Recovering Invisible Signals by Two-Field NMR Spectroscopy

Article abstract of DOI:10.1002/anie.201602978

Nuclear magnetic resonance (NMR) studies have benefited tremendously from the steady increase in the strength of magnetic fields. Spectacular improvements in both sensitivity and resolution have enabled the investigation of molecular systems of rising complexity. At very high fields, this progress may be jeopardized by line broadening, which is due to chemical exchange or relaxation by chemical shift anisotropy. In this work, we introduce a two-field NMR spectrometer designed for both excitation and observation of nuclear spins in two distinct magnetic fields in a single experiment. NMR spectra of several small molecules as well as a protein were obtained, with two dimensions acquired at vastly different magnetic fields. Resonances of exchanging groups that are broadened beyond recognition at high field can be sharpened to narrow peaks in the low-field dimension. Two-field NMR spectroscopy enables the measurement of chemical shifts at optimal fields and the study of molecular systems that suffer from internal dynamics, and opens new avenues for NMR spectroscopy at very high magnetic fields.

Full text of DOI:10.1002/anie.201602978

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201602978
German Edition: DOI: 10.1002/ange.201602978
NMR Spectroscopy
Recovering Invisible Signals by Two-Field NMR Spectroscopy
+
+
Samuel F. Cousin , Pavel Kade rˇ ꢀvek , Baptiste Haddou, Cyril Charlier, Thorsten Marquardsen,
Jean-Max Tyburn, Pierre-Alain Bovier, Frank Engelke, Werner Maas, Geoffrey Bodenhausen,
Philippe Pelupessy, and Fabien Ferrage*
[
2]
Abstract: Nuclear magnetic resonance (NMR) studies have
benefited tremendously from the steady increase in the strength
of magnetic fields. Spectacular improvements in both sensitiv-
ity and resolution have enabled the investigation of molecular
systems of rising complexity. At very high fields, this progress
may be jeopardized by line broadening, which is due to
chemical exchange or relaxation by chemical shift anisotropy.
In this work, we introduce a two-field NMR spectrometer
designed for both excitation and observation of nuclear spins
in two distinct magnetic fields in a single experiment. NMR
spectra of several small molecules as well as a protein were
obtained, with two dimensions acquired at vastly different
magnetic fields. Resonances of exchanging groups that are
broadened beyond recognition at high field can be sharpened
to narrow peaks in the low-field dimension. Two-field NMR
spectroscopy enables the measurement of chemical shifts at
optimal fields and the study of molecular systems that suffer
from internal dynamics, and opens new avenues for NMR
spectroscopy at very high magnetic fields.
and biological systems. Such line-broadening effects are
often exacerbated at high fields. Hence, chemists and
biologists face a dilemma: They must choose between good
sensitivity and resolution at high fields and more favorable
linewidths and relaxation rates at lower fields.
Magnetic-field-dependent properties can be probed by
a broad range of NMR techniques that explore two or more
fields in a single experiment, as in fast field-cycling relaxom-
[
3]
[4]
[5]
etry, zero-field NMR, dynamic nuclear polarization, and
[6]
other methods. Such systems have enabled the character-
ization of a host of properties of magnetic resonance
imaging, and particularly of contrast agents, materials,
[
7]
[8]
[9]
[
10]
as well as macromolecules. However, to the best of our
knowledge, no experiment has ever been proposed that allows
one to correlate chemical shifts at two different fields. The
requirement that both fields should be homogeneous has thus
far been a formidable obstacle. Herein, we introduce a two-
field NMR spectrometer and illustrate its potential for high-
resolution two-field NMR spectroscopy. The benefits of high
fields (sensitivity and resolution) and low fields (line narrow-
ing despite chemical exchange) can thus be combined. This
approach can yield high-resolution chemical-shift correlations
between high and low fields, for example, in heteronuclear
(e.g., H– C) spin systems in small molecules and proteins
alike. We show that signals of methyl groups that cannot be
detected at 14.1 T because of chemical exchange can be
recovered by two-field correlation spectroscopy. This work
shows how a wide range of molecules and biomolecules prone
to excessive broadening by chemical exchange can be studied
by NMR spectroscopy. Two-field NMR spectroscopy paves
T
he ability of nuclear magnetic resonance (NMR) to probe
the chemical and physical properties of matter at atomic
resolution renders it a universally applicable spectroscopic
method for molecular chemistry, material science, structural
biology, and medicine. The ubiquity of NMR spectroscopy has
greatly benefited from the enhanced resolution and sensitivity
offered by high magnetic fields and the introduction of two-
1
13
[1]
and multidimensional NMR experiments.
broadening, which is due to micro- to millisecond dynamics
known as “chemical exchange”), occurs in many chemical
Severe line
(
[
+]
[+]
[
*] S. F. Cousin, Dr. P. Kade ˇr ꢀvek, B. Haddou, Dr. C. Charlier,
Prof. Dr. G. Bodenhausen, Dr. P. Pelupessy, Prof. Dr. F. Ferrage
Department of Chemistry, Ecole Normale Supꢁrieure
PSL Research University
2
and
J.-M. Tyburn
Bruker BioSpin
34 rue de l’Industrie BP 10002, 67166 Wissembourg Cedex (France)
Dr. P.-A. Bovier
Bruker BioSpin AG
Industriestrasse 26, 8117 Fꢂllanden (Switzerland)
4 rue Lhomond, 75005 Paris (France)
Sorbonne Universitꢁs, UPMC Univ Paris 06, LBM
place Jussieu, 75005 Paris (France)
E-mail: Fabien.Ferrage@ens.fr
Dr. W. Maas
Bruker BioSpin
Billerica, MA 01821 (USA)
4
[
+]
[+]
S. F. Cousin, Dr. P. Kade ˇr ꢀvek, Dr. C. Charlier,
Prof. Dr. G. Bodenhausen, Dr. P. Pelupessy, Prof. Dr. F. Ferrage
CNRS, UMR 7203 LBM
Dr. C. Charlier
Present address: Laboratory of Chemical Physics
NIDDK, NIH, Bethesda, MD 20892 (USA)
7
5005 Paris (France)
+
[
] These authors contributed equally to this work.
B. Haddou
CNRS, UMR
8
Supporting information and the ORCID identification numbers for
the authors of this article can be found under http://dx.doi.org/10.
640 Pasteur, 75005 Paris (France)
1002/anie.201602978.
T. Marquardsen, Dr. F. Engelke
Bruker BioSpin GmbH
Silberstreifen 4, 76287 Rheinstetten (Germany)
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

Angewandte
Communications
Chemie
the way to a new generation of NMR spectrometers, where
multiple fields can be explored in the course of a single
experiment to achieve an optimal combination of sensitivity,
resolution, and spectral information.
Figure 1. The two-field NMR spectrometer. Ferroshims that are placed
LF
in the bore of the magnet provide a plateau at B0 =0.33 T. In addition
HF
to a classical probe positioned at the high-field center, B0 =14.1 T,
a second probe is introduced from the top of the bore and positioned
LF
at the plateau B0 . The sample is shuttled between the two magnetic
centers by a pneumatic system.
Figure 2. Two-field heteronuclear correlation spectrum. a) Pulse
sequence for 2F-HZQC. For details see the Supporting Information.
Narrow black and wide open rectangles represent 908 and 1808 pulses,
respectively. All pulses were applied along the x axis of the rotating
frame unless otherwise indicated. The phases are cycled as follows:
f =x, Àx; f =4{x}, 4{y}; f =2{x}, 2{Àx}, 2{y}, 2{Ày}; with the
Our two-field NMR spectrometer is based on a commer-
cial NMR spectrometer at 14.1 T (600 MHz for protons) with
a series of new accessories (Figure 1). First, a system was
designed to obtain a reasonably homogeneous field “plateau”
at 0.33 T in the stray field of the 14.1 T superconducting
magnet, with a homogeneity of about 10 ppm over the 2 cm
length of the sample (see the Supporting Information,
Figure S1). A patterned structure of soft magnetic materials,
1
2
3
receiver phase f =x, Àx, Àx, x. The phase f was incremented
acq
3
[
17]
following the TPPI scheme. The delays were t =1/(4J ) with
a
CH
JCH =125 Hz. d comprises the length of the gradient and the gradient
recovery delay, t =(t +n Dt )c, t =(t +n Dt )(1Àc),
1
0
1
1
2
0
1
1
[5a,11]
t =t (2cÀ1)+n Dt (cÀ0.5), t =t (1Àc), and t =t c. Dt is the time
3
0
1
1
4
0
5
0
1
known as ferroshims, was optimized.
A low-field triple
1
5
13
1
increment in the indirect dimension, and n is the index of the time
1
resonance probe ( N, C, H) equipped with a single-axis
gradient was designed and combined with radiofrequency (rf)
synthesizers and amplifiers operating at 1.42, 3.52, and
increment. c=(g /g +1)/2=0.6257. The gradients G , G , and G of
C
H
1
2
3
0
.9 ms duration were applied along the z axis with respective ampli-
À1
À1
tudes of 10 Gcm , 15 Gcm , and G =2(g /g )G GARP composite
pulse decoupling was applied on the C channel during detection
with w1GARP/2p=2.08 kHz. b) Natural-abundance { CLF, HHF} 2F-
HZQC spectrum of a sample containing 0.5m acetone and 0.5m tert-
butyl alcohol. Low-frequency one-dimensional C spectra (vertical
cross-sections) extracted from the 2F-HZQC spectrum at d( HHF)=
1
shearing transformation was used to obtain the CLF chemical shift in
the indirect dimension.
3
C
H
1.
[
3f]
[18]
13
1
4.1 MHz. Finally, a pneumatically driven sample shuttle
1
3
1
ensures fast transfer between the two magnetic centers. The
time required for transfer between the high and low fields is
about 100 ms.
1
3
1
High-resolution zero-quantum spectra can be obtained
for heteronuclear systems provided that the effective chem-
1
.146 ppm (tert-butyl alcohol) and d( H )=2.128 ppm (acetone). A
HF
13
[
12]
ical shift evolution is carefully tailored.
Herein, we
introduce a two-field heteronuclear zero-quantum correlation
1
3
1
experiment (2F-HZQC), where a zero-quantum C– H
1
3
1
coherence evolves under the difference of the offsets of
C
the H offset down by the ratio of the gyromagnetic ratios, g /
C
1
and H with respect to the relevant carrier frequencies at low
gH = 0.2514, in the indirect dimension. The evolution of the
coherence at low field is thus determined by a combination of
1
field, while single-quantum H coherences are detected at
1
3
1
eff
13
high field. The experiment is based on a simple sequence for
the
C
and
H
chemical shifts: wLF = w ( C)À
LF
[
13]
1
heteronuclear multiple quantum coherence (HMQC) and
(g /g )w ( H). This combination is immune to line broad-
C H LF
ening that is due to magnetic-field inhomogeneities.
The contributions of the latter to the linewidth in the
indirect dimension is significantly reduced by the tailored
zero-quantum sequence of Figure 2a (Figure 2b). In the
[
14]
related to methyl HZQC experiments with the following
modifications (Figure 2a): A two-spin order 2H C is gener-
z
z
ated before each transfer between the high- and low-field
1
3
1
centers, a zero-quantum C– H coherence is selected by
[15]
1
13
phase cycling at low field, and a 1808 pulse is used to scale
natural-abundance high-resolution [ H, C] 2F-HZQC spec-
2
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
These are not the final page numbers!

Angewandte
Communications
Chemie
trum of 0.5m tert-butanol and 0.5m acetone in D O (Fig-
2
ure 2b), the methyl signals of both butanol and acetone have
full linewidths at half height below 1 ppm. The single-
1
3
quantum C chemical shifts at low field can be obtained
after a shearing transformation of the spectrum, as the proton
chemical shifts do not depend on the magnetic field. The
spectrum has a similar appearance as a conventional high-
field heteronuclear single-quantum coherence (HSQC) cor-
[
16]
relation spectrum (Figure S3).
The generality of two-field NMR spectroscopy was
further demonstrated with a protein in solution (Figure 3).
Figure 4. a) The two exchanging methyl groups of the triazene com-
pound shown in orange and red swap their positions on a sub-
millisecond timescale at 318C. The non-exchanging methoxy group
shown in blue provides a reference signal. Top row (b–d): Conventional
high-field HSQC spectra with increasingly broad lines at 21, 26, and
318C. The lowest contour corresponds to 1/403 of the intensity of the
methoxy signal at 318C to show the weak signal of the exchanging
methyl groups. Bottom row (e–g): Two-field HZQC spectra with sharp
lines regardless of the temperature. The lowest contour corresponds
to 1/8 of the intensity of the methoxy signal at 318C.
1
13
Figure 3. 2D H, C correlation spectrum human ubiquitin labeled with
[
23]
1
3
1
Carlo (MCMC) methods
provides a k (218C) value of
ex
C H groups on the isoleucine d positions. An HSQC spectrum
3
1
À1
recorded at 14.1 T (red) was superimposed onto the two-field HZQC
spectrum recorded on the two-field spectrometer (blue) with the
sequence of Figure 2a (additional pulsed field gradients flank all 1808
pulses at both high and low field).
1700 s (Figure 4b and Figure S5). The system approaches
coalescence at 268C (Figure 4c). At 318C, the resonances of
the exchanging methyl groups are broadened beyond detec-
tion (Figure 4d). For comparison, the peak of the resonance
of the non-exchanging methoxy group is at least 1000 times
1
3
more intense (Figure S6). The decay rate of the C coherence
À1
A high-resolution 2F-HZQC spectrum was obtained for
was estimated to be about 1000 s at 14.1 T for an estimated
À1
a sample of human ubiquitin (1.5 mm, pH 4.5) with specific
k (318C) value of 3400 s . At 0.33 T, the exchange contri-
ex
1
3
1
À1
isotope labeling on the d1 C H methyl groups of its seven
bution is predicted to be dramatically reduced to 1.9 s ,
3
2
12
[19]
isoleucine residues with a H, C background. The narrow
linewidths in the indirect dimension are likely due to
relaxation interference effects, in the manner of transverse
corresponding to a line narrowing by a factor of 500.
Remarkably, the signal that is due to the exchanging methyl
groups is readily observable in the two-field correlation
spectra and barely affected over the whole temperature range
(Figure 4e–g). These spectra were recorded with a 2F-HZQC
sequence (see Figure S2).
Very high fields above 1 GHz will soon become available,
but their possible drawbacks are hard to predict. Chemical
exchange can lead to severe line broadening, and carbon-13
relaxation by chemical shift anisotropy (CSA) can become
a major limitation. Many multidimensional NMR experi-
ments include delays for evolution under chemical shifts that
may not be optimal at one and the same field for all nuclei. In
some cases, the best fields may be extremely high (e.g., to
reduce the effects of second-order quadrupolar couplings).
For single-quantum evolution of carbonyl C nuclei in large
proteins, the optimal magnetic field lies below 14 T. The
transverse C relaxation rates in proteins have been pre-
dicted to be minimal between about 2 and 5 T. On the other
hand, the optimal field may be close to 1 GHz for TROSY of
amide N– H pairs. Two-field NMR spectroscopy offers
the possibility of manipulating spins such as N, C, or P at
a field that is most appropriate for them, and detecting signals
of other nuclei such as H at a higher field where the best
[
20]
optimized relaxation spectroscopy (TROSY). The sensitiv-
ity is sufficient to obtain such a spectrum in nine hours. Weak
artifacts appear 2.7 ppm away from the most intense peaks in
the indirect dimension. The details of this spectrum will be the
subject of a forthcoming study.
Most chemical and biological systems suffer from
exchange broadening. In many organic molecules and bio-
molecules, this effect can be subtle and merely gives rise to
slight perturbations in the NMR spectra. In other instances,
for example, when labile protons undergo exchange with
[
21]
a solvent, or in so-called molten globules,
chemical
[24]
exchange may lead to dramatic spectral broadening, which
can prevent any NMR measurement. The line broadening
that is due to R strongly depends on the magnetic field B , so
1
3
[
25]
ex
0
1
3
that reducing the field may have dramatic effects on the
linewidths.
[
26]
Here, we combined the excellent sensitivity and resolu-
tion achieved at high fields with the dramatic reduction of
exchange-induced line broadening at low fields. At 14.1 T, the
1
5
1
[27]
1
5
13
31
1
3
C signals of the two methyl groups of the dimethyl triazene
[22]
1
compound
shown in Figure 4a are in slow exchange at
2
18C. Line-shape analysis based on Markov chain Monte
resolution and sensitivity can be achieved. The effects of field
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

Angewandte
Communications
Chemie
inhomogeneities can be greatly reduced by exploiting zero-
quantum coherences. The superb resolution and sensitivity
that can be achieved at high fields can thus be combined with
the favorable properties offered by low fields. Most high-field
NMR systems may be turned into two-field spectrometers
with a plateau at 0.33 T. The design of the ferroshims requires
a simple optimization for each type of superconducting
magnet. Two-field systems with a low-field center above
[8] a) S. Laurent, L. V. Elst, R. N. Muller, Contrast Media Mol.
Imaging 2006, 1, 128 – 137; b) S. Aime, M. Botta, S. G. Crich,
G. B. Giovenzana, R. Pagliarin, M. Piccinini, M. Sisti, E.
Terreno, J. Biol. Inorg. Chem. 1997, 2, 470 – 479; c) D. H.
Powell, O. M. Ni Dhubhghaill, D. Pubanz, L. Helm, Y. S.
Lebedev, W. Schlaepfer, A. E. Merbach, J. Am. Chem. Soc.
1996, 118, 9333 – 9346; d) S. R. Banerjee, E. J. Ngen, M. W. Rotz,
S. Kakkad, A. Lisok, R. Pracitto, M. Pullambhatla, Z. P. Chen, T.
Shah, D. Artemov, T. J. Meade, Z. M. Bhujwalla, M. G. Pomper,
Angew. Chem. Int. Ed. 2015, 54, 10778 – 10782; Angew. Chem.
2015, 127, 10928 – 10932; e) Y. Gossuin, Z. Serhan, L. Sandiford,
D. Henrard, T. Marquardsen, R. T. M. Rosales, D. Sakellariou, F.
Ferrage, Appl. Magn. Reson. 2016, 47, 237 – 246.
0
.5 T should rely on other technologies that are currently
[
28]
available to obtain a second magnetic-field plateau. This
study marks the beginning of a new generation of multiple-
field NMR experiments, and opens the way to the character-
ization of a wide variety of systems.
[
9] a) J. P. Korb, M. Whaley-Hodges, R. G. Bryant, Phys. Rev. E
1997, 56, 1934 – 1945; b) S. Stapf, R. Kimmich, R. O. Seitter,
Phys. Rev. Lett. 1995, 75, 2855 – 2858.
[
10] a) R. Kimmich, N. Fatkullin, NMR 3D Analysis Photopolyme-
rization, Vol. 170, 2004, pp. 1 – 113; b) E. Persson, B. Halle, J.
Am. Chem. Soc. 2008, 130, 1774 – 1787; c) M. F. Roberts, Q. Z.
Cui, C. J. Turner, D. A. Case, A. G. Redfield, Biochemistry 2004,
Acknowledgements
We thank Kaushik Dutta (New York Structural Biology
Center) for the ubiquitin plasmid as well as Lei Bruschweiler-
Li and Rafael Brꢀschweiler (the Ohio State University) for
the method for ubiquitin purification. This work was funded
by the European Research Council (ERC) under the Euro-
pean Community Seventh Framework Program (FP7/2007–
4
3, 3637 – 3650; d) C. Luchinat, G. Parigi, J. Am. Chem. Soc.
2007, 129, 1055 – 1064; e) G. Diakova, Y. A. Goddard, J.-P. Korb,
R. G. Bryant, Biophys. J. 2010, 98, 138 – 146; f) M. W. Clarkson,
M. Lei, E. Z. Eisenmesser, W. Labeikovsky, A. Redfield, D.
Kern, J. Biomol. NMR 2009, 45, 217 – 225.
[
[
[
11] P. A. Bovier, R. Schauwecker, D. Eckert, US2010171495 (A1),
2
010.
12] S. L. Duce, L. D. Hall, T. J. Norwood, J. Magn. Reson. 1990, 89,
73 – 286.
13] L. Mueller, J. Am. Chem. Soc. 1979, 101, 4481 – 4484.
2
013), ERC Grant Agreement 279519 (2F4BIODYN).
2
Keywords: chemical exchange · high-field NMR spectroscopy ·
kinetics · nuclear magnetic resonance ·
low-field NMR spectroscopy
[14] V. Tugarinov, R. Sprangers, L. E. Kay, J. Am. Chem. Soc. 2004,
126, 4921 – 4925.
[
[
[
[
[
[
[
15] G. Bodenhausen, H. Kogler, R. R. Ernst, J. Magn. Reson. 1984,
8, 370 – 388.
16] G. Bodenhausen, D. J. Ruben, Chem. Phys. Lett. 1980, 69, 185 –
88.
17] D. Marion, K. Wꢀthrich, Biochem. Biophys. Res. Commun. 1983,
13, 967 – 974.
18] A. J. Shaka, P. B. Barker, R. Freeman, J. Magn. Reson. 1985, 64,
47 – 552.
19] V. Tugarinov, V. Kanelis, L. E. Kay, Nat. Protoc. 2006, 1, 749 –
54.
20] V. Tugarinov, P. M. Hwang, J. E. Ollerenshaw, L. E. Kay, J. Am.
Chem. Soc. 2003, 125, 10420 – 10428.
21] a) B. A. Schulman, P. S. Kim, C. M. Dobson, C. Redfield, Nat.
Struct. Biol. 1997, 4, 630 – 634; b) P. A. Jennings, P. E. Wright,
Science 1993, 262, 892 – 896.
5
1
[
1] R. R. Ernst, G. Bodenhausen, A. Wokaun, Principles of
Magnetic Resonance in One and Two Dimensions, Clarendon
Press, Oxford, 1987.
1
5
[
[
2] A. G. Palmer, Chem. Rev. 2004, 104, 3623 – 3640.
3] a) R. Kimmich, E. Anoardo, Prog. Nucl. Magn. Reson. Spec-
trosc. 2004, 44, 257 – 320; b) S. Wagner, T. R. J. Dinesen, T.
Rayner, R. G. Bryant, J. Magn. Reson. 1999, 140, 172 – 178;
c) A. N. Pravdivtsev, A. V. Yurkovskaya, H.-M. Vieth, K. L.
Ivanov, J. Chem. Phys. 2014, 141, 155101; d) A. G. Redfield, J.
Biomol. NMR 2012, 52, 159 – 177; e) C. Y. Chou, M. L. Chu, C. F.
Chang, T. H. Huang, J. Magn. Reson. 2012, 214, 302 – 308; f) C.
Charlier, S. N. Khan, T. Marquardsen, P. Pelupessy, V. Reiss, D.
Sakellariou, G. Bodenhausen, F. Engelke, F. Ferrage, J. Am.
Chem. Soc. 2013, 135, 18665 – 18672; g) A. S. Kiryutin, A. N.
Pravdivtsev, K. L. Ivanov, Y. A. Grishin, H.-M. Vieth, A. V.
Yurkovskaya, J. Magn. Reson. 2016, 263, 79 – 91.
7
[
[
22] C. S. Rondestvedt, S. J. Davis, J. Org. Chem. 1957, 22, 200 – 203.
23] H. Haario, M. Laine, A. Mira, E. Saksman, Stat. Comput. 2006,
16, 339 – 354.
[
[
[
[
[
24] Z. H. Gan, P. Gorꢁkov, T. A. Cross, A. Samoson, D. Massiot, J.
Am. Chem. Soc. 2002, 124, 5634 – 5635.
25] V. Tugarinov, R. Muhandiram, A. Ayed, L. E. Kay, J. Am. Chem.
Soc. 2002, 124, 10025 – 10035.
[
4] D. P. Weitekamp, A. Bielecki, D. Zax, K. Zilm, A. Pines, Phys.
Rev. Lett. 1983, 50, 1807 – 1810.
26] C. Charlier, S. F. Cousin, F. Ferrage, Chem. Soc. Rev. 2016, 45,
[
5] a) M. Reese, M. T. Turke, I. Tkach, G. Parigi, C. Luchinat, T.
Marquardsen, A. Tavernier, P. Hofer, F. Engelke, C. Griesinger,
M. Bennati, J. Am. Chem. Soc. 2009, 131, 15086 – 15087; b) J.
Leggett, R. Hunter, J. Granwehr, R. Panek, A. J. Perez-Linde,
A. J. Horsewill, J. McMaster, G. Smith, W. Koeckenberger, Phys.
Chem. Chem. Phys. 2010, 12, 5883 – 5892; c) A. Bornet, S. Jannin,
G. Bodenhausen, Chem. Phys. Lett. 2011, 512, 151 – 154.
6] R. Hecht, A. G. Redfield, Phys. Rev. 1963, 132, 972 – 977.
7] S. H. Koenig, R. D. Brown, Prog. Nucl. Magn. Reson. Spectrosc.
2410 – 2422.
27] K. Pervushin, R. Riek, G. Wider, K. Wꢀthrich, Proc. Natl. Acad.
Sci. USA 1997, 94, 12366 – 12371.
28] C.-Y. Chou, F. Ferrage, G. Aubert, D. Sakellariou, Sci. Rep. 2015,
5, 12200.
[
[
Received: March 25, 2016
1990, 22, 487 – 567.
Published online: && &&, &&&&
4
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
NMR Spectroscopy
S. F. Cousin, P. Kade rˇ ꢁvek, B. Haddou,
C. Charlier, T. Marquardsen, J.-M. Tyburn,
P.-A. Bovier, F. Engelke, W. Maas,
G. Bodenhausen, P. Pelupessy,
F. Ferrage*
&&&& — &&&&
A two-field NMR spectrometer was
designed to excite and observe nuclear
spins in two distinct magnetic fields in
a single experiment. This approach ena-
bles the detection of narrow resonances
that are broadened beyond recognition at
high fields because of internal dynamics.
Recovering Invisible Signals by Two-Field
NMR Spectroscopy
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!