WO2006019105A1 - Agents de marquage fluorescent - Google Patents

Agents de marquage fluorescent Download PDF

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Publication number
WO2006019105A1
WO2006019105A1 PCT/JP2005/014983 JP2005014983W WO2006019105A1 WO 2006019105 A1 WO2006019105 A1 WO 2006019105A1 JP 2005014983 W JP2005014983 W JP 2005014983W WO 2006019105 A1 WO2006019105 A1 WO 2006019105A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
fluorescent labeling
labeling agent
fluorescent
compound
Prior art date
Application number
PCT/JP2005/014983
Other languages
English (en)
Japanese (ja)
Inventor
Tetsuo Nagano
Yasuteru Urano
Tomoko Mineno
Tasuku Ueno
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.
Priority to JP2006531814A priority Critical patent/JP5068535B2/ja
Publication of WO2006019105A1 publication Critical patent/WO2006019105A1/fr

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Classifications

    • 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
    • C09B11/06—Hydroxy derivatives of triarylmethanes in which at least one OH group is bound to an aryl nucleus and their ethers or esters
    • C09B11/08—Phthaleins; Phenolphthaleins; Fluorescein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present invention relates to a fluorescent labeling agent.
  • Fluorescence labeling is the detection of a target substance by combining a labeling agent such as an organic fluorescent dye with a biological substance that is the target substance, and utilizing the fluorescence properties such as excitation and fluorescence spectrum specific to the labeling agent. 'This is a method that enables quantification.
  • the fluorescent labeling agent should have a fluorescent group with strong fluorescence and a highly reactive active group (reactive functional group) that can react rapidly with the target biological substance. It is.
  • Fluorescein is a fluorescent dye having an absorption maximum of 492 nm and a fluorescence maximum of 515 nm, and is widely used as a parent nucleus of a fluorescent labeling agent with a high molecular yield of 0.85. Fluorescein is normally excited by a 488 nm argon laser and detected at 530 nm. Carboxyfluorescein is known as the most widely used fluorescent labeling agent to which this fluorescein is applied. This substance has a chemical structure in which a carboxyl group is introduced as a labeling site (reactive functional group) for binding to highly fluorescent fluorescein to proteins, etc. Are present (5-carboxyfluorescein and 6-carboxyfluorescein).
  • fluorescent labeling agents are often provided as succinimidyl esters or isothiocyanates for the purpose of modifying amino groups such as lysine residues in proteins.
  • the present inventors can further increase the sensitivity by optimizing the chemical structure of carboxyfluoresceins that have been widely used as fluorescent labeling agents from the viewpoint of photophysicochemistry. Research was conducted to develop a new fluorescent labeling agent.
  • the present inventors have found that the fluorescence quantum yield of carboxyfluorescein may decrease when it is converted to an ester or amide to label proteins using carboxyfluorescein. I found it. This may be due to photoinduced electron transfer.
  • R 4 each independently represents a hydrogen atom or a monovalent substituent, and at least one of them represents a reactive functional group that can be bonded to a labeled substance via a covalent bond
  • R 5 represents hydrogen.
  • R 6 and R 7 each independently represent a hydrogen atom or a halogen atom
  • R 8 represents a hydrogen atom, an alkyl carbo group, or an alkyl carbo group.
  • the fluorescent labeling agent R 5 is an alkyl group or an alkoxy group; the reactive functional groups, the fluorescent labeling agent is a carboxyl group or a reactive derivative thereof;
  • the fluorescent labeling agent in which the reactive functional group is an active ester group; the fluorescent labeling agent in which the labeling substance is a protein, nucleic acid, or lipid is provided.
  • the combination of Ri to R 5 is derived, for example, from the compound represented by the formula (I) after covalently bonding to the labeling substance. This is a combination in which the reduction potential of the benzene ring to which they are bonded is -2.1 V or less so that the residues are substantially highly fluorescent.
  • the present invention provides a method for fluorescently labeling a labeling target substance, which comprises the step of reacting the above fluorescent labeling agent with the labeling target substance.
  • the invention's effect [0009]
  • the fluorescent labeling agent of the present invention overcomes the decrease in fluorescence quantum yield due to labeling seen with carboxyfluorescein, and as a fluorescent labeling agent having a high quantum yield, protein or organic Useful for labeling compounds.
  • FIG. 1 is a graph showing an absorption spectrum of BSA modified with the fluorescent labeling agent of the present invention and a control carboxyfluorescein-SE.
  • FIG. 2 is a diagram showing the fluorescence spectrum (excitation wavelength: 490 nm) of BSA modified with the fluorescent labeling agent of the present invention and a control carboxyfluorescein-SE.
  • fluorescent labeling agent of the present invention is shown below.
  • a carboxyl group to be substituted on the benzene ring reacts with an amino group, a hydroxyl group, a thiol group, etc. present in the label target substance, whereby the label target substance can be fluorescently labeled.
  • each of the above compounds uses a carboxyl group on the benzene ring, and even if it is fluorescently labeled through a covalent bond such as an ester bond or an amide bond with the hydroxyl group-amino group of the label target substance. It has an excellent feature that the fluorescence intensity does not decrease and canoleboxoxif
  • the labeling agent may be a derivative of a carboxyl group on the benzene ring that can be bonded to the labeling substance via a covalent bond in the above compound.
  • the carboxyl group may be an acid neurogen. Or a mixed acid anhydride, or may form an active ester with nitro or halogen-substituted phenol, N-hydroxysuccinimide.
  • the carboxyl group that replaces the benzene ring is an active ester (for example, N-hydro A compound converted into xysuccinimidino estenole, p-nitropheneno estenole, pentafunololeorophenol ester, etc.) is preferred as the fluorescent labeling agent of the present invention.
  • an isocyanato group or an isothiocyanate group may be used as a functional group on the benzene ring that can be bonded to the labeling substance via a covalent bond.
  • Fluoroores and Their Amine— Reactive Derivatives 3 ⁇ 43 ⁇ 41 ⁇ (Thioto Reactive Probes), 5th Reagents for Reagents for Molecular Probes catalog (Handoook of Fluorescent Probes and Research Chemicals, Ninth Edition) Reactive functional groups described in Modifying Groups Other Than Tniols and A mines) can also be used.
  • an alkyl group or an alkoxy group is preferable, and a methyl group or a methoxy group can be used more preferably.
  • this new fluorescent labeling agent is advantageous from a synthetic point of view. That is, it is possible to obtain a single isomer with a limited substitution position of the force lpoxyl group in a high yield, and this point is also more useful than carboxyfluoresceins.
  • the fluorescent labeling agent of the present invention is a label for labeling substances (for example, various organic low molecules or organic polymer compounds in addition to biomolecules such as proteins, nucleic acids and lipids). It can be used for conversion.
  • the labeling method is not particularly limited, and can be performed according to the labeling method using carboxyfluorescein, which is a conventional fluorescent labeling agent.
  • tert-butyl ester 6 (271 mg, 1 mmol) was dissolved in tetrahydrofuran (20 mL) under an argon atmosphere. At -100 ° C, tert-butyllithium (1.48 M, 0.7 mL, 1 mmol) was slowly added and stirred at the same temperature for 30 minutes. To this solution was slowly added dropwise a solution of 3,6-bis- (tert-butyldimethylsila-loxy) -xanthen-9-one (91 mg, 0.2 mmol) in tetrahydrofuran (10 mL), and the container was aluminum. It was covered with foil and stirred for another hour at the same temperature. To the reaction solution, 2 N HC1 (5 mL) was stirred for 10 minutes. The reaction solution was diluted with saturated aqueous NaH 3 PO and extracted with ethyl acetate (3 X 15 mL).
  • tert-butyl ester 10 (287 mg, 1 mmol) was dissolved in tetrahydrofuran (20 mL) under an argon atmosphere. At -100 ° C, tert-butyllithium (1.48 M, 0.7 mL, 1 mmol) was slowly added and stirred at the same temperature for 30 minutes. In this solution, 3,6-bis- (tert- A solution of tildimethylsilyloxy) xanthen-9-one (91 mg, 0.2 mmol) in tetrahydrofuran (10 mL) was slowly added dropwise, the container was covered with aluminum foil, and the mixture was further stirred at the same temperature for 1 hour. To the reaction solution, 2 N HC1 (5 mL) was stirred for 10 minutes. The reaction solution was diluted with saturated aqueous NaH 3 PO and extracted with ethyl acetate (3 X 15 mL).
  • Pyrrolidine amide 14 (284 mg, 1 mmol) was dissolved in tetrahydrofuran (20 mL) in a well-dried container under an argon atmosphere. At -100 ° C, tert-butyllithium (1.48 M, 0.7 mL, 1 mmol) was slowly added and stirred at the same temperature for 30 minutes. To this solution was slowly added dropwise a solution of 3,6-bis- (tert-butyldimethylsila-loxy) -xanthen-9-one (91 mg, 0.2 mmol) in tetrahydrofuran (10 mL), and the container was covered with aluminum foil. The mixture was further stirred at the same temperature for 1 hour.
  • reaction solution 2 N HC1 (5 mL) was stirred for 10 minutes.
  • the reaction solution is diluted with saturated aqueous NaHPO solution, extracted with ethyl acetate (3 X 15 mL), and washed with saturated brine.
  • Example 6 Relationship between Fluorescence Quantum Yield of Compounds 11, 17, and 19, and 4-Methoxycarbo-fluorescein and Reduction Potential of Benzene Ring Site
  • Table 3 shows the relationship between the fluorescence quantum yields of compounds 11, 17, and 19, and 4-methoxycarbo-fluorescein and the reduction potential of the benzene ring moiety.
  • the reduction potential of the benzene ring moiety is a value measured in acetonitrile with A, B, and C, and in D aqueous solution with a saturated calomel electrode (SCE) as a reference electrode.
  • SCE saturated calomel electrode
  • the fluorescence quantum yield of compound 19 is high (0.673), compared to the fluorescence quantum yield (0.203) of 4-methoxycarbofluorescein.
  • the level of reduction potential at the benzene ring site of both compounds was similar. From these results, compounds with high electron density and which are not easily reduced are unlikely to cause photo-induced electron transfer (d-PeT) from the excited fluorophore (difficult to become a pet acceptor) and have high fluorescence quantum. This was considered to indicate the yield.
  • the reduction potentials of methyl ester of 4_carboxyl-sol and 3_carboxyl-anol were confirmed to be -2.54V and -2.34V, respectively. It was thought that it was less likely to be a PeT acceptor than the reduction potential of 2-methoxycarbo-naphthalene (C) at the benzene ring site of compound 19.
  • the fluorescent quantum yields of the esters of compounds 12 and 16 having boxyl-sole and 3-carboxyl-sole as the benzene ring moiety are 0.842 and 0.558, respectively, and the fluorescent quantum yield of 6-carboxyfluorescein methyl ester is Higher than rate. In this way, it was found that a combination of substituents that give a reduction potential to the benzene ring so as to be substantially high and fluorescent can be selected.
  • Compounds 12 and 16 are suitable as fluorescent labeling agents with high quantum yield.
  • Example 8 Fluorescence modification of BSA using the fluorescent labeling agent of the present invention
  • the fluorescent labeling agent of the present invention overcomes the decrease in the fluorescence quantum yield due to the labeling observed with carboxyfluorescein, and as a fluorescent labeling agent having a high quantum yield, proteins such as organic compounds Useful for labeling.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Organic Chemistry (AREA)
  • Pyrane Compounds (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

La présente invention décrit des agents de marquage fluorescent de formule générale (I) : (I) où : les groupements R1 à R4 représentent soit un hydrogène, soit un substituant monovalent, étant entendu que ces quatre groupements peuvent être différents les uns des autres, et à la condition qu'au moins l’un des groupements R1 à R4 soit un groupement fonctionnel réactif capable de se lier de façon covalente à la substance que l’on cherche à marquer. R5 représente un hydrogène ou un groupement monovalent autre que carboxy ou sulfo. R6 et R7 représentent soit un hydrogène, soit un halogène, ces deux groupements pouvant être différents l’un de l’autre. R8 représente un hydrogène, un alkylcarbonyle, ou un alkylcarbonyloxyméthyle. Les groupements R1 à R5 doivent être choisis de telle sorte que le potentiel de réduction du cycle benzénique auquel ils sont attachés soit suffisamment bas pour que la fluorescence de l’agent de marquage le formule générale (I) soit significativement élevée, lorsque ce dernier est lié de façon covalente au substrat que l’on cherche à marquer.
PCT/JP2005/014983 2004-08-17 2005-08-17 Agents de marquage fluorescent WO2006019105A1 (fr)

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Application Number Priority Date Filing Date Title
JP2006531814A JP5068535B2 (ja) 2004-08-17 2005-08-17 蛍光ラベル化剤

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US60198604P 2004-08-17 2004-08-17
US60/601,986 2004-08-17

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011149109A1 (fr) 2010-05-25 2011-12-01 三菱レイヨン株式会社 Substrat fluorescent pour la détection de l'activité enzymatique d'une enzyme agissant sur les nitriles
CN103224483A (zh) * 2013-05-06 2013-07-31 西北工业大学 用于标记寡糖的荧光化合物及其制备方法
US9051597B2 (en) 2010-05-25 2015-06-09 Mitsubishi Rayon Co., Ltd. Fluorescent substrate for detection of enzymatic activity of nitrile-related enzyme
US9657048B2 (en) 2014-08-04 2017-05-23 Auburn University Enantiomers of the 1′,6′-isomer of neplanocin A
CN106749153A (zh) * 2016-12-19 2017-05-31 华东理工大学 硝基还原酶的特异性荧光探针及其制备和用于肿瘤靶向荧光成像和监测肿瘤缺氧程度的应用
CN113999247A (zh) * 2021-11-03 2022-02-01 安徽理工大学 一种荧光酮类试剂的制备方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004005917A1 (fr) * 2002-07-08 2004-01-15 Daiichi Pure Chemicals Co., Ltd. Sonde fluorescente

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004005917A1 (fr) * 2002-07-08 2004-01-15 Daiichi Pure Chemicals Co., Ltd. Sonde fluorescente

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FEDIN A.V. ET AL: "Crystallographic characteristics of 2,3,7-trihydroxy-6-fluorone", KRISTALLOGRAFIYA, vol. 20, no. 1, 1975, pages 163 - 166, XP002995043 *
FOGL J. E AL: "Acid-base equilibriums in solutions of 2,6,7-trihydroxy-3H-xanhen-3-one derivatives", SBORNIK VYSOKE SKOLY CHEMICKO-TECHNOLOGICKE V PRAZE, H: ANALYTICKA CHEMIE, vol. H-16, 1981, pages 17 - 39, XP002995044 *
KULT K. ET AL: "Optimization of the spectrophotometric determination of germanium with 2-hydroxy-3-carboxyphenylfluorone in the presence of Septonex", SBORNIK VYSOKE SKOLY CHEMICKO-TECHNOLOGICKE V PRAZE H: ANALYTICKA CHEMIE, vol. H-21, 1986, pages 71 - 81, XP002995045 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011149109A1 (fr) 2010-05-25 2011-12-01 三菱レイヨン株式会社 Substrat fluorescent pour la détection de l'activité enzymatique d'une enzyme agissant sur les nitriles
US8697383B2 (en) 2010-05-25 2014-04-15 Mitsubishi Rayon Co., Ltd. Fluorescent substrate for detection of enzymatic activity of nitrile-related enzyme
US9051597B2 (en) 2010-05-25 2015-06-09 Mitsubishi Rayon Co., Ltd. Fluorescent substrate for detection of enzymatic activity of nitrile-related enzyme
CN103224483A (zh) * 2013-05-06 2013-07-31 西北工业大学 用于标记寡糖的荧光化合物及其制备方法
US9657048B2 (en) 2014-08-04 2017-05-23 Auburn University Enantiomers of the 1′,6′-isomer of neplanocin A
US10227373B2 (en) 2014-08-04 2019-03-12 Auburn University Enantiomers of the 1′,6′-isomer of neplanocin A
US10787478B2 (en) 2014-08-04 2020-09-29 Auburn University Enantiomers of the 1′,6′-isomer of neplanocin A
CN106749153A (zh) * 2016-12-19 2017-05-31 华东理工大学 硝基还原酶的特异性荧光探针及其制备和用于肿瘤靶向荧光成像和监测肿瘤缺氧程度的应用
CN106749153B (zh) * 2016-12-19 2020-10-09 华东理工大学 硝基还原酶的特异性荧光探针及其制备和用于肿瘤靶向荧光成像和监测肿瘤缺氧程度的应用
CN113999247A (zh) * 2021-11-03 2022-02-01 安徽理工大学 一种荧光酮类试剂的制备方法

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JPWO2006019105A1 (ja) 2008-05-08

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