JP2007314444A - Zinc fluorescence probe - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound useful as a zinc fluorescence probe. <P>SOLUTION: The compound represented by the general formula (I) ( wherein, R<SP>1</SP>and R<SP>2</SP>are each H or a 1-6C alkyl; and R<SP>3</SP>is a 1-6C hydroxyalkyl ) or a salt thereof is provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a zinc fluorescent probe that specifically captures zinc ions to form a complex and emits fluorescence as the complex is formed.

  Zinc is an essential metal element having the second highest content in the human body after iron, and most zinc ions present in cells are firmly bound to proteins and are involved in protein structure maintenance and function expression. Various reports have also been made on the physiological role of free zinc ions (usually below the μmol / L level) in cells, including enzyme catalysis, gene expression, apoptosis, and neurotransmission. There is. In living cells, by measuring zinc ions in real time and visualizing the distribution of zinc ions, the function of zinc ions in in vivo processes can be further elucidated.

Conventionally, in order to measure zinc ions in a tissue, a compound (zinc fluorescent probe) that specifically captures zinc ions to form a complex and emits fluorescence as the complex is formed has been used. The following compounds have been put to practical use as zinc fluorescent probes. For example, compounds using the fluorescein skeleton as a partial structure (specific fluorophore) that emits fluorescence upon complex formation include ZnAF-2 (Patent Document 1: International Publication WO2001 / 62755), Newport Green (Non-Patent Document 1). : a Molecular Probes, Inc. of the catalog "Handbook of Fluorescent Probes and Research Products " 8 th edition by Richard P. Haugland, pp.805-817) and the like have been put to practical use. Moreover, as a compound using a quinoline skeleton as a specific fluorophore, TSQ (Non-patent Document 2: Biol. Res., 27, 49 (1994)), Zinquin ethyl ester (Non-patent Document 3: Neurosci., 17, 6678 (1997)) has been put to practical use, and as a compound using a dansyl skeleton as a specific fluorophore, Dansylaminoethylcyclen (Non-patent Document 4: J. Am. Chem. Soc., 118, 12686 (1996)) Has been put to practical use.

  On the other hand, when a fluorescent probe is applied to a cell, the concentration of the fluorescent probe introduced into the cell may vary depending on the type of cell, and even in the same type of cell, the fluorescent probe is located in a highly hydrophobic part such as a membrane. There are many factors that affect the measurement, such as when the fluorescence is localized or the fluorescence intensity may be different at the measurement site due to the difference in the thickness of the cell membrane.

  A zinc ion fluorescent probe that can be applied to a ratio measurement method capable of reducing measurement errors due to these factors and performing quantitative analysis has also been developed (Patent Document 2: International Publication WO2002 / 102795). This method includes the steps of measuring the fluorescence intensity using two different wavelengths, either the fluorescence wavelength or the excitation wavelength, and detecting the ratio thereof, and the influence of the concentration of the fluorescent probe itself and the excitation light intensity can be ignored. At the same time, it is possible to eliminate measurement errors due to the localization, concentration change, or fading of the fluorescent probe itself that occurs when observation is performed at one wavelength.

Recently, Henary et al. Show that 2- (2'-hydroxyphenyl) benzimidazole derivatives selectively form complexes with zinc ions, and the excitation wavelength peak shifts to the longer wavelength side and the fluorescence wavelength peak shifts to the lower wavelength side. (Non-Patent Document 5: J. Phys. Chem. A., 106, 5210 (2002)), and further, 2- (2′-benzenesulfonamidophenyl) benzimidazole derivative is zinc fluorescence by the ratio method. It has been reported that it can be used as a sensor (Non-Patent Document 6: Chem. Eur. J., 10, 3015 (2004)).
Thus, the 2-phenylbenzimidazole derivative is useful as a zinc fluorescent probe, but there is a high demand for development of a derivative capable of detecting zinc ions with higher sensitivity.
International Publication WO2001 / 62755 International Publication WO2002 / 102795 Molecular Probes catalog "Handbook of Fluorescent Probes and Research Products" 8th edition by Richard P. Haugland, pp.805-817 Biol. Res., 27, 49 (1994) Neurosci., 17, 6678 (1997) J. Am. Chem. Soc., 118, 12686 (1996) J. Phys. Chem. A., 106, 5210 (2002) Chem. Eur. J., 10, 3015 (2004)

  An object of the present invention is to provide a compound or a salt thereof that can be a highly selective and highly sensitive zinc fluorescent probe, and to provide a zinc ion measurement method using the zinc fluorescent probe. More specifically, a zinc ion measurement method using 2- (2'-hydroxyphenyl) benzimidazole derivative that selectively captures zinc ions in aqueous solution and has excellent fluorescence characteristics of the complex after capture is provided. This is the subject of the present invention. Still another object of the present invention is to provide a compound that causes a wavelength shift in the excitation spectrum or the peak of the fluorescence spectrum by capturing zinc ions and significantly increases the fluorescence intensity, and includes a compound having the above characteristics. A zinc fluorescent probe and a method for measuring zinc ions using the zinc fluorescent probe are provided.

  As a result of intensive studies to solve the above problems, the present inventors have found that 2- (2′-hydroxy-3′-hydroxymethyl-phenyl) benzimidazole derivatives are selectively selected from zinc ions in the presence of zinc ions. When a 1: 1 complex is formed, the maximum absorption wavelength and the maximum excitation wavelength are significantly shifted, and when excited with excitation light around 350 nm, the maximum fluorescence wavelength shifts with a short wavelength as the zinc ion concentration increases and 430 nm. It was found that the fluorescence intensity in the vicinity increased significantly. The present invention has been completed based on these findings.

That is, according to the present invention, the following general formulas (I) and (II):
(Wherein R ¹ and R ² each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R ³ represents a hydroxyalkyl group having 1 to 6 carbon atoms)
(Wherein R ¹¹ and R ¹² each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R ¹³ represents a hydroxyalkyl group having 1 to 6 carbon atoms) or a compound thereof Salt is provided.
As a preferred embodiment of the above invention, there is provided a compound or a salt thereof in which R ¹ is a methyl group, R ² is a hydrogen atom, and R ³ is a hydroxymethyl group in the above general formula (I).

  From another point of view, according to the present invention, a zinc ion fluorescent probe comprising a compound represented by the above general formula (I) or (II) or a salt thereof; a compound represented by the above general formula (I) or (II) Or the zinc complex formed from the salt and zinc ion is provided.

According to the present invention, there is also provided a method for measuring zinc ions, comprising the following steps:
(a) reacting the compound represented by the above general formula (I) or (II) or a salt thereof with zinc ion; and
(b) A method comprising a step of measuring the fluorescence intensity of the zinc complex produced in the step (a); and the method described above wherein the measurement is carried out by a ratio method.

  The compound represented by the above general formula (I) or (II) or a salt thereof selectively forms a 1: 1 complex with zinc ion in the presence of zinc ion, and when excited with excitation light around 350 nm, zinc ion As the concentration increases, the fluorescence intensity around 430 nm increases remarkably, so that it can be used for zinc ion measurement. In addition, the compound represented by the above general formula (I) or (II) or a salt thereof shifts the maximum absorption wavelength and the maximum excitation wavelength to the longer wavelength side as the zinc ion concentration increases and shortens the maximum fluorescence wavelength. Since it has the property of shifting to the wavelength side, it can also be used for measuring in vivo zinc ions by the ratio method.

  In the present specification, the “alkyl group” means an alkyl group composed of a straight chain, a branched chain, a ring, or a combination thereof. Examples of the alkyl group having 1 to 6 carbon atoms include methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclo A propylmethyl group, n-pentyl group, n-hexyl group and the like can be mentioned. The same applies to the alkyl part of the hydroxyalkyl group. The number of hydroxyl groups present in the hydroxyalkyl group is preferably one and is preferably located at the end of the alkyl group.

In the general formula (I), among the alkyl groups having 1 to 6 carbon atoms represented by R ¹ , an alkyl group having 1 to 3 carbon atoms is preferable, and a methyl group is particularly preferable. The alkyl group having 1 to 6 carbon atoms represented by R ² is preferably a methyl group. The hydroxyalkyl group represented by R ³ is preferably a hydroxymethyl group. It is particularly preferable that R ¹ is a methyl group, R ² is a hydrogen atom, and R ³ is a hydroxymethyl group. In the above general formula (II), R ¹¹ , R ¹² and R ¹³ are the same as R ¹ , R ² and R ³ in the general formula (I).

  The compound of the present invention represented by the above general formula (I) or (II) can exist as an acid addition salt or a base addition salt. Examples of the acid addition salt include mineral acid salts such as hydrochloride, sulfate, and nitrate, or organic acid salts such as methanesulfonate, p-toluenesulfonate, oxalate, citrate, and tartrate. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt, organic amine salts such as ammonium salt, and triethylamine salt. In addition to these, a salt with an amino acid such as glycine may be formed. The compound of the present invention or a salt thereof may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention.

  The compound of the present invention represented by the above general formula (I) or (II) may have one or more asymmetric carbons depending on the type of substituent, but one or two or more asymmetric carbons may be present. In addition to stereoisomers such as optically active substances based on asymmetric carbon and diastereoisomers based on two or more asymmetric carbons, any mixture of stereoisomers, racemates, etc. are all within the scope of the present invention. Is included.

  A method for producing a representative compound of the compound represented by the general formula (I) of the present invention is shown in the following scheme. In the examples of the present specification, the production method described in this scheme is shown in more detail and specifically. Accordingly, a person skilled in the art appropriately selects reaction raw materials, reaction conditions, reaction reagents, and the like based on these descriptions, and modifies and modifies these methods as necessary, thereby adding the above general formula (I). Any of the compounds of the present invention represented by In order to produce the compound of the present invention represented by the general formula (II), 2,3-diaminonaphthalene may be used in place of o-phenylenediamine in the following scheme.

  The compound of the present invention represented by the above general formula (I) or (II) or a salt thereof of the present invention can be used for zinc ion measurement. The compound of the present invention represented by the above general formula (I) or (II) or a salt thereof selectively captures zinc ions to form a 1: 1 complex with zinc ions, but the complex is formed. When excited with excitation light near 350 nm, the fluorescence intensity near 430 nm increases about 18 times, and the peak of the excitation spectrum shifts significantly to the longer wavelength side. This wavelength shift can usually be observed in the range of about 20 nm depending on the zinc ion concentration, and is not affected by other metal ions (for example, sodium ion, calcium ion, potassium ion, or magnesium ion). It can be observed as a wavelength shift specific to zinc ions. Furthermore, the fluorescence spectrum peak can be observed as a short wavelength shift, usually in the range of about 17 nm depending on the zinc ion concentration.

  Therefore, the zinc ion can be measured by the ratio method by using the compound of the present invention as a zinc fluorescent probe, for example, by selecting and exciting two different wavelengths and measuring the ratio of the fluorescence intensity at that time. Become. The two different wavelengths are such that when excited at one wavelength, the fluorescence intensity increases with increasing zinc ion concentration, and when excited at the other wavelength, the fluorescent intensity decreases with increasing zinc ion concentration. You can choose. The ratio method is described in detail in Mason WT's book (Mason WT in Fluorescent and Luminescent Probes for Biological Activity, Second Edition, Edited by Mason WT, Academic Press). Specific examples of the measuring method using the compound of the invention are shown.

  In addition, the compound of the present invention represented by the above general formula (I) or (II) or a salt thereof has a characteristic that it can specifically capture zinc ions. Therefore, the compound of the present invention represented by the above general formula (I) or (II) or a salt thereof is extremely useful as a zinc fluorescent probe for measuring zinc ions in living cells and living tissues under physiological conditions. is there. The term “measurement” used in this specification should be interpreted in the broadest sense including quantitative and qualitative.

  The method of using the zinc fluorescent probe of the present invention is not particularly limited, and can be used in the same manner as conventionally known zinc fluorescent probes. Usually, the above general formula (I) is used for an aqueous medium such as physiological saline or buffer, or a mixture of an aqueous medium such as methanol, ethanol, acetone, ethylene glycol, dimethyl sulfoxide, dimethylformamide and an aqueous medium. ) Or a substance selected from the group consisting of (II) and a salt thereof, this solution is added to an appropriate buffer solution, and excited by two different wavelengths appropriately selected, What is necessary is just to measure the fluorescence intensity. In addition, for example, when measuring zinc ions in cells and tissues, the compounds represented by the above general formula (I) or (II) and the like in the tissues and cells by using microinjection or a surfactant and the like A substance selected from the group consisting of the salts can be introduced, and zinc ions in cells and tissues can be measured by a bioimaging technique.

  For example, the excitation wavelength of compound 3 shown in the above scheme and in the following examples is 332 nm, the fluorescence wavelength is 450 nm, and zinc ions up to a concentration of about 2500 μmol / L when used as a zinc fluorescent probe at 5 μmol / L. The fluorescence intensity increases depending on the concentration of zinc ions. Moreover, the peak of the excitation spectrum is blue shifted by about 20 nm depending on the zinc ion concentration. Therefore, ratio measurement is also possible by using this compound as a probe and using, for example, 332 nm and 352 nm as excitation wavelengths and measuring the intensity of fluorescence at each excitation wavelength to determine the intensity ratio. The zinc fluorescent probe of the present invention may be used in the form of a composition in combination with an appropriate additive. For example, it can be combined with additives such as a buffer, a solubilizing agent, and a pH adjuster.

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. The compound numbers in the examples correspond to the compound numbers in the above scheme.
Example 1: Synthesis of compound 3
(a) Synthesis of Compound 1 Compound 1 was synthesized according to the method described in Journal of the American Chemical Society, 74, 6257 (1952). Sodium hydroxide 4.44 g (0.11 mol) was dissolved in 10 mL of water, and 11.01 g (0.10 mol) of p-cresol was added. Further, 18.37 g (0.22 mol) of an aqueous formaldehyde solution (37%) was added, and the mixture was stirred at room temperature for 7 days. Subsequently, 200 mL of isopropyl alcohol was added to the reaction solution and stirred for 15 minutes, and the resulting white solid was filtered off with a glass filter. The solid separated by filtration was transferred to a beaker, 150 mL of 1N hydrochloric acid was added and ice-cooled for 10 minutes, and then the undissolved white solid was filtered off with a G5 glass filter to obtain Compound 1. (Yield 9.66 g, Yield 57%)
¹ H-NMR (CDCl ₃ , 400 MHz): 7.79 (s, 1H), 6.89 (s, 1H), 4.79 (d, 4H, J = 6.4 Hz), 2.32 (t, 2H, J = 12 Hz), 2.25 (s, 3H)

(b) Synthesis of Compound 2 Compound 2 was synthesized by applying the method described in Bulletin of the Chemical Society of Japan, 57, 2683 (1984). Compound 1, 5.08 g (30 mmol) was dissolved in 150 mL of chloroform, and 20.03 g (230 mmol) of manganese dioxide (IV) was added, followed by stirring at room temperature for 48 hours. The reaction solution was filtered using a glass filter, and the filtrate was evaporated under reduced pressure to obtain Compound 2 (white solid). (Yield 1.96 g, Yield 39%)
¹ H-NMR (CDCl ₃ , 400 MHz): 11.13 (s, 1H), 9.83 (s, 1H), 7.39 (d, 1H, J = 2.0 Hz), 7.26 (d, 1H, J = 1.6 Hz), 4.70 (s, 2H), 2.32 (s, 3H)

(c) Synthesis of Compound 3 Compound 3 was synthesized by applying the method described in Spectrochimica Acta, Part A, 63, 343 (2006). Compound 2, 0.53 g (3.2 mmol) was dissolved in ethanol 30 mL and dimethylformamide 3 mL, and sodium hydrogen sulfite 0.33 g (3.2 mmol) was added thereto, followed by stirring at room temperature for 4 hours. Subsequently, a solution prepared by dissolving 0.35 g (3.2 mmol) of o-phenylenediamine in 15 mL of dimethylformamide was added, and the mixture was refluxed at 130 ° C. for 4 hours. After completion, the solvent was distilled off under reduced pressure, a large amount of water was added thereto, and the precipitated solid was filtered off using a glass filter. The obtained solid was recrystallized with acetone to obtain Compound 3 (white powder). (Yield 0.69 g, Yield 86%)
¹ H-NMR (CDCl ₃ , 400 MHz): 13.27 (s, 1H), 13.19 (s, 1H), 7.75 (s, 1H), 7.68 (br s, 1H), 7.59 (br s, 1H), 7.32 (S, 1H), 7.28 (br s, 2H), 5.07 (br s, 1H), 4.59 (s, 2H), 2.34 (s, 3H)
Elemental Analysis:
_{Calcd for C 15 H 14 N 2} O 2: C, 70.85; H, 5.55; N, 11.02.
Found: C, 70.46; H, 5.50; N, 10.70.

Example 2: Optical properties of Compound 3 In order to examine the properties of Compound 3 as a zinc fluorescent probe, an absorption spectrum, an excitation spectrum, and a fluorescence spectrum were measured. The sample for spectrum measurement is compound 3 at 50 mmol / L, HEPES buffer (pH 7.4, I = 0.05 (50 mmol / L, in potassium nitrate aqueous solution), containing 0.5% methanol as a co-solvent) to 5 μmol / L. It was prepared by dissolving.

(a) Measurement conditions / Measurement conditions of absorption spectrum Measuring equipment: SHIMADZUUV-1600PC Ultraviolet / visible spectrophotometer Scanning speed: Medium scanning range: 250 to 500 nm
Sampling pitch: 0.2 nm
Measurement temperature: 25 ℃
・ Excitation spectrum and fluorescence spectrum measurement equipment: SHIMADZURF-5300PC Spectrofluorophotometer
Scan speed: Medium
Scan range: 250-600 nm
Excitation bandwidth: 10 nm
Fluorescence bandwidth: 10 nm
Sensitivity: Low
Measurement temperature: 25 ℃

  Table 1 shows the optical properties of Compound 3 before addition of zinc ion and after addition of zinc ion (2500 μmol / L, zinc sulfate). In addition, the change in zinc ion concentration when the concentration of compound 3 is 5 μmol / L (0 times (no addition), 10 times, 50 times, 100 times, 200 times, 400 times, 500 times the concentration of compound 3) Absorption spectrum and excitation spectrum for double concentration, but only for excitation spectrum measurement, 0 times (no addition), 10 times, 100 times, 200 times, and 500 times added zinc sulfate) (Fluorescence wavelength: 433 nm) and fluorescence spectrum (excitation wavelength: 355 nm) are shown in FIGS. 1 (a), (b), and (c). As a result of coordination between compound 3 and zinc ions, the excitation maximum wavelength of compound 3 was shifted to the 20 nm long wavelength side. In addition, the maximum fluorescence wavelength shifted to the 17 nm short wavelength side.

Example 3: Optical properties of compound 3 when various cations were added to compound 3 Changes in fluorescence intensity were examined when compound 3 was loaded with metal ions other than zinc ions. Metal ions include sodium ion Na ⁺ (sodium chloride), potassium ion K ⁺ (potassium chloride), calcium ion Ca ²⁺ (calcium chloride), magnesium ion Mg ²⁺ (magnesium sulfate), iron ion Fe ²⁺ (sulfuric acid) Iron, cobalt ion Co ²⁺ (cobalt sulfate), nickel ion Ni ²⁺ (nickel sulfate), manganese ion Mn ²⁺ (manganese chloride), and copper ion Cu ²⁺ (copper sulfate) were used. The fluorescence characteristics (excitation wavelength 355 nm) of compound 3 before and after the addition of each metal ion were measured, and the fluorescence intensity ratio before and after the addition of each metal ion (fluorescence intensity after addition of metal / fluorescence intensity when no metal was added) It is shown in FIG. Each metal ion is 5000 μmol / L for Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Mn ²⁺ , 2500 μmol / L for Co ²⁺ , Ni ^{2+, and} about Fe ^2+. 100 μmol / L and Cu ²⁺ were added to the compound 3 solution so as to be 25 μmol / L. The measurement conditions of the fluorescence spectrum are the same as in Example 2.

When each metal ion is added, in the case of Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , the fluorescence intensity hardly changes, and Fe ²⁺ , Co ²⁺ , Ni ²⁺ , Mn ²⁺ , Cu ^{2 In} the case of ⁺ , the fluorescence intensity was weakened. From the above, it was concluded that Compound 3 is a fluorescent probe specific for zinc ions, since the fluorescence intensity greatly increased only when zinc ions were added.

Example 4: Determination of acid dissociation constant The acid dissociation constant of compound 3 was determined by the absorption spectrum method. 2 mmol / L HEPES buffer (pH = 7.5, 8.0, I = 0.002 (in 2 mmol / L sodium chloride aqueous solution), containing 0.5% methanol as a co-solvent), 2 mmol / L CHES buffer (pH = 8.5, 9.0, 9.5, 10.0, I = 0.002 (in 2 mmol / L sodium chloride aqueous solution), containing 0.5% methanol as a co-solvent), 2 mmol / L CAPS buffer (pH = 10.5, 11.0, 12.0, I = 0.002 ( Compound 3 was dissolved in 9 buffers having different concentrations of 2 mmol / L sodium chloride aqueous solution and 0.5% methanol as a co-solvent, and the absorption spectrum was measured. The measurement conditions of the absorption spectrum are the same as in Example 2.

FIG. 3 (a) shows the measurement result of the absorption spectrum. As the pH increased, the absorbance near 360 nm increased and the absorbance near 310 nm decreased. Iso-absorption points were observed at 303 nm and 338 nm. This spectrum change is considered to be caused by the concentration change of the HL species and the L species shown in FIG.
The relationship between pH and pKa is:
pH = pKa + log ([L] / [HL])
Therefore, the concentrations of both species were determined from the absorbance at a wavelength of 359 nm. Since pKa is 9.58 from the intercept of the plot of FIG. 3 (c), the acid dissociation constant Ka of Compound 3 was determined to be 2.63 × 10 ⁻¹⁰ M.

Example 5: Determination of apparent complex formation constant Kapp between compound 3 and zinc ion The complex formation constant Kapp of compound 3 at pH 7.4 was determined by the absorption spectrum method. Compound 3 is dissolved in 50 mmol / L HEPES buffer (pH 7.4, I = 0.05 (in 50 mmol / L potassium nitrate aqueous solution), 0.5% methanol as a co-solvent) to 5 μmol / L, and zinc ion The absorption spectrum was measured by adjusting the solution so that the concentration was 0, 50, 250, 500, 1000, 2000, 2500 μmol / L. The measurement conditions of the absorption spectrum are the same as in Example 2.
From the measurement result of the absorption spectrum shown in FIG. 1 (a), when the zinc ion concentration increases, the concentration of the HL species shown in FIG. 3 (b) decreases, which is similar to the L species shown in FIG. 4 (a). The spectrum of the species (ML species) appears and its concentration increases. In addition, since isosbestic points appear at 262 nm and 341 nm, it can be seen that when compound 3 coexists with zinc ions, L species become ML species bonded to zinc ions 1: 1.
The relationship between zinc ion concentration [M] and Kapp is:
-Log [M] = logKapp + log ([HL] / [ML])
Therefore, the concentrations of both species were determined using absorbance at a wavelength of 352 nm. Since pKapp is 3.58 from the intercept in the plot of FIG. 4B, the apparent complex formation constant Kapp was determined to be 3.76 × 10 ³ M ⁻¹ .

Example 6: Measurement of fluorescence intensity by the ratio method
Zinc sulfate was added to a 5 μmol / L solution of Compound 3 using 50 mmol / L HEPES buffer (pH 7.4, I = 0.05 (50 mmol / L in potassium nitrate aqueous solution), containing 0.5% methanol as a co-solvent). The zinc ion concentration was adjusted to 0 to 2500 μmol / L. Changes in the ratio of fluorescence intensity at excitation wavelengths of 320 nm and 355 nm when the fluorescence wavelength is fixed at 433 nm are shown (FIG. 5 (a)).
In 50 mmol / L HEPES buffer (pH 7.4, I = 0.05 (50 mmol / L in potassium nitrite aqueous solution), containing 0.5% methanol as a co-solvent) Sodium chloride, potassium chloride, calcium chloride, and magnesium sulfate were added so that the ion concentration, potassium ion concentration, calcium ion concentration, and magnesium ion concentration were 0, 2500, and 5000 μmol / L, respectively. Changes in the ratio of fluorescence intensity at excitation wavelengths of 320 nm and 355 nm when the fluorescence wavelength is fixed at 433 nm are shown (FIG. 5 (b)).
When zinc ions are added, the ratio changes depending on the concentration of zinc ions, but when other ions are added, the ratio change is so small that it can be ignored. It was found that there is a possibility that can be quantified with high selectivity.

Example 7: Application of compound 3 to ratio method
Compound 3 5 μmol / L, zinc sulfate 2500 μmol in 50 mmol / L HEPES buffer (pH 7.4, I = 0.05 (in 50 mmol / L potassium nitrite aqueous solution), containing 0.5% methanol as co-solvent) The solution was adjusted to / L. Subsequently, sodium chloride, potassium chloride, calcium chloride, and magnesium sulfate were added to a concentration of 5000 μmol / L. FIG. 6 shows changes in the ratio of fluorescence intensity at excitation wavelengths of 320 nm and 355 nm when the fluorescence wavelength is fixed at 433 nm. No change in the ratio was observed when only zinc sulfate was added to compound 3 and when sodium chloride, potassium chloride, calcium chloride, and magnesium sulfate were added in addition to zinc sulfate. Therefore, even when sodium ion, potassium ion, calcium ion, and magnesium ion coexist, it was found that zinc ion can be quantified with high selectivity using Compound 3.

(A) absorption spectrum, (b) excitation spectrum (fluorescence wavelength: 433 nm), and (c) fluorescence spectrum (excitation wavelength: 355 nm) depending on the zinc ion concentration of compound 3. The zinc ion selectivity of compound 3 is shown. (a) shows the change in absorption spectrum accompanying the change in pH of Compound 3, (b) shows the change in the structure of Compound 3 accompanying the change in pH, and (c) shows the pH and log ([L] / [LH] of Compound 3. ]) Is plotted. [L] represents the concentration of the L species of (b), and [LH] represents the concentration of the LH species of (b). (a) shows the complex formation between zinc ion-free compound 3 and zinc ion, (b) plots the relationship between -log [M] and log ([M] / [ML]) in the presence of zinc ion The results are shown. [M] represents the zinc ion concentration, and [ML] represents the concentration of ML species in which the compound 3 of (a) is complexed with the zinc ion. (a) shows the ratio of zinc ion concentration to the fluorescence intensity of compound 3 (355 nm / 320 nm), and (b) shows the ratio of various metal ion concentrations to the fluorescence intensity of compound 3 (355 nm / 320 nm). . The ratio (355 nm / 320 nm) of compound 3 when zinc ions and various metal ions coexist is shown.

Claims (10)

The following general formula (I):
(Wherein R ¹ and R ² each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R ³ represents a hydroxyalkyl group having 1 to 6 carbon atoms) or a salt thereof . The following general formula (II):
(Wherein R ¹¹ and R ¹² each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R ¹³ represents a hydroxyalkyl group having 1 to 6 carbon atoms) or a salt thereof . 2- (2′-hydroxy-3′-hydroxymethyl-5′-methylphenyl) benzimidazole or a salt thereof. A zinc fluorescent probe comprising the compound according to any one of claims 1 to 3 or a salt thereof. Use of the compound according to any one of claims 1 to 3 or a salt thereof in zinc ion measurement. A reagent for measuring zinc ions comprising the compound or salt thereof according to any one of claims 1 to 3. A zinc complex formed from the compound according to any one of claims 1 to 3 or a salt thereof and zinc ions. A method for measuring zinc ions, comprising the following steps:
(a) reacting the compound according to any one of claims 1 to 3 or a salt thereof with zinc ions; and
(b) A method comprising a step of measuring the fluorescence intensity of the zinc complex produced in the above step. The method according to claim 8, wherein the fluorescence intensity is measured by a ratio method. The method according to claim 8, wherein the fluorescence intensity is measured by imaging.