KR20220124953A - 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. Probe PS, which is a novel compound, can selectively discriminate mercury ions (Hg^(2+)) among various metal ions, has a significantly lower detection limit (3.0375 × 10^(-8)M, 30.37 nM) than existing compounds, and thus can detect not only mercury ions contained in drinking water or tap water, but also mercury ions in cells without cytotoxicity, so that it is possible to be usefully used in various fields for confirming the presence of Hg^(2+) metal ions.

Description

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

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

With the development of modern industry, human beings are exposed to the risk of poisoning of various heavy metals due to environmental pollution caused by industrial wastewater and waste. In particular, mercury is a deadly substance that, when poisoned, causes abnormalities in the nervous system, resulting in speech and movement disorders and, in severe cases, paralysis of the limbs. As such, mercury ions are biologically important heavy metals because they have a detrimental effect on human health. Therefore, highly sensitive and selective detection and measurement of Hg(II) ions present in aqueous solutions are 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 research and understanding of supramolecule chemistry has shown great promise in the design of host compounds that can selectively bind ions or various other types of guest compounds. , which is of great help in research on the development of fluorescent chemosensors that can more easily observe selective binding with guest compounds using fluorescence changes. 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 limit of fluorescence.

Recently, interest in rhodamine derivatives has increased in designing chemical sensors for metal ions. While rhodamine derivatives are colorless and non-fluorescent, ring opening of spirolactam generates strong fluorescence. Based on 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 an efficient chemical sensor with excellent mercury selectivity and sensitivity.

Korean 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⁺ ) including 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 above object of the present invention, the present invention provides a compound represented by the following formula (1).

[Formula 1]

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

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

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

In addition, the present invention provides a method for detecting mercury ions (Hg ²⁺ ) comprising reacting the compound represented by Formula 1 with a sample.

In addition, the present invention comprises the steps of (a) reacting a compound represented by the following formula (2) and a compound represented by the following formula (3) to prepare a compound represented by the following formula (4);

(b) reacting the compound represented by Formula 4 obtained in step (a) and the compound represented by Formula 5 below to prepare a compound represented by Formula 1 below, providing a method for preparing the compound of Formula 1 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) compared to conventional compounds. Since it can detect not only mercury ions contained in drinking water or tap water, but also mercury ions in cells without 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 the absorbance of a chemical sensor PS (10 μM) in the absence and presence of Hg ²⁺ (50 μM).
2 shows an absorption spectrum (A) and a fluorescence emission spectrum (B) of the probe PS in the presence of various metal ions.
3 shows the selectivity and competition of probe PS (10 μM) against Hg ²⁺ (50 μM). The column in the front row shows the fluorescence response of the probe PS (10 μM) to the irradiated metal ion, and the column in the back row is Hg ² in a solution containing PS (10 μM) and another metal ion (50 μM) (λ _ex = 530 nm), respectively. A case in which ⁺ (50 μM) is continuously added is shown.
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) versus the concentration of Hg ²⁺ (0-10 μM) at 558 nm.
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 chemical sensor PS-Hg ²⁺ complex.
Figure 8 is a ¹ H NMR spectrum, (A) in DMSO-d ₆ with 1.0 equivalent of chemical sensor PS, (B) DMSO-d ₆ with chemical sensor PS and 0.5 equivalent of Hg ²⁺ , (C) shows the case with the chemical sensor PS and 1.0 equivalent of Hg ²⁺ .
9 (A) to (D) show bright field images of live MDA-MB-231 cells, (A) is a control, (B) is a probe in DMSO (PS), (C) is a PS and 5 μM Hg ²⁺ , (D) is PS and 10 μM Hg ²⁺ treated, (E) to (H) show fluorescence images of live MDA-MB-231 cells, (E) is control , (F) is a 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 control, (B) is probe in DMSO (PS), (C) is PS and 5 μM Hg ^{2 +} , (D) are PS and 10 μM Hg ²⁺ treated, (E) to (H) show fluorescence images of live A375 cells, (E) control, (F) probe in DMSO (PS), (G) is PS and 5 μM Hg ²⁺ , (H) is PS and 10 μM Hg ²⁺ treated.
11 shows the results of cell viability measurement 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 the chemical sensor PS.
13 shows ¹³ C NMR data of the chemical sensor PS.
14 shows IR spectrum data of the chemical sensor PS.
15 shows LCMS spectral data of the chemical sensor PS.
16 shows GC-MS spectral data of the chemical sensor PS.

The present invention provides a compound represented by the following formula (1).

[Formula 1]

The compound represented by Formula 1 is a novel compound, and the 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, which is hereinafter referred to as PS in the present specification.

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

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

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

The composition may have a pH of 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 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 a method commonly used in the art.

In another aspect, the present invention relates to (a) reacting a compound represented by the following formula (2) and a compound represented by the following formula (3) to prepare a compound represented by the following formula (4) and (b) the formula obtained in step (a) It provides a method for preparing a compound of Formula 1, comprising the step of reacting a compound represented by 4 and a compound represented by Formula 5 to prepare a compound represented by Formula 1 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. For absorption spectra, Shimadzu spectrophotometer (Scinco, Korea) was used, and FS-2 fluorescence spectrometer (Scinco, Korea) was used for fluorescence study. For all samples, absorption and fluorescence analyzes were performed at room temperature, and in fluorescence studies the samples were excited at 530 nm. LC-MS was checked 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 as internal standard. Melting points were measured using a Fisher Scientific (USA) MPA 160 instrument. The fluorescence time attenuation 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 ions (1 mM) stock solutions such as ³⁺ , Cr ³⁺ , Fe ³⁺ , Na ⁺ , K ⁺ , Cu ⁺ and Ca ⁺ were prepared by dissolving the corresponding salt in distilled water. Before the spectroscopic measurement, a known amount of probe PS stock solution is added to a 10 mL volumetric flask, followed by addition of a known volume of the corresponding metal ion and pH 7 solution, and finally a THF:water (8:2, v/v) system was used to dilute to the same concentration. Absorption and fluorescence intensity were recorded after 10 min for all samples. All samples were excited at 530 nm for fluorescence measurements. Both the excitation and emission slits were set to 5 nm.

Examples 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 conventionally known methods (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 reflux for 5 h 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 h, the heating was stopped and the reaction was gradually cooled to room temperature. In addition, the reactant was cooled to 0-5 °C, quenched slowly with distilled water, and then refluxed again for 5 hours. The reaction mixture was then cooled slowly to ambient temperature and then to 0-5 °C and basified (pH = 8) with 50% NaOH. The suspended solid material was stirred at 0-5° C. for an additional 1 hour, then filtered and 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 (0.5 gm, 1.042 mmol) and 2-Amino-5-{4-bromophenyl}-1,3,4-thiadiazole (0.32 gm, 1.042 mmol) in ethanol (5 ml) 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 thereto, and the reaction was continued in a nitrogen atmosphere until the reaction was completed (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 (3×15 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)

Examples 1-4. cell imaging

Cell culture and processing

Human melanoma cancer cell line A375 and breast cancer cell line MDA-MB-231 used in this study were obtained from KCLB (Korea 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 an incubator humidified with 5% CO ₂ .

The probe (10 μM) was dissolved in dimethyl sulfoxide (DMSO), and a Hg ²⁺ solution was prepared in distilled water. Cells already grown in this experiment (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 results of probe PS and PS-Hg ²⁺ complex on cell viability were analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2-5diphenyl tetrazolium bromide) assay. A375 melanoma and MDA-MB-231 cells were seeded into each well of a 96-well plate at a density of 0.4 x 10 ⁵ and 0.2 x 10 ⁵ cells, respectively. Cells were treated with 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 h. Inhibition of cell viability was scrutinized by 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

The fluorescence intensity of A375 and MDA-MB-231 cells was confirmed by fluorescence microscopy (BX51, Olympus, Tokyo, Japan) as previously described. Briefly, MDA-MB-231 and A375 cells were seeded in 35 mm culture plates at 1.5× ^{10 5} cells, and then the cells were irradiated under green light (wavelength 532 nm).

<Example 2: Confirmation of effect according to pH>

A UV-vis absorption experiment was performed to confirm the output of the probe PS according to the pH range. Various pH solutions at pH 3-10 were prepared and the best working range for the chemical sensor PS was identified. In a pH titration experiment, the metal-free chemical sensor PS was found to exhibit low absorbance values at pH 8-10. Absorbance values increase at lower pH because protonation causes ring opening. However, after Hg ²⁺ metal was mixed with 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 large basic conditions such as pH 9-10. The low absorbance values obtained at high pH values suggest that the Hg ²⁺ metal ions can be hydrolyzed to the hydroxide form and cannot form a complex with the chemical sensor PS. Therefore, the entire experiment of the present invention was performed at a pH of 7.0.

<Example 3: Confirmation of spectroscopic properties>

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 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 the probe PS (10 μM) in the 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 phases.

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 Hg ²⁺ . However, a large deformation was observed in the absorption spectrum after addition of 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 mixed organic and aqueous systems (THF:water, 8:2, V/V, pH=7). As indicated in Fig. 2B, the probe PS is non-fluorescent due to the closed five-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⁺ When combined with a series of numerous metal salts (5.0 equivalents) such as , Cr ³⁺ , Fe ³⁺ , Na ⁺ , K ⁺ , Cu ⁺ and Ca ⁺ , the emission spectrum did not change substantially. However, a 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⁺ , the probe PS existed in the form of spirolactam open ring (fluorescence), and a noticeable change was observed.

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

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

To investigate the effect of Hg ²⁺ (50 μM)-induced probe PS (10 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 others including Ca ⁺ (50 μM) Metal ions were incorporated into organic and aqueous systems combined with PS and Hg ²⁺ .

The results of the fluorescence emission intensity of the probe PS with each metal ion (red pillars) and the probe PS+Hg ²⁺ ions with other metal ions (green pillars) are shown in FIG. 3 . The irradiated background metal ions did not interfere with Hg ²⁺ metal ion recognition. In comparison, as shown in FIG. 3 (red column), Co ²⁺ showed slightly more fluorescence response than other metal ions. However, even in the presence of Co ²⁺ metal ions 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

The fluorescence intensity of the probe PS with and without Hg ²⁺ metal ions within the 0-10 μM concentration range is presented in FIG. 4 . The fluorescence emission intensity of the probe PS was found to increase linearly with Hg ²⁺ metal ion addition within the 0-10 μM range. The fluorescence intensity graph for [Hg ²⁺ ] prepared through the above results follows a linear equation as shown in FIG. 5 . The linear equation above fits well with fluorescence further enhancement results in the 0-10 μM range. The limit of the 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 with respect to the Hg ²⁺ metal ion concentration.

The calculated LOD in the present invention is much lower than previously reported methods. A comparison table for comparing the present invention with other reported methods for Hg ²⁺ metal ion detection is as follows (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 by simply changing the mole fraction of the ligand (Hg ²⁺ ). 6 shows a Job's plot and shows the corresponding absorption values for all solutions with a mole fraction of 0.1-0.9 for Hg ²⁺ metal ions. In this experiment, the maximum absorbance was found at about 0.5 mole fraction for Hg ²⁺ metal ions. The above result means that the chemical sensor PS and Hg ²⁺ metal ion form a 1:1 complex. In addition, the binding 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 represent 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 chemical sensor 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 probes 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 5-membered cyclic amide function (spirolactum) exhibits non-fluorescence behavior, and the open-ring form of 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 opens the spirocyclic ring, and as a result, these probes show more fluorescence in fluorescence studies or strong fluorescence emission intensity. Therefore, it can be suggested that the spirocyclic ring opening mechanism operates through chelation. The chelation is an 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 the rhodamine-based spirolactam chemical sensor, and the Hg ²⁺ -stimulated fluorescence enhancement of the chemical sensor PS is a very promising result of the spirolactam ring opening mechanism. Therefore, to closely examine the chelation mechanism, the fluorescence of the probe PS was enhanced by the addition of Hg ²⁺ metal ions actuated 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 titration solution shows an inflection point at about 0.5, which is the probe PS and Hg ²⁺ metal ions (eg PS-) in mixed organic and aqueous systems (8:2, THF: water). It means that a 1:1 coordination class 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, the probe PS (1.0 equiv.) was coupled with various concentrations of Hg ²⁺ metal ions, such as 0.5 and 1.0 equiv. Figure 8 consists of three distinct ¹ H NMR spectra (A/B & C), with spectrum A recorded only for probe PS whereas spectra B and C are probe PS with 0.5 and 1.0 equivalents of Hg ²⁺ , respectively (Fig. 1.0 equivalent).

As can be seen from 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 that no complex is formed after the addition of 0.5 equivalents of Hg ²⁺ metal ion-containing probe PS. However, when 1.0 equivalent of Hg ²⁺ was added, the metal ion combined with the probe PS to form a complex (PS-Hg ²⁺ ) and disappeared after the ring was opened. The binding mode between the Hg ²⁺ metal ion and the probe PS is shown in Scheme 2.

[Scheme 2]

Excited state complexation of probe PS-Hg ²⁺ yielded 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 certified 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 the addition of Hg ²⁺ metal ions within the concentration range of 0-10 μM, the fluorescence lifetime values increased with the addition of Hg ²⁺ metal ions, respectively, to 3.13, 3.53, and 3.91.

The above results indicate improvements in fluorescence intensity and excited single-state lifetime 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 the fluorescence method>

Example 5-1. Hg in environmental samples using 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 was found in drinking and tap water. Samples were collected at university regional campuses. To carry out this experiment, three different concentration solutions of Hg ²⁺ metal ions were prepared by spiking a standard solution 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 shown in FIG. 4 . Actual water sample analysis data are shown in Table 3.

[Table 3]

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

Example 5-2. Hg Utilizing 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 substances according to the WHO. Inhalation of mercury can cause serious health problems, including 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.

Bioimaging research was conducted to verify the reliability and functionality of the current chemical sensor. The synthesized chemical sensor PS was detected in two cancer cell lines, MDA-MB-231 and A375, respectively, with or without Hg ²⁺ . 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 . The 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. ) shows the treated MDA-MB-231 cells.

Fluorescence images were evaluated using fluorescence microscopy measurements at an excitation wavelength of 532 nm (green light). The fluorescence and brightfield transmission images of the test sample were the same as those of the control 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 the probe PS and Hg ²⁺ metal ions both exhibit high fluorescence when they interact with each other. Specifically, both cancer cell lines did not display fluorescence (MDA-MB-231) or showed very low fluorescence when already grown cancer cells were treated with the probe PS (A375). 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 Hg ²⁺ metal ions and exhibited strong fluorescence while increasing the concentration of Hg ²⁺ metal ions. Therefore, based on these experimental results and observations, probe PS can be the best product for checking the presence of Hg ²⁺ metal ions in cells.

cytotoxicity

The cytotoxic effect of probe PS in the presence or absence of Hg ²⁺ metal ions was investigated using two cancer cells, MDA-MB-231 and A375, respectively, using the MTT assay. Dimethyl sulfoxide (DMSO), which has non-toxic properties on the cell surface, was used as a solvent in the entire experiment. In addition, 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 both cancer cells are presented in Figure 11 (1 & 2). The degree of non-toxicity of all samples tested was within the controllable range. All samples containing the PS probe and 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 the probe PS and Hg ²⁺ ions may be useful for intracellular detection of Hg ²⁺ metal ions.

Claims (6)

A compound represented by the following formula (1).
[Formula 1]

A composition for detecting mercury ions (Hg ²⁺ ), comprising the compound represented by Formula 1 of claim 1 .
The composition of claim 2, wherein the mercury ions (Hg ²⁺ ) are present in cells.
The composition of claim 2, wherein the pH of the composition is 6 to 8.
A method for detecting mercury ions (Hg ²⁺ ) comprising reacting the compound represented by Formula 1 of claim 1 with a sample.
(a) reacting a compound represented by the following formula (2) and a compound represented by the following formula (3) to prepare a compound represented by the following formula (4);
(b) reacting the compound represented by Formula 4 obtained in step (a) and the compound represented by Formula 5 below to prepare a compound represented by Formula 1 below.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

Images