Biphasic Liquid Crystal and the Simultaneous Measurement of Isotropic and Anisotropic Parameters by Spatially Resolved NMR Spectroscopy

Article abstract of DOI:10.1002/chem.201702126

Residual dipolar couplings and other anisotropic NMR parameters are powerful tools for molecular structure elucidation when conventional techniques do not suffice. With current liquid crystalline preparations it is necessary to prepare two samples to extr

Full text of DOI:10.1002/chem.201702126

A Journal of
Accepted Article
Title: Biphasic Liquid Crystal and the Simultaneous Measurement of
Isotropic and Anisotropic Parameters by Spatially Resolved NMR
Spectroscopy
Authors: Malin Reller, Svenja Wesp, Martin R. M. Koos, Michael Knut
Reggelin, and Burkhard Luy
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To be cited as: Chem. Eur. J. 10.1002/chem.201702126
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Biphasic Liquid Crystal and the Simultaneous Measurement of
Isotropic and Anisotropic Parameters by Spatially Resolved NMR
Spectroscopy
Malin Reller,[a] Svenja Wesp,[b] Martin R.M. Koos,[a] Michael Reggelin,*[b] and Burkhard Luy*[a]
Abstract: Residual dipolar couplings and other anisotropic
NMR parameters are powerful tools for molecular structure
elucidation when conventional techniques do not suffice. With
current liquid crystalline preparations, it is necessary to prepare
two samples to extract isotropic and anisotropic data from
spectra and to derive the residual dipolar couplings. Here, we
present the preparation, measurement, and interpretation of a
novel biphasic liquid crystalline phase where a single sample
can be used to generate both isotropic and anisotropic data.
First, we introduce the synthesis of the chiral polymer leading
to the biphasic liquid crystal. Second, we present two
To obtain RDCs, the analyte must be measured in an
isotropic and anisotropic environment. This is usually done
by preparing two separate samples, one using
a
conventional isotropic solvent, and one using a so-called
alignment medium. The alignment medium provides a
weak anisotropic environment and consists either of a
3g, 5]
crosslinked polymer gel[3f,
or a lyotropic liquid
crystalline phase (LLC-phase).[1e, 1f, 4, 6]
Of particular importance are chiral LLC-phases due to their
potential ability to orient the enantiomers of a chiral
compound differently (DOE: differential order effect).[6]
approaches to measure spatially selective CLIP-HSQC spectra. Apart from the homopolypeptide based LLC-phases
From these spectra, we extracted the couplings, performed an
assignment of diastereotopic protons and achieved the
enantiomeric discrimination of isopinocampheol as a well-
studied test molecule.
derived from poly--benzyl-L/D-glutamate (PBLG/PBDG),
poly--ethyl-L-glutamate (PELG/PEDG), and poly--
carboxybenzoyl-L/D-lysin (PCBLL/PCBDL),[7] an increasing
number of alternatives has been developed. These
alternatives rely on polymers with helically chiral
backbones such as polyguanidines[8], polyisocyanides[9] or
polyacetylenes.[10]
Techniques to avoid the preparation of two separate
samples rely either on the application of a stretching
apparatus and measuring the sample at different stretching
strengths or on techniques, that orient the axis of alignment
at varying angles to the static magnetic field.[11] In addition,
the compressing apparatus by Gil et al.[12] represents an
effective way to measure both isotropic and anisotropic
parameters in a single sample.
Introduction
Residual dipolar couplings (RDCs) are important
anisotropic NMR parameters for the structure
determination of organic molecules.[1] They have been
3]
used in many conformational,[2] configurational[2l,
and
constitutional[4] studies and regularly led to success when
conventional methods failed.
Here, we introduce a stable biphasic lyotropic liquid
[a]
M.Sc. Malin Reller, Dr. Martin R.M. Koos, Prof. Dr. Burkhard Luy*
Institut für Organische Chemie and Institut für Biologische
Grenzflächen 4 – Magnetische Resonanz
Karlsruher Institut für Technologie (KIT)
Fritz-Haber-Weg 6,
76131 Karlsruhe (Germany)
E-mail: Burkhard.Luy@kit.edu
Homepage: http://www.ioc.kit.edu/luy
crystalline system
polyisocyanide with an anisotropic and an approximately
isotropic component in single sample. With
based
on
a
helically-chiral
a
a
correspondingly designed NMR pulse sequence the
spatially separated phases can be used to determine both
1
1TCH and JCH couplings simultaneously in different sub-
spectra, leading to the desired RDC-based splitting
contribution (1DCH
= −
1TCH 1JCH) in a single sample
[b]
M.Sc. Svenja Wesp, Prof. Dr. Michael Reggelin*
Organische Chemie
Technische Universität Darmstadt
Alarich-Weiss Str. 4
64287 Darmstadt (Germany)
E-mail: re@chemie.tu-darmstadt.de
Homepage: http://www.chemie.tu-darmstadt.de/reggelin/
measurement. We will first give a detailed description of the
synthesis and preparation of the biphasic LLC-phase,
before two different spatially resolved NMR pulse
sequences are introduced that allow the simultaneous and
separate measurement of isotropic and anisotropic
couplings. Experimental proof on both a biphasic isotropic
test sample used for initial pulse sequence implementation
and a biphasic LLC sample is given, before finally the
Supporting information for this article is given via a link at the end of
the document
measurement of RDCs from
a
single sample is
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demonstrated as well as its use for the assignment of
diastereotopic protons and the distinction of enantiomers.
displaying a doublet, split by the quadrupolar coupling
(∆Q(300 K) = 322 Hz) and the lower layer as being nearly
isotropic with a very small quadrupolar splitting (∆Q(300 K)
 1 Hz).
Moreover, spectra were recorded over an extended
temperature range revealing a temperature dependence of
the quadrupolar splitting in the anisotropic part (Figure 1).
Results and Discussion
Liquid Crystal Synthesis
The synthesis of monomer 1 was achieved in five steps by
modified procedures as published earlier[13] with an overall
yield of 47% (Scheme 1; for details see SI). As described
by Yashima, the Ni-initiated polymerisation yielded a yellow
polymer, poly-1, with a slight excess of a left-handed helical
structure as judged from the comparison of the chiroptical
data with published data.[13] Because of the high helix
inversion barriers of polyisocyanides[14] and the fact that the
polymerisation initially results in a non-uniform helical
conformation (kinetically controlled conformation: KCC), it
is necessary to anneal[14-15] the polymer for eight days in
toluene at 100°C to reach the thermodynamically stable
conformation (thermodynamically controlled conformation:
TCC). As already described by Yashima[13] this treatment
results in a predominantly left-handed helical structure with
Figure 1. 2H-Imaging spectra of a LLC-phase of poly-1 in CDCl3
(11.0% (m/m)) at different temperatures.
a
strong negative Cotton-Effect at  = 360 nm
(Θmax(360 nm) = −25511.47° cm2 dmol−1).
As Figure 2 shows, the changes in the quadrupolar splitting
are completely reversible. Obviously, the biphasic system
is rather stable towards temperature changes, which may
be due to the high molecular weight of the polymer, slowing
down diffusional processes considerably. Therefore, the
system’s thermal relaxation is slow on the timescale of the
NMR-measurements. This is
a beneficial behavior,
because it offers the opportunity to scale the alignment
strength in the anisotropic phase by exploiting its
temperature dependence. At the same time, the lower
quasi-isotropic phase remains nearly unaffected.
Scheme 1. Synthesis and polymerisation of the isocyanide monomer 1.
The polymer forms a lyotropic liquid crystalline phase in
CDCl3 with a critical concentration of approximately
13% (m/m) at room temperature.
The phase transition was monitored by measuring 2H-NMR
spectra of the deuterated solvent in the LLC phases
containing different amounts of polymer. The complete
replacement of the initial singlet by a doublet, split by the
quadrupolar coupling indicated the transition of the
isotropic to the anisotropic phase.
Preparation of Biphasic Liquid Crystal Sample
In the course of these experiments to determine the critical
concentration, we observed the formation of a biphasic
system when the concentration is slightly below this point
(11% (m/m)). To learn more about this system, we applied
the deuterium imaging experiment published by two of the
authors in 2013 (pulse sequence in Figure 3A).[16] This way,
we identified the upper layer as being anisotropic,
Figure 2. Temperature dependence of the solvent quadrupolar splitting
of the anisotropic part of a biphasic solution of poly-1 (11% (m/m) in
CDCl3). Squares symbolize data points during cooling; spheres
symbolize data points during the heating process.
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Indeed, varying the temperature from 250 K to 320 K was
possible without loss of the biphasic properties with the
quadrupolar splitting changing from 588 Hz down to 255 Hz
(Figure 2). To confirm the anticipated possibility to measure
both, isotropic and anisotropic data from the same sample,
we next studied the stability of the biphasic system towards
the presence of an analyte. To our delight, the addition of
26.5 mg (−)-isopinocampheol ((−)-IPC) does not change
the behavior of the system as described above. Therefore,
the measurement of scalar one bond couplings (1JCH) and
residual dipolar couplings (1DCH) from the same sample
should be possible by a combination of the already
mentioned imaging experiment with a CLIP-HSQC or more
elaborate derivatives thereof.[17]
spectrometer (G) by
푛
∙휋
1
훿 = 퐺∙훾
.
푧
(1)
∙푟
푁
For example, the spectrum in Figure 4B was measured with
the parameters set to n1 = 256, rz = 5 cm, γN = γD
41.07 rad s−1 T−1 and G = 4.295 mT cm−1.
=
Experiments for Sample-Phase Selective Measurements
For the measurement of scalar one bond (1JCH) and
residual dipolar couplings (1DCH), one of the established
methods is the measurement of CLIP-HSQC spectra and
the extraction of the desired couplings from the resulting
splittings.[17a] In contrast to e.g. a gel-based compression
device with isotropic and anisotropic contributions
distributed over the whole sample,[12] the liquid crystal in
our case forms a biphasic sample consisting of a strongly
oriented phase clearly separated from a nearly isotropic
phase. Normally, the whole sample volume is measured
which sometimes would lead to a sum of signals of the
isotropic and the anisotropic phase resulting in many cases
in spectra where coupling extraction might be impeded.
However, in the case of two separated phases the
application of spatially resolved or spatially selective NMR
techniques is possible.
Two corresponding approaches for the measurement of
spectra for the extraction of one-bond couplings have been
designed and will be introduced in the following. Both
experiments are based on the different, already mentioned
deuterium imaging experiments, which nowadays are
frequently used to control the homogeneity of alignment in
partially aligned samples.[16]
Figure 3. NMR experiments used in this article for spatial encoding
and/or spatial selection. Narrow black bars correspond to 90° pulses,
wide open bars to 180° pulses. The selective 180° degree pulses
indicated by wavy shapes and applied simultaneously with rectangular
gradients correspond to Q3 Gaussian cascade shaped 180° pulses[19]
[16]
(A) Pulse sequence for phase encoded 2H spectra (see Ref.
for
details). (B) Pulse sequence for spatially selective excited and phase
encoded 2H spectra. Gradient strengths were G1 = 11%, G2 = 18.75%,
G3 = 31%, G4 was incremented and the duration of the gradient pulse
was calculated via Equation 1. (C) Pulse sequence of a phase-encoded
CLIP-HSQC; phase cycling: Φ1 = 4(y), 4(−y); Φ2 = x; Φ3 = 2(x), 2(−x);
Φ4, = Φ5, = Φ6 = x, −x; Φrec = x, −x, x, −x, −x, x, −x, x. If 16 or more scans
are acquired Φ4 may also be cycled independently with Φ4 = 8(x), 8(−x).
Gradient strengths were G1 = 80%, G2 = 30%, G3 = 20.1%; G4 is
incremented to achieve spatial encoding in the indirect dimension. The
duration δ of G4 is calculated via Equation 1. Phase sensitive detection
for the indirect dimension was achieved by incrementing the phase of
Φ1 by 90° in States-TPPI fashion. (D) Spatially selectively excited CLIP-
HSQC with the corresponding phase cycling: Φ1 = x Φ2 = Φ6 = x, −x;
Φ3 = 2(x), 2(−x) Φ4 = Φ5= 4(x), 4(−x); Φrec = x, −x, x, −x, −x, x, −x, x. For
better performance of the sequence all 180° pulses on carbon were
Spatially Resolved CLIP-HSQC Experiments
The first technique uses phase encoding for spectral
resolution in a separate dimension and the basic principle
is best understood on the original spatially resolved 2H-
experiment.[16] Based on a well-known phase encoding
scheme, the strength of a pulsed field gradient of constant
length is incremented to add a space-dependent frequency
to the Larmor frequency of the signals (see Figure 3A).[18]
After Fourier transform in both dimensions, the data result
in a two dimensional spectrum where the direct dimension
shows the deuterium chemical shifts and the indirect is the
spatially resolved dimension, corresponding to the position
along the z-axis in the NMR-tube. The pulsed field gradient
length (δ) is calculated depending on the desired steps of
the gradient pulse in the indirect dimension (n1), the spatial
range (rz), the gyromagnetic ratio of the measured nucleus
(γN) and the maximum gradient strength of the
replaced by BIBOP- and BURBOP-pulses[20]. Δ = (2 ∙ 1JCH −1. Gradients
)
were applied with the following strengths: G1 = 11%, G2 = 11.36%,
G3 = 31%, G4 = 80%, G5 = 20.1% The selectivity of the Q3 shaped pulse
as well as the gradient strength of G2 was adjusted using Equations 2
and 3 in the main text. Phase-sensitive acquisition in the indirect
dimension was achieved using echo-antiecho encoding on gradient G1.
TPPI-progression was used on Φ1, Φ2 and Φrec to shift artifacts from the
center to the edges of the spectrum.
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Figure 4. (A) Scheme of the isotropic biphasic test sample. Upper part: 100 mM Sucrose in D2O, lower part: 100 mM (−)-menthol in CDCl3.
(B) Phase-encoded deuterium spectrum of the test sample. (C) 3D-view of the phase-encoded CLIP-HSQC. (D) Extracted positive projections of C
as explained in more detail in the main text; light blue: CLIP-HSQC of sucrose, dark blue: CLIP-HSQC of (−)-menthol.
For the measurement and the extraction of the residual
dipolar couplings of a biphasic sample, the imaging
D2O. All peaks are clearly separated and one-bond scalar
coupling extraction is straightforward (data not shown).
dimension is added to the pulse sequence of a CLIP-HSQC. The 3D spectrum is acquired as a single experiment,
To achieve that, the imaging gradient is applied during the
last delay of the CLIP-HSQC sequence (see Figure 3C). To
obtain the same result as in the deuterium imaging, the
gradient strength has to be reduced by a factor γH/γD = 6.5.
After Fourier transform, we obtain a three dimensional
spectrum. The direct dimension is the proton dimension;
the two indirect dimensions are the spatially resolved and
the carbon dimension. The resulting 3D spectrum is best
visualized by using an isotropic biphasic test sample,
consisting for example of (−)-menthol and sucrose as
solutes, and CDCl3 and D2O as corresponding solvents
(Figure 4A). We certainly recommend the use of such an
easily prepared sample for pulse sequence implementation
as it clearly allows a well-defined setup under which the
proposed experiments should work straightforwardly.
Chloroform has a higher density compared to deuterated
water and forms the lower phase in the sample. The 64
phase-increments producing the spatially resolved
spectrum in Figure 4B clearly identify the two phases by
their characteristic chemical shift values of 7.26 ppm
(CDCl3) and 4.72 ppm (D2O). In Figure 4C the
corresponding three-dimensional phase-encoded CLIP-
HSQC of the isotropic biphasic sample with 64 HSQC
planes comprising the different positions along the z-axis is
shown (Figure 4C). Clearly, the spatial resolution of the
spectrum allows complete separation of the two solutes in
the different phases, with (−)-menthol being entirely
dissolved in the chloroform phase and equally sucrose
being dissolved in the aqueous part. The light and dark blue
planes in the 3D plot of Figure 4C highlight the region of the
CLIP-HSQC spectra shown as contour plots in Figure 4D.
The dark blue spectrum shows the positive projection of the
planes 16 to 30 of the spatially encoded dimension
representing the CLIP-HSQC of (−)-menthol in CDCl3. The
light blue spectrum originates from the planes 34 to 51 and
corresponds to the CLIP-HSQC spectrum of sucrose in
making a comparison of the state of the two phases most
reliable as all changes to the sample are detected
simultaneously. In addition, all FIDs contribute to the
spectrum, leading to maximum signal-to-noise-ratio for the
heteronuclear correlation. A further positive point for the
method is the addition and possible selection of planes,
which can be performed after processing without prior
knowledge of the phases. The major drawback of the
method is certainly the measurement time needed for
recording all three dimensions. Depending on the required
resolution in the carbon and the spatially resolved
dimensions, the number of increments might be reduced
either classically or by the use of non-uniform sampling
techniques resulting in an overall reduction of valuable
measurement time. The use of less than 16 increments in
the phase-encoded dimension is not recommended, as the
resolution in our experience is too low for a good phase
separation. The experiment worked equally well for the
biphasic LLC sample (see the supporting information).
Spatially Selective CLIP-HSQC Experiments
The second approach is based on the simultaneous
application of a shaped pulse and a pulsed field gradient
for spatial selection of a certain volume at a predefined
position along the gradient-axis.[21] Like in the first approach,
the pulsed field gradient adds a spatially dependent
frequency to the Larmor frequency of the nuclei. By using
a shaped, bandselective pulse with a defined bandwidth,
only a disc of the sample is excited. Depending on the
offset offs of the shaped pulse the excited disc is shifted
from the upper part (e.g. positive offset of the band
selective pulse) to the lower part (negative offset of the
band selective pulse) of the sample. To determine the z
displacement, the position z0 of the center of the selected
excitation band is calculated by
푧0 = 2휋∙휈
.
(2)
offs
퐺∙훾
푁
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Figure 5. (A) Scheme of the biphasic LLC sample. Upper part: anisotropic (−)-IPC (11% polymer (m/m); Q = 347.7 Hz), lower part: quasi-isotropic
(−)-IPC (Q = 2.4 Hz). (B) Phase encoded deuterium spectrum acquired using the sequence shown in Figure 3A. (C) Spatially selectively excited
and phase-encoded deuterium spectra, acquired using the sequence shown in Figure 3B. Two spectra are color-coded and overlaid with selectively
excited regions indicated in A. The excited regions along the z-axis correspond to the excited regions in the spatially selectively excited CLIP-HSQC
spectra shown in D and E. (D) Spatially selectively excited CLIP-HSQC of the lower part of the sample (quasi-isotropic part). (E) Spatially selectively
excited CLIP-HSQC of the upper part of the sample (anisotropic part).
of alignment are clearly distinguishable by their deuterium
splittings.
The width of the disc (Δz) depends on the gradient strength
(G), the gyromagnetic ratio of the excited nuclei (γN) and
the bandwidth of the pulse (Δω). The relation of these is
described by
In the quasi-isotropic phase, the average deuterium
splitting is less than 2.4 Hz; in the anisotropic phase, it is
347.7 Hz at the measurement temperature of 298 K. The
homogeneity over the sample is good, but not perfect.
Slightly inhomogeneous alignment, causing variations of
the deuterium splitting in the upper part of up to 4 Hz, is
observed, while magnetic field inhomogeneities up to
0.025 ppm are visible in the lower part. We therefore
decided to focus on areas with particularly high
homogeneity. The corresponding selectively excited
regions have been visualized experimentally in Figure 5C
using the pulse program shown in Figure 3B and Equations
2 and 3. The spectra directly display which part of the
sample is excited by the slice selective excitation. The dark
blue spectrum corresponds to the quasi-isotropic phase,
the light blue spectrum results from the anisotropic phase.
The spectra shown in Figure 5D and 5E represent the
corresponding CLIP-HSQC spectra of the isotropic and the
anisotropic phase, respectively.
The biggest advantage of the spatially selective experiment
is certainly the reduced minimum measurement time
compared to the phase-encoded approach, as no third
dimension has to be recorded. On the other hand, the size
and position of the selectively excited sample slice has to
be determined beforehand. The second disadvantage, the
recording of isotropic and anisotropic phases in two
separate, consecutive experiments, can be avoided by
interleaved acquisition, alternatingly acquiring scans for the
two spectra. As in most relevant cases, however, the solute
and the biphasic LLC sample used were stable over time
and measurements were performed consecutively. Due to
the fast acquisition scheme, we acquired the two spectra
with the relatively high 13C digital resolution provided by 512
data points in an overall measurement time of 1 hour. As
can be seen in Figure 5, the resulting spectra are of high
quality and corresponding 1JCH and 1TCH-couplings could be
extracted with high precision.
∆푧 = ∆휔∙2휋
.
(3)
퐺∙훾
푁
To evaluate the bandwidth and placement of the shaped
180° pulses used in the spatial excitation sequence, we
developed the experiment presented in Figure 3B. It
connects the spatial excitation with the spatially resolved
second dimension. The spatial excitation is achieved by
excitation sculpting,[22] adding a gradient during the band
selective 180° pulses. Afterwards, the phase encoding
gradient is applied and incremented. After Fourier
transform,
a two-dimensional spectrum is obtained
originating from the excited disc only. Corresponding
spectra from the deuterated solvent are shown in Figure 5C
with two different offsets for the shaped pulses that are also
used for subsequent coupling measurements.
For the spatially selectively excited CLIP-HSQC, the
excitation sculpting sequence replaces the 90° excitation
pulse of the CLIP-HSQC (Figure 3D). The excitation
sculpting sequence was used to reduce artifacts coming
from chemical shift evolution during the application of the
band selective pulse. To reduce strong coupling artifacts
and dispersive antiphase contributions, a perfect echo was
added to the sequence. For better tolerance against B1-
field inhomogeneity and to compensate large carbon
chemical shift offsets, broadband carbon pulses, as e.g.
24]
BEBOP,[23] BIBOP,[23c,
BURBOP[25] pulses and the
BEBEtr and BUBI pulse sandwiches,[26] can be used. For
the experimental verification of the spatially selective
excitation we used the biphasic LLC-phase consisting of
poly-1 in CDCl3 as described above (see Figure 2 for a
photo of the sample). The quasi-isotropic phase composes
the lower part of the tube, whereas the anisotropic phase
2
resides on top. The H-imaging experiment of the sample
is given in Figure 5B: the two phases with different degree
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RDCs from a Single Measurement
as an estimate from the residual quadrupolar splitting of the
deuterium solvent spectra shows that these errors should
be approximately one percent of the overall couplings.
As a first test, we verified the validity of the data by
determining the relative configuration of IPC and assigned
the diastereotopic protons at C4 and C7. Starting from the
known structure, the four possible relative configurations of
(−)-isopinocampheol were constructed and energy
optimized using the program Chem3D and Avogadro. In
addition, the consequences of exchanging the already
mentioned diastereotopic protons, indicated e.g. by X4 for
the change of the proton assignment at carbon C4, was
investigated. The MSpin fit clearly resulted in the lowest
Cornilescu quality factor[28] of 0.062 for the correct structure
and proton assignment at C4 and C7. Only the exchange
of assignment of the geminal protons at C7 resulted in
similarly low quality factors as the large error in the RDC of
C7-H7 does not allow a clear distinction.
Our original aim has been to show the measurement of
residual dipolar couplings from a single liquid crystalline
sample.
Using the spatially selective excited CLIP-HSQC spectra
introduced in the previous section, corresponding RDCs of
(−)-isopinocampheol have been extracted using the
procedure described in Reference [3h]
Performing singular value decomposition fit with
.
a
MSpin,[27] we can see that the measured RDCs fit very well
to the known structure of (−)-isopinocampheol (Figure 6).
The data is summarized in the supporting information.
Next, the ability of the LLC-phase of poly-1 to distinguish
enantiomers was demonstrated. RDCs were determined
also for (+)-isopinocampheol with the spatially selective
experiment shown in Figure 3D (RDCs in Table 1). The
RDCs for (+)- and (−)-isopinocampheol have different
values for each C-H-pair as can be seen in Figure 6E.
Additionally, the correlation coefficient R = 0.6524
indicates a significant difference in RDCs for the two
enantiomers.
Table 1. Splitting contributions due to residual dipolar couplings for (+)
and (−)-isopinocampheol. The corresponding quadrupolar splittings of
the weakly oriented spectra were ∆Q = 340±0.1 Hz for (−)-IPC and ∆Q
= 345±0.6 Hz for (+)-IPC.
(−)-isopinocampheol
(+)-isopinocampheol
1DCH [Hz]
Error [Hz]
1DCH [Hz]
Error [Hz]
C1
9.8
0.6
0.7
0.3
0.4
0.4
0.4
−8.3
21.9
39.2
24.2
14.5
−18.7
0.4
1.6
0.3
0.3
0.3
0.4
C2
34.2
21.6
56.2
−19.7
−25.2
C3
C4, H4s
C4, H4a
C5
Figure 6. (A) Structure of (−)-IPC with atom numbering.
(B) Experimental vs calculated RDCs. (C) Calculated Cornilescu
-
C6
Q factors for the different configurations of isopinocampheol and
exchange of the diastereotopic protons in the prochiral CH2-groups. Ref:
corresponds to the structure with the correct assignment of the
diastereotopic protons; X4: The RDC values for the protons on C4 were
exchanged; X7: The RDC values for the protons of C7 were exchanged;
X4,7: The RDC values for the protons of C4 and C7 were exchanged.
(D) Structure of (+)-IPC. (E) Plot of experimental RDCs (1DCH_exp) of (−)-
IPC and (+)-IPC showing the low correlation of both, thus demonstrating
the enantiodiscrimination evoked by the liquid crystal poly-1.
C7, H7s
C7, H7a
C8
−4.7
−0.5
5.6
0.5
3.0
0.4
0.6
0.3
−11.3
−7.7
0.4
0.4
0.2
0.9
0.3
10.1
C9
−8.7
11.9
−13.1
−4.6
C10
Although the part of the biphasic liquid crystal taken as
isotropic is only approximately isotropic, the additional
systematic error introduced by the slight residual alignment
does not seem to interfere with the fitting. This is expected
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quality and in principle allows the extraction of couplings
Conclusions
even in cases where the sample would normally have to be
redone. It should be noted that the spatial encoding and
spatial selecting approaches can equally be combined with
HSQC-type experiments that show the 1TCH splitting in the
indirect dimension.
We introduced the synthesis and preparation of a stable
biphasic chiral lyotropic liquid crystalline system composed
of the polyisocyanide poly-1 in CDCl3. The denser phase is
quasi-isotropic, showing only a negligible degree of
alignment in a 600 MHz NMR spectrometer. The less
dense phase shows significant but weak alignment that can
be used for the extraction of residual dipolar couplings of
small molecules. The degree of alignment can be adjusted
reversibly within a wide temperature range. For the
simultaneous measurement of one-bond couplings, we
introduced two experiments based on the 2D CLIP-
HSQC.[12] One experiment uses imaging-like spatial phase
encoding with z-gradients, resulting in spatial resolution
along the NMR-tube in a third experimental dimension (see
e.g. [16] and references therein).
The second approach is based on spatially selective
excitation using the simultaneous application of a constant
pulsed field gradient and band-selective excitation
sculpting[22] in a number of 2D experiments. While the first
approach allows more flexibility in data extraction and
better overall sensitivity as all scans contribute to the signal,
the second approach is significantly faster (≤ 1 hour as
compared to the first approach, which involves acquisition
times of ≤ 8 hours). We finally demonstrated on (−)-
isopinocampheol as a well-studied test molecule that
residual dipolar couplings extracted from a single biphasic
liquid crystal are well-suited for the determination of relative
configuration and prochiral assignment. Additionally, we
show the very good enantiomeric discrimination of (−)-
isopinocampheol and (+)-isopinocampheol via the
measured RDCs.
Next to applications in the field of partially aligned samples,
the spatial resolution can also be used favourably in a
multitude of other contexts. Chemical reactions could be
monitored with spatial resolution at the surface of multi-
phase systems. Equally, monitoring time evolutions and
mixture developments over time in a single sample will now
1
not only be possible with H-1D experiments, which has
been shown impressively by John et al.,[3a] but also by
HSQC-type experiments with carbon chemical shift
information. Here, even
a
combination with fast
30]
experiments like the ASAP-type sequences[17c,
will
speed up the acquisition further. Therefore, we foresee a
wide range of applications with both the biphasic LLC
system as well as the proposed spatially selective and
spatial-encoded pulse sequences.
Experimental Section
The spectra shown in Figure 4B and 4C were recorded on
a 600 MHz Bruker Avance II spectrometer equipped with a
room temperature BBI probehead with z-gradient coils. At
maximum strength, the gradient unit delivers 53.5 G cm-1.
For demonstration and implementation purposes, we used
an isotropic biphasic sample consisting of 300 µl of
100 mM sucrose in D2O and 300 µl 100 mM of (−)-menthol
in CDCl3.
The spectrum presented in Figure 4B was recorded with
2048 points in the direct and 256 points in the indirect
dimension and one scan per increment leading to an overall
experimental time of 3 minutes. The spectral width for the
direct dimension was set to 20 ppm. The measured z-range
was set to 5 cm. The phase encoding pulsed field gradient
had a duration of 911.9 µs and the strength was set to
100%. The spectrum was processed with 2048 × 256
points. No apodization function was applied to either of the
dimensions. In the imaging dimension, the gradient is
increased with every increment starting from the largest
negative gradient value. As the zero with respect to
gradient strength is in the center of the acquired data in this
dimension, a circular shift of data is necessary for a phased
spectrum. In Bruker TopSpin, this is achieved by a large
first order phase correction of n1 ∙ 180°.
In Figure 4C the resulting three-dimensional CLIP-HSQC-
type spectrum is shown. The spectrum has been recorded
with 8192 points in the direct dimension, 64 points in the
phase-encoded dimension, and 128 points in the carbon
dimension, respectively. We recorded 16 dummy scans
and 2 scans per increment, which led to an overall
experimental time of 7 h 45 min. The spectral width of the
proton dimension was set to 10 ppm and for the carbon
dimension to 120 ppm. The delay Δ was set to the standard
The biphasic LLC system of poly-1 in CDCl3 appears to be
very well suited for the determination of the relative
configurations of small chiral molecules. Moreover, if the
very pronounced enantiodifferentiating effect found for
isopinocampheol can be observed for other chiral analytes
too, then this system may be a promising basis for future
efforts to solve the problem of the determination of absolute
configurations by solution state NMR.
The achievable alignment strength is well suited for the
measurement of RDCs on small molecules, and having
both isotropic and anisotropic phases in a single sample
has practical advantages as preparation efforts are
reduced. In addition, the scaling of alignment strength of up
to a factor two within the easily accessible temperature
range in the stable biphasic system is exceptional. With the
spatially resolved and spatially selective experiments
introduced here, the determination of scalar and residual
dipolar couplings is straightforward with no compromise in
spectral resolution or accuracy in determination.
Moreover, it should be possible to measure alignment
media and other phases with discontinuities or partial
inhomogeneity. In broken gels or only partly homogeneous
samples only the homogeneous regions of the sample
could be selected. This will generally enhance spectral
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10.1002/chem.201702126
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value (2 ∙ 145 Hz)−1, and the constant rz was set to 1.5 cm.
The phase encoding gradient was calculated according to
Equation 1 and had a duration of 116.7 µs with a
corresponding maximum gradient strength of 50%. In
Figure 4D the extracted planes of the three dimensional
spectrum are shown. The spectrum was processed with
8192 × 64 × 128 points; the proton and the carbon
dimension were apodized with a squared cosine function.
No apodization function was applied along the phase-
encoded dimension, but the large first order phase
correction of 11315.5° was used for the circular shift as
described above. The light blue spectrum shown in
Figure 4D is the positive sum projection of the planes 34 to
51 resulting in a CLIP-HSQC spectrum of sucrose, the dark
blue spectrum is the positive projection of the planes 16 to
30, resulting in the CLIP-HSQC spectrum of (−)-menthol.
The biphasic liquid crystalline sample was prepared as
described in the main text using 88.9 mg of polymer poly-1,
26.5 mg (172 µmol) (−)-IPC and 463 µl CDCl3. The other
sample with (+)-IPC was prepared with 78.7 mg of polymer
poly-1, 29.6 mg (191 µmol) (+)-IPC and 410 µl CDCl3. To
improve shim and lock performance, a DMSO capillary was
placed in both samples.
The spectra shown in Figure 5B-E were recorded on a
600 MHz Bruker Avance III spectrometer equipped with a
cryogenically cooled TCI probehead. The maximum
gradient strength of the z-gradient in this case was
50.7 G cm−1.
The phase-encoded deuterium spectrum shown in
Figure 5B was recorded with 2048 points in the direct
dimension and 128 points in the indirect dimension with 4
dummy scans and one scan per increment. The overall
experimental time was 6 minutes. The spectral width was
set to 15 ppm. The z-range was set to 3 cm. The length of
the phase encoding pulsed field gradient was 759.9 µs.
The maximum gradient strength was set to 95%. The
spectrum was processed with 2048 × 128 points and an
exponential apodization function applied to the direct
dimension with an additional line broadening of 0.3 Hz. The
applied to the indirect dimension. To the direct dimension
an exponential apodization function with an additional line
broadening of 0.3 Hz was applied. The first order phase
correction for the indirect dimension was set to 11520° for
the light blue spectrum and to −23401.6° for the dark blue
spectrum.
The spectra in Figure 5D and 5E were recorded with
8192 × 512 real points, 16 dummy scans and 4 scans per
increment, leading to an overall experimental time of 1 hour.
The spectral width was set to 6 ppm in the direct dimension
and 100 ppm in the carbon dimension. The shaped
bandselective 180° pulse (Q3 Gaussian cascade) with a
length of 395 µs and a corresponding amplitude of 8462 Hz
results in a bandwidth Δω of 8000 Hz. The offset of the
shaped pulse was set to −10000 Hz for the measurement
of the isotropic phase and to +10000 Hz for the
measurement of the anisotropic phase. The pulsed field
gradient during the selective 180° pulses had a strength of
11.36%. This corresponds to a width of the excited disc in
the sample of 0.33 cm at positions of +0.41 and −0.41 cm
from the center of the probehead gradient coil. The delay Δ
was set to (2 ∙ 145 Hz)−1. The 180° hard pulses were
replaced by broadband inversion and refocussing pulses
for carbon as reported previously for similar pulse
sequence types: BIBOP (37.5 kHz, 10 kHz, 600 μs, ±5%,
1200) inversion pulses[20a] and BURBOP-180 (37.5 kHz,
10 kHz, 1100 μs, ±5%, 2200) pulses[20b] were applied. The
two spectra were processed to 16384 × 1024 data points
and apodized with a squared cosine function in both
dimensions.
Acknowledgements
M.R.M.K. thanks the Fonds der Chemischen Industrie for a
PhD fellowship. B.L. thanks the HGF program BIFTM and
the Deutsche Forschungsgemeinschaft (instrumentation
facility Pro2NMR, LU 835/13-1, and SFB 1176 project Q2)
for financial support.
first
order
phase
correction
was
set
to
127.7 ∙ 180°= 22986°.
Keywords: anisotropic NMR • biphasic liquid crystal •
spatial NMR • RDCs • chirality
The light blue spectrum in Figure 5C was recorded with
2048 points in the direct and 64 points in the indirect
dimension and one scan per increment leading to an overall
experimental time of 3 minutes. The z-range was set to
3 cm. We used a Q3 Gaussian cascade shaped pulse[19]
with a bandwidth of 2000 Hz, a duration of 1580 µs and a
corresponding rf-amplitude of 2125 Hz. The offset of the
pulse was set to 2500 Hz. The pulsed field gradient that
was applied during the shaped pulse had a strength of
18.75%. The gradient before and after the shaped 180°
pulses was set to 11% and 31%. The dark blue spectrum
in Figure 5C was recorded with 2048 × 128 real points and
one scan per increment, leading to an overall experimental
time of 6 min. We used the same pulse parameters as in
the light blue spectrum besides the offset of the selective
180° pulse, which was set to −2500 Hz. The spectra were
processed without zero filling. No apodization function was
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10.1002/chem.201702126
Chemistry - A European Journal
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Two for One! A chiral polymer
forming a biphasic lyotropic liquid
crystalline sample is introduced,
which allows measurement of both
isotropic and anisotropic NMR data
in a single sample. A
characterization of the biphasic
system, spatially discriminative NMR
pulse sequences, and an example
for prochiral assignment as well as
enantiodiscrimination is given.
Malin Reller, Svenja Wesp, Martin R.
M. Koss, Michael Reggelin*,
Burkhard Luy*
Page No. – Page No.
Biphasic Liquid Crystal and the
Simultaneous Measurement of
Isotropic and Anisotropic
Parameters by Spatially Resolved
NMR Spectroscopy
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