Design of a 13C magnetic resonance probe using a deuterated methoxy group as a long-lived hyperpolarization unit

Article abstract of DOI:10.1002/anie.201202885

How to live longer: A fully deuterated ¹³C methoxy group ( ¹³CD₃O) is presented as a new long-lived hyperpolarization unit for designing a sensitive ¹³C magnetic resonance probe. By utilizing the unit, a hyperpolarized magnetic resonance probe for sensing hypochlorous acid was successfully designed. Copyright

Full text of DOI:10.1002/anie.201202885

Angewandte
Chemie
DOI: 10.1002/anie.201202885
Magnetic Resonance Probes
13
Design of a C Magnetic Resonance Probe Using a Deuterated
Methoxy Group as a Long-Lived Hyperpolarization Unit**
Tomohiro Doura, Ryunosuke Hata, Hiroshi Nonaka, Kazuhiro Ichikawa, and Shinsuke Sando*
There has been long-term effort toward molecular analysis in
living systems. For such a purpose, magnetic resonance (MR)
is one of the most promising techniques. Because of the good
penetration of radio waves, a MR-based chemical probe (MR
probe) can be detected even in a deep site of opaque bodies.
Therefore, various MR probes have been designed.[1]
Although they achieved good results in some cases, MR
probes have an intrinsic drawback, namely, low sensitivity.
Hyperpolarization methods,[2] including dynamic nuclear
polarization (DNP),[3] have been studied to overcome this
drawback. The hyperpolarization methods allow polarization
of nuclear spin populations, resulting in a large enhancement
of MR sensitivity compared to the thermally equilibrated
nuclei. It has been applied for in-cell or in vivo metabolic
analyses using stable isotope-enriched natural compounds.[2]
Recently, it was demonstrated that the concept of hyper-
polarization can be extended to MR probes for surveying the
chemical status of living systems.[4–12] Because carbon atoms
are present in almost all chemical probes, and sophisticated
13C-isotope labeling procedures are available, 13C-labeled
probes could be a choice for designing a hyperpolarized MR
probe. In practice, 13C-carbon dioxide,[10] 13C-benzoylformic
acid,[11] and 13C-ascorbic acid[12] have been successfully
designed as hyperpolarized MR probes to sense pH, H2O2,
and redox status, respectively.
hyperpolarization 13C unit for designing a hyperpolarized 13
C
MR probe. We successfully demonstrated the utility of this
unit by designing a hyperpolarized 13C chemical probe to
target hypochlorous acid (HOCl).[14]
For the new hyperpolarized 13C unit, we focused on alkoxy
or aryloxy groups. This group is one of the key structures for
designing target or environment-responsive chemical probes,
where it could work as a leaving group (Figure 1a). For
example, when R2 in Figure 1a is a 3-methylindole quinone or
Figure 1. How a) alkoxy or aryloxy and b) deuterated 13C methoxy
groups can produce alcohol when R2 is a reactive moiety that releases
these groups by target or environment-responsive reactions.
Almost all of the hyperpolarized 13C MR probes, however,
utilized a carbonyl carbon as a hyperpolarized 13C unit,
because of its long hyperpolarization lifetime. In other words,
the 13C unit, having sufficient hyperpolarization lifetime and
thus availability for designing a hyperpolarized 13C MR probe,
is almost limited to a carbonyl carbon. Such a strict structural
limitation makes it difficult to design a variety of hyper-
polarized 13C MR probes. Therefore, a new hyperpolarization
13C unit is highly desirable.
4-nitrobenzyl moiety, a hypoxic condition induces electron
reduction by reductase, resulting in an alkoxy/aryloxy-to-
alcohol conversion.[15] When R2 is a photoresponsive group
such as the 2-nitrobenzyl moiety, photoirradiation induces
alcohol release.[16] When R2O has the galactopyranoside
structure, it works as a substrate of b-galactosidase reporter
protein to afford an alcohol R1OH.[17] A similar alkoxy/
aryloxy-to-alcohol conversion is utilized in many environ-
ment-responsive prodrug designs.[15,18] Therefore, if the
alkoxy/aryloxy group joins in the hyperpolarization unit, it
can expand the design strategy of target or environment-
responsive hyperpolarized chemical probes.
Deuterated 13C is one of the promising candidate for
hyperpolarization 13C units.[13] Herein, we report a fully
deuterated 13C methoxy group (13CD3O) as a new long-lived
[*] T. Doura, R. Hata, Dr. H. Nonaka, Prof. S. Sando
INAMORI Frontier Research Center, Kyushu University
744 Motooka, Nishi-ku, Fukuoka (Japan)
We selected a fully deuterated 13C methoxy group
(13CD3O) as a candidate for the hyperpolarized alkoxy unit
(Figure 1b). A long hyperpolarized 13C lifetime is achieved by
nonprotonated 13C because of the lack of dipole–dipole
interaction with neighboring protons. In this sense, deutera-
tion is one of the most straightforward ways to increase the
hyperpolarization lifetime, as clearly demonstrated by deu-
terated choline chloride.[13] Furthermore, the hyperpolariza-
tion lifetime is highly dependent on molecular size. A smaller
size typically produces a longer hyperpolarization lifetime.
Therefore, we anticipated that a nonprotonated and small
E-mail: ssando@ifrc.kyushu-u.ac.jp
Prof. K. Ichikawa
Innovation Center for Medical Redox Navigation, Kyushu University
3-1-1, Maidashi, Higashi-ku, Fukuoka (Japan)
[**] This work was supported by the NEXT Program and partly by Grant-
in-Aid number 22685018 from JSPS. We thank Mr. T. Abe of Oxford
Instruments for helpful discussion and technical assistance on the
DNP experiments.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201202885.
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13CD3O unit could retain its hyperpolarized state for a rela-
tively long time.
favorable effect of deuteration in the methoxy group. The
[13C,D3]-p-anisidine seems to exist stably in blood at least in
DNP analysis time range (typically < 10 min) (Supporting
Information, Figure S1), but the 13C T1 value of [13C,D3]-p-
anisidine was shortened to be 23 s (9.4 T, 378C) in serum.
Importantly however, the observed T1 values of probe and
product were comparable or longer than those of hyper-
polarized 13C MR probes utilizing carbonyl carbon as a hyper-
polarization unit (T1 = 24.4 s at 11.7 T for 13C-benzoylformic
acid[11] and 15.9 s at 9.4 T for [1-13C]ascorbic acid[12a]),
supporting our idea that 13CD3O can be used in MR probes
as a hyperpolarization unit.
The hyperpolarization lifetime of the 13CD3O unit was
evaluated by measuring the spin–lattice relaxation time (T1),
which is directly related thereto. The T1 values were
determined by inversion recovery method using thermally
equilibrated compounds. The T1 value of the methoxy carbon
in CD3OH (3m in H2O/D2O = 9:1, 13C natural abundance)
was observed as 32.9 s (9.4 T, 378C), which was almost the
same as that of the carbonyl carbon in CH3CO2H (33.3 s,
9.4 T, 378C, 13C natural abundance). These results indicate
that a fully deuterated methoxy group 13CD3O has high
potential to be used as a long-lived hyperpolarization unit.
With the promising hyperpolarization unit 13CD3O in
hand, we then evaluated its practical utility by designing
a new chemical probe. A hyperpolarized 13C MR probe must
satisfy the following prerequisites: The probe should 1) have
We then investigated the reaction of the probe with
HOCl. The 13C chemical shift of [13C,D3]-p-anisidine probe
was observed at 56.6 ppm (Figure 2a, top). After reaction
a
13C-labeled nucleus with long T1 for a long hyperpolariza-
tion; 2) react with the target species rapidly within the
hyperpolarization lifetime; and 3) induce a sufficiently large
chemical shift change upon reaction.
We designed [13C,D3]-p-anisidine as a hyperpolarized 13C
MR probe that targets HOCl, an important ROS biomarker
for inflammatory diseases (Scheme 1a). The [13C,D3]-p-anisi-
Figure 2. a) 13C NMR spectra of thermally equilibrated [13C,D3]-p-anisi-
dine without (top) or with (middle) 2 equiv of HOCl, and (bottom)
13CD3OH. b) Time course of absorption changes (287 nm) of [13C,D3]-
p-anisidine in the presence (blue) or absence (red) of 2 equiv of HOCl.
The arrow indicates the addition point of HOCl.
with 2 equiv of HOCl, the peak at 56.6 ppm completely
disappeared and a new peak was observed at 49.6 ppm
(middle), which was assigned as 13CD3OH by comparison with
an authentic sample (bottom). HPLC analyses showed the
concomitant production of 1,4-benzoquinone imine (Support-
ing Information, Figure S2). These results indicate an efficient
reaction of [13C,D3]-p-anisidine with HOCl and production of
13CD3OH via the ipso-substitution mechanism shown in
Scheme 1a. The observed 13C chemical shift change of
7.0 ppm was sufficient to be monitored by 13C NMR spec-
troscopy.
Scheme 1. a) Chemical structure of [13C,D3]-p-anisidine and its expected
reaction with HOCl. b) Synthesis of [13C,D3]-p-anisidine.
dine has the 13CD3O structure, which could be used as a long-
lived hyperpolarization unit, on a HOCl-responsive p-amino-
phenyl scaffold.[19] It was anticipated that the [13C,D3]-p-
anisidine would react effectively with HOCl to release
13CD3OH by O-dealkylation by an ipso-substitution mecha-
nism (Scheme 1a). Furthermore, the HOCl-responsive reac-
tion is expected to induce a 13C chemical shift change large
enough to be monitored by 13C NMR spectroscopy.
Furthermore, [13C,D3]-p-anisidine reacted with HOCl
very quickly. When HOCl (200 mm) was added to the
[13C,D3]-p-anisidine (100 mm) solution, a rapid enhancement
of the absorption was observed and reached a maximum
within about 30 s (Figure 2b). On the other hand, no
absorption change was observed without HOCl. The
observed fast reaction kinetics was appropriate for DNP
analysis.
First, we measured 13C T1 values for the probe and
product. The [13C,D3]-p-anisidine was synthesized from 4-
nitrophenol using 13CD3I as a [13C,D3]-labeling reagent
according to the procedure in Scheme 1b and the Supporting
Information. The T1 values of [13C,D3]-p-anisidine and
13CD3OH (50 mm in H2O/D2O = 9:1) were determined as
44.4 s and 32.6 s (9.4 T, 378C), respectively (Scheme 1a).
Non-deuterated p-anisidine showed much shorter T1 value of
6.3 s (9.4 T, 378C, 13C natural abundance), suggesting the
Along with sufficient reactivity, [13C,D3]-p-anisidine also
exhibited high selectivity for HOCl. [13C,D3]-p-Anisidine was
intact after incubation with most of the biologically important
ROS (H2O2, ROOC, O2CÀ, and COH) and reactive nitrogen
species (RNS, ONOOÀ) except for HOCl and CNO (Support-
ing Information, Figure S3). [13C,D3]-p-Anisidine reacted
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with CNO, but the main product was not 1,4-benzoquinone
imine. Thus, 13CD3OH, the target product of the present
probe, was generated only when [13C,D3]-p-anisidine reacted
with HOCl.
Polarization of 3.8% and about 6100-fold signal enhance-
ment were achieved for [13C,D3]-p-anisidine, which was
slightly lower but still in the comparable range with that of
pyruvic acid (22000-fold signal enhancement) under the same
experimental and instrument settings. A large 13C NMR
signal derived from hyperpolarized [13C,D3]-p-anisidine was
observed with one scan (Figure 3b, left, 908 pulse angle). In
marked contrast, no signal was obtained for thermally
equilibrated [13C,D3]-p-anisidine under the same NMR mea-
surement conditions (Figure 3b, right, 908 pulse angle).
The hyperpolarized [13C,D3]-p-anisidine showed a suffi-
ciently long hyperpolarization lifetime. 13C NMR spectra of
the hyperpolarized [13C,D3]-p-anisidine (Ernst pulse angle,
188) were measured every 4 s and are shown in Figure 3c. The
time-resolved data revealed that a signal of hyperpolarized
[13C,D3]-p-anisidine could be observed for about 100 s after
starting the 13C NMR measurement.
All of the data indicate that the designed [13C,D3]-p-
anisidine satisfies the prerequisites: long 13C T1, rapid reaction
with HOCl, and large enough chemical shift change. We then
proceeded with evaluation of hyperpolarized [13C,D3]-p-
anisidine.
Hyperpolarization was achieved by the DNP method
(details on DNP procedures, experimental conditions, and
instrument settings are given in the Supporting Information).
Briefly, [13C,D3]-p-anisidine was mixed with OX63 radical in
[D6]DMSO/D2O (1:1) and subjected to hyperpolarization at
1.4 K. After 1.5 h, the sample was dissolved rapidly using
heated aqueous buffer and recovered for subsequent
13C NMR experiments (Figure 3a). Although the deute-
rium-decoupling procedure eliminates 13C–D coupling to
give a 13C singlet as demonstrated previously,[13] in this
experiment, deuterium-decoupling conditions were not used
so as to identify the deuterated 13C nucleus.
Finally, we applied the hyperpolarized [13C,D3]-p-anisi-
dine probe to the detection of HOCl. The hyperpolarized
[13C,D3]-p-anisidine (1.5 h polarization) was mixed with
2 equiv of HOCl and subjected to 13C NMR measurements.
Figure 3d shows a spectrum (908 pulse angle) observed 4 s
after reaction with HOCl. Along with the signal of the
hyperpolarized probe (56.7 ppm), a new signal was observed
at 49.8 ppm. This new signal corresponds to 13CD3OH, which
was produced by reaction of the probe with HOCl. Because of
the fast reaction, a HOCl-responsive probe-to-product con-
version was successfully monitored within the hyperpolariza-
tion lifetime; in this case, the peak of the product 13CD3OH
was observed as the main peak even 4 s after addition of
HOCl. These results demonstrate that the designed [13C,D3]-
p-anisidine, which has a fully deuterated 13C methoxy group,
functioned as a HOCl-responsive hyperpolarized 13C MR
probe.
In conclusion, we have presented a fully deuterated 13C
methoxy group (13CD3O) as a new 13C hyperpolarization unit
for designing a hyperpolarized 13C MR probe. The signifi-
cance of the designed 13CD3O group may be summarized as
follows. First is its high performance: The non-protonated and
small 13CD3O group was achieved a high hyperpolarization
efficiency and sufficient lifetime to be used as a long-lived
hyperpolarization unit. Second is its applicability: The
practical utility of the unit was demonstrated successfully by
developing a hyperpolarized chemical probe using the
13CD3O unit. The designed compound worked as a selective
and quick-response chemical probe for sensing HOCl,
a biomarker of inflammation. Practical in vivo applications
must await further studies on detection limit, biostability,
toxicity, bio-orthogonality, and distribution, although these
are beyond the scope of this initial study. Third is its
versatility: The alkoxy group is found in a variety of target
or environment-responsive chemical probes, wherein the
group could work as a leaving moiety. Therefore, the
13CD3O unit might be utilized as a hyperpolarization unit in
diverse chemical probes apart from the present HOCl-
responsive probe. Fourth is its simplicity: The 13CD3O unit
can be easily incorporated into chemical probes using
commercially available 13CD3I as an isotope source. All of
these advantages show the high potential of the designed
Figure 3. a) Illustration of experimental procedures. b) 13C NMR spec-
tra of hyperpolarized (4 s after rapid dissolution, left) and thermally
equilibrated (right) [13C,D3]-p-anisidine (10 mm). c) Time-resolved
13C NMR data of hyperpolarized [13C,D3]-p-anisidine (1.7 mm). The
peak amplitude of [13C,D3]-p-anisidine was plotted from 4 to 120 s after
initiation of the NMR acquisition. d) 13C NMR spectrum of hyperpolar-
ized [13C,D3]-p-anisidine (10 mm) observed 4 s after addition of HOCl
(20 mm). NMR spectra were measured with a 7.05 T NMR instrument
with 908 (b,d) and 188 pulse angles (c).
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13CD3O group as a new hyperpolarization unit for designing
various hyperpolarized MR probes.
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Chem. Sci. 2012, 3, 800.
Received: April 16, 2012
Revised: June 26, 2012
Published online: && &&, &&&&
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Keywords: deuteration · dipole–dipole interactions ·
.
hyperpolarization · nuclear magnetic resonance · spin–
lattice relaxation
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Angew. Chem. Int. Ed. 2012, 51, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Magnetic Resonance Probes
How to live longer: A fully deuterated 13
C
methoxy group (13CD3O) is presented as
a new long-lived hyperpolarization unit
for designing a sensitive 13C magnetic
resonance probe. By utilizing the unit,
a hyperpolarized magnetic resonance
probe for sensing hypochlorous acid was
successfully designed.
T. Doura, R. Hata, H. Nonaka,
K. Ichikawa, S. Sando*
&&&& — &&&&
Design of a 13C Magnetic Resonance
Probe Using a Deuterated Methoxy
Group as a Long-Lived Hyperpolarization
Unit
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!