Synthesis and hyperpolarized 15N NMR studies of 15N-choline-d13

Article abstract of DOI:10.1016/j.tet.2013.03.043

¹⁵N-Choline-d₁₃ was synthesized as a new biomarker for dynamic nuclear polarization, and the lifetime of the polarized signal governed by the spin-lattice relaxation time T₁ was measured. ¹⁵N-Choline-d₁₃ exhibited a T₁ that was over twice as long as that of ¹⁵N-choline, and the hyperpolarized ¹⁵N signal could be observed for 1 h.

Full text of DOI:10.1016/j.tet.2013.03.043

Tetrahedron 69 (2013) 3896e3900
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15
15
Synthesis and hyperpolarized N NMR studies of N-choline-d13
Keiko Kumagai a, Kazuyasu Kawashima a, Mai Akakabe a, Masayuki Tsuda a,
,
,
*
Takamasa Abe b y, Masashi Tsuda c
a Science Research Center, Kochi University, Kochi 783-8506, Japan
b NanoScience Division, Oxford Instrument KK, Tokyo 135-0047, Japan
c Center for Advanced Marine Core Research, Kochi University, Kochi 783-8502, Japan
a r t i c l e i n f o
a b s t r a c t
15N-Choline-d13 was synthesized as a new biomarker for dynamic nuclear polarization, and the lifetime
of the polarized signal governed by the spinelattice relaxation time T1 was measured. 15N-Choline-d13
exhibited a T1 that was over twice as long as that of 15N-choline, and the hyperpolarized 15N signal could
be observed for 1 h.
Article history:
Received 12 February 2013
Received in revised form 1 March 2013
Accepted 9 March 2013
Available online 15 March 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Choline
Dynamic nuclear polarization
1. Introduction
hyperpolarized 2 and 3 may be insufficient for in vivo metabolic
imaging. During our investigation of useful substrates for DNP, we
Techniques to retain highly polarized spins in solution via dy-
namic nuclear polarization (DNP) have enabled highly sensitive
NMR spectroscopic measurements and magnetic resonance spec-
troscopy (MRS) imaging for nuclei with spin ½ quantum numbers.1
Particularly, greater than 10,000-fold signal enhancements of
hyperpolarized 13C nuclei in short acquisition times were accom-
plished when the nuclei had relatively long spinelattice relaxation
times T1.2e4 This technique has been applied in real-time metabolic
studies of tumor tissue and cancers with several 13C-labeled
substrates.5e9
obtained DNP 13C NMR measurements of deuterium-enriched
glucoses, and discovered that C-perdeuterated glucose (glucose-
C-d7) exhibited a longer T1 and higher polarization level for the 13
C
signals than those of glucose samples without deuterium or with
partial C-deuteration.20 Therefore, we synthesized the C-perdeu-
terated 15N-choline [15N-choline-d13 (1)], and measured 15N NMR
spectra of these substrates after hyperpolarization by a solution
DNP procedure. Here, we describe the synthesis of 15N-choline-d13
(1) and 15N NMR profiles of hyperpolarized 1 by DNP.
Choline is a precursor of cellular phospholipid metabolism as
well as the neurotransmitter acetylcholine, and it has been applied
as a biomarker for cancer detection and metabolic response using
1H and 31P MRI,10 PET,11,12 and SPECT.13 Recently, choline with
a relatively long T1 for the 15N nucleus has been of interest as
a useful substrate for DNP 15N NMR and MRS studies.14e18 Erykyn
and co-workers described the high polarization level, long T1, and
long lifetime for the 15N signal14 in hyperpolarized 15N-choline (2)
by DNP. Independently, Denisov and co-workers reported that
natural-abundance 15N-choline with deuterated methyl groups
[choline-trimethyl-d9 (3)] prolonged the T1 of the 15N signal more
than that of 2.15 Since tumor uptake of choline is reached a maxi-
mum at 3 min after injection,19 T1s and lifetimes of 15N signals for
2. Results and discussion
15N-Choline-d13 (1) was synthesized from ethylene glycol-d4 (4,
98% 2H-enrichment) in five steps, as shown in Scheme 1. Although
installation of a trimethyl ammonium group is possible via a one-step
substitution reaction with trimethylamine, 15N-trimethylamine-d9 is
* Corresponding author. Tel.: þ81 88 864 6720; fax: þ81 88 864 6713; e-mail
address: mtsuda@kochi-u.ac.jp (M. Tsuda).
y
Present address: Bruker BioSpin K.K., Kanagawa 221-0022, Japan.
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.03.043

K. Kumagai et al. / Tetrahedron 69 (2013) 3896e3900
3897
Scheme 1. Synthesis of 15N-choline-d13 (1).
relativelyexpensive. So, we optedfora three-stepconversionthrough
treatment of 6 with 15N-labeled potassium phthalimide, hydrazi-
nolysis, and methylation with iodomethane-d3. The monosilyl alco-
hol (5) prepared from 4 was converted into the tosylate (6), which
was treated with 15N-labeled potassium phthalimide (98% 15N-en-
richment) to afford compound 7. Reaction of 7 with hydrazine gave
the 15N-labeled primary amine (8). Treatment of 8 with iodo-
methane-d3 (99.5% 2H-enrichment) and then silver oxide generated
deuterated choline iodide through permethylation of the amino
group and deprotection of the silyl group. 15N-Choline-d13 chloride
(1), with calculated enrichments of 96% 2H and 98% 15N, was obtained
through an ion-exchange step in 43% overall yield.
The 15N NMR experiments of the cholines were carried out using
a 9.4 T wide-bore NMR spectrometer equipped with a 5 mm CH
dual probe. The conventional 15N NMR spectrum of 1 (13 mM in
D2O) at thermal equilibrium (Boltzmann) polarization was mea-
sured using 3072 scans with a 10ꢀ flip angle and 10 s repetition
time, as shown in Fig. 1a. Hyperpolarization of the cholines was
performed using a HyperSense DNP polarizer (Oxford Instruments)
employing OX63 as a radical source under microwave irradiation at
94 GHz in the DMSO-d6/D2O glassing solvent system at 1.4 K for 3 h.
After dissolution with D2O, the 15N NMR spectrum (Fig. 1b) of
hyperpolarized 1 (2.2 mM) was immediately recorded with a single
acquisition using a 10ꢀ flip angle at 298 K. The signal-to-noise ratio
(SNR) of the ammonium nitrogen signal (41.72 ppm) for thermal
equilibrium 1 was 5.65, while hyperpolarized 1 had an SNR of 285.
From comparison of these 15N signal intensities in the hyper-
polarized and thermal equilibrium NMR spectra, the enhancement
Fig. 1. (a) 15N NMR spectrum of thermal equilibrium 15N-choline-d13 (1, 13 mM)
measured with 3072 scans using a 10ꢀ flip angle and 10 s repetition period. SNR was
5.65. (b) 15N NMR spectrum of hyperpolarized 15N-choline-d13 (1, 2.2 mM) recorded
with a single acquisition using a 10ꢀ flip angle. SNR was 285. A small shoulder of the
signal may be derived from the residual 15N-choline-d12
.
factor for hyperpolarized 1 was found to be 1.68ꢁ104. As 3.3ꢁ10ꢂ6
%
Similarly, the T1 of hyperpolarized 15N-choline-d13 (1) was found
to be 580ꢃ10 s. The perdeuterated choline 1 exhibited 2.9- and 1.5-
fold increases in T1 versus those of 2 and 3, respectively, indicating
that perdeuteration of the carbon nuclei around the ammonium
nitrogen in choline was effective in prolonging T1. Comparing of the
T1 for 1 with that of 3, the T1 of choline seems to be proportional to
deuterium numbers.
was accounted for by the thermal polarization P(15N) at 298 K and
9.4 T, the enhanced polarization P(DNP) for 1 was found to be 5.5%,
which was nearly equal to that of 2 (5.4%), as calculated in the same
manner.
Fig. 2 shows the time-dependent decays of the 15N signal in-
tensities for the hyperpolarized cholines (1e3) recorded every 10 s
using a 10ꢀ flip angle. The T1 values were estimated from similarities
between these observed intensities and curves simulated on the
basis of the equation reported by Day et al.21 The T1s of the hyper-
polarized 15N-choline (2) and natural-abundance choline-tri-
methyl-d9 (3) were found to be 200ꢃ5 and 380ꢃ70 s, respectively,
which were agreed well with previously reported data.14,15
Fig. 3a and b show the time-dependent 15N NMR spectra of
hyperpolarized 1 and 2 (final concentration: 1.8 mM), respectively,
which were recorded using a 10 mm CH probe with a maximum of
180 repetitions of 30 s acquisition duration employing a 10ꢀ radio
frequency flip angle. Although the signal intensity of the ammo-
nium nitrogen of 1 at 20 min (40 repetitions, Fig. 3c) retained one-

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K. Kumagai et al. / Tetrahedron 69 (2013) 3896e3900
3. Conclusions
In the present study, perdeuterated 15N-labeled choline (1) was
synthesized and the properties of hyperpolarized 1 were measured.
This study showed that perdeuteration of the carbon nuclei at-
tached to the ammonium nitrogen in choline prolonged the 15N
spinelattice relaxation time T1. The 1 h survival of the hyper-
polarized signal for 1 was remarkable. This may have been derived
from an increase in the dipolar interaction. To the best our
knowledge, the lifetime of hyperpolarized signal for 1 is the longest
in the molecular probes for solution DNP reported up to now. These
results indicate that perdeuterated 15N-labeled choline may be
useful for extended observation of choline metabolism in cells,
ex vivo or in vivo.
Fig. 2. Time-dependent decays of 15N signal intensities for hyperpolarized cholines
(1e3). Filled red, green, and blue squares represent the observed signal intensities of
hyperpolarized 15N-choline-d13 (1), 15N-choline (2), and choline-d9 (3), respectively.
Dotted green, blue, and red lines represent the fitting curves calculated for T1 at 200 s,
380 s, and 580 s, respectively. The signal intensities of 3 showed uneven because of
natural abundance for 15N and high noise level.
4. Experimental
4.1. General information and materials
All NMR spectra were recorded on a Varian 400MRWB spec-
trometer. 1H NMR and 13C NMR chemical shifts were reported in
parts per million (ppm) from tetramethylsilane (TMS) as an internal
standard. 2H NMR chemical shifts were reported in parts per mil-
fifth of that at the first acquisition, the ammonium nitrogen signal
in 2 had completely disappeared (Fig. 3d). The ammonium nitrogen
signal for 1 was confirmed even at 60 min (3600 s, 120 repetitions,
Fig. 3e).
lion (ppm) from CDCl3
(d 7.26) or D2O (d 4.80) as an external
standard. Chemical shifts in the 15N NMR spectra were based on
Fig. 3. 15N NMR spectra of hyperpolarized 15N-labeled cholines (1 and 2) measured using 10 mm CH probe. Individual spectra were processed with 5.0 Hz Gaussian line broadening.
(a) and (b) Time-dependent 15N NMR spectra of hyperpolarized 15N-choline-d13 (1) and 15N-choline (2), respectively. A shoulder of the signal in Fig. 3a may be derived from the
residual 15N-choline-d12. (c) and (d) 15N NMR spectra of hyperpolarized 15N-choline-d13 (1) and 15N-choline (2), respectively, 20 min after the start of measurement. (e) 15N NMR
spectrum of hyperpolarized 15N-choline-d13 (1) 60 min after the start of measurement.

K. Kumagai et al. / Tetrahedron 69 (2013) 3896e3900
3899
parts per million from 15N-choline (3) (
d
43.37), as described in the
97% yield. Compound 7: colorless oil; 1H NMR (CDCI3)
d
0.98 (9H, s),
previous paper.14 Measurements of DNP 15N NMR spectra are de-
scribed below. Electrospray ionization-mass spectrometry (ESI-MS)
results were obtained on JEOL JMS-T100LC and Thermo Scientific
EXACTIVE spectrometers, while high-resolution field desorption
mass spectrometry (HRFDMS) measurements were obtained on
a JEOL JMS-T100GCV spectrometer. Elemental analyses were ob-
tained using a J-Science Lab MICRO CORDER JM10 instrument.
Ethylene-d4 glycol (99.5% D), methyl-d3 iodide (98% D), and po-
tassium 15N-phthalimide (98% 15N) were obtained from Sigma-
eAldrich. 15N-Choline chloride and choline-trimethyl-d9 chloride
were purchased from CIL. Commercially available materials were
used as received without further purification. All reaction experi-
ments were carried out under argon in flame-dried glassware using
standard inert atmosphere techniques for introducing reagents and
solvents, unless otherwise noted.
7.30 (4H, m), 7.37 (2H, m), 7.59 (4H, m), 7.71 (2H, m), 7.82 (2H, m); 13C
NMR (CDCl3)
d 19.1, 26.7 (3C), 39.3 (m), 60.0 (m), 123.2 (2C), 27.6 (4C),
129.7 (2C), 132.1, 132.2, 133.2 (2C), 133.8 (2C), 135.5 (4C), 168.2,
168.3; 2H NMR (CHCl3): 3.88; 15N NMR (CDCl3)
d d 152.8; ESI-MS m/z
457.2 (MþNa)þ; HRESI-MS m/z 457.18778 (MþH)þ, calcd for
C126H223H145NO3SiNa, 457.18738.
4.2.4. 2-tert-Butyldiphenylsilyloxy[2H4]ethan[15N]amine 8. To a so-
lution of compound 7 (384 mg, 884
mmol) in ethanol (8 mL) was
added hydrazine monohydrate (130
mL, 2.68 mmol), and the mix-
ture was refluxed for 1 h. After the precipitate was removed, the
solvent was evaporated in vacuo. The residue was purified by silica
gel column chromatography (CHCl3/MeOH, 30:1) to afford amine 8
(200 mg, 656 m
mol) in 74% yield. Compound 8: colorless oil; 1H
NMR (CDCI3) d 1.06 (9H, s), 7.37 (4H, m), 7.43 (2H, m), 7.67 (4H, m);
13C NMR (CDCl3)
2H NMR (CHCl3) 2.79, 3.66; 15N NMR (CDCl3)
d d 31.1; GCFDMS m/z
d 19.1, 26.9 (3C) 127.7 (4C), 129.7 (2C), 135.5 (4C);
4.2. Synthesis
305.2 (MþH)þ; HRGCFDMS m/z 305.20385 (MþH)þ, calcd for
4.2.1. 2-tert-Butyldiphenylsilyloxy[2H4]ethanol 5. To a stirred sus-
C118H221H145NO3Si, 305.20051.
pension of sodium hydride (60% oil dispersion, 70.0 mg, 1.74 mmol)
in THF (1 mL) was added ethylene-d4 glycol (4, 88.4
m
L, 1.58 mmol)
4.2.5. 15N-Choline-d13 chloride 1. A solution of compound 8 (88.6 mg,
in THF (34 mL), and the mixture was refluxed with stirring for 1 h.
291
(184
m
mol) in methanol (1 mL) was treated with methyl-d3 iodide
The reaction mixture was cooled to 0 ꢀC and treated with tert-
mL, 2.96 mmol) and potassium carbonate (450 mg, 3.25 mmol)
butyldiphenylsilyl chloride (452
m
L, 1.74 mmol) at room tempera-
at room temperature for 16 h. After the precipitate was removed, the
solvent was evaporated in vacuo. To the residue was added EtOAc,
and the insoluble materials were collected by filtration. These crude
materials were dissolved in methanol, and then, silver oxide
ture for 16 h. After evaporation of the solvent, the residue was
partitioned between EtOAc and water. The organic phase was
washed with saturated aqueous NaHCO3 and then brine, dried over
MgSO4, and evaporated in vacuo. The residue was purified by silica
gel column chromatography (hexane/EtOAc, 10:1) to afford the
crude monosilyl alcohol (5, 375 mg, 1.23 mmol, 78%). Compound 5:
(137 mg, 592
mmol) was added. After stirring for 15 h at room
temperature and removal of the precipitate, to the reaction mixture
was added, dropwise, 1 M aqueous hydrochloric acid until pH 4,
followed by evaporation of solvent. After the addition of EtOH and
removal of the insoluble material, the solvent was evaporated to
colorless oil; 1H NMR (CDCI3)
m), 7.68 (4H, m); 13C NMR (CDCl3)
d
1.07 (9H, s), 7.40 (4H, m), 7.44 (2H,
19.2, 26.8 (3C), 62.9 (1C, m),
d
64.1 (1C, m), 127.8 (4C), 129.8 (2C), 133.2 (2C), 135.5 (4C); 2H NMR
obtain 15N-choline-d13 (1, 36.2 mg, 236
mmol) in 81% yield. Com-
(CHCl3)
d
3.66, 3.75; GCFDMS m/z 247.1 (Mꢂt-Bu)þ; HRGCFDMS m/z
pound 1: colorless oil; 13C NMR (CD3OD)
d 54.4 (3C, m), 57.1 (m), 68.6
247.11264 (Mꢂt-Bu)þ, calcd for C114H211H4O2Si, 247.10924. Anal.
(m); 2H NMR (H2O)
d
2.96 (9ꢁ2H), 3.27 (2ꢁ2H), 3.84 (2ꢁ2H); 15N
Calcd for C118H220H4O2Si requires: C, 71.00%. Found: C, 70.95%.
NMR (D2O)
d
41.72; ESI-MS m/z 118.2 (MꢂCl)þ; HRESI-MS m/z
118.18569 (MꢂCl)þ, calcd for C15H2H NO, 118.18562.
15
13
4.2.2. 2-tert-Butyldiphenylsilyloxy[2H4]ethyl 4-methylbenzenesulfo-
nate 6. To a solution of compound 5 (328 mg, 1.08 mmol), trie-
4.3. DNP 15N NMR
thylamine (375 mL, 2.69 mmol), and trimethylammonium chloride
(103 mg, 1.08 mmol) in acetonitrile (2 mL) was added a solution of
p-chlorotoluenesulfonyl chloride (308 mg, 1.62 mmol) in acetoni-
trile (1 mL) at 0 ꢀC, and stirring was continued for 1 h. After the
solvent was evaporated, the reaction mixture was partitioned be-
tween EtOAc and saturated aqueous citric acid. The organic phase
was washed with brine, dried over MgSO4, and evaporated in
vacuo. The residue was purified by silica gel column chromatog-
raphy (hexane/EtOAc, 20:1). Tosylate 6 (470 mg, 1.02 mmol) was
obtained in 95% yield. Compound 6: colorless oil; 1H NMR (CDCI3)
4.3.1. Polarization of cholines by DNP. Samples were polarized in
a HyperSense DNP polarizer (Oxford Instruments Molecular Bio-
tools, U.K.). Each of 1 and 2 together with 15 mM of trityl free
radical (OX63, GE Healthcare) was dissolved in DMSO-d6/D2O. The
mixture was placed in the 3.35 T superconducting magnet of the
DNP polarizer, frozen at 1.4 K, and irradiated with 94 GHz micro-
waves for 3 h. Instantaneous dissolution of the polarized and frozen
sample was performed by the addition of heated and pressurized
D2O (3 mL) containing 0.025% EDTA (189 ꢀC, 10 bar). The sample
was then transferred immediately via a thin TeflonÒ tube into an
NMR tube positioned in a broadband probe in a 9.4 T wide-bore
magnet (Varian). Final concentrations of 1 and 2 were fixed at 2.2
and 2.4 mM, respectively. Natural-abundance choline-trimethyl-d9
(3, 100 mg) was hyperpolarized as described above, with a final
concentration of 134 mM.
d
1.00 (9H, s), 2.44 (3H, s), 7.31 (2H, d, J¼6.5 Hz), 7.36 (4H, m), 7.43
(2H, m), 7.60 (4H, m), 7.87 (2H, d, J¼6.5 Hz); 13C NMR (CDCl3)
d 19.1,
21.7, 26.7 (3C), 60.7 (1C, m), 70.2 (1C, m), 127.7 (4C), 128.0 (2C),
129.8 (4C), 132.9 (2C), 133.0, 135.6 (4C), 144.7; 2H NMR (CHCl3)
d
3.77, 4.07; ESI-MS m/z 481.2 (MþNa)þ; HRESI-MS m/z 481.17811
(MþNa)þ, calcd for C125H226H4O4SSiNa, 481.17773.
4.2.3. 2-tert-Butyldiphenylsilyloxy[2H4]ethyl 1H-[15N]isoindole-1,3(2H)-
dione 7. To a solution of compound 6 (419 mg, 913 mmol) in dime-
4.3.2. Time-dependent 15N NMR spectra and T1 calculation of hyper-
polarized cholines. The time-dependent 15N NMR spectra of the
hyperpolarized samples were recorded with 80 repetitions of a 10 s
acquisition employing a 10ꢀ radio frequency flip angle, when the
5 mm dual probe was used. Spectral width and data points were
5 kHz and 5000, respectively. The FID data were processed with
0.5 Hz Gaussian line broadening. SNRs of the ammonium nitrogens
of hyperpolarized 1 (2.4 mM) and 2 (2.1 mM) in the first 15NMR
spectra were 285 and 303, respectively. The thermal 15N NMR
thylformamide (4 mL) was added potassium 15N-phthalimide
(204 mg, 1.10 mmol), and the mixture was refluxed for 2 h. After the
solvent was evaporated, the reaction mixture was partitioned be-
tween EtOAc and saturated aqueous NaHCO3. The organic phase was
washed with brine, dried over MgSO4, and evaporated in vacuo. The
residue was purified by silica gel column chromatography (hexane/
EtOAc, 10:1), and compound 7 (384 mg, 884 mmol) was obtained in

3900
K. Kumagai et al. / Tetrahedron 69 (2013) 3896e3900
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studies; Dr. E. Fukushi, of the Graduate School of Agriculture,
Hokkaido University, for useful suggestions on NMR studies and
measurement of FDMS; Dr. J. Kurita and T. Hino of Agilent Tech-
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Center, Hokkaido University, for ESI-MS measurements; and M.
Kiuchi of the Equipment Management Center, Hokkaido University,
for elemental analysis support.
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References and notes
1. Ardenkjaer-Larsen, J. H.; Fridlund, B.; Gram, A.; Hansson, L.; Lerche, M. H.;
Servin, R.; Thaning, M.; Golman, K. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
10158e10163.
21. Day, I. J.; Mitchell, J. C.; Snowden, M. J.; Davis, A. L. J. Magn. Reson. 2007, 187,
216e224.