Papers - Hanaoka, Kenjiro

Trans-omics landscape of systemic vasculitis identified matrix metalloproteinase 12 as a novel signature molecule.

Matsumoto K, Suzuki K, Magi M, Onishi S, Yoshida H, Takeshita M, Kuramoto J, Yazawa M, Kato T, Shimizu H, Ito T, Yamada S, Sasaki E, Hanaoka K, Noguchi-Sasaki M, Matsumoto Y, Takeuchi T, Kaneko Y

Rheumatology (Oxford, England)  2025.03

ISSN  1462-0324

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Development of a near-infrared fluorescent probe for the selective detection of severe hypoxia

Kasai, T; Fujita, K; Komatsu, T; Ueno, T; Kojima, R; Hanaoka, K; Urano, Y

RSC CHEMICAL BIOLOGY 6 ( 3 ) 449 - 456 2025.03

ISSN  2633-0679

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Identification of Fungal Metabolite Gliotoxin as a Potent Inhibitor Against Bacterial <i>O</i>-Acetylserine Sulfhydrylase CysK and CysM

Rahman, A; Ono, K; Toyomoto, T; Hanaoka, K; Sawa, T

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 26 ( 3 )  2025.02

ISSN  1661-6596

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Heat-guided drug delivery via thermally induced crosslinking of polymeric micelles

Yamada, S; Sasaki, E; Ohno, H; Hanaoka, K

COMMUNICATIONS CHEMISTRY 7 ( 1 ) 287 2024.12

Research paper (scientific journal), Joint Work, Accepted,  ISSN  2399-3669

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In vitro selection of dye-fluorescence-enhancing peptide aptamer by cDNA display.

Kubo T, Koike T, Ouchi T, Khaliq N, Sasaki E, Kuroda K, Ueda M, Hanaoka K, Nemoto N

Analytical biochemistry 698   115722 2024.11

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0003-2697

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Development of a Fluorescence Probe for Detecting Nitroreductase Activity Based on Steric Repulsion-Induced Twisted Intramolecular Charge Transfer (sr-TICT)

Sugimoto, M; Sasaki, E; Ohno, H; Ikeno, T; Yamada, S; Hanaoka, K

CHEMICAL & PHARMACEUTICAL BULLETIN 72 ( 9 ) 810 - 816 2024.09

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted,  ISSN  0009-2363

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Recent advances in Si-rhodamine-based fluorescent probes for live-cell imaging

Ohno, H; Sasaki, E; Yamada, S; Hanaoka, K

ORGANIC & BIOMOLECULAR CHEMISTRY 22 ( 16 ) 3099 - 3108 2024.04

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted,  ISSN  1477-0520

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Thioester-Based Coupled Fluorogenic Assays in Microdevice for the Detection of Single-Molecule Enzyme Activities of Esterases with Specified Substrate Recognition

Ukegawa, T; Komatsu, T; Minoda, M; Matsumoto, T; Iwasaka, T; Mizuno, T; Tachibana, R; Sakamoto, S; Hanaoka, K; Kusuhara, H; Honda, K; Watanabe, R; Urano, Y

ADVANCED SCIENCE 11 ( 10 ) e2306559 2024.03

Research paper (scientific journal), Joint Work, Accepted

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A general fluorescence off/on strategy for fluorogenic probes: Steric repulsion-induced twisted intramolecular charge transfer (sr-TICT)

Hanaoka, K; Ikeno, T; Iwaki, S; Deguchi, S; Takayama, K; Mizuguchi, H; Tao, F; Kojima, N; Ohno, H; Sasaki, E; Komatsu, T; Ueno, T; Maeda, K; Kusuhara, H; Urano, Y

SCIENCE ADVANCES 10 ( 7 ) eadi8847 2024.02

Research paper (scientific journal), Joint Work, Lead author, Corresponding author, Accepted,  ISSN  2375-2548

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Whole-Body and Whole-Organ 3D Imaging of Hypoxia Using an Activatable Covalent Fluorescent Probe Compatible with Tissue Clearing

Sakamoto, DM; Tamura, I; Yi, B; Hasegawa, S; Saito, Y; Yamada, N; Takakusagi, Y; Kubota, SI; Kobayashi, M; Harada, H; Hanaoka, K; Taki, M; Nangaku, M; Tainaka, K; Sando, S

ACS NANO 18 ( 6 ) 5167 - 5179 2024.02

Research paper (scientific journal), Accepted,  ISSN  1936-0851

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Discovery of a cystathionine <i>γ</i>-lyase (CSE) selective inhibitor targeting active-site pyridoxal 5′-phosphate (PLP) via Schiff base formation

Echizen, H; Hanaoka, K; Shimamoto, K; Hibi, R; Toma-Fukai, S; Ohno, H; Sasaki, E; Komatsu, T; Ueno, T; Tsuchiya, Y; Watanabe, Y; Otsuka, T; Saito, H; Nagatoishi, S; Tsumoto, K; Kojima, H; Okabe, T; Shimizu, T; Urano, Y

SCIENTIFIC REPORTS 13 ( 1 ) 16456 2023.09

Research paper (scientific journal), Joint Work, Corresponding author, Accepted,  ISSN  2045-2322

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Development of pathway-oriented screening to identify compounds to control 2-methylglyoxal metabolism in tumor cells.

Yanagi K, Komatsu T, Fujikawa Y, Kojima H, Okabe T, Nagano T, Ueno T, Hanaoka K, Urano Y

Communications chemistry 6 ( 1 ) 68 2023.04

Research paper (scientific journal), Joint Work, Accepted,  ISSN  2399-3669

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Real-time in situ X-ray micro-computed tomography study of the effect of impurities on the crystallization of amorphous nifedipine.

Amano Y, Misawa T, Miyazaki T, Ando D, Koide T, Izutsu KI, Kanazawa H, Hanaoka K, Yamamoto E

Journal of pharmaceutical and biomedical analysis 226   115248 2023.01

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0731-7085

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Alkyl gallates inhibit serine O-acetyltransferase in bacteria and enhance susceptibility of drug-resistant Gram-negative bacteria to antibiotics.

Toyomoto T, Ono K, Shiba T, Momitani K, Zhang T, Tsutsuki H, Ishikawa T, Hoso K, Hamada K, Rahman A, Wen L, Maeda Y, Yamamoto K, Matsuoka M, Hanaoka K, Niidome T, Akaike T, Sawa T

Frontiers in microbiology 14   1276447 2023

Research paper (scientific journal), Joint Work, Accepted,  ISSN  1664-302X

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Folate receptor-targeted near-infrared photodynamic therapy for folate receptor-overexpressing tumors.

Aung W, Tsuji AB, Hanaoka K, Higashi T

World journal of clinical oncology 13 ( 11 ) 880 - 895 2022.11

Research paper (scientific journal), Joint Work, Accepted,  ISSN  2218-4333

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General Design Strategy to Precisely Control the Emission of Fluorophores via a Twisted Intramolecular Charge Transfer (TICT) Process

Hanaoka Kenjiro, Iwaki Shimpei, Yagi Kiyoshi, Myochin Takuya, Ikeno Takayuki, Ohno Hisashi, Sasaki Eita, Komatsu Toru, Ueno Tasuku, Uchigashima Motokazu, Mikuni Takayasu, Tainaka Kazuki, Tahara Shinya, Takeuchi Satoshi, Tahara Tahei, Uchiyama Masanobu, Nagano Tetsuo, Urano Yasuteru

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 144 ( 43 ) 19778 - 19790 2022.10

Research paper (scientific journal), Joint Work, Lead author, Corresponding author, Accepted,  ISSN  0002-7863

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RELATIONSHIP BETWEEN MOUSE WOUND HEALING AND KERATIN 17

Ito M., Imbe Y., Wang Q., Aramaki N., Okabe K., Sakai S., Takaya K., Kanazawa H., Hanaoka K., Kishi K.

WOUND REPAIR AND REGENERATION 30 ( 4 ) A9 - A9 2022.07

ISSN  1067-1927

SEARCH FOR THE FORMATION MECHANISM OF ACTIN CABLE FOR COMPLETE SKIN REGENERATION

Imbe Y., Ito M., Wang Q., Aramaki N., Okabe K., Sakai S., Takaya K., Kanazawa H., Hanaoka K., Kishi K.

WOUND REPAIR AND REGENERATION 30 ( 4 ) A9 - A9 2022.07

ISSN  1067-1927

Development of a fluorescent probe library enabling efficient screening of tumour-imaging probes based on discovery of biomarker enzymatic activities.

Kuriki Y, Yoshioka T, Kamiya M, Komatsu T, Takamaru H, Fujita K, Iwaki H, Nanjo A, Akagi Y, Takeshita K, Hino H, Hino R, Kojima R, Ueno T, Hanaoka K, Abe S, Saito Y, Nakajima J, Urano Y

Chemical science 13 ( 16 ) 4474 - 4481 2022.04

Research paper (scientific journal), Accepted,  ISSN  2041-6520

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Molecular design of near-infrared (NIR) fluorescent probes targeting exopeptidase and application for detection of dipeptidyl peptidase 4 (DPP-4) activity

Hoshino Yuki, Hanaoka Kenjiro, Sakamoto Kei, Yasunaga Masahiro, Kojima Takashi, Kotani Daisuke, Nomoto Ayumu, Sasaki Eita, Komatsu Toru, Ueno Tasuku, Takamaru Hiroyuki, Saito Yutaka, Seto Yasuyuki, Urano Yasuteru

RSC CHEMICAL BIOLOGY 3 ( 7 ) 859 - 867 2022.03

Research paper (scientific journal), Lead author, Corresponding author, Accepted,  ISSN  2633-0679

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Rapid Visualization of Deeply Located Tumors In Vivo by Intravenous Administration of a gamma-Glutamyltranspeptidase-Activated Fluorescent Probe

Nakada Akihiro, Maruyama Takuma, Kamiya Mako, Hanaoka Kenjiro, Urano Yasuteru

BIOCONJUGATE CHEMISTRY 33 ( 3 ) 523 - 529 2022.03

Research paper (scientific journal), Accepted,  ISSN  1043-1802

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Sulfide catabolism ameliorates hypoxic brain injury

Marutani E., Morita M., Hirai S., Kai S., Grange R.M.H., Miyazaki Y., Nagashima F., Traeger L., Magliocca A., Ida T., Matsunaga T., Flicker D.R., Corman B., Mori N., Yamazaki Y., Batten A., Li R., Tanaka T., Ikeda T., Nakagawa A., Atochin D.N., Ihara H., Olenchock B.A., Shen X., Nishida M., Hanaoka K., Kevil C.G., Xian M., Bloch D.B., Akaike T., Hindle A.G., Motohashi H., Ichinose F.

Nature Communications (Nature Communications)  12 ( 1 ) 3108 2021.12

Research paper (scientific journal), Joint Work, Accepted

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The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain’s sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury.

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Discovery of an F-actin-binding small molecule serving as a fluorescent probe and a scaffold for functional probes.

Takagi T, Ueno T, Ikawa K, Asanuma D, Nomura Y, Uno SN, Komatsu T, Kamiya M, Hanaoka K, Okimura C, Iwadate Y, Hirose K, Nagano T, Sugimura K, Urano Y

Science advances (Science Advances)  7 ( 47 ) eabg8585 2021.11

Research paper (scientific journal), Accepted,  ISSN  2375-2548

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Actin is a ubiquitous cytoskeletal protein, forming a dynamic network that generates mechanical forces in the cell. There is a growing demand for practical and accessible tools for dissecting the role of the actin cytoskeleton in cellular function, and the discovery of a new actin-binding small molecule is an important advance in the field, offering the opportunity to design and synthesize of new class of functional molecules. Here, we found an F-actin-binding small molecule and introduced two powerful tools based on a new class of actin-binding small molecule: One enables visualization of the actin cytoskeleton, including super-resolution imaging, and the other enables highly specific green light-controlled fragmentation of actin filaments, affording unprecedented control of the actin cytoskeleton and its force network in living cells.

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Recent advances in detection, isolation, and imaging techniques for sulfane sulfur-containing biomolecules

Echizen H., Sasaki E., Hanaoka K.

Biomolecules (Biomolecules)  11 ( 11 )  2021.11

Research paper (scientific journal), Accepted

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Hydrogen sulfide and its oxidation products are involved in many biological processes, and sulfane sulfur compounds, which contain sulfur atoms bonded to other sulfur atom(s), as found in hydropersulfides (R-S-SH), polysulfides (R-S-Sn-S-R), hydrogen polysulfides (H2Sn), etc., have at-tracted increasing interest. To characterize their physiological and pathophysiological roles, selective detection techniques are required. Classically, sulfane sulfur compounds can be detected by cyanolysis, involving nucleophilic attack by cyanide ion to cleave the sulfur–sulfur bonds. The generated thiocyanate reacts with ferric ion, and the resulting ferric thiocyanate complex can be easily detected by absorption spectroscopy. Recent exploration of the properties of sulfane sulfur compounds as both nucleophiles and electrophiles has led to the development of various chemical techniques for detection, isolation, and bioimaging of sulfane sulfur compounds in biological samples. These include tag-switch techniques, LC-MS/MS, Raman spectroscopy, and fluorescent probes. Herein, we present an overview of the techniques available for specific detection of sulfane sulfur species in biological contexts.

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A Sulfonyl Azide-Based Sulfide Scavenger Rescues Mice from Lethal Hydrogen Sulfide Intoxication

Miyazaki Y., Marutani E., Ikeda T., Ni X., Hanaoka K., Xian M., Ichinose F.

Toxicological Sciences (Toxicological Sciences)  183 ( 2 ) 393 - 403 2021.10

Research paper (scientific journal), Accepted,  ISSN  10966080

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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.

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Matrix metalloprotease–14 is a target enzyme for detecting peritoneal metastasis in gastric cancer

Ogawa S., Kubo H., Murayama Y., Kubota T., Yubakami M., Matsumoto T., Ohashi T., Okamoto K., Kuriki Y., Hanaoka K., Urano Y., Otsuji E.

Photodiagnosis and Photodynamic Therapy (Photodiagnosis and Photodynamic Therapy)  35   102420 2021.09

Research paper (scientific journal), Accepted,  ISSN  15721000

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Background: Accurate diagnosis of peritoneal metastasis in gastric cancer (GC) is important to determine the appropriate treatment. This study aimed to examine whether matrix metalloprotease–14 (MMP–14) was a candidate enzyme in fluorescence imaging for the diagnosis of peritoneal metastasis in GC. Methods: GC and normal peritoneal (NP) tissues from 96 and 20 patients, respectively were evaluated for MMP–14 expression. Live cell imaging of GC cell lines (NUGC4, MKN45, MKN74, HGC-27, and Kato-III) was performed using the MMP–14-activatable fluorescence probe; BODIPY-MMP. Furthermore, the overall survival (OS) was calculated in all patients (n = 96). Results: MMP–14 expression was significantly higher in GC tissues (median: 3.57 ng/mg protein; range:0.64–24.4 ng/mg protein) than in NP tissues (median: 1.34 ng/mg protein; median: 0.53–3.09 ng/mg protein) (P < 0.01). Receiver operating characteristic curves showed that the area under the curve, sensitivity, and specificity were 0.907, 84.4%, and 90.0%, respectively. In live cell imaging using the BODIPY-MMP, fluorescence was observed in five GC cell lines. In the analysis of OS, the high expression of the MMP–14 group had a significantly poorer OS rate than the low expression of the MMP–14 group (P = 0.02). In the multivariate analyses, MMP-14 expression was an independent risk factor for OS (hazard ratio: 2.33; 95 % confidence interval: 1.05–5.45; P = 0.04). Conclusion: MMP–14 is a promising enzyme in intraoperative fluorescence imaging for peritoneal metastasis in GC, especially in patients with poor prognosis.

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Establishment of live-cell-based coupled assay system for identification of compounds to modulate metabolic activities of cells

Yanagi K., Komatsu T., Ogihara S., Okabe T., Kojima H., Nagano T., Ueno T., Hanaoka K., Urano Y.

Cell Reports (Cell Reports)  36 ( 1 ) 109311 2021.07

Research paper (scientific journal), Accepted,  ISSN  2211-1247

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In this study, we present a live-cell-based fluorometric coupled assay system to identify the compounds that can regulate the targeted metabolic pathways in live cells. The assay is established through targeting specific metabolic pathways and using “input” and “output” metabolite pairs. The changes in the extracellular output that are generated and released into the extracellular media from the input are assessed as the activity of the pathway. The screening for the glycolytic pathway and amino acid metabolism reveals the activities of the present drugs, 6-BIO and regorafenib, that regulate the metabolic fate of tumor cells.

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Design, synthesis and evaluation of enzyme-responsive fluorogenic probes based on pyridine-flanked diketopyrrolopyrrole dyes

Jenni S., Ponsot F., Baroux P., Collard L., Ikeno T., Hanaoka K., Quesneau V., Renault K., Romieu A.

Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy (Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy)  248   119179 2021.03

Research paper (scientific journal), Accepted,  ISSN  13861425

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The ever-growing demand for fluorogenic dyes usable in the rapid construction of analyte-responsive fluorescent probes, has recently contributed to a revival of interest in the chemistry of diketopyrrolopyrrole (DPP) pigments. In this context, we have explored the potential of symmetrical and unsymmetrical DPP derivatives bearing two or one 4-pyridyl substituents acting as optically tunable group(s). The unique fluorogenic behavior of these molecules, closely linked to N-substitution/charge state of their pyridine unit (i.e., neutral pyridine or cationic pyridinium), has been used to design DPP-based fluorescent probes for detection of hypoxia-related redox enzymes and penicillin G acylase (PGA). In this paper, we describe synthesis, spectral characterization and bioanalytical validations of these probes. Dramatic differences in terms of aqueous stability and enzymatic fluorescence activation were observed. This systematic study enables to delineate the scope of application of pyridine-flanked DPP fluorophores in the field of enzyme biosensing.

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Recent advances in probe design to detect reactive sulfur species and in the chemical reactions employed for fluorescence switching

Echizen H., Hanaoka K.

Journal of Clinical Biochemistry and Nutrition (Journal of Clinical Biochemistry and Nutrition)  68 ( 1 ) 9 - 17 2021.01

Research paper (scientific journal), Accepted,  ISSN  09120009

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Reactive sulfur species, including hydrogen sulfide, hydropersulfide, and polysulfide, have many roles in biological systems. For example, hydrogen sulfide is involved in the relaxation of vascular smooth muscles and mediation of neurotransmission, while sulfane sulfur, which exists in cysteine persulfide/polysulfide, and glutathione persulfide/polysulfide, is involved in physiological antioxidation and cytoprotection mechanisms. Fluorescence imaging is well suited for real-time monitoring of reactive sulfur species in living cells, and many fluorescent probes for reactive sulfur species have been reported. In such probes, the choice of detection chemistry is extremely important, not only to achieve effective fluorescence switching and high selectivity, but also because the reactions may be applicable to develop other chemical tools, such as reactive sulfur species donors/scavengers. Here, we present an overview of both widely used and recently developed fluorescent probes for reactive sulfur species, focusing especially on the chemical reactions employed in them for fluorescence switching. We also briefly introduce some applications of fluorescent probes for hydrogen sulfide and sulfane sulfur.

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Fluorescent probes for detection of supersulfides

Sasaki E., Hanaoka K.

Seikagaku (Seikagaku)  93 ( 5 ) 604 - 612 2021

ISSN  00371017

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A cytosolically localized far-red to near-infrared rhodamine-based fluorescent probe for calcium ions

Numasawa K., Hanaoka K., Ikeno T., Echizen H., Ishikawa T., Morimoto M., Komatsu T., Ueno T., Ikegaya Y., Nagano T., Urano Y.

Analyst (Analyst)  145 ( 23 ) 7736 - 7740 2020.12

Research paper (scientific journal), Accepted,  ISSN  00032654

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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

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Hypoxia-inducible factor-1 alpha maintains mouse articular cartilage through suppression of NF-κB signaling

Okada K., Mori D., Makii Y., Nakamoto H., Murahashi Y., Yano F., Chang S.H., Taniguchi Y., Kobayashi H., Semba H., Takeda N., Piao W., Hanaoka K., Nagano T., Tanaka S., Saito T.

Scientific Reports (Scientific Reports)  10 ( 1 ) 5425 2020.12

Research paper (scientific journal), Accepted

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HIF-1α, an essential transcription factor under hypoxic condition, is indispensable for chondrocytes during skeletal development but its expression and roles in articular chondrocytes are yet to be revealed. We examined HIF-1α protein expression and the hypoxic condition during mouse osteoarthritis (OA) development using state of the art hypoxic probes and found that its expression decreased as OA progressed, coinciding with the change in hypoxic conditions in articular cartilage. Gain- and loss-of-function of HIF-1α in cell culture experiments showed that HIF-1α suppressed catabolic genes such as Mmp13 and Hif2a. We confirmed these anticatabolic effects by measuring glycosaminoglycan release from wild type and conditional knock-out mice femoral heads cultured ex vivo. We went on to surgically induce OA in mice with chondrocyte-specific deletion of Hif1a and found that the development of OA was exacerbated. Increased expression of catabolic factors and activation of NF-κB signalling was clearly evident in the knock-out mice. By microarray analysis, C1qtnf3 was identified as a downstream molecule of HIF-1α, and experiments showed it exerted anti-catabolic effects through suppression of NF-κB. We conclude that HIF-1α has an anti-catabolic function in the maintenance of articular cartilage through suppression of NF-κB signalling.

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Calciprotein particle-induced cytotoxicity via lysosomal dysfunction and altered cholesterol distribution in renal epithelial HK-2 cells

Kunishige R., Mizoguchi M., Tsubouchi A., Hanaoka K., Miura Y., Kurosu H., Urano Y., Kuro-o M., Murata M.

Scientific Reports (Scientific Reports)  10 ( 1 ) 20125 2020.12

Research paper (scientific journal), Accepted

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Dietary phosphate overload induces chronic kidney disease (CKD), and calciprotein particles (CPPs), a form of nanoparticle comprising calcium phosphate and serum proteins, has been proposed to cause renal toxicity. However, the mechanism of CPP cytotoxicity in renal tubular cells is unknown. Here we show that in renal proximal tubular epithelial HK-2 cells, endocytosed CPPs accumulate in late endosomes/lysosomes (LELs) and increase their luminal pH by ~ 1.0 unit. This results in a decrease in lysosomal hydrolase activity and autophagic flux blockage without lysosomal rupture and reactive oxygen species generation. CPP treatment led to vulnerability to H2O2-induced oxidative stress and plasma membrane injury, probably because of autophagic flux blockage and decreased plasma membrane cholesterol, respectively. CPP-induced disruption of lysosomal homeostasis, autophagy flux and plasma membrane integrity might trigger a vicious cycle, leading to progressive nephron loss.

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Design of spontaneously blinking fluorophores for live-cell super-resolution imaging based on quantum-chemical calculations

Tachibana R., Kamiya M., Morozumi A., Miyazaki Y., Fujioka H., Nanjo A., Kojima R., Komatsu T., Ueno T., Hanaoka K., Yoshihara T., Tobita S., Urano Y.

Chemical Communications (Chemical Communications)  56 ( 86 ) 13173 - 13176 2020.11

Research paper (scientific journal), Accepted,  ISSN  13597345

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Spontaneously blinking fluorophores are powerful tools for live-cell super-resolution imaging under physiological conditions. Here we show that quantum-chemical calculations can predict key parameters for fluorophore design. We applied this methodology to develop a spontaneously blinking fluorophore with yellow fluorescence for super-resolution imaging of microtubules in living cells. This journal is

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Antibody Clicking as a Strategy to Modify Antibody Functionalities on the Surface of Targeted Cells

Komatsu T., Kyo E., Ishii H., Tsuchikama K., Yamaguchi A., Ueno T., Hanaoka K., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  142 ( 37 ) 15644 - 15648 2020.09

Research paper (scientific journal), Accepted,  ISSN  00027863

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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.

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How Viscous Is the Solidlike Structure at the Interface of Ionic Liquids? A Study Using Total Internal Reflection Fluorescence Spectroscopy with a Fluorescent Molecular Probe Sensitive to High Viscosity

Nishi N., Yamazawa T., Sakka T., Hotta H., Ikeno T., Hanaoka K., Takahashi H.

Langmuir (Langmuir)  36 ( 35 ) 10397 - 10403 2020.09

Research paper (scientific journal), Accepted,  ISSN  07437463

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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.

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Real-time in vivo imaging of extracellular atp in the brain with a hybrid-type fluorescent sensor

Kitajima N., Takikawa K., Sekiya H., Satoh K., Asanuma D., Sakamoto H., Takahashi S., Hanaoka K., Urano Y., Namiki S., Iino M., Hirose K.

eLife (eLife)  9   1 - 18 2020.07

Research paper (scientific journal), Accepted

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Adenosine 5’ triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase e subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.

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A Fluorescent Probe for Rapid, High-Contrast Visualization of Folate-Receptor-Expressing Tumors In Vivo

Numasawa K., Hanaoka K., Saito N., Yamaguchi Y., Ikeno T., Echizen H., Yasunaga M., Komatsu T., Ueno T., Miura M., Nagano T., Urano Y.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  59 ( 15 ) 6015 - 6020 2020.04

Research paper (scientific journal), Accepted,  ISSN  14337851

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Folate receptors (FRs) are membrane proteins involved in folic acid uptake, and the alpha isoform (FR-α) is overexpressed in ovarian and endometrial cancer cells. For fluorescence imaging of FRs in vivo, the near-infrared (NIR) region (650–900 nm), in which tissue penetration is high and autofluorescence is low, is optimal, but existing NIR fluorescent probes targeting FR-α show high non-specific tissue adsorption, and require prolonged washout to visualize tumors. We have designed and synthesized a new NIR fluorescent probe, FolateSiR-1, utilizing a Si-rhodamine fluorophore having a carboxy group at the benzene moiety, coupled to a folate ligand moiety through a negatively charged tripeptide linker. This probe exhibits very low background fluorescence and afforded a tumor-to-background ratio (TBR) of up to 83 in FR-expressing tumor-bearing mice within 30 min. Thus, FolateSiR-1 has the potential to contribute to the research in the field of biology and the clinical medicine.

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Rational Design of a Near-infrared Fluorescence Probe for Ca²⁺ Based on Phosphorus-substituted Rhodamines Utilizing Photoinduced Electron Transfer

Takahashi S., Hanaoka K., Okubo Y., Echizen H., Ikeno T., Komatsu T., Ueno T., Hirose K., Iino M., Nagano T., Urano Y.

Chemistry - An Asian Journal (Chemistry - An Asian Journal)  15 ( 4 ) 524 - 530 2020.02

Research paper (scientific journal), Accepted,  ISSN  18614728

　View Summary

Fluorescence imaging in the near-infrared (NIR) region (650–900 nm) is useful for bioimaging because background autofluorescence is low and tissue penetration is high in this range. In addition, NIR fluorescence is useful as a complementary color window to green and red for multicolor imaging. Here, we compared the photoinduced electron transfer (PeT)-mediated fluorescence quenching of silicon- and phosphorus-substituted rhodamines (SiRs and PRs) in order to guide the development of improved far-red to NIR fluorescent dyes. The results of density functional theory calculations and photophysical evaluation of a series of newly synthesized PRs confirmed that the fluorescence of PRs was more susceptible than that of SiRs to quenching via PeT. Based on this, we designed and synthesized a NIR fluorescence probe for Ca2+, CaPR-1, and its membrane-permeable acetoxymethyl derivative, CaPR-1 AM, which is distributed to the cytosol, in marked contrast to our previously reported Ca2+ far-red to NIR fluorescence probe based on the SiR scaffold, CaSiR-1 AM, which is mainly localized in lysosomes as well as cytosol in living cells. CaPR-1 showed longer-wavelength absorption and emission (up to 712 nm) than CaSiR-1. The new probe was able to image Ca2+ at dendrites and spines in brain slices, and should be a useful tool in neuroscience research.

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Metabolic-Pathway-Oriented Screening Targeting S-Adenosyl- l -methionine Reveals the Epigenetic Remodeling Activities of Naturally Occurring Catechols

Ogihara S., Komatsu T., Itoh Y., Miyake Y., Suzuki T., Yanagi K., Kimura Y., Ueno T., Hanaoka K., Kojima H., Okabe T., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  142 ( 1 ) 21 - 26 2020.01

Research paper (scientific journal), Accepted,  ISSN  00027863

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Methyl transfer reactions play important roles in many biological phenomena, wherein the methylation cofactor S-adenosyl-l-methionine (SAM) serves as the important currency to orchestrate those reactions. We have developed a fluorescent-probe-based high-throughput screening (HTS) system to search for the compounds that control cellular SAM levels. HTS with a drug repositioning library revealed the importance of catechol-O-methyltransferase (COMT) and its substrates in controlling the SAM concentrations and histone methylation levels in colorectal tumor cells.

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Detection of singularity in immunity and cancer by novel imaging techniques Comment

Nagai Takeharu, Chikuma Shunsuke, Hanaoka Kenjiro

BIOPHYSICS AND PHYSICOBIOLOGY 17   98 - 99 2020

Research paper (international conference proceedings)

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Design strategy for germanium-rhodamine based pH-activatable near-infrared fluorescence probes suitable for biological applications

Koide Y., Kojima R., Hanaoka K., Numasawa K., Komatsu T., Nagano T., Kobayashi H., Urano Y.

Communications Chemistry (Communications Chemistry)  2 ( 1 )  2019.12

Research paper (scientific journal), Accepted

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Fluorescence probes that can detect pH are useful tools for biological research and clinical diagnosis. Here we report pH-activatable near-infrared fluorescence probes, based on hydroxymethyl germanium-rhodamine (HMGeR), that are suitable for a range of biological applications. The pKa, the ratio of the fluorescent form in an acidic environment, and the absorption/emission wavelengths can all be conveniently optimized. The most promising probe, 2-HM IGeR, offers significant advantages over currently available near-infrared pH probes, notably high quantum efficiency, appropriate pKa value for biological applications, and high photostability. Further, our molecular design strategy allows easy conjugation of the probes to biomolecules without loss of functionality. We illustrate the value of this strategy by developing probe-Herceptin® and probe-avidin conjugates to visualize pH change in cellular vesicles during endocytosis, and to visualize tumors in a mouse model, respectively. We believe 2-HM IGeR is currently among the best-in-class pH-activatable near-infrared probes for biological and medical research.

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Development of ratiometric carbohydrate sensor based on boron dipyrromethene (BODIPY) scaffold

Cheruthu N.M., Komatsu T., Ueno T., Hanaoka K., Urano Y.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  29 ( 22 ) 126684 2019.11

Research paper (scientific journal), Accepted,  ISSN  0960894X

　View Summary

We designed a ratiometric carbohydrate sensor consisting of the boron dipyrromethene fluorophore substituted with boronic acid at the 2-position, based upon the strong substituent dependency of the absorbance/fluorescence wavelengths of BODIPY. The substituent is in equilibrium between the boronic acid B(OH)2 and boronate (B(OH)3−) forms, which have different absorbance/fluorescence wavelengths in the visible region. Reaction of the boronic acid moiety with hydroxy groups of carbohydrate affords a cyclic ester and shifts the equilibrium in favor of the boronate (B(OR)3−) form, resulting in a carbohydrate-concentration-dependent change of the fluorescence ratio. Thus, the sensor, BA-BODIPY, can ratiometrically detect carbohydrate at a pH near the pKa of cyclic ester formation.

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Separation-Based Enzymomics Assay for the Discovery of Altered Peptide-Metabolizing Enzymatic Activities in Biosamples

Ichihashi Y., Komatsu T., Kyo E., Matsuzaki H., Hata K., Watanabe T., Ueno T., Hanaoka K., Urano Y.

Analytical Chemistry (Analytical Chemistry)  91 ( 18 ) 11497 - 11501 2019.09

Research paper (scientific journal), Accepted,  ISSN  00032700

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We have developed a novel method to globally monitor the enzymatic activities of biological samples based on performing the global activity analysis on a proteome separated by native electrophoresis. The study of the alteration in peptide-metabolizing enzymatic activity in colorectal tumor specimens led us to the discovery of elevated thimet oligopeptidase activity, which contributed to the faster consumption of immune-stimulating peptide neurotensin.

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Design and Synthesis of an Activatable Photoacoustic Probe for Hypochlorous Acid

Ikeno T., Hanaoka K., Iwaki S., Myochin T., Murayama Y., Ohde H., Komatsu T., Ueno T., Nagano T., Urano Y.

Analytical Chemistry (Analytical Chemistry)  91 ( 14 ) 9086 - 9092 2019.06

Research paper (scientific journal), Accepted,  ISSN  00032700

　View Summary

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.

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Design of Photostable, Activatable Near-Infrared Photoacoustic Probes Using Tautomeric Benziphthalocyanine as a Platform

Toriumi N., Asano N., Ikeno T., Muranaka A., Hanaoka K., Urano Y., Uchiyama M.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  58 ( 23 ) 7788 - 7791 2019.06

Research paper (scientific journal), Accepted,  ISSN  14337851

　View Summary

Near-infrared (NIR) imaging techniques have attracted significant attention for biological and medicinal applications due to the ability of NIR to penetrate deeply into tissues. However, there are very few stable, activatable molecular probes that can utilize NIR light in the wavelength range beyond 800 nm. Herein, we report a new activatable NIR system for photoacoustic imaging based on tautomeric benziphthalocyanines (BPcs). We found that the existence of a free hydroxyl group is crucial for NIR absorption of BPcs. Synthesized water-soluble hydroxy BPcs exhibited high photostability and no fluorescence, which are desirable features for photoacoustic imaging. We synthesized BPcs in which the free hydroxyl group was masked by an esterase-labile or an H2O2-labile group. The photoacoustic signals of these hydroxy-masked BPcs were increased upon NIR excitation at 880 nm in the presence of esterase or H2O2, respectively. These are rare examples of activatable probes utilizing NIR light at around 900 nm.

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Development of a platform for activatable fluorescent substrates of glucose transporters (GLUTs)

Takasugi T., Hanaoka K., Sasaki A., Ikeno T., Komatsu T., Ueno T., Yamada K., Urano Y.

Bioorganic and Medicinal Chemistry (Bioorganic and Medicinal Chemistry)  27 ( 10 ) 2122 - 2126 2019.05

Research paper (scientific journal), Accepted,  ISSN  09680896

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We have developed a platform for activatable fluorescent substrates of glucose transporters (GLUTs). We firstly conjugated fluorescein to glucosamine via an amide or methylene linker at the C-2 position of D-glucosamine, but the resulting compounds, FLG1 and FLG2, showed no uptake into MIN6 cells. So, we changed the fluorophore moiety to a fluorescein analogue, 2-Me TokyoGreen, which is less negatively charged. TokyoGreen-conjugated glucosamines TGG1 and TGG2 were successfully taken up into cells via GLUT. We further derivatized TGG1 and TGG2, and among the synthesized compounds, 2-Me-4-OMe TGG showed weak fluorescence under the acidic conditions of the extracellular environment inside tumors and in gastric cancers, and strong fluorescence at the intracellular physiological pH, under the control of a photoinduced electron transfer (PeT) process. This fluorogenic platform should be useful for developing a range of activatable fluorescent substrates targeting GLUTs, as well as derivatives that would be fluorescently activated by various intracellular enzymes, such as esterases, β-galactosidase and bioreductases.

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Red Fluorescence Probe Targeted to Dipeptidylpeptidase-IV for Highly Sensitive Detection of Esophageal Cancer

Ogasawara A., Kamiya M., Sakamoto K., Kuriki Y., Fujita K., Komatsu T., Ueno T., Hanaoka K., Onoyama H., Abe H., Tsuji Y., Fujishiro M., Koike K., Fukayama M., Seto Y., Urano Y.

Bioconjugate Chemistry (Bioconjugate Chemistry)  30 ( 4 ) 1055 - 1060 2019.04

Research paper (scientific journal), Accepted,  ISSN  10431802

　View Summary

We have developed an activatable red fluorescence probe for dipeptidylpeptidase-IV (DPP-IV) by precisely controlling the photoinduced electron transfer (PeT) process of a red fluorescent scaffold, SiR600. The developed probe exhibited an extremely low background signal and showed significant fluorescence activation upon reaction with DPP-IV, enabling sensitive detection of esophageal cancer in clinical specimens from cancer patients.

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Development of novel dark quenchers and their application to imaging probes

Hanaoka K.

Yakugaku Zasshi (Yakugaku Zasshi)  139 ( 2 ) 277 - 283 2019

Research paper (scientific journal), Accepted,  ISSN  00316903

　View Summary

Rhodamine dyes are among the most widely used fluorescent dyes for bioimaging due to their high fluorescence quantum yield and high photostability. Recently, novel far-red to near-infrared (NIR) fluorescent dyes have been developed: Si-rhodamines (SiRs), in which the O atom of conventional rhodamine dyes at the 10 position of the xanthene moiety is replaced with a Si atom. These SiRs are excellent long-wavelength fluorophores for bioimaging, as they retain the advantageous photophysical properties of conventional rhodamine dyes. Further, we focused on the QSY dark quenchers, which contain the rhodamine scaŠold bearing aromatic rings at the N atoms at the 3,6-positions of the xanthene moiety; these show no fluorescence, irrespective of solvent polarity and pH. NIR fluorescent probes based on the F orster resonance energy transfer (FRET) mechanism have various practical advantages, and their molecular design is generally based on the use of NIR dark quenchers as cleavable FRET acceptors. However, few NIR dark quenchers can quench fluorescence in the Cy7 region (over 780 nm). We successfully developed SiR-based NIR dark quenchers (SiNQs) which show broad absorption covering this region. To demonstrate their usefulness, we designed and synthesized a NIR fluorescence probe for matrix metalloproteinase (MMP) activity using SiNQs. The developed probe was able to detect MMP activity in terms of NIR fluorescence, not only in vitro, but also in cultured cells and in a tumor-bearing mouse, in which the tumor was clearly visualized.

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Development of fluorescent probes for detecting reactive sulfur species and their application to development of inhibitors for 3MST

Echizen H., Hanaoka K.

Folia Pharmacologica Japonica (Folia Pharmacologica Japonica)  154 ( 3 ) 121 - 127 2019

Research paper (scientific journal), Accepted,  ISSN  00155691

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Hydrogen sulfide (H2S) has been reported to play an important role in biological systems. More recently, sulfane sulfur (sulfur with 0 or −1 charge) molecules have been also reported to be involved in various biological phenomena such as regulation of redox signaling and antioxidant functions. Fluorescent probes are one of the important chemical tools because it is easy to use and enable the real-time detection of the target molecules in living cells and tissues. We have successfully developed a highly selective H2S-detecting fluorescent probe, HSip-1. HSip-1 has been designed on the basis of the facts that the macrocyclic polyamine ligands form a stable complex with Cu2+, and Cu2+ also reacts with H2S and make a stable CuS complex. SSip-1 is a fluorescent probe for detecting sulfane sulfur and this fluorescent probe is designed on the basis of the unique feature of sulfane sulfur to bind reversibly to other sulfur atoms and the intramolecular spirocyclization reaction of xanthene dyes. SSip-1 is a highly selective fluorescent probe and can detect sulfane sulfur reversibly. Both HSip-1 and SSip-1 were able to be used for the live-cell fluorescence imaging. Further, we applied HSip-1 to the high-throughput screening (HTS) for the inhibitors of 3-mercaptopyruvate sulfurtransferase (3MST), one of the reactive sulfur species (RSS)-generating enzymes. We successfully found new 3MST inhibitors by screening of 174,118 compounds. We expect that these fluorescent probes and inhibitors would be useful to elucidate new functions of RSS and RSS-generating enzymes.

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Rapid detection of metastatic lymph nodes of colorectal cancer with a gamma-glutamyl transpeptidase-activatable fluorescence probe

Kubo H., Hanaoka K., Kuriki Y., Komatsu T., Ueno T., Kojima R., Kamiya M., Murayama Y., Otsuji E., Urano Y.

Scientific Reports (Scientific Reports)  8 ( 1 ) 17781 2018.12

Research paper (scientific journal), Accepted

　View Summary

Rapid diagnosis of metastatic lymph nodes (mLNs) of colorectal cancer (CRC) is desirable either intraoperatively or in resected fresh specimens. We have developed a series of activatable fluorescence probes for peptidase activities that are specifically upregulated in various tumors. We aimed to discover a target enzyme for detecting mLNs of CRC. Among our probes, we found that gGlu-HMRG, a gamma-glutamyl transpeptidase (GGT)-activatable fluorescence probe, could detect mLNs. This was unexpected, because we have previously reported that gGlu-HMRG could not detect primary CRC. We confirmed that the GGT activity of mLNs was high, whereas that of non-metastatic lymph nodes and CRC cell lines was low. We investigated the reason why GGT activity was upregulated in mLNs, and found that GGT was induced under conditions of hypoxia or low nutritional status. We utilized this feature to achieve rapid detection of mLNs with gGlu-HMRG. GGT appears to be a promising candidate enzyme for fluorescence imaging of mLNs.

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Red-Shifted Fluorogenic Substrate for Detection of lacZ-Positive Cells in Living Tissue with Single-Cell Resolution

Ito H., Kawamata Y., Kamiya M., Tsuda-Sakurai K., Tanaka S., Ueno T., Komatsu T., Hanaoka K., Okabe S., Miura M., Urano Y.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  57 ( 48 ) 15702 - 15706 2018.11

Research paper (scientific journal), Accepted,  ISSN  14337851

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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.

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A protein-coupled fluorescent probe for organelle-specific imaging of Na⁺

Taguchi R., Terai T., Ueno T., Komatsu T., Hanaoka K., Urano Y.

Sensors and Actuators, B: Chemical (Sensors and Actuators, B: Chemical)  265   575 - 581 2018.07

Research paper (scientific journal), Accepted,  ISSN  09254005

　View Summary

Intracellular sodium concentration plays important roles in many cellular processes, but methods to analyze dynamic change of [Na+] in organelles of interest are still lacking. To address this problem, controlled localization of fluorescent Na+ probes is a key issue. In this work, we developed a protein-coupled Na+-sensitive fluorescent probe, HaloNa-1, which covalently labelled a HaloTag protein expressed at specific organelles in living cells. Fluorescence intensity of HaloNa-1 increased by approximately three-fold when complexed with Na+, and the dissociation constant was within physiological range. Notably, by conjugating a fluorescent protein to the HaloTag protein and using it as internal standard, one can reduce unwanted variation of probe fluorescence due to different cell volume, HaloTag expression level, etc. With this technique, we could observe elevation of intracellular [Na+] after ionophore stimulation with higher reliability than conventional sensors. We believe our probe is potentially useful to understand Na+ dynamics inside cells.

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Development of a Series of Practical Fluorescent Chemical Tools to Measure pH Values in Living Samples

Takahashi S., Kagami Y., Hanaoka K., Terai T., Komatsu T., Ueno T., Uchiyama M., Koyama-Honda I., Mizushima N., Taguchi T., Arai H., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  140 ( 18 ) 5925 - 5933 2018.05

Research paper (scientific journal), Accepted,  ISSN  00027863

　View Summary

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.

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N-Acetyl Cysteine Functions as a Fast-Acting Antioxidant by Triggering Intracellular H <inf>2</inf> S and Sulfane Sulfur Production

Ezeriņa D., Takano Y., Hanaoka K., Urano Y., Dick T.P.

Cell Chemical Biology (Cell Chemical Biology)  25 ( 4 ) 447 - 459.e4 2018.04

Research paper (scientific journal), Accepted,  ISSN  24519456

　View Summary

The cysteine prodrug N-acetyl cysteine (NAC) is widely used as a pharmacological antioxidant and cytoprotectant. It has been reported to lower endogenous oxidant levels and to protect cells against a wide range of pro-oxidative insults. As NAC itself is a poor scavenger of oxidants, the molecular mechanisms behind the antioxidative effects of NAC have remained uncertain. Here we show that NAC-derived cysteine is desulfurated to generate hydrogen sulfide, which in turn is oxidized to sulfane sulfur species, predominantly within mitochondria. We provide evidence suggesting the possibility that sulfane sulfur species produced by 3-mercaptopyruvate sulfurtransferase and sulfide:quinone oxidoreductase are the actual mediators of the immediate antioxidative and cytoprotective effects provided by NAC. N-Acetyl cysteine (NAC), by itself a poor scavenger of oxidants, is converted inside cells to yield sulfane sulfur species, which are very potent scavengers of oxidants. This conversion may account for many of the antioxidative effects provided by NAC which have hitherto remained unexplained.

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Establishment of Molecular Design Strategy to Obtain Activatable Fluorescent Probes for Carboxypeptidases

Kuriki Y., Kamiya M., Kubo H., Komatsu T., Ueno T., Tachibana R., Hayashi K., Hanaoka K., Yamashita S., Ishizawa T., Kokudo N., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  140 ( 5 ) 1767 - 1773 2018.02

Research paper (scientific journal),  ISSN  00027863

　View Summary

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.

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Development of a novel inhibitor for reactive sulfur species-producing enzymes

Echizen H., Hanaoka K.

Seikagaku (Seikagaku)  90 ( 3 ) 388 - 393 2018

Research paper (scientific journal), Accepted,  ISSN  00371017

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Synthesis of unsymmetrical Si-rhodamine fluorophores and application to a far-red to near-infrared fluorescence probe for hypoxia

Hanaoka K., Kagami Y., Piao W., Myochin T., Numasawa K., Kuriki Y., Ikeno T., Ueno T., Komatsu T., Terai T., Nagano T., Urano Y.

Chemical Communications (Chemical Communications)  54 ( 50 ) 6939 - 6942 2018

Research paper (scientific journal), Accepted,  ISSN  13597345

　View Summary

Si-Rhodamines are bright fluorophores with red to near-infrared (NIR) emission, and are widely used for fluorescence imaging of biological phenomena. Here, in order to extend the scope of Si-rhodamine fluorophores, we established a versatile synthesis of unsymmetrical Si-rhodamines. To illustrate its value, we used one of these new fluorophores to synthesize a far-red to NIR fluorescence probe for hypoxia, and showed that it can visualize hepatic ischemia in mice in vivo.

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Molecular imaging of aberrant crypt foci in the human colon targeting glutathione S-transferase P1-1

Muguruma N., Okamoto K., Nakagawa T., Sannomiya K., Fujimoto S., Mitsui Y., Kimura T., Miyamoto H., Higashijima J., Shimada M., Horino Y., Matsumoto S., Hanaoka K., Nagano T., Shibutani M., Takayama T.

Scientific Reports (Scientific Reports)  7 ( 1 )  2017.12

Research paper (scientific journal)

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Aberrant crypt foci (ACF), the earliest precursor lesion of colorectal cancers (CRCs), are a good surrogate marker for CRC risk stratification and chemoprevention. However, the conventional ACF detection method with dye-spraying by magnifying colonoscopy is labor- A nd skill-intensive. We sought to identify rat and human ACF using a fluorescent imaging technique that targets a molecule specific for ACF. We found that glutathione S-transferase (GST) P1-1 was overexpressed in ACF tissues in a screening experiment. We then synthesized the fluorogenic probe, DNAT-Me, which is fluorescently quenched but is activated by GSTP1-1. A CRC cell line incubated with DNAT-Me showed strong fluorescence in the cytosol. Fluorescence intensities correlated significantly with GST activities in cancer cell lines. When we sprayed DNAT-Me onto colorectal mucosa excised from azoxymethane-treated rats and surgically resected from CRC patients, ACF with strong fluorescent signals were clearly observed. The ACF number determined by postoperative DNAT-Me imaging was almost identical to that determined by preoperative methylene blue staining. The signal-to-noise ratio for ACF in DNAT-Me images was significantly higher than that in methylene blue staining. Thus, we sensitively visualized ACF on rat and human colorectal mucosa by using a GST-activated fluorogenic probe without dye-spraying and magnifying colonoscopy.

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Development of an azo-based photosensitizer activated under mild hypoxia for photodynamic therapy

Piao W., Hanaoka K., Fujisawa T., Takeuchi S., Komatsu T., Ueno T., Terai T., Tahara T., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  139 ( 39 ) 13713 - 13719 2017.10

Research paper (scientific journal), Accepted,  ISSN  00027863

　View Summary

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.

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Fluorescent Probes and Selective Inhibitors for Biological Studies of Hydrogen Sulfide-and Polysulfide-Mediated Signaling

Takano Y., Echizen H., Hanaoka K.

Antioxidants and Redox Signaling (Antioxidants and Redox Signaling)  27 ( 10 ) 669 - 683 2017.10

Research paper (scientific journal), Accepted,  ISSN  15230864

　View Summary

Significance: Hydrogen sulfide (H2S) plays roles in many physiological processes, including relaxation of vascular smooth muscles, mediation of neurotransmission, inhibition of insulin signaling, and regulation of inflammation. Also, hydropersulfide (R-S-SH) and polysulfide (-S-Sn-S-) have recently been identified as reactive sulfur species (RSS) that regulate the bioactivities of multiple proteins via S-sulfhydration of cysteine residues (protein Cys-SSH) and show cytoprotection. Chemical tools such as fluorescent probes and selective inhibitors are needed to establish in detail the physiological roles of H2S and polysulfide. Recent Advances: Although many fluorescent probes for H2S are available, fluorescent probes for hydropersulfide and polysulfide have only recently been developed and used to detect these sulfur species in living cells. Critical Issues: In this review, we summarize recent progress in developing chemical tools for the study of H2S, hydropersulfide, and polysulfide, covering fluorescent probes based on various design strategies and selective inhibitors of H2S- and polysulfide-producing enzymes (cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase), and we summarize their applications in biological studies. Future Directions: Despite recent progress, the precise biological functions of H2S, hydropersulfide, and polysulfide remain to be fully established. Fluorescent probes and selective inhibitors are effective chemical tools to study the physiological roles of these sulfur molecules in living cells and tissues. Therefore, further development of a broad range of practical fluorescent probes and selective inhibitors as tools for studies of RSS biology is currently attracting great interest.

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An Activatable Photosensitizer Targeted to γ-Glutamyltranspeptidase

Chiba M., Ichikawa Y., Kamiya M., Komatsu T., Ueno T., Hanaoka K., Nagano T., Lange N., Urano Y.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  56 ( 35 ) 10418 - 10422 2017.08

Research paper (scientific journal),  ISSN  14337851

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We adopted a spirocyclization-based strategy to design γ-glutamyl hydroxymethyl selenorhodamine green (gGlu-HMSeR) as a photo-inactive compound that would be specifically cleaved by the tumor-associated enzyme γ-glutamyltranspeptidase (GGT) to generate the potent photosensitizer HMSeR. gGlu-HMSeR has a spirocyclic structure and is colorless and does not show marked phototoxicity toward low-GGT-expressing cells or normal tissues upon irradiation with visible light. In contrast, HMSeR predominantly takes an open structure, is colored, and generates reactive oxygen species upon irradiation. The γ-glutamyl group thus serves as a tumor-targeting moiety for photodynamic therapy (PDT), switching on tumor-cell-specific phototoxicity. To validate this system, we employed chick chorioallantoic membrane (CAM), a widely used model for preliminary evaluation of drug toxicity. Photoirradiation after gGlu-HMSeR treatment resulted in selective ablation of implanted tumor spheroids without damage to healthy tissue.

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Silicon-substituted Xanthene Dyes and Their Unique Photophysical Properties for Fluorescent Probes

Ikeno T., Nagano T., Hanaoka K.

Chemistry - An Asian Journal (Chemistry - An Asian Journal)  12 ( 13 ) 1435 - 1446 2017.07

Research paper (scientific journal), Accepted,  ISSN  18614728

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Polysulfides (H<inf>2</inf>S<inf>n</inf>) produced from the interaction of hydrogen sulfide (H<inf>2</inf>S) and nitric oxide (NO) activate TRPA1 channels

Miyamoto R., Koike S., Takano Y., Shibuya N., Kimura Y., Hanaoka K., Urano Y., Ogasawara Y., Kimura H.

Scientific Reports (Scientific Reports)  7 2017.04

Research paper (scientific journal), Accepted

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Hydrogen sulfide (H2S) exerts synergistic effects with another gaseous signaling molecule nitric oxide (NO) on ion channels and vasculature. However, the mechanism of the synergy is not well understood. Here, we show that the interaction between H2S and NO generates polysulfides (H2Sn), which activate transient receptor potential ankyrin 1 (TRPA1) channels. High performance liquid chromatography with tandem mass spectrometry analysis, along with the imaging of intracellular Ca2+ and H2Sn, showed that H2Sn and their effects were abolished by cyanolysis and by reducing substances such as dithiothreitol (DTT), cysteine, and glutathione (GSH). However, the effects of nitroxyl or nitrosopersulfide, other potential products of H2S and NO interaction, are not affected by cyanolysis or reducing substances. This study demonstrates that H2Sn are products of synergy between H2S and NO and provides a new insight into the signaling mechanisms.

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Discovery of Cell-Type-Specific and Disease-Related Enzymatic Activity Changes via Global Evaluation of Peptide Metabolism

Onagi J., Komatsu T., Ichihashi Y., Kuriki Y., Kamiya M., Terai T., Ueno T., Hanaoka K., Matsuzaki H., Hata K., Watanabe T., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  139 ( 9 ) 3465 - 3472 2017.03

Research paper (scientific journal),  ISSN  00027863

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Cellular homeostasis is maintained by a complex network of reactions catalyzed by enormous numbers of enzymatic activities (the enzymome), which serve to determine the phenotypes of cells. Here, we focused on the enzymomics of proteases and peptidases because these enzymes are an important class of disease-related proteins. We describe a system that (A) simultaneously evaluates metabolic activities of peptides using a series of exogenous peptide substrates and (B) identifies the enzymes that metabolize the specified peptide substrate with high throughput. We confirmed that the developed system was able to discover cell-type-specific and disease-related exo- and endopeptidase activities and identify the responsible enzymes. For example, we found that the activity of the endopeptidase neurolysin is highly elevated in human colorectal tumor tissue samples. This simple but powerful enzymomics platform should be widely applicable to uncover cell-type-specific reactions and altered enzymatic functions with potential value as biomarkers or drug targets in various disease states and to investigate the mechanisms of the underlying pathologies.

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Development of an Azoreductase-based Reporter System with Synthetic Fluorogenic Substrates

Shin N., Hanaoka K., Piao W., Miyakawa T., Fujisawa T., Takeuchi S., Takahashi S., Komatsu T., Ueno T., Terai T., Tahara T., Tanokura M., Nagano T., Urano Y.

ACS Chemical Biology (ACS Chemical Biology)  12 ( 2 ) 558 - 563 2017.02

Research paper (scientific journal), Accepted,  ISSN  15548929

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Enzyme/substrate pairs, such as β-galactosidase with chromogenic x-gal substrate, are widely used as reporters to monitor biological events, but there is still a requirement for new reporter systems, which may be orthogonal to existing systems. Here, we focused on azoreductase (AzoR). We designed and synthesized a library of azo-rhodamine derivatives as candidate fluorogenic substrates. These derivatives were nonfluorescent, probably due to ultrafast conformational change around the N=N bond after photoexcitation. We found that AzoR-mediated reduction of the azo bond of derivatives bearing an electron-donating group on the azobenzene moiety was followed by nonenzymatic cleavage to afford highly fluorescent 2-methyl-rhodamine green (2-Me RG), which was well retained in cells. We show that the AzoR/compound 9 reporter system can detect azoreductase-expressing live cells at the single cell level.

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Discovery and mechanistic characterization of selective inhibitors of H<inf>2</inf>S-producing Enzyme: 3-Mercaptopyruvate

Hanaoka K., Sasakura K., Suwanai Y., Toma-Fukai S., Shimamoto K., Takano Y., Shibuya N., Terai T., Komatsu T., Ueno T., Ogasawara Y., Tsuchiya Y., Watanabe Y., Kimura H., Wang C., Uchiyama M., Kojima H., Okabe T., Urano Y., Shimizu T., Nagano T.

Scientific Reports (Scientific Reports)  7 2017.01

Research paper (scientific journal), Accepted

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Very recent studies indicate that sulfur atoms with oxidation state 0 or -1, called sulfane sulfurs, are the actual mediators of some physiological processes previously considered to be regulated by hydrogen sulfide (H2S). 3-Mercaptopyruvate sulfurtransferase (3MST), one of three H2S-producing enzymes, was also recently shown to produce sulfane sulfur (H2S n). Here, we report the discovery of several potent 3MST inhibitors by means of high-throughput screening (HTS) of a large chemical library (174,118 compounds) with our H2S-selective fluorescent probe, HSip-1. Most of the identified inhibitors had similar aromatic ring-carbonyl-S-pyrimidone structures. Among them, compound 3 showed very high selectivity for 3MST over other H2S/sulfane sulfur-producing enzymes and rhodanese. The X-ray crystal structures of 3MST complexes with two of the inhibitors revealed that their target is a persulfurated cysteine residue located in the active site of 3MST. Precise theoretical calculations indicated the presence of a strong long-range electrostatic interaction between the persulfur anion of the persulfurated cysteine residue and the positively charged carbonyl carbon of the pyrimidone moiety of the inhibitor. Our results also provide the experimental support for the idea that the 3MST-catalyzed reaction with 3-mercaptopyruvate proceeds via a ping-pong mechanism.

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Development of Chemical Tools to Monitor and Control Isoaspartyl Peptide Methyltransferase Activity

Kimura Y., Komatsu T., Yanagi K., Hanaoka K., Ueno T., Terai T., Kojima H., Okabe T., Nagano T., Urano Y.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  56 ( 1 ) 153 - 157 2017.01

Research paper (scientific journal), Accepted,  ISSN  14337851

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We have established a coupled assay system targeting protein l-isoaspartyl methyltransferase (PIMT), a key enzyme in the metabolism of isoaspartyl peptides and proteins. The system utilizes a fluorogenic peptide probe containing an isoaspartyl residue at the P1′ position of the caspase-3 recognition sequence. Following PIMT-catalyzed methyl transfer reaction, the methylated probe is specifically cleaved by caspase-3 to give fluorescence activation. High-throughput screening of our chemical library with this assay system identified PIMT inhibitors that may be useful as leads in the design of chemical probes for controlling PIMT activity.

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Development of a reversible fluorescent probe for reactive sulfur species, sulfane sulfur, and its biological application

Takano Y., Hanaoka K., Shimamoto K., Miyamoto R., Komatsu T., Ueno T., Terai T., Kimura H., Nagano T., Urano Y.

Chemical Communications (Chemical Communications)  53 ( 6 ) 1064 - 1067 2017

Research paper (scientific journal), Accepted,  ISSN  13597345

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We report a reversible off/on fluorescent probe for monitoring concentration changes of sulfane sulfur by utilizing the unique ability of sulfane sulfur to bind reversibly to other sulfur atoms and the intramolecular spirocyclization reaction of xanthene dyes. It reversibly visualized sulfane sulfur in living A549 cells and primary-cultured hippocampal astrocytes.

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Fluorescence detection of serum albumin with a turnover-based sensor utilizing Kemp elimination reaction

Sakamoto S., Komatsu T., Ueno T., Hanaoka K., Urano Y.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  27 ( 15 ) 3464 - 3467 2017

Research paper (scientific journal), Accepted,  ISSN  0960894X

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The Kemp elimination reaction is a well-known chemical reaction that is facilitated on a protein surface microenvironment, and in particular is highly accelerated in a unique binding pocket of serum albumin. We have designed and synthesized a fluorescently activatable coumarin derivative with a benzisoxazole scaffold to enable monitoring of the Kemp elimination reaction in terms of fluorescence change for the first time. We show that this fluorescent sensor can sensitively and selectively quantitate serum albumin in blood samples. It also works in a dry-chemistry format.

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Development of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membrane permeability

Hirabayashi K., Hanaoka K., Egawa T., Kobayashi C., Takahashi S., Komatsu T., Ueno T., Terai T., Ikegaya Y., Nagano T., Urano Y.

Cell Calcium (Cell Calcium)  60 ( 4 ) 256 - 265 2016.10

Research paper (scientific journal), Accepted,  ISSN  01434160

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Fluorescence imaging of calcium ions (Ca2+) has become an essential technique for investigation of signaling pathways involving Ca2+ as a second messenger. But, Ca2+ signaling is involved in many biological phenomena, and therefore simultaneous visualization of Ca2+ and other biomolecules (multicolor imaging) would be particularly informative. For this purpose, we set out to develop a fluorescent probe for Ca2+ that would operate in a different color region (red) from that of probes for other molecules, many of which show green fluorescence, as exemplified by green fluorescent protein (GFP). We previously developed a red fluorescent probe for monitoring cytoplasmic Ca2+ concentration, based on our established red fluorophore, TokyoMagenta (TM), but there remained room for improvement, especially as regards efficiency of introduction into cells. We considered that this issue was probably mainly due to limited water solubility of the probe. So, we designed and synthesized a red-fluorescent probe with improved water solubility. We confirmed that this Ca2+ red-fluorescent probe showed high cell-membrane permeability with bright fluorescence. It was successfully applied to fluorescence imaging of not only live cells, but also brain slices, and should be practically useful for multicolor imaging studies of biological mechanisms.

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A Single Fluorescent Probe to Visualize Hydrogen Sulfide and Hydrogen Polysulfides with Different Fluorescence Signals

Chen W., Pacheco A., Takano Y., Day J.J., Hanaoka K., Xian M.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  55 ( 34 ) 9993 - 9996 2016.08

Research paper (scientific journal), Accepted,  ISSN  14337851

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Hydrogen sulfide (H2S) and hydrogen polysulfides (H2Sn, n>1) are endogenous regulators of many physiological processes. In order to better understand the symbiotic relationship and cellular cross-talk between H2S and H2Sn, it is highly desirable to develop single fluorescent probes which enable dual-channel discrimination between H2S and H2Sn. Herein, we report the rational design, synthesis, and evaluation of the first dual-detection fluorescent probe DDP-1 that can visualize H2S and H2Snwith different fluorescence signals. The probe showed high selectivity and sensitivity to H2S and H2Snin aqueous media and in cells.

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Unexpected Photo-instability of 2,6-Sulfonamide-Substituted BODIPYs and Its Application to Caged GABA

Takeda A., Komatsu T., Nomura H., Naka M., Matsuki N., Ikegaya Y., Terai T., Ueno T., Hanaoka K., Nagano T., Urano Y.

ChemBioChem (ChemBioChem)     1233 - 1240 2016.07

Research paper (scientific journal), Accepted,  ISSN  14394227

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Investigation of the unexpected photo-instability of 2,6-sulfonamide-substituted derivatives of the boron dipyrromethene (BODIPY) fluorophore led to the discovery of a photoreaction accompanied by multiple bond scissions. We characterized the photoproducts and utilized the photoreaction to design a caged γ-aminobutyric acid (GABA) derivative that can release GABA upon irradiation in the visible range (>450 nm). This allowed us to stimulate neural cells in mouse brain slices.

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Detection of NAD(P)H-dependent enzyme activity by time-domain ratiometry of terbium luminescence

Terai T., Ito H., Hanaoka K., Komatsu T., Ueno T., Nagano T., Urano Y.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  26 ( 9 ) 2314 - 2317 2016.05

Research paper (scientific journal), Accepted,  ISSN  0960894X

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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.

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Protein-Coupled Fluorescent Probe to Visualize Potassium Ion Transition on Cellular Membranes

Hirata T., Terai T., Yamamura H., Shimonishi M., Komatsu T., Hanaoka K., Ueno T., Imaizumi Y., Nagano T., Urano Y.

Analytical Chemistry (Analytical Chemistry)  88 ( 5 ) 2693 - 2700 2016.03

Research paper (scientific journal), Accepted,  ISSN  00032700

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K+ is the most abundant metal ion in cells, and changes of [K+] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K+] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K+, TLSHalo. TLSHalo is responsive to [K+] in the physiological range, with good selectivity over Na+ and retains its K+-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K+] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K+] change induced by K+ ionophores or by activation of a native Ca2+-dependent K+ channel (BK channel). Further, we show that K+ efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K+ dynamics with high spatiotemporal resolution.

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Chemical tools for the study of hydrogen sulfide (H<inf>2</inf>S) and sulfane sulfur and their applications to biological studies

Takano Y., Shimamoto K., Hanaoka K.

Journal of Clinical Biochemistry and Nutrition (Journal of Clinical Biochemistry and Nutrition)  58 ( 1 ) 7 - 15 2016.01

Research paper (scientific journal), Accepted,  ISSN  09120009

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Hydrogen sulfide (H2S) functions in many physiological processes, including relaxation of vascular smooth muscles, mediation of neurotransmission, inhibition of insulin signaling, and regulation of inflammation. On the other hand, sulfane sulfur, which is a sulfur atom with six valence electrons but no charge, has the unique ability to bind reversibly to other sulfur atoms to form hydroper sulfides (R-S-SH) and polysulfides (-S-Sn-S-). H2S and sulfane sulfur always coexist, and recent work suggests that sulfane sulfur species may be the actual signaling molecules in at least some biological phenomena. For example, one of the mechanisms of activity regulation of proteins by H2S is the S-sulfhydration of cysteine residues (protein Cys-SSH). In this review, we summarize recent progress on chemical tools for the study of H2S and sulfane sulfur, covering fluorescence probes utilizing various design strategies, H2S caged compounds, inhibitors of physiological H2S-producing enzymes (cystathionine γ-lyase, cystathionine β-synthase and 3-mercaptopyruvate sulfurtrans-ferase), and labeling reagents. Fluorescence probes offer particular advantages as chemical tools to study physiological functions of biomolecules, including ease of use and real-time, nondestructive visualization of biological processes in live cells and tissues.

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Development of silicon-substituted xanthene dyes and their application to fluorescent probes

Hanaoka K., Urano Y.

Yuki Gosei Kagaku Kyokaishi/Journal of Synthetic Organic Chemistry (Yuki Gosei Kagaku Kyokaishi/Journal of Synthetic Organic Chemistry)  74 ( 5 ) 512 - 520 2016

Research paper (scientific journal), Accepted,  ISSN  00379980

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Fluorescence imaging is one of the most powerful techniques for visualization of the temporal and spatial biological events in living cells. Many fluorescent probes have been developed so far and contributed greatly to biological and medical researches. For biological applications, xanthene dyes have many favorable characteristics, such as high water solubility and high fluorescence quantum yield, and have been utilized as the fluorescent core for a large number of fluorescent probes. In this manuscript, we introduce a novel red fluorescein analogue, TokyoMagenta (TM), in which the O atom at the 10 position of the xanthene moiety is replaced with a Si atom, and the absorption and emission wavelengths of TM are about 90 nm longer than those of fluorescein. These silicon-substituted xanthene dyes would be useful for multicolor imaging. Nowadays, we think these xanthene dyes have been becoming one of practical fluorophores for biological researches.

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Identification of lung inflammation-related elevation of acylamino acid releasing enzyme (APEH) activity using an enzymomics approach

Komatsu T., Yoshioka K., Hanaoka K., Terai T., Ueno T., Nagano T., Urano Y.

Chemical and Pharmaceutical Bulletin (Chemical and Pharmaceutical Bulletin)  64 ( 11 ) 1533 - 1538 2016

Research paper (scientific journal), Accepted,  ISSN  00092363

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Disease-associated alterations of enzymatic functions are potentially useful as disease biomarkers, and here we show that an enzymomics (omics of active enzymes) approach, in which enzymatic activities are screened with panels of substrates, can be an effective way to identify such alterations. In the present study, we used a panel of fluorogenic substrates to search for altered enzyme activities in bronchoalveolar lavage fluid (BALF) from a mouse model of lung inflammation. We found that acylamino acid releasing enzyme (APEH) activity was highly elevated, apparently reflecting the increased population of immune cells in the inflamed lung.

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Discovery of a pyruvylated peptide-metabolizing enzyme using a fluorescent substrate-based protein discovery technique

Yoshioka K., Komatsu T., Hanaoka K., Ueno T., Terai T., Nagano T., Urano Y.

Chemical Communications (Chemical Communications)  52 ( 23 ) 4377 - 4380 2016

Research paper (scientific journal), Accepted,  ISSN  13597345

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We employed a fluorescent substrate-based target discovery approach to screen the enzymome for metabolic activity towards pyruvyl-amidated peptides, and identified an acylamino acid-releasing enzyme (APEH). Cells overexpressing APEH exhibited higher metabolic activity towards the probe, N-pyruvyl-leucyl-7-amido-4-methylcoumarin (Pyr-Leu-AMC), while the selective APEH inhibitor AA74-1 blocked the reaction. Metabolism of various pyruvylated peptides in liver lysate was almost completely blocked by AA74-1. Pyruvyl peptides are synthesized in response to oxidative stress, but their biological role is poorly understood; identification of a key contributor to their metabolism should stimulate research on pathways leading from oxidative stress to protein modification and biological output.

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Development of a sensitive bioluminogenic probe for imaging highly reactive oxygen species in living rats

Kojima R., Takakura H., Kamiya M., Kobayashi E., Komatsu T., Ueno T., Terai T., Hanaoka K., Nagano T., Urano Y.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  54 ( 49 ) 14768 - 14771 2015.12

Research paper (scientific journal), Accepted,  ISSN  14337851

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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.

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Azobenzene-caged sulforhodamine dyes: A novel class of 'turn-on' reactive probes for hypoxic tumor cell imaging

Chevalier A., Piao W., Hanaoka K., Nagano T., Renard P.Y., Romieu A.

Methods and Applications in Fluorescence (Methods and Applications in Fluorescence)  3 ( 4 )  2015.12

Research paper (scientific journal), Accepted

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New sulforhodamine-based fluorescent 'turn-on' probes have been developed for the direct imaging of cellular hypoxia. Rapid access to this novel class of water-soluble 'azobenzene-caged' fluorophores was made possible through an easily-implementable azo-coupling reaction between a fluorescent primary arylamine derived from a sulforhodamine 101 scaffold (named SR101-NaphtNH2) and a tertiary aniline whose N-substituents are neutral, cationic, or zwitterionic. The detection mechanism is based on the bioreductive cleavage of the azo bond that restores strong far-red fluorescence (emission maximum at 625 nm) by regenerating the original sulforhodamine SR101-NaphtNH2. This valuable fluorogenic response was obtained for the three 'smart' probes studied in this work, as shown by an in vitro assay using rat liver microsomes placed under aerobic and then under hypoxic conditions. Most importantly, the probe namely SR101-NaphtNH2-Hyp-diMe was successfully applied for imaging the hypoxic status of tumor cells (A549 cells).

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Thiosulfate mediates cytoprotective effects of hydrogen sulfide against neuronal ischemia

Marutani E., Yamada M., Ida T., Tokuda K., Ikeda K., Kai S., Shirozu K., Hayashida K., Kosugi S., Hanaoka K., Kaneki M., Akaike T., Ichinose F.

Journal of the American Heart Association (Journal of the American Heart Association)  4 ( 11 )  2015.11

Research paper (scientific journal), Accepted

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Background-Hydrogen sulfide (H2S) exhibits protective effects in various disease models including cerebral ischemia-reperfusion (I/R) injury. Nonetheless, mechanisms and identity of molecules responsible for neuroprotective effects of H2S remain incompletely defined. In the current study, we observed that thiosulfate, an oxidation product of H2S, mediates protective effects of an H2S donor compound sodium sulfide (Na2S) against neuronal I/R injury. Methods and Results-We observed that thiosulfate in cell culture medium is not only required but also sufficient to mediate cytoprotective effects of Na2S against oxygen glucose deprivation and reoxygenation of human neuroblastoma cell line (SH-SY5Y) and murine primary cortical neurons. Systemic administration of sodium thiosulfate (STS) improved survival and neurological function of mice subjected to global cerebral I/R injury. Beneficial effects of STS, as well as Na2S, were associated with marked increase of thiosulfate, but not H2S, in plasma and brain tissues. These results suggest that thiosulfate is a circulating "carrier" molecule of beneficial effects of H2S. Protective effects of thiosulfate were associated with inhibition of caspase-3 activity by persulfidation at Cys163 in caspase-3. We discovered that an SLC13 family protein, sodium sulfate cotransporter 2 (SLC13A4, NaS-2), facilitates transport of thiosulfate, but not sulfide, across the cell membrane, regulating intracellular concentrations and thus mediating cytoprotective effects of Na2S and STS. Conclusions-The protective effects of H2S are mediated by thiosulfate that is transported across cell membrane by NaS-2 and exerts antiapoptotic effects via persulfidation of caspase-3. Given the established safety track record, thiosulfate may be therapeutic against ischemic brain injury.

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Identification of Tissue-Restricted Bioreaction Suitable for in Vivo Targeting by Fluorescent Substrate Library-Based Enzyme Discovery

Yoshioka K., Komatsu T., Nakada A., Onagi J., Kuriki Y., Kawaguchi M., Terai T., Ueno T., Hanaoka K., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  137 ( 38 ) 12187 - 12190 2015.09

Research paper (scientific journal), Accepted,  ISSN  00027863

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Tissue-restricted bioreactions can be utilized to design chemical-biological tools and prodrugs. We have developed a fluorescent-substrate-library-based enzyme discovery approach to screen tissue extracts for enzymatic activities of interest. Assay-positive candidate proteins were identified by diced electrophoresis gel assay followed by peptide mass fingerprinting. We discovered that pyruvyl anilide is specifically hydrolyzed by carboxylesterase 2 (CES2), which is predominantly localized in the liver and kidney. We show that the pyruvyl targeting group/CES2 enzyme pair can be used to deliver the 7-amino-4-methylcoumarin fluorophore specifically to the liver and kidney in vivo. Our screening approach should be useful to find other masking group/enzyme pairs suitable for development of fluorescent substrates and prodrugs.

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Analysis of Chemical Equilibrium of Silicon-Substituted Fluorescein and Its Application to Develop a Scaffold for Red Fluorescent Probes

Hirabayashi K., Hanaoka K., Takayanagi T., Toki Y., Egawa T., Kamiya M., Komatsu T., Ueno T., Terai T., Yoshida K., Uchiyama M., Nagano T., Urano Y.

Analytical Chemistry (Analytical Chemistry)  87 ( 17 ) 9061 - 9069 2015.09

Research paper (scientific journal), Accepted,  ISSN  00032700

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Fluorescein is a representative green fluorophore that has been widely used as a scaffold of practically useful green fluorescent probes. Here, we report synthesis and characterization of a silicon-substituted fluorescein, i.e., 2-COOH TokyoMagenta (2-COOH TM), which is a fluorescein analogue in which the O atom at the 10′ position of the xanthene moiety of fluorescein is replaced with a Si atom. This fluorescein analogue forms a spirolactone ring via intramolecular nucleophilic attack of the carboxylic group in a pH-dependent manner. Consequently, 2-COOH TM exhibits characteristic large pH-dependent absorption and fluorescence spectral changes: (1) 2-COOH TM is colorless at acidic pH, whereas fluorescein retains observable absorption and fluorescence even at acidic pH, and the absorption maximum is also shifted; (2) the absorption spectral change occurs above pH 7.0 for 2-COOH TM and below pH 7.0 for fluorescein; (3) 2-COOH TM shows a much sharper pH response than fluorescein because of its pK<inf>a</inf> inversion, i.e., pK<inf>a1</inf> > pK<inf>a2</inf>. These features are also different from those of a compound without the carboxylic group, 2-Me TokyoMagenta (2-Me TM). Analysis of the chemical equilibrium between pH 3.0 and 11.0 disclosed that 2-COOH TM favors the colorless and nonfluorescent lactone form, compared with fluorescein. Substitution of Cl atoms at the 4′ and 5′ positions of the xanthene moiety of 2-COOH TM to obtain 2-COOH DCTM shifted the equilibrium so that the new derivative exists predominantly in the strongly fluorescent open form at physiological pH (pH 7.4). To demonstrate the practical utility of 2-COOH DCTM as a novel scaffold for red fluorescent probes, we employed it to develop a probe for β-galactosidase. (Chemical Equation Presented).

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Artificial Ligands of Streptavidin (ALiS): Discovery, Characterization, and Application for Reversible Control of Intracellular Protein Transport

Terai T., Kohno M., Boncompain G., Sugiyama S., Saito N., Fujikake R., Ueno T., Komatsu T., Hanaoka K., Okabe T., Urano Y., Perez F., Nagano T.

Journal of the American Chemical Society (Journal of the American Chemical Society)  137 ( 33 ) 10464 - 10467 2015.08

Research paper (scientific journal), Accepted,  ISSN  00027863

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Artificial ligands of streptavidin (ALiS) with association constants of 106 M-1 were discovered by high-throughput screening of our chemical library, and their binding characteristics, including X-ray crystal structure of the streptavidin complex, were determined. Unlike biotin and its derivatives, ALiS exhibits fast dissociation kinetics and excellent cell permeability. The streptavidin-ALiS system provides a novel, practical compound-dependent methodology for repeated reversible cycling of protein localization between intracellular organella.

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Detection of NAD(P)H-dependent enzyme activity with dynamic luminescence quenching of terbium complexes

Ito H., Terai T., Hanaoka K., Ueno T., Komatsu T., Nagano T., Urano Y.

Chemical Communications (Chemical Communications)  51 ( 39 ) 8319 - 8322 2015.05

Research paper (scientific journal), Accepted,  ISSN  13597345

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We discovered that positively charged terbium complexes bearing 1,4,7,10-tetraazacyclododecane functionalized with amide ligands are highly sensitive to dynamic luminescence quenching by NAD(P)H. We exploited this phenomenon to establish a general time-resolved luminescence-based assay platform for sensitive detection of NAD(P)H-dependent enzyme activities. This journal is

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Fluorescent probes for hydrogen sulfide (H<inf>2</inf>S) and sulfane sulfur and their applications to biological studies

Shimamoto K., Hanaoka K.

Nitric Oxide - Biology and Chemistry (Nitric Oxide - Biology and Chemistry)  46   72 - 79 2015.04

Research paper (scientific journal), Accepted,  ISSN  10898603

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Hydrogen sulfide (H2S), a toxic gas with the smell of rotten eggs, plays key roles in many physiological processes, including relaxation of vascular smooth muscles, mediation of neurotransmission, inhibition of insulin signaling, and regulation of inflammation. The most commonly used methods or detecting H2S are the methylene blue method and the electrode method, but these methods require destructive sampling, e.g., homogenization of biological samples. On the other hand, the fluorescence detection method has been widely used in biological studies to study the physiological roles of H2S, because this technology provides real-time, easy-to-use, nondestructive detection in live cells or tissues. Many selective fluorescent probes for H2S have been reported. Sulfane sulfur compounds contain divalent sulfur atoms bonded to other sulfur atom(s), as in persulfides (RSSH) and polysulfides (RSSnSR). They are currently attracting increasing interest because one of the mechanisms of activity regulation of proteins by H2S is sulfhydration of cysteine residues (RSH→RSSH). Since H2S and sulfane sulfur are redox partners, they are very likely to coexist in biological systems, and from a reactivity point-of-view, sulfane sulfur seems likely to be much more effective than H2S in S-sulfhydration. Therefore, sulfane sulfur may be involved in mediating at least some of the biological activities of H2S. In this review, we summarize recent work on fluorescent probes selective for H2S and/or sulfane sulfur, and we briefly review their applications to biological studies.

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Development of a Series of Near-Infrared Dark Quenchers Based on Si-rhodamines and Their Application to Fluorescent Probes

Myochin T., Hanaoka K., Iwaki S., Ueno T., Komatsu T., Terai T., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  137 ( 14 ) 4759 - 4765 2015.04

Research paper (scientific journal), Accepted,  ISSN  00027863

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Near-infrared (NIR) fluorescent probes based on the Förster resonance energy transfer (FRET) mechanism have various practical advantages, and their molecular design is generally based on the use of NIR dark quenchers, which are nonfluorescent dyes, as cleavable FRET acceptors. However, few NIR dark quenchers can quench fluorescence in the Cy7 region (over 780 nm). Here, we describe Si-rhodamine-based NIR dark quenchers (SiNQs), which show broad absorption covering this region. They are nonfluorescent independently of solvent polarity and pH, probably due to free rotation of the bond between the N atom and the xanthene moiety. SiNQs can easily be structurally modified to tune their water-solubility and absorption spectra, enabling flexible design of appropriate FRET pair for various NIR fluorescent dyes. To demonstrate the usefulness of SiNQs, we designed and synthesized a NIR fluorescent probe for matrix metalloproteinase (MMP) activity using SiNQ780. This probe 1 could detect MMP activity in vitro, in cultured cells and in a tumor-bearing mouse, in which the tumor was clearly visualized, by NIR fluorescence. We believe SiNQs will be useful for the development of a wide range of practical NIR fluorescent probes. (Chemical Equation Presented).

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New class of bioluminogenic probe based on bioluminescent enzyme-induced electron transfer: BioLeT

Takakura H., Kojima R., Kamiya M., Kobayashi E., Komatsu T., Ueno T., Terai T., Hanaoka K., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  137 ( 12 ) 4010 - 4013 2015.04

Research paper (scientific journal), Accepted,  ISSN  00027863

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Bioluminescence imaging (BLI) has advantages for investigating biological phenomena in deep tissues of living animals, but few design strategies are available for functional bioluminescent substrates. We propose a new design strategy (designated as bioluminescent enzyme-induced electron transfer: BioLeT) for luciferin-based bioluminescence probes. Luminescence measurements of a series of aminoluciferin derivatives confirmed that bioluminescence can be controlled by means of BioLeT. Based on this concept, we developed bioluminescence probes for nitric oxide that enabled quantitative and sensitive detection even in vivo. Our design strategy should be applicable to develop a wide range of practically useful bioluminogenic probes.

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Gliotoxin suppresses NF-κB activation by selectively inhibiting linear ubiquitin chain assembly complex (LUBAC)

Sakamoto H., Egashira S., Saito N., Kirisako T., Miller S., Sasaki Y., Matsumoto T., Shimonishi M., Komatsu T., Terai T., Ueno T., Hanaoka K., Kojima H., Okabe T., Wakatsuki S., Iwai K., Nagano T.

ACS Chemical Biology (ACS Chemical Biology)  10 ( 3 ) 675 - 681 2015.03

Research paper (scientific journal), Accepted,  ISSN  15548929

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A linear ubiquitin chain, which consists of ubiquitin molecules linked via their N- and C-termini, is formed by a linear ubiquitin chain assembly complex (LUBAC) composed of HOIP, HOIL-1L, and SHARPIN, and conjugation of a linear ubiquitin chain on the NF-κB essential modulator (NEMO) is deeply involved in NF-κB activation induced by various signals. Since abnormal activation of NF-κB is associated with inflammatory disease and malignancy, we searched for an inhibitor of LUBAC by high-throughput screening (HTS) with a Tb3+-fluorescein FRET system. As a result, we found that the fungal metabolite gliotoxin inhibits LUBAC selectively by binding to the RING-IBR-RING domain of HOIP, the catalytic center of LUBAC. Gliotoxin has been well-known as an inhibitor of NF-κB activation, though its action mechanism has remained elusive. Here, we show that gliotoxin inhibits signal-induced NF-κB activation by selectively inhibiting LUBAC-mediated linear ubiquitin chain formation.

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Development of photo-controllable hydrogen sulfide donor applicable in live cells

Fukushima N., Ieda N., Kawaguchi M., Sasakura K., Nagano T., Hanaoka K., Miyata N., Nakagawa H.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  25 ( 2 ) 175 - 178 2015.01

Research paper (scientific journal), Accepted,  ISSN  0960894X

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Hydrogen sulfide (H2S) has multiple physiological roles, for example, in vasodilation and inflammation. It is a highly reactive gas under ambient conditions, so controllable H2S donors are required for studying its biological functions. Here, we describe the design, synthesis and application of a H2S donor (SPD-2) that utilizes xanthone photochemistry to control H2S release. H2S generation from SPD-2 was completely dependent on UVA-irradiation (325-385 nm), as confirmed by methylene blue assay and by the use of a H2S-selective fluorescent probe. SPD-2 was confirmed to provide controlled H2S delivery in live cells, and should be suitable for various biological applications.

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Synthesis of a photocontrollable hydrogen sulfide donor using ketoprofenate photocages

Fukushima N., Ieda N., Sasakura K., Nagano T., Hanaoka K., Suzuki T., Miyata N., Nakagawa H.

Chemical Communications (Chemical Communications)  50 ( 5 ) 587 - 589 2014.12

Research paper (scientific journal), Accepted,  ISSN  13597345

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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.

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Sodium thiosulfate attenuates acute lung injury in Mice

Sakaguchi M., Marutani E., Shin H.S., Chen W., Hanaoka K., Xian M., Ichinose F.

Anesthesiology (Anesthesiology)  121 ( 6 ) 1248 - 1257 2014.12

Research paper (scientific journal), Accepted,  ISSN  00033022

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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.

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A design strategy for small molecule-based targeted MRI contrast agents: Their application for detection of atherosclerotic plaques

Iwaki S., Hokamura K., Ogawa M., Takehara Y., Muramatsu Y., Yamane T., Hirabayashi K., Morimoto Y., Hagisawa K., Nakahara K., Mineno T., Terai T., Komatsu T., Ueno T., Tamura K., Adachi Y., Hirata Y., Arita M., Arai H., Umemura K., Nagano T., Hanaoka K.

Organic and Biomolecular Chemistry (Organic and Biomolecular Chemistry)  12 ( 43 ) 8611 - 8618 2014.11

Research paper (scientific journal), Accepted,  ISSN  14770520

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Gadolinium(III) ion (Gd3+) complexes are widely used as contrast agents in magnetic resonance imaging (MRI), and many attempts have been made to couple them to sensor moieties in order to visualize biological phenomena of interest inside the body. However, the low sensitivity of MRI has made it difficult to develop practical MRI contrast agents for in vivo imaging. We hypothesized that practical MRI contrast agents could be designed by targeting a specific biological environment, rather than a specific protein such as a receptor. To test this idea, we designed and synthesized a Gd3+-based MRI contrast agent, 2BDP3Gd, for visualizing atherosclerotic plaques by linking the Gd3+-complex to the lipophilic fluorophore BODIPY to stain lipid-rich environments. We found that 2BDP3Gd was selectively accumulated into lipid droplets of adipocytes at the cellular level. Atherosclerotic plaques in the aorta of Watanabe heritable hyperlipidemic (WHHL) rabbits were clearly visualized in T1-weighted MR images after intravenous injection of 2BDP3Gd in vivo.

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Fluorometric assay of integrin activity with a small-molecular probe that senses the binding site microenvironment

Komatsu T., Takeda A., Hanaoka K., Terai T., Ueno T., Tada Y., Nagano T., Urano Y.

Chemical Communications (Chemical Communications)  50 ( 100 ) 15894 - 15896 2014.11

Research paper (scientific journal), Accepted,  ISSN  13597345

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We have developed a small-molecular probe, consisting of cyclic RGD pentapeptides bearing a nitrobenzoxadiazole fluorophore at the 4′-residue, which detects integrin αVβ3 activity in terms of fluorescence intensity decrease due to quenching of the fluorophore by interaction with tyrosine-122 at the binding site of the protein. This probe appears to be suitable for a practical, high-throughput fluorescence assay to screen small-molecular modulators of integrin activity. © The Royal Society of Chemistry 2014.

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Practical fluorescence detection of acrolein in human plasma via a two-step tethering approach

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

Chemical Communications (Chemical Communications)  50 ( 95 ) 14946 - 14948 2014.11

Research paper (scientific journal), Accepted,  ISSN  13597345

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Acrolein, a cytotoxic α,β-unsaturated aldehyde and disease biomarker, was determined in plasma by means of a novel tethering strategy using Michael addition of the compound to a fluorescent dye, followed by immobilization of the product on microbeads via the aldehyde moiety. Elevation of blood acrolein was detected in mice treated with an anticancer agent cyclophosphamide, which releases acrolein upon activation. This method should be suitable for high-throughput diagnostic and clinical application. © the Partner Organisations 2014.

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Boron dipyrromethene as a fluorescent caging group for single-photon uncaging with long-wavelength visible light

Umeda N., Takahashi H., Kamiya M., Ueno T., Komatsu T., Terai T., Hanaoka K., Nagano T., Urano Y.

ACS Chemical Biology (ACS Chemical Biology)  9 ( 10 ) 2242 - 2246 2014.10

Research paper (scientific journal), Accepted,  ISSN  15548929

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Caged compounds are useful tools for precise spatiotemporal modulation of cell functions, but in most cases uncaging requires ultraviolet (UV) light, which is cytotoxic and has limited tissue penetration. Therefore, caged compounds that can be activated by longer-wavelength light are required. Here we describe a novel photoelimination reaction of 4-aryloxy boron dipyrromethene (BODIPY) derivatives and show that BODIPY can function as a caging group for phenol groups. We developed a novel BODIPY-caged histamine compound, which is photoactivatable with blue-green visible light to stimulate cultured HeLa cells in a spatiotemporally well-controlled manner. This caging strategy is expected to be widely applicable to develop tools for probing various cellular functions. (Chemical Equation Presented).

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Cytoprotective effects of hydrogen sulfide-releasing N-methyl-d-aspartate receptor antagonists mediated by intracellular sulfane sulfur

Marutani E., Sakaguchi M., Chen W., Sasakura K., Liu J., Xian M., Hanaoka K., Nagano T., Ichinose F.

MedChemComm (MedChemComm)  5 ( 10 ) 1577 - 1583 2014.10

Research paper (scientific journal), Accepted,  ISSN  20402503

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Hydrogen sulfide (H2S) exerts a host of biological effects ranging from cytotoxicity to cytoprotection. Cytotoxicity of H2S in neurodegenerative diseases may be mediated by N-methyl-d-aspartate receptor (NMDAR) activation. To exploit cytoprotective effects of H2S while minimizing its toxicity, we synthesized a series of H2S-releasing NMDAR antagonists and examined their effects against 1-methyl-4-phenylpyridinium (MPP+)-induced cell death, a cellular model of Parkinson's disease. We observed that cytoprotective effects of H2S-releasing NMDAR antagonists correlated with their ability to increase intracellular sulfane sulfur, but not H2S, levels. These studies suggest that H2S-donor compounds that increase intracellular sulfane sulfur levels are potentially useful neuroprotective agents against neurodegenerative diseases.

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Thermal or mechanical stimuli-induced photoluminescence color change of a molecular assembly composed of an amphiphilic anthracene derivative in water

Sagara Y., Komatsu T., Terai T., Ueno T., Hanaoka K., Kato T., Nagano T.

Chemistry - A European Journal (Chemistry - A European Journal)  20 ( 33 ) 10397 - 10403 2014.08

Research paper (scientific journal), Accepted,  ISSN  09476539

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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.

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Rapidly rendering cells phagocytic through a cell surface display technique and concurrent Rac activation

Onuma H., Komatsu T., Arita M., Hanaoka K., Ueno T., Terai T., Nagano T., Inoue T.

Science Signaling (Science Signaling)   ( 334 )  2014.07

Research paper (scientific journal), Accepted,  ISSN  19450877

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Cell surfaces represent a platform through which extracellular signals that determine diverse cellular processes, including migration, division, adhesion, and phagocytosis, are transduced. Techniques to rapidly reconfigure the surface properties of living cells should thus offer the ability to harness these cellular functions. Although the molecular mechanism of phagocytosis is well characterized, the minimal molecular players that are sufficient to activate this elaborate process remain elusive.We developed and implemented a technique to present a molecule of interest at the cell surface in an inducible manner on a time scale of minutes. We simultaneously induced the cell surface display of the C2 domain of milk fat globule epidermal growth factor factor 8 (MFG-E8) and activated the intracellular small guanosine triphosphatase Rac, which stimulates actin polymerization at the cell periphery. The C2 domain binds to phosphatidylserine, a lipid exposed on the surface of apoptotic cells. By integrating the stimulation of these two processes, we converted HeLa cells into a phagocytic cell line that bound to and engulfed apoptotic human Jurkat cells. Inducing either the cell surface display of the C2 domain or activating Rac alone was not sufficient to stimulate phagocytosis, which suggests that attachment to the target cell and actin reorganization together constitute the minimal molecular events that are needed to induce phagocytosis. This cell surface display technique might be useful as part of a targeted, cell-based therapy in which unwanted cells with characteristic surface molecules could be rapidly consumed by engineered cells.

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Selective ablation of β-galactosidase-expressing cells with a rationally designed activatable photosensitizer

Ichikawa Y., Kamiya M., Obata F., Miura M., Terai T., Komatsu T., Ueno T., Hanaoka K., Nagano T., Urano Y.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  53 ( 26 ) 6772 - 6775 2014.06

Research paper (scientific journal), Accepted,  ISSN  14337851

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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.

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Primary hepatocytes from mice lacking cysteine dioxygenase show increased cysteine concentrations and higher rates of metabolism of cysteine to hydrogen sulfide and thiosulfate

Jurkowska H., Roman H., Hirschberger L., Sasakura K., Nagano T., Hanaoka K., Krijt J., Stipanuk M.

Amino Acids (Amino Acids)  46 ( 5 ) 1353 - 1365 2014.05

Research paper (scientific journal), Accepted,  ISSN  09394451

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The oxidation of cysteine in mammalian cells occurs by two routes: a highly regulated direct oxidation pathway in which the first step is catalyzed by cysteine dioxygenase (CDO) and by desulfhydration-oxidation pathways in which the sulfur is released in a reduced oxidation state. To assess the effect of a lack of CDO on production of hydrogen sulfide (H2S) and thiosulfate (an intermediate in the oxidation of H2S to sulfate) and to explore the roles of both cystathionine γ-lyase (CTH) and cystathionine β-synthase (CBS) in cysteine desulfhydration by liver, we investigated the metabolism of cysteine in hepatocytes isolated from Cdo1-null and wild-type mice. Hepatocytes from Cdo1-null mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice. The greater flux of cysteine through the cysteine desulfhydration reactions catalyzed by CTH and CBS in hepatocytes from Cdo1-null mice appeared to be the consequence of their higher cysteine levels, which were due to the lack of CDO and hence lack of catabolism of cysteine by the cysteinesulfinate-dependent pathways. Both CBS and CTH appeared to contribute substantially to cysteine desulfhydration, with estimates of 56 % by CBS and 44 % by CTH in hepatocytes from wild-type mice, and 63 % by CBS and 37 % by CTH in hepatocytes from Cdo1-null mice. © 2014 Springer-Verlag Wien.

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Unbalanced excitability underlies offline reactivation of behaviorally activated neurons

Mizunuma M., Norimoto H., Tao K., Egawa T., Hanaoka K., Sakaguchi T., Hioki H., Kaneko T., Yamaguchi S., Nagano T., Matsuki N., Ikegaya Y.

Nature Neuroscience (Nature Neuroscience)  17 ( 4 ) 503 - 505 2014.04

Research paper (scientific journal), Accepted,  ISSN  10976256

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Hippocampal sharp waves (SWs)/ripples represent the reactivation of neurons involved in recently acquired memory and are crucial for memory consolidation. By labeling active cells with fluorescent protein under the control of an immediate-early gene promoter, we found that neurons that had been activated while mice explored a novel environment were preferentially reactivated during spontaneous SWs in hippocampal slices in vitro. During SWs, the reactivated neurons received strong excitatory synaptic inputs as opposed to a globally tuned network balance between excitation and inhibition. © 2014 Nature America, Inc. All rights reserved.

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

Sagara Y., Komatsu T., Ueno T., Hanaoka K., Kato T., Nagano T.

Journal of the American Chemical Society (Journal of the American Chemical Society)  136 ( 11 ) 4273 - 4280 2014.03

Research paper (scientific journal), Accepted,  ISSN  00027863

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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. © 2014 American Chemical Society.

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Development of azo-based fluorescent probes to detect different levels of hypoxia

Piao W., Tsuda S., Tanaka Y., Maeda S., Liu F., Takahashi S., Kushida Y., Komatsu T., Ueno T., Terai T., Nakazawa T., Uchiyama M., Morokuma K., Nagano T., Hanaoka K.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  52 ( 49 ) 13028 - 13032 2013.12

Research paper (scientific journal), Accepted,  ISSN  14337851

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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.

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A water-soluble mechanochromic luminescent pyrene derivative exhibiting recovery of the initial photoluminescence color in a high-humidity environment

Sagara Y., Komatsu T., Ueno T., Hanaoka K., Kato T., Nagano T.

Advanced Functional Materials (Advanced Functional Materials)  23 ( 42 ) 5277 - 5284 2013.11

Research paper (scientific journal), Accepted,  ISSN  1616301X

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Switching of the luminescence properties of molecular materials in response to mechanical stimulation is of fundamental interest and also has a range of potential applications. Herein, a water-soluble mechanochromic luminescent pyrene derivative having two hydrophilic dendrons is reported. This pyrene derivative is the first example of a mechanochromic luminescent organic compound that responds to relative humidity. Mechanical stimulation (grinding) of this pyrene derivative in the solid state results in a change of the photoluminescence from yellow to green. Subsequent exposure to water vapor induces recovery of the initial yellow photoluminescence. The color change is reversible through at least ten cycles. It is also demonstrated that this compound can be applied as a mechano-sensing material in frictional wear testing for grease, owing to its immiscibility in non-polar solvents and its non-crystalline behavior. Transmission electron microscope and atomic force microscope observations of samples prepared from dilute aqueous solutions of the pyrene derivative on suitable substrates, together with dynamic light scattering measurements for the compound in aqueous solution, indicate that this amphiphilic dumbbell-shaped molecule forms micelles in water. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Diced electrophoresis gel assay for screening enzymes with specified activities

Komatsu T., Hanaoka K., Adibekian A., Yoshioka K., Terai T., Ueno T., Kawaguchi M., Cravatt B., Nagano T.

Journal of the American Chemical Society (Journal of the American Chemical Society)  135 ( 16 ) 6002 - 6005 2013.04

Research paper (scientific journal), Accepted,  ISSN  00027863

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We have established the diced electrophoresis gel (DEG) assay as a proteome-wide screening tool to identify enzymes with activities of interest using turnover-based fluorescent substrates. The method utilizes the combination of native polyacrylamide gel electrophoresis (PAGE) with a multiwell-plate- based fluorometric assay to find protein spots with the specified activity. By developing fluorescent substrates that mimic the structure of neutrophil chemoattractants, we could identify enzymes involved in metabolic inactivation of the chemoattractants. © 2013 American Chemical Society.

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Red fluorescent probe for monitoring the dynamics of cytoplasmic calcium ions

Egawa T., Hirabayashi K., Koide Y., Kobayashi C., Takahashi N., Mineno T., Terai T., Ueno T., Komatsu T., Ikegaya Y., Matsuki N., Nagano T., Hanaoka K.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  52 ( 14 ) 3874 - 3877 2013.04

Research paper (scientific journal), Accepted,  ISSN  14337851

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We see red (and yellow and green): Probe 1 was developed for the visualization of cytoplasmic Ca2+, a pivotal second messenger in many biological responses. The new probe is suitable for multicolor imaging for the simultaneous detection of metal ions or proteins and is superior to the existing red fluorescent probe Rhod-2 for the monitoring of cytoplasmic Ca2+ oscillation in cultured cells (see fluorescence images of cells with 1 (top) and Rhod-2 (bottom)). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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TokyoGreen derivatives as specific and practical fluorescent probes for UDP-glucuronosyltransferase (UGT) 1A1

Terai T., Tomiyasu R., Ota T., Ueno T., Komatsu T., Hanaoka K., Urano Y., Nagano T.

Chemical Communications (Chemical Communications)  49 ( 30 ) 3101 - 3103 2013.03

Research paper (scientific journal), Accepted,  ISSN  13597345

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TokyoGreen (TG) derivatives were found to be efficient and specific substrates of an important drug-metabolizing enzyme, UDP-glucuronosyltransferase (UGT) 1A1. A rapid, specific, and sensitive assay of the enzyme was achieved simply by monitoring the change in fluorescence intensity. We also designed and developed the first “turn-on” fluorescent probes for UGTs. © 2001 The Royal Society of Chemistry.

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Rational design of highly sensitive fluorescence probes for protease and glycosidase based on precisely controlled spirocyclization

Sakabe M., Asanuma D., Kamiya M., Iwatate R.J., Hanaoka K., Terai T., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  135 ( 1 ) 409 - 414 2013.01

Research paper (scientific journal), Accepted,  ISSN  00027863

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We have synthesized and evaluated a series of hydroxymethyl rhodamine derivatives and found an intriguing difference of intramolecular spirocyclization behavior: the acetylated derivative of hydroxymethyl rhodamine green (Ac-HMRG) exists as a closed spirocyclic structure in aqueous solution at physiological pH, whereas HMRG itself takes an open nonspirocyclic structure. Ac-HMRG is colorless and nonfluorescent, whereas HMRG is strongly fluorescent. On the basis of these findings, we have developed a general design strategy to obtain highly sensitive fluorescence probes for proteases and glycosidases, by replacing the acetyl group of Ac-HMRG with a substrate moiety of the target enzyme. Specific cleavage of the substrate moiety in the nonfluorescent probe by the target enzyme generates a strong fluorescence signal. To confirm the validity and flexibility of our strategy, we designed and synthesized fluorescence probes for leucine aminopeptidase (Leu-HMRG), fibroblast activation protein (Ac-GlyPro-HMRG), and β-galactosidase (βGal-HMRG). All of these probes were almost nonfluorescent due to the formation of spirocyclic structure, but were converted efficiently to highly fluorescent HMRG by the target enzymes. We confirmed that the probes can be used in living cells. These probes offer great practical advantages, including high sensitivity and rapid response (due to regulation of fluorescence at a single reactive site), as well as resistance to photobleaching, and are expected to be useful for a range of biological and pathological investigations. © 2012 American Chemical Society.

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Reversible off-on fluorescence probe for hypoxia and imaging of hypoxia-normoxia cycles in live cells

Takahashi S., Piao W., Matsumura Y., Komatsu T., Ueno T., Terai T., Kamachi T., Kohno M., Nagano T., Hanaoka K.

Journal of the American Chemical Society (Journal of the American Chemical Society)  134 ( 48 ) 19588 - 19591 2012.12

Research paper (scientific journal), Accepted,  ISSN  00027863

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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. © 2012 American Chemical Society.

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Design strategy for a near-infrared fluorescence probe for matrix metalloproteinase utilizing highly cell permeable boron dipyrromethene

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

Journal of the American Chemical Society (Journal of the American Chemical Society)  134 ( 33 ) 13730 - 13737 2012.08

Research paper (scientific journal), Accepted,  ISSN  00027863

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Near-infrared (NIR) fluorescence probes are especially useful for simple and noninvasive in vivo imaging inside the body because of low autofluorescence and high tissue transparency in the NIR region compared with other wavelength regions. However, existing NIR fluorescence probes for matrix metalloproteinases (MMPs), which are tumor, atherosclerosis, and inflammation markers, have various disadvantages, especially as regards sensitivity. Here, we report a novel design strategy to obtain a NIR fluorescence probe that is rapidly internalized by free diffusion and well retained intracellularly after activation by extracellular MMPs. We designed and synthesized four candidate probes, each consisting of a cell permeable or nonpermeable NIR fluorescent dye as a Förster resonance energy transfer (FRET) donor linked to the NIR dark quencher BHQ-3 as a FRET acceptor via a MMP substrate peptide. We applied these probes for detection of the MMP activity of cultured HT-1080 cells, which express MMP2 and MT1-MMP, by fluorescence microscopy. Among them, the probe incorporating BODIPY650/665, BODIPY-MMP, clearly visualized the MMP activity as an increment of fluorescence inside the cells. We then applied this probe to a mouse xenograft tumor model prepared with HT-1080 cells. Following intratumoral injection of the probe, MMP activity could be visualized for much longer with BODIPY-MMP than with the probe containing SulfoCy5, which is cell impermeable and consequently readily washed out of the tissue. This simple design strategy should be applicable to develop a range of sensitive, rapidly responsive NIR fluorescence probes not only for MMP activity, but also for other proteases. © 2012 American Chemical Society.

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Highly activatable and environment-insensitive optical highlighters for selective spatiotemporal imaging of target proteins

Kobayashi T., Komatsu T., Kamiya M., Campos C., González-Gaitán M., Terai T., Hanaoka K., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  134 ( 27 ) 11153 - 11160 2012.07

Research paper (scientific journal), Accepted,  ISSN  00027863

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Optical highlighters are photoactivatable fluorescent molecules that exhibit pronounced changes in their spectral properties in response to irradiation with light of a specific wavelength and intensity. Here, we present a novel design strategy for a new class of caged BODIPY (4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene) fluorophores, based on the use of photoremovable protecting groups (PRPGs) with high reduction potentials that serve as both a photosensitive unit and a fluorescence quencher via photoinduced electron transfer (PeT). 2,6-Dinitrobenzyl (DNB)-caged BODIPY was efficiently photoactivated, with activation ratios exceeding 600-fold in aqueous solutions. We then combined this photoactivatable fluorophore with a SNAP (mutant of O 6-alkylguanine DNA alkyltransferase) ligand to obtain a small-molecule-based optical highlighter for visualization of protein dynamics, using the well-established SNAP tag technology. As proof of concept, we demonstrate spatiotemporal imaging of the fusion protein of epidermal growth factor receptor (EGFR) with SNAP tag in living cells. We also demonstrate highlighting of cells of interest in live zebrafish embryos, using the fusion protein of histone 2A with SNAP tag. © 2012 American Chemical Society.

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Red fluorescent scaffold for highly sensitive protease activity probes

Kushida Y., Hanaoka K., Komatsu T., Terai T., Ueno T., Yoshida K., Uchiyama M., Nagano T.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  22 ( 12 ) 3908 - 3911 2012.06

Research paper (scientific journal), Accepted,  ISSN  0960894X

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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.

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Near-infrared fluorescence probes for enzymes based on binding affinity modulation of squarylium dye scaffold

Oushiki D., Kojima H., Takahashi Y., Komatsu T., Terai T., Hanaoka K., Nishikawa M., Takakura Y., Nagano T.

Analytical Chemistry (Analytical Chemistry)  84 ( 10 ) 4404 - 4410 2012.05

Research paper (scientific journal), Accepted,  ISSN  00032700

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We present a novel design strategy for near-infrared (NIR) fluorescence probes utilizing dye-protein interaction as a trigger for fluorescence enhancement. The design principle involves modification of a polymethine dye with cleavable functional groups that reduce the dye's protein-binding affinity. When these functional groups are removed by specific interaction with the target enzymes, the dye's protein affinity is restored, protein binding occurs, and the dye's fluorescence is strongly enhanced. To validate this strategy, we first designed and synthesized an alkaline phosphatase (ALP) sensor by introducing phosphate into the squarylium dye scaffold; this sensor was able to detect ALP-labeled secondary antibodies in Western blotting analysis. Second, we synthesized a probe for β-galactosidase (widely used as a reporter of gene expression) by means of β-galactosyl substitution of the squarylium scaffold; this sensor was able to visualize β-galactosidase activity both in vitro and in vivo. Our strategy should be applicable to obtain NIR fluorescence probes for a wide range of target enzymes. © 2012 American Chemical Society.

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Development of hypoxia-sensitive Gd ³⁺-based MRI contrast agents

Iwaki S., Hanaoka K., Piao W., Komatsu T., Ueno T., Terai T., Nagano T.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  22 ( 8 ) 2798 - 2802 2012.04

Research paper (scientific journal), Accepted,  ISSN  0960894X

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Hypoxia occurs in various diseases, including cancer, ischemia, and acute and chronic vascular diseases. Here we describe the design and synthesis of the first hypoxia-sensitive MRI contrast agents, SAGds. SAGds showed a pH-dependent r 1 relaxivity change associated with intramolecular chelation of the nitrogen atom of the sulfonamide moiety to the Gd 3+ center. There was a correlation between the pK a of the r 1 relaxivity change and the sum of the Hammett σ constants of substituents on the aromatic ring. Among the synthesized compounds, 4NO 22MeOSAGd was selectively reduced to the amine by rat liver microsomes under hypoxic conditions, resulting in a 1.8-fold increment of the r 1 relaxivity owing to the change in pK a of the arylsulfonamide moiety. This enhancement of the r 1 relaxivity could be clearly detected in T 1-weighted MR images. Thus, 4NO 22MeOSAGd is a 'smart' MRI contrast agent for the detection of hypoxia under physiological conditions. © 2012 Elsevier Ltd. All rights reserved.

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Development of NIR fluorescent dyes based on Si-rhodamine for in vivo imaging

Koide Y., Urano Y., Hanaoka K., Piao W., Kusakabe M., Saito N., Terai T., Okabe T., Nagano T.

Journal of the American Chemical Society (Journal of the American Chemical Society)  134 ( 11 ) 5029 - 5031 2012.03

Research paper (scientific journal),  ISSN  00027863

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We have developed a series of novel near-infrared (NIR) wavelength-excitable fluorescent dyes, SiR-NIRs, by modifying the Si-rhodamine scaffold to obtain emission in the range suitable for in vivo imaging. Among them, SiR680 and SiR700 showed sufficiently high quantum efficiency in aqueous media. Both antibody-bound and free dye exhibited high tolerance to photobleaching in aqueous solution. Subcutaneous xenograft tumors were successfully visualized in a mouse tumor model using SiR700-labeled anti-tenascin-C (TN-C) antibody, SiR700-RCB1. SiR-NIRs are expected to be useful as labeling agents for in vivo imaging studies including multicolor imaging, and also as scaffolds for NIR fluorescence probes. © 2012 American Chemical Society.

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A reversible near-infrared fluorescence probe for reactive oxygen species based on Te-rhodamine

Koide Y., Kawaguchi M., Urano Y., Hanaoka K., Komatsu T., Abo M., Terai T., Nagano T.

Chemical Communications (Chemical Communications)  48 ( 25 ) 3091 - 3093 2012.02

Research paper (scientific journal), Accepted,  ISSN  13597345

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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.

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Selective two-step labeling of proteins with an off/on fluorescent probe

Hirabayashi K., Hanaoka K., Shimonishi M., Terai T., Komatsu T., Ueno T., Nagano T.

Chemistry - A European Journal (Chemistry - A European Journal)  17 ( 52 ) 14763 - 14771 2011.12

Research paper (scientific journal), Accepted,  ISSN  09476539

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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.

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Development of a highly selective fluorescence probe for hydrogen sulfide

Sasakura K., Hanaoka K., Shibuya N., Mikami Y., Kimura Y., Komatsu T., Ueno T., Terai T., Kimura H., Nagano T.

Journal of the American Chemical Society (Journal of the American Chemical Society)  133 ( 45 ) 18003 - 18005 2011.11

Research paper (scientific journal), Accepted,  ISSN  00027863

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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.

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Method for enhancing cell penetration of Gd ³⁺-based MRI contrast agents by conjugation with hydrophobic fluorescent dyes

Yamane T., Hanaoka K., Muramatsu Y., Tamura K., Adachi Y., Miyashita Y., Hirata Y., Nagano T.

Bioconjugate Chemistry (Bioconjugate Chemistry)  22 ( 11 ) 2227 - 2236 2011.11

Research paper (scientific journal), Accepted,  ISSN  10431802

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Gadolinium ion (Gd 3+) complexes are commonly used as magnetic resonance imaging (MRI) contrast agents to enhance signals in T 1-weighted MR images. Recently, several methods to achieve cell-permeation of Gd 3+ complexes have been reported, but more general and efficient methodology is needed. In this report, we describe a novel method to achieve cell permeation of Gd 3+ complexes by using hydrophobic fluorescent dyes as a cell-permeability-enhancing unit. We synthesized Gd 3+ complexes conjugated with boron dipyrromethene (BDP-Gd) and Cy7 dye (Cy7-Gd), and showed that these conjugates can be introduced efficiently into cells. To examine the relationship between cell permeability and dye structure, we further synthesized a series of Cy7-Gd derivatives. On the basis of MR imaging, flow cytometry, and ICP-MS analysis of cells loaded with Cy7-Gd derivatives, highly hydrophobic and nonanionic dyes were effective for enhancing cell permeation of Gd 3+ complexes. Furthermore, the behavior of these Cy7-Gd derivatives was examined in mice. Thus, conjugation of hydrophobic fluorescent dyes appears to be an effective approach to improve the cell permeability of Gd 3+ complexes, and should be applicable for further development of Gd 3+-based MRI contrast agents. © 2011 American Chemical Society.

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Development of a potassium ion-selective fluorescent sensor based on 3-styrylated BODIPY

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

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  21 ( 20 ) 6090 - 6093 2011.10

Research paper (scientific journal), Accepted,  ISSN  0960894X

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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.

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Rational design of boron dipyrromethene (BODIPY)-based photobleaching- resistant fluorophores applicable to a protein dynamics study

Komatsu T., Oushiki D., Takeda A., Miyamura M., Ueno T., Terai T., Hanaoka K., Urano Y., Mineno T., Nagano T.

Chemical Communications (Chemical Communications)  47 ( 36 ) 10055 - 10057 2011.09

Research paper (scientific journal), Accepted,  ISSN  13597345

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We studied the photobleaching of a library of boron dipyrromethene (BODIPY) derivatives with a range of electron densities, and found that the photobleaching rate is influenced by the electron-withdrawing capacity of the substituents. Electron-deficient BODIPYs generated less singlet oxygen, were less reactive to singlet oxygen, and were highly resistant to photobleaching. We confirmed the utility of one of these fluorophores, 2,6-diCO2R-BDP, for visualizing EGF receptor dynamics in cells expressing an SNAP-tagged EGF receptor. © The Royal Society of Chemistry 2011.

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Development of a far-red to near-infrared fluorescence probe for calcium ion and its application to multicolor neuronal imaging

Egawa T., Hanaoka K., Koide Y., Ujita S., Takahashi N., Ikegaya Y., Matsuki N., Terai T., Ueno T., Komatsu T., Nagano T.

Journal of the American Chemical Society (Journal of the American Chemical Society)  133 ( 36 ) 14157 - 14159 2011.09

Research paper (scientific journal), Accepted,  ISSN  00027863

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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.

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Development of a highly selective fluorescence probe for alkaline phosphatase

Kawaguchi M., Hanaoka K., Komatsu T., Terai T., Nagano T.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  21 ( 17 ) 5088 - 5091 2011.09

Research paper (scientific journal), Accepted,  ISSN  0960894X

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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.

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Fluorescence probe for lysophospholipase C/NPP6 activity and a potent NPP6 inhibitor

Kawaguchi M., Okabe T., Okudaira S., Hanaoka K., Fujikawa Y., Terai T., Komatsu T., Kojima H., Aoki J., Nagano T.

Journal of the American Chemical Society (Journal of the American Chemical Society)  133 ( 31 ) 12021 - 12030 2011.08

Research paper (scientific journal), Accepted,  ISSN  00027863

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Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that have a role in regulating extracellular nucleotide and phospholipid metabolism. Among the members of the NPP family, NPP1 and -3 act on nucleotides such as ATP, while NPP2, -6, and -7 act on phospholipids such as lysophosphatidylcholine and sphingomyelin. NPP6, a recently characterized NPP family member, is a choline-specific glycerophosphodiester phosphodiesterase, but its functions remain to be analyzed, partly due to the lack of highly sensitive activity assay systems and practical inhibitors. Here we report synthesis of novel NPP6 fluorescence probes, TG-mPC and its analogues TG-mPC 3C, TG-mPC 5C, TG-mPENE, TG-mPEA, TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr. Among the seven NPPs, only NPP6 hydrolyzed TG-mPC, TG-mPC 3C, and TG-mPENE. TG-mPC was hydrolyzed in the cell lysate from NPP6-transfected cells, but not control cells, showing that it is suitable for use in cell-based NPP6 assays. We also examined the usefulness of TG-mPC as a fluorescence imaging probe. We further applied TG-mPC to carry out high-throughput NPP6 inhibitor screening and found several NPP6-selective inhibitors in a library of about 80 000 compounds. Through structure-activity relationship (SAR) analysis, we identified a potent and selective NPP6 inhibitor with an IC 50 value of 0.21 μM. Our NPP6-selective fluorescence probe, TG-mPC, and the inhibitor are expected to be useful to elucidate the biological function of NPP6. © 2011 American Chemical Society.

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Development of a highly sensitive fluorescence probe for hydrogen peroxide

Abo M., Urano Y., Hanaoka K., Terai T., Komatsu T., Nagano T.

Journal of the American Chemical Society (Journal of the American Chemical Society)  133 ( 27 ) 10629 - 10637 2011.07

Research paper (scientific journal), Accepted,  ISSN  00027863

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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.

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Aminoluciferins as functional bioluminogenic substrates of firefly luciferase

Takakura H., Kojima R., Urano Y., Terai T., Hanaoka K., Nagano T.

Chemistry - An Asian Journal (Chemistry - An Asian Journal)  6 ( 7 ) 1800 - 1810 2011.07

Research paper (scientific journal), Accepted,  ISSN  18614728

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Firefly luciferase is widely used as a reporter gene in assays to study gene expression, gene delivery, and so on because of its extremely high signal-to-noise ratio. The availability of a range of bioluminogenic substrates would greatly extend the applicability of the luciferin-luciferase system. Herein, we describe a design concept for functional bioluminogenic substrates based on the aminoluciferin (AL) scaffold, together with a convenient, high-yield method for synthesizing N-alkylated ALs. We confirmed the usefulness of ALs as bioluminogenic substrates by synthesizing three probes. The first was a conjugate of AL with glutamate, Glu-AL. When Glu-AL, the first membrane-impermeable bioluminogenic substrate of luciferases, was applied to cells transfected with luciferase, luminescence was not observed; that is, by using Glu-AL, we can distinguish between intracellular and extracellular events. The second was Cy5-AL, which consisted of Cy5, a near-infrared (NIR) cyanine fluorescent dye, and AL, and emitted NIR light. When Cy5-AL reacted with luciferase, luminescence derived from Cy5 was observed as a result of bioluminescence resonance energy transfer (BRET) from AL to Cy5. The NIR emission wavelength would allow a signal to be observed from deeper tissues in bioluminescence in vivo imaging. The third was biotin-DEVD-AL (DEVD=the amino acid sequence Asp-Glu-Val-Asp), which employed a caspase-3 substrate peptide as a switch to control the accessibility of the substrate to luciferase, and could detect the activity of caspase-3 in a time-dependent manner. This generalized design strategy should be applicable to other proteases. Our results indicate that the AL scaffold is appropriate for a range of functional luminophores and represents a useful alternative substrate to luciferin. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Evolution of group 14 rhodamines as platforms for near-infrared fluorescence probes utilizing photoinduced electron transfer

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

ACS Chemical Biology (ACS Chemical Biology)  6 ( 6 ) 600 - 608 2011.06

Research paper (scientific journal), Accepted,  ISSN  15548929

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The absorption and emission wavelengths of group 14 pyronines and rhodamines, which contain silicon, germanium, or tin at the 10 position of the xanthene chromophore, showed large bathochromic shifts compared to the original rhodamines, owing to stabilization of the LUMO energy levels by σ *-π * conjugation between group 14 atom-C (methyl) σ * orbitals and a π * orbital of the fluorophore. These group 14 pyronines and rhodamines retain the advantages of the original rhodamines, including high quantum efficiency in aqueous media (φ fl = 0.3-0.45), tolerance to photobleaching, and high water solubility. Group 14 rhodamines have higher values of reduction potential than other NIR light-emitting original rhodamines, and therefore, we speculated their NIR fluorescence could be controlled through the photoinduced electron transfer (PeT) mechanism. Indeed, we found that the fluorescence quantum yield (φ fl) of Si-rhodamine (SiR) and Ge-rhodamine (GeR) could be made nearly equal to zero, and the threshold level for fluorescence on/off switching lies at around 1.3-1.5 V for the SiRs. This is about 0.1 V lower than in the case of TokyoGreens, in which the fluorophore is well established to be effective for PeT-based probes. That is to say, the fluorescence of SiR and GeR can be drastically activated by more than 100-fold through a PeT strategy. To confirm the validity of this strategy for developing NIR fluorescence probes, we employed this approach to design two kinds of novel fluorescence probes emitting in the far-red to NIR region, i.e., a series of pH-sensors for use in acidic environments and a Zn 2+ sensor. We synthesized these probes and confirmed that they work well. © 2011 American Chemical Society.

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Development of an Si-rhodamine-based far-red to near-infrared fluorescence probe selective for hypochlorous acid and its applications for biological imaging

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

Journal of the American Chemical Society (Journal of the American Chemical Society)  133 ( 15 ) 5680 - 5682 2011.04

Research paper (scientific journal), Accepted,  ISSN  00027863

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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.

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Positive correlation between the generation of reactive oxygen species and activation/reactivation of transgene expression after hydrodynamic injections into mice

Takiguchi N., Takahashi Y., Nishikawa M., Matsui Y., Fukuhara Y., Oushiki D., Kiyose K., Hanaoka K., Nagano T., Takakura Y.

Pharmaceutical Research (Pharmaceutical Research)  28 ( 4 ) 702 - 711 2011.04

Research paper (scientific journal), Accepted,  ISSN  07248741

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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.

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Development of a fluorescein analogue, TokyoMagenta, as a novel scaffold for fluorescence probes in red region

Egawa T., Koide Y., Hanaoka K., Komatsu T., CooTeraiper T., Nagano T.

Chemical Communications (Chemical Communications)  47 ( 14 ) 4162 - 4164 2011.03

Research paper (scientific journal), Accepted,  ISSN  13597345

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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.

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Rational design of ratiometric near-infrared fluorescent pH probes with various pK<inf>a</inf> values, based on aminocyanine

Myochin T., Kiyose K., Hanaoka K., Kojima H., Terai T., Nagano T.

Journal of the American Chemical Society (Journal of the American Chemical Society)  133 ( 10 ) 3401 - 3409 2011.03

Research paper (scientific journal), Accepted,  ISSN  00027863

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Novel ratiometric, near-infrared fluorescent pH probes with various pK a values have been designed and synthesized on the basis of aminocyanine bearing a diamine moiety, and their photochemical properties were evaluated. Under acidic conditions, these pH probes showed a 46- to 83-nm red shift of the absorption maximum. This change is sufficiently large to permit their use as ratiometric pH probes, and is reversible, whereas monoamine-substituted aminocyanines showed irreversible changes because of their instability under acidic conditions. Furthermore, the pKa values of these probes can be predicted from the calculated pKa values of the diamine moieties, obtained from the SciFinder database. This design strategy is very simple and flexible, and should be applicable to develop NIR pH probes for various applications. © 2011 American Chemical Society.

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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

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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

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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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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.

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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

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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

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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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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.

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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

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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

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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

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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

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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

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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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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.

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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

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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

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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

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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

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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

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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

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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

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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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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.

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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 2002.09

Research paper (scientific journal), Accepted,  ISSN  10745521

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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

Research paper (scientific journal), Accepted,  ISSN  03009580

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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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