Conformational rearrangements by water-soluble peptoid foldamers

Article abstract of DOI:10.1021/ol702207n

Peptoids are a family of N-substituted glycine oligomers that are capable of forming stable helical structures. We seek peptoid monomers that can establish a strong folding propensity in aqueous conditions. Here we utilize L-phenylalanine tert-butyl ester

Full text of DOI:10.1021/ol702207n

ORGANIC
LETTERS
2
007
Vol. 9, No. 24
003-5006
Conformational Rearrangements by
Water-Soluble Peptoid Foldamers
5
Sung Bin Y. Shin and Kent Kirshenbaum*
Department of Chemistry, New York UniVersity, 100 Washington Square East,
New York, New York 10003-6688
kent@nyu.edu
Received September 7, 2007
ABSTRACT
Peptoids are a family of N-substituted glycine oligomers that are capable of forming stable helical structures. We seek peptoid monomers that
can establish a strong folding propensity in aqueous conditions. Here we utilize -phenylalanine tert-butyl ester as a readily available reagent
L
for the synthesis of (S)-N-(1-carboxy-2-phenylethyl)glycine oligomers. The products form stable secondary structures in aqueous solution in
which the conformation is dramatically responsive to variations in pH and solvent composition.
Many biopolymers display the capability to interconvert
between distinct folded states. Such conformational switching
allows proteins to respond to their environment and to
secondary structure resembling that of the polyproline I type
helix. However, helical oligopeptoids predominantly com-
6
1
mediate signaling events. Through conformational rear-
(2) (a) Zimenkov, Y.; Dublin, S. N.; Ni, R.; Tu, R. S.; Breedveld, V.;
Apkarian, R. P.; Conticello, V. P. J. Am. Chem. Soc. 2006, 128, 6770. (b)
Awasthi, S. K.; Shankaramma, S. C.; Raghothama, S.; Balaram, P.
Biopolymers 2001, 58, 465. (c) Seebach, D.; Schreiber, J. V.; Abele, S.;
Daura, X.; van Gunsteren, W. F. HelV. Chim. Acta 2000, 83, 34. (d) Lee,
B.; Zuckermann, R. N.; Dill, K. A. J. Am. Chem. Soc. 2005, 127, 10999.
rangements, proteins can promote chemo-mechanical energy
transduction or exhibit allosteric control of their activities.
Extensive efforts have been made to design novel synthetic
peptides and peptidomimetic compounds whose conforma-
(e) Dolain, C.; Maurizot, V.; Huc, I. Angew. Chem., Int. Ed. 2003, 42, 2738.
tions are responsive to changes in pH, redox states, or solvent
(f) Okamoto, I.; Nabeta, M.; Hayakawa, Y.; Morita, N.; Takeya, T.; Masu,
H.; Azumaya, I.; Tamura, O. J. Am. Chem. Soc. 2007, 129, 1892. (g) Khan,
A.; Kaiser, C.; Hecht, S. Angew. Chem., Int. Ed. 2006, 45, 1878. (h)
Kolomiets, E.; Berl, V.; Odriozola, I.; Stadler, A.; Kyritsakas, N.; Lehn, J.
Chem. Commun. 2003, 23, 2868. (i) 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. (j) Zhao, Y.; Zhong,
Z.; Ryu, E. J. Am. Chem. Soc. 2007, 129, 218. (k) Ambroggio, X. I.;
Kuhlman, B. Curr. Opin. Struct. Biol. 2006, 16, 525.
composition.2
Our investigations are focused on a class of peptidomi-
metics called peptoids, which comprise N-substituted glycine
3
monomer units. Peptoids are an example of oligomers that
demonstrate a strong propensity to adopt well-ordered con-
formations. As such, they are among a family of compounds
(3) (a) Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.;
4
described as foldamers. Peptoids are broadly resistant to
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. (b) Patch, J.
A.; Kirshenbaum, K.; Seurynck, S. L.; Zuckermann, R. N. In Pseudo-
peptides in Drug DeVelopment; Nielson, P. E., Ed.; Wiley-VCH: Weinheim,
Germany, 2004; pp 1-35.
proteolytic degradation, and a variety of bioactive peptoid
5
compounds have been reported. Peptoid sequences incor-
porating bulky chiral side chains, such as (S)-N-(1-phenyl-
ethyl)glycine (Figure 1), have the capacity to adopt a compact
(4) (a) Appella, D. H.; Christianson, L. A.; Klein, D. A.; Powell, D. R.;
Huang, X.; Barchi, J. J.; Gellman, S. H. Nature 1997, 387, 381. (b) Hecht,
S.; Huc, I. In Foldamers; Wiley-VCH: Weinheim, Germany, 2007; pp xv.
(1) Gerstein, M.; Krebs, W. Nucleic Acids Res. 1998, 26, 4280.
1
0.1021/ol702207n CCC: $37.00
© 2007 American Chemical Society
Published on Web 10/26/2007

moieties are introduced upon the displacement of bromide
by diverse primary amine submonomer reagents. In this
study, we evaluate R-amino acids as readily accessible chiral
reagents for the solid-phase synthesis of peptoid oligomers.
We use L-phenylalanine tert-butyl ester as a submonomer
reagent for synthesis of a family of (S)-N-(1-carboxy-2-
phenylethyl)glycine (Nscp) oligomers (Scheme 1). The
carboxy phenylethyl side chains are anticipated to provide
both water solubility and structure-inducing elements. These
side chains endow the peptoid with a chiral center and steric
bulkstwo characteristics that have been described as im-
portant contributors in directing stable secondary structure
Figure 1. Model structure of previously studied helical peptoid
oligomers containing bulky chiral (S)-N-(1-phenylethyl)glycine
residues.
5
,9
formation. Many R-amino acids, including phenylalanine,
provide both these structural elements and additionally offer
carboxylic acid as an ionizable functional group. Foldamer
structures that incorporate ionizable groups may display
sensitivity toward pH conditions and act as elements that
trigger conformational rearrangements driven by electrostatic
interactions.
posed of (S)-N-(1-phenylethyl)glycine residues are generally
insoluble in water. Efforts to enhance the solubility of
structured peptoids have relied upon the additional presence
of monomers bearing polar side chains.5a,7 Because these
oligomers rely on one set of side chains for structure
formation and another set for water solubility, the diversity
of structured sequences is limited. Our aim in this study is
to demonstrate that R-amino acids can be used to afford
water-soluble peptoid secondary structures and provide the
constituents for conformational rearrangements that respond
to environmental influences such as pH, solvent polarity, and
ionic strength.
Oligopeptoids used in this study were synthesized on Rink
amide resin following the previously reported solid-phase
peptoid synthesis protocol (Scheme 1) with adjustments in
4
,6
reaction time and washing conditions.
The oligomers were cleaved from the resin with 95% TFA/
O. All compounds were purified to >95% homogeneity
H
2
Peptoid oligomers can be synthesized via efficient solid-
phase “submonomer chemistry” methods (Scheme 1) to
by reversed phase HPLC (Figure S1). Molecular weights
were confirmed by LC-MS and were uniformly in agreement
with expected values (Table S1).
The ability of the Nscp monomers to direct the formation
of stable secondary structure was initially evaluated by CD
analysis of a series of N-acetylated Nscp homo-oligomers
n
Scheme 1. Solid-Phase Submonomer Synthesis of AcNscp :
DIC, Diisopropylcarbodiimide; DMF, N,N-Dimethylformamide;
DIEA, Diisopropylethylamine; TFA, Trifluoroacetic Acid
(AcNscp
n
) ranging in length from a dimer to a 13-mer
(Scheme 1 and Figure 2). The magnitude of change in CD
intensity (per mole residue; in 5 mM phosphate and 5 mM
citric acid buffer, pH 2/40% acetonitrile) was substantial
between dimer to 7-mer. Little to no change in CD signature
was observed between 7-mer to 13-mer. Similar length-
dependent spectroscopic features have been considered to
be a hallmark for the presence of stable secondary structures
1
0
in synthetic foldamer systems. These results indicate that
Nscp homo-oligomers are capable of adopting stable second-
ary structures even in the absence of other structure-inducing
residues.
(6) (a) Kirshenbaum, K.; Barron, A. E.; Goldsmith, R. A.; Armand, P.;
Bradley, E. K.; Truong, K. T. V.; Dill, K. A.; Cohen, F. E.; Zuckermann,
R. N. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4303. (b) 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. (c) Fafarman,
A. T.; Borbat, P. P.; Freed, J. H.; Kirshenbaum, K. Chem. Commun. 2007,
incorporate specific sequences of chemically diverse N-
substituted glycine monomer units. This approach iterates
sequential steps of bromoacetylation and nucleophilic dis-
placement to construct each monomer unit. The side chain
8
4
, 377. (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,
1
25, 13525.
(5) (a) Barron, A. E.; Zuckermann, R. N. Curr. Opin. Chem. Biol. 1999,
(7) Sanborn, T. J.; Wu, C. W.; Zuckermann, R. N.; Barron, A. E.
3
, 681. (b) Patch, J. A.; Barron, A. E. Curr. Opin. Chem. Biol. 2002, 6,
Biopolymers 2002, 63, 12.
(8) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J. Am.
Chem. Soc. 1992, 114, 10646.
(9) Wu, C. W.; Sanborn, T. J.; Huang, K.; Zuckermann, R. N.; Barron,
A. E. J. Am. Chem. Soc. 2001, 123, 6778.
(10) (a) Wu, C. W.; Sanborn, T. J.; Zuckermann, R. N.; Barron, A. E. J.
Am. Chem. Soc. 2001, 123, 2958. (b) Violette, A.; Averlant-Petit, M. C.;
Semetey, V.; Hemmerlin, C.; Casimir, R.; Graff, R.; Marraud, M.; Briand,
J.; Rognan, D.; Guichard, G. J. Am. Chem. Soc. 2005, 127, 2156.
8
72. (c) Gibbons, J. A.; Hancock, A. A.; Vitt, C. R.; Knepper, S.; Buckner,
S. A.; Burne, M. E.; Milicic, I.; Kerwin, J. F.; Richter, L. S.; Taylor, E.
W.; Spear, K. L.; Zuckermann, R. N.; Spellmeyer, D. C.; Braeckman, R.
A.; Moos, W. H. J. Pharmacol. Exp. Ther. 1996, 277, 885. (d) Patch, J.
A.; Barron, A. E. J. Am. Chem. Soc. 2003, 125, 12092. (e) Wu, C. W.;
Seurynck, S. L.; Lee, K. Y. C.; Barron, A. E. Chem. Biol. 2003, 10, 1057.
(
2
f) Hara, T.; Durell, S. R.; Myers, M. C.; Appella, D. H. J. Am. Chem. Soc.
006, 128, 1995.
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Org. Lett., Vol. 9, No. 24, 2007

n
Figure 2. Length-dependent CD spectra of AcNscp (0.1 mg/mL;
dimer to 13-mer) in 5 mM sodium phosphate and 5 mM citric acid
buffer (pH 2)/40% acetonitrile at 25 °C.
AcNscp
8
was chosen for more thorough conformational
adopts
analysis. CD studies (Figure 3a) indicate that AcNscp
8
different secondary structures in aqueous and organic condi-
tions. Under neutral pH aqueous condition (5 mM sodium
phosphate buffer, pH 7), AcNscp exhibits an ellipticity
8
maximum at ca. 190 nm and two minima at ca. 198 and 218
nm. In organic solvent (100% acetonitrile), the CD signature
is significantly altered. The CD band intensity at ca. 198
nm is increased by almost 7-fold, and the higher wavelength
band is also enhanced. The ratio of these two CD bands is
altered from 0.6 (198:218 nm) in aqueous conditions to 1.8
(196:210 nm) for the sample in acetonitrile. The overall
magnitude of the CD ellipticity observed in organic solvent
2
-1
(θ
198 nm ) -93 000 deg cm dmol ) is among the most
intense observed in this family of peptidomimetics.
The results seen in Figure 3a were further analyzed by
adding acetonitrile to buffered aqueous sample (59 mM of
8
AcNscp in 5 mM sodium phosphate buffer, pH 7). The
results (Figure 3b) show a solvent-dependent alteration of
the CD spectra with an isodichroic point at ca. 200 nm,
indicative of a transition between two defined conformational
states. It should be noted that no CD spectral variations are
observed when acetonitrile was titrated to an aqueous sample
at pH 2 (Figure S2), which suggests that carboxylate anions
are required and play a dominant role in the observed
conformational rearrangements.
Figure 3. Circular dichroism spectra of (a) AcNscp
8
in aqueous
(5 mM sodium phosphate buffer, pH 7) and organic solvent (100%
acetonitrile) at 25 °C. (b) AcNscp in 5 mM sodium phosphate
8
buffer, pH 7, at 25 °C and various % acetonitrile compositions. (c)
AcNscp in 5 mM sodium phosphate buffer, pH 7/10% acetonitrile
8
at 25 °C and various NaCl concentrations.
In order to evaluate the electrostatic influence of carboxy-
late anions on the secondary structure, CD measurements
were made at varying ionic strengths at a fixed acetonitrile/
In order to further examine the influence of ionizable
groups on the secondary structure, the CD spectra of AcNscp
were evaluated under varying pH conditions (Figure 4). The
results show that AcNscp undergoes dramatic pH-dependent
conformational rearrangements in which two major sets of
CD signals are observed. At pH values below 4, AcNscp
8
H
2
O composition and pH. The data (Figure 3c) show that
8
screening charge-charge repulsive interactions leads to
enhanced CD signal intensity as the concentration of NaCl
is increased. The observed CD signal transition in this
experiment resembles the solvent-dependent CD transition
8
generates intense CD signatures that resemble the CD
characteristics seen in 100% acetonitrile (Figure 3a). At pH
(Figure 3b) between 10 and 30% acetonitrile/H
2
O at pH 7,
values greater than 6, the CD signal intensity of AcNscp
8
suggesting that these processes represent similar conforma-
tional transitions. The results provide additional evidence that
electrostatic interactions in AcNscp play a major role in
8
decreases dramatically. The CD spectra seen at this pH range
show a maximum at ca. 193 nm and a minimum at ca. 220
nm. The midpoint of the CD transition is observed around
promoting conformational rearrangements.
pH 4.9, near the pK
a
value of carboxylic acids. This suggests
Org. Lett., Vol. 9, No. 24, 2007
5005

8
At high pH, AcNscp displays multiple carboxylate anions,
in which charge-charge repulsive forces are expected to
generate more extended secondary structure, especially at
low ionic strength and high solvent polarity. At acidic pH
conditions, the side chains are neutralized, alleviating elec-
trostatic interactions, thereby allowing more compact sec-
ondary structure. In addition, these variations in charge
states may also influence the overall conformation by mod-
ulating the backbone carbonyl to side chain dipole interac-
1
1
tions.
In conclusion, we demonstrate how suitably modified
amino acids can readily be used as convenient synthons that
allow facile access to a broad range of biochemically relevant
side chains. These side chains direct the formation of stable
secondary structures and provide pH sensitivity that can
trigger conformational rearrangements. Spectroscopic studies
will be employed to elucidate the structural features of this
new class of peptidomimetic conformational rearrangements.
We seek to exploit these initial findings to enable the
1
2
regulation of functional synthetic foldamers.
Acknowledgment. This work was supported by a New
Investigator Award from the Alzheimer’s Association and
by a National Science Foundation CAREER Award. We
thank the NCRR/NIH for a Research Facilities Improvement
Grant (C06RR-165720) at NYU.
Figure 4. (a) Circular dichroism spectra of AcNscp
sodium phosphate and 5 mM citric acid buffer/40% acetonitrile at
5 °C and various pH conditions. (b) Per-residue molar ellipticity,
θ, versus pH for AcNscp in 5 mM sodium phosphate and 5 mM
citric acid buffer/40% acetonitrile at 25 °C. Solid squares, θ220 nm
open circles, θ198 nm
8
in 5 mM
Supporting Information Available: Full experimental
procedures and additional spectroscopic data are included.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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8
;
.
OL702207N
(
11) Gorske, B. C.; Bastian, B. L.; Geske, G. D.; Blackwell, H. E. J.
Am. Chem. Soc. 2007, 129, 8928.
12) (a) Vogel, K.; Wang, S.; Lee, R. J.; Chmielewski, J.; Low, P. S. J.
Am. Chem. Soc. 1996, 118, 1581. (b) Lee, H. M.; Chmielewski, J. J. Peptide
Res. 2005, 65, 355.
that the changes in the CD signals seen in Figure 4a are
representative of conformational changes triggered by varia-
tions in the ionization state of carboxylic acid side chains.
(
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Org. Lett., Vol. 9, No. 24, 2007