitrile, in contrast to the Nspe pentamer,
displayed a similar shape to that seen for a PPI peptide helix,
with a maximum near 210 nm and two shallow minima near
200 nm and near 225 nm.33 Although the X-ray crystallographic
data demonstrated that the Nrch pentamer had a helical
structure in the solid state, this peptoid, similar to the Nspe
pentamer above, was conformationally heterogeneous in solu-
tion, as exhibited by its low overall amide Kcis/trans value (∼1) in
acetonitrile (determined by 2D-NMR spectroscopy) and weak
CD spectrum.
The previous studies outlined above indicate that the gen-
eration of robust, conformationally homogeneous peptoid he-
lices remains a challenge. A more complete understanding of
sequenceꢀstructure relationships in peptoids would undoubt-
edly improve the design of well-defined peptoid helices a priori,
and further expand their utility as biomimetic agents capable of
performing specific functions. As highlighted above, cis/trans
isomerization of the tertiary backbone amides is a major con-
tributor to the conformational heterogeneity observed in many
peptoid oligomers.30 Strategies aimed at enforcing either cis- or
trans-amides within peptoid backbones have continued to evolve
as a more thorough understanding of the noncovalent forces
affecting peptoid tertiary amide cis/trans equilibria have emerged.34,35
Until recently, the highest reported overall backbone amide Kcis/trans
Subsequent investigations of Nrpe (the R variant of Nspe)
peptoid oligomers by Barron and co-workers detailed specific
sequence requirements for peptoid helix formation. In these
studies, peptoid helicity was promoted in sequences that con-
tained at least 50% α-chiral residues, or α-chiral aromatic side
chains every third residue (i, i + 3 positions), and an α-chiral
residue at the C-terminus.31 Additionally, increasing the oligo-
mer length resulted in peptoids with greater helical character.32 It
would be desirable, however, if fewer than 50% helix-inducing
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value for a peptoid was only approximately 3.2, and this was
adopted by (Nspe)15.32 We have since discovered that the α-
chiral aromatic side chain 1npe (Figure 1B) is highly effective for
exclusively promoting the cis-amide rotamer within a peptoid
backbone, primarily due to steric interactions.35 NMR analysis of
a model peptoid monomer containing this side chain revealed
that stereoelectronic effects, namely an nfπ*aromatic interaction
between the backbone carbonyl oxygen and side chain naphtha-
lene group, could also play a role in stabilizing the cis-amide
rotamer. 2D-NMR analysis of an Ns1npe trimer, Ac-(s1npe)3-
CONH2, revealed that this oligomer had an overall backbone
amide Kcis/trans of 9.7 in acetonitrile, which represented the
highest reported value for any peptoid oligomer to date.35 The
CD spectrum of Ac-(s1npe)3-CONH2 showed an intense max-
imum near 205 nm and an intense minimum near 220 nm, with a
magnitude nearly twice that of Nspe5. These exciting initial
results suggested that peptoid oligomers composed of the 1npe
side chain could potentially form well-folded PPI peptoid helices
with unprecedented conformational homogeneity in solution.
Herein, we report our efforts toward the generation of well-
defined peptoid helices through the systematic design, synthesis,
and structural characterization of a series of peptoid homo-
oligomers (1ꢀ13-mers) containing the s1npe side chain. X-ray
crystallographic analysis revealed that an Ns1npe monomer,
dimer, trimer, and tetramer each adopt solid-state structures
with dihedral angles similar to a PPI peptide helix. 2D-NMR
spectroscopic studies showed that these Ns1npe peptoids ex-
hibited very high overall amide Kcis/trans values, and CD studies
revealed intense and thermally stable CD spectra, both indicative
of exceptionally well-folded structures. The Ns1npe helix repre-
sents, to our knowledge, the most conformationally homoge-
neous peptoid helix reported to date. We anticipate that the
ability to generate such well-folded peptoid helices will provide
exciting opportunities for the exploration of structureꢀfunction
relationships in polypeptoids in the future.
Table 1. Peptoid N-Terminal Group, Side Chains, and
C-Terminal Groups in This Study
SCL-10Avp controller, an LC-10AT pump, an FCV-10ALvp solvent
mixer, and an SPD-10MAvp UVꢀvis diode array detector. All analytical
RP-HPLC work was performed using either a Restek Premier C18
column (5 μm, 4.6 mm ꢁ 250 mm) or a Restek C4 column (5 μm,
4.6 mm ꢁ 250 mm). Standard RP-HPLC conditions were as follows:
flow rate =1 mL/min; mobile phase A = 0.1% trifluoroacetic acid (TFA)
in water; mobile phase B = 0.1% TFA in acetonitrile. Purities were
determined by integration of peaks with UV detection at 220 nm.
LC-MS data were obtained using a Shimadzu LCMS-2010 system
equipped with two LC-10ADvp pumps, SCL-10Avp controller, SIL-
10ADvp autoinjector, SPD-M10Avp UVꢀvis diode array detector, and
single quadrupole analyzer (by ESI). A Supelco C18 wide-pore column
(15 cm ꢁ 2.1 mm) was used for all LC-MS work. Standard RP-HPLC
conditions for LC-MS were as follows: flow rate =200 μL/min; mobile
phase A = 0.1% formic acid in water; mobile phase B = 0.1% formic acid
in acetonitrile.
Attenuated total reflectance (ATR)-IR spectra were recorded with a
Bruker Tensor 27 spectrometer, outfitted with a single reflection
MIRacle horizontal ATR and a Ge crystal with a spectral range 5500ꢀ
700 cmꢀ1. Circular dichroism (CD) spectra were obtained on an Aviv
62A DS spectropolarimeter with Aviv CDS software.
Synthesis and Characterization of Peptoid Oligomers.
Peptoid 10 was synthesized using microwave-assisted solid-phase
methods on Rink-amide-derivatized polystyrene resin as previously
reported.38,39 The crude peptoid was purified by preparative RP-HPLC
and characterized by MS to confirm identity (see Table 2). Peptoids
1ꢀ13 were synthesized in solution using modified versions of previously
reported procedures.40 The crude peptoids were purified by flash silica
gel column chromatography and characterized by MS to confirm their
identities (see Table 2). See Supporting Information for detailed
synthetic procedures and complete compound characterization, includ-
ing HPLC and MS data, for peptoids 1ꢀ13 and their respective
synthetic intermediates.
’ EXPERIMENTAL SECTION
General. All reagents were purchased from commercial sources
(Aldrich and Acros) and used without further purification. Solvents were
purchased from commercial sources (Aldrich and Fisher) and used as is,
with the exception of dichloromethane (CH2Cl2) and n-hexane, which
were distilled immediately prior to use. Thin layer chromatography
(TLC) was performed on silica gel 60 F254 plates (E-5715ꢀ7, EMD).
SiliaFlash P60 silica gel (40ꢀ63 μm, Silicycle) was used for manual flash
column chromatography.36
1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were
recorded on either a Bruker AC-300 or a Varian MercuryPlus 300
spectrometer in deuterated solvents. Chemical shifts are reported in
parts per million (ppm, δ) using tetramethylsilane (TMS) as a reference
(0.0 ppm), unless otherwise noted. Couplings are reported in hertz.
Gradient correlation spectroscopy (gCOSY) and heteronuclear single
quantum coherence adiabatic (HSQCAD) NMR experiments were
performed on either a Varian Inova 500 or 600 MHz spectrometer
and were referenced to solvent. The data were processed using the
Varian VNMR software package (v. 6.1C) and visualized using SPARKY
software.37
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-
TOF) mass spectra were obtained on a Bruker RELEX II spectrometer
equipped with a 337 nm laser and a reflectron. In positive ion mode, the
acceleration voltage was 25 kV.
Reversed-phase high performance liquid chromatography (RP-
HPLC) was performed using a Shimadzu system equipped with an
NMR Analysis for Peptoid 1 and 10. Peptoids 1 and 10 were
dissolved in CD3CN to give a 10 mM solution. 1H NMR spectra were
acquired using 16 scans and a relaxation delay of 15 s. 1D-NOESY
spectra were acquired to verify previously reported guidelines for
distinguishing cis- and trans-amide rotamers.41 The Kcis/trans ratio was
calculated by averaging the integration ratios for three sets of rotamer-
related peaks.
NMR Analyses for Peptoid Oligomers 2ꢀ13. Peptoids 2ꢀ13
were dissolved in CD3CN to give 10 mM solutions and analyzed by
gCOSY and HSQCAD NMR experiments as detailed below. The
experiments were performed on either a Varian Inova 500 or 600 MHz
spectrometer using a 5 mm hcn probe. The gCOSY NMR spectra were
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Table 2. Structures, Purities, and Mass Spectral Data for
Table 3. Observed Torsion Angles of Peptoids 10 and 2ꢀ4 As
Peptoids 1ꢀ13, 10, and 50
Determined by X-ray Crystallography
monomer sequence
peptoid
residue
ω
j
ψ
χ1
(amino to carboxy
terminus)
%
puritya
calculated
mass
10
2
1
1
2
1
2
3
1
2
3
4
ꢀ9.4
ꢀ17.1
2.2
ꢀ79.0
ꢀ68.7
82.7
ꢀ149.7
ꢀ167.3
166.0
ꢀ125.7
ꢀ126.7
ꢀ135.7
ꢀ136.4
ꢀ132.4
ꢀ124.6
ꢀ83.6
ꢀ128.4
ꢀ120.3
ꢀ149.4
peptoid
observed massb
1
10
Ac-s1npe-COOtBu
99
99
99
99
98
98
99
98
96
97
95
98
95
96
95
327.2
270.1
538.4
749.4
960.5
328.2 [M + H]+
293.2 [M + Na]+
561.2 [M + Na]+
772.7 [M + Na]+
984.0 [M + Na]+
Ac-s1npe-CONH2
3
4
ꢀ13.9
ꢀ16.4
8.0
ꢀ71.7
ꢀ67.8
82.8
ꢀ160.6
ꢀ163.5
162.5
2
Ac-(s1npe)2-COOtBu
Ac-(s1npe)3-COOtBu
Ac-(s1npe)4-COOtBu
Ac-(s1npe)5-COOtBu
Ac-(spe)5-COOtBu
3
4
3.1
ꢀ88.8
ꢀ65.3
ꢀ66.8
75.7
ꢀ173.7
ꢀ178.8
177.2
5
50
1171.7 1195.6 [M + Na]+
921.5
944.6 [M + Na]+
ꢀ19.9
ꢀ11.7
ꢀ1.5
6
Ac-(s1npe)6-COOtBu
Ac-(s1npe)7-COOtBu
Ac-(s1npe)8-COOtBu
Ac-(s1npe)9-COOtBu
Ac-(s1npe)10-COOtBu
Ac-(s1npe)11-COOtBu
Ac-(s1npe)12-COOtBu
Ac-(s1npe)13-COOtBu
1382.7 1406.6 [M + Na]+
1594.8 1617.6 [M + Na]+
1805.9 1828.9 [M + Na]+
2017.0 2039.8 [M + Na]+
2228.1 2251.2 [M + Na]+
2439.2 2462.3 [M + Na]+
2650.3 2673.3 [M + Na]+
2862.5 2885.8 [M + Na]+
178.3
7
8
Table 4. Peptoid Structures 1ꢀ13, 10, and 50 and Their
Respective Overall Kcis/trans Values As Determined by
9
10
11
12
13
1H-COSY and 1Hꢀ C HSQCAD NMR in CD3CN at 24 °C
13
monomer sequence
a
peptoid oligomer
(amino to carboxy terminus)
overall Kcis/trans
a Determined by integration of the HPLC trace with UV detection at
220 nm. b Mass spectrometry data were acquired using either ESI or
MALDI-TOF techniques.
1
10
Ac-s1npe-COOtBu
4.9b
3.8b
10.8
16.0
>19
>19
3.3
Ac-s1npe-CONH2
2
Ac-(s1npe)2-COOtBu
Ac-(s1npe)3-COOtBu
Ac-(s1npe)4-COOtBu
Ac-(s1npe)5-COOtBu
Ac-(spe)5-COOtBu
3
used for assignment purposes, and the HSQCAD NMR spectra were
subsequently integrated to give the Kcis/trans values in Table 4. Assign-
ments of methine cis and trans peaks were based on previously observed
trends in chemical shifts.41
4
5
50
6
Ac-(s1npe)6-COOtBu
Ac-(s1npe)7-COOtBu
Ac-(s1npe)8-COOtBu
Ac-(s1npe)9-COOtBu
Ac-(s1npe)10-COOtBu
Ac-(s1npe)11-COOtBu
Ac-(s1npe)12-COOtBu
Ac-(s1npe)13-COOtBu
>19
>19
>19
>19
>19
>19
>19
>19
gCOSY NMR experiments were performed for peptoids 2ꢀ13 at
24 °C using the following parameter values: Spectral widths were 5252,
5571, 4667, 4783, 5561, 5804, 5533, 5504, 5513, 5697, 5388, 5320, 3938,
and 4139 Hz, respectively. The number of transients (nt) was 8. The
relaxation delays (d1) were 4.2, 4.2, 4.2, 4.2, 4.2, 5.9, 4.2, 3.4, 4.2, 4.2, 4.2,
4.2, 4.2, and 2.4, respectively. The numbers of increments (ni) were 350,
450, 400, 400, 450, 450, 500, 450, 500, 475, 450, 475, 400, and 350,
respectively. The number of points was 2048. The spectra were zero-
filled to generate matrices of 4096 ꢁ 4096 points.
7
8
9
10
11
12
13
a Weighted average of Kcis/trans for all residues. b Determined by integra-
HSQCAD NMR experiments42 were performed for peptoids 2ꢀ13
at 24 °C using the following parameter values: Spectral widths were
5252, 5571, 4667, 4783, 5561, 5804, 5533, 5504, 5513, 5697, 5388, 5320,
3938, and 4139 Hz, respectively, in the 1H dimension. Spectral widths
were 32428, 28582, 23509, 23795, 27972, 26464, 22844, 22792, 22008,
18286, and 19048 Hz, respectively, in the 13C dimension. The relaxation
delays (d1) were 2.8, 3, 2.8, 3, 2.8, 3.9, 2.8, 2.8, 3, 2.8, 2.8, 3, 2.8, and 2,
respectively. The numbers of transients (nt) were 6, 8, 6, 6, 6, 8, 8, 6, 8, 8,
8, 12, 8, and 6, respectively. The numbers of increments (ni) were 260,
256, 300, 300, 250, 250, 250, 250, 256, 250, 256, 256, 250, and 250,
respectively. The number of points was 2048. The spectra were zero-
filled to generate f1 ꢁ f2 matrices of 4096 ꢁ 4096 points.
tion of 1D-1H NMR spectrum.
X-ray Crystal Structure Data for 10 and 2ꢀ4. A ∼10 mg
sample was dissolved in HPLC-grade MeOH (10 and 2), HPLC-grade
acetone (3), or n-hexane in a minimal amount of CH2Cl2 (4). Slow
evaporation at room temperature afforded crystals suitable for X-ray
analysis after approximately one week. The X-ray crystal data for 10 and
2ꢀ4 have been deposited at the Cambridge Crystallographic Data
Centre with deposition numbers 787850ꢀ797853. See Supporting
Information for complete crystallographic details.
CD Analyses for Peptoids 1ꢀ13. Peptoid stock solutions were
prepared by dissolving at least 2 mg of each peptoid in spectroscopic
grade acetonitrile or methanol. The stock solutions then were diluted
with spectroscopic grade solvent to the desired concentration (∼60 μM)
by mass using a high-precision balance (Mettler-Toledo XS105). CD
spectra were obtained in a square quartz cell (path length 0.1 cm) at
24 °C using a scan rate of 100 nm/min. The spectrum of a solvent blank
was subtracted from the raw CD data, and the resulting data were
expressed in terms of per-amide molar ellipticity (deg cm2/dmol), as
calculated per mole of amide groups present and normalized by the
molar concentration of peptoid.
’ RESULTS AND DISCUSSION
Design of Peptoid Oligomers. We sought to investigate the
helix-forming propensity of Ns1npe peptoids relative to previous
studies of helical peptoids. As outlined above, these past inves-
tigations have used CD and NMR spectroscopy as primary
methods to determine overall peptoid helical character. In one
prominent study, Barron and co-workers synthesized a series of
Nrpe homo-oligomers with lengths varying from 1ꢀ20 residues
and utilized CD spectroscopy to probe the effects of chain length
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Figure 2. X-ray crystal structures of peptoids 10 and 2ꢀ4. (A) View perpendicular to the helical axis. (B) View parallel to the helical axis. Atom
designations: red = oxygen; blue = nitrogen; gray = carbon. Hydrogen atoms in parts A and B and the t-butyl methyl groups in part B have been removed
for clarity.
on peptoid secondary structure.32 They found that these α-chiral
aromatic side-chain-containing peptoids adopted largely helical
structures in acetonitrile (with few exceptions), and that the
degree of helicity became length-independent after 12 residues.
In order to compare to this past work, we chose to synthesize a
series of peptoid Ns1npe homo-oligomers with lengths varying
from 1 to 13 residues (Table 1). We hypothesized that these
peptoids would also adopt helical conformations in acetonitrile,
and that their CD signatures would display a similar length-
dependent increase. Moreover, we reasoned that the Ns1npe
peptoids would exhibit significantly higher overall amide Kcis/trans
values relative to Nrpe systems, based on our previously reported
influence peptoid secondary structure. Hydrogen-bonding inter-
actions between these two termini and multiple carbonyl oxygens
within the peptoid backbone play a significant role in stabilizing a
competing threaded loop structure at an oligomer length of nine
residues, and thereby disfavoring PPI helix formation.38,41 Ad-
ditionally, hydrogen-bonding interactions from carbonyl oxy-
gens within the peptoid backbone to the C-terminal amide
hydrogens are believed to contribute to conformational hetero-
geneity in peptoid helices by favoring the presence of trans-amide
bonds.30 These problems were avoided by using our solution-
phase synthetic approach. While solution-phase synthesis is
comparatively more labor-intensive than a solid-phase approach,
we were able to reproducibly obtain 50ꢀ100 mg of each peptoid
oligomer, which proved especially useful for performing a variety
of structural experiments on each compound.
We synthesized a series of peptoid Ns1npe homo-oligomers
with lengths varying from 1 to 13 residues (1ꢀ13, Table 1) with
t-butyl esters at the C-termini and acetyl groups at the N-termini
to mitigate hydrogen-bonding interactions. We found that the
presence of the t-butyl ester moiety was adventitious, as it greatly
facilitated the crystallization of Ns1npe peptoid dimer, trimer,
and tetramer (2ꢀ4) relative to their respective C-terminal
primary amide equivalents. Peptoid monomer 1 did not crystal-
lize despite repeated attempts, and we therefore include 10 for
crystallographic analysis purposes. The Nspe pentamer 50, which
contains an acetylated N-terminus and a t-butyl ester at the
C-terminus, was also synthesized as a key control to allow for
spectroscopic comparison to previously reported Nspe peptoids.
X-ray Crystallographic Study of N1npe Oligomers. Pep-
toids 10 and 2ꢀ4 readily crystallized, and we analyzed their
solid-state structures by X-ray crystallography (Figure 2). The back-
bone amide bonds in each peptoid all adopted cis-amide con-
formations. Notably, the X-ray crystal structure of tetramer 4
K
cis/trans values for an Ns1npe monomer (6.3), homodimer (7.0),
and homotrimer (9.7).35
Synthesis of Peptoid Oligomers. Peptoid oligomers are
typically synthesized on solid-phase resins using the highly
efficient submonomer approach.22 Using this method for the
synthesis of Ns1npe peptoids, however, resulted in low yields and
purities of the desired peptoid oligomers after the trimer stage,
presumably due to the increased steric congestion of the s1npe
side chain. We therefore utilized solution-phase synthetic tech-
niques to generate the peptoid oligomers, and used methods that
were similar to previously reported peptoid syntheses (see
Supporting Information for details).34,40 Because such synthetic
routes afford the opportunity to install unique functionalities at
the C- and N-termini, we selected C- and N-terminal moieties
that would be compatible with our investigations into peptoid
helix formation and obviate problems of residual functionality
associated with solid-phase synthesis. Most peptoid oligomers
synthesized on solid-phase resins are cleaved from Rink-amide
linkers under TFA-mediated conditions, affording a peptoid with
a primary amide at the C-terminus and an ammonium group at
the N-terminus. Notably, these functional groups can strongly
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revealed that this oligomer adopted a right-handed nascent helix
with approximately three residues per turn and a helical pitch of
approximately 6.0 Å. This structure is significant, as it represents
the first reported X-ray crystal structure of a peptoid with α-chiral
aromatic side chains. Analysis of the structures of 10, 2, and 3
shows the gradual progression of helix formation seen in tetramer
4 (Figure 2). The pitch observed in the solid-state structure of 4
is in agreement with the pitch of ∼6 Å previously reported for a
para-substituted Nspe pentamer, as experimentally determined
by 2D-NMR spectroscopy in methanol (see above).30 These
pitch distances deviate slightly from the pitch of ∼6.7 Å observed
in the solid-state structure of the related, aliphatic Nrch
pentamer.33 A possible reason for this difference could be the
smaller steric size of the Ns1npe planar aromatic rings relative to
the Nrch cyclohexyl ring, which may allow the helix to compress
into a more compact conformation.
The j and ψ backbone dihedral angles of peptoid 10 and 2ꢀ4
are listed in Table 3. Interestingly, at least one ω (amide)
dihedral angle in each peptoid deviates significantly from planar-
ity, ranging in value from ꢀ20° to 8° (equivalent to 340° to 8°).
Nonplanar amides have previously been observed in the X-ray
crystal structures of certain macrocyclic peptoids.43 In these
compounds, steric interactions upon macrocycle formation have
been hypothesized to lower the energetic barrier to amide bond
rotation and thus contribute to the amide bond distortion. Steric
interactions between amide side chains also could be a cause for
the observed amide distortions in 10 and 2ꢀ4.
Figure 3. CD spectra of peptoids 1ꢀ4 at ∼60 μM in acetonitrile.
Spectra were collected at 24 °C.
We also note that none of the naphthalene groups in these four
solid-state structures are oriented in such a way as to facilitate an
nfπ*aromatic backbone to side chain interaction. This is in
contrast to our understanding of the noncovalent interactions
involved in stabilizing cis-amides in Ns1npe systems in solution,
ascertained from our earlier NMR studies of a model Ns1npe-
type monomer (see above).35 However, crystal lattice interac-
tions within these peptoids may provide an explanation. Views of
the solid-state packing of peptoids 10 and 2ꢀ4 are provided in the
Supporting Information (Figures S-7ꢀS-11). These structures
show that the side chain naphthalene groups of adjacent peptoids
are in close contact with one another, and are aligned in orienta-
tions that allow for tight crystal packing. These packing interac-
tions may be preventing the peptoids from crystallizing with the
naphthalene groups involved in an intraresidue nfπ*aromatic
interaction. Importantly, we did not observe any specific, non-
covalent intermolecular interactions in the solid state that im-
pacted the backbone dihedral angles for 10 and 2ꢀ4, and therefore
the progressive formationof PPI peptoidstructure ismaintained in
all four solid-state structures.
2D-NMR Studies. We sought to examine the solution-phase
1
conformations of peptoids 1ꢀ13, and utilized H gCOSY and
13
1Hꢀ C HSQCAD NMR experiments to determine overall
Figure 4. CD spectra of peptoids 4ꢀ8 and 50 at ∼60 μM in acetonitrile.
Spectra were collected at 24 °C.
backbone amide Kcis/trans values in acetonitrile. These 2D-NMR
experiments are typically used to determine the presence of
different conformational isomers of peptoids in solution.31,33,38
In the case of PPI peptoid helices, a peptoid oligomer that exhibits
a high overall backbone amide Kcis/trans value is more confor-
mationally homogeneous relative to a peptoid oligomer with a
low Kcis/trans value. We were pleased to observe high Kcis/trans
values (4.9 to >19) for all of the Ns1npe peptoids (1ꢀ13;
Table 4). These values increase in a length-dependent fashion,
with trans-amide conformations becoming virtually undetect-
able at the tetramer (4) stage and beyond. To our knowledge,
the overall backbone amide Kcis/trans value of 19 for Ns1npe
peptoids 4ꢀ13 is the highest reported for any peptoid oligomer
to date.
To compare our Ns1npe systems to previously reported
Nspe peptoids, we examined the Kcis/trans values of the Ns1npe
pentamer 5 and the Nspe pentamer 50. These values were >19
and 3.3, respectively. A direct comparison to past reports is not
possible, as these Nspe-type peptoids had different functionality
at the N- and C-termini. However, the striking difference in
Kcis/trans between these two pentamers suggests that Ns1npe 5 is
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Figure 5. CD spectra of peptoids 8ꢀ13 at ∼60 μM in acetonitrile.
Spectra were collected at 24 °C.
Figure 6. CD spectra of peptoid 13 at ∼60 μM in acetonitrile. Data
were collected between 5ꢀ75 °C in 10° increments.
5ꢀ8 each have a spectral shape similar to tetramer 4 with intense
minima near 230 nm, shallow minima at 220 nm that decrease in
intensity with increasing chain length, and broad maxima near
205 nm. The intensities of the minima near 230 nm increase with
oligomer length, with peptoids 7 and 8 displaying nearly identical
intensities. Overall, the length-dependent increase in ellipticity
per amide in the CD spectra of peptoids 1ꢀ8 suggests that helix
folding could be a cooperative process,32,44 despite the lack of any
apparent long-range noncovalent interactions. It is reasonable to
suggest that the formation of N1npe secondary structure could
arise, in part, from the ability of the bulky N1npe residues to
sterically exclude space for adjacent residues to sample. However,
longer oligomers have a greater internal- to end-residue ratio
compared to shorter oligomers, and we cannot discount that this
ratio also contributes to the observed length dependent intensity
of the CD data for peptoids 1ꢀ8. Further experiments, including
thermal denaturation studies, are necessary to explore these
phenomena, and are ongoing.
As mentioned above, the CD spectrum of an Nspe pentamer in
methanol resembles that of an α-helix,30 while polypeptoids
containing Nrch (and related) residues display a distinct max-
imum at 210 nm and two shallow minima at 195 and 225 nm in
their CD spectra,33 which is characteristic of a PPI helix. We
observed that the CD spectrum for Nspe pentamer 50 in
acetonitrile (Figure 4) showed a weak α-helical signature with
two minima at 202 and 220 nm, which matches previously
reported CD spectra of peptoids containing this same α-chiral
and aromatic side chain, but was completely different from the
spectrum of Ns1npe pentamer 5. Indeed, the unique CD spectra
observed for Ns1npe peptoids in this study provide further
supporting evidence that the CD spectral shape of similar
peptoid structures can vary greatly depending on the identity
of the side chains within the peptoid oligomer. We note, however,
that Ns1npe, Nspe, and Nrch peptoids also have varying degrees
of conformational homogeneity, and this could alter their CD
spectra as well.
considerably more conformationally homogeneous than Nspe 50
in acetonitrile. We also observed that the distribution of cis-amide
methine peaks in the HSQC spectra of Ns1npe peptoids gradually
narrowed as oligomer length increased from 1 to 13 residues (see
Supporting Information). This reduction in the number of peaks in
the HSQC NMR spectra and a corresponding increase in CD signal
intensity (see below) is indicative of an increasingly ordered
structure.33 The reasons for this enhanced helix stability relative to
Nspe systems could be multifold, but we believe that steric interac-
tions between the bulky naphthalene-containing side chains play an
essential role. Additional spectroscopic and computational experi-
ments are necessary to further understand such interactions and
other related phenomena, and are ongoing.
CD Analysis of Peptoid Oligomers. We utilized CD spec-
troscopy to further analyze the conformations of peptoids 1ꢀ13
in solution. The CD spectra of peptoids 1ꢀ13 and 50 in acetonitrile
at room temperature are shown in Figures 3ꢀ5. Peptoids 1ꢀ3 had
similar CD spectra, with intense minima near 220 nm and intense
maxima near 205 nm (Figure 3). These CD spectra were
analogous in shape and intensity to the CD spectra that we
had previously reported for their primary amide analogues Ac-
(s1npe)1-CONH2 (10), Ac-(s1npe)2-CONH2, and Ac-(s1npe)3-
CONH2, respectively.35 Intriguingly, the CD spectrum of tetra-
mer 4 had a different spectral shape than peptoids 1ꢀ3. This
spectrum had a reduced minimum near 220 nm, and an increased
maximum near 205 nm, along with the concomitant appearance
of a new and intense minimum near 230 nm. We note that
tetramer 4 represents the first possible oligomer in the series in
which s1npe side chains could spatially overlap in one turn of a
helix, as indicated in the X-ray crystal structure of tetramer 4
(Figure 2). This i f i ( 3 overlap of the napthyl side chains could
contribute to the different CD spectral shapes observed for
tetramer 4 relative to the shorter peptoids 1ꢀ3.
The CD spectrum of tetramer 4 was then compared to the
spectra obtained for peptoids 5ꢀ8 and 50 (Figure 4). Peptoids
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polypeptoids of varying lengths from our series (3, 6, 9, and 12)
by CD in methanol at 24 °C. The CD spectra are shown in
Figure 7 and are nearly identical in intensity and shape as to those
observed in acetonitrile for these four peptoids (Figures 3ꢀ5).
These CD data suggest that Ns1npe polypeptoids can also adopt
PPI helices in polar protic solvents. Furthermore, this apparent
insensitivity to solvent type supports our hypothesis that steric
interactions are a primary cause for conformational restriction in
these systems. Maintenance of the Ns1npe helix in methanol is
particularly significant, as it bodes well for the future develop-
ment of Ns1npe helices for applications that demand aqueous
solubility.
’ SUMMARY AND OUTLOOK
We have discovered that Ns1npe peptoids can form extra-
ordinarily well-folded PPI helices. X-ray crystallographic studies
indicate that an Ns1npe peptoid monomer, dimer, trimer, and
tetramer each adopt structures with all cis-amide bonds and
dihedral angles similar to those seen in PPI peptide and peptoid
helices. The Ns1npe tetramer (4) adopts a right-handed helix in
the solid state, and the length-dependent set of Ns1npe peptoids
10 and 2ꢀ4 yielded the first X-ray crystal structures for any
peptoid with α-chiral aromatic side chains. 2D-NMR spectro-
scopic studies revealed that Ns1npe peptoids 1ꢀ13 showed very
high overall backbone amide Kcis/trans values in acetonitrile,
consistent with an all cis-amide, conformationally homogeneous
structure. Additionally, peptoids 1ꢀ13 exhibited intense, length-
dependent CD signatures in both acetonitrile and methanol. The
2D-NMR and CD spectroscopic data support the observed solid-
state structures of the Ns1npe monomer, dimer, trimer, and
tetramer, and indicate that Ns1npe peptoids adopt a PPI helix,
both in solution and in the solid state. We anticipate that the
ability to generate such well-folded peptoid helices via the
incorporation of Ns1npe residues will provide opportunities for
the exploration and delineation of new structureꢀfunction
relationships in peptoids. Most notably, these new structure-
promoting elements should prove especially useful for construct-
ing helical peptoid scaffolds with an expanded number of side
chain sites available for rational or combinatorial diversification
relative to Nspe heteropeptoids.31,32 Reducing the number of
required hydrophobic peptoid helix-inducing residues from 50%
should also promote enhanced water solubilities at longer residue
numbers.
In view of the results described here, an exciting prospect for
future peptoid design would involve the incorporation of the cis-
amide-promoting 1npe side chain in conjunction with the trans-
amide-promoting N-aryl side chain (that we and others have
reported)46,47 into peptoid oligomers. The resulting cis- and
trans-amide residues could be introduced in a variety of different
sequence patterns, alternating and otherwise, which could po-
tentially result in the formation of entirely unique secondary
structures, and thus expand the known conformational space
adopted by peptoids. Ongoing work in our laboratory is directed
at utilizing these and other evolving peptoid sequenceꢀstructure
relationships to design and generate novel polypeptoid
architectures.
Figure 7. CD spectra of peptoids 3, 6, 9, and 12 at ∼60 μM in
methanol. Spectra were collected at 24 °C.
Continuing in the length-dependent series, peptoids 9ꢀ13
exhibited CD spectra (Figure 5) that were slightly different from
the CD spectra of 4ꢀ8 (Figure 4). The maxima in 9ꢀ13
increased and were red-shifted from near 205 nm to near
210 nm, and the minima showed a slight red-shifting from near
229 nm to near 231 nm. Intriguingly, the minima near 231 nm in
9ꢀ13 decreased in a length-dependent manner. These results are
in contrast to the CD data presented above for 1ꢀ8 and to
previously reported length-dependent CD studies of Nspe
peptoids in which no decrease in CD signal intensity was observed
at increasing peptoid oligomer lengths up to 20 residues.32 An
interesting possibility for the observed trend in peptoids 9ꢀ13
could be the formation of self-associated multimeric structures,
which may be affecting the CD signal intensities at these longer
chain lengths and may be unique to the N1npe primary sequence.
However, lowering the concentration to 6 μM did not affect the
CD spectral shape or intensity of peptoid 6 or 13 (see Supporting
Information). Additional studies are needed to assess the nature
and significance of these interactions on the CD signal of Ns1npe
peptoids, if any.45
We next obtained variable-temperature CD spectra of the
Ns1npe peptoids to assess their thermal stability, and we selected
two peptoids for focused study (6 and 13). We chose hexamer 6
because it displayed the most intense minimum (near 229 nm) of
any of the peptoids 1ꢀ13, and 13 because it represents the
longest sequence in the series. The CD spectrum of 6 showed no
change in shape and only a moderate decrease in the CD signal
intensity over the temperature range 5ꢀ75 °C (see Supporting
Information). Peptoid 13 also maintained its spectral shape over
this temperature range, and displayed only a small reduction in
signal intensity (Figure 6). These CD data are in agreement with
previous studies on the thermal stability of Nspe peptoids,32,44
and demonstrate that the structures of Ns1npe peptoid oligo-
mers are thermally stable up to 75 °C.
’ ASSOCIATED CONTENT
Lastly, we sought to probe solvent effects on the conforma-
tions of Ns1npe peptoids by CD, and selected the polar protic
solvent methanol for study. We examined four Ns1npe
S
Supporting Information. Full characterization data for
b
peptoids 1ꢀ13 and their respective synthetic intermediates,
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HPLC and MS data for peptoid oligomers, HSQCAD spectra for
peptoid oligomers, and X-ray crystallographic data for peptoids
10 and 2ꢀ4, and a complete author list for the reference in
footnote 4. This material is available free of charge via the
Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
blackwell@chem.wisc.edu
(25) Kwon, Y.-U.; Kodadek, T. J. Am. Chem. Soc. 2007, 129,
1508–1509.
’ ACKNOWLEDGMENT
(26) Holub, J. M.; Garabedian, M. J.; Kirshenbaum, K. QSAR Comb.
Sci. 2007, 26, 1175–1180.
(27) Reddy, M. M.; Bachhawat-Sikder, K.; Kodadek, T. Chem. Biol.
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(30) Armand, P.; Kirshenbaum, K.; Goldsmith, R. A.; Farr-Jones, S.;
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We thank the NSF (CHE-0449959), ONR (N000140710255),
Greater Milwaukee Foundation, Burroughs Welcome Fund, and
Research Corporation for financial support of this work. Support
for the NMR facilities at UW—Madison by the NIH (1 S10
RR13866-01) and the NSF (CHE-9629688) is gratefully ac-
knowledged. X-ray crystal structures were visualized using the
UCSF Chimera package from the Resource for Biocomputing,
Visualization, and Informatics at the University of California, San
Francisco (supported by NIH P41 RR-01081). We thank Lara
Spencer for X-ray crystallographic work and Professors Samuel
Gellman and Benjamin Gorske for contributive discussions.
(31) Wu, C. W.; Sanborn, T. J.; Huang, K.; Zuckermann, R. N.;
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