Paramagnetic relaxation-based 19F MRI probe to detect protease activity

Article abstract of DOI:10.1021/ja077058z

A novel design principle for ¹⁹F MRI probes detecting protease activity was developed. This principle is based on ¹⁹F MRI signal quenching by the intramolecular paramagnetic effect from Gd³⁺. The intramolecular Gd³⁺

Full text of DOI:10.1021/ja077058z

Published on Web 12/23/2007
Paramagnetic Relaxation-Based 19F MRI Probe To Detect Protease Activity
Shin Mizukami,† Rika Takikawa,† Fuminori Sugihara,‡ Yuichiro Hori,† Hidehito Tochio,§
Markus Wa¨lchli, Masahiro Shirakawa,*,§,¶ and Kazuya Kikuchi*,†
DiVision of AdVanced Science and Biotechnology, Graduate School of Engineering, Osaka UniVersity, Osaka
565-0871, Japan, International Graduate School of Arts and Sciences, Yokohama City UniVersity, Kanagawa
230-0045, Japan, Department of Molecular Engineering, Graduate School of Engineering, Kyoto UniVersity, Kyoto
615-8510, Japan, Bruker BioSpin K.K., Ibaraki 305-0051, Japan, and CREST, Japan Science and Technology
Corporation, Saitama 332-0012, Japan
Received September 12, 2007; E-mail: kkikuchi@mls.eng.osaka-u.ac.jp; shirakawa@moleng.kyoto-u.ac.jp
Scheme 1. Design Principle of 19F MRI Probe Detecting a
Protease Activity
Real-time imaging of enzyme activities in vivo offers valuable
information in understanding living systems and in developing
medicine for various types of diseases. Currently, a variety of
fluorescent probes for detecting enzyme activities are generally used
for their high-sensitivity characteristics. However, general fluores-
cence imaging methods are not suitable for in vivo studies because
visible fluorescence scarcely transmits through animal bodies.
Although near-infrared fluorophores are useful for in vivo imaging
for tissue surfaces, they cannot be applied to the deep section of
Another class of 19F MRI probes is active agents.5 They undergo
the organ. On the other hand, magnetic resonance imaging (MRI)
chemical modification by the target molecules and then change their
is known as an imaging technique adequate for in vivo studies.
NMR parameters. Mason and co-workers have developed 19F MRI
probes detecting reporter enzyme activities by the chemical shift
change.8 These probes are hydrolyzed by the reporter enzyme and
change the 19F chemical shift. Although this approach is promising,
this is highly dependent on the magnitude of chemical shift changes
coupled with the target reaction, where sometimes the ranges of
the chemical shift changes are limited. We here propose a novel
design strategy for 19F MRI probes in detecting protease activity.
We constructed a design principle whereby the intramolecular
paramagnetic effect for T2 of the 19F NMR signal can be modulated
by protease activities. T2 is an important contrast factor for MRI,
as the apparent intensity of the MRI signal directly depends on T2
values. Generally, paramagnetic metal ions such as Fe3+, Gd3+, or
the paramagnetic molecules such as O2 shorten the T2 of samples
by paramagnetic relaxation enhancement (PRE).9 In particular, Gd3+
has a very strong relaxivity (T2-shortening activity) because of its
large electron spin quantum number. When 19F nuclei and a Gd3+
ion are attached to a short peptide, the 19F nuclei exhibit a strong
paramagnetic effect from the Gd3+. Thus, the T2 of the compound
in the 19F NMR would be shortened, and the MRI signal would be
attenuated. If the peptide has a substrate sequence which can be
cleaved by a certain protease, incubation of the compound with
the protease should induce the extension of the T2 and the
enhancement of the 19F MRI signal (Scheme 1).
According to the above strategy, we synthesized a 19F MRI probe,
Gd-DOTA-DEVD-Tfb, for detecting caspase-3 activity (Figure 1).
Caspase-3 is a marker enzyme of apoptosis and is used in the
evaluation of anticancer agents inducing apoptosis of tumor cells.
The probe consists mainly of three parts, which are a Gd3+ complex,
an enzyme substrate peptide, and a 19F-containing group. The
peptide sequence is DEVD because caspase-3 selectively cleaves
the C-terminal peptide bond of the sequence DXXD (X: optional).10
A macrocyclic metal ligand, DOTA, was attached via a â-alanine
linker with the N-terminus of 19F-containing peptide DEVD-Tfb,
which was synthesized by Fmoc solid-phase peptide synthesis. The
ligand-conjugated peptide, DOTA-DEVD-Tfb, was complexed with
Gd3+ ion and purified with a reversed-phase HPLC to yield Gd-
1H is a highly NMR-sensitive nuclide and abundant in living
1
bodies. Thus, H MRI is used as a powerful diagnostic imaging
technique for identifying many human pathologies or medical
conditions. Paramagnetic contrast agents such as several types of
Gd complexes are in clinical use for their abilities to enhance the
signal intensity by shortening the longitudinal relaxation time (T1)
of water protons. Presently, many scientists are interested in
modifying the structures of the contrast agents to be functional in
molecular imaging of biomolecules.1 MRI probes for pH,2 metal
ions,3 enzyme activities,4 and so on have been developed. However,
1H MRI often suffers from interference or low contrast due to the
background signals from intrinsic 1H, which hamper interpretation
of the resultant images. Therefore, non-proton MRI is currently
drawing a fair amount of attention.
One of the most promising nuclides for MRI is 19F.5 This nuclide
has a high gyromagnetic ratio (γ) of 40.05 MHz/T and a 100%
natural isotopic abundance ratio. Thus, the NMR sensitivity of 19
F
1
is 0.83 relative to H. Conveniently, due to their close γ values,
1
19F NMR can be measured with most H NMR instruments by
appropriately tuning the RF coils. In our bodies, 19F atoms are
concentrated in the form of solid salts mostly in bones and teeth.
Thus, the transverse relaxation time (T2) of the intrinsic 19F is
extremely shortened,6 and the MRI signal is hardly detectable. When
19F-containing compounds are treated in human or animals, only
the extrinsic 19F MRI signals can be monitored without interference
from background signals. For these above reasons, functional probes
for 19F MRI are very attractive for in vivo molecular imaging.
Known 19F MRI probes are roughly categorized into two groups;
one is the group of 19F-containing compounds which accumulate
in specific sites. Higuchi and co-workers synthesized a 19F-
containing thioflavin derivative that accumulates in amyloid â (Aâ)
aggregates and visualized Aâ plaques in living animals by 19F MRI.7
† Osaka University.
‡ Yokohama City University.
§ Kyoto University.
Bruker BioSpin K.K.
¶ CREST, JST.
9
794
J. AM. CHEM. SOC. 2008, 130, 794-795
10.1021/ja077058z CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 1. Structure of Gd-DOTA-DEVD-Tfb.
Figure 3. Time course of density-weighted 19F MR images of Gd-DOTA-
DEVD-Tfb (1 mM) with or without caspase-3 at 37 °C.
on MRI signal quenching from the intramolecular paramagnetic
effect of Gd3+. The intramolecular Gd3+ made the T2 of the probe
too short to be measured, with the paramagnetic effect which can
be cancelled by the probe hydrolyzation by caspase-3. T2 is a
parameter that can be used to generate contrasts in MR images.
Using this probe as a positive contrast agent, we demonstrated that
the probe could detect caspase-3 activity spatially from the phantom
image using 19F MRI. This method could be applied to the sensing
of not only other kinds of proteases but also other hydrolases such
as nucleases and phosphodiesterases. It is expected that this sensing
strategy might become the basis for the next stage of in vivo
molecular imaging techniques.
Figure 2. (a) 19F NMR spectra of DOTA-DEVD-Tfb (1 mM) and Gd-
DOTA-DEVD-Tfb (1 mM). Sodium trifluoroacetate was added as an internal
standard (0 ppm). (b) Time-dependent 19F NMR spectral change of Gd-
DOTA-DEVD-Tfb with caspase-3 at 37 °C.
DOTA-DEVD-Tfb. The probe structure was identified with ESI-
TOF MS, and its purity was confirmed by HPLC and 19F NMR.
Next, we measured the 19F T1 and T2 values of the probe free
from and in complex with Gd3+. The T1 and T2 of DOTA-DEVD-
Tfb (1 mM) were 1.910 and 0.326 s, respectively. In contrast, the
values of Gd-DOTA-DEVD-Tfb could not be measured, due to
markedly shorter relaxation times and the low signal intensity of
the 19F resonance. This result indicates that the 19F nucleus of Gd-
DOTA-DEVD-Tfb undergoes strong PRE by Gd3+. This effect was
explicitly shown in the one-dimensional 19F NMR spectrum, in
which the peak intensity of Gd-DOTA-DEVD-Tfb was largely
decreased with broadening as compared with that of DOTA-DEVD-
Tfb (Figure 2a). When we treated the Gd-DOTA-DEVD-Tfb with
caspase-3 at 37 °C, the 19F NMR peak became sharper and higher
in a time-dependent manner (Figure 2b). This indicated the
intramolecular paramagnetic effect from Gd3+ to 19F was cancelled
by the cleavage of the probe. The T1 and T2 of the 1 mM cleaved
product were extended to 0.122 and 0.032 s, respectively. These
values are still shorter than those of DOTA-DEVD-Tfb. An
additional experiment suggests that this is because of the intermo-
lecular PRE by the Gd3+ complex (Figure S4 in Supporting
Information).
Acknowledgment. This work was supported by MEXT of
Japan, by JST, by the Mitsubishi Foundation, by Kato Memorial
Bioscience Foundation, by Astellas Foundation for Research on
Metabolic Disorders, by the Uehara Memorial Foundation, by
Terumo Life Science Foundation, by Nagase Science and Technol-
ogy Foundation and by the Asahi Glass Foundation to K.K., by
the Cosmetology Research Foundation to S.M., by CREST (JST)
to M.S. We thank Dr. Tetsuro Kokubo at Yokohama City University
for the use of MRI instrument.
Supporting Information Available: Detailed experimental pro-
cedures and supplementary results. This material is available free of
charge via the Internet at http://pubs.acs.org.
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In conclusion, a novel design principle of 19F MRI probes
detecting protease activity was developed. This principle is based
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