KR20170120360A - Carboxylesterase-selective ratiometric two-photon fluorescent probe, and quantitative imaging method of carboxylesterase in vivo using the same - Google Patents

Abstract

The present invention relates to a two-photon fluorescence probe selectively responding to a carboxylesterase, and a quantitative imaging method of a carboxylesterase in vivo using the same. The novel two-photon fluorescence probe selectively displays a living cell and a biotissue It can be stained and react with a carboxyl esterase to exhibit a change in fluorescence color with high sensitivity. In addition, since the carboxylesterase can be selectively detected in living cells and biological tissues at depths of 300 μm or more, it is possible to quantitatively image the distribution and activity of carboxylesterase in living cells or whole body tissues.

Description

TECHNICAL FIELD The present invention relates to a two-photon fluorescence probe selectively responding to carboxylesterase, and a quantitative imaging method of a carboxylesterase in vivo using the same, and a quantitative imaging method of carboxylesterase in vivo,

The present invention relates to a two-photon fluorescence probe selectively responding to a carboxylesterase, and a quantitative imaging method of a carboxylesterase in vivo using the same, and more particularly, to a method for quantitative imaging of a living cell and a biotissue The present invention relates to a method for quantitatively irradiating the activity of a carboxyl esterase present at a deep place through a two-photon microscope with a low energy excitation source.

Estherase is an enzyme that hydrolyzes ester bonds and is an important enzyme involved in metabolism in vivo. Carboxylesterases (hereinafter referred to as 'CE') are directly linked to liver metabolism and fat syndrome, and regulation of CE is an important issue in pharmacological and clinical applications.

In particular, CE is an enzyme widely distributed in the human body to catalyze the hydrolysis of various endogenous and exogenous substrates. The deficiency of carboxylesterase causes diseases such as hepatic steatosis, obesity and hyperlipemia.

A number of single-photon fluorescence probes that are currently CE-sensitive have been developed, but a common problem with most single-photon fluorescence probes is their shallow penetration depth, their auto-fluorescence using their short excitation wavelength (<400 nm) And photo-bleaching phenomenon. Thus, there is a limit to the application to tissue imaging.

Two - photon microscope is used as a solution to this problem. By using two - photon microscope, it is possible to observe inside of living tissue of 100 ㎛ depth or more which is undamaged and to make a long time image in real time. However, there is no two-photon variable color fluorescence probe capable of selectively detecting the carboxyl esterase present in the biotissue.

Therefore, it is more stable to factors such as other nucleophiles and enzymes present in the environment of the body, and is more stable in the carboxyl esterase so that it can react with the carboxyl esterase to more accurately observe the activity of the carboxylesterase in vivo. There is an urgent need to develop a two-photon fluorescence probe that selectively responds.

Chinese Patent Publication No. 104342106

It is an object of the present invention to provide a method for selectively detecting a carboxylesterase using a novel two-photon fluorescent probe and quantitatively imaging the distribution and activity of the carboxylesterase in living cells or whole living tissue .

In order to achieve the above object, the present invention provides a two-photon fluorescence probe represented by the following formula (1).

[Chemical Formula 1]

Wherein R ₁ is C ₁ to C ₄ alkyl, C ₁ to C ₄ alkoxy, halogen, or aryl, and R ₂ or R ₃ are the same or different from each other and are hydrogen or methyl.

The present invention also relates to a method for producing a biosensor, comprising the steps of: injecting a two-photon fluorescent probe selectively responsive to a carboxylesterase represented by formula (1) in vivo; Wherein the two-photon fluorescence probe reacts with the in vivo carboxyl esterase to show fluorescence; And observing the fluorescence with a variable color two-photon microscope.

[Chemical Formula 1]

Wherein R ₁ is C ₁ to C ₄ alkyl, C ₁ to C ₄ alkoxy, halogen, or aryl, and R ₂ or R ₃ are the same or different from each other and are hydrogen or methyl.

The present invention also relates to a method for screening a candidate drug and a two-photon fluorescence probe in a sample separated from a human; Measuring the degree of activation of the carboxylesterase in the sample treated with the two-photon fluorescent probe; And selecting a candidate substance having an increased degree of activation of the carboxylesterase as compared to a sample not treated with the candidate drug.

The two-photon fluorescence probe according to the present invention is selectively stained in living cells and living tissue, and at the same time, reacts with a carboxyl esterase to exhibit a fluorescence change with high sensitivity. In a live cell and a living tissue having a depth of 300 μm or more Selectively detect the carboxylesterase and thus quantitatively visualize the distribution and activity of the carboxylesterase in living cells or whole living tissue.

Fig. 1 shows the enzyme reaction of estrase and SE1.
Fig. 2 shows the selectivity test of SE1.
Figure 3 is a diagram showing a pseudo-variable color TPM image of HepG2 cells cultured with SE1 (2 [mu] M) (a) and Compound 1 (2 [mu] M).
Figure 4 shows Western blot analysis (a, c) of CE1 antibody (a) and Hep2 cells cultured with CE2 antibody (c) using IgG antibody crosslinked protein-A / G agarose beads, CE1 antibody b) and CE2 antibody (d) after immunoprecipitation.
Figure 5 shows TPM (a, d, g), OPM (b, e, h) images and merged images (c, f, i) of HepG2 cells co- labeled with SE1 (2 [mu] M) Fig.
Fig. 6 shows the results of a pretreatment (a) and pretreatment (c) with 500 [mu] M BNPP for 1 hour before being labeled with SE1 and a pseudo-variable TPM image (F _yellow / B _blue ), (a) and (c).

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

The present inventors have found that the two-photon fluorescence probe selectively stains live cells and living tissues, and at the same time reacts with a carboxyl esterase to exhibit fluorescence color change with high sensitivity, thereby quantitatively determining the distribution and activity of carboxylesterase in living tissues And the present invention has been completed.

The present invention provides a two-photon fluorescence probe represented by the following general formula (1).

[Chemical Formula 1]

Wherein R ₁ is C ₁ to C ₄ alkyl, C ₁ to C ₄ alkoxy, halogen, or aryl, and R ₂ or R ₃ are the same or different from each other and are hydrogen or methyl.

In the two-photon fluorescence probe, R ₁ is selected from methyl or substituted or unsubstituted phenyl, and R ₂ and R ₃ may each be methyl, but are not limited thereto.

The two-photon fluorescence probe may be selectively sensitive to the carboxyl esterase.

A typical compound of the two-photon fluorescence probe according to the present invention may be a compound (SE1) represented by the following formula (2), wherein SE1 is a trimethyl-lock-based phenylacetate, 3- -4,6-dimethylphenyl) -3-methylbutyric acid derivative and a solubility group, and 2,5,8,11-tetraoxal tridecan-13-amine as an electron donor-acceptor two-

(2)

The compound represented by Formula 2 may be prepared by various methods. In one embodiment, Compound 1 is prepared by reacting Compound A and Compound B as shown in Reaction Scheme 1 below (Step 1); And a step of reacting Compound 1 with Compound C to prepare a compound of SE1 (Step 2).

[Reaction Scheme 1]

In the first step, N, N'-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole are added to the compound A solution, and the mixture is allowed to react at room temperature for 1 hour in a nitrogen gas atmosphere. To the reaction mixture, compound B And the reaction was further continued for 12 hours.

In the second step, N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide is added to the compound C solution, and the mixture is allowed to react at room temperature for 1 hour in a nitrogen gas atmosphere. Compound 1 is added to the reaction mixture And allowed to react for 48 hours.

The two-photon fluorescence probe represented by Formula 2 according to the present invention can react with an enzyme reaction product to sense a carboxylesterase based on an intermolecular charge transfer.

The present invention also relates to a method for producing a biosensor, comprising the steps of: injecting a two-photon fluorescent probe selectively responsive to a carboxylesterase represented by formula (1) in vivo; Wherein the two-photon fluorescence probe reacts with the in vivo carboxyl esterase to show fluorescence; And observing the fluorescence with a variable color two-photon microscope.

[Chemical Formula 1]

Wherein R ₁ is C ₁ to C ₄ alkyl, C ₁ to C ₄ alkoxy, halogen, or aryl, and R ₂ or R ₃ are the same or different from each other and are hydrogen or methyl.

The two-photon fluorescence probe may react with the in vivo carboxyl esterase to change the blue color emission of 420 to 470 nm to the yellow color light of 520 to 570 nm.

The carboxylesterase can be detected in living tissues at a depth of 250 to 350 μm.

The present invention also relates to a method for screening a candidate drug and a two-photon fluorescence probe in a sample separated from a human; Measuring the degree of activation of the carboxylesterase in the sample treated with the two-photon fluorescent probe; And selecting a candidate substance having an increased degree of activation of the carboxylesterase as compared to a sample not treated with the candidate drug.

The carboxylesterase deficiency disease may be any of fatty liver, hyperlipidemia, or obesity, but is not limited thereto.

The sample may be hepatocyte or liver tissue, but is not limited thereto.

The degree of activation of the carboxylesterase can be measured by colorimetric assay, immunoprecipitation, or Western blotting, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

Example 1 Synthesis of Compound 1

N, N'-dicyclohexylcarbodiimide (hereinafter abbreviated as 'DCC', 232 mg, 1.12 mmol) was added to a solution of A (250 mg, 0.75 mmol) dissolved in dry DMF 1-hydroxybenzotriazole (hereinafter referred to as 'HOBt', 152 mg, 1.12 mmol) was added thereto, and the reaction mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. The 2,5,8,11-tetraoxal tridecan-13-amine (B, 156 mg, 0.75 mmol) was then added to the reaction mixture and the reaction was allowed to proceed for another 12 hours.

After completion of reaction the solution was evaporated, the crude was dissolved in a mixture (crude mixture) in CH ₃ CN. The Precipitate dicyclohexylurea precipitated was removed by filtration, and the filtered liquid was concentrated under reduced pressure.

The crude product was purified by column chromatography using an eluent containing 5% methanol (CH ₃ OH) in EtOAc to give compound 1 as yellow semi-solid (Yield: 280 mg (72%)) ).

_{^{1 H NMR (CDCl 3, 400}} MHz): δ (ppm) 8.40 (s, 1H), 8.34 (s, 1H), 8.01 (d, J = 8.8 Hz, 1H), 8.00 (d, J = 8.8 Hz, (Dd, J = 8.4, 2.0 Hz, 1H), 7.66 (d, J = 8.8 Hz, 1H), 7.64 ), 6.87 (dd, J = 8.8,2.0 Hz, 1H), 6.72 (d, J = 2.0 Hz, 1H), 4.29 (brs, 4.8 Hz, 2H), 3.49 (t, J = 4.8 Hz, 2H), 3.31 (s, 3H), 2.92 (s, 3H); _{^{13 C NMR (CDCl 3, 100}} MHz): δ (ppm) 171.4, 167.2, 156.2, 148.7, 137.3, 135.1, 131.1, 130.1, 128.0, 126.8, 126.7, 126.6, 125.3, 125.0, 122.4, 121.5, 118.9, 103.4 , 72.1, 70.8, 70.7, 70.5, 70.2, 59.2, 40.4, 30.8; ^{HRMS (FAB +): m /} z calculated for [C 28 H 33 O 5 N 3 S] +: 524.2220, found: 524.2219.

Example 2 Synthesis of SE1

To a solution of 3- (2-acetoxy-4,6-dimethylphenyl) -3 (2-acetoxy-4,6-dimethylphenyl) -3-methylbutyric acid dissolved in 10 mL of a 1: 1 mixture of dry pyridine / N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride; N, N'-dimethylcarbodiimide hydrochloride was added to a solution of 4-methylbutyric acid 48 mg, 0.182 mmol, Aldrich, # 756377. 'EDC', 35 mg, 0.182 mmol] was added and the reaction mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere.

Compound 1 (48 mg, 0.092 mmol) was then added to the reaction mixture and additional reaction was allowed to proceed for 48 hours. After the reaction was complete, the solution was evaporated, washed with brine (30 mL), and DCM (30 mL × 3), and finally dried over anhydrous sodium sulfate.

The dried product was purified by column chromatography using CHCl ₃ with an eluent containing 10% methanol (CH ₃ OH) to obtain a pale yellow semi-solid SE1 (yield: 21 mg (30 %)).

_{^{1 H NMR (CDCl 3, 400}} MHz): δ (ppm) 8.59 (s, 1H), 8.53 (d, J = 1.6 Hz, 1H), 8.26 (dd, J = 8.8 Hz, 1.6Hz, 1H), 8.12 (d, J = 8.8 Hz, 1H), 7.98 (dd, J = 8.4, 1.6 Hz, 1H), 7.97 (s, 1 H), 7.38 (brs, NH-amide, 1 H), 7.25 (dd, J = 8.8, 1.6 Hz, 1H), 6.68 2H), 2.28 (s, 3H), 3.32 (s, 3H), 3.24 2.23 (s, 3 H), 2.01 (s, 3 H), 1.51 (s, 6 H); _{^{13 C NMR (CDCl 3, 100}} MHz): δ (ppm) 171.1, 170.1, 169.8, 166.9, 156.1, 149.6, 143.6, 138.5, 135.9, 135.4, 135.2, 134.1, 132.4, 132.0, 131.5, 130.6, 129.0, 127.6 , 127.1, 126.0, 125.5, 123.1, 121.8, 72.2, 70.8, 70.5, 70.1, 59.3, 46.1, 40.4, 40.1, 37.9, 32.4, 30.1, 25.7, 25.6, 22.2, 22.1, 20.7, 20.6; HRMS (FAB ⁺ ): m / z calculated for [C ₄₃ H ₅₁ O ₈ N ₃ S n a] ⁺ : 792.3289, found: 792.3289.

<Experimental Example 1> Analysis of physicochemical characteristics of SE1

1. Spectroscopy

The absorption spectrum was measured with a UV-Vis spectrophotometer (S-3100) and the fluorescence spectrum was measured with a fluorescence spectrophotometer (FluoroMate FS-2) in a 1 cm standard quartz cell (1 cm standard quartz cell). Fluorescence quantum yield was determined by literature method using 9,10-diphenylanthracene (0.93 in? = Cyclohexane) as a reference.

2. Enzyme Reaction Rate Analysis

Enzyme reaction rate measurements were performed using a fluorescence spectrophotometer (FluoroMate FS-2) with a 1 cm standard quartz cell. SE1 at various concentrations (0-40 [mu] M) was prepared by dissolving SE1 in PBS buffer (10 mM, pH = 7.4). The porcine liver esterase (PLE) enzyme was added to a final concentration of 0.88 μg / mL, and was incubated at 45 ° C (λ _ex = 373 nm) at 0 ° C to 30 minutes The strength was measured. The velocity parameter of the Michaelis-Menten equation was calculated using the hyperbolic function of the nonlinear fitting algorithm (OriginPro 8.0).

3. Two-photon fluorescence cross-sectional measurement

The two-photon cross-section (δ) was measured using femtosecond ('fs') fluorescence measurements. SE1 (1.0 × 10 ^-6 M) was dissolved in PBS buffer (10 mM, pH = 7.4) and the fluorescence intensity of the two-photon probe was measured at 720-880 nm using rhodamine 6G. The intensities of the two-photon-sensitive fluorescent spectrum of the reference and the sample were determined at the same excitation wavelength. The cross-sectional area of the two-photon fluorescence was calculated using the following equation.

[Equation 1]

Where s and r represent the sample and reference molecules, S represents the signal intensity collected from a CCD (Charge Coupled Device) detector, Φ represents the fluorescence quantum yield, and φ represents the total fluorescence collection efficiency , C represents the molecular density in solution, and [delta] _r represents the two-photon fluorescence cross-section of the reference molecule.

4. Selectivity evaluation of SE1

1 μM SE1 and various 200 μM ROS, 1 mM amino acid and glucose, 10 μg / L AChE, 20 U / L BChE, 0.1% human serum, and 5 μg / mL glucose in PBS buffer (10 mM, pH 7.4) hCE1 and hCE2 were added to obtain a fluorescence spectrum. Sample preparation is as follows.

At this time, ROS contained 30%, 70%, and 5% of hydrogen peroxide (H ₂ O ₂ ), tert-butylhydroperoxide (TBHP), and sodium hypochlorite (NaOCl) Aqueous solution. Hydroxyl radicals (OH) and tert-butoxy radicals (OtBu) were generated by reaction between 200 μM H ₂ O ₂ or TBHP and 1 mM Fe ^{2 +} .

Nitric oxide (NO) was prepared by purging PBS buffer (10 mM, pH 7.4) with nitrogen gas for 30 minutes, followed by nitric oxide (99.5%) for 30 minutes to prepare a stock solution (1.9 mM). Superoxide (O ^2- ) was purchased from KO ₂ . Peroxynitrile was prepared with a stock solution (10 mM in 0.3 M NaOH).

The amino acids were purchased from Sigma-Aldrich (LAA21), L-amino acids (Ala, Cys, Glu, Gly, Val, Thr, Tyr, Trp, Ser, Phe, Met, Leu, Ile, Asp, Lys, His) Was purchased from Sigma-Aldrich (G7528).

Acetylcholinesterase (AChE) was purchased from Sigma-Aldrich (C2888), butyrylcholinesterase ('BChE') was purchased from Sigma-Aldrich (C7512) Was purchased from Sigma-Aldrich (H4522), CE1 from Sigma-Aldrich (E0287), and CE2 from Sigma-Aldrich (E0412).

5. Inhibitor Analysis

HepG2 cells were washed with PBS and resuspended in 50 mM Tris-HCl (pH 7.4, Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich), 0.5% Triton X-100 (Sigma- 1 mM EDTA (Sigma-Aldrich), and protease inhibitor cocktail (Sigma-Aldrich) were homogenized in ice for 30 minutes using a bullet blender (NeXT Advance). The supernatant obtained by centrifugation of the homogeneous suspension at 10,000 rpm for 15 minutes was transferred to a new tube.

The supernatant containing 100 μg protein was incubated with each inhibitor [Donepezil hydrochloride monohydrate, the AChE inhibitor, ethopropazine hydrochloride, the BChE inhibitor, naringenin, PON2 inhibitor, PON2 Lead acetate and bis (4-nitrophenyl) phosphate (BNPP), which are CE inhibitors, were cultured in the presence of 10 [mu] M. The supernatant treated with the inhibitor was treated with 3 μM SE1 for 15 minutes, and the relative fluorescence intensity was obtained. The excitation wavelength was 405 nm.

6. Cell culture

All cells were transferred to glass bottoms (NEST) for two days before imaging. All of the cells were maintained in a humidified environment with a CO ₂ / air ratio of 5: 95 at 37 ° C. Cells were treated with 5% CO ₂ atmosphere for 30 min at 37 ° C, incubated with 2 μM SE1 and compound 1, washed three times with PBS (phosphate buffered saline; 'PBS', Gibco) And then imaged after culturing in colorless serum-free medium.

The culture medium for each cell is as follows. HepG2 cells (ATCC, Manassas, VA, USA) were cultured in MEM supplemented with 10% FBS (WelGene), penicillin (100 units / ml), and streptomycin (100 μg / ) Culture medium.

HeLa cervical cancer cells (ATCC, Manassas, Va., USA) were cultured in MEM supplemented with 10% FBS (WelGene), penicillin (100 units / ml), and streptomycin (100 μg / , Korea) medium.

Skin cancer (A431) cells (ATCC, Manassas, VA, USA) were cultured in RPMI1640 supplemented with 10% FBS (WelGene), penicillin (100 units / ml), and streptomycin (100 μg / ) Culture medium.

Raw 264.7 cells (ATCC, Manassas, VA, USA) were cultured in DMEM (WelGene Inc, Seoul, Korea) supplemented with 10% FBS (WelGene), penicillin (100 units / ml), and streptomycin Lt; / RTI &gt;

7. Immunoprecipitation and Western Blot Analysis

For immunoprecipitation, the HepG2 cells were washed with PBS and resuspended in 50 mM Tris-HCl (pH 7.4, Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich), 0.5% Triton X- mM EDTA (Sigma-Aldrich), and a protease inhibitor cocktail (Sigma-Aldrich) were homogenized in ice for 30 minutes using a bullet blender (NeXT Advance).

The supernatant obtained by centrifugation of the homogeneous suspension at 10,000 rpm for 15 minutes was transferred to a new tube.

The supernatant containing 100 μg of protein was used as a protein A / C strain using the major liver esterase enzyme CES1 antibody (sc-365248) or CES2 antibody (sc-100685) or control IgG (sc-2025) G agarose beads (GenDEPOT) overnight at 4 ° C. Antibody-conjugated agarose beads were centrifuged at 2000 rpm for 2 min and washed 3 times with homogenization buffer. The beads and supernatant proteins were used for western blot analysis. In Western blot analysis, proteins of SDS-PAGE were transferred to a PVDF membrane (Bio-Rad).

The PVDF membrane was treated with 5% non-fat milk for 2 hours and incubated with anti-CESl, anti-CES2, or anti-actin antibody overnight at 4 ° C. Goat anti-mouse IgG-HRP (sc-2005) was used as the secondary antibody and the band was visualized by Ez-Western Lumi Plus (ATTO). Antibodies used in Western blot analysis were purchased from Santa Cruz.

8. Two-Photon Fluorescence Microscopy (TPFM) analysis

Two-photon fluorescence microscope images of SE1-labeled cells and tissues were analyzed by spectral confocal and multiphoton microscopy with a numerical aperture of 0.30, 1.30, and 1.30 with a × 10 dry lens, a × 40 oil lens, and a × 100 oil objectives lens spectral confocal and multiphoton microscopes, Leica TCS SP8 MP).

The two-photon fluorescence microscope image was obtained by irradiating a two-photon fluorescence probe with a wavelength of 820 nm, 2901 mW (approximately 5.16 × in the focal plane) with a model-locked titanium-sapphire laser light source device (Mai Tai HP; Spectra Physics, 80 MHz, 100 fs pulse width) 10 ⁸ mW / cm ² average power) output and observed with a DMI 6000B microscope (Leica).

Live cell imaging using a live cell instrument (Chamlide IC) was performed by maintaining appropriate temperature, humidity, and pH values for a long period of time for a stable cell environment.

To acquire images at the 440-460 nm (F _blue ) and 500-550 nm (F _yellow ) ranges, we collect signals of 512 × 512 and 1024 × 1024 pixels of 8 bits at 400 Hz and 200 Hz scan rates, respectively Internal PMTs were used. Ratiometric image processing and analysis was performed using MetaMorph software.

9. Co-localization Experiments

HepG2, HeLa, A431, and Raw 264.7, which were appropriately combined with 1.0 μM of SE1 (2.0 μM) and a commercial cell organelle treater (LysoTracker Red DND-99 for Lysosome, MitoTracker Red FM for Mitochondria, ER-Tracker Red for Endoplasmic Reticulum) Cells were subjected to localization analysis using co-staining.

TPM and OPM images were obtained by collecting the wavelengths emitted at 520-570 nm (SE1, lambda _ex = 740 nm) and 600-650 nm (cellular organelle trekkers, lambda _ex = 552 nm).

The background images were collected, and the pixel distributions of the TPM and OPM images acquired from the green and red channels were compared using a scattergram. Pearson's colocalization coefficient (A) was calculated using the AutoQuant X2 program.

10. Preparation and dyeing of fresh rat liver fragments

Rat liver slices were prepared in the liver of 8 week old Sprague Dawley rats. The inter-rat fragments were cut into a thickness of 800 mu m in a PBS buffer using a vibrating-blade microtome.

The cleaved fragments were incubated with 20 μM SE1 in PBS, which was flushed with 95% O ₂ and 5% CO ₂ for 2 hours at 37 ° C.

The fragments were then washed three times with PBS, transferred to glass-bottomed dishes (NEST) and observed with a spectroscopic confocal microscope and a multi-photon microscope. The TPM image was observed at a depth of about 300 [mu] m.

11. Experimental results

Fig. 1 shows the enzymatic reaction between PEK and SE1. The fluorescence spectrum of SE1 was measured in PBS buffer (10 mM, pH 7.4, 37 ° C) measured every 1 minute before and after the addition of 0.2 unit PLE (b) of V ₀ / μM S ^-1 versus various concentrations of SE1 (0 μM to 40 μM) and SE1 (1.0 μM) after addition of human CE1 and CE2 (approximately 10 units) in PBS buffer (F _yellow / F _blue ) (c) according to the passage of time.

Referring to Figure 1 (a), when treated with PLE known to be similar to the activity of human CE1, an emission spectrum of SE1 in PBS buffer (10 mM, pH 7.4, 37 ℃) is a λ _fl for Compound 1 At 455 nm there was a gentle decrease and at 540 nm.

Referring to Figure 1 (b), Michaelis-Menten equation, depending on the speed, PLE- (specificity constant) can flight of SE1 for the catalyzed reaction, K _m = 4.33 ± 0.22 Μm one time, k _cat / K _m = 2.1 x 10 ⁵ M ^{- 1} s ^-1 . At this time, the value of the emergency number is more than 100 times greater than the value of the deacetylated trimethyllock including the rhodamine derivative.

In addition, the activity of SE1 by human CE1 (hCE1) and CE2 (hCE2) under similar conditions was very similar as shown in Fig. 1 (c).

To demonstrate that SE1 is a reliable CE substrate, the selectivity of SE1 was measured under various conditions. 2 is as for the selectivity of the test SE1, FIG. 2 (a) is a _{ROS (H 2 O 2, TBHP} , OCl -, O 2-, NO, ˙OtBu, ˙OH, ONOO -), amino acids (Cys, Glu (Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ ), glucose, AChE, BChE, Gly, Val, Thr, Tyr, Trp, Ser, Phe, Met, Leu, Ile, Asp, Lys, , Human plasma, hCE1, and hCE2. Bar shows the fluorescence ratio (F _yellow / F) integrated for 0 hours to 2 hours after addition of each species _blue .

Referring to FIG. 2 (a), SE1 is unaffected by other metabolites including various ROS, amino acids, sugars, and metal ions, and includes AChE, BChE, and paraoxonase ("PON") And exhibited excellent selectivity to CE over human serum.

Figure 2 (b) shows the effect of various inhibitors of the homogenized HepG2 cells (donepezil hydrochloride monohydrate, donepezil hydrochloride, doninenin, the PON1 inhibitor, lead acetate, and CE inhibitor, the AChE inhibitor, Bis (4-nitrophenyl) phosphate), and bar showed relative fluorescence intensity after 15 minutes of incubation with each of the above inhibitors.

Referring to FIG. 2 (b), while the other inhibitors of AChE, BChE, PON1, and PON2 showed minimal inhibition in the enzymatic reaction, the SE1 enzyme reaction after incubation with BNPP, a well known inhibitor for CE, .

Figure 3 shows TPM images of HepG2 cells cultured for 30 min with (a) SE1 (2 [mu] M) and (d) Compound 1 (2 [mu] M) (BNPP, 500 μM) and 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, 2 mM) were pre-treated in HepG2 cells. As a result, BNPP (F _yellow / F _blue ) in HepG2 cells reacted with SE1 by pretreatment with AEBSF decreased to about 50% and 25%, respectively.

Referring to FIGS. 4 (a) and 4 (c), CE1 and CE2 are two major CE isotypes well known in liver tissues. They are isolated from hepG2 cell lysate by immunoprecipitation using antibodies corresponding to CE1 and CE2 CE1 and CE2 can be selectively removed and confirmed by Western blot analysis.

4 (b) and FIG. 4 (d), SE1 fluorescence emission ratio (F _yellow / F _blue ) in HepG2 cell lysate following CE elimination was higher than CE1 and CE2 Respectively, by 45% and 47%, respectively.

Because CE1 and CE2 are known to be highly expressed and localized in the endoplasmic reticulum (ER), a high proportion of intracellular relevance within HepG2 cells is an issue, and the well known cell organelles, the variable bodies between the endoplasmic reticulum, mitochondria and lysosomes The images were compared as shown in FIG.

5, the excitation wavelengths for the TPM and OPM are 740 nm and 552 nm, respectively, and the scale bars are (a, d) = 19 μm and (g) = 23 μm and Pearson's simultaneous localization Because the Pearson's colocalization coefficients (A) for ER, mitochondria, and lysosomal trekker are 0.78, 0.23, and 0.09, respectively, SE1 images and ER trekker images are in good agreement, whereas mitochondria and lysosomal trekers do not I could not see.

From the above results, it can be concluded that SE1 is a selective substrate of CE in the cell, consistent with the CE activity of HepG2 cells mainly located in ER.

Referring to Figures 6 (a) and 6 (b), TPM images of SE1-labeled rat liver tissue clearly indicated the distribution of CE activity through individual cells at a thickness of about 300 [mu] m.

Referring to FIG. 6 (e), it was found that the average emission ratio (F _yellow / F _blue ) in the liver tissue was 1.04, which is considerably higher than the average emission ratio (F _yellow / F _blue ) have.

6 (c) to 6 (e), after the BNPP treatment for 1 hour, the value of the average emission ratio decreased to 0.85. From this data it can be seen that SE1 can selectively monitor CE activity deep inside the tissue using variable color TPM imaging. The image was obtained at a thickness of about 300 μm using a 740 nm excitation source and 420470 nm (F _blue ) and 520570 nm (F _yellow ) emission window. The scale bar shows (a, c) = 48 μm and (b, d) = 19 μm and (a, c) shows the average emission ratio (F _yellow / F _blue ).

Thus, SE1 is a new two-photon probe that quantitatively detects hCE activity in liver cells and liver tissue. The probe exhibits a considerable two-photon cross-sectional area, resulting in an emission color change from the labeled blue to yellow corresponding to hCE and is easy to carry out into the cell, high in physiological pH and high in the physiological range without affecting other metabolites including ROS and RNS Light stability and low cytotoxicity. In addition, the variable color TPM image using SE1 is an effective tool for accurately detecting hCE at intracellular level in living tissue.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is to be understood that various modifications and changes may be made without departing from the scope of the appended claims.

Claims (10)

A two-photon fluorescence probe represented by the following formula (1)
[Chemical Formula 1]

Wherein R ₁ is C ₁ to C ₄ alkyl, C ₁ to C ₄ alkoxy, halogen, or aryl,
R ₂ or R ₃ , equal to or different from each other, are hydrogen or methyl. The method according to claim 1, wherein the two-photon fluorescent probe is R ₁ is selected from methyl or a substituted or unsubstituted phenyl, two-photon fluorescent probe, characterized in that R ₂ and R ₃ are each methyl. The two-photon fluorescence probe according to claim 1 or 2, wherein the two-photon fluorescence probe is selectively sensitive to a carboxyl esterase. Injecting a two-photon fluorescent probe selectively responsive to a carboxylesterase represented by Formula 1 in vivo;
Wherein the two-photon fluorescence probe reacts with the in vivo carboxyl esterase to show fluorescence; And
And observing the fluorescence with a variable color two-photon microscope. A method of imaging a carboxylesterase comprising:
[Chemical Formula 1]

Wherein R ₁ is C ₁ to C ₄ alkyl, C ₁ to C ₄ alkoxy, halogen, or aryl,
R ₂ or R ₃ , equal to or different from each other, are hydrogen or methyl. [Claim 4] The method according to claim 4, wherein the two-photon fluorescence probe reacts with a carboxylesterase in vivo to change its color from 420 to 470 nm to yellow to 520 to 570 nm. / RTI &gt; 5. The method according to claim 4, wherein the carboxylesterase is detected in living tissue at a depth of 250 to 350 mu m. Treating the candidate drug and the two-photon fluorescent probe with a sample separated from the human;
Measuring the degree of activation of the carboxylesterase in the sample treated with the two-photon fluorescent probe; And
And selecting a candidate substance having increased degree of activation of the carboxylesterase as compared to a sample not treated with the candidate drug. [Claim 7] The method according to claim 7, wherein the carboxylesterase deficiency disease is any one of fatty liver, hyperlipidemia, and obesity. 8. The method of claim 7, wherein the sample is hepatocyte or liver tissue. [Claim 7] The method according to claim 7, wherein the degree of activation of the carboxylesterase is measured by a colorimetric assay, an immunoprecipitation method, or a Western blotting method.

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