CN104479396B - amino acids two-photon fluorescent dye - Google Patents

Abstract

Amino acid two-photon fluorescent dye, the fluorescent dye has the structure of general formula I. This type of two-photon fluorescent dye has a low fluorescence background in the non-nucleic acid region and non-tumor cells in the living tumor cells, has a strong fluorescent signal in the nucleic acid region in the living tumor cells, and has a strong effect on the nucleic acid in the living tumor cells. Strong specific markers. These compounds have a certain level of water solubility and good cell membrane permeability. And it also has a large effective two-photon absorption cross section. The compound of the present invention also has lower biological toxicity, phototoxicity and photobleaching. Its spectral range is sufficiently different from that of biological samples. .

Description

Amino Acid Two-Photon Fluorescent Dyes

technical field

The invention relates to a class of two-photon fluorescent dyes containing amino acid structure, a preparation method thereof, and the identification and quantitative detection of glutathione in living tumor cells or tissues by using the fluorescent dyes.

Background technique

Nucleic acid (Nucleic Acids) is a biological macromolecule that can carry and transmit the genetic information of organisms in cells. Nucleic acid can be divided into ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) according to its composition. DNA molecule is a kind of chain structure or double-stranded molecule with circular structure containing the genetic information of biological species, and RNA molecule is a kind of single-stranded molecule responsible for the translation and expression of DNA genetic information. Nucleic acid exists in all animal and plant cells and microorganisms. It is one of the most basic substances of life and plays an important role in determining the growth, inheritance, and variation of organisms. In recent years, in addition to the previously studied functions of nucleic acids, new functions have also been discovered. For example, the Institute of Industrial Technology Fusion of the Japan Industrial Technology Institute has developed artificial nucleic acids that can treat leukemia. This nucleic acid can be subjectively Discover the genetic gene that causes leukemia and cut it out. This kind of artificial nucleic acid is expected to become the main drug for treating leukemia in the future (Nature, 2000, 406, 473-474). Therefore, it is an important task to establish a simple, fast, effective, sensitive, specific recognition of nucleic acid in tumor cells and its quantitative imaging technology or method.

There are many kinds of methods for identifying and detecting nucleic acids, such as PCR technology, high-performance liquid chromatography for analysis, ultraviolet spectrophotometry, and the like. However, the above methods have the following defects in practical imaging applications: poor cell membrane permeability, inability to achieve selectivity for tumor cells, and the like. With the development of microscopic imaging technology, fluorescent dyes have become the most important attachment tools in microscopic imaging research. Fluorescence-labeled optical molecular imaging provides a good solution for basic research on nucleic acids. At present, fluorescent dyes such as phenanthridines (EB, PI), acridines (AO), imidazoles (Hoechst, DAPI) and cyanine family (Cy, TOTO, SYTO) have been applied to nucleic acid-related research. However, these dyes have some irreversible shortcomings, such as: ethidium bromide (EB) in phenanthridine dyes is carcinogenic, and GoldView is irritating to the skin and eyes. More importantly, compared with the newly developed two-photon fluorescent dyes, these traditional one-photon fluorescent dyes have the advantages of near-infrared excitation, dark-field imaging, avoidance of fluorescence bleaching and phototoxicity, targeted excitation, high lateral resolution and vertical Resolution, reducing the absorption coefficient of biological tissue and reducing the interference of tissue autofluorescence (Nature,1999,2:989-996.; Science,1997,275:530.; Angewandte Chemie International Edition,2001,40:2098.) has Certain flaws. Therefore, there are still great challenges in the construction and preparation of low/non-toxic, high membrane permeability, and selective recognition of nucleic acid-based two-photon dyes in tumor cells.

Contents of the invention

The purpose of the present invention, at first is to provide a class of amino acid two-photon fluorescent dyes, said dyes have the structure of general formula I:

In general formula I:

R is selected from:

L ₁ and L ₂ are each independently selected from C _1-8 alkyl, -O(CH ₂ ) _n -, -(CH ₂ ) _n O-, -O(CH ₂ ) _n O-, -NH(CH ₂ ) _n -, -(CH ₂ ) _n NH- and -NH(CH ₂ ) _n NH-, wherein n is an integer of 1-8.

In the structural formula of the above group, "---" is used to represent the free bond connecting the group with other parts of the compound.

The object of another aspect of the present invention is to provide a method for preparing the above-mentioned amino acid two-photon fluorescent dye, said method comprising the following steps:

1) HR-Br reacts with concentrated nitric acid in a molar ratio of 1:1-1:5 to prepare compound HR-NO ₂ (C ₁ );

The reaction temperature is 30-150°C, the reaction time is 1-20 hours, and the reaction solvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or a mixture thereof;

2) Compound HR-NO ₂ (C ₁ ) reacts with compound B ₁ in a molar ratio of 1:1-1:5 to prepare compound C ₂ :

The reaction temperature is 30-150°C, the reaction time is 1-10 hours, and the reaction solvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or a mixture thereof;

3) Compound C ₂ is reacted with a reducing agent at a molar ratio of 1:5-1:20 to prepare compound C ₃ :

The reaction temperature is 25-100°C, the reaction time is 1-10 hours, the reaction solvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or their mixture; the reducing agent is selected from zinc powder, iron powder , stannous chloride and palladium carbon;

4) Compound C ₃ is reacted with compound B ₂ in a molar ratio of 1:5-1:20 to prepare compound C ₄ :

The reaction temperature is 25-90°C, the reaction time is 1-10 hours, and the reaction solvent is selected from dichloromethane, ethanol, methanol, acetone, ethyl acetate or a mixture thereof;

5) compound _C4 is hydrolyzed to prepare compound I, and 1-10 times the molar equivalent of compound _C4 trifluoroacetic acid, acetic acid or sodium hydroxide is added to the reaction system;

The reaction temperature is 25-50° C., the reaction time is 1-6 hours, and the reaction solvent is selected from dichloromethane, ethanol, methanol, acetone, water or a mixture thereof.

In the above description about the preparation method of the amino acid-based two-photon fluorescent dye of the present invention, the definition of each substituent, ie the definition of R, L ₁ , and L ₂ , is the same as that in the above description of the compound.

The amino acid two-photon fluorescent dye of the present invention can be excited by two photons, and has a strong fluorescent signal for nucleic acid in tumor living cells and tissues, and can be used for specific ratio labeling and detection of nucleic acid in living tumor cells and tissues two-photon fluorescent dyes. And this kind of fluorescent dye has a certain level of water solubility, good cell membrane permeability, and has a large effective two-photon absorption cross section. At the same time, it has low biological toxicity, phototoxicity and photobleaching. Its spectral range is sufficiently different from that of biological samples.

Therefore, another object of the present invention is to provide the application of the above-mentioned amino acid two-photon fluorescent dye in biological detection and labeling. It is especially preferably applied to the detection and labeling of nucleic acids in tumor cells or tumor tissues.

Description of drawings

8 pieces of accompanying drawings of the present invention:

Fig. 1 is the characterization result of the solvation effect of the fluorescent dye compound A ₁ of the present invention in Example 1. Compound A ₁ was added to different solvents of each. Determination of UV absorption spectrum (a) and fluorescence emission spectrum (b) in different solvents

2 is the measurement result of the two-photon absorption cross section of the fluorescent dye compound A ₁ of the present invention in Example 1 in the presence and absence of nucleic acid. The determination solvent is: PBS buffer solution. The measurement method is as follows: using the femtosecond two-photon induced fluorescence method, using the NaOH solution of fluorescein (pH 11) as a reference, the concentration of the A ₁ solution used is 1×10 ^-4 M, the laser pulse width is 70fs, and the repetition frequency is 80MHz. The average output power of the laser is 1.5W (780nm), and the adjustable wavelength range is 700-980nm. In the experiment, the femtosecond laser wavelength is adjusted to the required test wavelength.

Figure 3 shows the fluorescent dye compound A _{2 synthesized in Example 4. Hela cells were incubated with A 2 at} a final concentration of 2.0 μM at 37° C. and 5% CO ₂ for 30 minutes. Then, shake and rinse with PBS for 5min×3, then add cell culture medium, and conduct two-photon laser confocal imaging. Select a representative area, observe with an oil lens (100×), and repeat three times. Imaging showed a strong fluorescent signal in Hela cells. The fluorescence collection band is 560-650nm.

Fig. 4 is the water solubility characterization result of the fluorescent dye compound A ₂ of the present invention in Example 4. Using aqueous solutions of different concentrations of compound A ₂ , the absorbance at the maximum absorption wavelength was measured. repeat three times.

Figure 5 shows the fluorescent dye compound A _{3 synthesized in Example 7. Hela cells were incubated with A 3 at} a final concentration of 2.0 μM at 37° C. and 5% CO ₂ for 30 minutes. Then, shake and rinse with PBS for 5min×3, then add cell culture medium, and conduct two-photon laser confocal imaging. Select a representative area, observe with an oil lens (100×), and repeat three times. Imaging showed a strong fluorescent signal in Hela cells. The fluorescence collection band is 520-550nm.

Fig. 6 is the measurement result of the two-photon absorption cross section of the fluorescent dye compound A ₃ of the present invention in the presence and absence of nucleic acid in Example 7. The determination solvent is: PBS buffer solution. The measurement method is as follows: using the femtosecond two-photon induced fluorescence method, using the NaOH solution of fluorescein (pH 11) as a reference, the concentration of the A ₁ solution used is 1×10 ^-4 M, the laser pulse width is 70fs, and the repetition frequency is 80MHz. The average output power of the laser is 1.5W (780nm), and the adjustable wavelength range is 700-980nm. In the experiment, the femtosecond laser wavelength is adjusted to the required test wavelength.

Fig. 7 shows the fluorescent dye compound A _{4 synthesized in Example 10. Hela cells were incubated with A 4 at} a final concentration of 2.0 μM at 37° C. and 5% CO ₂ for 30 minutes. Then, shake and rinse with PBS for 5min×3, then add cell culture medium, and conduct two-photon laser confocal imaging. Select a representative area, observe with an oil lens (100×), and repeat three times. Imaging showed a strong fluorescent signal in Hela cells. The fluorescence collection band is 560-650nm.

Fig. 8 is the water solubility characterization result of the fluorescent dye compound A ₄ of the present invention in Example 10. Using aqueous solutions of different concentrations of compound A ₄ , the absorbance at the maximum absorption wavelength was measured. repeat three times.

detailed description

Unless otherwise specified, the terms used herein have the following meanings.

The term "alkyl" as used herein includes straight chain alkyl, branched chain alkyl and cycloalkyl. When a specific alkyl group is mentioned, such as "propyl", "isopropyl" or "cyclopropyl", it refers specifically to straight-chain alkyl, branched chain alkyl and cycloalkyl with the specified number of carbon atoms. As described in a general form, such as "C _1-6 alkyl", it includes all groups with the number of carbon atoms that meet the requirements, "C _1-6 alkyl" includes but not limited to C _1-4 alkyl, C _{1- 3} Alkyl, methyl, ethyl, n-propyl, isopropyl, tert-butyl, cyclopropyl, cyclobutyl, methylcyclopropyl, etc. Similar rules apply to other groups used in this specification.

The invention discloses a class of end-functionalized two-photon fluorescent dyes with naphthalene as the matrix, which have the structure of general formula I:

In the general formula I, R is a fluorophore; L ₁ and L ₂ are connecting arms.

In the general formula I, the R is selected from:

Described R is preferably:

Said R is most preferably:

In general formula I, said L ₁ and L ₂ are each independently selected from: C _1-8 alkyl, -O(CH ₂ ) _n -, -(CH ₂ ) _n O-, -O(CH ₂ ) _n O-, -NH(CH ₂ ) _n -, -(CH ₂ ) _n NH- and -NH(CH ₂ ) _n NH-, wherein n is an integer of 1-8.

As a preferred embodiment, said L ₁ is selected from: C _1-8 alkyl, -O(CH ₂ ) _n -, -NH(CH ₂ ) _n -, -(CH ₂ ) _n NH- or -NH (CH ₂ ) _n NH-; preferably C _1-8 alkyl, -NH(CH ₂ ) _n -, -(CH ₂ ) _n NH- or -NH(CH ₂ ) _n NH-; more preferably -(CH ₂ ) _n -or -(CH ₂ ) _n NH-. In any specific embodiment, n is an integer of 1-8, preferably n is an integer of 1-4; especially preferably n is 2 or 3.

As a preferred embodiment, said L ₂ is selected from: C _1-8 alkyl, -(CH ₂ ) _n O-, -O(CH ₂ ) _n - or -O(CH ₂ ) _n O-, preferably C _1-8 alkyl; more preferably -(CH ₂ ) _n -. In any specific embodiment, n is an integer of 1-8, preferably n is an integer of 1-4; especially preferably n is 2 or 3.

The preferred technical features described above can be combined with each other, and the resulting technical solutions should be fully included in the scope of the present invention.

An example of a combination of preferred technical features is one of the specific embodiments of the present invention. The amino acid two-photon fluorescent dye is selected from compounds A ₁ -A ₄ :

On the other hand, the present invention provides the preparation method of the above-mentioned amino acid two-photon fluorescent dye of the present invention, comprising the following steps:

1) HR-Br reacts with concentrated nitric acid in a molar ratio of 1:1-1:5 to prepare compound HR-NO ₂ (C ₁ );

The reaction temperature is 30-150°C, the reaction time is 1-20 hours, and the reaction solvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or a mixture thereof;

In a specific embodiment, the reaction temperature in step 1) is preferably 30-130°C, more preferably 30-120°C, most preferably 30-110°C; the reaction time is preferably 1-18 hours, more preferably 1-17 hours , most preferably 1-16 hour; Described reaction solvent is preferably dichloromethane, ethanol, methanol, acetone, ethyl acetate, acetic acid or mixture thereof, more preferably dichloromethane, ethanol, methyl alcohol, ethyl acetate, acetic acid or its mixture Mixture, most preferably dichloromethane, ethyl acetate, acetic acid or its mixture; H-R-Br reacts with concentrated nitric acid preferably molar ratio 1:1-1:4.5, more preferably 1:1-1:4.3, most preferably 1: 1-1:4.

2) Compound HR-NO ₂ (C ₁ ) reacts with compound B ₁ in a molar ratio of 1:1-1:5 to prepare compound C ₂ :

The reaction temperature is 30-150°C, the reaction time is 1-10 hours, and the reaction solvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or a mixture thereof;

In a specific embodiment, the reaction temperature in step 2) is preferably 30-130°C, more preferably 30-120°C, most preferably 30-110°C; the reaction time is preferably 1-9.5 hours, more preferably 1-9 hours , most preferably 2-9 hours; described reaction solvent is preferably dichloromethane, ethanol, methanol, acetone, ethyl acetate, acetic acid or mixture thereof, more preferably dichloromethane, ethanol, methanol, ethyl acetate, acetic acid or its mixture A mixture, most preferably dichloromethane, ethanol, ethyl acetate, acetic acid or a mixture thereof; compound HR-NO ₂ (C ₁ ) reacts with compound B ₁ preferably in a molar ratio of 1:1-1:4.5, more preferably 1:1 -1:4.3, most preferably 1:1-1:4.

3) Compound C ₂ is reacted with a reducing agent at a molar ratio of 1:5-1:20 to prepare compound C ₃ :

The reaction temperature is 25-100°C, the reaction time is 1-10 hours, the reaction solvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or their mixture; the reducing agent is selected from zinc powder, iron powder , stannous chloride and palladium carbon;

In a specific embodiment, the reaction temperature in step 3) is preferably 30-100°C, more preferably 30-95°C, most preferably 30-90°C; the reaction time is preferably 1-9.5 hours, more preferably 1-9 hours , most preferably 2-9 hours; the reaction solvent is preferably dichloromethane, ethanol, methanol, acetone, glycerol, acetic acid or a mixture thereof, more preferably ethanol, methanol, acetone, glycerol, acetic acid or a mixture thereof, Most preferably ethanol, methyl alcohol, acetone, acetic acid or mixture thereof; Compound C reacts with reducing agent preferably molar ratio ₁ :7-1:20, more preferably 1:8-1:20, most preferably 1:10-1: 20. The reducing agent is preferably selected from zinc powder, iron powder, stannous chloride, more preferably zinc powder, stannous chloride, most preferably zinc powder.

4) Under the catalysis of DMAP, compound C ₃ , compound B ₂ and EDCl react at a molar ratio of 1:5:1-1:20:1 to prepare compound C ₄ :

The reaction temperature is 25-90°C, the reaction time is 1-10 hours, and the reaction solvent is selected from dichloromethane, ethanol, methanol, acetone, ethyl acetate or a mixture thereof;

In a specific embodiment, the reaction temperature in step 4) is preferably 25-85°C, more preferably 25-80°C, most preferably 25-75°C; the reaction time is preferably 1-9.5 hours, more preferably 1-9 hours , most preferably 2-9 hours; described reaction solvent is preferably dichloromethane, ethanol, acetone, ethyl acetate or a mixture thereof, more preferably dichloromethane, acetone, ethyl acetate or a mixture thereof, most preferably dichloromethane, Ethyl acetate or a mixture thereof; compound C ₃ , compound B ₂ react with EDCl preferably in a molar ratio of 1:6:1-1:20:1, more preferably 1:7:1-1:20:1, most preferably 1 :8:1-1:20:1.

5) compound _C4 is hydrolyzed to prepare compound I, and 1-10 times the molar equivalent of compound _C4 trifluoroacetic acid, acetic acid or sodium hydroxide is added to the reaction system;

The reaction temperature is 25-50° C., the reaction time is 1-6 hours, and the reaction solvent is selected from dichloromethane, ethanol, methanol, acetone, water or a mixture thereof.

In a specific embodiment, the reaction temperature in step 5) is preferably 25-45°C, more preferably 25-40°C, most preferably 25-35°C; the reaction time is preferably 1-5.5 hours, more preferably 1-5 hours , most preferably 2-5 hours; described reaction solvent is preferably dichloromethane, ethanol, acetone, water or a mixture thereof, more preferably dichloromethane, acetone, water or a mixture thereof, most preferably dichloromethane, water or a mixture thereof ; Trifluoroacetic acid, acetic acid or sodium hydroxide and compound C carry out the hydrolysis reaction, the preferred molar ratio is ₁ :1-1:9, more preferably 1:1-1:8, most preferably 1:1-1:7.

For the amino acid two-photon fluorescent dye compound synthesized by the above method, the structure of the amino acid two-photon fluorescent dye compound is confirmed by nuclear magnetic resonance spectrum or mass spectrum.

The amino acid two-photon fluorescent dye of the present invention has the following advantages:

The dye compound introduces a specific binding site for nucleic acid, which improves the specificity and specificity of the dye for nucleic acid detection and recognition in living tumor cells and tissues;

The dye compound has excellent two-photon performance, and has low biological photobleaching, photodamage and biological toxicity when applied to biological sample imaging;

The dye compound has low toxicity, easy-to-obtain raw materials, simple structure, easy preparation, and easy industrialization;

In view of this, the two-photon fluorescent dye of the present invention can be used for labeling nucleic acid in living tumor cells. In addition to being directly used for labeling nucleic acids in living tumor cells in the form described herein, the composition containing the two-photon fluorescent dye compound of the present invention can also be used for labeling nucleic acids in living tumor cells. The composition should contain an effective amount of one of the two-photon fluorescent dye compounds provided by the present invention. In addition, other components required for staining of biological samples, such as solvents, pH regulators, etc., may also be included. These components are all known in the art. The above-mentioned composition may exist in the form of an aqueous solution, or may exist in other suitable forms which are made into a solution with water just before use.

The present invention also provides a method for labeling nucleic acid in living tumor cells by using the above-mentioned two-photon fluorescent dye compound of the present invention, the method comprising the step of contacting the compound with a biological sample. The term "contacting" as used herein may include contacting in solution or in a solid phase.

The following non-limiting examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way.

Example 1

To prepare fluorescent dye compound A ₁ , the following synthetic route is adopted:

(1) Synthesis of compound 1-1

Add 20mmol of 4-nitro-1,8-naphthalene anhydride and 25mmol of N,N-dimethylethylenediamine into a round bottom flask containing 10ml of ethanol solution under nitrogen protection. The reaction was heated to reflux for 8h and then stopped. The mixture was poured into ice water, precipitated out, and was suction filtered to obtain a crude yellow solid powder, compound 1-1, with a yield of 90%.

(2) Synthesis of compound 1-2

Add 20mmol of compound 1-1 and 400mmol of zinc powder into a round bottom flask containing 10ml of ethanol solution, under nitrogen protection. The reaction was heated to reflux for 8h and then stopped. The mixture was poured into ice water, precipitated out, and was suction filtered to obtain a crude yellow solid powder, compound 1-2, with a yield of 95%.

(3) Synthesis of compound 1-3

Add 20mmol of compound _1-2 and 200mmol of E1 into a round-bottomed flask containing 20mmol of EDCl and a small amount of DMAP in 20ml of ethyl acetate, under nitrogen protection. The reaction was heated to reflux at 50° C. for 5 h and then stopped. The solvent was distilled off under reduced pressure, and compound 1-3 was obtained as a yellow solid powder with a yield of 32%.

(4) Synthesis of Compound A ₁

With the compound 1-3 of the previous step, the reaction temperature is 25 ° C, the reaction time is 5 hours, the reaction solvent is selected from dichloromethane, and 1.5 times the equivalent of trifluoroacetic acid is added. ): acetonitrile=10:1-1:10 as the eluent to prepare compound A ₁ in the liquid phase with a yield of 18%. ¹ H NMR (400MHz,DMSO)δ10.11(s,1H),7.97-7.83(m,4H),7.63(s,1H),6.65(s,1H),6.43(s,2H),3.48(s ,1H),3.08(s,2H),2.67–2.52(m,6H),2.27(s,6H).

Example 2

Solvation Effect Detection Test of Fluorescent Dye Compound A ₁

The fluorescent dye compound A1 synthesized in Example ₁ above was added into different solvents, and the ultraviolet absorption spectrum and fluorescence emission spectrum in different solvents were measured. The test results are shown in Figure 1. It can be seen that as the polarity of the solvent changes, the maximum absorption wavelength in the ultraviolet absorption spectrum moves accordingly, and the fluorescence emission spectrum also has a movement of the maximum emission wavelength. Figure 1(a) is the ultraviolet absorption spectrum of fluorescent dye compound A ₁ in different solvents, and Figure 1(b) is the fluorescence emission spectrum of fluorescent dye compound A ₁ in different solvents. The instruments used were Agilent 8453 UV Spectrophotometer and Agilent Cary Eclipse Fluorescence Spectrophotometer.

Example 3

Two _- photon effective absorption cross-section detection test of fluorescent dye compound A1:

Using the femtosecond two-photon-induced fluorescence method, using the NaOH solution (pH 11) of fluorescein as a reference, the fluorescent dye compound A1 synthesized in Example ₁ was added to the two-photon absorption cross section of the dimethyl sulfoxide solvent respectively. For the test, the concentration of the solution used is 1×10 ^-4 M, and the calculation formula is as follows:

${\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; the\; s</span>}}<span\; class="notranslate">\; =</span>{\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; r</span>}}\frac{{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; r</span>}}}{{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; the\; s</span>}}}\frac{{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; r</span>}}}{{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; the\; s</span>}}}\frac{{\mathrm{<span\; class="notranslate">\; f</span>}}_{\mathrm{<span\; class="notranslate">\; the\; s</span>}}}{{\mathrm{<span\; class="notranslate">\; f</span>}}_{\mathrm{<span\; class="notranslate">\; r</span>}}}\frac{{\mathrm{<span\; class="notranslate">\; \&Phi;</span>}}_{\mathrm{<span\; class="notranslate">\; r</span>}}}{{\mathrm{<span\; class="notranslate">\; \&Phi;</span>}}_{\mathrm{<span\; class="notranslate">\; the\; s</span>}}}$

C in the formula is the concentration of the solution, and n is the refractive index of the solvent, which can be obtained by looking up the table. F is the up-converted fluorescence intensity, measured by experiment. δ is the two-photon absorption cross section. The physical quantity of the reference solution is represented by the subscript r.

The two-photon effective absorption cross section (Φδ, as shown in FIG. 2 ) was determined in the presence and absence of nucleic acid. The excitation source of the two-photon excited fluorescence spectrum is a mode-locked femtosecond titanium sapphire laser, the laser pulse width is 70fs, the repetition frequency is 80MHz, the average output power of the laser is 1.5W (780nm), and the adjustable wavelength range is 700-980nm. The femtosecond laser wavelength is adjusted to the required test wavelength. As shown by the experimental results (Figure 2), when there is no nucleic acid, the two-photon effective absorption cross section of the fluorescent dye compound A ₁ is only 11 GM at 800nm; when there is nucleic acid in the test system, the two-photon effective absorption cross section of the fluorescent dye compound A ₁ The cross section is only 139GM at 770nm.

Example 4

Preparation of Probe Compound A ₂ :

(1) Synthesis of compound 2-1

Add 20mmol of 5-nitroacridin-1-amine and 30mmol of bromoethylenediamine into a round bottom flask containing 10ml of ethanol solution under nitrogen protection. The reaction was heated to reflux for 8h and then stopped. The mixture was poured into ice water, precipitated out, and suction filtered to obtain a crude yellow solid powder, Compound 2-1, with a yield of 79%.

(2) Synthesis of compound 2-2

Add 20mmol of compound 2-1 and 400mmol of zinc powder into a round bottom flask containing 10ml of ethanol solution, under nitrogen protection. The reaction was heated to reflux for 8h and then stopped. The mixture was poured into ice water, precipitated out, and suction filtered to obtain the crude product of yellow solid powder, compound 2-2, with a yield of 93%.

(3) Synthesis of compound 2-3

Add 20mmol of compound 2-2 and 200mmol of E ₁ into a round bottom flask containing 20mmol of EDCl and a small amount of DMAP in 20ml of ethyl acetate, under nitrogen protection. The reaction was heated to reflux at 50° C. for 5 h and then stopped. The solvent was distilled off under reduced pressure, and compound 2-3 was obtained as a yellow solid powder with a yield of 35% through column chromatography.

(4) Synthesis of fluorescent dye compound A ₂

With compound 2-3 at a reaction temperature of 25°C and a reaction time of 5 hours, the reaction solvent is selected from dichloromethane, and 1.5 times the equivalent of trifluoroacetic acid is added. After the reaction is completed, use water (containing 1% trifluoroacetic acid): Acetonitrile=10:1-1:10 was used as the eluent to prepare the liquid-phase fluorescent dye compound A ₂ with a yield of 15%. ¹ H NMR (400MHz,DMSO)δ10.14(s,1H),7.97(s,1H),7.63(s,2H),7.22(s,2H),6.95(s,2H),6.65(s,1H ),6.43(s,2H),4.41(s,1H),3.48(s,1H),3.08(s,2H),2.67–2.52(m,6H),2.27(s,6H).

Example 5

Recognition of Nucleic Acids in Live Tumor Cells by Fluorescent Dye Compound A ₂

Using the fluorescent dye compound A ₂ synthesized in Example 4, Hela cells were incubated with A ₂ at a final concentration of 2.0 μM at 37° C., 5% CO ₂ for 30 minutes. Then, shake and rinse with PBS for 5min×3, then add cell culture medium, and conduct two-photon laser confocal imaging. Select a representative area, observe with an oil lens (100×), and repeat three times. The experimental results show that the fluorescent dye compound A ₂ has a strong fluorescent signal in Hela cells. The fluorescence collection band in Figure 3 is 560-650nm.

Example 6

Water Solubility Detection Test of Fluorescent Dye Compound A ₂

The fluorescent dye _compound A2 synthesized in Example ₄ above was added to water, and the absorbance at the maximum absorption wavelength of aqueous solutions of A2 with different concentrations was measured. The test results are shown in Figure 4, and the results show that: when the concentration of Compound A ₂ is 4.0 μM, the absorbance value does not shift, that is, the solubility of the fluorescent dye Compound A ₂ in water is 4.0 μM. Figure ₄ shows the absorbance at the maximum absorption wavelength of different concentrations of probe A2. The instruments used were Agilent 8453 UV spectrophotometer.

Example 7

Preparation of Fluorescent Dye Compound A ₃

(1) Synthesis of compound 3-1

Add 20mmol of 4-nitro-1,8-naphthalene anhydride and 25mmol of N,N-dimethylethylenediamine into a round bottom flask containing 10ml of ethanol solution under nitrogen protection. The reaction was heated to reflux for 8h and then stopped. The mixture was poured into ice water, precipitated out, and suction filtered to obtain the crude product of yellow solid powder, compound 3-1, with a yield of 90%.

(2) Synthesis of compound 3-2

Add 20mmol of compound 3-1 and 400mmol of zinc powder into a round bottom flask containing 10ml of ethanol solution under nitrogen protection. The reaction was heated to reflux for 8h and then stopped. The mixture was poured into ice water, precipitated out, and suction filtered to obtain the crude product of yellow solid powder, compound 3-2, with a yield of 95%.

(3) Synthesis of compound 3-3

Add 20mmol of compound 3-2 and 200mmol of E ₂ into a round bottom flask containing 20mmol of EDCl and a small amount of DMAP in 20ml of ethyl acetate, under nitrogen protection. The reaction was heated to reflux at 50° C. for 5 h and then stopped. The solvent was distilled off under reduced pressure, and compound 3-3 was obtained as a yellow solid powder with a yield of 38% through column chromatography.

(4) Synthesis of Fluorescent Dye Compound A ₃

With compound 3-3 at a reaction temperature of 25°C and a reaction time of 5 hours, the reaction solvent is selected from dichloromethane, and 1.5 times the equivalent of trifluoroacetic acid is added. After the reaction is completed, use water (containing 1% trifluoroacetic acid): Acetonitrile=10:1-1:10 was used as the eluent to prepare the liquid-phase fluorescent dye compound A ₃ with a yield of 18%. ¹ H NMR (400MHz,DMSO)δ10.11(s,1H),7.95-7.81(m,4H),7.67(s,1H),6.55(s,1H),6.40(s,2H),3.45(s ,1H),3.08(s,2H),2.67–2.48(m,8H),2.27(s,6H).

Example 8

Labeling of Nucleic Acids in Living Cells by Fluorescent Dye Compound A ₃

Using the fluorescent dye compound A ₃ synthesized in Example 7, Hela cells were incubated with A ₃ at a final concentration of 2.0 μM at 37° C., 5% CO ₂ for 30 minutes. Then, shake and rinse with PBS for 5min×3, then add cell culture medium, and conduct two-photon laser confocal imaging. Select a representative area, observe with an oil lens (100×), and repeat three times. The results are shown in Figure 5, and the imaging results show that the fluorescent dye compound A ₃ has a strong fluorescent signal in Hela cells. The fluorescence collection band in Fig. 5 is 520-550nm.

Example 9

Two-photon effective absorption cross-section detection test of fluorescent dye compound A ₃ :

Adopt femtosecond two-photon induced fluorescence method, utilize the NaOH solution (pH 11) of fluorescein as reference, the test of the two-photon absorption cross section in the dimethyl sulfoxide solvent that the compound A3 synthesized in embodiment ₇ is added respectively, The concentration of the solution used is 1×10 ^-4 M, and the calculation formula is as follows:

${\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; the\; s</span>}}<span\; class="notranslate">\; =</span>{\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; r</span>}}\frac{{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; r</span>}}}{{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; the\; s</span>}}}\frac{{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; r</span>}}}{{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; the\; s</span>}}}\frac{{\mathrm{<span\; class="notranslate">\; f</span>}}_{\mathrm{<span\; class="notranslate">\; the\; s</span>}}}{{\mathrm{<span\; class="notranslate">\; f</span>}}_{\mathrm{<span\; class="notranslate">\; r</span>}}}\frac{{\mathrm{<span\; class="notranslate">\; \&Phi;</span>}}_{\mathrm{<span\; class="notranslate">\; r</span>}}}{{\mathrm{<span\; class="notranslate">\; \&Phi;</span>}}_{\mathrm{<span\; class="notranslate">\; the\; s</span>}}}$

C in the formula is the concentration of the solution, and n is the refractive index of the solvent, which can be obtained by looking up the table. F is the up-converted fluorescence intensity, measured by experiment. δ is the two-photon absorption cross section. The physical quantity of the reference solution is represented by the subscript r.

The two-photon effective absorption cross section (Φδ, as shown in FIG. 6 ) was measured in the presence and absence of nucleic acid. The excitation source of the two-photon excited fluorescence spectrum is a mode-locked femtosecond titanium sapphire laser, the laser pulse width is 70fs, the repetition frequency is 80MHz, the average output power of the laser is 1.5W (780nm), and the adjustable wavelength range is 700-980nm. The femtosecond laser wavelength is adjusted to the required test wavelength. As shown by the experimental results (Figure 6), when there is no nucleic acid, the two-photon effective absorption cross section of the fluorescent dye compound A ₁ is only 11 GM at 800nm; when there is nucleic acid in the test system, the two-photon effective absorption cross section of the fluorescent dye compound A ₁ The cross section is only 124GM at 770nm.

Example 10

Preparation of Fluorescent Dye Compound A ₄

(1) Synthesis of Compound 4-1

Add 20mmol 5-nitroacridine-1-carboxylic acid and 30mmol ethylenediamine into a round bottom flask containing 10ml ethanol solution, add 20mmol EDCl and a small amount of DMAP, and protect with nitrogen. Reflux reaction stopped after 5h. After the reaction was completed, the product was separated by column chromatography to obtain a crude orange solid powder, compound 4-1, with a yield of 67%.

(2) Synthesis of Compound 4-2

Add 20mmol of compound 4-1 and 400mmol of zinc powder into a round bottom flask containing 10ml of ethanol solution under nitrogen protection. The reaction was heated to reflux for 8h and then stopped. The mixture was poured into ice water, precipitated out, and suction filtered to obtain the crude product, compound 4-2, as an orange solid powder, with a yield of 96%.

(3) Synthesis of compound 4-3

Add 20mmol of compound 4-2 and 200mmol of E1 into _a round-bottomed flask containing 20mmol of EDCl and a small amount of DMAP in 20ml of ethyl acetate, under nitrogen protection. The reaction was heated to reflux at 50° C. for 5 h and then stopped. The solvent was distilled off under reduced pressure, and compound 4-3 was obtained as a yellow solid powder with a yield of 39% through column chromatography.

(4) Synthesis of fluorescent dye compound A ₄

With compound 4-3 at a reaction temperature of 25°C and a reaction time of 5 hours, the reaction solvent is selected from dichloromethane, and 1.5 times the equivalent of trifluoroacetic acid is added. After the reaction is completed, use water (containing 1% trifluoroacetic acid): Acetonitrile=10:1-1:10 was used as the eluent to prepare the liquid-phase fluorescent dye compound A ₄ with a yield of 15%. ¹ H NMR (400MHz,DMSO)δ10.14(s,1H),7.97(s,1H),7.63(s,2H),7.22(s,2H),6.95(s,2H),6.65(s,2H ),6.43(s,2H),3.48(s,1H),3.08(s,2H),2.67–2.52(m,6H),2.27(s,6H).

Example 11

Labeling of Nucleic Acids in Living Cells by Fluorescent Dye Compound A ₄

Using the fluorescent dye compound A ₄ synthesized in Example 10, Hela cells were incubated with A ₄ at a final concentration of 2.0 μM at 37° C., 5% CO ₂ for 30 minutes. Then, shake and rinse with PBS for 5min×3, then add cell culture medium, and conduct two-photon laser confocal imaging. Select a representative area, observe with an oil lens (100×), and repeat three times. The imaging results showed that the fluorescent dye compound A ₄ had a strong fluorescent signal in Hela cells. The fluorescence collection band in Fig. 7 is 560-650nm.

Example 12

Water Solubility Detection Test of Fluorescent Dye Compound A ₄

_The fluorescent dye compound A4 synthesized in Example ₁₀ above was added to water, and the absorbance at the maximum absorption wavelength of aqueous solutions of fluorescent dye compound A4 with different concentrations was measured. The test results are shown in Figure 8, which shows that when the concentration of the fluorescent dye compound A ₂ is 6.0 μM, the absorbance value does not shift, that is, the solubility of compound A ₄ in water is 6.0 μM. Figure ₈ shows the absorbance at the maximum absorption wavelength for different concentrations of probe A4. The instruments used were Agilent 8453 UV spectrophotometer.

The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be assumed that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, some simple deduction or replacement can be made, which should be regarded as belonging to the protection scope of the present invention. As a fluorescent dye is a use of the new compound of the present invention, it cannot be determined that the compound of the present invention is only used for fluorescent dyes, for those of ordinary skill in the technical field of the present invention, based on the same effect of the compound of the present invention as a fluorescent dye Under the consideration of the mechanism, some simple inferences can also be made, and it can be concluded that other applications of the compounds of the present invention should be considered as belonging to the protection scope of the present invention.

Claims (6)

1. amino acids Two-photon fluorescent dye, has a structure of formula I:

In formula I:

R is selected from:

L₁And L₂It is each independently selected from C_1-8Alkyl ,-O (CH₂)_n-、-(CH₂)_nO-、-O(CH₂)_nO-、-NH(CH₂)_n-、-(CH₂)_nNH-and-NH (CH₂)_nNH-, wherein n is the integer of 1-8.

2. the amino acids Two-photon fluorescent dye described in claim 1, it is characterised in that: described R is selected from:

3. the amino acids Two-photon fluorescent dye described in claim 1, it is characterised in that: described L₁Selected from C_1-8Alkyl ,-O (CH₂)_n-、-NH(CH₂)_n-、-(CH₂)_nNH-or-NH (CH₂)_nNH-。

4. the amino acids Two-photon fluorescent dye described in claim 1, it is characterised in that: described L₂Selected from C_1-8Alkyl ,-(CH₂)_nO-、-O(CH₂)_n-or-O (CH₂)_nO-。

5. the amino acids Two-photon fluorescent dye described in claim 1, selected from compound A₁-A₄:

。

6. the preparation method of the amino acids Two-photon fluorescent dye described in claim 1, comprises the steps:

1) H-R-Br and red fuming nitric acid (RFNA) 1:1-1:5 in molar ratio reacts, and prepares compound H-R-NO₂；

Reaction temperature is 30-150 DEG C, and the reaction time is 1-20 hour, and reaction dissolvent is selected from dichloromethane, ethanol, methyl alcohol, acetone, glycerine, ethyl acetate, acetic acid or its mixture；

2) compound H-R-NO₂With compound B₁1:1-1:5 reaction, prepares compound C in molar ratio₂:

Reaction temperature 30-150 DEG C, in 1-10 hour reaction time, reaction dissolvent is selected from dichloromethane, ethanol, methyl alcohol, acetone, glycerine, ethyl acetate, acetic acid or its mixture；

3) compound C₂React with reducing agent 1:5-1:20 in molar ratio, prepare compound C₃:

Reaction temperature 25-100 DEG C, in 1-10 hour reaction time, reaction dissolvent is selected from dichloromethane, ethanol, methyl alcohol, acetone, glycerine, ethyl acetate, acetic acid or its mixture；Reducing agent is selected from zinc powder, iron powder, stannous chloride and palladium carbon；

4) compound C₃With compound B₂1:5-1:20 reaction, prepares compound C in molar ratio₄:

Reaction temperature is 25-90 DEG C, and the reaction time is 1-10 hour, and reaction dissolvent is selected from dichloromethane, ethanol, methyl alcohol, acetone, ethyl acetate or its mixture；

5) compound C₄Preparing compound I through hydrolysis, reaction system adds 1-10 times of compound C₄The trifluoroacetic acid of molar equivalent, acetic acid or NaOH；

Reaction temperature is 25-50 DEG C, and the reaction time is 1-6 hour, and reaction dissolvent is selected from dichloromethane, ethanol, methyl alcohol, acetone, water or its mixture.