Highly efficient, water-soluble polarizing agents for dynamic nuclear polarization at high frequency

Article abstract of DOI:10.1002/anie.201304657

Well polarized: Two new polarizing agents PyPol and AMUPol soluble in glycerol/water mixtures are used for dynamic nuclear polarization (DNP) NMR spectroscopy. The enhancement factors (ε) are about 3.5 to 4 times larger than for the established agent TOTA

Full text of DOI:10.1002/anie.201304657

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Angewandte
Communications
DOI: 10.1002/anie.201304657
NMR Spectroscopy
Highly Efficient, Water-Soluble Polarizing Agents for Dynamic
Nuclear Polarization at High Frequency**
Claire Sauvꢀe, Melanie Rosay, Gilles Casano, Fabien Aussenac, Ralph T. Weber, Olivier Ouari,*
and Paul Tordo*
Dynamic nuclear polarization coupled to solid-state NMR
spectroscopy (DNP/ssNMR) is emerging as a very powerful
technique to overcome the limitations resulting from the
intrinsic poor sensitivity of NMR spectroscopy. The DNP
method exploits the microwave-driven transfer of polariza-
tion from the electron spin of a paramagnetic center (polar-
izing agent) to surrounding nuclei and has been demonstrated
to produce uniformly polarized samples. First discovered in
the 1950s with low magnetic fields,[1] high-field DNP NMR
experiments are now possible with the introduction of
gyrotron sources[2] which are capable of delivering high-
power high-frequency microwaves (MW) up to 527 GHz and
NMR probes that operate at approximately 100 K. DNP
experiments have also been performed at up to 263 GHz in
the 4–80 K temperature range with lower power sources.[3]
DNP experiments that achieve even a fraction of the
utions doped with high concentrations of mono-radical nitro-
xide species (40 to 100 mm), such as TEMPO.[6] In 2004, Hu[7]
et al. using a series of bis-TEMPO-n-ethyleneglycol (BTnE)
dinitroxides demonstrated that biradical polarizing agents
yielded significant improvements in the CE polarization
efficiency. Then, Song[8] et al. introduced the water-soluble
biradical TOTAPOL (Scheme 1) which has good solubility
1
theoretical maximum sensitivity enhancement (658 for H,
2617 for 13C) can allow the low sensitivity of NMR
spectroscopy to be overcome and to achieve breakthroughs
in the use of the technique to investigate previously inacces-
sible systems. In this respect, in the past few years DNP-
enhanced solid-state NMR spectroscopy under magic-angle
spinning (MAS) conditions has made great progress, and
DNP signal enhancement factors of up to approximately 50
and up to around 100 have been reported (at 9.4 T, 263 GHz,
100 K) for biological solids[4] and hybrid or inorganic materi-
als,[5] respectively.
Scheme 1. Structures of bTUrea, PyPol, AMUPol, bTbK, bCTbK, and
TOTAPOL.
and stability in aqueous media containing glycerol and salts at
concentrations typically found in protein solutions. Outstand-
ing results were obtained with TOTAPOL, which is today the
most commonly employed exogenous polarizing agent in
DNP/ssNMR applications to biological systems.[4] TOTAPOL
was shown, however, to have a relatively flexible structure[9]
which is detrimental to the frequency-matching conditions
required for an efficient CE mechanism, and the DNP
enhancements observed with TOTAPOL (e(1H) < 80 on
standard samples at 9.4 T, 263 GHz EPR, 100 K) remain far
from the theoretical limit, and decrease rapidly when T>
Initial high-field DNP experiments were performed with
cross-effect (CE) polarization transfer mechanism and sol-
[*] C. Sauvꢀe, Dr. G. Casano, Dr. O. Ouari, Prof. P. Tordo
Aix-Marseille Universitꢀ, CNRS, ICR UMR 7273
13397 Marseille cedex 20 (France)
E-mail: olivier.ouari@univ-amu.fr
paul.tordo@univ-amu.fr
Homepage: http://sites.univ-provence.fr/srep/
Dr. M. Rosay, Dr. R. T. Weber
Bruker BioSpin Corporation
15 Fortune Drive, Billerica, MA 01821 (USA)
100 K.[10] In addition, since CE DNP enhancements scale
relatively to the applied magnetic field as approximately B0
À1
,
Dr. F. Aussenac
Bruker BioSpin S.A.S.
34 rue de l’industrie, 67166 Wissembourg (France)
TOTAPOL is expected to yield even lower signal enhance-
ments[11] on high-field DNP/SSNMR spectrometers operating
at 14.1 (w0S = 395 GHz) or 18.8 T (w0S = 527 GHz), which are
required for optimal resolution.
[**] The research reported herein is based upon work supported by Aix-
Marseille Universitꢀ, CNRS and Bruker BioSpin. P. Tordo and O.
Ouari thank Prof. L. Emsley (ENS Lyon), Prof. Copꢀret (ETH Zurich)
and their co-workers for valuable discussions. We also thank Dr. W.
Maas (Bruker BioSpin) for his continued support and interest
towards the development of novel polarizing agents.
In 2009, in collaboration with Griffinꢀs group (MIT), we
achieved another step forward with the introduction of
bTbK[12] (Scheme 1), a biradical in which the two TEMPO
moieties are linked by a rigid tether inducing a quasi-
orthogonal relative orientation of the TEMPOs g tensors.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201304657.
1
For a glassy solution of bTbK, the H frequency-matching
10858
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Table 1: Values of e (1H), polarization build-up time (TDNP), T1 (1H), T1e
and T2e for bTUrea, TOTAPOL, PyPol and AMUPol.
condition for the CE mechanism is closely approached, and it
was demonstrated that bTbK provides DNP enhancement
factors 1.4 times higher than TOTAPOL under similar
conditions. Recently, in collaboration with Emsleyꢀs group
(ENS Lyon) we developed a series of bTbK analogues with
increased molecular weights and longer relaxation times T1e
and T2e, and we showed[5e] that in organic solvent glasses the
DNP enhancements obtained with bCTbK are 3 to 4 times
higher than those obtained with bTbK. However, bTbK and
bCTbK are insoluble in glycerol/water mixtures and only
sparingly soluble in DMSO/water, hence their use is almost
limited to DNP/ssNMR applications carried out in organic
solvents.[5d,e,i]
Compound e(1H)[a]
TDNP
[s]
T1(1H) T1e
[s]
T2e
[ms][e] [ms][e] [gmolÀ1
]
MW[d]
[b]
bTUrea[c]
TOTAPOL
62Æ3
70Æ3
(30Æ2)
207Æ7
16.5
5.6
(7.7)
5.7
17.3
7.1
(9.6)
6.5
(9.5)
3.3
(5.1)
306
342
1.8
3.2
368
399
PyPol
470
460
12
10
536
726
(128Æ9) (8.7)
AMUPol
235Æ5
3.5
(128Æ8) (4.9)
[a] DNP measurements in [D8]glycerol/D2O/H2O (60/30/10 volume
ratio) at 263 and 395 (in parenthesis) GHz (400 and 600 MHz 1H NMR
respectively), 97 K, 8 kHz MAS, 10 mm biradical concentration (except
for bTUrea, less than 3 mm) and 0.25m U-13C-15N proline. e(1H)
measured by 13C CP-MAS. [b] DNP polarization build-up time measured
with standard saturation recovery experiment with microwave irradi-
ation. [c] bTUrea sample was prepared without proline and DNP
enhancement measured on glycerol 13C natural abundance signal.
[d] Molecular weight. [e] At 9 GHz EPR frequency and 97 K with 0.1 mm
biradical concentration.
Two bTbK derivatives (bTbtk[13] and bTbtk-py[14]) show-
ing better water solubility and retaining the desirable
orientation of bTbK have been reported. However, the
increase in the DNP signal enhancements observed with
these polarizing agents compared to TOTAPOL remains
relatively modest (ebTbtk/eTOTAPOL ꢀ 1.1 and ebTbtk-py/eTOTAPOL
ꢀ 1.2). In the course of our search of new CE DNP polarizing
agents[5e,12,13,15] we investigated the influence of various
parameters (e–e dipolar interaction, g tensor orientations,
rigidity of the molecule, electron spin relaxation times) on the
DNP efficiency. Among the different dinitroxides that we
examined, bTUrea (Scheme 1) retained our attention.
bTUrea, first described in 1965,[16] has been tested as DNP
polarizing agent on a model system by Hu[9] et al. in DMSO/
water 60/40. It was described to be three times more efficient
than TEMPO but less efficient than TOTAPOL, poor
solubility was noted, and its use in DNP/ssNMR applications
has not been reported. We reexamined the performance of
bTUrea and TOTAPOL in DMSO/water and glycerol/water
solutions and noticed that at the same concentration bTUrea
was more efficient than TOTAPOL. We then synthesized
a series of bTUrea derivatives with the goals of improving
solubility in aqueous solution and also further enhancing
DNP efficiency. We report herein on two new biradical
polarizing agents PyPol and AMUPol (Scheme 1) which show
high water solubility and greatly enhanced DNP efficiency.
The general method to prepare PyPol and AMUPol is
shown in Scheme 2, and details on the synthesis are given in
the Supporting Information. DNP signal enhancements were
measured with AMUPol and PyPol with a proline standard
sample in water/glycerol at 263 and 395 GHz (400 and
600 MHz 1H frequency respectively), 97 K sample temper-
ature. Further instrumentation and experimental details are
given in the Supporting Information. DNP signal enhance-
ments e(1H), polarization build-up times (TDNP), and T1(1H)
values are shown in Table 1. A very strong signal enhance-
ment is observed (Figure 1) with these two new bTUrea
derivatives, with e of 235 and 207 at 263 GHz and 128 (both)
at 395 GHz for AMUPol and PyPol, respectively. The shorter
TDNP for AMUPol allows for faster signal averaging making it
the more favorable biradical for DNP/ssNMR applications.
Figure 1. 13C CPMAS spectra of 0.25m U-13C-15N proline with 10 mm
AMUPol in [D8]glycerol/D2O/H2O (60/30/10 volume ratio) with (top
trace) and without (bottom trace) microwave irradiation at 14 kHz
MAS, 97 K sample temperature, 8 scans, 1 dummy scan, 6.4 s recycle
delay, 9 W microwave power at end of probe waveguide.
A TOTAPOL sample was prepared and measured under
identical conditions for comparison and showed e of 70 and 30
at 263 and 395 GHz, respectively. The DNP efficiency for
AMUPol was investigated as a function of sample temper-
ature (Figure 2) and MAS spinning frequency (Supporting
Information). The temperature dependence of AMUPol is
almost linear from 100 K to over 180 K (e = 83 at 160 K)
whereas TOTAPOL shows a much steeper temperature
dependence (e < 10 at 160 K), especially below 130–
140 K.[10] This reduced temperature dependence allows for
DNP experiments with AMUPol at sample temperatures that
are significantly higher than typical DNP experiments and up
Scheme 2. Synthesis of PyPol and AMUPol.
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similar. Furthermore for the bTUrea derivatives the magni-
tude of the e–e dipole coupling (ca. 34 MHz) is larger than for
TOTAPOL (ca. 23 MHz). Therefore, we conclude that the
much higher efficiency of PyPol and AMUPol in comparison
with TOTAPOL results from their longer electron relaxation
times and larger e–e dipole coupling.[10c,20] In addition, owing
to the presence of the urea linker, the molecular structure of
bTUrea and its derivatives is much more rigid than that of
TOTAPOL[9] and more molecules are expected to closely
approach the CE frequency matching.
DNP on and off measurements were performed with 1.3 ꢁ
TDNP as the recycle delay which provides optimal sensitivity in
a multi-scan experiment. Since TDNP is not exactly equal to T1,
this can have a slight effect on the reported DNP signal
enhancement but best reflects to conditions under which
a DNP-enhanced solid-state NMR experiment would be run.
In conclusion, our experiments demonstrate the superior
performance at 263 and 395 GHz of the biradicals PyPol and
AMUPol when compared to TOTAPOL. The enhancement
factors obtained with AMUPol are about 3.5 times (at
263 GHz) and 4 times (at 395 GHz) larger than for TOTA-
POL under identical experimental conditions. These new
polarizing agents push back the barriers to the broadband
applicability of DNP/ssNMR to increasingly complex biolog-
ical systems or water soluble materials, currently not ame-
nable to NMR characterization.
1
Figure 2. Temperature dependence of H DNP signal enhancement for
AMUPol measured at 263 and 395 GHz. 1H DNP signal enhancement
measured by 13C CP-MAS experiment on 0.25m U-13C-15N proline with
10 mm AMUPol in [D8]glycerol/D2O/H2O (60/30/10 volume ratio) with
and without microwave irradiation for each data point. Microwave
power level optimized for each data point. 8 scans, 1 dummy scans,
recycle delay=1.3ꢁTDNP for each data point. Sample temperature
calibrated with KBr T1 measurements[21] in same rotor and active
volume as DNP sample.
to sample temperatures where spectral resolution may be
improved. The MAS dependence of the DNP signal enhance-
ment (Supporting Information) is relatively flat up to 14 kHz
spinning frequency, again in contrast to published data for
TOTAPOL which shows a noticeable drop in DNP efficiency
at MAS frequency above 3 kHz.[10a,c] This result is highly
encouraging for the development of faster spinning MAS
DNP probes and applications. Electron T1e and T2e relaxation
times were measured at 9 GHz EPR frequency and 97 K
(Table 1), and their temperature dependence as well as that of
T1(1H), are also shown in Supporting Information.
Received: May 29, 2013
Published online: August 16, 2013
Keywords: dinitroxides · dynamic nuclear polarization (DNP) ·
.
polarizing agents · solid-state NMR spectroscopy ·
synthesis (org.)
Many factors influence the efficiency of a dinitroxide CE
DNP polarizing agent, to name but a few: the e–e dipole
interaction, the electron relaxation times, the relative ori-
entation of the two g tensors. Furthermore the dinitroxide
must have a good solubility in the solvent system used for the
experiment and must be compatible with the formation of
a good glass at low temperatures. AMUPol and PyPol exhibit
good solubility (up to 30 mm) in glycerol/water mixtures (60/
40 v/v), and good frozen glasses are formed (T< 220 K). It has
been shown that lengthening T1e and T2e allows for better
saturation of the electron spins and results in higher CE
efficiency.[5e,17] For nitroxides in glassy organic solvents
around 100 K, on one hand it has been shown that T1e
depends on the molecular weight[18] of the radicals: the
heavier the radical, the slower the relaxation, on the other
hand, replacing the rotating methyl groups of TEMPO
moieties with spirocyclohexyl groups lengthen T2e.[5e,19]
Thus, AMUPol and PyPol are expected to have electron
relaxation times, significantly longer than those of bTUrea
and TOTAPOL. This assumption is confirmed with the EPR
measurements showing especially significant increase in T2e
for AMUPol and PyPol (Table 1). Preliminary DFT calcu-
lations (Supporting Information) show that for the major
conformer of bTUrea, PyPol and AMUPol, the relative
orientation of the TEMPO moieties and the e–e dipole
coupling (estimated from the point dipole approximation) are
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