Monitoring of biological one-electron reduction by 19F NMR using hypoxia selective activation of an 19F-labeled indolequinone derivative

Article abstract of DOI:10.1021/ja904953b

(Chemical Equation Presented) Biological reduction of fluorine-labeled indolequinone derivative (IQ-F) was characterized by 19F NMR for quantitative molecular understanding. The chemical shift change in 19F NMR allowed monitoring of the enzymatic reductio

Full text of DOI:10.1021/ja904953b

Published on Web 10/20/2009
Monitoring of Biological One-Electron Reduction by 19F NMR Using Hypoxia
Selective Activation of an 19F-Labeled Indolequinone Derivative
Kazuhito Tanabe,*,† Hiroshi Harada,‡,§ Michiko Narazaki,| Kazuo Tanaka,⊥ Kenichi Inafuku,⊥
Hirokazu Komatsu,† Takeo Ito,† Hisatsugu Yamada,†,§ Yoshiki Chujo,⊥ Tetsuya Matsuda,|
Masahiro Hiraoka,‡,§ and Sei-ichi Nishimoto*,†
Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto UniVersity, Katsura
Campus, Nishikyo-ku, Kyoto 615-8510, Japan, Department of Radiation Oncology and Image-applied Therapy,
Graduate School of Medicine, Kyoto UniVersity, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan, Nano-Medicine Merger
Education Unit, Kyoto UniVersity, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan, Department of Systems Science,
Graduate School of Informatics, Kyoto UniVersity, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan, and
Department of Polymer Chemistry, Graduate School of Engineering, Kyoto UniVersity, Katsura Campus,
Nishikyo-ku, Kyoto 615-8510, Japan
Received June 18, 2009; E-mail: tanabeka@scl.kyoto-u.ac.jp; nishimot@scl.kyoto-u.ac.jp
Intracellular reductases have been closely linked with activation
of certain drugs and probes in the tumor-specific microenviron-
ments.1 Among various enzymes, reductases that catalyze one-
electron reduction are involved in the selective activation of
functional compounds or materials under hypoxia,2 a well-known
pathophysiological characteristic of solid tumors.3 Such an enzy-
matic one-electron reduction has been recognized to be a useful
reaction applicable to the design of a tumor hypoxia targeting and
imaging strategy. Thus, further quantitative insights into the features
of this reaction are important.
IQ-F was synthesized by coupling 3-hydroxymethyl-5-methoxy-
1,2-dimethylindole-4,7-dione with F-OH (Scheme S1). We con-
ducted the enzymatic reduction of IQ-F in an Ar-purged aqueous
acetonitrile solution by means of NADPH:cytochrome P450 re-
ductase, which catalyzes the one-electron reduction of quinone
derivatives to semiquinone anion radicals.7a We incubated IQ-F
with NADPH:cytochrome P450 reductase and its cofactor ꢀ-NAD-
PH under hypoxic conditions. Figure 1 shows the reaction of IQ-F
monitored by 19F NMR. The appearance of a single new signal at
-73.6 ppm during hypoxic treatment is attributable to the formation
of F-OH, as confirmed by reference to authentic sample, while the
IQ-F starting compound almost completely disappeared (Figure 1B).
These results clearly indicate that IQ-F is activated to release the
corresponding alcohol F-OH by enzymatic reduction, thereby causing
a change in the 19F NMR spectra. In contrast, upon enzymatic treatment
under aerobic conditions, a substantial amount of IQ-F remained to
produce a negligible signal attributable to F-OH (Figure 1C). Thus,
enzymatic reduction of IQ-F occurred in a hypoxia-selective manner,
as can be monitored by 19F NMR.10
Here we demonstrate probing of the biological one-electron
reduction of a fluorine (F)-labeled indolequinone (IQ) derivative
by 19F NMR that gave us straightforward molecular information
even under complicated biological reduction conditions due to low
concentrations of endogenous F atoms and the absence of interfer-
ence with proton signals.4-6 The family of IQ compounds is well
characterized to be superior substrates for several reductases
expressed in tumor cells and readily undergoes enzymatic one-
electron reduction under hypoxic conditions. Consequently, IQ
derivatives have been employed to develop bioreductive prodrugs
and imaging probes targeting tumor hypoxia7,8 that are efficiently
activated by endogenous reductase to release a given functional
component selectively under hypoxic conditions. We prepared an 19
F
NMR signal supplier (IQ-F) consisting of a hypoxia-sensitive IQ parent
unit and a nonafluoro-tert-butyl group and monitored the change in
19F chemical shift during the bioreduction. One-electron reduction of
IQ-F by isolated or intracellular reductase under hypoxic conditions
released the nonafluoro-tert-butyl alcohol (F-OH) constituent. We
observed a new 19F signal due to the resultant F-OH at a characteristic
chemical shift, which differed from that of IQ-F. In contrast, the release
of F-OH was efficiently suppressed upon addition of O2 and thereby
the corresponding signal failed to appear. Kinetic studies indicated that
O2 prevented to a lesser extent the binding of IQ-F to reductase but
decreased the rate of the net reaction due to oxidation of a semiquinone
anion radical intermediate generated during the course of the one-
electron reduction into the parent IQ-F. In addition, the present reaction
could be monitored by chemical shift selective fast spin echo (FSE),9
leading to visualization of the hypoxia-selective reduction by signal
intensity using MR imaging.
Figure 1. (A) Bioreduction of IQ-F under hypoxic conditions to release
F-OH. (B, C, D) One-electron reduction of IQ-F monitored by 19F NMR.
IQ-F (0.93 mM) was incubated with NADPH:cytochrome P450 reductase
(11.4 µg) and ꢀ-NADPH (2 mM) at 37 °C in phosphate buffer; (B) incubated
for 15 min under hypoxic conditions; (C) incubated for 15 min under aerobic
conditions; and (D) before incubation.
The steady-state kinetic parameters, Vmax and Km, for the release of
F-OH were derived from a Lineweaver-Burk plot11 of the integra-
tion values evaluated in the 19F NMR spectra (Figure S1). The Vmax
value for F-OH formation under aerobic conditions (0.20 ( 0.04
pmol min-1) was considerably lower than that under hypoxic conditions
(3.1 ( 0.2 pmol min-1), while the Km value obtained from hypoxic
treatment (0.41 ( 0.04 mM) was similar to that from aerobic treatment
(0.59 ( 0.16 mM). Thus, binding of IQ-F to reductase was slightly
affected by the oxygen concentration, while the net reaction rate was
dramatically reduced in the presence of O2. These characteristics are
consistent with the conventional mechanism hitherto proposed for IQ
† Department of Energy and Hydrocarbon Chemistry.
‡ Department of Radiation Oncology and Image-applied Therapy.
§ Nano-Medicine Merger Education Unit.
| Department of Systems Science.
⊥ Department of Polymer Chemistry.
9
15982 J. AM. CHEM. SOC. 2009, 131, 15982–15983
10.1021/ja904953b CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
derivatives. A semiquinone anion radical intermediate is generated
under both hypoxic and aerobic conditions by enzymatic one-electron
reduction of IQ. The resulting intermediate is subject to oxidization
by O2 to regenerate the original IQ along with the formation of O2-•
under aerobic conditions, leading to significant suppression of the net
reaction. The enzymatic activation of IQ-F, which leads to an NMR
signal change, is likely to occur substantially under hypoxic conditions.
To better understand the function of IQ-F in living cells, we also
assessed the one-electron reduction of IQ-F in a human cell line of
lung carcinoma A549 cells that express NADPH:cytochrome P450
reductase in high amounts.12,13 A549 cells were cultured in the
presence of IQ-F for 12 h under hypoxic or aerobic conditions. The
culture medium and the cell lysate were individually harvested and
subjected to an NMR study. In a similar manner to the treatment of
IQ-F with isolated reductase, a signal originating form the formation
of F-OH was observed in the medium obtained upon incubation under
hypoxic conditions, as shown in Figure 2A. We also confirmed that a
weak F-OH signal was produced upon aerobic treatment. In a control
experiment, no signal was observed in the corresponding cell lysates
independent of oxygen concentrations (Figure S2). Therefore IQ-F
most likely penetrates into living cells where it is activated to release
F-OH by intracellular reductases in a hypoxic environment, while it
is promptly eliminated from cells due to the small size of the molecule,
resulting in the low concentration of the intracellular F-signal supplier,
which was below the detection limit.
In conclusion, the hypoxia-selective one-electron reduction of
IQ-F, which consists of a hypoxia-sensitive IQ parent unit and an
19F signal-transmitting molecular unit of nonafluoro-tert-butyl group,
was characterized by 19F NMR. During monitoring of the biological
reduction of IQ-F, hypoxia-selective activation occurred to induce
a chemical shift change of 19F signals attributable to the reductive
formation of F-OH. The disappearance of IQ-F to form F-OH could
be imaged simultaneously by 19F FSE, thus visualizing the
occurrence of the enzymatic one-electron reduction in a hypoxia-
selective manner by means of MR imaging.
The one-electron reduction of IQ derivatives has been widely
used for hypoxia targeting and imaging. The IQ-F activation system
could be applicable to MR imaging of tumor hypoxia. Optimization
of the chemical structure of IQ-F derivatives to increase water
solubility and intracellular retention and characterization of their
pharmacokinetic profiles are now in progress.
Acknowledgment. This work is partly supported by the
Innovative Techno-Hub for Integrated Medical Bioimaging Project
of the Special Coordination Funds for Promoting Science and
Technology, from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT), Japan and by the Program for
Promotion of Fundamental Studies in Health Sciences of the
National Institute of Biomedical Innovation (NIBIO), Japan.
Supporting Information Available: Detail of synthetic protocol
and enzymatic reduction of IQ-F. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Figure 2. (A) One-electron reduction of IQ-F in A549 cells. A549 cells
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evidence that incubation of IQ-F in a buffer resulted in a similar 19
F
image as in the sample incubated under aerobic conditions (Figure
S5), the hypoxia-selective one-electron reduction of IQ-F could be
clearly monitored by 19F MR imaging.
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