The canonical helix of urea oligomers at atomic resolution: Insights into folding-induced axial organization

Article abstract of DOI:10.1002/anie.200905592

(Figure Presented) Helical by nature: Urea-based peptidomimetics with proteinogenic side chains are fully helical in the crystalline state (see picture). Four acyclic residues are sufficient to drive complete helix formation with all complementary H-bonding sites being satisfied (up to 14 for a 8-mer). Helices pack head-to-tail to create infinite H-bonded networks with different topologies.

Full text of DOI:10.1002/anie.200905592

Angewandte
Chemie
DOI: 10.1002/anie.200905592
Helical Foldamers
The Canonical Helix of Urea Oligomers at Atomic Resolution: Insights
Into Folding-Induced Axial Organization**
Lucile Fischer, Paul Claudon, Nagendar Pendem, Emeric Miclet, Claude Didierjean,
Eric Ennifar, and Gilles Guichard*
Foldamers are discrete artificial oligomers with defined and
predictable folding patterns akin to naturally occurring
helices, turns, and linear strands.[1–3] Because of their diversity
in size, shape, and side chain appendages, and also their
resistance to enzymatic degradation, peptidomimetic helical
foldamers are unique scaffolds for use in a range of biological
and biomedical applications.[4] Characterizing such helical
folds at atomic resolution is of prime importance if molecules
are to be designed that can target biological surfaces and for
reliable structure–function analysis. To date, extensive crys-
tallographic data sets have been gathered on aliphatic (b- and
a/b-peptides) and aromatic oligoamides,[3] thus providing a
detailed picture of the structural diversity within these
foldamer families. Few other helical peptidomimetic back-
bones have been characterized by crystallographic analy-
sis.[5–10] Crystal structures are also central to gain precise
insight into axial[11] and lateral[12,13] self-assembling properties
of helical foldamers, en route to new tertiary and quaternary
structural motifs and more sophisticated self-assembled
nanostructures. Notable achievements include the atomic
structure determination of large (> 8 kDa) aromatic oligo-
amide foldamers[14] and helix-bundle quaternary structures
formed by designed b- and a/b-peptides.[12,13]
age,[21] (-NH-CH(Ri)-CH2N’H-CO)n-, have a remarkable
propensity to fold into helical secondary structures in
solution[22–26] and show promise for interaction with biologi-
cally relevant targets.[27,28] Compared to g-peptides,[5,29,30] helix
stabilization in oligoureas is promoted by the presence of
additional backbone conformational restriction and H-bond
donor sites. Although three-centered H-bonding between
Oligomers consisting of N,N’-linked urea bridging units
are receiving increasing attention as folding backbones.[9,15–20]
Peptidomimetic oligoureas belonging to the g-peptide line-
=
C O(i) and urea HN(iÀ3) and HN’(iÀ2) to form 12- and 14-
membered pseudo rings is likely to occur, detailed NMR
investigations provided some evidence for H-bond dissym-
À
À
metry with d(O(i) N(iÀ3)) < d(O(i) N’(iÀ2)). Notwith-
standing the high quality of structure calculations recently
achieved using NMR spectroscopy at 13C natural abun-
dance,[31] structural data at atomic resolution are clearly
needed to precisely describe the H-bonding pattern of the 2.5
helix and facilitate the design of functionally active oligoureas
and more complex structures. Herein, we report the first
crystal structures of enantiopure N,N’-linked oligoureas with
from 5 to 9 urea groups and containing exclusively acyclic
residues with proteinogenic side chains (that is, Me, iPr, iBu,
and Bn).
[*] Dr. L. Fischer, P. Claudon, Dr. N. Pendem, Dr. G. Guichard[+]
CNRS, Institut de Biologie Molꢀculaire et Cellulaire, Laboratoire
d’Immunologie et Chimie Thꢀrapeutiques
15 rue Renꢀ Descartes, 67000 Strasbourg (France)
E-mail: g.guichard@iecb.u-bordeaux.fr
Dr. E. Ennifar
Architecture et Rꢀactivitꢀ de l’ARN, Universitꢀ de Strasbourg, CNRS
IBMC, 15 rue Renꢀ Descartes, 67084 Strasbourg, (France)
Dr. C. Didierjean
CRM2, UMR-CNRS 7036, Groupe Biocristallographie
Universitꢀ Henri Poincarꢀ, BP 239, 54506 Vandœuvre (France)
Dr. E. Miclet
Oligoureas 1–4 have been prepared in solution by
stepwise assembly of N-Boc protected monomers.[32] The
nature of the capping groups at both ends of oligomers was
critical to grow high-quality crystals. The 4-bromophenyl
moiety in 3 and 4 was introduced to facilitate phasing. Crystal
structures of 1–4 were solved in the C2, C2, P41, and P21212
space groups, respectively.[32]
All four oligoureas, including short tetramer 1, are fully
helical in the solid state (Figure 1 and 2; Supporting Infor-
mation, Figure S1).[33] They adopt a right-handed 2.5-helical
fold that matches the conformation initially proposed from
Universitꢀ Pierre et Marie Curie-Paris 6, UMR 7613, Paris (France);
Case courrier 45, 4, place Jussieu, 75005 Paris (France)
[+] Present address: Institut Europꢀen de Chimie et Biologie
Universitꢀ de Bordeaux - CNRS UMR 5248
2 rue R. Escarpit, 33607 Pessac (France)
[**] This research was supported by the CNRS, ImmuPharma France
and by an ANR grant (NT05-4-42848). Fellowships from the
Universitꢀ de Strasbourg (N.P.) and ImmuPharma France (P.C.) are
gratefully acknowledged.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200905592.
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Overlay of the X-ray structure of octamer 3 and the
optimized structure of a related nona-urea determined by
NMR spectroscopy[31] provides precise insight into the
behavior of this helical fold in solution (Figure 1c). First,
the helix in solution is clearly distorted at both ends, which is a
direct consequence of an incomplete H-bond network.
Indeed, the carbonyl group of the residue 9 in the nonamer
only establishes an H-bond with the amide proton of the
residue 6. Compared to the crystal structure, the absence of
H-bonding between O(9) and HN’(7) modifies the orienta-
tion of the residue 9 urea plane and thus slightly distorts the
helix axis. The same feature is found for the residue 4, which is
not H-bonded to the HNꢁ amide of residue 2 but only to HN
of residue 1. As no H-bond occurs for the preceding residues,
the corresponding tail appears much more flexible than in the
solid state structure. The detailed conformations of residues 4
and 9 highlight the weaker H-bond character of the HNꢁ
amide compared to the HN amide. Such an observation is also
made for central residues of the helix, for which slightly
greater distances accompanied with smaller O-H-N angles are
found between O(i) and N’(iÀ2) than for O(i) and N(iÀ3).
This structural feature is in very good agreement with
experimental data recorded on oligoureas in pyridine, for
which temperature coefficients of well-structured regions
were all found to be greater for the HN amide (between À2.0
and À3.5 ppbKÀ1) than for HN’ (between À4.4 and
À5.6 ppbKÀ1),[25] but differs from the crystal structure of 3,
in which both H-bonds are equivalent. Torsion angles f, q1,
and q2 obtained over the 20 best NMR spectroscopically
determined structures for the well-defined segments are
À1008, 438, and 968, with RMSD values of 18, 68, and 78;
these values are comparable to those measured in the crystal
structure of 3. The difference of about 158 for q1 and q2 could
be related to the significant planarity deviations found for the
urea motifs in the crystal structure. Indeed, C-N’-C’-O
dihedral angles range from 58 to 188 and C-N-C’-O angles
from À238 to À68. As a consequence, whereas C-N’-N-C
dihedral angles are between 0 and 58 for all the calculated
structures determined from NMR spectroscopy, they range
between À238 and 188 in the X-ray structure. This result
shows that the double-bond character of the amide bond is
significantly reduced within urea motifs. Overall, slight
differences in H-bonding in NMR and X-ray structures and
in the distortion of urea planes observed in the crystal
structure account for the 2.5 ꢀ root-mean-square deviation
obtained when aligning solution and crystal structures
(Figure 1c).
Figure 1. Crystal structure of 3. a) View along helical axis. b) Detail of
the three-centered H-bonding. O(7)···HN(4) and O(7)···HN’(5) distan-
ces in ꢁ. c) Overlay with the 20 best NMR structures (in gray) of a
related nona-urea.[31] Side chains are omitted for clarity.
Figure 2. X-ray crystal structures of 1 and 4. a) Overlay of independent
molecules (blue and orange) in 1 (left) and 4 (right). Side chains in 1
have been removed for clarity. b) H-bond network at upper and lower
extremities of the helix in 4 (top) and 1 (bottom), showing conforma-
tional differences between independent molecules (carbon atoms in
blue and orange). D···A distances in ꢁ.
NMR spectroscopy studies of oligoureas in solution.[23–25]
Octamer 3, which has one single conformation in the crystal,
has a nearly perfect repeat of 2.5 residues per turn (Figure 1a)
and a rise per turn of 5.1 ꢀ. The average backbone torsion
Shorter oligoureas 1, 2, and 4 crystallized with two (1 and
2) or four (4) independent molecules in the asymmetric unit
(Figure 2a; Supporting Information, Figure S1). In all three
cases, the conformations of independent molecules differ
perceptibly, with the largest differences being localized at
both ends (Figure 2). These distortions, which are presumably
caused by packing effects and/or H-bonded interaction with
solvent molecules (H2O, MeOH), reflect conformational
plasticity of shorter helices. For example, in the crystal of
[34]
angles f, q1, and q2 are À1038, + 578, and + 808, and are
characterized by relatively low RMSD values (118, 58, 78).
Such a regular fold is allowed, as all possible intramolecular
12- and 14-membered H-bonded rings (i.e. 14) are present in
the crystal structure of 3 (Figure 1). Distances between O(i)
and H-bonded N’(iÀ2) and N(iÀ3) are very similar, and range
from 2.78 to 2.99 ꢀ and 2.82 to 3.00 ꢀ, respectively. The O(i)-
H-N’(iÀ2) and O(i)-H-N(iÀ3) angles are comparable with an
average value of 1398 (RMSD of 98 and 118 respectively).
À
tetra-urea 1, the O N distance between O(3) and NH(Me)
increases from 2.91 ꢀ (molecule A) to 3.67 ꢀ (molecule B)
owing to the insertion of a bridging water molecule (Fig-
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ure 2b, lower). Differences between 1(A) and 1(B) propagate
along the backbone, with a shift of O(4) from an equidistant
position between N(1) and N’(2) in 1(A) to a N’(2) distal
position with loss of the corresponding 12-membered
H-bonded ring in 1(B). This situation parallels that observed
in solution (see below), thus reflecting a hierarchy in the
formation of H-bonds. Distortions imposed by solvation at
the helix termini are even more pronounced in the structure
of hepta-urea 4. Whereas HN’(5) and HN(4) are directly
H-bonded to O(7) in 4(A) and 4(B), a bridging methanol
the head-to-tail region). In contrast to what is observed in 1, 2,
and 4, O(1) and O(2) are not H-bounded to the penultimate
and ultimate urea moieties of a neighboring helix, but rather
to the ultimate and penultimate ones, respectively (Figure 3c,
top). As a result, two neighboring helices are oriented at an
angle of ca 658 (Figure 3c, bottom), and crystal formation
generates an H-bonded right-handed superhelix (Figure 4).
À
molecule is intercalated in 4(C) and 4(D), thus the O(7)
À
N’(5) and O(7) N(4) distances increase to about 4.7 ꢀ and
4.3 ꢀ, respectively (Figure 2c). At the other end of the
molecule, residue 1 in 4(B) adopts a non-canonical geometry,
with a q2 angle value of + 1568 (compare with + 868 in 4(A);
Supporting Information, Figure S2).
Oligourea helices possess four free donor and two
acceptor groups that are capable of H-bonding at both ends
of the molecule. Head-to-tail arrangements of helices are
frequently observed in the crystal structures of DNA oligo-
nucleotides,[35] hydrophobic helical a-peptides,[36,37] and b-
peptide foldamers.[11] Generally these molecules form long
cylindrical helical columns.
By analogy, independent helices of pentaurea 1 and
hexaurea 2 in the asymmetric unit are aligned end-to-end with
formation of all possible three-centered H-bonds in the head-
to-tail regions (Figure 3a, top; 3b, left; Supporting Informa-
Figure 4. Supramolecular helix formed by H-bonded assembly of
helices 3. a) Side view in stereo. b) View along helical axis. Pitch: ca.
48.0 ꢁ, internal diameter: ca. 7 ꢁ, external diameter (with side chains):
ca. 28.0 ꢁ, groove: ca. 29.6 ꢁ.
The canonical 2.5 helical structure of oligoureas has been
characterized at atomic resolution. The similarity between the
structures reported herein and those deduced earlier from
NMR studies in solution is striking and underlines the
excellent complementarities of the two techniques to analyze
urea-based foldamers.[22–25,31,38] Our study also demonstrates
the robustness of the folding process: four acyclic residues are
sufficient to drive complete helix formation with all comple-
mentary H-bonding sites being satisfied. Overall, this crys-
tallographic data set provides the ground for the structure-
guided development of urea foldamers with function, such as
recognition of biomolecules and catalysis,[20,39,40] and also for
the elaboration of new tertiary and quaternary structural
motifs.
Received: October 7, 2009
Published online: December 28, 2009
Figure 3. Assembly of urea helices. a) H-bonding network between
pairs of helices in 2 (top) and 4 (bottom). b) Packing of helices in 2
(left) and 4 (right). c) Non-canonical intermolecular H-bonding pattern
(top) in 3 and orientation of neighboring helices at an angle (bottom).
Intermolecular H-bonds in green. D···A distances in ꢁ.
Keywords: foldamers · helical structures · peptidomimetics ·
.
self-assembly · X-ray diffraction
tion, Figure S3). In heptamer 4, the H-bonded network at the
boundary between neighboring helices differs substantially:
whereas complementary H-bonding sites between helices B
and C (A and D) are satisfied, assembly at the boundary
between helices C and A (D and B) is mediated in part by a
relay of methanol molecules (Figure 3a, bottom). Packing of
neighboring helices in 4 curves slightly (168 between A and C,
and 318 between C and B) (Figure 3b right) and forms a wavy
column. The bend is even more striking in nona-urea 3, in
which helices self-assemble by using a non-canonical water-
mediated intermolecular H-bonding pattern (6 H-bonds in
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