Communication
glass-forming solution containing a polarizing agent (usually
nitroxide biradicals such as TOTAPOL[10] or bTbK[11]) at a typical
concentration of 5–60 mm. The role of this solution, called
here the DNP-matrix, is to uniformly distribute the polarizing
agent in the sample. Accordingly, water-based DNP-matrices
used, for example, in biological applications, such as in the
context of lipid membranes, typically contain high concentra-
tions of glycerol or dimethyl sulfoxide (DMSO) (20 to 60%),
which may not be optimal for the integrity of the biological
sample. The DNP matrix composition is mainly dictated so that
it favors an efficient DNP mechanism. Even though the matrix
is chosen such that it is as compatible as possible with the
system of interest, the presence of the DNP matrix generally
poses several problems. First of all, it occupies a non-negligible
place in the fixed sample–rotor volume, thus decreasing signif-
icantly the amount of studied material and consequently the
absolute sensitivity of the experiment.[12,13] Secondly, the polar-
izing agents dissolved in the DNP matrix can potentially inter-
act with the system of interest leading to non-uniform distribu-
tion of radicals inside the sample,[14] radical reduction,[15] non-
uniform DNP enhancement, and paramagnetic-induced shifts
across the various resonances.[16]
Abstract: Magic-angle spinning dynamic nuclear polariza-
tion (MAS-DNP) has been proven to be a powerful tech-
nique to enhance the sensitivity of solid-state NMR
(SSNMR) in a wide range of systems. Here, we show that
DNP can be used to polarize lipids using a lipid-anchored
polarizing agent. More specifically, we introduce a C16-
functionalized biradical, which allows localization of the
polarizing agents in the lipid bilayer and DNP experiments
to be performed in the absence of excess cryo-protectant
molecules (glycerol, dimethyl sulfoxide, etc.). This consti-
tutes another original example of the matrix-free DNP ap-
proach that we recently introduced.
Since their discovery in the 60s,[1,2] liposomes have been exten-
sively studied and clearly appear as a unique nanostructure
with great promise because they can be used as synthetic bio-
logical membranes but also for drug delivery and more gener-
ally in the context of liposome–nanoparticle assemblies.[3]
More specifically, in the very challenging context of membrane
proteins or peptides embedded in phospholipid bilayers, solid-
state NMR has proven to be a key technique for the study of
their structural and dynamical properties.[4] However, the need
for a significant improvement in sensitivity of the technique is
crucial considering the dilution of the embedded molecule in
the sample.
Recently we introduced a “matrix-free” sample preparation
approach, designed to minimize the weight losses due to the
presence of the DNP matrix and thus optimize the absolute
sensitivity enhancement of the DNP experiment when com-
pared with standard NMR conditions.[12,17] Based on experi-
ments performed on microcrystalline (e.g., cellulose) and nano-
crystalline (Lyzozyme, a 14.3 kDa protein) samples, it was
shown that the presence of direct or indirect interaction (affini-
ty) between radicals and the sample of interest allows preserv-
High-field magic-angle spinning dynamic nuclear polariza-
tion (MAS-DNP) is currently emerging as a powerful technique
to strongly enhance the sensitivity of solid-state NMR and thus
to open new perspectives for the analysis and characterization
of a broad range of molecular systems in chemistry, biology,
and materials science.[5] The use of DNP for the study of bio-
molecules in lipid bilayers was pioneered in the Griffin group[6]
and further work in this direction has been recently present-
ed.[7–9]
ing
a uniform distribution of radical molecules in the
sample[12,17] without using an excess of glass-forming DNP
matrix. This cannot only yield an improved overall sensitivity
but also offer an additional advantage: More freedom to fulfill
specific sample requirements in terms of solvation, sample
state, and/or cryo-protection.
In these examples, as well as in other applications of MAS-
DNP, the sample preparation consists in dissolving, suspending,
or impregnating the system of interest with a cryo-protecting,
This particular aspect of matrix-free DNP is highlighted here
for the study of synthetic liposomes, as used for the investiga-
tion of membrane proteins or peptides.
[a] Dr. C. Fernꢀndez-de-Alba, Dr. H. Takahashi, A. Richard, Y. Chenavier,
Dr. L. Dubois, V. Maurel, Dr. D. Lee, Dr. S. Hediger, Dr. G. De Paꢁpe
Universitꢂ Grenoble Alpes
Following our previous work, in which we discussed direc-
tions to generalize the matrix-free DNP approach,[17] we used
here for the first time a system-driven radical design with the
aim to produce a new biradical with chemical properties allow-
ing a direct interaction with the system of interest. More spe-
cifically, we introduce a C16-functionalized biradical that allows
us to locate the polarizing agents in the lipid bilayer and to
perform DNP experiments in absence of usual cryo-protectant
molecules (glycerol, DMSO, etc.) used in DNP. Moreover, we
show that optimal DNP efficiency on liposomes (by using
a lipid-anchored biradical) can be obtained using commonly
used cryo-conditions described in other fields of research for
lipid membranes. It is then compared to results obtained with
standard DNP sample preparation based on a radical-contain-
ing glass-forming matrix, as used in the DNP literature up to
now. Finally, we discuss the minimal water content that needs
to be used to preserve a good DNP enhancement.
INAC, LCIB, 38000 Grenoble (France)
Fax: (+33)4-38-78-50-90
[b] Dr. C. Fernꢀndez-de-Alba, Dr. H. Takahashi, A. Richard, Y. Chenavier,
Dr. L. Dubois, V. Maurel, Dr. D. Lee, Dr. S. Hediger, Dr. G. De Paꢁpe
CEA, INAC, LCIB
38054 Grenoble (France)
[c] Dr. S. Hediger
CNRS, INAC, LCIB
38000 Grenoble (France)
[d] Dr. H. Takahashi
Present address:
ETH Zurich, Department of Physics
8093 Zurich (Switzerland)
[**] DNP=Dynamic nuclear polarization.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201404588.
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Chem. Eur. J. 2015, 21, 1 – 7
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ꢂ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!