CN115925667A - A ratio-type near-infrared fluorescent probe molecule and its preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of biological materials, in particular to a ratio-type near-infrared fluorescent probe molecule and its preparation method and application. The general structural formula of the fluorescent probe molecule of the present invention is as follows; the structural molecule can construct a ratiometric near-infrared second window fluorescent probe selectively activated by a specific substrate, which is used for deep tissue imaging and biosensing analyze.

Description

A ratio-type near-infrared fluorescent probe molecule and its preparation method and application

technical field

The invention belongs to the technical field of biological materials, and in particular relates to a ratio-type near-infrared fluorescent probe molecule, a preparation method thereof, and an application thereof.

Background technique

In recent years, near-infrared second window (1000-1700nm) in vivo fluorescence imaging has been rapidly developed in the field of biomedicine, thanks to the civilian use of InGaAs detectors (response range 900-1700nm). At present, the fluorescent probes in the second near-infrared window are mainly "off-on" type probes, which can be selectively activated by specific substrates in vivo to give signals, but they can only achieve " With and without signal changes, the use of "off-on" fluorescent probes can achieve qualitative and visual monitoring of specific substrates such as enzymes, reactive oxygen species/reactive nitrogen species (ROS/RNS) in the tumor microenvironment, but cannot Realize quantitative change monitoring.

In contrast, ratiometric fluorescent probes can collect signals through multiple channels and then correlate the signals of each channel, which has a self-calibration function and can realize quantitative analysis and monitoring. At present, there are relatively few near-infrared second-window ratiometric fluorescent probes and probe design strategies, and the types of substrates that can be detected are mainly strong oxidizing substances, which cannot meet the detection requirements for enzymes and other biomolecules. In order to realize quantitative visualization of the tumor microenvironment, it is urgent to construct a near-infrared second window ratiometric probe molecular platform and establish a reliable (semi)quantitative analysis method under deep tissue.

Contents of the invention

The object of the present invention is to provide a ratiometric near-infrared second window fluorescent probe molecule capable of visualizing and quantitatively monitoring the tumor microenvironment, its preparation method and application.

The ratio-type near-infrared fluorescent probe molecule provided by the present invention is denoted as Rap, and its compound structural formula is as follows:

Wherein, X is selected from O or S; Y is selected from ClO ₄ , PF ₆ , BF ₄ , Cl, Br, I, CF ₃ COO, CF ₃ SO ₃ , CH ₃ COO or CH ₃ SO ₃ ; R ₁ is selected from H , OH, OCH ₃ or N[(CH ₂ )nCH ₃ ] ₂ ; R ₂ and R ₃ are H, o-carboxyphenyl or phenyl or o-methylphenyl, R ₄ is substituted benzyloxy, which can be selected from 4-nitrobenzyloxy, 4-phosphobenzyloxy, 4-glutamyl transpeptidylbenzyloxy, 4-β-galactosylbenzyloxy, azidobenzyloxy, 2,4-di Nitrobenzyloxy, 4-boronic acid benzyloxy or 4-boronic acid benzyloxy, n is an integer of 0-6.

The present invention also provides the preparation method of above-mentioned ratiometric near-infrared fluorescent probe molecule, and its synthetic route is:

Wherein, X is selected from O or S; Y is selected from ClO ₄ , PF ₆ , BF ₄ , Cl, Br, I, CF ₃ COO, CF ₃ SO ₃ , CH ₃ COO or CH ₃ SO ₃ ; R ₁ is selected from H , OH, OCH ₃ or N[(CH ₂ )nCH ₃ ] ₂ ; R ₂ and R ₃ are H, o-carboxyphenyl or phenyl or o-methylphenyl, R ₄ is substituted benzyloxy, optional From 4-nitrobenzyloxy, 4-phosphobenzyloxy, 4-glutamyl transpeptidylbenzyloxy, 4-β-galactosylbenzyloxy, azidobenzyloxy, 2,4- Dinitrobenzyloxy, 4-boronic acid benzyloxy or 4-boronic acid benzyloxy, n is an integer from 0 to 6; compound 1 is substituted rhodamine 6G, compound 2 is substituted benzoyl, compound 3 is substituted acid chloride.

Concrete synthetic steps are:

(1) Synthesis of Intermediate 1:

Dissolve substituted rhodamine 6G (compound 1) in a mixed solution of sodium hydroxide and ethanol, and react at 150-170°C for 36-48 hours; after cooling, adjust the pH to 3-4 with hydrochloric acid solution, precipitate the solid and filter to obtain intermediate Product 1;

(2) Synthesis of Intermediate 2:

Dissolve intermediate 1 and cyclohexanone in concentrated sulfuric acid, react at 80-100°C for 1-3 hours, cool to room temperature, add ice water, add protonic acid, precipitate solid and filter to obtain intermediate 2;

Wherein, the protic acid can be selected from one of HClO ₄ , HPF ₆ , HBF ₄ , HCl, HBr, HI, CF ₃ COOH, CF ₃ SO ₃ H and CH ₃ SO ₃ H; wherein, the intermediate product 1 and The molar ratio of cyclohexanone is 1:(1～3);

(3) Synthesis of intermediate 3

Dissolve substituted benzoyl (compound 2) in concentrated sulfuric acid, react at 80-100°C for 1-3 hours, add ice water after cooling to room temperature, add protonic acid, precipitate solid and filter to obtain intermediate 3; intermediate 2 and The molar ratio of cyclohexanone is 1:(1～3);

Wherein, the protonic acid can be selected from one of HClO ₄ , HPF ₆ , HBF ₄ , HCl, HBr, HI, CF ₃ COOH, CF ₃ SO ₃ H and CH ₃ SO ₃ H;

(4) Synthesis of Intermediate 4:

Dissolve intermediate 3, malondialdehyde dianiline hydrochloride in a mixed solvent of acetic acid and acetic anhydride, and react at 90-110°C for 1-3 hours under nitrogen protection; after cooling to room temperature, concentrate the organic phase and use Column chromatography separation, obtains intermediate 4;

(5) Synthesis of Intermediate 5:

Under the protection of nitrogen, add intermediate 2 and intermediate 4 into a mixed solvent of toluene and n-butanol, and react at 80-120°C for 3-6 hours; after cooling to room temperature, the organic phase is concentrated and separated by column chromatography to obtain Intermediate 5; the molar ratio of intermediate 2 and intermediate product 4 is 1:(1～3);

(6) Synthesis of ratiometric near-infrared fluorescent probe molecules (product 1):

Dissolve intermediate 5 and substituted acid chloride (compound 3) in dichloromethane, add an organic base, and react at room temperature for 12 to 48 hours; after quenching the reaction, the organic phase is concentrated and separated by column chromatography to obtain product 1; intermediate 5 and substituted The feeding molar ratio of acid chloride is 1:(2~3).

Wherein, the organic base is selected from one of triethylamine, N,N-diisopropylethylamine and pyridine.

The ratio-type near-infrared second-window fluorescent probe molecules obtained in the present invention interact with enzymes and active oxygen/nitrogen (ROS/RNS) to generate ratio fluorescence signals.

The ratio-type near-infrared second window fluorescent probe molecule provided by the present invention has a large molar extinction coefficient, long absorption and emission wavelengths, good chemical stability and photostability, and also has a large range of acid and alkali. good stability. The platform has good molecular versatility and can be used to construct ratiometric near-infrared fluorescent probes that respond to various enzymes and reactive oxygen species. Therefore, it can realize (semi) quantitative and visual monitoring of biological small molecules (reactive oxygen species) and macromolecules in vivo. (enzymes) etc.

Furthermore, the ratiometric near-infrared fluorescent probe molecule (general formula 1) of the present invention has a maximum absorption peak at ~800nm and a maximum emission peak at ~950nm in a dichloromethane solution.

Furthermore, the molar extinction coefficient of the ratiometric near-infrared fluorescent probe molecule (general formula 1) of the present invention in a dichloromethane solution is ~95000M ^-1 cm ^-1 .

The ratiometric near-infrared fluorescent probe molecules provided by the present invention can be used for deep tissue imaging of organisms, biosensing analysis, etc., for example, to be prepared as reagents or micellar solutions.

Description of drawings

Fig. 1 is the absorption and fluorescence emission spectrum (corresponding to Example 1) of ratiometric near-infrared fluorescent probe Rap-N in PBS solution (PBS/glycerol/PEG400) excited at 808 nm.

Fig. 2 is the fluorescence ratio change spectrogram (corresponding to Example 1) of the ratiometric near-infrared fluorescent probe Rap-N in response to nitroreductase when excited at 808 nm.

Fig. 3 is the fluorescence ratio change spectrogram (corresponding to Example 2) of the ratiometric near-infrared fluorescent probe Rap-R in response to hydrogen peroxide excited at 808 nm.

Fig. 4 is the Rap-N probe used for tumor imaging in vivo (corresponding to Example 4).

Fig. 5 shows that the Rap-R probe is used for liver injury imaging in vivo (corresponding to Example 5).

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention is illustrated with the following specific examples, but the present invention is by no means limited to these examples. The following descriptions are only preferred embodiments of the present invention, and are only used to explain the present invention, and should not be construed as limiting the patent scope of the present invention. It should be noted that any modification, substitution or improvement made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Example 1:

Preparation of the ratiometric near-infrared fluorescent probe (referred to as Rap-N) of nitroreductase (NTR) response, the structural formula of the compound is as follows:

Concrete synthetic route is as follows:

Concrete synthetic steps are as follows:

(1) Synthesis of intermediate 1

Dissolve rhodamine 6G (compound 1) in a mixed solution of sodium hydroxide and ethanol, react at 150-170°C for 36-48 hours; after cooling, adjust the pH to 3-4 with hydrochloric acid solution, precipitate solids and filter to obtain intermediate product 1 ;

(2) Synthesis of Intermediate 2

Dissolve intermediate 1 and cyclohexanone in concentrated sulfuric acid, react at 80-100°C for 1-3 hours, add ice water after cooling to room temperature, and add HClO ₄ ; the precipitated solid is filtered to obtain intermediate 2; intermediate product 1 The molar ratio of feeding with cyclohexanone is 1:(1～3);

(3) Synthesis of intermediate 3

Dissolve compound 2 in concentrated sulfuric acid, react at 80-100°C for 1-3 hours, add ice water after cooling to room temperature, and add HClO ₄ ; the precipitated solid is filtered to obtain intermediate 3; the intermediate product 2 and cyclohexanone The molar ratio of feeding is 1:(1～3);

(4) Synthesis of Intermediate 4

Dissolve intermediate 3, malondialdehyde dianiline hydrochloride in a mixed solvent of acetic acid and acetic anhydride, and react at 90-110°C for 1-3 hours under nitrogen protection; after cooling to room temperature, concentrate the organic phase and use Column chromatography separation obtains intermediate 4;

(5) Synthesis of intermediate 5

Under the protection of nitrogen, add intermediate 2 and intermediate 4 into a mixed solvent of toluene and n-butanol, and react at 80-120°C for 3-6 hours; after cooling to room temperature, the organic phase is concentrated and separated by column chromatography to obtain intermediate body 5; wherein the molar ratio of intermediate product 2 and intermediate product 4 is 1:(1～3);

(6) Synthesis of Rap-N

Dissolve intermediate 5 and p-nitrobenzyl chloroformate in dichloromethane, add triethylamine, and react at room temperature for 12-48 hours; after quenching the reaction, the organic phase is concentrated and separated by column chromatography to obtain the final product Rap-N; , the molar ratio of intermediate 5 and p-nitrobenzyl chloroformate is 1:(2～3).

Concrete example:

(1) Synthesis of Intermediate 1

Rhodamine 6G (compound 1) was dissolved in a mixed solution of sodium hydroxide and ethanol, and reacted at 170°C for 36 hours; after cooling, the pH was adjusted to about 3-4 with hydrochloric acid solution, and the precipitated solid was filtered to obtain intermediate product 1;

(2) Synthesis of intermediate 2

Dissolve intermediate 1 and cyclohexanone in concentrated sulfuric acid, react at 90°C for 2 hours, add ice water after cooling to room temperature, and add HClO ₄ ; the precipitated solid is filtered to obtain intermediate 2; intermediate product 1 and cyclohexanone The feeding molar ratio is 1:1.2;

(3) Synthesis of intermediate 3

Dissolve compound 2 in concentrated sulfuric acid, react at 90°C for 2 hours, add ice water after cooling to room temperature, and add HClO ₄ ; the precipitated solid is filtered to obtain intermediate 3; wherein the molar ratio of intermediate product 2 and cyclohexanone is 1:1.2;

(4) Synthesis of Intermediate 4

Dissolve intermediate 3, malondialdehyde dianiline hydrochloride in a mixed solvent of acetic acid and acetic anhydride, and react at 100°C for 2 hours under nitrogen protection; after cooling to room temperature, the organic phase is concentrated and separated by column chromatography to obtain Intermediate 4;

(5) Synthesis of intermediate 5

Under nitrogen protection, intermediate 2 and intermediate 4 were added to a mixed solvent of toluene and n-butanol, and reacted at 110°C for 5 hours; after cooling to room temperature, the organic phase was concentrated and separated by column chromatography to obtain intermediate 5; The molar ratio of intermediate product 2 and intermediate product 4 is 1:1;

(6) Synthesis of Rap-N

Dissolve intermediate 5 and p-nitrobenzyl chloroformate in dichloromethane, add triethylamine, and react at room temperature for 24 hours; after quenching the reaction, the organic phase is concentrated and separated by column chromatography to obtain the final product Rap-N; wherein, The molar ratio of intermediate 5 and p-nitrobenzyl chloroformate is 1:1.

Under the excitation of 808nm, the change of fluorescence intensity of Rap-N ratiometric fluorescent probe in response to nitroreductase is shown in Figure 2.

Example 2:

The preparation of the ratiometric near-infrared fluorescent probe (referred to as Rap-R) of active oxygen response, the structural formula of the compound is as follows:

Concrete synthetic route is as follows:

Concrete synthetic steps are as follows:

(1) Synthesis of Intermediate 1

Dissolve rhodamine 6G (compound 1) in a mixed solution of sodium hydroxide and ethanol, react at 150-170°C for 36-48 hours; after cooling, adjust the pH to 3-4 with hydrochloric acid solution, precipitate solids and filter to obtain intermediate product 1 ;

(2) Synthesis of intermediate 2

Dissolve intermediate 1 and cyclohexanone in concentrated sulfuric acid, react at 80-100°C for 1-3 hours, add ice water after cooling to room temperature, and add HClO ₄ ; the precipitated solid is filtered to obtain intermediate 2; intermediate product 1 The molar ratio of feeding with cyclohexanone is 1:(1～3);

(3) Synthesis of intermediate 3

Dissolve compound 2 in concentrated sulfuric acid, react at 80-100°C for 1-3 hours, add ice water after cooling to room temperature, and add HClO ₄ ; the precipitated solid is filtered to obtain intermediate 3; the intermediate product 2 and cyclohexanone The molar ratio of feeding is 1:(1～3);

(4) Synthesis of Intermediate 4

Dissolve intermediate 3, malondialdehyde dianiline hydrochloride in a mixed solvent of acetic acid and acetic anhydride, and react at 90-110°C for 1-3 hours under nitrogen protection; after cooling to room temperature, concentrate the organic phase and use Column chromatography separation obtains intermediate 4;

(5) Synthesis of intermediate 5

Under the protection of nitrogen, add intermediate 2 and intermediate 4 into a mixed solvent of toluene and n-butanol, and react at 80-120°C for 3-6 hours; after cooling to room temperature, the organic phase is concentrated and separated by column chromatography to obtain intermediate body 5; wherein, the molar ratio of intermediate product 2 and intermediate product 4 is 1:(1～3);

(6) Synthesis of Rap-R

Intermediate 5 and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl chloroformate were dissolved in dichloromethane, Add triethylamine and react at room temperature for 12 to 48 hours; after quenching the reaction, the organic phase is concentrated and separated by column chromatography to obtain the final product Rap-R; wherein intermediate 5 and 4-(4,4,5,5-tetramethyl The molar ratio of -1,3,2-dioxaborolan-2-yl)benzyl chloroformate is 1:(2~3).

Concrete example:

(1) Synthesis of Intermediate 1

Rhodamine 6G (compound 1) was dissolved in a mixed solution of sodium hydroxide and ethanol, and reacted at 160°C for 48 hours; after cooling, the pH was adjusted to about 3-4 with hydrochloric acid solution, and the precipitated solid was filtered to obtain intermediate product 1;

(2) Synthesis of intermediate 2

Dissolve intermediate 1 and cyclohexanone in concentrated sulfuric acid, react at 95°C for 1.5 hours, add ice water after cooling to room temperature, and add HClO ₄ ; the precipitated solid is filtered to obtain intermediate 2; intermediate product 1 and cyclohexanone The feeding molar ratio is 1:1.3;

(3) Synthesis of intermediate 3

Dissolve compound 2 in concentrated sulfuric acid, react at 95°C for 1.5 hours, add ice water after cooling to room temperature, and add HClO ₄ ; the precipitated solid is filtered to obtain intermediate 3; wherein the molar ratio of intermediate product 2 and cyclohexanone is 1:1.3;

(4) Synthesis of Intermediate 4

Dissolve intermediate 3, malondialdehyde dianiline hydrochloride in a mixed solvent of acetic acid and acetic anhydride, and react at 110°C for 2 hours under the protection of nitrogen; after cooling to room temperature, the organic phase is concentrated and separated by column chromatography to obtain Intermediate 4;

(5) Synthesis of intermediate 5

Under nitrogen protection, intermediate 2 and intermediate 4 were added to a mixed solvent of toluene and n-butanol, and reacted at 100°C for 6 hours; after cooling to room temperature, the organic phase was concentrated and separated by column chromatography to obtain intermediate 5; , the molar ratio of intermediate product 2 and intermediate product 4 is 1:2;

(6) Synthesis of Rap-R

Intermediate 5 and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl chloroformate were dissolved in dichloromethane, Add triethylamine and react at room temperature for 24 hours; after quenching the reaction, the organic phase is concentrated and separated by column chromatography to obtain the final product Rap-R; wherein intermediate 5 and 4-(4,4,5,5-tetramethyl- The molar ratio of 1,3,2-dioxaborolan-2-yl)benzyl chloroformate is 1:3.

Under 808nm excitation, the change of fluorescence intensity of Rap-R ratiometric fluorescent probe in response to hydrogen peroxide is shown in Figure 3.

Example 3:

The ratio-type near-infrared fluorescent probe (Rap-R) of active oxygen response and the preparation method of phospholipid polyethylene glycol form micelles, the specific steps are as follows:

Dissolve the fluorescent probe Rap-R and DOPE-PEG ₂₀₀₀ in chloroform, the mass ratio of the two is 1:(20-100), after stirring for 0.5-2 hours, spin to dry the solvent, dry in vacuum, and heat to 50-80 After ℃, add deionized water to dissolve, sonicate, cool to room temperature, and then concentrate by ultrafiltration through an ultrafiltration tube to finally obtain Rap-R micellar solution.

Example 4:

Nitroreductase-responsive ratiometric near-infrared fluorescent probe Rap-N was used to monitor nitroreductase in tumors. Specific steps are as follows:

Inject 200 μL of Rap-N probe solution with a concentration of 100 μM into the tumor of anesthetized mice, and irradiate the mouse tumor with an external 808nm laser with a laser power density of 200mW/cm ² , using 900nm and 1000nm long-pass filters Fluorescence at 900-1700nm and 1000-1700nm were collected respectively and the intensity ratio of the two was calculated, and the content of nitroreductase was monitored according to the ratio of fluorescence signals of different tumors, as shown in Figure 4. In Fig. 4, the Rap-N probe was used to monitor the content of nitroreductase in 4T1 tumor and CT26 tumor, and the content of nitroreductase in the tumor was judged according to the ratio of fluorescence signals.

Example 5:

Rap-R, a ratiometric near-infrared fluorescent probe responsive to reactive oxygen species, was used to detect the content of reactive oxygen species (hydrogen peroxide) in inflamed tissues. Specific steps are as follows:

Inject 200 μL of Rap-R probe micellar solution with a concentration of 100 μM into the tail vein of anesthetized mice, and irradiate the mouse abdomen with an 808nm external laser with a laser power density of 200mW/cm ² , using 900nm and 1000nm long-pass Filters collect the fluorescence at 900-1700nm and 1000-1700nm respectively and calculate the intensity ratio of the two, and monitor the content of reactive oxygen species (hydrogen peroxide) in liver injury inflammation according to the ratio of non-fluorescence signals, as shown in Figure 5. In Figure 5, the Rap-R probe is used to monitor the content of reactive oxygen species (hydrogen peroxide) in liver injury in vivo, and the content of reactive oxygen species (hydrogen peroxide) is judged according to the ratio of the fluorescence signal. The content of active oxygen (hydrogen peroxide) after injury and drug treatment.

Claims (5)

1. a ratio type near-infrared fluorescent probe molecule is recorded as Rap, and it is characterized in that, its compound structural formula is as follows:

Wherein, X is selected from O or S; Y is selected from ClO ₄ , PF ₆ , BF ₄ , Cl, Br, I, CF ₃ COO, CF ₃ SO ₃ , CH ₃ COO or CH ₃ SO ₃ ; R ₁ is selected from H , OH, OCH ₃ or N[(CH ₂ )nCH ₃ ] ₂ ; R ₂ and R ₃ are H, o-carboxyphenyl or phenyl or o-methylphenyl, R ₄ is substituted benzyloxy, which can be selected from 4-nitrobenzyloxy, 4-phosphobenzyloxy, 4-glutamyl transpeptidylbenzyloxy, 4-β-galactosylbenzyloxy, azidobenzyloxy, 2,4-di Nitrobenzyloxy, 4-boronic acid benzyloxy or 4-boronic acid benzyloxy, n is an integer of 0-6. 2. a kind of preparation method of ratio type near-infrared fluorescent probe molecule as claimed in claim 1, is characterized in that, its synthetic route is as follows:

Wherein, X is selected from O or S; Y is selected from ClO ₄ , PF ₆ , BF ₄ , Cl, Br, I, CF ₃ COO, CF ₃ SO ₃ , CH ₃ COO or CH ₃ SO ₃ ; R ₁ is selected from H , OH, OCH ₃ or N[(CH ₂ )nCH ₃ ] ₂ ; R ₂ and R ₃ are H, o-carboxyphenyl or phenyl or o-methylphenyl, R ₄ is substituted benzyloxy, optional From 4-nitrobenzyloxy, 4-phosphobenzyloxy, 4-glutamyl transpeptidylbenzyloxy, 4-β-galactosylbenzyloxy, azidobenzyloxy, 2,4- Dinitrobenzyloxy, 4-boronic acid benzyloxy or 4-boronic acid benzyloxy, n is an integer from 0 to 6; compound 1 is substituted rhodamine 6G, compound 2 is substituted benzoyl, compound 3 is substituted acid chloride; The specific steps of preparation are as follows: (1) Synthesis of Intermediate 1: Compound 1 substituted rhodamine 6G was dissolved in a mixed solution of sodium hydroxide and ethanol, and reacted at 150-170°C for 36-48 hours; after cooling, the pH was adjusted to 3-4 with hydrochloric acid solution, and the precipitated solid was filtered to obtain intermediate product 1 ; (2) Synthesis of intermediate 2: Dissolve intermediate 1 and cyclohexanone in concentrated sulfuric acid, react at 80-100°C for 1-3 hours, add ice water after cooling to room temperature, add protonic acid, precipitate solid and filter to obtain intermediate 2; intermediate product 1 The molar ratio of feeding with cyclohexanone is 1:(1～3); (3) Synthesis of intermediate 3: Dissolve compound 2-substituted benzoyl in concentrated sulfuric acid, react at 80-100°C for 1-3 hours, add ice water after cooling to room temperature, add protonic acid, precipitate solid and filter to obtain intermediate 3; intermediate product 2 and cyclohexane The molar ratio of feeding ketones is 1:(1～3); (4) Synthesis of Intermediate 4: Dissolve intermediate 3, malondialdehyde dianiline hydrochloride in a mixed solvent of acetic acid and acetic anhydride, and react at 90-110°C for 1-3 hours under nitrogen protection; after cooling to room temperature, concentrate the organic phase and use Column chromatography separation, obtains intermediate 4; (5) Synthesis of Intermediate 5: Under the protection of nitrogen, add intermediate 2 and intermediate 4 into a mixed solvent of toluene and n-butanol, and react at 80-120°C for 3-6 hours; after cooling to room temperature, the organic phase is concentrated and separated by column chromatography to obtain Intermediate 5; the molar ratio of intermediate 2 and intermediate product 4 is 1:(1～3); (6) Synthesis of ratiometric near-infrared fluorescent probe molecules (product 1): Dissolve intermediate 5 and compound 3 substituted acid chloride in dichloromethane, add an organic base, and react at room temperature for 12 to 48 hours; after quenching the reaction, the organic phase is concentrated and separated by column chromatography to obtain product 1; intermediate 5 and substituted acid chloride The molar ratio of feeding is 1:(2~3). 3. The preparation method according to claim 2, characterized in that, the protonic acid described in step (2) and step (3) is selected from HClO ₄ , HPF ₆ , HBF ₄ , HCl, HBr, HI, CF ₃ COOH , CF ₃ SO ₃ H and CH ₃ SO ₃ H. 4. preparation method according to claim 2 is characterized in that, the organic base described in step (6) is selected from the one in triethylamine, N, N-diisopropylethylamine, pyridine. 5. The application of the ratiometric near-infrared fluorescent probe molecule as claimed in claim 1 in the preparation of biological deep tissue imaging, biosensing analysis reagents or micellar solutions.

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