Fine Tuning of β-Peptide Foldamers: A Single Atom Replacement Holds Back the Switch from an 8-Helix to a 12-Helix

Article abstract of DOI:10.1002/anie.201504126

Cyclic homologated amino acids are important building blocks for the construction of helical foldamers. N-aminoazetidine-2-carboxylic acid (AAzC), an aza analogue of trans-2-aminocyclobutanecarboxylic acid (tACBC), displays a strong hydrazino turn conform

Full text of DOI:10.1002/anie.201504126

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201504126
German Edition: DOI: 10.1002/ange.201504126
Foldamers
Fine Tuning of b-Peptide Foldamers: a Single Atom Replacement
Holds Back the Switch from an 8-Helix to a 12-Helix
Amandine Altmayer-Henzien, ValØrie Declerck, Jonathan Farjon, Denis Merlet, RØgis Guillot,
and David J. Aitken*
Abstract: Cyclic homologated amino acids are important
building blocks for the construction of helical foldamers. N-
aminoazetidine-2-carboxylic acid (AAzC), an aza analogue of
trans-2-aminocyclobutanecarboxylic acid (tACBC), displays
a strong hydrazino turn conformational feature, which is
proposed to act as an 8-helix primer. tACBC oligomers bearing
a single N-terminal AAzC residue were studied to evaluate the
ability of AAzC to induce and support an 8-helix along the
oligopeptide length. While tACBC homooligomers assume
a dominant 12-helix conformation, the aza-primed oligomers
preferentially adopt a stabilized 8-helix conformation for an
oligomer length up to 6 residues. The (formal) single-atom
exchange at the N terminus of a tACBC oligomer thus
contributes to the sustainability of the 8-helix, which resists
the switch to a 12-helix. This effect illustrates atomic-level
programmable design for fine tuning of peptide foldamer
architectures.
placed.[13] Further exploration of such modulating factors
could therefore enable the fine tuning of programmable
helical folding.
Oligomers of trans-2-aminocyclobutanecarboxylic acid
(tACBC) can adopt a 12-helix conformation both in solution
and in the solid state.[14] However, the preferred conformer of
a tACBC dipeptide is an 8-membered hydrogen-bonded ring
(C8),[15] and it has been suggested that a tACBC tetrapeptide
might display three consecutive C8 structural features.[16] This
corresponds to an 8-helix, which is a rarity for b-peptides.[17,18]
The 12-helix preference of tACBC oligomers raises the
question of the sustainability of an 8-helix (Figure 1);
F
oldamers are unnatural oligomers that adopt well-defined
folded patterns, and several factors that govern their con-
formational preferences have been identified.[1] b-Peptides
were amongst the first foldamers to be studied and they
provide benchmark helix-forming manifolds,[2,3] the diversity
of which has been enlarged through the study of a/b-, b/g-,
and other mixed peptides.[3,4] Oligomers of cyclic b-amino
acids adopt stabilized helices where the pitch depends largely
Figure 1. 8- vs. 12-membered H-bonded rings in tACBC oligomers and
the Hz-turn conformation of an AAzC residue.
b
a
=
on the backbone torsional angle q (N-C -C -C( O)), which in
turn is determined by the ring size and its stereochemistry.
Robust 14-helix[5,6] and 12-helix[7] foldamers can be con-
structed rationally from appropriate cyclic trans-b-amino
acids, while the alternating 10/12-helix[8] and regular 6-
ribbon strands[9] are observed for oligomers of cyclic cis-b-
amino acids. Nonetheless, helical folding may be influenced
by other factors, such as side-chain interactions,[10] the steric
bulk of the monomer,[11] solvent/concentration effects,[12] or
the oligomeric environment into which the monomer is
indeed, in previous cases where 12-helix conformers compete,
the 8-helix was only observed for small (ꢀ 4-mer) oligo-
mers.[17] N-Aminoazetidine-2-carboxylic acid (AAzC)[19] is an
aza analogue of tACBC and is characterized by a strong
tendency to form a bifurcated C8/5-ring H-bonded structure
known as a hydrazino (Hz) turn (Figure 1).[20,21] We therefore
examined the ability of a single AAzC residue to behave as an
“8-helix primer” when employed as the N-terminal residue in
an oligo-tACBC sequence.
Four oligopeptides (1–4; Figure 2) were prepared (see the
Supporting Information) in order to compare their conforma-
tional behavior with that of the corresponding homooligo-
mers Boc(tACBC)nOMe (5–8; n = 2, 4, 6, 8, respectively).[14]
The two series are identical with the exception of the formal
[*] Dr. A. Altmayer-Henzien, Dr. V. Declerck, Prof. Dr. D. J. Aitken
CP3A Organic Synthesis Group, ICMMO UMR 8182
UniversitØ Paris Sud
15 Rue George Clemenceau, 91405 Orsay cedex (France)
E-mail: david.aitken@u-psud.fr
Dr. J. Farjon, Prof. Dr. D. Merlet
LRMN, ICMMO UMR 8182, UniversitØ Paris Sud
15 Rue George Clemenceau, 91405 Orsay cedex (France)
Dr. R. Guillot
Services Communs, ICMMO UMR 8182, UniversitØ Paris Sud
15 Rue George Clemenceau, 91405 Orsay cedex (France)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201504126.
Figure 2. Structures of peptides 1–4.
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These observations were corroborated by NMR analysis
of the AAzC peptides 2–4 in CDCl3 solution. In each case, the
N4H 1H NMR signal appeared at d ꢁ 6.5 ppm and a group of
strongly deshielded signals at d ꢁ 9.0 ppm was observed for
the other amide groups, thus suggesting the latter to be
involved in strong hydrogen bonds. The intramolecular nature
of the H-bonds in 2 and 3 was confirmed by the absence of
a significant variation in chemical shift with concentration,
which is consistent with an 8-helix structure. For 4, only the
N10H signal (d = 8.99 ppm) was strongly deshielded and was
uninfluenced by concentration, thus suggesting the presence
of an Hz-turn. Other amide signals were moderately
deshielded (d = 7.86–8.70 ppm) and showed moderate con-
centration dependence, thus suggesting the presence of both
H-bonded and non-H-bonded conformations, which is in
agreement with a conformational equilibrium for 4. The N4H
signal was an exception, with significant concentration and
isotopic exchange effects suggesting that it remains essentially
non-H-bonded in 4.
Further evidence for the H-bond networks in peptides 2–4
in CDCl3 was acquired by using fast-pulsing high-field
SOFAST HMBC experiments.[22] The diagnostic N10H···N5
interaction of an Hz-turn was ascertained for all three
peptides by 1HN-15N SOAFST HMBC.
A
1HN-13CO
SOFAST HMBC experiment for 3 revealed a series of (i!
iÀ2) H-bonds, which together with the Hz-turn, constitute an
uninterrupted 8-helix (Figure 4). This is entirely consistent
Figure 3. IR absorption spectra (in CHCl3) of AAzC peptides 1–4 (top)
and tACBC oligomers 5–8 (bottom).
single-atom exchange (N for C) at the b-position of
the N-terminal residue in the former series.
À
In CHCl3 solution, the N H stretch absorptions
in the IR spectra were useful for tracking conforma-
tional changes with increasing peptide length in the
two series of peptides (Figure 3). In the reference
tACBC oligomer series, 5 showed an H-bond
absorption band at 3290 cmÀ1 consistent with a C8
conformer. This absorption appeared with progres-
sive red-shift in 6 and 7, although the spectrum of
the latter showed the presence of a second H-
bonded conformer. This second conformer was
predominant in the spectrum of 8. These data are
consistent with a predominant 8-helix conformation
for 6, a 12-helix conformer for 8, and the coexistence
Figure 4. Hydrogen bonding in peptides 3 (in CDCl3) and 4 (in [D5]pyridine)
observed by SOFAST HMBC NMR experiments.
of these two conformers for 7. In the AAzC peptide series 1–
with the IR data. A CDCl3 solution of 4 failed to give usable
2D NMR data, probably owing to the coexistence of two
interconverting conformers. NMR studies were carried out in
[D5]pyridine solution, thus allowing the identification of the
second conformer of 4. The Hz-turn was confirmed by 1H-15N
À1
À
4, the diagnostic small N H absorbance at 3360 cm
indicated the presence of an Hz-turn in all cases.[20] Peptides
1–3 showed a strong H-bond absorbance, which became
progressively red-shifted from 3300 to 3240 cmÀ1 with
increasing peptide length. The spectrum of 4 featured an
1
SOFAST HMBC, while H-13CO SOFAST HMBC revealed
additional strong H-bond absorption at 3300 cmÀ1 and an
four (i!iÀ3) H-bonds that define a 12-helical segment
involving all seven tACBC residues (Figure 4). Peptide 4
thus adopts a unique hybrid conformation in pyridine, starting
with an 8-membered Hz-turn and switching to a 12-helix for
the rest of the sequence. This conformer coexists with the 8-
helix for a solution of 4 in CHCl3.
The low solubility of peptides 1–4 in protic solvents
(< 5 mm in CD3OH) precluded similar NMR studies in such
media. However, the CD spectra were recorded in dilute
À1
À
absorption corresponding to free N H at 3440 cm . Com-
parison of the two series clearly suggests that in CHCl3
solution, the 12-helix is already prevalent in the tACBC
hexamer and predominant in the octamer, while for the
AAzC peptides, an Hz-turn-promoted 8-helix is predominant
up to the hexapeptide and remains a significant conformer for
the octapeptide; the second conformer is not clearly identi-
fied at this stage.
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Figure 5. CD spectra (in MeOH) of AAzC peptides 1–4 (top) and
tACBC oligomers 5–8 (bottom).
MeOH solution (0.2 mm) and compared with those of the
tACBC oligomers 5–8 (Figure 5). Comparison of AAzC
peptides 3 and 4 with oligomers 6 and 7, respectively, is
revealing. While the contribution from a 12-helix (max.
207 nm, min. 227 nm)[14] is already significant in tetramer 6,
this structure is only partially in evidence for hexapeptide 3.
Tetrapeptide 2 displayed a different curve (max. 203 nm, min.
223 nm): prevalence of an 8-helix conformation is an attrac-
tive hypothesis but this should be treated with caution since
the CD signal of the b-peptide 8-helix has not been calculated.
All CD curve intensities were only partially diminished over
the temperature range 5–558C, with no detectable conformer
change.[23]
A hybrid MCMM conformational search was carried out
on peptides 2–4 in vacuum by using MacroModel 10.6[24] and
the MMFF94 s force field without restraints. Low-energy
families were subjected to ab initio geometrical optimization
at the B3LYP 6-31G** level of theory in CHCl3 and in MeOH
using GAUSSIAN09.[25] The Hz-turn of AAzC dominated in
low-energy conformers, regardless of the other conforma-
tional features. Only two low-energy conformer families
emerged: the 8-helix and the Hz-turn/12-helix hybrid
(Figure 6). In CHCl3, the 8-helix was the most stable
conformer for 2 and 3, whereas for 4, the hybrid conformer
was only 0.7 kJmolÀ1 higher in energy. These results are in
complete agreement with the experimental data, thus sug-
gesting the predominance of the 8-helical conformer up to six
residues, and the presence of two conformers for octapeptide
4. In MeOH, the 8-helix is the lowest energy conformer for
tetrapeptide 2 by at least 8.6 kJmolÀ1. This would explain the
low CD ellipticity of 2, which can now be considered more
Figure 6. Calculated low-energy conformers of peptides 2–4.
confidently as the signature of an 8-helix. For 3, the 8-helix (3-
I) and the hybrid Hz-turn/12-helix (3-II) have closer energies
and for 4, the hybrid structure 4-II is more stable. These
observations account qualitatively for the increasing “12-
helix-like” contribution to CD data with increasing peptide
length.
Inspection of the backbone torsional angles (f, q, y)
revealed the remarkable simplicity of the helix pitch switch:
the AAzC residues in 4-I and 4-II are virtually superimpos-
able, with a y value (ca. 4.08) that is amenable to both 8-helix
and 12-helix progressions. In each helix type, the tACBC
residues have similar values for f (range 878 to 1008) and q
(range À958 to À1038); only the y values differ significantly
(ca. 358 in 4-I, ca. 958 in 4-II).
A single crystal of 4 was obtained through slow evapo-
ration of a CH2Cl2/MeOH solution and was analyzed by X-ray
diffraction (Figure 7).[26] The main characteristics of the
hybrid Hz-turn/12-helix conformer 4-II were in evidence:
the N-terminal AAzC shows the three-centered H-bonded
Hz-turn, while the tACBC segment adopted a 12-helix
structure into which a molecule of methanol had been
34
21
=
inserted between N H (residue 3) and O C (residue 6),
with minor distortion of the helical axis.[27]
From these studies, it emerges that replacement of the N-
terminal residue of a tACBC oligomer by AAzC, concep-
tually a single-atom substitution, has the effect of enhancing
8-helical folding prompted by the strong Hz-turn of AAzC.
The 8-helices described herein are the longest described to
date for b-amino acid oligomers. In CHCl3, the 8-helix is the
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[7] D. H. Appella, L. A. Christianson, D. A. Klein, D. R. Powell, X.
Huang, J. J. Barchi, Jr., S. H. Gellman, Nature 1997, 387, 381 –
384.
[8] a) D. Seebach, K. Gademann, J. V. Schreiber, J. L. Matthews, T.
Hintermann, B. Jaun, L. Oberer, U. Hommel, H. Widmer, Helv.
Chim. Acta 1997, 80, 2033 – 2038; b) T. A. Martinek, I. M.
Mµndity, L. Fülçp, G. K. Tóth, E. Vass, M. Hollósi, E. Forró, F.
Fülçp, J. Am. Chem. Soc. 2006, 128, 13539 – 13544.
[9] a) T. A. Martinek, G. K. Tóth, E. Vass, M. Hollósi, F. Fülçp,
Angew. Chem. Int. Ed. 2002, 41, 1718 – 1721; Angew. Chem. 2002,
114, 1794 – 1797; b) F. Rffla, S. Boussert, T. Parella, I. Díez-PØrez,
V. Branchadell, E. Giralt, R. M. OrtuÇo, Org. Lett. 2007, 9,
3643 – 3645.
Figure 7. X-ray diffraction structure of peptide 4: side view (left) and
view along the axis (right).
[10] a) I. M. Mµndity, A. Monsignori, F. Fülçp, E. Forró, F. Fülçp,
Chem. Eur. J. 2014, 20, 4591 – 4597; b) P. I. Arvidsson, M.
Rueping, D. Seebach, Chem. Commun. 2001, 649 – 650.
[11] A. HetØnyi, Z. Szakonyi, I. M. Mµndity, É. Szolnoki, G. K. Tóth,
T. A. Martinek, F. Fülçp, Chem. Commun. 2009, 177 – 179.
[12] É. Szolnoki, A. HetØnyi, T. A. Martinek, Z. Szakonyi, F. Fülçp,
Org. Biomol. Chem. 2012, 10, 255 – 259.
[13] I. M. Mµndity, E. WØber, T. A. Martinek, G. Olajos, G. K. Tóth,
E. Vass, F. Fülçp, Angew. Chem. Int. Ed. 2009, 48, 2171 – 2175;
Angew. Chem. 2009, 121, 2205 – 2209.
[14] C. Fernandes, S. Faure, E. Pereira, V. ThØry, V. Declerck, R.
Guillot, D. J. Aitken, Org. Lett. 2010, 12, 3606 – 3609.
[15] E. Torres, E. Gorrea, E. Da Silva, P. Nolis, V. Branchadell, R. M.
OrtuÇo, Org. Lett. 2009, 11, 2301 – 2304.
[16] E. Gorrea, G. Pohl, P. Nolis, S. Celis, K. K. Burusco, V.
Branchadell, A. Perczel, R. M. OrtuÇo, J. Org. Chem. 2012, 77,
9795 – 9806.
main conformer up to an octapeptide, whereas the corre-
sponding tACBC oligomers largely exist in 12-helical con-
formations.[14] In MeOH, the 8-helix makes a significant
contribution up to a 6-residue sequence when primed by
AAzC. When the 8-helix prompt is not followed, the tACBC
segment folds into a 12-helix, giving a hybrid Hz-turn/12-helix
conformer. These results contribute to our understanding of
the subtle factors that may govern the preferences for 8- or 12-
helix folding, and this understanding could be employed
advantageously to the design of polymorphic molecular
architectures.
Acknowledgements
[17] a) R. Threlfall, A. Davies, N. M. Howarth, J. Fisher, R. Cosstick,
Chem. Commun. 2008, 585 – 587; b) K. Gademann, A. Hane, M.
Rueping, B. Jaun, D. Seebach, Angew. Chem. Int. Ed. 2003, 42,
1534 – 1537; Angew. Chem. 2003, 115, 1573 – 1575; c) R. J.
Doerksen, B. Chen, J. Yuan, J. D. Winkler, M. L. Klein, Chem.
Commun. 2003, 2534 – 2535.
[18] a) J. D. Winkler, E. L. Piatnitski, J. Mehlmann, J. Kasparec, P. H.
Axelsen, Angew. Chem. Int. Ed. 2001, 40, 743 – 745; Angew.
Chem. 2001, 113, 765 – 767; b) S. Abele, P. Seiler, D. Seebach,
Helv. Chim. Acta 1999, 82, 1559 – 1571.
A. A.-H. is grateful for an Allocation SpØcifique pour
Normalien grant. J.F. thanks Dr. E. Guittet (ICSN, CNRS
Gif-sur-Yvette) for assistance with 950 MHz NMR experi-
ments and TGIR-RMN-THC Fr3050 CNRS for financial
support.
Keywords: foldamers · helical structures · hydrazino turn ·
[19] V. Declerck, D. J. Aitken, J. Org. Chem. 2011, 76, 708 – 711.
[20] A. Altmayer-Henzien, V. Declerck, D. Merlet, J.-P. Baltaze, J.
Farjon, R. Guillot, D. J. Aitken, J. Org. Chem. 2013, 78, 6031 –
6039.
[21] “Synthesis and Chemistry of a-Hydrazino Acids”: J. Vidal in
Origins and Synthesis of Amino Acids, Vol. 2 (Ed.: A. B.
Hugues), Wiley-VC, Weinheim, 2009, pp. 35 – 92.
[22] A. Altmayer-Henzien, V. Declerck, D. J. Aitken, E. Lescop, D.
Merlet, J. Farjon, Org. Biomol. Chem. 2013, 11, 7611 – 7615.
[23] The CD spectra of 3 and 4 could be taken in MeOH:H2O (1:1)
with no apparent change.
[24] MacroModel, version 10.6, Schrçdinger, LLC New York, NY,
2014.
hydrogen bonds · peptides
How to cite: Angew. Chem. Int. Ed. 2015, 54, 10807–10810
Angew. Chem. 2015, 127, 10957–10960
[1] a) Foldamers: Structure, Properties, and Applications (Eds.: S.
Hecht, I. Huc), Wiley-VCH, Weinheim, 2007; b) C. M. Good-
man, S. Choi, S. Shandler, W. F. DeGrado, Nat. Chem. Biol. 2007,
3, 252 – 262; c) A. Roy, P. Prabhakaran, P. K. Baruah, G. J.
Sanjayan, Chem. Commun. 2011, 47, 11593 – 11611; d) G.
Guichard, I. Huc, Chem. Commun. 2011, 47, 5933 – 5941.
[2] a) D. Seebach, J. Gardiner, Acc. Chem. Res. 2008, 41, 1366 –
1375; b) S. H. Gellman, Acc. Chem. Res. 1998, 31, 173 – 180.
[3] P. G. Vasudev, S. Chatterjee, N. Shamala, P. Balaram, Chem. Rev.
2011, 111, 657 – 687.
[4] a) L. K. A. Pilsl, O. Reiser, Amino Acids 2011, 41, 709 – 718;
b) A. D. Bautista, C. J. Craig, E. A. Harker, A. Schepartz, Curr.
Opin. Chem. Biol. 2007, 11, 685 – 692; c) M.-I. Aguilar, A. W.
Purcell, R. Devi, R. Lew, J. Rossjohn, A. I. Smith, P. Perlmutter,
Org. Biomol. Chem. 2007, 5, 2884 – 2890; d) M. Salwiczek, E. K.
Nyakatura, U. I. M. Gerling, S. Ye, B. Koksch, Chem. Soc. Rev.
2012, 41, 2135 – 2171; e) W. S. Horne, S. H. Gellman, Acc. Chem.
Res. 2008, 41, 1399 – 1408.
[25] Gaussian09, RevisionD.01, M. J. Frisch, et al., Gaussian, Inc.
Wallingford CT, 2009.
[26] CCDC 1051739 (4) contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
from The Cambridge Crystallographic Data Centre.
[27] A crystal of 3 was grown under similar conditions. The crystal
quality was poorer, but X-ray diffraction at 1.1 Š resolution
showed the structure was that of the hybrid conformation 3-II;
see the Supporting Information.
[5] D. H. Appella, L. A. Christianson, I. L. Karle, D. R. Powell, S. H.
Gellman, J. Am. Chem. Soc. 1996, 118, 13071 – 13072.
[6] D. Seebach, M. Overhand, F. N. M. Kühnle, B. Martinoni, Helv.
Chim. Acta 1996, 79, 913 – 941.
Received: May 6, 2015
Revised: June 18, 2015
Published online: July 23, 2015
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