Fluorescent zinc ion sensor
United States Patent 7541467
A compound represented by the following general formula (I) or a salt thereof:
wherein R1, R2, R3, and R4 independently represent 2-quinolyl group which may be substituted, 1-isoquinolyl group which may be substituted, or 3-isoquinolyl group whic m, n, p, and q independently represent 1 or 2; and L represents a single bond, or an alkylene group having 1 to 5 carbon atoms wherein said alkylene group may contain one or more heteroatoms in the main chain, and may have one or more substituents on the main chain, which is useful as a fluorescent zinc ion sensor.
Inventors:
Yano, Shigenobu (6-7-45-108, Ayameikeminami, Nara-shi, Nara 631-0033, JP)
Mikata, Yuji (4-6-5-2, Kunimidai, Kizu-cho, Soraku-gun, Kyoto 619-0216, JP)
Application Number:
Publication Date:
06/02/2009
Filing Date:
07/30/2004
Export Citation:
Yano, Shigenobu (Nara, JP)
Mikata, Yuji (Kyoto, JP)
Sekisui Medical Co., Ltd. (Tokyo, JP)
Primary Class:
Other Classes:
252/301.6R,
International Classes:
C07D215/20; G01N21/78; C07C229/00; C07D215/12; C07D217/14; C07D257/02; C07F3/06; C09K11/06; C09K11/54
Field of Search:
546/2, 546/176, 546/177, 252/301.6P, 546/140, 546/10
View Patent Images:
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US Patent References:
Nagano et al.6972182Colyer et al.6936687Komoriya et al.Yano et al.Nagano et al.Nagano et al.6903226Nagano et al.Nagano et al.Komatsu et al.6833386Nagano et al.Nagano et al.6756231Nagano et al.6753156Mathis et al.Nagano et al.6656927Nagano et al.Nagano et al.Nagano et al.Nagano et al.6569892Nagano et al.6525088Nagano et al.Elliott et al.6469051Nagano et al.6441197Nagano et al.6403625Nagao et al.6201134Nagano et al.6013802Hoyland et al.5874590Nagano et al.5863727Lee et al.5800996Lee et al.5656433Selvin et al.5648270Kuhn et al.5639615Selvin et al.5623080Neckers et al.5622821Selvin et al.5451343Neckers et al.5393514Pitner et al.5380880Pitner et al.5340716Ullman et al.5302731Pitner et al.5246867Lakowiez et al.5208148Haugland et al.5049673Tsien et al.5037615Kane4968631Dakubu4891075Dakubu et al.
Foreign References:
EP0314480Fluorescent indicator dyes for calcium working at long wavelengthsEP0515133Fluorone and pyronin Y derivatives.EP0582836Fluorescent pH indicatorsEP1069121REAGENT FOR SINGLET OXYGEN DETERMINATIONEP1260508REAGENTS FOR THE QUANTITATION OF ACTIVE OXYGENEP1260510FLUORESCENT PROBES FOR THE QUANTITATION OF ZINCJP6054381March, 1985JP6207112July, 1994JPAugust, 1994FLUORONE AND PYRONIN Y DERIVATIVESJPOctober, 1996NOVEL FLUORESCENT DYEJP9101262April, 1997JP1088124April, 1998JPAugust, 1998DIAMINOFLUORESCEIN DERIVATIVEJP5180773July, 1999JPANEW LABELLING REAGENTJPAFLUORESCENT PROBE FOR ZINCWOA1METHOD OF ASSAYWOA1USE OF A PHYCOBILIPROTEIN-BINDING PEPTIDE COMPLEX AS A FLUORESCENT TRACERWOA2HOMOGENOUS LUMINESCENCE ENERGY TRANSFER ASSAYSWOA1HOMOGENEOUS METHOD FOR DETECTING AND/OR DETERMINING PHOSPHORYLATING ACTIVITY IN A BIOLOGICAL MATERIALWOA1REAGENT FOR SINGLET OXYGEN DETERMINATIONWOA1ASSAY METHOD AND DEVICEWOA1FLUORESCENT PROBES FOR THE QUANTITATION OF ZINCWOA1MEASURING METHOD USING LONG LIFE FLUORESCENCE OF EXCITATION TYPEWOA1REAGENTS FOR THE QUANTITATION OF ACTIVE OXYGENWOA1REAGENTS FOR THE MEASUREMENT OF PEROXYNITRITESWOA1FLUORESCENT PROBEJPHAJPS6054381AJPHAJPH1088124AJPHAJPHAJPHAJPHA
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English Language Abstract of JP .
English Language Abstract of JP 9-101262.
English Language Abstract of JP 10-88124.
English Language Abstract of JP .
English Language Abstract of JP 60-54381.
English Language Abstract of JP 08-2714430.
English Language Abstract of JP 10-226688.
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An abstract of “Fluorescent Response of N, N, N′, N′-Tetrakis (2-quinolylmethyl) alkanediamines Toward Zinc Ion”: published on Aug. 1, 2003 for oral presentation in 21st ICP Satellite Symposium on Photochemistry and Photobiology of Complexes Including Supramolecular Systems and Coordination Compounds (Aug. 1-3, 2003), listed as P27.
An abstract “Fluorescent Response of Polyquinoline Derivatives toward Zinc Ion”, Abstracts 1st Joint symposium on Biofunctional Chemistry and Biotechnology, Oct. 12-13, 2003, Kumamoto University, listed as 2OA-09.
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An abstract of “Fluorescent Response of N, N, N′, N′-Tetrakis (2-quinolylmethyl) alkanediamines Toward Zinc Ion”, 10th International SPACC Symposium Nov. 25-27, 2003, listed as P17.
An abstract of “Fluorescent Response of Polyquinoline Derivatives toward Zinc Ion”, 23rd Organic Synthesis Seminar for the young - for organic synthesis in the future, Dec. 9, 2003, listed as P42.
Primary Examiner:
Aulakh, Charanjit S.
Attorney, Agent or Firm:
Greenblum & Bernstein, P.L.C.
What is claimed is:
1. A compound represented by the following general formula (I) or a salt thereof:
wherein R1, R2, R3, and R4 independently represent 2-quinolyl group which may be substituted with an alkoxy, 1-isoquinolyl group which may be substituted with an alkoxy, or 3-isoquinolyl group which may be substi m, n, p, and q independently represent 1 or 2; and L represents a single bond, or an alkylene group having 1 to 5 carbon atoms.
2. The compound or a salt thereof according to claim 1, wherein R1, R2, R3, and R4 represent 2-quinolyl group which may be substi m, n, p, and q represent 1; and L represents methylene group.
3. A fluorescent zinc ion sensor, which comprises the compound represented by the general formula (I) or a salt thereof according to claim 1.
4. A zinc complex formed by the compound represented by the general formula (I) or a salt thereof according to claim 1 and a zinc ion.
5. A method for measuring zinc ions, which comprises: (a) reacting a compound represented by the general formula (I) or a salt thereof according to claim 1 and a zinc ion to produce a zinc complex, and (b) measuring fluorescent intensity of the zinc complex.
6. A fluorescent zinc ion sensor, which comprises the compound represented by the general formula (I) or a salt thereof according to claim 2.
7. A zinc complex formed by the compound represented by the general formula (I) or a salt thereof according to claim 2 and a zinc ion.
8. A method for measuring zinc ions, which comprises: (a) reacting a compound represented by the general formula (I) or a salt thereof according to claim 2 and a zinc ion to produce a zinc complex, and (b) measuring fluorescent intensity of the zinc complex.
Description:
TECHNICAL FIELDThe present invention relates to a compound or salt thereof that can be used as a highly selective fluorescent zinc ion sensor.BACKGROUND ARTZinc ions play important roles in intracellular processes including enzyme catalysis, gene expression, apoptosis, neurotransmission and the like. Therefore, development of a zinc ion sensor is one of attractive fields for researchers of biological inorganic chemistry. Real time measurement of zinc ions and visualization of zinc ion distribution in living cells enable further elucidation of functions of zinc ions in bio-processes in vivo.As compounds that specifically bind to zinc ions, N,N,N′,N′-tetrakis(2-pyridyl-methyl)ethylenediamine (TPEN) as a cell membrane-permeable zinc chelator is known (Helv. Chim. Acta, 50, ). Further, zinc ion sensors based on the photoinduced electron transfer (PET) mechanism have been extensively developed in recent years. In these sensors, a zinc ion binds to a heteroatom located near a fluorescent dye to inhibit PET, thereby a fluorescent property of the sensor molecule is switched on. As compounds wherein a zinc chelator moiety is introduced into a fluorescent fluorescein derivative, ZnAF-2 (J. Am. Chem. Soc., 122, 1), Newport Green (the catalogue of Molecular Probes, Inc., “Handbook of Fluorescent Probes and Research Products” 8th edition, by Richard P. Haugland, pp. 805-817), Zinpyr-1 (J. Am. Chem. Soc., 122, ) and the like have been developed so far. Furthermore, various zinc ion sensors based on quinoline chromophores have also been developed. These sensors utilize fluorescence induced by complexation (Chelation Enhanced Fluorescence, CHEF), and light emission mechanism thereof has been studied in detail by using 2,6-bis(quinolinecarboxy)methylpyridine (P2Q)/zinc complex (J. Comput. Chem. Jpn., 2, pp.57-62, 2003). As zinc ion sensors based on this quinoline chromophores, TSQ (J. Neurosci. Methods, 20, 91, 1987), Zinquin (Biochem. J., 296, 403, 1993) and the like have already been practically used. However, since these compounds form both fluorescent 1:1 complex and 1:2 complex with zinc ions, quantitative analysis may sometimes become difficult when they are used for the measurement of zinc ions. Further, these zinc ion sensors, except for a few of them such as ZnAF-2, suffer from problems in that they have a high level of background fluorescence, and they are readily affected by pH in the physiological pH range, i.e., pH from 6 to 8. Moreover, these zinc ion sensors also have a problem in that multiple steps are required for their synthesis, and therefore, they cannot be manufactured in a large scale at low cost. DISCLOSURE OF THE INVENTIONAn object of the present invention is to provide a compound or salt thereof that can be used as a highly selective fluorescent zinc ion sensor. More specifically, the object of the present invention is to provide a compound or salt thereof that can specifically capture a zinc ion wherein the complex after the capture has a superior fluorescent characteristic. Further, another object of the present invention is to provide a compound that is not affected by physiological pH and can be used as a zinc ion sensor that can be produced at low cost.The inventors of the present invention conducted various researches to solve the foregoing objects. As a result, they found that a compound obtained by replacing the pyridine ring of the cell membrane-permeable zinc chelator, TPEN, with a quinoline ring successfully formed a 1:1 complex with a zinc ion in the presence of zinc ions, and the resulting complex emitted intense fluorescence, and that the compound had superior advantageous characteristics as a fluorescent zinc ion sensor. The present invention was achieved on the basis of these findings.The present invention thus provides a compound represented by the following general formula (I) or a salt thereof:
wherein R1, R2, R3, and R4 independently represent 2-quinolyl group which may be substituted, 1-isoquinolyl group which may be substituted, or 3-isoquinolyl group whic m, n, p, and q independently represent 1 or 2; and L represents a single bond, or an alkylene group having 1 to 5 carbon atoms wherein said alkylene group may contain one or more heteroatoms in the main chain, and may have one or more substituents on the main chain. From other aspects, the present invention also provides a fluorescent zinc ion sensor containing a compound represented by the aforementioned general formula (I) and a zinc complex formed with a compound represented by the aforementioned general formula (I) or a salt thereof and a zinc ion. This fluorescent zinc ion sensor can be used for the measurement of zinc ions in tissues or cells.From further aspects, the present invention also provides a method for measuring zinc ions, which uses a compound represented by the aforementioned general formula (I) or a salt thereof as a fluores a method for measuring zinc ions, which comprises (a) the step of reacting a compound represented by the aforementioned general formula (I) or a salt thereof and a zinc ion, and (b) the step of measuring fluorescence intensity of a zinc complex produced
and use of a compound represented by the aforementioned general formula (I) or a salt thereof as a fluorescent zinc ion sensor.BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 shows fluorescent characteristics of the fluorescent zinc ion sensor (TQEN) of the present invention.FIG. 2 shows results of job plot analyses of complex formation of TQEN with zinc ions.FIG. 3 shows fluorescent responses of TQEN in the presence of various metal ions.FIG. 4 shows results of titration of TQEN with zinc ions where changes were monitored by ultraviolet and visible absorption spectra.FIG. 5 shows results of titration of TQEN with zinc ions by means of NMR.FIG. 6 shows influence of pH on fluorescence intensity of a TQEN/zinc complex.FIG. 7 shows a result of X-ray structural analysis of a TQEN/zinc complex.FIG. 8 shows results of titration of 6OMeTQEN with zinc ions where changes in absorption were monitored by ultraviolet and visible absorption spectra.FIG. 9 shows fluorescent characteristics of the fluorescent zinc ion sensor (6OMeTQEN) of the present invention.BEST MODE FOR CARRYING OUT THE INVENTIONR1, R2, R3, and R4 independently represent 2-quinolyl group, 1-isoquinolyl group or 3-isoquinolyl group, and the 2-quinolyl group, 1-isoquinolyl group, and 3-isoquinolyl group may have one or more of any kinds of substituents at any positions. Each of R1, R2, R3, and R4 may be a different group. Alternatively, any two or more of them may be the same group.When the 2-quinolyl group, 1-isoquinolyl group, or 3-isoquinolyl group is substituted, types and number of the substituents and the substituting positions are not particularly limited. Preferred substituting positions are one or more positions selected from the 5-, 6-, and 7-positions of 2-quinolyl group, 1-isoquinolyl group, or 3-isoquinolyl group. Although it is not intended to be bound by any specific theory, when one or more substituents are present in the aforementioned position(s), each of the substituents may not inhibit formation of a zinc complex of a compound represented by the general formula (I) with a zinc ion. The number of the substituents of the 2-quinolyl group, 1-isoquinolyl group, or 3-isoquinolyl group may preferably be about 1 to 3. Unsubstituted 2-quinolyl group, 1-isoquinolyl group, or 3-isoquinolyl group may also be preferred.Examples of substituents that can be present on the 2-quinolyl group, 1-isoquinolyl group, or 3-isoquinolyl group include an alkyl group (the alkyl group may be a linear, branched, or cyclic alkyl, or a combination thereof, and has about 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms, which also applies to an alkyl moiety of other substituents having the alkyl moiety, e.g., alkoxy group and the like), an alkoxy group, hydroxyl group, an alkylcarbonyloxy group, an alkyloxycarbonylalkyloxy group, carboxyl group, an alkyloxycarbonyl group, succimidyloxycarbonyl group, an alkylcarbonyloxymethyloxycarbonyl group, amino group, an alkylamino group, a halogen group (the halogen atom may be any of fluorine atom, chlorine atom, bromine atom, and iodine atom), thiol group, an alkylthio group, a halogenated alkyl group, nitro group, sulfo group and an ester thereof, phosphono group and an ester thereof and the like. However, the substituents are not limited to these examples. One or more substituents may further be present on the aforementioned substituents.In the general formula (I), m, n, p, and q independently represent 1 or 2. When m, n, p, or q is 1, methylene group is meant to be present. When m, n, p, or q is 2, ethylene group is meant to be present. It is preferred that each of m, n, p, and q represents 1. Symbol “L” represents a single bond, or an alkylene group having 1 to 5 carbon atoms. The alkylene group may contain one or more heteroatoms in the main chain or may have one or more substituents on the main chain. In the present specification, the term “heteroatom” means a nonmetal atom except carbon atom, such as nitrogen atom, oxygen atom or sulfur atom. The alkylene group may have a branched chain, however, a linear alkylene group is preferred. The term “main chain” means an atomic chain in “L,” with minimum number of atoms, that connect each of the nitrogen atom and the carbon atom bound at each end of L. Examples of L containing a heteroatom in the main chain include —CH2—O—CH2—, —CH2—S—CH2—, —C2(CH3)—C2—O—CH2—, —CH2—CH2—O— and the like. However, L is not limited to these examples. The number, substituting positions ,and types of substituents that can be present on the main chain of L are not particularly limited. As substituents, such as those exemplified above as substituents that can be present on the 2-quinolyl group, 1-isoquinolyl group, or 3-isoquinolyl group can be used.Preferred examples of the compounds represented by the general formula (I) will be mentioned below. However, the compounds of the present invention are not limited to these examples.Among the compounds exemplified, the compounds where methoxy groups substitute on the quinoline ring have a property of a shift of an excitation wavelength to the side of longer wavelength, and therefore, they are highly useful for measurement of zinc ions in vivo because cell injury by the excitation light can be reduced. The compounds of the present invention represented by the aforementioned general formula (I) can exist as acid addition salts, and can also exist as base addition salts depending on type of substituents. Examples of the acid addition salts include mineral acid salts such as hydrochlorides, sulfates, and nitrates, and organic acid salts such as methanesulfonates, p-toluenesulfonates, oxalates, citrates, and tartrates and the like. Base addition salts are formed, for example, when a substituent of carboxyl group or the like exists, and examples thereof include metal salts such as sodium salts, potassium salts, calcium salts, and magnesium salts, ammonium salts, and organic amine salts such as triethylamine salts and the like. In addition to these examples, they may form salts with an amino acid such as glycine. The compounds of the present invention or salts thereof may also exist as hydrates or solvates, and any of these substances falls within the scope of the present invention.The compounds of the present invention represented by the aforementioned general formula (I) may have one or more asymmetric carbons depending on type of substituent. In addition to stereoisomers such as optically active substances based on one or more asymmetric carbons or diastereoisomers based on two or more asymmetric carbons, any of arbitrary mixtures of stereoisomers, racemates and the like also fall within the scope of the present invention.Among the compounds of the present invention represented by the general formula (I), compounds wherein R1, R2, R3, and R4 represent 2-quinolyl group, m, n, p and q represent 1, and L represents methylene group can be produced by one step, for example, by reacting 2-chloromethylquinoline and ethylenediamine. Details of this synthetic method will be described in the examples. Those skilled in the art can also readily synthesize other compounds that fall within the scope of the general formula (I) by referring to the methods described in the examples and by suitably choosing reaction raw materials, reaction conditions, reaction reagents and the like, and further adding modifications or alterations to these methods as required.The compounds of the present invention represented by the aforementioned general formula (I) or salts thereof are useful as fluorescent zinc ion sensors. Although the compounds of the present invention represented by the aforementioned general formula (I) or salts thereof themselves do not have a property of emitting intense fluorescence, they come to emit intense fluorescence when they capture zinc ions to form zinc complexes. The aforementioned compounds or salts thereof have characteristic features that they can specifically capture zinc ions and the formation of the complex is extremely fast. The compounds of the present invention also have characteristic features that they have substantially no background fluorescence, and the zinc complexes formed are hardly affected by pH within neutral range (pH 6 to 8) and emit stable and intense fluorescence. Therefore, the compounds of the present invention represented by the aforementioned general formula (I) or salts thereof are extremely useful as fluorescent zinc ion sensors for the measurement of zinc ions in living cells and living tissues under physiological condition. The term “measurement” used in the present specification should be construed in the broadest sense including quantification and qualification.Methods for using the fluorescent zinc ion sensors of the present invention are not particularly limited, and they can be used in the same manner as those for known zinc ion sensors. Ordinarily, a single kind of substances selected from the group consisting of the compounds represented by the aforementioned general formula (I) and salts thereof may be dissolved in an aqueous medium such as physiological saline or buffer, a mixture of water-miscible organic solvent such as ethanol, acetone, ethylene glycol, dimethyl sulfoxide, or dimethylformamide and the aqueous medium or the like, and the resulting solution may be added to the suitable buffer containing cells or tissue to measure fluorescence spectra. The fluorescent zinc ion sensor of the present invention may be combined with suitable additives and used in the form of compositions. For example, they can be combined with additives such as buffering agent, solubilizing agent, pH regulator and the like.EXAMPLESThe present invention will be explained more specifically with reference to the following examples. However, the scope of the present invention is not limited to these examples.Example 1 Synthesis of N,N,N′,N′-tetrakis(2-quinolylmethyl)ethylenediamine (TQEN)A mixture of 2-chloromethylquinoline hydrochloride (1.07 g, 5 mmol), ethylenediamine (75.0 mg 1.25 mmol), potassium carbonate (2.07 g, 15 mmol) and acetonitrile (10 mL) was refluxed by heating for 48 hours. The solvent was evaporated under reduced pressure, and then the residue was separated by phase separation using chloroform and water. The organic layer was dried, and the solvent was evaporated. The residue was washed with acetone to obtain the objective compound as white powder (0.70 g, 89%).mp 196 to 198° C. 1H NMR (CDCl3) δ (ppm): 2.89 (s, 4H), 3.96 (s, 8H), 7.49-7.54 (m, 8H), 7.66-7.71 (q, 8H), 7.87 (d, 4H), 7.99 (d, 4H) 13C NMR (CDCl3) δ (ppm): 52.58, 61.56, 120.85, 125.95, 127.16, 127.34, 128.88, 129.23, 136.08, 147.40, 160.29 ESI-MS calcd. for C42H37N6 ([M+H]+): 625.3074; Found: 625.3070 Anal. calcd. for C42H36N6: C, 80.61; H, 5.96; N, 13.43, Found: C, 80.69; H, 5.92; N, 13.41 Example 2 Synthesis of [Zn(TQEN)](ClO4)2 A mixture of TQEN (0.31 g, 0.50 mmol) and zinc perchlorate (0.18 g, 0.49 mmol) was stirred in acetonitrile at room temperature for 4 days. A small amount of unnecessary substances were removed by filtration, and then the solvent was evaporated under reduced pressure. The obtained residue was recrystallized from acetonitrile/ether to obtain a single crystal suitable for X-ray crystal structure analysis.1H NMR (CD3CN) δ (ppm): 3.18 (s, 4H), 4.1 (br., 4H), 4.43 (d, 4H), 7.3 (br., 4H), 7.46 (d, 4H), 7.5-7.7 (m, 4H), 8.07 (d, 4H), 8.56 (d, 4H) 13C NMR (CD3CN) δ (ppm): 57.00, 63.18, 123.17, 126.45, 129.19, 130.45, 130.79, 132.83, 142.94, 145.72, 160.78 ESI-MS calcd. for C42H36N6ZnClO4 ([Zn(TQEN)]ClO4): 787.1773; Found: 787.1766 Anal. calcd. for C42H38N6ZnCl2O9([Zn(TQEN)](ClO4)2.H2O): C, 55.61; H, 4.22; N, 9.26, Found: C, 55.85; H, 4.18; N, 9.35Example 3 Synthesis of 6-methoxy-2-bromomethylquinolineA mixture of 6-methoxy-2-methylquinoline (999 mg, 5.77 mmol), NBS (1.03 g, 5.77 mmol), AIBN (153 mg) and chlorobenzene (10 mL) was stirred at 120° C. for 2 hours, then added with AIBN (153 mg) again and further stirred for 4 hours with heating. The solvent was evaporated under reduced pressure, and then the residue was extracted with ether. The solvent was evaporated, and the residue was recrystallized from ethanol to obtain 6-methoxy-2-bromomethylquinoline as white powder (yield: 400 mg, 1.59 mmol, 28%).1H NMR (CDCl3) δ (ppm): 3.94 (s, 3H), 4.70 (s, 2H), 7.08 (d, 1H), 7.39 (q, 1H), 7.53 (d, 1H), 7.97 (d, 1H), 8.07 (d, 1H) 13C NMR (CDCl3) δ (ppm): 34.70, 55.69, 105.22, 121.74, 122.94, 128.68, 130.95, 136.25, 143.87, 154.62, 158.52Example 4 Synthesis of N,N,N′,N′-tetrakis(6-methoxy-2-quinolylmethyl)-ethylenediamine (6OMeTQEN)A mixture of 6-methoxy-2-bromomethylquinoline (0.076 g, 0.302 mmol), ethylenediamine (5 μL, 0.0755 mmol), potassium carbonate (0.125 g, 0.904 mmol) and acetonitrile (6 mL) was refluxed by heating for 14 hours. The solvent was evaporated under reduced pressure, and then the residue was separated by phase separation using chloroform and water. The organic layer was dried, and then the solvent was evaporated. The residue was washed with ethanol to obtain the objective compound as white powder (yield: 18.7 mg, 33%).mp 168 to 170° C. 1H NMR (CDCl3) δ (ppm): 2.84 (s, 4H), 3.90 (d, 20H), 6.94 (d, 4H), 7.30 (q, 4H), 7.48 (d, 4H), 7.78 (d, 4H), 7.88 (d, 4H) 13C NMR (CDCl3) δ (ppm): 52.78, 55.26, 55.50, 61.55, 105.05, 105.34, 121.35, 121.70, 121.81, 128.20, 130.21, 130.50, 134.87, 135.15, 143.51, 157.49, 157.94 ESI-MS calcd. for C46H45N6O4 ([M+H]+): 745.35; Found: 745.25 Test Example 1 When TQEN was irradiated with light of 317 nm in dimethylformamide (DMF) or a water-containing DMF solution (DMF:water=1:1), substantially no fluorescence was emitted. Whilst, fluorescence from TQEN was observed when zinc ions were added (fluorescence of 383 nm, FIG. 1). Further, job plot analyses revealed that TQEN formed a complex with 1 equivalent of zinc ions (FIG. 2).Test Example 2 Fluorescent responses of TQEN in the presence of various metal ions are shown in FIG. 3. TQEN emitted intense fluorescence in the presence of zinc ions or cadmium ions (about 60% of the fluorescence intensity obtained in the presence of zinc ions). Whilst, other metal ions gave almost no effect on the fluorescence of TQEN. None of sodium, potassium, magnesium, calcium, nickel, and manganese ions affected the fluorescence of the TQEN/zinc complex. However, 1 equivalent of copper, cobalt, cadmium, or silver ions and excess amount of iron ions inhibited fluorescence emission by TQEN with zinc, so it is possible that these metal ions form stronger complexes than zinc ions.Test Example 3 TQEN was titrated with zinc ions, and changes of ultraviolet and visible absorption spectra were monitored. The changes of absorption had an isosbestic point and stopped when 1 equivalent of zinc ions were added (FIG. 4). The results suggest that a TQEN complex containing more than 1 equivalent of zinc ions has the same absorbance as that of the 1:1 TQEN/zinc complex. In a competition experiment, metals in the former class of ions (sodium, potassium and the like) gave no change in absorption spectra of TQEN, whilst those of the latter class of ions (copper, cobalt and the like) induced the same spectral changes as those induced by zinc.Test Example 4 When TQEN was titrated with zinc and monitored by NMR, different peaks of TQEN and the TQEN/zinc complex were observed. The result suggests that an exchange rate between TQEN and TQEN bound with zinc is slower than the time scale of NMR (FIG. 5). In the zinc complex, the hydrogen atom at the benzyl position was observed as an AB quartet due to restricted degree of freedom of the conformation of the hydrogen atom in the complex. Further, in a competition experiment in the presence of 1 equivalent of TPEN, fluorescence from the TQEN/zinc complex completely disappeared. The result suggests that the zinc ion in the TQEN/zinc complex is completely removed by the stronger zinc ligand, TPEN. The zinc ion was not completely removed from the TQEN/zinc complex in an experiment using 1 equivalent of EGTA (ethylene glycol-bis(2-aminomethyl)-N,N,N′,N′-tetraacetic acid, KZn=not over 109 M), and only about 40% of fluorescence of the TQEN/zinc complex was quenched by EGTA in DMF-containing aqueous solution.Test Example 5 Influences by pH on fluorescent intensity of the TQEN/zinc complex was shown in FIG. 6. Stable and intense fluorescent intensities were obtained within the range of pH 4 to 10, which clearly indicate that the TQEN/zinc complex can be used as a fluorescent zinc ion sensor in a broad pH range.Test Example 6 A single crystal of [Zn(TQEN)](ClO4)2.2.5CH3CN was coated with paraffin oil and mounted on glass fibers. Measurements were performed at 173 K. For the measurements, monochromated MoKa of 50 kV/40 mA was used as a radiation source, and Rigaku Mercury CCD Detector was used. Substantially no decrease in peaks was observed during the measurements. The data were analyzed on PC by using CrystalClear Software (Rigaku). The structure was optimized by the direct method (SIR-92) using the least square method with respect to F2. Hydrogen atoms were considered as a riding model. The crystallographic data are summarized in Table 1. Major bond distances and angles are summarized in Table 2.TABLE 1CompoundsZn(TQEN)](ClO4)2.2.5CH3CNFormulaC47H43.5Cl2N8.5O8ZnFW 991.70Space groupP-1a, ?
12.581(3)b, ?
19.334(2)c, ?
21.051(5)α, deg
75.849(9)β, deg
72.6470(13)γ, deg
71.045(11)V, ?3 )Z 4Dcalc, g cm-3 1.444μ, cm-1 7.202θmax, deg
55.0temp, K 173no. reflns measd19595no. reflns used19595no. of params 1275final R1 (I & 2θ(I)) 0.078wR2 (all data) 0.207GOF 1.22R1 = Σ||Fo| - |Fc||/Σ|Fo|.wR2 = [Σw[(Fo2 - Fc2)2]/Σ[w(Fo2)2]]1/2. TABLE 2Zn(TQEN)](ClO4)2Zn—N(1)2.149(3), 2.147(3)Zn—N(2)2.169(3), 2.168(3)Zn—N(3)2.149(3), 2.150(3)Zn—N(4)2.393(3), 2.393(3)Zn—N(5)2.130(3), 2.131(3)Zn—N(6)2.369(3), 2.370(3)N(1)-Zn—N(2)82.15(11), 82.15(11)N(1)-Zn—N(3)76.15(11), 76.18(11)N(1)-Zn—N(4)77.98(10), 78.03(10)N(1)-Zn—N(5)159.48(10), 159.48(11)N(1)-Zn—N(6)92.23(10), 92.21(10)N(2)-Zn—N(3)157.67(11), 157.65(11)N(2)-Zn—N(4)90.64(10), 90.74(10)N(2)-Zn—N(5)77.72(10), 77.75(10)N(2)-Zn—N(6)77.94(10), 77.70(10)N(3)-Zn—N(4)89.99(10), 90.04(10)N(3)-Zn—N(5)124.24(10), 124.21(10)N(3)-Zn—N(6)97.48(10), 97.58(10)N(4)-Zn—N(5) 98.14(9), 98.03(10)N(4)-Zn—N(6)165.96(10), 165.87(10)N(5)-Zn—N(6)87.49(10), 87.53(10) The TQEN/zinc complex exists as two kinds of crystallographically independent molecules, one of which is shown in FIG. 7. TQEN binds to a zinc ion by means of 6 nitrogen atoms and provides an irregular octahedral coordination environment and twisted structure surrounding the zinc ion. All the 4 quinoline rings in the TQEN/zinc complex are in propeller-like positions due to steric hindrance between two quinoline rings (twisting angle: 21 degrees). This characteristic bonding scheme is considered to provide the desirable fluorescence feature of the TQEN/zinc complex.Test Example 7 In the same manner as in Test Example 3, solution of 6OMeTQEN (concentration of 6OMeTQEN: 34 μM) in water-containing DMF (DMF:water=1:1) was titrated with zinc ions (0 to 68 μM), and changes in absorption were monitored by measuring ultraviolet and visible absorption spectra (FIG. 8). As a result, it was confirmed that absorption wavelength was extended to around 350 nm, which is longer by about 20 nm than that of TQEN.Test Example 8 When 6OMeTQEN was irradiated with light having a wavelength of 340 nm, which was longer than the excitation wavelength (317 nm) of TQEN, in a water-containing DMF solution (DMF:water=1:1, concentration of 6OMeTQEN: 34 μM) in the same manner as that in Test Example 1, substantially no fluorescence was emitted. Whilst an increase in fluorescence emitted from 6OMeTQEN was observed when zinc ions (0 to 68 μM) were added (fluorescence of 410 nm, FIG. 9). These results suggest that 6OMeTQEN enables measurement of zinc ion concentrations using excitation light having a longer wavelength compared with TQEN, thereby successful reduction of cell injury due to excitation light is achievable when zinc ions in vivo, for example, are measured.INDUSTRIAL APPLICABILITYThe compounds represented by the aforementioned general formula (I) or salts thereof can be synthesized by one step in high yield by reacting ethylenediamine with 2-chloromethylquinoline or the like, and are characterized by successfully achieving specific capture of a zinc ion. Further, the complexes formed by the capture of zinc ions have superior fluorescent characteristics and are not affected by a physiological pH. Therefore, the compounds represented by the aforementioned general formula (I) or salts thereof are useful as fluorescent zinc ion sensors.
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