Cis -trans amide bond rotamers in β-peptoids and peptoids: Evaluation of stereoelectronic effects in backbone and side chains

Article abstract of DOI:10.1021/ja312532x

Non-natural peptide analogs have significant potential for the development of new materials and pharmacologically active ligands. One such architecture, the β-peptoids (N-alkyl-β-alanines), has found use in a variety of biologically active compounds but h

Full text of DOI:10.1021/ja312532x

Article
pubs.acs.org/JACS
Cis−Trans Amide Bond Rotamers in β‑Peptoids and Peptoids:
Evaluation of Stereoelectronic Effects in Backbone and Side Chains
Jonas S. Laursen, Jens Engel-Andreasen, Peter Fristrup, Pernille Harris, and Christian A. Olsen*
Department of Chemistry, Technical University of Denmark, Kemitorvet 207, DK-2800, Kongens Lyngby, Denmark
S
* Supporting Information
ABSTRACT: Non-natural peptide analogs have significant potential for
the development of new materials and pharmacologically active ligands.
One such architecture, the β-peptoids (N-alkyl-β-alanines), has found use in
a variety of biologically active compounds but has been sparsely studied
with respect to folding propensity. Thus, we here report an investigation of
the effect of structural variations on the cis−trans amide bond rotamer
equilibria in a selection of monomer model systems. In addition to various
side chain effects, which correlated well with previous studies of α-peptoids,
we present the synthesis and investigation of cis−trans isomerism in the first
examples of peptoids and β-peptoids containing thioamide bonds as well as
trifluoroacetylated peptoids and β-peptoids. These systems revealed an
increase in the preference for cis-amides as compared to their parent
compounds and thus provide novel strategies for affecting the folding of peptoid constructs. By using NMR spectroscopy, X-ray
crystallographic analysis, and density functional theory calculations, we present evidence for the presence of thioamide−aromatic
2
interactions through Csp −H···Samide hydrogen bonding, which stabilize certain peptoid conformations.
presence of tertiary backbone amide bonds gives rise to
increased flexibility due to a low-energy barrier between cis and
trans configurations. Thus, a high degree of cis-amide bonds
may occur in peptoids, which is almost exclusively observed at
proline in natural peptides and proteins (Figure 1B)5 and have
been enhanced by introduction of synthetic proline derivatives.6
The effect of various N-alkyl side chain functionalities on this
cis−trans equilibrium in peptoids has been studied by NMR
spectroscopy.7−10 Despite the inherent flexibility of peptoids,
secondary structures of oligomeric and cyclic peptoids have
been studied in some detail in solution by NMR spectrosco-
py11−13 and in the solid state by X-ray crystallography, and
some requirements for the formation of secondary peptoid
structure have been identified.13−16 For instance, the handed-
ness of a helical conformation depends on the enantiomeric
nature of α-chiral N-alkyl side chains, and the helix formation is
favored by the presence of bulky and aromatic substitu-
ents.11,16−18 Electronic n → π* interactions19 have also been
proposed to take part in the stabilization of secondary
structures of peptoids.8,9 These interactions involve donation
of a lone pair from a carbonyl oxygen atom into an empty π*
orbital of carbon atom of another carbonyl or an aromatic ring
INTRODUCTION
■
The 20 canonical α-amino acids constitute the fundamental set
of building blocks necessary for human ribosomal synthesis of
the major class of biopolymers comprised of proteins and
peptides. In traditional medicinal chemistry, this class of
compounds has not been considered suitable for drug
development, due to susceptibility to proteolytic degradation
in cellular environments and often poor cell permeability
properties. Nevertheless, recent tendencies in the pharmaceut-
ical industry have revealed an increased interest in the
development of so-called biologics. This may, at least in part,
be due to the successful approval and marketing of several
monoclonal antibodies as therapeutics during the past decade.
In order to circumvent the inherent stability problems,
however, extensive research in the field of peptidomimetic
designs has been undertaken. In addition to the nature of the
functional groups themselves, bioactive α-peptides realize their
high potency and selectivity due to stabilized secondary
structure formation, which displays these functionalities
accurately in three-dimensional space. Non-natural compounds
that are capable of adopting stabilized three-dimensional
structures mimicking or complementing those found in nature
are therefore of great interest, and as a class of compounds,
these various chemotypes have been coined “foldamers”.1 A
wide variety of foldamers have been developed and extensively
studied,2 with some of the prominent peptidomimetic examples
being β-peptides3 and peptoids (N-alkylglycines)4 (Figure 1A).
The tertiary amide backbone architecture in peptoids renders
them unable to stabilize putative folded structures by forming
intramolecular hydrogen-bond networks. Furthermore, the
(Figure 1C)20 and are optimal when mimicking the Burgi−
̈
Dunitz trajectory for nucleophilic attack.21 The β-peptides
(Figure 1A), on the other hand, retain the capability to form
intramolecular hydrogen-bond networks to stabilize secondary
Received: December 22, 2012
Published: January 23, 2013
© 2013 American Chemical Society
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an oligomer structure. In this way the effect of side chains may
be investigated with respect to steric and stereoelectronic
interactions. Furthermore, it was the scope of this work to
assess whether changes in the electronic properties of the
backbone would alter the conformational preferences of the
residues.
The first array of monomers was designed to include a
structurally diverse set of N-alkyl side chains accommodating
variations in steric bulk, α-branching, aromatic vs saturated
substituents and finally including an N-aryl substituent
(phenyl). The chosen set of eight different side chains (a−h)
was installed in two different monomer series: (1) 3a−h
containing a C-terminal ester functionality and (2) 4a−h
containing a C-terminal tertiary amide functionality thought to
better mimic the local environment of a single residue within an
oligomeric structure (Scheme 1A). The tert-butylester series
was prepared since these can be readily deprotected, which
allows for further coupling reactions as well as installation of
additional C-teminal functionalities. To probe the effects of the
various side chains on the rotameric preference of the β-peptoid
amide bond (the cis−trans equilibrium), acetyl groups were
installed to give N-terminal tertiary amides, as also investigated
in α-peptoid model systems.8−10 Analogous to those studies,
the trans−cis isomerism in our compounds could then be
1
determined by integration of the H NMR peaks assigned to
each rotamer.
Syntheses of the β-peptoid monomers were achieved by aza
Michael addition of a primary amine to acrylester (1) or
acrylamide (2) in MeOH,24 which has turned out to be an ideal
solvent for this tranformation as opposed to the originally
reported reactions in DMSO (Scheme 1A).23 This was
followed by acetylation to give the two series of monomer
model compounds (5a−h and 6a−h) for evaluation by NMR
spectroscopy.
In addition to the various N-alkyl side chains and differences
in C-terminal functionality, we were also interested in probing
the possibility of local n → π* interactions by altering the
electronic properties of amide carbonyls. The N-terminal
amides were therefore modified by introduction of trifluor-
oacetyl groups in place of the acetyl groups in selected
compounds (Scheme 1B). These were readily prepared from
3a,e,f and 4a,e,f by treatment with trifluoroacetic anhydride
(Scheme 1B).
Finally, we substituted carbonyl oxygen atoms with sulfur in a
selection of compounds to achieve introduction of minimal
peptide bond surrogates with altered electronic properties.27
Both amides in compounds 6a,e,f were individually mutated to
thioamides to give 10a,e,f and 12a,e,f, respectively (Scheme
1C,D). For their preparation, we utilized Lawesson’s reagent,28
which selectively converts amides to thioamides in the presence
of esters. Preparation of the C-terminal thioamides 10a,e,f,
were achieved by treating precursors 4a,e,f with Lawesson’s
reagent to give 9a,e,f, which were then acetylated to give the
target compounds (Scheme 1C). The N-terminal thioamides
12a,e, f, on the other hand, were synthesized by treating 5a,e,f
with Lawesson’s reagent to give 11a,e,f, followed by tert-
butylester cleavage and coupling to morpholine to yield the
target compounds (Scheme 1D). These changes were thus
quite efficiently introduced from common precursors to alter
the donor and acceptor capabilities of the two carbonyl groups.
NMR Spectroscopy of Acetylated Monomers. In order
to take possible solvent effects into consideration in our
evaluation of the monomers, we recorded NMR spectra in six
Figure 1. (A) Generic structures of the backbone architectures of
peptides (i.e., α-peptides), peptoids (i.e., α-peptoids), β3-peptides (β2-
and disubstituted β-peptides are not shown), and β-peptoids. (B)
Depiction of the equilibrium of trans- and cis-amide conformations in
proline and peptoid residues. (C) Examples of n → π* interactions
previously reported to exist in peptoids.8
structures, while the geometry of known helices is unlikely to
be stabilized by n → π* interactions.2,3
By combining the features of β-peptides and peptoids, the
ensembles of available foldameric scaffolds may be expanded
with β-peptoids, and several examples of biologically active
compounds containing this motif have been reported.22 The
structural properties of compounds with a β-peptoid backbone
architecture, however, have been studied to a far lesser extent
than its parent compounds since the first examples were
reported by Hamper et al. in 1998.23 The first three-
dimensional structure of a β-peptoid, which was achieved for
a cyclic tetramer, was thus reported by Taillefumier and co-
workers in 2008.24 Computational studies of linear oligomeric
β-peptoids have predicted several possible helical conforma-
tions,25 containing both the cis- and trans-amides, but studies
based on circular dichroism (CD) spectroscopy have been
inconclusive.26 To obtain experimental data regarding the
folding propensity of these molecules, we decided to prepare a
series of β-peptoid monomers and evaluate the structural
influence on cis−trans amide bond isomerization by NMR
spectroscopy under various conditions. Our collection of model
compounds was designed to investigate how stereoelectronic
effects and substituent bulk affect the conformational
preferences of β-peptoid monomers.
RESULTS AND DISCUSSION
■
Design and Synthesis. All our model compounds were
based on acylated β-peptoid monomers. This minimal design
was chosen to mimic the local interactions of a residue within
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a
Scheme 1. Synthesis of β-Peptoid Model Compounds
a
(A) Acetylated monomers 5a−h and 6a−h. Reagents and conditions: (a) MeOH, 50 °C, 16 h; (b) (i) for esters Ac2O (2 equiv), pyridine (2 equiv),
DMF, 0 °C → rt, 4 h or (ii) for amides AcCl (2 equiv), pyridine (2 equiv), DMF, CH2Cl2, 0 °C, 1 h. (B) Synthesis of trifluoroacetylated monomers
7a,e,f and 8a,e,f. Reagents and conditions: (c) Trifluoroacetic anhydride (2 equiv), pyridine (2 equiv), CH2Cl2, 0 °C, 4 h. (C and D) Synthesis of
thioamide-containing β-peptoid monomeric model compounds. Reagents and conditions: (d) Lawesson’s reagent (1.5 equiv), toluene, 110 °C, 3 h;
(e) AcCl (2 equiv), i-Pr2NEt (2 equiv), CH2Cl2, 0 °C, 3 h; (f) Lawesson’s reagent (0.6 equiv), toluene, 110 °C, 1 h; (g) 1 M LiOHaq−DMF 1:1, rt,
16 h; (h) morpholine (2 equiv), HBTU (2 equiv), i-Pr2NEt (2 equiv) CH2Cl2, rt, 16 h. (E) Abbreviations for N-alkyl side chains used are as follows:
spe = (S)-1-phenylethyl, sce = (S)-1-cyclohexylethyl, bn = benzyl, sbu = sec-butyl, s1npe = (S)-1-(1-naphthyl)ethyl, npm = 1-naphthylmethyl, bnz =
benzhydryl, ph = phenyl.
a
Table 1. Rotamer Equilibrium Constants (Kcis/trans) for Acetylated β-Peptoid Monomers in Various Solvents and Their
b
Corresponding Differences in Free Energy (ΔG values given in kJ × mol−1)
D2O
Kcis/trans
DMSO-d6
Kcis/trans
CD3OD
Kcis/trans
C-terminal esters
CD3CN
Kcis/trans
CDCl3
Kcis/trans
C6D6
Kcis/trans
compd
side chain
ΔG
ΔG
ΔG
ΔG
ΔG
ΔG
5a
5b
5c
5d
5e
5f
spe
sce
0.8
0.5
1.0
0.5
0.5
1.7
0
0.8
0.6
0.5
1.2
0.3
1.7
−3.1
0.9
0.3
−
0.7
0.4
0.9
2.2
0.3
2.2
−4.2
0.3
1.2
−
0.8
0.5
0.5
1.7
0.3
2.2
−3.1
0.9
0.3
−
0.9
0.4
0.3
2.2
0.9
1.7
−4.1
0.3
0.9
−
0.8
0.5
0.5
1.7
0.9
2.2
−4.5
0.3
1.7
−
bn
0.9
0.9
0.9
0.7
0.7
sbu
1.7
0.5
0.4
0.4
0.5
0.4
c
s1npe
npm
bnz
ph
n.s.
n.s.
n.s.
−
−
−
3.9
3.6
5.6
3.6
5.3
6.3
c
c
0.7
0.9
0.7
0.9
0.9
5g
5h
0.9
0.6
0.9
0.7
0.5
0.2
all trans
all trans
all trans
all trans
all trans
C-terminal amides
6a
6b
6c
6d
6e
6f
spe
sce
0.7
0.3
1.0
0.2
2.9
0.9
0.8
0.9
2.9
0
0.8
0.5
0.5
1.7
0.3
1.7
−2.7
1.2
0.3
−
0.5
0.3
1.7
2.9
0.9
3.9
−2.7
0.3
1.7
−
0.8
0.4
0.5
2.2
0.3
2.2
−2.8
0.5
0.3
−
0.4
0.4
2.2
2.2
2.9
5.6
−2.6
2.2
2.2
−
0.4
0.3
0.3
0.1
3.5
0.5
0.5
2.2
2.9
2.9
5.6
−3.1
1.7
1.7
−
bn
0.9
0.7
0.9
0.3
sbu
3.9
−2.6
0.3
0.5
−
0.5
0.2
0.4
0.1
s1npe
npm
bnz
ph
3.0
3.0
3.1
2.9
0.6
0.9
0.8
0.4
6g
0.9
0.5
0.9
0.4
c
6h
n.s.
all trans
all trans
all trans
all trans
all trans
a
b
c
Determined by integration of 1H NMR spectra of 12 mM compound solutions at ambient temperature. ΔG = −RT × ln(Kcis/trans). Not soluble.
different deuterated solvents of varying polarities (Table 1).
First, we looked at compound 5a containing the (S)-1-
phenylethyl side chain, which is one of the most well-studied
functionalities with respect to folding propensity of α-
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a
Table 2. Rotamer Equilibrium Constants (Kcis/trans) for Trifluoroacetylated β-Peptoid Monomers in Various Solvents and Their
b
Corresponding Differences in Free Energy (ΔG values given in kJ × mol−1)
D2O
Kcis/trans
DMSO-d6
CD3OD
Kcis/trans
C-terminal esters
CD3CN
Kcis/trans
CDCl3
Kcis/trans
C6D6
Kcis/trans
compd
side chain
ΔG
Kcis/trans
ΔG
ΔG
ΔG
ΔG
ΔG
c
c
c
7a
7e
7f
spe
s1npe
npm
n.s.
n.s.
n.s.
−
−
−
0.4
6.8
0.8
2.2
−4.7
0.5
0.3
6.3
0.8
2.9
−4.3
0.5
0.4
6.6
0.8
2.2
−4.6
0.5
0.3
6.3
0.9
2.9
−4.3
0.3
0.3
6.3
1.0
2.9
−4.3
0
C-terminal amides
8a
8e
8f
spe
s1npe
npm
0.4
2.2
−
−
0.4
5.6
0.8
2.2
−4.2
0.5
0.3
5.0
1.0
2.9
−3.9
0
0.4
5.5
0.8
2.2
−4.1
0.5
0.2
4.8
0.7
3.9
−3.8
0.9
0.2
4.5
0.6
3.9
−3.7
1.2
c
n.s.
c
n.s.
a
b
c
Determined by integration of 1H NMR spectra of 12 mM compound solutions at ambient temperature. ΔG = −RT × ln(Kcis/trans). Not soluble.
peptoids17 and has been studied briefly by CD spectroscopy in
β-peptoids.26 This model β-peptoid exhibited a slight
preference for the trans-amide configuration without any
notable solvent effect, and expectedly, we further showed that
the chiral identity had no influence on the conformational
distribution, by synthesizing the corresponding racemate and
the (R)-enantiomer (see Figure S1Supporting Information). As
no significant effects of the concentration on the Kcis/trans had
previously been reported for peptoids,9 we gratifyingly found
that to be true for β-peptoid solutions in CDCl3 between 6−
200 mM as well (Figure S2). Finally, we performed rotating
frame Overhauser effect spectroscopy (ROESY) experiments
on selected compounds (e.g., see Figure S3) in order to show
that the methyne group exhibiting the most downfield chemical
shift arises from the cis-amide conformation, which is in
agreement with previously published peptoid studies.8−10
Next, we compared the Kcis/trans data for 5a with those
obtained for 5b, which is a nonaromatic, fully saturated version
of 5a. The loss of aromaticity with only a slight increase in
steric bulk gave rise to an increase in the preference for the
trans conformation. This, in turn, indicates that the aromatic
moiety may cause a slight shift toward the cis-amide, which is
consistent with previous findings reported by Blackwell and co-
workers for peptoids.9 Likewise, this was also the case when we
decreased the steric congestion of the side chain by introducing
a benzyl group (5c), as the recorded Kcis/trans values were
comparable to those of 5a in all the tested solvents. We then
evaluated a combination of decrease in steric bulk further and
removal of aromaticity using the sec-butyl side chain (5d).
Similar equilibrium constants were observed for 5b and 5d,
which was in accordance with α-peptoid findings,9 as was the
significant preference for cis-amide configuration induced by the
(S)-1-(1-naphthyl)ethyl side chain (5e).15
Interestingly, introduction of a 1-naphtylmethyl side chain
(5f) resulted in approximately a 1:1 mixture of rotamers as
observed for 5a and 5b, showing that the naphthyl group itself
is not sufficient to induce a predominant amount of the cis-
amide. Thus, it would seem that the lack of α-branching enables
the naphthyl group to avoid structure inducing steric
interactions. Furthermore, we altered the bulk of the side
chain by introducing the disubstituted benzhydryl group (5g),
which, perhaps somewhat suprisingly, also gave rise to similar
Kcis/trans values as found for compound 5a. This shows that α-
branching only in combination with a very bulky group will
promote induction of the cis-conformation, which apparently is
uniquely well represented in the (S)-1-(1-naphthyl)-ethyl side
chain. However, alternative constructs, taking advantage of a
putative n → π*aryl interaction (Figure 1C) by introducing
electron-deficient aromatic substituents instead of increasing
the steric bulk, have been reported for peptoids as well.9,29
Finally, peptoid studies have also shown that direct
attachment of a phenyl group to the nitrogen atom (i.e.,
prepared from aniline subunits) leads to a very strong
preference for trans-amides.13 A single β-peptoid model system
of this type (5h) was evaluated and exhibited the expected
selectivity, by virtually giving rise to single sets of signals in all
1
tested solvents when analyzed by H NMR. As mentioned, we
also evaluated the entire series of side chains a−h in model
systems having C-terminal amides (6a−6h) instead of esters, to
mimic the environment of a β-peptoid residue within an
oligomer more appropriately. The data are shown in the lower
panel of Table 1, and inspection of the results reveals the same
trends as discussed for the C-terminal tert-butylesters.
Taken together, our side chain investigations indicate that
there may be a slight intrinsic preference for the trans-amide
rotamer in β-peptoid model systems (5d and 6d), which is in
agreement with results from peptoids containing methyl or
ethyl side chains.9 The equilibrium then shifts toward the cis-
amide rotamer to approximately 1:1 mixtures when adding
aromatic functionalities as substituents in the α-position of the
side chains. Based on the results of 5a vs 5b as well as 6a vs 6b
(in the polar solvents), it seems plausible that an n → π*aryl
interaction could play a role. Though, this interaction, in the
case of a phenyl or naphthyl group, is too weak to induce the
cis-amide as the preferred conformation. However, it is not
possible to unambiguously attribute this effect of the
aromaticity on Kcis/trans to an n → π*aryl interaction based on
our side chain experiments alone. Furthermore, the only
examples of a strong preference for the cis-amide required quite
specific steric properties of the side chain (5e and 6e). In an
attempt to gain further insight regarding possible stereo-
electronic effects on Kcis/trans in β-peptoids, we turned our
attention to model systems containing carbonyls with altered
electronic properties.
NMR Spectroscopy of Trifluoroacetylated Analogs. As
we found that the β-peptoid model systems display the same
behavior as peptoids upon side chain substitutions, we turned
our attention to backbone modifications. Examples of such
investigations have been reported for proline but have not, to
the best of our knowledge, been utilized for interrogation of
peptoid structure and conformational preference.
Noncovalent n → π*amide (Figure 1C) interactions contribute
to stabilization of protein secondary structures30 and have been
studied extensively in relation to collagen polyproline type-II
helical conformations.31 The presence of this type of interaction
has also been suggested in certain peptoid model systems.8
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a
Table 3. Rotamer Equilibrium Constants (Kcis/trans) for Thioamide-Containing β-Peptoid Monomers in Various Solvents and
b
Their Corresponding Differences in Free Energy (ΔG values given in kJ × mol−1)
D2O
Kcis/trans
0.4
DMSO-d6
CD3OD
Kcis/trans
CD3CN
Kcis/trans
CDCl3
Kcis/trans
C6D6
Kcis/trans
compd
side chain
ΔG
Kcis/trans
ΔG
ΔG
ΔG
ΔG
ΔG
10a
10e
10f
12a
12e
12f
spe
s1npe
npm
spe
2.2
−
−
0.5
2.0
0.9
0.9
5.4
1.0
1.7
−1.7
0.3
0.5
3.4
0.8
0.4
4.7
0.6
1.7
−3.0
0.5
0.5
2.8
0.9
0.7
4.5
0.9
1.7
−2.5
0.3
0.2
2.2
0.3
0.5
3.3
0.3
3.9
−1.9
2.9
0.2
2.6
0.3
0.5
5.2
0.3
3.9
−2.3
2.9
c
n.s.
n.s.
c
0.7
0.9
0.3
2.2
0.9
1.7
1.7
c
s1npe
n.s.
−
−
−4.1
0
−3.8
1.2
−3.7
0.3
−2.9
2.9
−4.0
2.9
c
npm
n.s.
a
b
c
Determined by integration of 1H NMR spectra of 12 mM compound solutions at ambient temperature. ΔG = −RT × ln(Kcis/trans). Not soluble.
Although such interactions would not be expected to have a
stabilizing effect on β-peptoid secondary structure due to
unfavorable geometry,25 we were interested in testing whether
the Kcis/trans values in our model systems were sensitive to this
type of interaction.
NMR Spectroscopy of Thioamide Analogs. Inspired by
another study of prolines by Raines and co-workers,27 we next
altered the carbonyl-donor capabilities by individually sub-
stituting the oxygen atoms with sulfur to increase the
“nucleophilicity”. If any carbonyl−carbonyl interactions (in
the N → C or C → N directionality) were to be playing a
significant role on the β-peptoid conforms, these sulfur
substitutions should give rise to differences in the Kcis/trans
values as compared to the corresponding oxygen-containing
compounds. Evaluating first the thioacetylated compounds
(12a,e,f), we found that they behaved similar to the acetylated
compounds. The only difference was observed in the (S)-1-(1-
napthyl)ethyl system (12e), which showed increased fractions
of the cis-amide. This would indicate that the sulfur is
interacting with the aromatic ring rather than the C-terminal
carbonyl. On the other hand, substitution of the C-terminal
oxygen atom with sulfur (10a,e,f) resulted in Kcis/trans values
very similar to those recorded for their acetylated parent
monomers (6a,e,f) in all cases (Table 3). This indicates that an
n → π*amide interaction in the C → N directionality, which in
theory should stabilize the cis configuration, is highly unlikely.
These are the first examples of thioamides in peptoids, and our
results show that this minimal amide bond surrogate may be
valuable for interrogation of higher oligomers and possibly also
in N-alkylglycine-based peptoids.
First we reasoned that substitution of the N-terminal acetyl
group for a trifluoroacetyl group would significantly alter the
electronic properties of the carbonyl through the strong
inductive electron-withdrawing effect of fluorine. This should
thus decrease the electronegativity of the N-terminal carbonyl,
which would render this position weaker as donor of a lone pair
from oxygen, whereas the carbonyl carbon atom would become
a better acceptor. Since the alkyl side chains exhibited
disfavoring of the cis-conformer, we chose to investigate
trifluoroacetylated analogs containing aromatic side chains
exclusively. We thus evaluated model compounds containing
(S)-1-phenylethyl (7a), (S)-1-(1-napthyl)-ethyl (7e), and
naphtylmethyl (7f) side chains (Table 2).
Surprisingly, at first glance, opposite effects were observed for
7a vs 5a and 7e vs 5e with a decrease and an increase in
Kcis/trans, respectively, while compounds 7f and 5f behaved alike
each other. Retrospectively, however, we hypothesize that a
decrease of the cis-amide fraction in the (S)-1-phenylethyl
system may in fact be explained by a weakened n → π*aryl
interaction, whereas the opposite trend in the (S)-1-(1-
napthyl)ethyl system is most likely of entirely steric nature.
The trifluoromethyl group is more sterically demanding than
the methyl, which may indeed be of particular significance in
the already congested amide bond of 7e. In support of this
hypothesis, we recently became aware of a study by Raines and
co-workers, in which it was shown that the rotamer equilibrium
of a trifluoroacetylated proline derivative was governed by
sterics, while the corresponding mono- and difluorinated
analogs were affected by the electron-withdrawing inductive
effect of fluorine.29 It was also suggested by Raines and co-
workers that fluorine may act as donor of an electron pair to an
antibonding π* orbital of the adjacent carbonyl, which would
then results in the opposite of the anticipated inductive effect.
Such interactions are indeed precedented in the literature, for
example, by using molecular torsion balance double mutant
systems.32
Peptoids. To address the effects of fluorination or
thioamide introduction in peptoids as well, we finally prepared
compounds 13−15 (Chart 1). These syntheses were achieved
by applying published methods for solution-phase peptoid
Chart 1. Structures and Kcis/trans Values for the Investigated
N-Alkylglycine Peptoids
We also tested the morpholine analogs (8a,e,f), and again
these exhibited trends that were similar to the tert-butylesters.
Thus, it seems unlikely that the C-terminal carbonyl should be
involved in the stabilization of monomer conformations.
Although we are not able to propose unequivocal guidelines
for the effects of introducing fluorine atoms in peptoid or
peptide backbones, we believe that this could prove to be a
useful addition to the arsenal of strategies for future design of
peptide mimics.
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Figure 2. Solid-state structures of compound 5g (A and B) and compound 8e (C−E) determined by X-ray crystallography. Atom coloring: gray,
carbon; white, hydrogen; red, oxygen; blue, nitrogen; and turquoise, fluorine. Green arrows indicate N → C directionality (D), and the dashed
magenta colored line indicates an edge to face aromatic interaction (E). The hydrogen atoms have been removed for clarity in D and E.
synthesis33 in combination with the protocols described for β-
peptoid functionalization vide supra (Scheme S1).
interactions (Figure 2E), the presence of n → π*aryl interactions
in solution cannot be definitively excluded.
The X-ray crystal structure of the N-terminal thioamide
peptoid analog 15 also revealed the presence of a cis-amide
configuration, as would be expected from the NMR data
(Figure 3). The distance between the C-terminal carbonyl and
Compound 13, which has been investigated previously,
exhibited the same Kcis/trans values as reported in CD3CN and
CDCl3 9 and an intermediate value in DMSO-d6, suggesting the
presence of a solvent effect in this system. Comparing these
values to the ones obtained for β-peptoid 8e revealed a similarly
lowered Kcis/trans value in CD3CN as compared to the other
tested solvents (Table 1). For compound 14, an even higher
preference for the cis-amide conformation was observed, and
this was affected to a much lesser extent by a change in the
solvent polarity. In analogy to the arguments presented for the
trifluoroacetylated β-peptoids, we hypothesize that this
equilibrium is primarily dictated by sterics, but also note that
the additional stabilization of the cis-amide conformation in the
DMSO
peptoid (e.g.,
K
cis/trans
for 14 vs 8e = 7.1 and 5.6,
respectively) may involve the aforementioned possibility of an
interaction between fluorine and the C-terminal carbonyl.
However, compelling evidence for the latter point would
require further experimentation.
Finally, the thioamide analog 15, like β-peptoid 12e,
exhibited higher Kcis/trans values than its oxoamide analog
(13) in polar solvents, and a significant decrease in the cis-
amide fraction in CDCl3 (Chart 1). This again indicates that
there is an interaction between the sulfur and the aromatic
residue, which results in favoring of the cis-amide conformation.
X-ray Crystallography. We were able to obtain diffraction
quality crystals for two β-peptoids, 5g and 8e, by slow
evaporation of chloroform solutions as well as the peptoid 15
by slow evaporation from an AcOEt solution. Thus, the solid-
state crystal structures of these model compounds were solved
by X-ray structure determination. The structure of 5g revealed
an extended backbone conformation (Figure 2A) with trans-
amide configuration, which is consistent with the obtained
Figure 3. Solid-state structure of compound 15 determined by X-ray
crystallography. Stick representations showing the COi+1···CiS
distance in green (A) and the distances between sulfur and its two
closest hydrogen atoms in magenta (B). Space-filling representations
showing hydrophobic packing of the naphthyl and the piperidine
groups (C) and (D). The hydrogen atoms have been removed for
clarity in A and B (except for the two hydrogens in close proximity to
sulfur).
the carbon of the thioamide is consistent with the presence of
an n → π*amide interaction (Figure 3A),19,30 which may explain
the higher Kcis/trans values recorded for the glycine-based
peptoids compared to the β-alanine-based peptoids. As was also
the case for compound 8e, the solid-state structure did not
provide any evidence of an n → π*aryl interaction. Interestingly,
however, the distance between one of the naphthyl hydrogen
atoms and the sulfur shown in Figure 3B (2.9 Å) is consistent
with an overlap of their orbitals to give rise to an aromatic C−
H···Samide interaction. This could offer an alternative explan-
ation of the stabilizing effect on the cis-amide conformation
obtained by introduction of the N-terminal thioamide
functionality. In order to shed more light on the identity of
the putative noncovalent carbonyl−aryl interaction in this
system, we performed density functional theory (DFT)
calculations on selected compounds (vide infra). We also
Kcis/trans = 0.7 in CDCl3. Notably, the two phenyl groups adopt
a periplanar relationship and are both pointed away from the
acetyl CH3 group (Figure 2B), which may also explain the
relatively high trans-amide ratio observed in solution despite its
significant steric bulk.
Compound 8e adopted the cis-amide conformation in the
solid state (Figure 2C), as would also be expected judging from
its Kcis/trans values. We had suspected that the attenuated
electron lone-pair donor capabilities of the N-terminal carbonyl
in the trifluoroacetylated compounds compared to acetylated
analogs would result in a decreased n → π*aryl effect. The
crystal structure of 8e shows no such interaction, and there are
no signs of fluorine−carbonyl interactions either. However, due
to the very dense crystal packing with antiparallel β-peptoid
backbones (Figure 2D) and edge to face aromatic π−π
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note that the proximity of the side chain methyne hydrogen,
and the carbonyl in this crystal structure (2.5 Å) as well as in
the structure of 8e described above are consistent with the
downfield shift observed in 1H NMR for this proton in the cis-
amide conformations.
stabilizing interaction between the sulfur and the naphthyl
group. This is again consistent with the trends of Kcis/trans
observed by NMR, and the preferred geometry is the same as
we found in the solid-state for compound 15 revealing close
proximity of the proton in position eight of the naphthyl
functionality with the carbonyl (Figure 4A,C).
Evidence for Aromatic C−H···Samide Interactions. To
gain further insight into the molecular features responsible for a
C−H···Samide interaction and its effect on the observed
preference for the cis-amide configuration in the presence of
the (S)-1-(1-napthyl)ethyl side chain, a computational study
was carried out. Initially, the peptoids (6e, 8e, 12e, 13−15)
were built in either the cis or the trans configuration and
subjected to a conformational search running 1000 steps using
the OPLS-2005 force-field34 and a GB/SA solvation model35
for water as incorporated in Macromodel version 9.6.36 The cis-
or trans-amide conformations were retained by applying a
constraint of 100 kJ × mol−1 × radian−2 to those particular
dihedral angles. Furthermore, to prevent irrelevant rotamers of
the morpholine headgroup to appear in the conformational
search, additional dihedral constraints were applied to the N-
terminal part of the molecules. The conformational search was
carried out using a combination of Monte Carlo multiple
minimum (MCMM) algorithm37,38 and the “Low-Mode”
search algorithm,39 with an energy window of 21 kJ × mol−1.
After this initial conformational search all of the generated
conformations were submitted to a further optimization with
DFT using the B3LYP functional.40 We used the 6-31G* basis
set41 along with the polarized continuum solvent model (PCM-
SCRF)42 with parameters suitable for water.
To further investigate the electronic properties responsible
for the observed cis-amide preference in the thioamide series,
we carried out natural bond order (NBO) analyses.43 By
inclusion of the trifluoroacetylated compounds 8e and 14 we
would be able to pinpoint the effect of this substitution in both
peptoid and β-peptoid backbones. For this purpose, super-
imposable, low-energy conformations of both cis- and trans-
isomers of 6e, 8e, 12e, 13−15 were selected. When comparing
the two cis-conformations of 6e and 12e, it is notable that while
the longer CS compared to CO (1.7 vs 1.2 Å) caused the
distance to the hydrogen of the naphtyl group to increase from
2.9 to 3.2 Å, the NBO analysis clearly showed that the
interaction is stronger in the thioamide case.
First of all, the natural charge on the aromatic hydrogen in
the thioamide (12e) is lower than in the amide compound
(0.2436 au for 12e vs 0.2455 au for 6e), although both
hydrogens are more electron deficient than their neighboring
hydrogen, which does not have such intramolecular interactions
(0.2503 au for 12e and 0.2487 au for 6e). In addition, second-
order perturbation analyses of 12e and 6e revealed calculated
stabilizing energies of this interaction to be 0.86 kcal × mol−1
and below the 0.5 kcal × mol−1 threshold, respectively.
In the trifluoroacetylated compound 8e, the amide oxygen is
less negatively charged as expected (−0.657 au in 8e vs −0.716
au in 6e). As a consequence, the electrostatic interaction with
the naphthyl hydrogen is expected to be even smaller than for
6e, however, in this case it is also below the threshold of 0.5
kcal × mol−1. This suggests that the increased cis−trans ratio
upon change of methyl to trifluoromethyl likely is caused by the
increased steric congestion of the larger fluorine atoms rather
than arising from an increased electrophilicity of the amide
carbonyl carbon. Finally, the three trans configured structures
featured a fully extended backbone with neither n → π*amide
nor electrostatic C−H···Samide interactions.
The lowest energy conformations of both 6e and 12e
contained the cis-amide configuration in agreement with our
Kcis/trans data from NMR as well as the X-ray diffraction data
(Figure 4). Notably, when visualizing the ensemble of
conformations with energies within 21 kJ × mol−1 (Figure
4B,D), the more homogeneous positioning of the N-alkyl side
chain in the thioamide analog indicates that there may be a
Next, we turned our attention to the peptoid series (13−15)
where the closer proximity of the other carbonyl group may
allow for the possibility of n → π*amide interactions in addition
to the electrostatic C−H···Samide interaction. For all of these
compounds, the C−H···Samide interaction shows up in the
second-order perturbation analysis part of the NBO analysis,
and it is only slightly stronger for the thioamide 15 (0.63 kcal ×
mol−1) compared to the amide 13 (0.58 kcal × mol−1). For the
trifluoroacetylated peptoid 14, the value is even higher at 0.65
kcal × mol−1, but the small energies considered, these
differences may well be within the inaccuracy of the method.
These effects on peptoid structure are currently under further
investigation in our laboratories.
A comparison of the chemical shifts assigned to the naphthyl
H-8 hydrogen in the cis-amide conformations of compounds
with altered electronic properties of the carbonyl support the
presence of the proposed interaction in solution as well (Figure
5). Thus, attenuation of the electron density of the oxygen by
introduction of fluorine atoms should render the hydrogen less
shielded and cause an upfield shift of the signal, which was
indeed what the spectra showed (6e vs 8e). Substitution of
oxygen with sulfur (6e vs 12e) should in principle affect this
putative interaction in the same manner. However, the opposite
Figure 4. Calculated structures of compounds 6e (A and B) and 12e
(C and D). All structures within 21 kJ × mol−1 of the global minimum
were superimposed.
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analysis, this structure indicated the presence of a stabilizing
2
effect through thioamide−aromatic interactions by Csp −
H···Samide “hydrogen bonds”. Whereas aromatic−sulfur inter-
actions have been described for proteins as well as in other
systems,45 the present work, to the best of our knowledge,
provides evidence for the first examples of intramolecular
conformation-stabilizing effects by introduction of thioamides,
which is in contrast to the destabilizing effect of thioamide
introduction in α-helical peptides.46
Importantly, this work shows that minimal peptide bond
surrogates like thioamides as well as fluorinated backbone
analogs are useful for investigation of peptoid and β-peptoid
structure. These modifications should therefore be considered
valuable for other types of peptidomimetics as well.
Thioamides, in particular, have recently found use in peptide
ligands and have been site-specifically introduced into proteins
to probe folding.47 We envision that the straight forward
methodology presented herein may encourage further studies
of thioamide-containing peptoid and β-peptoid oligomeric
systems.
Figure 5. Aromatic region of the 1H NMR spectra of 6e, 8e, and 12e
recorded in CD3CN. The asterisk denotes the signal of the position
eight proton of the naphthyl group (see, Figure S5 for signal
assignments by COSY and HMBC NMR). The same trend was
observed when comparing compounds 13−15 (data not shown).
effect was observed with a downfield shift of the signal (Figure
5), which gratifyingly is consistent with the calculated
ensembles and the Kcis/trans values that indicate a stronger
interaction for sulfur. Although hydrogen bonds to oxoamides
should be stronger than thioamides, we speculate that the
geometric restraint required for formation of the eight-
membered ring in our system does not allow for an optimal
hydrogen-bond distance, and therefore the larger radius of the
sulfur enables a higher degree of orbital overlap than the
oxygen. This is supported by the NBO analysis on the β-
peptoids 6e and 12e (see above). Additionally, the difference in
polarizability of thioamides as compared to oxoamides may play
a role44 and could also provide arguments to help explain the
solvent effects observed on Kcis/trans for some thioamide
compounds (vide supra).44a
ASSOCIATED CONTENT
* Supporting Information
■
S
Supplementary figures , experimental methods, charaterization
data, 1H NMR and 13C NMR spectra for all synthesized
compounds, selected 2D NMR spectra, and crystallographic
data (CIF). Coordinates (X, Y, Z) and solution phase SCF
energies for global minimum found in each conformational
search along with most favorable structure calculated using
DFT/B3LYP, and tables of data from the NBO analyses. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
cao@kemi.dtu.dk
■
CONCLUSIONS
■
To get a better understanding of the amide bond isomerization
in peptoids, we have synthesized and evaluated several series of
monomer β-peptoid model systems with varying electronic and
steric properties as well as two novel N-alkylglycine (peptoid)
model compounds containing a trifluoroacetyl group or an N-
terminal thioamide, respectively. Our studies show that some of
the trends found in peptoids are directly applicable to β-
peptoids. As such, the (S)-1-(1-napthyl)ethyl side chain
strongly induces the cis-amide conformation, while N-aryl
gives rise to trans. We thus found that a bulky substituent like
naphthyl in combination with α-branching is required for a cis-
amide preference, as a diphenyl-substituted benzhydryl side
chain was not sufficiently sterically demanding. In addition to
the investigation of various side chain effects, we prepared
model systems containing trifluoroacetyl groups as well as
thioamides to probe the electronic effects of the carbonyl
donor−acceptor capabilities. The NMR-based studies of these
compounds provided evidence for an interaction of the N-
terminal carbonyl/thiocarbonyl lone pair with the aromatic side
chain, but we saw no evidence for conformational stabilization
through noncovalent carbonyl−carbonyl interactions. The X-
ray crystal structures of two β-peptoid model compounds were
solved, which revealed one trans- and one cis-amide,
respectively. Those rotamer conformations were both in
agreement with the NMR experiments.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Ms. Anne Hector and Dr. Charlotte H. Gotfredsen
for assistance with NMR spectroscopy and Ms. Tina Gustafsson
for technical assistance with UPLC-MS and HRMS. This work
was supported by funds from DTU Chemistry and the Danish
Independent Research Council, Natural Sciences (Steno grant
no. 10-080907, C.A.O.). C.A.O. is a Lundbeck Foundation
Fellow.
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