Redox-activated manganese-based MR contrast agent

Article abstract of DOI:10.1021/ja312610j

Here we report a simple Mn coordination complex with utility as a redox-sensitive MR probe. The HBET ligand stabilizes both the Mn²⁺ and Mn³⁺ oxidation states. In the presence of glutathione (GSH), low relaxivity Mn^III-HBET is converted to high relaxivity Mn ^II-HBET with a 3-fold increase in relaxivity, and concomitant increase in MR signal. Alternately, hydrogen peroxide can convert Mn ^II-HBET to Mn^III-HBET with a reduction in MR signal.

Full text of DOI:10.1021/ja312610j

Communication
pubs.acs.org/JACS
Redox-Activated Manganese-Based MR Contrast Agent
Galen S. Loving,‡ Shreya Mukherjee,‡ and Peter Caravan*
A. A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, 149 13th Street, Suite 2301,
Charlestown, Massachusetts 02129, United States
S
* Supporting Information
MR is an attractive modality to image redox dynamics as it
ABSTRACT: Here we report a simple Mn coordination
complex with utility as a redox-sensitive MR probe. The
HBET ligand stabilizes both the Mn2+ and Mn3+ oxidation
states. In the presence of glutathione (GSH), low relaxivity
MnIII-HBET is converted to high relaxivity MnII-HBET
with a 3-fold increase in relaxivity, and concomitant
increase in MR signal. Alternately, hydrogen peroxide can
convert MnII-HBET to MnIII-HBET with a reduction in
MR signal.
allows the interrogation of intact, opaque organisms in three
dimensions at cellular resolution (∼10 μm) on high field
systems and submillimeter resolution on clinical scanners. The
deep tissue penetration and high resolution of MR make it
possible to directly translate findings from cells to mice to
humans.
One approach to a redox-sensitive MR probe is to employ a
redox-active metal ion. Gd(III), used in the majority of MR
probes, has only one stable oxidation state in aqueous media.
Manganese, however, can exist stably in a number of oxidation
states depending on the ligand field. High spin Mn(II)
complexes can also yield relaxivities that are comparable to
the best Gd(III) complexes,18,19 and there has been much
recent work on Mn(II) complexes as MR probes.20−23 Here we
propose a new class of redox-activated MR probes based on the
Mn(II)/Mn(III) redox couple.
There are several requirements for a useful redox-activated
MR probe: (1) a redox half-cell potential that is accessible to
biologically relevant reducing agents like glutathione (GSH);
(2) a ligand that stabilizes both oxidation states such that
reduction or oxidation does not result in decomposition; (3)
strong signal change upon activation, and ideally signal increase
in the presence of pathology, i.e., a turn-on probe; and (4)
kinetics that are rapid with respect to the imaging time scale.
A key design feature of a redox-active probe is to identify a
ligand that stabilizes both oxidation states. EDTA forms a very
stable 7-coordinate Mn (II) complex with one coordinated
water co-ligand, and the 2+ oxidation state is strongly favored,
Figure 1.24−26 By contrast, changing two carboxylato donors to
phenolato donors as in HBED or EHPG strongly favors
Mn(III) complexes27,28 with coordination number 6 and no
inner-sphere water, Figure 1.29 We hypothesized that HBET
(Figure 1) with a ligand structure that is intermediate between
EDTA and HBED, containing only one phenolato donor, could
oth intracellular and extracellular redox environments are
B
tightly regulated in healthy tissues and are closely
correlated with the physiological state of the cell.1,2 However,
this regulation is often disrupted during periods of cellular
stress, damage or cell death.3 While the dynamics of local redox
status play an important role in mediating various biological
processes such as cell-cycle progression, immune response and
wound-healing,3,4 persistent dysregulation of the extracellular
redox environment has been linked to a number of pathologies
including chronic inflammation,3 neoplastic growth,4 and
cancer cell aggressiveness.1,5,6 Indeed, the role of redox in
tumor biology continues to be an area of active research and is
the focus of many redox-activated prodrugs that are currently in
devolpment.7,8 New imaging methods will certainly facilitate
these endeavors while further advancing our basic under-
standing of redox dynamics in relation to disease.
There have been several redox-activated molecular imaging
probes reported, each of which employs a unique mechanism of
activation to address specific aspects of redox environment.
Some of these probes target tissues that are hypoxic (low
oxygen tension),9−12 as sometimes occurs in tumors. Positron
emission tomography (PET) probes 18F-fluoromisonidazole
(18F-MISO) and 64Cu-diacetyl-bis(N4-methylthiosemicarba-
zone) (64Cu-ATSM) have been used clinically to image
tumor hypoxia.10,12 Electron paramagnetic resonance (EPR)
imaging and spectroscopy probes based on redox-sensitive
nitroxides have proven effective at detecting the relative
abundance of thiols as well as other reducing species.13,14
Magnetic resonance (MR) probes have also been reported.
Examples include Gd(III) complexes in which the ligands are
capable of forming reversible disulfide linkages with cysteine
side chains of extracellular proteins thereby providing an
indirect view of local redox conditions,15,16 a Mn(II)-porphyrin
which undergoes oxidation to a Mn(III) species with lower
relaxivity under normoxic conditions,9 and recently, a pair of
Eu(III) complexes that can be activated in the presence of β-
NADH through reduction of the pendant arms of the ligands.17
Figure 1. Ligand structure strongly influences the redox potential of
the MnIII/MnII couple. Conversion of carboxylato to phenolato donors
shifts the half-cell potential by 617 mV in favor of the higher oxidation
state.
Received: December 26, 2012
Published: March 19, 2013
© 2013 American Chemical Society
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Communication
potentially exhibit metastability toward either oxidation state.
The preferred oxidation state of such a species would therefore
be highly sensitive to changes in redox environment. To test
this hypothesis, we prepared manganese complexes of HBED,
HBET, and EDTA to examine how the number of phenolate
groups contributes to the half-cell potentials of the Mn(III)/
Mn(II) redox couple. Cyclic voltammetry of MnII-HBET in
TRIS buffer (Figure 2) shows a reversible oxidation peak at
Figure 3. (Left) T1-weighted MR image recorded at 4.7 T of tubes
containing pure water, 0.5 mM MnIII-HBET and 0.5 mM MnII-HBET
in pH 7.4 TRIS buffer. (Right) Relaxivities measured at room
temperature at 4.7 T.
Figure 2. (a) Cyclic voltammogram at 100 mV/s for 25 mM MnII-
HBET in 25 mM pH 7.4 Tris buffer, 500 mM KNO3, and K4Fe(CN)6
as an internal standard. Potentials reported vs NHE. (b) Cottrell plot
of Mn2+/Mn3+ couple: ia (green) and ic (blue) vs √ν, where ν = scan
rate.
brighter image. T1 measurements at 4.7 T revealed a 3.3-fold
higher relaxivity for the reduced MnII-HBET form, Figure 3.
The higher relaxivity of MnII-HBET at 4.7 vs 1.4 T is likely due
to the difference in temperature for these measurements. The
slower tumbling rate at room temperature (4.7 T) results in a
higher relaxivity.
0.356 V (vs NHE), indicating the potential for using this
molecule as a redox probe. Anodic (ia) and cathodic current (ic)
are linear with the square root of scan rate (ν) over the range of
50−600 mV/s (Figure 2b), indicating this is a diffusion
controlled process.30 On the other hand, MnII-EDTA and
MnIII-HBED display irreversible oxidation and quasi-reversible
reduction peaks with half-cell potentials (MnIII/MnII) of 0.633
and 0.016 V respectively.
HBET (hydroxybenzylethylenediamine triacetic acid) was
synthesized from reductive amination of mono BOC-protected
ethylene diamine with salicaldehyde, followed by alkylation
with t-butyl bromoacetic acid, and then acid deprotection to
give the free ligand in overall 45% yield. The reaction of 1 equiv
of ligand with MnCl2 led to MnII-HBET in 84% isolated yield.
The MnIII-HBET complex was synthesized in 38% yield by
aerial oxidation of MnCl2 in the presence of 1 equiv of the
ligand under basic conditions, followed by RP-HPLC
purification. The HBET ligand stabilizes both oxidation states
to the extent that MnII-HBET and MnIII-HBET can be easily
separated and isolated in their pure forms using a standard RP-
HPLC system. MnII-HBET showed slight oxidation to MnIII-
HBET and no decomposition when incubated in phosphate
(5% oxidation) or carbonate (35% oxidation) buffer, but no
oxidation in citrate buffer for 1 h at 37 °C, pH 7.4. MnIII-HBET
was inert under the same conditions. However, we observed
complete ligand-exchange for both complexes when challenged
with EDTA.
The MnIII-HBET complex is easily reduced to the MnII state
in the presence of low millimolar quantities of GSH such that
the equilibrium lies strongly in favor of the reduced form. This
conversion appears to proceed directly to the product without
the accumulation of any long-lived reaction intermediates or
the formation of byproducts. This is demonstrated by the time-
course experiment shown in Figure 4b where the emergence of
the MnII-HBET complex occurs simultaneously with the
consumption of MnIII-HBET. The progress of this reaction
can also be followed in real-time by relaxometry (Figure 4a)
using conditions relevant for clinical imaging (1.4 T, 37 °C), or
by UV absorbance. Since the relaxivity of the Mn(II) complex is
greater than that of the Mn(III) complex, the longitudinal
relaxation rate of water protons (1/T1) will increase in
proportion to the concentration of MnII-HBET in the reaction.
Direct conversion of the Mn(III) complex to the Mn(II)
complex is further evidenced by the observation that the rate of
formation of the MnII-HBET species is effectively equivalent to
the consumption rate of the oxidized form as measured by UV
absorbance (Figure 4a).
On the other hand, MnII-HBET could not be oxidized by
GSSG, but it could be converted to MnIII-HBET by H2O2
(Figure S10). We observed that the oxidation of 0.5 mM MnII-
HBET with 1 mM H2O2 reaches equilibrium within 4 min at
70% conversion. This result opens the additional possibility of
imaging the production of reactive oxygen species, albeit
through a decrease of MR signal.
The T1 relaxivities of the MnII-HBET and MnIII-HBET
complexes were measured independently at 1.4 T in Tris buffer
(pH 7.4, 37 °C) and the values were 2.76 and 1.05 mM−1 s−1,
respectively. This difference in relaxivity is large considering
that the MnIII state still has four unpaired electrons, and may be
attributed to the presence of an inner-sphere water for MnII-
HBET (CN7), while MnIII-HBET may lack this coordinated
water (CN6). In Figure 3, we show a T1-weighted MR image at
4.7 T of glass inserts containing either pure water or equimolar
MnIII-HBET or MnII-HBET. The presence of the paramagnetic
complex results in higher signal intensity compared to pure
water. The higher relaxivity of MnII-HBET is apparent in its
A series of kinetic experiments were conducted at 37 °C (pH
7.4) to determine the empirical rate law for the reduction of
MnIII-HBET by glutathione. MnIII-HBET exhibits an absorb-
ance band at 375 nm (ε = 1.38 × 103 M−1 cm−1) in water that
is not present in the UV spectrum of the MnII-HBET complex.
This feature allowed us to easily monitor the reduction of
MnIII-HBET over time. We measured initial reaction rates at a
three different GSH concentrations (5, 10, and 20 mM). For
each GSH concentration, the reaction was performed at four
different initial concentrations of MnIII-HBET (0.3, 0.4, 0.5, and
0.6 mM). On the basis of these experiments, we determined
that the reaction was first-order in both [GSH] and [MnIII-
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Communication
diamine to increase stability,32 or with appropriate aryl
substituents to tune the redox potential. The ligand could
also be modified to incorporate a moiety that imparts specific
biological targeting. Finally, in vivo studies will require the need
to deconvolute differences in relaxivity from differences in
concentration to the MR signal; however, strategies exist that
rely on control probes33 or bimodal imaging.34 These avenues
are currently under investigation.
ASSOCIATED CONTENT
* Supporting Information
■
S
Full experimental details of complex synthesis and character-
ization, relaxivity, cyclic voltammetry, GSH reduction and H2O2
oxidation kinetics, and MR imaging. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
caravan@nmr.mgh.harvard.edu
■
Author Contributions
‡G.S.L. and S.M. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Dr. Anna Moore is gratefully acknowledged for providing
access to a spectrophotometer. This work was supported by
grants from the National Cancer Institute (CA161221 to PC
and a T32 post-doctoral fellowship to GSL, CA009502) and
instrumentation funded by the National Center for Research
Resources (RR14075).
Figure 4. (a) Reduction of 0.5 mM MnIII-HBET to MnII-HBET by 10
mM GSH in TRIS buffer (pH 7.4, 37 °C) results in increased proton
relaxation rate (1/T1, left axis) and concomitant decrease in mole
fraction of MnIII-HBET (right axis) as determined by characteristic UV
absorbance at 375 nm. (b) Reduction of 0.5 mM MnIII-HBET by 1
mM GSH at 26 °C monitored by LC-MS. No long-lived intermediate
species were observed in the reduction process.
REFERENCES
■
(1) Moriarty-Craige, S. E.; Jones, D. P. Annu. Rev. Nutr. 2004, 24,
481.
(2) Schafer, F. Q.; Buettner, G. R. Free Radical Biol. Med. 2001, 30,
1191.
(3) Rubartelli, A.; Lotze, M. T. Trends Immunol. 2007, 28, 429.
(4) Sarsour, E. H.; Kumar, M. G.; Chaudhuri, L.; Kalen, A. L.;
Goswami, P. C. Antioxid. Redox Signaling 2009, 11, 2985.
(5) Chaiswing, L.; Oberley, T. D. Antioxid. Redox Signaling 2010, 13,
449.
(6) Jonas, C. R.; Ziegler, T. R.; Gu, L. H.; Jones, D. P. Free Radical
Biol. Med. 2002, 33, 1499.
(7) Brown, J. M.; Wilson, W. R. Nat. Rev. Cancer 2004, 4, 437.
(8) Graf, N.; Lippard, S. J. Adv. Drug Delivery Rev. 2012, 64, 993.
(9) Aime, S.; Botta, M.; Gianolio, E.; Terreno, E. Angew. Chem., Int.
Ed. 2000, 39, 747.
(10) Eschmann, S.-M.; Paulsen, F.; Reimold, M.; Dittmann, H.; Welz,
S.; Reischl, G.; Machulla, H.-J.; Bares, R. J. Nucl. Med. 2005, 46, 253.
(11) Iwaki, S.; Hanaoka, K.; Piao, W.; Komatsu, T.; Ueno, T.; Terai,
T.; Nagano, T. Bioorg. Med. Chem. Lett. 2012, 22, 2798.
(12) Wood, K. A.; Wong, W. L.; Saunders, M. I. Nucl. Med. Biol.
2008, 35, 393.
(13) Bobko, A. A.; Eubank, T. D.; Voorhees, J. L.; Efimova, O. V.;
Kirilyuk, I. A.; Petryakov, S.; Trofimiov, D. G.; Marsh, C. B.; Zweier, J.
L.; Grigor’ev, I. A.; Samouilov, A.; Khramtsov, V. V. Magn. Reson. Med.
2012, 67, 1827.
(14) Kuppusamy, P.; Li, H.; Ilangovan, G.; Cardounel, A. J.; Zweier,
J. L.; Yamada, K.; Krishna, M. C.; Mitchell, J. B. Cancer Res. 2002, 62,
307.
(15) Menchise, V.; Digilio, G.; Gianolio, E.; Cittadino, E.; Catanzaro,
V.; Carrera, C.; Aime, S. Mol. Pharmaceutics 2011, 8, 1750.
(16) Raghunand, N.; Jagadish, B.; Trouard, T. P.; Galons, J. P.;
Gillies, R. J.; Mash, E. A. Magn. Reson. Med. 2006, 55, 1272.
HBET] with an overall second-order rate constant of (3.8
0.3) × 10−1 M−1 s−1. Concentrations of reduced glutathione
within the cell are typically 1−10 mM.31 In that range, the half-
life of the MnIII-HBET complex is roughly 3−30 min. By
comparison, the half-life in blood plasma, where glutathione
levels are approximately 3 orders of magnitude lower,31 would
be greater than one week.
In conclusion, the MnIII/MnII-HBET redox couple satisfies a
number of the key criteria required of a useful probe for redox
imaging. The redox half-cell potential is accessible to
biologically relevant reductants and oxidants like GSH and
H2O2, it displays good signal enhancement upon reduction to
the MR-active state, and the activation kinetics are sufficiently
rapid with respect to the imaging time scale. The redox
properties of this system are well behaved and the
interconversion between the two oxidation states occurs via a
simple and reversible one-electron process.
While the Mn-HBET system represents a useful prototype
for a redox sensitive probe, further work is necessary for in vivo
studies. The complexes were unstable to the EDTA challenge
and increased stability may be required. The redox kinetics
and/or potential may also need tuning to address specific
biological questions. However, we note that the HBET ligand
may readily be modified, e.g., by using a more preorganized
4622
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Communication
(17) Ratnakar, S. J.; Viswanathan, S.; Kovacs, Z.; Jindal, A. K.; Green,
K. N.; Sherry, A. D. J. Am. Chem. Soc. 2012, 134, 5798.
(18) Aime, S.; Anelli, L.; Botta, M.; Brocchetta, M.; Canton, S.;
Fedeli, F.; Gianolio, E.; Terreno, E. J. Biol. Inorg. Chem. 2002, 7, 58.
(19) Troughton, J. S.; Greenfield, M. T.; Greenwood, J. M.; Dumas,
S.; Wiethoff, A. J.; Wang, J.; Spiller, M.; McMurry, T. J.; Caravan, P.
Inorg. Chem. 2004, 43, 6313.
́
̌ ̌ ́
B.; Lukes, I.; Toth, E. Eur. J. Inorg. Chem. 2012, 2012,
(20) Drahos,
1975.
(21) Lee, T.; Zhang, X. A.; Dhar, S.; Faas, H.; Lippard, S. J.; Jasanoff,
A. Chem. Biol. 2010, 17, 665.
(22) Rolla, G. A.; Tei, L.; Fekete, M.; Arena, F.; Gianolio, E.; Botta,
M. Bioorg. Med. Chem. 2011, 19, 1115.
(23) Szabo, I.; Crich, S. G.; Alberti, D.; Kalman, F. K.; Aime, S. Chem.
Commun. 2012, 48, 2436.
(24) Oakes, J.; Smith, E. G. J. Chem. Soc., Faraday Trans. 1981, 77,
299.
(25) Richards, S.; Pedersen, B.; Silverton, J. V.; Hoard, J. L. Inorg.
Chem. 1964, 3, 27.
(26) Zetter, M. S.; Grant, M. W.; Wood, E. J.; Dodgen, H. W.; Hunt,
J. P. Inorg. Chem. 1972, 11, 2701.
(27) Frost, A. E.; Freedman, H. H.; Westerback, S. J.; Martell, A. E. J.
Am. Chem. Soc. 1958, 80, 530.
(28) Patch, M. G.; Simolo, K. P.; Carrano, C. J. Inorg. Chem 1983, 22,
2630.
(29) Bihari, S.; Smith, P. A.; Parsons, S.; Sadler, P. J. Inorg. Chim.
Acta. 2002, 331, 310.
(30) Bard, A. J.; Faulkner, L. R. Electrochemical Methods.
Fundamentals and Applications; 2nd ed.; Wiley: New York, 2001.
(31) Banerjee, R. J. Biol. Chem. 2012, 287, 4397.
(32) Kalman, F. K.; Tircso, G. Inorg. Chem. 2012, 51, 10065.
(33) Raghunand, N.; Howison, C.; Sherry, A. D.; Zhang, S.; Gillies,
R. J. Magn. Reson. Med. 2003, 49, 249.
(34) Frullano, L.; Catana, C.; Benner, T.; Sherry, A. D.; Caravan, P.
Angew. Chem., Int. Ed. 2010, 49, 2382.
4623
dx.doi.org/10.1021/ja312610j | J. Am. Chem. Soc. 2013, 135, 4620−4623