USRE40572E1 - Fluorescent probe for the quantitation of zinc - Google Patents

Fluorescent probe for the quantitation of zinc Download PDF

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USRE40572E1
USRE40572E1 US11/808,125 US80812503A USRE40572E US RE40572 E1 USRE40572 E1 US RE40572E1 US 80812503 A US80812503 A US 80812503A US RE40572 E USRE40572 E US RE40572E
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compound
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zinc
hydrogen atom
salt
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Tetsuo Nagano
Kazuya Kikuchi
Tomoya Hirano
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Sekisui Medical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/10—Amino derivatives of triarylmethanes
    • C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/10—Amino derivatives of triarylmethanes
    • C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
    • C09B11/245—Phthaleins having both OH and amino substituent(s) on aryl ring
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/28—Pyronines ; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a fluorescent probe for zinc that emits fluorescence by specifically trapping a zinc ion.
  • Zinc is an essential metallic element that is present in the human body in the largest amount next to iron. Most zinc ions in cells strongly couple to proteins and are involved in the maintenance of structure or in the expression of function of the protein. Various reports have been also made on the physiological role of free zinc ions, which are present in the cell in a very small quantity (generally at a level of ⁇ M or lower). In particular, zinc ions are considered to be significantly involved in one type of cell death, i.e., apoptosis, and it is reported that zinc ions accelerate senile plaque formation in Alzheimer's disease.
  • a compound (a fluorescent probe for zinc), which specifically traps a zinc ion to form a complex and emits fluorescence upon the formation of the complex, has been conventionally used to measure zinc ions in tissue.
  • TSQ Tin ethyl ester
  • Zinquin ethyl ester Tinylaminoethylcyclen
  • the measurement using TSQ, Zinquin, or Dansylaminoethylcyclen requires the use of a short wavelength excitation light (an excitation wavelength of 367 nm, 368 nm, and 323 nm, respectively). Accordingly, when these fluorescent probes for zinc are used for measurement in living systems, the short wavelength excitation light may cause damages of cells (Saibou Kougaku (Cell Technology), 17, pp. 584-595, 1998). A problem also arises that the measurement may be readily influenced by autofluorescence generated from cell systems, per se (fluorescence emitted by NADH or flavin).
  • Dansylaminoethylcyclen has a drawback in that the fluorescence intensity is significantly varied depending on different environments in which the agent exists at the time of measurement, e.g., differences in environments such as a type of a solvent, or extracellular, intracellular, or intramembrane water solubility or lipophilicity or the like (Tanpakushitsu*Kakusan*Kouso (Protein, Nucleic Acid and Enzyme), extra number, 42, pp. 171-176, 1997).
  • TSQ has a problem in that even distribution in the whole cell is difficult due to its high lipophilicity.
  • Newport Green has low affinity for zinc ions and fails to achieve practical measurement sensitivity, although the agent enables measurement with a long wavelength excitation light. Therefore, the development of a fluorescent probe for zinc has been desired that can measure zinc ions with high sensitivity without damaging cells.
  • the inventors of the present invention have conducted various studies to achieve the foregoing objects. As a result, they found that a compound having a cyclic amine or a polyamine as a substituent has high specificity with zinc ions, and by trapping zinc ions, the compound forms a complex which emits strong fluorescence with a excitation light in longer wavelength range (Japanese Patent Application No. (Hei) 11-40325). The inventors have further conducts studies and found that a compounds represented by general formula (I) can form a complex with zinc very rapidly and can emit strong fluorescence.
  • the present invention thus provides a compound represented by general formula (IA) or (IB) or a salt thereof: wherein R 1 and R 2 independently represent a hydrogen atom or a group represented by formula (A): wherein X 1 , X 2 , X 3 , and X 4 independently represent a hydrogen atom, an alkyl group, a 2-pyridylmethyl group, or a protective group for an amine group, and m and n independently represent 0 or 1, provided that R 1 and R 2 do not simultaneously represent hydrogen atoms; R 3 and R 4 independently represent a hydrogen atom or a halogen atom; R 5 and R 6 independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxymethyl group, and R 7 represents a hydrogen atom or an alkyl group.
  • R 1 and R 2 independently represent a hydrogen atom or a group represented by formula (A): wherein X 1 , X 2 , X 3 , and X 4
  • a compound represented by general formula (II) or a salt thereof wherein R 13 and R 14 independently represent a hydrogen atom or a halogen atom; R 17 represents a hydrogen atom or an alkyl group; and R 18 represents a hydrogen atom or a protective group for an amino group.
  • R 13 and R 14 independently represent a hydrogen atom or a halogen atom
  • R 17 represents a hydrogen atom or an alkyl group
  • R 18 represents a hydrogen atom or a protective group for an amino group.
  • R 17 and R 18 independently represent hydrogen atoms.
  • the aforementioned compound or a salt thereof in which a substituted amino group on the benzene ring binds in m-position or p-position relative to the group represented by —COOR 17 .
  • the present invention provides a compound represented by general formula (IIIA) or (IIIB) or a salt thereof: wherein R 21 and R 22 independently represent a hydrogen atom or a group represented by formula (B): wherein X 11 , X 12 , X 13 , and X 14 independently represent a hydrogen atom, an alkyl group, a 2-pyridylmethyl group, or a protective group for an amino group, and p and q are independently 0 or 1, provided that R 21 and R 22 do not simultaneously represent hydrogen atoms; Y represents —CO—NH— or —NH—CO—; R 23 and R 24 independently represent a hydrogen atom or a halogen atom; R 25 and R 26 independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxymethyl group; and R 27 represents a hydrogen atom or an alkyl group.
  • R 21 and R 22 independently represent a hydrogen atom or a group represented by formula (B): wherein X 11
  • Y on the benzene ring binds in m-position relative to the group represented by —COOR 27 (the corresponding carbonyl group when a lactone ring is formed).
  • the present invention provides a fluorescent probe for zinc which comprises a compound represented by the general formulas (I), (II), or (III) (excluding the compound wherein a protective group for an amino group is introduced) or a salt thereof; and a zinc complex constituted by a compound represented by the general formula (I), (II), or (III) (excluding the compound wherein a protective group for an amino group is introduced) or a salt thereof together with a zinc ion.
  • the aforementioned fluorescent probe for zinc can be used for measuring zinc ions in tissues or cells.
  • a method for measuring zinc ions wherein a compound represented by the general formula (I), (II), or (III) (excluding the compound wherein a protective group for an amino group is introduced) or a salt thereof is used as a fluorescent probe for zinc; a method for measuring zinc ions which comprises the steps of: (a) reacting a compound represented by the general formula (I), (II), or (III) (excluding the compound wherein a protective group for an amino group is introduced) or a salt thereof with zinc ions; and (b) measuring fluorescence intensity of the zinc complex produced in the above step; and the use of a compound represented by the general formula (I), (II), or (III) (excluding the compound wherein a protective group for an amino group is introduced) or a salt thereof as a fluorescent probe for zinc.
  • the compound represented by the general formula (I), (II), or (III) (limited to the compound wherein a protective group for an amino group is introduced) is useful as a synthetic intermediate for the aforementioned fluorescent probe for zinc.
  • FIG. 1 shows that the fluorescent probe for zinc according to the present invention (Compound 6) has excellent zinc ion selectivity.
  • FIG. 2 shows that the fluorescent probe for zinc according to the present invention (Compound 12) has excellent zinc ion selectivity.
  • FIG. 3 shows that the fluorescent probe for Zinc according to the present invention (Compound 21) has excellent zinc ion selectivity.
  • FIG. 4 shows results of a comparison of changes with time in fluorescence intensity between the fluorescent probes for zinc according to the present invention (Compound 6 and Compound 12) and ACF-1 having a cyclic polyamine moiety.
  • FIG. 5 shows a correlation between fluorescence intensity of the fluorescent probes for zinc according to the present invention (Compound 6 and Compound 12) and zinc ion concentration.
  • FIG. 6 shows changes in fluorescence intensity of Compound 12, Compound 21, and zinc complexes thereof with relation to pH changes.
  • FIG. 7 shows a result of investigation on changes in fluorescence intensity by ischemic stimulus using a rat hippocampal slice.
  • FIG. 8 shows a result of investigation on changes in fluorescence intensity in each of regions by ischemic stimulus using a rat hippocampal slice.
  • Alkyl group or an alkyl moiety of a substituent containing the alkyl moiety (for example, an alkylcarbonyl group or an alkylcarbonyloxymethyl group) used in the specification means, for example, a linear, branched, or cyclic alkyl group, or an alkyl group comprising a combination thereof having 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms, and more preferably 1 to 4 carbon atoms. More specifically, a lower alkyl group (an alkyl group having 1 to 6 carbon atoms) is preferred as an alkyl group.
  • Examples of the lower alkyl groups include methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, cyclopropylmethyl group, n-pentyl group, and n-hexyl group.
  • a halogen atom means any of a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, and preferably means a fluorine atom, a chlorine atom, or a bromine atom.
  • Types of protective groups for amino groups are not particularly limited.
  • a p-nitrobenzenesulfonic acid group, a trifluoroacetyl group, and a trialkylsily group can be suitably used.
  • the protective groups for amino groups reference can be made to, for example, “Protective Groups in Organic Synthesis,” (T. W. Greene, John Wiley & Sons, Inc. (1981)).
  • the positions of R 1 and R 2 substituted on the benzene ring are not particularly limited.
  • R 1 may preferably bind in Meta-position or para-position relative to the group represented by —COOR 7 (or corresponding carbonyl group when a lactone ring is formed).
  • the position of an amino group substituted on the benzene ring in general formula (II) is not particularly limited. Meta-Position or para-position relative to the group represented by —COOR 17 is preferred.
  • the position of Y substituted on the benzene ring is not particularly limited. Y may preferably bind in meta-position relative to the group represented by —COOR 27 (or corresponding carbonyl group when a lactone ring is formed).
  • R 1 and R 2 is preferably a hydrogen atom and the other is preferably a group represented by formula (A).
  • group represented by the formula (A) from X 1 to X 4 , preferably X 1 and X 2 , independently represent a 2-pyridylmethyl group.
  • X 1 and X 2 independently represent a 2-pyridylmethyl group.
  • m is 0, n is 1, and X 4 is hydrogen atom.
  • both of X 1 and X 2 are preferably 2-pyridylmethyl group.
  • R 5 and R 6 are preferably hydrogen atoms, and R 5 and R 6 are preferably acetyl groups or acetoxymethyl groups for imaging application. It is preferred that both of R 3 and R 4 are hydrogen atoms or chlorine atoms. R 7 is preferably a hydrogen atom.
  • both of R 13 and R 14 are preferably hydrogen atoms or chlorine atoms.
  • R 17 and R 18 are preferably hydrogen atoms.
  • R 21 and R 22 is preferably a hydrogen atom and the other is preferably a group represented by formula (B).
  • group represented by the formula (B) from X 11 to X 14 , preferably X 11 and X 12 , independently represent a 2-pyridylmethyl group.
  • X 11 and X 12 are preferably 2-pyridylmethyl groups.
  • both of X 11 and X 12 are preferably 2-pyridylmethyl groups.
  • Both of R 23 and R 24 are preferably hydrogen atoms or chlorine atoms.
  • R 25 and R 26 are preferably hydrogen atoms, and R 25 and R 26 are preferably acetyl groups or acetoxymethyl groups for imaging application.
  • R 27 is preferably a hydrogen atom.
  • the compounds of the present invention represented by the general formulas (I) to (III) can exist as acid addition salts or base addition salts.
  • the acid addition salts include: mineral acid salts such as hydrochloride, sulfate, and nitrate; and organic acid salts such as methanesulfonate, p-toluenesulfonate, oxalate, citrate, and tartrate.
  • the base addition salts include: metal salts such as sodium salts, potassium salts, calcium salts, and magnesium salts; ammonium salts; and organic amine salts such as triethylamine salts.
  • salts of amino acids such as glycine may be formed.
  • the compounds or salts thereof according to the present invention may exit as hydrates or solvates, and these substances fall within the scope of the present invention.
  • the compounds of the present invention represented by general formulas (IA), (IB), (II), (IIIA), and (IIIB) may have one or more asymmetric carbons depending on the types of the substituents.
  • Stereoisomers such as optically active substances based on one or more asymmetric carbons and diastereoisomers based on two or more asymmetric carbons, as well as any mixtures of the stereoisomers, racemates and the like fall within the scope of the present invention.
  • R 7 , R 17 , or R 27 is hydrogen atom, a carboxyl group may form a lactone, and such structural isomers also fall within the scope of the present invention.
  • a compound represented by general formula (IA) in which R 5 is a hydrogen atom and a compound represented by general formula (IB) in which R 7 is a hydrogen atom are tautomers
  • a compound represented by general formula (IIIA) in which R 25 is a hydrogen atom and a compound represented by general formula (IIIB) in which R 27 is a hydrogen atom are tautomers.
  • 4-Aminofluorescein, 5-aminofluorescein, and 6-aminofluorescein, which can be used as starting compounds, can be prepared by methods described in, for example, “Yuuki Gousei Kagaku (Synthetic Organic Chemistry) IX,” (Tetsuji Kametani, Nankodo Co., Ltd., p. 215 (1977)).
  • the compound represented by the general formula (III) can be prepared by, for example, a method shows in the following scheme by using a commercially available compound and the like as a reagent and a starting reaction material.
  • the compounds of the present invention represented by the general formulas (I), (II), and (III) are useful as fluorescent probes for zinc.
  • the compounds of the present invention represented by the general formulas (I), (II), or (III) or salts thereof, per se, do not emit strong fluorescence, whilst they come to emit strong fluorescence after the formation of zinc complexes by trapping zinc ions.
  • the above compounds or salts thereof are featured that they can specifically trap zinc ions and form the complex very rapidly.
  • the formed zinc complexes is featured to emit strong fluorescence under a long wavelength excitation light which does not cause any damage to living tissues or cells.
  • the compounds of the present invention represented by the general formula (I), (II), or (III) or salts thereof are very useful as a fluorescent probes for zinc for measurement of zinc ions in living cells or living tissues under a physiological condition.
  • the term “measurement” used in the specification should be construed in its broadest sense, including quantitative and qualitative measurement.
  • the method for using the fluorescent probe for zinc according to the present invention is not particularly limited, and the probe can be used in the same manner as conventional zinc probes.
  • a substance selected from the group consisting of the compounds represented by the general formula (I) and salts thereof is dissolved in an aqueous medium such as physiological saline or a buffered solution, or in a mixture of the aqueous medium and a water-miscible organic solvent such as ethanol, actone, ethylene glycol, dimethysulfoxide, and dimethylformamide, and then the resultant solution is added to a suitable buffered solution containing cells or tissues and a fluorescence spectrum can be measured.
  • the zinc complexes of Compound 6 and Compound 12 shown in the above scheme have the excitation wavelengths of 491 nm and 492 nm, and the fluorescence wavelengths of 513 nm and 514 nm, respectively.
  • the compound is used at a concentration of about 1 to 10 ⁇ M, zinc ions with a concentration of 10 ⁇ M or below can be measured.
  • the fluorescent probe for zinc according to the present invention may be combined with a suitable additive to use in the form of a composition.
  • the fluorescent probe for zinc can be combined with additives such as a buffering agent, a solubilizing agent, and a pH modifier.
  • Compound 22 for example, has lipophilicity such a degree that it can easily permeate cell membranes. After Compound 22 permeates cell membranes, the compound is hydrolyzed by an esterase present in the cytoplasm, thereby Compound 12 is produced. Compound 12 can hardly permeate cell membranes due to its water-solubility, and for this reason, Compound 12 can be retained intracellularly for a prolonged period of time. Accordingly, Compound 22 is very useful for measurement of zinc ions existing in an individual cell.
  • Compound 6 obtained in Example 1 and Compound 12 obtained in Example 2 were used to evaluate selectivity for zinc ions.
  • 5 ⁇ M of Compound 6 or Compound 12 was added in 100 mM HEPES buffer (pH 7.5) containing various metal ions (5 ⁇ M or 5 mM).
  • the fluorescence intensity was measured at the excitation wavelength of 491 nm and the fluorescence wavelength of 513 nm for Compound 6, and the excitation wavelength of 492 nm and the fluorescence wavelength of 514 nm for Compound 12. The results are shown in FIG. 1 (Compound 6) and FIG. 2 (Compound 12).
  • the fluorescence intensities on the ordinate axis are shown as numerical values with addition of each metal ion relative to the fluorescence intensity without addition of metal ion which is taken as 1. It is clearly understood that Compound 6 and Compound 12 of the present invention have extremely high selectivity for zinc ions, and the compound give absolutely no increase of fluorescence intensity even in the presence of sodium ions, potassium ions, calcium ions, and magnesium ions at high concentration (5 mM), which exist in a living organism in large amounts. It is also clearly understood that these metal ions do not affect the increase in fluorescence intensity by zinc ions.
  • Compound 21 exhibited high selectively for zinc.
  • sodium, potassium, calcium, and magnesium at high concentration (5 mM) which are metal ions present abundant in living organisms, gives almost no increase in fluorescence intensity.
  • These metal ions did not affect the increase in fluorescence intensity caused by zinc.
  • Zinc ion (final concentration 5 ⁇ M or 50 ⁇ M) was added in 100 mM HEPES (pH 7.5) containing 5 ⁇ M Compound 6, Compound 12, or ACF-1 (a compound having a cyclic polyamine moiety described as Compound (20) in Example 1 in Japanese Patent Applications No.(Hei) 11-40325) to measure fluorescence intensity.
  • the fluorescence intensity was measured at the excitation wavelength of 491 nm and the fluorescence wavelength of 513 nm for Compound 6, the excitation wavelength of 492 nm and the fluorescence wavelength of 514 nm for Compound 12, and the excitation wavelength of 495 nm and the fluorescence wavelength of 515 nm for ACF-1.
  • the results are shown in FIG. 4 .
  • the ordinate axis represents relative fluorescence intensity.
  • the fluorescence intensity is not instantly increased by ACF-1, whilst fluorescence intensities were instantly increased by Compound 6 and Compound 12 of the present invention. Accordingly, the use of the compound according to the present invention enables very quick detection of zinc, and also enables the detection of rapid change in the concentration of zinc.
  • Zinc ions at various concentrations were added in 100 mM HEPES buffer (pH 7.5) containing 5 ⁇ M Compound 6, Compound 12, ACF-1, or Newport Green (Handbook of Fluorescent Probes and Research Chemicals, 6th Edition by Richard P. Haugland, pp. 531-540), and changes in fluorescence intensity were measured.
  • the fluorescence intensity was measured at the excitation wavelength of 491 nm and the fluorescence wavelength of 513 nm for Compound 6, the excitation wavelength of 492 nm and the fluorescence wavelength of 514 nm for Compound 12, the excitation wavelength of 495 nm and the fluorescence wavelength of 515 nm for ACF-1, and the excitation wavelength of 505 nm and the fluorescence wavelength of 530 nm for Newport Green.
  • the results are shown in FIG. 5 .
  • the fluorescence intensities on the ordinate axis are shown as numerical values with addition of each metal ion relative to the fluorescence intensity without addition of metal ion which is taken as 1.
  • Compound 6 and Compound 12 of the present invention exhibited a high detection sensitivity.
  • the detection sensitivity of Compound 12 was very high, which verifies an optimum combination of the chelater moiety and the fluorescence-emitting moiety of the compound.
  • Buffers used are as follows.
  • the fluorescence intensity of Compound 21 was more stable than Compound 12 at pH of around 7.4 which is an intercellular pH, which indicates that the probe is hardly influenced by intercellular pH changes.
  • the change in fluorescence intentiy by ischemic stimulus was investigated using a rat hippocampal slice.
  • Ringer's solutions having the following formulations were used.
  • the Ringer's solutions used for the preparation and measurement of the slices were kept under constant bubbling of 95% O 2 /5% CO 2 .
  • a Wistar rat (200 to 250 g, male) was anesthetized with ether. After decapitation, the whole brain was rapidly extirpated and put into the ice-cooled chorline-Ringer's solution, and allowed to stand for 10 minutes. After the brain was cut into left and right hemispheriums or Shale loaded with the ice-cooled choline-Ringer's solution and the sorbet-like choline-Ringer's solution, the interbrain was removed and the hippocampus was taken out using a spatula. The hippocampus was placed on agar and fixed to the agar with pins, and sliced in the width of 300 ⁇ m using a rotary slicer. The sliced hippocampus was put into the Ringer's solution heated to 30° C. and allowed to stand for 30 minutes to 1 hour. The sliced hippocampus was kept in the Ringer's solution at room temperature until it was put to use.
  • a 10 mM solution of Compound 22 dissolved in DMSO was diluted to 10 ⁇ M with the Ringer's solution.
  • the sliced hippocampus was put into the resulting solution and incubated under a shaded condition at room temperature for 1.5 hour. After the sliced hippocampus was put into the fresh Ringer's solution and washed for about 30 minutes to 1 hour and 30 minutes, and then transferred into a chamber and subjected to measurement.
  • the warmed Ringer's solution was circulated (flow rate of 2 to 3 ml/minute) in the chamber to constantly keep the temperature at 33 to 34° C. Measurement was carried out using an inverted microscope (Olympus IX-70, objective lens: 4x, dichroic mirror: 505 nm).
  • Ischemic stimulus was carried out by exchanging the Ringer's solution circulated in the chamber in the following manner.
  • Ringer's solution (95% O 2 +5% CO 2 bubbling): 2 minutes (01 00.00 to 02 00. 00 in the figure) ⁇ Ringer's solution for ischemia (95% N 2 +5% CO 2 bubbling): 10 minutes (03 00. 00 to 12 00. 00 in the figure) ⁇ Ringer's solution (95% O 2 +5% CO 2 bubbling): 4 minutes (13 00. 00 to 16 00. 00 in the figure).
  • the fluorescence intensity was most remarkably increased in the CA1 region (1, 2, 3 in the drawing) after ischemic stimulus as shown in FIG. 8 .
  • the fluorescence intensity was also increased in the CA3 region (site 4 in the figure) and the dentate gyrus (sites 6 and 7 in the figure).
  • the ordinate axis of the graphs shows fluorescence intensity at the time of initiation of measurement (0.00 sec) which is taken as 1.00.
  • the compound of the present invention is useful as a fluorescent probe for measurement of zinc. More specifically, the compound of the present invention is characterized to form a complex with zinc very quickly and detection sensitivity is very high. Accordingly, the compound of the present invention is very useful as an agent for accurately measuring rapid changes in concentration of zinc ions in a living organism.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Luminescent Compositions (AREA)
  • Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
  • Pyridine Compounds (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Plural Heterocyclic Compounds (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Investigating Materials By The Use Of Optical Means Adapted For Particular Applications (AREA)
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PCT/JP2001/001503 WO2001062755A1 (fr) 2000-02-28 2001-02-28 Sondes fluorescentes pour la quantification du zinc

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Cited By (4)

* Cited by examiner, † Cited by third party
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US20090263910A1 (en) * 2003-05-14 2009-10-22 Tetsuo Nagano Method for measuring hypochlorite ion
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Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE397208T1 (de) * 2000-02-28 2008-06-15 Daiichi Pure Chemicals Co Ltd Messverfahren basierend auf fluoreszenz- energie- transfer mit einem donor langer fluoreszenzlebenszeit
US7087766B2 (en) * 2000-02-29 2006-08-08 Daiichi Pure Chemicals Co., Ltd. Reagents for the quantitation of active oxygen
EP1403268A4 (fr) * 2001-06-14 2004-11-24 Daiichi Pure Chemicals Co Ltd Sondes fluorescentes pour le zinc
AU2002365683A1 (en) * 2001-12-04 2003-06-17 Jensen Denmark A/S A rotary ironer for ironing essentially rectangular pieces of cloth
EP1553408A4 (fr) * 2002-07-08 2005-08-17 Tetsuo Nagano Sonde fluorescente
EP1553409A1 (fr) * 2002-10-16 2005-07-13 Daiichi Pure Chemicals Co., Ltd. Reactifs servant a mesurer des peroxynitrites
US7696245B2 (en) 2003-03-28 2010-04-13 Sekisui Medical Co., Ltd. Fluorescent probe for zinc
JP2004315501A (ja) * 2003-03-28 2004-11-11 Tetsuo Nagano 亜鉛蛍光プローブ
US8445702B2 (en) * 2003-05-05 2013-05-21 Life Technologies Corporation Zinc binding compounds and their method of use
US20050250214A1 (en) * 2004-05-05 2005-11-10 Gee Kyle R Zinc binding compounds and their method of use
JP4713343B2 (ja) 2003-09-05 2011-06-29 哲雄 長野 蛍光プローブ
JP2005194244A (ja) * 2004-01-09 2005-07-21 Shigenobu Yano 亜鉛イオン蛍光センサー
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5208148A (en) 1990-12-07 1993-05-04 Molecular Probes, Inc. Lipophilic fluorescent glycosidase substrates
US5874590A (en) 1997-02-19 1999-02-23 Tetsuo Nagano Diaminofluorescein derivative
US6013802A (en) 1995-02-06 2000-01-11 Molecular Probes, Inc. Fluorescent conjugates of metal-chelating nitrogen heterocycles
JP2000239272A (ja) 1999-02-18 2000-09-05 Tetsuo Nagano 亜鉛蛍光プローブ
US6201134B1 (en) 1997-07-02 2001-03-13 Tetsuo Nagano Diaminorhodamine derivatives
US6441197B1 (en) 2000-01-20 2002-08-27 Daiichi Pure Chemicals Co., Ltd. Diaminofluorescein derivative
US6525088B1 (en) 1998-03-31 2003-02-25 Tetsuo Nagano Agent for measurement of singlet oxygen
US20030157727A1 (en) 2000-02-28 2003-08-21 Tetsuo Nagano Measuring method using long life fluorescence of excitation type
US7087766B2 (en) 2000-02-29 2006-08-08 Daiichi Pure Chemicals Co., Ltd. Reagents for the quantitation of active oxygen

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US157727A (en) * 1874-12-15 Improvement in water-indicators for steam-boilers
US153027A (en) * 1874-07-14 Improvement in egg and fruit carriers
US37332A (en) * 1863-01-06 Improvement in machines for drawing bolts and spikes
WO2000075150A1 (fr) 1999-06-02 2000-12-14 Daiichi Pharmaceutical Co., Ltd. Ligands du recepteur ip¿3?
US7696245B2 (en) 2003-03-28 2010-04-13 Sekisui Medical Co., Ltd. Fluorescent probe for zinc

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5208148A (en) 1990-12-07 1993-05-04 Molecular Probes, Inc. Lipophilic fluorescent glycosidase substrates
US6013802A (en) 1995-02-06 2000-01-11 Molecular Probes, Inc. Fluorescent conjugates of metal-chelating nitrogen heterocycles
US5874590A (en) 1997-02-19 1999-02-23 Tetsuo Nagano Diaminofluorescein derivative
US6201134B1 (en) 1997-07-02 2001-03-13 Tetsuo Nagano Diaminorhodamine derivatives
US6469051B2 (en) 1997-07-02 2002-10-22 Tetsuo Nagano Diaminorhodamine derivative
US6525088B1 (en) 1998-03-31 2003-02-25 Tetsuo Nagano Agent for measurement of singlet oxygen
JP2000239272A (ja) 1999-02-18 2000-09-05 Tetsuo Nagano 亜鉛蛍光プローブ
US6441197B1 (en) 2000-01-20 2002-08-27 Daiichi Pure Chemicals Co., Ltd. Diaminofluorescein derivative
US6569892B2 (en) 2000-01-20 2003-05-27 Daiichi Pure Chemicals Co., Ltd. Diaminofluorescein derivatives
US6833386B2 (en) 2000-01-20 2004-12-21 Daiichi Pure Chemicals Co., Ltd. Diaminofluorescein derivatives
US20030157727A1 (en) 2000-02-28 2003-08-21 Tetsuo Nagano Measuring method using long life fluorescence of excitation type
US7087766B2 (en) 2000-02-29 2006-08-08 Daiichi Pure Chemicals Co., Ltd. Reagents for the quantitation of active oxygen

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
Bioorg. Khim., vol. 21, No. 10, pp. 795-801 (1995).
English language abstract of JP 2000-239272.
G. Walkup et al., "A New Cell-Permeable Fluorescent Probe for Zn<SUP>2+</SUP>", J. Am. Chem. Soc., vol. 122, pp. 5644-5645 (2000).
G. Walkup et al., "A New Cell-Permeable Fluorescent Probe for Zn2+", J. Am. Chem. Soc., vol. 122, pp. 5644-5645 (2000).
H. Godwin et al., "A Fluorescent Zinc-Probe Based on Metal-Induced Folding", J. Am. Chem. Soc., vol. 118, pp. 6514-6515 (1996).
H. Kojima et al., "Detection and Imaging of Nitric Oxide with Novel Fluorescent Indicators: Diaminofluoresceins", Anal. Chem. vol. 70, No. 13, pp. 2446-2453 (1998).
H. Kojima et al., "Fluorescent Indicators for Imaging Nitric Oxide Production", Angew. Chem., Int. Ed. vol. 38, No. 21, pp. 3209-3212 (1999).
J. Wagner et al., "Synthesis of Five Enantiomerically Pure Haptens Designed for In Vitro Evolution of Antibodies with Peptidase Activity", Bioorganic & Medicinal Chemistry, vol. 4, No. 6, pp. 901-916 (1996).
J.G. Reyes et al., "A Fluorescence Method to Determine Picomole Amounts of ZN (II) in Biological Systems", Biol. Res., vol. 27, pp. 49-56 (1994).
M. Tsuda et al., "Expression of Zinc Transporter Gene, ZnT-1, is Induced after Transient Forebrain Ischemia in the Gerbil", Neurosci., vol. 17, No. 17, pp. 6678-6684 (1994).
R. Hauglang, Handbook of Fluorescent Probes and Research Chemicals, 6th Edition pp. 503 and 531-540.
S. Zhu et al., "Study of Bulk Morphology of Polymer Latex Films using Laser Confocal Fluorescence Microscopy" Sci. China, Ser. B: Chem vol. 41, No. 5, pp. 549-555 (1998).
Saibou Kougaku (Cell Technology), 17, pp. 584-595, 1998.
T. Hirano et al., "Highly Zinc-Selective Fluorescent Sensor Molecules Suitable for Biological Applications", J. Am. Chem. Soc., vol. 122, No. 49, pp. 12399-12400 (2000).
T. Koike et al., "A Novel Biomimetic Zinc (II)-Fluorophore, Dansylamidoethyl-Pendant Macrocyclic Tetraamine 1,4,7,10-Tetraazacyclododecane (Cyclen)", J. Am. Chem. Soc., vol. 118, pp. 12696-12703, 1996.
T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, Inc. pp. v-xxi and 369-405.
Tanpakushitsu Kakusan Kouso (Protein, Nucleic Acid and Enzyme), extra No., 42, pp. 171-176, 1997.
Tetsuji Kametani, Nankodo Co., Ltd., p. 215, 1997.
U.S. Appl. No. 10/204,417, filed Aug. 28, 2002 (National Stage of PCT/JP01/01504 filed Feb. 28, 2001) having the title "Agent for Measurement of Reactive Oxygen" (Applicants: Tetsuo Nagano et al.).
U.S. Appl. No. 10/204,418, filed Aug. 28, 2002 (National Stage of PCT/JP01/01502 filed Feb. 28, 2001) having the title "Method for Measurement by Using Longlived Excitation Fluorescence" (Applicants: Tetsuo Nagano et al.).

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090263910A1 (en) * 2003-05-14 2009-10-22 Tetsuo Nagano Method for measuring hypochlorite ion
US20110117666A1 (en) * 2007-03-01 2011-05-19 The University Of Tokyo Fluorescent probe
US8465985B2 (en) 2007-03-01 2013-06-18 The University Of Tokyo Fluorescent probe
US20110159603A1 (en) * 2008-03-04 2011-06-30 The University Of Tokyo Fluorescent probe specific to hydrogen peroxide
US8394850B2 (en) * 2008-03-04 2013-03-12 The University Of Tokyo Fluorescent probe specific to hydrogen peroxide
US20110287552A1 (en) * 2008-12-05 2011-11-24 The University Of Tokyo Reagent for measuring active nitrogen
US8895317B2 (en) * 2008-12-05 2014-11-25 The University Of Tokyo Reagent for measuring active nitrogen

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US20030162298A1 (en) 2003-08-28
AU3599701A (en) 2001-09-03

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