研究者詳細 - 花岡　健二郎

論文 - 花岡　健二郎

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Design and synthesis of a highly sensitive off-on fluorescent chemosensor for zinc ions utilizing internal charge transfer

Hanaoka K., Muramatsu Y., Urano Y., Terai T., Nagano T.

Chemistry - A European Journal （Chemistry - A European Journal） 16 （ 2 ） 568 - 572 2010年01月

研究論文（学術雑誌）, 査読有り, ISSN 09476539

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 (Zn2+) is the second most abundant heavy metal ion in the human body after iron, and detection of chelatable Zn2+ in biological studies has attracted much attention. Herein, we present a novel, highly sensitive off-on fluorescent chemosensor for Zn2+ 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 Zn2+, 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 Zn2+. But even though the sensor shows a weak fluorescence in the absence of Zn2+, this fluorescence can be further suppressed by selecting an excitation wavelength that is barely absorbed by the Zn2+-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 Zn2+ 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 Zn2+-sensitive off-on fluorescent sensor molecule that employs the ICT mechanism; most off-on sensor molecules for Zn2+ employ the photoinduced electron transfer (PeT) mechanism. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

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Molecular design strategies for near-infrared ratiometric fluorescent probes based on the unique spectral properties of aminocyanines

Kiyose K., Aizawa S., Sasaki E., Kojima H., Hanaoka K., Terai T., Urano Y., Nagano T.

Chemistry - A European Journal （Chemistry - A European Journal） 15 （ 36 ） 9191 - 9200 2009年09月

研究論文（学術雑誌）, 査読有り, ISSN 09476539

In spite of the wide availability of various near-infrared (NIR) fluorophores as labeling reagents, there are few functional NIR fluorescent probes for which change in the absorption and/or fluorescence spectra upon specific reaction with biomolecules is seen. The widely used photoinduced electron-transfer mechanism is unsuitable for NIR fluorophores, such as tricarbocyanines, because their long excitation wavelength results in a small singlet ex-citation energy. We have reported the unique spectral properties of aminesubstituted tricarbocyanines, which were utilized to develop two design strategies. One approach was based on control of the absorption wavelength by using the difference in electron-do-nating ability before and after a specific reaction with a biomolecule, and the other approach was based on control of the fluorescence intensity by modulating the Förster resonance energytransfer efficiency through a change in the overlap integral that arises from the change in absorption under acidic conditions. These strategies were validated by obtaining tricarbocyaninebased ratiometric NIR fluorescent probes for esterase and for pH level. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA,.

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A simple and effective strategy to increase the sensitivity of fluorescence probes in living cells

Izumi S., Urano Y., Hanaoka K., Terai T., Nagano T.

Journal of the American Chemical Society （Journal of the American Chemical Society） 131 （ 29 ） 10189 - 10200 2009年07月

研究論文（学術雑誌）, 査読有り, ISSN 00027863

Noninvasive visualization and investigation of interactions among proteins, biomolecules, and enzymes in living cells is an important goal for biologists, and fluorescence probes are powerful tools for this purpose. Because many target molecules are present in only trace amounts, high sensitivity is very important, and it is common to improve the sensitivity of fluorescence probes by focusing on high reaction velocity, Kd. (Gee, K. R.; Archer, E. A.; Lapham, L. A.; Leonard, M. E.; Zhou, Z.; Bingham, J.; Diwu, Z. Bioorg. Med. Chem. Lett. 2000, 10, 1515-1518.) So far, we have designed and synthesized various highly sensitive fluorescence probes based on the above concepts. (Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2004, 126, 3357-3367. Komatsu, K.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 13447-13454.) Nevertheless, they were sometimes insufficiently sensitive to detect biomolecules in living cells, despite high chemical sensitivity in cuvette. In this report, we suggest a new approach to increase the sensitivity of fluorescence probes, focusing on their intracellular retention. Since calcein is well-retained, we investigated its structural, chemical, and optical characteristics and found that the iminodiacetic acid group (IAG) is a key structure for the intracellular retention. We next designed and synthesized novel fluorescence probes containing IAGs. They showed superior intracellular retention, making it possible to visualize low concentrations of target molecules that would be difficult to observe with conventional probes and permitting long-term observation in living cells. Improvement of intracellular retention of fluorescence probes holds great promise as a strategy for developing a wide range of highly sensitive probes for studies on various biological phenomena. © 2009 American Chemical Society.

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

Koide Y., Urano Y., Yatsushige A., Hanaoka K., Terai T., Nagano T.

Journal of the American Chemical Society （Journal of the American Chemical Society） 131 （ 17 ） 6058 - 6059 2009年05月

研究論文（学術雑誌）, 査読有り, ISSN 00027863

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.

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Development of 2,6-carboxy-substituted boron dipyrromethene (BODIPY) as a novel scaffold of ratiometric fluorescent probes for live cell imaging

Komatsu T., Urano Y., Fujikawa Y., Kobayashi T., Kojima H., Terai T., Hanaoka K., Nagano T.

Chemical Communications （Chemical Communications）（ 45 ） 7015 - 7017 2009年

研究論文（学術雑誌）, 査読有り, ISSN 13597345

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Design and synthesis of highly sensitive fluorogenic substrates for glutathione S-transferase and application for activity imaging in living cells

Fujikawa Y., Urano Y., Komatsu T., Hanaoka K., Kojima H., Terai T., Inoue H., Nagano T.

Journal of the American Chemical Society （Journal of the American Chemical Society） 130 （ 44 ） 14533 - 14543 2008年11月

研究論文（学術雑誌）, 査読有り, ISSN 00027863

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.

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The detection limit of a Gd<sup>3+</sup>-based T<inf>1</inf> agent is substantially reduced when targeted to a protein microdomain

Hanaoka K., Lubag A., Castillo-Muzquiz A., Kodadek T., Sherry A.

Magnetic Resonance Imaging （Magnetic Resonance Imaging） 26 （ 5 ） 608 - 617 2008年06月

研究論文（学術雑誌）, 査読有り, ISSN 0730725X

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.

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A Gd<sup>3+</sup>-based magnetic resonance imaging contrast agent sensitive to β-Galactosidase activity utilizing a receptor-induced magnetization enhancement (RIME) phenomenon

Hanaoka K., Kikuchi K., Terai T., Komatsu T., Nagano T.

Chemistry - A European Journal （Chemistry - A European Journal） 14 （ 3 ） 987 - 995 2008年02月

研究論文（学術雑誌）, 査読有り, ISSN 09476539

Magnetic resonance imaging (MRI) permits noninvasive three-dimensional imaging of opaque organisms. Gadolinium (Gd3+) complexes have become important imaging tools as MRI contrast agents for MRI studies, though most of them are nonspecific and report solely on anatomy. Recently, MRI contrast agents have been reported whose ability to relax water protons is triggered or greatly enhanced by recognition of a particular biomolecule. This new class of MRI contrast agents could open up the possibility of reporting on the physiological state or metabolic activity deep within living specimens. One possible strategy for this purpose is to utilize the increase in the longitudinal water proton r1 relaxivity that occurs upon slowing the molecular rotation of a small paramagnetic complex, a phenomenon which is known as receptor-induced magnetization enhancement (RIME), by either binding to a macro-molecule or polymerization of the agent itself. Here we describe the design and synthesis of a novel β-galactosidase-activated MRI contrast agent, the Gd3+ complex [Gd-5], by using the RIME approach. β-Galactosidase is commonly used as a marker gene to monitor gene expression. This newly synthesized compound exhibited a 57% increase in the r1 relaxivity in phosphate-buffered saline (PBS) with 4.5% w/v human serum albumin (HSA) in the presence of β-galactosidase. Detailed investigations revealed that RIME is the dominant factor in this increase of the observed r1 relaxivity, based on analysis of Gd3+ complexes [Gd-5] and [Gd-8], which is generated from [Gd-5] by the activity of β-galactosidase, and spectroscopic analysis of their corresponding Tb3+ complexes, [Tb-5] and [Tb-8]. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

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Time-resolved long-lived luminescence imaging method employing luminescent lanthanide probes with a new microscopy system

Hanaoka K., Kikuchi K., Kobayashi S., Nagano T.

Journal of the American Chemical Society （Journal of the American Chemical Society） 129 （ 44 ） 13502 - 13509 2007年11月

研究論文（国際会議プロシーディングス）, 査読有り, ISSN 00027863

Superior fluorescence imaging methods are needed for detailed studies on biological phenomena, and one approach that permits precise analyses is time-resolved fluorescence measurement, which offers a high signal-to-noise ratio. Herein, we describe a new fluorescence imaging system to visualize biomolecules within living biological samples by means of time-resolved, long-lived luminescence microscopy (TRLLM). In TRLLM, short-lived background fluorescence and scattered light are gated out, allowing the long-lived luminescence to be selectively imaged. Usual time-resolved fluorescence microscopy provides fluorescence images with nanosecond resolution and has been used to image interactions between proteins, protein phosphorylation, the local pH, the refractive index, ion or oxygen concentrations, etc. Luminescent lanthanide complexes (especially europium and terbium trivalent ions (Eu 3+ and Tb3+)), in contrast, have long luminescence lifetimes on the order of milliseconds. We have designed and synthesized new luminescent Eu3+ complexes for TRLLM and also developed a new TRLLM system using a conventional fluorescence microscope with an image intensifier unit for gated signal acquisition and a xenon flash lamp as the excitation source. When the newly developed luminescent Eu3+ complexes were applied to living cells, clear fluorescence images were acquired with the TRLLM system, and short-lived fluorescence was completely excluded. By using Eu 3+ and Tb3+ luminescent complexes in combination, time-resolved dual-color imaging was also possible. Furthermore, we monitored changes of intracellular ionic zinc (Zn2+) concentration by using a Zn2+-selective luminescent Eu3+ chemosensor, [Eu-7]. This new imaging technique should facilitate investigations of biological functions with fluorescence microscopy, complementing other fluorescence imaging methodologies. © 2007 American Chemical Society.

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Iron hydroxide nanoparticles coated with poly(ethylene glycol)-poly(aspartic acid) block copolymer as novel magnetic resonance contrast agents for in vivo cancer imaging

Kumagai M., Imai Y., Nakamura T., Yamasaki Y., Sekino M., Ueno S., Hanaoka K., Kikuchi K., Nagano T., Kaneko E., Shimokado K., Kataoka K.

Colloids and Surfaces B: Biointerfaces （Colloids and Surfaces B: Biointerfaces） 56 （ 1-2 ） 174 - 181 2007年04月

研究論文（学術雑誌）, 査読有り, ISSN 09277765

PEG-coated β-FeOOH nanoparticles were prepared through electrostatic complex formation of iron oxide nanoparticles with poly(ethylene glycol)-poly(aspartic acid) block copolymer [PEG-P(Asp)] in distilled water. By dynamic light scattering (DLS) measurement, the nanopaticle size was determined to be 70 nm with narrow distribution. The FT-IR and zeta potential experimental results proved that PEG-PAsp molecules bound to the surface of the iron oxide nanoparticles via the coordination between the carboxylic acid residues in the PAsp segment of the block copolymer and the surface Fe of the β-FeOOH nanoparticles. The PEG-coated nanoparticles revealed excellent solubility and stability in aqueous solution as well as in physiological saline. In vivo MRI experiments on tumor-bearing mice demonstrated that the PEG-coated nanoparticles prepared by the current approach achieved an appreciable accumulation into solid tumor, suggesting their potential utility as tumor-selective MRI contrast agents. © 2007 Elsevier B.V. All rights reserved.

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Design and synthesis of an enzyme activity-based labeling molecule with fluorescence spectral change

Komatsu T., Kikuchi K., Takakusa H., Hanaoka K., Ueno T., Kamiya M., Urano Y., Nagano T.

Journal of the American Chemical Society （Journal of the American Chemical Society） 128 （ 50 ） 15946 - 15947 2006年12月

研究論文（学術雑誌）, 査読有り, ISSN 00027863

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.

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Development of a zinc ion-selective luminescent lanthanide chemosensor for biological applications

Hanaoka K., Kikuchi K., Kojima H., Urano Y., Nagano T.

Journal of the American Chemical Society （Journal of the American Chemical Society） 126 （ 39 ） 12470 - 12476 2004年10月

研究論文（学術雑誌）, 査読有り, ISSN 00027863

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.

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Selective detection of zinc ions with novel luminescent lanthanide probes

Hanaoka K., Kikuchi K., Kojima H., Urano Y., Nagano T.

Angewandte Chemie - International Edition （Angewandte Chemie - International Edition） 42 （ 26 ） 2996 - 2999 2003年07月

研究論文（学術雑誌）, 査読有り, ISSN 14337851

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Design and synthesis of a novel magnetic resonance imaging contrast agent for selective sensing of zinc ion

Hanaoka K., Kikuchi K., Urano Y., Narazaki M., Yokawa T., Sakamoto S., Yamaguchi K., Nagano T.

Chemistry and Biology （Chemistry and Biology） 9 （ 9 ） 1027 - 1032 2002年09月

研究論文（学術雑誌）, 査読有り, ISSN 10745521

A series of new diethylenetriaminepentaacetic acid (DTPA)-bisamide chelators has been prepared and characterized for application as zinc sensors. We have designed and synthesized (GdLa)2-, which contains a DTPA-bisamide moiety. The R1 relaxivity of (GdLa)2- solution decreased monotonically on the addition of Zn2+. Moreover, (GdLa)2- showed high selectivity for Zn2+ against Ca2+ and Mg2+. We also measured the UV-visible spectra and the coldspray ionization (CSI) MS spectra and concluded that the 1-to-1 Zn2+ complex of (GdLa)2- is stable at higher concentrations of Zn2+. These complexes should provide the basis for creating a superior Zn2+-sensitive MRI contrast agent and are excellent candidates for incorporation into sensors designed for selective detection of Zn2+ in biological applications.

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Selective sensing of zinc ions with a novel magnetic resonance imaging contrast agent

Hanaoka K., Kikuchi K., Urano Y., Nagano T.

Journal of the Chemical Society. Perkin Transactions 2 （Journal of the Chemical Society. Perkin Transactions 2） 1 （ 9 ） 1840 - 1843 2001年

研究論文（学術雑誌）, 査読有り, ISSN 03009580

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.

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花岡 健二郎

論文 - 花岡 健二郎

花岡　健二郎

論文 - 花岡　健二郎