FULL PAPER
DOI: 10.1002/chem.201300817
Specific Detection and Imaging of Enzyme Activity
19
by Signal-Amplifiable Self-Assembling F MRI Probes
Kazuya Matsuo,[a] Rui Kamada,[a] Keigo Mizusawa,[a] Hirohiko Imai,[b] Yuki Takayama,[b]
Michiko Narazaki,[b] Tetsuya Matsuda,[b] Yousuke Takaoka,[a] and Itaru Hamachi*[a, c]
Abstract: Specific turn-on detection of
enzyme activities is of fundamental im-
portance in drug discovery research, as
well as medical diagnostics. Although
magnetic resonance imaging (MRI) is
one of the most powerful techniques
for noninvasive visualization of enzyme
activity, both in vivo and ex vivo,
promising strategies for imaging specif-
ic enzymes with high contrast have
been very limited to date. We report
herein a novel signal-amplifiable self-
assembling 19F NMR/MRI probe for
turn-on detection and imaging of spe-
cific enzymatic activity. In NMR spec-
troscopy, these designed probes are
“silent” when aggregated, but exhibit a
disassembly driven turn-on signal
change upon cleavage of the substrate
part by the catalytic enzyme. Using
these 19F probes, nanomolar levels of
two different target enzymes, nitrore-
ductase (NTR) and matrix metallopro-
teinase (MMP), could be detected and
visualized by 19F NMR spectroscopy
and MRI. Furthermore, we have suc-
ceeded in imaging the activity of endo-
genously secreted MMP in cultured
media of tumor cells by 19F MRI, de-
pending on the cell lines and the cellu-
lar conditions. These results clearly
demonstrate that our turn-on 19F
probes may serve as a screening plat-
form for the activity of MMPs.
Keywords: enzyme activity · imag-
ing agents · magnetic resonance
imaging · NMR spectroscopy · self-
assembly · signal amplification
Introduction
strategies for MRI are as yet quite limited. As a pioneering
example, 1H magnetic resonance (MR)-type switching
probes have been reported as powerful tools for in vivo
enzyme imaging by Meadeꢀs group.[4a] The switching mecha-
nisms are mainly based on paramagnetic relaxation en-
hancement (PRE) using paramagnetic metal complexes and
their coordinated water molecules.[5] For target-specific
imaging, heteronuclear MRI techniques have also attracted
considerable attention. In particular, 19F MRI is anticipated
to be a promising method because 19F has a high NMR sen-
sitivity approaching that of 1H (83% relative to 1H) and
there are no background signals even in vivo.[6] Despite the
potential importance of 19F MRI for medical diagnostics,
promising strategies for imaging specific enzymes with high
contrast are as yet very limited.[7] There are also some prob-
lems associated with 19F MRI that have yet to be overcome,
namely low sensitivity and poor efficiency of probe delivery.
Therefore, 19F MRI is an immature technique and both
more fundamental studies on 19F probes and improvement
of the instrumentation are needed to establish it as an in
vivo imaging tool.
There are many uses for the specific detection and imaging
of enzyme activity in vivo in both drug discovery research
and medical diagnostics. Chemical “switchable” probes,
which induce detectable signal changes in response to spe-
cific enzyme reactions, are particularly useful for imaging
enzyme activities because they allow selective and sensitive
detection with high signal-to-noise ratios.[1,2] Many fluores-
cent switching probes for detecting enzyme activities have
now been developed.[3] However, fluorescence techniques
are, in general, not suitable for in vivo imaging due to the
limited light transmission and its scattering in animal bodies.
Although magnetic resonance imaging (MRI) is known to
be an adequate technique for in vivo studies,[4] switching
[a] K. Matsuo, Dr. R. Kamada, Dr. K. Mizusawa, Dr. Y. Takaoka,
Prof. Dr. I. Hamachi
Department of Synthetic Chemistry and Biological Chemistry
Graduate School of Engineering, Kyoto University
Katsura, Nishikyo-Ku, Kyoto 615-8510 (Japan)
Fax : (+81)75-383-2759
Recently, we have developed a unique self-assembling
turn-on 19F probe for the specific detection of target pro-
teins composed of a hydrophilic protein ligand and a hydro-
phobic 19F detection modality.[8] These ligand-tethered
probes clearly exhibit a disassembly driven turn-on signal
change upon specific binding to target proteins.[8,9] More re-
cently, we have established that moderate stability of the ag-
gregates is critical for obtaining an ideal off/on response,
and the stability can be controlled by the hydrophobicity/hy-
[b] Dr. H. Imai, Dr. Y. Takayama, Dr. M. Narazaki, Prof. Dr. T. Matsuda
Department of System Science, Graduate School of Informatics
Kyoto University, 361-1 Yoshida-Honmachi
Sakyo-ku, Kyoto 606-8501 (Japan)
[c] Prof. Dr. I. Hamachi
Japan Science and Technology Agency (JST), CREST
5 Sanbancho, Chiyoda-ku, Tokyo 102-0075 (Japan)
Supporting information for this article is available on the WWW
Chem. Eur. J. 2013, 00, 0 – 0
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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