Details of a Researcher - Hanaoka, Kenjiro

Papers - Hanaoka, Kenjiro

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Design and synthesis of a novel fluorescence probe for Zn2+ based on the spirolactam ring-opening process of rhodamine derivatives

Sasaki H., Hanaoka K., Urano Y., Terai T., Nagano T.

Bioorganic and Medicinal Chemistry (Bioorganic and Medicinal Chemistry) 19 ( 3 ) 1072 - 1078 2011.02

Research paper (scientific journal), Accepted, ISSN 09680896

The spirolactam ring-opening process of rhodamine derivative is one of the most useful mechanisms for controlling fluorescence properties. However, the open/closed equilibrium reaction of rhodamine spirolactam has not been well characterized. Therefore, we examined the relationship between the spirolactam ring-opening process of rhodamine derivatives and the structure of the xanthene moiety. Based on the results of this investigation, we selected a candidate xanthene moiety for a Zn2+ sensor, and successfully developed a new fluorescence probe for Zn2+. © 2010 Elsevier Ltd. All rights reserved.

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A time-resolved fluorescence probe for dipeptidyl peptidase 4 and its application in inhibitor screening

Kawaguchi M., Okabe T., Terai T., Hanaoka K., Kojima H., Minegishi I., Nagano T.

Chemistry - A European Journal (Chemistry - A European Journal) 16 ( 45 ) 13479 - 13486 2010.12

Research paper (scientific journal), Accepted, ISSN 09476539

The prevalence of type 2 diabetes is increasing dramatically throughout the world. Recently, dipeptidyl peptidase 4 (DPP4) was identified as a potential antidiabetes target. Many DPP4 inhibitors, such as sitagliptin and vildagliptin, have been developed and marketed, but superior therapeutic agents are still required. Therefore, we have developed new methodology for screening of DPP4 inhibitors. Absorption-based measurements with para-nitroaniline or fluorescence-based measurements with the coumarin derivative 7-amino-4-methylcoumarin are often used for the screening of protease inhibitors, including DPP4 inhibitors, but these strategies are not sufficiently sensitive because of interfering background absorption and fluorescence, thus giving rise to many false-positive and false-negative results. Therefore, we have designed and synthesised a novel DPP4 probe (Gly-Pro-BCD-Tb; Gly=glycine, Pro=proline, andBCD defines the backbone of the probe comprising an aniline derivative as on/off switch, a 7-amino-4-methyl-2(1H)-quinolinone (cs-124) as antenna moiety, and a diethylenetriamine-N,N,N',N'',N''-pentaacetic acid (DTPA) as chelator moiety, Tb=terbium) for time-resolved fluorescence (TRF) measurements. TRF measurements with Gly-Pro-BCD-Tb showed high sensitivity and reliability in the inhibitory assay relative to Gly-Pro-MCA (MCA=4- methylcoumarin-7-amide), a conventional fluorescence probe for DPP4. Further, we employed our probe for high-throughput DPP4 inhibitor screening with 3841 randomly selected compounds and found that epibestatin, an epimer of bestatin (a well-known anticancer drug and general aminopeptidase inhibitor), showed dose-dependent DPP4 inhibitory activity. Interestingly, bestatin did not exhibit DPP4 inhibitory activity. We believe that this screening system will be useful for the discovery of DPP4 inhibitors with novel structural scaffolds. Reliable and highly sensitive inhibitor screening has been realised through the synthesis of a novel dipeptidyl peptidase 4 (DPP4) probe (see picture). The inhibitor screening employed high-throughput time-resolved fluorescence (TRF) measurements and identified epibestatin as a DPP4 inhibitor. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Hypoxia-sensitive fluorescent probes for in vivo real-time fluorescence imaging of acute ischemia

Kiyose K., Hanaoka K., Oushiki D., Nakamura T., Kajimura M., Suematsu M., Nishimatsu H., Yamane T., Terai T., Hirata Y., Nagano T.

Journal of the American Chemical Society (Journal of the American Chemical Society) 132 ( 45 ) 15846 - 15848 2010.11

Research paper (scientific journal), Accepted, ISSN 00027863

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Development of responsive lanthanide-based magnetic resonance imaging and luminescent probes for biological applications

Hanaoka K.

Chemical and Pharmaceutical Bulletin (Chemical and Pharmaceutical Bulletin) 58 ( 10 ) 1283 - 1294 2010.10

Research paper (scientific journal), Accepted, ISSN 00092363

Lanthanide complexes have unique chemical characteristics compared with typical organic complexes, and have recently attracted much interest because of the expanding need for new bioanalytical sensors. For example, magnetic resonance imaging (MRI) permits noninvasive three-dimensional imaging inside opaque organisms, and gadolinium ion (Gd3+) complexes have become important tools as MRI contrast agents. However, most of them are nonspecific, and report solely on anatomy. Therefore, responsive MRI contrast agents, so-called "smart" MRI contrast agents whose ability to relax water protons is greatly enhanced by recognition of a particular biomolecule, have great potential for elucidating biological phenomena. On the other hand, lanthanide complexes such as europium (Eu3+) and terbium (Tb 3+) complexes have excellent luminescence properties for biological applications, i.e., long luminescence lifetime of the order of milliseconds and a large Stoke's shift of >200 nm. Their long-lived luminescence is especially suitable for time-resolved measurements, because the interference from short-lived background fluorescence and scattered light rapidly decays to a negligible level after a pulse of excitation light is applied, and the emitted light can be collected after an appropriate delay time. These luminescent lanthanide complexes have already found commercial use as highly sensitive luminescent probes in heterogeneous and homogeneous assays. This paper reviews our research on the design and synthesis of responsive lanthanide-based MRI and luminescent probes for advanced bioimaging. © 2010 Pharmaceutical Society of Japan.

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Development of luciferin analogues bearing an amino group and their application as BRET donors

Takakura H., Sasakura K., Ueno T., Urano Y., Terai T., Hanaoka K., Tsuboi T., Nagano T.

Chemistry - An Asian Journal (Chemistry - An Asian Journal) 5 ( 9 ) 2053 - 2061 2010.09

Research paper (scientific journal), Accepted, ISSN 18614728

We systematically synthesized bioluminogenic substrates bearing an amino group on benzothiazole, quinoline, naphthalene, and coumarin scaffolds. They emit bioluminescence in various colors: red, orange, yellow, and green. An amino-substituted cou-marylluciferin derivative, coumarylami-noluciferin (CAL), showed the shortest bioluminescence wavelength among substrates reported so far. Further, the fluorescence of CAL did not exhibit solvatochromism, which suggests that its bioluminescence is not susceptible to environmental factors. We applied CAL as an energy-donor substrate for a bioluminescence resonance energy transfer (BRET) system with click beetle red luciferase (CBRluc), a mutant of firefly luciferase, as the energy-donor enzyme and yellow fluorescent protein (YFP) as the energy-acceptor fluorophore, and obtained a clearly bimodal bioluminescence spectrum. Stable bioluminescence that is not influenced by environmental factors is highly desirable for reliable measurements in biological assays. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Sensitive detection of acrolein in serum using time-resolved luminescence

Togashi M., Urano Y., Kojima H., Terai T., Hanaoka K., Igarashi K., Hirata Y., Nagano T.

Organic Letters (Organic Letters) 12 ( 8 ) 1704 - 1707 2010.04

Research paper (scientific journal), Accepted, ISSN 15237060

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Development and application of a near-infrared fluorescence probe for oxidative stress based on differential reactivity of linked cyanine dyes

Oushiki D., Kojima H., Terai T., Arita M., Hanaoka K., Urano Y., Nagano T.

Journal of the American Chemical Society (Journal of the American Chemical Society) 132 ( 8 ) 2795 - 2801 2010.02

Research paper (scientific journal), Accepted, ISSN 00027863

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.

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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 2010.01

Research paper (scientific journal), Accepted, 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 2009.09

Research paper (scientific journal), Accepted, 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 2009.07

Research paper (scientific journal), Accepted, 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 2009.05

Research paper (scientific journal), Accepted, 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

Research paper (scientific journal), Accepted, 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 2008.11

Research paper (scientific journal), Accepted, 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 Gd3+-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 2008.06

Research paper (scientific journal), Accepted, 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 Gd3+-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 2008.02

Research paper (scientific journal), Accepted, 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 2007.11

Research paper (international conference proceedings), Accepted, 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 2007.04

Research paper (scientific journal), Accepted, 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 2006.12

Research paper (scientific journal), Accepted, 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 2004.10

Research paper (scientific journal), Accepted, 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 2003.07

Research paper (scientific journal), Accepted, ISSN 14337851

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