Hyperpolarization of cis-15N2-Azobenzene by Parahydrogen at Ultralow Magnetic Fields**

Article abstract of DOI:10.1002/cphc.202100160

The development of nuclear spins hyperpolarization, and the search for molecules that can be efficiently hyperpolarized is an active area in nuclear magnetic resonance. In this work we present a detailed study of SABRE SHEATH (signal amplification by reversible exchange in shield enabled alignment transfer to heteronuclei) experiments on ¹⁵N₂-azobenzene. In SABRE SHEATH experiments the nuclear spins of the target are hyperpolarized through transfer of spin polarization from parahydrogen at ultralow fields during a reversible chemical process. Azobenzene exists in two isomers, trans and cis. We show that all nuclear spins in cis-azobenzene can be efficiently hyperpolarized by SABRE at suitable magnetic fields. Enhancement factors (relative to 9.4 T) reach up to 3000 for ¹⁵N spins and up to 30 for the ¹H spins. We compare two approaches to observe either hyperpolarized magnetization of ¹⁵N/¹H spins, or hyperpolarized singlet order of the ¹⁵N spin pair. The results presented here will be useful for further experiments in which hyperpolarized cis-¹⁵N₂-azobenzene is switched by light to trans-¹⁵N₂-azobenzene for storing the produced hyperpolarization in the long-lived spin state of the ¹⁵N pair of trans-¹⁵N₂-azobenzene.

Full text of DOI:10.1002/cphc.202100160

Articles
doi.org/10.1002/cphc.202100160
ChemPhysChem
Hyperpolarization of cis-15N2-Azobenzene by Parahydrogen
at Ultralow Magnetic Fields**
Kirill F. Sheberstov,*[a, b] Vitaly P. Kozinenko,[c, d] Alexey S. Kiryutin,[c, d] Hans-Martin Vieth,[e]
Herbert Zimmermann,[f] Konstantin L. Ivanov+,[c, d] and Alexandra V. Yurkovskaya[c, d]
Dedicated to the memory of Konstantin L’vovich Ivanov (1977–2021).
The development of nuclear spins hyperpolarization, and the
search for molecules that can be efficiently hyperpolarized is an
active area in nuclear magnetic resonance. In this work we
present a detailed study of SABRE SHEATH (signal amplification
by reversible exchange in shield enabled alignment transfer to
heteronuclei) experiments on 15N2-azobenzene. In SABRE
SHEATH experiments the nuclear spins of the target are
hyperpolarized through transfer of spin polarization from para-
hydrogen at ultralow fields during a reversible chemical
process. Azobenzene exists in two isomers, trans and cis. We
show that all nuclear spins in cis-azobenzene can be efficiently
hyperpolarized by SABRE at suitable magnetic fields.
Enhancement factors (relative to 9.4 T) reach up to 3000 for 15N
spins and up to 30 for the 1H spins. We compare two
approaches to observe either hyperpolarized magnetization of
15N/1H spins, or hyperpolarized singlet order of the 15N spin pair.
The results presented here will be useful for further experiments
in which hyperpolarized cis-15N2-azobenzene is switched by
light to trans-15N2-azobenzene for storing the produced hyper-
polarization in the long-lived spin state of the 15N pair of
trans-15N2-azobenzene.
1. Introduction
of magnitude.[1,2] This method is applicable when a pairwise
hydrogenation reaction occurs; the singlet spin order (popula-
tion imbalance between the singlet and triplet states of the
spin pair) of parahydrogen can be transferred into and
redistributed across the nuclear spin system of the target
molecule. Of particular interest are reversible hydrogenation
reactions happening in catalytic complexes: in this case a
dihydrogen molecule H2 that is dissolved in a solution can
reversibly bind to a metal centre. It is possible to transfer its
nuclear spin order to another target molecule that binds to the
same complex simultaneously with the hydrogens. The method
is called SABRE (Signal Amplification By Reversible Exchange),[3]
and it enables the polarization of a wide variety of substrates,[4]
including important biomarkers that can be used as contrast
agents for bio-imaging.[5] Importantly, this technique allows for
many repetitions of the hyperpolarization process with the
same target molecules since all the chemical processes are
cyclic.
The search for new targets for SABRE is challenging, as it
not easy to predict in advance the molecules that are suitable.
The catalytic complexes used for SABRE are usually based on
iridium (III), thus the target molecules should be able to
reversibly bind to iridium. Therefore, the molecule must contain
an electron-donating atom, such as nitrogen. There are
currently more than a hundred compounds known that can be
hyperpolarized by SABRE, and most of them contain a nitrogen
atom.[6]
Parahydrogen can be used to hyperpolarize nuclear spins
allowing enhancement of signals in nuclear magnetic resonance
(NMR) and magnetic resonance imaging (MRI) by up to 5 orders
[a] Dr. K. F. Sheberstov
Institut für Physik, Johannes Gutenberg Universität-Mainz
55128 Mainz, Germany
E-mail: sheberst@uni-mainz.de
[b] Dr. K. F. Sheberstov
Helmholtz-Institut Mainz, GSI Helmholtzzentrum für Schwerionenforschung
55128 Mainz, Germany
[c] V. P. Kozinenko, Dr. A. S. Kiryutin, Prof. K. L. Ivanov,+ Dr. A. V. Yurkovskaya
International Tomography Center SB RAS
Novosibirsk, 630090, Russia
[d] V. P. Kozinenko, Dr. A. S. Kiryutin, Prof. K. L. Ivanov,+ Dr. A. V. Yurkovskaya
Novosibirsk State University
Novosibirsk, 630090, Russia
[e] Prof. H.-M. Vieth
Freie Universität Berlin, Fachbereich Physik
14195 Berlin, Germany
[f] H. Zimmermann
Department of Biomolecular Mechanisms, Max-Planck-Institut für Medizini-
sche Forschung
69120 Heidelberg, Germany
[+] Deceased
[**] A previous version of this manuscript has been deposited on a preprint
server: https://arxiv.org/abs/2103.00289
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cphc.202100160
An invited contribution to a Special Collection on Parahydrogen En-
hanced Resonance
The most efficient way to transfer the spin order of
parahydrogen to the target molecule exploits a coherent
transfer through the J-coupling network; this process depends
strongly on the magnetic field at which SABRE is performed.
Heteronuclear spins such as 15N and 13C can be hyperpolarized
© 2021 The Authors. ChemPhysChem published by Wiley-VCH GmbH.
This is an open access article under the terms of the Creative Commons
Attribution Non-Commercial License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited
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by SABRE in zero- to ultralow-fields (ZULF). In ZULF the
difference between the Larmor frequency of protons and the
heteronuclear spin is comparable to the J-coupling between
them, making spontaneous coherent polarization transfer
through J-couplings possible. This method is called SABRE
SHEATH (shield enables alignment transfer to heteronuclei).[7,8]
Relaxation times of 15N and 13C spins are typically much
1
longer than those of H spins, making it possible to store the
hyperpolarization up to several minutes. An alternative possi-
bility is to store hyperpolarization in a so-called long-lived state
(LLS),[9] a state that is immune to certain relaxation mechanisms.
The longest relaxation times are observed for singlet order
formed within pairs of 15N spins[2,10–14] or 13C spins,[15] and
nonequilibrium spin states can exist for up to tens of minutes in
liquids at room temperature. The possibility to store hyper-
polarization expands the field of applications and has intensi-
fied the development of SABRE experiments producing
LLS.[13,14,16,17] Here we report on SABRE SHEATH experiments with
15N2-azobenzene (ABZ), a molecular target with LLS properties.
ABZ exists in two isomeric forms, trans-ABZ and cis-ABZ,
and the prevailing form in solution can be controlled by light
(see Figure 1a). Recently, it was discovered that the singlet state
of the 15N spin pair in trans-15N2-ABZ is long-lived, which enables
sustaining non-thermal spin order for up to 50 minutes even at
high magnetic field (>10 T).[12] An illustration of the SABRE
process of ABZ is shown in Figure 1b.
Figure 1. a) Chemical structure and photochromic property of azobenzene
(ABZ). b) Scheme of SABRE with cis-ABZ showing polarization transfer from a
parahydrogen molecule to cis-ABZ within a reversibly formed Ir-IMes
activated complex. IMes stands for 1,3-dimesitylimidazol-2-ylidene, L stands
for another possible ligands in the complex, either azobenzene, methanol, or
ClÀ .
reference.[19] Deuterated methanol-d4 (99.8% atom %D) was
purchased from Carl Roth.
The samples consisted of 50 mM of 15N2-ABZ and of 3 mM of the
precatalyst, [Ir(IMes)(COD)Cl] dissolved in deuterated methanol. The
sample was irradiated by near UV light (~380 nm) to convert
trans-15N2-ABZ into cis-15N2-ABZ. After irradiation, the cis-15N2-ABZ to
trans-15N2-ABZ ratio reached 1:1. Next, the parahydrogen gas was
bubbled through the solution to activate the [Ir(IMes)(COD)Cl]
complex. This process was performed inside the NMR spectrometer
The possibility to hyperpolarize cis-15N2-ABZ with SABRE was
reported previously,[4] however it was a large scale study of
many compounds, information about SABRE of 15N2-ABZ was
limited, and the polarization level was not determined
accurately. Here we present a detailed study on SABRE SHEATH
of 15N2-ABZ and compare experiments, in which hyperpolarized
magnetization of 15N and 1H is observed to experiments in
which hyperpolarized singlet order of the 15N spins pair is
observed.
1
and monitored by H NMR. The activation of the complex took up
to 10 minutes without addition of any substrates except for 15N2-
°
ABZ. The temperature in experiments was set to 20 C.
Chemical shifts of 15N are referenced on the Ξ-scale utilizing the
absolute Larmor frequency of TMS and ΞN =10.1329118 for liquid
ammonia to obtain the absolute frequency for 0 ppm of the 15N
spins.[20]
Experimental Section
The precatalyst, [Ir(IMes)(COD)Cl] (COD stands for 1,5-cycloocta-
diene), was synthesized using the procedure described in
reference[18] and 15N-enriched ABZ was synthesized as described in
Parahydrogen was enriched up to 90% using a Bruker para-
hydrogen generator, and a pressure of 3 bar was used for all
experiments. The bubbling time t was optimized by measuring the
“build up curve”, where the intensity of the hyperpolarized signals
is plotted as function of t (not shown here). After bubbling in a
magnetic field of 500 nT for 30 s, the intensity reached a plateau
with the level of hyperpolarization about 1.7 times higher as
compared to a bubbling time of 10 s. For all subsequent experi-
ments the bubbling period was fixed at t ¼10 s – this made it
possible to achieve a high degree of polarization with a high
experiment repetition rate. Further details of the experimental
setup are given in reference.[21]
Konstantin L’vovich Ivanov passed away on
5th of March, 2021 at the age 44, during the
review process of this manuscript. In his early
career, he made major contributions to the
theory of chemical reaction kinetics in liquid
phase. Later, he contributed to unravelling
mechanisms of light-induced nuclear hyper-
polarization in liquids and solids using the
concept of level anti-crossing (LAC). In recent
years, his main research efforts, both theoret-
ical and experimental, were concentrated on
spin and chemical dynamics in PHIP and
SABRE methods using LAC; for this, he was
awarded the Günther Laukien Prize in 2020.
The work on SABRE of azobenzene was one
of the last of his project.
The spin system of cis-15N2-ABZ includes 12 coupled spins; all of
them can be hyperpolarized by SABRE. There are slightly different
experimental protocols allowing different ways to observe the
hyperpolarization. These methods are presented in Figure 2. Most
of the steps are identical: the sample containing 15N2-ABZ and
precatalyst was placed in a magnetic shield; the required ultralow
field was set up using a set of coils, and the parahydrogen gas was
bubbled through the solution. The supply of gas was then stopped
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Figure 2. Experimental protocols: a) Field cycling in a SABRE SHEATH experiment with consecutive (i) destruction of equilibrium magnetization at the high
field of the NMR spectrometer, followed by transfer of the sample into the magnetic shield, (ii) bubbling parahydrogen at ZULF conditions in a variable
magnetic field for a time t, and (iii) return to high field and excitation of the NMR signals by a hard pulse. b) Field cycling in a SABRE SHEATH experiment to
observe hyperpolarized singlet order of the 15N spin pair in cis-15N2-ABZ. In this case, excitation is performed by using a SLIC pulse (see text). The T00 block
°
indicates a filter based on 90 pulses with pulsed field gradients; for details see reference [23].
by immediate equilibration of pressure at the inlet and outlet of
the 15N spin pair. The ability to hyperpolarize singlet order is
the NMR tube at a flow rate about 40 sccm; then the magnetic field
reminiscent of the performance of SABRE SHEATH methods in
was instantaneously switched to 25 μT. Such an instantaneous
jump of the magnetic field is not necessary, but it allowed better
diazarines,[16,17] diazines,[14] and pyridazine derivatives.[13]
The large size of the spin system makes it difficult to model
the experiments: the full spin system in the complex with one
15N2-ABZ molecule contains 14 coupled spins. Nevertheless,
some of the details can be understood from exploring the
magnetic field dependencies of SABRE and by modeling the
process with simplified spin systems.
reproducibility of experiments. Then the sample was transferred
inside the NMR spectrometer (9.4 T, 400 MHz H Larmor frequency)
where different detection sequences were applied.
1
°
The first possibility is to apply a hard 90 pulse (Figure 2a),
detecting magnetization of the hyperpolarized spins. Because the
1
spin system of 15N2-ABZ contains both 15N and H spins, detection
can be performed either by using the 15N or the 1H channel.
The relevant J-couplings in the Ir-IMes complex with 15N2-
ABZ are shown in Figure 3a, while the J-couplings in cis-15N2-
ABZ are shown in Figure 3b. The values for free cis-15N2-ABZ are
taken from reference,[19] while the J-couplings in Ir-IMes
The second possibility is to detect hyperpolarized singlet order of
the 15N spin pair in cis-15N2-ABZ. It is worth noting that the two 15N
spins in 15N2-ABZ are close to magnetic equivalence, the eigenstates
of this pair are close to the singlet and triplet states, hence in most
cases singlet order is not observable after applying the usual hard
NMR pulses. Established techniques for converting singlet order
into magnetization were used to convert singlet order into magnet-
ization; here we use a method that is called “spin-lock induced
crossing” (SLIC).[22] SLIC pulses were adjusted to convert singlet
order of the 15N spin pair of 15N2-ABZ into observable
magnetization.[12,19] SLIC can be used to convert the singlet order
into either 15N magnetization or 1H magnetization of the ortho-
protons in 15N2-ABZ. Consequently, the SLIC pulse was applied
either at the 15N resonance frequency (525 ppm for cis-15N2-ABZ) or
1
complex were determined from the line splittings in H and 15N
NMR spectra. These spectra were acquired after bubbling
°
parahydrogen in high field at 5 C, which slowed down the
exchange and made the splitting visible (see Supporting
Information).
2
Cis and trans JHN-couplings were distinguished in accord-
ance with previous studies of 2JHN-couplings in Rh-based
1
complexes.[25] Interestingly, the JNN-coupling in cis-15N2-ABZ is
different for the free form (~22 Hz) and for the molecule bound
in equatorial position (~18 Hz). This is because the electron
density is shifted from the attached site towards the metal
center.
1
at the H-ortho frequency (6.85 ppm for cis-15N2-ABZ). The nutation
1
frequency matched the JNN-coupling, which is 22 Hz, and the pulse
duration was 0.3 s.
2. Results and Discussion
Polarization transfer in the spin network of 15N2-ABZ is a
sophisticated process. Hyperpolarization propagates through
the strongly coupled nuclei eventually polarizing remote spins.
As we show further, the protons in the phenyl rings of 15N2-ABZ
can be polarized only when the SABRE process is performed at
ultralow fields. This is similar to previous studies of metronida-
zole where remote spins are hyperpolarized through the J-
coupling network.[24] Also, different spin orders are polarized; of
particular interest are 15N magnetization and singlet order of
Figure 3. J-couplings in the Ir-IMes complex (a) and in cis-15N2-ABZ (b). IMes
stands for 1,3-dimesitylimidazol-2-ylidene, L stands for another possible
ligands in the complex, either azobenzene, methanol, or ClÀ .
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2.1. Magnetization Formed by SABRE SHEATH
correlation between the 15N signals (see Supporting Informa-
tion).
A typical 15N spectrum obtained after SABRE SHEATH of 15N2-
ABZ at 500 nT (protocol of Figure 2a) is shown in Figure 4. In
this figure the strongest signal near 525 ppm corresponding to
the free cis-15N2-ABZ is cut. There are several other hyper-
polarized signals, which otherwise are invisible in the thermal
equilibrium spectrum. All 15N nuclei in the sample solution
belong to 15N2-ABZ, so all these signals are attributed to
different signals of either cis- or trans-15N2-ABZ attached in
equatorial or axial sites of the Ir-IMes complex. Detailed
assignment of these signals was not our goal here, but we
determined the signals corresponding to cis-15N2-ABZ attached
to Ir-IMes in equatorial position. These signals are highlighted in
Figure 4. They are broadened due to chemical exchange. The
signal at ~420 ppm corresponds to the 15N position coordinated
with the Ir, whereas the remote 15N nitrogen has almost the
same chemical shift as in the free cis-15N2-ABZ. The assignment
was confirmed by correlation experiments showing that there is
Only the free form of cis-15N2-ABZ and not the trans-ABZ is
hyperpolarized as can be concluded from comparison of the
thermal and the hyperpolarized spectra shown in Figure 5.
Probably, absence of the hyperpolarization for the free
trans-15N2-ABZ can be explained by steric restrictions. For the
shown spectrum (Figure 4 and Figure 5a) the enhancement
factor was jɛj�3000. It was calculated as ratio between the
integral intensity of the SABRE SHEATH spectrum and the
intensity observed in the thermal equilibrium polarized spec-
trum. The same sample was used to acquire both spectra, but
the thermally polarized spectrum is sum of 128 acquisitions to
get an acceptable signal-to-noise ratio (SNR).
1H signals of cis-15N2-ABZ were also hyperpolarized (Fig-
ure 5b). The enhancement factors were jɛj�10 for each of the
proton groups. This is two orders of magnitude less than the
enhancement factors for the 15N signal. The difference can be
1
partially explained by the fact that H spins have a ~10 times
a
2JHN-coupling between the attached 15N and one of the
higher absolute value of the gyromagnetic ratio, and therefore
thermal polarization of ptotons is ~10 times larger compare to
nitrogen nuclei. Another order of magnitude may be explained
parahydrogen protons in the Ir-IMes complex as well as
1
by the weak J-couplings between the H and 15N spins, making
polarization transfer to be inefficient. Another possible explan-
ation is that the relaxation times of 1H and 15N spins are
substantially different at 500 nT, or during sample transport,
but they were not measured here.
The integral intensities of the hyperpolarized 1H signals
correspond to the stoichiometric ratio of protons in 15N2-ABZ,
when SABRE SHEATH is performed at 500 nT. Also, in this case,
the detected spin order of protons corresponds to the integral
magnetization which changes when SABRE is performed at
another magnetic field.
The field dependence of the enhancements produced by
SABRE SHEATH is shown in Figure 6. Let us first consider the 15
N
Figure 4. Fragments of the 15N NMR spectrum showing signals of 15N2-ABZ in
Ir-IMes complexes, polarized via SABRE SHEATH at 500 nT. The phase of the
spectrum was adjusted to have absorptive signals. The experimental
protocol is shown in Figure 2a.
signals (Figure 6a). The maximal enhancement for the free
cis-15N2-ABZ was achieved in the field range 200–500 nT (Fig-
Figure 5. 15N NMR spectra (a) and 1H NMR spectra (b) showing comparison of 15N2-ABZ signals in thermal equilibrium (blue, absorptive peaks) and after SABRE
SHEATH hyperpolarization at 500 nT (red, emissive peaks). The phases in SABRE SHEATH spectra were set with respect to corresponding thermal spectrum.
The 15N NMR spectrum at thermal equilibrium shown in (a) was summed for 128 times. Otherwise, all the compared spectra were acquired with identical
acquisition and processing parameters. The experimental protocol for the SABRE experiments is shown in Figure 2a.
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Figure 6. Field dependence of 15N NMR signals (a) and 1H NMR signals (b) hyperpolarized by SABRE SHEATH. The experimental values of the signal integrals
are shown by scatter plots; the calculation results are shown by the solid lines. Details of the calculations are given in the text. All the functions are normalized
by the integral intensity of the corresponding signal intensity at thermal equilibrium. Note that the Y-scale is different for each subplot. Top row in (a) shows
the data for free cis-15N2-ABZ. Middle row in (a) shows the data for the 15N spin of the cis-15N2-ABZ complex in equatorial position that is directly attached to Ir-
IMes. Bottom row in (a) shows the data for the 15N position of cis-15N2-ABZ remote from the Ir-IMes complex. Top row in (b) shows data for the ortho-1H spins
in cis-15N2-ABZ. Middle row in (b) shows data for the meta-1H spins in cis-ABZ. Bottom row in (b) shows data for the para-1H spins in cis-15N2-ABZ. The
experimental protocol is shown in Figure 2a.
ure 6a, top row). The field dependences of 15N signals
corresponding to cis-15N2-ABZ in equatorial position of the Ir-
IMes complex behave similarly with the signal corresponding to
the free cis-15N2-ABZ (compare rows in Figure 6a). This appear-
ance may be explained by the fast chemical exchange of
cis-15N2-ABZ between its free form and the form bound in
equatorial position: by the time the sample arrives inside the
NMR spectrometer (stage iii in Figure 2a) all the cis-15N2-ABZ
molecules might have experienced the chemical exchange
many times and the observed polarization of 15N spins in the
complex is secondary. It is formed by the hyperpolarized
molecules that came from the bulk.
thermal equilibrium and additionally multiplied by a factor of 2
due to stoichiometry. This gave us the enhancements shown in
Figure 6a.
This field dependence of 15N signals is reminiscent of the
analogous field dependence for other substrates, for example,
for 15N-enriched pyridine.[21] Good agreement is seen between
experiments and calculated curves shown as the solid lines in
Figure 6a. The experimental function is somewhat broader,
probably, due to the presence of additional spins in the system
that were not considered in the simulation or due to limited
lifetime of the active complex where polarization transfer
happens. The calculation was done for a simplified spin system
with just 6 spins: 2 1H spins coming from the parahydrogen and
The enhancement factors ɛ for the 15N signals in the
complex are difficult to measure because it would take too
much time to acquire thermally polarized signals for them.
Therefore, the presented enhancements were estimated using
the following procedure: first, the amount of complexes with
4
15N spins, corresponding to 2 cis-15N2-ABZ molecules in the
equatorial position of the Ir-IMes complex. The calculation was
done using the program iRelax available online;[26] the detailed
description of the calculation can be found in reference.[21] We
note that this calculation does not consider the spin dynamics
of the polarization transfer, nor the lifetime of the complex.
First, the projection of the initial singlet state of parahydrogen
onto the eigen states of the full spin system was calculated. All
coherences in the obtained density matrix were set to zero and
1
cis-15N2-ABZ was determined from the H spectrum. To do that
we integrated the signals of cis-15N2-ABZ and the signals
corresponding to hydrides at ~À 20 ppm in the 1H NMR
spectrum of thermally polarized sample. This was done for a
sample pressurized with 3 bar (same as in SABRE experiments)
of thermally polarized H2 gas. We assume that the 1H NMR
signal of the hydrides at around À 20 ppm can be used to
quantify the amount of the Ir-IMes complex with cis-15N2-ABZ
bound in the equatorial position (see Supporting Information).
The ratio between free cis-15N2-ABZ with respect to cis-15N2-ABZ
bound in the equatorial position determined in such a way was
found to be about 34. This ratio was then multiplied by the
ratio of the integrals in the 15N spectra of the hyperpolarized
signal with respect to the signal of the free cis-15N2-ABZ in
b
then the Liouville bracket of the obtained state with the Iz
operator of all 15N spins was taken. Interestingly, when larger
spin systems with several 1H spins of cis-15N2-ABZ were
calculated, they showed poor agreement between experimental
and calculated data. We believe that this discrepancy is due to
1
the weak polarization levels observed for the H spins. Hence,
the hyperpolarization of the 15N magnetization in cis-15N2-ABZ
was not strongly affected by the presence of 1H spins.
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The magnetic fields that were applied inside the magnetic
so-called T00 filter[23] was applied to destroy all spin orders
except for the singlet order. The following SLIC pulse converted
hyperpolarized singlet order of the 15N spin pair into either 15N
coherence (Figure 7a) or into coherence of the ortho-protons of
cis-15N2-ABZ (Figure 7b). Due to the T00 filter and the selective
excitation, these NMR spectra contain only the hyperpolarized
signal. A specific spectral feature was observed in the 15N
spectrum: the main line was surrounded by weak satellites with
the line splitting between them equal to approximately two
2JNN-couplings. These satellites correspond to enhanced outer
singlet-triplet coherences that sometimes can be observed in
singlet NMR experiments with nearly equivalent spin pairs.[28,29]
These experiments clearly show that apart from the 15N and
1H magnetization, singlet order of the 15N spin pair is populated
during SABRE SHEATH experiments. We note that equilibrium
population of the singlet order is usually zero at any magnetic
field. The observed lines are 400 times (for 15N) or 30 times (for
1H) stronger than the normal NMR signals.
shield (Figure 2a, stage ii) were always collinear to the field
inside the NMR spectrometer. The observed sign of the 15N
signal of the free cis-15N2-ABZ in SABRE SHEATH experiments
was always opposite to the thermally polarized one. This
observation confirms that the sign of the 1JHH-coupling between
the parahydrogen protons in the Ir-IMes complex is negative:
1
simulation with a positive JHH-coupling inverts the sign of 15N
polarization. This is also in agreement with the values for similar
complexes, but with different substrates.[27]
1
The field dependence of the H spin hyperpolarization in
cis-15N2-ABZ is shown in Figure 6b. Typically, 1H spins of
substrates are hyperpolarized by SABRE in a few mT magnetic
field.[27] However, the 1H spins of cis-15N2-ABZ were hyper-
polarized by SABRE only in the ultralow fields. This means that
they cannot be directly polarized by the parahydrogen protons
in the Ir-IMes complex, but instead their hyperpolarization arises
as result of polarization transfer from the hyperpolarized 15N
spins. Calculation of the field dependencies of the 1H spins
should take into account propagation of the polarization
averaged over the lifetime of the complex; such simulations go
beyond the scope of this work. There are two fields at which all
curves of the 1H spins peak: at 150 nT and ~500 nT. Data for the
ortho-protons look similar to the data of para-protons. Both
groups change sign of their polarization in the field range from
200 to 400 nT. When the field is lowered to almost zero no net
magnetization can be formed. However, some hyperpolariza-
tion is still observed; it corresponds to antiparallel orientation
between ortho- and meta-protons. Upon increasing the mag-
netic field beyond 1 uT no hyperpolarization is generated.
The field dependence of the hyperpolarization of 15N singlet
order was measured via the 15N channel (Figure 8a) and via the
1H channel (Figure 8b). Both curves shown in Figure 8 have a
similar appearance, which is expected as they correspond to
two different ways of observing the same hyperpolarized state.
Singlet order is formed most efficiently at zero magnetic field;
with increasing field the polarization level gradually drops to
zero. There is a wide range of magnetic fields (10-5 to 10À 2 T)
where the effect is roughly constant. The dependence is
somewhat similar to the SABRE experiments on singlet order of
the 15N spin pair in diazirines.[16,17] There is a substantial
formation of singlet order observable up to 10À 2 T. Theis et al.
explained this feature by observing that singlet order-enrich-
ment happens due to mixing of spin levels that do not depend
on the magnetic field.[16]
2.2. 15N Singlet Order Formed by SABRE SHEATH
We also measured the field dependence of the relaxation
times of the hyperpolarized 15N singlet order. The lifetime was
below 10 s at every field and thus not longer than the
corresponding longitudinal relaxation time of 15N spins in
Using the experimental scheme shown in Figure 2b, we
acquired NMR spectra detecting only the singlet order of the
15N spin pair of free cis-15N2-ABZ. Before the SLIC excitation,[22]
a
Figure 7. 15N NMR spectra (a) and 1H NMR spectra (b) showing a comparison of 15N2-ABZ signals in thermal equilibrium (blue) and after SABRE SHEATH
hyperpolarization at 10 nT of the singlet order of the 15N spin pair (red). The thermal spectrum in (a) was acquired in 4 scans with proton decoupling. This
narrowed the line and increased SNR. Hyperpolarized 15N spectrum exhibits outer singlet-triplet coherences. The experimental protocol for the SABRE
experiments is shown in Figure 2b.
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and bubbling of hydrogen is enough. Only cis-15N2-ABZ, but not
trans-15N2-ABZ, is hyperpolarized in its free form. On the other
hand, we observe complicated 15N spectra hyperpolarized by
SABRE SHEATH. There are many hyperpolarized signals corre-
sponding to 15N2-ABZ bound to Ir-IMes, and further research is
necessary to make the proper assignment of these complexes
and to measure their properties. Here, we focussed on studying
in detail SABRE SHEATH of free cis-15N2-ABZ and of cis-15N2-ABZ
bound to the equatorial position of the Ir-IMes complex.
Our study provides details about optimal fields to perform
SABRE SHEATH experiments with cis-15N2-ABZ in methanol.
Further interest in this molecule is caused by the presence of a
long-lived state in trans-15N2-ABZ. The lifetime of the singlet
order of the 15N spin pair in trans-15N2-ABZ reaches tens of
minutes even at high magnetic fields (>10 T),[12] thus making it
interesting for high-field MRI applications. Experiments combin-
ing SABRE hyperpolarization of cis-15N2-ABZ, its photoswitching
to trans-15N2-ABZ and storage of the hyperpolarization in the
LLS of the 15N spin pair are currently underway in our
laboratory.
Here we compared SABRE SHEATH experiments where the
15N magnetization is observed (Figure 2a) to those where the
15N singlet order is preserved and subsequently converted into
detectable signals (Figure 2b) in cis-15N2-ABZ. The spectral
enhancement is in the latter case reduced by a factor of about
10. The optimal field range to polarize 15N and 1H magnetization
lies between 200 and 500 nT, while the optimal magnetic fields
to polarize singlet order of the 15N spin pair are in the range
between À 100 to 100 nT, though even at relatively high
magnetic fields of 10À 2 T some hyperpolarized singlet order can
still be generated.
Figure 8. Field dependence of 15N NMR signals (a) and 1H NMR signals (b)
corresponding to singlet order of the 15N spin pair in cis-15N2-ABZ hyper-
polarized by SABRE SHEATH. The experimental protocol for the SABRE
experiments is shown in Figure 2b. All the functions were normalized by the
integral intensity of the corresponding signal intensity at thermal equili-
brium.
The achievable hyperpolarization level at optimal field that
is observed via the H channel is highest for the experiments
1
cis-15N2-ABZ. The absence of long-living character is however
expected for cis-15N2-ABZ and is in accordance with previous
observations.[19] One reason is that cis-15N2-ABZ experience
chemical exchange between the free and bound forms, with
the later form having in general much shorter relaxation times
and active mechanisms that may cause relaxation of the 15N
singlet order. The second reason is that even in the free form
the molecular geometry of cis-ABZ is not symmetric, making it
possible for dipolar interactions with the protons in the phenyl
rings to drive relaxation of the 15N singlet order. This was
thoroughly analysed by Stevanato et al. for a cis- and trans-
derivative of 2,3-13C2-butenedioic acid.[30]
where singlet order of the 15N spin pair is observed. The
enhancement ɛ~30 means that for achieving the same SNR in
the thermally polarized 1H NMR spectrum of cis-15N2-ABZ it
would take about 900 times longer acquisition time. Detection
of the hyperpolarized singlet order of the 15N spin pairs via the
1H channel seems to be an interesting perspective for several
reasons. First, the detection via the 1H channel is more sensitive
by ~2 orders of magnitude than detection via the 15N channel.
Second, this channel is preferable for potential MRI applications,
as most of MRI instruments are designed to detect the 1H
magnetization. Finally, we note that the scheme shown in
Figure 2b achieves highly selective excitation of the hyper-
polarized signals of free cis-15N2-ABZ. This selectivity is achieved
through the pulse sequence where first the gradient filter is
applied and then selective excitation by SLIC pulse is perfomed.
This allows observation of only the hyperpolarized component,
thus making cis-15N2-ABZ an interesting molecule, that can be
used in singlet-contrast MRI experiments.[31]
The enhancement factor for the observation of singlet order
1
via the 15N channel reaches ɛ~400 and ~30 via the H channel.
1
A factor of 10 between these values is expected, because H
spins in thermal equilibrium have an approximately 10 times
higher polarization than 15N spins.
3. Conclusions
In this work we studied SABRE SHEATH experiments of 15N2-ABZ
and have shown that this molecule is a suitable substrate. There
is no need to add other substrates (e.g. pyridine) to the solution
to activate the [Ir(IMes)(COD)Cl] precatalyst. Addition of cis-ABZ
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Manuscript received: February 27, 2021
Revised manuscript received: April 2, 2021
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SABRE SHEATH experiments on 15N2-
azobenzene are presented. It is
Dr. K. F. Sheberstov*, V. P. Kozinenko,
Dr. A. S. Kiryutin, Prof. H.-M. Vieth, H.
Zimmermann, Prof. K. L. Ivanov,
Dr. A. V. Yurkovskaya
shown that all nuclear spins in cis-
azobenzene can be hyperpolarized
by SABRE at suitable magnetic fields.
Enhancement factors (relative to 9.4
T) reach up to 3000 for 15N spins and
up to 30 for the 1H spins. Two ap-
proaches are compared to observe
either hyperpolarized magnetization
of 15N/1H spins, or hyperpolarized
singlet order of the 15N spin pair.
1 – 9
Hyperpolarization of cis-15N2-Azo-
benzene by Parahydrogen at
Ultralow Magnetic Fields