A hydrogen peroxide-responsive hyperpolarized 13C MRI contrast agent

Article abstract of DOI:10.1021/ja111589a

We report a new reaction-based approach for the detection of hydrogen peroxide (H₂O₂) using hyperpolarized ¹³C magnetic resonance imaging (¹³C MRI) and the H₂O ₂-mediated oxidation of α-ketoacids to carboxylic acids. ¹³C-Benzoylformic acid reacts selectively with H₂O ₂ over other reactive oxygen species to generate ¹³C- benzoic acid and can be hyperpolarized using dynamic nuclear polarization, providing a method for dual-frequency detection of H₂O₂. Phantom images collected using frequency-specific imaging sequences demonstrate the efficacy of this responsive contrast agent to monitor H₂O ₂ at pre-clinical field strengths. The combination of reaction-based detection chemistry and hyperpolarized ¹³C MRI provides a potentially powerful new methodology for non-invasive multi-analyte imaging in living systems.

Full text of DOI:10.1021/ja111589a

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pubs.acs.org/JACS
A Hydrogen Peroxide-Responsive Hyperpolarized 13C MRI Contrast
Agent
Alexander R. Lippert,† Kayvan R. Keshari,§ John Kurhanewicz,§ and Christopher J. Chang*,†,‡
†Department of Chemistry and ‡Howard Hughes Medical Institute, University of California, Berkeley, California 94720, United States
§Department of Radiology and Biomedical Imaging, University of California, San Francisco, California 94158, United States
S Supporting Information
b
magnetized samples and signal enhancements approaching 105-
fold. The development of rapid dissolution procedures16 follow-
ABSTRACT: We report a new reaction-based approach for
ing DNP has allowed the use of hyperpolarized [1-13C]-
the detection of hydrogen peroxide (H2O2) using hyperpo-
larized 13C magnetic resonance imaging (13C MRI) and the
H2O2-mediated oxidation of R-ketoacids to carboxylic acids.
13C-Benzoylformic acid reacts selectively with H2O2 over
other reactive oxygen species to generate 13C-benzoic acid
and can be hyperpolarized using dynamic nuclear polariza-
tion, providing a method for dual-frequency detection of
H2O2. Phantom images collected using frequency-specific
imaging sequences demonstrate the efficacy of this respon-
sive contrast agent to monitor H2O2 at pre-clinical field
strengths. The combination of reaction-based detection
chemistry and hyperpolarized 13C MRI provides a poten-
tially powerful new methodology for non-invasive multi-
analyte imaging in living systems.
pyruvate,17 [2-13C]-fructose,18 [5-13C]-glutamine,19 13C-
HCO3,20 [1,4-13C2]-fumarate,21 and 15N-choline,22 alone and
in combination,23 as metabolic probes.24 These hyperpolarized
probes ultimately report on the flux of specific enzymes by
monitoring the conversion of isotopically labeled natural sub-
strates to their metabolic products. On the other hand, our
interest in the detection of molecular species such as H2O2,
which can simultaneously originate from multiple cellular
sources, requires the use of probes that are not prone to rapid
metabolism, so as to avoid false positives. In addition to being
metabolically inert and non-toxic, an ideal 13C MRI probe of this
type should also possess favorable physical properties for DNP,
produce an observable chemical shift upon rapid and selective
reaction with the analyte of interest, and provide a system where
both reactants and products have 13C-labeled nuclei with long
spin-lattice relaxation times (T1).
eactive oxygen species (ROS) are intimately involved in the
To this end, observations of the rapid oxidative decarboxylations
of R-ketoacids25 led us to investigate these species as hyperpolariz-
able 13C MRI contrast agents using a reaction-based scheme for
H2O2 detection. Initial studies focused on benzoylformic acid
(BFA) because this R-ketoacid is non-toxic and undergoes minimal
metabolism in both cells26 and animals.27 Kinetics measurements
reveal that the reaction between BFA and H2O2 proceeds rapidly to
produce benzoic acid (BA) within minutes with a second-order rate
constant of 10.31 ( 0.26 M-1 min-1 (Supporting Information,
R
genesis and progression of numerous ailments1 ranging from
cancer2 to neurodegeneration3 to diabetes.4 The relative stability
and diffusibility of H2O2 poise this ROS to act as a potential
diagnostic marker for the presence and progression of a wide
range of pathological states. Indeed, the measurement of H2O2 in
exhaled breath condensates has been used as a clinical marker for
illnesses associated with lung inflammation,5 including asthma,6
chronic obstructive pulmonary diseases,7 and cystic fibrosis.8
The clinical use of this direct marker for oxidative stress and
inflammation in deep tissues is hampered in large part by a lack
of methods to non-invasively detect H2O2 in thicker, non-
transparent specimens.9 As part of a larger program in our
laboratory to develop novel ways to study the chemistry and
biology of H2O2 in livingcells and animals by molecular imaging,10
wenow introducea new type of reaction-basedhyperpolarized13C
MRI contrast agent for the detection of H2O2.
Figure S1). Moreover, the 13C ketone nucleus of BFA and the 13
C
carbonyl nucleus of BA are expected to have long T1 values (on the
order of tens of seconds), and the chemical shift difference between
this carbon atom upon conversion of BFA to BA, the product of
oxidative decarboxylation, is ca. 20 ppm. Encouraged by these initial
findings, we synthesized BFA with a 13C-label on the ketone carbon
(13C-BFA) using sulfur ylide acylation and oxidation chemistry
(Scheme 1).25 Reaction of neat bromoacetonitrile with tetra-
hydrothiophene provided the cyanosulfonium bromide 1. The
cyanosulfur ylide formed in situ was then coupled with 13C-benzoic
acid (13C-BA) using HATU to yield the 13C-cyanosulfur ylide 2.
Finally, oxone oxidation afforded 13C-BFA after HPLC purification.
With this compound in hand, we proceeded to test its ability to
undergo DNP under clinically relevant conditions. In these
standard protocols, DNP results from the transfer of polarization
from a stable organic radical species to a spin S = 1/2 nucleus by
MRI offers a promising approach for precise non-invasive
molecular imaging of deep tissues in real time. However, its full
potential as an imaging modality has yet to be realized because of
a low sensitivity11 that restricts most MRI experiments to
imaging highly abundant endogenous protons in water and
lipids. To overcome this limitation, hyperpolarization methods12
such as optical pumping,13 para-H2-induced polarization,14 and
dynamic nuclear polarization (DNP)15 can drastically enhance
an MRI signal by manipulating spin states to produce samples
with large non-equilibrium spin populations, leading to highly
Received: December 23, 2010
Published: March 02, 2011
r
2011 American Chemical Society
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|

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Scheme 1. Design of a Hyperpolarized 13C MRI Contrast
Agent for Detection of H2O2 through H2O2-Mediated R-
Ketoacid Oxidative Decarboxylation and Synthesis of 13C-
BFA and Its H2O2-Mediated Conversion to 13C-BA
microwave irradiation at 1-2 K in a solid-state glass.15 Efficient
transfer of polarization from the free radical to the nuclei of
interest relies heavily on the formation of a uniform glass, which
is typically achieved with highly concentrated solutions; for
biological use, water is an appropriate solvent so that no residual
toxic organic solvents are present after rapid dissolution of the
hyperpolarized sample. We found that 13C-BFA is extremely
soluble in water at concentrations >5 M, with solutions repro-
ducibly forming a glassy solid upon flash-freezing in liquid N2.
Representative preparations utilize 6.0 M solutions of 13C-BFA
and 15 mM OX063 radical in water and were hyperpolarized at
1.33 K, irradiating at 94.094 GHz, using a HyperSense instru-
ment (Oxford Instruments). Under these unoptimized condi-
tions, 13C-BFA polarizes with a build-up time constant of 899 s,
providing 5.2% polarization and a ∼5500-fold signal enhance-
ment. The T1 values of the labeled 13C2 ketone were measured to
be 24.4 ( 0.4 s at 11.7 T and 18.6 ( 0.3 s at 14.1 T (Figure S3).
We then evaluated hyperpolarized samples of 13C-BFA for
spectroscopic detection of H2O2 (Figure 1). After hyperpolar-
ization, the sample was rapidly dissolved in 100 mM phosphate
buffered to pH 7.8 with 0.3 mM EDTA to a final concentration of
5 mM and reacted with various concentrations of H2O2. Spectra
were acquired with a single scan in the presence of H2O2
concentrations ranging from 10 to 1000 μM. Figure 1a shows
a set of spectra 21 s after rapid dissolution and reaction with
H2O2. The labeled carboxylate 13C1 resonance of the 13C-BA
product exhibits a chemical shift of 176 ppm, and the unlabeled
carboxylate C1 carbon of the starting 13C-BFA appears as a
doublet at 173.5 ppm; the ratio of the integrated 13C-BA peak to
the unlabeled 13C-BFA peak displayed a maximum at 21 s and a
good linear correlation with increasing concentrations of H2O2
(Figure 1b), indicating that hyperpolarized 13C-BFA can readily
detect concentrations of H2O2 at high micromolar levels in vitro
that are within the range implicated in states of oxidative stress
that lead to cellular senescence,28 despite trace amounts of 13C-
benzoic acid (<0.1%) produced during synthetic manipulation
and rapid dissolution procedures that can reduce the accuracy of
such measurements. Although we were able to detect these levels
of H2O2 using this first-generation probe, ongoing efforts are
geared toward improving sensitivity to lower physiological levels
that might be encountered in vivo through newer probes and
optimized hyperpolarization protocols.
Figure 1. (a) 13C NMR spectra of hyperpolarized 13C-BFA after 21 s of
reaction with 10, 100, 250, 500, 750, and 1000 μM H2O2 at 11.7 T.
Spectra were acquired with a single scan every 3 s with a 5° pulse, except
for 10 and 100 μM, which were acquired after 21 s with a 90° pulse. (b)
Linear correlation of the ratio of integrated peak intensities of the C1
(carboxylate carbon) of 13C-BA to the C1 (carboxylate carbon) of
13C-BFA versus the concentration of H2O2; R2 = 0.988.
Figure 2. Response of 50 μM BFA to various ROS. All ROS were added
at 5 mM, except for O2-, which was generated enzymatically at a rate of
24 μmol/min for 120 min (2.9 mM total). Concentrations of benzoic
acid were measured by HPLC after 0, 5, 10, 15, and 20 min of reaction
except for that with O2-, which was measured after 0, 30, 60, 90, and 120
min of reaction.
exceedingly high, non-physiological concentrations does OCl-
or ONOO- (500 mM) show reactivity with BFA (Figure S2),
producing 20% and 70% NMR conversions, respectively. We
speculate that the selectivity for H2O2 over OCl- arises from an
increased nucleophilicity of the former, whereas the selectivity
over ONOO- is most likely due to the short lifetime of the latter
under physiological conditions.29
Finally, we established the ability of 13C-BFA to image H2O2
at pre-clinical field strengths. Specifically, we attained phantom
images using a 14.1 T micro-imager equipped with 100 G/cm
gradients and millipede 1H and quadrature birdcage 13C.RF coils.
Figure 3 displays phantoms of samples containing 20 mM
hyperpolarized 13C-BFA with 0, 25, 50, 100, and 200 mM added
H2O2, thermally polarized 5 M 13C-BA in dimethyl acetamide
(DMA), and thermally polarized 5 M 13C-BFA in H2O. First, we
The oxidative decarboxylation of BFA is relatively selective for
H2O2 over other biologically relevant ROS as monitored using
analytical HPLC. After reacting BFA for 20 min at room
temperature, only samples treated with H2O2 show significant
conversion to benzoic acid (Figure 2). Only upon addition of
1
acquired H spin echo images of all the tubes to indicate tube
placement (Figure 3a). Next, we acquired 13C MRI images using
frequency-specific excitation pulses to obtain images of thermally
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COMMUNICATION
selectivity assays, polarization procedures, spectroscopy, and
imaging. This material is available free of charge via the Internet
at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
chrischang@berkeley.edu
’ ACKNOWLEDGMENT
This work has been supported by the National Institutes of
Health (GM 79465), Amgen, Astra Zeneca, Novartis, and the
Packard and Sloan Foundations. C.J.C. is an Investigator of the
Howard Hughes Medical Institute. We gratefully acknowledge
Dr. Hikari Yoshihara, Mr. Martin Ipuy, and Mr. Bertram Koelsch
for experimental assistance.
Figure 3. Phantom images of 5 M thermally polarized 13C-BFA in H2O,
5 M thermally polarized 13C-BA in DMA, and 20 mM hyperpolarized
13C-BFA in 100 mM phosphate, 0.3 mM EDTA buffered at pH 7.8 with
0, 25, 50, 100, and 200 mM H2O2. (a, left side) 1H spin echo image. (b-
e, right) Frequency-specific images with selective excitation of the
resonance of (b, top left) 5 M 13C-BA in DMA, (c, top right) 5 M
13C-BFA in H2O, (d, bottom left) 13C-BA buffered at pH 7.8, and (e,
bottom right) 13C-BFA buffered at pH 7.8. Images (b)-(e) were
acquired after ∼37 s of reaction with H2O2 with a TR = 150 ms, FOV
40 Â 40 Â 40 mm, 16 Â 12 Â 12 matrix, and zero-filled to a final
resolution of 1.25 mm isotropic.
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