Large-Amplitude Conformational Changes in Self-Assembled Multi-Stranded Aromatic Sheets

Article abstract of DOI:10.1002/anie.202014670

The orchestration of ever larger conformational changes is made possible by the development of increasingly complex foldamers. Aromatic sheets, a rare motif in synthetic foldamer structures, have been designed so as to form discrete stacks of intercalated

Full text of DOI:10.1002/anie.202014670

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Accepted Article
Title: Large amplitude conformational changes in self-assembled multi-
stranded aromatic sheets
Authors: Joan Atcher, Pedro Mateus, Brice Kauffmann, Frédéric Rosu,
Victor Maurizot, and Ivan Huc
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Large amplitude conformational changes in self-assembled multi-
stranded aromatic sheets*
Joan Atcher,[a,b] Pedro Mateus,[b] Brice Kauffmann,[c] Frédéric Rosu,[c] Victor Maurizot[b] and Ivan Huc*[a]
[a]
Dr. J. Atcher, Prof. Dr. I. Huc
Department of Pharmacy and Center for Integrated Protein Science
Ludwig-Maximilians-Universität,
Butenandtstr. 5-13, 81377 München, Germany.
E-mail: ivan.huc@cup.lmu.de
URL: https://huc.cup.uni-muenchen.de/
[b]
[c]
Dr. J. Atcher, Dr. P. Mateus, Dr. V. Maurizot
Université de Bordeaux, CNRS, Bordeaux Institut National Polytechnique, CBMN (UMR 5248)
Institut Européen de Chimie et Biologie
2 rue Robert Escarpit, 33600 Pessac, France.
Dr. B. Kauffmann, Dr. F. Rosu
Université de Bordeaux, CNRS, Inserm, IECB (UMS 3033 – US001)
Institut Européen de Chimie et Biologie
2 rue Robert Escarpit, 33600 Pessac, France.
Supporting information for this article is given via a link at the end of the document.
Abstract: The orchestration of ever larger conformational changes is
made possible by the development of increasingly complex foldamers. There is obvious interest in mastering and implementing such
protein-protein associations, induced by e.g. domain swapping.[3]
Aromatic sheets, a rare motif in synthetic foldamer structures, have
been designed so as to form discrete stacks of intercalated aromatic
strands through the self-assembly of two identical subunits. Ion-
mobility ESI-MS confirms the formation of compact dimers. X-ray
crystallography reveals the existence of two distinct conformational
dimeric states that require large changes to interconvert. Molecular
dynamics simulation validates the stability of the two conformations
and the possibility of their interconversion.
processes in synthetic chemical systems and important progress
is being made in this direction using foldamers’ inherent
conformational equilibria, including conformational changes
induced by ligands, light or electrons.[4] These efforts mark a
transition from the mere control of molecular structures to the
orchestration of dynamics.[5] As improvements in foldamer design
and synthesis give access to increasingly large and complex
objects,[6,7] one may expect novel types of conformational
dynamics to emerge. Here, we report the unexpected discovery
of a complex conformational change in a multi-stranded aromatic
sheet foldamer. While aromatic foldamers have been known as
notoriously rigid folded molecules,[8] we find that increasing their
size gives access to new types of deformation.
In biopolymers, conformational changes of large amplitude or with
long range effects are associated with important molecular
functions. They mediate cooperative binding between distant
sites, for example the dioxygen binding sites of hemoglobin
tetramers;[1] they allow for the remote transfer of information, as
in G protein-coupled receptors;[2] and they give rise to distinct
We have reported on the folding of aromatic sheets
stabilized by interactions between  systems and by rigid turns
such as TS (Figure 1b) that hold aromatic groups at a distance
Figure 1. a) Chemical structure and schematic representation of two-stranded aromatic sheet 1. b-d) Structures defining the letter code of TS, A, TL and PNO, and
association mode of the latter two (dashed lines depict hydrogen bonds while hashes show aromatic stacking). e) Schematic representation of a self-assembled
aromatic sheet. f) Structure and schematic representation of 2. (R = -CH2CH(CH2CH3)2).
1
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suitable for face-to-face stacking.[9] Interest for these systems
stemmed from the low occurrence of sheets among foldamer
structures when compared to helices, and the apparent greater
difficulty to design discrete sheets that do not further aggregate
and precipitate.[10] Compound 1 (Figure 1a) was prepared as a
representative of earlier designs. Its structure was fully
characterized in solution through a complete assignment of its
NMR spectra (Figure 2b, Figures S16-S20) and in the solid state
(Figure 2a) to establish that the strand curvature associated with
the A units (Figure 1c) gives rise to a head-to-head arrangement
experimental and calculated CCS distribution of 22 are both narrow and
indicative of a very compact conformation.
The synthesis of 2 was carried out using classical aromatic
amide coupling steps and is presented in the supporting
information. Its NMR spectrum in toluene-d8 showed sharp
resonances indicating the presence of well-defined species
(Figure 2c). However, the number of peaks was larger than
expected for a C2 symmetrical dimer. In other nonpolar solvents,
the lines tended to broaden, indicating faster dynamics, but the
spectrum remained complex regardless of temperature (Figures
S23-S28). Dilution experiments down to 0.54 mM[12] did not
produce any change, suggesting that if an aggregate was present,
its dissociation constant was below this value (Figure S21). The
ESI mass spectrum clearly showed the formation of a 22 dimer.
Furthermore, an ion mobility analysis[13] generated a collision
cross-section profile that matched well with the calculated profile
of a compact interdigitated sheet conformation (Figure 2d, Figure
S38). However, these data alone did not allow to ascertain the
very structure of 2 and reasons for NMR signal multiplicity.
Essential evidence came from two crystal structures of 2,
which validated the initial design but also uncovered
unanticipated structural variations (Figure 3). Both structures
showed the expected dimerization as depicted in Figure 1e, in
particular the reciprocal intercalation, the hydrogen bonding of
PNO N-oxide function of one strand to the TL amide NH of the other
strand (dO-HN ~ 2.1 Å, Figure 3e,j), and the formation of a face-to-
face stack of six A2 segments. In addition, the structures also
demonstrated two conformations, C1 (Figure 3a-e, crystallized
from CH2Cl2/hexane) and C2 (Figure 3f-j, crystallized from
CHCl3/MeOH) that display considerable differences, as
highlighted by dashed lines throughout Figure 3. Through a
complex and large amplitude sliding of the various A2 segments
with respect to one another (Figure 3d,i), the TS, TL and pivaloyl
(piv) units undergo a large swing, while the overall architecture is
preserved. Among notable differences between C1 and C2 is the
fact that A2 segments are perfectly flat and stacked perpendicular
to the structure main axis in C1 (Figure 3c), whereas they are
tilted and slightly M-helically twisted in C2 (Figure 3h).[14] The
complex (Figure 2c) or broad (Figure S23-S28) NMR spectra of 2
are consistent with the large conformational changes required to
interconvert C1 and C2. Furthermore, one cannot exclude the
existence of intermediate or alternate conformational states, for
example a P-helically twisted diastereomeric analogue of C2.[14]
We endeavored to investigate C1 and C2 using molecular
dynamics simulations (MD) to assess their stability (Figure S29-
S37). Energy minimization of the C1 and C2 structures from the
crystal data did not give rise to notable changes and produced the
starting coordinates for 10 ns MD runs performed every 100
degrees from 200 K up to 900 K. Several parameters were
systematically monitored over time: the hydrogen bond distances
at each NO-TL contact, as well as the TS-TS, TL-TL, and the piv-piv
distances (Figure S29). Up to 400 K both C1 and C2 were found
to be stable (Figure 4a, Figures S30, S31a,b, movies S1, S2).
Intermolecular hydrogen bonds did not disrupt and limited
fluctuations allowed for the discrimination of two distinct C1 and
C2 conformational ensembles. As can also be seen in the crystal
structures (Figure 3), C2 is characterized by a TS-TS distance
about half the piv-piv distance, whereas these two parameters are
comparable in C1. These features are well reproduced by MD.
Perhaps the most characteristic difference between the two is the
of stacked aromatics within a curved two-stranded sheet.[9c]
A
marker of the stability of the sheet conformation is the slow
rotation on the NMR timecale of the dimethyl-para-
phenylenediamine rings of TS (S22).
As an extension of this work, we devised that turn TL (Figure
1d) might promote the intercalation of an aromatic group and thus
give access to unprecedented discrete self-assembled aromatic-
sheets (Figure 1e). Derivatives of N,N’-dibenzyl 2,6-pyridine-
dicarboxamide have indeed been shown to bind to aryl
compounds, including within macrocyclic structures, through both
hydrogen bonding and aromatic stacking.[11] Using a pyridine N-
oxide PNO as a hydrogen bond acceptor, we thus designed
sequence 2 (Figure 1f), comprised of both a short turn TS and a
long turn TL. Based on energy-minimized molecular models, 2
was expected to dimerize through a reciprocal intercalation.
Figure 2. a) Crystal structure of 1. b) Part of the 1H 400 MHz NMR spectrum of
1 in CDCl3 at 298 K. c) Part of the 1H 700 MHz NMR spectrum of 2 in toluene-
d8 at 298 K. d) Collision cross section (DTCCSHe) of 22 measured by drift tube
ion mobility ESI-MS (black circles) in He and calculated from snapshots
sampled from a molecular dynamics simulation (green bars). A molecular model
of 22 with some fraying at one end (red arrow) is shown at right. The
corresponding calculated CCS is indicated on the graph, illustrating that the
2
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relative positionning of the TL units: the one that is "above" the
other in C1, is "below" in C2 (Figure 3b vs 3g). However, the TL-
TL distance alone does not discriminate between these two states.
concerted manner, in particular the piv-piv and TS-TS distances.
Overall, the MD simulations are suggestive of sharp transition
between C1 and C2, without dissociation, supporting the
existence of (at least) two distinct ensembles of conformations.
The C1<->C2 transition entails that the two TL units pass each
other. During this process, the isobutoxy side chains that they
carry (Figure 4b) undergo extensive contacts that may then
constitute a barrier. For future developments, one may take
advantage of the proximity between these side chains to install
functionalities conducive of attractive or repulsive forces, that
would stabilize one or the other conformer. A step further would
be to introduce functionalities that would allow for the controlled
switching between the two. The concept of aromatic sheet self-
assembly may also be extended using turn units that do not allow
for the intercalation of just one aromatic, such as TL, but of two or
more.[15] Furthermore, the original half-pipe architecture may be
exploited for the purpose of molecular recognition, particularly
with regards to the fact that functional groups in position 9 of the
A monomers (here only methyl groups) all converge towards the
interior of the cavity.
Figure 4. a) Overlay of ten snapshots of the 10 ns MD simulation of C1 at 400K
showing limited fluctuations at this temperature. b) Snapshots of a C1->C2
transition during the MD simulation of C1 at 500K. Side chains and hydrogen
atoms have been omitted for clarity except the isobutoxy side chains of TL in b).
Acknowledgements
This work was supported by the European Research Council
under the European Union’s Seventh Framework Programme
(grant agreement no. ERC-2012-AdG-320892). It benefited from
the facilities and expertise of the Biophysical and Structural
Chemistry platform at IECB, CNRS UMS3033, INSERM US001,
Bordeaux University, France. We thank Dr. L. Sebaoun for
preliminary experiments.
Figure 3. Top views (a,f), front views (b,g), side views (c,h), details of
intramolecular - stacking (d,i) and hydrogen bonding (e,j) of two crystal
structures of 22 demonstrating distinct conformers C1 (a-e) and C2 (f-j).[14] In a-
d and f-i, TS, TL and the terminal t-butyl groups are shown as orange, green and
black space filling models, respectively. The rest of the molecules are shown in
tube representation. The A units are shown in red or blue to distinguish the two
molecules within each 22 dimer. Included solvent molecules, side chains, and
hydrogen atoms (except amide NH in e and j) have been omitted for clarity.
Keywords: foldamers • sheets • aromatic stacking • self-
assembly • conformational change
MD runs performed at higher temperatures (500-900 K) led
to larger fluctuations. At the highest temperatures, disruption of
the hydrogen bonds and local unfolding occurred frequently yet
the structures remained integral. During these runs the
fluctuations were large enough to create junctions between the
C1 and C2 conformational spaces, and some C1->C2 or C2->C1
transitions occurred (Figure 4b, Figures S31c,d, S32b, movie S3).
All the parameters that discriminate C1 and C2 then changed in a
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4
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Entry for the Table of Contents
Unexpected conformational changes involving a global concerted rearrangement of the structure have been evidenced in large
multistranded aromatic sheets. Increasing foldamer size thus gives access to new and defined conformational trajectories.
5
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