A fluorescent dye, its preparation method and application

Abstract

The application provides a fluorescent dye and a preparation method and application thereof, and the fluorescent dye has the following chemical structural formula: wherein R1 and R2 are independently selected from H, Cl, Br and I; R3 is selected from alkyl,, and wherein n is a positive integer. The problems of low binding selectivity and overlapping of life fingerprints of traditional dyes in a complex lipid system are solved, and high-resolution identification of lipids such as CE, TG and PL in a serum environment is realized. Meanwhile, the lipids can be classified and detected in situ without extraction, and when the fluorescent dye is used as a probe, the dependence on chromatography and mass spectrometry technology for complicated pretreatment can be broken based on the synergistic effect of the fluorescent probe and the fluorescent lifetime imaging technology, so that the spatial distribution analysis and relative quantification of serum lipids are realized. In addition, a standardized classification criterion can be established, and a quantifiable lipid subtype analysis tool is provided for clinical use.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical detection materials and analysis technology, and particularly relates to a fluorescent dye, a preparation method and application thereof. BACKGROUND

[0002] Serum lipid subtype typing has key significance for the accurate diagnosis of cardiovascular diseases and metabolic syndrome. In the related technology, the dye in the application of fluorescence lifetime imaging (FLIM) is not sensitive enough to the lipid microenvironment, resulting in weak lifetime difference and highly overlapped distribution after binding with different lipids (typical as the characteristic lifetime values of cholesterol ester (CE) and triglyceride (TG) are close), and it is difficult to establish an effective classification threshold system. SUMMARY

[0003] The technical purpose of the present application is to provide a fluorescent dye, a preparation method and application thereof, aiming to solve the technical problem that the dye in the related technology is not sensitive enough to the lipid microenvironment, resulting in weak lifetime difference and highly overlapped distribution after binding with different lipids, and it is difficult to establish an effective classification threshold system.

[0004] To solve the above technical problems, the present application provides a fluorescent dye, which has the following chemical structure:

[0005] , wherein R1 and R2 are independently selected from H, Cl, Br and I; R3 is selected from alkyl, , and , wherein n is a positive integer.

[0006] Further, in some embodiments, the alkyl includes CH3 and C12-18 alkyl.

[0007] The second aspect of the present application provides a preparation method of the fluorescent dye, comprising:

[0008] Step 1, phenothiazine and a first substance are added to an organic solvent for stirring; a first intermediate is obtained by column chromatography separation; the first substance is selected from any one of halogenated alkyl, 2-(2-(2-methoxyethoxy)ethoxy)ethyl p-toluenesulfonate, 1,3-propane sulfolane, brominated PEG2000 and a second substance; the second substance includes 3-bromo-1-propanol and phosphorus oxychloride;

[0009] Step 2, under an inert atmosphere, phosphorus oxychloride is cooled to -5-5℃ by ice bath, an equal amount of DMF is added, stirred for 30 min, then the DMF solution of the first intermediate is added, warmed to 80-100℃, and stirred until the first intermediate is completely reacted; ice water is added to the mixture and the pH is adjusted to 7-8; extracted, washed and concentrated; the second intermediate is obtained by column chromatography separation;

[0010] Step 3, under inert atmosphere, dissolve the second intermediate in dichloromethane, drop trifluoroacetic acid, add 2,4-dimethylpyrrole in dichloromethane solution under light-proof condition, stir the solution; add 2,3-dichloro-5,6-dicyano-benzoquinone and stir; under ice bath, add triethylamine and boron trifluoride in ether solution and stir; after concentration, column chromatography is used to separate to obtain the fluorescent dye.

[0011] Further, in some embodiments, when the first substance in step 1 is selected from any one of halogenated alkyl, 2-(2-(2-methoxyethoxy)ethoxy)ethyl p-toluenesulfonate, 1,3-propane sulfone lactone and bromo-PEG2000, the step 1 comprises: placing phenothiazine, the first substance and KOH solution in dimethyl sulfoxide, heating to 55-65℃, stirring until the phenothiazine is completely consumed; adding ice water, extracting, washing and concentrating; purifying by column chromatography to obtain the first intermediate.

[0012] Further, in some embodiments, when the first substance in step 1 is 3-bromo-1-propanol and phosphorus oxychloride, the step 1 comprises: placing phenothiazine, 3-bromo-1-propanol and KOH solution in dimethyl sulfoxide, heating to 55-65℃, stirring until the phenothiazine is completely consumed; adding ice water, extracting, washing and concentrating; purifying by column chromatography to obtain the third intermediate; dissolving the third intermediate and pyridine in chloroform and cooling to-20--10℃; adding chloroform solution of phosphorus oxychloride, stirring and heating to 10-30℃; distilling under reduced pressure and washing to obtain the fourth intermediate; adding the fourth intermediate to deionized water, stirring the solution; extracting, washing and drying; distilling under reduced pressure to obtain the first intermediate.

[0013] Further, in some embodiments, before the operation step of placing phenothiazine, the first substance and KOH solution in dimethyl sulfoxide in step 1, it further comprises: drying dimethyl sulfoxide by a drying agent or activated molecular sieve.

[0014] Further, in some embodiments, before the operation step of dissolving the second intermediate in dichloromethane under inert atmosphere in step 3, it further comprises: drying dichloromethane by re-distillation.

[0015] Further, in some embodiments, the step 2 uses saturated sodium carbonate to adjust the pH value.

[0016] The third aspect of the embodiment of the present application provides application of the fluorescent dye to in-situ detection and analysis of lipids in serum.

[0017] The fourth aspect of the embodiment of the present application provides application of the fluorescent dye to preparation of a fluorescent probe targeting lipids.

[0018] The fluorescent dye, the preparation method and the application thereof in the application have the beneficial effects that, compared with related technologies, the problems of low binding selectivity and overlapping of life fingerprints of traditional dyes in a complex lipid system are solved, high-resolution recognition of lipids such as cholesteryl ester (CE), triglyceride (TG) and phospholipid (PL) in a serum environment is achieved. Meanwhile, the lipids can be classified and detected in situ without extraction, when the fluorescent dye is used as a probe, based on the synergistic effect of the fluorescent probe and fluorescence lifetime imaging (FLIM) technology, the dependence of chromatography and mass spectrometry technology on complicated pretreatment is broken through, the spatial distribution analysis and relative quantification of serum lipids are achieved. In addition, a standardized classification criterion can be established, the classification failure problem of the FLIM technology in related technologies due to overlapping of life distribution is solved by presetting a life threshold, and a quantifiable lipid subtype analysis tool is provided for clinical treatment. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a fluorescence spectrum diagram of the fluorescent dye in the embodiment of the application in tetrahydrofuran-methanol solutions with different volume ratios;

[0020] Figure 2 is a fluorescence lifetime decay diagram of the fluorescent dye in the embodiment of the application in tetrahydrofuran-methanol solutions with different volume ratios;

[0021] Figure 3 is a flowchart of the fluorescent dye for pure lipid detection in the embodiment of the application;

[0022] Figure 4 is a life comparison of the fluorescent dye and traditional dyes in combination with pure lipids in the embodiment of the application;

[0023] Figure 5 is a flowchart of the fluorescent dye for serum detection in the embodiment of the application;

[0024] Figure 6 is a serum FLIM diagram and data result diagram of normal people and hyperlipidemia patients in the embodiment of the application. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the application more clear, the application is further described in detail below in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the application, and are not used to limit the application.

[0026] Serum lipid subtype classification is of great significance for the precise diagnosis of cardiovascular diseases and metabolic syndrome. The related detection technology still has systematic limitations. Traditional biochemical methods (such as enzyme method and colorimetric method) are only limited to total amount analysis (such as total cholesterol and total triglyceride), and cannot realize specific analysis of lipid components; although the combination of chromatography and mass spectrometry (LC-MS / GC-MS) can provide component information at the molecular level, it relies on a complicated sample pretreatment process (including lipid extraction and derivatization steps), and the equipment cost is high and the analysis period is long, which is difficult to meet the clinical instant detection demand. The alternative solution based on optical imaging, especially the fluorescence analysis technology, also faces multiple bottlenecks: first, the emission spectrum of the traditional lipid probe (such as Nile red and conventional BODIPY series dye) after binding with different lipids is highly overlapped, which makes the classification strategy based on the fluorescence emission wavelength invalid; second, the fluorescence intensity is significantly dependent on the concentration, which is easily disturbed by the local enrichment concentration gradient and the light bleaching effect, and does not have the quantitative ability; third, the complex matrix of serum (such as albumin, apolipoprotein and metabolites) causes non-specific adsorption of the probe, resulting in a sharp increase in background noise and a decrease in signal-to-noise ratio; it is particularly important to note that in the related technology, the dye in the fluorescence lifetime imaging (FLIM) application is not sensitive enough to the lipid microenvironment, so that the lifetime difference after binding with different lipids is weak and the distribution is highly overlapped (such as the characteristic lifetime values of cholesterol ester (CE) and triglyceride (TG) tend to be close), which makes it difficult to establish an effective classification threshold system.

[0027] The first aspect of the embodiment of the present application provides a fluorescent dye, which has the following chemical structural formula:

[0028] , wherein R1 and R2 are independently selected from H, Cl, Br and I; R3 is selected from alkyl, , and , wherein n is a positive integer.

[0029] In the embodiment of the present application, the superiority of the fluorescent dye is embodied in the following aspects:

[0030] Strong microenvironment responsiveness: a phenothiazine-BODIPY fluorescent molecular system is constructed, so that the microenvironment responsiveness of the fluorescent dye can be strengthened, wherein the intramolecular electronic structure of phenothiazine can be used to adjust the intramolecular electronic push-pull ability, the sensitivity of the fluorescence lifetime to the local polarity and viscosity can be significantly enhanced, and the lifetime value τ becomes a specific reporter factor of the lipid microenvironment.

[0031] Serum compatibility regulation: different straight chains (alkyl chains, PEG) and ionized groups (sulfonic acid groups, phosphate groups) are modified on the periphery, so that the non-specific adsorption of serum proteins can be inhibited by the steric hindrance effect and electrostatic repulsion, and the anti-serum interference ability is strong; compared with traditional dyes, the signal-to-noise ratio is at least improved by 3 times.

[0032] Target structure direction optimization: the rigid hydrophobic skeleton of large plane is constructed by adopting phenothiazine derivatives, the dense alkyl chain area of cholesteryl ester is embedded by Van der Waals force, so that the lifetime value tau can be greater than 3.5 ns, and the long lifetime characteristic can be realized; and the BODIPY derivative containing C12-C18 alkyl chain can be designed in the fluorescent dye, and the flexible structure of the C12-C18 alkyl chain can be adapted to the liquid core of the triglyceride, so that the lifetime value tau can be between 2.5-3 ns, so that the medium lifetime response is generated; furthermore, the fluorescent dye can also construct an amphiphilic structure (such as a phosphatidylcholine mimic), wherein the hydrophilic head group (phosphocholine) can anchor the surface polarity region of the lipid bilayer, and induce a short lifetime state (tau < 2.0 ns); in this way, the problem of insufficient lipid FLIM resolution in serum can be solved, and the lifetime difference is significant.

[0033] Excitation spectrum synergy: by adjusting the power supply group and the type of branch, the fluorescence lifetime response range and biocompatibility of the fluorescent dye are extended, so that when the fluorescent dye is used as a probe, the maximum absorption peaks of all fluorescent probes converge in the range of 530-560 nm, which is suitable for the standard laser light source of 488 nm, and the fluorescence lifetime of the fluorescent probe is adjusted in the range of 1.7-5 ns.

[0034] Therefore, the embodiment of the present application solves the problems of low binding selectivity and overlapping lifetime fingerprint of traditional dyes in a complex lipid system, and realizes high-resolution identification of cholesterol ester (CE), triglyceride (TG), phospholipid (PL) and other lipid categories in serum environment. At the same time, the lipid can be classified and detected in situ without extraction, and when the fluorescent dye is used as a probe, the synergistic effect of the fluorescent probe and the fluorescence lifetime imaging (FLIM) technology can be used to break through the dependence of chromatography and mass spectrometry technology on complicated pretreatment, realize spatial distribution analysis and relative quantification of serum lipid components. In addition, a standardized classification criterion can be established, and the classification failure problem caused by overlapping lifetime distribution in the related technology can be solved by presetting the lifetime threshold, so as to provide a quantifiable lipid subtype analysis tool for clinical use.

[0035] The second aspect of the embodiment of the present application provides a preparation method of a fluorescent dye, comprising:

[0036] Step 1, phenothiazine and a first substance are added to an organic solvent for stirring; a first intermediate is obtained by column chromatography separation; the first substance is selected from any one of halogenated alkyl, 2-(2-(2-methoxyethoxy)ethoxy)ethyl p-toluenesulfonate, 1,3-propane sulfolane, bromo-PEG2000 and a second substance; the second substance includes 3-bromo-1-propanol and phosphorus oxychloride;

[0037] Step 2, under inert atmosphere, cool phosphorus oxychloride to -5~5°C by ice bath, add DMF (N,N-dimethylformamide) and stir for 30 min, then add the DMF (N,N-dimethylformamide) solution of the first intermediate, warm to 80~100°C, and stir until the first intermediate reaction is complete; add ice water to the mixture and adjust the pH to 7~8; extract, wash and concentrate; column chromatography to separate the second intermediate;

[0038] Step 3, under inert atmosphere, dissolve the second intermediate in dichloromethane, drop trifluoroacetic acid, add 2,4-dimethylpyrrole in dichloromethane solution under light-free conditions, stir the solution; add 2,3-dichloro-5,6-dicyanoquinone and stir; under ice bath, add triethylamine and boron trifluoride ether solution and stir; after concentration, column chromatography to separate the fluorescent dye.

[0039] The fluorescent dye can be obtained by the above preparation method, which can solve the problems of low binding selectivity and overlapping of lifetime fingerprints of traditional dyes in complex lipid systems, and realize high-resolution recognition of lipid categories such as cholesterol ester (CE), triglyceride (TG), phospholipid (PL) and the like in serum environment. At the same time, the lipid can be classified and detected in situ without extraction, and when the fluorescent dye is used as a probe, based on the synergistic effect of the fluorescent probe and fluorescence lifetime imaging (FLIM) technology, the dependence on tedious pretreatment of chromatography and mass spectrometry technology can be broken through, and the spatial distribution analysis and relative quantification of serum lipids can be realized. In addition, a standardized classification criterion can be established, and the classification failure problem caused by the overlapping of lifetime distribution in the related technology can be solved by presetting the lifetime threshold, thereby providing a quantifiable lipid subtype analysis tool for clinical use.

[0040] Further, in some embodiments, the first substance in step 1 can be selected from any one of halogenated alkyl, 2-(2-(2-methoxyethoxy)ethoxy)ethyl p-toluenesulfonate, 1,3-propane sulfolane and brominated PEG2000, at this time, the step 1 specifically comprises: placing phenothiazine, the first substance and KOH solution in dimethyl sulfoxide, warming to 55~65°C, and stirring until the phenothiazine is completely consumed; add ice water, extract, wash and concentrate; purify by column chromatography to obtain the first intermediate.

[0041] Specifically, step 1 is a step of preparing the first intermediate. The phenothiazine, the first substance and dimethyl sulfoxide can be mixed in an alkaline environment, and the temperature is raised at the same time, so that the whole reaction is carried out at 55-65°C, and the specific reaction temperature can be 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65°C, etc. In the actual preparation process, the reaction can be monitored by TLC until the phenothiazine is completely consumed, that is, the reaction is completed. In addition, after the reaction is completed, it can be cooled to room temperature (10-30°C) first, and then the reaction mixture is poured into ice water, extracted with dichloromethane, washed with water and concentrated. In addition, the crude product obtained after concentration is purified by silica gel column chromatography to obtain the first intermediate.

[0042] It can be understood that the first intermediate prepared by the above steps is a fluorescent dye, and the corresponding fluorescent dye can be:

[0043]

[0044] Further, in some embodiments, when the first substance of step 1 is 3-bromo-1-propanol and phosphorus oxychloride, step 1 comprises: placing the phenothiazine, 3-bromo-1-propanol and KOH solution in dimethyl sulfoxide, heating to 55-65°C, and stirring until the phenothiazine is completely consumed; add ice water, extract, wash and concentrate; purify the third intermediate by column chromatography; dissolve the third intermediate and pyridine in chloroform and cool to -20 to -10°C; slowly add a chloroform solution of phosphorus oxychloride, stir and heat to 10-30°C; distill under reduced pressure and wash to obtain the fourth intermediate; add the fourth intermediate to deionized water, stir the liquid; extract, wash and dry; distill under reduced pressure to obtain the first intermediate.

[0045] Specifically, step 1 is a step of preparing the first intermediate, which includes three processes.

[0046] The first process: the phenothiazine, the first substance and dimethyl sulfoxide can be mixed in an alkaline environment, and the temperature is raised at the same time, so that the whole reaction is carried out at 55-65°C, and the specific reaction temperature can be 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65°C, etc. In the actual preparation process, the reaction can be monitored by TLC until the phenothiazine is completely consumed, that is, the reaction is completed. In addition, after the reaction is completed, it can be cooled to room temperature (10-30°C) first, and then the reaction mixture is poured into ice water, extracted with dichloromethane, washed with water and concentrated. In addition, the crude product obtained after concentration is purified by silica gel column chromatography to obtain the third intermediate.​​​​​​​

[0047] The second process: the third intermediate and phosphorus oxychloride are further reacted, the third intermediate and pyridine can be first dissolved in chloroform and cooled to -20~ -10℃, which can be -20, -19, -18, -17, -16, -15, -14, -13, -12, -11 or -10℃, etc.; then slowly add the chloroform solution of phosphorus oxychloride, keep stirring at the cooling temperature for a period of time; finally, slowly warm the whole system to room temperature, continue to stir; remove chloroform by reduced pressure distillation, and wash the crude product with toluene to obtain the fourth intermediate.

[0048] The third process: the fourth intermediate is added to deionized water, and the solution is stirred at room temperature; the obtained crude product is extracted with ethyl acetate; after washing with water, it is dried with anhydrous sodium sulfate; then remove ethyl acetate by reduced pressure distillation to obtain the first intermediate.

[0049] It can be understood that the first intermediate obtained by the above steps is used to prepare a fluorescent dye, and the corresponding fluorescent dye can be: .

[0050] Further, in some embodiments, before the phenothiazine, the first substance and the KOH solution are placed in dimethyl sulfoxide in step 1, the dimethyl sulfoxide can be dried by a desiccant or activated molecular sieve, so that the water and impurities in the dimethyl sulfoxide can be removed, so that the dimethyl sulfoxide can be kept dry and pure, and the subsequent steps will not be disturbed by water and impurities, which is beneficial to the preparation of fluorescent dyes.

[0051] Further, in some embodiments, in step 2, under an inert atmosphere, the phosphorus oxychloride is cooled to -5~5℃ by ice bath, DMF (N, N-dimethylformamide) is added and stirred for 30 min, the reaction system is discolored, then the DMF solution of the first intermediate is added, and the temperature is raised to 80~100℃ and stirred until the first intermediate is completely reacted; add ice water to the mixture and adjust the pH to 7~8; extract, wash and concentrate; column chromatography is used to separate the second intermediate.

[0052] Specifically, DMF and phosphorus oxychloride are reacted at low temperature to generate Vilsmeier reagent, which is then subjected to electrophilic substitution with the first intermediate, followed by hydrolysis to generate the formylated product. Additionally, by way of example, the inert gas can be argon, helium, or the like. Understandably, in some embodiments, the reaction of Step 2 can also be carried out under nitrogen atmosphere. Step 2 can be carried out under ice bath condition with phosphorus oxychloride cooling, which can be at -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, or 5°C, or the like. DMF is added dropwise at low temperature until color change; the temperature is then slowly increased and the DMF solution of the first intermediate is added dropwise, which can be at 80, 80, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 100°C, or the like, and the reaction is carried out at the increased temperature, which can be monitored by thin layer chromatography (TLC) until the first intermediate is completely reacted. After the reaction is completed, the mixture is poured into ice water while hot, which can be adjusted with saturated sodium carbonate solution; dichloromethane is used to extract the product, which is then washed with water and concentrated. The crude product after concentration is purified by silica gel column chromatography to obtain the second intermediate.

[0053] It should be understood that the TLC monitoring of the reaction is a routine technique in the art and will not be described here.

[0054] Further, in some embodiments, the operation of adding triethylamine and boron trifluoride ether solution under ice bath in Step 3 includes: slowly adding triethylamine under ice bath, stirring for 15-45 min, and continuously adding boron trifluoride ether solution, stirring for 8-12 h.

[0055] Specifically, triethylamine and boron trifluoride ether solution can be added in Step 3 in sequence, so that the triethylamine added first can neutralize the acid in the reaction system and form an alkaline environment in the reaction system, promoting the coordination of the substrate and boron trifluoride. At this time, Step 3 can be specifically as follows: under inert atmosphere, the second intermediate is dissolved in dichloromethane, trifluoroacetic acid is added, 2,4-dimethylpyrrole in dichloromethane is slowly added dropwise under light shielding condition, and the liquid is stirred; 2,3-dichloro-5,6-dicyanoquinone (DDQ) is added and stirred; triethylamine is slowly added dropwise under ice bath, stirring for 15-45 min, boron trifluoride ether (BF3·Et2O) solution is continuously added dropwise, stirring for 8-12 h; after concentration, column chromatography is used for separation to obtain the fluorescent dye. By way of example, the inert gas can be argon, helium, nitrogen, or the like.

[0056] In addition, in step 3, before the second intermediate is dissolved in dichloromethane under inert atmosphere, the dichloromethane is dried by re-evaporation. In this way, the water and impurities in the dichloromethane can be removed, so that the dichloromethane can be kept dry and pure, and the subsequent steps will not be disturbed by water and impurities, which is beneficial to the preparation of the fluorescent dye.

[0057] Further, for the preparation method of the fluorescent dye, the following is further illustrated by specific examples, and it should be understood that the specific examples described herein are only used to explain the present application, but not to limit the present application.

[0058] Example 1

[0059] Step i, 1 mmol of phenothiazine (A0), 1.5 mmol of iodomethane and 3 mmol of KOH were stirred in 50 ml of dry dimethyl sulfoxide, and the reaction was monitored by TLC until the phenothiazine was completely consumed. After cooling to room temperature, it was poured into ice water, extracted with dichloromethane, washed with water and concentrated. The crude product was purified by silica gel column chromatography (a mixed system of dichloromethane and petroleum ether mixed at a volume ratio of 1:2) to obtain the first intermediate (A1).

[0060] Step ii, under argon, 10 mmol of phosphorus oxychloride was cooled to 0°C in an ice bath, 50 mmol of DMF was slowly added dropwise, and the reaction system was stirred for 30 minutes until it turned light yellow. 1 mmol of the first intermediate was slowly added dropwise in DMF, and the temperature was slowly raised to 90°C. The reaction was monitored by TLC until the first intermediate was completely consumed. The mixture was quickly poured into ice water, the pH was adjusted to 7.5 with saturated sodium carbonate solution, extracted with dichloromethane, washed with water and concentrated. The crude product was purified by silica gel column chromatography (a mixed system of dichloromethane and petroleum ether mixed at a volume ratio of 1:2) to obtain the second intermediate (A2).

[0061] Step iii, under argon, 1 mmol of the second intermediate was dissolved in dry dichloromethane, 3 drops of trifluoroacetic acid (TFA) were added, 2.1 mmol of 2,4-dimethylpyrrole in dichloromethane was slowly added dropwise in the dark, and the solution was stirred at room temperature. 1 mmol of 2,3-dichloro-5,6-dicyanoquinone was added and stirred for 4 hours. 5 mL of triethylamine (TEA) was slowly added dropwise under ice bath, stirred for 30 minutes, and then 5 mL of boron trifluoride etherate (BF3·Et2O) solution was added dropwise. The reaction mixture was concentrated and purified by silica gel column chromatography (a mixed system of dichloromethane and petroleum ether mixed at a volume ratio of 1:2) to obtain the fluorescent dye (PTZ-BDP-C1).

[0062] In which, the chemical reaction formula involved in Example 1 is as follows:

[0063]

[0064] Example 2

[0065] Step i, 1 mmol of phenothiazine (A0), 1.5 mmol of bromododecane and 3 mmol of KOH were stirred in 50 mL of dry dimethyl sulfoxide, and the temperature was raised to 60°C. The reaction was monitored by TLC until the phenothiazine was completely consumed. After cooling to room temperature, it was poured into ice water, extracted with dichloromethane, washed with water and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the first intermediate (B1).

[0066] Step ii, 10 mmol of phosphorus oxychloride was cooled to 0°C under argon, and 50 mmol of DMF was slowly added dropwise. After stirring for 30 minutes until the reaction system turned light yellow, 1 mmol of the first intermediate was added dropwise in DMF, and the temperature was slowly raised to 90°C. The reaction was monitored by TLC until the first intermediate was completely consumed. The mixture was quickly poured into ice water, and the pH was adjusted to 7 with saturated sodium carbonate solution. After extraction with dichloromethane, washing with water and concentration, the crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the second intermediate (B2).

[0067] Step iii, 1 mmol of the second intermediate was dissolved in dry dichloromethane, and 3 drops of trifluoroacetic acid (TFA) were added dropwise. 2.1 mmol of 2,4-dimethylpyrrole in dichloromethane was slowly added dropwise in the dark, and the solution was stirred at room temperature. 1 mmol of 2,3-dichloro-5,6-dicyanoquinone was added, and stirring was continued for 4 hours. 5 mL of triethylamine (TEA) was slowly added dropwise under ice bath, and stirring was continued for 30 minutes. 5 mL of boron trifluoride etherate (BF3·Et2O) solution was added dropwise, and stirring was continued for 10 hours. The reaction mixture was concentrated and purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the fluorescent dye (PTZ-BDP-C12).

[0068] Among them, the chemical reaction formula involved in Example 2 is as follows:

[0069]

[0070] Example 3

[0071] Step i, 1 mmol of phenothiazine (A0), 1.5 mmol of bromooctadecane and 3 mmol of KOH were stirred in 50 mL of dry dimethyl sulfoxide, and the temperature was raised to 60°C. The reaction was monitored by TLC until the phenothiazine was completely consumed. After cooling to room temperature, the mixture was poured into ice water, extracted with dichloromethane, washed with water and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the first intermediate (C1).

[0072] Step ii, 10 mmol of phosphorus oxychloride was cooled to 0°C under argon, and 50 mmol of DMF was slowly added dropwise. After stirring for 30 minutes until the reaction system turned light yellow, 1 mmol of the first intermediate was added dropwise in DMF, and the temperature was slowly raised to 90°C. The reaction was monitored by TLC until the first intermediate was completely consumed. The mixture was quickly poured into ice water, and the pH was adjusted to 7.5 with saturated sodium carbonate solution. After extraction with dichloromethane, washing with water and concentration, the crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the second intermediate (C2).

[0073] Step iii, 1 mmol of the second intermediate was dissolved in dry dichloromethane, and 3 drops of trifluoroacetic acid (TFA) were added dropwise. 2.1 mmol of 2,4-dimethylpyrrole in dichloromethane was slowly added dropwise in the dark, and the solution was stirred at room temperature. 1 mmol of 2,3-dichloro-5,6-dicyanoquinone was added, and stirring was continued for 4 hours. 5 mL of triethylamine (TEA) was slowly added dropwise under ice bath, and stirring was continued for 30 minutes. 5 mL of boron trifluoride etherate (BF3·Et2O) solution was added dropwise, and stirring was continued for 10 hours. The reaction mixture was concentrated and purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the fluorescent dye (PTZ-BDP-C18).

[0074] In Example 3, the chemical reaction formula is as follows:

[0075]

[0076] Example 4

[0077] Step i, stir 1 mmol phenothiazine (A0), 1.5 mmol 2-(2-(2-methoxyethoxy)ethoxy)ethyl p-toluenesulfonate and 3 mmol KOH in 50 mL dry dimethyl sulfoxide, warm up to 60 °C, monitor the reaction by TLC until the phenothiazine is consumed completely. Cool down to room temperature, pour into ice water, extract with dichloromethane, wash with water and concentrate. Purify the crude product by silica gel column chromatography (a mixture of dichloromethane and petroleum ether mixed in a volume ratio of 1:2) to obtain the first intermediate (D1).

[0078] Step ii, cool 10 mmol phosphorus oxychloride to 0 °C under argon, slowly add 50 mmol DMF, stir for 30 minutes until the reaction system turns light yellow, slowly add 1 mmol DMF solution of the first intermediate, and slowly warm up to 90 °C. Stir the reaction until the first intermediate is completely consumed by TLC monitoring. Quickly pour the mixture into ice water, adjust the pH to 7.5 with saturated sodium carbonate solution, extract with dichloromethane, wash with water and concentrate. Purify the crude product by silica gel column chromatography (a mixture of dichloromethane and petroleum ether mixed in a volume ratio of 1:2) to obtain the second intermediate (D2).

[0079] Step iii, dissolve 1 mmol of the second intermediate in dry dichloromethane, add 3 drops of trifluoroacetic acid (TFA), slowly add 2.1 mmol 2,4-dimethylpyrrole in dichloromethane solution under light protection, stir the solution at room temperature. Add 1 mmol 2,3-dichloro-5,6-dicyano-benzoquinone and continue stirring for 4 hours, slowly add 5 mL triethylamine (TEA) under ice bath, stir for 30 minutes, continue to add 5 mL boron trifluoride etherate (BF3·Et2O) solution, stir for 10 hours. Concentrate the reaction mixture and purify by silica gel column chromatography (a mixture of dichloromethane and petroleum ether mixed in a volume ratio of 1:2) to obtain the fluorescent dye (PTZ-BDP-Ether).

[0080] Wherein, the chemical reaction formula involved in Example 4 is as follows:

[0081]

[0082] Example 5

[0083] Step i, 1 mmol of phenothiazine (A0), 1.5 mmol of 1,3-propane sultone and 3 mmol of KOH were stirred in 50 mL of dry dimethyl sulfoxide, and the temperature was raised to 60°C. The reaction was monitored by TLC until the phenothiazine was consumed. After cooling to room temperature, the mixture was poured into ice water, extracted with dichloromethane, washed with water and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the first intermediate (E1).

[0084] Step ii, 10 mmol of phosphorus oxychloride was cooled to 0°C under argon, and 50 mmol of DMF was slowly added dropwise. After stirring for 30 minutes until the reaction system turned light yellow, 1 mmol of the first intermediate was added dropwise in DMF, and the temperature was slowly raised to 90°C. The reaction was monitored by TLC until the first intermediate was consumed. The mixture was quickly poured into ice water, and the pH was adjusted to 7.5 with saturated sodium carbonate solution. After extraction with dichloromethane, washing with water and concentration, the crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the second intermediate (E2).

[0085] Step iii, 1 mmol of the second intermediate was dissolved in dry dichloromethane, and 3 drops of trifluoroacetic acid (TFA) were added dropwise. 2.1 mmol of 2,4-dimethylpyrrole in dichloromethane was slowly added dropwise in the dark, and the solution was stirred at room temperature. 1 mmol of 2,3-dichloro-5,6-dicyanoquinone was added, and stirring was continued for 4 hours. 5 mL of triethylamine (TEA) was slowly added dropwise under ice bath, and stirring was continued for 30 minutes. 5 mL of boron trifluoride etherate (BF3·Et2O) solution was added dropwise, and stirring was continued for 10 hours. The reaction mixture was concentrated and purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the fluorescent dye (PTZ-BDP-SO3).

[0086] In Example 5, the chemical reaction formula is as follows:

[0087]

[0088] Example 6

[0089] Step i, 1 mmol of phenothiazine (A0), 3 mmol of KOH and 1.5 mmol of 1,3-propane sultone (3-Bromo-1-propanol) were stirred in 50 mL of dry dimethyl sulfoxide, and the temperature was raised to 60°C. The reaction was monitored by TLC until the phenothiazine was completely consumed. After cooling to room temperature, the mixture was poured into ice water, extracted with dichloromethane, washed with water and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the third intermediate (F1).

[0090] Step ii, 1 mmol of the first intermediate and 1 mL of pyridine were dissolved in 50 mL of dry chloroform and cooled to -15°C. A mixture of 4.5 mL of phosphorus oxychloride and 40 mL of chloroform was slowly added dropwise over 30 min. The reaction was stirred at -15°C for 2 h, and then the temperature was slowly raised to room temperature. The stirring was continued for 1.5 h. The chloroform was removed by distillation under reduced pressure, and the crude product was washed with toluene to obtain the fourth intermediate as a green oil (F2).

[0091] Step iii, the crude product remaining from step ii was added to 50 mL of deionized water, and the solution was stirred at room temperature. The crude product was extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate, and the ethyl acetate was removed by distillation under reduced pressure to obtain the first intermediate as a white solid (F3).

[0092] Step iv, 10 mmol of phosphorus oxychloride was cooled to 0°C under an argon atmosphere in an ice bath, and 50 mmol of DMF was slowly added dropwise. The reaction was stirred for 30 min until the reaction mixture turned light yellow. Then, 1 mmol of the fourth intermediate in DMF was slowly added dropwise, and the temperature was slowly raised to 90°C. The reaction was monitored by TLC until the first intermediate was completely consumed. The mixture was quickly poured into ice water, and the pH was adjusted to 8 using a saturated sodium carbonate solution. The mixture was extracted with dichloromethane, washed with water, and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the second intermediate (F4).

[0093] Step v, under argon, 1 mmol of the second intermediate was dissolved in dry dichloromethane, 3 drops of trifluoroacetic acid (TFA) was added, 2.1 mmol of 2,4-dimethylpyrrole in dichloromethane was added dropwise slowly under dark, the solution was stirred at room temperature. 1 mmol of 2,3-dichloro-5,6-dicyano-benzoquinone was added and stirred for 4 hours, 5 mL of triethylamine (TEA) was added dropwise slowly under ice bath, stirred for 30 minutes, 5 mL of boron trifluoride etherate (BF3·Et20) solution was added dropwise, stirred for 10 hours. The reaction mixture was concentrated and purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the fluorescent dye (PTZ-BDP-PO3).

[0094] The chemical reaction formula involved in Example 6 is as follows:

[0095]

[0096] Example 7

[0097] Step i, 1 mmol of phenothiazine (A0), 1.5 mmol of bromo-PEG2000 (PEG 2000 Br) and 3 mmol of KOH were stirred in 50 mL of dry dimethyl sulfoxide, and the temperature was raised to 60°C. The reaction was monitored by TLC until the phenothiazine was completely consumed. After cooling to room temperature, it was poured into ice water, extracted with dichloromethane, washed with water and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the first intermediate (G1).

[0098] Step ii, under argon, 10 mmol of phosphorus oxychloride was cooled to 0°C under ice bath, 50 mmol of DMF was added dropwise slowly, stirred for 30 minutes until the reaction system turned light yellow, 1 mmol of the first intermediate in DMF was added dropwise slowly, and the temperature was slowly raised to 90°C. The reaction was stirred until the first intermediate was completely consumed by TLC monitoring. The mixture was quickly poured into ice water, the pH was adjusted to 7.5 with saturated sodium carbonate solution, extracted with dichloromethane, washed with water and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the second intermediate (G2).

[0099] Step iii, under argon, dissolve 1 mmol of the second intermediate in dry dichloromethane, add 3 drops of trifluoroacetic acid (TFA), slowly add 2.1 mmol of 2,4-dimethylpyrrole in dichloromethane solution under dark, stir the solution at room temperature. Add 1 mmol of 2,3-dichloro-5,6-dicyano-benzoquinone, continue to stir for 4 hours, slowly add 5 mL of triethylamine (TEA) in an ice bath, stir for 30 minutes, continue to add 5 mL of boron trifluoride etherate (BF3·Et2O) solution, stir for 10 hours. Concentrate the reaction mixture and purify by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the fluorescent dye (PTZ-BDP-PEG).

[0100] The chemical reaction formula involved in Example 7 is as follows:

[0101]

[0102] Example 8

[0103] The prepared fluorescent dyes PTZ-BDP-C1, PTZ-BDP-C12, and PTZ-BDP-C18 were dissolved in DMSO to obtain a 1 mM stock solution. The stock solution was further diluted with n-hexane, tetrahydrofuran, DMSO, and methanol to obtain a 1 μM test solution. These solutions were tested for absorption, emission spectrum, and fluorescence lifetime. The absorption spectrum was measured by a UV-Vis spectrophotometer, and the fluorescence spectrum was measured by a fluorescence spectrometer. For example, the UV-Vis spectrophotometer can be an Agilent Cary 4000 UV-Vis spectrophotometer, and the fluorescence spectrometer can be a HORIBA FluoroMax+ fluorescence spectrometer. As shown in Table 1, the maximum absorption wavelength (λabs), the maximum emission wavelength (λem), and the fluorescence lifetime (τ) of the fluorescent dyes PTZ-BDP-C1, PTZ-BDP-C12, and PTZ-BDP-C18 in different solvents are specifically listed in Table 1. From the data in Table 1, it can be seen that the maximum emission wavelength of the fluorescent dyes PTZ-BDP-C1, PTZ-BDP-C12, and PTZ-BDP-C18 is between 500-600 nm, and they have a fluorescence lifetime response to different polar solvents and a positive correlation between the fluorescence lifetime and the polarity of the solvent.

[0104] Table 1. Spectral properties of PTZ-BDP-C1, PTZ-BDP-C12, and PTZ-BDP-C18

[0105]

[0106] Example 9

[0107] Fluorescent dyes PTZ-BDP-C1, PTZ-BDP-C12, and PTZ-BDP-C18 were dissolved in DMSO to obtain 1 mM stock solutions. The stock solutions were diluted with tetrahydrofuran-methanol mixtures at different volume ratios (total volume 3 mL, concentration 10 μM, volume percentage of tetrahydrofuran in the mixtures 0%, 20%, 40%, 60%, 80%, and 100%) to obtain test solutions. The fluorescence spectra and fluorescence lifetime decay spectra of these test solutions were obtained using a fluorescence spectrometer, exemplarily a HORIBA FluoroMax+ fluorescence spectrometer. Figure 1 and Figure 2 As shown, fluorescence intensity and fluorescence lifetime change with increasing volume percentage of tetrahydrofuran. Utilizing the excellent power supply properties of phenothiazine, the intramolecular charge transfer (ICT) of the fluorescent dye is effectively improved, significantly enhancing the fluorescence lifetime of the fluorescent dye molecule's responsiveness to the microenvironment. Compared to the 2-3 ns of conventional BODIPY dyes, the fluorescent dye of this invention achieves a lifetime of 1-5 ns.

[0108] Example 10

[0109] Dissolve the fluorescent dye PTZ-BDP-C1 in DMSO to obtain a 1 mM stock solution, and proceed as follows: Figure 3 The procedure shown involves mixing the stock solution with pure lipid cholesterol, triglycerides, and phospholipids at a ratio of 1:50, incubating for 2 minutes, spotting each sample onto a glass slide, mounting with a neutral mounting medium, and then imaging using a fluorescence lifetime imaging system. Data were statistically analyzed using 5 samples per group and 5 fields of view per sample. Specific results are recorded in Table 2 and... Figure 4 In the middle. At the same time, according to... Figure 5 The steps shown are as follows: blood samples are centrifuged to obtain serum, and the serum samples are statistically analyzed following the same steps as those for processing pure lipids, including sample preparation, imaging, and analysis. For lifetime analysis of the serum samples, a phasor plot is used for analysis and classification. Specific results are recorded in Table 2. Additionally, Nile Red dye is used as a control group, and the same procedure is followed. Figure 3 The steps shown and as Figure 5 The steps shown involve mixing the dye Nile Red with pure lipids to obtain the corresponding data results, and simultaneously mixing the dye Nile Red with serum samples to obtain the corresponding data results. The specific results are recorded in Table 2.

[0110] According to Table 2 and Figure 4The results show that the fluorescent dye of the present invention can solve the problems of low binding selectivity and overlapping lifetime fingerprints of traditional dyes in complex lipid systems, and achieve high-resolution identification of lipid categories such as cholesterol esters (CE), triglycerides (TG), and phospholipids (PL) in the serum environment; at the same time, it has an extremely high signal-to-noise ratio and high reliability.

[0111] Table 2 Dye Performance Verification

[0112]

[0113] Example 11

[0114] according to Figure 5 The sample preparation process described involves preparing serum samples from 10 normal individuals (N) and 10 patients with hyperlipidemia (P), and staining them with PTZ-BDP-C1, PTZ-BDP-C12, and PTZ-BDP-C18 dyes. Simultaneously, data are acquired and statistically analyzed using a FLIM imaging system. Figure 6 As shown in Figure a, FLIM images reveal large lipid aggregates (>5 μm) in the patient's serum; further analysis of FLIM images using phasor plots yielded... Figure 6 As shown in Figure b, the proportion of CE-enriched areas increased by 35% (p<0.01) and the proportion of TG-enriched areas increased by 42% (p<0.001) in the patient group.

[0115] Furthermore, a third aspect of the present invention provides the application of fluorescent dyes for in-situ detection and analysis of lipids in serum.

[0116] In this embodiment of the invention, fluorescent dye and serum sample are mixed, stained, and then mounted as a slide for imaging using a fluorescence lifetime imaging system. The volume ratio of the fluorescent dye solution to the serum sample is 1:50.

[0117] The specific operation process can be: the serum is pretreated, diluted or low-speed centrifuged to remove particulate matter; a fluorescent dye is added, and incubated for 2-45 minutes; the mixture is evenly spotted on a glass slide, dried, and then sealed with a neutral resin to obtain a sealed slide; the sealed slide is used for pulsed laser excitation, photon time information is collected, 2D / 3D fluorescence lifetime data are obtained, and preliminary noise filtering and background subtraction processing are performed. Due to the strong anti-serum interference ability of the fluorescent dye and the high signal-to-noise ratio, lipid extraction is not required, and the test can be fast and simple, and the whole process can be completed in less than 1 hour; at the same time, CE / TG / PL subtypes can also be accurately distinguished, and the accuracy is more than 90%, which provides a new lipid subtype diagnostic index (such as CE / TG ratio) for cardiovascular diseases, fatty liver and the like; and the test stability is high, the fluorescence lifetime is not affected by the concentration and / or light bleaching, and the RSD (relative standard deviation) is less than 5%; in addition, as shown in Figure 6 the lipid distribution and lipoprotein structure can also be intuitively presented.

[0118] In addition, it can be understood that in some embodiments, the serum sample can be replaced by a whole blood sample; in addition, the fluorescent dye can also be extended to lipid analysis of cells and tissues and the like.

[0119] Further, in some embodiments, the concentration of the fluorescent dye and the incubation conditions can be optimized, and the optimal staining concentration range of the fluorescent dye can be found for different types of serum samples (normal, high-fat, disease-related, etc.). By performing staining experiments at a series of concentration gradients and combining FLIM measurement, the optimal concentration that can ensure sufficient signal intensity while avoiding dye aggregation and non-specific binding can be found. At the same time, different incubation conditions, including temperature, time and pH value, etc. can be explored to affect the binding efficiency of the fluorescent dye and the lipid. By designing an experimental matrix, the most suitable incubation parameter combination can be determined to ensure that the dye and serum lipid are fully combined in a short time and stable characteristic fluorescence lifetime signals are generated.

[0120] Further, the fourth aspect of the embodiment of the present application provides the application of the fluorescent dye for preparing a lipid analysis diagnostic product. Specifically, according to the characteristics of the fluorescent dye, related products can be made according to the fluorescent dye, which can be used for lipid detection and analysis diagnosis, and the practicability of the fluorescent dye is improved.

[0121] Further, the fifth aspect of the embodiment of the present application provides the application of the fluorescent dye for preparing a fluorescent probe for targeting lipid. Specifically, according to the characteristics of the fluorescent dye, a fluorescent probe for targeting lipid can be made according to the fluorescent dye, which can be used for monitoring lipid in serum, playing a role in rapid detection of lipid and prevention of diseases, and improving practicability.

Claims

1. A fluorescent dye, characterized in that, The fluorescent dye has the following chemical structural formula: wherein R1, R2are independently selected from H, Cl, Br and I; R3is selected from C12-18 alkyl.

2. The method for preparing the fluorescent dye according to claim 1, characterized in that, Comprising: Step 1, the phenothiazine, the first substance and KOH solution are placed in dimethyl sulfoxide, heated to 55~65℃, stirring until the phenothiazine is consumed completely; add ice water, extract, wash and concentrate; The first intermediate is purified by column chromatography; the first substance is selected from halogenated alkyl; Step 2, under inert atmosphere, the phosphorus oxychloride is cooled to-5~5℃ by ice bath, add equal amount of DMF and stir for 30 minutes, then add the DMF solution of the first intermediate, heat to 80~100℃, and stir until the first intermediate is completely reacted; add ice water to the mixture and adjust the pH to 7~8; extract, wash and concentrate; The second intermediate is obtained by column chromatography separation; Step 3, under inert atmosphere, the second intermediate is dissolved in dichloromethane, drop in trifluoroacetic acid, add 2,4-dimethylpyrrole dichloromethane solution under dark condition, stir the solution; add 2,3-dichloro-5,6-dicyanoquinone and stir; under ice bath, add triethylamine and boron trifluoride ether solution and stir; after concentration, column chromatography separation is carried out to obtain the fluorescent dye.

3. The preparation method according to claim 2, characterized in that, Before the operation step of placing phenothiazine, the first substance and KOH solution in dimethyl sulfoxide in step 1, it further comprises: drying dimethyl sulfoxide by desiccant or activated molecular sieve.

4. The preparation method according to claim 2, characterized in that, Before the operation step of dissolving the second intermediate in dichloromethane under inert atmosphere in step 3, it further comprises: drying dichloromethane by re-distillation.

5. The preparation method according to claim 2, characterized in that, The step 2 uses saturated sodium carbonate to adjust the pH value.

6. Use of a fluorescent dye for the differential identification of lipid classes of cholesteryl esters, triglycerides and phospholipids in serum environment, and for the in situ classification analysis of lipids in serum without extraction, characterized in that, The fluorescent dye has the following chemical structural formula: wherein R1, R2are independently selected from H, CI, Br and I; R3is selected from CH3, C12-18 alkyl.

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