KR102105939B1 - Fluorescent probe compound for detecting tyrosinase in live cells and a tyrosinase detection fluorescence sensor comprising the same - Google Patents

Abstract

.The present invention relates to a fluorescent probe compound for detecting tyrosine oxidase represented by the following [Formula 1] and a fluorescent sensor for detecting tyrosine oxidase comprising the same:
[Formula 1]

.

Description

Fluorescent probe compound for detecting tyrosinase in live cells and a tyrosinase detection fluorescence sensor comprising the same}

The present invention relates to a fluorescent probe compound for detecting tyrosine oxidase in cells and a fluorescent sensor for detecting tyrosine oxidase containing the same.

The urgency to gain initial knowledge of the pathogenic dysfunction of multiple living systems motivates us to develop diagnostic tools. Non-invasiveness, selectivity, and sensitivity are key characteristics that are very desirable in tools for diagnosing disease in living specimens (Non-Patent Document 1). Of the various imaging methods for diagnosing disease, fluorescent probes have become a very attractive strategy because of their ability to classify and identify desired chemicals based on molecular reactivity differences and the ability to obtain spatial images with minimal disturbance ( Non-Patent Document 2).

Tyrosine oxidase (Tyrosinase) is a copper-containing enzyme that catalyzes the oxidation and oxidation of phenol derivatives to quinone in the presence of oxygen molecules (Non-Patent Document 3). Because melanin is an essential component of melanoma progression, oxidative chemical changes from phenol to melanin in living organisms cause melanoma epithelial carcinoma. Melanoma is the most lethal skin carcinoma with poor prognosis and high metastasis (Non-Patent Document 4). Additionally, tyrosinase causes dopamine neurotoxicity and neurodegeneration associated with Parkinson's disease. Therefore, finding a suitable marker for such biochemical presence is desirable in understanding the activity of tyrosinase in various biochemical and pathological processes, and ultimately enables diagnosis of tyrosinase-related diseases. Although there have been many efforts to visualize melanoma according to the degree of overexpression of tyrosinase, it is still difficult to develop a fast and selective fluorescent probe capable of monitoring tyronase in a living system (Non-Patent Document 5). Conventional fluorescent probes exhibit a fluorescence reaction when not only tyrosinase but also reactive oxygen species (ROS) such as HOCl, H ₂ O ₂ , and NO ₃ (Non-Patent Document 6), due to unwanted interference of ROS There is a problem that tyrosine oxidase cannot be selectively detected.

As described above, several probes for detecting tyrosinase in cells have been reported, but little has been reported about the development of probes and sensors that detect tyrosinase specific and effectively at a satisfactory level in cells and tissues.

 S. K. Arya and S. Bhansali, Chem. Rev., 2011, 111, 6783.  K. Fujimoto, S. Yamada and M. Inouye, Chem. Commun., 2009, 7164.  C. M. Kumar, U. V. Sathisha, S. Dharmesh, A. G. Rao and S. A. Singh, Biochimie, 2011, 93, 562.  I. J. Fiddler, Cancer Control, 1995, 2, 398.  T. I. Kim, J. Park, S. Park, Y. Choi and Y. Kim, Chem. Commun., 2011, 47, 12640.  J. Kim and Y. Kim, Analyst, 2014, 139, 2986.

In the present invention, to provide a fluorescent probe compound for detecting tyrosine oxidase having high selectivity to tyrosine oxidase and a fluorescent sensor for detecting tyrosine oxidase containing the same.

The present invention to solve the above problems,

To provide a fluorescent probe compound for tyrosine oxidase detection represented by the following [Formula 1]:

[Formula 1]

.

.

In addition, the present invention provides a fluorescent sensor for detecting tyrosine oxidase containing the fluorescent probe compound.

The fluorescent probe compound according to the present invention reacts with excellent selectivity to tyrosine oxidase, and has excellent photophysical properties and low cytotoxicity, so that it can effectively image tyrosine oxidase.

1 (A) is a result of observing the time-dependent fluorescence change of tyrosinase (100 U mL ^-1 ) and Tyro-1 (a compound represented by [Formula 1]) (5 μM) in culture, (B) It is the result of observing the concentration-dependent fluorescence change of Tyro-1 (5 μM) under various tyrosinase concentrations (0-100 U mL ^-1 ). Incubation was carried out in PBS buffer (pH = 7.4, 1% DMSO) at 37 DEG C for 1 hour. (C) shows tyrosinase activity inhibition assay results using Tyro-1 (5 μM) in the presence of PBS buffer (pH = 7.4, 1% DMSO) and various concentrations of kojic acid at 37 ° C. . Tyrosinase (100 U mL ^{- 1} ) was incubated with inhibitors at 37 ° C. for 20 minutes. (D) shows a fluorescent reaction result of the Tyro-1 (5 μM) under various ^{ROS: (a) NO 2- 500} μM in PBS buffer (pH 7.4, 1% DMSO) in 37 ℃; (b) ClO ^- 500 μM; (c) H ₂ O ₂ 500 μM; (d) TBHP 500 μM; (e) ^· OH 100 μM; (f) TBO ^· 100 μM; And (g) tyrosinase (100 U mL ^-1 ). All fluorescence reactions were obtained at 452 nm; The excitation wavelength is 400 nm, and the slit set is 3/5 nm.
2 is a one-photon laser microscope image of Tyro-1 (10 μM) as a function of time in B16F10 cells (magnification, ⅹ40). λ _ex = 405 nm; λ _em = 420-500 nm. Scale bar = 50 μM.
FIG. 3 is a two-photon laser microscope image of Tyro-1 (10 μM) over time in B16F10 cells (magnification, ⅹ100). Cells were cultured at 37 ° C. with Tyro-1 (10 μM) for various times (0, 2, 3 hours). λ _ex = 740 nm; λ _em = 420-520 nm. Scale bar = 20 μM.
4 is a two-photon laser scanning microscope image of probe Tyro-1 (10 μM) in B16F10 cells (magnification, ⅹ100). (A) Cell only (control); (B) cells incubated with Tyro-1 (10 μM) for 2 hours; (C) Cells cultured for 2 hours with Tyro-1 after pretreatment with kojic acid (100 μM) for 2 hours; (D) cells pre-treated with α-MSH (200 nM) for 2 hours and cultured for 2 hours with Tyro-1; (E) Cells incubated with Tyro-1 for 2 hours after pre-treatment with α-MSH and kojic acid for 2 hours. λ _ex = 740 nm; λ _em = 420-520 nm. Scale bar = 20 μM.
5 shows fluorescence images of probe Tyro-1 (10 μM) and tyrosinase in B16F10 cells (magnification, ⅹ100). (A) A two-photon laser scanning microscope image of cells that are not probe treated; (B) cells incubated with Tyro-1 (10 μM), tyrosinase (100 U mL ^{- 1} ) for 2 hours; (C) cells pre-treated with H ₂ O ₂ (50 μM) for 2 hours, and then incubated with probe Tyro-1 for 2 hours; (DF) was pre-treated with H ₂ O ₂ and kojic acid (10, 50 and 100 μM) for 2 hours, and then incubated with Tyro-1 for 2 hours. λ _ex = 740 nm; λ _em = 420-520 nm. Scale bar = 20 μM. The relative fluorescence intensity of a two-photon laser scanning microscope image is set to a common threshold for all images using ImageJ (* p <0.05), the average value of each treatment group, and then a total of nine cells per treatment condition (A-F) It was quantified in.
6 shows the UV-Vis spectrum of Tyro-1 (5 μM) in the absence (black line) and presence (red line) of tyrosinase (200 U / mL) in PBS buffer (pH 7.4, 1% DMSO). Data were recorded after incubation at 25 ° C. for 30 minutes.
7 shows a linear correlation between ΔF and tyrosinase concentration (20-80 U / mL). ΔF means the difference in fluorescence intensity of Tyro-1 (5 μM) before and after tyrosinase culture.
8 shows an estimate of the reaction rate constant between Tyro-1 (5 μM) and tyrosinase (100 U / mL) by applying a regression equation; K = 0.004S ^-1
FIG. 9 shows changes in fluorescence intensity of Tyro-1 (5 μM) at 452 nm for various concentrations of Tyrosinase (0-0.1 U / mL) in PBS buffer (pH 7.4, 1% DMSO) with a slit width of 3/5 nm. Shows.
Figure 10 shows the results of the fluorescence reaction of Tyro-1 (5 μM) when incubated with tyrosinase (100 U / mL) in different pH buffers (PBS with 1% DMSO) at 37 ° C. The fluorescence intensity of each point was excited at 400 nm with a slit set at 3/5 nm, and recorded at 452 nm.
11 shows the fluorescence response of Tyro-1 (5 μM) to various amino acids (500 μM): a) alanine in PBS buffer (pH 7.4, 1% DMSO) at 37 ° C .; b) arginine; c) asparagine; d) aspartic acid; e) cysteine; f) glutamine; g) glutamic acid; h) glycine; i) histidine; j) homo cysteine; k) isoleucine; l) leucine; m) lysine; n) methionine; o) phenylalanine; p) proline; q) serine; r) threonine; s) tryptophan; t) tyrosine; u) valine and v) tyrosinase (100 U / mL). All fluorescence reactions were recorded at 452 nm by excitation at 400 nm with slits set at 3/5 nm.
12 shows the fluorescence response of Tyro-1 (5 μM) to various metal ions (500 μM): a) Ag ⁺ dissolved in PBS buffer (pH 7.4, 1% DMSO) at 37 ° C .; b) Al ^{3 +;} c) Ba ^{2 +} ; d) Ca ^{2 +;} e) Cd ^{2 +} ; f) Co ^{2 +;} g) Cu ^{+ 2;} h) Fe ^{2 +} ; i) Fe ^{3 +;} j) K ⁺ ; k) Mg ^{2 +;} l) Mn ^{2 +;} m) Ni ²⁺ ; n) ^{2 +} Pb; o) Zn ^{2 +} ; p) ascorbic acid; q) GSH and r) tyrosinase (100 U / mL). All fluorescence reactions were recorded at 452 nm by excitation at 400 nm with slits set at 3/5 nm.
FIG. 13 is LC-MS data of tyrosinase-treated Tyro-1.
14 is a ¹ H-NMR spectrum of Tyro-1.
15 is a result of ¹³ C-NMR spectrum of Tyro-1.
16 shows the HRMS results of Tyro-1.
17 shows the cell viability of probe Tyro-1. Cells were incubated with 0, 1,5,10 and 50 μM probes Tyro-1 for 12 hours in B16F10 (A) and HeLa (B) cells. The results represent the mean ± SE of 3 independent experiments. * p <0.05.
18 shows a daylight image of Tyro-1 (10 μM) in HeLa cells. (A) Cell only (control); Cells incubated with Tyro-1 (10 μM) for 2 hours; (C) Cells incubated with Tyro-1 for 2 hours after pretreatment with kojic acid (100 μM) for 2 hours. λ _ex = 405 nm, λ _em = 420-500 nm. Scale bar = 20 μM.
19 shows daylight laser scanning microscopy images of Tyro-1 (10 μM) in B16F10 cells (magnification, × 40). (A) Cell only (control); (B) cells incubated with Tyro-1 (10 μM) for 2 hours; (C) Cells cultured for 2 hours with Tyro-1 after pretreatment with kojic acid (100 μM) for 2 hours; (D) cells pre-treated with α-MSH (200 nM) for 2 hours and cultured for 2 hours with Tyro-1; (E) Cells incubated with Tyro-1 for 2 hours after pre-treatment with α-MSH and kojic acid for 2 hours. λ _ex = 405 nm, λ _em = 420-500 nm. Scale bar = 20 μM.

Hereinafter, the present invention will be described in more detail.

The present invention is to provide a probe compound capable of selectively detecting tyrosine oxidase in a cell and a sensor including the same.

Accordingly, the present invention provides a fluorescent probe compound for tyrosine oxidase detection represented by the following [Formula 1]:

[Formula 1]

.

.

The compound represented by [Formula 1], as shown in [Tyrosinase detection mechanism], strong fluorescence by inhibiting the photoinduced electron transfer effect (Photoinduced Electron Transfer, PET) while the phenol structure is changed to a quinone structure in the presence of Tyrosinase It characterized in that it represents.

[Tyrosinase detection mechanism]

In addition, the present invention provides a fluorescent sensor for detecting tyrosine oxidase containing the fluorescent probe compound.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples and the like are intended to illustrate the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited thereby.

Experimental method

Materials, methods and equipment

All reactants and solvents were purchased from Sigma-Aldrich, Avra, Loba chem and used without further purification. Flash column chromatography was performed using silica gel (100-200 mesh) and analytical thin layer chromatography was performed using silica gel 60 (pre-coated sheet with 0.25 mm thickness). Mass spectra were recorded on an anionic SpecHiResESI mass spectrometer. NMR spectra were collected on a 400 MHz spectrometer (Bruker, Germany).

Compound synthesis

A compound represented by [Chemical Formula 1] (Tyro-1) according to the present invention was synthesized according to the following synthetic route.

[Synthetic route]

a) (i) Pyrrole, triphosgene, 1,2-dichloroethane, 0 ° C., 2 h; (ii) Pyrrole, 80 ° C., 30 min; b) (i) POCl ₃ , 1,2-dichloroethane, 80 ° C., 2 h. (ii) Et ₃ N, BF ₃ E _t2 O; c) Dopamine. HCl, Et ₃ N, Dichloromethane, 25 ° C, 6 h.

Synthesis of Compound B and 1

Compound B and Compound 1 are conventionally described in Y. Zhang, X. Shao, Y. Wang, F. Pan, R. Kang, F. Peng, Z. Huang, W. Zhang and W. Zhao Chem . Commun ., 2015 , 51, 4245-4548.).

Synthesis of Tyro-1

Compound 1 (150 mg, 0.664 mmol) was added to the solution with dopamine stirred. HCl (377 mg, 1.99 mmol) and triethylamine (0.28 mL, 1.99 mmol) in DCM (20 mL) at 0 ° C. was added and stirred at room temperature for 6 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (3 x 25 mL). The organic layer was washed with water (2 x 50 mL), brine (1 x 50 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (100-200 mesh) using ethyl acetate in pet ether DCM (5: 5) as eluent to give Tyro-1 (90 mg; 64.47%) of a light brown tongue. .

¹ H-NMR (400 MHz, DMSO- d 6): δ 9.70 (s, 1H); 8.89 (s, 1 H); 8.76 (s, 1 H); 7.58 (s, 1 H); 7.45 (d, J = 3.60 Hz, 1H); 7.35 (s, 1H); 7.32 (d, J = 4.0 Hz, 2H); 6.69 (q, 2H); 6.57 (q, 2H); 6.38 (q, 1 H); 3.92 (t, J = 7.20 Hz, 2H); 2.95 (q, 2H); ¹³ C-NMR (100 MHz, DMSO- d 6): 149.41, 145.99, 144.01, 134.21, 131.82, 130.21, 124.41, 122.45, 120.01, 117.65, 114.75, 112.89, 47.12, 32.75; ESI-HRMS m / z (M + H): calcd. 344. 13, found 344.1375.

UV- Vis  And Fluorescence Spectroscopy

Absorption spectra were recorded on a UV-1800 spectrophotometer (Shimadzu), and fluorescence spectra were recorded using an RF-5301 fluorescence spectrophotometer (Shimadzu) in a 300 cm volume of 1 cm standard quartz cell. A 500 μM probe stock solution was prepared by dissolving the probe in DMSO. A stock solution of 100 U / mL tyrosinase was prepared by dissolving in PBS buffer (pH = 7.4). In an Eppendorf tube, 3 μL of 500 μM Tyro-1 and an appropriate volume of tyrosinase or other reagents were mixed with PBS buffer. The final 300 μL volume of reaction solution was incubated at 37 ° C. Thereafter, it was transferred to a quartz cell for fluorescence measurement. Fluorescence emission spectra were recorded at an excitation wavelength of 400 nm and emission was monitored in the range of 400-600 nm (λem = 452 nm) with a slit width set at 3/5 nm.

Cell culture

Mouse melanoma (B16F10) and human cervical cancer (HeLa) cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea). B16F10 and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) ((Welgene, Gyeongsangbuk-do, Korea)) with 10% FBS (VWR International, Radnor, PA, USA) and 1% penicillin-streptomycin (Welgene) All cultured cells were maintained at 37 ° C. in a humidified atmosphere containing 5% CO ₂ .

Two-photon fluorescence microscopy

1 × 10 ⁵ cells were dispensed into a 35 mm glass bottom confocal dish (SPL Life Science, Gyeonggi-do, Korea) and stabilized for 1 day. When the cells reached 80% confluency, they were treated with probe Tyro-1 (10 μM in DMSO) at 37 ° C., 5% CO ₂ for 0-3 hours. To evaluate the tyrosinase stimulation effect, cells were pretreated for 2 hours in the absence or presence of α-MSH (200nM) and H ₂ O ₂ (50 μM), and then probe Tyro-1 (10 μM at 37 ° C., 5% CO ₂₎ . ) And incubated for 2 hours. To evaluate the tyrosinase inhibitory effect, cells were pretreated for 2 hours with or without kojic acid (100 μM) and then incubated for 2 hours with probe Tyro-1 (10 μM) at 37 ° C., 5% CO ₂ . Cells were then washed with PBS. Two-photon fluorescence microscopy images of the B16F10 cell labeled probe were obtained with a spectral confocal and two-photon microscope (Leica TCS SP2) with an objective lens of × 100 (NA = 1.30 OIL). Two-photon fluorescence microscopy images were obtained by exciting the probe with a mode-fixed titanium sapphire laser source (coherent chameleon, 90 MHz, 200 fs) set to a wavelength of 740 nm using a DM IRE2 microscope (Leica). Signals were collected with an 8-bit unsigned 512 X 512 pixels at 400 Hz scan rate using an internal PMT to obtain images ranging from 420-520 nm.

Daylight  One-photon fluorescence microscopy

The fluorescence images were taken using a confocal laser scanning microscope (Carl-Zeiss LSM 700 Exciter, Oberko chen, Germany) under the same conditions. Fluorescence channels were excited with a Si laser at 405 nm and emission was collected with a 420-500 nm band pass filter.

cell Survival rate

Approximately 2 × 10 ⁴ cells were seeded in 96 well microplates (SPL Life Science) and cultured for one day. After incubation, cells were treated with DMSO and Tyro-1 was treated for 12 hours. To analyze the cell viability of the probe in the cells, cell viability analysis was performed using the CytoTox96® non-radioactive cytotoxicity assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Fluorescence levels were analyzed using a SPECTRA MAX GEMINI EM microplate reader (Molecular Devices, Sunnyvale, CA, USA). The wavelength was set to 490 nm. Cell viability was expressed as a percentage of absorbance measured in treated wells compared to control wells.

Statistical analysis

Statistical significance was analyzed by comparison with control values using Student's t-test. The data represent the mean ± SE, and were considered significant when the P-values were less than 0.05.

Linear range and detection limits

The limit of detection was calculated using fluorescence titration. The fluorescence emission spectrum of the probe Tyro-1 (2.0 × 10-6 M) was measured 10 times to obtain a standard deviation of the sample. The fluorescence intensity at 452 nm was expressed as tyrosinase concentration. The detection limit was calculated using the equation (3σ / k: where σ is the standard deviation of the blank measurement, k is the slope between fluorescence intensity versus tyrosinase concentration).

Results and Discussion

BODIPY is a versatile fluorophore that facilitates large Stokes shift, relatively high quantum yield, and derivative synthesis suitable for the application (Y. Gabe, Y. Urano, K. Kikuchi, H. Kojima and T. Nagano, J. Am. Chem. Soc., 2004, 126, 3357.). In addition, it has the advantage of showing a systematic fluorescence change by controlling the relative Gibbs free energy (thermodynamic potential) through the intramolecular light-induced electron transfer (PeT) process.

The fluorescent probe compound (Tyro-1) for tyrosine oxidase detection according to the present invention is composed of a phenol structure that reacts to Tyrosinase and a bodyy phosphor that emits 452 nm fluorescence, and the phenol structure is changed to a quinone structure in the presence of Tyrosinase. While suppressing the photo-induced electron transfer effect (Photoinduced Electron Transfer, PET) is characterized by showing a strong fluorescence.

The potential of Tyro-1 was estimated by observing photophysical properties in a phosphate buffer at 37 ° C, an artificial physiological environment. As shown in FIG. 6, UV absorbance of Tyro-1 at 400 nm wavelength increased when tyrosinase of 100 U / mL was present. On the other hand, as a result of the fluorescence intensity of Tyro-1 as a function of the incubation time and tyrosinase concentration, the fluorescence intensity gradually increased over time after the addition of Tyrosinase, and eventually the light emission amount up to 12.5 times at 452 nm. It has been shown to reach an increased plateau (Figure 1A). Fluorescence change of Tyro-1 under artificial physiological environment shows excellent linear correlation with variable concentration of tyrosinase in the concentration range of 20-80U / mL (Fig. 1B, Fig. 7). Tyro-1 reacted with tyrosinase at a rate of 4 × 10 ^-3 s ^-1 , which means a pseudo-unimolecular reation (Fig. 8). In addition, as a result of calculating the detection limit of tyrosinase by applying the regression equation, the detection limit value was found to be 2.5 × 10 ^-2 U / mL (FIG. 9), confirming that Tyro-1 has high sensitivity to tyrosinase. Did.

Next, to confirm that Tyro-1 is suitable for bioimaging, fluorescence reaction of Tyro-1 was confirmed when cultured with or without tyrosinase at 37 ° C. and a physiological pH range (pH 6.0-8.0). Considering photoelectron transfer (PeT), the absence of Tyrosinase shows little Tyro-1 fluorescence, and the fact that the fluorescence intensity of Tyro-1 hardly changes for 2 hours indicates the excellent stability of Tyro-1. After incubation with Tyrosinase, Tyro-1 showed a high fluorescence reaction at a pH in the range of 6.0-8.0 as in the physiological environment (FIG. 10). These results indicate that Tyro-1 produces a strong and stable fluorescent signal when exposed to tyrosinase under physiological conditions. This is because photoelectron transport is significantly blocked by oxidation of the dopamine moiety.

In addition, inhibition of tyrosinase activity was evaluated by measuring the degree of fluorescence attenuation of Tyro-1 in the presence of kojic acid, a tyrosinase inhibitor, at physiological conditions of pH 7.4 PBS and 37 ° C. As shown in FIG. 1C, a significant decrease in fluorescence intensity was observed in the solution containing kojic acid and cultured Tyro-1 and tyrosinase as compared to the control group. In addition, the fluorescence reaction of Tyro-1 gradually decreased as the concentration of Kojic acid increased. Through this, it was confirmed that tyrosinase oxidizes Tyro-1 to regain fluorescence suppressed by photoelectron transfer.

Next, the chemical selectivity of Tyro-1 to tyrosinase was evaluated using other related ROS amino acids and metal ions. 1D and 11-12, it was confirmed that Tyro-1 shows fluorescence enhancement only when tyrosinase is present and no fluorescence enhancement when other analytes are present. The stability of Tyro-1 to various oxidizing agents, including HOCl, is clearly due to the redox potential mismatch between Tyro-1 and the oxidizing agents. The fact that there is oxidation FerriBRIGHT by catechol portion of the Fe ^{3 +} (DP Kennedy, CM Kormos and SC Burdette, J. Am. Chem. Soc., 2009, 131, 8578.) , while the analog has been oxidized by tyrosinase Supported by previous reports (TI Kim, J. Park, S. Park, Y. Choi and Y. Kim, Chem. Commun., 2011, 47, 12640.). In addition, Tyro-1 only shows a small amount of fluorescence when exposed to 500 μM of H ₂ O ₂ , which is 150 times higher than the H ₂ O ₂ concentration in melanoma (J. Bustamante, L. Guerra, L. Bredeston, J. Mordoh and A. Boveris, Exp. Cell Res., 1991, 196, 172.). These results strongly suggest that Tyro-1 can detect tyrosinase in living systems without being disturbed by other biological analytes, including oxidizing agents.

To understand the mechanism of Tyro-1 fluorescence enhancement, Tyro-1 pretreated with tyrosinase was analyzed using LC-MS analysis. As shown in Figure 13, the MS value was found to be 93.12% abundance at 342.2 (M + 1), which means that the catechol portion of Tyro-1 was oxidized to o-quinone. These results support the fact that tyrosinase plays a decisive role in oxidizing the catechol portion of Tyro-1 to o-quinone, thereby nullifying the phototransfer effect, and thus the oxidized form of Tyro-1 becomes fluorescent.

To demonstrate the activity of tyrosinase in cells, the cytotoxicity of Tyro-1 was evaluated by analyzing the cell viability of the B16F10 and HeLa cell lines using the CytoTox96® nonradioactive cytotoxicity assay kit before adding Tyro-1. Up to 10 μM (Fig. 17), Tyro-1 did not show significant cytotoxicity, which means that Tyro-1 is suitable for in vitro imaging based on tyrosinase expression.

Next, in vitro cell imaging was used to investigate tyrosinase activity in living cells using Tyro-1. As is well known, tyrosinase is overexpressed in the B16F10 murine melanoma cell line (R. White and F. Hu, J. Invest. Dermatol., 1977, 68, 272.) and insignificantly expressed in the HeLa cell line. Therefore, B16F10 and HeLa were selected as positive and negative controls, respectively. Next, it was observed that the confocal microscopic fluorescence of Tyro-1 (10 μM) and cultured B16F10 increased significantly as a function of time (FIG. 2). This clearly shows that Tyro-1 can detect the activity of tyrosinase in living systems. In HeLa of tyrosinase negative, there was little fluorescence improvement even under the same experimental conditions (Fig. 18).

Next, the two-photon signaling ability for tracking tyrosinase activity of Tyro-1 was investigated using a two photon laser scanning microscopy. As a result of the measurement, similar to the results of the daylight confocal microscopy experiment (FIG. 2), Tyro-1 and B6F10 cells cultured showed improved fluorescence with time (FIG. 3). In addition, it was confirmed that Tyro-1 was stable for at least 3 hours after treatment on living cells, and thus it was confirmed that Tyro-1 can be usefully used as a two-photon probe for evaluating endogenous tyrosinase activity.

Next, the degree of cell labeling according to tyrosinase expression in B16F10 cells was observed using a two-photon laser scanning microscope (FIG. 4). To evaluate the effect of tyrosinase inhibition on the fluorescence activity of Tyro-1, B16F10 cells were pretreated with tyrosinase inhibitor kojic acid. As a result of the measurement, the fluorescence intensity of B16F10 cells pretreated with kojic acid was significantly reduced compared to Tyro-1 treated B16F10 as a control (FIG. 4B) (FIG. 4C). On the other hand, it was confirmed that α-melanocyte (α-MSH), a stimulating hormone that up-regulates endogenous tyrosinase activity, and cultured Tyro-1 treated B16F10 cells showed higher fluorescence intensity than the control group. In addition, a pattern similar to that observed in two-photon fluorescence imaging was confirmed in daylight microscopy imaging results (FIG. 19). All of these results support that Tyro-1 can detect tyrosinase regulation in the cellular environment based on the degree of fluorescence labeling of living cells.

Finally, we investigated whether Tyro-1 can track the up-regulation of tyrosinase activity induced by H2O2 in the cell. For this, B16F10 cells were pre-treated with H ₂ O ₂ (50 μM) and then cultured with or without kojic acid. And after Tyro-1 treatment, the cells were cultured for 2 hours to observe the change in fluorescence intensity. The fluorescence intensity of the cells was calculated using ImageJ software. As shown in FIG. 5C, the fluorescence intensity from B16F10 cells cultured with H ₂ O ₂ (50 μM) was 3 times higher than that from B16F10 cells cultured without H ₂ O ₂ (FIG. 5B). On the other hand, the fluorescence intensity of B16F10 cells dropped significantly depending on the concentration of kojic acid (Fig. 5D-F). Through these results, it was confirmed that Tyro-1 can be used as a non-invasive two-photon fluorescent probe for detecting endogenous H ₂ O ₂ mediated tyrosinase activity in the course of cancer as well as in a general physiological environment.

conclusion

Based on the photophysical properties measured through the above-described examples, it was confirmed that the compound (Tyro-1) according to the present invention can effectively detect tyrosine oxidase in cells due to its low detection limit and high selectivity. Specifically, when 200 U / mL of tyrosinase was present under artificial physiological conditions, the UV absorbance of Tyro-1 improved up to 8 times at 400 nm wavelength. Moreover, under the same conditions, the fluorescence intensity of Tyro-1 at a wavelength of 400 nm was improved up to 12.5 times. In addition, Tyro-1 showed a low detection limit for tyrosinase, and the limit was 2.5 × 10 ^-2 U / mL. In addition, Tyro-1 is very stable within the physiological pH range and was able to detect tyrosinase without being disturbed by substances commonly present in biological systems. In addition, since Tyro-1 showed high cell viability, it was confirmed that Tyro-1 is a suitable candidate for in vitro mapping of tyrosinase expression.

As a result, the compound (Tyro-1) according to the present invention emits fluorescence very stably under physiological conditions and shows high selectivity to tyrosine oxidase even under high concentrations of free radical species, and hardly reacts with hydrogen peroxide. It is possible to successfully monitor tyrosinase in both daylight and di-photon. Therefore, the compound according to the present invention can be utilized as an efficient tool for tyrosine oxidase detection, and can be usefully used in various biological and clinical applications, such as research on the physiological and pathological roles of tyrosine oxidase in cells. You can.

Claims (2)

Fluorescent probe compound for tyrosine oxidase detection represented by the following [Formula 1]:
[Formula 1]

. A fluorescent sensor for detecting tyrosine oxidase comprising the fluorescent probe compound according to claim 1.

Images