Robust helix formation in a new family of oligoureas based on a constrained bicyclic building block

Article abstract of DOI:10.1002/anie.201205842

BAC for more: A constrained bicyclic building block with urea linkages is an efficient combination for the formation of a highly rigid helical system. This type of bicyclic amino carbamoyl (BAC) foldamer was studied both in solution (see scheme) and in the solid state. A robust H-bond (dotted line) network was found between the carbonyl oxygen atoms (red) and the amino groups (dark blue) within the helix. Copyright

Full text of DOI:10.1002/anie.201205842

Angewandte
Chemie
DOI: 10.1002/anie.201205842
Oligourea Foldamers
Robust Helix Formation in a New Family of Oligoureas Based on
a Constrained Bicyclic Building Block**
Baptiste Legrand, Christophe Andrꢀ, Emmanuel Wenger, Claude Didierjean, Marie
Christine Averlant-Petit, Jean Martinez, Monique Calmes,* and Muriel Amblard*
The field of foldamers has become an important area of
chemistry over the years because of the particular structural
and functional properties that foldamers display. The design,
structural properties, and activities of different families of
foldamers, including aromatic polyamides or structures
closely related to peptides such as peptoids, b-peptides, and
[1–8]
g-peptides, have been exhaustively reviewed.
Among the
foldamers based on natural peptide sequences, b-peptides are
the most widely studied system. In this system, cyclic b-amino
acids are used as building blocks, allowing stabilization of
various secondary structures in oligomers by strongly pro-
[
3,9–12]
moting gauche-type torsion angles.
Our group has
Figure 1. Chemical formulas of (S)-ABOC 1, oligoureas 2, 3, 4, and
N-Boc-aminobicyclo[2.2.2]octane-2-succinimidyl carbamate 5.
recently described a highly constrained bicyclic b-amino
acid, named (S)-ABOC 1, (S)-aminobicyclo[2.2.2]octane-2-
carboxylic acid (Figure 1). This b -trisubstituted bicyclic
[
13]
2,3,3
amino acid, able to induce a turn in peptides both in solution
Although many cyclic b-amino acid oligomers and acyclic
urea oligomers have been reported for more than ten years,
their combination in a single structure has never been
explored. However, such a combination should be a promising
approach in the development of a highly stable helix
structure, even with few motifs. Thus, we focused on the
design of oligomers combining both the benefit of conforma-
tionally constrained building blocks and of the bifidic hydro-
gen-bond stabilization from the urea linkages. In addition, the
use of the bicyclo[2.2.2]octane system afforded the first
[14]
and in the solid state,
displays drastically reduced con-
formational freedom and a q angle locked at approximately
1
5
58. Therefore, this motif is particularly attractive for the
design of new foldamers. To stabilize the helical system, other
parameters should be considered. Indeed, Guichard and co-
[
15,16]
workers
linkages for oligourea foldamers that are g-peptide mimet-
ics.
provided a useful tool by introducing urea
[
17,18]
The presence of additional nitrogen atoms in the urea
linkage promotes helix stabilization by introducing additional
conformational restriction to the backbone and hydrogen-
bond donor sites.
2
3,3
example of a C ,C -trisubstitution pattern in the construction
of structured oligomers.
To study the progressive folding of bicyclic amino
carbamoyl oligomers (BAC oligomers), we synthesized
three oligoureas of different lengths (n = 2, 4, 6). The target
oligoureas 2, 3, and 4 (Figure 1) were synthesized in solution
by stepwise assembly using a standard Boc/Bzl strategy (see
Supporting Information for full details). For this purpose, the
succinimidyl carbamate derivative 5 (Boc-BAC-OSu), as
a precursor of oligourea synthesis, was prepared from Boc-
(S)-ABOC-OH according to a previously reported proce-
[
+]
[
*] C. Andrꢀ, Prof. J. Martinez, Dr. M. Calmes, Dr. M. Amblard
Institut des Biomolꢀcules Max Mousseron (IBMM), UMR 5247
CNRS, Universitꢀs Montpellier 1 et 2
1
5 avenue Charles Flahault, 34000 Montpellier (France)
E-mail: monique.calmes@univ-montp2.fr
muriel.amblard@univ-montp1.fr
Homepage: http://www.ibmm.univ-montp1.fr/
[
+]
Dr. B. Legrand, Dr. M. C. Averlant-Petit
Laboratoire de Chimie-Physique Macromolꢀculaire,
LCPM—UMR 7568 CNRS Universitꢀ de Lorraine, 1 rue Grandville,
[
19]
dure.
For all oligomers, we used two capping groups, the N-
5
4001 Nancy Cedex 1 (France)
[20]
benzhydrylglycolamide ester (OBg ester), for its ability to
E. Wenger, Dr. C. Didierjean
[21]
induce a folded conformation in short peptides
and its
Laboratoire de Cristallographie, Rꢀsonance Magnꢀtique et Modꢀ-
lisation, UMR 7063 CNRS Universitꢀ de Lorraine, Boulevard des
Aiguillettes, 54506 Vandoeuvre-lꢁs-Nancy Cedex (France)
ability to ease oligomer synthesis, and the 4-bromophenyl
group, to favor crystal formation and facilitate phasing. The
OBg ester was introduced at the C-terminal end as a b-Ala-
OBg residue. This group is orthogonal to Boc and selectively
removed under mild alkaline conditions. In addition, it
afforded an excellent chromophore that allowed for control
of the oligomer synthesis. The 4-bromophenyl group was
introduced by way of an isocyanate derivative for capping the
N-terminus of oligoureas at the end of the synthesis.
+
[
] These authors contributed equally to this work.
[
**] We thank the CNRS, MESR and ANR (ANR-08-BLAN-0066-01) for
financial support, and the SCBIM and Universitꢀ de Lorraine for
NMR and XRD facilities.
Supporting information for this article (experimental details) is
available on the WWW under http://dx.doi.org/10.1002/anie.
01205842.
2
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Angewandte
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The first coupling was performed by adding equimolar
amounts of b-Ala-OBg to the activated monomer 5 in the
presence of N,N-diisopropylethylamine (DIEA). Then, oli-
goureas 2–4 were prepared by successive Boc deprotection,
trifluoroacetate salt (TFA salt) neutralization, and coupling
using the activated monomer 5. After the last coupling and
deprotection of the oligomers, one equivalent of 4-bromo-
phenyl isocyanate was added for the final capping step.
NMR spectroscopy studies were conducted on dimer 2,
tetramer 3, and hexamer 4 in CD OH. Despite the repetitive
3
nature of the sequences and the numerous methylene groups
on the BAC moiety, the NMR signals were well dispersed
(
Supporting Information, Figures S2–4). Therefore, nearly all
1
13
15
H, C, and N resonances could be assigned by combining
homonuclear COSY, TOCSY, ROESY experiments and
1
5
13
heteronuclear N-HSQC, C-HSQC, and HSQC-TOCSY at
1
5
13
N and C natural abundance (Tables S1–6). The high
1
dispersion of the H NMR signals of the methylene groups
of the six BAC residues in peptide 4 is illustrated in the
TOCSY spectra (Figures S5–6).
For the three oligoureas, all measurable J(N’H, CH)
coupling constants were > 8 Hz, mostly distributed around 9–
3
a
1
0 Hz, which are typical of an anti-periplanar arrangement
a
between the N’H and the CH protons (Table S7). They
a
Figure 2. a) Superimposition of the NH/ CH region of the TOCSY
black) and ROESY (red) NMR spectra of the hexamer 4 recorded in
a
[15]
indicated a restricted rotation around the N’À C bond.
(
a
Numerous non-sequential NOEs were detected all along the
sequence and provided strong evidence for the highly
structured nature of the three oligoureas. Characteristic
long range NOEs between i,i + 2 and i,i + 3 residues could
CD OH at 298 K. Long range NH,N’H(i+2)/ CH(i) characteristic
3
NOEs are reported. Other NOE cross-peaks are intra-residual HN’/
a
a
a
CH and HN/ CH, and sequential HN(i+1)/ CH(i). Numbers shown
in the spectra correspond to the BAC residue number shown in (b).
b) Unambiguously assigned characteristic long-range NOEs along the
hexaurea sequence.
g1
a
be unambiguously assigned. Strong
medium or weak NH(i+2)/ CH(i) and N’H(i+2)/ CH(i), and
CH(i+2)/ CH(i),
a
a
g2
g2
weak N’H(i+3)/ CH(i) and NH(i+2)/ CH(i) long range
NOE correlations involving BAC methylene protons were
detected (Figure 2, Tables S8–10). Interestingly, the NH(i+2)/
a
a
CH(i) and N’H(i+2)/ CH(i) NOEs had been previously
assigned, for oligoureas composed of acyclic motifs, to
a minor alternative conformation in methanol but no
[22]
structure was elucidated.
NOEs were used as constraints for the NMR solution
structure calculations of 2, 3, and 4 using a simulated
[
23,24]
annealing procedure with AMBER 10 (Figure 3).
For
these three oligomers, 26, 49, and 110 unambiguous distance
restraints were respectively introduced (inter-residue NOEs
are shown in Tables S8–10).
The resulting lowest-energy conformations, considered as
the most representative structures for tetramer 3 and hexamer
4, exhibited a well-defined right-handed 2.5-helix with a pitch
around 5.1 ꢀ. Moreover, although dimer 2 was too short to
achieve a complete helix turn, numerous NOEs were detected
and the calculated structures superimposed well with low
RMSD values (< 0.3 ꢀ) when the flexible N-terminal 4-BrPh
and C-terminal b-Ala-OBg moieties were omitted
Figure 3. Superimposition of the heavy atoms of the backbone of the
20 lowest energy NMR solution structures of dimer 2 (yellow),
tetramer 3 (green), and hexamer 4 (cyan). The disordered C-terminal
moiety and protons have been omitted for clarity. Dotted lines=hy-
drogen bonds.
(
Table S11). The poly-BAC oligomer was stabilized by
a regular bifidic intramolecular 12- and 14-membered H-
bond pseudo-ring network, resulting from C=O(i)···HN(i+1)
and C=O(i)···HN’(i+2) hydrogen bonds, forming a right-
handed 12/14-helix. The simultaneous presence of 12- and 14-
size of the sequences, f, q , and q BAC dihedral angles of the
1
2
membered pseudocycles was previously shown in N,N’-linked
oligoureas with proteinogenic side chains. Regardless of the
three compounds shared remarkably close values, except for
[
15]
the q value of the N-terminal BAC residue of each oligourea.
2
1
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Omitting the N-terminal distortion, the typical average f, q1,
The crystal structure of 3’ showed a right-handed 2.5-helix
and q values in solution were 81 Æ 148, 51 Æ 68, and À102 Æ
with a C- to N-terminal macrodipole. Except for the f angle
which exhibited a slight deviation, the dihedral angles of BAC
2
1
38, respectively (Tables S13–15). The solution structures of
dimer 2, tetramer 3, and hexamer 4 showed great similarity
when superimposed (Figure S7). The poly-BAC helix was
homogeneous and no critical sequence size was needed to
initiate the characteristic 2.5-helical fold, demonstrating the
high helical propensity of this sequence type.
residues in the crystal structures (f= 63 Æ 68, q = 54 Æ 98, and
1
q = À109 Æ 218) were close to those measured in solution
2
(f= 81 Æ 148, q = 51 Æ 68, and q = À102 Æ 138; Figure S8,
1
2
Table S15). Notably, the q angle value is comparable to that
1
described in the literature for a canonical oligourea helix,
[
25,26]
In addition, no inconsistent correlations in CD OH were
while the f and q angle values were permuted.
2
3
found in the ROESY spectra of the oligoureas 3 and 4, as has
been observed for the oligoureas described by Guichard and
co-workers and were assigned to urea-bond cis–trans isomer-
In addition, a significant difference between the structures
of compound 3 in solution and compound 3’ in solid-state
could be noticed. The N-terminal moiety of 3’, because of the
[
25]
ization.
This result suggested that no isomerization oc-
insertion of a CH OH molecule, was rotated of approximately
3
curred (or could not be detected) showing that the BAC
residues induced a high level of rigidity and robustness into
the poly-BAC sequences. The bulky bicyclo[2.2.2]octane
motif probably prevented the local isomerization of the
urea bond by inducing unfavorable steric clashes between the
BAC methylene groups and the urea amide protons.
We determined the crystal structures of dimer 2 and
tetramer 3’, corresponding to the methyl ester of 3 (see
crystallographic data, Table S12). Partial transesterification of
1008 (f (BAC 3) = 167 Æ 48) in the solid-state structure. This
rotation produced a disruption of the 12- and 14-membered
H-bonded ring through the formation of H-bonds between
the N-terminal urea and the urea carbonyl group linking BAC
moieties 2–3 and the inserted CH OH molecule.
3
In the crystal structures of both dimer 2 and tetramer 3’,
the molecules stacked into cylindrical columns to maximize
the interactions between the boundaries of the helices. This
arrangement is generally observed in the solid state of
[
25]
3
in CH OH during the long crystallization process occurred
molecules with helices.
In the crystal of 3’, all of the
3
and led to a crystalline structure of the corresponding methyl
ester 3’. The asymmetric unit of 2 consisted of one molecule
while that of 3’ contained two independent molecules
exhibiting quite similar structures. The solid-state structures
of the dimer and tetramer were globally similar when
superimposed on those structures obtained in solution
columns were oriented in the same direction and their axes
coincided with the crystallographic screw axes of the P21
space group. Within a column, the stacking of the helical
molecules was sinusoidal and a connection between two
successive molecules was made through bifurcated hydrogen
bonds (Figure S10). In the crystal of 2, two contiguous
columns were upside down in relation to each other (Fig-
ure S9). They were held together by bifurcated hydrogen
bonds and water molecules. Within a pillar, hydrogen bonds
between the carbonyl group of the second urea moiety and
the OBg amide group linked the molecules together.
(
Figure 4). A similar distortion of the N-terminal end of
dimer 2 was observed in both the solution and solid-state
structures. Nevertheless, the C-terminal b-Ala-OBg group
was disordered in the NMR structures, while a complete turn
of the helix could be achieved through a subsequent bifidic
hydrogen bond, implicating the carbonyl group of the b-Ala
in the crystal structure of 2 (Figure S9).
Finally, far-UV (190–260 nm) CD spectra for compounds
2, 3, and 4 were recorded in CH OH (Figure 5 and Fig-
3
ure S11). Their CD spectra shared the same shape, with
maxima around 205 nm. Interestingly, similar spectra with
slightly shifted maxima around 203 nm were obtained for
folded oligoureas in an uncapped acyclic series containing at
[
22]
least five residues. This CD signature highlighted a similar
folding property of both series. However, the CD profile of
Figure 4. a) Superimposition of the NMR (yellow and green) and X-ray
crystal (purple) structures of dimer 2 and tetramer 3/3’. The H-bond
network of one X-ray crystal structure of oligomer 3’ is shown at the
right. A co-crystallized MeOH molecule is represented by green and
red spheres. b) Typical hydrogen-bond pattern defining the oligourea
hexamer 4 (12/14 helix).
Figure 5. a) CD spectra of dimer 2 (yellow), tetramer 3 (green), and
hexamer 4 (cyan) recorded at 208C in CH OH. b) CD signals at
3
lmax =204.8 nm at variable temperature from 0 to 558C.
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the tetramer with acyclic residues had a broad negative band
at approximately 202 nm and did not exhibit the typical
profile of folded oligoureas, in contrast to the BAC dimer 2
and tetramer 3. These results point out again the propensity of
the BAC motif to induce a helical structure in oligomers.
For all oligomers, a gradual increase of the temperature,
up to 558C, induced a slight linear decrease of the maxima of
molar ellipticity without variation in the shape of the spectra.
When the temperature was returned to 208C, a total recovery
of the CD signal was observed, suggesting that the limited
thermal loss of structure induced by higher temperatures was
non-cooperative and fully reversible.
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along the helix surface could be easily controlled.
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Received: July 24, 2012
Published online: October 4, 2012
[
Keywords: bicyclo[2.2.2]octane · foldamers · helical structures ·
.
NMR spectroscopy · oligoureas
[
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