Yuichiro Koide

Development of an Si-Rhodamine-Based Far-Red to Near-Infrared Fluorescence Probe Selective for Hypochlorous Acid and Its Applications for Biological Imaging

A far-red to near-infrared (NIR) fluorescence probe, MMSiR, based on Si-rhodamine, was designed and synthesized for sensitive and selective detection of HOCl in real time. MMSiR and its oxidized product SMSiR have excellent properties, including pH-independence of fluorescence, high resistance to autoxidation and photobleaching, and good tissue penetration of far-red to NIR fluorescence emission. The value of MMSiR was confirmed by real-time imaging of phagocytosis using a fluorescence microscope. wsMMSiR, a more hydrophilic derivative of MMSiR, permitted effective in vivo imaging of HOCl generation in a mouse peritonitis model. This probe is expected to be a useful tool for investigating the wide range of biological functions of HOCl.

Evolution of Group 14 Rhodamines as Platforms for Near-Infrared Fluorescence Probes Utilizing Photoinduced Electron Transfer

The absorption and emission wavelengths of group 14 pyronines and rhodamines, which contain silicon, germanium, or tin at the 10 position of the xanthene chromophore, showed large bathochromic shifts compared to the original rhodamines, owing to stabilization of the LUMO energy levels by σ*-π* conjugation between group 14 atom-C (methyl) σ* orbitals and a π* orbital of the fluorophore. These group 14 pyronines and rhodamines retain the advantages of the original rhodamines, including high quantum efficiency in aqueous media (Φ(fl) = 0.3-0.45), tolerance to photobleaching, and high water solubility. Group 14 rhodamines have higher values of reduction potential than other NIR light-emitting original rhodamines, and therefore, we speculated their NIR fluorescence could be controlled through the photoinduced electron transfer (PeT) mechanism. Indeed, we found that the fluorescence quantum yield (Φ(fl)) of Si-rhodamine (SiR) and Ge-rhodamine (GeR) could be made nearly equal to zero, and the threshold level for fluorescence on/off switching lies at around 1.3-1.5 V for the SiRs. This is about 0.1 V lower than in the case of TokyoGreens, in which the fluorophore is well established to be effective for PeT-based probes. That is to say, the fluorescence of SiR and GeR can be drastically activated by more than 100-fold through a PeT strategy. To confirm the validity of this strategy for developing NIR fluorescence probes, we employed this approach to design two kinds of novel fluorescence probes emitting in the far-red to NIR region, i.e., a series of pH-sensors for use in acidic environments and a Zn(2+) sensor. We synthesized these probes and confirmed that they work well.

Development of NIR Fluorescent Dyes Based on Si–rhodamine for in Vivo Imaging

We have developed a series of novel near-infrared (NIR) wavelength-excitable fluorescent dyes, SiR-NIRs, by modifying the Si-rhodamine scaffold to obtain emission in the range suitable for in vivo imaging. Among them, SiR680 and SiR700 showed sufficiently high quantum efficiency in aqueous media. Both antibody-bound and free dye exhibited high tolerance to photobleaching in aqueous solution. Subcutaneous xenograft tumors were successfully visualized in a mouse tumor model using SiR700-labeled anti-tenascin-C (TN-C) antibody, SiR700-RCB1. SiR-NIRs are expected to be useful as labeling agents for in vivo imaging studies including multicolor imaging, and also as scaffolds for NIR fluorescence probes.

Development of a Far-Red to Near-Infrared Fluorescence Probe for Calcium Ion and its Application to Multicolor Neuronal Imaging

To improve optical imaging of Ca(2+) and to make available a distinct color window for multicolor imaging, we designed and synthesized CaSiR-1, a far-red to near-infrared fluorescence probe for Ca(2+), using Si-rhodamine (SiR) as the fluorophore and the well-known Ca(2+) chelator BAPTA. This wavelength region is advantageous, affording higher tissue penetration, lower background autofluorescence, and lower phototoxicity in comparison with the UV to visible range. CaSiR-1 has a high fluorescence off/on ratio of over 1000. We demonstrate its usefulness for multicolor fluorescence imaging of action potentials (visualized as increases in intracellular Ca(2+)) in brain slices loaded with sulforhodamine 101 (red color; specific for astrocytes) that were prepared from transgenic mice in which some neurons expressed green fluorescent protein.

Red Fluorescent Probe for Monitoring the Dynamics of Cytoplasmic Calcium Ions

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

Design and development of enzymatically activatable photosensitizer based on unique characteristics of thiazole orange.

A novel design strategy for controlling the fluorescence and photosensitizing ability of thiazole orange (TO) has been developed. The validity of this approach was demonstrated by the synthesis of a beta-galactosidase-activatable photosensitizer, PhoTO-Gal, in which fluorescence is simultaneously activated. PhoTO-Gal was demonstrated to kill HEK293 lacZ(+) cells, which express beta-galactosidase, but not HEK293 lacZ(-) cells, under light illumination. Such activatable photosensitizers should allow more refined PDT without the side effect of prolonged light sensitivity and should also be useful as tools for reporter enzyme expression-specific cell ablation.

Activatable Optical Imaging with a Silica-Rhodamine Based Near Infrared (SiR700) Fluorophore: A comparison with cyanine based dyes

Optical imaging is emerging as an important tool to visualize tumors. However, there are many potential choices among the available fluorophores. Optical imaging probes that emit in the visible range can image superficial tumors with high quantum yields; however, if deeper imaging is needed then near-infrared (NIR) fluorophores are necessary. Most commercially available NIR fluorophores are cyanine based and are prone to nonspecific binding and relatively limited photostability. Silica-containing rhodamine (SiR) fluorophores represent a new class of NIR fluorophores, which permit photoactivation via H-dimer formation as well as demonstrate improved photostability. This permits higher tumor-to-background ratios (TBRs) to be achieved over longer periods of time. Here, we compared an avidin conjugated with SiR700 (Av-SiR700) to similar compounds based on cyanine dyes (Av-Cy5.5 and Av-Alexa Fluor 680) in a mouse tumor model of ovarian cancer metastasis. We found that the Av-SiR700 probe demonstrated superior quenching, enabling activation after binding-internalization to the target cell. As a result, Av-SiR700 had higher TBRs compared to Av-Cy5.5 and better biostability compared to Av-Alexa Fluor 680.