Building β-peptide H10/12 foldamer helices with six-membered cyclic side-chains: Fine-tuning of folding and self-assembly

Article abstract of DOI:10.1021/ol102494m

The ability of the β-peptidic H10/12 helix to tolerate side-chains containing six-membered alicyclic rings was studied. cis-2-Aminocyclohex-3-ene carboxylic acid (cis-ACHEC) residues afforded H10/12 helix formation with alternating backbone configuration.

Full text of DOI:10.1021/ol102494m

ORGANIC
LETTERS
2010
Vol. 12, No. 23
5584-5587
Building ꢀ-Peptide H10/12 Foldamer
Helices with Six-Membered Cyclic
Side-Chains: Fine-Tuning of Folding and
Self-Assembly
Istva´n M. Ma´ndity,† Livia Fu¨lo¨p,‡ Eleme´r Vass,§ Ga´bor K. To´th,‡
Tama´s A. Martinek,*,† and Ferenc Fu¨lo¨p†
Institute of Pharmaceutical Chemistry, UniVersity of Szeged, H-6720 Szeged, Eo¨tVo¨s u.
6, Hungary, Department of Medical Chemistry, UniVersity of Szeged, H-6720 Szeged,
Do´m t. 8, Hungary, and Institute of Chemistry, Department of Organic Chemistry,
Eo¨tVo¨s Lora´nd UniVersity, H-1117 Budapest, Pa´zma´ny P. s. 1/A, Hungary
martinek@pharm.u-szeged.hu
Received October 15, 2010
ABSTRACT
The ability of the ꢀ-peptidic H10/12 helix to tolerate side-chains containing six-membered alicyclic rings was studied. cis-2-Aminocyclohex-
3-ene carboxylic acid (cis-ACHEC) residues afforded H10/12 helix formation with alternating backbone configuration. Conformational polymorphism
was observed for the alternating cis-ACHC hexamer, where chemical exchange takes place between the major left-handed H10/12 helix and
a minor folded conformation. The hydrophobically driven self-assembly was achieved for the cis-ACHC-containing helix which was observed
as vesicles ∼100 nm in diameter.
The ꢀ-peptide foldamers are among the most interesting
artificial polymers.1 The importance of ꢀ-peptides in chem-
istry and biology is due to their promising biological
applications.2 The stable secondary structure patterns avail-
able to ꢀ-peptides are efficiently controllable via the side-
chain chemistry,3 and it is possible to construct helices that
mimick the overall geometry of the natural R-helix: H14,4
H12,5 the alternating H10/12,6 and the de noVo designed
(2) (a) Tew, G. N.; Scott, R. W.; Klein, M. L.; Degrado, W. F. Acc.
Chem. Res. 2010, 43, 30. (b) Seebach, D.; Gardiner, J. Acc. Chem. Res.
2009, 41, 1366. (c) Gardiner, J.; Mathad, R. I.; Jaun, B.; Schreiber, J. V.;
Flogel, O.; Seebach, D. HelV. Chim. Acta 2009, 92, 2698. (d) Mowery,
B. P.; Lindner, A. H.; Weisblum, B.; Stahl, S. S.; Gellman, S. H. J. Am.
Chem. Soc. 2009, 131, 9735. (e) Michel, J.; Harker, E. A.; Tirado-Rives,
J.; Jorgensen, W. L.; Schepartz, A. J. Am. Chem. Soc. 2009, 131, 6356.
(3) (a) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. ReV. 2001,
101, 3219. (b) Martinek, T. A.; Fu¨lo¨p, F. Eur. J. Biochem. 2003, 270, 3657.
(c) Fu¨lo¨p, F.; Martinek, T. A.; To´th, G. K. Chem. Soc. ReV. 2006, 35, 323.
(4) (a) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.;
Gellman, S. H. J. Am. Chem. Soc. 1996, 118, 13071. (b) Seebach, D.;
Overhand, M.; Ku¨hnle, F. N. M.; Martinoni, B.; Oberer, L.; Hommel, U.;
Widmer, H. HelV. Chim. Acta 1996, 79, 913. (c) Hete´nyi, A.; Ma´ndity,
I. M.; Martinek, T. A.; To´th, G. K.; Fu¨lo¨p, F. J. Am. Chem. Soc. 2005,
127, 547.
† Institute of Pharmaceutical Chemistry, University of Szeged.
‡ Department of Medical Chemistry, University of Szeged.
§ Eo¨tvo¨s Lora´nd University.
(1) (a) Goodman, C. M.; Choi, S.; Shandler, S.; DeGrado, W. F. Nat.
Chem. Biol. 2007, 3, 252. (b) Horne, W. S.; Gellman, S. H. Acc. Chem.
Res. 2008, 41, 1399. (c) Wu, Y. D.; Han, W.; Wang, D. P.; Gao, Y.; Zhao,
Y. L. Acc. Chem. Res. 2008, 41, 1418. (d) Horne, W. S.; Price, J. L.;
Gellman, S. H. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 9151.
10.1021/ol102494m  2010 American Chemical Society
Published on Web 11/04/2010

H14/16 helices.7 The controlled self-assembly of these
foldameric helices leads to helix bundles,8 vesicle-forming
membranes of vertically amphiphilic helices,9 and lyotropic
liquid crystals,10 all of importance for future applications.
These self-assembling phenomena have been reported only
for H14 helices, and in the solution phase, only for those
containing segments of trans-2-aminocyclohexanecarboxylic
acids (trans-ACHC). While the H10/12 helix has been stated
to be inherently stable,11 its controlled self-assembly has not
been observed. Even H10/12 helices formed by the enatio-
meric pairs of cis-2-aminocyclopentanecarboxylic acids (cis-
ACPC) escape self-association in solution. Following the
design principle of the alternating backbone configuration,
Boc-protected ꢀ-peptide tetramers and hexamers were syn-
thetized through use of a bulky and strongly hydrophobic
cis-ACPC derivative, diexo-3-aminobicyclo[2.2.1]hept-5-ene-
2-carboxylic acid enantiomers (diexo-ABHEC).12 In contrast
with the expected H10/12 helix, this oligomer did not exhibit
a helical fold; its secondary structure is banana-shaped and
aggregation in a polar solvent was not reported.
therefore, free ꢀ-peptide amides were synthetized by using
the strongly hydrophobic bicyclic diexo-3S,4R-ABHEC and
diexo-3R,4S-ABHEC monomers. The structures studied are
shown in Scheme 1.
Scheme 1. Structures with Alternating Back-Bone
Configurations Made from the cis-ACHC (1, 2), cis-ACHEC
(3, 4), diexo-ABHEC (5, 6), and cis-ACPC (7)
The present work focuses on the ability of the H10/12
helix to tolerate side-chains containing six-membered ali-
cyclic rings. While the helix-stabilizing effect of the ACHC
residues for the H14 helix4 and the H12 helix formation of
the trans-ACPC oligomers5 are well-known, the role of the
six membered side-chain in the alternating H10/12 helix has
not been studied. Through the increased hydrophobicity of
the side chain, a further aim was the induction of the self-
assembly for the ꢀ-peptide H10/12 helix.
Enantiopure cis-1R,2S-ACHC, cis-1S,2R-ACHC, cis-1R,2S-
ACHEC and cis-1S,2R-ACHEC were utilized as building
blocks.14 The effects of N- and C-terminal protecting groups
on the self-assembly ꢀ-peptides have been described;4c
The foldamers were synthetized on a solid support by
means of Fmoc technology, leading to unprotected sequences.
The saturated oligomers 1 and 2 were prepared in two ways:
by the catalytic reduction of 3 and 4 in an H-Cube
apparatus15 and by direct coupling of the Fmoc-cis-ACHC
monomers. The foldamers were characterized through the
use of HPLC, ESI-MS, and various NMR methods, with
different solvents: 4 mM solutions in CD3OH, DMSO-d6,
and water (H2O/D2O 90:10). The NMR signal dispersions
were good for most of the compounds in these solvents; no
signal broadening was observed and signal assignment could
be performed. Interestingly, significant signal broadening was
detected for 2 in all solvents. Cooling the sample to 245 K
in CDCl3 furnished well-resolved signals where a new set
of signals was frozen out and signal assignment was
achievable for the major conformer. This finding suggests
that 2 has two stable conformers that undergo chemical
exchange.
For the hexameric sequences 2, 4, and 6, NH/ND exchange
studies in CD3OD resulted in decay plots with low slopes.
For 4 and 6, the proton resonances relating to the terminal
nitrogen, the amide NH2 and the C-terminal amide disap-
peared immediately after dissolution, while the remainder
of the signals persisted even 1 h after dissolution (see
Supporting Information). Despite the phenomenon of chemi-
cal exchange at an intermediate rate, the average signal
intensity measured for the amide protons indicated similar
decay for 2. The corresponding amide hydrogens of the
hexamers are significantly shielded from the solvent in
consequence of H-bonding interactions, which may be an
indication of the existing folded secondary structure. The
shorter sequences 1, 3, and 5 exhibited considerably faster
exchange; all the signals were practically lost within 40 min.
(5) Appella, D. H.; Christianson, L. A.; Klein, D. A.; Powell, D. R.;
Huang, X.; Barchi, J. J.; Gellman, S. H. Nature 1997, 387, 381.
(6) (a) Seebach, D.; Gademann, K.; Schreiber, J. V.; Matthews, J. L.;
Hintermann, T.; Jaun, B. HelV. Chim. Acta 1997, 80, 2033. (b) Seebach,
D.; Abele, S.; Gademann, K.; Guichard, G.; Hintermann, T.; Jaun, B.;
Matthews, J. L.; Schreiber, J. V.; Oberer, L.; Hommel, U.; Widmer, H.
HelV. Chim. Acta 1998, 81, 932. (c) Sharma, G. V. M.; Reddy, K. R.;
Krishna, P. R.; Sankar, A. R.; Jayaprakash, P.; Jagannadh, B.; Kunwar,
A. C. Angew. Chem., Int. Ed. 2004, 43, 3961. (d) Sharma, G. V. M.; Reddy,
K. R.; Krishna, P. R.; Sankar, A. R.; Narsimulu, K.; Kumar, S. K.;
Jayaprakash, P.; Jagannadh, B.; Kunwar, A. C. J. Am. Chem. Soc. 2003,
125, 13670. (e) Martinek, T. A.; Ma´ndity, I. M.; Fu¨lo¨p, L.; To´th, G. K.;
Vass, E.; Hollo´si, M.; Forro´, E.; Fu¨lo¨p, F. J. Am. Chem. Soc. 2006, 128,
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(7) Ma´ndity, M. I.; We´ber, E.; Martinek, T. A.; Olajos., G.; To´th, G. K.;
Vass, E.; Fu¨lo¨p, F. Angew. Chem., Int. Ed. 2009, 48, 2171.
(8) (a) Molski, M. A.; Goodman, J. L.; Craig, C. J.; Meng, H.; Kumar,
K.; Schepartz, A. J. Am. Chem. Soc. 2010, 132, 3658. (b) Daniels, D. S.;
Petersson, E. J.; Qiu, J. X.; Schepartz, A. J. Am. Chem. Soc. 2007, 129,
1532. (c) Qiu, J. X.; Petersson, E. J.; Matthews, E. E.; Schepartz, A. J. Am.
Chem. Soc. 2006, 128, 11338.
(9) Martinek, T. A.; Hete´nyi, A.; Fu¨lo¨p, L.; Ma´ndity, I. M.; To´th, G. K.;
De´ka´ny, I.; Fu¨lo¨p, F. Angew. Chem., Int. Ed. 2006, 45, 2396.
(10) (a) Pomerantz, W. C.; Yuwono, V. M.; Pizzey, C. L.; Hartgerink,
J. D.; Abbott, N. L.; Gellman, S. H. Angew. Chem., Int. Ed. 2008, 47, 1241.
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Org. Lett., Vol. 12, No. 23, 2010
5585

This finding suggests that increasing chain length leads to
increased order.
Electronic circular dichroism (ECD) fingerprints were
recorded at room temperature in methanol and water (Figure
1). The ECD spectra of 2 and 4 in methanol exhibit features
Figure 2. Long-range NOE interactions for (a) 2 in CDCl3 and for
(b) 4 and (c) 6 found in DMSO and CD3OH.
Figure 1. ECD curves of 1 mM solutions of 2 (red), 4 (blue), 6
(black), and 7 (cyan, taken from ref 6e and corrected for opposite
handedness) in methanol. Data are normalized to unit chromophore.
3a).17 The secondary structure of the minor conformer of 2
could not be assigned experimentally because of its low
similar to those of 7, which has been assigned as a H10/12
helix earlier.6e The signal intensities for 2 and 4 are lower
indicating a destabilizing effect of the 6-membered side
chains. The ECD curve of 2 in water exhibited a significant
drift, possibly due to the light scattering of the large
aggregates (see DLS and TEM results), and thus this data
set was not interpretable. The H10/12 helical feature was in
part observed for 4 in water as well (see Supporting
Information). For 6, the ECD curve in methanol displayed a
minimum at ∼215 nm and a maximum at ∼200 nm. This
clear difference from the typical ECD curve of the H10/12
helix and also from the known ECD patterns of ꢀ-peptide
strand-like structures6e,16 supports the view that 6 does not
fold into the H10/12 helix in the solution phase, but the
overall structure exhibits helicity. The ECD curve of 6 in
water demonstrated a maximum at ∼210 nm and a minimum
at ∼190 nm; these significant differences from the results
in methanol indicating a solvent-driven structural change.
As mentioned above, 2 exhibited good signal dispersion
in CDCl3 at 245 K, where a mixture of two conformers was
observed. For the major conformer, the NOESY spectrum
at 245 K exhibited a long-range NOE pattern characteristic
of the left-handed H10/12 helix: NH1-CꢀH3, CꢀH2-NH4, NH3-
CꢀH5 and CꢀH4-NH6 (Figure 2a). On the basis of the NMR
restraints, structure refinement was performed. The resulting
low-energy conformational cluster corresponds to the left-
handed H10/12 helical structure. The lowest energy confor-
mation was subjected to ab initio quantum chemical opti-
mization. At both the RHF/3-21G and B3LYP/6-311G**
levels of theory, the left-handed H10/12 proved stable (Figure
Figure 3. (a) H10/12 helix geometry for 2, (b) the H18/20 helix
geometry for 2, (c) and the circular fold for 6, obtained through
NMR restrained structure refinement and by final optimization at
the B3LYP/6-311G** level of theory.
relative abundance (∼6%). The low overall NH/ND ex-
change rate for 2 indicates that the conformational equilib-
rium can not involve an unfolded minor conformer or a
significant fraction of unfolded intermediate states. Molecular
modeling at the B3LYP/6-311G** level suggested that the
H-bond-stabilized conformation closest in energy is the left
handed H18/20 (∆E ) 4 kcal mol-1) (Figure 3b). The left-
handed H18/20 helix is proposed, which can occur through
a rapid shift between the H-bonds, without the overall
disassembly of the helical structure. For longer sequences
this helix could be the dominant conformation.
Importantly, for 4 the CꢀH-NH vicinal couplings exhibited
an alternating pattern (values given for CD3OH): 9.8 Hz for
NH3--CꢀH3 and NH5-CꢀH5, and 8.7 Hz for NH2-CꢀH2, NH4-
(16) (a) Martinek, T. A.; To´th, G. K.; Vass, E.; Hollo´si, M.; Fu¨lo¨p, F.
(17) Frisch, M. J. Gaussian 03, reVision E.1; Gaussian, Inc.: Pittsburgh,
PA, 2003; http://www.Gaussian.com. For full citation, see Supporting
Information.
Angew. Chem., Int. Ed. 2002, 41, 1748. (b) Segman, S.; Lee, M. R.; Vaiser,
V.; Gellman, S. H.; Rapaport, H. Angew. Chem., Int. Ed. 2010, 49, 716
.
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Org. Lett., Vol. 12, No. 23, 2010

CꢀH4 and NH6-CꢀH6. For 4, long-range NOEs were recorded
from the ROESY spectra in CD3OH and DMSO-d6. These
NOE interactions are typical for the H10/12 helix and the
structure refinement resulted in the left-handed H10/12 in
the same way as found for 2 (Figure 2b) which is in
accordance with the CꢀH-NH vicinal couplings as well.
In order to test the secondary structure of 6, ROESY
measurements were run in CD3OH, DMSO-d6 and water
(H2O/D2O 90:10). A series of NOEs were observed in these
solvents for the pairs NHi-C4Hi and NHi-C1Hi-1 (Figure 2c),
but i - i+2 interactions could not be detected, which is
evidence against the formation of the alternating H10/12 helix
and lends support to a bent conformation.12 For 6, the CꢀH-
NH vicinal couplings did not show an alternating pattern.
The same structure refinement based on NMR-derived
distance restraints, converged to a circle-like fold (Figure
3c). This conformation was corroborated by the absence of
C7Hi-C1Hi-1 and C7Hi-C4Hi+1 NOE interactions in the ROESY
spectra, which should appear for an elongated strand.
Transmission electron microscopy (TEM) and dynamic
light scattering (DLS) were employed to analyze the self-
association phenomena. For a 4 mM solution of 2 in water,
vesicles with an average diameter of 100 nm were observed
in the TEM images immediately after dissolution, sonication
and filtration. The vesicles tended to associate into clusters.
This result was supported by the DLS measurements,
indicated particles with diameters in the range 100-1000
nm. For 1 and 3-6, no self-association was observed under
the same conditions (Figure 4). The morphology of the
associates did not change after 1 week of incubation.
Small changes in the side-chain chemistry resulted in
significantly different behavior in the association properties
of the ꢀ-peptide foldamers studied. Solvophobically driven
self-assembly of the H10/12 helix was observed only the
cyclohexane side-chain (2). This suggests that the pronounced
hydrophobic nature of the cyclohexane side-chain promotes
self-assembly, given the tendency of the H10/12 helix to fold.
On the other hand, the strongly hydrophobic cis-ABHEC
residue in itself is not sufficient to initiate aggregation,
indicating the necessity of folding into the compact helical
structure. These findings indicate that the interplay of the
stable compact helical conformation and the adequately
hydrophobic side-chain is necessary for the creation of self-
assembling foldameric helices.
For an assessment of the hydrophilic/hydrophobic char-
acter of the studied foldamers in their folded state (lowest
energy ab initio geometries), hydrophobic and hydrophilic
surface areas were calculated (see Table S4, Supporting
Information). The values indicate that the self-assembling
foldamer 2 exhibits the largest hydrophilic/hydrophobic
surface ratio in its folded state.
The ꢀ-peptidic alternating H10/12 helix tolerates the six-
membered side-chain topology. Both the cis-ACHC and the
cis-ACHEC afforded the H10/12 helix. Based on the ECD
results, we can conclude that the six membered ring topology
slightly destabilizes the H10/12 helix as compared with the
alternating cis-ACPC oligomers. Moreover, the cis-ACHC-
containing foldamer 2 exhibited conformational polymor-
phism with two folded conformational states that underwent
chemical exchange. A noteworthy finding was that an
apparently subtle change in the hybridization of a carbon
atom pair in the side-chain can tune the conformational
behavior to such an extent. The bicyclic diexo-ABHEC
prevented the formation of a small-diameter helix; the
experimental results pointed to a circle-like fold which was
sufficiently stable in methanol to maintain considerably
shielded H-bonds. For the ꢀ-peptide helices studied, minor
changes in the side-chain topology, size and shape resulted
in large effects on the self-assembly process. It emerged,
that the H10/12 helix is capable of self-association in a polar
medium if the helix is built up from cis-ACHC residues.
Our observations strongly suggest that the hydrophobically
driven helix association is a result of the combination of the
hydrophobic nature of the side-chains and the presence of a
stable secondary structure. These results serve as an important
starting point for the design of helix assemblies involving
the H10/12 helix.
Acknowledgment. We thank the Hungarian Research
Foundation (NK81371) and COST (CM0803) for financial
support. L.F. and T.A.M. acknowledge the Ja´nos Bolyai
Fellowship from the HAS. The computational resources of
HPC U-Szeged (ALAP4-00092/2005) are acknowledged.
Supporting Information Available: NMR signal assign-
ments, NMR spectra, NH/ND exchange, ECD, MS, HPLC
characterization and modeling data. This material is available
free of charge via the Internet at http://pubs.acs.org.
Figure 4. TEM images of (a) 2, (c) 4, and (e) 6 and DLS curves
for (b) 2, (d) 4, and (f) 6 as 4 mM solutions in water.
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