KR20130089491A - Zn2+ ion selective fluorescent probe and method for preparing the same - Google Patents

Abstract

PURPOSE: A Zn^2+ ion selective fluorescence probe is provided to selectively combine with a Zn^2+ ion without an interference phenomenon about other metal ions and excel as an imaging probe of intracellular Zn^2+ ions for biological use by high cytopermeability. CONSTITUTION: A Zn^2+ selective fluorescence probe of a chemical formula 1 is provided. A manufacturing method of the Zn^2+ selective fluorescence probe manufacture a compound of the chemical formula 1 by reacting a compound of a chemical formula 3 with trifluoroacetic acid. The compound of the chemical formula 3 is manufactured by reacting a compound of a chemical formula 4 with t-butylbromoacetate. The compound of the chemical formula 4 is manufactured by reacting a compound of a chemical formula 5 with triphenylphosphine (PPh_3) and pyridine.

Description

Zn2 + ion selective fluorescent probe and method for preparing the same}

The present invention relates to a fluorescent probe and a method of manufacturing the same as, more particularly, to a metal chelating group is a poly-amino carboxylic naphthalenol having a mid-butyl-based water-soluble Zn ^{2 +} as a sensor on which a Zn ^{2 +} selectivity.

Metal ions perform a wide range of specific functions in biological systems. While metal ions have an important effect on cell metabolism, sophisticated devices of cells and local tissues actively control the biological activity of metal ions in the form of enzymes and cofactors. Thus genetic abnormalities or environmental factors that misregulate the concentration or activity of metal ions may lead to dysfunction of tissues or organs [J.J.R. Frausto da Silva, R. J. P. Williams, The biological chemistry of the elements, Oxford University Press, Oxford, 1991].

Zinc is one of the most abundant divalent ionic metals in living organisms and acts as an essential cofactor for many metabolic enzymes and transcription factors, and in humans it has an average of 2.3 g of zinc [B.L. Vallee, D. S Auld, Zinc coordination, function, and structure of zinc enzymes and other proteins, Biochemistry 29 (1990) 5647-5659]. It is also known that these essential ingredient elements in the form of inorganic salts cause fatal organ disorders and induce copper deficiency [M.S. Willis, S.A. Monaghan, M.L. Miller, R. W. McKenna, W.D. Perkins, B.S. Levinson, V. Bhushan, S.H. Kroft, Am. J. Clin. Pathol. 123 (2005) 125-131].

To date, few ligands have been found to recognize zinc ions in aqueous media [Y. Zhou, H.N. Kim, J. Yoon, Bioorg. Med. Chem. Lett. 20 (2010) 125-128. However, in most cases, organic solvents and surfactants are used to dissolve ligands in an aqueous environment, and thus there is a problem in that unwanted cytotoxicity is exhibited both inside and outside the body. Thus, there is an increasing demand for a technology capable of selectively detecting zinc ions in vivo accurately and selectively without showing cytotoxicity. In particular, there is a growing need for the development of fluorescent probes that are selective for zinc ions and present in the presence of other metal ions.

The first aspect of the present invention, shows selective binding affinity for Zn ^{2 +} in HEPES buffer, does not receive the interference for other metal ions, the cell permeability is high imaging intracellular Zn ^{2 +} ions to provide a fluorescent probe having a Zn ^{2 +} ion selective capable.

The second technical problem to be achieved by the present invention is to provide a method for manufacturing the fluorescent probe.

The third aspect is to provide a Zn ^{2 +} ion selective corresponding ethyl ester derivative of the fluorescent probe with and a method of manufacturing another object of the present invention.

In order to solve the above problems, the present invention provides a fluorescent probe having zinc divalent ion selectivity of the general formula (1):

≪ Formula 1 >

The present invention to achieve the second technical problem,

A method for preparing a fluorescent probe having zinc divalent ion selectivity comprising the step of preparing a compound of Formula 1 by reacting a compound of Formula 3 with a trifluoroacetic acid:

<Formula 3>

According to an embodiment of the present invention, the compound of Formula 3 may be prepared by reacting a compound of Formula 4 with a t-butylbromoacetate mixture in an organic solvent:

&Lt; Formula 4 >

According to an embodiment of the present invention, the compound of formula 4 may be prepared by reacting a compound of formula 5 with triphenylphosphine (PPh ₃ ) and pyridine:

&Lt; Formula 5 >

According to an embodiment of the present invention, the compound of formula 5 may be prepared by reacting a compound of formula 6 with triphenylphosphine (PPh ₃ ) and sodium azide:

(6)

According to an embodiment of the present invention, the compound of Formula 6 may be prepared by reacting a compound of Formula 7 with 2-methoxyethanol and diethanolamine:

&Lt; Formula 7 >

The present invention provides a corresponding ethyl ester derivative of a fluorescent probe having zinc divalent ion selectivity of the formula (2) in order to achieve the third technical problem:

<Formula 2>

According to an embodiment of the present invention, the compound of Formula 2 may be prepared by reacting the compound of Formula 4 with ethyl bromoacetate:

Fluorescent probe having a Zn ^{2 +} selectivity according to the present invention is selectively coupled to Zn ^{2 +} ions and with no interference for other metal ions, indicates a non-sensitive to pH. Further, the higher the cell permeability exhibits excellent characteristics as an imaging probe of the effective intracellular Zn ^{2 +} ions in biological applications.

1 is a mechanism of a compound of formula 2 that is hydrolyzed by an esterase and converted to a compound of formula 1. FIG.
Figure 2 (a) is a fluorescence spectrum of 1 (λex = 346 nm) in the presence of various metal ions (5equiv.) In HEPES buffer (10 mM, pH = 7.4). 2 (b) is of the ^{Na +, K +, Ca 2} +, Mg 2 + 1.0 mM , and ^{Mn 2 +, Fe 2 +,} Fe 3 +, Co 2 +, Ni 2 + HEPES buffer (pH = 7.4), under Cu ^{2 +} 10 μM there appears when (white bars) of 10 μM was added to Zn ^{2 +} (black bars) 10 μM is a relative fluorescence intensity of the formula (I).
3 is a fluorescence spectrum of HEPES buffer (10 mM, pH = 7.4) for different amounts of Zn ^{2 +} presence of the formula 1 (λex = 346 nm). The turning insert is a graph of light emission adequate Zn ^{2 +} at 447 nm.
4 is an optimized structure of the first and Zn + 1 ^{+ 2} calculated in the B3LYP / 6-31G (d) level of theory, the hydrogen atoms have been omitted for clarity.
5 (top) is the profile of the frontier molecular orbital of 1. Green and red correspond to different stages of molecular wave function for HOMO and LUMO, with a classification of 0.02 au. Figure 5 (bottom) is a profile in the 1 ⁺ Zn ² + frontier orbital of a molecule.
6 (a) is a confocal fluorescence image of HeLa cells incubated with 2 μM Formula 2 for 30 minutes at λ _ex = 458 nm. (b) is bright field. (c) is a Zn ^{2 +} / pyrithione: as (20 μM, 1 1 ratio) is an image after the processing and 40 minutes. (d) is an image after addition of 0.1 mM TPEN.

The present invention provides a fluorescent probe having zinc divalent ion selectivity of the formula (1):

&Lt; Formula 1 >

Among various phosphors, 1,8-naphthalimide is commonly used as a phosphor due to its high light stability, large Stokes shift, and high quantum yield. The compound of Formula 1 is a naphthalimide-based water-soluble Zn ^{2 +} ion sensor having a polyamino carboxylate as a metal chelating group, and exhibits selective binding affinity for Zn ^{2 +} ions, thereby inducing intracellular Zn ^{2 +} ion imaging. Works effectively on

As a result of the quantum calculation, the electron density in the chelating group was not present at all in the compound of Formula 1 + Zn ^{2 +} , whereas it was present in the chelating group of the compound of Formula 1. In addition, the confocal image of HeLa cells cultured with the compound of Formula 2, an ethyl ester derivative, revealed that the fluorescence sensor was hydrolyzed by esterase to form the compound of Formula 1 of the present invention. Indicated. It can be seen that the increased cell permeability The properties as effective intracellular Zn ^{2 +} imaging probe for biological applications.

The present invention also provides a method of preparing a fluorescent probe having zinc divalent ion selectivity comprising the step of preparing a compound of Formula 1 by reacting a compound of Formula 3 with a trifluoroacetic acid:

<Formula 3>

The reaction can be carried out by stirring the compound of formula 3 and trifluoroacetic acid in an organic solvent such as methylene chloride.

Preferably, the compound of formula 3 may be prepared by reacting a compound of formula 4 with a t-butylbromoacetate mixture in an organic solvent:

&Lt; Formula 4 >

For example, dimethylformamide (DMF) may be used as the organic solvent, and the compound of Chemical Formula 3 may be prepared by extracting with ethyl acetate, removing water and organic solvent, and then separating the mixture by silica gel column chromatography. can do.

Preferably, the compound of formula 4 may be prepared by reacting a compound of formula 5 with triphenylphosphine (PPh ₃ ) and pyridine:

 &Lt; Formula 5 >

The compound of Formula 5 may be prepared by reacting a compound of Formula 6 with triphenylphosphine (PPh ₃ ) and sodium azide in an organic solvent:

(6)

For example, dimethylformamide (DMF) may be used as the organic solvent, and the compound of Formula 5 may be a mixture of the compound of Formula 6, triphenylphosphine (PPh ₃ ), and sodium azide. The temperature was lowered to 0 ° C., tetrabromomethane (CBr ₄ ) was added dropwise slowly and converted to azide and further converted to amine by Staudinger reaction, extracted with ethyl acetate, After removing the water and the organic solvent, it can be prepared by the process of separation by silica gel column chromatography.

Meanwhile, the compound of Formula 6 may be prepared by reacting the compound of Formula 7 with 2-methoxyethanol and diethanolamine.

&Lt; Formula 7 >

The present invention also provides a corresponding ethyl ester derivative of a fluorescent probe having zinc divalent ion selectivity of the formula (2) and a method for preparing the same:

<Formula 2>

The compound of Formula 2 may be prepared by reacting the compound of Formula 4 with ethyl bromoacetate.

For reference, the following scheme shows an exemplary schematic reaction diagram for preparing the compound of formula 1 and its corresponding ethyl ester compound from 4-bromo- Nn -butyl-1,8-naphthalimide.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to preferred embodiments for better understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited thereto.

Preparation Example 1 Synthesis of Compound 6

Diethanolamine (20 mL) and compound 7 (3.80 g, 11.4 mmol) were added to 2-methoxyethanol (20 mL), and the mixture was refluxed for 24 hours. After the reaction was completed, water was added to the reaction mixture, and extracted four times with 50 ml of ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained crude compound was separated by silica gel column chromatography using methylene chloride added with 3-5% methanol, and then the solvent was dried to give 1.56 g (yield 38.4%) of compound 6. ¹ H NMR (CDCl ₃ , 400 MHz): δ 8.87 (d, 1 H, J = 8.51 Hz); 8.46 (d, 1 H, J = 7.24 Hz); 8.41 (d, 1 H, J = 8.10 Hz); 7.62 (t, 1H, J = 7.85 Hz); 7.34 (d, 1H, J = 8.18 Hz); 4.11 (t, 2H, J = 7.55 Hz); 3.86 (br s, 4 H); 3.61 (t, 4H, J = 5.18 Hz); 3.09 (s, 2H); 1.67 (m, 2H); 1.42 (m, 2H); 0.96 (t, 3H, J = 7.34 Hz). ¹³ C NMR (CDCl ₃ , 100 MHz): 164.5, 164.1, 154.7, 132.0, 131.3, 131.2, 130.3, 127.2, 125.6, 122.9, 117.0, 116.3, 59.8, 55.4, 40.3, 30.3, 20.5, 14.1 ppm.

Preparation Example 2 Synthesis of Compound 5

Compound 6 (200 mg, 0.56 mmol), PPh ₃ (441 mg, 1.68 mmol) and NaN ₃ (182 mg, 2.80 mmol) were dissolved in anhydrous DMF (10 mL), the solution was then lowered to 0 ° C. and CBr ₄ ( 558 mg, 1.68 mmol) was slowly added dropwise over 10 minutes. Thereafter, the temperature of the reactant was raised to room temperature, followed by stirring for 12 hours. After the reaction was completed, water was added to the reaction mixture, and extracted four times with 50 ml of ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained crude compound was separated by silica gel column chromatogram (solvent; 1: 9 = EA: Hexane), and the solvent was dried to give 198 mg (yield 87.0%) of compound 5. ¹ H NMR (CDCl ₃ , 400 MHz): δ 8.60 (d, 1H, J = 7.34 Hz); 8.56 (d, 1 H, J = 8.47 Hz); 8.54 (d, 1H, J = 8.02 Hz); 7.75 (t, 1H, J = 7.88 Hz); 7.43 (d, 1 H, J = 7.99 Hz); 4.17 (t, 2H, J = 7.53 Hz); 3.66 (t, 4H, J = 5.80 Hz); 3.44 (t, 4H, J = 5.75 Hz); 1.71 (m, 2H); 1.45 (m, 2H); 0.98 (t, 3H, J = 7.34 Hz). ¹³ C NMR (CDCl ₃ , 100 MHz): 164.5, 164.0, 152.5, 131.8, 131.7, 130.5, 130.3, 127.9, 126.6, 123.6, 118.9, 118.5, 53.5, 49.2, 40.3, 30.4, 20.6, 14.1 ppm.

Preparation Example 3 Synthesis of Compound 4

Compound 5 (190 mg, 0.467 mmol) and PPh ₃ (736 mg, 2.81 mmol) were dissolved in 5 mL of pyridine and allowed to stand at room temperature for 1 hour. Concentrated aqueous ammonia (1 mL) was added to the solution, which was then kept at room temperature for 2 hours. After pyridine was removed under reduced pressure, methylene chloride and 1 N HCl were added to the reaction mixture. The organic layer was further washed with 1 N HCl, the water layers were combined, neutralized with KOH, and extracted three times with methylene chloride. The extracted organic layer was washed with water and brine, the water was removed with anhydrous sodium sulfate, and then the solvent was dried to give 120 mg (72.5%) of compound 4. ¹ H NMR (CDCl ₃ , 400 MHz): δ 8.52 (d, 1H, J = 7.05 Hz); 8.40 (d, 1 H, J = 8.42 Hz); 8.18 (d, 1 H, J = 8.35 Hz); 7.56 (t, 1H, J = 7.88 Hz); 6.63 (d, 1H, J = 8.56 Hz); 4.15 (t, 2H, J = 7.59 Hz); 3.43 (t, 2H, J = 5.62 Hz); 3.08 (t, 2H, J = 5.62 Hz); 2.88 (t, 2H, J = 5.54 Hz); 2.76 (t, 2H, J = 5.62 Hz); 1.70 (m, 2H); 1.44 (m, 2H); 1.25 (s, 2H); 0.97 (t, 3H, J = 7.31 Hz). ¹³ C NMR (CD ₃ OD, 100 MHz): 164.5, 164.0, 150.7, 134.2, 130.5, 129.3, 127.6, 123.9, 121.7, 120.2, 108.3, 103.6, 51.2, 47.3, 42.6, 40.6, 39.6, 30.1, 20.3, 13.1 ppm.

Preparation Example 4 Synthesis of Compound 3

After dissolving compound 4 (250 mg, 0.705 mmol) and KHCO ₃ (353 mg, 3.53 mmol) in anhydrous DMF (10 mL), the solution was lowered to 0 ° C. and t-butylbromoacetate (1.24) diluted in 5 mL of DMF. g, 3.53 mmol) was added slowly over 20 minutes. Then, the temperature of the reactant was raised to room temperature, followed by stirring for 60 hours. After the reaction was completed, water was added to the reaction mixture, and extracted four times with 50 ml of ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained crude compound was separated by silica gel column chromatography using methylene chloride added with 3-5% methanol, and then the solvent was dried to give 373 mg (yield 75.9%) of compound 3. ¹ H NMR (CDCl ₃ , 400 MHz): δ 8.60-8.56 (m, 2H); 8.44 (d, 1H, J = 8.30 Hz); 7.59 (t, 1H, J = 7.83 Hz); 7.32 (s, 1H); 6.61 (d, 1 H, J = 8.51 Hz); 4.17 (t, 2H, J = 7.57 Hz); 3.42 (s, 6H); 3.37 (br s, 2H); 3.02 (br s, 2H); 2.84 (m, 4 H); 1.72 (m, 2H); 1.50 (s, 9 H); 1.45 (m, 2H); 1.39 (s, 18 H); 0.97 (t, 3H, J = 7.38 Hz). ¹³ C NMR (CDCl ₃ , 100 MHz): 172.0, 170.7, 165.2, 164.5, 150.7, 134.9, 131.2, 130.2, 128.0, 124.4, 122.9, 120.8, 109.4, 104.0, 81.9, 81.3, 56.5, 56.4, 52.5, 52.2 , 51.8, 41.4, 40.1, 30.6, 28.4, 28.3, 20.7, 14.1 ppm.

Preparation Example 5 Synthesis of Compound 1

Compound 3 (157 mg, 0.225 mmol) was dissolved in dried methylene chloride (5.0 mL), trifluoroacetic acid (2.0 mL) was added at 0 ° C, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated and then evaporated under reduced pressure again with dimethyl ether. The residue was solidified with dimethyl ether and dried to give 55 mg (yield 46.2%) of compound 1. ¹ H NMR (DMSO-d ₆ , 400 MHz): δ 8.54 (t, 2H, J = 8.22 Hz); 8.24 (d, 1 H, J = 8.43 Hz); 7.89 (t, 1H, J = 7.87 Hz); 7.83 (d, 1 H, J = 7.76 Hz); 4.05 (t, 2H, J = 7.31 Hz); 3.54 (s, 6H); 3.48 (br s, 2H); 3.11 (br s, 2H); 2.96 (br s, 2H); 2.74 (br s, 2H); 1.62 (m, 2H); 1.35 (m, 2H); 0.92 (t, 3H, J = 7.31 Hz). ¹³ C NMR (DMSO-d ₆ , 100 MHz): 173.3, 166.4, 163.9, 163.5, 145.3, 131.8, 131.7, 130.3, 128.9, 128.5, 127.1, 123.2, 122.4, 56.5, 56.1, 54.7, 50.6, 49.8, 49.3 , 30.2, 20.4, 14.4 ppm.

Comparative Example. Synthesis of Compound 2

After dissolving compound 4 (520 mg, 1.47 mmol) and KHCO ₃ (735 mg, 7.34 mmol) in anhydrous DMF (10 mL), the solution was lowered to 0 ° C. and ethylbromoacetate (1.23 g 7.34) diluted in 5 mL of DMF. mmol) was added slowly over 20 minutes. Thereafter, the temperature of the reactant was raised to room temperature, followed by stirring for 60 hours. After the reaction was completed, water was added to the reaction mixture, and extracted four times with 50 ml of ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained crude compound was separated by silica gel column chromatogram using methylene chloride added with 3-5% methanol, and then the solvent was dried to give 210 mg (yield 23.3%) of compound 2. ¹ H NMR (CDCl ₃ , 400 MHz): δ 8.57 (d, 1H, J = 7.19 Hz); 8.51 (d, 1 H, J = 8.31 Hz); 8.43 (d, 1 H, J = 8.38 Hz); 7,61 (t, 1H, J = 7.86 Hz); 7.15 (s, 1 H); 6.60 (d, 1 H, J = 8.52 Hz); 4.24 (q, 2H, J = 7.14 Hz); 4.17 (t, 2H, J = 7.50 Hz); 4.06 (q, 4H, J = 7.12 Hz); 3.54 (s, 6H); 3.37 (br s, 2H); 3.02 (br s, 2H); 2.87 (m, 4H); 1.72 (m, 2H); 1.45 (m, 2H); 1.30 (t, 3H, J = 7.15 Hz); 1.18 (t, 6H, J = 7.15 Hz); 0.97 (t, 3H, J = 7.33 Hz). ¹³ C NMR (CDCl ₃ , 100 MHz): 172.5, 171.3, 165.1, 164.4, 150.5, 134.8, 131.2, 130.1, 127.7, 124.6, 122.9, 120.7, 109.6, 104.1, 61.2, 60.7, 55.5, 55.4, 52.7, 52.2 , 51.9, 41.2, 40.1, 30.5, 20.7, 14.4, 14.3, 14.1 ppm.

Example 1. Fluorescence Characterization

Of HEPES buffer (10 mM, pH = 7.4) Li +, Na +, K +, Mg 2 +, Ca 2 +, Sr 2 +, Ba 2 +, Cu 2 +, Zn 2+, Hg 2 +, Co 2 ^+, it was examined the absorption and fluorescence changes that appear when the Mn ^{+ 2} and the chloride salt of a wide range of metal ion containing Ni ^{2 +} was added, exhibited a change in the fluorescence in Fig.

Was made the addition of Zn ^{2 +} ions with a 100% aqueous solution of the compound 1 having a carboxylate group as the metal chelating induced fluorescence enhancement was significantly. In contrast, no change in the spectra was observed when other metal ions were added, and the fluorescence properties of Compound 1 + Zn ^{2 + in} the presence of other metal ions were investigated and shown in FIG. 2 (b). . Of 10 μM, depending on the usage rate of the physiological conditions ^{Mn 2 +, Fe 2 +,} Fe 3 +, Co 2 +, Ni 2 +, Cu 2 + and 1.0 mM of ^{Ca 2 +, Mg 2+, Na} +, K + when the addition, the subsequent addition of metal they did not lower the fluorescence intensity of a compound 1 + Zn ^{2 +,} which means that high selectivity of the compound 1 of the Zn ^{2 +} ions compared to other competing metal ions from an aqueous medium.

Zn ^{2 +} was measured fluorescence changes of the compounds (1) according to the concentration increase in the 10 mM HEPES buffer, which is shown in Fig. Compound 1 exhibited excellent fluorescence enhancement upon addition of Zn ^{2 +} in the physiological pH range. Low pH values, the carboxylate is protected Zn ^{2 +} because they can be a weakly coordinating ability of the I did not have fluorescence notable change in pH is less than 3, a satisfactory when was increased the pH from 4 to 10 Zn ^{2 +} detection capability Indicated. Fluorescence intensity of a compound 1 was saturated when increasing by the addition of Zn ^{2 +} ions of 1.0 equivalent of the compound [1]. Compound 1 and Zn ^{2 +} dissociation constant (K _d) the fluorescence titration curves and Job's graph analysis results between, 9.00 X 10 ^-7 M were determined to be, the stoichiometric ratio between in HEPES buffer, compound 1 with Zn ^{2 +} Was confirmed to be 1: 1.

Example 2 DFT and TD-DFT Calculation

By performing both calculations confirmed the compound 1 and its zinc complexes of Compound 1 + fluorescence change in Zn ^{+ 2.} Ground state using Density Functional Theory (DFT) using Becke's 3-parameter hybrid exchange function combined with Lee-Yang-Parr gradient-corrected correlation function (B3LYP) and base set 6-31G * the optimum of structure of the compound 1 and compound 1 + Zn ^{+ 2} about, it is shown in Figure 43. Electron transition wavelengths and oscillator intensities were calculated using the time-dependent density functional theory (TD-DFT) using the same functional set and basis set. When Zn ^{2 +} is present, it was confirmed that the three carboxyl groups in the compound 1 and the central nitrogen (N 1 in FIG. 4) are coordinated with Zn ^{2 +} . The calculated bond distances were 2.02 Å (Zn-O3), 2.08 Å (Zn-O1), 2.09 Å (Zn-O2) and 2.10 Å (Zn-N1), respectively. Among the other two nitrogen atoms in the chelating group, N 2 bonds relatively weakly (2.27 kPa), and the interaction of N ³ with Zn ²⁺ is very weak (4.02 kPa). As a result of the TD-DFT calculation, in compound 1, HOMO is the boundary trajectory function occupied by the lowest three electron transitions (S ₀ → S ₁ , S ₀ → S ₂ and S ₀ → S ₃ ) from the ground state to the singlet excited state. It can be seen that -3, HOMO-2, HOMO-1, HOMO and LUMO as a non-occupied boundary orbital function (FIG. 5, top). Similarly, in compound 1 + Zn ²⁺ , HOMO-3, HOMO-1, and HOMO are occupied boundary orbitals, and LUMO and LUMO + 1 are unoccupied boundary orbitals (FIG. 5, bottom). This is accompanied by.

Compound 1 and 1 + occupancy bounds main difference in orbital of Zn ^{2 +} whereas that within the available electron density chelating groups that may be involved in the PET or ICT process Compound 1 + Zn ^{2 +,} which do not exist at all compound 1 It exists. This difference eliminates the possibility of fluorescent quenching through PET or ICT in compound 1 + Zn ²⁺ . In 1 + Zn ²⁺ , the lowest electron transition with oscillator intensity greater than 0.01 (> 0.01) is S ₂ , which contributes significantly to the LUMO + 1 transition (calculated wavelength is 379 nm) in HOMO. Here is the state. In compound 1 the transition has oscillator strength well below 0.01 (<< 0.01) and contributes significantly to the LUMO transition (calculated wavelength 366 nm) in HOMO.

It is noteworthy that, as can be seen in the HOMO → LUMO transition, photo-excited charge transfer (PICT) from the chelating group to the phosphor moiety in compound 1 can lead to fluorescence quenching in compound 1. However, one + Zn chelating groups of ^{2 +} I is the electron density is lacking in the first Zn + ^{2 +} PICT If so happens that the addition of Zn ²⁺ to a solution of 1, the fluorescence of 1 is enhanced.

Example 3. Confocal Fluorescence Image

Ion forming denied pyrithione then with (2-mercapto-pyridine -N- oxide) was added to Zn ^{+ 2} from the outside for the fluorescence imaging of Zn ²⁺ in cultured cells (HeLa cells) as the biological use of a probe of the present invention Tested. In early experiments, Compound 1 was found to be hydrophilic and apparently impermeable to cell membranes. The ester functional group was prepared based on the fact that the cell membrane permeability is good and is transformed into the carboxylate sensor compound 1 by the action of the esterase. Confocal fluorescence images of HeLa cells incubated with 2 μM of the ester derivative 2 is Zn ^{2 +} / pyrithione (1: 1) of 20 μM bungan 40 were obtained by treatment with a sharp, showing them in Fig. Fluorescence images of cells exposed to prolonged treatment with N, N, N, N, -tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a cell-permeable powerful metal ion chelating agent, were restored to their original intensity. , and via these results it confirms that compound 1 is effective intracellular Zn ^{2 +} imaging probe for biological applications.

Claims (7)

A fluorescent probe having zinc divalent ion selectivity of Formula 1
&Lt; Formula 1 >

A method for preparing a fluorescent probe having zinc divalent ion selectivity, characterized by preparing a compound of Formula 1 by reacting a compound of Formula 3 with trifluoroacetic acid:
(3)

The method of claim 2, wherein the compound of Chemical Formula 3 is prepared by reacting a compound of Chemical Formula 4 with t-butylbromoacetate.
&Lt; Formula 4 >

The method of claim 3, wherein the compound of Chemical Formula 4 is prepared by reacting the compound of Chemical Formula 5 with triphenylphosphine (PPh ₃ ) and pyridine.
&Lt; Formula 5 >

The method of claim 4, wherein the compound of Formula 5 is prepared by reacting the compound of Formula 6, triphenylphosphine (PPh ₃ ) and sodium azide to prepare a fluorescent probe having zinc divalent ion selectivity Way:
(6)

The method of claim 5, wherein the compound of Formula 6 is prepared by reacting a compound of Formula 7 with 2-methoxyethanol and diethanolamine.
&Lt; Formula 7 >

The corresponding ethyl ester derivative of the fluorescent probe having zinc divalent ion selectivity of Formula 2:
(2)

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