A benzyl alcohol derivative of the BDPA radical for fast dissolution dynamic nuclear polarization NMR spectroscopy

Article abstract of DOI:10.1039/c4ob02356k

The synthesis, structural characterization and the successful application of a carbon centered radical derived from 1,3-bisdiphenylene-2-phenylallyl (BDPA), its benzyl alcohol derivative (BA-BDPA), as a polarizing agent for Dynamic Nuclear Polarization (DNP) are described. The reported BA-BDPA radical meets all the requirements to become a promising candidate for its use in in vivo DNP-NMR experiments: it is soluble in neat [1-¹³C]pyruvic acid, insoluble in the dissolution transfer solvent and is effective as a polarizing agent in fast dissolution DNP-NMR applications, without the need for using glassing agents. Moreover, it enables a simple but effective in-line radical filtration to obtain hyperpolarized solutions of [1-¹³C]pyruvic acid free of radicals that offers a better polarization performance. This journal is

Full text of DOI:10.1039/c4ob02356k

Organic &
Biomolecular Chemistry
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A benzyl alcohol derivative of the BDPA radical for
fast dissolution dynamic nuclear polarization NMR
spectroscopy†
Cite this: Org. Biomol. Chem., 2015,
13, 2689
J. L. Muñoz-Gómez,a,b E. Monteagudo,c V. Lloveras,a,b T. Parella,c J. Veciana*a,b and
J. Vidal-Gancedo*a,b
The synthesis, structural characterization and the successful application of a carbon centered radical
derived from 1,3-bisdiphenylene-2-phenylallyl (BDPA), its benzyl alcohol derivative (BA-BDPA), as a polar-
izing agent for Dynamic Nuclear Polarization (DNP) are described. The reported BA-BDPA radical meets
all the requirements to become a promising candidate for its use in in vivo DNP-NMR experiments: it is
soluble in neat [1-13C]pyruvic acid, insoluble in the dissolution transfer solvent and is effective as a polariz-
ing agent in fast dissolution DNP-NMR applications, without the need for using glassing agents. Moreover,
it enables a simple but effective in-line radical filtration to obtain hyperpolarized solutions of [1-13C]-
pyruvic acid free of radicals that offers a better polarization performance.
Received 6th November 2014,
Accepted 2nd January 2015
DOI: 10.1039/c4ob02356k
www.rsc.org/obc
challenging approaches based on the application of hyper-
polarization methods on modern liquid-state NMR spectro-
Introduction
NMR spectroscopy has become a fundamental analytical tech- scopy are an ongoing field of research, namely optical
nique1 widely used for the identification and characterization pumping,5,6 para-hydrogen induced polarization7,8 (PHIP) and
of chemical and biochemical molecules under both solution dynamic nuclear polarization9,10 (DNP). In particular, fast dis-
and solid-state conditions, in terms of chemical structure, solution of highly-polarized small molecules combined with
dynamics, and intra- and intermolecular interactions as well DNP11 is one of the most efficient approaches. This technique
as for the complex mixture analysis. In addition, magnetic reso- is based on cooling down a solution sample, usually formed
nance imaging (MRI) is one of the best non-invasive clinical by (i) the analyte, (ii) a glassing solvent agent and (iii) a radical
imaging methods currently used in medicine.2 However, the (typically a trityl radical), into a high magnetic field (3.35 T).
progress of both NMR and MRI techniques is still limited Under such conditions and applying microwave irradiation at
owing to their relatively poor sensitivities due to the inherent an appropriate frequency of the radical resonance, the strong
low thermal-equilibrium Boltzmann polarization of some of polarization of unpaired electrons on free radical molecules,
the involved nuclear spins, such as 13C and 15N. During the homogeneously distributed within the solution, can be trans-
last few years, enormous efforts have been devoted to enhance ferred to nearby nuclei of the analyte. Then, the hyper-
the NMR sensitivity, including the hardware development of polarized frozen solution is automatically dissolved in a warm
cryogenically cooled probes3 and the availability of super- solvent (water or methanol) and quickly transferred to the
conducting magnets with higher magnetic fields.4 Other NMR spectrometer, where the enhanced signals from the
analyte must be quickly detected by conventional NMR tech-
niques. To mention a few examples, fast dissolution DNP
studies have been successfully reported in several in vitro and
aInstitut de Ciència de Materials de Barcelona, (ICMAB-CSIC) Campus Universitari
de Bellaterra, E-08193 Cerdanyola del Vallès, Spain. E-mail: j.vidal@icmab.es
bNetworking Research Center on Bioengineering, Biomaterials and Nanomedicine
in vivo applications such as, for instance, for structural eluci-
dation,3,12 to monitor chemical and biochemical reactions,13,14
(CIBER-BBN), Barcelona, Spain
cServei de Ressonància Magnètica Nuclear, Facultat de Ciències i Biociències,
to study molecular interactions,15 to develop new and ultrafast
NMR experiments,16,17 to perform in vivo 13C imaging of
Universitat Autònoma de Barcelona, E-08193 Bellaterra, Catalonia, Spain
†Electronic supplementary information (ESI) available: Synthesis and character-
metabolic reactions2,18,19 or seeking for new biomarkers by
real-time molecular imaging.11,20,21
ization of free radicals 3–8, EPR spectra of BA-BDPA, polarization build-up
curves for BA-BDPA at different concentrations, removal of BA-BDPA from the
Currently, the water-soluble trityl-based stable free radical
dissolution, UV-Visible and EPR of in-line filtered dissolution, cleaning of the
OX6322 (structure not shown) is the most frequently used
polarizer dissolution line and experimental linewidths. See DOI: 10.1039/
polarizing agent in dissolution DNP for low-γ nuclei such as
c4ob02356k
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ELEXSYS E500 X-band spectrometer equipped with
a
field-frequency (F/F) lock accessory and built in NMR Gauss-
meter. A rectangular (TE 102) cavity was used for the
measurements.
Dynamic nuclear polarization
Sample preparations. (a) BDPA solutions: As described pre-
viously,26 4.0 mg BDPA were completely dissolved in 100 μl of
sulfolane, then 100 μl of [1-13C]pyruvic acid were added to
achieve a final sample concentration of 40 mM of BDPA. (b)
Radical 8 solutions: 1.8 mg of 8 were completely dissolved in
100 μl of [1-13C]pyruvic acid and 0.36 μl of benzene were
added. The final concentration of both BA-BDPA 8 and
benzene was 40 mM. The same procedure was followed to
obtain all sample solutions of 8 at different concentrations
(20, 60 and 80 mM). (c) OX63 solutions: 2.4 mg of OX63 were
completely dissolved in 100 μl of [1-13C]pyruvic acid yielding a
final concentration of 15 mM of OX63.
DNP experiments. Aliquots of 20 μl were polarized using the
HyperSense® (Oxford Instruments Molecular Biotools, Oxford,
UK) commercial polarizer working at 3.35 T, 1.4 K. Samples
were polarized for approximately 90 minutes irradiating at the
frequency of the corresponding optimum positive peak P(+),
determined by the 13C microwave sweep spectrum. The hyper-
polarized sample was then dissolved in 4 ml of water and auto-
matically transferred (10 s) 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 a 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 3 × 4 ml methanol
and 3 × 4 ml water, see the ESI.†
Fig. 1 Structures of BDPA and BA-BDPA free radicals.
13C or 15N, mainly because it presents a narrow electron para-
magnetic resonance (EPR) linewidth, which is among the nar-
rowest linewidths found in free radicals. This narrow linewidth
translates into a low electron heat capacity and yields a lower
spin temperature for the electron spin–spin interaction reser-
voir that is in thermal contact with the nuclear Zeeman
system. Alternatively, nitroxide-based radicals and biradicals23
present a wider EPR linewidth and, as a consequence, DNP of
the nitroxide doped 13C substrates, which also proceeds pre-
dominantly via thermal mixing, yields a relatively lower
polarization. Recently, the BDPA radical (Fig. 1), which has a
comparable linewidth to OX63, has been demonstrated to be a
versatile polarizing agent under a variety of conditions,24 from
solid-state DNP experiments25 to fast dissolution DNP of
[1-13C]pyruvic acid.26 In the latter case, although the residual
BDPA radical traces can be removed from the hyperpolarized
solution by filtration, its insolubility into neat [1-13C]pyruvic
acid requires the use of sulfolane as a glassing agent, hamper-
ing its application in in vivo experiments.
Herein we present the synthesis of a novel benzyl alcohol
derivative of the BDPA (BA-BDPA, 8) which is a carbon-centered
free radical soluble in [1-13C]pyruvic acid. The DNP sample
preparation conditions are similar to those for OX63 which do
not require glassing agents. Besides, it is shown that the
residual traces of the BA-BDPA radical can be rapidly removed
during the transfer process, as in the case of the non-functio-
nalized commercially available BDPA, becoming a promising
polarizing agent for fast dissolution DNP-enhanced in vivo
applications.
Liquid-state 13C DNP-NMR measurements: the 13C DNP-
NMR spectra were acquired 10 s after the sample dissolution
using a conventional 1D broadband heterodecoupled 13C pulse
scheme employing a 90° 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 37 878 Hz in a single scan. After
Fourier transformation, the frequency-domain spectra were
then manually phased and baseline corrected.
After the complete decay of polarization, a conventional 1D
13C NMR thermal equilibrium spectrum was acquired with an
acquisition time of 0.47 s and a recycle delay of 2 s. The data
were collected into 32 K data points, with a spectral width of
34 722 Hz and as the sum of 32 transients using a 30° degree
flip angle, unless otherwise stated.
The liquid-state enhancements were calculated, as
described previously,27 by the division of the signal intensity
of the hyperpolarized signal over the intensity of the thermal
Material and methods
1,3-Bisdiphenylene-2-phenylallyl (BDPA) radical–benzene clath-
rate (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 (see ESI, Fig. S1†) were recorded at equilibrium signal multiplied by the square root of the
room temperature in THF (HPLC quality) using a Bruker number of scans.
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Fig. 2 13C microwave DNP spectrum of 50 μl [1-13C] pyruvic acid
doped with 40 mM 8 (solid circles) at 3.35 T and 1.4 K. The arrows indi-
cate P(+) = 94.087 GHz and P(−) = 94.117 GHz polarization peaks,
respectively. The microwave sweep spectrum of 50 μl of 1 : 1 (v/v) [1-13C]
pyruvic acid : sulfolane doped with 40 mM BDPA (open circles) is also
shown. It is noteworthy that as the amount of [1-13C]pyruvic acid in the
BDPA sample is half, the intensity of the signal is lower.
Scheme 1 Synthesis of free radical 8. Conditions: (i) tBuOK, EtOH,
reflux 16 h; (ii) Br2 in CCl4, room temperature, 5 min; (iii) NaOH in EtOH,
reflux, 30 min; (iv) fluorene, tBuOK in DMA, room temperature, 1 h; (v)
DIBALH, anhydrous THF, room temperature, 16 h; (vi) DBU in DCM, then
AgNO3.
The optimum positive P(+) and negative P(−) peaks were deter-
mined for both samples by scanning from 94.000 to 94.200
GHz for 3 minutes at each frequency every 5 MHz. The
optimum P(+) was set at 94.077 and 94.087 GHz for BDPA and
8, respectively. The separation between positive and negative
polarization peaks |P(+) − P(−)| for BDPA and 8 was 50 and
30 MHz, respectively. These close values and the comparable
EPR linewidths for both radicals,29 extracted from the micro-
wave DNP spectra, suggest that the same dominant thermal
mixing polarization transfer mechanism is operative in both
cases.
In order to determine the optimum concentration of
radical 8 to be dissolved in [1-13C]pyruvic acid, 20 μl sample
aliquots with concentrations of 20, 40, 60 and 80 mM were
polarized at an optimal frequency of 94.087 GHz, for approxi-
mately 90 minutes (see Fig. S2 in ESI†), and automatically
transferred to the NMR spectrometer through a home-made
in-line filtration system that consists of a high pressure adapter
connected to a syringe filled with cotton (see Fig. S4 in ESI†).
This filtration completely removed the radical from the dissol-
ution down to 10−7 M, as confirmed by UV-Vis and EPR spec-
troscopy (see the final UV-Vis and EPR solution spectra in ESI,
Fig. S5†).
The optimum concentration of radical 8 found was 40 mM
(Fig. S2†). In Fig. 3 we show the 13C polarization build-up
curve for the sample with radical 8 in the optimum concen-
tration found and the ones corresponding to BDPA (40 mM)
and OX63 (15 mM), for comparison. As can be observed in
Fig. 3, the 40 mM solution of 8 provides better solid-state
polarization levels with an important reduction in the polariz-
ation time with respect to the BDPA radical. This can be
related to the reduction in the build-up time constant (Tc)
which is three times lower. It can also be observed that com-
parable polarization levels are achieved between 8 and the
Results and discussion
The synthesis of BDPA carboxylic acid 6 (see ESI†) is shown in
Scheme 1.28 This multistep synthesis consisted of conden-
sation between fluorene and 4-carboxybenzaldehyde followed
by a double bond bromination to obtain compound 4. The
latest step was improved by changing the previously reported
reaction conditions: in CCl4 and for only five minutes
(Y: 88%). The next steps were the elimination of hydrogen
bromine, which generates compound 5, and, finally, the allylic
substitution which adds the final fluorene ring that completes
the BDPA structure. The reduction reaction of the carboxylic
group of 6 to the corresponding benzyl alcohol 7 was per-
formed with diisobutylaluminium hydride (DIBALH) in excess.
The acid reduction without anion formation was possible
because of the big size of this reducing agent. This process
could be naked-eye controlled by the absence of the typical
blue colour corresponding to the BDPA anion. Finally, radical
8 was isolated by the treatment of the benzilic alcohol deriva-
tive with 1,8-diazabicycloundec-7-ene (DBU) followed by
AgNO3 as an oxidant agent in dichloromethane (DCM). The
total yield of the synthesis of 8 was 40%.
The DNP experiments were carried out using freshly pre-
pared solutions of radical 8 in [1-13C]pyruvic acid.‡ The micro-
wave sweep or 13C microwave DNP spectrum (Fig. 2) shows the
13C NMR signal intensity against the microwave irradiation fre-
quency of 40 mM solution of BDPA and BA-BDPA radical 8.
‡The radical is stable in solution at least for some hours. The EPR spectrum
intensity and shape of a freshly prepared solution of the radical did not change
after 2 hours.
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Table 1 Liquid-state NMR enhancements (ε) measured 10 s after dissol-
ution of [1-13C]pyruvic acid samples doped with different concentrations
of 8, BDPA and OX63 radicals
Radical
conc. (mM)
Hyperpolarized
compound
Dissolution
solvent
Liquid-state
enhancement
BA-BDPA 20 mM
BA-BDPA 40 mM
BA-BDPA 60 mM
BA-BDPA 80 mM
BDPA 40 mM
[1-13C]pyruvic acid
[1-13C]pyruvic acid
[1-13C]pyruvic acid
[1-13C]pyruvic acid
[1-13C]pyruvic acid
[1-13C]pyruvic acid
Water
Water
Water
Water
Water
Water
2191
2886
2726
1387
2454
2658
OX63 15 mM
It is worth highlighting that radical 8 shows a great advan-
tage over OX63 in the transfer process. Indeed, radical 8 is
insoluble in the solvent transfer medium (H2O) and hence it is
totally retained in the filtering system (see Fig. S5 in ESI†).
Thus, the transferred dissolution only contains the hyper-
polarized pyruvic acid. In contrast, the radical OX63 remains
in solution since it is soluble in the solvent used, making
the relaxation of the hyperpolarized pyruvic acid solution
faster, with the consequent loss of hyperpolarization due to
the interaction of the free radical with the hyperpolarized
nuclei.26,30,31
Fig. 3 13C polarization build-up curve of 20 µl [1-13C]pyruvic acid
doped with 40 mM 8 (open triangles). The curves of 20 µl 1 : 1 (v/v)
[1-13C]pyruvic acid : sulfolane doped with 40 mM BDPA (open circles)
and 20 µl [1-13C]pyruvic acid doped with 15 mM OX63 (crosses) are also
shown for comparison. It is noteworthy that the amount of [1-13C]
pyruvic acid in the BDPA sample is half of the others because of its for-
mulation, [1-13C]pyruvic acid : sulfolane (1 : 1). The polarization build-up
time constants (Tc) are also shown for each corresponding curve.
Conclusions
A benzyl alcohol derivative of BDPA, 8, has been synthesized,
characterized and tested as a polarizing agent for fast dissol-
ution DNP. This radical shows some advantages with respect
to the commercially available BDPA, such as: (a) sulfolane is
avoided as the glassing agent in the sample preparation, (b)
better polarization levels are achieved under solid state DNP
conditions and (c) better 13C signal NMR enhancements are
reached under liquid NMR conditions. Moreover, the required
polarization time has been substantially reduced by a factor of
2 when [1-13C]pyruvic acid is polarized. Additionally, it has
been shown that radical 8 can be easily removed from the
hyperpolarized solution in the transfer process, minimizing
the loss of hyperpolarization for the presence of the para-
magnetic free radical in the solution.
Fig. 4 Hyperpolarized, Hyp (θ = 90°, 1 scan) and thermal equilibrium,
TH (θ = 30°, 32 scans) 13C DNP-NMR spectra of [1-13C1]pyruvic acid
after filtration. The TH spectrum is enlarged 50 times.
Acknowledgements
OX63 trityl radical. The polarizing build-up curves (Fig. 3) were
recorded in the solid state and the average liquid-state NMR
enhancements (ε) were calculated after acquisition of the
corresponding thermal equilibrium 13C spectrum (Fig. 4 and
S6†). Table 1 shows the liquid-state NMR enhancements
measured 10 s after dissolution of [1-13C]pyruvic acid samples
doped with different concentrations of the studied radical: 8
(20, 40, 60 and 80 mM), BDPA (40 mM) and OX63 (15 mM).
The best polarization for 8 remains the same than in the solid
state (40 mM), although the time of polarization (and with it
the build-up time constant) is not too much longer than in the
60 mM concentration (see Fig. S2 and S3 in ESI†).
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 Nanomedicine (CIBER-BBN),
Consejo Superior de Investigaciones Científicas by the JAE
Grand, and Amable Bernabé for his work in MALDI spec-
trometry. NMR studies were carried out at the joint NMR facil-
ity of the Universitat Autònoma de Barcelona and CIBER-BBN
(Cerdanyola del Vallès).
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