JP4171834B2 - Sulfonate compound and fluorescent probe using the same - Google Patents

Description

  The present invention relates to a sulfonate compound and a fluorescent probe using the same.

  In recent years, it has become clear that various diseases are caused by the increased generation of reactive oxygen species in vivo. Therefore, in vivo dynamic analysis of reactive oxygen species is important in elucidating the etiology, pathology and the like. In the analysis of reactive oxygen species, a bioimaging method using a fluorescent probe plays a central role. As fluorescent probes for hydrogen peroxide, for example, compounds such as dichlorofluorescin (reduced form of dichlorofluorescein), dihydrorhodamine 123, and Amplex Red represented by the following formula have been developed and used widely. Their fluorescence mechanism is based on an oxidation reaction with hydrogen peroxide in the presence of peroxidase or peroxidase-like enzyme.

  However, not only hydrogen peroxide but many reactive oxygen species act as oxidants. As a result, when the conventional fluorescent probe as described above is used, it is considered that the total generation amount of the in-vivo oxidizing agent containing various active oxygen species is evaluated, not the hydrogen peroxide generation amount. In fact, it has been reported that these probes also respond to reactive oxygen species other than hydrogen peroxide (singlet oxygen, hydroxyl radical, peroxynitrite, etc.) and various heme proteins with oxidizing ability. Furthermore, it has been pointed out that these probes may overestimate hydrogen peroxide because they generate hydrogen peroxide by air oxidation.

  A compound that generates a fluorescent compound not by a redox reaction but by a deprotection reaction with hydrogen peroxide is also known. That is, acylated resorufin (non-fluorescent compound) is converted to fluorescent compound resorufin by a deprotection reaction with hydrogen peroxide (see Non-Patent Documents 1 and 2). However, they suffer from the disadvantage of not giving a selective response to hydrogen peroxide because they undergo simple hydrolysis in the cell system.

From the above, development of a fluorescent probe that responds to hydrogen peroxide with high selectivity is highly desired from the viewpoint of cell physiology and the like.
H. Maeda, S. Matsu-ura, M. Nishida, T. Semba, Y. Yamauchi, H. Ohmori, Chem. Pharm. Bull. 2001, 49, 294-298; H. Maeda, S. Matsu-ura, M. Nishida, Y. Yamauchi, H. Ohmori, Chem. Pharm. Bull. 2002, 50, 169-174.

  Accordingly, an object of the present invention is to provide a novel organic compound that can be used for a fluorescent probe or the like that responds highly selectively to hydrogen peroxide.

  In order to solve the above-described problems, the present invention provides a sulfonate compound having a structure represented by the following general formula (I).

［前記式中、Ｘ ¹ 、Ｘ ² 、Ｘ ³ 、およびＸ ⁴ は、それぞれ水素原子またはハロゲンであり、同一でも異なっていても良い。］
Ｂは、ペンタフルオロフェニル基または２，４，６−トリフルオロフェニル基である。
In the formula (I), A is
It is a formula represented by formula (X-1), (X-2) or (X-3)

[Wherein, X ¹ , X ² , X ³ , and X ⁴ are each a hydrogen atom or halogen, and may be the same or different. ]
B is a pentafluorophenyl group or a 2,4,6-trifluorophenyl group.

  The sulfonic acid ester compound of the present invention is represented by the above general formula (I), so that, for example, it does not respond to superoxide, hydroxyl radical, and singlet oxygen, but responds only to hydrogen peroxide. Can be used for various fluorescent probes.

  Conventional fluorescent probes such as dichlorofluoresin (reduced form of dichlorofluorescein), dihydrorhodamine 123, Amplex Red and the like utilize an oxidation-reduction reaction. In the present invention, the compound of the general formula (I) is an atomic group A. Utilizes a completely different mechanism in which —O exhibits fluorescence by cleaving a covalent bond with a sulfonyl group to deprotect it. As a result of intensive studies, the present inventors have found this mechanism. In addition, acylated resorufin is a compound that exhibits fluorescence upon deprotection with hydrogen peroxide. However, as described above, it does not give a selective response to hydrogen peroxide because it undergoes simple hydrolysis in the cell system. There were drawbacks. However, the present inventors have intensively studied this point as well, and as a result of designing various molecules, the present inventors have found the structure of the general formula (I) and arrived at the present invention. This makes it possible to provide a highly selective fluorescent probe that does not respond to superoxide, hydroxyl radicals, and singlet oxygen but responds only to hydrogen peroxide.

  Hereinafter, embodiments of the present invention will be described.

In the formula (I), the atomic group B is a pentafluorophenyl group or a 2,4,6-trifluorophenyl group . This group is preferable from the viewpoints of sensitivity of the fluorescent probe and H ₂ O ₂ selectivity .

In the formula (I), A is
It is a formula represented by Formula (X-1), (X-2) or (X-3).

[In the above formula, X ¹ , X ² , X ^Three , And X ^Four Are each a hydrogen atom or a halogen, which may be the same or different. ]

  As a particularly preferable example of the sulfonic acid ester compound of the present invention, for example, there is a compound represented by any one of the following formulas 1, 2, 4 and 5.

In formulas 1 and 2, X ^{1 to} X ⁴ are each a hydrogen atom or halogen, and may be the same or different.

  The method for producing the sulfonic acid ester compound of the present invention represented by the general formula (I) is not particularly limited, and a known method for producing a sulfonic acid ester compound and the like can be appropriately used. ) And a compound of the following formula (III) can be produced by a production method comprising a step of combining them.

In formula (III), X ⁵ is halogen, and A and B are the same as those in formula (I). Moreover, as said compound of Formula (II), the said fluorescent compound etc. can be used, for example, A preferable fluorescent compound is as above-mentioned, for example. That is, as the compound of the formula (II), for example, fluorescein represented by the following formula and its derivatives, resorufin, 7-hydroxy-4- (trifluoromethyl) coumarin, 7-hydroxy-4-methylcoumarin, etc. Although there is a pigment, it is not limited to these, and various compounds can be used.

  In the present invention, “halogen” refers to any halogen element, and examples thereof include fluorine, chlorine, bromine and iodine. The alkyl group is not particularly limited, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group. The same applies to a group containing an alkyl group in its structure (for example, an alkylcarbamoyl group, an alkoxycarbonyl group, etc.). Although it does not specifically limit as an alkanoyl group, For example, a formyl group, an acetyl group, a propionyl group, an isobutyryl group, a valeryl group, an isovaleryl group etc. are mention | raise | lifted, The C1 alkanoyl group shall point to a formyl group.

  When isomers such as tautomers, stereoisomers, and optical isomers exist in the compound represented by the formula (I), these isomers are also included in the compound of the present invention. Furthermore, when the compound of the above formula (I) and other compounds according to the present invention can form a salt, the salt is also included in the compound of the present invention. The salt is not particularly limited, and may be, for example, an acid addition salt or a base addition salt. Further, the acid forming the acid addition salt may be an inorganic acid or an organic acid, and the base forming the base addition salt may be an inorganic base. An organic base may be used. Although it does not specifically limit as said inorganic acid, For example, a sulfuric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, etc. are mention | raise | lifted. The organic acid is not particularly limited, and examples thereof include p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid. The inorganic base is not particularly limited, and examples thereof include ammonium hydroxide, alkali metal hydroxides, alkaline earth metal hydroxides, carbonates and hydrogen carbonates, and more specifically, for example, Examples thereof include sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, calcium hydroxide and calcium carbonate. The organic base is not particularly limited, and examples thereof include ethanolamine, triethylamine, and tris (hydroxymethyl) aminomethane.

  The method for producing the salt of the compound of the present invention is not particularly limited, and for example, it can be produced by a method such as appropriately adding the above acid or base to the compound of the present invention by a known method.

  Although the use of the sulfonate ester of the present invention is not particularly limited, it is suitable for a fluorescent probe, and particularly suitable for a fluorescent probe for detecting hydrogen peroxide. The fluorescent probe of the present invention exhibits high selectivity for hydrogen peroxide by containing the sulfonate compound of the present invention.

  The specificity (selectivity) of the fluorescent probe using the sulfonic acid ester compound of the present invention is not particularly limited, but is preferably as follows, for example. That is, the fluorescence response to hydrogen peroxide is preferably 5 times or more of the fluorescence response to superoxide, more preferably 10 times or more, particularly preferably 50 times or more, and the upper limit is particularly limited. Although not, for example, it is less than 100 times the fluorescence response to superoxide. The fluorescence response to hydrogen peroxide is preferably 5 times or more, more preferably 10 times or more, particularly preferably 50 times or more, and particularly preferably an upper limit value for hydroxyl radicals. It is not, for example, less than 100 times the fluorescence response to hydroxyl radicals. The fluorescence response to hydrogen peroxide is preferably 5 times or more, more preferably 10 times or more of the fluorescence response to glucose, ascorbic acid, 1,4-hydroquinone, propylamine, or diethylamine, It is particularly preferably 50 times or more, and the upper limit is not particularly limited. For example, it is 100 times or less of the fluorescence response to any of these substances. The fluorescence response to hydrogen peroxide is preferably 5 times or more, more preferably 10 times or more, particularly preferably 50 times or more when compared with the fluorescence response to cytochrome P450 reductase + NADPH system or diaphorase + NADH system, The value is not particularly limited, but is, for example, 100 times or less of the fluorescence response for any of these systems. These are values obtained by comparing hydrogen peroxide and each of the above substances in equimolar amounts, the measurement temperature is 37 ° C., the excitation wavelength is 485 ± 20 nm, and the emission wavelength is 530 ± 25. Suppose that it is nm. However, the measurement conditions using the fluorescent probe of the present invention are not limited to this, and any measurement conditions are possible.

  The use of the fluorescent probe of the present invention is not particularly limited and can be used for any application. For example, it can be used for bioimaging, and a kit including the fluorescent probe of the present invention is appropriately constructed to be a bioimaging kit. Can be provided. The hydrogen peroxide detection method using the fluorescent probe of the present invention is excellent in sensitivity and accuracy, and is suitable for application to bioimaging methods and the like. Therefore, the sulfonate compound of the present invention is also suitable for uses such as clinical analysis reagents.

The present invention also provides a labeled antibody, wherein the label is the hydrogen peroxide detection probe of the present invention. As such a labeled antibody of the present invention, for example, if an antibody is bound to the atomic group A in the sulfonic acid ester compound of the general formula (I), the atomic group B-SO ₂ — is cleaved to cause fluorescence. A responding labeled antibody is obtained. As an example of such a labeled antibody, for example, a compound such as compound 1 ′ in the following scheme 1 can be mentioned, and it is changed to fluorescent compound 3 ′ by cleaving the sulfonate bond. However, Scheme 1 is merely an example and does not limit the present invention.

  Furthermore, the fluorescent probe for detecting hydrogen peroxide according to the present invention includes, for example, an assay method using blotting (transcription), an assay method using an antigen-antibody reaction, a catalase quantification method, a hydrogen peroxide scavenging ability of a functional food or medicine. It can be used for measurement methods, clinical analysis methods, and the like. Hereinafter, these will be described more specifically.

  First, the assay method using the blotting (transcription) refers to the substance blotted (transcribed) on a support, the fluorescent probe for detecting hydrogen peroxide of the present invention or the labeled antibody of the present invention labeled by the fluorescent probe. It is an assay method including the process detected using. Although the said substance is not specifically limited, For example, protein is preferable. The kit of the present invention used for the assay method using blotting (transcription) includes the fluorescent probe for detecting hydrogen peroxide of the present invention or an antibody labeled with the fluorescent probe, and the support, and if necessary, the rest. These reagents may be included as appropriate.

  Specific operations, steps, reagents used, etc. in the assay method using blotting are not particularly limited except that the fluorescent probe for detecting hydrogen peroxide or labeled antibody of the present invention is used, and known reagents, operations, etc. Can be used as appropriate. For example, as the support, an agarose gel, polyacrylamide gel, nitrocellulose membrane, nylon membrane, PVDF (polyvinylidene difluoride) membrane or the like can be appropriately used depending on the purpose.

  When the substance is a protein, the assay method using the blotting includes, for example, a step of reacting a protein blotted on the support with a glucose oxidase-labeled antibody to produce a conjugate, and the hydrogen peroxide of the present invention. Comparing the fluorescence reaction in the presence and absence of the conjugate with the fluorescence response in the absence of the conjugate and the step of performing the reaction between the fluorescent probe for detection and hydrogen peroxide in the presence and absence of the conjugate, respectively. By doing so, it can be performed by a method including a step of quantifying the bound substance.

  As a protein assay method using blotting, there is a method conventionally known as a so-called Western blot method, but the detection sensitivity is improved by using the hydrogen peroxide detection probe of the present invention as described above. High effects can be obtained. In the above assay method, when a protein is reacted with a glucose oxidase-labeled antibody to produce a conjugate, for example, as in the prior art, first, the protein is first bound to the primary antibody, and then the glucose is used as the secondary antibody. A method such as binding to an oxidase-labeled antibody can be used.

  The protein assay method using the blotting may include, for example, a step of reacting the protein blotted on the support with the labeled antibody to produce a conjugate when the labeled antibody of the present invention is used. The method can include a step of reacting the bound product with hydrogen peroxide and a step of measuring a fluorescence response after the reaction. That is, for example, when a glucose oxidase labeled antibody and a protein are bound in a conventional Western blotting method, the labeled antibody of the present invention may be used in place of the glucose oxidase labeled antibody. Thus, using the labeled antibody of the present invention has advantages such as high detection sensitivity.

  As an assay method using an antigen-antibody reaction using the probe for detecting hydrogen peroxide of the present invention, there are the following methods in addition to the above.

  That is, the assay method using the antigen-antibody reaction is, for example, an assay method including a step of binding and reacting a glucose oxidase-labeled antibody and a substance to be detected. Comparing the reaction with hydrogen in the presence and absence of a conjugate produced by the binding reaction with the fluorescence response in the presence of the conjugate and in the absence of the conjugate, respectively. And an assay method including the step of quantifying the bound substance.

  As an assay method using a glucose oxidase-labeled antibody, for example, an assay method such as so-called ELISA has been widely used. However, by using the fluorescent probe for detecting hydrogen peroxide of the present invention in such an assay method, advantages such as high detection sensitivity of a substance to be detected can be obtained. The kit used in these assays includes, for example, the fluorescent probe for detecting hydrogen peroxide of the present invention and a glucose oxidase-labeled antibody, and may optionally include other reagents as necessary.

  The assay method using the antigen-antibody reaction may be, for example, an assay method using the labeled antibody of the present invention as a labeled antibody. In such a method, for example, a step of reacting the labeled antibody with a substance to be detected to generate a bound product, a step of reacting the bound product with hydrogen peroxide, and a fluorescence response after the reaction are measured. It is more preferable to include a process. That is, for example, in the conventional ELISA method, the labeled antibody of the present invention may be used instead of the glucose oxidase labeled antibody. In the above assay method, when a substance to be detected is reacted with the labeled antibody to generate a conjugate, for example, the substance to be detected is first bound to a primary antibody, and then this is labeled with the label as a secondary antibody. A method of reacting with an antibody can be used. The kit used for such an assay contains the labeled antibody of the present invention, and may further contain other reagents as necessary.

  Next, the catalase quantification method using the fluorescent probe for detecting hydrogen peroxide according to the present invention is a method for determining the fluorescence response after reacting the fluorescent probe for detecting hydrogen peroxide according to the present invention with hydrogen peroxide. This is done by comparing the case in the presence of the sample with the case in the absence. For example, the catalase concentration in the human or animal body fluid can be measured by performing the catalase quantification method using the human or animal body fluid or a sample extracted from the body fluid as the catalase-containing sample. Furthermore, the catalase determination method can be applied to, for example, measurement of hydrogen peroxide scavenging ability of functional foods or pharmaceuticals. That is, a step of measuring the concentration of catalase in the body fluid of a human or animal using the method for quantifying catalase, a step of administering a functional food or a medicament to the human or animal thereafter, and a method of quantifying the catalase again after administration. The method of measuring the concentration of catalase in the body fluid of the human or animal can measure the hydrogen peroxide scavenging ability of the functional food or medicine. A kit suitable for these measurement methods can be obtained by appropriately designing a kit including the hydrogen peroxide detection probe of the present invention.

  As described above, human or animal using an assay method using blotting (transcription), an assay method using an antigen-antibody reaction, a catalase quantification method, or a method for measuring the hydrogen peroxide scavenging ability of a functional food or pharmaceutical. Various clinical analysis methods can be performed by performing an assay for. For this reason, the sulfonate compound of the present invention is suitable for clinical analysis reagents as described above, and for clinical analysis suitable for various applications by appropriately setting a kit containing this clinical analysis reagent. A kit is obtained.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, this embodiment is merely an example of the present invention, and the present invention is not limited to this.

  In this example, O-pentafluorobenzene sulfonated products 1a to 1c (non-fluorescent compounds) of the fluorescent compounds fluoresceins 3a to 3c shown in Scheme 2 above were synthesized. And 1a to 1c are based on a completely new fluorescence reaction consisting of simple deprotection, and do not respond to superoxide, hydroxyl radical and singlet oxygen, but highly selective fluorescence responding only to hydrogen peroxide It was clarified that it functions as a probe. In addition, 1a to 1c acetylated compounds 2a to 2c (Scheme 3) were synthesized. 2a to 2c, especially 2a and 2c, selectively detect intracellular oxidative stress caused by hydrogen peroxide in the freshwater green alga Chlamydomonas rheinhardi as a fluorescent probe for detecting hydrogen peroxide generated in cells. Clarified that it can be detected. Furthermore, the compounds 4 and 5 were also synthesized.

(Measurement conditions, etc.)
The nuclear magnetic resonance (NMR) spectrum was measured using EX-270 (trade name) manufactured by JEOL (270 MHz at ¹ H measurement) as a measuring instrument. Chemical shifts are expressed in parts per million (ppm). Tetramethylsilane (TMS) was used for the internal standard of 0 ppm. Coupling constants (J) are shown in hertz and the abbreviations s, d, t, q, m and br are singlet, doublet, triplet, quadruple, respectively. Represents a quartet, a multiplet, and a broad line. Mass spectrometry (MS) was performed by high resolution (HRMS) / fast atom bombardment method (FAB) using JMS-700 (trade name) manufactured by JEOL. Infrared absorption (IR) spectrum was performed by KBr method using VALOR-III (trade name) manufactured by JASCO Corporation. Elemental analysis was performed using CHN CORDER MT-5 (trade name) manufactured by Yanaco. The melting point was measured using MP-S3 (trade name) manufactured by Yanaco Corporation. For column chromatography separation, silica gel (manufactured by Merck, silica gel 60 (trade name)) was used. All chemicals were reagent grade and were purchased from Aldrich, Lancaster, Tokyo Kasei, Nacalai Tesque, and Wako Pure Chemicals.

(Synthesis)
[Synthesis of 1a to 1c]
Compounds 1a to 1c were all synthesized by the same method. That is, first, tetrafluorobenzenesulfonyl chloride (1.1 eq) was added to a suspension of 2,6-lutidine (5.0 mL) -dichloromethane (20 mL) of a fluorescein derivative (1.0 g) corresponding to any of the target compounds 1a to 1c. Was added at 0 ° C. The resulting mixture was stirred overnight at room temperature. The reaction mixture was diluted with dichloromethane (200 mL), washed with 1M hydrochloric acid (200 mL x 2) and saturated brine (200 mL), and dried over magnesium sulfate. The residue obtained by evaporating the solvent under reduced pressure was purified by silica gel column chromatography (dichloromethane-acetone = 20: 1). The instrumental analysis values of 1a to 1c are shown below.

1a: 0.69 g (41%) yellow solid. Melting point. 100-116 ° C (dec). ¹ H-NMR (270 MHz, CDCl ₃ , TMS): δ = 8.04 (d, ³ J _{H, H} = 6.9 Hz , 1H; aromatic ring), 7.73-7.62 (m, 2H), 7.21 (d, ⁴ J _{H, H} = 2.1 Hz, 1H; aromatic ring), 7.17 (d, ³ J _{H, H} = 7.4 Hz, 1H; Aromatic ring), 6.88 (dd, ³ J _{H, H} = 8.7 Hz, ⁴ J _{H, H} = 2.1 Hz, 1H; aromatic ring), 6.81 (d, ³ J _{H, H} = 8.7 Hz, 1H; aromatic ring) , 6.76 (d, ⁴ J _{H, H} = 2.1 Hz, 1H; aromatic ring), 6.65 (d, ³ J _{H, H} = 8.6 Hz, 1H; aromatic ring), 6.59-6.55 (m, 1H), 5.76 ( FTIR (KBr): ν = 3340 (OH, br) 1765 (CO, s), 1736 (CO, s) cm ^-1 . FAB HRMS calculated C ₂₆ H ₁₂ F ₅ O ₇ S (MH ⁺ ): 563.0224; found: 563.0223.

1b: 0.36 g (23%) yellow solid. Melting point. 107-125 ° C (dec). ¹ H-NMR (270 MHz, CDCl ₃ , TMS): δ = 8.07 (d, ³ J _{H, H} = 6.6 Hz , 1H; aromatic ring), 7.78-7.68 (m, 2H), 7.46 (s, 1H; aromatic ring), 7.18 (d, ³ J _{H, H} = 6.8 Hz, 1H; aromatic ring), 6.98 (s, 1H ; Aromatic ring), 6.83 (s, 1H; aromatic ring), 6.75 (s, 1H; aromatic ring), 5.99 (brs, 1H; OH). FTIR (KBr): ν = 3324 (OH, br) 1768 (CO , s), 1736 (CO, s) cm -1 FAB HRMS calcd _{C 26 H 10 Cl 2 F 5} O 7 S (MH +):. 630.9444; Found: 630.9417.

1c: 0.37 g (23%) yellow solid. Melting point. 90-104 ° C (dec). ¹ H-NMR (270 MHz, CDCl ₃ , TMS): δ = 8.05 (d, ³ J _{H, H} = 6.8 Hz , 1H; aromatic ring), 7.77-7.66 (m, 2H), 7.39 (d, ³ J _{H, H} = 6.3 Hz, 1H; aromatic ring), 7.18 (d, ⁴ J _{H, F} = 7.1 Hz, 1H; Aromatic ring), 6.95 (d, ⁴ J _{H, F} = 7.4 Hz, 1H; aromatic ring), 6.57 (d, ³ J _{H, F} = 10.1 Hz, 1H; aromatic ring), 6.49 (d, ³ J _{H, F} = 10.4 Hz, 1H; aromatic ring), 5.79 (brs, 1H; OH). FTIR (KBr): ν = 3310 (OH, br) 1768 (CO, s), 1748 (CO, s) cm- ¹ . FAB HRMS calculated C ₂₆ H ₁₀ F ₇ O ₇ S (MH ⁺ ): 599.0035; found: 599.0027.

[Synthesis of 2a to 2c]
2a to 2c were all synthesized by the same method. That is, first, a compound corresponding to any one of the target compounds 2a to 2c among the compounds 1a to 1c was synthesized as described above, and triethylamine (0.5 g) was added to a dichloromethane solution of 0.5 g of this compound and acetyl chloride (1.1 eq). 1.1 eq) was added at 0 ° C. The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with dichloromethane (200 mL), washed with 1M hydrochloric acid (200 mL) and saturated brine (200 mL), and dried over magnesium sulfate. The residue obtained by evaporating the solvent under reduced pressure was purified by silica gel column chromatography (dichloromethane). The instrumental analysis values 2a to 2c are shown below.

2a: 0.51 g (95%) White solid. Melting point. 209-210 ° C. ¹ H-NMR (270 MHz, [D ₆ ] DMSO, TMS): δ = 8.06 (d, ³ J _{H, H} = 7.3 Hz , 1H; aromatic ring), 7.86-7.74 (m, 2H), 7.51 (d, ⁴ J _{H, H} = 2.1 Hz, 1H; aromatic ring), 7.41 (d. ³ J _{H, H} = 7.3 Hz, 1H; Aromatic ring), 7.27 (s, 1H; aromatic ring), 7.07 (dd, ³ J _{H, H} = 8.7 Hz, ⁴ J _{H, H} = 2.1 Hz, 1H; aromatic ring), 6.97-6.89 (m, 3H) , 2.29 (s, 3H; CH ₃ ). FTIR (KBr): ν = 1771 (CO, s) cm ^-1 . Elemental analysis (%) Calculated C ₂₈ H ₁₃ F ₅ O ₈ S: C 55.64, H 2.17; found: C 55.63, H 2.36.calculated FAB HRMS C ₂₆ H ₁₄ F ₅ O ₈ S (MH ⁺ ): 605.0330; found: 605.0331.

2b: 0.49 g (92%) white solid. Melting point. 120-124 ° C (dec). ¹ H-NMR (270 MHz, [D ₆ ] DMSO, TMS): δ = 8.06 (d, ³ J _{H, H} = 7.3 Hz, 1H; aromatic ring), 7.87-7.76 (m, 2H), 7.76 (s, 1H; aromatic ring), 7.53 (s, 1H; aromatic ring), 7.49 (s, 1H; aromatic ring), 7.24 (s, 1H; aromatic ring), 7.08 (s, 1H; aromatic ring), 2.36 (s, 3H; CH ₃ ). FTIR (KBr): ν = 1775 (CO, s) cm ^-1 . Elemental analysis ( %) Calculated C ₂₈ H ₁₁ Cl ₂ F ₅ O ₈ S: C 49.94, H 1.65; Found: C 49.96, H 1.84.FAB HRMS calculated C ₂₈ H ₁₂ Cl ₂ F ₅ O ₈ S (MH ⁺ ) : 672.9550; Found: 672.9549.

2c: 0.47 g (87%) White solid. Melting point. 213-214 ° C. ¹ H-NMR (270 MHz, [D ₆ ] DMSO, TMS): δ = 8.05 (d, ³ J _{H, H} = 7.1 Hz , 1H; aromatic ring), 7.85-7.74 (m, 2H), 7.76 (d, ⁴ J _{H, F} = 6.4 Hz, 1H; aromatic ring), 7.47 (d. ⁴ J _{H, F} = 6.4 Hz, 1H; Aromatic ring), 7.43 (d, ³ J _{H, H} = 7.4 Hz, 1H; aromatic ring), 7.13 (d, ³ J _{H, F} = 10.2 Hz, 1H; aromatic ring), 6.95 (d, ³ J _{H, F} = 10.2 Hz, 1H; aromatic ring), 2.35 (s, 3H; CH ₃ ). FTIR (KBr): ν = 1778 (CO, s) cm ^-1 . Elemental analysis (%) Calculated C ₂₈ H ₁₁ F ₇ O ₈ S: C 52.51, H 1.73; Found: C 52.60, H 1.99.FAB HRMS calculated C ₂₈ H ₁₂ F ₇ O ₈ S (MH ⁺ ): 641.0141; Found: 641

[Composition of 4 and 5]
Compounds 4 and 5 were synthesized by the same method. Specifically, first, pentafluorobenzenesulfonyl chloride or 2,4,6-trifluorobenzenesulfonyl chloride (1.1 eq) was added to a suspension of resorufin sodium salt (2.0 g) in pyridine (20 mL) at −40 ° C. . The resulting mixture was stirred at −40 to −20 ° C. for 6 hours. The reaction mixture was diluted with dichloromethane (200 mL), washed with 1M hydrochloric acid (200 mL x 2) and saturated brine (200 mL), and dried over magnesium sulfate. The residue obtained by distilling off the solvent under reduced pressure was produced by silica gel column chromatography (dichloromethane-acetone = 20: 1). The instrumental analysis values of compounds 4 and 5 are shown below.

4: 1.7 g (45%) orange crystals. Melting point. 200-201 ° C (from AcOEt). ¹ H-NMR (270 MHz, CDCl ₃ , TMS): δ = 7.82 (d, ³ J _{H, H} = 8.6 Hz, 1H; aromatic ring), 7.43 (d, ³ J _{H, H} = 9.9 Hz, 1H; aromatic ring), 7.24-7.19 (m, 2H; aromatic ring), 6.88 (dd, ³ J _{H, H} = 9.9 Hz, ³ J _{H, H} = 2.0 Hz, 1H; aromatic ring), 6.34 (d, ⁴ J _{H, H} = 2.0, 1H; aromatic ring), 6.49 (dd, ³ J _{H, F} = 10.1 Hz, ⁴ J _{H, F} = 2.4 Hz, 2H; aromatic ring). FTIR (KBr): ν = 1624 (CO, s) cm ^-1 . Elemental analysis (%) Calculated C ₁₈ H ₆ F ₅ NO ₅ S: C 48.77 , H 1.36, N 3.16, S 7.23, F 21.43; Found: C 48.69, H 1.54, N 3.02, S 7.00, F 21.38.

5: 2.2 g (64%) orange crystals. Melting point. 204-207 ° C (from AcOEt). ¹ H-NMR (270 MHz, CDCl3, TMS): δ = 7.79 (d, ³ J _{H, H} = 8.2 Hz , 1H; aromatic ring), 7.42 (d, ³ J _{H, H} = 9.9 Hz, 1H; aromatic ring), 7.22-7.18 (m, 2H; aromatic ring), 6.91-6.84 (m, 3H; aromatic ring), 6.32 (d, ⁴ J _{H, H} = 2.0 Hz, 1H; aromatic ring). FTIR (KBr): ν = 1624 (CO, s) cm ^-1 . Elemental analysis (%) Calculated C ₁₈ H ₈ F ₃ NO ₅ S: C 53.08, H 1.98, N 3.44; Found: C 53.07, H 2.14, N 3.17.FAB HRMS calculated C ₁₈ H ₉ F ₃ NO ₅ S (MH ⁺ ): 408.0154; Found: 408.0158.

(H ₂ O ₂ selectivity evaluation of compound 1)
Compounds 1a, 1b and 1c in Scheme 2 all showed a H ₂ O ₂ concentration dependent response in a 96 well microplate assay. In the assay, first, a 1a DMSO solution (10 mM), a 1b DMSO solution (2 mM), and a 1c DMSO solution (2 mM) were prepared, and these were respectively prepared in HEPES buffer (pH 7.4, 10 mM). The probe solution of each compound was prepared by diluting 400 times. The concentration of each compound in these probe solutions is 25 μM for 1a, 5 μM for 1b, and 5 μM for 1c. Further, 150 μL of each probe solution was reacted with H ₂ O ₂ water (0.92 mM, 10 μL) at 25 ° C. for 60 minutes. The detection limits for 1a, 1b, and 1c are 46.0 (RSD, n = 8; 1.2%), 23.1 (0.5%), and 4.6 (1.0%) pmol, respectively, which are values that can be used for cell level analysis. there were. A linear calibration curve with a correlation coefficient of 0.999 or more was obtained in the range of 92.3 nmol or less. The slopes were 172, 215 and 591 au / nmol for 1a, 1b and 1c, respectively.

The fluorescence responses of 1a, 1b and 1c thus obtained to H ₂ O ₂ were compared with the fluorescence responses to HO · (hydroxyl radical) and O ₂ ⁻ · (superoxide). The HO · sources, using a Fenton reaction with _{H 2 O 2 (1 mM,} 10 μL) and ^{Fe 2+ (5 mM, 10 μL} ) (Fenton reaction), and, O ₂ ^- · hypoxanthine ( HPX) (1 mM, 10 μL) and xanthine oxidase (XO) (0.26 U / mL, 10 μL) were generated by an enzymatic reaction. Table 1 shows the results.

Table 1 compares the fluorescence responses observed by reacting 1a, 1b or 1c with H ₂ O ₂ , HO · or O ₂ ⁻ ·, respectively. The numbers in the table represent fluorescence intensity (RFU). However, RFU from measurements is a value obtained by subtracting the fluorescence intensity obtained in the absence of H ₂ O ₂ or HPX.

[Table 1]
Fluorescence intensity (RFU)
1a 1b 1c
H ₂ O ₂ 1150 2193 4375
H ₂ O ₂ + Fe ²⁺ 28 195 184
HPX + XO 566 1733 3580
HPX + XO + catalase −49 −51 −44
HPX + XO + SOD 899 1733 3340

The reaction of 1a, 1b and 1c with HO. Showed a very small response compared to the reaction with H ₂ O ₂ . The fluorescence response of reactions 1a, 1b and 1c in the HPX-XO system was completely eliminated by the addition of catalase (5000 U / mL, 10 μL), but superoxide dismutase (SOD) (1000 U / mL, 10 maintained or increased in the presence of μL). These results support that 1a, 1b and 1c are highly selective fluorescent probes for H ₂ O ₂ compared to HO · and O ₂ ⁻ .

(Evaluation of H ₂ O ₂ selectivity of compound 2 in algal cells)
Light stimulation can cause oxidative stress in green algae. Stimulation with Cu ²⁺ causes intracellular formation of various ROS such as superoxide, hydrogen peroxide and hydroxyl radical. The cells are further specifically activated by paraquat (PQ) or methylene blue (MB), thereby causing oxidative stress due to the production of superoxide or ¹ O ₂ . Therefore, an experimental model using the freshwater green alga Chlamydomonas reinharadtii was useful for evaluating the adaptation of the probe of the present invention to cell lines. Their acetylated derivatives 2a, 2b and 2c (Scheme 3) were used to load 1a, 1b and 1c into algal cells. It was confirmed by the microplate assay similar to the above that esterase is necessary for 2a, 2b and 2c to function as a hydrogen peroxide detection probe.

That is, first, each of compounds 2a, 2b, and 2c was dissolved in DMSO to obtain 10 mM stock solutions of each compound. Next, Chlamydomonas reinhardtii (IAM C-238) cultured according to a conventional method is inoculated into 3 mL of MBM culture solution, and 7.5 μL of the compound 2a, 2b or 2c stock solution is used. A probe-loaded cell suspension was prepared by loading for 30 minutes at 25 ° C. in the dark. In addition, prepare a 96-well tissue culture plate, and add 50 μL of the indicated concentrations MBM solution of CuCl ₂ , PQ or MV to each well, and inoculate the probe-loaded cell suspension (50 μL) there. And incubated under light irradiation or in the dark. After 60 minutes, the fluorescence of the cells was measured using a fluorescence measuring device (trade name: CytoFluor II multiwell fluorescence plate reader, PerSeptive Biosystems, USA). At this time, the excitation and emission filters were set to 485 ± 20 and 530 ± 25 nm, respectively.

FIG. 1 shows the fluorescence response results in the cell line. The figure shows that Chlamydomonas reinhardi cells were loaded with 2a, 2b, 2c (25 μM) or DCFH-DA (50 μM) in the dark for 30 minutes at 25 ° C, in 96-well microplates under light irradiation or It is a graph which shows the correlation of a density | concentration (Concentration) versus fluorescence increment (Fluorescence increment) when it incubated in presence of Cu ^<2+ >, PQ, or MB for 60 minutes in the dark place, and measured the fluorescence response. White circles in the figure represent results under light irradiation, and black circles represent results in a dark place.

From the results shown in FIG. 1, it can be seen that cells loaded with 2a, 2b, and 2c show a fluorescence response depending on the stimulation concentration when incubated with Cu ²⁺ and measured under light irradiation. Among them, 2a and 2c in particular were found to have a high selectivity for the above conditions and a large fluorescence increment. This result, 2a and 2c are, as a result of Cu ²⁺ stimulation and light irradiation, O ₂ ^- that the oxidative stress derived from the cells, and ^1, O ₂ without H ₂ O ₂ formation was detected Show. Given that compounds 1a and 1c have a high degree of H ₂ O ₂ selectivity, it is assumed that 2a and 2c penetrated the cells and were converted to 1a and 1c, respectively. In contrast to the behavior of 2a, 2b and 2c, DCFH-DA effectively detected oxidative stress due to PQ and MB and also detected ROS generated by Cu ²⁺ stimulation. This result indicates that DCFH is not useful as a probe to provide an indication of total oxidant. Thus, it was confirmed that Compound 2, especially 2a and 2c, act as probes without loss of selectivity in the cell line.

These results, 1a, 1b and 1c are, by non-oxidative mechanisms, O ₂ ^- ·, functions as a novel fluorescent probe having a high degree of selectivity for H ₂ O ₂ compared with HO · and ¹ O ₂ Indicates to do. These new probes and their homologues facilitate the measurement of cell-derived H ₂ O ₂ and provide dynamic functions not only for algal cells but also for phagocytes and vascular endothelium cells. ) Can be expected to help elucidate

  As shown by the above data, 1a to 1c in the hydrogen peroxide detection showed selectivity that could not be achieved with fluorescent probes based on the oxidative fluorescence mechanism. Therefore, 1a to 1c and analogs thereof are expected to be used as fluorescent probes having the selectivity required for analysis at the cellular level. As fluorescent compounds having a phenolic hydroxyl group, those having a wide variety of structures and fluorescent properties are readily available. These O-pentafluorobenzene sulfonylated forms are expected to be used as hydrogen peroxide fluorescent probes as well. That is, for example, as shown in Scheme 4 below. Therefore, it is expected that a fluorescent probe (and a reagent for clinical analysis) having characteristics suitable for the purpose of use can be designed and developed by utilizing the fluorescence mechanism developed by the present inventors.

  As described above, the sulfonic acid ester compound of the present invention is suitable for the use of a fluorescent probe that responds with high selectivity to hydrogen peroxide. Fluorescent probes are important reagents in current cell physiology research. Furthermore, considering the recent hard and soft developments in image analysis systems and flow cytometry (cell sorters) using fluorescent microscopes, fluorescent probes will become increasingly popular in the future. The market is expected to expand. The present invention is suitable for, for example, bioimaging and clinical analysis, and can be used favorably for developing new treatments and drug discovery seeds for diseases associated with reactive oxygen species. Furthermore, the use of the sulfonic acid ester compound of the present invention is not limited to a fluorescent probe, and can be used for any application.

It is a graph which shows the fluorescence response result of compound 2a-2c in a cell system.

Claims (15)

The sulfonate compound containing the structure represented by the following general formula (I).

In the formula (I), A is
It is a formula represented by formula (X-1), (X-2) or (X-3)

[Wherein, X ¹ , X ² , X ³ , and X ⁴ are each a hydrogen atom or halogen, and may be the same or different. ]
B is a pentafluorophenyl group or a 2,4,6-trifluorophenyl group. The sulfonic acid ester compound according to claim 1 represented by any one of the following formulas 1, 2, 4 and 5.

In formulas 1 and 2, X ^{1 to} X ⁴ are each a hydrogen atom or halogen, and may be the same or different. The manufacturing method of the sulfonate compound of Claim 1 including the process of combining the compound of following formula (II), and the compound of following formula (III).

In the formula (II), A is a formula represented by the formula (X-1), (X-2) or (X-3).

[Wherein, X ¹ , X ² , X ³ , and X ⁴ are each a hydrogen atom or halogen, and may be the same or different. ]
In formula (III), X ⁵ is a halogen, and B is a pentafluorophenyl group or a 2,4,6-trifluorophenyl group. A fluorescent probe comprising the sulfonate compound according to claim 1 . The fluorescent probe according to claim 4 , which is used for hydrogen peroxide detection. The fluorescent probe according to claim 4 or 5 , which is used for bioimaging. Kits for Bioimaging containing a fluorescent probe according to claim 6. At least one selected from the group consisting of an assay method using blotting (transcription), an assay method using an antigen-antibody reaction, a catalase quantification method, a method for measuring hydrogen peroxide scavenging ability of a functional food or pharmaceutical, and a clinical analysis method The fluorescent probe according to claim 5 , which is used in one method. An antibody that is a labeled antibody, wherein the label is the fluorescent probe according to claim 5 . A kit for use in an assay method using blotting (transcription), comprising the fluorescent probe according to claim 5 or the antibody according to claim 9 and a support . An assay kit comprising the fluorescent probe according to claim 5 and a glucose oxidase-labeled antibody. An assay kit comprising the antibody according to claim 9 . Including fluorescent probe according to claim 5, a kit for measuring catalase concentration in the human or animal body fluids, in the catalase concentration measurement, with hydrogen peroxide fluorescent probe according to claim 5 reaction A kit for quantifying catalase by comparing the fluorescence response after the reaction is performed in the presence or absence of a human or animal body fluid or a sample extracted from the body fluid . A reagent for clinical analysis comprising the sulfonic acid ester compound according to claim 1 or 2 . A clinical analysis kit comprising the clinical analysis reagent according to claim 14 .

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