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A sensitive bioluminogenic probe for highly reactive oxygen species (hROS), SO3 H-APL, was developed based on the concept of dual control of bioluminescence emission by means of bioluminescent enzyme-induced electron transfer (BioLeT) and modulation of cell-membrane permeability. This probe enables non-invasive visualization of physiologically relevant amounts of hROS generated deep inside the body of living rats for the first time. It is expected to serve as a practical analytical tool for investigating a wide range of biological functions of hROS in vivo. The design concept should be applicable to other in vivo bioluminogenic probes.

The development of sophisticated fluorescent probes has contributed to the elucidation of the molecular mechanisms of many complex biological phenomena. In particular, fluorescence imaging of the calcium ion (Ca) has become an essential technique for the investigation of signaling pathways involving Ca as a second messenger. For example, changes in the intracellular Ca concentration have been found to be related to physiological responses in obesity, as well as immune responses and pathological responses in Alzheimer s disease. Because Ca signaling is involved in so many biological phenomena, it is expected that the simultaneous visualization of Ca and other biomolecules, that is, multicolor imaging, would be particularly informative. For this purpose, we require a fluorescent probe for Ca that operates in a different color window from that of probes for other molecules. Fluorescent Ca sensors can be categorized into twomain classes: those based on genetically encoded fluorescent proteins and those based on fluorescent small organic molecules. Although both types of sensors have certain advantages and drawbacks, small-molecule-based probes have the particular advantage that their AM ester form (cell-permeable acetoxymethyl ester derivative) can be readily bulk loaded into live cells with no need for transfection. Most currently used small-molecular fluorescent probes for Ca are fluorescein-based, such as Fluo-3, Fluo-4, Calcium Green-1, and Oregon Green 488 BAPTA-1, and emit green fluorescence (ca. 527 nm). There are also some redemitting fluorescent probes for Ca, such as Rhod-2 (ca. 576 nm), which is based on the rhodamine scaffold. These red-emitting fluorescent probes for Ca, including Rhod-2, are also widely used for biological studies; however, the cationic nature of the rhodamine scaffold generally causes Rhod-2 AM to localize into mitochondria. Although this behavior is useful for monitoring the Ca dynamics of mitochondria, the visualization of cytoplasmic Ca is much more important for research on Ca signaling. The influx of Ca into the cytoplasm from the extracellular environment and/or from intracellular stores (including the endoplasmic reticulum) triggers numerous cellular responses mediated by the interaction of Ca with various Ca-binding proteins, such as calmodulin and troponin C. Fura Red is a representative near-infrared fluorescent probe for Ca that is often used in biological research. However, it has extremely low fluorescence quantum efficiency (Ffl 0.013). Accordingly, the fluorescence signal is very small unless a high concentration of Fura Red or a high-powered laser is used. However, the use of a high dye concentration has a buffering effect on Ca, whereas the use of a high laser power causes rapid photobleaching of the dye and phototoxicity to the cells. Thus, a novel fluorescent probe for cytoplasmic Ca with strong emission in the long-wavelength region would be extremely useful, especially for multicolor imaging. In the present study, we designed and synthesized a novel and practical red-fluorescence-emitting probe suitable for monitoring cytoplasmic Ca and confirmed its usefulness for the visualization of stimulus-induced Ca oscillation in HeLa cells. As a fluorophore that emits in the red region, we chose TokyoMagenta (TM). The absorption and fluorescence wavelengths of this fluorescein analogue are 90 nm longer than those of fluorescein. TM was also expected to retain the advantages of the fluorescein scaffold, including cytoplasmic localization. For the development of the red fluorescent probe, we chose a combination of 2-Me-substituted TM as the fluorescent moiety and 1,2-bis(o-aminophenoxy)ethaneN,N,N’,N’-tetraacetic acid (BAPTA) as a specific chelator for Ca, and synthesized CaTM-1 (Figure 1; see also Scheme S1 in the Supporting Information). The fluorescence-activation ratio of CaTM-1 in the presence/absence of Ca is 5.6:1 (Figure 2a,b, Table 1). To further improve this ratio, we decided on the strategy of decreasing the energy of the highest occupied molecular orbital (HOMO) of the fluorophore to obtain a high level of [*] T. Egawa, K. Hirabayashi, Dr. Y. Koide, C. Kobayashi, Dr. N. Takahashi, Dr. T. Terai, Dr. T. Ueno, Dr. T. Komatsu, Dr. Y. Ikegaya, Prof. N. Matsuki, Prof. T. Nagano, Dr. K. Hanaoka Graduate School of Pharmaceutical Sciences The University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan) E-mail: khanaoka@mol.f.u-tokyo.ac.jp