Published papers

We have developed a novel red fluorescent dye, 2Me SiR600 (λem = 613 nm), in which the O atom of Rhodamine Green at the 10 position of the xanthene moiety is replaced with a Si atom, as a scaffold for probes to detect protease activity with extremely high S/N ratio. As proof of concept, we designed and synthesized probes for caspase-3 activity (Z-DEVD-SiR600) and leucine aminopeptidase activity (Leu-SiR600). Caspase-3-mediated cleavage of Z-DEVD-SiR600 resulted in a large bathochromic shift (93 nm) of the absorption maximum and a 432-fold fluorescence enhancement. © 2012 Elsevier Ltd. All rights reserved.

The Escherichia coli lacZ gene encoding β-galactosidase is a widely used reporter, but few synthetic substrates are available for detecting its activity with single-cell resolution in living samples. Our recently reported fluorogenic substrate SPiDER-βGal is suitable for this purpose, but its hydrolysis product shows green fluorescence emission, and a red-shifted analogue is therefore required for use in combination with green fluorescent protein (GFP) markers. Herein, we describe the development of a red-shifted fluorogenic substrate for β-galactosidase, SPiDER-Red-βGal, based on a silicon rhodol scaffold and a carboxylic group as the intramolecular nucleophile. LacZ-positive cells were successfully labeled with SPiDER-Red-βGal at single-cell resolution in living samples, which enabled us to visualize different cell types in combination with GFP markers.

We have developed an activatable photosensitizer capable of specifically inducing the death of β-galactosidase-expressing cells in response to photoirradiation. By using a selenium-substituted rhodol scaffold bearing β-galactoside as a targeting substituent, we designed and synthesized HMDESeR-βGal, which has a non-phototoxic spirocyclic structure owing to the presence of the galactoside moiety. However, β-galactosidase efficiently converted HMDESeR-βGal into phototoxic HMDESeR, which exists predominantly in the open xanthene form. This structural change resulted in drastic recovery of visible-wavelength absorption and the ability to generate singlet oxygen (1O2). When HMDESeR-βGal was applied to larval Drosophila melanogaster wing disks, which express β-galactosidase only in the posterior region, photoirradiation induced cell death in the β-galactosidase-expressing region with high specificity. Bull's eye! An activatable photosensitizer capable of specifically inducing the death of β-galactosidase-expressing cells in response to light irradiation was developed. Reaction with the enzyme resulted in a dynamic structural change to the phototoxic open form (see scheme), thus enabling the specific ablation of cells of interest in living tissues. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A remarkable enhancement of luminescence is observed in a solution of the TbIII complex 1 upon adding ZnII ions (see picture). Complex 1 has a high selectivity for ZnII and a long luminescence lifetime of the order of milliseconds, thus is an excellent lead compound for the development of ZnII-sensitive luminescent chemosensors with long luminescence lifetimes. A = light absorption, ET = energy transfer, E = light emission.

Light-based microscope imaging techniques using fluorescence sensor molecules suffer from photobleaching and light scattering, but magnetic resonance imaging (MRI) can provide three-dimensional imaging without these problems. Recently, “smart” MRI contrast agents which modulate the access of water to a chelated gadolinium (Gd3+) ion in the presence or absence of a specific trigger have been reported. Zinc (Zn2+) is an essential component of many enzymes, transcription factors and synaptic vesicles in excitatory nerve terminals, so imaging of chelatable Zn2+is of interest. We have designed and synthesized the Gd3+DTPA bisamide complex 7a as a Zn2+-sensitive MRI contrast agent. Compound 7a shows a dose-dependent change in the R1relaxivity in the presence of Zn2+. We investigated this relaxation behavior, and for this purpose we also synthesized the Gd3+DTPA amide ethyl ester complex 7b. It was shown that binding between 7a and Zn2+caused a change in the relaxation time. Moreover, 7a had high selectivity for Zn2+against Ca2+and Mg2+. Compound 7a may have practical problems for in vivo usage, since the R1relaxivity is reduced with increased Zn2+concentration. However, this report demonstrates new approaches to the design and synthesis of Gd3+complexes with R1values that change with variation in Zn2+concentration. © 2001 The Royal Society of Chemistry.

We present a novel design strategy for off/on fluorescent probes suitable for selective two-step labeling of proteins. To validate this strategy, we designed and synthesized an off/on fluorescent probe, 1-Ni 2+, which targets a cysteine-modified hexahistidine (His) tag. The probe consists of dichlorofluorescein conjugated with nitrilotriacetic acid (NTA)-Ni 2+ as the His-tag recognition site and a 2,4-dinitrophenyl ether moiety, which quenches the probe's fluorescence by photoinduced electron transfer (PeT) from the excited fluorophore to the 2,4-dinitrophenyl ether (donor-excited PeT; d-PeT) and also has reactivity with cysteine. His-tag recognition by the NTA-Ni 2+ moiety is followed by removal of the 2,4-dinitrophenyl ether quencher by proximity-enhanced reaction with the cysteine residue of the modified tag; this results in a marked fluorescence increase. Addition of His-tag peptide bearing a cysteine residue to aqueous probe solution resulted in about 20-fold fluorescence increment within 10 min, which is the largest fluorescence enhancement so far obtained with a visible light-excitable fluorescent probe for a His-based peptide tag. Further, we successfully visualized CysHis 6-peptide tethered to microbeads without any washing step. The probe also showed a large fluorescence increment in the presence of His 6Cys-tagged enhanced blue fluorescent protein (EBFP), but not His 6-tagged EBFP. We consider this system is superior to large fluorescence tags (e.g., green fluorescent protein: 27 kDa), which can perturb protein folding, trafficking and function, and also to existing small tags, which generally show little fluorescence increase upon target recognition and therefore require a washout step. This strategy should also be applicable to other tags. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Background: Acute lung injury is characterized by neutrophilic inflammation and increased lung permeability. Thiosulfate is a stable metabolite of hydrogen sulfide, a gaseous mediator that exerts antiinflammatory effects. Although sodium thiosulfate (STS) has been used as an antidote, the effect of STS on acute lung injury is unknown. The authors assessed the effects of STS on mice lung and vascular endothelial cells subjected to acute inflammation.

We report the design, synthesis and application of a directly photocontrollable hydrogen sulfide (H2S) donor, which releases H2S proportionally to the intensity and duration of photoirradiation. Photocontrolled H2S release from this donor was also demonstrated in bovine serum. This H2S donor should be suitable for use in various biological systems. © 2013 The Royal Society of Chemistry.

Simple low molecular weight (MW) chelates of Gd3+ such as those currently used in clinical MRI are considered too insensitive for most molecular imaging applications. Here, we evaluated the detection limit (DL) of a molecularly targeted low MW Gd3+-based T1 agent in a model where the receptor concentration was precisely known. The data demonstrate that receptors clustered together to form a microdomain of high local concentration can be imaged successfully even when the bulk concentration of the receptor is quite low. A GdDO3A-peptide identified by phage display to target the anti-FLAG antibody was synthesized, purified and characterized. T1-weighted MR images were compared with the agent bound to antibody in bulk solution and with the agent bound to the antibody localized on agarose beads. Fluorescence competition binding assays show that the agent has a high binding affinity (KD=150 nM) for the antibody, while the fully bound relaxivity of the GdDO3A-peptide/anti-FLAG antibody in solution was a relatively modest 17 mM-1 s-1. The agent/antibody complex was MR silent at concentrations below ∼9 μM but was detectable down to 4 μM bulk concentrations when presented to antibody clustered together on the surface of agarose beads. These results provided an estimate of the DLs for other T1-based agents with higher fully bound relaxivities or multimeric structures bound to clustered receptor molecules. The results demonstrate that the sensitivity of molecularly targeted contrast agents depends on the local microdomain concentration of the target protein and the molecular relaxivity of the bound complex. A model is presented, which predicts that for a molecularly targeted agent consisting of a single Gd3+ complex with bound relaxivity of 100 mM-1 s-1 or, more reasonably, four tethered Gd3+ complexes each having a bound relaxivity of 25 mM-1 s-1, the DL of a protein microdomain is ∼690 nM at 9.4 T. These experimental and extrapolated DLs are both well below current literature estimates and suggests that detection of low MW molecularly targeted T1 agents is not an unrealistic goal. © 2008 Elsevier Inc. All rights reserved.

Molecular assemblies that change photoluminescence color in response to thermal or mechanical stimulation without dissociation into the monomeric states in water are described herein. A dumbbell-shaped amphiphilic compound forms micellar molecular assemblies in water and exhibits yellow photoluminescence derived from excimer formation of the luminescent core, which contains a 2,6-diethynylanthracene moiety. Annealing of the aqueous solution induces a photoluminescence color change from yellow to green (λ em, max=558→525nm). The same photoluminescence color change is also achieved by rubbing the yellow-photoluminescence-emitting molecular assemblies adsorbed on glass substrates with cotton wool in water. The observed green photoluminescence is ascribed to micelles that are distinct from the yellow-photoluminescence-emitting micelles, on the basis of transmission electron microscopy observations, atomic force microscopy observations, and dynamic light scattering measurements. We examined the relationship between the structure of the molecular assemblies and the photophysical properties of the anthracene derivative in water before and after thermal or mechanical stimulation and concluded that thermal or mechanical stimuli-induced slight changes of the molecular-assembled structures in the micelles result in the change in the photoluminescence color from yellow to green in water. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

NAD(P)H-dependent oxidoreductases play important roles in biology. Recently, we reported that the luminescence lifetime of some Tb3+ complexes is sensitive to NAD(P)H, and we used this phenomenon to detect activities of these enzymes. However, conventional time-resolved luminescence assays are susceptible to static quenchers such as ATP. Herein we describe a detection methodology that overcomes this issue: the intensity of the sample is measured twice with different delay times and the intensity ratio value is used as an index of NAD(P)H concentration. The method is more robust than single-point measurement, and is compatible with high-throughput assays using conventional microplate readers.

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. © 2011 American Chemical Society.

Hydrogen sulfide (H 2S) has recently been identified as a biological response modifier. Here, we report the design and synthesis of a novel fluorescence probe for H 2S, HSip-1, utilizing azamacrocyclic copper(II) ion complex chemistry to control the fluorescence. HSip-1 showed high selectivity and high sensitivity for H 2S, and its potential for biological applications was confirmed by employing it for fluorescence imaging of H 2S in live cells. © 2011 American Chemical Society.

We have developed a red-emitting fluorescent K + probe, B3TAC, which also shows a wavelength shift upon binding to K +. The probe was synthesized by conjugating a cryptand-based chelator, 2-triazacryptand [2,2,3-1-(2-methoxyethoxy)benzene (TAC), to position 3 of the BODIPY fluorophore through a styryl linker. In water-acetonitrile mixed solvent, it responded to K + in the physiological concentration range with high selectivity over Na + and other metal ions. B3TAC is potentially useful for measuring cellular K + ion concentration, as well as for simple, naked-eye detection of K + in solution. © 2011 Elsevier Ltd. All rights reserved.

A sensitive bioluminogenic probe for highly reactive oxygen species (hROS), SO3H-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. You light up my life: A sensitive bioluminogenic probe for highly reactive oxygen species (hROS) 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.

In biological systems, the pH in intracellular organelles or tissues is strictly regulated, and differences of pH are deeply related to key biological events such as protein degradation, intracellular trafficking, renal failure, and cancer. Ratiometric fluorescence imaging is useful for determination of precise pH values, but existing fluorescence probes have substantial limitations, such as inappropriate pKa for imaging in the physiological pH range, inadequate photobleaching resistance, and insufficiently long excitation and emission wavelengths. Here we report a versatile scaffold for ratiometric fluorescence pH probes, based on asymmetric rhodamine. To demonstrate its usefulness for biological applications, we employed it to develop two probes. (1) SiRpH5 has suitable pKa and water solubility for imaging in acidic intracellular compartments; by using transferrin tagged with SiRpH5, we achieved time-lapse imaging of pH in endocytic compartments during protein trafficking for the first time. (2) Me-pEPPR is a near-infrared (NIR) probe; by using dextrin tagged with Me-pEPPR, we were able to image extracellular pH of renal tubules and tumors in situ. These chemical tools should be useful for studying the influence of intra- and extracellular pH on biological processes, as well as for in vivo imaging.

Detection of chelatable zinc (Zn2+) in biological studies has attracted much attention recently, because chelatable Zn2+ plays important roles in many biological systems. Lanthanide complexes (Eu 3+, Tb3+, etc.) have excellent spectroscopic properties for biological applications, such as long luminescence lifetimes of the order of milliseconds, a large Stoke's shift of >200 nm, and high water solubility. Herein, we present the design and synthesis of a novel lanthanide sensor molecule, [Eu-7], for detecting Zn2+. This europium (Eu3+) complex employs a quinolyl ligand as both a chromophore and an acceptor for Zn2+. Upon addition of Zn2+ to a solution of [Eu-7], the luminescence of Eu3+ is strongly enhanced, with high selectivity for Zn2+ over other biologically relevant metal cations. One of the important advantages of [Eu-7] is that this complex can be excited with longer excitation wavelengths (around 340 nm) as compared with previously reported Zn2+-sensitive luminescent lamthanide sensors, whose excitation wavelength is at too high an energy level for biological applications. The usefulness of [Eu-7] for monitoring Zn2+ changes in living HeLa cells was confirmed. This novel Zn2+-selective luminescent lanthanide chemosensor [Eu-7] should be an excellent lead compound for the development of a range of novel luminescent lanthanide chemosensors for biological applications.

Photodynamic therapy (PDT) utilizes photoirradiation in the presence of photosensitizers to ablate cancer cells via generation of singlet oxygen (1O2), but it is important to minimize concomitant injury to normal tissues. One approach for achieving this is to use activatable photosensitizers that can generate 1O2 only under specific conditions. Here, we report a novel photosensitizer that is selectively activated under hypoxia, a common condition in solid tumors. We found that introducing an azo moiety into the conjugated system of a seleno-rosamine dye effectively hinders the intersystem crossing process that leads to 1O2 generation. We show that the azo group is reductively cleaved in cells under hypoxia, enabling production of 1O2 to occur. In PDT in vitro, cells under mild hypoxia, within the range typically found in solid tumors (up to about 5% O2), were selectively ablated, leaving adjacent normoxic cells intact. This simple and practical azobased strategy should be widely applicable to design a range of activatable photosensitizers.

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. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.