Published on Web 02/05/2010
Development and Application of a Near-Infrared Fluorescence
Probe for Oxidative Stress Based on Differential Reactivity of
Linked Cyanine Dyes
Daihi Oushiki,†,‡ Hirotatsu Kojima,‡,§ Takuya Terai,†,‡ Makoto Arita,†
Kenjiro Hanaoka,†,‡ Yasuteru Urano,† and Tetsuo Nagano*,†,‡,§
Graduate School of Pharmaceutical Sciences and Chemical Biology Research InitiatiVe, The
UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, and CREST, Japan Science and
Technology Agency, 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012, Japan
Received November 30, 2009; E-mail: tlong@mol.f.u-tokyo.ac.jp
Abstract: Reactive oxygen species (ROS) operate as signaling molecules under various physiological
conditions, and overproduction of ROS is involved in the pathogenesis of many diseases. Therefore,
fluorescent probes for visualizing ROS are promising tools with which to uncover the molecular mechanisms
of physiological and pathological processes and might also be useful for diagnosis. Here we describe a
novel fluorescence probe, FOSCY-1, operating in the physiologically favorable near-infrared region. The
probe consists of two differentially ROS-reactive cyanine dyes connected by a linker; reaction of the more
susceptible dye with ROS releases intramolecular fluorescence quenching of the less susceptible dye. We
successfully applied this probe to detect ROS produced by HL60 cells and porcine neutrophils and for
imaging oxidative stress in a mouse model of peritonitis.
Introduction
ROS. However, for imaging in tissues or individuals, fluoro-
chromes whose absorption and emission maxima are in the near-
Reactive oxygen species (ROS) are important signaling
molecules which regulate a wide range of physiological func-
tions, but overproduction of ROS results in oxidative stress and
is involved in the pathogenesis of many diseases, including
cardiovascular disease, cancer, and neurological disorders.1-3
Therefore, methods for visualizing ROS would be powerful tools
to elucidate the molecular mechanisms that underlie such
physiological and pathological conditions and might also be
useful for diagnosis.
infrared (NIR) region, 650-900 nm, are preferred, because they
offer low phototoxicity to cells, minimal interference from
hemoglobin absorption, low autofluorescence, and good tissue
penetration.15,16 From these points of view, existing probes have
severe limitations for in vivo applications. Therefore, we
attempted to develop a novel NIR fluorescence probe for
imaging of ROS in vivo.
Among NIR fluorochromes, cyanine dyes have attracted con-
siderable attention, and several functional cyanine probes have
recently been developed.17 Akkaya et al. reported an NIR fluo-
rescence probe for Ca2+, whose fluorescence intensity is controlled
by photoinduced electron transfer (PeT).18 Our group also devel-
oped NIR fluorescent probes (DACs) for nitric oxide based on the
PeT mechanism.19 A ratiometric NIR probe for Zn2+, DIPCY, was
also designed by our group.20 Fluorescence modulation of DIPCY
is controlled by the difference in the electron-donating ability of
Various methods for visualizing ROS have been developed.4-7
Among them, fluorescence imaging methods are generally
superior in terms of sensitivity, selectivity, and ease of use.
Several groups, including ours, have reported fluorescence
probes for ROS based on fluorescein, rhodamine, or BODIPY,
which are fluorochromes emitting in the visible region.8-14
These probes have been widely used for cellular imaging of
† Graduate School of Pharmaceutical Sciences, The University of Tokyo.
‡ Japan Science and Technology Agency.
(10) Kenmoku, S.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc.
2007, 129, 7313–7318.
§ Chemical Biology Research Initiative, The University of Tokyo.
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10.1021/ja910090v 2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 2795–2801 2795