15 results on '"T. Nagano"'
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2. Thermal or mechanical stimuli-induced photoluminescence color change of a molecular assembly composed of an amphiphilic anthracene derivative in water.
- Author
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Sagara Y, Komatsu T, Terai T, Ueno T, Hanaoka K, Kato T, and Nagano T
- Subjects
- Friction, Luminescence, Micelles, Temperature, Water chemistry, Anthracenes chemistry, Luminescent Agents chemistry, Surface-Active Agents chemistry
- Abstract
Molecular assemblies that change photoluminescence color in response to thermal or mechanical stimulation without dissociation into the monomeric states in water are described herein. A dumbbell-shaped amphiphilic compound forms micellar molecular assemblies in water and exhibits yellow photoluminescence derived from excimer formation of the luminescent core, which contains a 2,6-diethynylanthracene moiety. Annealing of the aqueous solution induces a photoluminescence color change from yellow to green (λem, max =558→525 nm). The same photoluminescence color change is also achieved by rubbing the yellow-photoluminescence-emitting molecular assemblies adsorbed on glass substrates with cotton wool in water. The observed green photoluminescence is ascribed to micelles that are distinct from the yellow-photoluminescence-emitting micelles, on the basis of transmission electron microscopy observations, atomic force microscopy observations, and dynamic light scattering measurements. We examined the relationship between the structure of the molecular assemblies and the photophysical properties of the anthracene derivative in water before and after thermal or mechanical stimulation and concluded that thermal or mechanical stimuli-induced slight changes of the molecular-assembled structures in the micelles result in the change in the photoluminescence color from yellow to green in water., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2014
- Full Text
- View/download PDF
3. Additive effects of amines on asymmetric hydrogenation of quinoxalines catalyzed by chiral iridium complexes.
- Author
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Nagano T, Iimuro A, Schwenk R, Ohshima T, Kita Y, Togni A, and Mashima K
- Subjects
- Amines chemistry, Catalysis, Hydrogenation, Molecular Structure, Amines chemical synthesis, Iridium chemistry, Organometallic Compounds chemistry, Quinoxalines chemistry
- Abstract
The additive effects of amines were realized in the asymmetric hydrogenation of 2-phenylquinoxaline, and its derivatives, catalyzed by chiral cationic dinuclear triply halide-bridged iridium complexes [{Ir(H)[diphosphine]}(2)(μ-X)(3)]X (diphosphine = (S)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl [(S)-BINAP], (S)-5,5'-bis(diphenylphosphino)-4,4'-bi-1,3-benzodioxole [(S)-SEGPHOS], (S)-5,5'-bis(diphenylphosphino)-2,2,2',2'-tetrafluoro-4,4'-bi-1,3-benzodioxole [(S)-DIFLUORPHOS]; X = Cl, Br, I) to produce the corresponding 2-aryl-1,2,3,4-tetrahydroquinoxalines. The additive effects of amines were investigated by solution dynamics studies of iridium complexes in the presence of N-methyl-p-anisidine (MPA), which was determined to be the best amine additive for achievement of a high enantioselectivity of (S)-2-phenyl-1,2,3,4-tetrahydroquinoxaline, and by labeling experiments, which revealed a plausible mechanism comprised of two cycles. One catalytic cycle was less active and less enantioselective; it involved the substrate-coordinated mononuclear complex [IrHCl(2)(2-phenylquinoxaline){(S)-BINAP}], which afforded half-reduced product 3-phenyl-1,2-dihydroquinoxaline. A poorly enantioselective disproportionation of this half-reduced product afforded (S)-2-phenyl-1,2,3,4-tetrahydroquinoxaline. The other cycle involved a more active hydride-amide catalyst, derived from amine-coordinated mononuclear complex [IrCl(2)H(MPA){(S)-BINAP}], which functioned to reduce 2-phenylquinoxaline to (S)-2-phenyl-1,2,3,4-tetrahydroquinoxaline with high enantioselectivity. Based on the proposed mechanism, an Ir(I)-JOSIPHOS (JOSIPHOS = (R)-1-[(S(p))-2-(dicyclohexylphosphino)ferrocenylethyl]diphenylphosphine) catalyst in the presence of amine additive resulted in the highest enantioselectivity for the asymmetric hydrogenation of 2-phenylquinoxaline. Interestingly, the reaction rate and enantioselectivity were gradually increased during the reaction by a positive-feedback effect from the product amines., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2012
- Full Text
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4. Salicylic-acid derivatives as antennae for ratiometric luminescent probes based on lanthanide complexes.
- Author
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Terai T, Ito H, Kikuchi K, and Nagano T
- Subjects
- Fluorescent Dyes, Hydrolases metabolism, Luminescence, Molecular Structure, Organometallic Compounds chemistry, Lanthanoid Series Elements chemistry, Salicylates chemistry, Terbium chemistry
- Abstract
Long-lived ratiometric sensors: Luminescent lanthanide complexes are widely used in time-resolved assays of biomolecules, but most of the sensors with these complexes rely on single-point intensity measurements. Herein, we introduce a simple strategy to create ratiometric probes by using salicylic-acid derivatives as the antenna moiety of Tb(3+) complexes. As an example, a probe for alkaline phosphatase (ALP) was developed (see scheme)., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2012
- Full Text
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5. Visible-light-triggered release of nitric oxide from N-pyramidal nitrosamines.
- Author
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Karaki F, Kabasawa Y, Yanagimoto T, Umeda N, Firman, Urano Y, Nagano T, Otani Y, and Ohwada T
- Subjects
- Cyclization, Models, Molecular, Molecular Structure, Light, Nitric Oxide chemistry, Nitrosamines chemistry
- Abstract
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., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2012
- Full Text
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6. Selective two-step labeling of proteins with an off/on fluorescent probe.
- Author
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Hirabayashi K, Hanaoka K, Shimonishi M, Terai T, Komatsu T, Ueno T, and Nagano T
- Subjects
- Binding Sites, Fluorescence, Molecular Structure, Organometallic Compounds chemistry, Fluorescent Dyes chemical synthesis, Fluorescent Dyes chemistry, Green Fluorescent Proteins chemistry, Nickel chemistry, Nitrilotriacetic Acid chemistry, Oligopeptides chemistry, Peptides chemistry, Proteins chemistry
- Abstract
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., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2011
- Full Text
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7. Dye-sensitized solar cells based on donor-π-acceptor fluorescent dyes with a pyridine ring as an electron-withdrawing-injecting anchoring group.
- Author
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Ooyama Y, Nagano T, Inoue S, Imae I, Komaguchi K, Ohshita J, and Harima Y
- Abstract
A new-type of donor-acceptor π-conjugated (D-π-A) fluorescent dyes NI3-NI8 with a pyridine ring as electron-withdrawing-injecting anchoring group have been developed and their photovoltaic performances in dye-sensitized solar cells (DSSCs) are investigated. The short-circuit photocurrent densities and solar energy-to-electricity conversion yields of DSSCs based on NI3-NI8 are greater than those for the conventional D-π-A dye sensitizers NI1 and NI2 with a carboxyl group as the electron-withdrawing anchoring group. The IR spectra of NI3-NI8 adsorbed on TiO(2) indicate the formation of coordinate bonds between the pyridine ring of dyes NI3-NI8 and the Lewis acid sites (exposed Ti(n+) cations) of the TiO(2) surface. This work demonstrates that the pyridine rings of D-π-A dye sensitizers that form a coordinate bond with the Lewis acid site of a TiO(2) surface are promising candidates as not only electron-withdrawing anchoring group but also electron-injecting group, rather than the carboxyl groups of the conventional D-π-A dye sensitizers that form an ester linkage with the Brønsted acid sites of the TiO(2) surface., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2011
- Full Text
- View/download PDF
8. A time-resolved fluorescence probe for dipeptidyl peptidase 4 and its application in inhibitor screening.
- Author
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Kawaguchi M, Okabe T, Terai T, Hanaoka K, Kojima H, Minegishi I, and Nagano T
- Subjects
- Diabetes Mellitus, Type 2 drug therapy, Dipeptides chemistry, Dipeptidyl Peptidase 4 analysis, Dipeptidyl-Peptidase IV Inhibitors chemistry, Dipeptidyl-Peptidase IV Inhibitors pharmacology, Leucine analogs & derivatives, Leucine pharmacology, Metalloproteins chemistry, Molecular Structure, Spectrometry, Fluorescence, Terbium chemistry, Thermodynamics, Time Factors, Dipeptides chemical synthesis, Dipeptidyl Peptidase 4 metabolism, Dipeptidyl-Peptidase IV Inhibitors chemical synthesis, Metalloproteins chemical synthesis
- Abstract
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.
- Published
- 2010
- Full Text
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9. Design and synthesis of a highly sensitive off-on fluorescent chemosensor for zinc ions utilizing internal charge transfer.
- Author
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Hanaoka K, Muramatsu Y, Urano Y, Terai T, and Nagano T
- Subjects
- 1-Naphthylamine chemical synthesis, 1-Naphthylamine chemistry, Fluorescent Dyes chemistry, Molecular Structure, Naphthalimides chemistry, Quinolones chemistry, Spectrometry, Fluorescence methods, 1-Naphthylamine analogs & derivatives, Fluorescent Dyes chemical synthesis, Naphthalimides chemical synthesis, Quinolones chemical synthesis, Zinc analysis
- Abstract
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.
- Published
- 2010
- Full Text
- View/download PDF
10. Unprecedented halide dependence on catalytic asymmetric hydrogenation of 2-aryl- and 2-alkyl-substituted quinolinium salts by using Ir complexes with difluorphos and halide ligands.
- Author
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Tadaoka H, Cartigny D, Nagano T, Gosavi T, Ayad T, Genêt JP, Ohshima T, Ratovelomanana-Vidal V, and Mashima K
- Published
- 2009
- Full Text
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11. Total synthesis and biological evaluation of the cytotoxic resin glycosides ipomoeassin A-F and analogues.
- Author
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Nagano T, Pospísil J, Chollet G, Schulthoff S, Hickmann V, Moulin E, Herrmann J, Müller R, and Fürstner A
- Subjects
- Antineoplastic Agents chemistry, Antineoplastic Agents toxicity, Catalysis, Cell Line, Tumor, Crystallography, X-Ray, Glycoconjugates chemistry, Glycoconjugates toxicity, Humans, Molecular Conformation, Ruthenium chemistry, Stereoisomerism, Antineoplastic Agents chemical synthesis, Glycoconjugates chemical synthesis
- Abstract
A multitasking C-silylation strategy using the readily available compound 26 as a surrogate for cinnamic acid represents the key design element of a total synthesis of all known members of the ipomoeassin family of resin glyosides. This protecting group maneuver allows the unsaturated acids decorating the glucose subunit of the targets to be attached at an early phase of the synthesis, prevents their participation in the ruthenium-catalyzed ring-closing metathesis (RCM) used to form the macrocyclic ring, and protects them against reduction during the hydrogenation of the resulting cycloalkene over Wilkinson's catalyst. As the C-silyl group can be concomitantly removed with the O-TBS substituent using tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF) in acetonitrile, no separate protecting group manipulations were necessary in the final stages, thus contributing to a favorable overall "economy of steps". In addition to the naturally occurring ipomoeassins, a small set of synthetic analogues has also been prepared by "diverted total synthesis". The cytotoxicity of these compounds was assayed with two different cancer cell lines. The recorded data confirm previous findings that the acylation- and oxygenation pattern of these amphiphilic glycoconjugates is highly correlated with their biological activity profile. Ipomoeassin F turned out to be the most promising member of the series, showing IC(50) values in the low nanomolar range.
- Published
- 2009
- Full Text
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12. Molecular design strategies for near-infrared ratiometric fluorescent probes based on the unique spectral properties of aminocyanines.
- Author
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Kiyose K, Aizawa S, Sasaki E, Kojima H, Hanaoka K, Terai T, Urano Y, and Nagano T
- Subjects
- Esterases metabolism, Fluorescent Dyes chemical synthesis, Hydrogen-Ion Concentration, Molecular Structure, Spectroscopy, Near-Infrared, Carbocyanines chemistry, Fluorescent Dyes chemistry
- Abstract
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.
- Published
- 2009
- Full Text
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13. A Gd3+-based magnetic resonance imaging contrast agent sensitive to beta-galactosidase activity utilizing a receptor-induced magnetization enhancement (RIME) phenomenon.
- Author
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Hanaoka K, Kikuchi K, Terai T, Komatsu T, and Nagano T
- Subjects
- Animals, Binding Sites, Cattle, Contrast Media chemical synthesis, Enzyme Activation, Humans, Kinetics, Luminescence, Organometallic Compounds chemical synthesis, Quantum Theory, Rabbits, Rats, Sensitivity and Specificity, Species Specificity, Spectrophotometry, Ultraviolet methods, Time Factors, Contrast Media chemistry, Gadolinium chemistry, Magnetic Resonance Imaging, Organometallic Compounds chemistry, Serum Albumin chemistry, beta-Galactosidase chemistry
- Abstract
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].
- Published
- 2008
- Full Text
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14. Total synthesis and evaluation of the actin-binding properties of microcarpalide and a focused library of analogues.
- Author
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Fürstner A, Nagano T, Müller C, Seidel G, and Müller O
- Subjects
- 4-Butyrolactone pharmacology, Alkanes pharmacology, Alkenes pharmacology, Binding Sites, Bridged Bicyclo Compounds, Heterocyclic pharmacology, Fungi metabolism, Heterocyclic Compounds, 1-Ring pharmacology, Ketones pharmacology, Molecular Structure, Stereoisomerism, Thiazolidines pharmacology, Actins metabolism, Alkanes chemical synthesis, Alkenes chemistry, Heterocyclic Compounds, 1-Ring chemical synthesis, Ketones chemistry
- Abstract
A comparative investigation shows that hydroxylated 10-membered lactones modeled around the fungal metabolites microcarpalide (1) and pinolidoxin (2) are endowed with selective actin-binding properties. Although less potent than the marine natural product latrunculin A, which represents the standard in the field, nonenolides of this type are significantly less toxic and accommodate substantial structural editing. Most notable is the fact that even an intramolecular transesterification with formation of a hydroxylated butanolide skeleton does not annihilate their microfilament disrupting capacity. This finding calls for a reinvestigation of the biological profile of other fungal metabolites that embody a similar motif. Microcarpalide (1) serving as the calibration point for this comparative study was prepared by total synthesis based on ring-closing metathesis (RCM) as the key step. The chosen route favorably compares to previous approaches to this target and provides further support for the notion that the (E,Z)-configuration of a medium-sized cycloalkene can be controlled by proper choice of the catalyst as previously outlined by our group. 9-epi-Microcarpalide 26 and furanone 27 as representative examples of the "natural productlike" compounds investigated herein have been characterized by crystal structure analysis.
- Published
- 2007
- Full Text
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15. A novel design method of ratiometric fluorescent probes based on fluorescence resonance energy transfer switching by spectral overlap integral.
- Author
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Takakusa H, Kikuchi K, Urano Y, Kojima H, and Nagano T
- Subjects
- Cell Membrane Permeability, Coumarins chemical synthesis, Coumarins pharmacokinetics, Cytosol enzymology, Endothelium, Vascular cytology, Endothelium, Vascular enzymology, Endothelium, Vascular metabolism, Fluoresceins chemical synthesis, Fluoresceins pharmacokinetics, Fluorescence Resonance Energy Transfer methods, Fluorescent Dyes chemical synthesis, Fluorescent Dyes pharmacokinetics, Humans, Kinetics, Protein Tyrosine Phosphatases chemistry, Static Electricity, Coumarins chemistry, Fluoresceins chemistry, Fluorescent Dyes chemistry, Protein Tyrosine Phosphatases metabolism
- Abstract
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.
- Published
- 2003
- Full Text
- View/download PDF
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