Tasuku Ueno

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.

Fluorescent probes for sensing and imaging

A diverse array of small molecule–based fluorescent probes is available for many different types of biological experiments. Here we examine the history of these probes and discuss some of the most interesting applications.

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.

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.

A Photocleavable Rapamycin Conjugate for Spatiotemporal Control of Small GTPase Activity

We developed a novel method to spatiotemporally control the activity of signaling molecules. A newly synthesized photocaged rapamycin derivative induced rapid dimerization of FKBP (FK-506 binding protein) and FRB (FKBP-rapamycin binding protein) upon UV irradiation. With this system and the spatially confined UV irradiation, we achieved subcellularly localized activation of Rac, a member of small GTPases. Our technique offers a powerful approach to studies of dynamic intracellular signaling events.

Organelle-specific, rapid induction of molecular activities and membrane tethering

Using new chemically inducible dimerization probes, we generated a system to rapidly target proteins to individual intracellular organelles. Using this system, we activated Ras GTPase at distinct intracellular locations and induced tethering of membranes from two organelles, endoplasmic reticulum and mitochondria. Innovative techniques to rapidly perturb molecular activities and organelle-organelle communications at precise locations and timing will provide powerful strategies to dissect spatiotemporally complex biological processes.

Rapid and orthogonal logic gating with a gibberellin-induced dimerization system

Using a newly synthesized gibberellin analog containing an acetoxymethyl group (GA(3)-AM) and its binding proteins, we developed an efficient chemically inducible dimerization (CID) system that is completely orthogonal to existing rapamycin-mediated protein dimerization. Combining the two systems should allow applications that have been difficult or impossible with only one CID system. By using both chemical inputs (rapamycin and GA(3)-AM), we designed and synthesized Boolean logic gates in living mammalian cells. These gates produced output signals such as fluorescence and membrane ruffling on a timescale of seconds, substantially faster than earlier intracellular logic gates. The use of two orthogonal dimerization systems in the same cell also allows for finer modulation of protein perturbations than is possible with a single dimerizer.

Reversible Off–On Fluorescence Probe for Hypoxia and Imaging of Hypoxia–Normoxia Cycles in Live Cells

We report a fully reversible off-on fluorescence probe for hypoxia. The design employs QSY-21 as a Förster resonance energy transfer (FRET) acceptor and cyanine dye Cy5 as a FRET donor, based on our finding that QSY-21 undergoes one-electron bioreduction to the radical under hypoxia, with an absorbance decrease at 660 nm. At that point, FRET can no longer occur, and the dye becomes strongly fluorescent. Upon recovery of normoxia, the radical is immediately reoxidized to QSY-21, with loss of fluorescence due to restoration of FRET. We show that this probe, RHyCy5, can monitor repeated hypoxia-normoxia cycles in live cells.

Covalent attachment of mechanoresponsive luminescent micelles to glasses and polymers in aqueous conditions.

Covalent attachment of mechanoresponsive luminescent organic or organometallic compounds to other materials is a promising approach to develop a wide variety of mechanoresponsive luminescent materials. Here, we report covalently linkable mechanoresponsive micelles that change their photoluminescence from yellow to green in response to mechanical stimulation under aqueous conditions. These micelles are composed of a dumbbell-shaped amphiphilic pyrene derivative having amine groups at the peripheral positions of its dendrons. Using a well-established cross-linker, the micelles were covalently linked via their peripheral amine groups to the surface of glass beads, polylactic acid (PLA) beads, and living cells under aqueous conditions. Vortexing of glass beads bearing the micelles in a glass vial filled with water caused a photoluminescence color change from yellow to green. PLA beads bearing the micelles showed no change in photoluminescence color under the same conditions. We ascribe this result to the lower density and stiffness of the PLA beads, because the color of the PLA beads changed on vortexing in the presence of bare glass beads. HeLa cells and HL-60 cells bearing the micelles showed no obvious photoluminescence color change under vortexing. The structure, photophysical properties, and mechanism of photoluminescence color change of the micellar assemblies were examined.

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