Highly sensitive near-infrared fluorescent probes for nitric oxide and their application to isolated organs

Article abstract of DOI:10.1021/ja042967z

Novel near-infrared (NIR) fluorescent probes for nitric oxide (NO) have been designed, synthesized, and evaluated. Their NIR fluorescence was increased in an NO concentration-dependent manner under physiological conditions, and their reaction efficiency w

Full text of DOI:10.1021/ja042967z

Published on Web 02/24/2005
Highly Sensitive Near-Infrared Fluorescent Probes for Nitric Oxide and Their
Application to Isolated Organs
Eita Sasaki, Hirotatsu Kojima, Hiroaki Nishimatsu, Yasuteru Urano, Kazuya Kikuchi,†,§
†
†
‡
†,§
‡
,†
Yasunobu Hirata, and Tetsuo Nagano*
Graduate School of Pharmaceutical Sciences, and Faculty of Medicine, The UniVersity of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan, and PRESTO, Japan Science and Technology Corporation,
Kawaguchi, Saitama, Japan
Received November 22, 2004; E-mail: tlong@mol.f.u-tokyo.ac.jp
Nitric oxide (NO), which is synthesized through conversion of
L-arginine to L-citrulline by NO synthase in vivo, is an important
signaling molecule involved in the regulation of a wide range of
physiological and pathophysiological mechanisms, and many
disorders related to NO signaling impairment have been reported.1
Therefore, methods for visualizing NO would be powerful tools to
examine in detail the NO signaling mechanisms3 and might
eventually be useful for diagnosis as well.
Scheme 1
,2
Fluorescence imaging methods are generally superior in terms
of sensitivity, selectivity, and ease of use. Recently, we have
successfully developed several fluorescent probes for NO,4 and
these probes have been widely used in biological applications.
However, they have severe limitations with regard to ex vivo
applications to detect NO in isolated organs since their fluorescence
lies in the visible region around 500-550 nm, which cannot
penetrate deeply into human tissues. While visible light is highly
absorbed by biological substances, such as hemoglobin, near-
infrared (NIR) light at around 650-900 nm is less absorbed by
such molecules and can penetrate more deeply into tissues.
Moreover, it has the further advantage that autofluorescence is not
observed upon NIR excitation. For these reasons, NIR fluorescence
,5
Then, we examined the spectral properties of the probes. As we
expected, the NIR fluorescence intensity of DACs greatly increased
in an NO concentration-dependent manner (Figure 1). The fluo-
6
imaging is potentially very attractive for in vivo imaging. In this
report, we present novel NIR fluorescent probes which are highly
sensitive to NO and establish their utility by imaging NO in isolated
intact rat kidneys.
Figure 1. (A) Absorbance spectra and (B) emission spectra (excitation at
750 nm) of DAC-S (3 µM) in the presence of various amounts of NO at 37
°C in 0.1 M sodium phosphate buffer, pH 7.4. The spectra were obtained
15 min after the addition of a saturated NO aqueous solution (2 mM; 0-12.5
µL) to a solution of DAC-S (3 mL).
The probes are composed of two moieties: tricarbocyanine as
the NIR fluorophore, which has a high extinction coefficient of
5
-1
-1
about 1.5-2.0 × 10 M cm , and o-phenylenediamine as the
NO-sensitive fluorescence modulator. Our strategy for detecting
NO is based on the change of the electron-donating ability of
o-phenylenediamine upon selective NO-mediated transformation of
diamine into triazole under aerobic conditions. We predicted that
o-phenylenediamine should quench the fluorescence of tricarbocya-
nine because electron transfer should occur from o-phenylenedi-
amine to the excited fluorophore. On the other hand, the corre-
sponding triazole formation should recover its NIR fluorescence
since the triazole ring should not have sufficient electron-donating
ability for such photoinduced electron transfer (PeT) to occur
rescence quantum yields of DAC-Ts were 14-fold higher than that
of DACs. We next evaluated the effect of pH on the fluorescence.
The fluorescence of DACs was quenched at pH above 6, and that
of DAC-Ts were independent of pH from 2 to 12. That is to say,
DACs work well under physiological pH conditions, although the
fluorescence intensities of DACs were relatively high at pH below
6. We can explain this recovery of the fluorescence under acidic
conditions in terms of protonation of the amino group reducing
the electron-donating ability of o-phenylenediamine and abolishing
7
a
the PeT; the pK value of protonated o-phenylenediamine is 4.47.
These spectral properties indicate that PeT is successfully regulated
(Scheme 1). We named these new probes diaminocyanines, DACs,
8
in these molecules. According to the Rehm-Weller equation, the
and the corresponding triazole compounds DAC-Ts. They can be
classified into two types; DAC-P with two propyl groups was
designed to penetrate cellular membranes without any modification,
and DAC-S with sulfonate groups was expected to be highly soluble
in water. Consequently, the appropriate type can be selected for
particular purposes.
longer the wavelength of fluorophore is, the more difficult it is for
PeT to occur. As far as we know, there is only one reported NIR
fluorescent probe in which the fluorescence is controlled by the
9
PeT mechanism. Therefore, it is noteworthy that we could design
these fluorescent probes and control PeT in the NIR region.
Furthermore, we compared the reactivity with NO of DAC-S
4
with that of DAF-2, which is a widely used NO fluorescent probe
†
Graduate School of Pharmaceutical Sciences.
‡
(Figure 2A). It was previously shown that the reaction rate of
Faculty of Medicine.
§
5
PRESTO.
o-phenylenediamine with NO depends on electron density. In the
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10.1021/ja042967z CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. (A) Structure of DAF-2. (B) The extent of formation of the
triazoles of DAC-S and DAF-2 (3 µM, 0.1% DMSO) after addition of
various amounts of NO at 37 °C in 0.1 M sodium phosphate buffer, pH
7
.4. The fluorescence intensity was measured 15 min after the addition of
a saturated NO aqueous solution (2 mM; 0-24 µL) to a solution of DAC-S
and DAF-2 (3 mL), and the extent of formation of the corresponding
triazoles was calculated from the fluorescence quantum yields.
case of DAC-S, the electron density is increased by an electron-
donating oxygen atom, while DAF-2 has an electron-withdrawing
carboxylate group. Thus, DAC-S is expected to react faster than
DAF-2. The result of competitive reaction of DAC-S and DAF-2
with NO is shown in Figure 2B. DAF-2 did not react with NO
until almost all of the DAC-S was converted into DAC-S-T. When
Figure 3. (A) Increase of the fluorescence intensity of DAC-P in rat kidney
upon administration of NOC13. The fluorescence intensity is an averaged
value calculated from an entire picture plane. The rat kidney was perfused
with Krebs-Henseleit buffer at 5 mL/min. After the loading of DAC-P (5
µM) for 4 min, NOC13 (0.1 or 1 mM) was administered for 3 min (shown
by arrows). (B) The captured NIR fluorescence image of a part of a rat
kidney after the loading of DAC-P. (C) The image after the administration
of NOC13 (0.1 mM). (D) The image after the administration of NOC13 (1
mM). All images are reproduced in pseudocolor.
8
µL of a saturated NO aqueous solution was added, 44% of DAC-S
and 0.83% of DAF-2 were converted to the corresponding triazoles.
Namely, the reaction efficiency of DAC-S with NO is at least 53
times higher than that of DAF-2 under an equimolar condition. This
means that, although DAF-2 has functioned well in many kinds of
cells so far, these new probes can potentially detect NO more
effectively in biological tissues in the presence of endogenous
competitors, such as thiols.
Finally, we applied DAC to isolated rat kidneys to examine
whether it worked in ex vivo biological systems and whether we
could observe the fluorescence change from outside the kidney
without making sections. We selected DAC-P because it should
be loaded more readily than DAC-S. Kidneys from male Wistar
rats were isolated and perfused as described previously.10 A diagram
of the system is shown in the Supporting Information. As we
expected, DAC-P was easily loaded into the kidneys simply by
administering it into the right renal artery with the perfusate for
rat kidneys. Because the reaction rate of DACs with NO is fast
and the observation of their NIR fluorescence is less subject to
interference by biological substances, our NO-detecting probes are
expected to be applicable to not only cellular but also in vivo NO
imaging, and work along this line is proceeding.
Acknowledgment. This work was supported in part by the
Ministry of Education, Culture, Sports, Science and Technology
of Japan (Grants for The Advanced and Innovational Research
Program in Life Sciences to T.N., 15790070 to H.K.). H.K. was
also supported by the Nissan Science Foundation, by the Nakatomi
Foundation, and by the Konica Imaging Science Foundation.
Supporting Information Available: Full experimental procedures,
synthesis, and characterization data for all compounds, spectral proper-
ties of DACs and DAC-Ts, pH profiles of DAC-S and DAC-S-T, a
diagram of the perfused rat kidney system, and an NIR fluorescence
image of a renal section. This material is available free of charge via
the Internet at http://pubs.acs.org.
several minutes and was hardly washed out throughout the
observation. Then, NOC13,11 which is an NO donor with a half-
life of 4.7 min in aqueous buffer solution at pH 7.4 and 37 °C,
was administered in the same manner. The NIR fluorescence images
were captured every 20 s with a fluorescence stereomicroscope.
We observed a fluorescence increase during the administration of
NOC13 (Figure 3A). That is to say, DAC-P functions in ex vivo
biological systems, and we could detect its fluorescence from
outside the kidney. We noticed many circular patterns with a
diameter of approximately 0.2 mm on the fluorescence images
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