論文

Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins. However, methodology to monitor the activities of CPs is poorly developed. Here, we present the first versatile design strategy to obtain activatable fluorescent probes for CPs by utilizing intramolecular spirocyclization of rhodamine to translate the "aliphatic carboxamide to aliphatic carboxylate" structural conversion catalyzed by CPs into dynamic fluorescence activation. Based on this novel strategy, we developed probes for carboxypeptidases A and B. One of these probes was able to detect pancreatic juice leakage in mice ex vivo, suggesting that its suitability for intraoperative diagnosis of pancreatic fistula. This design strategy should be broadly applicable to CPs, as well as other previously untargetable enzymes, enabling development of fluorescent probes to study various pathological and biological processes.

Aiming at the evaluation of the viscosity of the interfacial solidlike structure of ionic liquids (ILs), we performed total internal reflection fluorescence (TIRF) spectroscopy for N,N-diethyl-N′-phenyl-rhodamine (Ph-DER), a fluorescent probe that is sensitive to viscosity in a high-viscosity range. TIRF spectra at the glass interface of trioctylmethylammonium bis(nonafluorobutanesulfonyl)amide (TOMAC4C4N), a hydrophobic IL, showed that the fluorescence intensity of Ph-DER increases with the decrease of the evanescence penetration depth, suggesting that there exists a high-viscosity region at the interface. In contrast, glycerol, which is a molecular liquid with a bulk viscosity similar to that of TOMAC4C4N, did not show such a fluorescence increase, supporting that the formation of a highly viscous solidlike structure at the interface is intrinsic to ILs. A model analysis suggested that the high viscous region at the glass interface of TOMAC4C4N is at least twice thicker than the ionic multilayers at the air interface, implying that the solid substrate enhances the ordering of the interfacial structure of ILs. The viscosity at the glass interface of TOMAC4C4N was found to be at least 40 times higher than that of the liquid bulk.

Ca2+ is one of the most important second messengers in cells. A far-red to near-infrared (NIR) Ca2+ fluorescent probe is useful for multi-color imaging in GFP or YFP-expressing biosamples. Here we developed a cytosolically localized far-red to NIR rhodamine-based fluorescent probe for Ca2+, CaSiR-2 AM, while rhodamine dyes are basically localized to mitochondria or lysosomes in cells. This journal is

Exposure to hydrogen sulfide (H2S) can cause neurotoxicity and cardiopulmonary arrest. Resuscitating victims of sulfide intoxication is extremely difficult, and survivors often exhibit persistent neurological deficits. However, no specific antidote is available for sulfide intoxication. The objective of this study was to examine whether administration of a sulfonyl azide-based sulfide-specific scavenger, SS20, would rescue mice in models of H2S intoxication: Ongoing exposure and post-cardiopulmonary arrest. In the ongoing exposure model, SS20 (1250 μmol/kg) or vehicle was administered to awake CD-1 mice intraperitoneally at 10 min after breathing 790 ppm of H2S followed by another 30 min of H2S inhalation. Effects of SS20 on survival were assessed. In the post-cardiopulmonary arrest model, cardiopulmonary arrest was induced by an intraperitoneal administration of sodium sulfide nonahydrate (125 mg/kg) in anesthetized mice. After 1 min of cardiopulmonary arrest, mice were resuscitated with intravenous administration of SS20 (250 μmol/kg) or vehicle. Effects of SS20 on survival, neurological outcomes, and plasma H2S levels were evaluated. Administration of SS20 during ongoing H2S inhalation improved 24-h survival (6/6 [100%] in SS20 vs 1/6 [17%] in vehicle; p =. 0043). Post-arrest administration of SS20 improved 7-day survival (4/10 [40%] in SS20 vs 0/10 [0%] in vehicle; p =. 0038) and neurological outcomes after resuscitation. SS20 decreased plasma H2S levels to pre-arrest baseline immediately after reperfusion and shortened the time to return of spontaneous circulation and respiration. These results suggest that SS20 is an effective antidote against lethal H2S intoxication, even when administered after cardiopulmonary arrest.

We established a methodology for initiating cross-linking of antibodies selectively on the cell surface through intermolecular copper-free click reactions facilitated by increased effective concentrations of antibodies binding to target antigens. Upon cross-linking of tetrazine- and bicyclononyne-modified trastuzumab on the surface of HER2-overexpressing cells, increased antibody uptake and activation of intracellular signaling were observed. Our findings demonstrate that the cross-linking reaction can significantly alter the biophysical properties of proteins, activating their unique functionalities on targeted cells to realize an increased cargo delivery and synthetic manipulation of cellular signaling.

Photoacoustic (PA) imaging is a novel imaging modality that combines the high contrast of optical imaging and the deep tissue penetration of ultrasound. PA imaging contrast agents targeting various biological phenomena have been reported, but the development of activatable PA probes, which show a PA signal only in the presence of target molecules, remains challenging in spite of their potential usefulness for real-time PA imaging of specific biomolecules in vivo. To establish a simple design strategy for activatable PA probes, we first designed and synthesized a silicon-rhodamine based near-infrared nonfluorescent dye, wsSiNQ660 (water-soluble SiNQ660), as a scaffold and demonstrated that it offers a high conversion efficiency from light to ultrasound compared to typical near-infrared fluorescent dyes. Importantly, absorption off/on strategies previously established for rhodamine-based fluorescent probes are also applicable to this nonfluorescent dye scaffold. We validated this approach by synthesizing an activatable PA probe for hypochlorous acid (HOCl) and confirmed that it enables three-dimensional imaging of HOCl in mouse subcutis.

Methods of covalent labeling of a specific tag protein with small-molecular dyes play an important role in studying dynamic behaviors of proteins in living cells. On the basis of quinone methide chemistry, we designed and synthesized a β-galactosidase labeling probe, CMFβ-gal, which shows a fluorescence wavelength change accompanying the labeling reaction, owing to fluorescence resonance energy transfer (FRET). Since the FRET efficiency changes accompanying the labeling reaction, fluorescence of labeled protein can be observed separately from that of the unreacted probe, so immediate detection of the target protein is possible. This is the first report of a protein labeling probe which features a change of fluorescence wavelength upon reaction, allowing the labeled protein to be detected even in the presence of unreacted probe. Copyright © 2006 American Chemical Society.

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. © 2008 American Chemical Society.

Ratiometric fluorescent probes based on boron dipyrromethene (BODIPY) were developed based on a novel design strategy, in which a change of the electron-withdrawing character of the 2,6-substituents resulting from reaction with a target molecule generates a fluorescence wavelength change. © The Royal Society of Chemistry 2009.

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 5-galactosidase-activatable photosensitizer, PhoTO-Gal, in which fluorescence is simultaneously activated. PhoTO-Gal was demonstrated to kill HEK293 lacZ(+) cells, which express 5-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. © 2009 American Chemical Society.

Reactive oxygen species (ROS) operate as signaling molecules under various physiological conditions, and overproduction of ROS is involved in the pathogenesis of many diseases. Therefore, fluorescent probes for visualizing ROS are promising tools with which to uncover the molecular mechanisms of physiological and pathological processes and might also be useful for diagnosis. Here we describe a novel fluorescence probe, FOSCY-1, operating in the physiologically favorable near-infrared region. The probe consists of two differentially ROS-reactive cyanine dyes connected by a linker; reaction of the more susceptible dye with ROS releases intramolecular fluorescence quenching of the less susceptible dye. We successfully applied this probe to detect ROS produced by HL60 cells and porcine neutrophils and for imaging oxidative stress in a mouse model of peritonitis. © 2010 American Chemical Society.

A novel lanthanide probe was designed, synthesized, and employed for a sensitive and reliable assay of acrolein based on time-resolved luminescence measurement, which suppresses the background signal of serum. © 2010 American Chemical Society.

Based on the findings that the azo functional group has excellent properties as the hypoxia-sensor moiety, we developed hypoxia-sensitive near-infrared fluorescent probes in which a large fluorescence increase is triggered by the cleavage of an azo bond. The probes were used for fluorescence imaging of hypoxic cells and real-time monitoring of ischemia in the liver and kidney of live mice. © 2010 American Chemical Society.

We present a design strategy for fluorescence probes with a high off/on activation ratio in the red wavelength region, based on a novel fluorescein analogue in which the O atom at the 10 position of the xanthene chromophore is replaced with a Si atom. To demonstrate the usefulness of this strategy, we designed and synthesized a red-fluorescent probe for β-galactosidase, and showed that it works in live HEK293 cells. © 2011 The Royal Society of Chemistry.

Purpose: Hydrodynamic injection has been shown to reactivate silenced transgene expression in mouse liver. In this study, the roles of inflammatory cytokines and reactive oxygen species (ROS) in the reactivation were examined. Methods: Production of inflammatory cytokines and ROS by hydrodynamic injection of saline was examined in mice that had received a hydrodynamic injection of a plasmid expressing Gaussia luciferase. The level of reporter gene expression was used as an indicator of the reactivation. The involvement of cytokines and ROS was examined by depleting Kupffer cells or by pre-administration of antioxidants, respectively. Results: A hydrodynamic injection of saline induced a significant production of interleukin (IL)-6. Depleting Kupffer cells using clodronate liposomes markedly reduced the IL-6 production but had no significant effect on the transgene expression. On the other hand, an injection of catalase or N-acetylcysteine significantly inhibited the hydrodynamic injection-induced reactivation of silenced transgene expression. The silenced expression was also reactivated by carbon tetrachloride, an inducer of oxidative stress in the liver, in a dose-dependent manner, and this reactivation was significantly inhibited by catalase. Conclusions: These findings show a positive correlation between the generation of ROS and the reactivation of silenced transgene expression after hydrodynamic injections. © 2010 Springer Science+Business Media, LLC.

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. © 2011 American Chemical Society.

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. © 2011 American Chemical Society.

We have developed the first highly selective fluorescence probe for alkaline phosphatase (ALP), TG-mPhos. This probe shows selectivity for ALP over protein tyrosine phosphatase and protein serine/threonine phosphatase. Our previously developed TG-Phos, which has a phenolic phosphate linkage in place of the alcoholic phosphate linkage of TG-mPhos, lacks this selectivity. TG-mPhos should enable precise fluorescence imaging of ALP activity in biological applications. © 2011 Elsevier Ltd. All rights reserved.

We have designed and synthesized a reversible near-infrared (NIR) fluorescence probe, 2-Me TeR, for reactive oxygen species (ROS), utilizing the redox properties of the tellurium (Te) atom. 2-Me TeR is oxidized to fluorescent 2-Me TeOR by various ROS, while the generated 2-Me TeOR is quickly reduced in the presence of glutathione to regenerate 2-Me TeR. This redox-induced reversible NIR-fluorescence response of 2-Me TeR allowed us to detect the endogenous production of ROS and subsequent homeostatic recovery of the intracellular reductive environment in hydrogen peroxide-stimulated HL-60 cells. This probe is expected to be useful for monitoring the dynamics of ROS production continuously in vivo © 2012 Royal Society of Chemistry.