US20060275912A1 - Fluorescent probes - Google Patents

Fluorescent probes Download PDF

Info

Publication number
US20060275912A1
US20060275912A1 US10/547,305 US54730506A US2006275912A1 US 20060275912 A1 US20060275912 A1 US 20060275912A1 US 54730506 A US54730506 A US 54730506A US 2006275912 A1 US2006275912 A1 US 2006275912A1
Authority
US
United States
Prior art keywords
group
substituted
alkyl group
compound
alkyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/547,305
Other languages
English (en)
Inventor
Tetsuo Nagano
Yu Gabe
Yasuteru Urano
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Daiichi Pure Chemicals Co Ltd
Original Assignee
Daiichi Pure Chemicals Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Daiichi Pure Chemicals Co Ltd filed Critical Daiichi Pure Chemicals Co Ltd
Assigned to TETSUO NAGANO, DAIICHI PURE CHEMICALS CO., LTD. reassignment TETSUO NAGANO ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GABE, YU, NAGANO, TETSUO, URANO, YASUTERU
Publication of US20060275912A1 publication Critical patent/US20060275912A1/en
Priority to US12/688,625 priority Critical patent/US20100120160A1/en
Priority to US13/425,802 priority patent/US8673957B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/022—Boron compounds without C-boron linkages
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
    • C09K2211/1055—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms with other heteroatoms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/145555—Hetero-N
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/17—Nitrogen containing
    • Y10T436/177692—Oxides of nitrogen

Definitions

  • Diaminorhodamine derivatives that can efficiently react with nitrogen monoxide under a neutral condition, and give a triazole derivative having superior fluorescence intensity have been proposed (U.S. Pat. No. 6,201,134).
  • the fluorescence of the diaminorhodamine derivatives shifts to a longer wavelength side compared with the aforementioned fluorescein derivatives, thus the fluorescence does not substantially overlaps the autofluorescence of cells, and the fluorescence does not attenuate even in an acidic region. Therefore, the fluorescence of the diaminorhodamine derivatives does not damage living tissues and cells, and enable measurement of nitrogen monoxide in a fluorescent region of a longer wavelength than in the autofluorescent region of cells.
  • the inventors of the present invention conducted various researches to provide a fluorescent probe that specifically and efficiently captures nitrogen monoxide to emit fluorescence. As a result, they successfully provided an agent for nitrogen monoxide measurement that can efficiently react with nitrogen monoxide under a neutral condition to give a fluorescent substance having superior fluorescence intensity by using fluorescence chromophores of indacene derivatives (Japanese Patent Application No. 2002-80230).
  • the inventors of the present invention further conducted researches, and as a result, they found that an agent for nitrogen monoxide measurement further having superior water-solubility was successfully provided by introducing a substituent such as a carboxy-substituted alkyl group into the fluorescence chromophore of the indacene derivatives, and that an fluorescence intensity of the fluorescent triazole derivative, produced by trapping of nitrogen monoxide by the agent, was subjected to almost no influence by pH fluctuation.
  • the present invention was achieved on the basis of these findings.
  • R 1 and R 2 represent amino groups that substitute at adjacent positions on the benzene ring, wherein one of the amino groups may have one alkyl group which may be substituted;
  • R 3 and R 4 independently represent hydrogen atom, a C 1-6 alkyl group, or a C 1-6 alkoxy group,
  • R 5 and R 8 independently represent a C 1-6 alkyl group which may be substituted,
  • R 6 and R 9 independently represent a carboxy-substituted C 1-6 alkyl group, an alkoxycarbonyl-substituted C 1-6 alkyl group, a sulfo-substituted C 1-6 alkyl group, or an alkyl sulfonate-substituted C 1-6 alkyl group, and
  • R 7 and R 10 independently represent a C 1-6 alkyl group which may be substituted, an aryl group which may be substituted, a C 1-6 alkoxycarbonyl group which
  • the aforementioned compound or a salt thereof wherein R 6 and R 9 are 2-carboxy-1-ethyl groups, and R 5 , R 7 , R 8 , and R 10 are methyl groups is provided.
  • the present invention also provides an agent for measurement of nitrogen monoxide comprising the aforementioned compound or a salt thereof.
  • the present invention provides a compound represented by the following formula (II): wherein, R 11 and R 12 combine together to represent a group represented by —N ⁇ N—NR 30 — which forms a ring structure at adjacent positions on the benzene ring wherein R 30 represents hydrogen atom, or an alkyl group which may be substituted, or R 11 and R 12 represent a combination of an amino group (which may have an alkyl group which may be substituted or a protective group for amino group) and nitro group that substitute at adjacent positions on the benzene ring; R 13 and R 14 independently represent hydrogen atom, a C 1-6 alkyl group, or a C 1-6 alkoxy group, R 15 and R 18 independently represent a C 1-6 alkyl group which may be substituted, R 16 and R 19 independently represent a carboxy-substituted C 1-6 alkyl group, an alkoxycarbonyl-substituted C 1-6 alkyl group, a sulfo-substituted
  • FIG. 1 shows the results of the measurement of changes of excitation and fluorescence spectra of Compound (7) after addition of NOC 13.
  • (A) represent the excitation spectrum (Em: 535 nm)
  • (B) represents the fluorescence spectrum (Ex: 520 nm).
  • the curves indicate the results for the NOC 13 concentrations of 5.0 ⁇ M, 2.0 ⁇ M, 1.0 ⁇ M, 0.5 ⁇ M, and 0 ⁇ M in the order that the fluorescence intensities shown by the curves decrease from the highest.
  • FIG. 2 shows the results of HPLC measurement of the reaction product obtained by adding NOC 13 to Compound (7).
  • FIG. 3 shows the results of measurement of fluorescence intensity of Compounds (7) and (8) at various pH values.
  • FIG. 4 shows changes of fluorescence intensity observed when a solution of NO in chloroform was added to a solution of Compound (9) in chloroform.
  • the alkyl group may be a linear, branched, or cyclic alkyl group, or a combination thereof, unless otherwise specifically mentioned.
  • An alkyl moiety of other substituents containing the alkyl moiety e.g. alkoxy group
  • the type, number, and substitution position of the substituent are not particularly limited.
  • the functional group may have, for example, a halogen atom (any of fluorine atom, chlorine atom, bromine atom, and iodine atom), hydroxy group, amino group, carboxy group, sulfo group, an alkyl sulfonate group, or the like as the substituent.
  • a halogen atom any of fluorine atom, chlorine atom, bromine atom, and iodine atom
  • the carboxy-substituted C 1-6 alkyl group represented by R 6 or R 9 is preferably a monocarboxy-substituted C 1-6 alkyl group.
  • the carbon number of the C 1-6 alkyl moiety of the carboxy-substituted C 1-6 alkyl group is preferably 1 to 4, more preferably 2 or 3, and most preferably 2. It is most preferred that R 6 and R 9 are 2-carboxy-1-ethyl groups.
  • Examples of the alkoxycarbonyl-substituted C 1-6 alkyl group represented by R 6 or R 9 include a C 1-6 alkyl ester of the aforementioned carboxy-substituted C 1-6 alkyl group.
  • Preferred examples include an ethoxycarbonyl-substituted C 1-6 alkyl group, and the like.
  • a monosulfo-substituted C 1-6 alkyl group is preferred.
  • a monoalkyl sulfonate-substituted C 1-6 alkyl group is preferred.
  • alkyl sulfonate group in the alkyl sulfonate-substituted C 1-6 alkyl group a C 1-6 alkyl sulfonate (C 1-6 alkyl-O—SO 2 —) is preferred.
  • R 6 and R 9 are monocarboxy-substituted C 1-6 alkyl groups, especially when R 6 and R 9 are 2-carboxy-1-ethyl groups, superior effects are obtained in that water-solubility of the compounds remarkably increases, and fluorescence intensity of the compounds represented by the formula (II) produced by a reaction with nitrogen monoxide is not subjected to influence of pH fluctuation.
  • a phenyl group is preferred.
  • the phenyl group has a substituent, sulfo group, a sulfonate group, and the like are preferred as the substituent, and a sulfo group is particularly preferred.
  • the C 1-6 alkoxycarbonyl group represented by R 7 or R 10 ethoxycarbonyl group is preferred.
  • the substituent which exists on the vinyl group represented by R 7 or R 10 include a phenyl group, monoaminophenyl group, and a diaminophenyl group (for example, 3,4-diaminophenyl group).
  • R 6 and R 9 are 2-carboxy-1-ethyl groups.
  • R 5 , R 7 , R 8 , and R 10 are C 1-6 alkyl groups which may be substituted.
  • R 15 , R 16 , R 17 , R 18 , R 19 , and R 20 in the formula (II) should be understood in the same manner as R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 .
  • R 1 and R 2 represent amino groups that substitute at adjacent positions on the benzene ring. Both of R 1 and R 2 may be unsubstituted amino groups. Alternatively, either of R 1 and R 2 may be substituted with one alkyl group, and the alkyl group may have one or more substituents. Examples of the alkyl group which substitutes on the amino group include, for example, a linear or branched C 1-18 alkyl group (preferably a C 1-6 alkyl group). Specifically, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, tert-butyl group and the like can be used.
  • alkyl group which has a substituent examples include, for example, a C 1-6 alkyl group substituted with a substituted or unsubstituted aryl group (aralkyl group) and the like.
  • aryl-substituted alkyl group for example, benzyl group, phenethyl group, paramethoxybenzyl group, paraethoxycarbonylbenzyl group, paracarboxybenzyl group, and the like can be used.
  • R 11 and R 12 combine together to represent a —N ⁇ N—NR 30 — group which forms a ring structure at adjacent positions on the benzene ring.
  • R 30 represents hydrogen atom, or an alkyl group which may be substituted. Examples of this alkyl group include a linear or branched C 1-18 alkyl group (preferably a C 1-6 alkyl group), and examples of the alkyl group which has a substituent include, for example, a substituted or unsubstituted aralkyl group.
  • the aralkyl group for example, benzyl group, phenethyl group, paramethoxybenzyl group, paraethoxycarbonylbenzyl group, paracarboxybenzyl group, and the like can be used.
  • R 11 and R 12 also represent a combination of an amino group (which may have one substituent) and nitro group that substitute at adjacent positions on the benzene ring, wherein either of R 11 and R 12 represents an amino group, and the other represents nitro group.
  • the amino group represented by either R 11 or R 12 may be unsubstituted, or the group may have one alkyl group, for example, a C 1-18 alkyl group, preferably a C 1-6 alkyl group. Further, the alkyl group may be substituted, and for example, a substituted or unsubstituted aralkyl group or the like may substitute on the amino group.
  • the amino group may have a protective group for amino group, for example, an acyl group such as acetyl group, trifluoroacetyl group, and benzoyl group; an alkylsilyl group such as trimethylsilyl groups, or the like.
  • An aralkyl group such as benzyl group may also be used as the protective group.
  • the compounds of the present invention represented by the formulas (I) and (II) may form a salt.
  • Type of the salt is not particularly limited, and the salt may be an acid addition salt or base addition salt.
  • the acid addition salt include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as methanesulfonate, citrate, p-toluenesulfonate, and oxalate.
  • Examples of the base addition salt include metal salts such as sodium salt, potassium salt, and calcium salt, ammonium salts, and organic amine salts such as methylamine salt, and triethylamine salt.
  • the compounds may form a salt with an amino acid such as glycine.
  • salts of the compounds of the present invention are not limited to these specific examples.
  • the compounds of the present invention represented by the formula (I) or (II) may have one or more asymmetric carbon. Any of optical isomers in an optically pure form, arbitrary mixtures of optical isomers, racemates, diastereoisomers in a pure form, mixtures of diastereoisomers, and the like based on one or more asymmetric carbon atoms fall within the scope of the present invention.
  • the compounds of the present invention may exist as hydrates or solvates, and it should be understood that these substances also fall within the scope of the present invention.
  • the compounds represented by the aforementioned formula (II) are useful as synthetic intermediates of the compounds represented by the formula (I).
  • the compounds represented by the formula (II) the compounds wherein R 11 and R 12 combine together to represent the —N ⁇ N—NR 30 — group, which forms a ring structure at adjacent positions on the phenyl ring, can be prepared by reacting a compound represented by the aforementioned formula (I) with nitrogen monoxide. These compounds have strong fluorescent property as described later, and are useful for measurement of nitrogen monoxide.
  • the compounds represented by the formula (I) of the present invention have a property that they efficiently react with nitrogen monoxide under a neutral condition and provide compounds of the formula (II) wherein R 11 and R 12 combine together to form the group —N ⁇ N—NR 30 — which forms a ring structure at adjacent positions on the benzene ring in a good yield.
  • the compounds represented by the formula (I), per se, emit almost no fluorescence when irradiated with excitation light of around 485 nm under a neutral condition, whereas the compounds of the above formula (II) have the property of emitting extremely strong fluorescence under the same condition.
  • nitrogen monoxide in living tissues or cells can be measured by introducing the compound represented by the formula (I) into a living tissue or a cell to allow the compound to react with nitrogen monoxide to form the fluorescent compound of the above formula (II), and measuring the fluorescence of the compound.
  • the compounds of the formula (I) of the present invention have superior reactivity with nitrogen monoxide, and thus have an outstanding characteristic that they enable measurement of nitrogen monoxide with high sensitivity and accuracy.
  • the term “measurement” used in the specification should be construed in its broadest sense, which includes various measurement purposes such as, for example, detection, quantification, qualitative analysis and the like.
  • the above reaction can preferably be carried out under a neutral condition, for example, in a range of from pH 6.0 to 8.0, preferably in a range of from pH 6.5 to 7.8, and more preferably in a range of from pH 6.8 to 7.6.
  • a neutral condition for example, in a range of from pH 6.0 to 8.0, preferably in a range of from pH 6.5 to 7.8, and more preferably in a range of from pH 6.8 to 7.6.
  • the measurement of nitrogen monoxide using the compounds of the present invention is not limited to the measurements under the neutral range or weakly acidic range.
  • measurement can also be performed under a strongly acidic condition such as in gastric mucosal cells.
  • the compounds represented by the formula (II) of the present invention have an extremely superior characteristic that fluorescence intensity does not change in a wide range of pH, and thus they enable accurate measurement without being influenced by pH fluctuation.
  • the measurement of fluorescence can be carried out according to a known fluorometry method (see, for example, Wiersma, J. H., Anal. Lett., 3, pp. 123-132, 1970; Sawicki, C. R., Anal. Lett., 4, pp. 761-775, 1971; Damiani, P. and Burini, G., Talanta, 8, pp. 649-652, 1986; Damiani, P. and Burini, G., Talanta, 8, pp. 649-652, 1986; and Misko, T. P., Anal. Biochem., 214, pp. 11-16, 1993, and the like).
  • the nitrogen monoxide measurement according to the present invention for example, irradiation with light of about 520 nm as excitation light, and measurement of fluorescence of about 535 nm may preferably be performed.
  • the compounds represented by the formula (II) of the present invention have a superior characteristic that they give sufficient fluorescence intensity even with an excitation light having a long wavelength, and therefore they can reduce damages on the living body, tissues, cells, and the like. Further, by using the light having such a wavelength, efficient cut off can be obtained by using a fluorescence filter provided on an ordinary fluorescence microscope, and measurement with high sensitivity can be achieved without using an unordinary filter.
  • the aforementioned measurement of nitrogen monoxide may be carried out in the presence of an oxygen source.
  • an oxygen source for example, oxygen, ozone, oxide compounds or the like can be used.
  • oxygen dissolved oxygen can generally be used, and if desired, oxygen gas may be introduced into the reaction system, or an agent that can generate oxygen (e.g., hydrogen peroxide) may be added.
  • the oxide compounds are not particularly limited so long as they have an oxide bond that can easily be cleaved, e.g., N—O, S—O, or P—O.
  • PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, Maeda, H., et al., J. Leuk. Biol., 56, pp. 588-592, 1994; and Akaike, T., et al., Biochemistry, 32, pp. 827-832, 1993
  • carboxy-PTIO which has carboxyl group introduced at the para-position of the phenyl group of PTIO and the like
  • triphenylphosphine oxide triethylamine oxide or the like
  • PTIO and derivatives thereof are particularly preferred compounds, and they can be readily obtained by those skilled in the art (listed in, for example, Organic Chemicals Catalog, 32, 1994, Tokyo Kasei Co., Ltd.).
  • the oxide compounds, per se, may be used as a reaction agent, or those encapsulated in liposomes or other may also be used.
  • An amount of the oxygen source is not particularly limited. A preferable amount may be at least 1 ⁇ mol or more, preferably 10 to 30 ⁇ mol, and more preferably about 10 to 20 ⁇ mol, based on nitrogen monoxide to be measured.
  • the oxygen source For the measurement of a sample from a living body, from about 10 to 20 ⁇ mol of the oxygen source may preferably be added to the samples.
  • a required amount of the oxygen source is generally supplied by dissolved oxygen. If the amount of oxygen source is extremely small, measuring sensitivity may sometimes be lowered, and if an extremely large amount of oxygen source exists, emission of fluorescence may be disadvantageously affected. Therefore, it is preferred that an amount of nitrogen monoxide to be measured is predicted by a preliminary experiment or a known method so that the oxygen source within an appropriate concentration range can be added.
  • the reaction can be carried out at a temperature of from 10 to 25° C.
  • a mixed acid was prepared by adding fuming nitric acid (1 mL), which was added 3 times as divided portions, to concentrated sulfuric acid (6 mL) under ice cooling, and added portionwise with 4-acetamidobenzaldehyde (1.91 g, 11.7 mmol). After the addition, the reaction mixture was immediately poured onto ice, and the solids were collected by filtration. The solids were sufficiently washed with cold water, dried, purified by silica gel column chromatography (developing solvent: CH 2 Cl 2 ), and then recrystallized from water to obtain the target compound as pale yellow needlelike solid (1.57 g, yield: 64%).
  • Methyl 5-(benzyloxycarbonyl)-2,4-dimethyl-3-pyrrolepropionate (3.1 g, 9.8 mmol) was dissolved in acetone (100 mL). This solution was added with 10% Pd—C, and stirred at room temperature under hydrogen gas. When the starting material disappeared, the reaction mixture was filtered, and the filtrate was distilled under reduced pressure. The residue was added with trifluoroacetic acid (10 mL), and heated at 40° C. for 10 minutes under an argon flow. The reaction mixture was added with chloroform, and washed once with water, and the aqueous layer was extracted twice with chloroform.
  • 6-(4-Amino-3-nitrophenyl)-4,4′-bis(2-methoxycarbonylethyl)-3,3,5,5′-tetramethylpyrromethene (213 mg, 0.42 mmol) was dissolved in dichloromethane (20 mL). The solution was added with diisopropylethylamine (DIEA, 1 mL, 5.7 mmol) under an argon flow, and stirred at room temperature for 10 minutes. The solution was further added with boron trifluoride diethyl etherate (1 mL, 7.9 mmol), and stirred for 40 minutes. A little after the addition, fluorescence appeared.
  • DIEA diisopropylethylamine
  • the reaction mixture was washed once with water and twice with aqueous 2 N NaOH, and the aqueous layer and the NaOH layer were combined, and extracted 3 times with dichloromethane.
  • the fluorescence characteristics of Compound (7) and Compound (8) were measured.
  • the quantum yield was measured by using F-4500 (Hitachi), and the other spectra were measured by using LS50B (Perkin Elmer). The measurement was performed at 20° C. for a solution of a sample dissolved in 0.1 M sodium phosphate buffer (pH 7.4) using dimethyl sulfoxide of less than 0.2% as a cosolvent.
  • the quantum yield was calculated relative to that of fluorescein, which was 0.85 in aqueous 0.1 M NaOH. The results are shown in Table 1.
  • NOC 13 which is a nitrogen monoxide generating agent, was added to Compound (7), and changes of excitation and fluorescence emission spectra were measured.
  • the compound (7) at 5 ⁇ M (cosolvent: 0.1% DMSO) and NOC 13 were incubated at 37° C. for 1 hour in 0.1 M sodium phosphate buffer (pH 7.4), and then excitation and fluorescence spectra were measured. The measurement was performed with slit widths of Ex/Em 2.5/2.5 nm, excitation wavelength of 520 nm, and emission wavelength of 535 nm. The results are shown in FIG. 1 . Increase of fluorescence depending on the concentration of added NOC 13 was observed.
  • FIG. 2 The results obtained by incubating Compound (7) at 5 ⁇ M (cosolvent: 0.1% DMSO) and NOC 13 (20 ⁇ M) at 37° C. for 1 hour in 0.1 M sodium phosphate buffer (pH 7.4) and then analyzing the product by HPLC are shown in FIG. 2 .
  • a solution of DAMBO-P Me at 1 ⁇ M in chloroform was added with 5 ⁇ L/minute of a solution of NO in chloroform (prepared by bubbling argon gas in chloroform for 10 minutes and then bubbling NO gas in the same for 3 minutes), and the measurement was performed for 10 minutes.
  • FIG. 4 shows that measurement of NO in an organic solvent is also possible by using p-DAMBO-P Me . From the result, it can be understood that the compound of the present invention has cell membrane permeability, and achieves efficient detection of NO even in a liposoluble environment such as biomembranes.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Materials Engineering (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
US10/547,305 2003-02-28 2004-02-27 Fluorescent probes Abandoned US20060275912A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US12/688,625 US20100120160A1 (en) 2003-02-28 2010-01-15 Fluorescent Probe
US13/425,802 US8673957B2 (en) 2003-02-28 2012-03-21 Fluorescent probe

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2003052256 2003-02-28
JP2003-052256 2003-02-28
PCT/JP2004/002407 WO2004076466A1 (ja) 2003-02-28 2004-02-27 蛍光プローブ

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/688,625 Continuation US20100120160A1 (en) 2003-02-28 2010-01-15 Fluorescent Probe

Publications (1)

Publication Number Publication Date
US20060275912A1 true US20060275912A1 (en) 2006-12-07

Family

ID=32923393

Family Applications (3)

Application Number Title Priority Date Filing Date
US10/547,305 Abandoned US20060275912A1 (en) 2003-02-28 2004-02-27 Fluorescent probes
US12/688,625 Abandoned US20100120160A1 (en) 2003-02-28 2010-01-15 Fluorescent Probe
US13/425,802 Expired - Fee Related US8673957B2 (en) 2003-02-28 2012-03-21 Fluorescent probe

Family Applications After (2)

Application Number Title Priority Date Filing Date
US12/688,625 Abandoned US20100120160A1 (en) 2003-02-28 2010-01-15 Fluorescent Probe
US13/425,802 Expired - Fee Related US8673957B2 (en) 2003-02-28 2012-03-21 Fluorescent probe

Country Status (4)

Country Link
US (3) US20060275912A1 (ja)
EP (1) EP1604994A4 (ja)
JP (1) JP4522365B2 (ja)
WO (1) WO2004076466A1 (ja)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070298507A1 (en) * 2004-02-23 2007-12-27 Tetsuo Nagano Fluorescent Probe
US20090258434A1 (en) * 2005-11-14 2009-10-15 The University Of Tokyo Fluorescent probe for peroxynitrite
US8258171B2 (en) 2006-11-15 2012-09-04 The University Of Tokyo pH-sensitive fluorescent probe

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006306752A (ja) * 2005-04-27 2006-11-09 Kanagawa Acad Of Sci & Technol 化学発光性化合物及びそれから成る標識剤
JP2007238489A (ja) * 2006-03-07 2007-09-20 Kanagawa Acad Of Sci & Technol 化学発光性化合物及びそれから成る標識剤
US20100231125A1 (en) * 2009-03-12 2010-09-16 Sheng Li Organic light emitting device to emit in near infrared
KR101397530B1 (ko) 2012-08-10 2014-05-20 한국과학기술원 비대칭이고 치환 가능한 벤즈아줄렌 형태의 형광 화합물 및 그의 제조방법
CN103194214A (zh) * 2013-04-11 2013-07-10 武汉大学 一种用于一氧化氮检测的荧光探针
CN104194773B (zh) * 2014-08-26 2016-08-17 武汉大学 一种细胞膜靶向一氧化氮荧光探针及其制备方法和应用
CN106220664B (zh) * 2016-08-10 2018-06-22 浙江大学 一种检测自噬流的荧光探针及其制备和应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5248782A (en) * 1990-12-18 1993-09-28 Molecular Probes, Inc. Long wavelength heteroaryl-substituted dipyrrometheneboron difluoride dyes
US5874590A (en) * 1997-02-19 1999-02-23 Tetsuo Nagano Diaminofluorescein derivative
US5981746A (en) * 1997-05-30 1999-11-09 Avl Medical Instruments Luminescence indicator
US6201134B1 (en) * 1997-07-02 2001-03-13 Tetsuo Nagano Diaminorhodamine derivatives

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3967943B2 (ja) * 2002-03-22 2007-08-29 哲雄 長野 蛍光プローブ
FR2882056B1 (fr) * 2005-02-15 2007-06-01 Centre Nat Rech Scient Dipyrromethenes-bore borocarbones insatures
JP5228190B2 (ja) * 2005-11-14 2013-07-03 国立大学法人 東京大学 パーオキシナイトライト蛍光プローブ
EP2098529B1 (en) * 2006-11-15 2014-06-18 The University of Tokyo Ph-sensitive fluorescent probe

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5248782A (en) * 1990-12-18 1993-09-28 Molecular Probes, Inc. Long wavelength heteroaryl-substituted dipyrrometheneboron difluoride dyes
US5874590A (en) * 1997-02-19 1999-02-23 Tetsuo Nagano Diaminofluorescein derivative
US5981746A (en) * 1997-05-30 1999-11-09 Avl Medical Instruments Luminescence indicator
US6001999A (en) * 1997-05-30 1999-12-14 Avl Medical Instruments Luminescence indicator including ionophoric moiety
US6201134B1 (en) * 1997-07-02 2001-03-13 Tetsuo Nagano Diaminorhodamine derivatives

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070298507A1 (en) * 2004-02-23 2007-12-27 Tetsuo Nagano Fluorescent Probe
US7939330B2 (en) 2004-02-23 2011-05-10 Tetsuo Nagano Fluorescent probe
US20090258434A1 (en) * 2005-11-14 2009-10-15 The University Of Tokyo Fluorescent probe for peroxynitrite
US8178669B2 (en) 2005-11-14 2012-05-15 The University Of Tokyo Fluorescent probe for peroxynitrite
US8258171B2 (en) 2006-11-15 2012-09-04 The University Of Tokyo pH-sensitive fluorescent probe

Also Published As

Publication number Publication date
US8673957B2 (en) 2014-03-18
US20100120160A1 (en) 2010-05-13
JP4522365B2 (ja) 2010-08-11
EP1604994A1 (en) 2005-12-14
EP1604994A4 (en) 2009-03-25
US20120178174A1 (en) 2012-07-12
WO2004076466A1 (ja) 2004-09-10
JPWO2004076466A1 (ja) 2006-06-01

Similar Documents

Publication Publication Date Title
US8673957B2 (en) Fluorescent probe
EP1000941B1 (en) Diaminorhodamine derivatives
US8178669B2 (en) Fluorescent probe for peroxynitrite
US8143069B2 (en) Fluorescent probe and method of measuring hypochlorite ion
EP3636653A1 (en) Asymmetrical si rhodamine and rhodol synthesis
US7524876B2 (en) Luciferin derivatives
US7939330B2 (en) Fluorescent probe
EP1674579A1 (en) Fluorescent probe
JP3967943B2 (ja) 蛍光プローブ
US9688857B2 (en) Fluorescent probe
US7524974B2 (en) Fluorescent probe
CA2218641C (en) Diaminofluorescein derivative
EP1314730B1 (en) Reagent for determining singlet oxygen
US9250232B2 (en) Fluorescent probe
JP4279065B2 (ja) 亜鉛蛍光プローブ
US20040235902A1 (en) Fluorescent probes for zinc
CN118126032A (zh) 荧光探针及其制备方法和应用

Legal Events

Date Code Title Description
AS Assignment

Owner name: TETSUO NAGANO, JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:NAGANO, TETSUO;GABE, YU;URANO, YASUTERU;REEL/FRAME:018016/0020

Effective date: 20060509

Owner name: DAIICHI PURE CHEMICALS CO., LTD., JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:NAGANO, TETSUO;GABE, YU;URANO, YASUTERU;REEL/FRAME:018016/0020

Effective date: 20060509

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION