Kenjiro Hanaoka

Development of a Highly Selective Fluorescence Probe for Hydrogen Sulfide

Hydrogen sulfide (H(2)S) has recently been identified as a biological response modifier. Here, we report the design and synthesis of a novel fluorescence probe for H(2)S, HSip-1, utilizing azamacrocyclic copper(II) ion complex chemistry to control the fluorescence. HSip-1 showed high selectivity and high sensitivity for H(2)S, and its potential for biological applications was confirmed by employing it for fluorescence imaging of H(2)S in live cells.

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.

Development of a Highly Sensitive Fluorescence Probe for Hydrogen Peroxide

Hydrogen peroxide is believed to play a role in cellular signal transduction by reversible oxidation of proteins. Here, we report the design and synthesis of a novel fluorescence probe for hydrogen peroxide, utilizing a photoinduced electron transfer strategy based on benzil chemistry to control the fluorescence. The practical value of this highly sensitive and selective fluorescence probe, NBzF, was confirmed by its application to imaging of hydrogen peroxide generation in live RAW 264.7 macrophages. NBzF was also employed for live cell imaging of hydrogen peroxide generated as a signaling molecule in A431 human epidermoid carcinoma cells.

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.

Design and Synthesis of a Highly Sensitive Off–On Fluorescent Chemosensor for Zinc Ions Utilizing Internal Charge Transfer

Fluorescence imaging is a powerful tool for the visualization of biological molecules in living cells, tissue slices, and whole bodies, and is important for elucidating biological phenomena. Furthermore, zinc (Zn(2+)) is the second most abundant heavy metal ion in the human body after iron, and detection of chelatable Zn(2+) in biological studies has attracted much attention. Herein, we present a novel, highly sensitive off-on fluorescent chemosensor for Zn(2+) by using the internal charge transfer (ICT) mechanism. The rationale of our approach to highly sensitive sensor molecules is as follows. If fluorescence can be completely quenched in the absence of Zn(2+), chemosensors would offer a better signal-to-noise ratio. However, it is difficult to quench the fluorescence completely before Zn(2+) binding, and most sensor molecules still show very weak fluorescence in the absence of Zn(2+). But even though the sensor shows a weak fluorescence in the absence of Zn(2+), this fluorescence can be further suppressed by selecting an excitation wavelength that is barely absorbed by the Zn(2+)-free sensor molecule. Focusing on careful control of ICT within the 4-amino-1,8-naphthalimide dye platform, we designed and synthesized a new chemosensor (1) that shows a pronounced fluorescence enhancement with a blueshift in the absorption spectrum upon addition of Zn(2+). The usefulness of 1 for monitoring Zn(2+) changes was confirmed in living HeLa cells. There have been several reports on 4-amino-1,8-naphthalimide-based fluorescent sensor molecules. However, 1 is the first Zn(2+)-sensitive off-on fluorescent sensor molecule that employs the ICT mechanism; most off-on sensor molecules for Zn(2+) employ the photoinduced electron transfer (PeT) mechanism.

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.

Design and Synthesis of Highly Sensitive Fluorogenic Substrates for Glutathione S-Transferase and Application for Activity Imaging in Living Cells

Here we report the development of fluorogenic substrates for glutathione S-transferase (GST), a multigene-family enzyme mainly involved in detoxification of endogenous and exogenous compounds, including drug metabolism. GST is often overexpressed in a variety of malignancies and is involved in the development of resistance to various anticancer drugs. Despite the medical significance of this enzyme, no practical fluorogenic substrates for fluorescence imaging of GST activity or for high-throughput screening of GST inhibitors are yet available. So, we set out to develop new fluorogenic substrates for GST. In preliminary studies, we found that 3,4-dinitrobenzanilide (NNBA) is a specific substrate for GST and established the mechanisms of its glutathionylation and denitration. Using these results as a basis for off/on control of fluorescence, we designed and synthesized new fluorogenic substrates, DNAFs, and a cell membrane-permeable variant, DNAT-Me. These fluorogenic substrates provide a dramatic fluorescence increase upon GST-catalyzed glutathionylation and have excellent kinetic parameters for the present purpose. We were able to detect nuclear localization of GSH/GST activity in HuCCT1 cell lines with the use of DNAT-Me. These results indicate that the newly developed fluorogenic substrates should be useful not only for high-throughput GST-inhibitor screening but also for studies on the mechanisms of drug resistance in cancer cells.

Development of Azo‐Based Fluorescent Probes to Detect Different Levels of Hypoxia

Let it shine: New hypoxia-sensitive fluorescent probes were developed; they consist of a rhodamine moiety with an azo group directly conjugated to the fluorophore. Because of an ultrafast conformational change around the NN bond, the compounds are nonfluorescent under normoxia. However, under hypoxia, the azo group is reduced, and a strongly fluorescent rhodamine derivative is released.

Rational Design of Highly Sensitive Fluorescence Probes for Protease and Glycosidase Based on Precisely Controlled Spirocyclization

We have synthesized and evaluated a series of hydroxymethyl rhodamine derivatives and found an intriguing difference of intramolecular spirocyclization behavior: the acetylated derivative of hydroxymethyl rhodamine green (Ac-HMRG) exists as a closed spirocyclic structure in aqueous solution at physiological pH, whereas HMRG itself takes an open nonspirocyclic structure. Ac-HMRG is colorless and nonfluorescent, whereas HMRG is strongly fluorescent. On the basis of these findings, we have developed a general design strategy to obtain highly sensitive fluorescence probes for proteases and glycosidases, by replacing the acetyl group of Ac-HMRG with a substrate moiety of the target enzyme. Specific cleavage of the substrate moiety in the nonfluorescent probe by the target enzyme generates a strong fluorescence signal. To confirm the validity and flexibility of our strategy, we designed and synthesized fluorescence probes for leucine aminopeptidase (Leu-HMRG), fibroblast activation protein (Ac-GlyPro-HMRG), and β-galactosidase (βGal-HMRG). All of these probes were almost nonfluorescent due to the formation of spirocyclic structure, but were converted efficiently to highly fluorescent HMRG by the target enzymes. We confirmed that the probes can be used in living cells. These probes offer great practical advantages, including high sensitivity and rapid response (due to regulation of fluorescence at a single reactive site), as well as resistance to photobleaching, and are expected to be useful for a range of biological and pathological investigations.