Synthesis and Characterization of Nitroaromatic Peptoids: Fine Tuning Peptoid Secondary Structure through Monomer Position and Functionality

Article abstract of DOI:10.1021/jo8023363

N-Substituted glycine oligomers, or peptoids, have emerged as an important class of foldamers for the study of biomolecular interactions and for potential use as therapeutic agents. However, the design of peptoids with well-defined conformations a priori remains a formidable challenge. New approaches are required to address this problem, and the systematic study of the role of individual monomer units in the global peptoid folding process represents one strategy. Here, we report our efforts toward this approach through the design, synthesis, and characterization of peptoids containing nitroaromatic monomer units. This work required the synthesis of a new chiral amine building block, (S)-1-(2-nitrophenyl)ethanamine (s2ne), which could be readily installed into peptoids using standard solid-phase peptoid synthesis techniques. We designed a series of peptoid nonamers that allowed us to probe the effects of this relatively electron-deficient and sterically encumbered a-chiral side chain on peptoid structure, namely, the peptoid threaded loop and helix. Circular dichroism spectroscopy of the peptoids revealed that the nitroaromatic monomer has a significant effect on peptoid secondary structure. Specifically, the threaded loop structure was disrupted in a nonamer containing alternating V-(S)-1-phenylethylglycine (VVspe) and VVs2ne monomers, and the major conformation was helical instead. Indeed, placement of a single Ns2ne at the N-terminal position of (VVspe)₉ resulted in a destabilized form of the threaded loop structure relative to the homononamer (VVspe)9. Conversely, we observed that incorporation of V-(S)-1-(4-nitrophenyl)ethylglycine (VVsnp, a p-nitro monomer) at the VV-terminal position stabilized the threaded loop structure relative to (VVspe)₉. Additional experiments revealed that nitroaromatic side chains can influence peptoid nonamer folding by modulating the strength of key intramolecular hydrogen bonds in the peptoid threaded loop structure. Steric interactions were also implicated for the VVs2ne monomer. Overall, this study provides further evidence that aromatic side-chain structure, even if perturbed in a single monomer unit, can strongly influence local peptoid backbone conformation.

Full text of DOI:10.1021/jo8023363

Synthesis and Characterization of Nitroaromatic Peptoids: Fine
Tuning Peptoid Secondary Structure through Monomer Position
and Functionality
Sarah A. Fowler, Rinrada Luechapanichkul, and Helen E. Blackwell*
Department of Chemistry, UniVersity of WisconsinsMadison, 1101 UniVersity AVenue, Madison,
Wisconsin 53706-1322
blackwell@chem.wisc.edu
ReceiVed October 22, 2008
N-Substituted glycine oligomers, or peptoids, have emerged as an important class of foldamers for the
study of biomolecular interactions and for potential use as therapeutic agents. However, the design of
peptoids with well-defined conformations a priori remains a formidable challenge. New approaches are
required to address this problem, and the systematic study of the role of individual monomer units in the
global peptoid folding process represents one strategy. Here, we report our efforts toward this approach
through the design, synthesis, and characterization of peptoids containing nitroaromatic monomer units.
This work required the synthesis of a new chiral amine building block, (S)-1-(2-nitrophenyl)ethanamine
(s2ne), which could be readily installed into peptoids using standard solid-phase peptoid synthesis
techniques. We designed a series of peptoid nonamers that allowed us to probe the effects of this relatively
electron-deficient and sterically encumbered R-chiral side chain on peptoid structure, namely, the peptoid
threaded loop and helix. Circular dichroism spectroscopy of the peptoids revealed that the nitroaromatic
monomer has a significant effect on peptoid secondary structure. Specifically, the threaded loop structure
was disrupted in a nonamer containing alternating N-(S)-1-phenylethylglycine (Nspe) and Ns2ne monomers,
and the major conformation was helical instead. Indeed, placement of a single Ns2ne at the N-terminal
position of (Nspe)9 resulted in a destabilized form of the threaded loop structure relative to the
homononamer (Nspe)9. Conversely, we observed that incorporation of N-(S)-1-(4-nitrophenyl)ethylglycine
(Nsnp, a p-nitro monomer) at the N-terminal position stabilized the threaded loop structure relative to
(Nspe)9. Additional experiments revealed that nitroaromatic side chains can influence peptoid nonamer
folding by modulating the strength of key intramolecular hydrogen bonds in the peptoid threaded loop
structure. Steric interactions were also implicated for the Ns2ne monomer. Overall, this study provides
further evidence that aromatic side-chain structure, even if perturbed in a single monomer unit, can strongly
influence local peptoid backbone conformation.
Introduction
features have prompted the development of peptoids as tools
to probe a diverse range of biological processes and as
therapeutic agents. Recent examples include peptoid-derived
Oligomers of N-substituted glycine, or peptoids, represent a
versatile class of peptidomimetics for the study of biomolecular
phenomena.1 Peptoids are attractive for at least three reasons,
including (1) their ease of synthesis, (2) their proteolytic
stability, and (3) the wide variety of non-native functionality
that can be incorporated into their amide side chains. These
(1) (a) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J. Am.
Chem. Soc. 1992, 114, 10646–10647. For a recent review of peptoids, see: (b)
Patch, J. A.; Kirshenbaum, K.; Seurynck, S. L.; Zuckermann, R. N.; Barron,
A. E. In Pseudopeptides in Drug DeVelopment; Nielsen, P. E., Ed.; Wiley-VCH:
Weinheim, Germany, 2004; pp 1-31.
1440 J. Org. Chem. 2009, 74, 1440–1449
10.1021/jo8023363 CCC: $40.75 2009 American Chemical Society
Published on Web 01/21/2009

Synthesis and Characterization of Nitroaromatic Peptoids
FIGURE 1. (A) The peptoid helix, shown here as the structure of (Nspe)10 (1). Structure generated by molecular mechanics from the calculated
structure of (Nspe)8.6a Peptoid backbone highlighted in green. (B) The peptoid threaded loop, shown here as the structure of (Nspe)9 (2). Structure
generated by solution-phase 2D NMR analyses.10 Peptoid backbone highlighted in green and residues numbered sequentially from N- to C-terminus;
intramolecular hydrogen bonds shown in cyan. 3D-images for helix and loop generated using Chimera (v. 1.2199).11
transcription factor mimics,2 protein-protein interaction inhibi-
tors,3 antimicrobial agents,4 and lung surfactant mimics.5 An
underlying theme in the majority of these studies has been the
connection between peptoid function and peptoid structure.6 For
certain applications, well-folded peptoids are essential for
activity, while unstructured peptoids appear to suffice, or even
are superior, for other applications.3,4 These structure-function
connections are largely made after the design, synthesis, and
characterization process. The future development of peptoids
as chemical tools will require an ability to determine the
applications for which structured peptoids are or are not required,
and simultaneously, the development of straightforward de novo
design strategies to generate structured peptoids. Conformational
heterogeneity arising from backbone amide isomerism, however,
has significantly complicated the design of well-folded peptoid
structural motifs, and the current understanding of the peptoid
folding process remains at a formative stage.
approach has been particularly fruitful for the construction of
peptoid helices.7 Namely, the inclusion of R-chiral aromatic or
R-chiral aliphatic amide side chains in peptoids enforces a helical
conformation reminiscent of a polyproline type-I helix, with
all cis-amide bonds and three residues per turn (Figure 1A).
The peptoid helix was first reported by Barron, Kirshenbaum,
Dill, Zuckermann, and co-workers and has been characterized
by computational techniques, circular dichroism (CD) spectros-
copy, X-ray crystallography, and 2D NMR.7,8 Most peptoid
helices are composed primarily of (S)-N-(1-phenylethyl)glycine
units (Nspe, Figure 1) and are commonly identified by a
diagnostic CD signature with peaks at 192, 202, and 218 nm.
Through the analysis of Nspe-type oligomers of various lengths
and composititons,7b,9 Barron and co-workers developed a set
of predictive rules for peptoid helix formation. In brief,
oligomers containing 50% R-chiral side chains or an “aromatic
face” running down the longitudinal face of the helix (i.e.,
aromatic side chains in a 3-fold periodic pattern) are predicted
to form the most stable helices, and these helices are further
stabilized when the C-terminal residue is R-chiral and at
oligomer lengths beyond 12 residues. These rules have proven
valuable in the recent design of peptoid mimics of various
naturally occurring R-peptide helices.4,5
In contrast to the R-peptide backbone, peptoid backbones lack
both chiral centers and hydrogen bond donors, and this, coupled
with facile tertiary amide isomerism, reduces the conformational
rigidity of peptoids. One strategy to enforce more predictable
peptoid folding is the “reinstallation” of chiral centers or
hydrogen-bond donors into peptoid side chains, and the former
Through careful study of the peptoid helix, Barron and co-
workers uncovered a new peptoid structure in 2006, the threaded
(7) (a) Kirshenbaum, K.; Barron, A. E.; Goldsmith, R. A.; Armand, P.;
Bradley, E. K.; Truong, K. T.; Dill, K. A.; Cohen, F. E.; Zuckermann, R. N.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4303–4308. (b) Wu, C. W.; Sanborn,
T. J.; Zuckermann, R. N.; Barron, A. E. J. Am. Chem. Soc. 2001, 123, 2958–
2963.
(2) Xiao, X. S.; Yu, P.; Lim, H.-S.; Sikder, D.; Kodadek, T. J. Comb. Chem.
2007, 9, 592–600.
(3) Hara, T.; Durell, S. R.; Myers, M. C.; Appella, D. H. J. Am. Chem. Soc.
2006, 128, 1995–2004.
(4) Chongsiriwatana, N. P.; Patch, J. A.; Czyzewski, A. M.; Dohm, M. T.;
Ivankin, A.; Gidalevitz, D.; Zuckermann, R. N.; Barron, A. E. Proc. Natl. Acad.
Sci. U.S.A. 2008, 105, 2794–2799.
(5) Seurynck-Servoss, S. L.; Dohm, M. T.; Barron, A. E. Biochemistry 2006,
45, 11809–11818.
(6) (a) Armand, P.; Kirshenbaum, K.; Falicov, A.; Dunbrack, R. L., Jr.; Dill,
K. A.; Zuckermann, R. N.; Cohen, F. E. Fold. Des. 1997, 2, 369–375. (b) Lee,
B.-C.; Chu, T. K.; Dill, K. A.; Zuckermann, R. N. J. Am. Chem. Soc. 2008, 130,
8847–8855.
(8) (a) Armand, P.; Kirshenbaum, K.; Goldsmith, R. A.; Farr-Jones, S.;
Barron, A. E.; Truong, K. T. V.; Dill, K. A.; Mierke, D. F.; Cohen, F. E.;
Zuckermann, R. N.; Bradley, E. K. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4309–
4314. (b) Wu, C. W.; Kirshenbaum, K.; Sanborn, T. J.; Patch, J. A.; Huang, K.;
Dill, K. A.; Zuckermann, R. N.; Barron, A. E. J. Am. Chem. Soc. 2003, 125,
13525–13530.
(9) Wu, C. W.; Sanborn, T. J.; Huang, K.; Zuckermann, R. N.; Barron, A. E.
J. Am. Chem. Soc. 2001, 123, 6778–6784.
(10) Huang, K.; Wu, C. W.; Sanborn, T. J.; Patch, J. A.; Kirshenbaum, K.;
Zuckermann, R. N.; Barron, A. E.; Radhakrishnan, I. J. Am. Chem. Soc. 2006,
128, 1733–1738.
(11) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt,
D. M.; Meng, E. C.; Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605–1612.
J. Org. Chem. Vol. 74, No. 4, 2009 1441

Fowler et al.
loop (shown in Figure 1B).10 This discovery was significant,
as up until this point, the peptoid helix was the only known,
defined secondary structure for peptoids. A peptoid nonamer
of R-chiral aromatic side chains (Nspe)9 (2, evaluated as the
trifluoroacetic acid salt) was found to display a unique CD
signature in acetonitrile, with a single broad peak of significant
intensity at 203 nm.7b Further analysis of 2 by 2D NMR
techniques in acetonitrile-d3 revealed that it adopted a well-
defined looplike structure. The loop was stabilized by three
intramolecular hydrogen bonds from backbone carbonyls (resi-
dues 5, 7, and 9) to the N-terminal secondary ammonium and
one intramolecular hydrogen bond from a backbone carbonyl
(residue 2) to the C-terminal primary amide (Figure 1B).10 In
contrast to the peptoid helix (Figure 1A), the threaded loop
structure has four cis and four trans amide bonds. As only the
Nspe nonamer 2 and nonamers with closely related R-chiral
residues are able to form the threaded loop, this oligomer length
appears poised to compact itself and form the four intramolecular
hydrogen bonds. Indeed, Nspe pentamers to octamers and
decamers to longer oligomers all show a heightened propensity
for helix formation over threaded loop formation,7b indicating
that the molecular contacts necessary for threaded loop forma-
tion are unique to nine residue long peptoids. Notably, the
threaded loop in 2 can be converted back to a helix by addition
of a solvent capable of disrupting the intramolecular hydrogen
bonds (e.g., methanol), suggesting that the loop and helix are
end points on a conformational continuum for this nonamer
class.10
Beyond the threaded loop and helix, turn motifs have been
recently reported in peptoids, achieved either by macrocycliza-
tion12 or by incorporation of a turn-inducing triazole unit.13
These motifs certainly expand the repertoire of known peptoid
structures, and their applications in peptoid research are now
being delineated. Additional research is necessary, however, to
expand our understanding of the molecular level interactions
that are necessary for peptoid folding into helices, loops, turns,
and beyond. These structures are currently reserved to a
relatively small set of peptoid primary sequence space. A major
goal of our research is to develop new and robust approaches
to construct discretely folded peptoids through the strategic
installation of functionality capable of noncovalent interac-
tions,14 starting at the most basic monomer level15 and building
to more complex, oligomeric structures.16,17 We report herein
our recent efforts toward this goal through a systematic study
of peptoids containing R-chiral nitroaromatic monomers.
In 2005, we reported the design and synthesis of a peptoid
nonamer of alternating R-chiral aromatic (Nspe) and achiral
nitroaromatic (N-1-(2-nitrophenylethyl)glycine, N2nb) monomer
units.14 The CD signature of this alternating heterononamer
indicated that it adopted a helical structure in acetonitrile, with
intensities analogous to that of helical Nspe octamer and decamer
peptoids (evaluated as the mirror image Nrpe systems).7b Again,
the Nspe homononamer 2 adopts a threaded loop as opposed to
a helix. We initially postulated that the pattern of more electron-
rich (Nspe) and more electron-poor aromatic side chains (N2nb)
along each of the heterononamer helix faces could stabilize a
helical conformation relative to the threaded loop due to
potential intramolecular quadrupolar interactions between side
chains on each face of the helix. Later studies, however,
indicated that such quadrupolar interactions are most likely not
a major contributor to helix stabilization,16 and we sought to
further investigate the origin of helix stabilization in this peptoid
system. We reasoned that a more direct comparison could be
made between this nitroaromatic heterononamer and (Nspe)9 2
if the N2nb side chains were also R-chiral. Therefore, to perform
this experiment, we required (S)-1-(2-nitrophenyl)ethanamine
(s2ne) as a new chiral amine building block for peptoid
synthesis.
Simultaneous to our analysis of nitroaromatic peptoids, our
laboratory also has been studying the effects of single amide
side chain mutations on peptoid helix and threaded loop
structure in Nspe-type systems. We recently reported the
incorporation of (S)-N-(1-pentafluorophenylethyl)glycine
(Nsfe) at specific positions in R-chiral peptoid nonamers,16
and observed that either helical or threaded loop structure
could be promoted depending on the position at which the
substituted side chain was incorporated. This outcome was
attributed to the electron-withdrawing effect of the pentafluo-
roaromatic side chain on adjacent backbone amide carbonyls
and the ammonium terminus, and thus the ability to weaken
or strengthen hydrogen bonds in the threaded loop structure
(at positions 3, 6, and 8 or 1, respectively; Figure 1B). In
addition, other sites were found to be perturbed by pen-
tafluoroaromatic substitution (positions 2 and 5), suggesting
that additional amide carbonyls also may participate in
hydrogen bonding in the threaded loop. We desired to
examine the generality of this “mutational” approach, and
within the context of the current study, sought to incorporate
electron-withdrawing nitroaromatic side chains at key posi-
tions to further probe the stability of the hydrogen bonds in
the threaded loop structure. Specifically, we were interested
in enhancing the hydrogen-bonding capacity of the am-
monium terminus (position 1) in peptoid nonamers, as this
was shown to significantly stabilize the threaded loop
structure.16
Herein, we report the synthesis of (S)-1-(2-nitrophenyl)etha-
namine (s2ne, 5b), the design and construction of peptoid
nonamers containing this chiral amine, and the structural
characterization of the nonamer products by CD and NMR
spectroscopy. First, we synthesized a heteropeptoid of alternating
Nspe and Ns2ne units to compare to our previous work.
Characterization of this nonamer by CD spectroscopy revealed
that a helical conformation induced by nitroaromatic side chains
(12) Shin, S. B. Y.; Yoo, B.; Todaro, L. J.; Kirshenbaum, K. J. Am. Chem.
Soc. 2007, 129, 3218–3225.
(13) Pokorski, J. K.; Miller Jenkins, L. M.; Feng, H.; Durell, S. R.; Bai, Y.;
Appella, D. H. Org. Lett. 2007, 9, 2381–2383.
(14) Gorske, B. C.; Jewell, S. A.; Guerard, E. J.; Blackwell, H. E. Org. Lett.
2005, 7, 1521–1524.
(15) Gorske, B. C.; Bastian, B. L.; Geske, G. D.; Blackwell, H. E. J. Am.
Chem. Soc. 2007, 129, 8928–8929.
(16) Gorske, B. C.; Blackwell, H. E. J. Am. Chem. Soc. 2006, 128, 14378–
14387.
(17) Fowler, S. A.; Stacy, D. M.; Blackwell, H. E. Org. Lett. 2008, 10, 2329–
2332.
(18) We utilize the previously established nomenclature for this peptoid
residue (Nsnp) for clarity. See ref 7a.
1442 J. Org. Chem. Vol. 74, No. 4, 2009

Synthesis and Characterization of Nitroaromatic Peptoids
SCHEME 1. Synthesis of (S)-1-(2-Nitrophenyl)ethanamine (s2ne, 5b)
is further stabilized when all side chains are R-chiral. This result
provides further support for the set of rules for helix stabilization
developed by Barron and co-workers9 and extends it to a nitro-
functionalized aromatic side chain. Second, we synthesized and
studied Nspe heterononamers with Ns2ne and the 4-substituted
analogue, (S)-1-(4-nitrophenyl)ethylglycine (Nsnp),18 at the
N-termini. These single monomer mutations allowed us to probe
the effects of a nitroaromatic side chain on the hydrogen bond
from the N-terminal ammonium to residues 5, 7, and 9 in the
threaded loop structure. Both of these nonamers were observed
to adopt a threaded loop conformation in acetonitrile. However,
the inclusion of Ns2ne caused the nonamer structure to be
significantly destabilized relative to the canonical threaded loop
for (Nspe)9 (2), while the Nsnp-containing nonamer was
stabilized. Overall, our findings suggest that peptoid folding can
be controlled by subtle structural alterations to individual
monomer units, some as simple as the position of a functional
group on one aromatic ring (ortho vs para). These results
suggest a pathway not only for the stabilization of known
peptoid structures but also for the future design of new structural
motifs.
starting material acetamide, however.22 We therefore sought a
more reactive nitrating reagent, and in turn, an alternate
protecting group for spe. Our final route proceeded as follows.
First, spe was protected as the more acid-stable trifluoroaceta-
mide (3) under standard conditions (Scheme 1).23 Thereafter,
phenyl nitration was performed by treatment of 3 with the highly
+
reactive electrophile CF3SO3-NO2 generated from nitric and
triflic acid. The nitration product was obtained in 92% yield as
a 3:2 ratio of para and ortho isomers (4a and 4b). (We note
that nitro compounds 4-6 are susceptible to degradation by
light and were handled and stored in the dark throughout this
study.) The separation of regioisomers 4a and 4b by column
chromatography proved difficult, and we therefore examined
replacing the trifluoroacetyl24 with other amine protecting groups
to aid separation (e.g., Boc, Fmoc, and acetyl). We found that
the acetylated mixture of 5a and 5b could be easily separated
by recrystallization from ethyl acetate, allowing for the isolation
of the o-nitro acetamide (6) in nearly quantitative yield and
>95% purity.25 To complete the synthesis of s2ne, acetamide
6 was deprotected to yield the amine hydrochloride salt 5b in
35% overall yield from spe. We found this synthetic route to
be highly robust, and we isolated 5b routinely on a 200 mg
scale. Notably, nitroaromatic amine 5b should prove useful not
only for peptoid synthesis but also as new chiral building block
for other synthetic applications.
During the course of our synthetic work, we obtained an
X-ray crystal structure of o-nitro acetamide 6 (shown in Figure
2). The molecules were arranged in the P43 space group, a tetrad
repeat in which each molecule is rotated 90° relative to the
neighboring molecule. Intermolecular hydrogen bonds were
present between adjacent amide protons and amide oxygens.
The crystal structure not only confirmed the identity of
acetamide 6, but also highlighted the hydrogen-bonding capabil-
ity of this class of nitro aromatic compound. We reasoned this
property could influence the folding of peptoid oligomers
containing s2ne (5b), most notably those with a propensity to
adopt a threaded loop.
Results and Discussion
Synthesis of (S)-1-(2-Nitrophenyl)ethanamine (s2ne). Ni-
trophenylethylamines have been used for the chiral resolution
of organic compounds for over 30 years,19 and several synthetic
routes have been pursued for their preparation. Syntheses of
racemic 1-(2-nitrophenyl)ethanamine, either via reductive ami-
nation of a ketone20 or borane reduction of an oxime,21 have
been reported previously. A route to enantiopure s2ne or r2ne,
to our knowledge, is yet to be reported. Perry et al.19a reported
a straightforward synthetic route to the p-nitro analogue, (S)-
1-(4-nitrophenyl)ethylamine, and this amine (snp) is now
commercially available. This method involved the nitration of
spe with HNO3, and only the para-nitration product was isolated.
We sought to modify this nitration method to yield appreciable
quantities of the ortho-nitration product, s2ne (5b), from spe.
Our optimized route is shown in Scheme 1.
Peptoid Design. We previously reported that an alternating
heterononamer of Nspe and N2nb (7) exhibited a helical CD
Similar to Perry et al.,19a our initial route to 5b involved
protecting spe as the acetamide, and then subsequent nitration
with either HNO3 or HNO3/H2SO4. This procedure only returned
(22) We also were able to synthesize s2ne (5b) from 2′-nitroacetophenone
via a selective reduction of the ketone to the R-alcohol, conversion to the S-azide,
and reduction to the S-amine, similar to the work of Gorske et al. (see ref 16).
However, the overall yield of 5b was significantly lower using this approach
(12%, data not shown).
(19) (a) Perry, C. W.; Brossi, A.; Deitcher, K. H.; Tautz, W.; Teitel, S.
Synthesis 1977, 492–494. (b) Nieuwenhuijzen, J. W.; Grimbergen, R. F. P.;
Koopman, C.; Kellogg, R. M.; Vries, T. R.; Pouwer, K.; van Echten, E.; Kaptein,
B.; Hulshof, L. A.; Broxterman, Q. B. Angew. Chem., Int. Ed. 2002, 41, 4281–
4286.
(20) Walker, J. W.; McCray, J. A.; Hess, G. P. Biochemistry 1986, 25, 1799–
1805.
(23) Coe, J. W.; Brooks, P. R.; Vetelino, M. G.; Wirtz, M. C.; Arnold, E. P.;
Huang, J.; Sands, S. B.; Davis, T. I.; Lebel, L. A.; Fox, C. B.; Shrikhande, A.;
Heym, J. H.; Schaeffer, E.; Rollema, H.; Lu, Y.; Mansbach, R. S.; Chambers,
L. K.; Rovetti, C. C.; Schulz, D. W.; Tingley, F. D., III; O’Neill, B. T. J. Med.
Chem. 2005, 48, 3474–3477.
(24) Bergeron, R. J.; McManis, J. S. J. Org. Chem. 1988, 53, 3108–3111.
(25) The quantity of acetamide 6 isolated by crystallization equaled the
quantity of trifluoroacetamide 4b formed. Purity was assessed by 1H NMR
(>95%) and HPLC (96% purity at 220 nm).
(21) Salerno, C. P.; Magde, D.; Patron, A. P. J. Org. Chem. 2000, 65, 3971–
3981.
J. Org. Chem. Vol. 74, No. 4, 2009 1443

Fowler et al.
TABLE 1. Mass Spectrometry Data for Peptoids 2 and 8-10a
peptoid
calcd mass
obsd mass (m/z)
2
8
9
10
1466.8
1646.7
1511.7
1511.7
1468.3 [M + H]+
846.4 [M + 2Na]2+
1535.5 [M + Na]+
1535.4 [M + Na]+
a Peptoids purified to >95% purity before analysis by CD and NMR.
Mass spectrometry data acquired using MALDI-TOF.
monomer Nsnp replacements at the N-termini (Ns2ne(Nspe)8 9
and Nsnp(Nspe)8 10, respectively). Electron density would be
removed from the N-terminal ammonium in both nonamers.
However, the ortho-nitroaromatic group should engender stron-
ger inductive effects in peptoid 9 relative to those in p-
nitroaromatic peptoid 10. We reasoned that the o-nitro group
in peptoid 9 could also have altered steric contacts in the
threaded loop structure relative to peptoid 10 and the homonon-
amer 2, and this could play an additional role in the conforma-
tion adopted by peptoid 9.26
FIGURE 2. X-ray crystal structure of acetamide 6. The unit cell is
indicated; intermolecular hydrogen bonds are shown in dashed gray.
SCHEME 2. Microwave-Assisted Peptoid Submonomer
Synthesisa
Peptoid Synthesis. We synthesized (Nspe)9 2 (for use as a
control) and peptoid nonamers 8-10 on Rink-amide linker
derivatized polystyrene resin using a modified version of our
previously reported, microwave (µW)-assisted peptoid synthesis
method (shown in Scheme 2).14 Heterononamer 7 was synthe-
sized using a similar submonomer synthesis method; we utilized
a sample of 7 from our earlier work as a control for this study.14
To build each peptoid monomer unit, bromoacetic acid was
coupled to the resin-supported amine (µW 30 s, 35 °C), followed
by displacement of the bromide with a primary amine (spe, s2ne
(5b), or snp; µW 90 s, 95 °C; Scheme 2). Notably, we observed
decreased yields and crude purities for the peptoids constructed
from R-chiral nitroaromatic amines (s2ne (5b) and snp), most
probably due to the decreased nucleophilicities of these
amines.14 Careful reaction optimization revealed that µW heating
for 1 h at a reduced temperature (60 °C) was beneficial for
incorporation of these amines; for example, the crude purity of
nonamer 9 was 13% using standard µW conditions and increased
to 20% with a longer heating time. This modified amination
method was therefore used to incorporate the nitroaromatic
amines into peptoids 8-10. After acid-mediated resin cleavage,
peptoids 2 and 8-10 were purified to homogeneity (>95%) by
preparative RP-HPLC to yield ∼2-5 mg of each peptoid. The
purities and identities of the purified peptoids were confirmed
by analytical RP-HPLC (see the Supporting Information) and
mass spectrometry (Table 1). Peptoids containing nitroaromatic
side chains were manipulated and stored in the dark throughout
this study to minimize the chance of photodecomposition.
CD Characterization of Peptoids 7 and 8. The secondary
structures of the alternating nonamer peptoids 7 and 8 were
evaluated by CD spectroscopy in the first phase of this study
(Figure 3). We observed that peptoid 8 containing all R-chiral
residues (Nspe and Ns2ne) displayed a CD signature indicative
of helicity in acetonitrile, and this signature was qualitatively
similar in shape and intensity to that for peptoid 7 containing
a Notes: SN2 reaction conditions are for (a) amine spe, (b) s2ne and snp.
Oligomers are cleaved from the solid support by stirring in 95% TFA/H2O
for 20 min at rt. µW ) microwave irradiation.
signature in acetonitrile (see above).14 However, the comparison
of heterononamer 7 to (Nspe)9 (2) was not ideal, as 7 contains
only five R-chiral side chains as opposed to the nine in 2. The
number of chiral centers in a molecule will influence the
intensity of its CD spectrum, and the inclusion of R-chiral side
chains in peptoids is well documented to rigidify their structures,
whether helical or threaded loop (see above). Therefore, our
first design goal was to generate an alternating heterononamer
of Nspe and Ns2ne units, i.e., Nspe(Ns2neNspe)4 (8), to compare
to homononamer 2 and heterononamer 7 by CD spectroscopy.
The second goal of our peptoid design was to further probe
the effects of tuning the hydrogen-bonding capability of the
N-terminal ammonium in the peptoid threaded loop. We
reasoned that inductive withdrawal of electron density at this
terminus by a pendant nitroaromatic group could likely increase
the acidity of the ammonium group, and thus increase its
hydrogen bonding potential. We consequently designed Nspe
nonamers with single o-nitro monomer Ns2ne or p-nitro
(26) We synthesized and characterized an o-methyl analogue of peptoid 9
as a steric control. As (S)-1-(2-methylphenyl)ethylamine was not commercially
available, we utilized 2-methylbenzylamine (2mb) for this control peptoid
(N2mb(Nspe)8). The removal of the R-methyl group on the N-terminal side chain
in this peptoid (and in similar nonamers we have studied) appears to strengthen
the threaded loop (data not shown). We believe this may be due to an electronic
effect, as the lack of an R-methyl group should decrease electron density at the
N-terminal ammonium. Since we were unable to make direct comparisons
between the control N2mb(Nspe)8 and peptoid 9, we omit this relatively complex
analysis here for brevity.
1444 J. Org. Chem. Vol. 74, No. 4, 2009

Synthesis and Characterization of Nitroaromatic Peptoids
FIGURE 3. CD spectra of peptoids 7 and 8 at 60 µM in acetonitrile.
Spectra were collected at 24 °C.
FIGURE 4. CD spectra of peptoids 2, 9, and 10 at 60 µM in
acetonitrile. Spectra were collected at 24 °C.
only ∼50% R-chiral residues (i.e., five Nspe and four N2nb).
We note that both peptoids 7 and 8 have o-nitroaromatic amide
side chains at positions 2, 6, and 8 and that the adjacent amide
carbonyls (at positions 1, 5, and 7) act as hydrogen bond
acceptors in the canonical threaded loop structure (see Figure
1B). The electron-withdrawing nitroaromatic groups could be
weakening these adjacent hydrogen bonds and thus destabilizing
threaded loop structure and, in turn, enforcing helical structure
in peptoids 7 and 8. The spectrum of nonamer 8 more closely
resembled the spectrum of a peptoid helix than that of a loop,
with a minimum at 218 nm of slightly greater intensity than
the minimum at 202 nm. In fact, the shape and intensity of the
spectrum of nonamer 8 was very similar to the previously
reported spectrum of the Nrpe heptamer, which was interpreted
as a representation of multiple conformations, of which the
major conformer was the peptoid helix.7b
resonances than the spectrum in acetonitrile-d3, with ap-
proximately the same level of resolution. The HSQC spectrum
of peptoid 7 in DMSO-d6 contained multiple resonances for the
benzylic protons, and we observed an approximately 5:1 cis/
trans ratio of amide rotamers (by integration of benzylic protons;
see the Experimental Section for details of the NMR experi-
ments). These data suggested that more than one conformer of
7 was present in both acetonitrile and DMSO solutions. Based
on these CD and NMR results, we deduce that peptoid 7 has a
moderately lower propensity for a helical conformation relative
to peptoid 8. This result is in accord with the helix stabilization
rules outlined by Barron and co-workers: that is, the higher the
percentage of R-chiral residues in a peptoid helix, the more
stable the helical structure.7b,9 Another important outcome from
this study is our demonstration that the inclusion of multiple
o-nitroaromatic amide side chains, whether R-chiral or not, can
stabilize helical structure relative to loop structure in peptoid
nonamers.
CD Characterization of Peptoids 9 and 10. We initiated
the second phase of this study by characterizing peptoid
heterononamers 9 and 10 by CD spectroscopy. We expected to
see a strengthening of the threaded loop structure in peptoids 9
and 10, each with an R-chiral nitroaromatic amide side chain
at position 1, due to strengthening of hydrogen bonds to the
presumably more acidic N-terminal ammoniums in these pep-
toids. In accord with previous interpretations of CD spectra in
the peptoid field, we looked for an increase in CD peak intensity
as an indication of increased structural stability.7,9 We compared
the CD spectra of peptoids 9 and 10 to the CD spectrum of the
canonical threaded loop, (Nspe)9 (2), in acetonitrile (Figure 4).
Indeed, the spectrum of nonamer 10, containing Nsnp at position
1, showed a 30% increase in intensity at 203 nm relative to the
spectrum of 2 (albeit slightly shifted to lower wavelength (∼201
nm)),28 suggesting that the threaded loop structure was stabilized
in this peptoid relative to 2. We attributed this increase in loop
stability in 10 to strengthened hydrogen bonding to the
N-terminal ammonium, as a result of the electron-withdrawing
p-nitroaromatic side chain at position 1. Such a stabilization
effect is congruent with our previous results for the incorporation
The CD spectrum for peptoid 7, in contrast to that for 8, had
a minimum at 218 nm that was moderately lower in intensity
relative to the minimum at ∼202 nm (Figure 3). This pattern
of intensities is not a common observation for peptoid helices.
Indeed, a single broad peak of significant intensity at 203 nm
is actually indicative of the threaded loop (see above),7b
suggesting that peptoid 7 could also sample this conformation
in acetonitrile. To gain further insight into the structure of
peptoid 7, we evaluated this nonamer by NMR spectroscopic
techniques in acetonitrile-d3 at 24 °C (see Supporting Informa-
tion). We observed many overlapping resonances that were
1
poorly resolved in the 1D H NMR spectrum of 7, suggesting
multiple conformations were present in solution. We next sought
to evaluate the ratio of cis and trans amide bonds in 7 using
heteronuclear single quantum coherence (HSQC) NMR; unfor-
tunately, the low solubility of 7 in acetonitrile-d3 precluded us
from obtaining an acceptable HSQC spectrum due to low signal
strength. Instead, we chose to evaluate 7 in DMSO-d6, which
allowed us to prepare a more concentrated sample.27 The 1D
1H NMR spectrum of 7 in DMSO-d6 displayed more overlapping
(27) We note that DMSO may be able to disrupt hydrogen bonding in a
threaded loop-type structure (based on a qualitative comparison of the NMR
spectra of 2 in acetonitrile-d3 and DMSO-d6; see the Supporting Information).
Therefore, the cis/trans ratio determined for 7 may be higher in DMSO relative
to acetonitrile. We include the DMSO-d6 data here to provide our most
comprehensive analysis of peptoid 7.
(28) Similar blue shifts in CD spectra for peptoids containing Nsnp residues
have been observed previously; see ref 7a.
J. Org. Chem. Vol. 74, No. 4, 2009 1445

Fowler et al.
FIGURE 5. CD spectra of peptoid 9 at 60 µM in acetonitrile/methanol
solutions (0-100% methanol). Spectra were collected at 24 °C.
FIGURE 6. Portion of a 2D NOESY spectrum of peptoid 9 showing
correlations between benzylic H (ω1) and CH3 (ω2). Ratio of amide
rotamers ) 1.2:1 trans/cis.
of a pentafluoroaromatic amide side chain (Nsfe) at position 1
in an Nspe nonamer (see above).16
exist in acetonitrile, and addition of a sufficient quantity of
methanol results in conversion of the threaded loop to a helical
structure (see above). Our titration study of peptoid 9 revealed
a helical CD signature in solutions of g10% methanol/
acetonitrile. For comparison, a 50% methanol/acetonitrile solu-
tion is required to fully disrupt the hydrogen bonds in 2 and
observe a helical CD signature.10,16 Addition of just 10%
methanol, however, was sufficient to disrupt the structure of 9
in acetonitrile. Observing this solvent-induced transition, we
contend that the structure of peptoid 9 in acetonitrile is similar
to the threaded loop, and that it contains intramolecular hydrogen
bonds. However, because only a small percentage of methanol
was required to disrupt these hydrogen bonds, we infer that they
are substantially weakened in peptoid 9 relative to 2, potentially
due to lengthened bonds and/or contorted bond angles.
In contrast to peptoid 10, we observed a weakening of the
CD signature for nonamer 9 containing the o-nitroaromatic
monomer (Ns2ne) at position 1 relative to nonamer 2 (Figure
4). The minimum was shifted to 208 nm and was ∼4-fold less
intense than 10, and the overall signature was significantly
broader than that for either 10 or 2. A rationale for loop
destabilization in peptoid 9 due to an inductive effect was not
immediately apparent. Careful examination of the reported 3D
structure of the threaded loop for 2 (see Figure 1B) revealed
that an ortho-substituent in the position 1 amide side chain may
have unfavorable steric interactions with both the backbone and
side chains in the loop (e.g., the amide carbonyls at residues 5
and 7 and R-methyl at residue 9).29 Unfavorable steric interac-
tions between the o-nitro group and the R-methyl group within
the Ns2ne monomer should also be considered. The side chain
could adopt a dihedral angle that avoids this interaction, which
could potentially torque the ammonium hydrogens away from
the backbone acceptor carbonyls and thereby weaken threaded
loop structure in 9. In addition to these potential interactions,
we cannot discount the potential for the formation of a H-bond
between the o-nitro and the N-terminal ammonium, which would
again limit the ammonium’s ability to stabilize the threaded loop.
But, interestingly, we did not observe a CD spectrum for 9
indicating helical structure in acetonitrile. If the o-nitro group
was preventing peptoid 9 from folding into a threaded loop, it
was not reverting to a helical conformation. Rather, the overall
shape of the CD spectrum for 9 suggested a threaded loop,
though the lowered intensity led us to postulate that the structure
was not highly stable.
We performed NMR experiments on nonamer 9 in acetoni-
trile-d3 to further examine its structure. The 1D 1H NMR
1
spectrum of 9 was highly similar to the H NMR spectrum of
(Nspe)9 (2) in acetonitrile-d3; the dispersion of resonances
suggested unique chemical environments for each amide side
chain and that 9 adopts one major conformation (see the
Supporting Information). Integration of the benzylic resonances
indicated that there were an approximately equal number of cis
and trans amide bonds in peptoid 9.30 The 2D NOESY spectrum
for 9 likewise showed resonances for one major conformer (a
portion of the spectrum is shown in Figure 6). Integration of
the benzylic resonances in the NOESY spectrum indicated a
1.2:1 trans/cis amide ratio, or 4.4 trans-amides and 3.6 cis-
amides on average, for peptoid 9 in acetonitrile. This ratio is
comparable to that of the canonical threaded loop, 2, which has
approximately four cis and four trans amides.10 Based on these
NMR and CD experiments, we believe heterononamer 9 adopts
a destabilized threaded loop structure in acetonitrile, in which
the oligomer is perhaps not as tightly coiled as 2 due to the
presence of the o-nitroaromatic substituent at the N-terminus.
This “unwinding” of the threaded loop is congruent with our
To obtain more insight into the structure of Ns2ne(Nspe)8
(9), we acquired CD spectra of 9 in solutions of varying
compositions of methanol/acetonitrile (Figure 5). Such solvent
titration experiments have been performed previously on non-
amer peptoids that adopt the threaded loop structure to probe
the relative strength of the intramolecular hydrogen bonds.10,16
Methanol competes for the intramolecular hydrogen bonds that
1
(30) Assignments in the 1D H NMR spectrum for 9 were corroborated by
2D NOESY data. See the Supporting Information for the 1D 1H NMR spectrum
(29) For a space-filling model of the threaded loop structure adopted by
nonamer 2, see Figure 4 in ref 10.
of peptoid 9.
1446 J. Org. Chem. Vol. 74, No. 4, 2009

Synthesis and Characterization of Nitroaromatic Peptoids
CD solvent titration data for peptoid 9, indicating significantly
weakened intramolecular hydrogen bonding in the nonamer
relative to 2. Such destabilized threaded loop conformations have
been proposed previously for homononamers of R-chiral
aliphatic side chains.10
patterns and functionality on their aromatic rings. Strategies to
stabilize peptoid architectures through minimal side-chain
perturbations could also prove valuable for the development of
functional peptoids. This could permit the installation of a higher
percentage of side chains that endow the peptoid with desirable
properties (e.g., solubility, cell permeability, or biological
activity), as opposed to being restricted largely to those that
only enforce structure. Ongoing work in our laboratory is
directed toward applying these design strategies to further refine
and expand the existing repertoire of peptoid secondary
structures and will be reported in due course.
Together, the results of the second phase of this study
indicated that both the structure and position of a substituent
on the N-terminal aromatic side chain can either dramatically
destabilize (o-nitro) or stabilize (p-nitro) the threaded loop.
While inductive effects can explain the stabilizing effect of the
p-nitroaromatic side chain in peptoid 10, the reasons behind
the destabilization of loop structure in the o-nitroaromatic
analogue (9) are less clear. Our current hypothesis is that this
is largely due to a steric effect and/or formation of an H-bond
between the nitro group and the N-terminus, but we cannot
discount other possible inductive effects or electrostatic interac-
tions also. Overall, these results further underscore the dynamic
nature of the threaded loop structure, and again, the ability to
alter peptoid secondary structure through seemingly minor
structural perturbations to their primary sequence.
Experimental Section
2,2,2-Trifluoro-N-((S)-1-phenylethyl)acetamide (3). (S)-(-)-
R-Methylbenzylamine (spe, 2.130 mL, 16.50 mmol) was dissolved
in CH2Cl2 (30 mL) and cooled to 0 °C. Pyridine (3.325 mL, 41.26
mmol, 2.5 equiv) was added to the stirring solution of amine,
followed by dropwise addition of trifluoroacetic anhydride (2.910
mL, 20.64 mmol, 1.25 equiv) over 30 min.23 The reaction was
allowed to warm to room temperature (rt) and stirred for 3 h. The
reaction mixture was poured into 0.5 N HCl (30 mL) and vigorously
stirred for 5 min. The layers were separated, and the aqueous layer
was extracted with CH2Cl2 (3 × 10 mL). The combined organic
layers were washed with 0.5 N HCl (20 mL), H2O (2 × 20 mL),
and saturated NaHCO3 (20 mL) and dried through a cotton plug.
The organic phase was concentrated and dried in vacuo to give
trifluoroacetamide 3 as a white powdery solid (3.556 g, 16.37 mmol,
99% yield). TLC in CH2Cl2: Rf ) 0.66; 1H NMR (300 MHz, CDCl3)
δ 7.33 (m, 5H), 6.39 (bs, 1H), 5.15 (p, J ) 7.2 Hz, 1H), 1.59 (d,
Conclusions
We have discovered two new design principles for peptoid
folding based on the incorporation of nitroaromatic amide side
chains into peptoids. This work was facilitated by the develop-
ment of a new synthetic route to (S)-1-(2-nitrophenyl)ethanamine
(s2ne, 5b), which could be readily incorporated into peptoids
using solid-phase methods. First, we showed that the presence
of Ns2ne, or its achiral variant N2nb, can destabilize the peptoid
threaded loop secondary structure in alternating Nspe heter-
ononamers (7 and 8). CD and NMR experiments revealed that
the major conformations of these peptoids were helical, and we
postulate that the origin of this effect is destabilization of key
hydrogen bonds in the threaded loop structure due to the
electron-withdrawing capabilities of the nitroaromatic groups.
Second, we demonstrated that the incorporation of a single
Ns2ne at the N-terminus of an Nspe nonamer (9) also could
destabilize threaded loop structure. Peptoid 9 exhibited a unique
CD spectrum in acetonitrile, which further CD and NMR studies
indicated to be that of a loosely coiled, threaded loop. In contrast,
the p-nitro-substituted analogue of 9 (nonamer 10) exhibited a
significantly stabilized threaded loop structure relative to 9 and
the canonical threaded loop (2), as determined by CD analysis.
This latter result is in accord with the strengthened hydrogen
bonding capability of the N-terminal ammonium in peptoid 10,
and this outcome was also originally anticipated for peptoid 9.
The reasons behind this dramatic shift in loop stability for
isomeric peptoids 9 and 10 remain unclear, although we believe
that the differing steric effects of o-nitro versus p-nitro groups
may play a major role.
To close, this study provides further evidence that peptoid
side chain structure, even if perturbed in a single monomer unit,
can strongly influence local peptoid backbone conformation. Our
results serve to further delineate the direct connections between
primary sequence and structure in R-chiral peptoids. Moreover,
we demonstrate, to our knowledge, the most exquisite control
of peptoid secondary structure reported to date, achievable
through highly subtle alterations of amide side chain structure
(i.e., the placement of a single functional group on one amide
side chain). These results have important implications for the
design of new peptoid structures and for the analysis of
previously reported R-chiral peptoids with different substitution
J ) 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 156.6 (q, JCF
)
36.8 Hz), 141.0, 129.2, 128.4, 126.4, 116.1 (q, JCF ) 286.7 Hz),
50.0, 21.1; EI MS calcd 217.0, obsd [M+] 217.1.
2,2,2-Trifluoro-N-((S)-1-(4-nitrophenyl)ethyl)acetamide (4a)
and 2,2,2-Trifluoro-N-((S)-1-(2-nitrophenyl)ethyl)acetamide (4b).
Trifluoromethanesulfonic acid (CF3SO3H; 2.9 mL, 32.69 mmol, 2
equiv) was dissolved in CH2Cl2 (110 mL) and cooled to 0 °C under
N2. HNO3 (0.74 mL, 16.34 mmol, 1 equiv) was added, and the
reaction mixture was stirred for 15 min at 0 °C, after which time
it was cooled to -78 °C.23 Trifluoroacetamide 3 (3.55 g, 16.34
mmol) was dissolved in CH2Cl2 (30 mL) and slowly added to the
cooled reaction mixture over 1 h, followed by a CH2Cl2 rinse (10
mL) of the addition funnel. The reaction mixture was stirred at
-78 °C for an additional 30 min and then at -40 °C for 16 h. The
yellow-orange reaction mixture was poured into ice (100 g) and
stirred vigorously for 5 min. The layers were separated, and the
aqueous layer was extracted with CH2Cl2 (3 × 25 mL). The organic
layers were combined, washed with H2O (3 × 100 mL), saturated
NaHCO3 (100 mL), and H2O (100 mL), and dried through a cotton
plug. The organic phase was concentrated and dried in vacuo to
give a ∼3:2 mixture of 4a and 4b as a pale orange solid (4.026 g,
15.35 mmol, 94% combined yield; 57% 4a and 43% 4b as
1
determined by H NMR integration): TLC in 20% diethyl ether/
toluene, 4a Rf ) 0.44, 4b Rf ) 0.50; 1H NMR (300 MHz, CDCl3)
(4a) δ 8.24 (d, J ) 8.9 Hz, 2H), 7.50 (d, J ) 8.9 Hz, 2H), 6.65
(bs, 1H), 5.22 (p, J ) 7.2 Hz, 1H), 1.63 (d, J ) 7.2 Hz, 3H); (4b)
δ 7.97 (dd, J ) 7.8, 1.2 Hz, 1H), 7.65 (m, 1H), 7.49 (m, 1H), 7.32
(m, 1H), 7.12 (bs, 1H) 5.54 (p, J ) 7.2 Hz, 1H), 1.65 (d, J ) 7.2
Hz, 3H); 13C NMR (75 MHz, CDCl3) (4a) δ 156.9 (q, JCF ) 37.5
Hz), 148.6, 136.5, 127.2, 124.4, 115.8 (q, JCF ) 286.7 Hz), 49.3,
21.5; (4b) δ 156.7 (q, JCF ) 37.0 Hz), 147.7, 134.2, 132.8, 129.2,
128.7, 125.5, 47.6, 20.8; ESI MS calcd 262.0, obsd [M + Na]+
285.3.
(S)-1-(4-Nitrophenyl)ethanamine (5a) and (S)-1-(2-Nitrophe-
nyl)ethanamine (5b). The mixture of trifluoroacetamides 4a and
4b (0.215 g, 0.82 mmol) was dissolved in a 16:1 MeOH/H2O
solution (33 mL). K2CO3 (0.572 g, 4.10 mmol, 5 equiv) was added,
and the reaction mixture was refluxed with stirring for 16 h.24 The
J. Org. Chem. Vol. 74, No. 4, 2009 1447

Fowler et al.
orange reaction mixture was concentrated in vacuo to yield an
orange solid, which was dissolved in H2O (30 mL) and extracted
with CHCl3 (3 × 15 mL). The organic phase was washed with 1
N NaOH (15 mL). The aqueous layers were combined, adjusted to
pH 11 by the addition of 10% NaOH (3 mL), and extracted with
CHCl3 (3 × 15 mL). The organic layers were combined, dried over
MgSO4, filtered, and concentrated in vacuo to give a mixture of
amines 5a and 5b as an orange oil (0.136 g, 0.819 mmol, 100%
yield): TLC in CH2Cl2 Rf ) 0.10 (one spot); 1H NMR (300 MHz,
CDCl3) (5a) δ 8.18 (d, J ) 8.8 Hz, 2H), 7.53 (d, J ) 8.4 Hz, 2H),
4.26 (q, J ) 6.5 Hz, 1H), 1.42 (d, J ) 6.5 Hz, 3H); (5b) δ 7.79
(dd, J ) 5.0, 1.4 Hz, 1H), 7.76 (dd, J ) 5.0, 1.4 Hz, 1H), 7.60
(ddd, J ) 7.8, 7.6, 1.0 Hz, 1H), 7.36 (ddd, J ) 8.1, 7.3, 1.4 Hz,
1H), 4.60 (q, J ) 6.5 Hz, 1H), 1.45 (d, J ) 6.5 Hz, 3H); 13C NMR
(75 MHz, CDCl3) (5a) δ 149.0, 147.0, 126.8, 123.8, 50.9, 25.8;
(5b) δ 155.2, 142.0, 133.2, 127.7, 127.6, 124.0, 46.1, 24.7; ESI
MS calcd 166.0, obsd [M + H]+ 167.1.
20 equiv) in DMF (1 mL) was added to the resin, and the mixture
was heated with stirring in the µW reactor to 95 °C in 90 s for spe
or at 60 °C for 1 h (with 1 min ramp time) for s2ne and snp. The
reaction mixture was filtered, and the resin was washed with DMF
(5 × 1 mL) and drained. This two-step sequence of acylation and
amination was repeated for the coupling of subsequent peptoid
residues to generate peptoid nonamers. The resin-bound nonamers
were washed with CH2Cl2 (4 × 1 mL) and drained. The peptoids
were cleaved from the resin by stirring in 95% trifluoroacetic acid/
H2O (0.5 mL) for 20 min at rt. The resin was filtered and washed
with CH2Cl2 (2 × 0.5 mL), and the filtrate was concentrated under
N2 to yield the peptoids as pale pink oils. Purity was determined
by HPLC (UV detection at 220 nm), and the product identity was
confirmed by MALDI-TOF MS. The peptoids were purified by
preparatory HPLC to >95% purity before CD and NMR analyses.
X-ray Crystallography. X-ray crystallography was performed
on a CCD diffractometer with Mo KR (λ ) 0.71073 Å) radiation.
Crystal data for acetamide 6: C10H12N2O3, M ) 208.22, tetragonal,
a ) 7.9423(19) Å, b ) 7.9423(19) Å, c ) 16.974(9) Å, U )
1070.7(6) Å3, T ) 100(2) K, space group P43, Z ) 4, µ(Mo KR)
N-((S)-1-(2-Nitrophenyl)ethyl)acetamide (6). A mixture of
amines 5a and 5b (3.23 g, 19.48 mmol) and diisopropylethylamine
(10 mL, 58.4 mmol, 3 equiv) was stirred at 0 °C, and acetic
anhydride (4.60 mL, 48.7 mmol, 2.5 equiv) was added dropwise
to the solution over 20 min. The reaction mixture was stirred for
3 h, during which time it was allowed to come to rt. The reaction
mixture was cooled to 0 °C, acidified by the addition of 1 N HCl
(15 mL), and extracted with EtOAc (3 × 50 mL). The combined
organic layers were washed with saturated NaHCO3 (3 × 30 mL)
and saturated NaCl (2 × 30 mL), dried over Na2SO4, and
concentrated in vacuo to yield a red-brown oil (4.02 g, 19.30 mmol,
99%). This material was 40% ortho isomer 6 (as determined by
1H NMR integration). The ortho isomer was isolated by partial
recrystallization from EtOAc. Briefly, the 4 g mixture of isomers
was repeatedly dissolved in EtOAc (4 × 4 mL). The para isomer
was fully soluble in this volume of EtOAc, while the ortho isomer
(6) was insoluble and formed pale yellow needles that could be
isolated in 96% purity (as determined by HPLC integration at 220
) 0.097 mm-1, 2954 reflections measured, 1114 unique (Rint
)
0.1140) which were used in all calculations. The final wR(F2) was
0.0892 (all data). See the Supporting Information for full details.
CD Analyses. Circular dichroism (CD) spectra were obtained
on a digital spectropolarimeter at 24 °C. Peptoid stock solutions
were prepared by dissolving at least 2 mg of each peptoid in
spectroscopic grade acetonitrile. The stock solutions were diluted
with spectroscopic grade acetonitrile to the desired concentration
(60 µM) by mass using a high-precision balance. CD spectra were
obtained in a square quartz cell (path length 0.1 cm) 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 Microsoft Excel 2004.
NMR Analyses. The heteronuclear single quantum coherence
adiabatic (HSQCAD) NMR experiment31 for 7 was performed on
a 600 MHz NMR spectrometer using a 5 mm hcn probe. The data
were processed using the Varian VNMR software package (v. 6.1C).
Peptoid 7 was dissolved in dimethyl sulfoxide-d6 to give a ∼4 mM
solution and transferred to a 5 mm NMR tube. The experiment
was performed at 24 °C using the following parameter values:
1
nm): TLC in EtOAc, Rf ) 0.38; H NMR (300 MHz, CDCl3) δ
7.86 (dd, J ) 8.3, 1.3 Hz, 1H), 7.57 (ddd, J ) 7.7, 7.0, 1.3 Hz,
1H), 7.50 (dd, J ) 7.7, 1.5 Hz, 1H), 7.39 (ddd, J ) 8.3, 7.0, 1.5
Hz, 1H), 6.15 (bs, 1H), 5.48 (p, J ) 7.1 Hz, 1H), 1.96 (s, 3H),
1.54 (d, J ) 7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 169.5,
148.8, 138.9, 133.5, 128.8, 128.2, 125.1, 46.8, 23.3, 21.5; ESI MS
calcd 208.0, obsd [M + H]+ 209.2.
1
spectral widths were 6913.8 Hz in the H dimension and 28622.5
Hz in the 13C dimension. The number of transients (nt) and number
of increments (ni) were 24 and 400, respectively. The number of
points (np) was 2048. Square cosine window functions were applied
in both dimensions. The spectra were zero-filled to generate f1 ×
f2 matrices of 4096 × 2048 points.
(S)-1-(2-Nitrophenyl)ethanamine Hydrochloride (5b). Aceta-
mide 6 (0.206 g, 0.989 mmol) was refluxed in 5% aqueous HCl
(10 mL) for 14 h. The reaction mixture was concentrated in vacuo
to a yellow oil, which crystallized upon addition of diethyl ether.
The diethyl ether was removed, and the residue was dried in vacuo
to give 5b as a yellow crystalline solid (0.200 g, 0.988 mmol, 99%
yield): 1H NMR (300 MHz, CD3OD) δ 8.101 (dd, J ) 7.6, 0.9 Hz,
1H), 7.838 (m, 2H), 7.685 (ddd, J ) 8.4, 6.4, 2.3 Hz, 1H), 5.010
(q, J ) 6.7 Hz, 1H), 1.730 (d, J ) 6.8 Hz, 3H); 13C NMR (75
MHz, CD3OD) δ 150.3, 135.6, 134.0, 131.6, 129.0, 126.6, 47.5,
20.2; IR (cm-1): 3406 (broad), 2885, 1601, 1534, 1341, 1096, 857;
ESI MS calcd 167.1, obsd [M]+ 167.1; mp (HCl salt) 184-188
The 2D NOESY NMR experiment for 9 was performed on a
600 MHz NMR spectrometer using a 5 mm hcn probe. The data
were processed using the Varian VNMR software package (v. 6.1C)
and visualized using SPARKY software.32 Peptoid 9 was dissolved
in acetonitrile-d3 to give a ∼1 mM solution and placed in a 5 mm
Shigemi NMR tube that was susceptibility-matched for dimethyl-
sulfoxide. The 2D NOESY experiment was performed at 24 °C
using the following parameter values: spectral widths were 8200.1
Hz in both 1H dimensions. The number of transients (nt) and number
of increments (ni) were 24 and 325, respectively. The number of
points (np) was 2086, and 512 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 × 2048 points.
°C; [R]25 (CH3OH, c ) 0.016) +2.812.
D
Peptoid Synthesis. Peptoids were synthesized on polystyrene
resin with an Fmoc-protected Rink amide linker (0.69 mmol/g)
using a modified version of our previously reported, µW-assisted
peptoid synthesis method.14 The resin Fmoc group was deprotected
by soaking the resin (60 mg, 0.041 mmol) in a 20% piperidine/
DMF solution (1 mL) for 20 min at rt, after which time the resin
was washed with DMF (5 × 1 mL) and drained. This deprotection
procedure was repeated (1×). A solution of bromoacetic acid (0.10
g, 0.81 mmol, 20 equiv) and DIC (0.12 mL, 0.81 mmol, 20 equiv)
in DMF (1 mL) was added to the deprotected resin (60 mg, 0.041
mmol, 1 equiv). The reaction mixture was heated with stirring in
a commercial µW reactor to 35 °C in 30 s. The reaction mixture
was filtered, and the resin was washed with DMF (5 × 1 mL) and
drained. A solution of amine (spe, s2ne (5b), or snp; 0.81 mmol,
Acknowledgment. We thank the NSF (CHE-0449959),
Burroughs Wellcome Fund, Research Corporation, Johnson &
Johnson, and 3M for financial support of this work. H.E.B is
(31) Boyer, R. D.; Johnson, R.; Krishnamurthy, K. J. Magn. Reson. 2003,
165, 253–259.
(32) Goddard, T. D.; Kneller, D. G. SPARKY, V. 3.112; University of
California: San Francisco, CA.
1448 J. Org. Chem. Vol. 74, No. 4, 2009

Synthesis and Characterization of Nitroaromatic Peptoids
an Alfred P. Sloan Foundation fellow. S.A.F. acknowledges the
ACS Division of Medicinal Chemistry for a predoctoral
fellowship (sponsored by Sanofi-Aventis) and Pfizer Global
R&D for a Diversity in Organic Chemistry Fellowship. R.L.
thanks the Faculty of Science at Mahidol University (Thailand)
for an Undergraduate Student Degree Honor Program scholar-
ship. Support for the NMR facilities at UWsMadison 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 Prof. Annelise Barron for the coordinates for the
peptoid images in Figure 1; these molecular graphics images
were produced using the UCSF Chimera package from the
Resource for Biocomputing, Visualization, and Informatics at
the University of California, San Francisco (supported by NIH
P41 RR-01081). We also thank Dr. Charlie Fry and Dr. Monika
Ivancic for assistance with NMR experiments, Dr. Ilia Guzei
for X-ray crystallographic analyses, and Dr. Benjamin Gorske
and Dr. Matthew Bowman for contributive discussions.
Supporting Information Available: General experimental
information, crystallographic information file for acetamide 6,
full X-ray data for 6, HPLC data for peptoids 2 and 8-10, and
NMR data for amine 5b and peptoids 2, 7, and 9. This material
is available free of charge via the Internet at http://pubs.acs.org.
JO8023363
J. Org. Chem. Vol. 74, No. 4, 2009 1449