Solid state NMR studies of oligourea foldamers: Interaction of 15N-labelled amphiphilic helices with oriented lipid membranes

Article abstract of DOI:10.1039/c1ob06278f

Synthetic oligomers that are derived from natural polypeptide sequences, albeit with unnatural building blocks, have attracted considerable interest in mimicking bioactive peptides and proteins. Many of those compounds adopt stable folds in aqueous environments that resemble protein structural elements. Here we have chemically prepared aliphatic oligoureas and labeled them at selected positions with ¹⁵N for structural investigations using solid-state NMR spectroscopy. In the first step, the main tensor elements and the molecular alignment of the ¹⁵N chemical shift tensor were analyzed. This was possible by using a two-dimensional heteronuclear chemical shift/dipolar coupling correlation experiment on a model compound that represents the chemical, and thereby also the chemical shift characteristics, of the urea bond. In the next step ¹⁵N labeled versions of an amphipathic oligourea, that exert potent antimicrobial activities and that adopt stable helical structures in aqueous environments, were prepared. These compounds were reconstituted into oriented phospholipid bilayers and the ¹⁵N chemical shift and ¹H-¹⁵N dipolar couplings of two labeled sites were determined by solid-state NMR spectroscopy. The data are indicative of an alignment of this helix parallel to the membrane surface in excellent agreement with the amphipathic character of the foldamer and consistent with previous models explaining the antimicrobial activities of α-peptides.

Full text of DOI:10.1039/c1ob06278f

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PAPER
Solid state NMR studies of oligourea foldamers: Interaction of 15N-labelled
amphiphilic helices with oriented lipid membranes†
Christopher Aisenbrey,a Nagendar Pendem,b Gilles Guichard*b and Burkhard Bechinger*a
Received 28th July 2011, Accepted 25th October 2011
DOI: 10.1039/c1ob06278f
Synthetic oligomers that are derived from natural polypeptide sequences, albeit with unnatural building
blocks, have attracted considerable interest in mimicking bioactive peptides and proteins. Many of
those compounds adopt stable folds in aqueous environments that resemble protein structural elements.
Here we have chemically prepared aliphatic oligoureas and labeled them at selected positions with 15
for structural investigations using solid-state NMR spectroscopy. In the first step, the main tensor
N
elements and the molecular alignment of the 15N chemical shift tensor were analyzed. This was possible
by using a two-dimensional heteronuclear chemical shift/dipolar coupling correlation experiment on a
model compound that represents the chemical, and thereby also the chemical shift characteristics, of
the urea bond. In the next step 15N labeled versions of an amphipathic oligourea, that exert potent
antimicrobial activities and that adopt stable helical structures in aqueous environments, were
prepared. These compounds were reconstituted into oriented phospholipid bilayers and the 15
N
chemical shift and 1H-15N dipolar couplings of two labeled sites were determined by solid-state NMR
spectroscopy. The data are indicative of an alignment of this helix parallel to the membrane surface in
excellent agreement with the amphipathic character of the foldamer and consistent with previous
models explaining the antimicrobial activities of a-peptides.
Foldamers are endowed with properties (structural predictability,
Introduction
extended contact areas, diversity and resistance to proteolysis)
that make them potentially useful as pharmacological tools to
address some of the limitations of natural a-peptides. Reported
applications of peptidomimetic foldamers range from inhibitors
of protein–protein interaction to host-defense peptide mimics.
Antimicrobial foldamers inspired by host-defense peptides include
amphiphilic helical oligoamides such as b-peptides17–19 and pep-
a-Peptides exert many important biological activities and have
therefore been subject to a wide variety of biophysical and
structural investigations. In particular, they are known to act
as neurotransmitters, have antimicrobial and cell penetrating
activities, or act as nucleic acid transfection agents.1–5 Many of
these, in particular the hydrophobic and amphipathic sequences,
interact with phospholipid membranes where they exert a variety
of activities in a lipid composition-dependent manner.6,7 Such
interactions result in the transmembrane passage of hydrophilic
macromolecules, the formation of pores, transient openings or
the stabilization of the membrane,8,9 properties that are also of
biomedical importance.10,11
Over the last 15 years, unnatural oligomers with folding
patterns (i.e. foldamers 12) akin to protein structure elements have
gained considerable interest in the context of mimicking bioactive
peptides and interacting with biomolecules (proteins, nucleic acids,
phospholipid components of biological membranes, . . . ).13–16
21
toids (oligomers of N-alkyl glycine),20 extended arylamides as
well as non oligoamide foldamers such as aromatic and aliphatic
N,N¢-linked oligoureas.22 Antibacterial oligoureas such as 8-mer 1
(Fig. 1) display significant antimicrobial activities against a broad
spectrum of Gram-positive and Gram-negative bacteria.23,24 They
have been designed assuming an idealized 2.5-helical structure
(i.e. 2.5 residues per turn), by sequestration of cationic residues
on one face of the helix. The 2.5-helical structure of aliphatic
urea foldamers has been thoroughly investigated for oligomers
of various lengths in solution by diverse spectroscopic methods
(NMR,25–27 electronic circular dichroism (ECD),28 FT-IR 29) and in
the crystal state by X-ray diffraction.30,31 The helix is characterized
by a (+)-synclinal arrangement around the main chain C–C bonds
and is stabilized by a collection of three centered H-bonds closing
12- and 14-membered pseudo-rings.
aUniversite´ de Strasbourg/CNRS, UMR7177, Institut de Chimie, Faculte´ de
chimie, 4 rue Blaise Pascal, 67070 Strasbourg, France. E-mail: bechinger@
unistra.fr; Fax: +33 3 68 85 51 51; Tel: +33 3 68 85 51 50
bUniversite´ de Bordeaux-CNRS UMR5248, Institut Europe´en de Chimie et
Biologie, 2 rue Robert Escarpit, 33607 Pessac, France. E-mail: g.guichard@
iecb.u-bordeaux.fr
Although steady-state fluorescence anisotropy measurements
provided a first hint that antibacterial oligoureas such as 1
interact with phospholipid membranes,24 the precise mechanism
of their interaction with the membrane is still not well understood.
† This article is part of an Organic & Biomolecular Chemistry web theme
issue on Foldamer Chemistry.
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Fig. 1 Formula of reference antibacterial oligourea 1 and related oligomers 2 and 3 investigated in this study specifically 15N-labelled at residue 5 and 8,
respectively.
NMR spectroscopy is a proven method for the biophysical
characterization of peptides, including their structure, dynamics
and interactions in the presence of membranes.32 This technique
allows one to not only determine their conformation in many
different environments, such as aqueous buffers, solvent mixtures
or in the presence of micelles, but also to monitor their dynamics
at high resolution and therefore their conformational plasticity.3,32
The detailed description of the interaction properties of the
NMR nuclei in the magnetic field of the NMR spectrometer in
terms of interaction tensors has allowed for the development of
elaborate solution state NMR techniques (e.g. residual dipolar
couplings and residual chemical shift anisotropy) and for the
detailed analysis of the dynamics and conformation of these
polypeptides.33–36 Furthermore, the knowledge of the size and
molecular alignment of the tensor elements is a key requirement for
the structural and dynamic analysis of solid-state NMR spectra.
The nuclei located in the polypeptide backbone, such as the 15N of
the amide bond, have been proven to be particularly useful during
the analysis of the structure and topology of labeled polypeptides
when interacting with membranes.32 Consequently, a large number
of studies have been performed using this nucleus, with efforts to
characterize the 15N chemical shift tensor.37 In particular, when
investigated by static oriented solid-state NMR spectroscopy the
15N chemical shift of membrane associated polypeptides has been
shown to be directly correlated to the bilayer topology.38
By analogy to antibacterial a-peptides,32 solid state NMR is
likely to provide insight into the three-dimensional structure,
dynamics and topology of amphiphilic oligoureas in lipid bilayers.
Towards this end, in this paper we first determine the 15N
chemical shift tensor of the urea bond and thereafter present up-
to-date solid-state NMR investigations revealing the membrane
interactions of 15N-labelled antimicrobial oligourea sequences
(compounds 2 and 3, Fig. 1) reconstituted into oriented phos-
pholipid bilayers.
Scheme 1 Synthesis of 15N-labelled succinimidyl carbamate 5 and model
diurea4. BOP= benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorophosphate; DIPEA
= diisopropylethylamine; DIAD =
diisopropyl azodicarboxylate; DSC = N,N¢-disuccinimidyl carbonate;
TFA = trifluoroacetic acid.
15N-valine which was converted to [15N]-(R)-succinimidyl-(2-(tert-
butoxycarbonylamino)-3-methylbutyl)carbamate 5 with an over-
all yield of 49% (5 steps). The coupling of 5 with methylamine
hydrochloride in the presence of diisopropylethylamine followed
by Boc removal and reaction with benzyl isocyanate afforded 4
with a 81% yield.
The size of the main tensor elements of a labelled site within
a polypeptide39 or within an oligourea can be read from the
discontinuities that are obvious in the one-dimensional chemical
shift powder pattern (or the envelope describing the magic angle
sample spinning (MAS) side band intensities) and the values
obtained after the simulation of the spectrum obtained from 4
(Fig. 2B) are s11 = 39 ppm, s22 = 89 ppm and s33 = 152 ppm
(Fig. 2A). Determining their alignment within the molecular
coordinate system is more demanding, and in the case of the
15N amide bonds, this has been achieved by studies of single
crystals, comparison to model compounds, quantum mechani-
cal calculations40,41 and more recently through two-dimensional
Results and discussion
NMR determination of the 15N chemical shift tensor of a model
diurea
In order to perform NMR spectroscopic investigations of
oligourea foldamers, the tensor of the 15N labelled backbone site
was first determined using a simple 15N-labelled diurea (4). The
synthesis of 4 (Scheme 1) started from commercially available
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the 1H-15N dipolar coupling of the individual molecules.43 In
this sample all molecular alignments are present in a random
fashion (spherical distribution) and by adding up the spectral
contributions, a two-dimensional powder pattern line shape is
obtained; where the intensity distribution is a function of the
relative alignment of the inter-atomic 1H-15N vector of the dipolar
coupling and the 15N chemical shift tensor.43 The powder pattern
line shape is characterized by two symmetry-related ellipsoid-like
contributions, which add up into a “wing-like” distribution of
1
resonance intensities (Fig. 2C).44 The angle between the H-15N
bond and s33 is reflected in the eccentricity of the ellipsoidal
resonance distribution, i.e. for s33 parallel to the N–H vector the
two-dimensional spectrum reduces to lines that cross each other at
the isotropic chemical shift and dipolar coupling (93 ppm/0 kHz),
whereas for s33 perpendicular to 1H-15N a round spectral line shape
is obtained. Computer simulations indicate that the observed
spectral line shape (Fig. 2C) fits well to an angle of 25◦ covered
1
by the H-15N bond and s33. The main tensor elements and this
geometrical arrangement are illustrated in Fig. 2E. Although other
symmetry-related arrangements cannot a priori be excluded from
the spectral analysis alone, the close chemical relationship between
the amide and urea bonds suggests that the urea bond tensor
alignment is related to that of the peptide bond (Fig. 2D, E).
In the latter case the angle covered by s33 and the N–H vector
amounts on average to 18◦ (recently reviewed in reference 37).
The modest difference in the average tensor alignment between
the two compounds ranges within the variance observed when the
tensors of a large variety of peptide bonds have been analyzed.
However, when compared to the peptide bond (Fig. 2D) the
higher shielding in the plane of the urea bond (Fig. 2E) and the
concomitant reduction of the main tensor elements s11 and s33
is probably due to the extra electron density carried by the non-
bonding electrons of the nitrogen which takes a position equivalent
to the Cai-1 atom of the peptide bond.
Solid-state NMR analysis of 15N-labelled antibacterial oligoureas
Fig. 2 A: Simulation of a 1D powder pattern using the principal values
for the 15N chemical shift tensor of s11 = 39 ppm, s22 = 89 ppm and s33
In the next step, octamer 2, an analogue of antibacterial oligourea
1 specifically 15N-labelled at residue 5 as indicated in Fig. 1, was
reconstituted into phospholipid bilayers that are mechanically
oriented along glass surfaces with their membrane normal parallel
to the magnetic field direction.45 The resulting separated local field
spectrum representing the resonances of residue 5 is shown in
Fig. 3B (black line) and compared to the powder pattern line
shape of model compound 4 (Fig. 3B gray lines and Fig. 2C). The
=
152 ppm, and the orientation of the tensor as derived from the experiment
shown in panel C and graphically presented in E. B: Proton-decoupled
15N solid-state NMR spectrum of the model 15N-labelled diurea 4. C: The
separated local field spectrum of 4 correlating the 15N chemical shift and
the 1H-15N dipolar coupling is shown by solid lines. The grey shading
represents the simulated spectrum with a = 25◦, s11 = 39 ppm, s22
=
89 ppm and s33 = 152 ppm. Only the upper half of the symmetric spectrum
is shown. D: representation of the 15N chemical shift tensor of the peptide
bond and E: of diurea 4. In panels D and E the peptide bond and the
corresponding structure in the urea bond are shown in dark grey. In panel
E the areas in dark and light grey are shown as a continuous planar
structure although the possibility of small deviations has been detected in
more recent structural work.30 For both molecules (D and E) the s11 and
s33 main tensor elements are aligned within this plane.
1
spectrum consists of a single peak pair at 59 ppm and a H-15N
dipolar coupling of 5.2 kHz. Oligourea 3 labeled at position 8
leads to a similar spectrum with a peak at 83 ppm and a dipolar
splitting of 4.4 kHz (not shown).
In order to determine the oligourea alignments relative to the
membrane normal/Bo field that are in agreement with these two
experimental parameters, the analysis of topological constraints,
that has already proven highly successful with L-amino acid
polypeptides,32,46,47 was adapted by taking into consideration the
structure and tensor of the oligourea bond (Fig. 2E). To evaluate
which of the membrane alignments agree with the experimental
measurements, the backbone of the previously solved crystal
structure of a helical octaurea with benzyl side chains (CCDC
750017)30 was used as a template onto which was modeled the
solid-state NMR techniques on non-oriented samples (reviewed
in references 37,42).
Fig. 2C shows the separated local field spectrum of the powder
of the model compound 4. The compound was designed in such a
manner to represent all the chemical characteristics, and thereby
the chemical shift properties, of an urea bond (Scheme 1). This
two-dimensional spectrum correlates the 15N chemical shift and
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Fig. 3 15N separated local field spectrum of the labelled octaurea 2
reconstituted into uniaxially oriented 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine (POPC) bilayers. Whereas panel B shows the two-dimen-
sional spectrum, panel A presents the trace at 4800 Hz (dotted line in B).
The membrane normal is aligned parallel to the magnetic field direction
of the NMR spectrometer. The experimental powder pattern line shape of
compound 4 is shown in light grey for comparison (Fig. 2C).
amphipathic distribution of side chains of the antibacterial
oligourea 1. As a starting configuration, the helix long axis was
oriented parallel to the z-axis, which coincides with the membrane
normal and Bo, thereby following previous protocols.48,49 There-
after the oligomer was rotated first around the z-axis (thereby
changing the pitch angle) and then around the y-axis (thereby
changing the helical tilt angle) (Fig. 4A). For each alignment thus
obtained the corresponding chemical shift and dipolar coupling of
the Val-type residue at position 5 was calculated and compared to
the experimental spectrum. In Fig. 4B the orientations which agree
with the experimental chemical shift of the 15N labeled site within
residue 5 (59 1 ppm) are represented by the black solid lines and
alignments that agree with the dipolar splitting of 5.1 0.1 kHz
are shown by the dashed traces. When the two restriction plots are
combined with each other, only three intersections are obtained
(marked 1, 2 and 3 in Fig. 4B) and these are the only oligourea
alignments that are consistent with both 15N chemical shift and
the 15N-1H dipolar coupling of the residue 5 labeled site.
The configurations relative to the membrane surface corre-
sponding to these alignments are shown in Fig. 5 as molecular
models. Whereas alignment 1 (Fig. 5A) results in a perfect
placement of the hydrophilic and hydrophobic residues in the
corresponding environments, orientations 2 and 3 (Fig. 5B, C)
place an amino alkyl (lysine type) side chain (shown in blue)
in the membrane interior. Therefore the configuration 1 is likely
to be the energetically more probable orientation. At the same
time this in-planar configuration agrees with the physico-chemical
properties of this amphipathic molecule and provides qualitative
confirmation of the tensor characterization (Fig. 2).
Fig. 4 Analysis of the possible membrane alignments of octaurea 2 that
agree with the experimental solid-state NMR spectrum shown in Fig. 3. A:
The initial positioning of the helix axis of 2 (z-axis) parallel to the magnetic
field direction and the definition of the rotation axes. (B) Orientational
constraints of 2 that arise from the experimental 15N chemical shift (solid
line) and the 15N-1H dipolar coupling (dashed line).
compound (CCDC 750017)30 and that the label at position 8 is
very close to the terminus of the helical structure, a region of the
oligourea where structural differences are likely to occur when the
conformations in the crystal and the membrane are compared to
each other. This is also supported by previous findings showing
that the helix tends to fray at this end.26,30
In the case of a-peptides, it has been shown that due to unique
tensor properties, including the alignment of the unique tensor
element s33 almost parallel to the long axis of a-helical domains,
the 15N chemical shift provides a direct indicator of the membrane
alignment of these helices.38 As a consequence, chemical shifts
around 200 ppm (i.e. close to s33) are indicative of transmembrane
helices and values <100 pm (i.e. in the range of s11 and s22)
are characteristic of helices alignments close to parallel to the
surface.38,50
Therefore, we simulated the possible 15N chemical shift/1H-
15N dipolar coupling spectra that result from oligourea helix
alignments of different tilt angles. We focused on alignments that
are within 15◦ from perfect in-planar and transmembrane configu-
rations (Fig. 6). Notably, as with labeled peptide bonds for each tilt
angle a wheel-like distribution of chemical shift/dipolar couplings
is obtained, the exact resonance position being dependent on
the position of the 15N label and the rotational pitch angle (i.e.
rotation around z in Fig. 4A). As with a-peptides, transmembrane
alignments exhibit chemical shift values close to s33 whereas
When the NMR spectra of compound 3 (not shown) are
taken into consideration, the topological analysis of this site
agrees with an in-planar alignment but different tilt/pitch angular
pairs are obtained when compared to the labeled residue 5. It
should be noted however that the topological calculations use the
pdb-coordinates from the crystal structure of a related octaurea
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where these two parameters combined in most cases do not provide
information about the rotational pitch angle (Fig. 2D in 32).
Finally, it should be noted that cationic amphipathic antimi-
crobial peptides have inspired the design of the promising helical
peptidomimetics presented also in this work.23,51 Both classes of
compounds are characterized by amphipathic helical structures
that align parallel to the membrane surface, which suggests that
they also share a closely related mechanism for their antimicrobial
action.51,52
Conclusions
Here, for the first time, antimicrobial oligoureas23,24 carrying 15N
labeled backbone sites have been prepared and the 15N chemical
shift tensor of a related model compound has been determined.
Knowledge of the main tensor elements and their alignment within
the molecular coordinate system is a prerequisite to perform NMR
experiments in which the structure, dynamics, interactions and
topology of the oligourea foldamer can be investigated.
Using this information the association of an antimicrobial
amphipathic oligourea with oriented lipid membranes was studied
successfully and a surface alignment was obtained analogous
to observations made of amphipathic helical polypeptides such
as model sequences,53,54 magainins7,50,55 or other antimicrobial
peptides.56–59 Both topologies that agree with the experimental
constraints (Fig. 4 and 5) correspond to (approximately) in-
planar orientations, and are in agreement with previous studies
on amphipathic polypeptide helices.
Fig. 5 Illustration of the molecular alignments that are consistent with
15N chemical shift and 15N-1H dipolar coupling (cf. Fig. 3 and 4). Panel A
corresponds to a stereo view of alignment 1 in Fig. 4, panels B and C to
alignments 2 and 3, respectively. The amino alkyl (Lys type) side chains are
displayed in blue, the isopropyl (Val type) side chains in red and indole (Trp
type) in yellow. The hydrogen binding network is indicated by yellow lines.
The lipid membranes are symbolized by a box with a semi-transparent
surface.
Experimental
1. General
Thin layer chromatography (TLC) was performed on silica gel 60
F254 (Macherey-Nagel) with detection by UV light and charring
with 1% w/w ninhydrin in ethanol followed by heating. Flash
column chromatography was carried out on silica gel (0.063–
0.200 nm). Reverse phase (RP) HPLC analysis was performed on
˚
a Nucleosil C18 column (4.6 ¥ 150 mm, 100 A, 5 mm) (Macherey
Nagel) using a linear gradient of A (0.1% TFA in H2O) and B
(0.08% TFA in CH3CN) at a flow rate of 1.2 mL min-1 with
UV detection at 214 nm. Unless specified, the HPLC gradient
Fig. 6 Simulations of the polarization inversion spin exchange at the
magic angle (PISEMA) spectra for an oligourea helix in in-planar and
transmembrane configurations. The lines represent the possible peak
positions, the exact resonance being a function of the position of the
15N label relative to the rotational pitch angle. The black dot and line
correspond to perfect transmembrane and in-plane alignments, respec-
tively. The interrupted lines show the changes in the spectral properties
when the tilt angle deviates from these perfect alignments in steps of 5◦.
Tilt angle of the transmembrane conformation: solid black: 0◦, dotted
grey: 5◦, dashed grey: 10◦, dashed-dotted grey: 15◦. Tilt angle for the
in-planar configuration: solid black: 90◦, dotted grey: 85◦, dashed grey:
80◦, dashed-dotted grey: 75◦.
1
used is 30–100% B in 20 min. H NMR and 13C NMR spectra
were recorded using a Bruker Advance DPX-300. Mass spectra
were recorded using an LCQ Advantage Max (Thermo Fisher
Scientific) mass spectrometer coupled to a surveyor plus HPLC
(Thermo Fisher Scientific) system using a Nucleodur C18 (2 ¥
˚
100 mm, 100 A, 3 mm) column (Macherey Nagel) with a linear
gradient of A (0.1% formic acid in H2O) and B (CH3CN LC/MS
grade). When not specified, the gradient used is 30–100% B in
4
10 min. Boc-g -Val-OH was prepared as described.60 Activated
monomers with side chains of Val, Trp and Lys were prepared
in-plane orientations show 15N chemical shifts in the range of
s11 and s22. Fig. 6 shows that indeed the 15N chemical shift alone
allows one to deduce the membrane alignment of oligoureas in a
semi-quantitative manner. When the angular restraints from 15N
chemical shift and/or 1H-15N dipolar coupling are combined with
each other the two parameters exhibit good complementarity and
only a few well-defined angular pairs of tilt and rotational pitch
angles are obtained (Fig. 4). This is in contrast to a-peptides
using a previously described procedure.24,61
2. Experimental procedure for the synthesis of 15N labelled
activated building block with isopropyl (Val type) side chain
N-Boc protected [15N]-L-Valine (6). To (S)-15N-2-amino-3-
◦
methyl butanoic acid (1.0 g, 8. 46 mmol) at 0 C, a 4 N NaOH
(2.13 mL) solution was added followed by (Boc)2O (2.03 mL,
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9.31 mmol). The reaction was stirred overnight. The reaction
mixture was acidified with 1 N KHSO4 solution, diluted with
EtOAc and the organic phase was dried over the NaSO4 and
evaporated to give Boc-[15N]-Val-OH 6 (1.8 g, 98%) as white
solid.
3. Experimental procedure for the synthesis of 15N labelled model
diurea (4)
[15N]-(R)-tert-butyl (3-methyl-1-(3-methylureido)butan-2-yl)car-
bamate (9). To a stirred solution of HCl·NH2CH3 (0.058 g,
0.872 mmol) in CH3CN (4 mL) at 0◦ C, was added DIPEA (0.3 mL,
1.74 mmol), followed by 5 (0.2 g, 0.581 mmol) after 5 min. The
reaction mixture was diluted with EtOAc and the organic layer
was washed with a saturated NaHCO3 solution, 1 N KHSO4,
water and brine. The organic layer was dried over NaSO4 and
[15N]-2-tert-Butoxycarbonyl-L-valinol (7). To a cold solution of
6 (1.8 g, 8.25 mmol) in THF (20 mL) were successively added
benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hex-
afluorophosphate (BOP) (4.01 mL, 9.08 mmol) and diisopropy-
lethylamine (DIPEA) (1.68 mL, 9.9 mmol). After 15 min, sodium
borohydride (0.312 g, 8.25 mmol) was added and the reaction
mixture was stirred for 20 min. THF was evaporated from the
reaction mixture and diluted with EtOAc. The organic layer was
washed with 2 ¥ 20 mL saturated NaHCO3 solution, dried over
NaSO4, and evaporated. The residue was purified by column
chromatography (silica gel, 30% EtOAc in cyclohexane) to give
7 (1.42 g, 84%) as a white solid; 1H NMR (300 MHz, CDCl3) d =
4.83 (d, J = 7.9 Hz, 15NH), 4.53 (d, J = 7.9 Hz, 15NH), 3.73–3.54
(m, 2H), 3.49–3.32 (m, 1H), 2.23 (br s, OH), 1.90–1.73 (m, 1H),
1.44 (s, 9H), 0.94 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H).
1
evaporated to yield 9 (0.135 g, 89%) as a white solid; H NMR
(300 MHz, CDCl3) d = 5.14–5.01 (m, 1NH), 4.85 (d, J = 9.1 Hz,
15NH), 4.76–4.67 (m, 1NH), 4.55 (d, J = 9.1 Hz, 15NH), 3.51–3.39
(m, 1H), 3.35–3.12 (m, 2H), 2.76–2.73 (m, 3H), 1.81–1.69 (m, 1H),
1.42 (s, 9H), 0.94 (d, J = 7.0 Hz, 3H), 0.92 (d, J = 7.0 Hz, 3H).
[15N]-(R)-1-benzyl-3-(3-methyl-1-(3-methylureido)butan-2-yl)
urea (4). Compound 9 (0.095 g, 0.37 mmol) was treated with
trifluoroacetic acid (TFA) (1 mL) at 0◦ C for 30 min. Concentration
under reduced pressure and co-evaporation with cyclohexane left
a residue which was dried under high vacuum. To a stirred solution
of the resulting TFA salt in CH3CN (2 mL) was added DIPEA
(0.13 mL, 0.73 mmol), and after 10 min the reaction mixture
was treated with benzyl isocyanate (0.051 g, 0.38 mmol) and
stirred for 1 h. The reaction was concentrated and upon addition
of a saturated NaHCO3 solution, a precipitate formed. It was
collected and washed with saturated NaHCO3 solution, water,
1 N KHSO4, water and further washed with cyclohexane and
n-pentane to remove the non-polar impurities and finally dried
[15N]-(R)-tert-butyl(1-(1,3-dioxoisoindolin-2-yl)-3-methylbutan-
2-yl)carbamate (8). To a solution of 7 (1.42 g, 6.96 mmol) in
dry THF (15 mL) at 0 ◦C, were added phthalimide (1.12 g,
7.65 mmol), and triphenylphosphine (1.92 g, 7.3 mmol), followed
by diisopropyl azodicarboxylate (DIAD) (1.47 mL, 7.3 mmol).
After 2 h, THF was evaporated from the reaction mixture and
diluted with CH2Cl2 and cyclohexane. The yellow precipitate
which formed was filtered and the residue was washed with Et2O
and purified by column chromatography (silica gel, 15% EtOAc in
cyclohexane) to give 8 (2.1 g, 92%) as a white solid; 1H NMR (300
MHz, CDCl3) d = 7.87–7.78 (m, 2H), 7.71–7.65 (m, 2H), 4.67 (d,
J = 9.7 Hz, 15NH), 4.37 (d, J = 9.7 Hz, 15NH), 3.92–3.80 (m, 1H),
3.75–3.64 (m, 2H), 1.91–1.74 (m, 1H), 1.15 (s, 9H), 1.0 (d, J = 7.0
Hz, 3H), 0.98 (d, J = 7.0 Hz, 3H).
1
under high vacuum to give 4 (0.097 g, 91%) as a white solid; H
NMR (300 MHz, CD3OH) d = 7.34–7.18 (m, 5H), 6.44–6.34 (m,
NH), 5.94–5,84 (m, 2NH), 5.62 (d, J = 9.4 Hz, 1NH), 4.34–4.26
(m, 2H), 3.64–3.51 (m, 1H), 3.24–3.06 (m, 2H), 2.65 (d, J = 4.6
Hz, 3H), 1.83–1.65 (m, 1H), 0.93 (d, J = 7.0 Hz, 3H), 0.90 (d, J =
7.0 Hz, 3H).
4. Synthesis of 15N labelled urea oligomer 2 and 3
General Procedure. 15N labelled urea oligomer 2 and 3 were
synthesized using Boc chemistry on a multichannel synthesizer
with a semi-automatic mode on a 100 mmol scale starting from 4-
methylbenzhydrylamine (MBHA) resin (0.62 mmol g-1). The first
[15N]-(R)-O-succinimidyl-(2-(tert-butoxycarbonylamino)-3-me-
thylbutyl)carbamate (5). To a warm (~70◦ C) solution of 8 (2.1 g,
6.3 mmol) in MeOH (20 mL), was added hydrazine hydrate
(0.93 mL, 18.9 mmol) and the reaction mixture was stirred
overnight. The reaction mixture was filtered through a sintered
funnel, the MeOH evaporated and the residue was diluted with
CH2Cl2. The organic layer was washed with a saturated NaHCO3
solution and dried over Na2SO4. The solvent was evaporated under
reduced pressure to give the free amine (1.04 g) as a yellow syrup
which was used as such for the next step without purification. A
solution of the amine in CH2Cl2 (10 mL) was added to a solution
of N,N¢-disuccinimidyl carbonate (1.57 g, 6.14 mmol) in CH2Cl2
(20 mL) and the reaction mixture was stirred for 15 min. The
precipitate which formed was filtered and the residue was washed
with CH2Cl2 and 3 ¥ 20 mL of 1 N KHSO4. The organic layer was
dried over NaSO4 and evaporated to give 5 (1.4 g, 65%) as a white
solid; 1H NMR (300 MHz, CDCl3) d = 6.17–6.07 (m, 1NH), 4.58
(d, J = 9.1 Hz, 15NH), 3.61–3.49 (m, 1H), 3.44–3.19 (m, 2H), 2.80
(s, 4H), 1.84–1.71 (m, 1H), 1.44 (s, 9H), 0.96 (d, J = 7.0 Hz, 3H),
0.93 (d, J = 7.0 Hz, 3H).
4
coupling step was performed with a solution of Boc-protected g -
Val-OH (1.5 eq.) in DMF, with BOP (1.5 eq.), HOBt (1.5 eq.)
and DIPEA (5 eq.) and the suspension was mixed under periodic
nitrogen bubbling for 30 min. The coupling was performed twice
and completion was monitored by a Kaiser ninhydrin test. For
each following coupling step, a solution of the succinimidyl
carbamate building block with a side chain of either Val, Trp
or Lys(2-Cl-Z) (3 eq.) or 15N labelled succinimidyl carbamate
derivative 5 (3 eq.) and DIPEA (6 eq.) in DMF was added
on the resin, and the suspension was mixed under periodic
nitrogen bubbling for 120 min. A double coupling was performed
systematically. The Boc group was removed using TFA (5 and
10 min) under nitrogen bubbling. The resin was then filtered and
washed with DMF (5 ¥ 1 min). Acylation of the terminal amino
group of the oligourea was performed in DMF, in the presence of
isopropyl isocyanate (3 eq.) and DIPEA (5 eq.) and the suspension
was mixed under periodic nitrogen bubbling for 30 min. At the
end of the synthesis, the resin was washed with CH2Cl2, Et2O
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and dried under nitrogen. Side chain deprotection and cleavage
of the oligomer from the resin were performed simultaneously by
treatment with HF (containing 10% p-cresol as a scavenger) for
60 min at 0 ◦C. The crude oligomers were finally purified by RP-
HPLC (linear gradient, 20–80% B, 30 min) to a final purity ≥95%
and lyophilized.
7. Analysis and simulation of NMR spectra using Mathematica
and SIMPSON
Contour plots of the type in Fig. 4 were created using Mathematica
3.1 (Wolfram Research, Champaign, Il, USA). The tensor for the
labeled nitrogen was placed inside the coordinate system of the
pdb file. Rotations of the tensors first around the z-axis and then
around the y-axis were performed with the angles as variables in
an analytical form. The chemical shift or dipolar coupling were
calculated for a B-field in the z-direction as a function of the
rotation angles. Then the contour plot function of Mathematica
was used to show the orientations, where the chemical shift values
agree with the measured value.
The simulations of the 1D- and the PISEMA powder patterns
were performed using SIMPSON.65 In the case of the 1D simula-
tion a simple 90◦- acquisition program was used, whereas for the
simulation of the 2D-PISEMA spectra the full pulse-program was
used. Powder averaging was active by using “powder-crystal files”
(zcw986 for the PISEMA and zcw28656 for the 1D).
[15N] labelled oligourea 2. Synthesis on 100 mmol scale. Purity
of crude product >55% (C18 RP-HPLC). Yield after purification:
(35 mg, 31%); White powder; RP-HPLC tR 14.48 min (linear
gradient, 20–80% B, 30 min); MS (ES+) m/z 1388.3 [M + H]+.
[15N] labelled oligourea 3. Synthesis on 100 mmol scale. Purity
of crude product >70% (C18 RP-HPLC). Yield after purification:
(35 mg, 33%); White powder; RP-HPLC tR 14.21 min (linear
gradient, 20–80% B, 30 min); MS (ES+) m/z 1388.3 [M + H]+.
5. Preparation of oriented membranes for solid-state NMR
measurement
Uniaxially oriented samples for solid-state NMR spectroscopy
were prepared, as described in detail previously,45 by co-dissolving
10 mg of 15N labelled oligourea 2 or 3 and 200 mg of 1-palmitoyl-
2-oleoyl-sn-glycero-3-phosphocholine (POPC) in ~1 mL trifluo-
roethanol. Most of the organic solvent was slowly evaporated
with a stream of nitrogen and the remainder volume of about
200 mL was spread onto ultra-thin cover glasses (8 ¥ 11 mm from
Marienfeld, Lauda-Ko¨nigshofen, Germany). After drying on air,
the samples were exposed to high vacuum overnight to remove all
traces of the organic solvents. The membranes were equilibrated
at 93% relative humidity, before stacking the glass-plates on top
of each other. Finally, the stacks were stabilized with Teflon tape,
sealed with a polyethylene plastic wrapping45 and inserted into a
commercial flat coil62 e-free double-resonance probe head (Bruker
Biospin Rheinstetten, Germany) in such a manner that the glass
plate normal is oriented parallel to the magnetic field direction of
the NMR spectrometer (Bo).
Acknowledgements
We are grateful for the financial support from the Agence
National de la Recherche (TRANSPEP project ANR-07-PCVI-
0018), Vaincre la Mucoviscidose, the University of Strasbourg
(PPF RMN), the University of Bordeaux, the Re´gion Aquitaine,
the French Ministry of Research and the CNRS.
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