WO2006019105A1 - Fluorescent labeling agents - Google Patents

Abstract

Fluorescent labeling agents represented by the general formula (I): (I) wherein R1 to R4 are each independently hydrogen or a monovalent substituent, with the proviso that at least one of R1 to R4 is a reactive functional group capable of binding to a substance to be labeled through a covalent bond; R5 is hydrogen or a monovalent group exclusive of carboxy and sulfo; R6 and R7 are each independently hydrogen or halogeno; and R8 is hydrogen, alkylcarbonyl, or alkylcarbonyloxymethyl, with the proviso that R1 to R5 must be a combination which can give the benzene ring to which they are bonded such a low reduction potential that the compound represented by the general formula (I) can exhibit substantially high fluorescence in a state bound to the substrate to be labeled through a covalent bond.

Description

 Specification

 Fluorescent labeling agent

 Technical field

 [0001] The present invention relates to a fluorescent labeling agent.

 Background art

 [0002] The detection and quantification of minute amounts of biological substances such as proteins and physiologically active substances with high sensitivity and high accuracy is an important research subject in the development of chemical biology and biotechnology. For this purpose, fluorescent labeling technology is widely used. 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. For this purpose, 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.

[0003] 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). However, it is difficult to separate and synthesize these two isomers, and in most cases they are used as fluorescent labeling agents in a mixture. In addition, fluorescent labeling agents are often provided as succinimidyl esters or isothiocyanates for the purpose of modifying amino groups such as lysine residues in proteins.

 Fluorescein 5-Carboxyfluorescein (5CF) 6-Carboxyfluorescein (6CF)

 Disclosure of the invention

 Problems to be solved by the invention

 [0004] 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.

 [0005] On the other hand, the present inventors have recently found that the 2-position carboxyl group, which has been considered to be essential for the fluorescence properties of fluorescein, can be replaced with other substituents. (WO2004 / 005917). The fluorescence properties of the compound in which the carboxyl group of fluorescein is substituted with a methyl group or a methoxy group show the same excitation wavelength and fluorescence quantum yield as fluorescein! did.

 Combined with the above findings, we adopted a method that considers fluorescein divided into a xanthene moiety, which is a fluorophore, and a benzene ring moiety perpendicular to this, and the carboxyl group at the 2-position of the benzene ring part of fluorescein is a methyl group or a methoxy group. To increase the electron density at the benzene ring site, making it less likely to cause photo-induced electron transfer (d-PeT) from the excited fluorophore, and is not observed with carboxyfluorescein. We succeeded in developing a novel fluorescent labeling agent that overcomes the decrease in fluorescence quantum yield due to improved labeling.

[0006] According to the present invention, the following formula (I): [Chemical 2]

R⁴はそれぞれ独立に水素原子又は一価の置換基を示すが 、これらのうち少なくとも 1個は、被ラベル化物質に共有結合を介して結合可能な反 応性官能基を示し; R⁵は水素原子、カルボキシル基またはスルホン酸基以外の一価 の基を示し; R⁶及び R⁷はそれぞれ独立に水素原子又はハロゲン原子を示し; R⁸は水 素原子、アルキルカルボ-ル基、又はアルキルカルボ-ルォキシメチル基を示し、た だし、

R⁴、及び R⁵の組み合わせは、式 (I)で表される化合物が被ラベル 化物質に共有結合した後、実質的に高い蛍光性になるように、それらが結合するべ

、還元電位を与える組み合わせである）で表される蛍光ラベル化剤が 提供される。 (

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. Represents a monovalent group other than an atom, a carboxyl group or a sulfonic acid group; 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. -Represents a ruoxymethyl group, provided that

The combination of R ⁴ and R ⁵ should be bound so that the compound represented by formula (I) becomes substantially highly fluorescent after covalently binding to the labeling substance.

, A combination that gives a reduction potential).

[0007] According to a preferred embodiment of the invention, 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.

In the compound represented by the formula (I), 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.

 From another viewpoint, 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.

 Brief Description of Drawings

 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.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0011] Representative examples of the fluorescent labeling agent of the present invention are shown below. In these compounds, 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.

 [Chemical 3]

特に、上記の各化合物は、ベンゼン環上のカルボキシル基を使用し、被ラベルイ匕 物質の水酸基ゃァミノ基との間でエステル結合又はアミド結合などの共有結合を介し て蛍光ラベルイ匕しても実質的に蛍光強度が低下しないという優れた特徴を有しており 、カノレボキシフ

In particular, 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

 Highly sensitive labeling of target substances is possible compared to lourescein.

[0012] 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. For example, 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. In particular, 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. Further, for example, 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. In addition, Fluoroores and Their Amine— Reactive Derivatives, ¾¾1 皁 (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.

 As the substituent at the 2-position on the benzene ring, for example, an alkyl group or an alkoxy group is preferable, and a methyl group or a methoxy group can be used more preferably.

 As shown below, when a conventional carboxyfluorescein is used as a fluorescent labeling agent for a hydroxyl group, a remarkable decrease in the fluorescence quantum yield is observed, but the compound 12 of the present invention was used as a fluorescent labeling agent. In some cases, a high quantum yield is maintained after labeling.

[Chemical 4]

 6CF 6CF-Ester

 φ = 0.87 φ = 0.20 Furthermore, 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.

Example

 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples.

Example 1: Production of the fluorescent labeling agent of the present invention

<Scheme 1>

[Chemical 5]

(In the scheme, Me represents a methyl group, and represents a tert-butyl group. The same shall apply hereinafter.) (A) 3-bromo-4-methylbenzoic acid tert-butyl ester (2)

 Dissolve 3-bromo-4-methylbenzoic acid (1) (430 mg, 2 mmol) in tert-butanol (10 mL) in a well-dried container and add Boc 0 (Boc = tert-butoxycarbo) under an argon atmosphere. -Le, 0

 2

.46 mL, 2 mmol) and dimethylaminopyridine (DMAP, 73 mg, 0.6 mmol) were added. The reaction was carried out overnight with stirring at 40 ° C. After diluting the reaction solution with water, D /:-邈 彔 '¾ ^-ε- rci i- κα) [8ioo]

· Ϊ́060 "^ ε punoj O260" ^ S Ο Η D ^ ορο ₊ [Η +] (S -IS3

) S Η '(HS' ΐ0 '(Hf' ω) 9-8Γ9 '(0 ・ 6 = f' ΗΖ 'Ρ) 86 · 9' (ε ・ 8 = f 'Ηΐ' Ρ) WL

'(9 · ΐ = [' Ηΐ 'Ρ) Ϊ8 "Ζ' (Ζ · ΐ '6" Ζ = [' Ηΐ 'ΡΡ) 80 · 8 9 (OS' ζ 00S) Wake up Η _τ

. (% 86 ¾τ) §ω is ^^) ^^^ ((2 ^; 8)

 / Be! ^ ： 瀚 缀 ffi 缀) つ Het ^ — ^: ^^ mu! ^ / F, ¾¾ ί ^

 Ι

 ^) TOO]

• SISrSO punoj '9 rS0 OH. . ₊ [H +] (S -IS3) S HH '(H6' ^s ) 8

S'l '(HS' s) 0Z-9-Z9 "9 '(0 · 6 = f' HS 'Ρ) 20"Z' (ΐ · 8 = f Ήΐ 'Ρ) 6S'Z' (8 • ΐ = ΓΗΐ 'Ρ) 08 "Ζ' (8 · ΐ 'ΐ · 8 = ΓΗΐ' ΡΡ) 0Γ8 9 (' ^ζ 00S) Awakening Η _τ

、つ（ΐ:6= /. ¾¾ (% S6 ¾τ) §ω 9

, One (ΐ: 6 = /

— ^ / Be ^ /! ^: 瀚 缀 ffi 缀) joy e ^ — ^: ^^ mu! ^ / F, ¾¾

 ¾ 爵 单 斜 单 i ^^^^ os ^ N 氺 雜 etsu ¾ ^ 氺 ^ Tough

、ェ 醫D Π ¹¹¹ eixs) 邈, Π 缀 氺 od Η¾Να ^ ¾ ¾¾¾¾ίΗ. · Π

ェ

, / Ma-/ ¾ 醬, Stopping; C)> ^ (τιι Οΐ) ^ ^^ Q) (ΐο

UIUI z'O 'Sui 16) Be-6-Begin ^ (^^ / ^ ^:--9 Co 5 继 缀 °: · Πφί ί ¾ ^^ 0ε "¾ ^ ίΤ6)> ^ Φ¾α ^ο111111 ΐ Ζ' Ma ^ (Η,

 douiui τ 'Sin HZ) Ζ ： # D ^:-Stop ^ 蹈 Beco) 醬 兹 つ 1ί¾> Τ

 s ^^ Ke) [9 TOO]

• (H6 ^<s ) 8S'I '(HS' s) ZVZ (Γ8 = f 'HI' P) 9

Z'L '(Ζ · ΐ' 6 "Ζ = f 'Ηΐ' ΡΡ) Ϊ8" Ζ '(Ζ · ΐ = f' Ηΐ 'Ρ) 2Γ8 9 ODOD' ^ Η 00S) WiH Η _τ

ΟΪ χε) / . % ΐ9? §Ω τεε z-^^ m ^, (σ: 6) 邈 4§ / Be ^ ^ 萆. ^

ΟΪ χε) /

C86M0 / S00Zdf / X3d L S0T610 / 900Z OAV Dissolve 4-bromo-3-methylbenzoic acid (5) (430 mg, 2 mmol) in tert-butanol (10 mL) in a well-dried container and boc 0 (0.46 mL, 2 mmol) under argon. And DMAP (73

 2

 mg, 0.6 mmol) was added. The reaction was carried out overnight with stirring at 40 ° C. The reaction solvent was diluted with water, extracted with ethyl acetate (3 × 10 mL), washed with water and saturated brine, and dried over anhydrous Na 2 SO 4. The organic solvent was distilled off and the resulting residue was applied to a silica gel column.

 twenty four

 Purification (elution solvent: hexane / ethyl acetate (9: 1)) gave colorless oily compound 6 342 mg (yield 63%).

^J H NMR (300 MHz, CDC1) δ 7.83 (d, 1Η, J = 2.0), 7.64 (dd, 1H, J = 8.3, 2.0), 7.5

 Three

 6 (d, 1H, J = 8.3) 2.43 (s, 3H), 1.58 (s, 9H).

[0019] (E) Compound 7

 In a well-dried container, 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).

 twenty four

 Washed with water and dried over anhydrous Na 2 SO 4. The organic solvent was distilled off, and the resulting residue was

 twenty four

 Purification by gel chromatography (elution solvent: dichloromethane / methanol (9: 1)) gave orange solid 768 mg (yield 85%).

 1H NMR (300 MHz, methanol—d) δ 8.05 (s, 1H), 8.01 (d, 1H, J = 8.1), 7.36 (d, 1

 Four

H, J = 7.9), 7.03 (d, 2H, J = 9.2), 6.68-6.71 (m, 4H), 2.12 (s, 3H), 1.65 (s, 9H); HR MS (ESI-MS) (M + H] ⁺ calcd for CHO 403.1546, found 403.1501.

 25 23 5

 [0020] (F) Compound 8

Compound 7 (60 mg, 0.15 mmol) was dissolved in trifluoroacetic acid / dichloromethane (1: 1, 10 mL) in a well-dried container and stirred at room temperature for 1 hour under an argon atmosphere. The organic solvent was distilled off, and the resulting residue was purified by silica gel chromatography (elution solvent: dichloromethane / methanol (8: 2)) to give an orange solid compound 8 49 mg (yield 94%) Got. H NMR (300 MHz, DMSO- d) δ 8.05 (s, IH), 7.98 (d, IH, J = 8.0), 7.43 (d, IH, J

6

 = 7.9), 6.96 (d, 2H, J = 9.2), 6.77-6.73 (m, 4H), 2.07 (s, 3H); HRMS (ES to MS) [M + H] + calcd for CHO 347.0920, found 347.0897.

 21 15 5

Example 2: Fluorescence characteristics

 The quantum yield of each compound is dissolved in 0.1M phosphate buffer at pH 7.4 (pH 9.0 for 6CFs) so that the absorbance at 490 nm is 0.02 or less, and fluorescein in 0.1N NaOH aqueous solution. Was determined by a relative method as a control (fluorescence quantum yield = 0.85).

[table 1]

例 3 :本発明の蛍光ラベル化剤の製造

Example 3: Production of fluorescent labeling agent of the present invention

<Scheme 2>

[Chemical 6]

 17

 [0023] (A) 3-bromo-4-methoxybenzoic acid tert-butyl ester (10)

 Dissolve 3-bromo-4-methoxybenzoic acid (9) (462 mg, 2 mmol) in tert-butanol (10 mL) in a well-dried container and boc 0 (0.46 mL, 2 mmol) under an argon atmosphere. And DMAP (73

 2

 mg, 0.6 mmol). The mixture was refluxed for 10 hours with stirring. The reaction solution was diluted with water, extracted with ethyl acetate (3 X 10 mL), washed with water and saturated brine, and dried over anhydrous Na 2 SO 4.

 2 4 dried. The organic solvent was distilled off, and the resulting residue was purified by silica gel chromatography (elution solvent: hexane / ethyl acetate (9: 1)) to give a white solid compound 10 356 mg (yield 62%) Got.

^J H NMR (300 MHz, CDCl) δ 8.15 (d, IH, J = 2.0), 7.93 (dd, IH, J = 8.6, 2.0), 6.8

 Three

 9 (d, IH, J = 8.6) 3.94 (s, 3H), 1.58 (s, 9H).

[0024] (B) Compound 11

In a well-dried container, 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).

twenty four

 Washed and dried over anhydrous Na 2 SO 4. The organic solvent was distilled off, and the resulting residue was

 twenty four

 Purification by oral chromatography (elution solvent: dichloromethane / methanol (9: 1)) gave 48 mg of orange solid compound 11 (yield 57%).

^J H NMR (300 MHz, methanol—d) δ 8.24 (dd, 1Η, J = 8.8, 2.2), 7.82 (d, 1H, J = 2.

 Four

2), 7.35 (d, 1H, J = 8.8), 7.13 (d, 2H, J = 9.3), 6.74-6.70 (m, 4H), 3.82 (s, 3H), 1.5 8 (s, 9H); HRMS (ES MS) [M + H] ⁺ calcd for CHO 419.1495, found 419.1461.

 25 23 6

 [0025] (C) Compound 12

 Compound 11 (63 mg, 0.15 mmol) was dissolved in trifluoroacetic acid / dichloromethane (1: 1, 10 mL) in a well-dried container and stirred at room temperature for 1 hour in an argon atmosphere. The organic solvent was distilled off, and the resulting residue was purified by silica gel chromatography (elution solvent: dichloromethane / methanol (8: 2)) to give orange solid compound 12 45 mg (yield 83%) Got.

 JH NMR (300 MHz, DMSO— d) δ 8.20 (dd, 1H, J = 8.8, 2.2), 7.82 (d, 1H, J = 2.0),

 6

7.43 (d, 1H, J = 8.8), 7.09 (d, 2H, J = 9.7), 6.76-6.73 (m, 4H), 3.78 (s, 3H); HRMS (ESI— MS) [M + H] ⁺ calcd for CHO 363.0869, found 363.0840.

 21 15 6

 [0026] (D) Compound 14

 In a well-dried container, 4-bromo-3-methoxybenzoic acid (13) (462 mg, 2 mmol) is dissolved in dimethylformamide (10 mL), and carbodiimide (CDI, 486 mg, 3 mmol), DMAP (367 mg, 3 mmol) and pyrrolidine (0.33 mL, 4 mmol) were added. The reaction was carried out overnight with stirring at room temperature. The reaction solution was diluted with water, extracted with ethyl acetate (3 × 10 mL), washed with water and saturated brine, and dried over anhydrous Na 2 SO 4. Organic solvent

 twenty four

 The residue obtained was purified by silica gel chromatography (elution solvent: hexane / ethyl acetate (2: 1 to 1: 1)), and white solid compound 14 488 mg (yield 86 %). JH NMR (300 MHz, CDC1) δ 7.40 (d, 1H, J = 8.1), 6.96 (d, 1H, J = 1.7), 6.83 (dd,

 Three

1H, J = 8.0, 1.8) 3.77 (s, 3H), 3.47 (t, 2H, J = 6.6), 3.28 (t, 2H, J = 6.4), 1.86—1.71 ( m, 4H).

[0027] (E) Compound 15

 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. To the 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.

twenty four

 Clean and dry with anhydrous Na 2 SO 4. The organic solvent was distilled off, and the resulting residue was

 twenty four

 Purification by matrix chromatography (elution solvent: dichloromethane / methanol (9: 1)) gave 15 35 mg of orange solid compound (yield 42%).

^J H NMR (300 MHz, methanol—d) δ 7.37 (s, 1H), 7.32 (s, 2H), 7.00 (d, 2H, J = 9.

 Four

 0), 6.56-6.52 (m, 4H), 3.79 (s, 3H), 3.68—3.60 (m, 4H), 2.07—1.99 (m, 4H); HRMS (ESI-MS) [MH] ”calcd for CHNO 414.1342, found 414.1332.

 25 20 1 5

 [0028] (F) Compound 16

 Compound 15 (62 mg, 0.15 mmol) was dissolved in 1 N NaOH (10 mL) and refluxed for 2 hours. The reaction solution was neutralized with 2 N HC1, extracted with ethyl acetate (3 × 15 mL), washed with saturated brine, and dried over anhydrous Na 2 SO 4. The organic solvent was distilled off, and the resulting residue was

 twenty four

 Purification by GRAPHI (elution solvent: dichloromethane / methanol (8: 2)) gave 1649 mg (yield 90%) of an orange solid compound.

 1H NMR (300 MHz, methanol-d) δ 7.82 (d, 1H, J = 1.1), 7.75 (dd, 1H, J = 7.7, 1.

 Four

 3), 7.21 (d, 1H, J = 7.9), 7.02 (d, 2H, J = 9.0), 6.56-6.51 (m, 4H), 3.79 (s, 3H); HR MS (ESI-MS) (MH ] "calcd for CHO 361.0712, found 361.0704.

 21 13 6

 [0029] (G) Compound 17

 Dissolve Compound 16 (36 mg, 0.10 mmol) ert-butanol (5 mL) in a well-dried container and add Boc 0 (0.11 mL, 0.50 mmol) and DMAP (18 mg, 0.15 mmol) under an argon atmosphere.

 2

Yeah. The reaction was carried out overnight with stirring at room temperature. The reaction solution was diluted with water Then, extract with ethyl acetate (3 X 5 mL), wash with saturated brine, and dry over anhydrous Na SO.

 2 4 The organic solvent was distilled off, and the resulting residue was purified by silica gel chromatography (eluent: dichloromethane / methanol (9: 1)) to give an orange solid compound 17 12 mg (yield 29%) Got.

 1H NMR (300 MHz, methanol-d) δ 7.79-7.77 (m, 2H), 7.33 (d, 1H, J = 8.3), 6.94

 Four

(d, 2H, J = 9.2), 6.56-6.50 (m, 4H), 3.80 (s, 3H); HRMS (ESI-MS) [M + H] ⁺ calcd for CHO 419.1495, found 419.1489.

 25 23 6

 [0030] Example 4: Fluorescence properties

 The quantum yield of each compound is dissolved in 0.1M phosphate buffer at pH 7.4 (pH 9.0 for 6CFs) so that the absorbance at 490 nm is 0.02 or less, and fluorescein in 0.1N NaOH aqueous solution. Was determined by a relative method as a control (fluorescence quantum yield = 0.85).

 [Table 2]

 [0031] Example 5: Synthesis of Compound 19

 [Chemical 7]

化合物 18は J. Am. Chem. So ， 123, pp.2530-2536(2001)に記載の方法で合成し た。

Compound 18 was synthesized by the method described in J. Am. Chem. So, 123, pp.2530-2536 (2001).

 Compound 18 was dissolved in methanol, a few drops of sulfuric acid were added, and the mixture was refluxed for 20 hours. Next, the reaction solution was poured into ice, extracted with ethyl acetate, and dried over anhydrous Na 2 SO 4. Organic solvent

 twenty four

The residue was purified by silica gel chromatography and recrystallized from methanol / dichloromethane to obtain Compound 19. H NMR (300 MHz, DMSO— d): δ 3.63 (s, 3H); 6.30 (br, 2H); 6.84 (m, 4H); 7.76 (m

 6

 , 2H); 8.06 (m, 2H); 8.30 (m, IH); 8.89 (s, IH); 11.04 (br, IH) .HRMS (ESI +): calcd for M ++ 1, 397.107; found, 397.105. Mp: 325 ° C dec. Anal. Calcd for CHO -0.75

 25 16 5

H O: N; 0, C; 73.25, H; 4.30. Found: N, 0; C; 73.43, H; 4.37.

 2

 [0032] 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]

[0033] 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.

 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.

[0034] As another example of the benzene ring site, the reduction potentials of methyl ester of 4_carboxyl-sol and 3_carboxyl-anol (A and B) 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. 4-cal 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 7: Synthesis of succinimidyl esters (SE)

[Chemical 8]

3 eq. NHS

 3 eq. WSCD

-D 2 t

10 eq. NHS

 10 eq. WSCD

 DMF

 r. t., 1 h

 18%

16 (47b) Compound 4 (2la), Compound 8 (2lb), Compound I2 (47a), Compound I6 (47b), l mmol, N-hydroxysuccinimide (NHS) 2, 3 or 10 mmol each in anhydrous dimethylformamide 5 mL Into these solutions, add 2, 3 or 10 mmol of water-soluble carbodiimide (WSCD: N- (3-dimethylaminopropyl) -Ν'-ethyl carpositimide) under ice-cooling. . C for 1 or 2 hours. After the reaction, an aqueous phosphoric acid solution was added, extracted with methylene chloride and dried. The solvent was distilled off under reduced pressure to obtain the desired succinimidyl ester. If necessary, the target product was purified using a silica gel force ram.

[0037] 21aSE

^J H NMR (300 MHz, methanol—d) δ 8.28 (dd, 1H, J = 8.2, 1.8), 8.02 (d, 1H, J = 1.

 Four

7), 7.74 (d, 1H, J = 8.2), 7.06 (d, 2H, J = 9.3), 6.77-6.73 (m, 4H), 2.89 (s, 4H), 2.1 9 (s, 3H); HRMS (ES MS) [M + H] ⁺ calcd for CH NO + 444.1083, found 444.1117.

 25 18 7

 21bSE

^J H NMR (300 MHz, methanol—d) δ 8.26 (s, 1H), 8.19 (d, 1H, J = 9.2), 7.52 (d, 1

 Four

H, J = 7.9), 7.06 (d, 2H, J = 9.0), 6.77-6.73 (m, 4H), 2.94 (s, 4H), 2.17 (s, 3H); HR MS (ESI-MS) (M + H] ⁺ calcd for CH NO ⁺ 444.1083, found 444.1041.

 25 18 7

 [0038] 47aSE

^J H NMR (300 MHz, CD CN) δ 8.37 (dd, 1H, J = 8.8, 2.2), 7.97 (d, 1H, J = 2.2), 7.

 Three

39 (d, 1H, J = 8.8), 7.03 (d, 2H, J = 9.5), 6.60—6.57 (m, 4H), 3.82 (s, 3H), 2.83 (s, 4 H); HRMS (ESI- MS) [M + H] ⁺ calcd for CH NO + 460.1032, found 460.0989.

 25 17 8

 47bSE

^J H NMR (300 MHz, CD CN) δ 7.94 (dd, 1H, J = 7.9, 1.5), 7.87 (d, 1H, J = 1.5), 7.

 Three

44 (d, 1H, J = 7.9), 7.01 (d, 2H, J = 9.7), 6.58-6.55 (m, 4H), 3.79 (s, 3H), 2.89 (s, 4 H); HRMS (ESI- MS) [M + H] ⁺ calcd for CH NO + 460.1032, found 460.1009.

 25 17 8

 [0039] Example 8: Fluorescence modification of BSA using the fluorescent labeling agent of the present invention

Dissolve 15 mg of urine serum albumin (BSA) in 500 μL of 100 mmol / L borate buffer (ρΗ9.0), and stir 50 μL of 20 mM dimethylformamide solution of the fluorescent labeling agent of the present invention. While adding. The mixture was allowed to react at room temperature for 1.5 hours, and then purified with 1.5 g of Sephadex G25. The eluent used was 1 × PBS (100 mmol / L NaCl, 40 mmol / LN aPi). Figure 1 shows the absorption spectrum of BSA labeled with the fluorescent labeling agent of the present invention, and Fig. 2 shows the fluorescence spectrum of BSA labeled with the fluorescent labeling agent of the present invention. Torr (excitation wavelength: 490 nm). From the results shown in the figure, it was shown that the fluorescent labeling agent of the present invention enables highly fluorescent modification of BSA.

Industrial applicability

 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.

Claims

Claims Formula (I) below:  [Chemical 1]

(Where

And R ⁴ each independently represent a hydrogen atom or a monovalent substituent, and at least one of these represents a reactive functional group that can be bonded to the labeled substance via a covalent bond; R ⁵ represents Represents a monovalent group other than a hydrogen atom, a carboxyl group or a sulfonic acid group; 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 Represents a carbo-loxymethyl group, provided that

And the combination of R ⁵ is low in the benzene ring to which the compound represented by the formula (I) is covalently bonded to the labeling substance and then becomes substantially highly fluorescent, and is reduced by 1, A fluorescent labeling agent represented by a combination that gives an electric potential). [2] R ⁵ is an alkyl group or an alkoxy group, a fluorescent labeling agent according to claim 1. [3] The fluorescent labeling agent according to claim 1 or 2, wherein the reactive functional group is a carboxyl group or a reactive derivative thereof. 4. The fluorescent labeling agent according to claim 3, wherein the reactive functional group is an active ester group. [5] The fluorescent labeling agent according to any one of claims 1 to 4, which is S protein, nucleic acid, or lipid. [6] A method for fluorescently labeling a substance to be labeled, comprising the step of reacting the fluorescent labeling agent according to any one of claims 1 and 5 and the labeling substance. .