Synthesis and evaluation of 13C-labeled 5-5-dimethyl-1-pyrroline-N-oxide aimed at in vivo detection of reactive oxygen species using hyperpolarized 13C-MRI

Article abstract of DOI:10.1016/j.freeradbiomed.2018.11.013

Effective means to identify the role of reactive oxygen species (ROS) mediating several diseases including cancer, ischemic heart disease, stroke, Alzheimer's and other inflammatory conditions in in vivo models would be useful. The cyclic nitrone 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) is a spin trap frequently used to detect free radicals in vitro using Electron Paramagnetic Resonance (EPR) spectroscopy. In this study, we synthesized ¹³C-labeled DMPO for hyperpolarization by dynamic nuclear polarization, in which ¹³C NMR signal increases more than 10,000-fold. This allows in vivo ¹³C MRI to investigate the feasibility of in vivo ROS detection by the ¹³C-MRI. DMPO was ¹³C-labeled at C5 position, and deuterated to prolong the T₁ relaxation time. The overall yield achieved for 5–¹³C-DMPO-d₉ was 15%. Hyperpolarized 5–¹³C-DMPO-d₉ provided a single peak at 76 ppm in the ¹³C-spectrum, and the T₁ was 60 s in phosphate buffer making it optimal for in vivo ¹³C MRI. The buffered solution of hyperpolarized 5–¹³C-DMPO-d₉ was injected into a mouse placed in a 3 T scanner, and ¹³C-spectra were acquired every 1 s. In vivo studies showed the signal of 5–¹³C-DMPO-d₉ was detected in the mouse, and the T₁ decay of ¹³C signal of hyperpolarized 5–¹³C-DMPO-d₉ was 29 s. ¹³C-chemical shift imaging revealed that 5–¹³C-DMPO-d₉ was distributed throughout the body in a minute after the intravenous injection. A strong signal of 5–¹³C-DMPO-d₉ was detected in heart/lung and kidney, whereas the signal in liver was small compared to other organs. The results indicate hyperpolarized 5–¹³C-DMPO-d₉ provided sufficient ¹³C signal to be detected in the mouse in several organs, and can be used to detect ROS in vivo.

Full text of DOI:10.1016/j.freeradbiomed.2018.11.013

Author’s Accepted Manuscript
13
Synthesis and evaluation of C-labeled 5-5-
dimethyl-1-pyrroline-N-oxide aimed at in vivo
detection of reactive oxygen species using
13
hyperpolarized C-MRI
Keita Saito, Deepak Sail, Kazutoshi Yamamoto,
Shingo Matsumoto, Burchelle Blackman, Shun
Kishimoto, Jeffery Brender, Rolf E. Swenson,
James B. Mitchell, Murali C. Krishna
www.elsevier.com
PII:
S0891-5849(18)31572-7
https://doi.org/10.1016/j.freeradbiomed.2018.11.013
FRB14035
DOI:
Reference:
To appear in:
Free Radical Biology and Medicine
Received date: 10 September 2018
Revised date: 30 October 2018
Accepted date: 13 November 2018
Cite this article as: Keita Saito, Deepak Sail, Kazutoshi Yamamoto, Shingo
Matsumoto, Burchelle Blackman, Shun Kishimoto, Jeffery Brender, Rolf E.
Swenson, James B. Mitchell and Murali C. Krishna, Synthesis and evaluation of
13
C-labeled 5-5-dimethyl-1-pyrroline-N-oxide aimed at in vivo detection of
13
reactive oxygen species using hyperpolarized C-MRI, Free Radical Biology
and Medicine, https://doi.org/10.1016/j.freeradbiomed.2018.11.013
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Synthesis and evaluation of 13C-labeled 5-5-dimethyl-1-pyrroline-N-oxide aimed at in vivo
detection of reactive oxygen species using hyperpolarized 13C-MRI
Keita Saitoa,1, Deepak Sailb,1, Kazutoshi Yamamotoa, Shingo Matsumotoc, Burchelle Blackmanb,
Shun Kishimotoa, Jeffery Brendera, Rolf E. Swensonb, James B. Mitchella, and Murali C.
Krishnaa
aRadiation Biology Branch, National Cancer Institute, Bethesda, MD, USA
bImaging Probe Development Center, National Heart, Lung, and Blood Institute, Rockville, MD,
USA
cGraduate School of Information Science and Technology, Hokkaido University, Sapporo,
Hokkaido, Japan
*Corresponding author: Murali C. Krishna, Radiation Biology Branch, Center for Cancer
Research, National Cancer Institute, Building 10, Room B3B69, NIH, 9000 Rockville Pike,
Bethesda, MD 20892-1002. Phone: 301-496-7511; Fax: 301-480-2238; E-mail:
murali@helix.nih.gov.
ABSTRACT
Effective means to identify the role of reactive oxygen species (ROS) mediating several
diseases including cancer, ischemic heart disease, stroke, Alzheimer’s and other inflammatory
conditions in in vivo models would be useful. The cyclic nitrone 5,5-Dimethyl-1-pyrroline-N-
oxide (DMPO) is a spin trap frequently used to detect free radicals in vitro using Electron
13
Paramagnetic Resonance (EPR) spectroscopy. In this study, we synthesized C-labeled DMPO
13
for hyperpolarization by dynamic nuclear polarization, in which C NMR signal increases more
13
than 10000-fold. This allows in vivo C MRI to investigate the feasibility of in vivo ROS
13
13
detection by the C-MRI. DMPO was C-labeled at C5 position, and deuterated to prolong the
1 both contributed equally

T1 relaxation time. The overall yield achieved for 5-13C-DMPO-d9 was 15%. Hyperpolarized 5-
13
13C-DMPO-d9 provided a single peak at 76 ppm in the C-spectrum, and the T1 was 60 s in
phosphate buffer making it optimal for in vivo 13C MRI. The buffered solution of hyperpolarized
5-13C-DMPO-d9 was injected into a mouse placed in a 3 T scanner, and 13C-spectra were acquired
every 1 s. In vivo studies showed the signal of 5-13C-DMPO-d9 was detected in the mouse, and
the T1 decay of 13C signal of hyperpolarized 5-13C-DMPO-d9 was 29 s. 13C-chemical shift imaging
revealed that 5-13C-DMPO-d9 was distributed throughout the body in a minute after the
intravenous injection. A strong signal of 5-13C-DMPO-d9 was detected in heart/lung and kidney,
whereas the signal in liver was small compared to other organs. The results indicate
13
hyperpolarized 5-13C-DMPO-d9 provided sufficient C signal to be detected in the mouse in
several organs, and can be used to detect ROS in vivo.
Graphical Abstract:
Key words: hyperpolarized 13C-MRI; spin trapping; DMPO: reactive oxygen species
INTRODUCTION:
Nitrone and nitroso based spin traps have been used to trap short lived free radicals with
life times in the order of nanoseconds to stabilize as radical spin-adducts with longer half-lives
(minutes) which can be detected by Electron Spin Resonance (EPR) spectroscopy (1). From the

EPR spectra of the spin-adducts which accumulate to detectable levels, the identity of the short-
lived intermediates can be confirmed (2). The EPR-spin trapping methodology has been used to
detect, characterize and quantify short-lived intermediates generated in various radiolytic,
photochemical, sonochemical, and enzymatic reactions (3-6). Nitrone based spin traps such as
DMPO, PBN (N-tert-Butyl-α-phenylnitrone)
and POBN (α-(4-Pyridyl N-oxide)-N-tert-
butylnitrone) have been widely used in chemical and cellular studies to detect free radical
adducts using EPR spectroscopy (1,7-9). Newer derivatives of DMPO such as 5-
(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), 2-ethoxycarbonyl-2-methyl-
3,4-dihydro-2H-pyrrole-1-oxide (EMPO), 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide
(BMPO) have been developed for enhanced sensitivity in detecting radicals where the spin
adducts are longer lived (4,10-16). Nitrones have been shown to have minimal to no toxicity in
vitro and in vivo (~200 mg/kg in mice, rats) and were effective as antioxidants in vitro and in
vivo against various types of oxidative stress (5,17-20). Free radical adducts of nitrones such as
PBN and POBN were detected after in vivo administration in mice which were exposed to whole
body ionizing radiation and the protective effects were attributed to their radical scavenging
ability (18,19,21). While EPR spectroscopy has been widely used to detect free radical
intermediates, NMR based techniques and immune-spin trapping techniques have enabled the
use of nitrones using NMR (22-24), immunology and MRI methods for the detection of free
radical based end products in vitro and in vivo (23-28). Of the nitrones developed for spin
trapping applications, the PBN derivative NXY-059 has been evaluated for clinical use in
protecting against post ischemic reperfusion injury in ischemic stroke in brain (21,29-31).
Carmichael et al., conducted a systematic study to quantify the EPR-detectable radical
adducts formed when a known quantity of free radicals were generated using ionizing radiation
(32). Free radicals with poor reactivity such as superoxide were trapped with > 90% efficiency

generating the corresponding spin-adduct (4,14) (33). Superoxide radical reactions occur
primarily at the nitrone bond of DMPO yielding the DMPO-O2-. spin adducts which themselves
are short lived and are converted to the DMPO-OH radical which is a more persistent radical (Eq
1, 2).
DMPO + O2-.
b
DMPO-OOH
DMPO-OH
(Eq 1)
(Eq 2)
DMPO-OOH b
Conversely, nitrone traps yielded radical adducts of the hydroxyl radical in the range of 14 –
35% in spite of diffusion limited rates of reaction (Eq 3).
.
DMPO + OH b DMPO-OH
(Eq 3)
The low yield of the spin adducts of these highly reactive radicals, regardless of the efficient
rates of reaction were attributed to non-selective reaction on the spin trap other than at the
nitrone bond, which are not detectable by EPR (Eq 4).
.
DMPO + OH b
DMPO. + H2O
(Eq 4)
Greater than 60% of the reactions of short lived radicals with nitrones result in
intermediateswhich are also transient and ultimately form stable diamagnetic end products (Eq 5)
R 2.6
(32).
DMPO. b Diamagnetic end products
(Eq 5)
To monitor such transient intermediates of DMPO reaction with highly reactive radicals,
which are rapidly converted to diamagnetic end products, hyperpolarized MRI methods may be

applied since the hyperpolarized spin states in DMPO may survive the brief paramagnetic state
of the intermediates. While the paramagnetic intermediates cannot be detected, the diamagnetic
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end-products produced in reactive oxygen species (ROS) can be detected by C MRI. The
13
injected 5-13C-DMPO and the ROS mediated diamagnetic end-products will have distinct C
chemical shifts from which are distinguishable.
Hyperpolarization of the nuclear spin states using dynamic nuclear polarization (DNP) in
13C labeled at sites with T1 ~ 30 s provided > 10,000 fold enhancement in sensitivity for 13C MRI
and allowed metabolic studies of the conversion of these substrates to their products in vivo in
pre-clinical and clinical settings. Requirements for a candidate molecule suitable for in vivo
hyperpolarized MRI include: a) to be non-toxic at high bolus concentrations for in vivo
administration; b) have a carbon atom in the molecule with T1 longer than 20 s; c) high aqueous
solubility (> 5 M); d) form a glass when mixed with the paramagnetic trityl radicals and frozen
to enable DNP. Molecules which satisfy these conditions can be detected with high sensitivity
after DNP by in vivo 13C MRI. Endogenous molecules such as pyruvate, fumarate, a-
ketoglutarate etc., are examples of molecules where the carbonyl carbon atoms have been
13
enriched with C and polarized to provide 4 orders of magnitude increase in sensitivity. This
enhanced sensitivity allowed detection and imaging of these substrates and their metabolic
products in vivo. With the 4-5 orders of sensitivity enhancement provided by DNP, it is possible
in practice to detect and image ~ 5 uM of ROS-induced diamagnetic end products in vivo.
DMPO is liquid at room temperature and it forms glass when it is frozen with trityl
radicals, making it suitable for DNP. In this study, we identified C-5 as a site in the molecule
with a long T1 for C atom, synthesized 13C-labeled DMPO, and investigated feasibility of
13
hyperpolarized C- MRI and established conditions to image its biodistribution in vivo in a

mouse. In vivo models of oxidative stress where high levels of reactive oxygen species (ROS)
are generated focally, hyperpolarized MRI methods may become practical for the detection and
imaging of free radical processes.
MATERIALS AND METHODS
Reagents
DMPO was purchased from Dojindo Laboratories. OX063 and the symmetric trityl
radical (commonly known as the “Finland radical”) were purchased from GE Healthcare. Tris,
ethylendiaminetetraacetic acid (EDTA), hypoxanthine, Fe-EDTA, SOD, and catalase were
purchased from Sigma-Aldrich. Xanthine oxidase (XO) was purchased from Roche Molecular
Biochemicals (Indianapolis, IN, USA). Isotopically labeled compounds were obtained from
Cambridge Isotope Laboratories, Inc., MA. All other reagents and solvents were obtained from
Sigma Aldrich, Inc. and used as received unless otherwise noted. Flash chromatography was
performed on a Teledyne Isco CombiFlash Companion instrument with UV detection at 220 and
254nm. Analytical HPLC analysis was performed on an Agilent 1200 Series instruments
equipped with multi wavelength detectors using an Agilent SB C18 column (4.6 × 50mm, 3.5
µm) with a flow rate of 1 mL/min. Solvent A was 0.05 % TFA in water, solvent B was 0.05 %
TFA in ACN, and a linear gradient of 5% B to 95% B over 10 min was used. APCI mass
spectrometry was performed on 6130 Quadrupole LC/MS Agilent Technologies instrument
equipped with diode array detector. HRMS data were acquired using Waters XEVO G2-XS Q-
1
Tof in ESI positive mode. H and 13C NMR spectra were recorded with a Varian spectrometer
operating at 400MHz and 101 MHz respectively. Chemical shifts are reported in parts per
million (δ) and are referenced to tetramethylsilane (TMS).
Synthesis of 5-13C-DMPO-d9

Schemes of 5-13C-DMPO-d9 synthesis are summarized in Figure 1.
Acetone-[2-13C, d6] (3): A 3N LiOD solution in D2O (0.625 mL) was added to Acetone-[2-13C]
(10.0 g, 170 mmol) and D2O (20.0 mL). The reaction mixture was stirred for 72h at RT. The
acetone was distilled via short path distillation. The distillate was subjected to the same reaction
conditions except that the stirring was for 48 h. The process was repeated after second and third
distillation. At the end of four cycles, 98.6 % deuteration of compound 3 (9.87 g, 152 mmol) was
obtained in a 89% yield. 13C NMR (101 MHz, D2O) δ215.5 (C–2)
Isopropanol-[2-13C, d7] (4): Ruthenium on carbon (5%, 4.8 g) was added to a solution of
acetone-[2-13C, d6] compound 3 (9.8 g, 150.6 mmol) in D2O (40.0 mL) in a Parr reactor vessel.
Air from the vessel was evacuated and replaced with deuterium gas at 45 PSI pressure. The
reaction vessel was shaken for 16 h keeping the pressure of deuterium at 45 PSI. A small aliquot
was analyzed by 13C NMR and the reaction was found to be complete. The reaction mixture was
filtered over a celite pad and the residue washed with deionized water. The combined filtrate was
distilled via a short path distillation. The distillate obtained was the pure desired product 4 (9.11
g, 136 mmol) in an 90% yield. 13C NMR (101 MHz, D2O) δ63.51 (C–2, t, J = 22.2 Hz)
2-Bromopropane-[2-13C, d7] (5): A solution of 48 % DBr in D2O (50.0 mL) was slowly added to
Isopropanol-[2-13C, d7] compound 4 (9.10 g, 136 mmol) at 0°C. The reaction mixture was
stirred for 16h at RT. After a short path distillation the product 5 (13.2 g, 103 mmol) was
obtained in a 76% yield. 13C NMR (101 MHz, CDCl3) δ44.85 (C–2, t, J = 23.3 Hz)
2-Nitropropane-[2-13C, d7] (6): Silver nitrite (16.20 g, 105.3 mmol) was added to a mixture of
molecular sieves (4Å, 5g) and 2-bromopropane-[2-13C, d7] compound 5, (11.5 g, 88.4 mmol) in
1,4-dioxane (100.0 mL). The mixture was stirred in the dark for 48 h. It was then analyzed by 13C
NMR and found to be complete. The reaction mixture was filtered and the residue washed with

ether. The filtrates were combined and the solvent concentrated at reduced pressure, while
keeping the temperature of water bath below 20°C. The dried product 6 (5.10 g, 53.6 mmol) was
obtained in a 60% yield. 13C NMR (101 MHz, DMSO) δ78.21 (C–2, t, J = 22.8 Hz)
Methyl-4-methyl-4-nitro pentanoate-[2-13C, d9] (8): Methyl acrylate-2,3,3-d3 compound 7 (5.0
g; 53.6 mmol) was added to a solution of 2-nitropropane-[2-13C, d7] compound 6, (5.1 g; 52.5
mmol) and Triton-B (40% in H2O, 2.0 mL) in 1,4-dioxane (100.0 mL) at 70°C for 60 min. The
temperature of the reaction was then raised to 85°C and maintained for 3 h. The solution was
cooled to RT, diluted with 200 mL DCM and washed with 1 N HCl (300ml). The aqueous phase
was then washed with DCM (3x100 mL). All organic fractions were combined, dried with
magnesium sulfate and concentrated. The oil 8 (5.39g, 29.1 mmol) was obtained in a 52% yield.
δ13C NMR (101 MHz, CDCl3)15 δ86.8 (C–4), 51.72(–OCH3), 28.4 (C–3), 24.8 (C–2)
4-Methyl-4-nitro pentanal-[2-13C, d9] (9): DIBAL-H (1M in DCM, 32 mL, 32 mmol) was
added over 30 min. to a mixture of 4 Å molecular sieves (2.0 g), and nitropentanoate 8 (4.74 g;
25.6 mmol) in DCM (45 mL) at –78°C. The reaction mixture was stirred for 1h at –78°C under
an atmosphere of Ar. It was then quenched with addition of wet silica (20 g silica + 10 g water).
The temperature was raised to RT. The reaction mixture was then filtered and the residue washed
with 3 x 50 mL DCM. All DCM fractions were combined and washed with 300 mL 1N HCl
solution. Aqueous phases were extracted with 2 x 100 mL DCM. Organic fractions were
combined, dried with MgSO4, and concentrated provide product 9 (3.59 g, 23.1 mmol) in an 90%
13
13
yield. C NMR (101 MHz, CDCl3) δ199.8 (–CHO), 86.70 (C–4); Lit16: C NMR (75.47 MHz,
CDCl3) δ25.61 (–CH3), 32.09 (C–3), 38.60 (C–2), 87.09 (C–4), 199.68 (–CHO).
Dimethyl pyrroline-N-oxide-[5-13C, d9] (10): Acetic acid-d4 (5.98 g, 93.5 mmol) was added over
1 h to a mixture of activated zinc dust (3.05 g; 47.7 mmol, 2.1 eq.) and pentanal (3.45 g, 22.2
mmol, 1.0 eq.) in ethanol-d (50 mL) at 0°C under Ar. The reaction mixture was then stirred at

4°C for 44 h. It was then diluted with cold ethanol (20 mL) and filtered. The residue was washed
with 4x75 mL cold ethanol. The ethanol phases were combined and concentrated. The crude
syrup obtained was chromatographed twice on a normal phase silica (100 % DCM to 9:1 DCM-
MeOH) to provide DMPO 10 (0.891 g, 7.23 mmol) in a 32% yield. 13C NMR (101 MHz, CDCl3)
δ73.1 (C–5); Lit17 : 13C NMR (101 MHz, CDCl3): δ23.9 (C–3), 25.2 (–CH3), δ33.8 (C–4), δ72.8
(C–5), 129.0 (nitronyl). ESI-MS: 124.1 (M+).
1
The NMR data is as follows: H NMR (400 MHz) CDCl3 δ6.79 (dd, J = 6.7, 2.7 Hz, 1H), 2.55
(d, J = 4.4 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 73.1 (C–5);Lit17 : 13C NMR (101 MHz,
CDCl3): δ23.9 (C–3), 25.2 (–CH3), δ33.8 (C–4), δ72.8 (C–5), 129.0 (nitronyl). HRMS (ESI/Q-
TOF) m/z: [M+H]+ Calcd for C513CH3D9NO+ 124.1512; Found 124.1513
Animal studies
All animal experiments were carried out in compliance with the Guide for the care and
use of laboratory animal resources (National Research Council, 1996) and approved by the
National Cancer Institute Animal Care and Use Committee (NCI-CCR-ACUC (Bethesda).
Female C3H/Hen mice were supplied by the Frederick Cancer Research Center, Animal
Production (Frederick, MD).
The mice were anesthetized with isoflurane (4% for induction and 1.5-2.5 % for
maintaining anesthesia) in medical air (500 mL/min) and were positioned prone in a MRI
scanner. During MRI scans, the breathing rate of the mouse was monitored with a pressure
transducer (SA Instruments Inc., Stony Brook, NY) and was maintained at 70 15 breaths per
minute. Core body temperature was also monitored with a non-magnetic rectal temperature
probe (FISO Technologies, INC., Quebec, Canada) and was maintained at 36 1°C with a flow
of warm water. For administration of 5-13C-DMPO-d9, a 30-gauge needle was cannulated into the
tail vein and was extended using polyethylene tubing (PE-10).

Hyperpolarized 13C-MRI studies
MRI scans were performed on a 3 T scanner controlled with EasyScan and PowerScan
(MR Solutions, Acton, MA), a 4.7 T scanner controlled with ParaVision 5.1 (Bruker Bio-Spin
MRI GmbH), or a 3 T scanner (Achieva 3T-TX, Philips Healthcare, Best, Nertherland). A 17
mm home-built 13C solenoid coil placed inside of a saddle coil for 1H and a 35 mm 13C-1H saddle
coil was used for phantom and a mouse, respectively. In the MRI scans of mice body, T2-
weighted anatomical images were obtained using a fast spin echo sequence (FSET2) with TE of
13 ms, TR of 2,500 ms, 8 slices, 2 mm thickness, resolution of 0.25 × 0.25 mm. 5-13C-DMPO-d9
(30 μL) containing 15 mM symmetric trityl and 2.5 mM gadolinium chelate ProHance (Bracco
Diagnostics, Milano, Italy) was hyperpolarized using the Hypersense DNP polarizer (Oxford
Instruments, Abingdon, UK) as described previously. After 60–90 min, the hyperpolarized
sample was rapidly dissolved in 4.5 mL of PBS containing 100 mg/L EDTA. The hyperpolarized
5-13C-DMPO-d9 solution (60 mM) was intravenously injected through a catheter placed in the tail
vein of the mouse (12 μL/g body weight). For the dynamic study, 13C spectra in mouse body were
acquired every 1s for 240 s. 13C two-dimensional spectroscopic images were also acquired 25-47
s after the beginning of the DMPO injection, with a 32 × 32 mm, field of view in a 8 mm coronal
slice through the body, a matrix size of 16 × 16, spectral width of 3330 Hz, repetition time of 85
ms, and excitation pulse with a flip angle of 10º. The total time required to acquire each image
was 23 s. In the MRI scans of mice head, 5-13C-DMPO-d9 was hyperpolarized using the SPINlab
13
(GE Healthcare) for 3–4 hours, and the scans were performed using the Philips 3 T scanner. C
two-dimensional spectroscopic images were acquired 30 s after the beginning of the DMPO
injection, with a 28 × 28 mm, field of view in a 10 mm axial slice through the head, a matrix size
of 14 × 14, spectral width of 3333 Hz, repetition time of 86 ms, and excitation pulse with a flip
angle of 3º.

RESULTS
Synthesis of 13C-labeled DMPO (Deepak)
Preliminary 13C NMR spectroscopy studies conducted using a 4. 7 T MRI scanner
identified that of all carbon atoms, the carbon atom at the 5-position of DMPO (Figure 2A) with
a chemical shift of 76 ppm (Figure 2B) had the longest T1 (Figure 2C). Since DMPO is
amphipathic, we tested the polarization efficiency using, the Finland-COOH radical, which is
also an amphipathic paramagnetic probe. A frozen glass mixture of the DMPO and the Finland-
COOH radical was polarized for 60 minutes and rapidly dissolved to room temperature and
measuring its NMR spectrum immediately. A significant enhancement of the signal intensity of
13
the C NMR signal was observed (Figure 2D) compared to the signals from DMPO polarized
13
with OX063 (Figure 2B). Based on these observations, DMPO enriched with C-labeled at C5
position and deuterated was synthesized to further prolong the T1 relaxation time.
Since the initial synthesis of unlabeled DMPO (34,35) several other researchers have
synthesized DMPO and its analogs using various routes (36-42). Based on works of Beth et al.
(43) and Janzen et al. (38), synthesis of labeled DMPO as a spin trap agent was first explored by
13
Pou et al. (37). In order to prepare DMPO with C at position C5 and deuterated protons on
neighboring carbons (to prolong the T1 relaxation times) we followed the procedure of Le et al.
13
(41) and Leinisch et al. (42). We found that the C-C2 deuterated three carbon starting material
13
could be effectively prepared from, the h-d exchange on acetone C-C2 as described in the
literature (44) under basic conditions. Per-deuterated acetone-13C-C2 was then reduced to
isopropanol-13C-C2 with Pd/C under D2 atmosphere. Labeled isopropanol was brominated with
DBr, converted to 2-nitropropane with silver nitrite and then alkylated with methyl acrylate-
2,3,3-d3.

The resulting nitropentanoate was reduced to aldehyde with DIBAL-H. This was
followed by treatment with activated zinc and acetic acid to yield the cyclic nitrone, DMPO.
However, the resulting DMPO was not fully deuterated due to the DIBAL reduction. The overall
yield achieved for DMPO-d9 was 15%. Figure 1 shows the overall synthetic scheme followed.
Hyperpolarization of 13C-DMPO
For successful polarization, the molecule to be polarized should dissolve at high
concentrations (~ 5 M) and form a homogenous glass with the trityl radical when frozen. Of the
13
four trityl radical analogs for hyperpolarization of C-DMPO, namely OX063, Finland trityl,
OX063-HCl, Finland-COOH, Finland-COOH was found to be the most suited for glass
formation and polarization of DMPO. Figure 3A shows the polarization build-up of DMPO with
13
natural abundance C (open circles) and 5-13C-DMPO-d9 (closed circles) with 15 mM Finland-
COOH and 2.5 mM Gd chelate ProHance. The time constant of polarization build-up in the
hyperpolarizer was found to be ~ 23 min. After the polarization was maximal, the hyperpolarized
samples were dissolved in PBS containing EDTA, and 13C-spectra were measured every 1 s using
the 4.7 T scanner with 10 deg flip angle. The T1 relaxation time of non-enriched/non-deuterated
DMPO and 5-13C-DMPO-d9 was 26 s and 60 s, respectively, suggesting that deuteration of
DMPO extended the T1 significantly (Figure 3B). The T1 of 5-13C-DMPO-d9 was similar to that
13
of 1-13C-pyruvic acid, which is the most frequently used probe in hyperpolarized C-MRI in
13
vivo, suggesting C-DMPO has sufficiently long T1 to permit in vivo studies. With the known
toxicological data of DMPO from prior studies where large bolus doses have no adverse effects
(20) and with the long T1 of the 5-C atom, the results suggest that imaging of hyperpolarized
DMPO in vivo monitoring its biodistribution and its transformation is feasible.
In Vivo 13C MRI of hyperpolarized 5-13C-DMPO-d9

The in vivo behavior of DMPO was tested in a mouse where the DMPO after dissolution
of the hyperpolarized mixture was administered through a tail vein cannula of a mouse placed in
1
13
a double tuned H/13C coil and the C signal monitored every 1 s using the 3 T scanner with 10
deg flip angle pulse. The signal of 5-13C-DMPO-d9 was detected in the mouse body immediately
after its injection and detectable for more than 150 s (Figure 4A). Figure 4B shows the signal
intensity curve of 5-13C-DMPO-d9 after iv administration of the hyperpolarized sample in the
mouse. From the signal intensity curve, the T1 decay of 5-13C-DMPO-d9 in mouse was estimated
to be 29s. The persistence of the hyperpolarized signal in vivo for this duration points to the
feasibility of imaging DMPO biodistribution and possible use for monitoring its transformation
either enzymatically or through reactive oxygen species.
13
We carried out C-chemical shift imaging in a mouse body after iv administration of
hyperpolarized 5-13C-DMPO-d9. Figure 5A shows consecutive anatomical slices (2 mm thickness
each) of the mouse body. Figure 5B shows the 13C-CSI overlaid on the anatomic image obtained
from 8mm slice thickness of the mouse body 25-47 s after hyperpolarized 5-13C-DMPO-d9
injection. The DMPO map obtained from the chemical shift image (Figure 5C) revealed that 13C-
DMPO was distributed through the mouse body within a minute of its injection. The signal was
higher in the heart/lung region and kidney, whereas the signal from liver was relatively low
compared to the other region (Figure 5D, E). To obtain a higher signal in the mouse liver, we did
slice selected 13C-MRS by averaging 100 transients (Figure 5 F). Signal to noise ratio was much
13
better compared to C-CSI. The relative levels of DMPO in the heart, kidney and liver regions
are shown in Figure 5G. No metabolites were detected in the normal mouse liver suggesting that
enzymatic conversion of DMPO was minimal.
Several studies showed that DMPO exhibits anti-oxidative and anti-inflammatory effects
in vivo (45,46). A recent study showed that DMPO injected directly into the intranigral region in

rats afforded protection against 6-OH-Dopamine mediated neurotoxicity (10). With the enhanced
sensitivity provided by hyperpolarization, it is possible to examine the biodistribution of DMPO
in the brain following systemic administration intravenously. A tail vein cannula was placed in
an anesthetized mouse placed in a doubly tuned resonator encompassing the head region. Figure
6A shows the 13C-CSI overlayed on the anatomic image in an axial profile.
DMPO
biodistribution in the brain was monitored immediately after hyperpolarized DMPO was
13
administered (Figure 6B). The C image shows evidence of DMPO accumulation in the brain
region suggesting that it penetrates the blood-brain barrier rapidly and accumulates at effective
concentrations while retaining polarization. This image represents the first imaging evidence for
the localization of DMPO in the brain and may explain the observed antioxidant effects it exerts.
DISCUSSION:
DNP of low g nuclei in molecules enriched with 13C, 15N has allowed imaging of
13
endogenous molecules and their biochemical transformations in vivo. C enriched pyruvate,
fumarate, urea, glucose, gluconolactate, bicarbonate, have been successfully imaged in vivo in
various animal models to obtain information pertaining to metabolism, perfusion and pH.
Exogenous molecules such as ascorbate, glycerate, methyl glutamine were also developed and
tested for probe for perfusion, pH. Most probes report on biochemical, physiologic, and
microenvironmental features. Dehydroascorbate has been used to probe the levels of ROS by
monitoring its transformation to ascorbate and determining the ration of DHA/AA (47).
However, this transformation being a 2-electron process, the ratio may also be significantly
influenced by the tissue redox status. The search for hyperpolarizable antioxidants which can
report on oxidative stress and provide noninvasive capabilities to monitor these reactions led to
considering nitrones as candidate probes for the following reasons: a) Nitrones can have carbon

13
atoms which can have potentially long C T1s. b) Nitrones are non-toxic even at high bolus
doses typically used for HP MRI; c) NXY-059, a nitrone derivative has been characterized to
have anti-oxidant activity and has been developed and used for human applications to limit post-
ischemic reperfusion injury (21). DMPO shares many of these features and has been successfully
polarized. DMPO has long been used as a spin trap to scavenge reactive oxygen species, identify
the intermediates and quantify them in simple chemical systems using EPR spectroscopy.
However, in cellular studies, it was of limited use since the spin adducts undergo rapid reduction
to diamagnetic products which are EPR invisible. NMR-Spin trapping overcame some of these
limitations. In vivo detection of ROS was demonstrated by EPR by administering nitrone spin
traps and analyzing the tissue extracts (22,24). Direct in vivo detection of spin adducts was also
demonstrated using POBN by using low-frequency EPR by exposing tumors to high radiation
doses (19). These approaches however, cannot be translated to in vivo imaging because of poor
sensitivity.
HP-MRI with 5-13C-DMPO-d9 has the potential to provide imaging assessment of reactive
oxygens species because: a) the signals of the original molecule can be significantly enhanced by
the DNP method; b) the molecule biodistribution in vivo is effective to have adequate global
concentrations in organs/regions of interest; c) appropriate models of ROS mediated conversion
of the DMPO to stable diamagnetic products proceeds with sufficient efficiency to have
detectable levels of products with characteristic chemical shifts. The hyperpolarized DMPO
13
biodistribution monitored by C MRI revealed that significant levels were attained in heart,
kidney, brain with detectable levels in the liver and muscle. The demonstration of the in vivo
13
biodistribution of DMPO, by C MRI using hyperpolarization techniques sets the stage for
oxidative stress evaluation in vivo. With suitable in vivo models of focal ROS generation at high

levels such as post-ischemic reperfusion injury or systemic ROS generation such as with
lipopolysaccharide challenge, this method can be applied for in vivo imaging of oxidative stress.
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Figure 1: Summary of schemes of 5-13C-DMPO-d9 synthesis.
Figure 2: (A) Position of 13C-labeled in DMPO which has the longest T1. (B) 13C-MR spectrum of
unlabeled DMPO hyperpolarized with OX063. Single peak was detected at 76 ppm which was
from 13C atom at the 5-position of DMPO. (C) The signal decay after dissolution in PBS. T1 was
13
calculated to be 26 s from the decay curve. (D) C-MR spectrum of unlabeled DMPO with
13
natural abundance C, hyperpolarized with Finland-COOH. The signal to noise ratio of DMPO
peak was remarkably increased by using Finland-COOH.
13
Figure 3: (A) Hyperpolarization build-up of unlabeled DMPO (○) and C-labeled DMPO-d9 (●)
with 15 mM Finland-COOH and 2.5 mM Gd chelate ProHance. The build-up was monitored by
an NMR spectroscopy in the hyperpolarizer. (B) The decay of 13C-signal at 76 ppm of unlabeled
DMPO (○) and 13C-labeled DMPO-d9 (●). Deuteration of DMPO extended the T1 of the 13C signal
from 26 s to 60 s.
13
Figure 4: (A) Dynamic C-MR spectra in a mouse body after hyperpolarized 5-13C DMPO-d9
injection. The spectra were acquired every 1 s for 240 s. DMPO peak (76 ppm) was obserbed.
(B) Signal intensity curve of DMPO calculated from the spectra. The T1 was estimated to be 29 s
from the curve.
Figure 5: (A) T2-weighted anatomical images of a mouse body (4 adjacent slices). The thickness
13
of each slice was 2 mm. (B) C-chemical shift image (CSI) in the mouse body acquired 25 s
13
after hyperpolarized 5-13C-DMPO-d9 injection. The C-CSI was obtained from 8 mm thickness

slice, and the size of each pixel was 2 mm × 2 mm. (C) Distribution of DMPO in the mouse body
13
calculated from the C-CSI. The positions of heart/lung, liver, and kidney were obtained from
13
13
the T2-weighted anatomy. (D, E) C-MR spectrum of heart and left kidney obtaied from the C
13
CSI. (F) C-MR spectrum from a mouse liver with 100 times averaging. (G) Averaged signal
inteisty of 13C-DMPO in each organ calculated form the DMPO distribution map.
13
Figure 6: (A) C-CSI in the mouse head acquired 30 s after hyperpolarized 5-13C-DMPO-d9
injection. The CSI was obtained from 10 mm thickness slice, and overlaied on a 2 mm thickness
of T2-weighted anatomical image. (B) Distribution of DMPO in the mouse head calculated from
the 13C-CSI.
Figure S1: 1H NMR spectra of DMPO.
Figure S2: 13C NMR spectra of DMPO
Highlights
ꢀ
ꢀ
ꢀ
ꢀ
Spin traps/radical scavengers cannot be currently imaged by MRI
13C labeled spin trap DMPO can be imaged in vivo after hyperpolarization
Its biodistribution was followed in vivo after hyperpolarization.
13C MRI with hyperpolarized DMPO can be used for in vivo free radical
detection/imaging.

Scheme 1: Synthesis of 2-Nitropropane-[2-13C;d7].
Scheme 2: Synthesis of Methyl 4-methyl-4-nitropentanoate-[2-13C; 2,3,3-d3]
Scheme 3: Synthesis of DMPO-[5-13C, d9]
Figure 1

A
N+
O-
D
200
180
160
140
120
100
80
60
40
20
Chemical shift (ppm)
Figure 2

(A)
(B)
100
16000
Non-labeled DMPO
13C-labeled DMPO
Non-labeled DMPO
13C-labeled DMPO
90
80
70
60
50
40
30
20
10
0
14000
12000
10000
8000
6000
4000
2000
0
0
10
20
30
40
50
60
70
0
50
100
150
200
-10
Time (min)
Time (sec)
Figure 3

20000
15000
10000
5000
0
(A)
(B)
0
50
100
150
200
Time (sec)
Figure 4

(A)
(B)
Heart/Lung
(C)
Liver
Kidney
160
140
120
100
80
160
140
120
100
80
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
Liver
(100 Average)
Heart
Left kidney
(E)
(D)
(F)
60
60
40
40
20
20
0
0
-20
-20
110
100
90
80
70
60
50
40
110
100
90
80
70
60
50
40
110
100
90
80
70
60
50
40
Chemical shift (ppm)
Chemical shift (ppm)
Chemical shift (ppm)
800
700
600
500
400
300
200
100
0
(G)
Heart
Liver
Left kidney
Figure 5

(A)
(B)
100
29
0
Figure 6