Orthogonality of Calcium Concentration and Ability of 4,5-diaminofluorescein to Detect NO

The Journal of Biological Chemistry. Jan, 2002  |  Pubmed ID: 11641405

We have developed diaminofluoresceins (DAFs) and diaminorhodamines as fluorescent indicators for NO based on the specific reaction of the aromatic vicinal diamines with NO. Among them, 4,5-diaminofluorescein (DAF-2) is widely used for real-time biological imaging of NO in cultured cells or tissues by many researchers. Contrary to a recent report of divalent cation sensitivity and photoactivation of DAF-2 (Broillet, M. C., Randin, O., and Chatton, J. Y. (2001) FEBS Lett. 491, 227-232), our study using NO gas itself reveals that the reaction of DAF-2 and NO is completely independent of Ca2+ and Mg2+ at physiological concentrations. Ca2+ enhances not the conversion of DAF-2 into its fluorescent product (DAF-2 triazole) but the release of NO from NO donors. Therefore it is concluded that DAF-2 can provide reliable information on NO production in biological systems regardless of the dynamic changes of Ca2+ concentration.

Activation Mechanism of Gi and Go by Reactive Oxygen Species

The Journal of Biological Chemistry. Mar, 2002  |  Pubmed ID: 11781308

Reactive oxygen species are proposed to work as intracellular mediators. One of their target proteins is the alpha subunit of heterotrimeric GTP-binding proteins (Galpha(i) and Galpha(o)), leading to activation. H(2)O(2) is one of the reactive oxygen species and activates purified Galpha(i2). However, the activation requires the presence of Fe(2+), suggesting that H(2)O(2) is converted to more reactive species such as c*OH. The analysis with mass spectrometry shows that seven cysteine residues (Cys(66), Cys(112), Cys(140), Cys(255), Cys(287), Cys(326), and Cys(352)) of Galpha(i2) are modified by the treatment with *OH. Among these cysteine residues, Cys(66), Cys(112), Cys(140), Cys(255), and Cys(352) are not involved in *OH-induced activation of Galpha(i2). Although the modification of Cys(287) but not Cys(326) is required for subunit dissociation, the modification of both Cys(287) and Cys(326) is necessary for the activation of Galpha(i2) as determined by pertussis toxin-catalyzed ADP-ribosylation, conformation-dependent change of trypsin digestion pattern or guanosine 5'-3-O-(thio)triphosphate binding. Wild type Galpha(i2) but not Cys(287)- or Cys(326)-substituted mutants are activated by UV light, singlet oxygen, superoxide anion, and nitric oxide, indicating that these oxidative stresses activate Galpha(i2) by the mechanism similar to *OH-induced activation. Because Cys(287) exists only in G(i) family, this study explains the selective activation of G(i)/G(o) by oxidative stresses.

Design and Synthesis of an Enzyme-cleavable Sensor Molecule for Phosphodiesterase Activity Based on Fluorescence Resonance Energy Transfer

Journal of the American Chemical Society. Feb, 2002  |  Pubmed ID: 11853439

Ratiometric measurement is a technique that can provide precise data and even quantitative detection. To carry out ratiometric measurements, it is necessary that the sensor molecule exhibits a large shift in its emission or excitation spectrum after reaction with the target molecule. Fluorescence resonance energy transfer (FRET) is one mechanism used to obtain a large spectral shift. In this study, our aim was to develop a ratiometric fluorescent sensor molecule for phosphodiesterase activity based on FRET. We designed and synthesized CPF4 with a coumarin donor, a fluorescein acceptor, and two phenyl linkers having the phosphodiester moiety interposed between them. In the emission spectrum of CPF4 in aqueous buffer excited at 370 nm, the emission of the coumarin donor was strongly quenched and the emission of the fluorescein acceptor was observed. This emission spectrum demonstrates that energy transfer from the coumarin donor to the fluorescein acceptor proceeds efficiently. Addition of a phosphodiesterase to an aqueous solution of CPF4 resulted in an increase in the donor fluorescence and a decrease in the acceptor fluorescence. CPF4 exhibited a large shift in its emission spectrum after the hydrolysis of the phosphodiester group by the enzyme. This large shift of the emission spectrum indicates that ratiometric measurements can be made by using CPF4. The method described in this paper for designing enzyme-cleavable sensor molecules based on FRET should be readily applicable to other hydrolytic enzymes.

Impurity Profiling of Ephedrines in Methamphetamine by High-performance Liquid Chromatography

Journal of Chromatography. A. Feb, 2002  |  Pubmed ID: 11873994

Separation of the enantiomers and diastereomers of ephedrines was investigated for impurity profiling of methamphetamine. We describe a method for the analysis of (1S,2R)-(+)-ephedrine, (1R,2S)-(-)-ephedrine, (1S,2S)-(+)-pseudoephedrine and racemic methylephedrine in bulk methamphetamine by HPLC using two different columns: a phenyl-beta-cyclodextrin-type column and an ODS-type column. The analytes were detected by UV absorbance measurement at 210 nm. As little as 0.05% of each ephedrine in bulk methamphetamine could be determined. In the impurity profiling of methamphetamine, the identification of ephedrines may provide valuable information about the precursor. This method was confirmed to be sufficiently sensitive to identify trace amounts of (1R,2S)-(-)-ephedrine and (1S,2S)-(+)-pseudoephedrine in bulk methamphetamine synthesized by the Emde method.

Bioimaging of Nitric Oxide

Chemical Reviews. Apr, 2002  |  Pubmed ID: 11942795

A Fluorescent Anion Sensor That Works in Neutral Aqueous Solution for Bioanalytical Application

Journal of the American Chemical Society. Apr, 2002  |  Pubmed ID: 11942829

Anion recognition and anion sensing are of interest because anions play many important roles in living organisms. Most currently known anion sensors work only in organic solution, but sensors for biological applications are required to function in neutral aqueous solution. We have designed and synthesized a novel fluorescent sensor for anions. The sensor molecule 1-Cd(II) contains 7-amino-4-trifluoromethylcoumarin as a fluorescent reporter and Cd(II)-cyclen (1,4,7,10-tetraazacyclododecane) as an anion host. In neutral aqueous solution, Cd(II) of 1-Cd(II) is coordinated by the four nitrogen atoms of cyclen and the aromatic amino group of coumarin. When various anions are added to 100 mM HEPES buffer solution (pH 7.4) containing 1-Cd(II), the aromatic amino group of coumarin is displaced from Cd(II), causing a change of the excitation spectrum. While pyrophosphate and citrate were detected with high sensitivity, fluoride and perchlorate produced no response. Among organic anions, ATP and ADP gave strong signals, while cAMP showed little signal. By utilizing the different affinities of the sensor for AMP and cAMP, the activity of phosphodiesterase, which cleaves cyclic nucleotide, was monitored in real-time. The sensor should have many biochemical and analytical applications and the sensing principle should be widely applicable to the sensing of other molecules.

Hydrophobic Modifications at 1-phosphate of Inositol 1,4,5-trisphosphate Analogues Enhance Receptor Binding

Bioorganic & Medicinal Chemistry Letters. Mar, 2002  |  Pubmed ID: 11958992

Inositol 1,4,5-trisphosphate (IP(3)) analogues were synthesized in order to investigate the importance of the environment of 1-phosphate of IP(3) for strong binding to the IP(3) receptor. Our results show that hydrophobic modifications of the 1-phosphate moiety enhance the binding affinity, with considerable latitude of substituent structure.

Improvement and Biological Applications of Fluorescent Probes for Zinc, ZnAFs

Journal of the American Chemical Society. Jun, 2002  |  Pubmed ID: 12047174

The development and cellular applications of novel fluorescent probes for Zn2+, ZnAF-1F, and ZnAF-2F are described. Fluorescein is used as a fluorophore of ZnAFs, because its excitation and emission wavelengths are in the visible range, which minimizes cell damage and autofluorescence by excitation light. N,N-Bis(2-pyridylmethyl)ethylenediamine, used as an acceptor for Zn2+, is attached directly to the benzoic acid moiety of fluorescein, resulting in very low quantum yields of 0.004 for ZnAF-1F and 0.006 for ZnAF-2F under physiological conditions (pH 7.4) due to the photoinduced electron-transfer mechanism. Upon the addition of Zn2+, the fluorescence intensity is quickly increased up to 69-fold for ZnAF-1F and 60-fold for ZnAF-2F. Apparent dissociation constants (K(d)) are in the nanomolar range, which affords sufficient sensitivity for biological applications. ZnAFs do not fluoresce in the presence of other biologically important cations such as Ca2+ and Mg2+, and are insensitive to change of pH. The complexes with Zn2+ of previously developed ZnAFs, ZnAF-1, and ZnAF-2 decrease in fluorescence intensity below pH 7.0 owing to protonation of the phenolic hydroxyl group of fluorescein, whose pKa value is 6.2. On the other hand, the Zn2+ complexes of ZnAF-1F and ZnAF-2F emit stable fluorescence around neutral and slightly acidic conditions because the pKa values are shifted to 4.9 by substitution of electron-withdrawing fluorine at the ortho position of the phenolic hydroxyl group. For application to living cells, the diacetyl derivative of ZnAF-2F, ZnAF-2F DA, was synthesized. ZnAF-2F DA can permeate through the cell membrane, and is hydrolyzed by esterase in the cytosol to yield ZnAF-2F, which is retained in the cells. Using ZnAF-2F DA, we could measure the changes of intracellular Zn2+ in cultured cells and hippocampal slices.

Mossy Fiber Zn2+ Spillover Modulates Heterosynaptic N-methyl-D-aspartate Receptor Activity in Hippocampal CA3 Circuits

The Journal of Cell Biology. Jul, 2002  |  Pubmed ID: 12119362

Although Zn2+ is contained in large amounts in the synaptic terminals of hippocampal mossy fibers (MFs), its physiological role in synaptic transmission is poorly understood. By using the newly developed high-sensitivity Zn2+ indicator ZnAF-2, the spatiotemporal dynamics of Zn2+ was monitored in rat hippocampal slices. When high-frequency stimulation was delivered to the MFs, the concentration of extracellular Zn2+ was immediately elevated in the stratum lucidum, followed by a mild increase in the stratum radiatum adjacent to the stratum lucidum, but not in the distal area of stratum radiatum. The Zn2+ increase was insensitive to a non-N-methyl-d-aspartate (NMDA) receptor antagonist but was efficiently attenuated by tetrodotoxin or Ca2+-free medium, suggesting that Zn2+ is released by MF synaptic terminals in an activity-dependent manner, and thereafter diffuses extracellularly into the neighboring stratum radiatum. Electrophysiological analyses revealed that NMDA receptor-mediated synaptic responses in CA3 proximal stratum radiatum were inhibited in the immediate aftermath of MF activation and that this inhibition was no longer observed in the presence of a Zn2+-chelating agent. Thus, Zn2+ serves as a spatiotemporal mediator in imprinting the history of MF activity in contiguous hippocampal networks. We predict herein a novel form of metaplasticity, i.e., an experience-dependent non-Hebbian modulation of synaptic plasticity.

Multiple Active Intermediates in Oxidation Reaction Catalyzed by Synthetic Heme-thiolate Complex Relevant to Cytochrome P450

Journal of the American Chemical Society. Aug, 2002  |  Pubmed ID: 12167058

We have studied oxidation reactions using a synthetic heme-thiolate (SR complex) in order to ascertain the contributions of multiple intermediates derived from heme-thiolate to the oxygen atom transfer reaction to substrate. First, degradation of peroxyphenylacetic acid (PPAA) was examined in the presence of various substrates. The O-O bond cleavage mode of PPAA was clearly dependent on the reactivity of the substrate, and an easily oxidizable substrate enhanced heterolytic O-O bond cleavage. Second, competitive oxidations of cyclooctane and cyclooctene were carried out with various peroxybenzoic acids containing a series of substituents at the para-position as an oxygen source. The ratios of alkane hydroxylation rate/alkene epoxidation rate were dependent on the nature of the para-substituent of the oxidant. We conclude that substrate and oxidant interact with each other during the oxygen atom transfer reaction, that is, oxidation reaction occurs before O-O bond cleavage, even in the reaction catalyzed by heme-thiolate, which is considered to promote O-O bond cleavage. The results of an (18)O-incorporation study that is frequently performed to determine the active intermediates derived from iron porphyrins were consistent with this conclusion.

A Novel, Cell-permeable, Fluorescent Probe for Ratiometric Imaging of Zinc Ion

Journal of the American Chemical Society. Sep, 2002  |  Pubmed ID: 12207508

Zn(2+) plays important roles in various biological systems; as a result, the development of tools that can visualize chelatable Zn(2+) has attracted much attention recently. We report here newly synthesized fluorescent sensors for Zn(2+), ZnAF-Rs, whose excitation maximum is shifted by Zn(2+) under physiological conditions. Thus, these sensors enable ratiometric imaging, which is a technique to reduce artifacts by minimizing the influence of extraneous factors on the fluorescence of a probe. Ratiometric measurement can provide precise data, and some probes allow quantitative detection. ZnAF-Rs are the first ratiometric fluorescent sensors for Zn(2+) that enable quantitative analysis under physiological conditions. ZnAF-Rs also possess suitable K(d) for applications, and high selectivity against other biologically relevant cations, especially Ca(2+). Using these probes, changes of intracellular Zn(2+) concentration in cultured cells were monitored successfully. We believe that these probes will be extremely useful in studies on the biological functions of Zn(2+).

Design and Synthesis of a Novel Magnetic Resonance Imaging Contrast Agent for Selective Sensing of Zinc Ion

Chemistry & Biology. Sep, 2002  |  Pubmed ID: 12323377

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

Development of Novel Fluorescence Probes That Can Reliably Detect Reactive Oxygen Species and Distinguish Specific Species

The Journal of Biological Chemistry. Jan, 2003  |  Pubmed ID: 12419811

We designed and synthesized 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2- [6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) as novel fluorescence probes to detect selectively highly reactive oxygen species (hROS) such as hydroxyl radical (*OH) and reactive intermediates of peroxidase. Although HPF and APF themselves scarcely fluoresced, APF selectively and dose-dependently afforded a strongly fluorescent compound, fluorescein, upon reaction with hROS and hypochlorite ((-)OCl), but not other reactive oxygen species (ROS). HPF similarly afforded fluorescein upon reaction with hROS only. Therefore, not only can hROS be differentiated from hydrogen peroxide (H(2)O(2)), nitric oxide (NO), and superoxide (O2*-) by using HPF or APF alone, but (-)OCl can also be specifically detected by using HPF and APF together. Furthermore, we applied HPF and APF to living cells and found that HPF and APF were resistant to light-induced autoxidation, unlike 2',7'-dichlorodihydrofluorescein, and for the first time we could visualize (-)OCl generated in stimulated neutrophils. HPF and APF should be useful as tools to study the roles of hROS and (-)OCl in many biological and chemical applications.

Light-harvesting Ionic Dendrimer Porphyrins As New Photosensitizers for Photodynamic Therapy

Bioconjugate Chemistry. Jan-Feb, 2003  |  Pubmed ID: 12526693

Photodynamic therapy (PDT) is a promising therapeutic modality for treatment of solid tumors. In this study, third-generation aryl ether dendrimer porphyrins (DPs) with either 32 quaternary ammonium groups (32(+)DPZn) or 32 carboxylic groups (32(-)DPZn) were evaluated as a novel, supramolecular class of photosensitizers for PDT. DPs showed a different cell-association profile depending on the positive or negative charge on the periphery, and both DPs eventually localized in membrane-limited organelles. In contrast, protoporphyrin IX (PIX), which is a hydrophobic and relatively low molecular weight photosensitizer used as a control in this study, diffused through the cytoplasm except the nucleus. Confocal fluorescent imaging using organelle-specific dyes indicated that PIX induced severe photodamage to disrupt membranes and intracellular organelles, including the plasma membrane, mitochondrion, and lysosome. On the other hand, cells treated with DPs kept the characteristic fluorescent pattern of such organelles even after photoirradiation. However, notably 32(+)DPZn achieved remarkably higher (1)O(2)-induced cytotoxicity against LLC cells than PIX. Furthermore, both dendrimer porphyrins had far lower dark toxicity as compared with PIX, demonstrating their highly selective photosensitizing effect in combination with a reduced systemic toxicity.

Nitric Oxide (NO) Synthase MRNA Expression and NO Production Via Muscarinic Acetylcholine Receptor-mediated Pathways in the CEM, Human Leukemic T-cell Line

Life Sciences. Mar, 2003  |  Pubmed ID: 12628472

Nitric oxide (NO) is synthesized from L-arginine by neuronal, endothelial and inducible isoforms of NO synthase (nNOS, eNOS and iNOS, respectively) and is involved in the regulation of a variety of physiological functions, including immune activity. In vascular endothelial cells, stimulation of M(3) subtype of muscarinic acetylcholine receptors (mAChRs) triggers NO synthesis by eNOS. Human lymphocytes express several mAChR subtypes and their stimulation increases the intracellular free Ca(2+) concentration and up-regulates c-fos gene expression. While the above findings suggest involvement of the lymphocytic cholinergic system in the regulation of immune function, little is known on NOS expression and NO synthesis in T-lymphocytes. In the present study, using reverse transcription-polymerase chain reaction, we found that CEM cells express mRNAs encoding iNOS and nNOS, but not for eNOS. In addition, using quantitative fluorescence microscopy and a novel NO-sensitive fluorescent indicator, DAF-2, we found that oxotremorine-M (Oxo-M) (100 microM), a non-selective mAChR agonist, enhances NO production in the cells. This effect of Oxo-M was antagonized by pirenzepine (10 microM), an antagonist acting preferentially at M(1) mAChR and by atropine (10 microM). Also 4-DAMP (10 microM), an antagonist acting preferentially at M(3) mAChR, reduced significantly the effect of Oxo-M, while AFDX-116 (10 microM), an antagonist acting preferentially at M(2) mAChR, was ineffective. These findings suggest that T-lymphocytes express functional mAChRs linked to NO synthesis by nNOS and/or iNOS.

A Novel Design Method of Ratiometric Fluorescent Probes Based on Fluorescence Resonance Energy Transfer Switching by Spectral Overlap Integral

Chemistry (Weinheim an Der Bergstrasse, Germany). Apr, 2003  |  Pubmed ID: 12658644

A ratiometric measurement, namely, simultaneous recording of the fluorescence intensities at two wavelengths and calculation of their ratio, allows greater precision than measurements at a single wavelength, and is suitable for cellular imaging studies. Here we describe a novel method of designing probes for ratiometric measurement of hydrolytic enzyme activity based on switching of fluorescence resonance energy transfer (FRET). This method employs fluorescent probes with a 3'-O,6'-O-protected fluorescein acceptor linked to a coumarin donor through a linker moiety. As there is no spectral overlap integral between the coumarin emission and fluorescein absorption, the fluorescein moiety cannot accept the excitation energy of the donor moiety and the donor fluorescence can be observed. After cleavage of the protective groups by hydrolytic enzymes, the fluorescein moiety shows a strong absorption in the coumarin emission region, and then acceptor fluorescence due to FRET is observed. Based on this mechanism, we have developed novel ratiometric fluorescent probes (1-3) for protein tyrosine phosphatase (PTP) activity. They exhibit a large shift in their emission wavelength after reaction with PTPs. The fluorescence quenching problem that usually occurs with FRET probes is overcome by using the coumarin-cyclohexane-fluorescein FRET cassette moiety, in which close contact of the two dyes is hindered. After study of their chemical and kinetic properties, we have concluded that compounds 1 and 2 bearing a rigid cyclohexane linker are practically useful for the ratiometric measurement of PTPs activity. The design concept described in this paper, using FRET switching by spectral overlap integral and a rigid link that prevents close contact of the two dyes, should also be applicable to other hydrolytic enzymes by introducing other appropriate enzyme-cleavable groups into the fluorescein acceptor.

Attenuation of Neuropathic Pain by the Nociceptin/orphanin FQ Antagonist JTC-801 is Mediated by Inhibition of Nitric Oxide Production

The European Journal of Neuroscience. Apr, 2003  |  Pubmed ID: 12713641

At the spinal level, the involvement of nociceptin/orphanin FQ (N/OFQ) in pain transmission is controversial. JTC-801, a selective nonpeptidergic N/OFQ antagonist, is a good tool to examine the involvement of endogenous N/OFQ in pathophysiological conditions. In the present study, we studied the effect of JTC-801 on neuropathic pain induced by L5 spinal nerve transection in mice. Thermal hyperalgesia was evident on day 3 postsurgery and maintained during the 10-day experimental period. Oral administration of JTC-801 relieved the thermal hyperalgesia in neuropathic mice in a dose-dependent manner. Following L5 nerve transection, the increase in nitric oxide synthase (NOS) activity was observed in the superficial layer of dorsal horn and around the central canal in the spinal cord by NADPH diaphorase histochemistry. Using the novel fluorescent nitric oxide (NO) detection dye diaminofluorescein-FM, we confirmed that NO production increased in the spinal slice prepared from neuropathic mice and that the increase was more prominent in the ipsilateral side to the nerve transection than in the contralateral side. These increases in NOS activity and NO production in neuropathic mice were blocked by pretreatment of oral JTC-801. Although intraperitoneal injection of the nonselective NOS inhibitor NG.-nitro-L-arginine methyl ester transiently, but significantly, attenuated neuropathic hyperalgesia, inducible NOS-deficient mice showed neuropathic pain after L5 spinal nerve transection. These results suggest that N/OFQ is involved in the maintenance of neuropathic pain and that the analgesic effect of JTC-801 on neuropathic pain is mediated by inhibition of NO production by neuronal NOS.

[Statistics on Operations at the Department of Urology, Nippon Telegraph and Telephone West Osaka Hospital During a Ten-year Period from 1992 to 2001]

Hinyokika Kiyo. Acta Urologica Japonica. Mar, 2003  |  Pubmed ID: 12728535

A 10-year clinical statistic survey was made on the operations performed at the department of urology, NTT West Osaka Hospital between 1992 and 2001. The total number of operations was 2,540, comprising 1,899 males and 641 females, and a total of 1,559 ESWL was performed. The number of operations per year was chronologically constant. Major operations were transurethral resection of prostate (299 cases). The number of operations for malignant tumors, for examples radical nephrectomy and enucleation for renal cancer, nephroureterectomy for upper urinary tract cancer and transurethral resection of bladder tumor, has increased since 1997 year after year. The number of ESWL has decreased since 1994. Renal transplantation was introduced in 1995, and laparoscopic adrenalectomy also in 1997.

Production of Singlet Oxygen on Irradiation of a Photodynamic Therapy Agent, Zinc-coproporphyrin III, with Low Host Toxicity

Biometals : an International Journal on the Role of Metal Ions in Biology, Biochemistry, and Medicine. Dec, 2003  |  Pubmed ID: 12779244

Zinc-coproporphyrin III (Zincphyrin) acts efficiently as a photodynamic therapy (PDT) agent in mice, while it shows no tumor cell-killing activity in vitro and has a high LD50 (low toxicity) in mice. It appears to have advantages over other porphyrins as a practical PDT reagent. In order to examine the action mechanism of Zincphyrin in PDT, we evaluated the photochemical characteristics of Zincphyrin by measurement of the near-infrared emission at 1268 nm, which provides direct evidence for formation of 1O2. Intense emission was observed in the presence of Zincphyrin, and was completely inhibited by NaN3, a 1O2 scavenger. Based on a quenching study, the rate constant of the reaction of 1O2 with NaN3 was determined to be 1.5-3.5 M(-1) s(-1), which is close to the reported value (3.8 x 10(8) M(-1) s(-1)). The intensity of the 1O2-specific emission was proportional to both the laser power and the concentration of Zincphyrin. The fluorescence quantum yield of Zincphyrin was 0.004 in phosphate buffer (100 mM, pH 7.4), which indicates that the excited state decays via other pathway(s) faster than through the fluorescence emission pathway. The lifetime of the triplet state of Zincphyrin (210 micros) was relatively long compared to that of other porphyrins, such as hematoporphyrin (Hp) (40 micros), coproporphyrin I (50 gs), or coproporphyrin III (36 gs). These results demonstrate the photodynamic generation of 1O2 by Zincphyrin.

Spatiotemporal Laser Inactivation of Inositol 1,4,5-trisphosphate Receptors Using Synthetic Small-molecule Probes

Chemistry & Biology. Jun, 2003  |  Pubmed ID: 12837383

A malachite green-conjugated inositol 1,4,5-trisphosphate (MGIP(3)) induces specific inactivation of IP(3) receptor (IP(3)R) in tissue samples upon laser irradiation. To verify potential usefulness of the method for studies of cellular Ca(2+) signaling, we conducted laser inactivation at the single-cell level and show that IP(3)R was inactivated with extremely high spatiotemporal resolution. In the presence of MGIP(3), the Ca(2+) release function of IP(3)R in single B lymphoma cells decayed exponentially with increasing duration of laser irradiation with a time constant of 3.4 s. Moreover, by confining laser irradiation to a spatially distinct region of differentiated PC12 cells, subcellular inactivation of IP(3)R was attained, as revealed by a loss of local Ca(2+) signal. Such real-time inactivation of IP(3)R only within a subcellular region may provide a powerful method for investigating spatiotemporal dynamics of Ca(2+) signaling.

Rational Design Principle for Modulating Fluorescence Properties of Fluorescein-based Probes by Photoinduced Electron Transfer

Journal of the American Chemical Society. Jul, 2003  |  Pubmed ID: 12848574

Fluorescence properties of fluorescein-based probes are shown to be finely controlled by the rate of photoinduced electron transfer from the benzoic acid moiety (electron donor) to the singlet excited state of the xanthene moiety (electron acceptor fluorophore). The occurrence of photoinduced electron transfer is clearly evidenced by transient absorption spectra showing bands due to the radical cation of the electron donor moiety and the radical anion of the xanthene moiety, observed in laser flash photolysis experiments. The photoinduced electron transfer rates and the rates of back electron transfer follow the Marcus parabolic dependence of electron transfer rate on the driving force. Such a dependence provides for the first time a quantitative basis for a rational design principle which has high efficiency in modulating fluorescence properties of fluorescein-based probes.

Selective Detection of Zinc Ions with Novel Luminescent Lanthanide Probes

Angewandte Chemie (International Ed. in English). Jul, 2003  |  Pubmed ID: 12851952

Development of Selective, Visible Light-excitable, Fluorescent Magnesium Ion Probes with a Novel Fluorescence Switching Mechanism

The Analyst. Jun, 2003  |  Pubmed ID: 12866894

Novel fluorescent Mg2+ probes, 2'-carboxyfluorescein (2'-CF) and its derivatives, that are excitable by visible light, were designed, synthesized and characterized. After complexation with Mg2+, the absorption and emission maxima of these fluorescent probes were red-shifted and the fluorescence intensity was increased 11-fold at neutral pH. The apparent dissociation constant (Kd) of the Mg2+-2'-CF complex was 15.8 mM at neutral pH. The Kd of the Ca2+-2'-CF complex was about 10 times larger than that of the Mg2+ complex and the other derivatives showed similar characteristics. In contrast, all the commercially available fluorescent Mg2+ probes have a higher affinity for Ca2+ than Mg2+. 2'-CF fluoresced in alkaline solution (pH > 8) and the pK, value was 8.8. The pKa value of the Mg2+-2'-CF complex was 6.8, and the fluorescence intensity was increased in the neutral conditions. Thus, the addition of Mg2+ resulted in a lowering of the pKa, and also an increase of the fluorescence intensity.

Active Oxygen Species Generated from Photoexcited Fullerene (C60) As Potential Medicines: O2-* Versus 1O2

Journal of the American Chemical Society. Oct, 2003  |  Pubmed ID: 14558828

To characterize fullerenes (C(60) and C(70)) as photosensitizers in biological systems, the generation of active oxygen species, through energy transfer (singlet oxygen (1)O(2)) and electron transfer (reduced active oxygen radicals such as superoxide anion radical O(2)(-)* and hydroxyl radical *OH), was studied by a combination of methods, including biochemical (DNA-cleavage assay in the presence of various scavengers of active oxygen species), physicochemical (EPR radical trapping and near-infrared spectrometry), and chemical methods (nitro blue tetrazolium (NBT) method). Whereas (1)O(2) was generated effectively by photoexcited C(60) in nonpolar solvents such as benzene and benzonitrile, we found that O(2)(-)* and *OH were produced instead of (1)O(2) in polar solvents such as water, especially in the presence of a physiological concentration of reductants including NADH. The above results, together with those of a DNA cleavage assay in the presence of various scavengers of specific active oxygen species, indicate that the active oxygen species primarily responsible for photoinduced DNA cleavage by C(60) under physiological conditions are reduced species such as O(2)(-)* and *OH.

Highly Sensitive Fluorescence Probes for Nitric Oxide Based on Boron Dipyrromethene Chromophore-rational Design of Potentially Useful Bioimaging Fluorescence Probe

Journal of the American Chemical Society. Mar, 2004  |  Pubmed ID: 15012166

Boron dipyrromethene (BODIPY) is known to have a high quantum yield (phi) of fluorescence in aqueous solution but has not been utilized much for biological applications, compared to fluorescein. We developed 8-(3,4-diaminophenyl)-2,6-bis(2-carboxyethyl)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (DAMBO-P(H)), based on the BODIPY chromophore, as a highly sensitive fluorescence probe for nitric oxide (NO). DAMBO-P(H) had a low phi value of 0.002, whereas its triazole derivative (DAMBO-P(H)-T), the product of the reaction of DAMBO-P(H) with NO, fluoresced strongly (phi = 0.74). The change of the fluorescence intensity was found to be controlled by an intramolecular photoinduced electron transfer (PeT) mechanism. The strategy for development of DAMBO-P(H) was as follows: (1) in order to design a highly sensitive probe of NO, the reactivity of o-phenylenediamine derivatives as NO-reactive moieties was examined using 4,5-diaminofluorescein (DAF-2, a widely used NO fluorescence probe), (2) in order to avoid pH-dependency of the fluorescence intensity, the PeT process was controlled by modulating the spectroscopic and electrochemical properties of BODIPY chromophores according to the Rehm-Weller equation based on measurement of excitation energies of chromophores, ground-state reduction potentials of PeT acceptors (BODIPYs), and calculation of the HOMO energy level of the PeT donor (o-phenylenediamine moiety) at the B3LYP/6-31G level, (3) in order to avoid quenching of fluorescence by stacking of the probes and to obtain probes suitable for biological applications, hydrophilic functional groups were introduced. This strategy should be applicable for the rational design of other novel and potentially useful bioimaging fluorescence probes.

Zinc Sensing for Cellular Application

Current Opinion in Chemical Biology. Apr, 2004  |  Pubmed ID: 15062780

Numerous tools for Zn2+ sensing in living cells have become available in the past three years. Among them, fluorescence imaging using fluorescent sensor molecules has been the most popular approach. Some of these sensor molecules can be used to visualize Zn2+ in living cells. Some of the biological functions of Zn2+ have been clarified using these sensor molecules, especially in neuronal cells, which contain a high concentration of free Zn2+.

Mitochondrial Signal Lacking Manganese Superoxide Dismutase Failed to Prevent Cell Death by Reoxygenation Following Hypoxia in a Human Pancreatic Cancer Cell Line, KP4

Antioxidants & Redox Signaling. Jun, 2004  |  Pubmed ID: 15130279

One of the major characteristics of tumor is the presence of a hypoxic cell population, which is caused by abnormal distribution of blood vessels. Manganese superoxide dismutase (MnSOD) is a nuclear-encoded mitochondrial enzyme, which scavenges superoxide generated from the electron-transport chain in mitochondria. We examined whether MnSOD protects against hypoxia/reoxygenation (H/R)-induced oxidative stress using a human pancreas carcinoma-originated cell line, KP4. We also examined whether MnSOD is necessarily present in mitochondria to have a function. Normal human MnSOD and MnSOD without a mitochondrial targeting signal were transfected to KP4 cells, and reactive oxygen species, nitric oxide, lipid peroxidation, and apoptosis were examined as a function of time in air following 1 day of hypoxia as a H/R model. Our results showed H/R caused no increase in nitric oxide, but resulted in increases in reactive oxygen species, 4-hydroxy-2-nonenal protein adducts, and apoptosis. Authentic MnSOD protected against these processes and cell death, but MnSOD lacking a mitochondrial targeting signal could not. These results suggest that only when MnSOD is located in mitochondria is it efficient in protecting against cellular injuries by H/R, and they also indicate that mitochondria are primary sites of H/R-induced cellular oxidative injuries.

A Novel Fluorescent Probe for Zinc Ion Based on Boron Dipyrromethene (BODIPY) Chromophore

Chemical & Pharmaceutical Bulletin. Jun, 2004  |  Pubmed ID: 15187390

ZnAB has the combined structure of N,N-bis(2-pyridylmethyl)ethylenediamine as a specific chelater for Zn(2+) and 1,3,5,7-tetramethyl-8-phenyl-boron dipyrromethene as a fluorophore. Complexation of ZnAB with Zn(2+) produces a remarkable enhancement of fluorescence intensity. ZnAB has the advantages of less sensitivity to solvent polarity and pH than fluorescein-based Zn(2+) probes. Furthermore, it is not influenced by other cations, such as Na(+), K(+), Ca(2+), and Mg(2+), which exist at high concentrations under physiological conditions, even at 2.5 mM. The results show that ZnAB is a Zn(2+) probe suitable for biological applications.

Determination of Origin of Ephedrine Used As Precursor for Illicit Methamphetamine by Carbon and Nitrogen Stable Isotope Ratio Analysis

Analytical Chemistry. Jul, 2004  |  Pubmed ID: 15253668

The sale of ephedrine, one of the precursors of methamphetamine, is strictly controlled and monitored in various countries to prevent the production of illicit methamphetamine. There are three kinds of production scheme for ephedrine manufacture, and it is very useful for precursor control to investigate the origin of ephedrine used for the synthesis of illicit methamphetamine. By means of stable isotope ratio mass spectrometry (IR-MS), we investigated the origin of ephedrine based on the delta(13)C and delta(15)N values. The various origins of ephedrine (biosynthetic, semisynthetic, or synthetic) could be discriminated clearly by using these values. The delta(15)N values of synthetic ephedrine were more negative than those of ephedrine from other sources. By the repeated distillation of methylamine in our laboratory, we confirmed that this could be due to isotope separation during distillation for the purification of methylamine used for ephedrine synthesis. The values for ephedrine used as the precursor were well-correlated with those for methamphetamine synthesized from it. This drug characterization analysis should be useful to illuminate the origin of the precursors used for clandestine methamphetamine and to trace the diversion of medicinal ephedrine for illicit manufacture of methamphetamine.

AMP-activated Protein Kinase Inhibits Angiotensin II-stimulated Vascular Smooth Muscle Cell Proliferation

Circulation. Jul, 2004  |  Pubmed ID: 15262850

AMP-activated protein kinase (AMPK) is a stress-activated protein kinase that works as a metabolic sensor of cellular ATP levels. Here, we investigated whether AMPK signaling has a role in the regulation of the angiotensin II (Ang II)-induced proliferation signal in rat vascular smooth muscle cells (VSMCs).

Modification of Intracellular Ca2+ Dynamics by Laser Inactivation of Inositol 1,4,5-trisphosphate Receptor Using Membrane-permeant Probes

Chemistry & Biology. Aug, 2004  |  Pubmed ID: 15324806

A membrane-permeant malachite green-conjugated IP3 analog (MGIP3/PM) was synthesized as a probe for small molecule-based CALI (smCALI), and its effect on the Ca2+ signaling in intact DT40 chicken B cells was examined. In DT40 B cells treated with the smCALI probe, laser irradiation inhibited IP3-induced Ca2+ oscillations in response to B cell receptor stimulation, demonstrating that IP3R was acutely inactivated. We then applied smCALI to clarify the mechanism of capacitative Ca2+ entry (CCE), in which involvement of IP3R has been suggested. Despite the inactivation of IP3R by smCALI, thapsigargin-induced CCE remained unaffected, providing evidence that functional IP3R is not required for CCE in DT40 cells. These results demonstrate the potency of the smCALI technique for the study of the roles of IP3R in complex intracellular Ca2+ dynamics.

[Fluorescence Spectroscopy]

Tanpakushitsu Kakusan Koso. Protein, Nucleic Acid, Enzyme. Aug, 2004  |  Pubmed ID: 15377003

Development of a Zinc Ion-selective Luminescent Lanthanide Chemosensor for Biological Applications

Journal of the American Chemical Society. Oct, 2004  |  Pubmed ID: 15453781

Detection of chelatable zinc (Zn(2+)) in biological studies has attracted much attention recently, because chelatable Zn(2+) plays important roles in many biological systems. Lanthanide complexes (Eu(3+), Tb(3+), 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 Zn(2+). This europium (Eu(3+)) complex employs a quinolyl ligand as both a chromophore and an acceptor for Zn(2+). Upon addition of Zn(2+) to a solution of [Eu-7], the luminescence of Eu(3+) is strongly enhanced, with high selectivity for Zn(2+) 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 Zn(2+)-sensitive luminescent lamthanide sensors, whose excitation wavelength is at too high an energy level for biological applications. The usefulness of [Eu-7] for monitoring Zn(2+) changes in living HeLa cells was confirmed. This novel Zn(2+)-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.

Rational Principles for Modulating Fluorescence Properties of Fluorescein

Journal of the American Chemical Society. Nov, 2004  |  Pubmed ID: 15506772

Rational design strategies based on practical fluorescence modulation mechanisms would enable us to rapidly develop novel fluorescence probes for target molecules. Here, we present a practical and general principle for modulating the fluorescence properties of fluorescein. We hypothesized that (a) the fluorescein molecule can be divided into two moieties, i.e., the xanthene moiety as a fluorophore and the benzene moiety as a fluorescence-controlling moiety, even though there is no obvious linker structure between them, and (b) the fluorescence properties can be modulated via a photoinduced electron transfer (PeT) process from the excited fluorophore to a reducible benzene moiety (donor-excited PeT; d-PeT). To evaluate the relationship between the reduction potential of the benzene moiety and the fluorescence properties, we designed and synthesized various derivatives in which the reduction potential of the benzene moiety was fine tuned by introducing electron-withdrawing groups onto the benzene moiety. Our results clearly show that the fluorescence properties of fluorescein derivatives were indeed finely modulated depending upon the reduction potential of the benzene moiety. This information provides a basis for a practical strategy for rational design of novel functional fluorescence probes.

Characterization of N-methyl-D-aspartate Receptor Subunits Involved in Acute Ammonia Toxicity

Neurochemistry International. Jan, 2004  |  Pubmed ID: 12971910

Rapid administration of large doses of ammonia leads to death of animals, which is largely prevented by pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists. The present study focuses on a subunit(s) of NMDA receptor involved in ammonia-induced death by use of NMDA receptor GluRepsilon subunit-deficient (GluRepsilon(-/-)) mice and the selective GluRepsilon2 antagonist CP-101,606. Acute ammonia intoxication was induced in mice (eight per group) by a single intraperitoneal (i.p.) injection of ammonium chloride. Appearance of neurological deteriorations depended on the doses of ammonium chloride injected. While wild-type, GluRepsilon1(-/-), GluRepsilon4(-/-), and GluRepsilon1(-/-)/epsilon4(-/-) mice all died by ammonium chloride at 12 mmol/kg during the first tonic convulsions, two of eight GluRepsilon3(-/-) mice survived. Pretreatment of wild-type mice with CP-101,606 prevented two mice from ammonia-induced death. Pretreatment of GluRepsilon3(-/-) mice with CP-101,606 prevented the death of three mice and prolonged the time of death of non-survivors. Similarly, the neuronal form of nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI) as well as the nonselective NOS inhibitor L-NMMA, but not the inducible NOS inhibitor 1400W, partially prevented the death of mice and prolonged the period of death. Furthermore, ammonium chloride prolonged the increase in intracellular free Ca2+ concentration ([Ca2+]i) and subsequent NO production induced by NMDA in the cerebellum. These results suggest that activation of NMDA receptor containing GluRepsilon2 and GluRepsilon3 subunits and following activation of neuronal NOS are involved in acute ammonia intoxication which leads to death of animals.

Design and Synthesis of Zinc-selective Chelators for Extracellular Applications

Journal of the American Chemical Society. Jan, 2005  |  Pubmed ID: 15656603

Zinc (Zn2+) is found in every cell in human bodies. A few millimolar of free Zn2+ exists in the vesicles of presynaptic neurons in the mammalian brain and is released by synaptic activity or depolarization, modulating the function of certain ion channels and receptors. Although various chemical tools for measuring Zn2+ in biological samples, such as fluorescent probes for Zn2+, have been developed, Zn2+-selective chelators have room to be improved. Research on Zn2+ signals in the brain has traditionally employed several chelators, which have several shortcomings for biological applications. Here we report the design, synthesis, and properties of new membrane-impermeable chelators selective for Zn2+ and describe biological applications in hippocampal slices. As a result, our newly designed chelator revealed the first biological implication that presynaptic Zn2+ can be released in the CA1 region. This confirms the utility of these new chelatotrs as extracellular Zn2+ chelators for biological applications.

Enhanced Excitability of Hippocampal Mossy Fibers and CA3 Neurons Under Dietary Zinc Deficiency

Epilepsy Research. Feb, 2005  |  Pubmed ID: 15716032

On the basis of the evidence that susceptibility to kainate-induced seizures is enhanced by zinc deficiency and that glutamate concentrations in hippocampal extracellular fluid are excessively increased during seizures, excitability of hippocampal mossy fibers and CA3 neurons was examined using hippocampal slices, which were prepare from mice fed a zinc-deficient diet for 4 weeks. The spatio-temporal dynamics of zinc and calcium was monitored using their indicators, membrane-impermeable ZnAF-2 and membrane-permeable fura-2 AM, respectively. When the molecular layer of dentate gyrus was stimulated with 100mM KCl for 1s, the increased percentages of extracellular zinc in the stratum lucidum and CA3 pyramidal cell layer were higher in zinc-deficient mice than in the control mice, implying that glutamate release from the mossy fibers of the dentate granular cells is enhanced by zinc deficiency. Judging from the increased percentages, however, the amount of zinc released was estimated to be less in zinc-deficient mice. On the other hand, the basal calcium concentrations in the stratum lucidum and CA3 pyramidal cell layer detected with fura-2 were higher in zinc-deficient mice than in the control mice, indicating that hippocampal calcium homeostasis is affected by zinc deficiency. Furthermore, the increased percentage of intracellular calcium in the stratum lucidum by stimulation with high K+ was enhanced by the zinc deficiency. The alteration of hippocampal calcium homeostasis seems to enhance excitability of dentate granular cells in zinc deficiency, following by an enhanced excitability of postsynaptic structures in CA3 neurons.

Inhibition of Autotaxin by Lysophosphatidic Acid and Sphingosine 1-phosphate

The Journal of Biological Chemistry. Jun, 2005  |  Pubmed ID: 15769751

Autotaxin (ATX) or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is an NPP family member that promotes tumor cell motility, experimental metastasis, and angiogenesis. ATX primarily functions as a lysophospholipase D, generating the lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine. ATX uses a single catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters, but its regulation is not well understood. Using a new fluorescence resonance energy transfer-based phosphodiesterase sensor that reports ATX activity with high sensitivity, we show here that ATX is potently and specifically inhibited by LPA and sphingosine 1-phosphate (S1P) in a mixed-type manner (Ki approximately 10(-7) M). The homologous ecto-phosphodiesterase NPP1, which lacks lysophospholipase D activity, is insensitive to LPA and S1P. Our results suggest that, by repressing ATX activity, LPA can regulate its own biosynthesis in the extracellular environment, and they reveal a novel role for S1P as an inhibitor of ATX, in addition to its well established role as a receptor ligand.

Highly Sensitive Near-infrared Fluorescent Probes for Nitric Oxide and Their Application to Isolated Organs

Journal of the American Chemical Society. Mar, 2005  |  Pubmed ID: 15771488

Novel near-infrared (NIR) fluorescent probes for nitric oxide (NO) have been designed, synthesized, and evaluated. Their NIR fluorescence was increased in an NO concentration-dependent manner under physiological conditions, and their reaction efficiency with NO was at least 53 times higher than that of a widely used NO probe, DAF-2. They were confirmed to function in isolated intact rat kidneys. Because NIR light can penetrate deeply into tissues, these probes may have potential for in vivo NO imaging.

Evolution of Fluorescein As a Platform for Finely Tunable Fluorescence Probes

Journal of the American Chemical Society. Apr, 2005  |  Pubmed ID: 15796553

Fluorescence imaging is the most powerful technique currently available for continuous observation of dynamic intracellular processes in living cells. Suitable fluorescence probes are naturally of critical importance for fluorescence imaging, but only a very limited range of biomolecules can currently be visualized because of the lack of flexible design strategies for fluorescence probes. At present, design is largely empirical. Here we show that the carboxylic group of traditional fluorescein dyes, formerly considered indispensable, has been replaced with other substituents, affording various kinds of new fluoresceins. Further, by breaking out of the traditional structure of fluorescein, we developed the first and totally rational design strategy for novel fluorescence probes based on a strict photochemical basis. The value of this approach is exemplified by its application to develop a novel, highly sensitive, and membrane-permeable fluorescence probe for beta-galactosidase, which is the most widely used reporter enzyme.

Selective Zinc Sensor Molecules with Various Affinities for Zn2+, Revealing Dynamics and Regional Distribution of Synaptically Released Zn2+ in Hippocampal Slices

Journal of the American Chemical Society. Jul, 2005  |  Pubmed ID: 16028930

We have developed a series of fluorescent Zn(2+) sensor molecules with distinct affinities for Zn(2+), because biological Zn(2+) concentrations vary over a wide range from sub-nanomolar to millimolar. The new sensors have K(d) values in the range of 10(-8)-10(-4) M, compared with 2.7 nM for ZnAF-2. They do not fluoresce in the presence of other biologically important metal ions such as calcium or magnesium, and they can detect Zn(2+) within 100 ms. In cultured cells, the fluorescence intensity of ZnAF-2 was saturated at low Zn(2+) concentration, while that of ZnAF-3 (K(d) = 0.79 muM) was not saturated even at relatively high Zn(2+) concentrations. In hippocampal slices, we measured synaptic release of Zn(2+) in response to high-potassium-induced depolarization. ZnAF-2 showed similar levels of fluorescence increase in dentate gyrus (DG), CA3 and CA1, which were indistinguishable. However, ZnAF-3 showed a fluorescence increase only in DG. Thus, by using a combination of sensor molecules, it was demonstrated for the first time that a higher Zn(2+) concentration is released in DG than in CA3 or CA1 and that we can easily visualize Zn(2+) concentration over a wide range. We believe that the use of various combinations of ZnAF family members will offer unprecedented versatility for fluorescence-microscopic imaging of Zn(2+) in biological applications.

Extension of the Applicable Range of Fluorescein: a Fluorescein-based Probe for Western Blot Analysis

Angewandte Chemie (International Ed. in English). Aug, 2005  |  Pubmed ID: 16044483

[Analysis of Intracellular Membrane Protein Function by Small Molecule-based Chromophore-assisted Laser Inactivation]

Tanpakushitsu Kakusan Koso. Protein, Nucleic Acid, Enzyme. Aug, 2005  |  Pubmed ID: 16104611

Highly Efficient and Photostable Photosensitizer Based on BODIPY Chromophore

Journal of the American Chemical Society. Sep, 2005  |  Pubmed ID: 16131160

Photosensitizers are reagents that produce reactive oxygen species upon light illumination and are commonly used to study oxidative stress or for photodynamic therapy. There are many available photosensitizers, but most have limitations, such as low photostability, structural instability, or a limited usable range of solvent conditions. Here, we describe a novel photosensitizer scaffold (2I-BDP) based on the unique characteristics of the BODIPY chromophore (i.e., high extinction coefficient, high photostability, and insensitivity to solvent environment). 2I-BDP shows stronger near-infrared singlet oxygen luminescence emission and higher photostability than the well-known photosensitizer, Rose Bengal. Unlike other photosensitizers, this scaffold is widely applicable under various conditions, including lipophilic and aqueous environments. HeLa cells loaded with 2I-BDP could be photosensitized by light illumination, demonstrating that 2I-BDP is potentially useful as a reagent for cell photosensitization, oxidative stress studies, or PDT.

The Detection and Quantification of Highly Reactive Oxygen Species Using the Novel HPF Fluorescence Probe in a Rat Model of Focal Cerebral Ischemia

Neuroscience Research. Nov, 2005  |  Pubmed ID: 16168507

A novel fluorescence probe, 2-[6-(4'-hydroxy) phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (HPF) was used to investigate the generation of highly reactive oxygen species (hROS) under ischemia both in vitro and in vivo. In the in vitro study, HT 22 cells were used to demonstrate that was predominantly detected in the cytoplasm, which coincides with the location of the mitochondria and then its HPF fluorescence gradually increased from 6 to 24 h due to glutamate induced oxidative stress. In the in vivo study, the permanent and transient middle cerebral artery occlusion (MCAO) was induced in rats. Brain slices were incubated in an artificial medium containing HPF. The area of enhanced HPF fluorescence existed in both the ischemic core and the peri-infarct area at 4h after MCAO in both permanent and transient MCAO models. The area extended beyond the boundary of the ischemic damage into biochemically viable tissue. The enhanced fluorescent intensity following transient MCAO was higher than that observed in the permanent MCAO model. Hydroxyl radical scavenger, MCI-186 significantly suppressed the enhanced fluorescence intensity. This study demonstrated that HPF has a high sensitivity and specificity for the detection of hROS in focal cerebral ischemia as well as in a cellular model of oxidative stress.

Evidence for PH Dependent Zn2+influx in K562 Erythroleukemia Cells: Studies Using ZnAF-2F Fluorescence and 65Zn2+ Uptake

Archives of Biochemistry and Biophysics. Oct, 2005  |  Pubmed ID: 16183033

Using both ZnAF-2F (a Zn2+ specific fluorophore) and 65Zn2+, we determined the rate of transporter mediated Zn2+ influx (presumably mediated by the SLC39A1 gene product, protein name hZIP1) under steady state conditions and studied the effects of extracellular acidification. When K562 erythroleukemia cells were placed in Zn2+ containing buffers (1-60 microM), the initial rate of 65Zn2+ accumulation mirrored the apparent rise in free intracellular Zn2+ concentrations sensed by ZnAF-2F. Therefore, newly transported Zn2+ equilibrated with the free intracellular Zn2+ pool sensed by ZnAF-2F. A new steady state with elevated free intracellular Zn2+ was established after about 30 min. An estimate of 11 microM for the Km and 0.203 nmol/mg/s for the Vmax were obtained for Zn2+ influx. 65Zn2+ uptake and ZnAF-2F fluorescent changes were inhibited by extracellular acidification (range tested: pH 8-6, IC50 = pH 6.34). The IC50 for proton effects was close to the pKa for histidine, suggesting conserved histidine residues present in SLC39A1 play a critical role in Zn2+ influx and are involved in the pH effect.

Charge Separation in a Nonfluorescent Donor-acceptor Dyad Derived from Boron Dipyrromethene Dye, Leading to Photocurrent Generation

The Journal of Physical Chemistry. B. Aug, 2005  |  Pubmed ID: 16852949

Boron dipyrromethene (BODIPY), which is commonly used as an energy absorbing and transferring antenna molecule, has been modified to contain an electron donor moiety, 8-(2,4,5-trimethoxyphenyl)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (MEOPHBDP). The photoinduced electron transfer from a 2,4,5-trimethoxyphenyl moiety to a BODIPY moiety of MEOPHBDP in acetonitrile was observed by femtosecond laser flash photolysis measurements. The lifetime of the charge-separated state of MEOPHBDP was 59 ps at 298 K. The dye-sensitized solar cells (DSSC) were prepared using MEOPHBDP with carboxylic acid (MEOPHBDP-COOH) and a reference BODIPY dye having no electron donor moiety. The photovoltaic measurements were performed using a standard two-electrode system consisting of a working electrode and a Pt sputtered electrode in methoxyacetonitrile containing 0.5 M iodide and 0.05 M I(2). The photoelectrochemical properties of DSSC with MEOPHBDP are compared with those with a reference BODIPY dye.

Fluorescence-based Zinc Ion Sensor for Zinc Ion Release from Pancreatic Cells

Analytical Chemistry. Aug, 2006  |  Pubmed ID: 16906726

In this paper, we describe the synthesis and characterization of analytical properties of fluorescence-based zinc ion-sensing glass slides and their application in monitoring zinc ion release from beta pancreatic cells in cell cultures. To fabricate the sensors, the zinc ion indicator ZnAF-2 {6-[N-[N',N'-bis(2-pyridinylmethyl)-2-aminoethyl]amino-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one} was modified to include a sufficiently long linking aliphatic chain with a terminal carboxyl functional group. The recently synthesized ZnAF-2 zinc ion indicator provided high zinc ion selectivity in physiological solutions containing millimolar levels of calcium and other possible interfering cations. The carboxyl-modified ZnAF-2 was conjugated to the activated surface of glass slides, which then served as zinc ion sensors. It was possible to grow pancreatic cells directly on the zinc-sensing glass slide or on a membrane placed on these glass slides. The sensors were used to monitor zinc ion release events from glucose-stimulated pancreatic cells. The study showed that the zinc ion sensors responded effectively to the release of zinc ions from pancreatic cells at the nanomolar level with high selectivity and rapid subsecond response time.

Mechanism-based Molecular Design of Highly Selective Fluorescence Probes for Nitrative Stress

Journal of the American Chemical Society. Aug, 2006  |  Pubmed ID: 16910633

Nitrative stress is implicated in various pathogenic processes, including neurodegenerative disorders, but there is no practical fluorescence probe which can monitor the generation of nitrative stress with high selectivity. To design a suitable fluorescence probe, we have first focused on the fluorescence quenching mechanism of the nitro group, which has been believed to be a unique quencher of fluorescent dyes. We found that nitro group-based fluorescence quenching could be explained in terms of an electron transfer process, from the excited fluorophore to the electron-deficient aromatic nitro moiety. By utilizing this result, we succeeded in developing novel fluorogenic probes, NiSPYs, which can selectively monitor the generation of nitrative stress based on aromatic nitration. NiSPYs showed strong fluorescence enhancement upon the reaction with nitrating agents, including peroxynitrite, but showed little or no fluorescence augmentation in the presence of other reactive oxygen species. NiSPYs should be potentially useful as tools to study the role of nitrative stress in various biological applications.

Tunable Design Strategy for Fluorescence Probes Based on 4-substituted BODIPY Chromophore: Improvement of Highly Sensitive Fluorescence Probe for Nitric Oxide

Analytical and Bioanalytical Chemistry. Oct, 2006  |  Pubmed ID: 16924384

4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) is a well-known fluorophore, with a high molar extinction coefficient and high fluorescence quantum efficiency (Phi(fl)). Furthermore, its structure can be modified to change its excitation and emission wavelengths. However, little work has been done on the structural modification of fluorines at the B-4 position with other functional groups. We synthesized 4-methoxy-substituted BODIPY derivatives in satisfactory yields, and found that they exhibited improved solubility in aqueous solution. Moreover, their oxidation and reduction potentials were greatly decreased without any change in their absorbance and fluorescence properties. These features of 4-substituted BODIPYs may be useful for developing novel fluorescence probes based on the intramolecular photoinduced electron transfer (PeT) mechanism, because it is possible to optimize the PeT process precisely by modulating the electrochemical properties of the fluorophore. The value of this approach is exemplified by its application to the development of a highly sensitive and pH-independent fluorescence probe for nitric oxide.

[Development and Biological Applications of Various Bioimaging Probes]

Yakugaku Zasshi : Journal of the Pharmaceutical Society of Japan. Oct, 2006  |  Pubmed ID: 17016020

Fluorescence imaging is the most powerful technique currently available for continuous observation of dynamic intracellular processes in living cells. However, only a very limited range of biomolecules can be visualized because of the lack of flexible design strategies for fluorescence probes. In our laboratory, it was elucidated that fluorescein which has been widely employed as a core of fluorescence probes could be understood as a directly linked electron donor/fluorophore acceptor system. Fluorescence properties of fluorescein derivatives could be easily anticipated and modulated by controlling the rate of photoinduced electron transfer (PeT) from the donor moiety to the xanthene fluorophore. Further, we found that the opposite direction of PeT from the singlet excited fluorophore to the electron acceptor moiety could be occurred. More than twenty probes for imaging of nitric oxide, beta-galactosidase, highly reactive oxygen species, zinc ion et al. have been developed according to precise and rational design strategies based on PeT mechanism.

Design and Synthesis of an Enzyme Activity-based Labeling Molecule with Fluorescence Spectral Change

Journal of the American Chemical Society. Dec, 2006  |  Pubmed ID: 17165702

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 beta-galactosidase labeling probe, CMFbeta-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.

Creation of Superior Carboxyfluorescein Dyes by Blocking Donor-excited Photoinduced Electron Transfer

Organic Letters. Dec, 2006  |  Pubmed ID: 17165905

[Structure: see text] Carboxyfluoresceins are widely utilized as fluorescence labeling reagents, but we recently found that their emission intensity is markedly decreased after esterification. On the basis of our hypothesis that the fluorescence decrease is due to a donor-excited photoinduced electron transfer (d-PeT) process, we have developed novel carboxyfluorescein derivatives in which the d-PeT process is hampered, and the emission intensity is not decreased upon esterification. These novel dye derivatives display high quantum yields and are expected to be useful as labeling agents.

Inhibition of Presynaptic Activity by Zinc Released from Mossy Fiber Terminals During Tetanic Stimulation

Journal of Neuroscience Research. Jan, 2006  |  Pubmed ID: 16342122

Zinc exists in high densities in the giant boutons of hippocampal mossy fibers. On the basis of the evidence that zinc decreases extracellular glutamate concentration in the hippocampus, the presynaptic action of zinc released from mossy fibers during high-frequency (tetanic) stimulation was examined using hippocampal slices. The increase in zinc-specific fluorescent signals was observed in both extracellular and intracellular compartments in the mossy fiber terminals during the delivery of tetanic stimuli (100 Hz, 1 sec) to the dentate granule cell layer, suggesting that zinc released from mossy fibers is immediately retaken up by mossy fibers. When mossy fiber terminals were preferentially double-stained with zinc and calcium indicators and tetanic stimuli (100 Hz, 1 sec) were delivered to the dentate granule cell layer, the increase in calcium orange signal during the stimulation was enhanced in mossy fiber terminals by addition of CaEDTA, a membrane-impermeable zinc chelator, and was suppressed by addition of zinc. The decrease in FM4-64 signal (vesicular exocytosis) during tetanic stimulation (10 Hz, 180 sec), which induced mossy fiber long-term potentiation, was also enhanced in mossy fiber terminals by addition of CaEDTA and was suppressed by addition of zinc. The present study demonstrates that zinc released from mossy fibers may be a negative-feedback factor against presynaptic activity during tetanic stimulation.

Development of a Ratiometric Fluorescent Zinc Ion Probe in Near-infrared Region, Based on Tricarbocyanine Chromophore

Journal of the American Chemical Society. May, 2006  |  Pubmed ID: 16704241

Novel ratiometric fluorescent probes for Zn2+ in the near-infrared region, based on a tricarbocyanine chromophore, have been designed, synthesized, and evaluated. Upon addition of Zn2+, a 44 nm red shift of the absorption maximum was observed, which indicates that this probe could work as a ratiometric probe for Zn2+. This change is due to the difference in the electron-donating ability of the amine substituent before and after reaction with Zn2+. This fluorescence modulation of amine-substituted tricarbocyanines should be applicable to dual-wavelength measurement of various biomolecules or enzyme activities.

Modulation of Luminescence Intensity of Lanthanide Complexes by Photoinduced Electron Transfer and Its Application to a Long-lived Protease Probe

Journal of the American Chemical Society. May, 2006  |  Pubmed ID: 16719474

Luminescent lanthanide complexes (Tb(3+), Eu(3+), etc.) have excellent properties for biological applications, including extraordinarily long lifetimes and large Stokes shifts. However, there have been few reports of lanthanide-based functional probes, because of the difficulty in designing suitable complexes with a luminescent on/off switch. Here, we have synthesized a series of complexes which consist of three moieties: a lanthanide chelate, an antenna, and a luminescence off/on switch. The antenna is an aromatic ring which absorbs light and transmits its energy to the metal, and the switch is a benzene derivative with a different HOMO level. If the HOMO level is higher than a certain threshold, the complex emits no luminescence at all, which indicates that the lanthanide luminescence can be modulated by photoinduced electron transfer (PeT) from the switch to the sensitizer. This approach to control lanthanide luminescence makes possible the rational design of functional lanthanide complexes, in which the luminescence property is altered by a biological reaction. To exemplify the utility of our approach to the design of lanthanide complexes with a switch, we have developed a novel protease probe, which undergoes a significant change in luminescence intensity upon enzymatic cleavage of the substrate peptide. This probe, combined with time-resolved measurements, was confirmed in model experiments to be useful for the screening of inhibitors, as well as for clinical diagnosis.

[Chemical Biology: Overview]

Tanpakushitsu Kakusan Koso. Protein, Nucleic Acid, Enzyme. Oct, 2007  |  Pubmed ID: 18051372

[Development of Fluorescent Probes for Nitric Oxide]

Tanpakushitsu Kakusan Koso. Protein, Nucleic Acid, Enzyme. Oct, 2007  |  Pubmed ID: 18051375

[Mechanism-based Molecular Design of Highly Selective Peroxynitrite Probe]

Tanpakushitsu Kakusan Koso. Protein, Nucleic Acid, Enzyme. Oct, 2007  |  Pubmed ID: 18051376

[In-vivo Cancer Fluorescence Imaging with Novel Precisely-designed Fluorescence Probes]

Tanpakushitsu Kakusan Koso. Protein, Nucleic Acid, Enzyme. Oct, 2007  |  Pubmed ID: 18051385

[How to Develop Custom-designed Fluorescence Probes for Molecular Imaging]

Nihon Rinsho. Japanese Journal of Clinical Medicine. Feb, 2007  |  Pubmed ID: 17302268

Fluorescence imaging is the most powerful technique currently available for continuous observation of dynamic intracellular processes in living cells. However, only a very limited range of biomolecules can be visualized because of the lack of flexible design strategies for fluorescence probes. In our laboratory, it was elucidated that fluorescein which has been widely employed as a core of fluorescence probes could be understood as a directly linked electron donor/fluorophore acceptor system. Fluorescence properties of fluorescein derivatives could be easily anticipated and modulated by controlling the rate of photoinduced electron transfer (PeT) from the donor moiety to the xanthene fluorophore. Further, we found that the opposite direction of PeT from the singlet excited fluorophore to the electron acceptor moiety could be occurred. More than twenty probes for imaging of nitric oxide, beta-galactosidase, highly reactive oxygen species, zinc ion et al. have been developed according to precise and rational design strategies based on PeT mechanism.

Iron Hydroxide Nanoparticles Coated with Poly(ethylene Glycol)-poly(aspartic Acid) Block Copolymer As Novel Magnetic Resonance Contrast Agents for in Vivo Cancer Imaging

Colloids and Surfaces. B, Biointerfaces. Apr, 2007  |  Pubmed ID: 17324561

PEG-coated beta-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 beta-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.

An Enzymatically Activated Fluorescence Probe for Targeted Tumor Imaging

Journal of the American Chemical Society. Apr, 2007  |  Pubmed ID: 17352471

Beta-galactosidase is a widely used reporter enzyme, but although several substrates are available for in vitro detection, its application for in vivo optical imaging remains a challenge. To obtain a probe suitable for in vivo use, we modified our previously developed activatable fluorescence probe, TG-betaGal (J. Am. Chem. Soc. 2005, 127, 4888-4894), on the basis of photochemical and photophysical experiments. The new probe, AM-TG-betaGal, provides a dramatic fluorescence enhancement upon reaction with beta-galactosidase, and further hydrolysis of the ester moiety by ubiquitous intracellular esterases affords a hydrophilic product that is well retained within the cells without loss of fluorescence. We used a mouse tumor model to assess the practical utility of AM-TG-betaGal, after confirming that tumors in the model could be labeled with an avidin-beta-galactosidase conjugate. This conjugate was administered to the mice in vivo, followed by AM-TG-betaGal, and subsequent ex vivo fluorescence imaging clearly visualized intraperitoneal tumors as small as 200 microm. This strategy has potential clinical application, for example, in video-assisted laparoscopic tumor resection.

Design and Synthesis of a Library of BODIPY-based Environmental Polarity Sensors Utilizing Photoinduced Electron-transfer-controlled Fluorescence ON/OFF Switching

Journal of the American Chemical Society. May, 2007  |  Pubmed ID: 17425310

We systematically examined the mechanism of the solvent polarity dependence of the fluorescence ON/OFF threshold of the BODIPY (boron dipyrromethene) fluorophore and the role of photoinduced electron transfer (PeT). In a series of BODIPY derivatives with variously substituted benzene moieties at the 8-position, the oxidation potential of the benzene moiety became more positive and the reduction potential of the BODIPY fluorophore became more negative as the solvent polarity was decreased; consequently, the free energy change of PeT from the benzene moiety becomes larger in a more nonpolar environment. Utilizing this finding, we designed and synthesized a library of probes in which the threshold of fluorescence ON/OFF switching corresponds to different levels of solvent polarity. These environment-sensitive probes were used to examine bovine serum albumin (BSA) and living cells. The polarity at the surface of albumin was concluded to be similar to that of acetone, while the polarity of the internal membranes of HeLa cells was similar to that of dichloromethane.

Highly Activatable and Rapidly Releasable Caged Fluorescein Derivatives

Journal of the American Chemical Society. May, 2007  |  Pubmed ID: 17474746

Development of a Highly Specific Rhodamine-based Fluorescence Probe for Hypochlorous Acid and Its Application to Real-time Imaging of Phagocytosis

Journal of the American Chemical Society. Jun, 2007  |  Pubmed ID: 17506554

The tetramethylrhodamine (TMR) fluorophore is a useful platform for fluorescence probes, being applicable, for example, to biological investigations utilizing fluorescence microscopy, owing to its excellent photochemical properties in aqueous media. We have developed new TMR derivatives that show different dependences of their behavior upon the environment. Among them, HMTMR showed unique characteristics, and its putative spirocyclic structure was confirmed by X-ray crystallography. Utilizing this discovery, we have established a strategy to modulate the fluorescence of TMR by regulating the spirocyclization, and we have obtained a new fluorescence probe that can detect hypochlorous acid specifically. This probe, HySOx, can work in 99.9% aqueous solution at pH 7.4 and was confirmed to be able to detect hypochlorous acid being generated inside phagosomes in real time. HySOx is tolerant to autoxidation and photobleaching under bioimaging conditions. Regulation of the spirocyclization of rhodamines, as we describe here, provides a new approach to the rational development of novel fluorescence probes.

A Thiol-reactive Fluorescence Probe Based on Donor-excited Photoinduced Electron Transfer: Key Role of Ortho Substitution

Organic Letters. Aug, 2007  |  Pubmed ID: 17645349

We designed and synthesized a novel thiol-reactive fluorescence probe based on the BODIPY fluorophore. The fluorescence of this probe is strongly quenched by donor-excited photoinduced electron transfer (d-PeT) from BODIPY to maleimide, but after reaction with thiol, the fluorescence of BODIPY is restored, affording a 350-fold intensity increase.

Design and Synthesis of Fluorescent Probes for Selective Detection of Highly Reactive Oxygen Species in Mitochondria of Living Cells

Journal of the American Chemical Society. Aug, 2007  |  Pubmed ID: 17672465

Site-specific Oxidative Stress Induction

Chemistry & Biology. Aug, 2007  |  Pubmed ID: 17719486

In this issue of Chemistry & Biology, Kelly and colleagues describe the development of two novel ROS-generating compounds [1] that specifically localize in the nucleus or mitochondrion. Their application reveals that nuclei and mitochondria respond differently to oxidative stress, in terms of gene expression and survival pathway activation.

Development of an Iminocoumarin-based Zinc Sensor Suitable for Ratiometric Fluorescence Imaging of Neuronal Zinc

Journal of the American Chemical Society. Nov, 2007  |  Pubmed ID: 17927174

Ratiometric imaging is a technique to reduce artifacts by minimizing the influence of extraneous factors on the fluorescence of a sensor and is particularly useful for cellular imaging studies. Here we characterized the iminocoumarin fluorophore as a new scaffold for sensors for ratiometric imaging. The iminocoumarin 4 showed a high quantum yield in aqueous media on excitation in the visible wavelength region, while its coumarin analogue showed little fluorescence. We therefore developed a novel fluorescence probe, ZnIC, for ratiometric imaging of Zn2+, using iminocoumarin as a fluorophore and (ethylamino)dipicolylamine as a Zn2+ chelator. ZnIC exhibited almost the same fluorescence properties as 4, and the emission spectrum of this probe was red-shifted on addition of Zn2+ under physiological conditions. ZnIC is selective for Zn2+ over other biologically important metal ions, such as Ca2+ and Mg2+, and has high affinity for Zn2+. To confirm the suitability of ZnIC for biological applications, we employed it for the ratiometric detection of changes in intracellular Zn2+ in cultured cells and in rat hippocampal slices. The results indicate that iminocoumarin is a useful fluorophore for fluorescence microscopic imaging and that ZnIC should be useful for studies on the biological functions of Zn2+.

Time-resolved Long-lived Luminescence Imaging Method Employing Luminescent Lanthanide Probes with a New Microscopy System

Journal of the American Chemical Society. Nov, 2007  |  Pubmed ID: 17927176

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 (Eu3+ 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 Eu3+ 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.

A Gd3+-based Magnetic Resonance Imaging Contrast Agent Sensitive to Beta-galactosidase Activity Utilizing a Receptor-induced Magnetization Enhancement (RIME) Phenomenon

Chemistry (Weinheim an Der Bergstrasse, Germany). 2008  |  Pubmed ID: 17992679

Magnetic resonance imaging (MRI) permits noninvasive three-dimensional imaging of opaque organisms. Gadolinium (Gd(3+)) 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 r(1) 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 macromolecule or polymerization of the agent itself. Here we describe the design and synthesis of a novel beta-galactosidase-activated MRI contrast agent, the Gd(3+) complex [Gd-5], by using the RIME approach. beta-Galactosidase is commonly used as a marker gene to monitor gene expression. This newly synthesized compound exhibited a 57% increase in the r(1) relaxivity in phosphate-buffered saline (PBS) with 4.5% w/v human serum albumin (HSA) in the presence of beta-galactosidase. Detailed investigations revealed that RIME is the dominant factor in this increase of the observed r(1) relaxivity, based on analysis of Gd(3+) complexes [Gd-5] and [Gd-8], which is generated from [Gd-5] by the activity of beta-galactosidase, and spectroscopic analysis of their corresponding Tb(3+) complexes, [Tb-5] and [Tb-8].

Selective Photoinactivation of Protein Function Through Environment-sensitive Switching of Singlet Oxygen Generation by Photosensitizer

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2008  |  Pubmed ID: 18172220

Chromophore-assisted light inactivation is a promising technique to inactivate selected proteins with high spatial and temporal resolution in living cells, but its use has been limited because of the lack of a methodology to prevent nonspecific photodamage in the cell owing to reactive oxygen species generated by the photosensitizer. Here we present a design strategy for photosensitizers with an environment-sensitive off/on switch for singlet oxygen ((1)O(2)) generation, which is switched on by binding to the target, to improve the specificity of protein photoinactivation. (1)O(2) generation in the unbound state is quenched by photoinduced electron transfer, whereas (1)O(2) generation can occur in the hydrophobic environment provided by the target protein, after specific binding. Inositol 1,4,5-trisphosphate receptor, which has been suggested to have a hydrophobic pocket around the ligand binding site, was specifically inactivated by an environment-sensitive photosensitizer-conjugated inositol 1,4,5-trisphosphate receptor ligand without (1)O(2) generation in the cytosol of the target cells, despite light illumination, demonstrating the potential of environment-sensitive photosensitizers to allow high-resolution control of generation of reactive oxygen species in the cell.

Evaluation of Characteristic Deuterium Distributions of Ephedrines and Methamphetamines by NMR Spectroscopy for Drug Profiling

Analytical Chemistry. Feb, 2008  |  Pubmed ID: 18271510

We have established a method for quantitative analysis of the deuterium contents (D/H) at the phenyl, methine, benzyl, N-methyl and methyl groups of l-ephedrine/HCl, d-pseudoephedrine/HCl and methamphetamine/HCl by 2H NMR spectroscopy. Comparison of the 5 position-specific D/H values of l-ephedrine/HCl and d-pseudoephedrine/HCl prepared by three methods (chemical synthesis, semichemical synthesis, and biosynthesis) showed that chemically synthesized ephedrines and semisynthetic ephedrines have highly specific distributions of deuterium at the methine position and at the benzyl position, compared with the other positions. The classification of several methamphetamine samples seized in Japan in terms of the D/H values at these two positions clearly showed that the methamphetamine samples had been synthesized from ephedrines extracted from Ephedra plants or semisynthetic ephedrines but not from synthetic ephedrine. This isotope ratio analysis method should be useful to trace the origins of seized methamphetamine in Southeast Asia.

Angiotensin II and Tumor Necrosis Factor-alpha Synergistically Promote Monocyte Chemoattractant Protein-1 Expression: Roles of NF-kappaB, P38, and Reactive Oxygen Species

American Journal of Physiology. Heart and Circulatory Physiology. Jun, 2008  |  Pubmed ID: 18441197

We examined whether ANG II and TNF-alpha cooperatively induce vascular inflammation using the expression of monocyte chemoattractant protein (MCP)-1 as a marker of vascular inflammation. ANG II and TNF-alpha stimulated MCP-1 expression in a synergistic manner in vascular smooth muscle cells. ANG II-induced MCP-1 expression was potently inhibited to a nonstimulated basal level by blockade of the p38-dependent pathway but only partially inhibited by blockade of the NF-kappaB-dependent pathway. In contrast, TNF-alpha-induced MCP-1 expression was potently suppressed by blockade of NF-kappaB activation but only modestly suppressed by blockade of p38 activation. ANG II- and TNF-alpha-induced activation of NF-kappaB- and p38-dependent pathways was partially inhibited by pharmacological inhibitors of ROS production. Furthermore, ANG II- and TNF-alpha-stimulated MCP-1 expression was partially suppressed by ROS inhibitors. We also examined whether endogenous ANG II and TNF-alpha cooperatively promote vascular inflammation in vivo using a wire injury model of the rat femoral artery. Blockade of both ANG II and TNF-alpha further suppressed neointimal formation, macrophage infiltration, and MCP-1 expression in an additive manner compared with blockade of ANG II or TNF-alpha alone. These results suggested that ANG II and TNF-alpha synergistically stimulate MCP-1 expression via the utilization of distinct intracellular signaling pathways (p38- and NFkappaB-dependent pathways) and that these pathways are activated in ROS-dependent and -independent manners. These results also suggest that ANG II and TNF-alpha cooperatively stimulate vascular inflammation in vivo as well as in vitro.

Calcineurin is Critical for Sodium-induced Neointimal Formation in Normotensive and Hypertensive Rats

American Journal of Physiology. Heart and Circulatory Physiology. Jun, 2008  |  Pubmed ID: 18456738

It is well known that excessive intake of sodium chloride (sodium) is a risk factor for cardiovascular disease because it raises blood pressure. However, sodium loading reportedly promotes cardiovascular disease independently of its effect on blood pressure. To examine the mechanisms by which sodium loading promotes vascular inflammation independently of its effect on blood pressure, we examined the role of calcineurin in sodium loading-induced vascular inflammation using a wire injury model of the rat femoral artery. Calcineurin mRNA expression in the wire-injured femoral artery was significantly higher in sodium-loaded normotensive rats, such as Wistar-Kyoto (WKY) rats, than that in control WKY rats. Neointimal formation was also significantly enhanced in sodium-loaded WKY rats compared with control WKY rats. Gene transfer of an adenovirus expressing a dominant negative mutant of calcineurin (AdCalADeltaC92Q) significantly suppressed neointimal formation in sodium-loaded WKY rats to a level similar to that observed in control WKY rats. Calcineurin expression and neointimal formation were more significantly enhanced in hypertensive rats, such as spontaneously hypertensive rats (SHRs), than those in control WKY rats. AdCalADeltaC92Q infection significantly suppressed neointimal formation in SHRs to a level similar to that observed in control WKY rats. These results suggest that sodium loading promotes neointimal formation, even in normotensive rats, and that hypertension further stimulates neointimal formation. These results also suggest that calcineurin plays a pivotal role in this process.

Development of a Novel Fluorescent Probe for Fluorescence Correlation Spectroscopic Detection of Kinase Inhibitors

Bioorganic & Medicinal Chemistry Letters. Jul, 2008  |  Pubmed ID: 18524589

We have developed a fluorescently labeled probe for high-throughput screening of kinase inhibitors using fluorescence correlation spectroscopy. With this probe, we have successfully evaluated the inhibitory activities of known inhibitors of a model kinase, ASK1. Because the probe contains a general kinase inhibitor, staurosporine, we believe that this homogeneous, high-throughput, and simple method can be applied to the inhibitor screening of other kinases as well.

[Cellular Imaging by Using Molecular Probes]

Nihon Yakurigaku Zasshi. Folia Pharmacologica Japonica. Jul, 2008  |  Pubmed ID: 18622096

Blockade of Endogenous Proinflammatory Cytokines Ameliorates Endothelial Dysfunction in Obese Zucker Rats

Hypertension Research : Official Journal of the Japanese Society of Hypertension. Apr, 2008  |  Pubmed ID: 18633186

To study the role of endogenous proinflammatory cytokines in endothelial dysfunction in diabetes, we administered semapimod, an inhibitor of proinflammatory cytokine production, to obese Zucker (OZ) rats, and examined its effect on endothelium-dependent vasorelaxation. Endothelium-dependent vasorelaxation induced by acetylcholine and adrenomedullin (AM) was significantly reduced in OZ rats compared to a control group of lean Zucker rats. Semapimod significantly restored endothelium-dependent vasorelaxation in OZ rats. This effect of semapimod was well correlated with the reduction in the serum concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-6, and C-reactive protein, as well as with the recovery of AM-induced Akt phosphorylation and cGMP production. Furthermore, acute administration of TNF-alpha significantly suppressed endothelium-dependent vasorelaxation and AM-induced cGMP production. These results implicate endogenous proinflammatory cytokines, especially TNF-alpha, in endothelial dysfunction in diabetes, and indicate that blockade of these cytokines will be a promising strategy for inhibiting the progression of vascular inflammation.

Fluorescent Probes for Bioimaging Applications

Current Opinion in Chemical Biology. Oct, 2008  |  Pubmed ID: 18771748

Fluorescent probes based on small organic molecules have become indispensable tools in modern biology because they provide dynamic information concerning the localization and quantity of the molecules of interest, without the need of genetic engineering of the sample. In this review, following a brief outline of the principle of fluorescence imaging, we recount some recent achievements in the field of small-molecular fluorescent probes. First, probes for metal cations, including those suitable for two-photon imaging, are introduced. Next, methodologies to visualize proteases are discussed, with special emphasis on activity-based probes for use in vivo. All these probes have been confirmed to be applicable to cellular or in vivo imaging.

Design and Synthesis of Highly Sensitive Fluorogenic Substrates for Glutathione S-transferase and Application for Activity Imaging in Living Cells

Journal of the American Chemical Society. Nov, 2008  |  Pubmed ID: 18841967

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.

Selective Molecular Imaging of Viable Cancer Cells with PH-activatable Fluorescence Probes

Nature Medicine. Jan, 2009  |  Pubmed ID: 19029979

A long-term goal of cancer diagnosis is to develop tumor-imaging techniques that have sufficient specificity and sensitivity. To achieve this goal, minimizing the background signal originating from nontarget tissues is crucial. Here we achieve highly specific in vivo cancer visualization by using a newly designed targeted 'activatable' fluorescent imaging probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel acidic pH-activatable probes based on the boron-dipyrromethene fluorophore were synthesized and then conjugated to a cancer-targeting monoclonal antibody. As proof of concept, ex vivo and in vivo imaging of human epidermal growth factor receptor type 2-positive lung cancer cells in mice was performed. The probe was highly specific for tumors with minimal background signal. Furthermore, because the acidic pH in lysosomes is maintained by the energy-consuming proton pump, only viable cancer cells were successfully visualized. The design concept can be widely adapted to cancer-specific, cell surface-targeting molecules that result in cellular internalization.

A New Constitutively Active Mutant of AMP-activated Protein Kinase Inhibits Anoxia-induced Apoptosis of Vascular Endothelial Cell

Hypertension Research : Official Journal of the Japanese Society of Hypertension. Feb, 2009  |  Pubmed ID: 19262472

The inhibition of apoptotic changes in vascular endothelial cells is important for preventing vascular damage from hypoxia. AMP-activated protein kinase (AMPK) has recently been identified as playing a role in vascular protection. Although the chemical reagent 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) has been used to stimulate AMPK activity, AICAR has been associated with several nonspecific reactions. We therefore constructed a new constitutively active mutant of AMPK alpha 1 (NcaAMPK), which lacks the autoinhibitory domain in AMPK alpha 1 and in which threonine 172 has been replaced with aspartate. We investigated whether NcaAMPK has an anti-apoptotic effect in vascular endothelial cells under anoxic conditions. NcaAMPK, or green fluorescent protein (GFP) as a control, was overexpressed in human umbilical vein endothelial cells (HUVECs). After HUVECs were incubated for 40 h under normoxic or anoxic conditions, we examined cell viability, caspase 3/7 activity, and expression and phosphorylation levels of apoptosis-related proteins. Cell viabilities under anoxic conditions were improved in NcaAMPK-overexpressing cells. Anoxia increased caspase 3/7 activity, but NcaAMPK reduced this increase significantly. NcaAMPK overexpression increased protein kinase B/Akt Ser473 and endothelial nitric oxide synthase Ser1177 phosphorylation, but pretreatment with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) did not decrease the viability of NcaAMPK-overexpressing HUVECs. Furthermore, co-expression of a dominant-negative Akt reduced the improvement in cell viability and the suppression of poly (ADP-ribose) polymerase cleavage by NcaAMPK under anoxic conditions. In conclusion, NcaAMPK inhibited anoxia-induced apoptosis in vascular endothelial cells through Akt activation, suggesting that activation of AMPK might protect against ischemic vascular injury.

Design and Development of Enzymatically Activatable Photosensitizer Based on Unique Characteristics of Thiazole Orange

Journal of the American Chemical Society. May, 2009  |  Pubmed ID: 19364098

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 beta-galactosidase-activatable photosensitizer, PhoTO-Gal, in which fluorescence is simultaneously activated. PhoTO-Gal was demonstrated to kill HEK293 lacZ(+) cells, which express beta-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.

Use of Stable Isotope Ratios for Profiling of Industrial Ephedrine Samples: Application of Hydrogen Isotope Ratios in Combination with Carbon and Nitrogen

Forensic Science International. Aug, 2009  |  Pubmed ID: 19428199

The utility of hydrogen stable isotope ratio measurement by IR-MS for establishing the origin of ephedrine and pseudoephedrine (ephedrines), precursors of methamphetamine, was evaluated. There are two kinds of commercial semisynthetic ephedrines, one produced from molasses and the other from pyruvic acid. While the semisynthetic ephedrines derived from pyruvic acid cannot be discriminated from biosynthetic ephedrines and synthetic ephedrines based on delta(13)C and delta(15)N values, they could be identified from the delta(2)H values. The low deuterium content of biosynthetic ephedrines (delta(2)H: -193 to -151 per thousand) allows a clear distinction from synthetic ephedrines (delta(2)H: -73 to -30 per thousand), semisynthetic ephedrines derived from pyruvic acid (delta(2)H: +75 to +148 per thousand) and semisynthetic ephedrines derived from molasses (delta(2)H: -74 to +243 per thousand). The wide range of delta(2)H values of semisynthetic ephedrines is therefore very useful for the detailed classification of ephedrines, in combination with the measurement of delta(13)C and delta(15)N values as described in our previous work. This study was carried out on a limited number of samples reflecting the various routes of ephedrines manufacture. But it has become apparent that this stable-isotope analysis is an appropriate means by which to screen for manufacturing process of ephedrines. This approach should be useful for worldwide precursor control of methamphetamine.

Anion Sensor-based Ratiometric Peptide Probe for Protein Kinase Activity

Organic Letters. Jul, 2009  |  Pubmed ID: 19552460

A new fluorescent sensor consisting of Cd(II)-cylcen appended aminocoumarin and a substrate peptide for protein kinase A (PKA) has been designed. Upon phosphorylation by PKA, the metal complex moiety binds to a phosphorylated residue, which in turn displaces the coumarin fluorophore, and this event results in ratiometric change of excitation spectrum in neutral aqueous solution.

A Simple and Effective Strategy to Increase the Sensitivity of Fluorescence Probes in Living Cells

Journal of the American Chemical Society. Jul, 2009  |  Pubmed ID: 19572714

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, K(d). (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.

Molecular Design Strategies for Near-infrared Ratiometric Fluorescent Probes Based on the Unique Spectral Properties of Aminocyanines

Chemistry (Weinheim an Der Bergstrasse, Germany). Sep, 2009  |  Pubmed ID: 19650089

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 excitation energy. We have reported the unique spectral properties of amine-substituted 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-donating 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 energy-transfer efficiency through a change in the overlap integral that arises from the change in absorption under acidic conditions. These strategies were validated by obtaining tricarbocyanine-based ratiometric NIR fluorescent probes for esterase and for pH level.

Fluorescence Imaging of Tumors with "smart" PH-activatable Targeted Probes

Methods in Molecular Biology (Clifton, N.J.). 2009  |  Pubmed ID: 19685299

One goal of molecular imaging is to establish a widely applicable technique for specific detection of tumors with minimal background originated from non-target tissues. In this study, a "smart" activatable strategy for specific tumor imaging is proposed in which pH-activatable targeted probes specifically detect tumors after binding to the target cell surface proteins, internalization, and eventual acidic pH activation within the acidic organelles. We successfully visualized submillimeter-sized tumors using this strategy in two different tumor mouse models. Since the design of pH-activatable targeted probes can be applied to any target molecules on the cell surface that are to be internalized after ligand binding, this imaging strategy can afford a general and powerful method to diagnose and monitor the target tumors.

Squalene As a Target Molecule in Skin Hyperpigmentation Caused by Singlet Oxygen

Biological & Pharmaceutical Bulletin. Sep, 2009  |  Pubmed ID: 19721223

Based on our previous finding (Biochem. Biophys. Res. Commun., 223, 578-582, 1996) of singlet oxygen generation from coproporphyrin excreted on the skin surface from Propionibacterium acnes, we hypothesized that singlet oxygen formed in this way under UV exposure would promote peroxidation of skin surface lipids. We found that squalene was oxidized efficiently by singlet oxygen derived from coproporphyrin under UV exposure, and that the rate constant of squalene peroxidation by singlet oxygen was ten-fold higher than that of other skin surface lipids examined. The reaction was promoted more efficiently by UVA than by UVB. Furthermore, we found that topical application of squalene peroxide induced skin hyperpigmentation through increasing prostaglandin E(2) release from keratinocytes in guinea pigs. These results suggest that squalene peroxide formation by singlet oxygen plays a key role in photo-induced skin damage.

Bioimaging Probes for Reactive Oxygen Species and Reactive Nitrogen Species

Journal of Clinical Biochemistry and Nutrition. Sep, 2009  |  Pubmed ID: 19794917

Reactive oxygen species (ROS) play key roles in many pathogenic processes, including carcinogenesis, inflammation, ischemia-reperfusion injury and signal transduction. Also, reactive nitrogen species (RNS) cause various biological events such as neurodegenerative disorders. Sensitive and specific detection methods for ROS and RNS in biological samples should be useful for elucidation of biological events both in vitro and in vivo. Fluorescent probes based on small organic molecules have become indispensable tools in modern biology because they provide dynamic information concerning the localization and quantity of biological molecules of interest, without the need of genetic engineering of the sample. In this review, we recount some recent achievements in the field of small molecular fluorescent probes. First, the probes for nitric oxide and peroxynitrite as RNS are introduced and the probes of hydroxyl radical, hydrogen peroxide, hypochlorous and singlet oxygen as ROS are discussed, based on the fluorescence off/on switching mechanisms including photoinduced electron transfer and spirocyclization processes, and with some applications for in vitro and in vivo systems.

Development of 2,6-carboxy-substituted Boron Dipyrromethene (BODIPY) As a Novel Scaffold of Ratiometric Fluorescent Probes for Live Cell Imaging

Chemical Communications (Cambridge, England). Dec, 2009  |  Pubmed ID: 19904379

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.

[Integration Research of Structural Biology and Chemical Biology Focused on Drug Discovery]

Tanpakushitsu Kakusan Koso. Protein, Nucleic Acid, Enzyme. Sep, 2009  |  Pubmed ID: 21089565

Development of Luciferin Analogues Bearing an Amino Group and Their Application As BRET Donors

Chemistry, an Asian Journal. Sep, 2010  |  Pubmed ID: 20661993

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 coumarylluciferin derivative, coumarylaminoluciferin (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.

A Time-resolved Fluorescence Probe for Dipeptidyl Peptidase 4 and Its Application in Inhibitor Screening

Chemistry (Weinheim an Der Bergstrasse, Germany). Dec, 2010  |  Pubmed ID: 20938933

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.

Development of Fluorescent Probes for Bioimaging Applications

Proceedings of the Japan Academy. Series B, Physical and Biological Sciences. 2010  |  Pubmed ID: 20948177

Fluorescent probes, which allow visualization of cations such as Ca(2+), Zn(2+) etc., small biomolecules such as nitric oxide (NO) or enzyme activities in living cells by means of fluorescence microscopy, have become indispensable tools for clarifying functions in biological systems. This review deals with the general principles for the design of bioimaging fluorescent probes by modulating the fluorescence properties of fluorophores, employing mechanisms such as acceptor-excited Photoinduced electron Transfer (a-PeT), donor-excited Photoinduced electron Transfer (d-PeT), and spirocyclization, which have been established by our group. The a-PeT and d-PeT mechanisms are widely applicable for the design of bioimaging probes based on many fluorophores and the spirocyclization process is also expected to be useful as a fluorescence off/on switching mechanism. Fluorescence modulation mechanisms are essential for the rational design of novel fluorescence probes for target molecules. Based on these mechanisms, we have developed more than fifty bioimaging probes, of which fourteen are commercially available. The review also describes some applications of the probes developed by our group to in vitro and in vivo systems.

Hypoxia-sensitive Fluorescent Probes for in Vivo Real-time Fluorescence Imaging of Acute Ischemia

Journal of the American Chemical Society. Nov, 2010  |  Pubmed ID: 20979363

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.

Design and Synthesis of a Highly Sensitive Off-on Fluorescent Chemosensor for Zinc Ions Utilizing Internal Charge Transfer

Chemistry (Weinheim an Der Bergstrasse, Germany). Jan, 2010  |  Pubmed ID: 19918808

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 (Zn(2+)) is the second most abundant heavy metal ion in the human body after iron, and detection of chelatable Zn(2+) in biological studies has attracted much attention. Herein, we present a novel, highly sensitive off-on fluorescent chemosensor for Zn(2+) 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 Zn(2+), 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 Zn(2+). But even though the sensor shows a weak fluorescence in the absence of Zn(2+), this fluorescence can be further suppressed by selecting an excitation wavelength that is barely absorbed by the Zn(2+)-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 Zn(2+) 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 Zn(2+)-sensitive off-on fluorescent sensor molecule that employs the ICT mechanism; most off-on sensor molecules for Zn(2+) employ the photoinduced electron transfer (PeT) mechanism.

Adipose Tissue-derived Stem Cells Inhibit Neointimal Formation in a Paracrine Fashion in Rat Femoral Artery

American Journal of Physiology. Heart and Circulatory Physiology. Feb, 2010  |  Pubmed ID: 19940081

Subcutaneous adipose tissue contains a lot of stem cells [adipose-derived stem cells (ASCs)] that can differentiate into a variety of cell lineages. In this study, we isolated ASCs from Wistar rats and examined whether ASCs would efficiently differentiate into vascular endothelial cells (ECs) in vitro. We also administered ASCs in a wire injury model of rat femoral artery and examined their effects. ASCs expressed CD29 and CD90, but not CD34, suggesting that ASCs resemble bone marrow-derived mesenchymal stem cells. When induced to differentiate into ECs with endothelial growth medium (EGM), ASCs expressed Flt-1, but not Flk-1 or mature EC markers such as CD31 and vascular endothelial cadherin. ASCs produced angiopoietin-1 when they were cultured in EGM. ASCs stimulated the migration of EC, as assessed by chemotaxis assay. When ASCs that were cultured in EGM were injected in the femoral artery, the ASCs potently and significantly inhibited neointimal formation without being integrated in the endothelial layer. EGM-treated ASCs significantly suppressed neointimal formation even when they were administered from the adventitial side. ASC administration significantly promoted endothelial repair. These results suggested that although ASCs appear to have little capacity to differentiate into mature ECs, ASCs have the potential to secrete paracrine factors that stimulate endothelial repair. Our results also suggested that ASCs inhibited neointimal formation via their paracrine effect of stimulation of EC migration in situ rather than the direct integration into the endothelial layer.

Development and Application of a Near-infrared Fluorescence Probe for Oxidative Stress Based on Differential Reactivity of Linked Cyanine Dyes

Journal of the American Chemical Society. Mar, 2010  |  Pubmed ID: 20136129

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.

Sensitive Detection of Acrolein in Serum Using Time-resolved Luminescence

Organic Letters. Apr, 2010  |  Pubmed ID: 20232900

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.

A Fluorescence Correlation Spectroscopy-based Assay for Fragment Screening of Slowly Inhibiting Protein-peptide Interaction Inhibitors

Analytical Biochemistry. Jul, 2010  |  Pubmed ID: 20298671

A fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen fragment-size compounds that weakly and slowly inhibit protein-peptide interactions was established. The interactions were detected by the increased diffusion time of a fluorescently labeled peptide probe after binding to its interacting protein. We analyzed the interactions between the c-Cbl TKB domain and phosphopeptides derived from ZAP-70, APS, and EGFR with the FCS assay and obtained 6 hit fragments that bound to the c-Cbl interaction sites. The binding amounts of the fragments were measured by direct binding measurements using surface plasmon resonance, and 5 fragments were found to bind selectively. The effect of 2 of the 5 fragments on the interaction with c-Cbl and the peptide exhibited strong time dependency. Furthermore, the inhibition by the selected 5 fragments on the protein-peptide interaction was confirmed by their effect on pull-down assays of c-Cbl with the biotin-conjugated interaction peptides. These results indicate the advantage of our FCS-based assay to study the time-dependent binding of compounds to their target protein.

Establishment of an Indicator Cell System for Hepatitis C Virus

Microbiology and Immunology. Apr, 2010  |  Pubmed ID: 20377749

Although a cell culture system for HCV JFH-1 strain has been developed, no robust cell culture system for serum-derived HCV is available. In this study, we have established systems capable of monitoring infection with JFH-1 virus based on specific reporter gene expression through proteolysis of chimeric transcription factors by HCV NS3/4A protease. We utilized a transcriptional factor Gal4-TBP that synergistically enhances transcription of the GAL4UAS and HIV-1 LTR tandem promoter with the Tat protein. We constructed chimeric Tat and Gal4-TBP transcription factors containing the HCV NS3/4A cleavage sequence of a mitochondria-resident IPS-1, but not those of the HCV polyprotein, and manipulated them to localize in the ER. Upon infection with JFH-1 virus, the transcription factors were efficiently cleaved by HCV protease, migrated into the nucleus and activated the reporter gene under the tandem promoter. Upon infection with JFH-1 virus, the Huh7OK1/TG-Luc cell line carrying the transcription factors and a luciferase gene under the promoter expressed luciferase in a dose-dependent manner in close correlation with HCV RNA replication. Huh7OK1/TG-LNGFR cells carrying the transcription factors and a cDNA of human low affinity nerve growth factor receptor under the promoter were selectively concentrated by immunomagnetic cell sorting upon infection with JFH-1 virus. These results indicate that the chimeric constructs bearing the ER-resident IPS-1 sequence are specifically recognized and efficiently cleaved by HCV protease and are harnessed for detection of HCV replication and for recovery of HCV-infected cells. This strategy may be applicable for the establishment of cell culture systems for the isolation of serum-derived HCV.

Development of Enzyme-activated Photosensitizer Based on Intramolecular Electron Transfer

Bioorganic & Medicinal Chemistry Letters. Aug, 2010  |  Pubmed ID: 20615703

Photosensitizers produce cytotoxic reactive oxygen species (ROS) upon light illumination, but it is difficult to ablate cells of a specific type (e.g., tumor cells) in the presence of other cell populations, because of the limited precision with which light illumination can be directed to small areas. Here, we report a strategy to achieve cell type-specific ablation by using an enzyme-activated off/on switch for oxidative stress induction. In the unactivated photosensitizer, induction of oxidative stress is quenched by intramolecular electron transfer. However, the target cells express an enzyme that hydrolyzes a substrate moiety of the photosensitizer and the activated photosensitizer induces oxidative stress. As proof of concept, we designed and synthesized a xanthene-based photosensitizer, TGI-betaGal, whose oxidative stress induction ability is switched on following hydrolysis reaction with beta-galactosidase, a widely used gene marker. TGI-betaGal could selectively ablate lacZ-positive cells, whereas it showed no toxicity to lacZ-negative cells, upon light illumination.

Design and Synthesis of a Novel Fluorescence Probe for Zn2+ Based on the Spirolactam Ring-opening Process of Rhodamine Derivatives

Bioorganic & Medicinal Chemistry. Feb, 2011  |  Pubmed ID: 20620067

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 Zn(2+) sensor, and successfully developed a new fluorescence probe for Zn(2+).

Positive Correlation Between the Generation of Reactive Oxygen Species and Activation/reactivation of Transgene Expression After Hydrodynamic Injections into Mice

Pharmaceutical Research. Apr, 2011  |  Pubmed ID: 21116691

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.

Identification of Novel ASK1 Inhibitors Using Virtual Screening

Bioorganic & Medicinal Chemistry. Jan, 2011  |  Pubmed ID: 21134755

Apoptosis signal-regulating kinase 1 (ASK1, also called MAP3K5) is a mitogen-activated protein kinase kinase kinase (MAP3K) that plays important roles in stress-induced cell death and inflammation, and is expected as a new therapeutic target for cancer, cardiovascular diseases, and neurodegenerative diseases. We identified novel ASK1 inhibitors by virtual screening from the public chemical library collected by Chemical Biology Research Initiative (CBRI) at the University of Tokyo.

A Photocleavable Rapamycin Conjugate for Spatiotemporal Control of Small GTPase Activity

Journal of the American Chemical Society. Jan, 2011  |  Pubmed ID: 21142151

We developed a novel method to spatiotemporally control the activity of signaling molecules. A newly synthesized photocaged rapamycin derivative induced rapid dimerization of FKBP (FK-506 binding protein) and FRB (FKBP-rapamycin binding protein) upon UV irradiation. With this system and the spatially confined UV irradiation, we achieved subcellularly localized activation of Rac, a member of small GTPases. Our technique offers a powerful approach to studies of dynamic intracellular signaling events.

Phosphatidylinositol 4-kinase III Beta is a Target of Enviroxime-like Compounds for Antipoliovirus Activity

Journal of Virology. Mar, 2011  |  Pubmed ID: 21177810

Enviroxime is an antienterovirus compound that targets viral protein 3A and/or 3AB and suppresses a step in enterovirus replication by unknown mechanism. To date, four antienterovirus compounds, i.e., GW5074, Flt3 inhibitor II, TTP-8307, and AN-12-H5, are known to have similar mutations in the 3A protein-encoding region causing resistance to enviroxime (a G5318A [3A-Ala70Thr] mutation in poliovirus [PV]) and are considered enviroxime-like compounds. Recently, antienterovirus activity of a phosphatidylinositol 4-kinase III beta (PI4KB) inhibitor, PIK93, was reported, suggesting that PI4KB is an important host factor targetable by antienterovirus compounds (N. Y. Hsu et al., Cell 141:799-811, 2010). In this study, we analyzed the inhibitory effects of previously identified enviroxime-like compounds (GW5074 and AN-12-H5) and a newly identified antienterovirus compound, T-00127-HEV1, on phosphoinositide (PI) kinases. We found that T-00127-HEV1 inhibited PI4KB activity with a higher specificity for than other PI kinases, in contrast to GW5074, which had a broad specificity for PI kinases. In contrast, AN-12-H5 showed no inhibitory effect on PI4KB activity and only moderate inhibitory effects on PI 3-kinase activity. Small interfering RNA (siRNA) screening targeting PI kinases identified PI4KB is a target of GW5074 and T-00127-HEV1, but not of AN-12-H5, for anti-PV activity. Interestingly, T-00127-HEV1 and GW5074 did not inhibit hepatitis C virus (HCV) replication, in contrast to a strong inhibitory effect of AN-12-H5. These results suggested that PI4KB is an enterovirus-specific host factor required for the replication process and targeted by some enviroxime-like compounds (T-00127-HEV1 and GW5074) and that enviroxime-like compounds may have targets other than PI kinases for their antiviral effect.

A Dual Inhibitor Against Prolyl Isomerase Pin1 and Cyclophilin Discovered by a Novel Real-time Fluorescence Detection Method

Biochemical and Biophysical Research Communications. Mar, 2011  |  Pubmed ID: 21333629

Pin1, a peptidyl prolyl cis/trans isomerase (PPIase), is a potential target molecule for cancer, infectious disease, and Alzheimer's disease. We established a high-throughput screening method for Pin1 inhibitors, which employs a real-time fluorescence detector. This screening method identified 66 compounds that inhibit Pin1 out of 9756 compounds from structurally diverse chemical libraries. Further evaluations of surface plasmon resonance methods and a cell proliferation assay were performed. We discovered a cell-active inhibitor, TME-001 (2-(3-chloro-4-fluoro-phenyl)-isothiazol-3-one). Surprisingly, kinetic analyses revealed that TME-001 is the first compound that exhibits dual inhibition of Pin1 (IC₅₀=6.1 μM) and cyclophilin, another type of PPIase, (IC₅₀=13.7 μM). This compound does not inhibit FKBP. This finding suggests the existence of similarities of structure and reaction mechanism between Pin1 and cyclophilin, and may lead to a more complete understanding of the active sites of PPIases.

Rational Design of Ratiometric Near-infrared Fluorescent PH Probes with Various PKa Values, Based on Aminocyanine

Journal of the American Chemical Society. Mar, 2011  |  Pubmed ID: 21341656

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 pK(a) values of these probes can be predicted from the calculated pK(a) 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.

Development of a Fluorescein Analogue, TokyoMagenta, As a Novel Scaffold for Fluorescence Probes in Red Region

Chemical Communications (Cambridge, England). Apr, 2011  |  Pubmed ID: 21359330

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.

In Situ Evaluation of Kinetic Resolution Catalysts for Nitroaldol by Rationally Designed Fluorescence Probe

The Journal of Organic Chemistry. May, 2011  |  Pubmed ID: 21370849

Development of effective chemical catalysts is a key concern in organic chemistry. Therefore, convenient screening systems for chemical catalysts are required, and although some fluorescence-based HTS systems have been developed, little attempt has been made to apply them to asymmetric catalysts. Therefore, we tried to develop a chiral fluorescence probe which can evaluate the reactivity and enantioselectivity of asymmetric catalysts. We focused on kinetic resolution catalysts as a target of our novel fluorescence probe, employing β-elimination following acylation of nitroaldol. Once the hydroxyl group of nitroaldol is acylated, β-elimination occurs immediately, affording nitro olefin. Therefore, we designed and synthesized a fluorescence probe with an asymmetric nitroaldol moiety. Its fluorescence intensity decreases dramatically upon β-elimination, so the fluorescence decrease is an indicator of the reaction yield. Thus, the enantioselectivity of kinetic resolution catalysts can be assessed simply by measuring the fluorescence intensities of the reaction mixtures of the two enantiomers; it is not necessary to purify the product. This fluorescence probe revealed that benzotetramisole is a superior catalyst for kinetic resolution of nitroaldol. Furthermore, we established an HTS system for asymmetric catalysts, using a fluorescence probe and benzotetramisole. To our knowledge, this is the first fluorescence-based HTS system for asymmetric catalysts.

Evolution of Group 14 Rhodamines As Platforms for Near-infrared Fluorescence Probes Utilizing Photoinduced Electron Transfer

ACS Chemical Biology. Jun, 2011  |  Pubmed ID: 21375253

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.

Aminoluciferins As Functional Bioluminogenic Substrates of Firefly Luciferase

Chemistry, an Asian Journal. Jul, 2011  |  Pubmed ID: 21416616

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.

Development of an Si-rhodamine-based Far-red to Near-infrared Fluorescence Probe Selective for Hypochlorous Acid and Its Applications for Biological Imaging

Journal of the American Chemical Society. Apr, 2011  |  Pubmed ID: 21443186

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.

Real-time Measurements of Protein Dynamics Using Fluorescence Activation-coupled Protein Labeling Method

Journal of the American Chemical Society. May, 2011  |  Pubmed ID: 21473619

We present a fluorescence activation-coupled protein labeling (FAPL) method, which employs small-molecular probes that exhibit almost no basal fluorescence but acquire strong fluorescence upon covalent binding to tag-proteins. This method enables real-time imaging of protein labeling without any washout process and is uniquely suitable for real-time imaging of protein dynamics on the cell surface. We applied this method to address the spatiotemporal dynamics of the EGF receptor during cell migration.

Development of a Highly Selective Fluorescence Probe for Alkaline Phosphatase

Bioorganic & Medicinal Chemistry Letters. Sep, 2011  |  Pubmed ID: 21482108

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.

Virtual Screening and Further Development of Novel ALK Inhibitors

Bioorganic & Medicinal Chemistry. May, 2011  |  Pubmed ID: 21515061

Anaplastic lymphoma kinase (ALK) has been in the spotlight in recent years as a promising new target for therapy of non-small-cell lung cancer (NSCLC). Since the identification of the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene in some NSCLC patients was reported in 2007, various research groups have been seeking ALK inhibitors. Above all, crizotinib (PF-02341066) has been under clinical trial, and its therapeutic efficacy of inhibiting ALK in NSCLC has been reported. Among anticancer drugs, drug resistance appears frequently necessitating various kinds of inhibitors. We identified novel ALK inhibitors by virtual screening from the public chemical library collected by the Chemical Biology Research Initiative (CBRI) at the University of Tokyo, and inhibitors that are more potent were developed.

Development of a Highly Sensitive Fluorescence Probe for Hydrogen Peroxide

Journal of the American Chemical Society. Jul, 2011  |  Pubmed ID: 21692459

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.

Fluorescence Probe for Lysophospholipase C/NPP6 Activity and a Potent NPP6 Inhibitor

Journal of the American Chemical Society. Aug, 2011  |  Pubmed ID: 21721554

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(3)C, TG-mPC(5)C, TG-mPENE, TG-mPEA, TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr. Among the seven NPPs, only NPP6 hydrolyzed TG-mPC, TG-mPC(3)C, 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.

β-Galactosidase Fluorescence Probe with Improved Cellular Accumulation Based on a Spirocyclized Rhodol Scaffold

Journal of the American Chemical Society. Aug, 2011  |  Pubmed ID: 21786797

We identified a rhodol bearing a hydroxymethyl group (HMDER) as a suitable scaffold for designing fluorescence probes for various hydrolases. HMDER shows strong fluorescence at physiological pH, but phenolic O-alkylation of HMDER results in a strong preference for the spirocyclic form, which has weak fluorescence. As a proof of concept, we utilized this finding to develop a new fluorescence probe for β-galactosidase. This probe has favorable characteristics for imaging in biological samples: it has good cellular permeability, and its hydrolysis product is well-retained intracellularly. It could rapidly and clearly visualize β-galactosidase activity in cultured cells and in Drosophila melanogaster tissue, which has rarely been achieved with previously reported fluorescence probes.

Fluorescent Probes for Sensing and Imaging

Nature Methods. Aug, 2011  |  Pubmed ID: 21799499

Rational Design of Boron Dipyrromethene (BODIPY)-based Photobleaching-resistant Fluorophores Applicable to a Protein Dynamics Study

Chemical Communications (Cambridge, England). Sep, 2011  |  Pubmed ID: 21818482

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-diCO(2)R-BDP, for visualizing EGF receptor dynamics in cells expressing an SNAP-tagged EGF receptor.

Development of a Far-red to Near-infrared Fluorescence Probe for Calcium Ion and Its Application to Multicolor Neuronal Imaging

Journal of the American Chemical Society. Sep, 2011  |  Pubmed ID: 21827169

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.

Development of a Potassium Ion-selective Fluorescent Sensor Based on 3-styrylated BODIPY

Bioorganic & Medicinal Chemistry Letters. Oct, 2011  |  Pubmed ID: 21906942

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.

Method for Enhancing Cell Penetration of Gd3+-based MRI Contrast Agents by Conjugation with Hydrophobic Fluorescent Dyes

Bioconjugate Chemistry. Nov, 2011  |  Pubmed ID: 21985057

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.

Development of a Highly Selective Fluorescence Probe for Hydrogen Sulfide

Journal of the American Chemical Society. Nov, 2011  |  Pubmed ID: 21999237

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

Activatable Optical Imaging with a Silica-rhodamine Based Near Infrared (SiR700) Fluorophore: a Comparison with Cyanine Based Dyes

Bioconjugate Chemistry. Dec, 2011  |  Pubmed ID: 22034863

Optical imaging is emerging as an important tool to visualize tumors. However, there are many potential choices among the available fluorophores. Optical imaging probes that emit in the visible range can image superficial tumors with high quantum yields; however, if deeper imaging is needed then near-infrared (NIR) fluorophores are necessary. Most commercially available NIR fluorophores are cyanine based and are prone to nonspecific binding and relatively limited photostability. Silica-containing rhodamine (SiR) fluorophores represent a new class of NIR fluorophores, which permit photoactivation via H-dimer formation as well as demonstrate improved photostability. This permits higher tumor-to-background ratios (TBRs) to be achieved over longer periods of time. Here, we compared an avidin conjugated with SiR700 (Av-SiR700) to similar compounds based on cyanine dyes (Av-Cy5.5 and Av-Alexa Fluor 680) in a mouse tumor model of ovarian cancer metastasis. We found that the Av-SiR700 probe demonstrated superior quenching, enabling activation after binding-internalization to the target cell. As a result, Av-SiR700 had higher TBRs compared to Av-Cy5.5 and better biostability compared to Av-Alexa Fluor 680.

Rational Development of Caged-biotin Protein-labeling Agents and Some Applications in Live Cells

Chemistry & Biology. Oct, 2011  |  Pubmed ID: 22035795

Biotin-(strept)avidin complex is widely used in biotechnology because of its extremely high binding constant, but there is no report describing spatiotemporally controlled formation of the complex in live cells. Here, based on X-ray crystal structure analysis and calorimetric data, we designed and synthesized photoreleasable biotins, which show greatly reduced affinity for (strept)avidin, but recover native affinity after UV irradiation. For application at the cell surface, we introduced an amine-reactive moiety into these "caged" biotin molecules. Specific fluorescence imaging of live cells that had been labeled with these agents and then UV-irradiated, was accomplished by addition of streptavidin conjugated with a fluorophore. We also demonstrated the applicability of these compounds for UV-irradiated-cell-specific drug delivery by using caged-biotin-labeled cells, a prodrug, and streptavidin conjugated with a prodrug-activating enzyme.

Selective Two-step Labeling of Proteins with an Off/on Fluorescent Probe

Chemistry (Weinheim an Der Bergstrasse, Germany). Dec, 2011  |  Pubmed ID: 22106092

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(6)Cys-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.

Rapid Cancer Detection by Topically Spraying a γ-glutamyltranspeptidase-activated Fluorescent Probe

Science Translational Medicine. Nov, 2011  |  Pubmed ID: 22116934

The ability of the unaided human eye to detect small cancer foci or accurate borders between cancer and normal tissue during surgery or endoscopy is limited. Fluorescent probes are useful for enhancing visualization of small tumors but are typically limited by either high background signal or the requirement for administration hours to days before use. We synthesized a rapidly activatable, cancer-selective fluorescence imaging probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), with intramolecular spirocyclic caging for complete quenching. Activation occurs by rapid one-step cleavage of glutamate with γ-glutamyltranspeptidase (GGT), which is not expressed in normal tissue, but is overexpressed on the cell membrane of various cancer cells, thus leading to complete uncaging and dequenching of the fluorescence probe. In vitro activation of gGlu-HMRG was evident in 11 human ovarian cancer cell lines tested. In vivo in mouse models of disseminated human peritoneal ovarian cancer, activation of gGlu-HMRG occurred within 1 min of topically spraying the tumor, creating high signal contrast between the tumor and the background. The gGlu-HMRG probe is practical for clinical application during surgical or endoscopic procedures because of its rapid and strong activation upon contact with GGT on the surface of cancer cells.

Visible-light-triggered Release of Nitric Oxide from N-pyramidal Nitrosamines

Chemistry (Weinheim an Der Bergstrasse, Germany). Jan, 2012  |  Pubmed ID: 22179925

Although many organic/inorganic compounds that release nitric oxide (NO) upon photoirradiation (phototriggered caged-NOs) have been reported, their photoabsorption wavelengths mostly lie in the UV region, because X-NO bonds (X=heteroatom and metal) generally have rather strong π-bond character. Thus, it is intrinsically difficult to generate organic compounds that release NO under visible light irradiation. Herein, the structures and properties of N-pyramidal nitrosamine derivatives of 7-azabicyclo[2.2.1]heptanes that release NO under visible light irradiation are described. Bathochromic shifts of the absorptions of these nitrosamines, attributed to HOMO (n)-LUMO (π*) transitions associated with the nonplanar structure of the N-NO moiety, enable the molecules to absorb visible light, which results in N-NO bond cleavage. Thus, these compounds are innate organic caged-NOs that are uncaged by visible light.

A Practical Strategy to Create Near-infrared Luminescent Probes: Conversion from Fluorescein-based Sensors

Chemical Communications (Cambridge, England). Mar, 2012  |  Pubmed ID: 22222313

Luminescent lanthanide complexes incorporating Yb(3+) and Nd(3+) are attracting much attention as imaging agents, but there have been few practical methods to make responsive sensors with these complexes. Here, we introduce a general strategy to synthesize near-infrared luminescent probes by conjugating a Yb(3+) chelate to established fluorescein-based probes. As the first demonstration, we present a complex, based on the green-emitting probe DAF-4, that responds to nitric oxide (NO) in aqueous solution with a significant increase in luminescence intensity at 980 nm.

A Long-lived Luminescent Probe to Sensitively Detect Arylamine N-acetyltransferase (NAT) Activity of Cells

Chemical Communications (Cambridge, England). Feb, 2012  |  Pubmed ID: 22252439

Arylamine N-acetyltransferase (NAT) is an important phase II metabolizing enzyme that influences drug efficacy and adverse effects. Here, we report a long-lived luminescent lanthanide complex as a probe for NAT, employing an intraligand photoinduced electron transfer strategy. The probe shows approximately 100-fold increase of luminescence upon N-acetylation catalyzed by NAT, with relatively high specificity for NAT2 over NAT1. It is the first NAT probe that is suitable for sensitive, homogeneous, and rapid detection of NAT activity of recombinant enzyme or cell lysate, and is expected to be useful for drug discovery and clinical diagnosis.

A Novel Pim-1 Kinase Inhibitor Targeting Residues That Bind the Substrate Peptide

Journal of Molecular Biology. Mar, 2012  |  Pubmed ID: 22306408

A new screening method using fluorescent correlation spectroscopy was developed to select kinase inhibitors that competitively inhibit the binding of a fluorescently labeled substrate peptide. Using the method, among approximately 700 candidate compounds selected by virtual screening, we identified a novel Pim-1 kinase inhibitor targeting its peptide binding residues. X-ray crystal analysis of the complex structure of Pim-1 with the inhibitor indicated that the inhibitor actually binds to the ATP-binding site and also forms direct interactions with residues (Asp128 and Glu171) that bind the substrate peptide. These interactions, which cause small side-chain movements, seem to affect the binding ability of the fluorescently labeled substrate. The compound inhibited Pim-1 kinase in vitro, with an IC(50) value of 150 nM. Treatment of cultured leukemia cells with the compound reduced the amount of p21 and increased the amount of p27, due to Pim-1 inhibition, and then triggered apoptosis after cell-cycle arrest at the G(1)/S phase. This screening method may be widely applicable for the identification of various new Pim-1 kinase inhibitors targeting the residues that bind the substrate peptide.

Myeloperoxidase Exacerbates Secondary Injury by Generating Highly Reactive Oxygen Species and Mediating Neutrophil Recruitment in Experimental Spinal Cord Injury

Spine. Feb, 2012  |  Pubmed ID: 22322369

Study Design. An animal study using myeloperoxidase-knockout (MPO-KO) mice to examine the in vivo role of myeloperoxidase (MPO) in spinal cord injury (SCI).Objective. To clarify the influence of MPO on inflammatory cell infiltration, tissue damage, and functional recovery after SCI.Summary of Background Data. MPO is considered to be important in spreading tissue damage after SCI because it generates strong neurotoxic oxidant hypochlorite acid (HOCl). However, the direct involvement of MPO in the pathophysiology of SCI remains to be elucidated.Methods. To compare the inflammatory reaction, tissue damage, and neurological recovery after SCI, a moderate contusion injury was created at the 9 thoracic level in MPO-KO mice and wild-type mice. A HOCl-specific probe solution was injected into the lesion epicenter to assess the spatiotemporal production of MPO-derived HOCl. Inflammatory reactions were quantified by flow cytometry and quantitative real time PCR, and tissue damage was evaluated by an immunohistochemical analysis. The motor function recovery was assessed by the open-field locomotor score.Results. Prominent production of HOCl was observed during the hyper-acute phase of SCI at the lesion site in the wild type mice, however, little expression was observed in the MPO-KO mice. In this phase, the number of infiltrated neutrophils was significantly reduced in the MPO-KO mice compared to the wild-type mice. In addition, significant differences were observed in the expression levels of pro-inflammatory cytokines and apoptosis-related genes between two groups. In the histological sections, fewer TUNEL-positive apoptotic cells and more spared myelin were observed at the lesion site in MPO-KO mice. Consistent with these results, better functional recovery was observed in the MPO-KO mice than in the wild-type mice after SCI.Conclusion. These results clearly indicated that MPO exacerbated secondary injury and impaired the functional recovery by not only generating strong oxidant HOCl, but also by.

A Reversible Near-infrared Fluorescence Probe for Reactive Oxygen Species Based on Te-rhodamine

Chemical Communications (Cambridge, England). Feb, 2012  |  Pubmed ID: 22344329

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.

Development of NIR Fluorescent Dyes Based on Si-rhodamine for in Vivo Imaging

Journal of the American Chemical Society. Mar, 2012  |  Pubmed ID: 22390359

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.