New strategies for the design of folded peptoids revealed by a survey of noncovalent interactions in model systems

Article abstract of DOI:10.1021/ja907184g

Controlling the equilibria between backbone cis- and trans-amides in peptoids, or N-substituted glycine oligomers, constitutes a significant challenge in the construction of discretely folded peptoid structures. Through the analysis of a set of monomeric peptoid model systems, we have developed new and general strategies for controlling peptoid conformation that utilize local noncovalent interactions to regulate backbone amide rotameric equilibria, including n→π*, steric, and hydrogen bonding interactions. The chemical functionalities required to implement these strategies are typically confined to the peptoid side chains, preserve chirality at the side chain N-α-carbon known to engender peptoid structure, and are fully compatible with standard peptoid synthesis techniques. Our examinations of peptoid model systems have also elucidated how solvents affect various side chain-backbone interactions, revealing fundamental aspects of these noncovalent interactions in peptoids that were largely uncharacterized previously. As validation of our monomeric model systems, we extended the scope of this study to include peptoid oligomers and have now demonstrated the importance of local steric and n→π* interactions in dictating the structures of larger, folded peptoids. This new, modular design strategy has guided the construction of peptoids containing 1-naphthylethyl side chains, which we show can be utilized to effectively eliminate trans-amide rotamers from the peptoid backbone, yielding the most conformationally homogeneous class of peptoid structures yet reported in terms of amide rotamerism. Overall, this research has afforded a valuable and expansive set of design tools for the construction of both discretely folded peptoids and structurally biased peptoid libraries and should shape our understanding of peptoid folding.

Full text of DOI:10.1021/ja907184g

NIH Public Access
Author Manuscript
J Am Chem Soc. Author manuscript; available in PMC 2011 September 18.
Published in final edited form as:
J Am Chem Soc. 2009 November 18; 131(45): 16555–16567. doi:10.1021/ja907184g.
New Strategies for the Design of Folded Peptoids Revealed by a
Survey of Noncovalent Interactions in Model Systems
Benjamin C. Gorske, Joseph R. Stringer, Brent L. Bastian, Sarah A. Fowler, and Helen E.
*
Blackwell
Department of Chemistry, University of Wisconsin–Madison, 1101 University Avenue, Madison,
WI 53706-1322
Abstract
Controlling the equilibria between backbone cis- and trans-amides in peptoids, or N-substituted
glycine oligomers, constitutes a significant challenge in the construction of discretely folded
peptoid structures. Through the analysis of a set of monomeric peptoid model systems, we have
developed new and general strategies for controlling peptoid conformation that utilize local
noncovalent interactions to regulate backbone amide rotameric equilibria, including n→π*, steric,
and hydrogen bonding interactions. The chemical functionalities required to implement these
strategies are typically confined to the peptoid side chains, preserve chirality at the side chain N-α-
carbon known to engender peptoid structure, and are fully compatible with standard peptoid
synthesis techniques. Our examinations of peptoid model systems have also elucidated how
solvents affect various side chain-backbone interactions, revealing fundamental aspects of these
noncovalent interactions in peptoids that were largely uncharacterized previously. As validation of
our monomeric model systems, we extended the scope of this study to include peptoid oligomers
and have now demonstrated the importance of local steric and n→π* interactions in dictating the
structures of larger, folded peptoids. This new, modular design strategy has guided the
construction of peptoids containing 1-naphthylethyl side chains, which we show can be utilized to
effectively eliminate trans-amide rotamers from the peptoid backbone, yielding the most
conformationally homogeneous class of peptoid structures yet reported in terms of amide
rotamerism. Overall, this research has afforded a valuable and expansive set of design tools for the
construction of both discretely folded peptoids and structurally-biased peptoid libraries, and
should shape our understanding of peptoid folding.
Introduction
The family of oligomers comprising poly-N-substituted glycines, or “peptoids”, was
originally conceived to address the early demands of combinatorial chemistry for efficient
1, 2
library synthesis. This legacy is manifest in the peptoid submonomer synthesis method,
which is capable of delivering sizable and structurally diverse peptoid libraries derived from
3
a continually expanding pool of commercially available primary amines. As such,
thousands of peptoid monomers are now readily accessible via this protocol, rivaling if not
exceeding the number of amino acids available for the construction of analogous peptide
4
5
libraries. Resistance to proteolysis and rapid cellular uptake have further rendered peptoids
attractive candidates for biological applications. Screening of peptoid libraries, both random
6
and focused, has yielded numerous bioactive compounds, including antimicrobial agents,
*Corresponding author. Phone: 608/262-1503; Fax: 608/265-4534, blackwell@chem.wisc.edu.
Supporting Information Available: Full characterization data for model peptoids, HPLC data for peptoid pentamers, NMR spectra for
model systems and pentamers, computational data, and a complete author list for the reference in footnote 1. This material is available
free of charge via the Internet at http://pubs.acs.org.

Gorske et al.
Page 2
7
8
9
neutralizers of bacterial endotoxins, lung surfactants, drug delivery vehicles, and ligands
10
for pharmaceutically relevant targets such as the HDM2 protein, G-protein-coupled
receptors, and the SH3 domains of signaling proteins. Apart from biological
11
12
13
applications, screening of peptoids has also produced new DNA drag tags, chemical
14
15, 16
sensors, and organocatalysts.
The decoration of discretely folded oligomeric scaffolds with chemical functionality in a
17–19
combinatorial fashion has proven an attractive strategy for library focusing.
The design
of such libraries requires a thorough understanding of the folding propensities of the chosen
oligomeric scaffolds. This view, in part, has motivated enormous efforts to decipher the
relationships between sequence and secondary structure in biopolymers such as peptides and
20
proteins. These relationships have already been used to efficiently bias peptide libraries
and have also greatly benefited the understanding of synthetic foldamers, but compared to
peptides and proteins, the folding of peptoids has not been as extensively studied. Indeed,
considering the tremendous potential demonstrated by peptoids in the short time since their
inception, the intramolecular interactions that direct their folding remain largely unclear,
21
even in comparison to other foldamer systems such as β-peptides. A deeper understanding
of the peptoid folding process would certainly contribute to the rational design of peptoid
structures competent for a wide range of applications, and likely increase the utility of future
peptoid libraries.
Early research on peptoid secondary structure revealed the propensity of peptoids to adopt
22–24
helical structures analogous to the cis-amide polyproline I (PPI) helix.
N-α-chiral side
25
chains were shown to promote the folding of these structures in both solution and the solid
state, despite the lack of main chain chirality and secondary amide hydrogen bond donors
26
crucial to the formation of many α-peptide secondary structures (Figure 1). Although
computational studies initially suggested that steric interactions between N-α-chiral aromatic
27
side chains and the peptoid backbone primarily dictated helix formation, both intra- and
intermolecular aromatic stacking interactions have also been proposed to participate in
23, 25, 27
stabilizing such helices.
Other classes of peptoid side chains have been designed to
introduce dipole-dipole, hydrogen bonding, and electrostatic interactions that stabilize the
peptoid helix, although their development typically has not encompassed scrutinizing the
24, 28–30
individual noncovalent interactions involved.
The majority of these strategies rely
on incorporation of N-α-chiral centers in order to impart helix handedness. However, a facile
equilibration between the cis- and trans-conformations of the tertiary backbone amides often
counters these organizing influences by giving rise to significant populations of both
25
rotamers, leading to disruption and fraying of peptoid helices, especially at the termini.
The inherent flexibility of peptoids in general can lead to coexisting and potentially inter-
converting conformers in solution, making analysis of peptoid structure less than
25
straightforward. Moreover, this conformational heterogeneity complicates the de novo
design of new peptoid secondary structures.
A recent report describing a distinctive threaded loop secondary structure adopted by several
peptoid homononamers composed of N-α-chiral amide side chains has provided new
31
insights into how peptoid amide isomerization might be controlled. Notably, this loop
structure contains both cis- and trans-amides, and is stabilized by hydrogen bonds between
the peptoid backbone and the N-terminal ammonium group. These hydrogen bonds are
apparently of sufficient strength as to overwhelm the helical propensities resulting from
local interactions between the side chains and the backbone. This finding — that a relatively
small number of noncovalent interactions can stabilize both cis- and trans-amides within the
backbones of large peptoid structures — suggests that strategically positioned noncovalent
interactions could be harnessed to rationally control amide isomerism and generate new
peptoid structural motifs. In this context, peptoids stand apart from typical peptides and
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Gorske et al.
Page 3
other secondary amide-based foldamers in that their tertiary amide backbones are devoid of
hydrogen bond donors, creating a unique opportunity to rationally control the distribution of
these donors by selection of appropriate hydrogen bonding side chains. Such engineered
hydrogen bonding networks could conceivably act as templates for both inter- and
intramolecular structural assembly. Alternatively, the elimination of hydrogen bonding
motifs in peptoids would greatly facilitate investigations of other more subtle noncovalent
interactions that, while relatively weak individually, might collectively exert significant
influences on peptoid structure.
We have undertaken a research program focused on characterizing the roles of hydrogen
bonding and other noncovalent interactions in directing peptoid amide isomerism, and
thereby, peptoid folding in general. The recent focus of these studies has been on small,
peptoid model systems. The principal objective of these studies was to isolate and
characterize only the local interactions between peptoid side chains and the backbone, in the
hope that this restriction might yield highly modular and easily applicable design criteria for
controlling the structures of larger peptoid oligomers. We recently reported the initial
findings of these studies, which showed that n→π* interactions can regulate peptoid amide
32, 33
isomerism in monomeric peptoid model systems.
However, the roles that these
interactions might play in determining the folded structures of actual oligomeric peptoids
remained unexplored. Here, we present a comprehensive overview of our investigations in
this area to date that encompass a much wider range of side chain structures, noncovalent
interaction classes, and solvents. We have developed several new and general strategies for
controlling peptoid conformation that utilize local noncovalent interactions to regulate
backbone amide isomerism. The chemical functionalities required to implement these
strategies typically are confined to the peptoid side chains, preserve the chirality of the side
chain at the N-α-carbon, and are fully compatible with standard peptoid synthesis
techniques. Our examinations of peptoid model systems have also elucidated how various
side chain-backbone interactions are affected by solvent, revealing fundamental aspects of
these noncovalent interactions in peptoids that were largely uncharacterized previously in
the literature. As validation of these monomeric model systems, we extended the scope of
the study to include longer peptoid oligomers that demonstrate the importance of such
noncovalent interactions within the context of larger, structured peptoids. This modular
approach to peptoid design culminated in the installation of 1-napthylethyl side chains into a
series of peptoid oligomers, which were found to virtually suppress trans-amide rotamers in
the backbone and consequently should prove to be valuable structure-promoting elements in
peptoids. Indeed, our preliminary structural characterization suggests that these oligomers
represent the most conformationally defined, acyclic cis-peptoids yet reported. Collectively,
this work has afforded a valuable new set of approaches for the rational design of both
folded peptoids and structurally biased peptoid libraries, and should contribute to the
growing understanding of peptoid folding.
Experimental Section
General
All reagents were purchased from commercial sources (Alfa-Aesar, Aldrich, Advanced
ChemTech, and Acros) and used without further purification. Solvents were purchased from
commercial sources (Aldrich and J. T. Baker) and used as is, with the exception of
dichloromethane (CH Cl ) and n-hexane, which were distilled immediately prior to use.
2
2
Thin layer chromatography (TLC) was performed on silica gel 60 F plates (E-5715-7,
254
®
EMD). SiliaFlash P60 silica gel (40–63 μm, Silicycle) was used for manual flash column
chromatography. High-pressure flash chromatography (HPFC) was performed using
34
prepacked silica gel columns (KP-SIL, Biotage) on a Biotage SP system.
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Gorske et al.
Page 4
1
13
19
H NMR (500 MHz), C NMR (125 MHz), and F NMR (470 MHz) spectra were
recorded on either a Varian Unity or an Inova 500 MHz spectrometer in deuterated
1
13
19
solvents. H NMR (300 MHz), C NMR (75 MHz), and F NMR (282 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
tetramethyl silane (TMS) as a reference (0.0 ppm), unless otherwise noted. Couplings are
reported in hertz. Nuclear Overhauser effect spectroscopy (NOESY), gradient double
quantum filtered correlation spectroscopy (gDQCOSY), and heteronuclear single quantum
coherence adiabatic (HSQCAD) NMR experiments were performed on a Varian Inova 500
or 600 MHz spectrometer, and were referenced to solvent. The data were processed using
35
the Varian VNMR software package (v. 6.1C) and visualized using SPARKY software.
™
Electrospray ionization (ESI) mass spectra were obtained on a Waters (Micromass) LCT
spectrometer equipped with a time-of-flight analyzer. Samples were dissolved in methanol
and sprayed with a sample cone voltage of 20. Electron impact (EI) mass spectra were
™
obtained on a Waters (Micromass) AutoSpec spectrometer equipped with a magnetic
sector. The samples were placed in a glass capillary and inserted directly into the source, and
the resulting vapor was bombarded with 70 eV electrons. Perfluorokerosene (PFK) was used
for calibration. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)
mass spectra were obtained on a Bruker REFLEX 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 SCL-10Avp controller, a LC-10AT pump, an
FCV-10ALvp solvent mixer, and an SPD-10MAvp UV/vis diode array detector. A Restek
Premier C18 column (5 μm, 4.6 mm × 250 mm) was used for all analytical RP-HPLC work.
A Vydac protein & peptide C18 column (10 μm, 22 mm × 250 mm) was used for all
preparative RP-HPLC work. Standard RP-HPLC conditions were as follows: flow rates
were 1 mL/min for analytical separations and 9 mL/min for preparative separations; 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 equipped with two LC-10ADvp
pumps, an SCL-10Avp controller, an SIL-10ADvp autoinjector, an SPD-M10Avp UV/vis
diode array detector, and a single quadrupole analyzer (for ESI). A Supelco 15 cm × 2.1 mm
C18 wide-pore column 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 = 0.4% formic acid in
water; mobile phase B = 0.2% formic acid in acetonitrile.
Attenuated total reflectance (ATR)-IR spectra were recorded with either a Bruker Tensor 27
spectrometer outfitted with a single reflection MIRacle Horizontal ATR by Pike
−1
Technologies and a ZnSe crystal with a spectral range of 20,000 to 650 cm , or a Bruker
Equinox 55 spectrometer outfitted with a single reflection MIRacle Horizontal ATR and a
−1
Ge crystal with a spectral range of 5500 to 600 cm . Circular dichroism (CD) spectra were
obtained on an Aviv 62A DS spectropolarimeter with Aviv CDS software.
Synthesis and characterization of peptoid model systems 1–32
All peptoid model systems were synthesized in solution according to previously reported
32
32
procedures. Peptoid model systems 1–15 and 30 have been characterized previously.
Full characterization data for peptoid model systems 16–29, 31, and 32 are provided in the
Supporting Information.
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Gorske et al.
Page 5
Synthesis and characterization of peptoid oligomers 33–38
Peptoids 33–38 were synthesized using microwave-assisted solid-phase methods on Rink-
36, 37
amide-derivatized polystyrene resin as previously reported.
The crude peptoids were
purified by preparative RP-HPLC and characterized by MS to confirm their identity (see
Table 1). See Supporting Information for HPLC data for peptoids 33–38.
Determination of cis/trans rotamer ratios (K
) for the peptoid model systems
cis/trans
1
Each model peptoid was dissolved in the desired solvent to give a 15 mM solution. H NMR
spectra were acquired using 16 scans and a relaxation delay of 25 s. 1D-NOESY spectra
were acquired to verify previously reported guidelines for distinguishing cis- and trans-
31, 33
amide rotamers.
In cases where these guidelines appeared to be inapplicable, additional
1D-NOESY spectra were acquired to distinguish cis- and trans-amide rotamers. Cis/trans
ratios were calculated by averaging the integration ratios for at least two sets of rotamer-
related peaks. The cis/trans ratios of the hydrophobic model peptoid 4 were statistically
identical at 1, 5, 15, 60, and 180 mM concentrations in the polar solvent CD CN, and the
3
cis/trans ratios of the more hydrophilic model peptoid 27 at 1, 5, 10, 15, 25, 50, and 75 mM
were statistically identical in the nonpolar solvent CDCl , suggesting that solvophobic
3
aggregation is insignificant under the conditions examined in this study.
NMR analyses for peptoid model systems 27 and 30
Peptoids 27 and 30 were dissolved in CD CN to give 15 mM solutions and analyzed by
3
NOESY as detailed below. The experiments were performed on a Varian Inova 600 MHz
spectrometer using a 5 mm hcn probe.
NOESY experiments on 27 in CD CN were performed at 24 °C using the following
3
parameter values: mix time = 700 ms; spectral width = 8000 Hz; number of transients (nt) =
16; number of increments (ni) = 64. The number of points (np) was 2048, and 192 points
were obtained by linear prediction in the f1 dimension. Square cosine window functions
were applied in both dimensions. The spectra were zero-filled to generate f1 × f2 matrices of
4096 × 4096 points. Parameters for NOESY experiments on 30 in CD CN were reported
3
32
previously.
NMR analyses for peptoid oligomers 33–38
Peptoids 33–38 were dissolved in CD CN to give 15 mM solutions and analyzed by
3
gDQCOSY and HSQCAD NMR experiments as detailed below. The experiments were
performed on a Varian Inova 600 MHz spectrometer using a 5 mm hcn probe. The
gDQCOSY spectra were used for assignment purposes, and the HSQCAD NMR spectra
were subsequently integrated to give the K
methyne cis and trans peaks were based on previously observed trends in chemical shift,
values in Table 6. Assignments of
cis/trans
37
and verified by NOESY NMR, unless otherwise noted.
gDQCOSY NMR experiments were performed for peptoids 33–36 at 24 °C using the
following parameter values: Spectral widths were 5323, 5287, 4727, and 6611 Hz,
respectively. The numbers of transients (nt) were 8, 16, 16, and 16, respectively. The
relaxation delays (d1) were 1.20, 1.12, 1.68, and 2.30, respectively. The numbers of
increments (ni) were 300, 300, 300, and 256, respectively. The number of points (np) was
4096, with 1200 points obtained by linear prediction in the f1 dimension. Square cosine
window functions were applied in both dimensions. The spectra were zero-filled to generate
f1 × f2 matrices of 8192 × 8192 points.
38
HSQCAD NMR experiments were performed for peptoids 33–38 at 24 °C using the
following parameter values: Spectral widths were 5323, 5212, 4727, 6611, 5914, and 5914
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Gorske et al.
Page 6
1
Hz, respectively, in the H dimension. Spectral widths were 28787, 28684, 30234, 36199,
13
30948, and 25983 Hz, respectively, in the C dimension. The relaxation delays (d1) were
1.50, 0.84, 1.68, 2.30, 3.36, and 2.94, respectively. The number of transients (nt) were 8, 8,
8, 8, 2, and 2, respectively. The number of increments (ni) was 256, with 1200 points
obtained by linear prediction in the f1 dimension. The number of points was 2048. Square
cosine window functions were applied in both dimensions. The spectra were zero-filled to
generate f1 × f2 matrices of 8192 × 2048 points.
CD analyses for peptoids 26, 37, and 38
Peptoid stock solutions were prepared by dissolving at least 2 mg of each peptoid in
spectroscopic grade acetonitrile. The stock solutions then were diluted with spectroscopic
grade acetonitrile 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 25 °C using a scan rate of 100 nm/min, with five averaged scans per
spectrum. The spectrum of an acetonitrile blank was subtracted from the raw CD data, and
the resulting data were plotted using Kaleidagraph software (v. 4.03).
Results and Discussion
Design and synthesis of peptoid model systems
We designed monomeric peptoid model systems that mimicked the local environments of
isolated amide side chains within a peptoid oligomer in order to dissect short-range (i· · ·i or
i· · ·i±1) interactions. Since we were primarily interested in elucidating the impact of side
chain structure on backbone amide isomerism, our initial monomeric models consisted of
two tertiary amides positioned opposite the side chain, as in a typical peptoid backbone. We
developed two straightforward syntheses of these diamide models (Scheme 1) that were
easily adapted for the installation of other C-terminal and N-terminal capping groups besides
32
amides, such as esters. This flexibility of design permitted us to probe the role of either
terminus in any side chain-backbone interaction of interest in our model system.
The peptoid model system side chains and terminal capping groups examined in this study
are shown in Table 2. N-α-chiral amide side chains are heavily represented, as these have
been extensively investigated previously and found to promote peptoid helix and loop
folding (see above). In addition to this consideration, side chain functionalities were selected
to explore the effects of four key types of noncovalent interactions on peptoid amide cis/
trans equilibria: (1) n→π* interactions between an amide and an aromatic ring (n→π* ),
Ar
(2) n→π* interactions between two carbonyls (n→π* ), (3) side chain-backbone steric
C=O
interactions, and (4) side chain-backbone hydrogen bonding interactions. Our analyses of
each type of interaction in a series of model systems are described sequentially below. The
1
cis/trans ratio of the N-terminal amide in each model system was determined by H NMR
and NOESY and used to assess the influence of the noncovalent interactions on peptoid
amide isomerism.
Investigations of the impact of n→π* interactions on peptoid amide rotamer equilibria
Ar
In earlier work, we observed that the incorporation of electron-deficient aromatic rings into
37,39
peptoids could have a profound impact on their structure.
Analysis of related, small
model systems suggested that these structural perturbations arise in part from an interaction
between a lone pair of the proximal amide oxygen and a π* orbital of the electron-deficient
40, 41
aromatic ring, that is, an n→π* interaction (also referred to as an lp-π interaction).
Ar
This type of interaction has been loosely defined by a carbonyl oxygen-to-ring centroid
distance between 2.8 and 3.8 Å, and a dihedral angle between the aryl plane and the
42
carbonyl plane (X C=O) of 90° or less. We reasoned that an interaction between an N-α-
2
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Gorske et al.
Page 7
chiral aromatic peptoid side chain and the nearest backbone carbonyl would easily meet
these geometrical constraints and, if operative, would exclusively stabilize the cis-rotamer of
the participating amide, increasing K (Figure 2). We tested this possibility by
cis/trans
1
constructing peptoid model systems 1–3 and examining them using H NMR, initially
focusing on the acetamide cis/trans ratios obtained in acetonitrile (
CD CN
3
K
), since
cis/trans
this solvent has been commonly used in structural studies of peptoids (Figure 3, Table
22, 23, 25, 37
3).
Furthermore, this solvent minimizes inter-amide interactions that can also
K
affect cis/trans; these interactions and their solvent-dependence are discussed in the next
section.
We hypothesized that model compounds 1 and 2 would exhibit higher cis/trans ratios than 3,
since electron-withdrawing groups on aromatic rings are known to increase the strength of
41
n→π* interactions. In accord with our hypothesis, we observed a nearly two-fold
Ar
CD CN
3
increase in
K
for the compounds containing the electron-deficient benzyl side
cis/trans
chains fe and np (1 and 2) compared to the control compound containing the unsubstituted
benzyl side chain pe (3) (Table 3). Replacement of the aromatic ring with a cyclohexyl ring
CD CN
3
(cy, to yield 4) lowered
K
, as expected. Although these model systems
cis/trans
validated the possibility of n →π
*
interactions in a typical peptoid, an n →π *
i i Ar
i
i+1 Ar
interaction involving the C-terminal amide could not be excluded. We thus constructed
control compounds 5–7, in which the C-terminal amide was replaced with a methyl group
CD CN
3
(Figure 3). This series of compounds exhibited nearly identical
K
values to the
cis/trans
piperidinyl series (2–4), suggesting that n →π * interactions do not significantly regulate
i
i Ar
peptoid amide isomerism, and establishing that the C-terminal amide has little impact
CD CN
3
on
systems (pyridinium 8 (4mpy) and phenolate 9 (mph), Figure 3) to further explore the
possibility of controlling K by tuning the electronics of N-α-chiral aromatic side
K
in these systems (Table 3). We also synthesized formally charged model
cis/trans
cis/trans
chains. As expected, the presence of mph in 9 substantially demoted the cis-amide rotamer
relative to pe in 6, while the 4mpy side chain in 8 strongly promoted the cis-amide.
Comparison of NOESY NMR data for model compounds 6 and 8 indicated that the
conformation in which the aromatic ring and the amide carbonyl are eclipsed is significantly
more populated by 8 than by 6 (Figure 4), implying that carbonyl-aromatic interactions
dictate K
in these systems.
cis/trans
Our previously reported calculations of Mulliken atomic charges suggested that electrostatic
interactions between the atoms directly participating in the n→π* interaction do not
Ar
contribute considerably to K
in this class of peptoid model systems (5, 6, 8, and
cis/trans
32, 43
9).
On the contrary, the calculations indicated that K
correlates closely with the
cis/trans
LUMO energy, and even more strikingly with the LUMO density at the carbon in the
aromatic ring connected to the side chain. We sought to decouple, as much as possible, the
contributions of electrostatic and stereoelectronic influences on n→π* interactions to
Ar
complement these computational data. Solvent modulation is a useful approach for probing
44–46
noncovalent interactions,
even though its impact on a particular interaction of interest
can be somewhat obscured by solvent-charge screening and/or by perturbations elsewhere in
the molecule. We thus proceeded to determine K values for the structurally simplest
cis/trans
series — the methyl-capped compounds 5–7 — in a range of solvents (Table 3).
K
K
for compound 7 (ch), which served as a baseline indicator of solvent effects on
in the absence of n→π* interactions, generally tracked with solvent polarity but
cis/trans
cis/trans
displayed only slight sensitivity to the choice of solvent (Table 3). The aromatic side chain
of 6 (pe) did not significantly sensitize K to solvent polarity compared to 7, as
cis/trans
indicated by the similar (differing by less than 0.1 kcal) differences in ΔG
between solvents for 6 and 7. However, 5 (np) diverged significantly (ΔG
observed
differing
cis/trans
cis/trans
by more than 0.2 kcal) from this trend, exhibiting a larger K
in CDCl . Chloroform
cis/trans
3
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Gorske et al.
Page 8
would not be expected to enhance withdrawal of electron density from the aromatic ring by
the nitro group (thereby lowering the aryl LUMO energy and increasing K ), as the
cis/trans
resulting polarization would be less stabilized by such a nonpolar solvent. Therefore, this
divergence may derive from electrostatic/dipole-dipole interactions between the nitro group
and the acetamide that reinforce the n→π* interaction in CDCl . These electrostatic
Ar
3
interactions would likely be more effectively screened in more polar solvents such as
CD OD
3
methanol; however,
Δ G
for 5 remained significantly lower than for 6, and
cis/trans
ΔG for 5 and 6 were similarly affected by switching the solvent from acetonitrile to
cis/trans
methanol, suggesting that electrostatic interactions are less important than the n→π*
Ar
orbital interactions in this case. Indeed, even in aqueous solution, we observed that the cis-
D O
2
amide rotamer of 8 (4mpy) is dominant (
K
= 3.92), demonstrating that the side
cis/trans
chain-backbone interactions in these highly polarized systems are robust, even in strongly
polar solvents that would be expected to significantly screen electrostatic interactions. This
finding suggests that the inclusion of these charged aromatic monomers could produce
water-soluble peptoid helices with enhanced conformational stability relative to pe
homooligomers; such scaffolds could be valuable for biological applications. The impact of
n→π* interactions in general on the structures of peptoid oligomers is examined further in
Ar
the last section of this paper.
Investigations of the impact of n→π*
interactions on peptoid amide rotamer equilibria
C=O
Having scrutinized n→π* interactions in model systems containing a single amide, we
Ar
turned our attention to diamide peptoid model systems that might more accurately reproduce
the local environment experienced by a peptoid monomer unit within a larger peptoid
oligomer. As discussed above, we observed that the presence of a piperidinyl amide at the
CD CN
3
C-terminus did not significantly perturb
relative to the single amide models (e.g., 2–4 vs. 5–7). Nonetheless, we were intrigued by
recent literature reports by Raines, Wennemers, and co-workers of n→π* interactions
K
for the N-terminal acetamide
cis/trans
C=O
between tertiary prolyl amides and their considerable importance in stabilizing the trans-
47–50
amides of polyproline II (PPII) helices.
Similar to the n→π* interactions discussed
Ar
above, these interactions are characterized by donation of electron density from an amide
carbonyl lone pair into the π* orbital of an adjacent carbonyl. Due to geometric and
stereoelectronic differences between the π* orbitals of aromatic rings and amides, a
somewhat more restricted set of geometric constraints has been demarcated for n→π*
C=O
47
interactions (O …C =O distance ≤ 3.2 Å, angle = 109° ± 10°; Figure 5) relative to
i−1
i
i
n→π* interactions. These constraints preclude cis-amides from acting as donors in i· · ·i+1
Ar
n→π*
interactions along the backbone, and n→π*
interactions are thus observed to
C=O
C=O
exclusively stabilize trans-amide donors in polyprolines.
The strong similarity between the tertiary amide backbones of peptoids and polyprolines
caused us to speculate that these n→π* interactions could also be exploited to stabilize
C=O
trans-amides in peptoids, providing a design strategy complementary to the cis-amide-
stabilizing n→π* interactions described above. Previous studies have shown that esters are
Ar
excellent acceptors for these n→π*
delocalization into the carbonyl π* orbital.
the carbonyl lowers the energy of the π* orbital, enhancing n→π*
interactions due to significantly reduced π-electron
C=O
33, 51, 52
This reduction of electron donation into
interactions in esters
C=O
relative to amides, just as removing substituents that donate electrons into aromatic rings
(e.g., 9 vs. 6 above) enhances n→π* interactions. We hypothesized that amides
Ar
disfavoring π-electron delocalization might also enhance n→π*
interactions, albeit to a
C=O
lesser extent than esters. To test this hypothesis and probe for the presence of n→π*
C=O
interactions within peptoids in general, we constructed model systems possessing methyl
esters and dimethylamides at the C-terminus (10–15, Figure 3). As noted in the previous
section, piperidinyl amide-capped compounds 2–4 did not exhibit any evidence of these
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Gorske et al.
Page 9
CD CN
3
interactions in acetonitrile, giving nearly identical
K
values to those of the
cis/trans
methyl-capped compounds 5–7. However, we expected that the dimethylamide would be
somewhat less capable of stabilizing positive charge at the nitrogen compared to the
piperidinyl amide, and thus might participate in an n→π*
interaction with the N-terminal
C=O
acetamide. Analogous to the studies above, we also examined these systems in a range of
solvents (Table 3).
We were pleased to observe that the nature of the C-terminal cap dramatically modulated the
acetamide cis-trans equilibrium, with the ester group (in 10–12) producing the largest
reductions in K
relative to the methyl- and piperidinyl-amide-capped systems (2–4),
cis/trans
as predicted. Although stabilization of the trans-amide in the dimethylamide systems (13–
15) relative to compounds 2–4 was modest, the former demonstrated the feasibility of inter-
amide n→π*
interactions within larger peptoid oligomers. We were especially intrigued
C=O
by the large increases in ΔG
observed upon decreasing the solvent polarity for the
cis/trans
piperidinyl and dimethyl amide series, relative to the ester series. Since previous studies
have shown that n→π*
electrostatic in nature,
interactions are primarily hyperconjugative rather than
we hypothesized that the greater solvent sensitivity of ΔG
C=O
53, 54
cis/trans
for the amides (2–4, 13–15) versus the esters (10–12) may be due to reduced charge
separation within the more polarizable piperidinyl and dimethylamides in nonpolar solvents.
Such suppression of amide resonance would be expected to increase the strength of
33
n→π*
interactions by lowering the energy of the π* orbital. Although some debate
C=O
55, 56
over the significance of amide resonance has emerged,
the greater polarizability of
amides, defined here as their capacity to accommodate internal charge separation via
resonance, is commonly cited to explain their significantly higher rotational barriers (~18
57–60
kcal/mol) compared to esters (~10 kcal/mol) and carbamates (~15 kcal/mol),
Indeed,
the greater sensitivity of amide rotational barriers to solvent polarity, compared to that of
carbamates, has been attributed to the less polarizable “ester-type resonance” in the latter.
59
In the context of this argument, the absence of n→π*
interactions for compounds 2–4 in
C=O
acetonitrile can be ascribed to the high degree of amide polarization facilitated by the both
the piperidinyl substituent and the polar solvent.
Interestingly, the amide-capped systems (2–4, 13–15) showed a marked increase in
ΔG
in methanol as compared to acetonitrile, deviating from the solvent polarity trend
cis/trans
above, while ΔG
for the methyl- and ester-capped systems (5–7, 10–12) remained
cis/trans
relatively constant in both solvents. This implies that amide-amide n→π*
interactions,
C=O
but not amide-ester interactions or isolated amide rotameric equilibria, are strongly affected
by solute-solvent hydrogen bonding in these systems. Since esters are known to be poorer
61
hydrogen bond acceptors than amides, these data suggest that hydrogen bonds to the
amide acceptors of n→π*
interactions may enhance these interactions. Further
C=O
corroboration of these hypotheses in the context of peptoids containing amide and ester side
chains is described later in this paper.
Investigations of the impact of steric interactions on peptoid amide rotamer equilibria
Steric interactions play an extremely important role in peptoid folding by constraining the
rotations of bonds within the peptoid backbone. For example, it is these interactions that
likely transmit chirality from the N-α-chiral peptoid side chains to the backbone, imparting
27
“handedness” to the peptoid helix. Steric interactions in peptoids are expected to be highly
24
robust in that they are relatively unaffected by solvation. This property also greatly
simplifies their computational simulation and facilitates their utilization for rational design
applications, as aptly illustrated by the successful computational prediction of the peptoid
27
helical structure. Although computations predict that steric interactions restrict all three
27
peptoid backbone torsions (φ, ψ, and ω), the present study focuses on the roles of these
interactions in regulating peptoid amide isomerism (ω) by systematically probing the steric
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Gorske et al.
Page 10
profiles of peptoid side chains in a series of model systems typically containing a C-terminal
piperidinyl amide (16–26; Figure 6, Table 4).
In general, increasing the steric bulk of the peptoid side chain in the model systems should
increase the population of the cis-amide isomer, since the relatively small carbonyl oxygen,
as compared to the acetamide methyl group, can more easily accommodate steric
27
crowding. Comparison of the ΔG
values within each solvent demonstrates that
cis/trans
these systems largely conform to this paradigm. The solvent-dependent trends observed
largely mirror those of piperidinyl amides 1–4 (Table 3) and are consistent with the action of
n→π* interactions, as discussed in the previous sections. The sole exception is compound
23, which lacks a C-terminal carbonyl and exhibits little solvent-dependent change in
K
, as expected. We observed that complete removal of all N-α-substituents (i.e., Me,
cis/trans
AVG
16) produced a very low average K
value for the solvents examined (
K
), in
cis/trans
cis/trans
keeping with this steric argument (Table 4) and in agreement with previous studies of
62
similar N,N-dialkyl amides. Although a single appended methyl group (Et, 17) gave a
AVG
similarly low value for
K
, further substitution at the N-α-carbon (ip, 18)
cis/trans
AVG
increased
K
by two-fold, again commensurate with side chain steric bulk. In
cis/trans
contrast, increasing the steric bulk at the N-β-carbon, as with sb (19) and ipe (20), did not
AVG
have any significant effect on
K
, giving values similar to those observed for ip
cis/trans
(18) and the cyclohexyl side chain (ch, 4; Table 3). We also examined a
(trimethylsilyl)methyl side chain (tmsm, 21) as part of an effort to determine whether a lack
of steric bulk at the N-α-position might be compensated for by an even larger increase in
bulk at the N-β-position relative to the ipe side chain. Although the K
values for 21
cis/trans
were indeed higher than those of the aliphatic side chain model systems, analysis of this
system is complicated by the possibility of dative oxygen-silicon bonding between the side
chain silyl group and the acetamide oxygen. We suggest that this class of interactions could
also be useful for controlling amide isomerism in peptoids, and is worthy of study in the
future.
Turning our attention to the sterics of aromatic side chains, we appended a methyl group to
the well-characterized pe side chain to determine if such modifications could be used to
increase K
(1pp, 22; Table 4). As expected, we observed a modest (20%) increase
relative to pe (3); this increase was nearly identical in each solvent tested,
cis/trans
AVG
in
strongly suggesting a steric origin for the increase. Similar reasoning explains the lower
values determined for the N-benzyl compounds pm-Me (23) and pm-Pip (24), as
K
cis/trans
K
cis/trans
compared to the pe-Me (6) and pe-Pip (3) systems containing an N-α-methyl group (Table
2). Intriguingly, the absence of an N-α-methyl group in the side chain of pm-Pip (24), in
conjunction with the presence of a piperidinyl amide at the C-terminus, especially promotes
the trans-acetamide, since K
for pm-Pip (24) is dramatically lower (by 50%, on
cis/trans
average) than that for pm-Me (23) in all solvents examined, while pe-Me (6) and pe-Pip (3)
exhibit more similar K values.
cis/trans
In an effort to more dramatically regulate amide isomerism using steric interactions, we
constructed model systems containing naphthylethyl side chains (2npe-Pip, 25 and 1npe-
Pip, 26; Table 4). We conjectured that the expanded size of these side chains could multiply
steric interactions with nearby atoms, thereby restricting bond rotations. Furthermore, the
electronic perturbation resulting from expansion of the aromatic ring system might
simultaneously engender strong n→π* interactions. Although the model system
Ar
containing the 2-substituted naphthyl group (25) displayed only a slight increase in K
cis/trans
compared to the related aromatic side chain pe (in 3), the 1-substituted naphthyl group (in
26) largely suppressed the trans-acetamide rotamer. Preliminary molecular mechanics
calculations suggest that steric interactions between the naphthyl side chain and the
backbone of 26 restrict rotation of the naphthyl group such that n→π* interactions
Ar
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Gorske et al.
Page 11
stabilizing the cis-rotamer are favored; these steric interactions are calculated to be
significantly less severe in 25 (see Supporting Information.) The strong cis-rotameric
preference elicited by the 1-naphthylethyl side chain, if representative of that within a
polypeptoid, suggests that this side chain could prove exceptionally useful for the
construction of peptoid helices. We return to this possibility below.
Investigations of the impact of hydrogen bonding interactions on peptoid amide rotamer
equilibria
Hydrogen bonds are ubiquitous in peptide and protein structures, and are also essential to the
21
folding of many non-natural foldamer classes. Peptoids offer the distinctive opportunity to
engineer the locations of all hydrogen bond donors via side chain selection, while
conserving structural elements that promote folding. The strength of typical hydrogen bonds
in organic solvents (up to 3 kcal/mol) relative to other noncovalent interactions makes them
attractive architectural elements, and they have been exploited previously to stabilize
24, 28, 31
peptoid structures (e.g., the threaded loop, vide supra).
We therefore undertook a
study of a set of peptoid model systems containing side chains capable of hydrogen bonding
to the peptoid backbone (27–32; Figure 7).
We commenced our investigations of hydrogen bonding by examining the primary amide
side chain fipa (27), derived from phenylalanine (Figure 7). We initially considered the
possibility that a hydrogen bond could form between this side chain amide and the N-
terminal acetamide, stabilizing the cis-acetamide isomer (Figure 8A), even though such γ-
63
turn motifs are rare in peptides. However, K
for 27 (Table 5) increased in the
cis/trans
hydrogen bonding solvent methanol and decreased in the nonpolar solvent chloroform,
implying that a side chain-acetamide hydrogen bond does not significantly affect the
rotameric ratio of 27. Furthermore, the downfield chemical shift of the amide side chain
1
proton in the trans-acetamide relative to the cis-acetamide in the H NMR spectrum of 27
(in acetonitrile and chloroform) suggests that any hydrogen bonds formed are stronger in the
64
trans-acetamide than in the cis-acetamide (see Supporting Information).
We reasoned that modulating the strength of the putative hydrogen bonds in the peptoid
model systems might inform our understanding of how they affect K
in these systems.
cis/trans
To this end, we examined additional model systems in which the secondary amide
substituent on the side chain was systematically altered (28–30; Figure 7, Table 5). (We note
that NMR spectroscopy did not detect any trans-rotamers (as defined in Table 2) of the side
CD CN
3
chain secondary amides of this series.) The
K
values obtained increased
cis/trans
concomitantly with the expected acidity of the secondary amide proton; however, a direct
hydrogen bond to the cis-acetamide seemed unlikely (Figure 8A; see discussion of 27
32
above). Based on these observations and NOESY data for 27 and 30, we hypothesized that
a hydrogen bond preferentially forms between the side chain and the piperidinyl amide
instead, leaving K
to be determined by the relative energies of competing backbone-
cis/trans
side chain (Figure 8B; favoring cis) and backbone-backbone (Figure 8C; favoring trans)
n→π* interactions. Convincing solid state evidence for this hypothesis was provided by
C=O
our previously reported X-ray crystal structure of the fpnan system (30), which exhibits both
32
the hydrogen bond and the n→π*
interaction shown in Figure 8B. The strong (≥10:1)
C=O
preference for the cis-acetamide in acetonitrile, coupled with the NOESY data, also
corroborates this model for stabilization of the cis-acetamide. As with the previously
CD OD
3
examined systems containing the C-terminal piperidinyl amide (vide supra), the
CD CN
3
K
values for 27–29 and 30 were typically lower than the
K
values,
cis/trans
CDCl
cis/trans
3
while
K
values were uniformly low as compared to the other solvents
cis/trans
examined, consistent with stronger backbone-backbone n→π*
interactions (Figure 8C)
C=O
in this solvent. This solvent trend suggested that the tertiary piperidinyl amide, being more
polarizable than the side chain secondary amide, might be more sensitive to solvent polarity
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Gorske et al.
Page 12
and could out-compete the side chain amide as the acceptor of the n →π *
interaction
C=O
with the acetamide in chloroform; this solvent-dependent, divergent behavior of secondary
65
and tertiary amides has been reported previously.
In order to further evaluate this theory, we replaced the amide hydrogen bond donor in the
side chain with an ester (31, Table 5), an excellent acceptor of n→π*
interactions (see
C=O
CD CN
3
above). As expected,
ΔG
significantly decreased (by 0.33 kcal/mol) versus
cis/trans
amide 27, confirming that any hydrogen bonding that occurs is not required for stabilizing
the cis-acetamide, and further supporting our hypothesis that backbone-to-side chain n→π
*
interactions (Figure 8B) stabilize the cis-acetamide rotamer in this series. Moreover,
C=O
ΔG
for 31 in chloroform also decreased by ~0.3 kcal/mol compared to 27, signifying
cis/trans
that this backbone-side chain n→π*
interaction is mostly unaffected by solvent polarity
C=O
per se, again in line with expectations for the relatively non-polarizable ester (vide supra).
However, ΔG values for amides 29 and 30 decreased by varying amounts in the
cis/trans
hydrogen bonding solvent methanol relative to acetonitrile, and were generally lower than
for ester 31, even though esters are typically better acceptors of n→π* interactions.
C=O
These findings, in conjunction with the X-ray crystal structure of 30, suggest that a
hydrogen bond can stabilize a conformation (observed in the solid state) that predisposes the
backbone and side chain amides to participate in an n→π*
interaction (Figure 8B). In
C=O
this respect, the hydrogen bond appears to perform a similar function to that of the steric
interactions in 1-naphthylethyl system 26, in that it serves to enforce a conformational bias
that engenders an n→π interaction. The significance of this hydrogen bond is underscored
by the fact that a significant decrease of ΔG
in methanol vs. acetonitrile is observed
cis/trans
only for the compounds with amide side chains, which would be susceptible to hydrogen
bond disruption by the solvent. (Note, solvent-mediated enhancement of backbone-backbone
n→π*
interactions in methanol, such as that observed for 2–4 and 13–15 above, could
C=O
also play a role in lowering ΔG
.) On the other hand, strengthening of the backbone-
cis/trans
backbone n→π*
interactions (Figure 8C) due to reduced polarization of the piperidinyl
C=O
amide in chloroform again appears to be the major factor contributing to the reduction of
in chloroform versus acetonitrile for 28–30 (vide supra). Further confirmation of
K
cis/trans
this finding was obtained by examining 32, which lacked the piperidinyl amide cap and
therefore exhibited uniformly higher (as compared to 31) and nearly identical K
cis/trans
values for 32 in the three solvents examined, including chloroform. Indeed, the nearly
CD CN
3
identical
ΔG
values for 31 and 32 indicate that backbone-backbone n→π *
cis/trans C=O
interactions (Figure 8C) are virtually nonexistent in acetonitrile. These data also eliminate
the possibility of significant n→π *
interactions between the backbone piperidinyl amide
C=O
and the side chain ester.
Overall, the data acquired for this series of model systems (27–32) demonstrate that this
class of side chain amide hydrogen bond donors does not directly interact with the N-
terminal amide to dictate K
(Figure 8A). Rather, our results suggest that solvent-
cis/trans
dependent hydrogen bonding and amide polarization affect the energies of the competing
backbone-side chain (Figure 8B, favoring cis) and backbone-backbone (Figure 8C, favoring
trans) n→π*
interactions that determine K
. In light of the data above,
cis/trans
C=O
incorporation of amide side chains into peptoids could constitute a highly effective strategy
for promoting cis-amides in the peptoid backbone in polar solvents, as evidenced by the
large cis/trans ratios exhibited by acetanilide 29 (~5:1), and especially 30 (≥10:1), in
acetonitrile. Intriguingly, this same side chain class might also be used to construct new and
optically active, trans-amide peptoid secondary structures in nonpolar solvents, since its
30
capacity to stabilize trans-amides in chloroform rivals the other side chains in this report.
In general, the wide range of K
values attainable using side chain amides, coupled
cis/trans
with their synthetic accessibility via standard coupling techniques, suggests that this side
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Gorske et al.
Page 13
chain class could be extremely useful for rationally tuning amide isomerism in the peptoid
backbone.
Studies of Peptoid Oligomers Derived from the Model Systems
The peptoid model systems discussed above were designed to investigate interactions
between peptoid side chains and the backbone, and thus typically consisted of a single side
chain flanked by two backbone carbonyls. In a peptoid oligomer, however, each backbone
carbonyl is in turn typically surrounded by two peptoid side chains, which may act
cooperatively or competitively to influence amide rotameric equilibria. Therefore, peptoids
containing at least two side chains could validate a role for the noncovalent interactions
discussed above in directing amide isomerism within larger peptoid oligomers. To this end,
we synthesized several polypeptoids (33–38, Figure 9) that were designed to probe these
1
noncovalent interactions within peptoid oligomers, and analyzed their H-COSY and
HSQCAD NMR spectra in CD CN to determine backbone amide cis/trans ratios (Table 6).
3
At the outset, we were most interested in examining n→π* interactions in polypeptoids,
Ar
since these were expected to promote peptoid helix formation by stabilizing backbone cis-
amides. We therefore incorporated a single snp residue (the S-enantiomer of the np side
chain) within an spe pentamer at the central position away from the termini, in an attempt to
minimize interference with its cis/trans ratio by hydrogen bonding to the C-terminus and to
more accurately simulate the structural environment within longer peptoids. We were
gratified to observe that this snp side chain, which exhibited a K
of ~3.3 in the methyl
cis/trans
(5) and piperidinyl (2) model systems, significantly increased the population of cis-amides
in 33 relative to the spe homopentamer 34 (Table 6). We subsequently examined two
pentamers containing the sfe residue (35 and 36), which exhibited excellent resolution of
37
signals by residue type in their HSQCAD spectra. Once again, we found that our model
systems were qualitatively predictive of K
in peptoid 35, with the sfe residues
value than the corresponding model system
cis/trans
CD CN
3
exhibiting an even higher (46%)
K
cis/trans
CD CN
3
(1), and a six-fold higher
K
than the spe residues. Interestingly, the overall cis/
cis/trans
trans ratios for the homopentamers 34 and 36 were found to be somewhat lower than those
of the corresponding monomeric model systems. Since previous studies have demonstrated
that hydrogen bonding at peptoid termini in related systems can profoundly affect
23, 25, 31, 37
conformation, especially by locally promoting trans-amides,
we suggest that
66
hydrogen bonding at the termini of these pentamers may have a similar impact. This
assertion is supported by the low K values obtained for the spe residues of the mixed
cis/trans
oligomer 35, two of which constitute the terminal residues. Notwithstanding such hydrogen
bonding-derived effects, these findings demonstrate that n→π* interactions can indeed
Ar
regulate amide isomerism at internal residues of N-α-chiral aromatic polypeptoids, and can
be used to rationally control peptoid structure.
We next constructed homooligomers composed of (S)-1-naphthylethyl (s1npe) side chains
(37 and 38, Table 6), since the consistently high K
values obtained using this side
cis/trans
chain suggested that it could prove particularly useful for constructing robust peptoid
helices. We were pleased to observe that the very high K observed for the monomeric
cis/trans
system (26; 6.27) in acetonitrile was amplified in the s1npe homooligomers in a length-
CD CN
3?
dependent manner, delivering a
(38), respectively – to our knowledge, the highest overall amide cis/trans ratios observed for
any acyclic peptoid oligomers to date. This length-dependent amplification of K is
K
of 7.0 and 9.7 in the dimer (37) and trimer
cis/trans
cis/trans
indicative of cooperatively stabilized structure — a hallmark of well-folded peptoids —
although the nature of such cooperative effects has not been examined extensively so
23, 67
far.
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Page 14
To gain additional insight into the structures of 37 and 38, we obtained their circular
dichroism (CD) spectra in acetonitrile. The CD spectra of 37, and of 38 in particular,
contained very intense extrema (Figure 10), especially near 220 nm, which has been
23, 31
25, 31
associated with the presence of cis-amides
and helical peptoids in general.
Comparison of these spectra with those of the corresponding optically active model system
(S-26) reveals a striking length-dependent increase in ellipticity per amide, again suggestive
29
of a cooperatively folded secondary structure. In conjunction with the NMR data, these
findings establish that 1npe is highly effective for inducing local cis-amide conformations in
polypeptoids. Moreover, a reduction in the number of correlations in the HSQCAD NMR
(see Supporting Information) and concomitant increase in CD signal strength data with
increasing oligomer length, as observed in these systems, have been cited as evidence for
26
increasing conformational homogeneity as a function of oligomer length. Given that spe
23
pentamer 34 has been shown to be more structured than the analogous spe trimer, the
significantly higher (more than 6-fold) overall K
measured for 1npe trimer 38 as
cis/trans
compared to spe pentamer 34 (Table 6) suggests that incorporation of 1npe may permit the
construction of extremely robust peptoid helices. Indeed, the intensity of the CD signal at
37
220 nm for trimer 38 is more than twice that reported for pentamer 34 and its
23
enantiomer. In light of these data, a much higher degree of side chain diversity than was
previously possible in designed peptoid helices may be achievable by using the 1npe side
22
chain to reduce the number of residues dedicated to maintaining structural integrity.
Summary
This comprehensive study has revealed several new strategies for controlling peptoid amide
isomerism using noncovalent interactions, including n→π*, steric, and hydrogen bonding
interactions. These strategies were developed through the design, synthesis, and
characterization of small, model peptoid systems, which were shown to be largely predictive
of the rotameric equilibria of the backbone amides in N-α-chiral aromatic peptoid oligomers,
an important and often utilized class of peptoids. One of the most significant findings of this
study is that cis-amides in the peptoid backbone can be exclusively promoted by select N-α-
chiral acetanilide and N-1-naphthylethyl side chains, putatively by n →π *
/hydrogen
C=O
bonding interactions and n→π * /steric interactions, respectively. In the former case, the
Ar
relative polarizabilities of the side chain- versus backbone-amides appear to give rise to the
dramatic solvent effects observed, with nonpolar solvents reversing the cis-amide preference
in the peptoid backbone. This phenomenon evokes an intriguing new possibility for peptoid
structural design entailing the rational tuning of backbone- and side chain-amide electronics
to stabilize either cis- or trans-amides, potentially generating novel peptoid structures that
28
can also fold in a solvent-dependent manner. The subtle and solvent-dependent interplay
of n→π*, steric, and hydrogen bonding interactions in peptoids has not been previously
investigated; these findings could thus provide a framework for further development of new
peptoid design strategies.
The application of one of the new design tactics reported herein – the incorporation of N-1-
naphthylethyl side chains into peptoids – yielded peptoid oligomers that exhibited
unprecedented conformational homogeneity. In this context, the structure-promoting
elements presented here should prove especially useful for constructing well-folded peptoid
scaffolds with an expanded number of side chain sites available for rational or combinatorial
diversification. In addition to elucidating aspects of the regulation of peptoid amide
isomerism via noncovalent interactions, these findings may also be useful in other foldamer
regimes employing tertiary amides. As a whole, this work significantly expands the
available repertoire of peptoid design strategies, and should enable the construction of both
familiar and as of yet uncharacterized peptoid architectures.
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Gorske et al.
Page 15
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank the NSF (CHE-0449959), ONR (N000140710255), Burroughs Wellcome Fund, Research Corporation,
Dreyfus Foundation, Johnson & Johnson, and 3M for financial support of this work. H.E.B is an Alfred P. Sloan
Foundation fellow. S.A.F. acknowledges the ACS Division of Medicinal Chemistry for a pre-doctoral fellowship
(sponsored by Sanofi-Aventis) and Pfizer Global R&D for a Diversity in Organic Chemistry Fellowship. Support
for the NMR facilities at UW-Madison by the NIH (1 S10 RR13866-01 and 1 S10 RR08389-01) and the NSF
(CHE-0342998 and CHE-9629688) is gratefully acknowledged. We thank Professors Ronald Raines and Samuel
Gellman for many contributive discussions, and Dr. Grant Geske for computational experiments.
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Figure 1.
Comparison of the peptoid and α-peptide primary structures.
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Figure 2.
Left: The n→π* interaction (indicated by the red arrow) proposed to increase K
for
cis/trans
Ar
peptoid backbone amides. Right: Newman projection depicting the n→π* interaction.
Ar
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Figure 3.
Structures of the peptoid model systems (1–15) designed to probe n→π* interactions.
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Figure 4.
Left: Structures of model compounds 6 and 8. Right: Newman projections depicting the
most populated conformations for cis-6 and cis-8, as indicated by the NOEs shown:
red=stronger for 8 v. 6, black=identical for 6 and 8.
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Figure 5.
Left: The n→π*
interaction (indicated by the red arrow) proposed to reduce K
for
C=O
cis/trans
C=O
the donating amide in peptoids. Right: Newman projection depicting the n→π*
interaction
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Figure 6.
Structures of the peptoid model systems (16–26) designed to probe steric interactions.
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Figure 7.
Structures of the peptoid model systems (27–32) designed to probe hydrogen bonding
interactions.
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Figure 8.
Possible noncovalent interactions affecting K
for 27. A) Direct hydrogen bond (red
cis/trans
dashes) stabilizing the cis-acetamide. B) Backbone-side chain n→π*
interaction (green
C=O
arrow) stabilizing the cis-acetamide. C) Backbone-backbone n→π*
arrow) stabilizing the trans-acetamide.
interaction (blue
C=O
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Figure 9.
Structures of the polypeptoids (33–37) designed to examine noncovalent interactions in
peptoid oligomers.
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Page 27
Figure 10.
CD spectra of Ac-s1npe-Pip (S-26), Ac-(s1npe) -CONH (37), and Ac-(s1npe) -CONH
2
2
3
2
(38) at 60 μM concentration in acetonitrile. Spectra were collected at 25 °C.
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Scheme 1.
The generic synthetic routes (eqs 1 and 2) used in the construction of the peptoid model
1
systems. Reagents and conditions: a. 0.9 equiv. R NH , 1 equiv. triethylamine, CH Cl , 0
2
2
2
2
°C, 60 min. b. 0.85 equiv. R NH , DMF, 0–24 °C, 12 h. c. 4 equiv. (CH CO) O, 2.1 equiv.
2
3
2
(i-Pr) EtN, CH Cl , 25 °C, 30 min. d. 2.1 equiv. (CH CO) O, CH Cl , 25 °C, 30 min. e. 2
2
2
2
3
2
2
2
equiv. NaH, 1.2 equiv. CH I, DMF, 0 °C, 5 min. Details of representative syntheses of these
3
32
compound classes have been reported previously.
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Table 1
Structures, purities, and calculated and observed masses for peptoid pentamers 33–38.
a
b
c
observed mass
peptoid pentamer monomer sequence (amino to carboxy terminus)
% purity
calculated mass
909.4
932.1 [M+Na+]+
887.1 [M+Na+]+
1067.1 [M+Na+]+
1337.1 [M+Na+]+
504.1[M+Na+]+
715.4 [M+Na+]+
33
34
35
36
37
38
Ac-(spe)2-snp-(spe)2-CONH2
Ac-(spe)5-CONH2
95
99
99
99
99
98
864.5
Ac-(sfe-spe)2-spe-CONH2
Ac-(sfe)5-CONH2
1044.4
1314.2
Ac-(s1npe)2-CONH2
Ac-(s1npe)3-CONH2
481.2
692.3
a
b
c
See text for definitions of peptoid monomer abbreviations.
Determined by integration of the HPLC trace with UV detection at 220 nm.
Mass spectrometry data were acquired using either ESI or MALDI-TOF techniques.
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Table 2
Structure of the generic peptoid model system (top), and the structures and abbreviated names of the C-termini
(R ) and side chains (R ) examined in this study.a
1
2
a
Single stereocenters do not influence K
in these systems; enantiomeric excesses were therefore not determined. The cis-rotamer was
cis/trans
defined as that which orients the side chain and the oxygen atom on the same side of the amide bond.
J Am Chem Soc. Author manuscript; available in PMC 2011 September 18.