Optimized polarization build-up times in dissolution DNP-NMR using a benzyl amino derivative of BDPA

Article abstract of DOI:10.1039/c6ra00635c

The synthesis of two novel BDPA-like radicals, a benzyl amino (BAm-BDPA, 7) and a cyano (CN-BDPA, 5) derivative, is reported and their behaviour as polarizing agents for fast dissolution Dynamic Nuclear Polarization (DNP) is evaluated. The radical 7 is a

Full text of DOI:10.1039/c6ra00635c

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DOI: 10.1039/C6RA00635C
ARTICLE
Optimized Polarization Build-Up Times in
Dissolution DNP-NMR Using a Benzyl Amino
Derivative of BDPA.
J. L. Muñoz-Gómez,a,‡ E. Monteagudo,b,‡ V. Lloveras,a T. Parella,b J. Veciana,a,*
and J. Vidal-Gancedoa,*
Cite this: DOI: 10.1039/x0xx00000x
The synthesis of two novel BDPA-like radicals, a benzyl amino (BAm-BDPA,
7) and a cyano
Received 00th January 2012,
Accepted 00th January 2012
(CN-BDPA, ) derivatives, is reported and their behaviour as polarizing agents for fast
5
dissolution Dynamic Nuclear Polarization (DNP) is evaluated. The radical
7 is a promising
candidate for DNP studies since it is soluble in neat [1-13C]pyruvic acid (PA), and therefore
the use of an additional glassing agent for sample homogeneity is avoided. In addition, a 60
DOI: 10.1039/x0xx00000x
www.rsc.org/
mM sample of
polarization times (about 1800 s). It is shown that DNP-NMR measurements using
7
offers optimum 13C NMR signal enhancements by using fairly short
7
can be
performed much efficiently in terms of signal enhancement by polarization build-up time unit
than using the reference OX63 or BDPA radicals. These enhanced features are translated to a
substantial reduction of polarization times that represent an optimum temporary use of the
DNP polarizer and allow economize liquid helium consumption.
methodology for obtaining strongly polarized solutions for its
use in both in-vitro and in-vivo applications;10 for structural
Introduction
Nuclear Magnetic Resonance (NMR) spectroscopy is an
essential analytical technique widely used for the structural
characterization and quantification of chemical compounds. On
the other hand, Magnetic Resonance Imaging (MRI) has
become a very important non-invasive tool in medicine for
early diagnostic of many diseases; for instance to obtain
metabolic maps, anatomical and functional information, flux
perfusion and diffusion studies, among others.1 The main
drawback of NMR/MRI techniques is their poor sensitivities
due to the inherent low thermal-equilibrium Boltzmann
polarization of some interesting nuclear spins, such as the low
natural-abundance 13C and 15N. Different hyperpolarization
methods2 have been developed to enhance the NMR signal,
such as optical pumping,3,4 para-hydrogen induced
polarization5,6 (PHIP) and dynamic nuclear polarization7,8,9
(DNP). Among them, dissolution DNP has become a powerful
elucidation,11,12 to monitor chemical and biochemical
reactions,13,14 to study molecular interactions,15 to develop new
and ultrafast NMR experiments,16,17 to perform in-vivo 13C
imaging of metabolic reactions10,18 or looking for new
biomarkers by real-time molecular imaging.19,20 Recently,
dissolution DNP has allowed real time assessment of low
sensitive nuclei such as 13C by in-vivo NMR21 and the
development of new pH-sensitive MRI agents.22 DNP
experiments involve a sample containing the analyte, a source
of free electrons, typically a radical molecule, and often a
glassing agent solvent to ensure the sample homogeneity,
leading the proper polarization transfer from the unpaired
electrons of the organic radical to the nearby nuclei of the
analyte. Radicals such as TEMPO,23 BDPA,24 or trityl,25 have
been widely used as mono- and/or biradicals as DNP polarizing
agents.26 Nowadays, the most extended radical used for in-vivo
DNP applications is the trityl-based stable free radical OX63,27
mainly because it presents a narrow electron paramagnetic
a
Institut de Ciència de Materials de Barcelona, (ICMAB-CSIC) Campus
resonance (EPR) linewidth which enable to polarize low-

Universitari de Bellaterra, E-08193 Cerdanyola del Vallès, Spain and Networking
Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-
BBN), Barcelona, Spain.
nuclei. However, the OX63 radical is a very expensive
commercial compound and soluble in the most common
buffered solution conditions, hampering the translation of its
hyperpolarizing features into human subject experiments.
b
Servei de Ressonància Magnètica Nuclear, Facultat de Ciències i Biociències,
Universitat Autònoma de Barcelona, E-08193 Bellaterra, Catalonia, Spain.
‡ These authors have contributed equally.
†Electronic Supplementary Information (ESI) available: [Synthesis and
characterization of free radicals
This journal is © The Royal Society of Chemistry 2013
See DOI: 10.1039/x0xx00000x
1-7, EPR spectra of BAm-BDPA and CN-BDPA.
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Additionally, it presents relatively long polarization build-up Radical
8
was firstly synthesized by the Koelsch's method.32
times (typically 60-90 min.), with the consequent time- Then, Kuhn and Neugebeauer developed an alternative method
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consuming measurements and high helium consumption.
Recently, the carbon centered BDPA radical,24
which involved fewer steps.34 Efforts DtOoI: 1s0y.n10th39e/sCiz6eRA0B0D63P5AC
8
in Chart 1) derivatives have been carried out by few research groups,35,36
(
and the benzyl alcohol derivative of BDPA (BA-BDPA)28 have like Kuhn34 and Fox33, and recently we have reported the
been reported as new feasibly polarizing agents for dissolution synthesis of a benzyl alcohol derivative of BDPA (BA-
DNP. The original radical
8
, which has a comparable EPR BDPA).28 On the other hand, biradicals containing BDPA units
linewidth to OX63, is a versatile polarizing agent under a have been limited only to molecules containing two BDPA
variety of sample conditions,29 from solid-state DNP30 to fast radicals linked through the phenyl ring.34
dissolution DNP of [1-13C]pyruvic acid (PA).31 In the latter In this work, the synthesis of the cyano BDPA derivative at the
case, although the residual BDPA radical traces can be removed 4-position of the phenyl ring (4) was carried out following the
from the hyperpolarized solution by filtration, its insolubility Kuhn and Neugebeauer methodology,34 because it requires
into neat PA requires the use of sulfolane as a glassing agent, fewer steps and provides higher yields (Scheme 1). This
limiting its application for in-vivo experiments. On the other multistep synthesis consisted on a condensation between
hand, the BA-BDPA radical is soluble in PA and can be easily fluorene and 4-cyanobenzaldehyde followed by a double bond
removed from the hyperpolarized solution by in-line filtration.
Herein, we report the synthesis and application as polarizing improved by changing the previously reported reaction
agents for dissolution DNP of two new BDPA derivatives: the conditions using sonication of suspended compound in CCl4
benzyl amino BDPA (BAm-BDPA, , Chart 1) and the cyano for only five minutes. The next step was the elimination of
(CN-BDPA, , Chart 1) radicals. Considering that a key step in hydrogen bromine to produce the conjugated acceptor , in a
dissolution DNP-NMR studies is the required polarization short reaction time (15 min.) to avoid the possible hydrolysis of
build-up time for an optimum signal enhancement, an the cyano group. Finally, the allylic substitution of added the
experimental factor ( /saturation time) to measure the final fluorene ring that completes the BDPA structure ( ).
bromination to obtain compound 2. The latest step was
1
7
5
3
3


4
efficiency of the dissolution DNP-NMR process has been
introduced. The general polarizing behavior and efficiency of
CN
CN
CN
CN
radicals
5 and 7 in front PA are analyzed and compared with
Br
CH2NH2
6
Br
i)
ii)
iii)
Br
the standards BDPA and OX63.
O
H
3
1
2
iv)
CH2NH2
CN
vii)
vi)
7
4
v)
CN
Chart 1 Structures of BDPA radical derivatives: CN-BDPA (5), BAm-BDPA (7)
and the reference BDPA (8).
5
Scheme 1. Synthetic scheme of BDPA derivatives. Reagents and conditions: i)
Results and discussion
tBuOK, EtOH, reflux 16 h.; ii) Br2 in CCl4, room temperature, 5 min.; iii) NaOH in
t
EtOH, reflux, 30 min.; iv) Fluorene, BuOK in DMA room temperature, 15 min.;
vi) LiAlH4 THF anhydrous; v) and vii) DBU in DCM, then AgNO3.
Synthesis and characterization
The reference radical
8 is remarkably stable to oxygen in the
solid state32 and has been reported to be stable to oxygen in
solution with the exclusion of light.33 However, to achieve the
highest persistance in solution in the dark it is necessary the
presence of benzene which forms a complex with the radical.33
On the other hand, BDPA radical is reduced to give the
corresponding carbanion when exposed to strong bases, such as
hydroxide or alkoxide, and also reacts with strong acids.32
By reduction of the cyano derivative
anhydrous THF at low temperature, the benzylamino derivative
was isolated after treatment in acid media (Scheme 1). It
appeared as a slightly red colored solid suggesting the presence
of minimum concentrations of radical, as confirmed later on by
the EPR spectrum. Although the sample contained such radical
traces, the NMR spectra could be registered without problems.
4 with LiAlH4 in
6
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5 and 7
were isolated by a treatment with 1,8- commercial BDPA radical does.31 The optimal frequency of
Finally, radicals
diazabicycloundec-7-ene (DBU), as a base, followed by work was determined as the positive peak polarization of P(+) =
View Article Online
AgNO3, as an oxidant agent in dichloromethane (DCM). The 94,080 GHz.
total yields of and were 74 % and 52 %, respectively. The Several samples were prepared (20, 40, 60 and 80 mM) to
carbanion formation and its oxidation to radical were monitored determine the optimal concentration of radical for DNP
by UV-Vis due to the different absorbance of the anion and experiments. The samples were made dissolving the appropriate
radical forms. BDPA carbanion derivatives show an absorption quantity of radical into PA and adding an equimolar quantity
DOI: 10.1039/C6RA00635C
5
7
7
7
band at around 600 nm and the radical form at around 495 nm. of benzene. A 20 µl aliquot of each concentration was polarized
Thus, solutions of BDPA carbanion derivatives are in general at the optimal frequency until saturation, except for the 20 mM
blue colored, as is the case of radical
7, but in the case of sample. As can be observed in Fig. 1b, the best polarization
radical it was green due to the electron-withdrawing cyano level in solid-state conditions was achieved for the 60 mM
5
group. However, the radical solutions were red in both cases, as sample. The time needed to saturate the different samples was
for all reported BDPA radicals, because the electron density of quite different depending on radical concentration, decreasing
the SOMO orbital is mainly located over the fluorenyl rings at higher concentrations. It could be observed how the curve
with null perturbation of the substituted phenyl ring.37,38 The corresponding to the 20 mM is not saturated after 5400 s of
EPR spectra at room temperature and in frozen solution of
and , as well as their corresponding simulations are depicted in reach the maximum solid-state polarization level. In the case of
Figs. S1-S3 of the ESI.
5
polarization while the 60 mM sample only needs 1800 s to
7
the most concentrated 80 mM sample, saturation was achieved
faster (1100 s) at expense to a slightly lower enhancement.
These saturation times are also reflected as an exponential
decay in the build-up time constants (Tc, Fig. 2). The Tc value
is lower at higher concentrations which could be associated to a
thermal mixing mechanism.39
Dissolution DNP studies using BAm‐BDPA (7) and CN‐
BDPA (5) radical derivatives.
Due to the insolubility of radical
prepared using sulfolane as a co-solvent, as previously
described for the BDPA radical.31 Thus, once the radical
was
5 in neat PA, the samples were
5
completely dissolved in 100 µl of sulfolane, the same amount
of PA (100 µl) was added, obtaining a final 40 mM
concentration of the radical in the mixture. The 13C microwave
DNP spectrum (Fig. S4a), acquired with 100 µl of the
previously prepared solution, showed a positive polarization
peak at P(+) = 94.080 GHz (frequency of work) and the
negative one at P(-) = 94.120 GHz. The polarization build-up
curve (Fig. S4b) was performed with a 30 µl aliquot of the
frozen sample (40 mM) irradiated at the optimal frequency. A
slow polarization build-up curve with low polarization level
was obtained, and, in addition, the sample was not stable with
Fig. 1 a) 13C microwave DNP spectrum of 50 µl [1-13C]pyruvic acid doped with
40 mM radical 7 at 3.35 T and 1.4 K. b) Solid-state 13C polarization build-up
curves of 20 µl [1-13C]pyruvic acid doped with different concentrations of radical
7.
time (see ESI). Since the decomposition of the radical
5 could
be the origin of such behavior,33 as occurs for the BDPA
radical, several experiments with equimolar quantities of
radical and benzene (see ESI) were repeated. However, the data
obtained by the build-up curves were similar to those obtained
in the previous case. Thus, it could be concluded that
good polarizing agent of PA due to its low stability.
5 is not a
As noted before, radical BAm-BDPA
enabling thereby its use without any glassing agent. The sample
was prepared by dissolving into PA and then an equimolar
quantity of benzene was added giving a 40 mM concentration
of
. The acquisition of the 13C microwave DNP spectrum was
7 is soluble in neat PA
7
7
carried out with an aliquot of 50 µl of the previous described
sample and showed a positive polarization peak at 94.080 GHz
and a negative one at 94.125 GHz (Fig. 1a). The separation
between both polarization peaks (P(+) - P(-)= 94080 - 94125)
was 45 MHz, which is an intermediate value between the non-
functionalized BDPA radical24 (50 MHz) and the cyano
Fig. 2 Build-up time constants (Tc) versus concentration of radical 7, OX63 and
BDPA, performed at their optimal sample conditions in PA. Notice that BDPA
includes sulfolane in a 1:1 ratio.
derivative
5 (40 MHz). This could fulfill the requirements to
polarize 13C nuclei by the thermal mixing mechanism as
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After polarization, all the samples were fast dissolved into hot previously described: PA:sulfolane (1:1) doped with 40 mM of
water dissolution of Na2EDTA (743 µM) and automatically BDPA and PA doped with 15 mM of OX63. Fig. 3 shows the
View Article Online
transferred to a 600 MHz NMR spectrometer to perform the polarization build-up curves recordedDOinI: 10a.103H9y/Cp6eRrAS0e0n6s3e5®C
corresponding 13C NMR measurements. The overall transfer polarizer working at 3.35 T and 1.4 K, whereas Table 1
time was 8.5 s (6 s of transfer between DNP and NMR displays the corresponding liquid-state NMR signal
instruments, 2 s of sample stabilization and 500 ms of enhancement factorsIn addition, we introduce an efficient
measurement time). The liquid-state signal enhancement (
was calculated by the acquisition of a conventional 1D 13C enhancement achieved by DNP saturation time afforded during
NMR experiment with a single scan and using an excitation flip the complete dissolution DNP-NMR process ( /saturation

)
polarization factor () to assess the liquid-state signal

= 
angle θ = 90º, for all four hyperpolarized and the thermal time [s], last column in Table 1).
equilibrium samples (Entries 1-4, Table 1). To measure the
thermal sample, the hyperpolarized sample was maintained 15
minutes outside the magnet to ensure the complete relaxation. It
can be observed in Table 1 that the maximum signal
enhancement in liquid-state conditions is achieved for the 60
mM sample, in strong agreement with the solid-state
measurements.

Table 1 Liquid-state NMR enhancement ratio () measured 8.5 s after the
dissolution in hot water solution of Na2EDTA (743 M) of PA samples doped
with different concentrations of radical 7, BDPA and OX63.
%
13C polarization
after sample dissolution was measured at 298 K into and a magnetic field of
14.1 T.
%pol
13C
after

Liquid-state
enhancement
()
Sat.
Pol.
/sat.
time)
[s-1]
Entry
Radical
timea [s] time [s]
Fig. 3 13C polarization build-up curves at 3.35 T and 1.4 K of 20 µl PA doped
with 60 mM of BAm-BDPA 7 (squares), 20 µl PA doped with 15 mM OX63
(crosses) and 20 µl 1:1 (v/v) PA:sulfolane doped with 40 mM BDPA (circles). It
should be noticed that the quantity of PA in the sample with BDPA is half of the
others because of its formulation.
dissol.
BAm-
BDPA
20 mM
1
2
3
4
-
5400
3600
2400
2600
872
1.1
8.7
---
BAm-
BDPA
40 mM
3500
1800
1100
7109
9358
6623
2.0
5.2
6.0
As can be observed in Fig. 3, the polarization time required to
reach the saturation plateau with a 60 mM sample of
7 is two
times lower than with OX63 radical and three times lower than
with BDPA (Entries 3, 5 and 7 in Table 1). However, the ε
factor and the percentage of 13C polarization are partially lower
with respect to OX63 and BDPA radicals, mainly because the
different radical concentrations could lead to different
BAm-
BDPA
60 mM
11.4
8.1
BAm-
BDPA
80 mM
hyperpolarization decay rates. On the other hand, although
 at
a 80 mM concentration is not too much high, we must not
ignore that the saturation time is even shorter (1100 s; solid-
state curve shown in Fig. 1b, Entry 4 in Table 1), specifically
more than three times lower than OX63 and five times lower
BDPA
40 mM
5
6
7
8
5400
5400
3600
3600
5400
1800
3600
1800
11583
4478
14.1
5.5
2.1
0.8
3.4
2.0
BDPA
40 mM
than BDPA. Thus, although the
show partially lower enhancements, the use of radical

at both 60 mM and 80 mM
as
OX63 15
mM
7
12369
7228
15.1
8.8
polarizing agent seems to be advantageous since it can reduce
the overall experimental time with respect to OX63 and BDPA
radicals, resulting in a very efficient polarizing agent. This is
OX63 15
mM
a
clearly observed comparing the different
 factors in Table 1
The saturation time has been taken as the time corresponding to the
(Entries 3, 5 and 7). Typically, the time needed to polarize a
sample in a dissolution DNP experiment using the OX63 trityl
98% solid-state polarization percentage estimated by the HyperSense®
during the polarization build-up curve acquisition.
radical is 60-90 min. whereas with for a 60 mM solution of
7 it
can be significantly reduced to 30 min. This leads to economize
instrumentation time and minimize the consumption of liquid
helium. Comparison of build-up time constant (Tc) of BAm-
In order to compare these results with those with the non-
functionalized BDPA radical and the golden standard OX63
radical, dissolution DNP experiments were also performed
using these radicals in their optimal concentrations as was
BDPA
7
with the ones for OX63 and BDPA radicals performed
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at their optimal concentration in PA (Fig. 2) shows the same µl of sulfolane and benzene (80 mM). Once the radical was
tendency. Indeed, the build-up time constant for radical is completely dissolved, 100 µl of PA were added, obtaining a
lower than for OX63 and BDPA radicals, and hence the final solution where the concentrationDOofI: 1b0.o10th3V9ie/rCwa6dARritcAica0lel0O6an3ln5inCde
polarization time needed to polarize PA is also inferior. benzene was 40 mM. (b) Radical solutions: 1.8 mg of were
Taking into account the 13C polarization build-up curves at completely dissolved in 100
l of PA and 0.36 l of benzene
solid-state (Fig. 3), a comparison of the liquid-state DNP were added. The final concentration of both and benzene was
enhancements of all three samples at short polarization times 40 mM. The same protocol was followed in order to prepare
was performed. Thus, after 1800 s of polarization, all the solutions of at all different concentrations (20, 60 and 80
samples were fast dissolved into hot water dissolution of mM). (c) BDPA solutions: 40 mM BDPA solutions were
Na2EDTA (743 µM) and automatically transferred to a 600 prepared by dissolving 4.0 mg of into 100 l of sulfolane,
MHz NMR spectrometer to perform the corresponding NMR then 100 of PA were added yielding the desired
measurements (Entries 3, 6 and 8 in Table 1). It is worth noting concentration. (d) OX63 solutions: 15 mM solutions of OX63
that the highest signal enhancement ( ) is obtained by (60 were prepared by completely dissolving 2.4 mg of radical into
mM), which is 1.3 and 2 times higher than with OX63 and 100 l of PA.
BDPA radicals, respectively. We could also observe that is Microwave sweep: The optimum positive P(+) and negative
the most efficient polarizing agent as it has the highest factor P(-) polarization peaks for each radical sample were determined
meaning an optimal compromise between a reasonable signal by irradiating for 3 minutes at each frequency in a range from
7
7
7


7
7
8
8


l

7

7

enhancement and a relatively short polarization time.
94.000 to 94.200 GHz unless otherwise is stated.
Dissolution DNP Experiments: HyperSense® (Oxford
A
Instruments Molecular Biotools, Oxford, UK) commercial
polarizer working at 3.35 T and 1.4 K was used to
Conclusions
Two novel BDPA radical derivatives have been synthesized
and studied as polarizing agents for fast dissolution DNP-NMR
hyperpolarize 20
l aliquots of each different sample. These
were polarized by irradiation at the corresponding optimum
positive peak P(+) determined by the 13C microwave DNP
spectrum for each radical used until polarization saturation. The
hyperpolarized sample was then dissolved with 4 ml water and
automatically transferred into an empty NMR tube placed
inside a Bruker AVANCE-III 600 MHz spectrometer (Bruker
Biospin, Rheinstetten, Germany), previously locked and
shimmed with a reference sample. The spectrometer was
equipped with a 5 mm broadband TXI inverse probehead
incorporating a z-gradient coil and working at field strength of
14.1 T (600.13 and 150.03 MHz, 1H and 13C frequencies
respectively). After dissolution, the polytetrafluoroethylene
(PTFE) transfer pipe line was cleaned with with methanol (3 x
4 ml) and then with Water/Na2EDTA (743 µM) (3 x 4 ml), after
each DNP experiment.
Liquid-state 13C DNP-NMR measurements: The 13C DNP-NMR
spectra were acquired 8.5 s after the sample dissolution using a
conventional 1D broadband, heterodecoupled 13C spectrum was
recorded employing a 90º RF pulse. The data were collected
into 32 K data points during an acquisition time of 0.43 s and
using a spectral width of 37878 Hz in a single scan. Prior to
Fourier transformation an exponential function with a line
broadening of 1 Hz was applied, the frequency-domain spectra
were then manually phased and baseline corrected.
applications. The radical CN-BDPA,
unstable with time and hence it is not a good polarizing agent of
PA. On the other hand, the radical BAm-BDPA, , has proved
to be a quick and efficient polarizing agent of PA without need
of glassing agent, showing optimum levels of 13C signal NMR
enhancements per polarization time unit. Its use as polarizing
agent in dissolution DNP studies can represent important saves
in measurement time, optimized instrument usage and liquid
helium consumption.
5, has shown to be
7
Material and methods
Tris{8-carboxyl-2,2,6,6-tetra[2-(1-hydroxyethyl)]-benzo(1,2-
d:4,5-d’)bis(1,3)dithiole-4-yl}methyl sodium salt (OX63,
Oxford Instruments Molecular Biotools, Oxford, UK), 1,3-
bisdiphenylene-2-phenylallyl (BDPA) radical/benzene clathrate
(1:1), [1-13C]pyruvic acid, sulfolane and all the solvents were
purchased from Sigma-Aldrich Company, Ltd. (Tres Cantos,
Madrid, Spain) and were used without further purification.
X-band EPR
The EPR spectra were recorded in a Bruker ELEXYS E500 X-
band spectrometer equipped with a TE102 microwave cavity, a
Bruker variable temperature unit, a field-frequency (F/F) lock
system Bruker ER 033 M; line positions were determined with
an NMR Gaussmeter Bruker ER 035 M. The modulation
amplitude was kept well below the line width, and the
microwave power was well below saturation.
After complete decay of polarization, a conventional 1D 13C
NMR thermal equilibrium spectrum was acquired with an
acquisition time of 0.43 s. The data were collected into 32 K
data points, with a spectral width of 37878 Hz in a single and
using a 90º degree flip angle, unless otherwise stated. It was
processed in the same way than hyperpolarized 13C DNP-NMR
spectrum. The liquid-state enhancements were calculated, by
the division of the signal integral of the hyperpolarized signal
over the integral of the thermal equilibrium.
Dynamic Nuclear Polarization Sample Preparations: (a)
Radical
5 solutions: 3.54 mg of radical 5 were dissolved in 100
µl of sulfolane and then the same amount of PA (100 µl) was
added, obtaining a final 40 mM concentration of the radical in
the mixture. After the first trial: 3.54 mg were dissolved in 100
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Acknowledgements
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This research was supported by the DGI Grants "POMAs"
(CTQ2010−19501) and "Be-Well" (CTQ2013−40480-R),
MINECO (CTQ2012-32436), AGAUR Grant (2014SGR-0017)
and MINECO Grant (PTA 2011-5037-I) for the subprogram
Personal Técnico de Apoyo. We also thank the Networking
Research Center on Bioengineering, Biomaterials and
DOI: 10.1039/C6RA00635C
Nanomedicine
(CIBER-BBN,
Consejo
Superior
de
Investigaciones Científicas by the JAE Grant, Amable Bernabé
for his work in MALDI spectrometry. NMR studies were
carried out at the joint NMR facility of the Universitat
Autònoma de Barcelona and CIBER-BBN (Cerdanyola del
Vallès).
9 J. H. Ardenkjær-Larsen, B. Fridlund, A. Gram, G. Hansson, L. Hansson, M. H.
Lerche, R. Servin, M. Thaning and K. Golman, Proc. Natl. Acad. Sci. U.
S. A., 2003, 100, 10158.
10 F. A. Gallagher, M. I. Kettunen and K. M. Brindle, Prog. Nucl. Magn. Reson.
Spectrosc., 2009, 55, 285.
TOC
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