KR102573697B1 - Rhodamine-based novel compound and its use for detecting mercury ions - Google Patents

Abstract

The present invention relates to a novel rhodamine-based compound and a composition for detecting mercury ions using the same, wherein the novel compound, probe PS, can selectively discriminate mercury ions (Hg ²⁺ ) among various metal ions, and It has a significantly lower detection limit (3.0375 × 10 ^-8 M, 30.37 nM) than other compounds, and can detect not only mercury ions in drinking water or tap water, but also mercury ions in cells without cytotoxicity, so Hg ²⁺ It can be usefully used in various fields for confirming the presence of metal ions.

Description

Rhodamine-based novel compound and its use for detecting mercury ions}

The present invention relates to a novel rhodamine-based compound and a composition for detecting mercury ions using the same.

Along with modern industrial development, human beings are exposed to the risk of poisoning by various heavy metals due to environmental pollution caused by industrial wastewater and waste. In particular, mercury is a fatal substance that can cause abnormalities in the nervous system when poisoned, resulting in speech and motor disorders, and in severe cases paralysis of the limbs. As such, mercury ions are biologically important heavy metals because they have harmful effects on human health. Therefore, highly sensitive and selective detection and measurement of Hg(II) ions in aqueous phase is essential for human health, molecular biology and environmental safety.

Accordingly, many studies have been conducted to develop a selective and sensitive sensor for chemically or biochemically important ionic substances. Recent understanding and research on supramolecule chemistry has shown great potential in the design of host compounds that can selectively combine with ions or various other types of guest compounds. , It is of great help to research on the development of a fluorescent chemosensor that can more easily observe the selective binding with a guest compound using fluorescence change. Sensors based on metal ion-induced fluorescence changes have provided a particularly useful means for the detection of metal ions due to their simplicity and high detection limits of fluorescence.

Recently, interest in rhodamine derivatives has increased in designing chemical sensors for metal ions. While rhodamine derivatives are non-fluorescent and colorless, ring opening of spirolactam generates strong fluorescence. In view of these characteristics, attempts have been made to use rhodamine derivatives for the detection of metal ions. Nevertheless, there remains a need for the development of efficient chemosensors with excellent mercury selectivity and sensitivity.

Republic of Korea Patent Publication No. 10-2010-0019906

It is an object of the present invention to provide novel rhodamine-based compounds.

Another object of the present invention is to provide a composition for detecting mercury ions (Hg ²⁺ ) containing the compound.

Another object of the present invention is to provide a method for detecting mercury ions (Hg ²⁺ ) using the compound.

Another object of the present invention is to provide a method for preparing the novel compound.

In order to achieve the object of the present invention, the present invention provides a compound represented by Formula 1 below.

[Formula 1]

In addition, the present invention provides a composition for detecting mercury ions (Hg ²⁺ ) including the compound represented by Formula 1 above.

In one embodiment of the present invention, the mercury ion (Hg ²⁺ ) may be present in cells.

In one embodiment of the present invention, the pH of the composition may be 6 to 8.

In addition, the present invention provides a mercury ion (Hg ²⁺ ) detection method comprising the step of reacting the compound represented by Chemical Formula 1 with a sample.

In addition, the present invention is (a) reacting a compound represented by the formula (2) and a compound represented by the formula (3) to prepare a compound represented by the formula (4) and

(b) providing a method for preparing a compound of formula 1, comprising the step of preparing a compound represented by formula 1 by reacting the compound represented by formula 4 obtained in step (a) and the compound represented by formula 5 below do.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

Probe PS, a novel compound of the present invention, can selectively discriminate mercury ions (Hg ²⁺ ) among various metal ions, and has a significantly lower detection limit (3.0375 × 10 ^-8 M, 30.37 nM) than conventional compounds. Since it can detect not only mercury ions contained in drinking water or tap water, but also mercury ions in cells due to non-cytotoxicity, it can be usefully used in various fields for confirming the presence of Hg ²⁺ metal ions.

1 shows the effect of pH on absorbance of chemical sensor PS (10 μM) in the absence and presence of Hg ²⁺ (50 μM).
Figure 2 shows the absorption spectrum (A) and fluorescence emission spectrum (B) of the probe PS in the presence of various metal ions.
Figure 3 shows the selectivity and competition of probe PS (10 μM) to Hg ²⁺ (50 μM). The columns in the front row show the fluorescence response of the probe PS (10 μM) to the irradiated metal ions, and the columns in the back row show Hg ² in solutions containing PS (10 μM) and other metal ions (50 μM) (λ _ex = 530 nm), respectively. ⁺ (50 μM) is added continuously.
Figure 4 shows the change in fluorescence intensity (λ _ex = 530 nm) of PS (10 μM) in THF.
5 shows a plot of the emission intensity of PS (10 μM) at 558 nm versus the concentration of Hg ²⁺ (0-10 μM).
6 shows a Jobs plot suggesting a 1:1 stoichiometry of chemical sensors PS and Hg ²⁺ .
7 shows a modified Benesi-Hildebrand plot of the chemosensor PS-Hg ²⁺ complex.
8 is ^{a 1} H NMR spectrum, in which (A) is DMSO-d ₆ when 1.0 equivalent of chemical sensor PS is present, and (B) is in DMSO-d ₆ when chemical sensor PS and 0.5 equivalent of Hg ²⁺ are present. , (C) represents the case in which the chemical sensor PS and 1.0 equivalent of Hg ²⁺ are present.
9 (A) to (D) show bright field images of living MDA-MB-231 cells, (A) is a control, (B) is a probe (PS) in DMSO, (C) is PS and 5 μM of Hg ²⁺ , (D) is PS and 10 μM of Hg ²⁺ treated, (E) to (H) show fluorescence images of live MDA-MB-231 cells, (E) is a control group , (F) is the probe (PS) in DMSO, (G) is PS and 5 μM Hg ²⁺ , (H) is PS and 10 μM Hg ²⁺ treated.
10 (A) to (D) show bright field images of live A375 cells, (A) is a control, (B) is a probe (PS) in DMSO, (C) is PS and 5 μM Hg ^{2 +} , (D) is PS and 10 μM of Hg ²⁺ treated, (E) to (H) show fluorescence images of living A375 cells, (E) is a control, (F) is a probe in DMSO (PS) and (G) are treated with PS and 5 µM Hg ²⁺ , and (H) are treated with PS and 10 µM Hg ²⁺ .
11 shows the results of measuring cell viability using the MTT assay. (1) relates to MDA-MB-231 cells, control is untreated, Hg(II) is 10 μM Hg ²⁺ , B1 is probe PS, B2 is PS and 5 μM Hg ²⁺ , B3 is PS and 10 μM of Hg ²⁺ was treated for 24 hours. (2) relates to A-375 cells, control is untreated, Hg(II) is 10 μM Hg ²⁺ , M1 is probe PS, M2 is PS and 5 μM Hg ²⁺ , M3 is PS and 10 μM of Hg ²⁺ was treated for 24 hours.
12 shows ¹ H NMR data of chemical sensor PS.
13 shows ¹³ C NMR data of chemical sensor PS.
14 shows IR spectrum data of the chemical sensor PS.
15 shows LCMS spectrum data of chemical sensor PS.
16 shows GC-MS spectrum data of the chemical sensor PS.

The present invention provides a compound represented by Formula 1 below.

[Formula 1]

The compound represented by Formula 1 is a novel compound, and its name is 2-(5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl)-3',6'-bis(ethylamino)-2 ',7'-dimethylspiro [isoindoline-1,9'-xanthen] -3-one, and is referred to as PS hereafter.

PS can be prepared through a two-step synthesis as follows.

In another aspect, the present invention provides a composition for detecting mercury ions (Hg ²⁺ ) including the compound represented by Formula 1 above. When the mercury reacts with PS, the spirolactam ring opens and causes a fluorescence reaction, so mercury ions can be detected using PS.

The mercury ion (Hg ²⁺ ) may be present in cells. As it was experimentally confirmed that the PS of the present invention can effectively detect intracellular Hg ²⁺ without exhibiting cytotoxicity, it can be an effective compound for confirming the existence of intracellular Hg 2+ metal ions.

The pH of the composition may be 6 to 8, preferably pH 7. This is because it exhibits high absorbance at the above pH.

In another aspect, the present invention provides a method for detecting mercury ions (Hg ²⁺ ) comprising reacting the compound represented by Chemical Formula 1 with a sample. The detection may be confirmed through UV-vis absorption, observation of fluorescence intensity, etc., but is not limited thereto, and may be performed by methods commonly used in the art.

In another aspect, the present invention provides (a) reacting a compound represented by Formula 2 and a compound represented by Formula 3 to prepare a compound represented by Formula 4, and (b) a chemical formula obtained in step (a) Provided is a method for preparing a compound of Formula 1, including the step of preparing a compound represented by Formula 1 by reacting a compound represented by Formula 4 with a compound represented by Formula 5 below.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

Hereinafter, the present invention will be described in more detail through examples and test examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Example 1: Experimental method>

Example 1-1. Materials and Methods

Rhodamine 6G, Thiosemicarbazide, 4-bromo benzoic acid and potassium tert-butoxide were purchased from Sigma Aldrich. Tetrahydrofuran (THF), ethyl acetate (EtOAc), dichloromethane, and methanol were purchased from a commercial company (Samcheon Chemical Korea). All chemicals were used without purification. Absorption spectra were measured using a Shimadzu spectrophotometer (Scinco, Korea), and fluorescence studies were performed using a FS-2 fluorescence spectrometer (Scinco, Korea). For all samples, absorption and fluorescence assays were performed at room temperature, and samples were excited at 530 nm for fluorescence studies. LC-MS was examined with a 2795 / ZQ2000 (waters) spectrometer. FT-IR spectra were recorded on a Frontier IR (Perkin Elmer) spectrometer using KBr pellets. ¹ H NMR and ¹³ C NMR spectra were recorded on a Bruker Avance 400 MHz spectrophotometer (400 and 100 MHz for proton and carbon NMR) with TMS used as an internal standard. The melting point was measured using an MPA 160 instrument from Fisher Scientific (USA). The fluorescence time decay performance of the probe PS in the presence or absence of Hg ²⁺ was measured at each wavelength using a time-correlated single photon counting (TCSPC) spectrophotometer (HORIBA-Ihr320, Japan), and the obtained output was Hg ² To evaluate the fluorescence lifetime values of the probe PS in the presence and absence of ⁺ were investigated in the data station software provided with the instrument.

Example 1-2. Preparation of metal ion solutions for optical studies

A stock solution of probe PS (1 mM) was prepared in tetrahydrofuran (THF) solvent. and Hg ²⁺ , Ba ²⁺ , Ca ²⁺ , Cd ²⁺ , Co ²⁺ , Cu ²⁺ , Fe ²⁺ , Mg ²⁺ , Mn ²⁺ , Ni ²⁺ , Pb ²⁺ , Zn ²⁺ , Al Metal ion (1 mM) storage solutions such as ³⁺ , Cr ³⁺ , Fe ³⁺ , Na ⁺ , K ⁺ , Cu ⁺ and Ca ⁺ were prepared by dissolving the corresponding salt in double distilled water. Prior to spectroscopic measurements, a known volume of probe PS stock solution was added to a 10 mL volumetric flask, followed by a known volume of corresponding metal ion and pH 7 solution, and finally a THF:water (8:2, v/v) system. were diluted to equal concentrations using Absorption and fluorescence intensities were then recorded after 10 minutes for all samples. For fluorescence measurements, all samples were excited at 530 nm. Both the excitation and emission slits were set to 5 nm.

Example 1-3. synthesis

Step 1) Synthesis of 5-(4-bromophenyl)-1,3,4-thiadiazol-2-amine (1A)

As described in Scheme 1, intermediate 1A was prepared by a known method (L. Yan, M. Yang, X. Leng, M. Zhang, Y. Long, B. Yang, A new dual-function fluorescent probe of Fe3+ for bioimaging and probe-Fe3+ complex for selective detection of CN-, Tetrahedron. 72 (2016) 4361-4367).

[Scheme 1]

i) POCl ₃ , 1 h reflux; H ₂ O and 5 h reflux ii) Potassium tert-butoxide, ethanol, RT, 12 h. Potassium tert-butoxide.

POCl ₃ (3mL), 4-Bromo-Benzoic acid (0.5gm, 2.4mmol) and thiosemicarbazide (0.24gm, 2.73mmol) were added to a 100 ml round bottom flask (RBF). The reaction mixture was heated to 75-80 °C. After 1 hour, heating was stopped and the reaction was gradually cooled to room temperature. In addition, the reaction mass was cooled to 0-5 °C, quenched slowly with distilled water and then refluxed for another 5 h. The reaction mixture was then cooled slowly to ambient temperature and then to 0-5 °C and basified with 50% NaOH (pH = 8). The suspended solid material was stirred at 0-5° C. for another hour, then filtered, dried, and recrystallized from absolute ethanol to obtain compound 1A, which was further used in the next step.

Step 2) 2-(5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl)-3′,6′-bis (ethylamino)-2′,7′-dimethylspiro[isoindoline-1, Synthesis of 9′-xanthen]-3-one (PS)

Rhodamine 6G (0.5gm, 1.042mmol) and 2-Amino-5-{4-bromophenyl}-1,3,4-thiadiazole (0.32gm, 1.042mmol) were dissolved in ethanol (5ml) in a nitrogen atmosphere at ambient temperature. mixed. The reaction mixture was stirred at ambient temperature for 10 minutes, then potassium tert-butoxide (0.14 gm, 1.252 mmol) was slowly added, and the reaction was continued under a nitrogen atmosphere until the reaction was complete (12 hours). After completion of the reaction (monitored by TLC), the reaction mixture was quenched with cold water (15 mL) and extracted with ethyl acetate (3 x 15 mL). The ethyl acetate layer was washed with water (10 mL) followed by brine solution (15 mL). The organic layer was dried over anhydrous sodium sulfate, distilled under vacuum and purified by column chromatography using (dichloromethane / methanol) as eluent to give a pink solid.

Yield: 85%, M.P: > 260 °C;

¹ H NMR (DMSO-d ₆ , 400 MHz) δ (ppm): δ 8.58 - 6.78 (m, 8 H), 6.25 (m, 4 H), 5.76 (s, 2 H), 3.19 - 3.09 (q, 4 H), 1.82 (s, 6H), 1.20 (t, 6 H) (see Fig. 12).

¹³ C NMR (101 MHz, DMSO) δ 167.25, 147.77, 135.44, 132.63, 132.22, 131.52, 129.22, 128.90, 104.44, 37.39, 14.20 (see FIG. 13)

IR (KBr) v / cm ^-1 : 3022.19, 2889.70, 2299.44, 1946.44, 1693.56, 967.13, 903.90 (see Fig. 14)

LC-MS: 654.4 m/z . (M+2) (see Fig. 15)

GCMS: 653 m/z . (See Fig. 16)

Example 1-4. cell imaging

Cell culture and processing

The human melanoma cancer cell line A375 and the breast cancer cell line MDA-MB-231 used in this study were obtained from KCLB (Korean Cell Line Bank). A375 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.,) and MDA-MB-231 cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.,). Both media contained 10% heat-inactivated fetal bovine serum (FBS; Tissue Culture Biologicals. Long Beach, CA, USA), streptomycin (50 μg/mL; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and penicillin ( 50 units/mL; Sigma-Aldrich). The cells were grown at 37° C. in a humidified incubator with 5% CO ₂ .

A probe (10 μM) was dissolved in dimethyl sulfoxide (DMSO) and a Hg ²⁺ solution was prepared in distilled water. In this experiment, already grown cells (MDA-MB-231 and A375) were either untreated (control) or treated with probe PS (10 μM) in growth medium for 3 hours and then washed twice with PBS. Thereafter, the cells were incubated with different concentrations of Hg ²⁺ (5 and 10 μM) for 3 hours and then washed twice with PBS at 37 °C. Images of both cells were observed using a fluorescence microscope.

Check cell viability

The effect of probe PS and PS-Hg ²⁺ complex on cell viability was performed by MTT (3-(4,5-dimethylthiazol-2-yl)-2-5diphenyl tetrazolium bromide) assay. A375 melanoma and MDA-MB-231 cells were seeded in each well of a 96-well plate at 0.4 x 10 ⁵ and 0.2 x 10 ⁵ cell densities, respectively. Cells were treated in 10 μM of probe PS and 10 μM of Hg ²⁺ and co-treated with various concentrations of probe (5 and 10 μM) for 24 hours. Inhibition of cell viability was scrutinized by the MTT assay (10 μL MTT/100 μL cells) and optical absorbance was measured at 570 nm using a microplate reader.

In vitro fluorescence image observation

Fluorescence intensities of A375 and MDA-MB-231 cells were checked by fluorescence microscopy (BX51, Olympus, Tokyo, Japan) as previously described. Briefly, MDA-MB-231 and A375 cells were seeded at 1.5×10 ⁵ cells in a 35 mm culture plate, and then the cells were irradiated under green light (wavelength 532 nm).

<Example 2: Confirmation of effect according to pH>

In order to confirm the output of the probe PS according to the pH range, a UV-vis absorption experiment was performed. Various pH solutions were prepared at pH 3-10 and the best working range for the chemical sensor PS was identified. In pH titration experiments, the metal-free chemical sensor PS was found to exhibit low absorbance values at pH 8-10. The absorbance value increases at low pH because protonation causes ring opening. However, after mixing Hg ²⁺ metal into the chemical sensor PS, the absorbance was higher in the pH range of 6-8, and then, as shown in FIG. 1, the absorbance immediately decreased under very basic conditions such as pH 9-10. The low absorbance values obtained at high pH values suggest that Hg ²⁺ metal ions can be hydrolyzed to the hydroxide form and cannot form complexes with the chemical sensor PS. Therefore, the entire experiment of the present invention was carried out at pH 7.0.

<Example 3: Confirmation of spectroscopic characteristics>

Example 3-1. Selectivity of probe PS through absorption and fluorescence studies

Ba ²⁺ , Ca ²⁺ , Cd ²⁺ , Co ²⁺ , Cu 2+ , Fe ²⁺ , Mg ^{2+ , Mn 2+ , Ni 2+ , Pb 2+ , Zn 2+ , Al 3+ , Cr 3} In order to explore the sensitivity of the probe PS to various metal ions, such as ⁺ , Fe ³⁺ , Na ⁺ , K ⁺ , Cu ⁺ and Ca ^{+ ,} etc., in a THF:water system (8:2, v/v, pH = 7.0) UV-vis absorbance and fluorescence studies were performed.

In the UV-vis absorption experiment, the absorption spectrum of probe PS (10 μM) in mixed organic and aqueous systems did not show a response exceeding 500 nm (Fig. 2A). This shows that the probe PS exists in a closed spirocyclic form (non-fluorescence) rather than an open ring (fluorescence) in the mixed organic and aqueous solvent phase.

In addition, there was no substantial change due to the addition of 5.0 equivalents of various metal ions (alkali, alkaline earth metal group, transition, non-transition metal, etc.) except for Hg ²⁺ . However, a large change was observed in the absorption spectrum after adding Hg ²⁺ , indicating that the probe PS can selectively discriminate Hg ²⁺ metal ions.

Likewise, the selectivity of the probe PS for Hg ²⁺ metal ions was further examined using fluorescence spectroscopy in a mixed organic and aqueous system (THF:water, 8:2, V/V, pH = 7). As shown in Fig. 2B, probe PS is non-fluorescent due to the closed 5-membered spirolactam framework. Probe PS is Ba ²⁺ , Ca ²⁺ , Cd ²⁺ , Co ²⁺ , Cu ²⁺ , Fe ²⁺ , Mg ²⁺ , Mn ²⁺ , Ni ²⁺ , Pb ²⁺ , Zn ²⁺ , Al ³⁺ , Cr ³⁺ , Fe ³⁺ , Na ⁺ , K ⁺ , Cu ⁺ and Ca ⁺ , etc. When combined with a number of metal salts (5.0 eq.), the emission spectra did not undergo substantial change. However, significant fluorescence enhancement was observed in the emission spectrum at 558 nm after combining the probe PS with Hg ²⁺ metal ions. This makes the probe PS an “on” fluorescent chemical sensor for the detection of Hg ²⁺ metal ions ( FIG. 2B ). After interaction with Hg ²⁺ , probe PS was present in the spirolactam open-ring form (fluorescence), and marked changes were observed.

Example 3-2. Confirmation of the effect of different metal ions on PS-Hg2+

Competing metal ion experiments were performed to investigate the effect of coexisting other metal ions on the emission intensity.

To investigate the effect of probe PS (10 μM) induced by Hg ²⁺ (50 μM) on fluorescence intensity, Ba ²⁺ , Ca ²⁺ , Cd ²⁺ , Co ²⁺ , Cu ²⁺ , Fe ²⁺ , Mg ²⁺ , Mn ²⁺ , Ni ²⁺ , Pb ²⁺ , Zn ²⁺ , Al ³⁺ , Cr ³⁺ , Fe ³⁺ , Na ⁺ , K ⁺ , Cu ⁺ and Ca ⁺ (50 μM) Metal ions were incorporated into organic and aqueous systems in which PS and Hg ²⁺ were combined.

The fluorescence emission intensity results of the probe PS with each metal ion (red column) and the probe PS+Hg ²⁺ ion with another metal ion (green column) are shown in FIG. 3 . Irradiated background metal ions did not interfere with recognition of Hg ²⁺ metal ions. In comparison, as shown in FIG. 3 (red column), Co ²⁺ exhibited slightly more fluorescence response than other metal ions. However, even if Co ²⁺ metal ions were present in the same solution, the fluorescence intensity of the PS+Hg ²⁺ complex was not significantly affected. The above result means that the probe PS has selectivity and sensitivity to Hg ²⁺ .

<Example 4: Statistical Data Analysis>

Example 4-1. Fluorescence titration and limit of detection (LOD) determination

Fluorescence intensities of probe PS with and without Hg ²⁺ metal ions within the concentration range of 0-10 μM are presented in FIG. 4 . The fluorescence emission intensity of the probe PS was found to increase linearly with the addition of Hg ²⁺ metal ions within the range of 0-10 μM. The fluorescence intensity graph for [Hg ²⁺ ] prepared through the above results follows a linear equation as shown in FIG. 5 . The above linear equation fits well with fluorescence additive enhancement results in the range of 0-10 μM. The limit of detection (LOD) was calculated using Equation 1 below and was observed to be 3.0375 × 10 ^-8 M (30.37 nM).

[Equation 1]

where σ and K represent the slope between the standard deviation of the blank measurement and the fluorescence intensity versus Hg ²⁺ metal ion concentration.

The LOD calculated in the present invention is much lower than previously reported methods. A comparison table for comparing the present invention with other reported methods for detecting Hg ²⁺ metal ions is shown below (Table 1).

[Table 1]

Example 4-2. Determination of binding stoichiometry and binding constants of complexes

A Job's plot experiment was performed to determine the binding stoichiometry between the chemical sensor PS-Hg ²⁺ complexes. In this experiment, the final sum of the mole fractions of the ligand (PS) and the metal (Hg ²⁺ ) was kept constant while simply changing the mole fraction of the ligand (Hg ²⁺ ). Figure 6 shows Job's plot and shows the corresponding absorption values of all solutions having a mole fraction of Hg ²⁺ metal ion of 0.1-0.9. The maximum absorbance in this experiment was found at about 0.5 mole fraction for Hg ²⁺ metal ion. The above result means that the chemical sensor PS and the Hg ²⁺ metal ion form a 1:1 complex. In addition, the coupling constant between the probe PS and Hg ²⁺ from the fluorescence intensity data (558 nm) is expressed by Equation 2 below.

[Equation 2]

Here, F ₀ , F and F _max denote the maximum fluorescence intensity in the absence of Hg ²⁺ , in the presence of different concentrations of Hg ²⁺ and in the presence of Hg ²⁺ , respectively. K _B is the binding constant and n is the number of Hg ²⁺ metal ions bound to the chemosensor PS and the Hg ²⁺ complex, where n = 1. In addition, the modified Benesi-Hildebrand (BH) graph is shown in FIG. 7 . The slope value obtained from the linear BH plot was used to calculate the binding constant, confirming a high binding constant value of K _B = 6 × 10 ⁴ M ^-1 for 1 : 1 complexation between the probe PS and Hg ²⁺ .

Example 4-3. sensing mechanism

Rhodamine or its derivatives are the ultimate model in chemical sensor research due to their unique structural properties. The closed five-membered cyclic amide function (spirolactum) exhibits non-fluorescent behavior, and the open-ring form of spirolactam (spirolactum) induces strong fluorescence emission intensity. Moreover, in the absence of heavy metal ions or cations, these probes retain their colorless and non-fluorescent spirocyclic conformation. However, the presence of metal cations causes the spirocyclic ring to open, resulting in these probes exhibiting more fluorescence or strong fluorescence emission intensity in fluorescence studies. Therefore, it can be proposed that the spirocyclic ring-opening mechanism works through chelation. Chelation is the Hg ²⁺ metal ion from the complex coordinated with the oxygen atom of the amide functional group present in the probe PS. There are many studies on rhodamine-based spirolactam chemosensors, and Hg ²⁺ -stimulated fluorescence enhancement of chemical sensor PS is a highly anticipated result of the spirolactam ring opening mechanism. Therefore, to further investigate the chelation mechanism, the fluorescence of the probe PS was enhanced by adding Hg ²⁺ metal ions, which were operated via a spirocyclic ring-opening approach.

Specifically, several experiments including Jobs plot, ¹ H NMR titration and fluorescence lifetime were performed. In the Jobs plot experiment (Fig. 6), the absorbance value of the titrant solution shows an inflection point at about 0.5, which indicates that the probe PS and Hg ²⁺ metal ions (e.g. PS- It means that a 1:1 coordination rank is formed between Hg2+). Likewise, the stoichiometry and coordination between probe PS and Hg ²⁺ metal ions were confirmed by ¹ H NMR titration experiments. Here, probe PS (1.0 equiv.) was combined with various concentrations of Hg ²⁺ metal ions, such as 0.5 and 1.0 equiv. Figure 8 consists of three separate ¹ H NMR spectra (A / B & C), wherein spectrum A was only recorded for probe PS, whereas spectra B and C were probe PS with 0.5 and 1.0 equivalents of Hg ²⁺ respectively ( 1.0 equivalent).

As shown in A and B of FIG. 8, a peak with a chemical shift value of 5.04 ppm for NH protons appeared, suggesting that the 5-membered spirocyclic ring still exists in a closed form. This means no complex formation after addition of 0.5 eq of Hg ²⁺ metal ion containing probe PS. However, when 1.0 equivalent of Hg ²⁺ was added, the metal ion bonded to the probe PS to form a complex (PS-Hg ²⁺ ) and disappeared after the ring was opened. The binding mode of the Hg ²⁺ metal ion and the probe PS is shown in Scheme 2.

[Scheme 2]

Excited-state complexation of probe PS-Hg ²⁺ was performed to obtain fluorescence lifetime values in the presence and absence of Hg ²⁺ metal ions for probe PS in a mixed THF:water (V/V, 8:2, pH = 7) system. It was further verified by inspection and is shown in Table 2.

[Table 2]

First, the fluorescence lifetime was calculated for the probe PS detected at 1.63 ns. Then, after adding Hg ²⁺ metal ions within the concentration range of 0-10 μM, the fluorescence lifetime value increased with the addition of Hg ²⁺ metal ions and was 3.13, 3.53, and 3.91, respectively.

The above results indicate that fluorescence intensity and excited single-state lifetime are improved through complexation by freezing of the non-radiative pathway generated by the non-flexible PS-Hg ²⁺ complex rather than the relatively flexible probe PS.

<Example 5: Application of fluorescence method>

Example 5-1. Hg in environmental samples using the chemical sensor PS ²⁺ quantitative measurement of

The practical applicability of the present chemical sensor PS was evaluated using a spike and recovery approach. Hg ²⁺ contamination has been found in drinking and tap water. Samples were collected from the university's local campus. To perform this experiment, three different concentration solutions of Hg ²⁺ metal ions were prepared by spiking standard solutions of Hg ²⁺ metal ions. The test was performed by spiking a known amount of a standard Hg ²⁺ metal ion solution. The fluorescence emission intensities of these three testers were evaluated and correlated with the fluorescence emission intensities within the 0–10 μM range presented in Fig. 4. The actual water sample analysis data is shown in Table 3.

[Table 3]

For the analysis of real water samples, the calculated recovery for the known amount of added Hg ²⁺ was between 96 and 100 %. The above results show that the present invention can be applied to the evaluation of real water samples.

Example 5-2. Hg using chemical sensors (PS) and cytotoxicity studies ²⁺ intracellular imaging of

cell imaging

Mercury is a naturally occurring element found in air, soil and water. Mercury is one of the top 10 most dangerous chemicals according to the WHO. Inhalation of mercury can cause serious human health problems such as lethality to the nervous system, immune system, digestive system, kidneys and lungs. Therefore, in order to overcome these concerns, the present inventors synthesized and developed a chemical sensor having high selectivity as well as sensitivity to Hg ²⁺ metal ions.

A bioimaging study was conducted to verify the reliability and functionality of the current chemical sensor. The synthesized chemical sensor PS was screened in two cancer cell lines, MDA-MB-231 and A375, with or without Hg ²⁺ treatment, respectively. Cells cultured in this experiment were treated with various concentrations of probe PS and Hg ²⁺ metal ions, and fluorescence images obtained for various models are shown in FIGS. 9 and 10 . Fluorescence images shown in FIG. 9 are the probes PS (10 μM) (B1), PS (10 μM) + 5 μM Hg ²⁺ (B2), and PS (10 μM) + 10 μM Hg ²⁺ (B3) in DMSO. ) represents the treated MDA-MB-231 cell.

Fluorescence images were evaluated using fluorescence microscopy measurements at an excitation wavelength of 532 nm (green light). Fluorescence and bright field transmission images of the test samples were identical to those of the controls for live MDA-MB-231 and A375 cells, as shown in FIGS. 9 and 10 . As a result of the experiment, it was confirmed that both probe PS and Hg ²⁺ metal ions showed high fluorescence when they interacted with each other. Specifically, both cancer cell lines showed no fluorescence (MDA-MB-231) or very low fluorescence (A375) when already grown cancer cells were treated with the probe PS. In addition, different concentrations (5 & 10 μM) of probe PS (10 μM) and Hg ²⁺ were collectively treated on grown MDA-MB-231 and A375 cells. It was found that the probe PS strongly coordinated with the Hg ²⁺ metal ion and exhibited strong fluorescence while increasing the concentration of the Hg ²⁺ metal ion. Therefore, based on these experimental results and observations, probe PS can be the best product for confirming the presence of Hg ²⁺ metal ions in cells.

cytotoxicity

The cytotoxic effect of the probe PS in the presence or absence of Hg ²⁺ metal ions was investigated using the MTT assay using two cancer cells, MDA-MB-231 and A375, respectively. Dimethyl sulfoxide (DMSO), which is non-toxic to the cell surface, was used as a solvent in the whole experiment. In addition, the two cancer cells were treated with different concentrations of Hg ²⁺ (5 and 10 μM) for probe PS (10 μM), Hg ²⁺ (10 μM) and probe PS (10 μM) for 24 hours. Cell viability measurements performed on all cancer cells are presented in Figure 11 (1 & 2). The degree of non-toxicity of all samples tested was within a controllable range. All samples containing the PS probe and the PS-Hg ²⁺ complex were found to be non-toxic to breast cancer cells MDA-MB-231 and human melanoma A375 cells. From the above results, it can be inferred that the complex of probe PS and Hg ²⁺ ions can be useful for detecting Hg ²⁺ metal ions in cells.

Claims (6)

A composition for detecting mercury ions (Hg ²⁺ ) comprising a compound represented by Formula 1 below.
[Formula 1]

delete According to claim 1, wherein the mercury ion (Hg ²⁺ ) is present in cells, the composition.
The composition according to claim 1, wherein the pH of the composition is 6 to 8.
delete delete