WO2016108316A1 - Two-photon fluorescent probe, preparation method therefor, and ph imaging method using same - Google Patents

Abstract

The present invention relates to a two-photon fluorescent probe for imaging pH. The two-photon fluorescent probe of the present invention senses the pH in an acidic region and a variation thereof on the basis of a benzimidazole-structured derivative. The probe has an intrinsic isotropic point, low toxicity and high photostability, and can measure the pH in thousandths of a pH unit while exhibiting a color change of two-photon fluorescence at a pH of 4-7. According to the present invention, the two-photon fluorescent probe is selectively dyed inside living cells and biotissue and simultaneously exhibits a color change (blue-green) of strong fluorescence by a reaction with an acidic pH. In addition, the two-photon fluorescent probe can be easily loaded on cells due to a high water solubility and a low molecular weight. Additionally, the distribution of pH in biological cells or gentle biotissue and the activity thereof can be investigated by a ratio image since the pH can be selectively detected in biological cells and biotissue of a depth of 100-200 μm for at least 60 minutes. Therefore, the present invention can greatly contribute to pH-associated life science research, the early diagnosis of diseases, the development of a diagnostic reagent and a therapeutic agent, and the like since the pH can be quantitatively analyzed and different samples can be compared and analyzed.

Description

Two-photon fluorescent probe, preparation method thereof, and PH imaging method using the same

The present invention relates to a two-photon fluorescent probe for imaging the pH, and more particularly, to a two-photon fluorescent probe for measuring and imaging the pH by quantifying the concentration of hydrogen ions in the acidic region in the cell or cell tissue, a method of manufacturing the same and pH using the same It relates to an imaging method.

Numerous cellular metabolisms, including endoctic processes, signaling, cell death and defense, are associated with acidic environments. The acidic environment activates the function of enzymes and the breakdown of proteins. In eukaryotic cells, acidic organs such as lysosomes (pH 4.5-5.5) and endosomes (pH 4.5-6.8) contain many enzymes and proteins with various activities and functions. On the other hand, when these organs have a neutral pH, the function of the cells is impaired, causing many diseases such as cancer and degenerative diseases. Thus, to understand the role of the acidic environment physiologically and pathologically, there is a need to measure intracellular pH values at the cellular, tissue and organ level.

To understand this function, numerous single-photon fluorescent probes, such as green fluorescent proteins (GFPs) or Lyso Sensor DND-160, have been developed, but have a shallow transmission depth (<100 nm) due to the short excitation wavelength (<500 nm). Μm), photobleaching and cell autofluorescence, etc., limiting the application to tissue imaging. A solution to this problem is two-photon microscopy (TPM). Two-photon microscopy offers several advantages over single-photon microscopy because it uses two near-infrared photons with low excitation energy as excitation sources, specifically with increased transmission depth, localized excitation, and long-term imaging. have.

Recently, studies have been reported that a variable color two-photon fluorescent probe containing 2-aminoflorene can be used in a region close to pH 7 (Non-Patent Document 1). In addition, small molecule two-photon fluorescence probes such as DND-160 have been reported to measure pH in living tissues through variable color two-photon fluorescence microscopy. However, the two-photon fluorescent probe comprising pyridine as a proton binding site has a problem in that the fluorescence expression rate is low, especially in the acidic region, to 0.1 or less. In addition, in order to measure the pH of the acidic region, the two-photon fluorescent probe must have a pKa value at 4.5 to 6.5 pH. There is also the inconvenience of having to be able to easily modify it to bind the target material to measure the pH in a particular organelle, and to find a dye that is sensitive to pH.

[Preceding technical literature]

[Non-Patent Documents]

Yao, S .; Schafer-Hales, K. J .; Belfield, K. D. Org. Lett. 2007, 9, 5645.

Therefore, the first problem to be solved by the present invention is the pH of the acidic region in the cell, in particular the acid organelle of the endosomes or lysosomes present in living cells and selectively located inside and outside the oligomer or protein, so that the pH can be directly and in real time. It is to provide a two-photon fluorescent probe to measure and image it concretely and clearly.

In addition, a second problem to be solved by the present invention provides a method for manufacturing the two-photon fluorescent probe.

In addition, the last problem to be solved by the present invention provides a method for quantifying the intracellular pH by the two-photon fluorescence microscope using the two-photon fluorescence probe to image.

The present invention provides a two-photon fluorescent probe represented by the following formula (1) to in order to solve the above problems:

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

Wherein X ₁ and X ₂ are each independently H, F, Cl, Br, OCH ₃ , OH, NH ₂ , N (CH ₃ ) _2, NHCH _3, NCS, or CO ₂ H, and R ₁ and R ₂ Are each independently H, CH ₃ , CH ₂ (CH ₂ ) _n CH ₃ , CH ₂ (CH ₂ ) _n CO ₂ H, CH ₂ (CH ₂ ) _n OH, or CH ₂ (CH ₂ ) _n SO ₃ H, n is an integer of 0-10, and the same also applies below.

In addition, the present invention provides a method for producing a two-photon fluorescent probe represented by the formula (1), comprising the step of mixing and reacting a compound represented by the formula (A), a compound represented by the formula (B) and p-toluenesulfonic acid monohydrate It is characterized by.

[Formula A]

[Formula B]

Wherein X ₁ and X ₂ are each independently H, F, Cl, Br, OCH ₃ , OH, NH ₂ , N (CH ₃ ) _2, NHCH _3, NCS, or CO ₂ H, and R ₁ and R ₂ Are each independently H, CH ₃ , CH ₂ (CH ₂ ) _n CH ₃ , CH ₂ (CH ₂ ) _n CO ₂ H, CH ₂ (CH ₂ ) _n OH, or CH ₂ (CH ₂ ) _n SO ₃ H, which is also the same below.

In addition, the present invention provides a method for producing a two-photon fluorescent probe represented by the formula (2) comprising the following steps:

(a) mixing a compound represented by the following Formula B, a compound represented by the following Formula C, and p-toluenesulfonic acid monohydrate to react to prepare a compound represented by the following Formula D;

(b) preparing a compound represented by the following Chemical Formula E by mixing and reacting the compound represented by Chemical Formula D of step (a) with an aqueous LiOH solution; And

(c) reacting the compound represented by the formula (E) of step (b) with N, N-dimethylethylenediamine.

[Formula B]

[Formula C]

[Formula D]

[Formula E]

Wherein X ₁ and X ₂ are each independently H, F, Cl, Br, OCH ₃ , OH, NH ₂ , N (CH ₃ ) _2, NHCH _3, NCS, or CO ₂ H, and R ₁ and R ₂ Are each independently H, CH ₃ , CH ₂ (CH ₂ ) _n CH ₃ , CH ₂ (CH ₂ ) _n CO ₂ H, CH ₂ (CH ₂ ) _n OH, or CH ₂ (CH ₂ ) _n SO ₃ H, which is also the same below.

The present invention also provides a method of imaging the intracellular pH using the two-photon fluorescent probe.

The imageable depth may be 100-200 μm.

The cells may be living cells, organelles or cellular tissues of pH 4-7.

The two-photon fluorescent probe according to the present invention selectively stains inside living cells and biological tissues and simultaneously reacts with protons to show strong color change in fluorescence. Due to its high solubility in water and its low molecular weight, it can be easily loaded into cells and can also selectively detect pH in living cells and in 100-200 μm deep tissues over a time period of 60 minutes or more. The distribution and activity of pH within can be observed by imaging through a two-photon microscope. This enables quantitative analysis of pH and comparative analysis of different samples, which can greatly contribute to pH-related life science research, early diagnosis of disease, and development of diagnostic reagents and therapeutics.

Figure 1 (a) and (b) is the intensity of the pH change of the photon fluorescent spectrum of BH1 (a) and BH1L (b) in the standard buffer, (c) is BH1, BH2, BH3 in the photon mode And pH of BH1L versus I _green / I _iso (excitation wavelength is 360 nm).

(A) is a two-photon activity spectrum of BH1 in standard buffers (pH 3.5, 7.2 and 10), (b) is a graph showing the change in two-photon excitation fluorescence spectrum of BH1 with pH, and (c) is a two-photon mode in a graph showing the pH versus _green I / I _iso of BH1, BH2, BH3 and BH1L (excitation wavelength is 470 ㎚).

Figure 3. Survival graph of HeLa cells through MTS (Micro Technology Services) analysis. (a) BH1, (b) BH2, (c) BH3, and (d) BH1L.

Figure 4 (a) is a two-photon excitation fluorescence spectrum of ion-transmitter-Hell cells treated with BH1 3 μM, (b) is a two-photon I _green / I _IR titration curve according to the pH of the HeLa cells, (c) is Variable color TPM image of HeLa cells treated with BH1 3 μM, (d) is an enlarged photograph (arrow number is pH of measurement site, scale bar is (c) 30 μm, (d) 5 μm).

5. (a) and (b) are two-photon fluorescence microscope (a) and one-photon fluorescence microscope (b) images of HeLa cells treated with BH1L and LysoTracker Red. (c) is a composite photograph of (a) and (b) (excitation wavelength is 740 nm in a two-photon fluorescence microscope, 543 nm in a one-photon fluorescence microscope, 20 micrometers in a scale bar).

Figure 6 (a) is a two-photon excitation fluorescence spectrum of ion permeate-Hell cells treated with BH1 3 μM. (b) is two-photon I _green / I _IR titration curves according to pH of HeLa cells. (c) and (d) are variable color TPM images (c) and enlarged photographs (d) of HeLa cells treated with BH1 3 μM. (The number of arrows is the pH of the measurement site, the scale bar is 30 μm in (c), (d) 5 μm) (c) and (e) are variable color TPM images of HeLa cells treated with BH1 3 μM ( c) is before NH ₄ Cl addition and (e) is after addition. (d) and (f) are composite pictures corresponding to DIC images. (g) is an enlarged photograph of (c) showing the pH change with time (excitation wavelength 740 nm, scale bar is 20 micrometers in (c), 8 micrometers in (g)).

7. Hippocampal sections of rats Pseudocolored Ratiometric two-photon fluorescence microscopy images. (a) and (b) were treated with BH1L, and (c) and (d) were treated with BH1. (a) and (c) are 120 TPM images accumulated to visualize the pH along the Z-direction at a penetration depth of 90-180 μm. (b) and (d) 63 times magnification of the DG region (excitation wavelength is 740 nm, scale bar is 300 μm in (a) and (c), and 47 μm in (b) and (d)).

8. Schematic diagram showing the change in fluorescence color of the two-photon fluorescent probe and the image showing the intracellular pH.

Figure 9 is a graph showing the absorption intensity (right) according to the photon spectra (left) and probe concentration in standard buffer (pH 7.2, 3 mL). (a) and (b) are BH1, (c) and (d) are BH2, (e) and (f) are BH3, (g) and (h) are BH1L and (i) and (j) are P1 .

10. I photon (a, d and g), two photon (b, e and h) fluorescence spectra according to standard buffer pH and I _green / I _iso plots (c, f and i) according to pH. (a), (b) and (c) are BH2, (d), (e) and (f) are BH3, (g), (h) and (i) are BH1L. The excitation wavelength is (a), (d) and (g) is 360 nm, (b), (e) and (h) is 740 nm, and the results at (c), (f) and (i) are maximum. Standardized at I _green / I _iso .

Figure 11. Photon absorption (a) and emission (b) spectra of P1 in standard buffers (pH 3.5, 7.2 and 10).

12. Two-photon activity spectra of BH2, BH3 and BH1L in standard buffers (pH 3.5, 7.2 and 10). (a) is BH2, (b) is BH3 and (c) is BH1L (the error rate of the two-photon active cross-sectional area δΦ is 15%).

13. (a), (c), (e) and (g) are TPM images of HeLa cells treated with BH1 (a), BH2 (c), BH3 (e), and BH1L (g). (b), (d), (f) and (h) are relative two-photon excitation fluorescence (TPEF; two-photon) over time in the AC region shown in (a), (c), (e) and (g). excited fluorescence) Intensity is a graph showing intensity intensity (numerical intensity measured every 2 seconds for 1 hour in xyt mode, and two-photon excitation fluorescence was collected in femtosecond pulses at 400-600 nm with an excitation wavelength of 740 nm Scale bar is 75 μm).

14. Two-photon excitation fluorescence spectra obtained from HeLa cells treated with BH1 (a) and BH1L (b).

15. TPM images of HeLa cells treated with BH1 3 μM. Two-photon excitation fluorescence was collected as femtosecond pulses at (a) 440-460 nm ( I _IR ) and (b) 500-550 nm ( I _green ) when the excitation wavelength was 740 nm. (c) is pseudocolored variable color two-photon fluorescence microscopy image ( I _green / I _IR ) (scale bar is 30 μm).

Figure 16. TPM images of HeLa cells treated with BH1L before (a, b and c) and after (e, f and g) addition of NH ₄ Cl. Two-photon excitation fluorescence at femtosecond pulses at excitation wavelength 740 nm, (a) and (e) at 440-460 nm ( I _IR ), and (b) and (f) at 500-550 nm ( I _green ) Collected.

17. Real-time variable color two-photon fluorescence microscopy images ( I _green / I _IR ) of HeLa cells treated with BH1L before (a) and after (b) addition of NH ₄ Cl (scale bar is 8 μm).

18. TPM image of rat hippocampal sections. (a), (c) and (e) were treated with BH1L for 1 hour, and (b), (d) and (f) were treated with BH1 for 1 hour. Two-photon excitation fluorescence is collected with femtosecond pulses at 440 nm excitation wavelength, (a) and (b) at 440-460 nm ( I _IR ), (c) and (d) at 500-550 nm ( I _green ) It became. 120 TPM images were accumulated to visualize the pH along the Z-direction at a penetration depth of 90-180 μm.

19. TPM image of rat hippocampal sections. (a), (b) and (c) were treated with 30 μM BH1L for 1 hour, and (d), (e) and (f) were treated with BH1 for 1 hour. Two-photon excitation fluorescence at femtosecond pulses at excitation wavelength 740 nm, (a) and (b) at 440-460 nm ( I _IR ), and (c) and (d) at 500-550 nm ( I _green ) Collected. (c) and (f) are pseudocolored variable color two-photon fluorescence microscopy images ( I _green / I _IR ) (scale bar is 47 μm).

The present invention relates to a two-photon fluorescence probe for measuring the pH in the cell, and because it uses a low energy excitation source can be measured by quantifying the real-time pH of the cell in real time without destroying the cell.

Histidine is an essential amino acid containing imidazole groups that have buffering capacity in the living body, and benzimidazole is a derivative of imidazole having about 5.5 pKa in acidic organelles. In the present invention, the benzimidazole group is used as a proton acceptor, and is introduced at position 6 of 2-aminonaphthalene, which is a phosphor, to change the pH 4-7 region in the cell through color change (blue-green) according to the reaction with hydrogen ions. It provides a two-photon fluorescent probe that can be quantitatively measured and monitored in 0.001 pH units.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present invention provides a two-photon fluorescent probe represented by the following Chemical Formulas 1 to 6.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

Wherein X ₁ and X ₂ are each independently H, F, Cl, Br, OCH ₃ , OH, NH ₂ , N (CH ₃ ) _2, NHCH _3, NCS, or CO ₂ H, and R ₁ and R ₂ Are each independently H, CH ₃ , CH ₂ (CH ₂ ) _n CH ₃ , CH ₂ (CH ₂ ) _n CO ₂ H, CH ₂ (CH ₂ ) _n OH, or CH ₂ (CH ₂ ) _n SO ₃ H, n is an integer of 0-10, and the same also applies below.

The two-photon fluorescent probe represented by Chemical Formula 1 may image pH while reacting with hydrogen ions in an internal pH 4-7 region of a cell, a cell organelle, or a living tissue to exhibit fluorescence. The probe has a pKa 4-6.5 range by substituting an electron donor or electron acceptor with a benzimidazole group. Figure 8 is a schematic diagram showing the fluorescence color change of the two-photon fluorescent probe according to an embodiment of the present invention and the image showing the intracellular pH observed through a two-photon fluorescence microscope. When the cells are irradiated with a low excitation wavelength (740 nm), the two-photon fluorescence probe is protonated to cause a color change from blue to green, and the two-photon fluorescence microscope can observe the change and quantify the pH.

In addition, the two-photon fluorescent probe represented by Formula 2 and Formula 3 introduces a tertiary amine group to react with protons in an acidic organ to form a quaternary amine salt, and has about pKa 10. In particular, it can be selectively positioned inside the endosome or lysosome to sense the pH.

In addition, the two-photon fluorescent probe represented by Chemical Formulas 4 to 6 may introduce a maleimide group to selectively covalently bond with a thiol group of a biomolecule or a succinimdyl ester group to selectively introduce an amine group into a covalent bond. By selectively labeling oligomers or proteins, pH can be measured.

As can be seen in Figure 2a, a graph showing the two-photon active cross-sectional area (δΦ) versus the excitation wavelength of a two-photon fluorescent probe according to an embodiment of the present invention in buffers of pH 3.5, 7.2 and 10. It can be seen that the cross-sectional area is the largest at pH 3.5, which means that the two-photon fluorescent probe of the present invention has high pH measuring activity in the acidic region.

FIG. 2C is a calibration graph showing an isotropic point ( I _green ) reference emission ( I _iso ) ratio ( I _green / I _iso ) according to pH of a two-photon fluorescent probe according to an embodiment of the present invention. As shown in FIG. 2C, when plotting the plots, the plots show linearity at pH 4.5 to pH 7, which can be obtained from the pH 4.5 to pH 7 region representing a linear section through I _green / I _iso . Means.

Two-photon fluorescence probes of the present invention exhibit high fluorescence intensity two-photon emission spectra for regions with pH 4-7 in cells (FIGS. 4, 6). Thus, pH can be imaged in real time in the pH 4-7 region of the cell. More preferably, the pH can be imaged by quantitatively measuring in real time in the pH 4.5-7 region.

According to an embodiment of the present invention, the present invention is a two-photon fluorescent probe represented by the formula (1) prepared by mixing the compound represented by the formula (A), the compound represented by the formula (B) and p-toluenesulfonic acid monohydrate It provides a method of manufacturing.

[Formula A]

[Formula B]

Wherein X ₁ and X ₂ are each independently H, F, Cl, Br, OCH ₃ , OH, NH ₂ , N (CH ₃ ) _2, NHCH _3, NCS, or CO ₂ H, and R ₁ and R ₂ Are each independently H, CH ₃ , CH ₂ (CH ₂ ) _n CH ₃ , CH ₂ (CH ₂ ) _n CO ₂ H, CH ₂ (CH ₂ ) _n OH, or CH ₂ (CH ₂ ) _n SO ₃ H, which is also the same below.

According to another embodiment of the present invention, the present invention provides a method for producing a two-photon fluorescent probe represented by the formula (2) comprising the following steps:

(a) preparing a compound represented by the following formula (D) prepared by mixing and reacting a compound represented by formula (B), a compound represented by formula (C), and p-toluenesulfonic acid monohydrate;

(b) preparing a compound represented by the following Chemical Formula E by mixing and reacting the compound represented by Chemical Formula D of step (a) with an aqueous LiOH solution; And

(c) reacting the compound represented by the formula (E) of step (b) with N, N-dimethylethylenediamine.

[Formula B]

[Formula C]

[Formula D]

[Formula E]

Wherein X ₁ and X ₂ are each independently H, F, Cl, Br, OCH ₃ , OH, NH ₂ , N (CH ₃ ) _2, NHCH _3, NCS, or CO ₂ H, and R ₁ and R ₂ Are each independently H, CH ₃ , CH ₂ (CH ₂ ) _n CH ₃ , CH ₂ (CH ₂ ) _n CO ₂ H, CH ₂ (CH ₂ ) _n OH, or CH ₂ (CH ₂ ) _n SO ₃ H, which is also the same below.

Since the two-photon fluorescent probes represented by Chemical Formulas 1 to 6 of the present invention irradiate the cells with a low energy excitation wavelength (740 µm), the distribution of pH is selectively distributed to living cells and biological tissues of 100-200 µm depth without cell destruction. It can be seen and measured in real time for more than 60 minutes, so the pH change and activity can be imaged through a two-photon fluorescence microscope.

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

<Example>

The two-photon fluorescent probe represented by Chemical Formula 1 of the present invention can be prepared as shown in Scheme 1 below, but is not limited thereto.

Scheme 1

The following compound 1 was prepared by the method described in Document 1 (Document 1: Kim, HM; Jeong, BH; Hyon, Ju-Y .; An, MJ, Seo, MS; Hong, JH; Lee, KJ; Kim, CH Joo, T. Hong, Seok-C. Cho, BRJ Am. Chem. Soc. 2008, 130, 4246).

Example 1. Preparation of Two-photon Fluorescent Probe (BH1)

0.35 g, 1.9 mmol, o-phenylenediamine (0.31 g, 22.9 mmol) and p-tolunesulfonic acid monohydrate (0.072 g, 0.38 mmol) were added to 10 mL of DMF. The mixture was stirred for 6 hours under nitrogen atmosphere. After cooling to room temperature, 50 mL of water was added to the mixture and filtered. The precipitate was washed with distilled water and dried in vacuo. The crude product was purified by column chromatography (silica gel) using EtOAc / hexane (ethylene acetate / hexane, 1: 1 v / v) as eluent to give a light brown solid. Hereinafter referred to as BH1.

Yield: 0.16 g (31%); Melting point: 214-216 ° C .; ¹ H NMR (d6-DMSO): δ 12.82 (br s, 1 H), 8.46 (s, 1 H), 8.09 (d, J = 8.8 Hz, 1 H), 7.72-7.70 (m, 2 H), 7.56 (br s , 2H), 7.18-7.16 (m, 2H), 7.02 (dd, J = 8.8, 1.6 Hz, 1H), 6.70 (d, J = 1.6 Hz, 1H), 6.27 (d, J = 4.8 Hz, 1H) . 2.80 (d, J = 4.8 Hz, 3H). ¹³ C NMR (100 MHz, d6-DMSO): δ 151.9, 148.6, 135.8, 128.9, 125.7, 125.6, 123.9, 122.3, 121.6, 118.7, 101.4, 29.6 ppm; HRMS (FAB ⁺ ): m / z calcd for [C ₁₈ H ₁₅ N ³⁺ H ⁺ ]: 274.1344, found: 274.1343.

Example 2. Preparation of Two-photon Fluorescent Probe (BH2)

4,5-difluoro-1,2-phenylenediamine was used instead of o-phenyyenediamine, and a light yellow solid was obtained. Hereinafter referred to as BH2.

Yield: 33%; Melting point: 237-240 ° C .; ¹ H NMR (d6-DMSO): δ 13.01 (br s, 1H), 8.43 (s, 1H), 8.04 (d, J = 8.8 Hz, 1H), 7.71-7.69 (m, 2H), 7.60 (br s , 2H), 7.02 (d, J = 8.8, 1H), 6.70 (s, 1H), 6.29 (d, J = 4.8 Hz, 1H), 2.80 (d, J = 4.8 Hz, 3H). ¹³ C NMR (100 MHz, d6-DMSO): δ 153.9, 148.7, 147.6, 147.5, 145.1, 136.0, 128.9, 125.8, 125.7, 125.6, 123.7, 121.7, 118.8, 101.4, 29.6 ppm; HRMS (FAB ⁺ ): m / z calcd for [C ₁₈ H ₁₃ N ₃ F ²⁺ H ⁺ ]: 310.1156, found: 310.1156.

Example 3. Preparation of Two-photon Fluorescent Probes (BH3)

4,5-dimethoxy-1,2-phenylenediamine was used instead of o-phenyyenediamine, and a pale yellow solid was obtained. Hereinafter referred to as BH3.

Yield: 36%; Melting point: 235-238 ° C .; ¹ H NMR (d6-DMSO): δ 12.56 (br s, 1 H), 8.34 (d, J = 1.6 Hz, 1 H), 8.02 (dd, J = 8.8, 1.6 Hz, 1 H), 7.69-7.67 (m, 2H), 7.09 (br s, 2H), 7.00 (dd, J = 8.8, 1.6 Hz, 1H), 6.69 (d, J = 1.6 Hz, 1H), 6.20 (d, J = 4.4 Hz, 1H), 3.81 (s, 6H), 2.80 (d, J = 4.4 Hz, 3H); ¹³ C NMR (100 MHz, d6-DMSO): δ 150.4, 148.3, 146.2, 135.5, 128.8, 125.8, 125.7, 124.7, 123.7, 122.9, 118.7, 101.5, 55.9, 29.7 ppm; HRMS (FAB ⁺ ): m / z calcd for [C ₂₀ H ₁₉ O ₂ N ³⁺ H ⁺ ]: 334.1556, found: 334.1555.

Example 4. Preparation of Two-photon Fluorescent Probe (BH1L)

Two-photon fluorescent probe represented by the formula (2) can be prepared as shown in the following schemes 2 to 4, it is not limited thereto.

Scheme 2

Scheme 3

Scheme 4

Compound 2 was prepared by the method described in Document 2 (Document 2: Masanta, G .; Lim, CS; Kim, HJ; Han, JH; Kim, HM; Cho, BRJ Am. Chem. Soc. 2011, 133, 5698).

Compound 3 was the same as Example 1, except that Compound 2 was used instead of Compound 1, and a pale yellow solid was obtained at a yield of 51%.

Yield: 51%; Melting point: 163-165 ° C .; ¹ H NMR (d6-DMSO): δ 12.87 (br s, 1 H), 8.54 (s, 1 H), 8.16 (d, J = 8.8 Hz, 1 H), 7.84 (d, J = 9.2 Hz, 1 H), 7.79 (d, J = 8.8 Hz, 1H), 7.60 (br s, 2H), 7.23-7.19 (m, 3H), 7.00 (s, 1H), 4.40 (s, 2H), 3.65 (s, 3H), 3.12 (s, 3H); ¹³ C NMR (100 MHz, d6-DMSO): δ 170.6, 151.7, 147.6, 135.0, 129.2, 126.3, 125.6, 125.4, 124.0, 123.3, 116.0, 105.4, 53.1, 51.6, 39.0 ppm.

Compound A dissolved Compound 3 (0.03 g, 0.91 mmol) in 5 mL of THF and added 3 mL of an aqueous LiOH (0.22 g, 9.2 mmol) solution through a cannula. The reaction mixture was stirred at 25 ° C for 6 h. The solvent was evaporated and 5 mL of distilled water was added to the mixture. The mixture was acidified with 37% HCl solution until pH was 3. The precipitate was purified by filter, washed with distilled water, diethyl ether and dried in vacuo. Compound A was obtained as a yellow solid without further purification.

Yield: 0.27 g (90%); Melting point: 205-208 ° C .; ¹ H NMR (d6-DMSO): δ 12.80 (br s, 1H), 8.51 (s, 1H), 8.12 (dd, J = 8.4, 1.6 Hz, 1H), 7.82 (d, J = 9.2 Hz, 1H) , 7.76 (d, J = 8.4 Hz, 1H), 7.58 (br s, 2H), 7.22-7.16 (m, 3H), 6.96 (d, J = 2.4 Hz, 1H), 4.24 (s, 2H), 3.11 (s, 3 H). ¹³ C NMR (100 MHz, d6-DMSO): δ 171.7, 151.8, 147.9, 135.2, 129.1, 126.3, 125.5, 124.0, 123.0, 121.7, 116.1, 105.1, 53.5, 39.1 ppm.

Compound A (0.2 g, 0.06 mmol), 1,3-dichlorohexyl carbodiimide (1,3-dicyclohexyl carbodiimide, 0.14 g, 0.66 mmol) and 1-hydroxybenzotriazole (0.089 g, 0.66 mmol) 10 mL CH ₂ Cl _{2 was} added and stirred at room temperature under nitrogen atmosphere for 1 hour. N, N-dimethylethylenediamine ((N, N-dimethyl) ethylenediamine, 0.058 g, 0.66 mmol) was added to the mixture, which was stirred for 12 hours under a nitrogen atmosphere. The precipitate was purified and concentrated under reduced pressure. The obtained product was filtered by column chromatography (silica gel, CHCl ₃ / MeOH (10: 1-4: 1)) to give a light yellow solid. Hereinafter, it is called BH1L.

Yield: 0.17 g (71%); Melting point. 235-240 ° C; ¹ H NMR (d6-DMSO): δ 12.84 (br s, 1 H), 8.52 (s, 1 H), 8.13 (dd, J = 8.4, 1.6 Hz, 1 H), 7.89 (br t, J = 6.0 Hz, 1 H , amide-NH), 7.83 (d, J = 9.2 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 7.2 Hz, 1H), 7.51 (d J = 7.2 Hz, 1H), 7.19-7.15 (m, 3H), 6.95 (d, J = 2.4 Hz, 1H), 4.06 (s, 2H), 3.18 (q, J = 6.4 Hz, 2H), 3.12 (s, 3H), 2.29 (t, J = 6.4 Hz, 2H), 2.13 (s, 6H). ¹³ C NMR (100 MHz, d6-DMSO): δ 169.0, 151.7, 147.9, 135.1, 129.0, 126.2, 125.5, 125.4, 124.0, 123.1, 116.2, 105.2, 58.1, 55.7, 45.1, 39.1, 36.6 ppm HRMS (FAB ⁺ ): m / z calcd for [C ₂₄ H ₂₇ 0 ₁ N ⁵⁺ H ⁺ ]: 402.2294, found: 402.2293.

Comparative Example 1. Preparation of a two-photon fluorescent probe containing a pyridine group (P1)

Two-photon fluorescent probe represented by the formula (P1) was prepared as shown in Scheme 5.

Scheme 5

Compound 4 (6-bromo-N-methyl-2-naphthylamine) was prepared by the method described in Document 1 (Document 1: Kim, HM; Jeong, BH; Hyon, Ju-Y .; An, MJ, Seo, MS; Hong, JH; Lee, KJ; Kim, CH; Joo, T .; Hong, Seok-C .; Cho, BRJ Am. Chem. Soc. 2008, 130, 4246).

Compound 4 (0.2 g, 0.85 mmol), 4-pyridinylborinic acid (0.16 g, 1.28 mmol), kiss (triphenylphosphine) palladium (0) (Tetrakis (triphenylphosphine) palladium (0), 0.093 g, 0.034 mmol ) Was dissolved in 10 mL N, N-dimethylacetamide. To the mixture was added 1 mL of an aqueous solution of Cs ₂ CO ₃ (0.55 g, 1.69 mmol) in vacuo. The mixture was degassed with nitrogen for 15 minutes and stirred at 120 ° C. for 18 hours under a nitrogen atmosphere. After cooling to room temperature, the mixture was filtered with 50 mL of water. The precipitate was washed with distilled water and dried in vacuo. The obtained product was recrystallized from CH ₂ Cl ₂ to give a light brown solid.

Yield: 0.11 g (55%); Melting point: 223-226 ° C .; ¹ H NMR (d6-DMSO): δ 8.60-8.59 (m, 2H), 8.15 (s, 1H), 7.77-7.69 (m, 5H), 7.00 (dd, J = 8.8, 2.0 Hz, 1H), 6.69 (s, 1 H), 6.19 (d, J = 4.8 Hz, 1H), 2.80 (d, J = 4.8 Hz, 3H). ¹³ C NMR (100 MHz, d6-DMSO): δ 149.8, 148.4, 147.0, 135.4, 128.9, 128.7, 126.1, 125.7, 124.0, 120.4, 118.6, 101.1, 29.6 ppm HRMS (FAB ⁺ ): m / z calcd for [C ₁₆ H ₁₄ N ²⁺ H ⁺ ]: 235.1236, found: 235.1235.

<Preparation and test method of material>

1. Spectroscope device

Absorption spectra were measured on S-3100 UV-Vis spectrometer and fluorescence spectra on FluoroMate FS-2 fluorescence spectrometer in 1 cm standard quartz cells. Fluorescence quantum efficiency was calculated as 9,10-diphenylanthracene (Φ = 0.93 in cyclohexane).

2. pH value

3 μL of a two-photon fluorescent probe in DMSO (dimethylsulfoxide, 1.0 × 10 ⁻³ M) was added to a cuvette containing 3 mL of standard solution. The pHs of the two-photon fluorescent probes, BH 1-3 and BH1L prepared according to one embodiment of the present invention were obtained by the following formula.

R is the observed ratio of isotropic point ( I _iso ) and 500-550 nm ( I _green ) at a given pH. R _max and R _min are the maximum and minimum limit values of R, and C is the slope. I _a / I _b is the fluorescence intensity ratio of pH 3.5 to pH 10 at the selected wavelength to find the denominator of R. In this case, the correction is extinguished using the correct isotropic point. To obtain the pKa value in the two-photon mode, the two-photon excitation fluorescence spectra were shown on a DDC meter (Monora 320 spectrograph set with Andor iDus DV401A-BV). Two-photon fluorescent probes are excited at a 740 nm wavelength, 2510 mW (200 mW equivalent in focus) on a model-locked titanium-sapphire laser light source device (Mai Tai HP, Spectra Physics, 80 MHz pulses per second, 100 fs pulse width) It became.

3. Measurement of two-photon fluorescence cross section

Two-photon cross-sectional area (δ) was measured using femto second (fs) fluorescence measurement. Probes (1.0 × 10 ⁻⁶ M) were dissolved in standard buffers (pH 3.5, 7.2 and 10), respectively, and two photons indicating fluorescent soils were measured using rhodamine 6G at 720-880 nm. Two-photon excited fluorescence spectral intensities of the reference and the sample were determined at the same excited wavelength. The cross section of the two-photon fluorescent probe was calculated using Equation 1 below.

[Equation 1]

δ = δr (SsФrfrcr) / (SrФsfscs)

In Equation 1, s and r represent the sample and reference molecules, S represents the signal intensity collected from the CCD detector, Ф represents the fluorescence quantum yield, f represents the total fluorescence collection efficiency of the experimental instrument, c Represents the molecular density in the solution and δr represents the cross-sectional area of the two-photon fluorescent probe of the reference molecule.

4. Cell Culture

HeLa cervical cancer cells (ATCC, Manassas, VA, USA) were treated with 10% FBS (WelGene), penicillin (100 units / mL) and streptomycin (100 μg / mL) in DMEM (WelGene Inc, Seoul, Korea). Incubated while feeding. Two days prior to imaging, cells were transferred to a glass bottom dish (NEST). All cells were grown in a humidified environment with an air / CO ₂ ratio of 95: 5 at 37 ° C. Growth medium was removed for labeling and changed to serum-free DMEM. The cells were injected with 3 μM probe and incubated at 37 ° C. for 30 minutes.

5. Two-Photon Fluorescence Microscopy (TPFM)

Microscopic images of two-photon fluorescent probes labeled with cells and tissues were used with spectral confocal and multiphoton microscopes (Leica TCS SP8 MP). Two-photon fluorescence probe microscopy images excite two-photon fluorescence probes to 2510 mW output at 740 nm wavelength using a model-locked titanium-sapphire laser light source device (Mai Tai HP, Spectra Physics, 80 MHz pulses per second, 100 fs pulse width) Observed by DMI6000B microscope (Leica). To obtain images in the 440-460 nm (IR) and 500-550 nm (green) ranges, internal PMTs were used to collect the signals. Ratiometric image processing and analysis was performed using MetaMorph software.

6. Cell pH Calibration

The pH calibration curve is represented by I _green / I _IR of HeLa cells treated with ion permeate carriers and BH1 or BH1L. Cells were incubated for 30 minutes at 37 ° C., 5% CO ₂ in DMSO stock containing 3.0 μL of 1 mM BH1 or 1 mM BH1L, and the extracellular media was 1 mL of calibration buffer (125 mM KCl, 20 mM NaCl, 0.5 mM CaCl ₂ , 0.5 mM MgCl ₂ , 5 μΜ nigericin, 5 μΜ monensin, and 25 mM buffer; acetates at pH 3.5, 4.0, 4.3, 5.0, 5.2; MES at pH 5.5, 6.0; pH 6.5, 7.0 , HEPES of 8.0). Cells were then treated in calibration buffer for 15-20 minutes at room temperature. Two-photon fluorescent probe intensities at 440-460 nm ( I _IR ) and 500-550 nm ( I _green ) of BH1 or BH1L vary with pH, plotting plots of pH vs. I _green / I _IR with pH calibration curves. Shown in

7. Light stability

The photostability of BH 1-3 and BH1L was shown by monitoring the change in two-photon excitation fluorescence intensity over time at three designated points in the probe-labeled HeLa cells (FIG. 13). Two-photon excitation fluorescence intensity showed high light stability while maintaining almost the same value for one hour.

8. Cytotoxicity

In order to measure the toxicity to HeLa cells of the two-photon fluorescent probe of the present invention, it is shown in Figure 3, using the MTS assay (Cell Titer 96H; Promega, Madison, WI, USA) method.

9. Preparation and Pigmentation of Rat Hippocampus Tissue

Hippocampus fragments were prepared from hippocampus of 2 week old rats. The coronal fragments were in artificial cerebrospinal fluid (ACSF; 138.6 mM NaCl, 3.5 mM KCl, 21 mM NaHCO ₃ , 0.6 mM NaH ₂ PO ₄ , 9.9 mM D-glucose, 1 mM CaCl ₂ , and 3 mM MgCl ₂ ). Was cut into a 400 μm thickness using a vibrating-blade microtome. The pieces were incubated with 10 mM BCa1 and 20 mM BH1 and BH1L in ACSF exhaling 95% O 2 and 5% CO ₂ for 40 minutes at 37 ° C. The fragments were then washed three times with ACSF and transferred to glass-bottomed dishes, which were observed by electron microscopy. Two-photon microscopic images were observed at a depth of 90-180 μm.

Test Example 1. Solubility Measurement

A certain amount of the two-photon fluorescent probe prepared in the above example was dissolved in DMSO (dimethyl sulfoxide, 1.0 × 10 ⁻² M) and stored. Dilute the solution from 6.0 × 10 ⁻³ to 6.0 × 10 ⁻⁵ and buffer solution ((0.1 M citric acid, 0.1 M KH ₂ PO ₄ , 0.1 M Na ₂ B ₄ O ₇ , 0.1 M Tris, 0.1 M KCl , pH 7.2) was added by micro syringe to a cuvette containing 3 mL The concentration of DMSO in the buffer solution was maintained at 0.2%.

9 is a graph showing the absorption rate according to the concentration of the two-photon fluorescent probe prepared according to the Examples and Comparative Examples of the present invention. Plots in the graph were linear at low concentrations and downward curves at higher concentrations.

The maximum point in the straight line means solubility. The solubility of Examples (BH1, BH2, BH3, BH1L) and Comparative Example (P1) of the present invention is 6, 2, 10, 10 and 2 μM, respectively, in pH 7.2 buffer solution, which is sufficient for cell staining. .

Test Example 2 Evaluation of pH, pKa and Mineral Physical Properties

PKa and photophysical experimental results of the two-photon fluorescent probes prepared in Examples and Comparative Examples are shown in Table 1 below. All measurements were performed in standard buffer solutions (0.1 M citric acid, 0.1 M KH ₂ PO ₄ , 0.1 M Na ₂ B ₄ O ₇ , 0.1 M tris (hydroxymethyl) aminomethane, 0.1 M KCl).

Table 1

b: Maximum wavelength of photon absorption spectrum in nm.

c: maximum wavelength of the photon emission spectrum in nm.

d: Fluorescence quantum yield.

e: pKa measured in daylight mode. In parentheses are pKa. Measured in two-photon mode.

f: Maximum wavelength of two-photon absorption spectrum in nm.

g: Peak two-photon cross-sectional area per photon at 10 ^-50 cm ⁴ s / photon in GM.

h: two photon action cross-section in 10 ^-50 cm ⁴ s, margin of error ± 15%.

(1) photophysical properties in daylight mode

At pH 7.2, BH1 exhibited maximum absorption at 337 nm and maximum fluorescence emission at 118 nm, indicating relatively high Stokes shift of 118 nm (Table 1). Almost similar results were obtained at pH 10. On the other hand, as the pH changed from 7.2 to 3.5, the maximum absorption and maximum fluorescence emission of BH1 shifted to the longer wavelengths of 368, 494 nm (FIG. 1A and Table 1). This red shift seems to be due to the cationization of nitrogen in the benzimidazole group. Thus, it enhances molecular charge transfer (ICT). This means that the fluorescence quantum efficiencies (Φ) of BH1 and BH1-H ⁺ have the highest values of 1 and 0.76, respectively, and are suitable as pH probes in biological buffers. In addition, an isomission point (474 nm) was observed between 455 nm and 494 nm, which is λ _fl . Similar results were seen with BH2 and BH3 (FIG. 10 and Table 1). As expected, the spectrum of BH1L also appeared almost identical to BH1 (FIG. 1B and Table 1). On the other hand, P1 showed a negligible fluorescence quantum efficiency (Φ = 0.008) at pH 2 and no fluorescence at pH 3.5. In contrast, the maximum absorption (λ _abs ) gradually shifted from 333 nm to 395 nm (Table 1 and FIG. 11). Thus, BH1 to BH3 showed the highest fluorescence quantum efficiency in the biobuffer and the emission color change from blue to green as the pH changed from neutral to acidic.

(2) pH and pKa

The pKa of the conjugate acid of BH1 to 3 was obtained from a titration curve of the isotropic point ( I _iso ) and the emission ratio ( I _green / I _iso ) of 500-550 nm ( I _green ). The pKa 4.92-6.11 range means that acidic regions can be detected. The pKa shift is due to electron withdrawing (F in BH2) or electron donating (OMe in BH3). In addition, the pKa value of BH1L was found to be almost the same as BH1.

(3) photophysical properties in two-photon mode

The pH measuring capability in the two-photon mode of the two-photon fluorescent probe of the present invention was evaluated. The two-photon fluorescence spectra (Φδ) of BH1 at pH 7.2 and 3.5 showed 40 and 140 GM, respectively (Table 1 and FIG. 2A). The maximum two-photon fluorescence spectrum (Φδ _max ) of BH1-H ⁺ with electron withdrawing groups is 3.5 times larger than BH1, which enhances molecular charge transfer (ICT) between the electron acceptor and electron donor. Similar results were seen for BH2 and its cations, with BH3 showing a maximum Φδ that was about 2 times smaller than BH1 (Table 1 and FIG. 12). In particular, BH1L and its cations showed a maximum Φδ that was twice as large as BH1 (Table 1 and FIG. 12). Perhaps this is due to the surrounding substituted functional groups. In addition, the properties of the two-photon excitation fluorescence (TPEP) spectrum of BH1 versus pH appeared similar to the results of the one-photon mode (Table 1 and Figure 2b, c), while the two-photon fluorescence emission ratio ( I _green / I _iso ). As the pH value changed from 7.2 to 3.5, it increased about 10 times, which is more than 2 times higher than the value obtained in daylight mode. Since Φδ _max of BH1-H ⁺ is three times larger than BH1 (FIG. 2A), these results indicate that it may contribute to larger two-photon excitation fluorescence enhancement at 500-550 nm ( I _iso ) compared to the one-photon mode. (FIGS. 1A and 2B). Similar results were seen with BH2, BH3 and BH1L (FIG. 10).

Test Example 3 Cytotoxicity Evaluation

Intracellular pH was measured by TPM using BH1. BH1 was injected into HeLa cells treated with ionopheres at 740 nm two-photon excitation (TP exctation). As a result, the two-photon emission fluorescence spectra were emitted at lambda _fl values of 445 and 490 nm, respectively. This value almost coincides with the buffer and shift values in and around the 10 nm difference. The emission spectrum of BH1 shows a gradual shift with increasing solvent polarity, which means that the probes of the invention are more homogeneous and hydrophobic in the intracellular buffer. The variable color imaging ( I _green / I _IR ) results were obtained using an internal reference window of 440-460 nm ( I _IR ) and a pH recognition window of 500-550 nm ( I _green ). It was obtained by (Fig. 4a). In addition, the imaging values ( I _green / I _IR ) of HeLa cells treated with BH1 appeared in the pH range of 5.6-7.4 across the various compartments in the cells (FIGS. 4D and S7). As shown in FIG. 4B, the slope of the graph is almost linear, which means that the pH can be quantified using variable color imaging ( I _green / I _IR ) in the pH 4-7 region.

Example 5. pH Imaging in Lysosomes

The pH in lysosomes was measured using BH1L, a lysosomal target two-photon fluorescent probe. To determine if BH1L is located at a specific site in the lysosomes, HELL cells were treated with Lysotracker Red DND-99 (LTR), also known as BH1L and lysosomal photon fluorescent probes, respectively. 5 is a two-photon fluorescence microscopy image (FIG. 5A) and a one-photon fluorescence microscopy image (FIG. 5B) and superimposed images (FIG. 5C) (Pearson colocalization coefficient: 0.095). In addition, BH1L was injected and imaged into HeLa cells treated with ion permeate carriers at pH 8 and 3.5. As a result, λ _fl emitted two-photon excited fluorescence spectra at 445 and 485 nm (FIG. 6A). This is almost the same as the result measured in the buffer (FIG. 10). Therefore, it can be seen that pKa of HeLa cells treated with ion transporter and BH1L is 5.63 ± 0.08 similar to that of the buffer.

Variable color imaging ( I _green / I _IR ) of HeLa cells treated with BH1L shows various pH values in lysosomes (FIG. 6). This means that BH1L can be used to measure the pH in each zone in the pH 4.6-5.9 range (Figure 16).

In addition, pH change in lysosomes was observed in real time. A weak base NH ₄ Cl 5 mL was added to slowly increase the pH in the lysosome and reached pH 6.5-6.7 within 4 minutes as the pH value started to rise (FIGS. 6g and S9). Most characteristicly, small waves were noticeably observed every 0.1 pH (FIG. 17). This means that BH1L can measure the pH value in lysosomes in living cells.

Example 6. Brain Tissue pH Imaging in Rats

Using the two-photon fluorescent probe of the present invention, the micro flakes of the hippocampus of rats, which are responsible for learning and memory, were monitored. Since the structure of the brain tissue is heterogeneous, 120 variable color imaging ( I _green / I _IR ) were measured at a depth of 90-180 μm (FIG. 18). To demonstrate the usefulness of the BH1L of the present invention in imaging of cells in tissues, micro flakes of the hippocampus of rats were monitored.

The overall pH distribution was seen in the acidic pH regions CA1, CA3 and the dentate gyrus (DG) zone (FIG. 7A). In particular, the distinction between the DG and CA regions was well demonstrated in terms of cell types and functions responsible for neuronal and gene expression. Acidic pH in DG appears to be related to cellular metabolic processes in this zone, indicating the need for research to investigate biological function. In addition, high magnification images can clearly show the acidic portion of each cell in the DG at a depth of penetration of about 100 μm (FIGS. 7B and S11). Similar results were observed in tissues treated with BH1 (FIG. 7C, d).

Thus, the above experimental results indicate that the two-photon fluorescent probe of the present invention can clearly measure the pH value of the acidic compartment in the cells in living cells and tissues using two-photon microscopy.

Claims (12)

Two-photon fluorescent probe represented by the formula 1 to 6: [Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

Wherein X ₁ and X ₂ are each independently H, F, Cl, Br, OCH ₃ , OH, NH ₂ , N (CH ₃ ) _2, NHCH _3, NCS, or CO ₂ H, and R ₁ and R ₂ Are each independently H, CH ₃ , CH ₂ (CH ₂ ) _n CH ₃ , CH ₂ (CH ₂ ) _n CO ₂ H, CH ₂ (CH ₂ ) _n OH or CH ₂ (CH ₂ ) _n SO ₃ H, where n is an integer from 0-10. The two-photon fluorescent probe of claim 1, wherein the two-photon fluorescent probe reacts with hydrogen ions in a pH 4-7 region of a cell, organelle, or living tissue to fluoresce. The two-photon fluorescent probe of claim 1, wherein the two-photon fluorescent probes represented by the formulas (2) and (3) react with hydrogen ions in the endosome or in the lysosome to fluoresce. The two-photon fluorescent probe of claim 1, wherein the two-photon fluorescent probe represented by Chemical Formulas 4 to 6 reacts with hydrogen ions of the oligomer or protein to fluoresce. Method for preparing a two-photon fluorescent probe represented by the formula (1) prepared by mixing a compound represented by the formula (A), a compound represented by the formula (B) and p-toluenesulfonic acid monohydrate [Formula A]

[Formula B]

[Formula 1]

Wherein X ₁ and X ₂ are each independently H, F, Cl, Br, OCH ₃ , OH, NH ₂ , N (CH ₃ ) _2, NHCH _3, NCS, or CO ₂ H, and R ₁ and R ₂ Are each independently H, CH ₃ , CH ₂ (CH ₂ ) _n CH ₃ , CH ₂ (CH ₂ ) _n CO ₂ H, CH ₂ (CH ₂ ) _n OH or CH ₂ (CH ₂ ) _n SO ₃ H. (a) mixing a compound represented by the following Formula B, a compound represented by the following Formula C, and p-toluenesulfonic acid monohydrate to react to prepare a compound represented by the following Formula D; (b) preparing a compound represented by the following Chemical Formula E by mixing and reacting the compound represented by Chemical Formula D of step (a) with an aqueous LiOH solution; And (c) a process for preparing a compound represented by the following formula (2) comprising the step of reacting the compound represented by the formula (E) of step (b) and N, N-dimethylethylenediamine by mixing: [Formula B]

[Formula C]

[Formula D]

[Formula E]

[Formula 2]

Wherein X ₁ and X ₂ are each independently H, F, Cl, Br, OCH ₃ , OH, NH ₂ , N (CH ₃ ) _2, NHCH _3, NCS, or CO ₂ H, and R ₂ is H, CH ₃ , CH ₂ (CH ₂ ) _n CH ₃ , CH ₂ (CH ₂ ) _n CO ₂ H, CH ₂ (CH ₂ ) _n OH or CH ₂ (CH ₂ ) _n SO ₃ H. Method for imaging the intracellular pH through a two-photon fluorescence microscope using any one of two-photon fluorescent probe represented by the formula [Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

Wherein X ₁ and X ₂ are each independently H, F, Cl, Br, OCH ₃ , OH, NH ₂ , N (CH ₃ ) _2, NHCH _3, NCS, or CO ₂ H, and R ₁ and R ₂ Are each independently H, CH ₃ , CH ₂ (CH ₂ ) _n CH ₃ , CH ₂ (CH ₂ ) _n CO ₂ H, CH ₂ (CH ₂ ) _n OH or CH ₂ (CH ₂ ) _n SO ₃ H, where n is an integer from 0-10. The method of claim 7, wherein the depth of imaging is 100-200 μm. 8. The method of claim 7, wherein said cell is a live cell, organelle or cell tissue at pH 4-7. 10. The method of claim 9, wherein said organelle is an endosome or lysosome. The method of claim 10, wherein a two-photon fluorescent probe represented by Chemical Formula 2 or Chemical Formula 3 is used. According to claim 7, Method of imaging the intracellular pH containing the oligomer or protein using any one of the two-photon fluorescent probe represented by the formula 4 to 6.

Images