KR101000017B1 - Chromo-fluorescent Derivatives of Quinaldine capable of intracellular ZnII and HgII dynamic exchange and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a novel fluorescent compound comprising a chromogenic portion of nitroazobenzene and a fluorescent portion of benzenesulfonamidoquinaldine. In addition, by using the fluorescence color change generated when zinc and / or mercury ions are added to the compound according to the present invention, the presence and / or amount of the metal ions can be detected, and a sensitizer, a biosensor, etc. It can be used as. The compound according to the present invention has superior selectivity to zinc and mercury ions than other metal ions, and is characterized by supplying zinc ions to life and removing mercury ions.

NABQ, fluorescence, quinaldine, zinc, mercury ions.

Description

Chromo-fluorescent Derivatives of Quinaldine capable of intracellular Zn (II) and Hg (II) dynamic exchange and manufacturing method

The present invention relates to a fluorescent compound capable of exchanging metal ions in a cell, a preparation method thereof, and a use thereof. More specifically, the present invention relates to a fluorescent colorable quinaldine derivative capable of exchanging zinc and mercury ions in a cell, a method for preparing the same, and a use thereof.

Zinc is an important mineral needed to control protein synthesis and cellular production of the immune system. The deficiency of zinc thus breaks the balance of metabolism, which in turn causes growth retardation in young children, reproductive dysfunction in males and females, decreased blood sugar, poor bone growth, brain disease, and high blood cholesterol. Cause problems. Another important effect of zinc on our bodies can be seen in zinc-binding proteins such as metallothionein, which detoxify lead by sequestering lead in the intestinal cells.

Mercury, on the other hand, is highly toxic and therefore causes a wide range of environmental pollution due to a number of natural and artificial causes of this metal. Sustained accumulation of mercury in the liver, kidneys, and spleen also causes DNA damage and disorders in the nervous system and mitosis. Consideration of this fact by can ssolrigo is a lot of interest in Zn ^{2 + 2 +} Hg supply and development of technology for the purpose of removal of (hereinafter referred to as mercury ions) (hereinafter, zinc ion) within the organism.

Fluorescent biosensors are of great interest because of their simplicity, selectivity, and sensitivity to specific metal cations in living organisms. One of the various fluorescence sensing mechanisms, ICT (Intracellular Charge Transfer) has been used extensively for ion sensing, molecular exchange and fluorescent labeling because ICT-based molecules emit long wavelengths and are very sensitive to spectral changes. to be.

Therefore, in order to overcome the above-mentioned problems, the present inventors have come to make a new compound having excellent selectivity for specific cations or anions in aqueous solution, supplying beneficial minerals to life and removing harmful minerals.

An object of the present invention is to provide a quinaldine derivative compound having excellent selectivity to zinc and mercury ions.

Another object of the present invention is to provide a quinaldine derivative compound for supplying zinc ions to living organisms and removing mercury ions.

Still another object of the present invention is to provide a method for preparing a quinaldine derivative compound having excellent selectivity to zinc and mercury ions, and supplying zinc ions to a living body and removing mercury ions.

Another object of the present invention is to provide a use of a quinaldine derivative compound having excellent selectivity to zinc and mercury ions, and supplying zinc ions to a living body and removing mercury ions.

The above and other objects of the present invention can be achieved by the present invention described in detail.

The quinaldine derivative compound according to the present invention is characterized in that it is represented by the following formula (1) comprising a chromogenic portion of the nitroazobenzene and benzenesulfonamidoquinaldine (benzenesulfonamidoquinaldine). Method for producing a compound represented by the formula (1) according to the present invention is a method for producing NABQ according to the present invention 8-aminoquinaldine (8-aminoqunaldine) and benzene sulfide obtained by reducing 8-nitroquinaldine (8-nitroquinaldine) 1-step 8-benzenesulfonamidoquinaldine is prepared by mixing and stirring benzenesulfonyl chloride with pyridine and the 8-benzenesulfonamidoquinaldine and 4- Nitrobenzenediazonium tetra fluoroborate (4-nitrobenzenediazonium tetra fluoroborate) is characterized in that it comprises a second step of preparing a compound represented by the formula (1) by mixing with pyridine in an organic solvent and stirring.

The method of using the quinaldine derivative compound according to the present invention utilizes the color fluorescence change generated when zinc ions, mercury ions, or both are added to the compound represented by the formula (1), thereby the presence or absence of the metal ion, the metal ion It is characterized by detecting the amount of, or both.

The present invention is excellent in selectivity with respect to zinc and mercury ions, and has the effect of providing a quinaldine derivative, a manufacturing method and use thereof for supplying zinc ions to a living body and removing mercury ions.

The present invention is a novel ICT-based molecule 5- (4-nitrobenzene diazo) -8-benzenesulfonamidequinaldine (hereinafter referred to as NABQ: 5- (4-nitrobenzene diazo) -8-benzenesulfonamidoquinaldine, represented by the following formula 1) Compound). The plurality of reaction parts of NABQ is composed of a chromophore of nitro azobenzene and a fluorescence of benzenesulfonamidoquinaldine, which causes a change in color and fluorescence due to metal cation complexation. Fluorescent chromogenic changes of NABQ following metal ion complexing have a significant effect on biological systems such as cells.

[Formula 1]

NABQ according to the invention is synthesized from 8-nitroquinaldine via a three step procedure of reduction, sulfonation and diazoation. More specifically, in the method for producing NABQ according to the present invention, 8-benzenesulfonamidoquinaldine is prepared by mixing and stirring 8-aminoquinaldine and benzenesulfonyl chloride obtained by reducing 8-nitroquinaldine together with pyridine. And a second step of preparing NABQ by mixing and stirring the 8-benzenesulfonamidoquinaldine and 4-nitrobenzenediazonium tetrafluoroborate with pyridine in an organic solvent. do.

The 8-nitroquinaldine is a known compound. Furthermore, the method of chemically reducing this compound can be easily carried out by those skilled in the art. For example, 8-nitroquinaldine may be mixed with Pd-C and Dioxane under a hydrogen atmosphere, and stirred for 12 hours to prepare 8-aminoquinaldine.

The benzenesulfonyl chloride and pyridine are known compounds, and 8-benzenesulfonamidoquinaldine is prepared by mixing and stirring together with 8-aminoquinaldine. At this time, the reaction temperature is characterized in that -4 ~ 4 ℃, preferably 0 ℃. In addition, the stirring time is characterized in that 4 to 6 hours, preferably 5 hours. The step is a sulfonamide reaction step, the other reaction conditions and the method itself can be easily carried out by those skilled in the art.

The 8-benzenesulfonamidoquinaldine and 4-nitrobenzenediazonium tetra fluoroborate are mixed with pyridine in an organic solvent and stirred to prepare a compound represented by Chemical Formula 1. At this time, the reaction temperature is characterized in that -4 ~ 4 ℃, preferably 0 ℃. In addition, the stirring time is characterized in that 11 to 13 hours, preferably 12 hours. THF may be used as the organic solvent, but is not particularly limited thereto. The step is a diazotization step, the other reaction conditions and the method itself can be easily carried out by those of ordinary skill in the art.

As shown in Figure 8a, absorption spectra without NABQ show ICT band characteristics around 400 nm.

UV / Vis and fluorescence spectra according to the invention were recorded with an S-3100 spectrophotometer and an RF-5301PC spectrophotometer, respectively. Perchlorate solution (1.00 mM) of each metal was prepared in CH ₃ CN. NABQ solution (1.00 mM) was also prepared in CH ₃ CN. Measurement of all fluorescence spectra according to the present invention was made at 343 nm lifting, insect slit width 3.0 and 5.0 nm. UV / Vis and fluorescence titration experiments were performed by using 20 μM and 10 μM NABQ with varying concentrations of metal chlorate in CH ₃ CN / H ₂ O solution (1.1, v / v).

FIG. 8a shows the UV / Vis spectra of NABQ (20 μΜ) with the presence of various metal cations in CH ₃ CN / H ₂ O solution (1.1, v / v). The metal cations are Na ⁺ , Li ⁺ , K ⁺ , Cs ⁺ , Rb ⁺ , Sr ^{2 +} , Ba ^{2 +} , Cu ^{2 +} , Mg ^{2 +} , Ca ^{2 +} , Cd ^{2 +} , Hg ^{2 +} and Zn ^{2 +} 200 μM each was used.

FIG. 8b shows the NABQ (20 μM) by addition of various concentrations of HgCl ₂ (0, 2, 4, 8, 12, 16, 20 μM) in CH ₃ CN / H ₂ O solution (1.1, v / v). UV / Vis spectra are shown.

Figure 8c shows the NABQ (20 μM) with addition of various concentrations of ZnCl ₂ (0, 2, 4, 8, 12, 16, 20 μM) in CH ₃ CN / H ₂ O solution (1.1, v / v). UV / Vis spectra are shown.

3a shows various metal cations (Na ⁺ Li ⁺ , K ⁺ , Cs ⁺ , Rb ⁺ , Sr ^{2 +} , in a CH ₃ CN / H ₂ O solution (1.1, v / v) at pH 7.4 (0.02 M HEPES). ^{Ba 2 +, Cu 2 +,} Mg 2 +, Ca 2 +, Cd 2 +, Hg 2 + and Zn ^{2 +} -, respectively, when the hydrochloride is the addition of 100μM), will showing the fluorescence of NABQ (10μM), and the 3b turns pH 7.4 (0.02 M HEPES) in CH ₃ CN / H ₂ O solution (1.1, v / v) increase in the concentration of the Zn ^{2 +} in (0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8 and 10μM) is shown the fluorescence response of NABQ (10μM), it is excited at 365nm, and the fluorescence intensity of NABQ according to the concentration of zinc ions at 513nm.

As zinc ions were added, λ max moved 36 nm deeply at a new wavelength (436 nm) and the color of the solution changed from yellow to red (see FIG. 8A). Only zinc ions increased about 11 times more in fluorescence intensity at 513 nm and no other cations showed a marked increase in fluorescence. 9 shows that zinc ions can be selectively detected among the various cations tested. 3b shows that the addition of 0.2 μM of zinc ions increases the fluorescence intensity by 90%, and the addition of 0.5 μM of zinc ions causes the fluorescence intensity to reach its highest point. This indicates that NABQ is a zinc ion sensor with excellent sensitivity and selectivity for zinc ions, while enabling quantitative analysis of zinc ions.

9 shows the change in fluorescence of NABQ (10 μM) + metal cations with addition of zinc ions (10 μM) at 513 nm CH ₃ CN / H ₂ O solution (1/1, v / v). I represents the fluorescence intensity of NABQ + metal cation when zinc ions are present and I ₀ is not present.

As shown in FIG. 10, the plot analysis of Job shows the maximum when the mole fraction of zinc ions is 0.33, which means that the composition ratio of NABQ and zinc ions is 2: 1. As shown in FIG. 11B, FAB-MS with m / z = 958 (2NABQ + Zn 2+) also provides a solid basis for 2: 1 stoichiometry. 11A, 11B and 11C show FAB-MS of NABQ, NABQ + Zn (ClO ₄ ) ₂ , and NABQ + Hg (ClO ₄ ) ₂ , respectively. O'Halloran et al. ( J. Bio . Inort . Chem. 1999 , 4, 775-783, J. Am . Chem . Soc . 1999 , 121, 11448-11458) provide a single crystal structure of quinoline, namely 2: 1 It is reported that in complexing mode zinc ions are coordinated to sulfonamide groups as well as to nitrogen atoms of quinoline.

On the other hand, when mercury ions were added to the NABQ solution, it was found that the UV spectrum of λmax moved by 44 nm at a new wavelength centered at 444 nm. The fluorescence emission of NABQ was considerably quenched for mercury ions, as shown in Figure 3a. It is well known that mercury ions act as fluorescent quenching ions through enhanced spin-orbit coupling.

4a to turn the pH 7.4 (0.02 M HEPES) in CH3CN / H2O solution (1 / .1 v / v) in, deultteugo at ^{365nm, 2NABQ-Zn 2 + (} 5.0μM according to HgCl ₂ (0-10μM) with different concentrations of ) represents the change in the fluorescence, the degree 4b - ■ - represents the decrease in fluorescence intensity of _{^{2NABQ-Zn 2 + (I 0}} -I) at 510 nm in accordance with the increase of the concentration of Hg ^{2 +,} - ▲ - is in accordance with the increase of the concentration of Hg ^{+ 2} indicates a reduction in the fluorescence intensity of NABQ alone.

As shown in FIG. 4B, 2NABQ-Zn ²⁺ exhibited stronger quenching than NABQ in titration experiments of mercury ions. As shown in the Figure 12, when the unexpected NAB-Hg ^{2 +} complexes have been Zn ^{2 +} by the addition of 1 equivalent to a solution, it did not have a fluorescence emission recovery achieved. This means that the binding properties of NABQ Hg ^{+ 2} is greater than the Zn ^{+ 2.}

12 shows CH ₃ CN / H ₂ O solution (1/1, v / v), various concentrations of ZnCl ₂ (0, 2, 4, 6, 8, 10, 20, 30, 40 in a lift state of 365 nm). , it shows the change in fluorescence NABQ-Hg ^{2 +} (10μM) in accordance with the addition of 50 μM).

FIG. 23 depicts ¹ HNMR data of NABQ (20 μM) with or without Hg (ClO ₄ ) ₂ in CDCl ₃ / CD ₃ CN solution (9/1, v / v). FIG. 24 also shows ¹ HNMR data of NABQ (20 μM) with or without Zn (ClO ₄ ) ₂ in CDCl ₃ / CD ₃ CN solution (9/1, v / v).

In 2NABQ-Zn ^{2 +} solution results in Figure fluorescence quenching by applying different cations was not observed. In contrast, the as shown in FIG. 13 and FIG. 14, when adding the Hg ^{+ 2} in the above solution was the result of the complete fluorescence quenching. Therefore 2NABQ-Zn ^{2 +} complexes than NABQ indicates a more selective and sensitive to Hg ^{+ 2} ion, which means that it can be used as the Hg ^{+ 2} sensor.

Figure 13 shows the fluorescence of NABQ (10 μM) with the addition of CH ₃ CN / H ₂ O solution (1/1, v / v), zinc ion (10 μM) and one of the other metal cations (10 μM) at 513 nm. It is a change. I is, I ₀ shows the fluorescence intensity of NABQ + Zn ^{+ 2} when there is no in the presence of a metal cation.

FIG. 14 shows 2NABQ-Zn ²⁺ ( ¹ ) with the addition of a CH ₃ CN / H ₂ O solution (1/1, v / v), mercury ions (10 μM) and one of the other metal cations (10 μM) at 513 nm. 10 μM) of fluorescence change. I shows the fluorescence intensity of 2NABQ-Zn ^{2 + +} metal cation is when there is no, I ₀ when the mercury ions present.

By mixing NABQ-Hg the same amount under the hexane vapor divergent (hexane vapour diffusion) to an in-depth understanding of the binding mode for ^{2 +} 2NABQ-Zn ^{2 +} and Hg (NO _{3) 2} by inserting the CH ₃ CN solution One decision was obtained. Crystallographic data on the crystal structure was submitted to CCDC 651996 at the Cambridge Crystallographic Data Centre. As shown in FIG. 5, the crystal structure is 1 to coordinate interaction between NABQ and Hg ^{+ 2:} shows a first combination ratio. Further, Hg ^{2 +} ion is bonded to the pyridyl N atom of the sulfonamide with nitric acid. The fifth turning substituted atom ellipsoid structure of NABQ-Hg ^{+ 2} with the (atomic displacement ellipsoid) is shown with a 30% probability level.

^{NABQ-Hg 2 + (NO 3} -) diffraction data were collected on Bruker AXS SMART time the light receiver of the light beam is dansaeng Mo Kα (λ = 0.71073Å) mounted at 170 K season. The crystals were grown on glass fibers, CCD data were measured and built using the Bruker-SAINT software package, and the structure was calculated using SHEXTL V5.10. The hydrogen atoms were located where they were calculated. ^{NABQ-Hg 2 + (NO 3} -) specific crystallographic data for are set forth in Table 1 and Table 2 below.

Empirical formula C ₂₂ H ₁₆ Hg N ₆ O ₇ S Formula weight 709.06 Temperature 170 (2) K Wavelength 0.71073 Crystal system Monoclinic Space group C2 / c Unit cell dimensions a = 22.629 (2) Åα = 90.00 ° b = 15.2721 (16) Åβ = 113.893 (2) ° c = 15.4352 (16) Åγ = 90.00 ° Volume 4877.1 (9) Å ³ Z 8 Density (calculated) 1.931 Mg / m ³ Absorption coefficient 6.453 mm ^-1 F (000) 2736 Crystal size 0.10 x 0.05 x 0.03 mm ³ Reflections collected 12736 Independent reflections 4550 [R (int) = 0.0845] Data / restraints / parameters 4550/3/335 Goodness-of-fit on F2 0.905 Final R indices [I> 2σ (I)] R ₁ = 0.0458, wR ₂ = 0.1023 R indices (all data) R ₁ = 0.0717, w R ₂ = 0.1059 Largest diff. peak and hole 2.182 and -2.211 e.Å ^-3

Hg (1) -N (1) 2.136 (7) Hg (1) -O (5) 2.178 (10) Hg (1) -N (2) 2.477 (7) S (1) -O (2) 1.434 (6) S (1) -O (1) 1.444 (6) S (1) -N (1) 1.638 (7) S (1) -C (4) 1.777 (9) N (1) -Hg (1) -O (5) 161.8 (3) N (1) -Hg (1) -N (2) 70.6 (2) O (5) -Hg (1) -N (2) 127.5 (3)

Furthermore, 8-benzenesulfonamidoquinaldine and 4-nitrobenzenediazonium tetrafluoroborate according to the present invention are mixed to form NABQ. Single crystals of 8-benzenesulfonamidoquinaldine for X-ray diffraction analysis were produced in CH ₃ CN solution for one week under hexane vapor divergence. Crystallographic data on the crystal structure was submitted to CCDC as CCDC 665481.

Diffraction data of 8-benzenesulfonamidoquinaldine were collected at room temperature on a Bruker SMART AXS diffractometer equipped with a mono Kn of Mo Kα (λ = 0.71073 μs) light. The crystals were grown on glass fibers, CCD data were measured and built using the Bruker-SAINT software package, and the structure was calculated using SHEXTL V5.10. The hydrogen atoms were located where they were calculated. Specific crystallographic data for 8-benzenesulfonamidoquinaldine are shown in Tables 3 and 4 below.

Empirical formula C ₁₆ H ₁₄ N ₂ O ₂ S Formula weight 298.35 Temperature 293 (2) K Wavelength 0.71073 Crystal system Monoclinic Space group P2 ₁ / c Unit cell dimensions a = 10.0948 (14) Åα = 90.00 ° b = 19.406 (3) Åβ = 112.153 (2) ° c = 7.9672 (11) Åγ = 90.00 ° Volume 4877.1 (9) Å ³ Z 4 Density (calculated) 1.371 Mg / m ³ Absorption coefficient 0.229 mm ^-1 F (000) 624 Crystal size 0.10 x 0.05 x 0.01 mm ³ Reflections collected 8006 Independent reflections 2834 [R (int) = 0.0606] Data / restraints / parameters 2834/0/195 Goodness-of-fit on F2 0.845 Final R indices [I> 2σ (I)] R ₁ = 0.0477, w R ₂ = 0.1013 R indices (all data) R ₁ = 0.1240, w R ₂ = 0.1189 Largest diff. peak and hole 0.162 and -0.277 e.Å ^-3

S (1) -O (1) 1.425 (2) S (1) -O (2) 1.4300 (19) S (1) -N (1) 1.611 (3) S (1) -C (6) 1.754 (3) N (1) -C (7) 1.409 (4) N (1) -H (1N) 0.78 (3) N (2) -C (15) 1.320 (3) N (2) -C (12) 1.363 (3) O (1) -S (1) -O (2) 119.14 (13) O (1) -S (1) -N (1) 108.78 (15) O (2) -S (1) -N (1) 105.08 (14) O (1) -S (1) -C (6) 107.21 (14) O (2) -S (1) -C (6) 108.87 (13) N (1) -S (1) -C (6) 107.22 (13) C (7) -N (1) -S (1) 126.3 (3) C (7) -N (1) -H (1N) 109 (2) S (1) -N (1) -H (1N) 116 (2) C (15) -N (2) -C (12) 117.7 (3)

First turning on fluorescence-based on-off switching shows a coupling mechanism of NABQ having Zn ^{2 +} and Hg ^{2 +} ions. NABQ will emit weak fluorescence initially, but emits strong fluorescence Combined with Zn ^{+ 2.} Considering the complex shape of ^{2NABQ-Zn 2 +, Zn 2} + ions is a two previously mentioned to the nitrogen atom of the sulfonamide unit and quinoline: It is known that the coordination complex in 1 mode. When it based on crystal structure analysis of the above-2NABQ Zn ^{2 +,} Hg ^{+ 2} and is separated from the Zn ^{2 +} 2NABQ-Zn ^{2 +} complex, to form a new combined NABQ and fluorescence is missing.

Sensing specific molecules for selective monitoring of metal ions in living organisms is of great importance and has biological applicability. To see if NABQ could be used in this way, monkey kidney cells (LLC-MK2) were incubated with NABQ (50 μM) and fluorescence changes were observed by confocal microscopy. As shown in Figure 6b, the NABQ shows a weak fluorescence image, which is consistent with the previous results. Next, cells were incubated with NABQ for 5 minutes at 37 ° C. in growth solution under the same conditions and ZnCl ₂ (50 μM, only 1 equiv) was added. At this time, the cells could see a significant increase in fluorescence in the space, which means that the NABQ be used to detect the organism in Zn ^{+ 2.}

Sixth and turn shows a the confocal fluorescence image of LLC-MK2 cells in Zn ^{2 +} (excitation at 405nm. X20 objective lens Zeiss LSM 510 META confocal microscope), (a) is of LLC-MK2 cells with NABQ Bright field transfer image, (b) fluorescence image with NABQ (50 μM), (c) image immediately after ZN ^{2 +} (50 μM) is added, (d) the composite image of (a) and (c) Indicates.

Here, as shown in the Figure 7, Zn ^{2 +} in 2NABQ-Zn ^{2 +} complexes were found in LLC-MK2 cells in is replaced by Hg ^{2 +.} 7b, as the road and the road 7c shown in fluorescence of stronger initially 2NABQ-Zn ^{2 + 2 +} Hg disappeared as one equivalent of a gradually added. Furthermore, the AMC-HN3, Hep-2 and MDCK cell lines were tested and similar results were obtained as shown in FIGS. 15-20. Thus NABQ has applicability as Zn ^{+ 2,} and the medium within the imaging cells of the Hg ^{+ 2} Zn Based on the exchanged attribute ^{2 +} and / or Hg ^{+ 2.}

The LLC-MK2, AMC-HN3, Hep-2 and MDCK four cells were cultured (Dulbeccos modified Eagles medium: GibcoBRL, USA), 10% (v / v) Hyclone (heat-inactivated fetal calf serum), 100 Incubated in air containing u / ml penicillin, 100 ug / ml streptomycin, 0.25 mM L-glutamine, 37 ° C., 5% CO ₂ . When the cells reached a logarithmic functional state, the cell density was adjusted to 1 × 10 ⁵ per / well. The cells were also used to be planted in 12-well plates with 1.0 mL of cell suspension in each well. After cell fixation, the mediator was removed. Rinse the cell membrane twice with PBS (Phosphate Buffered Saline) and add 1.0 mL of medium to each well.

To determine the cell permeability of zinc ions first, the cells were incubated for 5 minutes at room temperature with 50 μM of NABQ sensor. As soon as 1 equivalent of zinc ions were added to the cells, fluorescence images of intracellular zinc ions were observed through a Zeiss LSM 510 META confocal microscope. The excitation wavelength of the laser is 405 nm and the emission spectra are in the range of 420-480 nm (single channel). LSM META was used as image analysis software. For all images, the settings of confocal microscopy such as transmission density, brightness, contrast ratio, scan speed, etc. were kept the same to compare the relative intensity of intracellular zinc ion fluorescence. Furthermore, the addition of one equivalent of zinc ions to the cells drastically reduced the release of zinc ion complexes. Fluorescence intensity was reduced and fluorescence images were taken in a similar manner to the first step.

FIG. 15 is a confocal fluorescence image of zinc ions in AMC-HN3 cells (wavelength at 405 nm, Zeiss LSM META confocal microscope, 20x magnification lens), (a) AMC- with NABQ (50 μM) a bright field image of a transmission HN3, (b) is a fluorescence image of the AMC-HN3 in NABQ (50 μM), (c ) shows an image immediately after the Zn ^{2 +} (50 μM) it added.

FIG. 16 is a confocal fluorescence image of mercury ions in AMC-HN3 cells (wavelength at 405 nm, Zeiss LSM META confocal microscope, 20x magnification lens), (a) 2NABQ / Zn ²⁺ (50 μM) a bright field image of a transmission-AMC with HN3, (b) shows an image immediately after the addition of Hg ^{2 +} (50 μM).

FIG. 17 is a confocal fluorescence image of zinc ions in Hep-2 cells (wavelength at 405 nm, Zeiss LSM META confocal microscope, 20x magnification lens), (a) Hep- with NABQ (50 μM) a bright field image of the transmission 2, (b) is a fluorescence image of the Hep-2 in NABQ (50 μM), (c ) shows an image immediately after the Zn ^{2 +} (50 μM) it added.

FIG. 18 is a confocal fluorescence image of mercury ions in Hep-2 cells (wavelength at 405 nm, Zeiss LSM META confocal microscope, 20x magnification lens), (a) 2NABQ / Zn ^{2 +} (50 μM) the bright-field image of a transmission in Hep-2, (b) shows an image immediately after the addition of Hg ^{2 +} (50 μM).

FIG. 19 is a confocal fluorescence image of zinc ions in MDCK cells (wavelength at 405 nm, Zeiss LSM META confocal microscope, 20x magnification lens), (a) bright field of MDCK with NABQ (50 μM) transmission image, (b) is a fluorescence image of a MDCK with NABQ (50 μM), (c ) shows an image immediately after the Zn ^{2 +} (50 μM) it added.

FIG. 20 is a confocal fluorescence image of mercury ions in MDCK cells (wavelength at 405 nm, Zeiss LSM META confocal microscope, 20x magnification lens), (a) with 2NABQ / Zn ^{2 +} (50 μM) a bright field image of a transmission MDCK, (b) shows an image immediately after the addition of Hg ^{2 +} (50 μM).

NABQ and 2NABQ-Zn ^{2 +} LLC-MK2 cells to know about the impact more chairs for the cytotoxicity of Hg ^{2 +} were handling the Hg ^{2 +,} Hg ^{2 +} and NABQ, and Hg ^{2 +} and 2NABQ-Zn ² + (Untreated cells served as controls). Post-treatment cell viability was taken as a measure of toxicity and is shown in Figure 21. As expected, it was treated with Hg ^{2 +} exhibited severe poisoning of the cell as indicated by the loss of 90% cell viability appeared after one day culture were added to the ion. However, cell viability when added to the Hg ^{+ 2} and NABQ surprisingly been or about 50%, up to Hg ^{+ 2} and the survival rate is about 77% when added to 2NABQ-Zn ^2+. Further, when there is Hg ^{+ 2} would be the 2NABQ-Zn ^{2 +} than NABQ be noted that it was more effective to increase cell viability.

21 survival (cells that are not treated in turn LLC-MK2 cells when the cells are treated with Hg ^{2 +,} Hg ^{2 +} and NABQ (50μM), and Hg ^{2 +,} or 2NABQ-Zn ^{2 +} (50μM) are 100% Assume survival rate). Cell viability was assayed by MTT method.

Next, ^{Hg 2 +, 2NABQ-Zn 2} +, or Hg ^{+ 2} and Zn ^{2 +-2NABQ} the Hg ²⁺ ion bonded to NABQ in metal ion exchange process by executing the emission spectroscopy on the supernatant of treated cells with that I saw that it was removed. As depicted in claim 22 also, Hg ^{2 +} ions was the fluorescence intensity of the supernatant of the only treatment with 2NABQ-Zn ^{2 +} in the absence of cells are slightly decreased, which is an upper layer being 2NABQ-Zn ^{2 +} is added in the cell This is because the concentration in the liquid is reduced. It was significant fluorescence quenching of 2NABQ-Zn ²⁺ in the supernatant in a state in which there are cells in the Hg ^{+ 2} ion, which is absorbed in the first cell into the Hg ^{+ 2} ions 2NABQ-Zn ^{2 +} Zn ^{2 +} ions in the complex It is released out of the cell replaced with the.

The turn 22a shows a change in fluorescence ^{2NABQ-Zn 2 + (25μM)} , the turns 22b 2NABQ-Zn ²⁺ (25μM) represents a change in the fluorescence remainder of LLC-MK2 of the addition, the turning 23c 2NABQ-Zn ²⁺ Fluorescence change of the residual of LLC-MK2 containing mercury ions with (25 μM) addition was shown, all of which were measured at an excited wavelength of 365 nm.

2 summarizes the NABQ metal ion exchange process in cells. In this model, NABQ provides a Zn ^{2 +} in cells, and plays a role of double-metal-sensitive ion-exchange medium to release the Hg ^{+ 2} ions.

After all, the compound according to the invention functions as a chromophore and a fluorescent moiety for the metal cation. NABQ is Zn ^{2 +} and Hg ^{+ 2} and 2, respectively, as deduced from the X-ray crystal structure: The coordination complexes by 1 stoichiometry: 1 and 1. As observed by confocal microscopy, NABQ fluorescence changes for metal cation complexes in living cells are consistent with the results of theoretical fluorescence studies. Confocal imaging may also results in emission of Hg ²⁺ ion out of the cell by executing the mutual (reciprocal) metal ion exchange with the Hg ^{+ 2} ions to penetrate into the cell. This suggests that NABQ may play a role in Zn ^{2 +} transport and Hg ^{2 +} removal in living cells, leading to an increase in cell viability in the presence of harmful Hg ^{2 +} ions.

The use method according to the present invention utilizes the fluorescence color change generated when zinc ions, mercury ions, or both are added to the compound represented by Chemical Formula 1, thereby providing the presence of the metal ions, the amount of the metal ions, or It is characterized by detecting all of them. As mentioned above, the present invention shows very good selectivity for zinc and mercury ions. Techniques for detecting metal ions using the fluorescence color change, and thus the technique for measuring the amount of metal ions, can be easily implemented by those skilled in the art.

In addition, the quinaldine derivative compound according to the present invention can be used as a sensitizer, the method of producing the sensitizer itself can be easily carried out by those skilled in the art. Furthermore, the quinaldine derivative compounds according to the present invention can also be used as a biosensor.

The invention can be better understood by the following examples, which are intended for the purpose of illustration of the invention and are not intended to limit the scope of protection defined by the appended claims.

Example

Example  1: Preparation of the compound represented by formula (1)

8-benzenesulfonmididoquinaldine (1.0 g, 3.35 mmol) and 4-nitrobenzenediazonium tetrafluroborate (0.87 g, 3.6 mmol) were added to 100 mL of THF and stirred at 0 ° C. for 30 minutes. Thereafter, 10 mL of pyridine is dropped dropwise and stirred at 0 ° C. for 12 hours. The stirred solution is washed with 300 mL of water and extracted with 300 mL of CH ₂ Cl ₂ . The organic layer was washed twice with 300 mL of water and dried over anhydrous magnesium sulfate. The dried result was purified by column chromatography on ethyl acetate using hexane acetate / hexanes (1/3, v / v), the purified final product was NABQ and had a yield of 33%.

Mp: 210 o C. ¹ H NMR (200 MHz, CDCl ₃ ): δ 9.6 (s, NHSO ₂ , 1H), 9.09 (d, ArH, 1H), 8.35 (d, ArH, 2H), 8.06-7.96 (m, ArH, 4H), 7.89 (d, ArH, 2H), 7.50-7.24 (m, ArH, 4H), 2.77 (s, CH ₃ , 3H); ¹³ C NMR (50 MHz; CDCl3) δ 158.82, 155.99,148.53, 142.07, 139.25, 137.43, 137.30, 133.27, 132.44, 129.12, 128.17, 127.2, 125.40, 124.78, 124.31, 123.4, 113.52, 113.21, 25.17, 25.17 FAB-MS calc. for C ₂₂ H ₁₇ N ₅ O ₄ S [M + H] ⁺ 448.1, found 448.

Simple modifications and variations of the present invention can be readily made by those skilled in the art, and such variations or modifications can be considered to be included within the scope of the present invention.

First turning on fluorescence-based on-off switching shows a coupling mechanism of NABQ having Zn ^{2 +} and Hg ^{2 +} ions.

2 shows the NABQ metal ion exchange process in cells.

3 shows the fluorescence intensity of NABQ when various metal ions are added.

The fourth turn shows the change in fluorescence 2NABQ-Zn ^{2 +} in accordance with the concentration of the mercury ion.

Fifth turn shows the structure of NABQ-Hg ^{2 +.}

Sixth turn shows the confocal fluorescence image of LLC-MK2 cells in Zn ^{+ 2.}

Seventh turn shows the confocal fluorescence image of LLC-MK2 cells in Zn ^{+ 2.}

Figure 8a shows the UV / Vis spectra of NABQ with the presence of various metal cations.

Figure 8b shows the UV / Vis spectra of NABQ with the addition of various concentrations of HgCl ₂ .

8c also shows the UV / Vis spectra of NABQ (20 μM) with the addition of various concentrations of ZnCl ₂ .

9 shows the change in fluorescence of NABQ + metal cations with the addition of zinc ions.

Claim 10 degrees to Tan Nana the Job's plot for NABQ and Zn ^{+ 2.}

11A, 11B and 11C show FAB-MS of NABQ, NABQ + Zn (ClO ₄ ) ₂ , and NABQ + Hg (ClO ₄ ) ₂ , respectively.

12th turn illustrates the fluorescence changes of NABQ-Hg ^{+ 2} in accordance with the addition of various concentrations of ZnCl _2.

Figure 13 shows the change in fluorescence of NABQ with the addition of one of zinc ions and other metal cations.

First it shows the change in fluorescence 2NABQ-Zn ^{2 +} in accordance with one of addition of the 14 degrees of mercury ions and other metal ions.

Figure 15 shows confocal fluorescence images of zinc ions in AMC-HN3 cells.

FIG. 16 shows confocal fluorescence images of mercury ions in AMC-HN3 cells.

Figure 17 shows confocal fluorescence images of zinc ions in Hep-2 cells.

18 shows confocal fluorescence images of mercury ions in Hep-2 cells.

19 shows confocal fluorescence images of zinc ions in MDCK cells.

20 shows confocal fluorescence images of mercury ions in MDCK cells.

21 to turn the cells shows the Hg ^{2 +,} Hg ^{+ 2} and NABQ (50μM), and Hg ^{2 +,} or survival rate of LLC-MK2 cells, when treated with 2NABQ-Zn ^{2 +.}

Article 22a turns mercury ions according to 2NABQ-Zn represents the fluorescence change of the ^{2 +,} the 22b turn indicates the fluorescence change in the remainder of LLC-MK2 according to 2NABQ-Zn ²⁺ addition, the 23c turns 2NABQ-Zn ^{2 +} addition Fluorescence change of the residual LLC-MK2 is shown.

FIG. 23 depicts ¹ HNMR data of NABQ with or without Hg (ClO ₄ ) ₂ .

FIG. 24 depicts ¹ HNMR data of NABQ (20 μM) with or without Zn (ClO ₄ ) ₂ .

Claims (8)

A quinaldine derivative compound, characterized in that represented by the formula [Formula 1]

A first step in which 8-benzenesulfonamidoquinaldine is prepared by mixing and stirring 8-aminoquinaldine and benzenesulfonyl chloride obtained by reducing 8-nitroquinaldine with pyridine; And A second step of preparing the compound represented by Chemical Formula 1 by mixing and stirring the 8-benzenesulfonamidoquinaldine and 4-nitrobenzenediazonium tetra fluoroborate together with pyridine in an organic solvent; Method of producing a quinaldine derivative comprising a. [Formula 1]

3. The method of claim 2, wherein the first step is performed at −4˜4 ° C. 4. The method of claim 2, wherein the first step is to prepare a quinaldine derivative, characterized in that stirring for 4 to 6 hours. 3. The method of claim 2, wherein the second step is performed at −4˜4 ° C. 4. The method of claim 2, wherein the second step is stirred for 11 to 13 hours. delete It is used to detect the presence of the metal ions, the amount of the metal ions, or both by utilizing the color fluorescence change generated when zinc ions, mercury ions, or both are added to the compound represented by Formula 1 A sensitizer comprising a compound represented by the formula (1). [Formula 1]

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