A versatile synthetic route to the preparation of 15N heterocycles

Article abstract of DOI:10.1002/jlcr.3699

A robust medium-scale (approximately 3 g) synthetic method for ¹⁵N labeling of pyridine (¹⁵N-Py) is reported based on the Zincke reaction. ¹⁵N enrichment in excess of 81% was achieved with approximately 33% yield. ¹⁵N-Py serves as a standard substrate in a wide range of studies employing a hyperpolarization technique for efficient polarization transfer from parahydrogen to heteronuclei; this technique, called SABRE (signal amplification by reversible exchange), employs a simultaneous chemical exchange of parahydrogen and a to-be-hyperpolarized substrate (e.g., pyridine) on metal centers. In studies aimed at the development of hyperpolarized contrast agents for in vivo molecular imaging, pyridine is often employed either as a model substrate (for hyperpolarization technique development, quality assurance, and phantom imaging studies) or as a co-substrate to facilitate more efficient hyperpolarization of a wide range of emerging contrast agents (e.g., nicotinamide). Here, the produced ¹⁵N-Py was used for the feasibility study of spontaneous ¹⁵N hyperpolarization at high magnetic (HF) fields (7 T and 9.4 T) of an NMR spectrometer and an MRI scanner. SABRE hyperpolarization enabled acquisition of 2D MRI imaging of catalyst-bound ¹⁵N-pyridine with 75 × 75 mm² field of view (FOV), 32 × 32 matrix size, demonstrating the feasibility of ¹⁵N HF-SABRE molecular imaging with 2.4 × 2.4 mm² spatial resolution.

Full text of DOI:10.1002/jlcr.3699

A versatile synthetic route to the preparation of 15N heterocycles
Nikita V. Chukanov,a,b Bryce M. Kidd,c Larisa M. Kovtunova,b,d Valerii I. Bukhtiyarov,d
Roman V. Shchepin,e Eduard Y. Chekmenev,*,e,g,h Boyd M. Goodson,c,f Kirill V.
Kovtunov,*,a,b Igor V. Koptyuga,b
aInternational Tomography Center SB RAS, Novosibirsk, 630090, Russia
bNovosibirsk State University, Novosibirsk, 630090, Russia
cDepartment of Chemistry and Biochemistry, fMaterials Technology Center, Southern Illinois
University, Carbondale, IL 62901, USA
dBoreskov Institute of Catalysis SB RAS, Novosibirsk, 630090, Russia
eDepartment of Biomedical Engineering and Physics, Vanderbilt-Ingram Cancer Center
(VICC), Vanderbilt Institute of Imaging Science (VUIIS), Department of Radiology,
Vanderbilt University Medical Center, Nashville, TN 37232, USA
gRussian Academy of Sciences, Moscow, 119991, Russia
hIbio, Department of Chemistry, Wayne State University, Karmanos Cancer Center, Detroit, MI
48083, USA
*kovtunov@tomo.nsc.ru
*chekmenev@wayne.edu
KEYWORDS: Parahydrogen; PHIP; Hyperpolarization; Contrast Agent; pyridine; 15N; MRI
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Abstract
A robust medium-scale (~3 g) synthetic method for 15N labeling of pyridine (15N-Py) is
reported based on the Zincke reaction. 15N enrichment in excess of 81% was achieved with
~33% yield. 15N-Py serves as a standard substrate in a wide range of studies employing a
hyperpolarization technique for efficient polarization transfer from parahydrogen to
heteronuclei; this technique, called SABRE (Signal Amplification by Reversible Exchange),
employs a simultaneous chemical exchange of parahydrogen and to-be-hyperpolarized
substrate (e.g. pyridine) on metal centers. In studies aimed at the development of
hyperpolarized contrast agents for in vivo molecular imaging, pyridine is often employed
either as a model substrate (for hyperpolarization technique development, quality assurance,
and phantom imaging studies) or as a co-substrate to facilitate more efficient
hyperpolarization of a wide range of emerging contrast agents (e.g. nicotinamide). Here, the
produced 15N-Py was used for the feasibility study of spontaneous 15N hyperpolarization at
high magnetic (HF) fields (7 T and 9.4 T) of an NMR spectrometer and a MRI scanner,
respectively. SABRE hyperpolarization enabled acquisition of 2D MRI imaging of catalyst-
bound 15N-pyridine with 7575 mm2 field of view (FOV), 3232 matrix size, demonstrating
the feasibility of 15N HF-SABRE molecular imaging with 2.42.4 mm2 spatial resolution.
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Introduction
Isotopic labelling with stable isotopes of hydrogen, carbon, oxygen, and nitrogen is
increasingly used in a wide range of applications.[1-4] 13C and 15N labels have also been
recently employed for synthesis of a wide range of biomolecules with an eye towards
magnetic resonance applications.[2] Indeed, for such applications NMR hyperpolarization
techniques are often employed to generate non-equilibrium nuclear spin polarization
(typically ~10-6/Tesla at room temperature) all the way to near unity (or 100%) nuclear spin
polarization.[5-8] The process of hyperpolarization (i.e., significant polarization enhancement
above the thermal polarization level induced by the applied static magnetic field) results in
corresponding gains (by several orders of magnitude) of NMR signals. These hyperpolarized
(HP) biomolecules can be employed as contrast agents for in vivo studies, which opens up
new opportunities for probing metabolism non-invasively by means of HP MRI.[9, 10]
Despite the MRI signal boost provided by hyperpolarization, biomedical applications of
HP contrast agents are often limited[2, 11] when isotopic labeling is not employed due to low
natural abundance of key biologically relevant spin-½ nuclei: e.g. 13C (1.1%) and 15N
(0.37%). Therefore, 13C and 15N labeling is required to enhance the MRI signals in the
context of biomedical applications, which are presently the main drivers behind the
development of hyperpolarization techniques in general.[2, 9, 12]
13
Although proton spins have a higher detection sensitivity (compared to that of C and
15N nuclei) and are very abundant (near 100%) in the context of MRI applications, they are
rarely employed for hyperpolarization of HP contrast agents owing to their relatively short
spin-lattice relaxation time constant (T1) values (typically in the range of a few seconds). As a
result, proton-based HP contrast agents[13, 14] undergo very rapid depolarization.[15-22] For
example, HP contrast agent loses ~95% of its initial polarization level (and effective potency)
within 3T1. 13C and 15N nuclear spins of biomolecules have T1 values of the order of tens of
13
15
seconds[2, 23-30] and several minutes[31, 32] (in vivo) respectively. Therefore, HP C and N
sites provide more efficient means of polarization storage in the context of preparation,
13
administration, and MRI imaging of HP contrast agents.[9, 33-40] Additionally, C and 15N HP
contrast agents do not suffer from competition with any significant in vivo background signal,
compared to the large water and lipid background signals in HP proton MRI.[11, 41-43]
Historically 13C-based HP contrast agents have been introduced first, due to more
readily available 13C-enriched compounds and efficient hyperpolarization processes using
dissolution Dynamic Nuclear Polarization (d-DNP)[44] in the context of 13C hyperpolarization
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of carboxyl groups in a wide range of biomolecules.[2] Isolated 13C carboxyl groups (i.e.
13
15
without directly attached protons, which typically decrease T1 of neighboring C and N
sites) demonstrate relatively long in vivo T1 values for HP contrast agents, often approaching
~1 minute.[2]
Nevertheless, 15N HP contrast agents have significantly longer T1 values of more than 2
minutes[31] in vivo. A number of HP compounds exhibit in vitro 15N T1 values on the order of
3-10 minutes.[33, 38, 45] Moreover, the use of long-lived spin states[46] offer an intriguing
possibility of extending the exponential decay constant to 20+ minutes for 15N sites.[40]
Furthermore, 15N has a significantly greater (by at least several fold) chemical shift dispersion
13
compared to that of C, in part because many relevant N-containing species possess lone
15
pairs on the nitrogen site; therefore, HP N sites can serve as molecular probes for sensing
characteristics of the local environment[37] such as pH.[43, 47]
Technical improvements of the d-DNP technique have recently allowed for more
efficient hyperpolarization of 15N sites.[43] Additionally, a second method for the HP contrast
agent production based on parahydrogen (a spin isomer of hydrogen) called Signal
Amplification By Reversible Exchange (SABRE)[48, 49] has been shown to be very efficient
for hyperpolarization of 15N sites in a number of molecular frameworks, including those
found in several biomolecules.[47, 50-55] A variant of SABRE, dubbed “SABRE-SHEATH”
(SABRE in SHield Enables Alignment Transfer to Heteronuclei)[56, 57] can produce 15N
polarization (%P15N) in excess of 20% in less than 1 minute.[52] Moreover unlike the case
with d-DNP, which requires expensive cryogenic and magnetic equipment, SABRE-
SHEATH is technologically undemanding, and therefore it is a relatively cheap
15
hyperpolarization technique.[53] As a result of these recent developments in d-DNP and N
SABRE-SHEATH, the interest for use of HP 15N labeled biomolecules has been rekindled.[38,
58-61]
Regardless of the technique employed (d-DNP or SABRE), the structure of a given
15
synthesized N-labeled compound is not altered during the hyperpolarization process. The
reader is directed to other review papers on d-DNP hyperpolarization,[2, 8, 62] which is outside
the scope of this work. In the SABRE approach, the to-be-hyperpolarized substrate and
parahydrogen (pH2) exchange reversibly with a metal complex (Figure 1).[63] The first
observation of the SABRE effect was realized for pyridine and in subsequent years it was
extended to many N-[40, 48, 52, 53, 63, 64] and S-[65] containing heterocycles and other N-based
functional groups.[40] Importantly, the SABRE approach allows the transfer of spin order not
only to the protons of the corresponding substrates but also to the heteronuclei; to achieve
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that step, pulse sequences[66, 67] or a magnetic shield[68] (SABRE-SHEATH)[56, 57] can be
successfully applied. Moreover, while SABRE-SHEATH (the most efficient technique in
terms of both speed of polarization and maximum demonstrated %P15N[52]) has also been
13
19
shown to successfully hyperpolarize C[69, 70] and F[71] sites (and potentially other spin ½
nuclei[72]) in biomolecular frameworks, it has been recognized that the presence of a spin ½
15N at the site for catalyst binding[69] (i.e., the N atom directly participating in exchangeable
13
binding to the metal center, Figure 1) enables significantly more efficient polarization of C
and other biologically relevant sites. To summarize, 15N-labeling is critical for both 15N-based
HP contrast agents as well as other (13C, 19F, etc.-based) HP contrast agents in the context of
the SABRE-SHEATH hyperpolarization technique.
Figure 1. A schematic diagram of Signal Amplification by Reversible Exchange (SABRE)[48,
49, 73, 74]
15
15
for the case of efficient hyperpolarization of the N site of N-pyridine using the
SABRE-SHEATH approach. Note the simultaneous chemical exchange of pH2 and the to-be-
hyperpolarized substrate (here, 15N-pyridine) in the equatorial positions of this IrIMes
hexacoordinate complex[75] and the spontaneous nature of polarization transfer via two-bond
spin-spin couplings when the process is performed at the matching magnetic field of
approximately 1 micro-Tesla (T).[56, 57, 76]
15
Recently, SABRE-SHEATH has been employed to hyperpolarize several N-enriched
15
molecules: N-pyridine,[56] 15N2-imidazole,[47, 59] 15N-nicotinamide,[56, 58] etc. Most of these
compounds in the 15N-labeled form are not available commercially or available at a very high
(>$500/0.1 g) cost not very well suitable for the development of biomedical applications on a
human scale requiring ~1 g of HP contrast agent.[9] Robust synthetic approaches are certainly
required to enable this field of molecular imaging with a broad range of 15N-labeled
biomolecules.
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There are two major synthetic approaches/strategies for preparation of 15N-labeled
heterocycles. The first one is de novo, i.e. from Latin “from scratch”, when the desired
molecular framework is built using reagents (components) that are smaller than and different
15
from the target product. We have recently demonstrated this approach in the context of N
SABRE-SHEATH for preparation of 15N2-imidazole.[59] The second strategy employs the
15
14
target contrast agent itself without N enrichment as a starting material, where the N-site
15
(natural abundance of nitrogen isotopes) is effectively “replaced” by the N isotope. While
both approaches have their merits, the second approach is attractive because a number of
complex biomolecules (including drugs) can be potentially enriched in just two steps. For
example, we have recently demonstrated this approach using a Zincke salt[77] on 15N-
nicotinamide, and demonstrated good yields along with ~98% 15N enrichment.[58]
The work presented here focuses on the synthesis of 15N-Py using the Zincke[77]
reaction. 15N-Py is an important molecular target in itself, because it is one of the most
studied molecule by SABRE hyperpolarization methods in general, and therefore, it can be
useful for mechanistic and phantom imaging studies.[67, 69, 78-82] Moreover, a variant of the
SABRE-SHEATH approach has been demonstrated where Py is added to enhance
polarization of other HP substrates (e.g. 15N-nicotinamide,[58] and 15N-acetonitrile[69]),
indicating that 15N-Py-mediated SABRE catalyst activation could be useful for preparation of
other HP contrast agents via SABRE-SHEATH. More importantly,15N-Py embodies a
molecular framework around which many other useful 15N HP contrast agents could be
15
developed in the future in the form of the substituted N-pyridines (which is an active and
15
on-going effort of our collaboration). While other synthetic approach for preparation of N-
15
pyridine has been reported,[83] it lacks versatility for preparation of substituted N-pyridine-
based biomolecules, e.g. nicotinamide, etc., which is of significant interest to hyperpolarized
MR community.[84] Therefore, the synthetic approach studied here would be of significant
interest for future synthetic efforts in this field. We note that the starting material of the
approach demonstrated here is relatively inexpensive 15NH4Cl (<$20/g ca. 2017), and the
product was produced with ~33% yield and >81% 15N enrichment, showing an efficient route
for 15N enrichment of pyridine-based (and potentially other) heterocycles. Finally, the
15
15
demonstrated labeled N-Py and potentially other N-labeled compounds enabled by this
approach can be hyperpolarized by both SABRE and d-DNP hyperpolarization techniques,
and therefore, a broad community of those working in the field of HP molecular imaging
would benefit from the work described here.
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15
Here in particular, we have employed the prepared N-Py material for the feasibility
study of spontaneous 15N hyperpolarization and 15N molecular imaging at high magnetic
field[85] of a 9.4 T MRI scanner and a 7.0 T NMR spectrometer.
Experimental
General
All solvents were purchased from common vendors and were used as received. All
13
15
NMR spectra and images (1H, C, N) were recorded on Bruker 300 MHz and Bruker 400
MHz Avance III NMR spectrometers. 1-chloro-2,4-dinitrobenzene was recrystallized from
ethanol. N-(2,4-Dinitrophenyl) pyridinium chloride was prepared by interaction of pyridine
with 1-chloro-2,4-dinitrobenzene in acetone according to a procedure similar to that reported
previously.[86]
Synthesis of 15N-Pyridine
First (small-scale) procedure. 150 mL of anhydrous methanol at 0 °C was placed in a
round bottom flask. Next sodium metal (0.43 g, 18.7 mmol) was added with continuous
stirring. As soon as the sodium has completely reacted (no hydrogen gas observed), 15NH4Cl
(1.00 g, 18.35 mmol) was added. Next, (i) a solution of N-(2,4-Dinitrophenyl) pyridinium
chloride (2.63 g, 9.34 mmol) in anhydrous methanol (20 mL) was added drop-by-drop to the
mixture under stirring and the mixture was stirred for one week at room temperature, and (ii)
activated carbon (2.00 g) was added and stirred for 30 minutes and then was filtered. The
supernatant solution was distilled, and a concentrated hydrochloric acid (8 mL) was added to
the distillate, and the resulting solution was evaporated to dryness under vacuum. The formed
pyridinium hydrochloride was placed in 2 mL flask using a minimal amount of water for the
quantitative collection of the formed hydrochloride, and excess of sodium hydroxide (1.5 g)
was then added. The mixture was cooled down by liquid nitrogen during neutralization, and
15
the reaction was deemed completed. The resulting mixture was distilled, and N-Py was
obtained as an aqueous solution (0.64 g) with concentration of about 25% (15N isotopic purity
is better than 60%), and the produced material was used in the subsequent NMR/MRI
experiments. The total yield of pyridine was about 22%.
Second (medium-scale optimized) procedure. Pyridine (60.00 mL, 744 mmol) and 2,4-
dinitro-chlorobenzene (25.4 g, 125 mmol) were placed in an oven-dried round bottom flask
(1 L) supplied with a magnetic stir bar. The solids were dissolved in anhydrous methanol and
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left to stir at room temperature for 2 days under inert (argon) atmosphere. The flask was
placed on a rotary evaporator to remove excess of unreacted pyridine, and anhydrous THF
(300 mL) was added to the reaction mixture. White amorphous residue was formed on the
bottom of the flask. THF was decanted carefully, leaving the residue on the bottom of the
flask. Another portion of anhydrous THF (100 mL) was added rapidly, and the flask was
rotated several times and left for several minutes before THF was carefully decanted from the
residue. The THF wash was repeated 5 times. The flask was placed on a rotary evaporator to
remove last traces of THF from the pyridine-Zincke salt. Anhydrous methanol (150 mL) was
added, and the flask was flashed with argon and put aside.
To an oven-dried round bottom flask (5 L) supplied with a stir bar, 15NH4Cl (574 mmol,
31.26 g) and anhydrous methanol (3 L) were added. The solution was cooled to 0 °C and
sodium methoxide methanolic solution (30 wt%, 516 mmol, 118 mL) was added drop-wise.
The methanolic solution of pyridine-Zincke salt (described above) was added dropwise to the
flask. The resulting solution was left to stir under an inert atmosphere (argon) for 2 days. The
supernatant solution was distilled, and 2 M solution of hydrochloric acid (250 mL) in Et2O
was added to the distillate (pH ~ 2), and the resulting solution was evaporated to dryness
under vacuum. The resulting crude pyridine hydrochloride was placed in 100 mL flask, and
an excess of solid sodium hydroxide (25 g) was added and the mixture was distilled.
Molecular sieves (4 Å, 5.7 g, Fisher chemical, P/N M514-500) were added to the obtained
15N-Py. A second distillation and drying by molecular sieves allowed the preparation of 3.3 g
of 15N-Py (yield: 33%; 15N isotopic purity is >81%), Figure 2.
Cl
NO2
Cl
N
15NH4Cl
NO2
NO2
MeOH
15N
N
MeONa
Yield ~33%
Isotopic purity (15N) >81%
NO2
15
15
Figure 2. The overall scheme of N-Py enrichment with 15N (from NH4Cl source) via
Zincke salt formation.
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Parahydrogen preparation, NMR and MRI experiments
Enrichment of parahydrogen (pH2) spin isomer was performed with a Bruker
parahydrogen generator, that allows filling of a 0.9 L-tank with 7 atm of ~90% pH2 in
approximately 100 minutes.
15
For the observation of N enhanced resonances the polarization transfer from pH2-
15
15
derived protons to N nuclei of N-Py (prepared via the first procedure described above)
was realized via high-field SABRE (HF-SABRE) and SABRE-SHEATH approaches.
Specifically, pH2 gas was bubbled (flow rate 30 mLmin-1) through a 1/16 in. (OD) Teflon
capillary extended to the bottom of a 5 mm medium-wall NMR tube containing a SABRE
catalyst solution and 15N-Py (10 mM of IrCl(COD)(IMes) (IMes = 1,3-bis(2,4,6-
trimethylphenyl)imidazol-2-ylidene;[75, 87] COD = cyclooctadiene; note this catalyst precursor
was synthesized in accord with a previously reported procedure[85]) in CD3OD. pH2 bubbling
occurred in the magnetic shield for the SABRE-SHEATH approach. When the gas flow was
15
halted, the sample was transferred to the high magnetic field, where N NMR spectra were
recorded. For the HF-SABRE method pH2 bubbling was performed at high magnetic field (7
T) of NMR spectrometer during the detection of 15N resonances.
The MRI investigations were carried out with a 400 MHz Bruker scanner equipped
with a 15N/1H 25 mm MRI probe. The standard EPI (echo-planar imaging) MRI pulse
sequence was utilized with 3232 matrix size, 4 averages, 11 ms echo time (TE), 7575mm2
field of view (FOV), and approximately 4 seconds’ total scan time. The MRI experiments
were performed using a 10-mm outer dimeter NMR tube.
Results and Discussion
To the best of our knowledge, there is only one method for the synthesis of 15N-Py: the
reaction of 2-ethoxy-3,4-dihydro-2H-pyran with 15N-NH4Cl in the presence of methylene
blue (Figure 3).[83] The yield was reported as 55% when the equivalent ratio of pyran
15
derivative and NH4Cl was used. In a similar manner, 4-methylpyridine was obtained from
2-ethoxy-4-methyl-3,4-dihydro-2H-pyran.[86] The main limitation of this synthetic approach
is the use of substituted pyridine derivatives, which can be complex in the context of future
SABRE biomedical applications.[5]
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methylene
blue
15NH4Cl
H+
15N
15N
15N
O
O
HO
O
OEt
15NH
O
Figure 3. Reaction scheme from the previous report[83] of 15N-pyridine synthesis.
An alternative synthetic approach involves the formation of a Zincke salt[77] and is
followed by reaction with 15NH4Cl (as a source of 15N label) (Figure 4). To date, this
approach has only been applied to the synthesis of 15N-nicotinamide.[58, 88, 89] In the first step,
the unlabeled pyridine derivative is reacted with dinitrochlorobenzene to form the Zincke salt
(Figure 4). Next, 15NH4Cl is used (as a source of 15N label) to enable heterocycle ring
15
opening, and dinitroaniline is replaced by N-labeled ammonia. After that, the cyclization
proceeds, and the labeled 15N-pyridine derivative is formed, Figure 4. The reactions shown in
Figure 3 and Figure 4 clearly indicate that preparation of 15N-pyridine derivatives would
[83]
become challenging using method described in Ref. #
(because a sophisticated precursor
15
would be required), whereas the Zincke-salt-based approach allows for straightforward N-
enrichment using non-labeled biomolecule, e.g. nicotinamide (vitamin B3) shown in Figure
4.
Cl
NO2
CONH2
CONH2
NO2
H215N
Cl
N
N
CONH2
CONH2
NO2
15NH4Cl
NO2
Et3N
15N
N
NO2
HCl
NO2
Figure 4. Reaction scheme and mechanism for the preparation of 15N-nicotinamide via
formation of Zincke salt.
The less reactive Zincke salts such as l-N-(2,4-dinitrobenzene) pyridinium chloride
have previously been employed to perform ring closure effectively and eliminate the
dinitroaniline.[90] Therefore, we have employed and investigated this strategy for its potential
of 15N labeling of pyridine.
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Figure 5. High-resolution mass spectrometry of the final product (15N-Py) performed by
direct liquid infusion using an Orbitrap mass spectrometer (Thermo-Finnigan, San Jose, CA)
equipped with an Ion-Max source housing and an atmospheric pressure chemical ionization
(APCI) probe in positive ion mode at a resolving power of 60,000 (at m/z 400). Note the
13
presence of the peak at ca. 82.05 due to contribution from C natural abundance of ~1.1%
(therefore, the probability of having one 13C in any of the five carbon positions is greater than
5%).
An initial quantity of N-(2,4-dinitrophenyl) pyridinium chloride was prepared by
interaction of pyridine with 1-chloro-2,4-dinitrobenzene in acetone.[86] This salt was used in
15
the reaction with a methanol solution of NH4Cl (Figure 2). Purification via filtration with
15
charcoal and the distilling of all volatile compounds including N-Py was performed under
atmospheric pressure. We assumed that the slight decomposition of pyridine-Zincke salt back
to the unlabeled pyridine during the synthesis and extraction could be responsible for the less-
than-ideal isotopic purity at this stage (i.e. ~81% vs. theoretical 98%). Next, the product was
extracted in the form of solid pyridinium hydrochloride, which is neutralized by NaOH, and
15
15
distilled. The final product of N-Py was formed as an aqueous solution with ~81% N
1
13
15
isotopic purity confirmed by mass spectrometry analysis, Figures 5. H, C, and N NMR
spectra are provided in the Supporting Information (Figures S1-S3).
While this 15N labeling efficiency is lower than that reported by 15N-nicotinamide
(98%), it is relatively high and certainly useful for future biomedical applications of 15N
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SABRE hyperpolarization. Of note, pyridine is one of the most studied molecules for the
observation of the hyperpolarized 1H and 15N NMR resonances via polarization transfer from
parahydrogen via SABRE hyperpolarization approaches, and as a result, this work would be
also of interest to those working in the field of fundamental advances of SABRE
hyperpolarization technology as well. Moreover, demonstration that the presented approach
works for preparation of 15N-Py (in addition to the previously reported 15N-nicotinamide
preparation[58]) opens the road for future 15N-enrichment of more-complex pyridine
derivatives using this relatively straightforward approach. The key synthetic results and their
comparison with method described in Ref. # [83] are provided in Table 1.
Table 1. The summary of 15N pyridine synthesis using presented here method and
method employed in Ref. # [83]
Method from Ref. # [83]
This work using Zincke-salt-
based method
~33%
Reaction Yield
15N isotopic purity
~55%
~98%
~81%
Straightforward applicability No
to preparation of substituted
15N-pyridines
Yes
15
Here, the feasibility of N high-field (HF) SABRE was investigated in the context of
an HP MRI application. In the case of HF-SABRE, pH2 bubbling through a water-methanol
solution of pyridine and homogeneous SABRE catalyst occurred directly inside the high field
15
of a 300 MHz NMR spectrometer along with the recording of N NMR spectra (Figure 6).
15
Under these conditions, the N signal enhancement of the free py resonance is negligible,
15
15
Figure 6b. The observable HP N NMR signal of the bound N-Py was detected only after
15
100 averages (~50 seconds of total experimental time). The N signal enhancement factor
(15N) of ca. 100-fold was estimated by comparison of the HP spectrum (Figure 6b) and a
15
15
thermally polarized spectrum of a N reference sample with known N concentration and
15
polarization (Figure 6a). N SABRE-SHEATH approach (wherein pH2 bubbling occurs at a
T magnetic field created by the magnetic shield followed by fast sample transfer to the high
magnetic field for analysis[56, 57]) allowed detection of both free and catalyst-bounded 15N-Py
resonances, Figure 6c. As with the case of the HF-SABRE approach the signal enhancement
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
15N (of approximately about 700-fold) was estimated via comparison of the 15N HP pyridine
15
15
resonances with the N signal reference provided by the thermal signal of NH4Cl (37%)
aqueous solution, Figure 6a, -- in line with previous studies at such concentrations.[56, 57]
Figure 6. 15N NMR spectra obtained for (a) 15N signal reference of 15NH4Cl (37%) aqueous
solution, (b) HF-SABRE, and (c) SABRE-SHEATH of HP 15N resonances of 15N-Py.
15
While N MRI of SABRE-SHEATH has been demonstrated before,[57] 15N MRI of
HF-SABRE has not been investigated. Therefore, the HF-SABRE polarization approach was
15
15
used to enable N MRI of N-Py. The EPI MRI pulse sequence was employed, and the
15
corresponding N 2D MRI image of the cross-section of the 10 mm NMR tube is shown in
Figure 7. The imaging spatial resolution was 2.42.4 mm2, which is similar to previous
resolution (22 mm2) reported for 15N SABRE-SHEATH imaging. However, we note that the
HF-SABRE MRI image here was recorded at ~10 times lower concentration of the HP
substrate (i.e., catalyst bound 15N-Py) and significantly lower (by at least one order of
15
15
magnitude) N polarization value of the HP N-Py. 15N MRI with similar spatial resolution
(despite nominally lower concentration and nuclear spin polarization) is possible, because 15N
polarization is continuously replenished by the HF-SABRE procedure.
The feasibility of HF-SABRE imaging is important in the context of potential
biomedical applications as a facile approach for preparation of HP phantoms to optimize
imaging protocols and studies. Moreover, SABRE-SHEATH primarily hyperpolarizes the
15
free N pool (Figure 6c), and therefore MRI imaging of SABRE-SHEATH hyperpolarized
solutions reports on spatial distribution of the free pool of HP 15N-Py. On the other hand, HF-
SABRE enhances primarily catalyst-bound species (Figure 6b), and therefore MRI imaging
This article is protected by copyright. All rights reserved.

of HF-SABRE hyperpolarized solutions provides visualization of the catalyst-bound HP 15N-
Py species. Moreover, since HF-SABRE hyperpolarization is quickly replenishable due to
continuous pH2 bubbling, the additional sensitivity boost due to continuous re-
hyperpolarization is achieved, which is advantageous for catalysis visualization via MRI
imaging.
Figure 7. 15N MRI image of high-field (HF) 15N SABRE of HP 15N-pyridine obtained using
an EPI pulse sequence with 11 ms echo time (TE), 3232 matrix size (2.42.4 mm2 pixel
size) over 7575 mm2 field of view (FOV), and 4 acquisitions (total experimental time was
approximately 4 seconds).
Conclusions
In summary, an efficient approach for 15N labeling of pyridine is reported based on the
formation of a Zincke salt intermediate. We demonstrated a relatively simple and cheap (1 g
15
15
of NH4Cl costs less than $20 ca. 2017) synthetic approach for the N-enriched pyridine
synthesis, which can be used as a robust sample for both SABRE catalyst testing and MRI
pulse sequence optimization and other applications. We envision that the relative simplicity
and high labeling efficiency (>81%) and overall good yield (>33%) will be of use for future
synthetic efforts of labeling of more complex pyridine derivatives in the context of SABRE
hyperpolarization and bio-imaging applications. The presented approach is versatile, because
it employs the exact non-enriched compound of interest as a precursor in non-15N-labeled
form. Therefore, complex pyridine-based compounds can be 15N-enriched using this
15
approach. For example, we have recently reported on synthesis of substituted N-pyridine
using the reported here approach for preparation of the corresponding Zincke salt.[91] The
This article is protected by copyright. All rights reserved.

prepared 15N-Py was employed for NMR signal hyperpolarization via SABRE technique with
15
signal enhancement of N free resonance of ~ 700-fold via the SABRE-SHEATH approach
and 15N catalyst-bound resonance of ~ 100-fold via the HF-SABRE approach.
Acknowledgements
The Russian team thanks the Russian Science Foundation (grant #17-73-20030) for the
support of 15N pyridine testing via NMR and MRI. N.V.C. thanks Dr. A. I. Taratayko for the
useful discussions. This work was supported by NSF under grants CHE-1416268, and CHE-
1416432, NIH 1R21EB018014, 1R21EB020323, and 1R21CA220137, DOD CDMRP BRP
W81XWH-12-1-0159/BC112431, DOD PRMRP awards W81XWH-15-1-0271 and
W81XWH-15-1-0272, and Exxon Mobil Knowledge Build.
Conflict of Interest
The authors did not report any conflict of interest.
Corresponding Authors
*For K.V.K e-mail, kovtunov@tomo.nsc.ru
*For E.Y.C.: phone, +1-(313) 577-2182; fax, +1-(313) 577-8822; e-mail,
chekmenev@wayne.edu
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