Tuning peptoid secondary structure with pentafluoroaromatic functionality: A new design paradigm for the construction of discretely folded peptoid structures

Article abstract of DOI:10.1021/ja065248o

Peptoids, or oligomers of N-substituted glycine, are an important class of non-native polymers whose close structural similarity to natural α-peptides and ease of synthesis offer significant advantages for the study of biomolecular interactions and the de

Full text of DOI:10.1021/ja065248o

Published on Web 10/18/2006
Tuning Peptoid Secondary Structure with Pentafluoroaromatic
Functionality: A New Design Paradigm for the Construction
of Discretely Folded Peptoid Structures
Benjamin C. Gorske and Helen E. Blackwell*
Contribution from the Department of Chemistry, UniVersity of WisconsinsMadison,
1101 UniVersity AVenue, Madison, Wisconsin 53706-1322
Received July 21, 2006; E-mail: blackwell@chem.wisc.edu
Abstract: Peptoids, or oligomers of N-substituted glycine, are an important class of non-native polymers
whose close structural similarity to natural R-peptides and ease of synthesis offer significant advantages
for the study of biomolecular interactions and the development of biomimetics. Peptoids that are N-substituted
with R-chiral aromatic side chains have been shown to adopt either helical or “threaded loop” conformations,
depending upon solvent and oligomer length. Elucidation of the factors that impact peptoid conformation
is essential for the development of general rules for the design of peptoids with discrete and novel structures.
Here, we report the first study of the effects of pentafluoroaromatic functionality on the conformational
profiles of peptoids. This work was enabled by the synthesis of a new, R-chiral amine building block, (S)-
1-(pentafluorophenyl)ethylamine (S-2), which was found to be highly compatible with peptoid synthesis
(delivering (S)-N-(1-(pentafluorophenyl)ethyl)glycine oligomers). The incorporation of this fluorinated
monomer unit allowed us to probe both the potential for π-stacking interactions along the faces of peptoid
helices and the role of side chain electrostatics in peptoid folding. A series of homo- and heteropeptoids
derived from S-2 and non-fluorinated, R-chiral aromatic amide side chains were synthesized and
characterized by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Enhance-
ment of π-stacking by quadrupolar interactions did not appear to play a significant role in stabilizing the
conformations of heteropeptoids with alternating fluorinated and non-fluorinated side chains. However,
incorporation of (S)-N-(1-(pentafluorophenyl)ethyl)glycine monomers enforced helicity in peptoids that
typically exhibit threaded loop conformations. Moreover, we found that the incorporation of a single (S)-
N-(1-(pentafluorophenyl)ethyl)glycine monomer could be used to selectively promote looped or helical
structure in this important peptoid class by tuning the electronics of nearby heteroatoms. The strategic
installation of this monomer unit represents a new approach for the manipulation of canonical peptoid
structure and the construction of novel peptoid architectures.
surfactants,7 high-density ligand arrays,8 activation domain
Introduction
mimics,9 inhibitors of protein-protein interactions,10 and ve-
Peptoids, or oligomers of N-substituted glycine, have emerged
as a versatile class of peptidomimetics for the study of
biomolecular interactions.1,2 Interest in peptoids as tools for
chemical biology and biotechnology continues to increase due
to the relative ease with which they can be synthesized,3,4 their
resistance to proteolytic degradation,5 and the wide variety of
non-native functionality that can be incorporated. These advan-
tages have motivated the development of peptoids for a broad
range of applications, including antimicrobial agents,6 lung
hicles for gene and drug delivery.11 In turn, this work has
prompted an examination of the role of secondary structure in
peptoid function.12 However, as with peptides and proteins, the
design and prediction of peptoid structure a priori presents a
(6) Patch, J. A.; Barron, A. E. J. Am. Chem. Soc. 2003, 125, 12092-12093.
(7) Seurynck, S. L.; Patch, J. A.; Barron, A. E. Chem. Biol. 2005, 12, 77-88.
(8) Li, S.; Bowerman, D.; Marthandan, N.; Klyza, S.; Luebke, K. J.; Garner,
H. R.; Kodadek, T. J. Am. Chem. Soc. 2004, 126, 4088-4089.
(9) Liu, B.; Alluri, P. G.; Yu, P.; Kodadek, T. J. Am. Chem. Soc. 2005, 127,
8254-8255.
(10) Hara, T.; Durell, S. R.; Myers, M. C.; Appella, D. H. J. Am. Chem. Soc.
2006, 128, 1995-2004.
(1) Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell, D.
A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe, C. K.;
Spellmeyer, D. C.; Tan, R.; Frankel, A. D.; Santi, D. V.; Cohen, F. E.;
Bartlett, P. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9367-9371.
(2) For reviews of peptoids, see: (a) 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.
(b) Patch, J. A.; Barron, A. E. Curr. Opin. Chem. Biol. 2002, 6, 872-877.
(3) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J. Am. Chem.
Soc. 1992, 114, 10646-10647.
(4) Alluri, P. G.; Reddy, M. M.; Bachhawat-Sikder, K.; Olivos, H. J.; Kodadek,
T. J. Am. Chem. Soc. 2003, 125, 13995-14004.
(5) Miller, S. M.; Simon, R. J.; Ng, S.; Zuckermann, R. N.; Kerr, J. M.; Moos,
W. H. Bioorg. Med. Chem. Lett. 1994, 4, 2657-2662.
(11) Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.; Steinman,
L.; Rothbard, J. B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13003-13008.
(12) (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. (c) Wu, C. W.; Sanborn, T. J.; Huang, K.; Zuckermann, R. N.; Barron,
A. E. J. Am. Chem. Soc. 2001, 123, 6778-6784. (d) 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. (e) 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.
9
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Tuning Peptoids with Pentafluoroaromatic Functionality
A R T I C L E S
lecular π-stacking interactions along these faces have been
proposed to play a role in stabilizing peptoid helices.20,21
Alternatively, intermolecular side chain interdigitation has also
been examined as a mechanism for helix stabilization.12b These
aromatic interactions, in conjunction with steric and stereoelec-
tronic repulsions between the side chains and the backbone, are
presumed to be critical for folding, as the peptoid backbone is
devoid of features known to otherwise dictate peptide and
protein folding, i.e., hydrogen-bond donors and main chain
chirality (Figure 1).
The potential significance of aromatic-aromatic interactions
in peptoid structures prompted our investigation of perfluori-
nated aromatic groups as components of peptoid side chains,
as they possess nearly equal, but opposite, quadrupole polarities
compared to non-fluorinated aryl rings.22 Numerous studies
suggest that favorable quadrupolar interactions between properly
aligned aromatic and perfluoroaromatic rings can enhance
π-stacking interactions.21,23 However, recent explorations of this
phenomenon using a peptide R-helix scaffold did not provide
evidence for aromatic-perfluoroaromatic attractions stronger
than those of the aryl-aryl type.24 The authors of this previous
study proposed that incorrect alignment and spacing of the
aromatic rings were likely responsible for the lack of additional
stabilization. As peptoids have substantially higher conforma-
tional flexibility relative to peptides,1,2 we reasoned that peptoids
would provide an improved scaffold for the further exploration
of aromatic-perfluoroaromatic interactions in foldamers. In-
deed, this flexibility would be essential for the observation of
face-to-face intramolecular aromatic interactions, as these typi-
cally require interplanar distances on the order of 3.5 Å25sfar
less than those observed between side chains in the solid-state
and NMR structures of peptoid helices (ca. 6 Å).12d,20a We
hypothesized that these interactions, if operative in peptoids,
could be exploited to enhance peptoid helix stability. Further-
more, perturbation of the side chain electronics and steric
contours by fluorination could illuminate other factors relevant
to peptoid folding, such as hydrophobic or inductive effects.
In this work, we report the synthesis of (S)-1-(pentafluoro-
phenyl)ethylamine (S-2) and the incorporation of this new,
chiral, perfluoroaromatic building block into a series of peptoids.
This synthetic work has enabled the first evaluation of the effects
of pentafluoroaromatic side chain functionality on the confor-
mational profiles of peptoids. These studies have revealed that
pentafluoroaromatic side chains can stabilize peptoid secondary
structures via a mechanism that is distinct from the quadrupole-
enhanced π-stacking mechanism that we had originally pro-
posed. Namely, this new peptoid synthon enables site-selective
tuning of hydrogen bonds to peptoid backbone heteroatoms,
Figure 1. (Left) R-Chiral aromatic amine building blocks S-1 and S-2.
(Right) A comparison of peptoids and R-peptides.
formidable challenge. At the root of this challenge lies the need
to further develop our understanding of noncovalent interactions
and how they affect the folding of both natural and synthetic
polymers.13 Hydrophobic,14 ionic,15 hydrogen bonding,16 and
aromatic interactions17 are among the many noncovalent interac-
tions implicated in ordering the structures of folded molecules.
We sought to examine these interactions within a peptoid
framework as part of an effort to develop new design strategies
for generating well-folded peptoids.18 Our initial investigations
have focused on the role of aromatic interactions in this context,
since these are believed to significantly influence the folding
of not only peptoids (Vide infra), but also a wide range of natural
and non-natural foldamers.17,19
Pioneering research by Barron, Zuckermann, Dill, and co-
workers revealed the first design strategy for the generation of
stable secondary structures in peptoids. In this past work, the
incorporation of certain R-chiral aromatic and aliphatic amide
side chains was found to enforce stable helical structure in
peptoids similar to that of the polyproline type I helix in
R-peptides.12 The majority of these studies have focused on
peptoids composed predominately of either (R)- or (S)-N-(1-
phenylethyl)glycine (designated Nrpe or Nspe, respectively),
which are constructed from commercially available (R)- or (S)-
1-phenylethylamine building blocks (S-1, Figure 1) using the
well-established, submonomer peptoid synthesis method.3 The
peptoid helix consists entirely of cis-amide bonds and has a
periodicity of ca. three residues per turn and a pitch of ca. 6 Å;
helix sense is determined by the handedness of the R-chiral side
chains, with S- and R-side chains giving right- and left-handed
peptoid helices, respectively.12 Helix formation is particularly
promoted in these systems by the incorporation of aromatic side
chains with 3-fold periodicity, which creates “aromatic faces”
along the length of the peptoid helix.12c Attractive, intramo-
(13) (a) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173-180. (b) Hill, D. J.;
Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S. Chem. ReV. 2001,
101, 3893-4012.
(14) Southall, N. T.; Dill, K. A.; Haymet, A. D. J. J. Phys. Chem. B 2002, 106,
521-533.
(15) Kumar, S.; Nussinov, R. ChemBioChem 2002, 3, 604-617.
(16) (a) Dill, K. A. Biochemistry 1990, 29, 7133-7155. (b) Appella, D. H.;
Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am.
Chem. Soc. 1996, 118, 13071-13072. (c) Meot-Ner, M. Chem. ReV. 2005,
105, 213-284.
(17) (a) Waters, M. L. Biopolymers 2004, 76, 435-445. (b) Waters, M. L. Curr.
Opin. Chem. Biol. 2002, 6, 736-741.
(18) Gorske, B. C.; Jewell, S. A.; Guerard, E. J.; Blackwell, H. E. Org. Lett.
2005, 7, 1521-1524.
(19) For selected examples, see: (a) Graciani, N. R.; Tsang, K. Y.; McCutchen,
S. L.; Kelly, J. W. Bioorg. Med. Chem. 1994, 2, 999-1006. (b) Prince, R.
B.; Saven, J. G.; Wolynes, P. G.; Moore, J. S. J. Am. Chem. Soc. 1999,
121, 3114-3121. (c) Gabriel, G. J.; Sorey, S.; Iverson, B. L. J. Am. Chem.
Soc. 2005, 127, 2637-2640. (d) Mahalakshmi, R.; Raghothama, S.;
Balaram, P. J. Am. Chem. Soc. 2006, 128, 1125-1138. (e) Hu, Z.-Q.; Hu,
H.-Y.; Chen, C.-F. J. Org. Chem. 2006, 71, 1131-1138.
(20) (a) Armand, P.; Kirshenbaum, K.; Goldsmith, R. A.; Farr-Jones, S.; Barron,
A. E.; Truong, K. T.; 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) Armand, P.; Kirshenbaum, K.; Falicov, A.; Dunbrack, R. L. Jr.; Dill,
K. A.; Zuckermann, R. N.; Cohen, F. E. Folding Des. 1997, 2, 369-375.
(21) For an excellent review of aromatic-aromatic interactions, see: Meyer,
E. A.; Castellano, R. K.; Diederich, F. Angew. Chem., Int. Ed. 2003, 42,
1210-1250.
(22) Battaglia, M. R.; Buckingham, A. D.; Williams, J. H. Chem. Phys. Lett.
1981, 78, 420-423.
(23) Coates, G. W.; Dunn, A. R.; Henling, L. M.; Dougherty, D. A.; Grubbs,
R. H. Angew. Chem., Int. Ed. Engl. 1997, 36, 248-251.
(24) Butterfield, S. M.; Patel, P. R.; Waters, M. L. J. Am. Chem. Soc. 2002,
124, 9751-9755.
(25) (a) Patrick, C. R.; Prosser, G. S. Nature (London) 1960, 187, 1021. (b)
Williams, V. E.; Lemieux, R. P.; Thatcher, G. R. J. J. Org. Chem. 1996,
61, 1927-1933. (c) Xu, R.; Gramlich, V.; Frauenrath, H. J. Am. Chem.
Soc. 2006, 128, 5541-5547.
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A R T I C L E S
Gorske and Blackwell
amine). The eluent was removed by careful distillation to afford 162.2
mg of a yellow liquid that was 68% amine S-2 by mass, as indicated
by 1H NMR analysis (remaining mass was residual pentane/EtOAc).26
49% yield overall. An ee of >99% was determined by reversed-phase
high performance liquid chromatography (RP-HPLC) using a Daicel
Chemical Industries Chiralcel OD-H column (flow rate ) 1 mL/min,
1% ethanol/hexanes; UV detection at 260 nm; retention time ) 20.9
min).28 TLC Rf ) 0.25 (1:1 pentane/EtOAc with 1% triethylamine);
1H NMR (CDCl3, 300 MHz) δ 4.47 (q, J ) 7.0 Hz, 1H), 1.54 (dt, J )
7.0, 0.9 Hz, 3H); 13C NMR (CDCl3, 1H broadband-decoupled, 75 MHz)
δ144.6 (multiplet), 139.9 (multiplet), 137.5 (multiplet), 120.3 (multi-
plet), 43.1 (s), 23.4 (s); 19F NMR (CDCl3) δ -145.1 (dd, J ) 23,
8 Hz, 2F), -157.1 (t, J ) 21 Hz, 1F), -162.2 (ddd, J ) 23, 23, 8 Hz,
2F); IR (ATR): 3394 (broad), 2984, 2938, 1738, 1652, 1521, 1500,
1460, 1376, 1300, 1245, 1132, 1115, 971, 939, 849 cm-1; EI-MS:
expected m/z 211.0, observed m/z 212.1 [M + H+]+; [â]D25) -12.2°
(CDCl3, c ) 0.017).
Peptoid Synthesis, Purification, and Characterization. Peptoids
5-20 were synthesized according to the microwave-assisted synthesis
procedure we reported previously (Table 1).18 Amine building blocks
(S)-1-phenylethylamine (S-1) and (S)-1-(pentafluorophenyl)ethylamine
(S-2) were used for peptoid synthesis and were either purchased from
Aldrich or synthesized as described above, respectively.29 After acid-
mediated cleavage from the resin, peptoid samples were analyzed by
RP-HPLC (with UV detection) using a Restek Premier C18 column (5
µm, 4.6 mm × 25 cm). Initial purities were determined by integration
at 220 nm and ranged from 42 to 84%. Peptoid oligomers 5-20 were
purified to homogeneity (>97%) by preparative RP-HPLC using a
Vydac protein and peptide C18 column (10 µm, 22 mm × 250 mm).
The purified peptoids were lyophilized to afford white powders. The
purities and molar masses of the purified peptoids were verified by
analytical RP-HPLC and mass spectrometry, respectively (Table 1).
Circular Dichroism Analyses. Circular dichroism (CD) spectra were
obtained on a Jasco J-715 spectropolarimeter with J-700 for Windows
Standard Analysis software (v. 1.50.01). CD data were analyzed and
plotted using Microsoft Excel 2004 and KaleidaGraph 4.0 software.
Peptoid stock solutions were prepared by dissolving at least 2 mg of
each peptoid in a precisely measured amount of spectroscopic grade
acetonitrile (ca. 830 mg). The stock solutions then were diluted with
spectroscopic grade acetonitrile to the desired concentration (ca. 60
µM) by mass using a Mettler-Toledo XS105 high-precision balance.
CD spectra were obtained in a square quartz cell (path length 0.2 cm)
at room temperature using a scan rate of 100 nm/min, with 20 averaged
scans per spectrum. The spectrum of an acetonitrile blank was subtracted
from the CD traces, and the resulting data were smoothed using the
weighting function in KaleidaGraph 4.0.
while simultaneously preserving a steric profile that supports
helix formation. Here, we demonstrate exquisite control over
peptoid nonamer folding by the strategic incorporation of a
single (S)-N-(1-(pentafluorophenyl)ethyl)glycine, or “Nsfe”,
monomer unit. This new design principle has facilitated the
construction of well-folded peptoids with unprecedented stability
and should significantly expand the potential of peptoids as
chemical tools in numerous applications. Furthermore, when
installed in conjunction with hydrogen bonding side chains, this
pentafluoroaromatic monomer could provide access to heretofore
unknown peptoid structures that are stabilized by additional,
tunable hydrogen bonds.
Experimental Section
General. All reagents were purchased from commercial sources
(Alfa-Aesar, Aldrich, Advanced ChemTech, and Acros) and used
without further purification. Solvents were purchased from commercial
sources (Aldrich and J. T. Baker) and used as is, with the exception of
dichloromethane (CH2Cl2), which was distilled over calcium hydride
immediately prior to use. Fmoc-protected, Rink amide linker-derivatized
polystyrene resin (100-200 mesh, loading ) 0.69 mmol/g; Advanced
ChemTech) was used for all solid-phase syntheses. Full details of the
instrumentation and analytical methods used in this work can be found
in the Supporting Information.
Synthesis of (S)-1-(Pentafluorophenyl)ethyl Azide (S-4). Diphen-
ylphosphoryl azide (DPPA, 1.12 mL, 5.19 mmol) and diethyl azodi-
carboxylate (DEAD, 1.00 mL, 5.19 mmol) were cannulated into a dry
50 mL flask containing 25 mL of dry tetrahydrofuran (THF) and
immersed in an ice-water bath. The solution was stirred under N2 for
15 min, after which a solution of (R)-(+)-1-(pentafluorophenyl)ethanol
(1.00 g, 4.72 mmol) in dry THF was cannulated into the flask. The
solution was stirred under N2 for an additional 15 min while immersed
in the ice-water bath. Triphenylphosphine (PPh3, 1.11 g, 4.25 mmol)
was dissolved in dry THF and cannulated into the flask. The reaction
was stirred for 15 min under N2, after which it was sealed and placed
in a freezer at -5 °C for 16 h. The THF was subsequently removed by
careful distillation to give a yellow, viscous residue that was further
purified by flash silica gel column chromatography (pentane). The
pentane was removed by careful distillation to afford 1.84 g of a clear
liquid that was 47% (S)-1-(pentafluorophenyl)ethyl azide (S-4) by mass,
as indicated by 1H NMR (remaining mass was residual pentane).26 77%
1
yield. TLC Rf ) 0.36 (pentane); H NMR (CDCl3, 300 MHz) δ 5.01
1
(q, J ) 7.1 Hz, 1H), 1.67 (d, J ) 7.1 Hz, 3H); 13C NMR (CDCl3, H
broadband-decoupled, 75 MHz) δ 145.0 (dddd, JCF ) 249.8, 17.0, 8.3,
4.6 Hz), 141.1 (dtt, JCF ) 256.7, 13.6, 5.3 Hz), 137.7 (ddddd, JCF
)
252.8, 16.5, 12.7, 4.9, 2.3 Hz); 19F NMR (CDCl3) δ -142.4 (dd, J )
21.5, 5.8 Hz, 2F), -153.7 (t, J ) 21.5 Hz, 1F), -161.1 (ddd, J )
21.5, 21.5, 6.5 Hz, 2F); IR (ATR): 3342, 2992, 2944, 2115, 1653,
Heteronuclear Single Quantum Coherence (HSQC) NMR Analy-
ses. HSQC NMR experiments30 were performed on a Varian Inova
600 MHz spectrometer using a 5 mm hpx probe. The data were
processed using the Varian VNMR software package (v. 6.1C) and
visualized using SPARKY software.31 Peptoids 5, 7, and 9 were
dissolved in acetonitrile-d3 to give ca. 6 mM solutions. For analysis,
400 µL aliquots of these solutions were placed in a 5 mm Shigemi
NMR tube that was susceptibility-matched for dimethyl sulfoxide. The
HSQC experiments were performed at 24 °C using the following
parameter values: spectral widths were 6794.6 Hz in the 1H dimension
and 27903.7 Hz in the 13C dimension. The number of transients (nt)
and number of increments (ni) were 24 and 512, respectively. The
1503, 1459, 1425, 1358, 1305, 1244, 1152, 1026, 971, 917, 848 cm-1
EI-MS: expected m/z 237.0, observed m/z 237.0 [M]+; [â]D25 ) +7.28°
(CDCl3, c ) 0.055).
;
Synthesis of (S)-1-(Pentafluorophenyl)ethylamine (S-2).27 Azide
S-4 (196.3 mg, 0.83 mmol) was dissolved in 10 mL of THF in a dry
25 mL flask. PPh3 (435.0 mg, 1.66 mmol) was dissolved in 15 mL of
THF and added dropwise to the stirred solution. After the bubbling of
N2 had subsided, distilled H2O (134 mL, 7.45 mmol) was added, and
the reaction mixture was stirred for 16 h at 45 °C. The solvent was
removed in Vacuo, and the product was purified by flash silica gel
chromatography (1:1 pentane/ethyl acetate (EtOAc) with 1% triethyl-
(28) Enantiomeric excess (99% ee) was determined by comparison of peak area
percentages with those of a sample of racemic amine 2.
(29) A 1.0 M solution of (S)-1-(pentafluorophenyl)ethylamine (S-2) in DMF
was used for peptoid synthesis and was reused in coupling reactions up to
10 times without seriously compromising yields. This procedural modifica-
tion allowed us to conserve this amine reagent.
(26) Amine S-2 and azide S-4 were unusually volatile when dissolved in organic
solvents. Careful distillation ameliorated this effect, but nevertheless, the
complete removal of solvents significantly reduced yields.
(27) Amine S-2 has been synthesized in racemic form previously using a
different, and lower yielding, method. See: Petrova, T. D.; Savchenko, T.
I.; Ardyukova, T. F.; Yakobson, G. G. IzV. Sib. Otd. Akad. Nauk SSSR,
Ser. Khim. Nauk 1970, 3, 119-122.
(30) Boyer, R. D.; Johnson, R.; Krishnamurthy, K. J. Magn. Reson. 2003, 165,
253-259.
(31) Goddard, T. D.; Kneller, D. G. SPARKY, version 3.110; University of
California: San Francisco, CA.
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Tuning Peptoids with Pentafluoroaromatic Functionality
A R T I C L E S
Table 1. Structures and Mass Spectrometry Data for Peptoid Oligomers 5-20a
peptoid oligomer
monomer sequence (amino to carboxy terminus)
calculated mass
observed mass
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Nspe-(Nsfe-Nspe)2
1002.4
1826.6
822.5
1003.3 [M + H+]+
1827.6 [M + H+]+
823.5 [M + H+]+
1467.8 [M + H+]+
1273.2 [M + H+]+
2112.7 [M + H+]+
1827.4 [M + H+]+
1579.8 [M + Na+]+
1557.2 [M + H+]+
1557.7 [M + H+]+
1557.4 [M + H+]+
1557.2 [M + H+]+
1579.5 [M + Na+]+
1557.8 [M + H+]+
1557.8 [M + H+]+
1558.0 [M + H+]+
Nspe-(Nsfe-Nspe)4
(Nspe)5
(Nspe)9
(Nsfe)5
(Nspe)13
1466.8
1272.2
2111.1
1826.6
1556.7
1556.7
1556.7
1556.7
1556.7
1556.7
1556.7
1556.7
1556.7
Nspe-Nsfe-(Nspe)2-(Nsfe)2-Nspe-Nsfe-Nspe
Nsfe-(Nspe)8
(Nspe)-(Nsfe)-(Nspe)7
(Nspe)2-(Nsfe)-(Nspe)6
(Nspe)3-(Nsfe)-(Nspe)5
(Nspe)4-(Nsfe)-(Nspe)4
(Nspe)5-(Nsfe)-(Nspe)3
(Nspe)6-(Nsfe)-(Nspe)2
(Nspe)7-(Nsfe)-(Nspe)
(Nspe)8-Nsfe
a All compounds were purified to >97% homogeneity before analysis by CD and NMR. Mass spectrometry data were acquired using either ESI or
MALDI-TOF techniques.
Scheme 1. Synthesis of (S)-1-(Pentafluorophenyl)ethylamine
We also desired to avoid racemization of the chiral starting
material and product. In an attempt to address these issues, we
(S-2)a
examined a variant of the Mitsunobu reaction using alcohol
R-3.33 We found that the initial displacement step did not occur
at an appreciable rate at low temperatures (-78 °C), while
modest racemization was observed at room temperature. A
systematic scan of reaction conditions revealed that the displace-
ment step proceeds with good yield and complete inversion of
configuration using 0.9 equiv of triphenylphosphine at 0 °C.
a Reagents and conditions: (a) 1.1 equiv of diphenylphosphoryl azide
(DPPA), 1.1 equiv of diethyl azodicarboxylate (DEAD), 0.9 equiv of PPh3,
Azide S-4 was then reacted with triphenylphosphine in the
presence of water to afford the desired (S)-1-(pentafluorophen-
yl)ethylamine (S-2) in good yield and high enantiopurity (99%).
During reaction optimization, we noted the propensities of the
azide (S-4) and amine (S-2) to evaporate when mixed with
common organic solvents, possibly due to the formation of
azeotropes.26 This behavior was most pronounced for the azide
(S-4) and warranted careful distillation for solvent removal. The
absolute configuration of S-2 was verified by X-ray crystal-
lographic analysis of a crystal of the (+)-2,3-dibenzoyl-D-tartrate
salt (see Figure S-1, Supporting Information).34 We found this
synthetic route to be reproducible and conductible on a gram
scale. Pentafluoroaromatic amine S-2 should prove valuable not
THF, -5 °C, 36 h, 77% yield. (b) 2 equiv of PPh3, 9 equiv of H2O, THF,
45 °C, 12 h, 63% yield.
number of points (np) was 2048, 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.
Results and Discussion
Synthesis of (S)-1-(Pentafluorophenyl)ethylamine (S-2).
Our two-step synthetic route to (S)-1-(pentafluorophenyl)-
ethylamine (S-2) is shown in Scheme 1.27 In devising a route
to amine S-2, we sought to overcome a number of potential
synthetic difficulties. Foremost of these was the potential for
nucleophilic aromatic substitution on the pentafluorophenyl ring;
this was indeed observed at elevated temperatures and has
proven to be a problematic side reaction in similar systems.32
(33) Thompson, A. S.; Humphrey, G. R.; Demarco, A. M.; Mathre, D. J.;
Grabowski, E. J. J. J. Org. Chem. 1993, 58, 5886-5888.
(34) An ORTEP diagram of the S-2 salt is shown in the Supporting Information
(Figure S-1). X-ray coordinates for S-2 have been deposited with the
Cambridge Crystallographic Database Centre (CCDC entry no. 281861).
These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/
data_request/cif.
(32) Dilman, A. D.; Belyakov, P. A.; Korlyukov, A. A.; Struchkova, M. I.;
Tartakovsky, V. A. Org. Lett. 2005, 7, 2913-2915.
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Scheme 2. Microwave-Assisted Peptoid Submonomer Synthesisa
three faces of the peptoid helix. We hypothesized that quadru-
polar π-stacking interactions in these heteropeptoids could
perturb, and potentially strengthen, helical conformations relative
to homooligomer peptoids (7-10). We also constructed hetero-
nonamer 11 in which an identical number of Nsfe units (relative
to nonamer 6) were arranged to maximize the alignment of like
monomers along each helical face. This peptoid allowed us to
probe for helix disruptions caused by weakened π-stacking
interactions. We reasoned that comparison of peptoids with
identical monomer compositions, such as 6 and 11, would most
convincingly demonstrate any sequence-dependent phenomena,
i.e., π-stacking, while minimizing interference by other sequence-
independent effects, such as local steric interactions. Peptoid
homopentamer 7, homononamer 8, homo-13-mer 10 (consisting
of Nspe monomers), and homopentamer 9 (consisting of Nsfe
monomers) served as important controls for these studies.
The relative helical propensities of peptoids with R-chiral side
chains can be assessed by circular dichroism (CD), a well-
established tool for the evaluation of folded structures in peptides
and proteins.36 For peptoids with R-chiral aromatic side chains,
the correlation of relatively intense CD spectra (i.e., diagnostic
bands at 192 and 202 nm for the π-π* transition, and at 218
nm for the n-π* transition) with helical secondary structures
has been supported by molecular modeling, NMR, and X-ray
crystallography studies.12 We therefore obtained the CD spectra
of peptoids 5-11 to gain further insight into the effect of Nsfe
units on peptoid secondary structures (shown in Figure 3).
Control Nspe homopentamer 7 has been shown previously
to display the canonical CD signature for the peptoid helix,12b
while control Nspe homononamer 8 exhibits an atypical, intense
CD signature (i.e., a broad, shouldered peak at 203 nm) that
corresponds to a nonhelical, “threaded loop” structure.12e
Reduction in CD intensity at 202 nm has been observed for
longer Nspe homopeptoids (11-20 units) such as 13-mer 10,
which exhibit enhanced helicity relative to shorter peptoids (5-8
units). Negative CD intensities near 202 nm have been suggested
to vary inversely with helical propensity in R-chiral aromatic
peptoids, with deeper minima indicating a higher population of
conformations containing trans-amide bonds.12b As expected,
we obtained CD spectra for control homopeptoids 7, 8, and 10
that were analogous to those previously reported (Figure 3).
Turning to fluoroaromatic peptoid pentamers 5 and 9, we
found that heteropentamer 5 displayed a CD signature similar
in shape to that of the Nspe homopentamer control (7), yet with
a 3-fold weaker intensity at 202 nm (Figure 3, left). The Nsfe
homopentamer 9 exhibited a further reduction in intensity at
202 nm compared to 5 in its CD signature and an almost
complete abolition of the band near 190 nm. These striking
effects may be attributable to the fluorination of the aromatic
side chains, which could perturb the π-π* transitions of the
amide chromophore and produce a blue shift of these bands.
Similar blue shifts have been reported in other peptoids with
deactivated aromatic side chains.12a The disappearance of the
190 nm band may be due to a blue shift below the observable
range of these experiments. In contrast, the bands near 215 nm
appear to be relatively unaffected by side chain modification,
and a comparison of intensities near this wavelength suggests
that the helicities of heteropentamer 5 and homopentamers 7
and 9 are similar. Overall, the CD data did not demonstrate
a Beads ) 200 mesh polystyrene with rink amide linker. Reagents and
conditions: (a) 2 M bromoacetic acid, 2 M N,N-diisopropylcarbodiimide,
DMF, microwave heating (30 s to 35 °C). (b) 1 M amine building block
NH2R1, DMF, microwave heating (90 s to 95 °C). For details of this
synthetic method, see ref 18.
Figure 2. Representations of the relative side chain alignments viewed
down the putative helical axes of peptoids 5-11. The N-terminal residue
is shown at the bottom of the downward-pointing spoke, with subsequent
residues displayed in a counterclockwise, inward-winding spiral, as shown
for 5. S ) Nspe, green F ) Nsfe.
only for peptoid synthesis but also as a chiral synthon in a
variety of synthetic applications.
Peptoid Synthesis. Peptoids are synthesized routinely via the
solid-phase submonomer synthesis method developed by Zuck-
ermann et al.3 This method entails coupling of bromoacetic acid
to a solid support, followed by nucleophilic displacement of
the bromide with a primary amine to produce peptoid monomer
units (Scheme 2). We recently reported a microwave-assisted
version of this procedure for the efficient synthesis of peptoids
incorporating deactivated benzyl amines and found it to be well
suited for the installation of pentafluoroaromatic amine S-2.18,35
This synthetic method was used to generate the subject peptoids
of this study (5-20, Table 1) in good to excellent crude purities,
typically in the range of 70-85%.28,29 Notably, we did not
observe substitution reactions on the fluoroaromatic ring using
this protocol. All peptoids were purified to homogeneity (>97%)
by preparative RP-HPLC prior to structural analyses. Purities
and molar masses of HPLC-purified samples were confirmed
by analytical RP-HPLC and mass spectroscopy (Table 1).
Investigations of π-Stacking in Peptoid Helices. A series
of heteropeptoids and homopeptoids containing (S)-N-(1-phen-
ylethyl)glycine (Nspe) and (S)-N-(1-(pentafluorophenyl)ethyl)-
glycine (Nsfe) units were synthesized in order to probe π-
stacking interactions in peptoid helices (5-11, Table 1). The
side chain positions viewed down the putative helical axes of
these peptoids are shown schematically in Figure 2. Hetero-
pentamer 5 and heterononamer 6 were designed to display
alternating aromatic and fluoroaromatic side chains along the
(35) Other researchers have investigated microwave-assisted peptoid synthesis.
See: (a) Olivos, H. J.; Alluri, P. G.; Reddy, M. M.; Salony, D.; Kodadek,
T. Org. Lett. 2002, 4, 4057-4059. (b) Fara, M. A.; Diaz-Mochon, J. J.;
Bradley, M. Tetrahedron Lett. 2006, 47, 1011-1014.
(36) Woody, R. W. Methods Enzymol. 1995, 246, 34-71.
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Tuning Peptoids with Pentafluoroaromatic Functionality
A R T I C L E S
Figure 3. CD spectra of peptoids 5, 7, and 9 (left) and peptoids 6, 8, 10, and 11 (right) at 60 µM in acetonitrile. Spectra were collected at 25 °C.
that quadrupole-enhanced, intramolecular π-stacking is operative
in these systems.
We next examined the impact of Nsfe units on the structures
of longer peptoid systems, i.e., nonamers 6, 8, and 11 (Figure
3, right). As peptoid nonamers composed largely of R-chiral,
aromatic monomers had been shown to significantly populate
alternative, nonhelical conformational states (Vide supra),12b
these systems provided latitude for enhancement of helical
structure. We were gratified to observe that the CD signature
of the alternating heterononamer 6 indicated a dramatic increase
in helical propensity relative to the Nspe homononamer 8 and
corresponded closely to that of the longer, helical Nspe 13-mer
10.12b However, heterononamer 11 exhibited a nearly identical
CD signature to peptoid 6, despite the altered arrangement of
aromatic quadrupoles along the faces of this peptoid. These
results suggested that intramolecular π-stacking is not the
dominant factor in the stabilization of canonical helices for this
peptoid class.
We also considered the possibility that intermolecular inter-
digitation of peptoid side chains mediated by aromatic interac-
tions could stabilize peptoid helices. Although prior work by
Wu et al. has demonstrated that the structure of an Nrpe hexamer
is independent of concentration between 0.8 and 64 µM in
acetonitrile,12b we reasoned that the distinctive quadrupole and/
or solubility characteristics of Nsfe monomers could facilitate
stabilization by this mechanism. Solutions of Nspe homopen-
tamer 7 and Nsfe homopentamer 9 (each 60 µM) were mixed,
and the CD spectrum of the mixture was recorded (Figure 4).
This trace was simply the average of the spectra of its
constituents, as would be expected if no aggregation-induced
folding was to occur. The peptoid mixture then was heated to
65 °C and subsequently left standing at room temperature in
an attempt to overcome potential kinetic barriers to interdigi-
tation by annealing; however, the CD spectrum of the mixture
remained unchanged. The mixture was finally diluted with an
equal volume of water to determine if increased solvent polarity
might induce interdigitation by hydrophobic aggregation, but
only a small increase (within the error range determined for
dilution by volume) in helicity was observed. We note that any
increase in helicity could also arise from the enhanced hydro-
Figure 4. CD spectra of 60 µM solutions of peptoids 7 and 9 in acetonitrile
(red and blue), a 1:1 mixture of 7 and 9 (total peptoid concentration was
60 µM in acetonitrile) before (green) and after (orange) attempted annealing,
and a 1:1 mixture of 7 and 9 (total peptoid concentration of 30 µM) in 1:1
acetonitrile/water (purple). Spectra were collected at 25 °C.
phobic collapse of unassociated, individual peptoids, as has been
previously suggested.37 Overall, these experiments did not
provide compelling evidence that intermolecular interdigitation
of the peptoid side chains stabilizes helical structure under these
conditions. Studies examining this phenomenon over a wider
range of conditions are ongoing in our laboratory, however, in
order to further assess the possibility of aggregation-induced
folding in these systems.
The studies above suggest that intramolecular, quadrupole-
enhanced π-stacking may not play an appreciable role in
stabilizing peptoid helices. The CD spectra of heterononamer
peptoids 6 and 11 containing identical numbers of Nsfe
monomers were the most notable in this regard; these were
(37) Bradley, E. K.; Kerr, J. M.; Richter, L. S.; Figliozzi, G. M.; Goff, D. A.;
Zuckermann, R. N.; Spellmeyer, D. C.; Blaney, J. M. Mol. DiVersity 1997,
3, 1-15.
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Gorske and Blackwell
Figure 5. Methyne regions in the HSQC NMR spectra of the peptoid pentamers 5, 7, and 9. Samples were prepared in acetonitrile-d3 (ca. 6 mM) and
analyzed at room temperature (24 °C). For full HSQC spectra, see Supporting Information (Figures S-2 to S-4).
nearly equal in intensity, regardless of the sequence of aromatic
and pentafluoroaromatic groups along the helical faces. These
data caused us to speculate that periodic incorporation of
R-chiral aromatic side chains in peptoids may stabilize helices
by other means, such as hydrophobic or cooperative steric
effects. However, we could not completely rule out the
possibility that intra- or intermolecular π-stacking may still be
operative but is not directly observable in these experiments.
We therefore obtained heteronuclear single quantum coherence
(HSQC) NMR data for pentamer peptoids 5, 7, and 9 (Figure
5) in an effort to obtain additional information about the
behavior of these systems. Analysis of the peptoid side chain
methyne correlations in this type of NMR experiment has been
used previously to assess the conformational distribution of
peptoids in solution.12d As the peptoid helix consists exclusively
of cis-amide bonds, the relative integrations of cis- and trans-
amide correlations have been used as secondary indicators of
peptoid helicity. Furthermore, the number of distinct methyne
correlations has also been used to qualitatively evaluate con-
formational homogeneity. We expected that the introduction of
strongly electron-withdrawing groups such as amine S-2 would
decrease the barrier for cis-trans isomerization of the tertiary
amides,38 resulting in increased backbone flexibility. We thus
analyzed samples in acetonitrile-d3 at 24 °C with peptoid
concentrations of ca. 6 mM.
A comparison of the HSQC NMR spectra revealed a reduced
number of correlations for Nsfe homopentamer 9 relative to
pentamers 5 and 7 (Figure 5), initially implying greater
conformational homogeneity in this system relative to the other
pentamers. However, the broader line widths in the Nsfe
homopentamer 9 spectrum suggested that this peptoid could be
subject to dynamic conformational interconversion on the NMR
experimental time scale at room temperature. This interconver-
sion of peptoid conformers has previously been observed in
similar peptoid systems, albeit at rates that presumably did not
complicate the assessment of conformational distributions.20
Alternatively, line broadening might also be caused by aggrega-
tion; however, data acquisition at concentrations approaching
the detection limit of the NMR spectrometer (below 1 mM)
did not improve spectral quality (data not shown). Regardless
of the cause, this signal broadening complicated the rigorous
quantification and integration of the methyne correlations for
9, although a semi-quantitative analysis of the pentamer spectra
revealed that the overall percentages of cis- and trans-amide
bonds were similar (see Table S-1, Supporting Information).
Notably, the Nsfe residues of 5 (at positions 2 and 4) exhibited
a significantly higher cis:trans-amide ratio than the Nspe
residues (approximately 3:1 versus 1:1). As “fraying” of the
peptoid helix at the C-terminus has been observed previously,20
this finding could be attributed to a higher degree of helicity at
internal positions within the oligomer, as opposed to the termini,
and suggests that peptoid 5 is indeed structured.
We also obtained rotating frame Overhauser spectroscopy
(ROESY) NMR of spectra of all three homopentamers (see
Figures S-5 to S-7, Supporting Information) in order to reinforce
the structural parity of this peptoid series. These spectra
exhibited similar contact patterns that suggest the spatial
relationships between protons are comparable among the
members of this peptoid class. Overall, the congruity of these
ROESY and HSQC NMR spectra, in conjunction with the well-
defined CD spectra exhibited by the pentamers, implies that
these systems are structurally similar. Ongoing studies in our
laboratory are directed at further characterizing the solution-
phase behavior of several of the peptoid systems introduced
herein by NMR. This work is expanding our understanding of
the folding of perfluoroaromatic peptoids and will be reported
in due course.
Influence of Pentafluoroaromatic Monomers on Peptoid
Nonamer Structure. We were intrigued by the dramatic
enforcement of helicity observed in the CD spectra of peptoid
nonamers upon Nsfe incorporation, e.g., peptoids 6 and 11.
However, the experiments outlined above suggested that in-
tramolecular π-stacking interactions between side chains were
not primarily responsible for this effect. During the course of
these investigations, Huang et al. reported an NMR structure
of the Nspe homononamer 8 that resembled a “threaded loop”
(shown in Figure 6, left).12e This structure is stabilized by
intramolecular hydrogen bonds between the peptoid termini
and several backbone amide carbonyls (residues 5, 7, and 9 in
Figure 6, left). The structural necessity of these contacts was
demonstrated by several studies in which the structure was
disordered upon titration with protic solvents. We hypothesized
(38) Eberhardt, E. S.; Panisik, N., Jr.; Raines, R. T. J. Am. Chem. Soc. 1996,
118, 12261-12266.
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Tuning Peptoids with Pentafluoroaromatic Functionality
A R T I C L E S
Figure 6. (Left) Representative NMR structure of the Nspe nonamer 8 in acetonitrile.40 Numbers indicate relative positions of amide side chains on the
peptoid backbone (from amino to carboxy terminus). Backbone atoms are shown only; red ) oxygen; blue ) nitrogen; gray ) carbon. Putative hydrogen
bonds are shown as green lines. (Right) CD spectra of peptoids 8 and 12-20 at 60 µM concentrations in acetonitrile. Trace colors correlate with those of
the Nsfe position numbers in structure at left. Spectra were collected at 25 °C.
that Nsfe pentafluoroaromatic rings could significantly deactivate
the proximal backbone amides as hydrogen bond acceptors,39
disrupting the threaded loop structure and enhancing the helicity
of peptoids such as 6 and 11. This behavior could be exploited
to selectively “tune” the hydrogen bonding capacities of each
backbone carbonyl, providing an approach for rationally con-
trolling peptoid structure while simultaneously retaining the
established steric profile for peptoid helix formation. We tested
this hypothesis by performing an Nsfe “scan” of the Nspe
nonamer 8, in which a series of nine peptoids were synthesized,
each incorporating the Nsfe monomer at a different location
(12-20, Table 1). The CD spectra of these peptoids were
obtained and analyzed to assess the structural impact of the
pentafluoroaromatic side chain at each location (Figure 6, right).
The position of the single Nsfe monomer in the nonamer
peptoid clearly had remarkable consequences for the folding
of this peptoid class. The intensities of the CD bands near 192,
202, and 218 nm have been used previously to qualitatively
assess the relative populations of helical and looped secondary
structures adopted by Nspe homononamer 8. As described
above, the threaded loop structure is characterized by a strong
minimum near 205 nm in its CD spectrum, whereas the helical
conformation is diagnosed by the appearance of several minima,
including an intense minimum at 218 nm.12e We analyzed the
CD spectra of peptoids 12-20 according to these criteria (Figure
6, right). As we predicted, the incorporation of fluoroaromatic
side chains near critical hydrogen bonding positions significantly
disrupted the threaded loop structure (positions 3, 6, and 8; 14,
17, and 19). We also discovered that the threaded loop is
disrupted by incorporation of fluoroaromatic side chains at two
amides not previously implicated in hydrogen bonding (positions
2 and 5; 13 and 16); this disruption is nearly absolute in the
former case. An examination of the three-dimensional structure
of the threaded loop did not reveal any significant steric
repulsion that might result from the increased size of the Nsfe
monomer relative to Nspe; however, the carbonyl of residue 1
is located four bonds from the N-terminal ammonium protons
and is ideally situated to form an intramolecular hydrogen bond
to the amine terminus. Our data suggest that the formation of
such a hydrogen bond may play a pivotal role in the formation
and/or stabilization of the loop structure. The cause of the
disruption resulting from installation of the fluoroaromatic side
chain at residue 5 is unclear, but we speculate that formation
of a hydrogen bond to this amide may also play a role during
folding. In contrast, fluoroaromatic side chains at positions 4,
7, and 9 (15, 18, and 20) produced inconsequential changes in
the CD spectra of these peptoids relative to homononamer 8.41
(39) Preliminary calculations at the RHF 6-31G* level show ca. 1.5 times more
electron density at the carbonyl oxygen of an Nspe peptoid monomer system
(A) relative to that of an analogous Nsfe monomer system (B) (see structures
below; carbonyls of interest indicated). The electron density ratio was
determined by comparing both the electrostatic potential and HOMO maps
of A and B. The calculations were performed using Spartan ‘04 for
Macintosh (Wavefunction, Inc., Irvine, CA; Kong et al. J. Comput. Chem.
2000, 21, 1532-1548; see Supporting Information for full author list).
Ongoing studies in our laboratory are focused on further examining the
electrostatics and conformational preferences of these and other monomeric
peptoid systems at higher levels of theory.
Most strikingly, we found that installation of the Nsfe
monomer at the amino terminus (in peptoid 12) significantly
stabilized the threaded loop structure relative to the helical
structure. We reasoned that inductive withdrawal of electron
density at this terminus by the pendant fluoroaromatic group
likely increases both the acidity and positive charge of the
ammonium group, with concomitant strengthening of its hy-
drogen bonding potential. We titrated an acetonitrile solution
(41) We note that excessive withdrawal of electron density from the backbone
amides would likely increase their susceptibility to hydrolysis and nucleo-
philic attack (e.g., imides); however, we did not observe any degradation
of Nsfe-amide bonds during the synthetic work and structural analyses
described herein.
(40) The atomic coordinates for this NMR structure were graciously provided
by the authors in ref 12e.
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Gorske and Blackwell
Figure 7. CD spectra of homononamers (Nspe)9 (8) (left) and Nsfe-(Nspe)8 (12) (right) at 60 µM concentrations in acetonitrile/methanol solutions. Spectra
were collected at 25 °C.
of 12 with the polar protic solvent methanol, analyzed these
solutions by CD to assess the resistance of 12 to denaturation,
and compared these data to those obtained using the Nspe
homononamer 8. As previously reported, the threaded loop
structure formed by 8 was completely lost upon titration to
approximately 50% methanol (Figure 7, left).12e In contrast, the
loop structure formed by heteropeptoid 12 was extremely
resistant to titration with methanol, withstanding total denatur-
ation until the titration was complete (Figure 7, right).
chains. This work was enabled by the development of an
efficient synthesis of (S)-1-(pentafluorophenyl)ethylamine (S-
2), which we found to be highly compatible as a building block
for peptoid construction. Systematic CD and NMR studies did
not suggest that incorporation of amine S-2 into peptoids
stabilizes helical structure by enhancing π-stacking interactions
along the helical face. Rather, we have discovered that the
judicious incorporation of this amine can effectively control the
folding of peptoids by modulating the hydrogen bonding
capacities of nearby heteroatoms. Moreover, the distinctive
threaded loop structure can be effectively promoted, even in
predominantly protic solvents, using this new pentafluoroaro-
matic peptoid building block.
These findings demonstrate that peptoid nonamer structure
can be controlled by the strategic incorporation of Nsfe
monomers. Our data suggest that these monomers serve to
control folding by modulating the hydrogen bonding potentials
of proximal amides.39 This hypothesis is supported by our
previous observation that the nucleophilicity, and presumably
also the electron density, of deactivated benzyl amines are
significantly reduced relative to those of nonsubstituted benzyl
amines.18 The stronger helical CD signatures displayed by
peptoids 6 and 11 (with Nsfe monomers at positions 2, 4, 6,
and 8, and 2, 4, 5, and 8, respectively), as well as other related
systems previously examined in our laboratory,18 can be
explained according to this rationale. Furthermore, incorporation
of this pentafluoroaromatic side chain at the amino terminus
dramatically enhances the capabilities of the latter as a hydrogen
bond donor, stabilizing the unique threaded loop structure while
simultaneously preserving the well-established steric profile
known to promote peptoid folding. Notably, this is accomplished
without introducing other heteroatoms such as oxygen or
nitrogen that could potentially compete for hydrogen bonds and
prevent proper folding. The stabilized, Nsfe threaded loop
structure largely persists in the presence of protic solvents, which
more closely mimic the aqueous media relevant to biological
function. As such, this research represents an initial step toward
the construction of new, water-stable peptoid architectures that
could be explored within this context.
We contend that the strategic incorporation of fluoroaromatic
and perhaps other electron-deficient aromatic side chains18 into
peptoids is a straightforward and effective strategy for control-
ling peptoid structure via hydrogen bonding interactions. This
design principle should significantly expand the scope of peptoid
design and enhance the utility of peptoids for a broad range of
applications. In the future, we envisage the co-introduction of
hydrogen bonding and fluoroaromatic peptoid side chains, which
could dramatically broaden this strategy by increasing the
number of tunable hydrogen bonds. These additional tunable
interactions could provide access to a diverse range of new
peptoid secondary structures, potentially with increased aqueous
solubility. Indeed, peptoids represent a unique foldamer class
with which to explore this tactic, as their distinguishing tertiary
amide backbones (as opposed to the secondary amide backbones
of typical R- and â-peptides) would not compete with hydrogen
bond donor groups installed on side chains. We also anticipate
that this hydrophobic side chain could facilitate the hydrophobic
collapse of water-soluble peptoids to generate tertiary structures,
as has been recently demonstrated.42 Ongoing work in our
laboratory is directed at further developing these design tactics
in an effort to expand the existing repertoire of peptoid folding
motifs beyond the established helix and threaded loop structures.
Conclusions
We have discovered a new design principle for peptoid
folding based on the incorporation of pentafluoroaromatic side
(42) Lee, B.-C.; Zuckermann, R. N.; Dill, K. A. J. Am. Chem. Soc. 2005, 127,
10999-11009.
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Tuning Peptoids with Pentafluoroaromatic Functionality
A R T I C L E S
Acknowledgment. We thank the NSF (CHE-0449959),
Research Corporation, the Greater Milwaukee Foundation Shaw
Scientist Program, CEM Corporation, and the UW-Madison for
financial support of this work, and the W. M. Keck Foundation
for support of instrumentation at the UW Keck Center for
Chemical Genomics. Support of the NMR facilities at UW-
Madison by the NIH (1 S10 RR13866-01, 1 S10 RR04981-01,
and 1 S10 RR0 8389-01) and the NSF (CHE-0342998, CHE-
9629688, CHE-8813550, and CHE-9208463) is gratefully
acknowledged. We thank Professors Annelise Barron and Ishwar
Radhakrishnan for providing the structural coordinates for the
peptoid threaded loop, Dr. Charles Fry and Dr. Monika Ivancic
for assistance with NMR spectroscopy, Dr. Ilia Guzei for X-ray
crystallographic analyses, Professor Thomas Brunold for use
of his CD instrumentation, Grant Geske for modeling studies,
and Professors Annelise Barron, Kent Kirshenbaum, and Samuel
Gellman for numerous helpful discussions.
Supporting Information Available: General experimental
information, details of microwave instrumentation, X-ray crystal
structure analysis of S-2/(+)-2,3-dibenzoyl-D-tartrate salt com-
plex, NMR spectra of a peptoid pentamers 5, 7, and 9, and
complete ref 39. This material is available free of charge via
the Internet at http://pubs.acs.org.
JA065248O
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