阅读说明：本技术 一类红光香豆素类聚集诱导发光荧光染料 (Fluorescent dye capable of inducing aggregation and luminescence of red-light coumarins ) 是由 王本花 宋相志 钟浩 韩少辉 乐秀秀 于 2020-12-31 设计创作，主要内容包括：本发明公开一类具有AIE效应的脂滴靶向香豆素类红光染料,该红光染料具有具有如式I所示的结构,其中R＝CN,COCH-3,COOEt,H。测试表明：该一系列染料具有大的斯托克斯位移,较适合生物应用的红光区域波长。其中R＝COOEt生物成像表明,该染料与商业染料BODIPY 493/503共染皮尔逊(氏)同域化达0.9032。(The invention discloses lipid drop targeted coumarin red-light dyes with AIE effect, which have a structure shown as a formula I, wherein R is CN or COCH 3 COOEt, H. The test shows that: the series of dyes have large Stokes shift and are more suitable for the red light region wavelength of biological application. Where R-COOEt bioimaging indicates that this dye co-stains pearson homodomization up to 0.9032 with the commercial dye BODIPY 493/503.)

1. A red coumarin aggregation-induced emission fluorescent dye has a structural general formula as shown in formula I:

wherein R is CN, COCH₃，COOEt，H。

Technical Field

The invention belongs to the field of fine chemical engineering, and particularly relates to synthesis and application of a red coumarin aggregation-induced emission fluorescent dye. Compared with other dyes, the coumarin dye generally has the advantages of higher fluorescence quantum yield, stability and simple preparation. These advantages make it widely applicable to fields such as biological imaging, biosensing, etc. The coumarin aggregation-induced emission fluorescent dye synthesized by the inventor has a larger conjugated system and a stronger ICT effect, so that the coumarin aggregation-induced emission fluorescent dye has larger Stokes shift and longer emission wavelength and can be used as a fluorescent dye for deep research. Cyano is used as an electron-withdrawing group, the electron-withdrawing ability of the cyano is stronger than that of acetyl and ester groups, and thus the emission wavelength and Stokes shift of the cyano are also larger than those of acetyl and ester groups. By combining the characteristics of the substances, the longer emission wavelength and the larger Stokes shift have greater advantages in the aspects of some biomarkers, fluorescence imaging and the like.

Background

In recent years, a variety of organic fluorescent dyes have been developed for cell and tissue imaging, such as nile red, BODIPY, rhodamine, and the like. These fluorescent dyes are used in solutions of low concentration because their fluorescence tends to be quenched at high concentration due to the quenching (ACQ) effect caused by aggregation. The ACQ effect greatly limits the working concentration of conventional dyes, thereby limiting their further applications in ultrasensitive assays and long-term monitoring. Recently, the Tang Benzhou subject group proposed a new photophysical phenomenon of Aggregation Induced Emission (AIE) in a class of luminophores. Compared to conventional ACQ dyes, luminophores with AIE properties are weakly emissive when the dye is dissolved in solution, but exhibit solid fluorescence in the aggregate state. This feature enables AIE molecules to act at high concentrations, making them promising for long-term tracking or identification of analytes. Therefore, the AIE dye is a novel dye having excellent sensitivity and light stability.

The traditional dye coumarin has the advantages of high fluorescence quantum yield, high stability and simple preparation, but most of coumarins in the current literature have aggregation-induced quenching effect, which greatly limits the application of the coumarin. Analytical approaches based thereon tend to undergo cumbersome solution preparation steps and are concentration limited, their fluorescence intensity tends to be low, and their fluorescence molecule lifetime also tends to be lower than that of AIE molecules. On the other hand, many biological samples fluoresce themselves, typically in the blue-green region of the spectrum, which interferes with the fluorescent signal generated by the fluorophore of coumarin and its derivatives. Therefore, the development of novel coumarins with long wavelength AIE properties is of particular importance.

Lipid droplets are unique organelles in biological systems that store neutral lipids, distributed in different cell lines in the cytoplasm. It not only promotes the transportation of lipid to other organelles, but also improves the transportation efficiency of protein. Second, some scientists have found that viruses are closely related to lipid droplets in biological systems. This indicates that the steps of viral assembly occur around the lipid droplet. Again, some studies have found that lipid droplets are not only a simple depot for lipid storage, but also a dynamic subcellular structure that regulates lipid metabolism. They are closely related to various metabolic diseases such as diabetes, cardiovascular diseases, hypertension, and the like. To facilitate the application of lipid droplets in the field of biomedical research, it is important to track and image intracellular lipid droplets.

Disclosure of Invention

In view of the above situation, one of the objects of the present invention is to provide a method for preparing an AIE fluorescent dye, which has the advantages of simple synthesis, mild reaction conditions and low cost; the second purpose is to provide an AIE fluorescent dye with long-wavelength emission and larger Stokes shift; the third object provides the use of the compounds to localize lipid droplet organelles.

The technical scheme is as follows: in order to achieve the purpose, the technical scheme of the invention is as follows: a coumarin AIE fluorescent dye compound has a structural formula as shown in formula I:

wherein R is one of cyano, acetyl, ester group and hydrogen.

When R in formula I is an ester group, the synthetic route of compound F of the present invention is as follows:

when R in formula I is cyano, the synthetic route of compound G of the present invention is shown below:

when R in formula I is acetyl, the synthetic route of the compound H is shown as follows:

when R in formula I is hydrogen, the synthetic route of the compound I of the invention is as follows:

the compound A is prepared by Vilsmeier-Haack reaction of a compound E and phosphorus oxychloride, and the synthetic route is as follows:

the compound E is prepared by reacting a compound J with boron tribromide, and the synthetic route is as follows:

maximum absorption lambda of compound F, G, H in toluene, dioxane, dichloromethane, dimethyl sulfoxide, tetrahydrofuran_abs(nm), maximum emission lambda_em(nm), relative fluorescence quantum efficiency Φ_f(reference is made to a solution of coumarin 7 in methanol) and the molar extinction coefficient ε λ_abs(M^-1cm^-1) Stokes shift Δ_ss(nm) is shown in the following table:

TABLE optical Properties of formula I in different solvents

The above table shows that: in addition to the compound I, the formula I (R is cyano, acetyl and ester) has the characteristics of large Stokes shift, long-wavelength emission and the like, and the result shows that the formula I is suitable for biological imaging.

Compound F bioimaging showed: it can localize to the lipid droplet (co-localization factor 0.90) and emit in the lipid droplet at 595 nm.

Drawings

FIG. 1 shows the UV-visible absorption spectrum of compound F of the present invention in toluene, dioxane, dichloromethane, dimethyl sulfoxide, tetrahydrofuran, and methanol.

FIG. 2 shows the UV-visible absorption spectrum of compound G of the present invention in toluene, dioxane, dichloromethane, dimethyl sulfoxide, tetrahydrofuran, and methanol.

FIG. 3 shows the UV-visible absorption spectrum of compound H of the present invention in toluene, dioxane, dichloromethane, dimethyl sulfoxide, tetrahydrofuran, and methanol.

FIG. 4 shows the UV-visible absorption spectrum of compound I of the present invention in toluene, dioxane, dichloromethane, dimethyl sulfoxide, tetrahydrofuran, and methanol.

FIG. 5 shows normalized fluorescence emission spectra of compound F of the present invention in toluene, dioxane, dichloromethane, dimethyl sulfoxide, tetrahydrofuran, and methanol, with excitation at 440nm, slit 5/5nm, and voltage at 500V. From the figure, it can be seen that the solvation shift is as high as 129nm from 571nm in toluene with minimum polarity to 700nm in DMSO with maximum polarity, indicating that compound F has the potential to image in organelles.

FIG. 6 shows normalized fluorescence emission spectra of compound G of the present invention in toluene, dioxane, dichloromethane, dimethylsulfoxide, tetrahydrofuran, and methanol, with excitation at 460nm, slit 5/5nm, and voltage at 500V. As can be seen, the solvation shift from 616nm in toluene, which is the least polar, to 750nm in DMSO, which is the most polar, is as high as 134nm, indicating that compound G has the potential to localize organelles.

FIG. 7 shows normalized fluorescence emission spectra of compound H of the present invention in toluene, dioxane, dichloromethane, dimethyl sulfoxide, tetrahydrofuran, and methanol, with excitation at 450nm, slit 5/5nm, and voltage at 500V. As can be seen, the solvation shift from 610nm in toluene, which is the least polar, to 722nm in DMSO, which is the most polar, is as high as 112nm, indicating that Compound H has the potential to localize organelles.

FIG. 8 shows normalized fluorescence emission spectra of compound I of the present invention in toluene, dioxane, dichloromethane, dimethylsulfoxide, and tetrahydrofuran, with excitation at 460nm, slit 5/5nm, and voltage at 500V. As can be seen, the solvation shift reaches 74nm from 487nm emission in toluene, which is the least polar, to 561nm emission in DMSO, which is the most polar.

FIG. 9 is a graph of fluorescence emission intensity of compound F of the present invention in DMSO solutions with water content of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, excitation at 440nm, slit 5/5nm, and voltage at 500V. As can be seen, when the water content is below 60%, the solution appears to be weakly emissive; from 60% to 97%, the solution was in an aggregate state with a fluorescence enhancement of 56.3 fold, indicating that compound F has AIE properties.

FIG. 10 is a line graph showing the fluorescence emission intensity ratio of the above compound F at different water contents to the initial fluorescence emission intensity.

FIG. 11 is a graph of fluorescence emission intensity of compound G of the present invention in DMSO solutions with water content of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 97%, 99%, excitation 466nm, slit 5/5nm, voltage 500V. As can be seen, when the water content is below 50%, the solution appears to be weakly emissive; when the water content is 60%, the solution is in an aggregation state, and the fluorescence is enhanced by 18.1 times; when the water content is more than 60%, the fluorescence intensity gradually decreases due to poor solubility of the compound in water. Indicating that compound G has AIE properties.

FIG. 12 is a line graph showing the fluorescence emission intensity ratio of the above compound G at different water contents to the initial fluorescence emission intensity.

FIG. 13 is a graph of fluorescence emission intensity of compound H of the present invention in DMSO solutions with water content of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 97%, 99%, excitation 453nm, slit 5/5nm, and voltage 500V. As can be seen, when the water content is below 50%, the solution appears to be weakly emissive; when the water content is 60-90%, the solution is in an aggregation state, and the fluorescence is enhanced by 51.86 times; when the water content is 97%, the fluorescence intensity gradually decreases due to poor solubility of the compound in water. Indicating that compound H has AIE properties.

FIG. 14 is a line graph showing the fluorescence emission intensity ratio of the above compound H at different water contents to the initial fluorescence emission intensity.

FIG. 15 is a graph of the fluorescence emission intensity of Compound I of the present invention in a methanol solution with a water content of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 97%, 99%, with excitation at 399nm, slit 5/5nm, and voltage 500V. As can be seen, when the water content is below 60%, the solution appears to be weakly emissive; when the water content is 60-90%, the solution is in an aggregation state, the fluorescence is enhanced by 39.47 times, and when the water content is higher than 90%, the fluorescence intensity is gradually reduced due to poor solubility of the compound in water. Indicating that compound I has AIE properties.

FIG. 16 is a line graph showing the fluorescence emission intensity ratio of the above compound I at different water contents to the initial fluorescence emission intensity.

FIG. 17 is a graph of normalized emission spectra of solid fluorescence of compounds F, G, H and I according to the present invention, wherein F is 401nm, G is 480nm, H is 466nm, I is 410nm, slit is 5/5nm, and voltage is 600V. The solid fluorescence shifts from the yellow-green red of compound I to the orange-red of compound G as R changes in formula I.

FIG. 18 shows the preparation of Compound F according to the invention¹H NMR spectrum.

FIG. 19 shows the preparation of Compound F according to the invention¹³C NMR spectrum.

FIG. 20 is a drawing of Compound G according to the invention¹H NMR spectrum.

FIG. 21 shows the preparation of compound G according to the invention¹³C NMR spectrum.

FIG. 22 shows the preparation of compound H according to the invention¹H NMR spectrum.

FIG. 23 shows a scheme for the preparation of compound H according to the invention¹³C NMR spectrum.

FIG. 24 shows the preparation of compound I according to the invention¹H NMR spectrum.

FIG. 25 shows the preparation of Compound I according to the invention¹³C NMR spectrum.

FIG. 26 is a drawing of Compound E according to the invention¹H NMR spectrum.

FIG. 27 is a drawing of Compound E according to the invention¹³C NMR spectrum.

FIG. 28 is a drawing of Compound A of the present invention¹H NMR spectrum.

FIG. 29 shows a scheme for the preparation of compound A according to the invention¹³C NMR spectrum.

FIG. 30 is Compound F (10)^-3M) and commercial lipid drop dye BODIPY 493/503(1ug/mL) and HeLa were incubated together for 30min, then the confocal imaging of cell laser was carried out, the excitation is 405nm, A is 500-550nm channel (the fluorescence of BODIPY 493/503 was collected); b is a channel of 570-620nm (collecting the fluorescence of the compound F); c is a bright field; d is a mixed image of A and B.

FIG. 31 is a Pearson homography of compound F above with BODIPY 493/503 with an overlap factor of up to 0.9032.

Detailed description of the preferred embodiment

Example 1: synthesis of Compound E

Compound J (3.416g, 10.51mmol) was charged to a 100mL three-necked flask under an argon atmosphere at-78 deg.C, 20mL anhydrous dichloromethane was added, and BBr was slowly added dropwise₃After dropwise addition, the reaction solution was reacted at room temperature for 3 hours, the reaction solution was poured into 250mL of ice water, extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and column chromatography was performed to obtain 3.2g of a product with a yield of 97.9%.¹H NMR(400MHz,DMSO)δ9.65(s,1H),7.67(t,J＝9.4Hz,2H),7.30(dd,J＝8.8,7.0Hz,4H),7.16(d,J＝2.2Hz,1H),7.08–6.99(m,6H),6.96(dd,J＝3.5,2.3Hz,1H),6.93(dd,J＝3.5,2.3Hz,1H),6.88(d,J＝2.3Hz,1H)。¹³C NMR(101MHz,DMSO)δ156.29,147.79,145.68,136.15,129.95,129.52,129.39,124.88,124.29,123.38,121.24,118.92,117.68,108.45。HRMS(ESI):m/z calcd for C₂₂H₁₇NO[M-H]^-310.1237,found 310.1233。

Example 2: synthesis of Compound F

Under the atmosphere of argon at 0 ℃, dropwise adding phosphorus oxychloride (1.56mL, 16.86mmol) into 3mL DMMF, turning the reaction liquid red after 30min, adding a compound E (2000g, 6.43mmol) into a flask, reacting for 1h at 80 ℃, turning the reaction liquid red and sticky, adding ice water to quench the reaction liquid, filtering, drying, and performing column chromatography to obtain a compound A220 mg with the yield of 10%. Recovery of compound E1200 mg can be used for the next repeated synthesis of compound A.¹H NMR(400MHz,DMSO)δ11.54(s,1H),10.55(s,1H),8.64(d,J＝2.2Hz,1H),7.98(d,J＝8.9Hz,1H),7.74(d,J＝8.8Hz,1H),7.42–7.29(m,4H),7.15–7.06(m,6H),7.05(d,J＝8.9Hz,1H),7.01(dd,J＝8.8,2.3Hz,1H)。¹³C NMR(101MHz,CDCl3)δ192.97,165.53,148.82,147.08,138.70,134.50,130.27,129.56,125.35,124.09,123.55,120.84,116.48,110.63,109.32。HRMS(ESI):m/z calcd for C₂₃H₁₇NO₂[M-H]^-338.1187,found 338.1179。

Example 3: synthesis of formula I

Synthesis of Compound F:

dissolving the compound A (0.15mmol) and the compound B (0.17mmol) in 3mL of absolute ethyl alcohol, adding 10uL of piperidine, refluxing for 15min, pouring into water, extracting with EA, drying an organic phase with anhydrous sodium sulfate, performing rotary evaporation on an organic solvent, and performing column chromatography to obtain a red solid compound F10mg with the yield of 15.13%.¹H NMR(400MHz,CDCl₃)δ8.91(s,1H),7.95(d,J＝8.9Hz,1H),7.76(d,J＝2.2Hz,1H),7.72(d,J＝8.8Hz,1H),7.38–7.29(m,5H),7.28(s,1H),7.25(s,1H),7.21–7.12(m,6H),4.39(q,J＝7.1Hz,2H),1.38(t,J＝7.1Hz,3H)。¹³C NMR(101MHz,CDCl₃)δ163.72,157.09,156.75,148.91,146.89,144.74,135.72,131.13,130.14,129.70,125.77,125.40,124.41,122.88,115.38,114.08,111.69,111.43,61.86,14.18。HRMS(ESI):m/z calcd for C₂₈H₂₁NO₄[M+Na]⁺458.1363,found 458.1411。

Synthesis of Compound G:

dissolving the compound A (0.15mmol) and the compound C (0.17mmol) in 2mL of absolute ethyl alcohol, adding 5uL of piperidine, refluxing for 15min, pouring into water, extracting with EA, drying an organic phase with anhydrous sodium sulfate, performing rotary evaporation on an organic solvent, and performing column chromatography to obtain a red solid compound G15mg with the yield of 25%.¹H NMR(400MHz,DMSO)δ9.51(s,1H),8.26(d,J＝8.9Hz,1H),8.14(s,1H),7.99(d,J＝8.8Hz,1H),7.46(d,J＝8.9Hz,1H),7.37(t,J＝7.7Hz,4H),7.25(d,J＝8.7Hz,1H),7.13(dd,J＝18.2,7.8Hz,6H)。¹³C NMR(101MHz,DMSO)δ157.75,156.25,149.96,148.92,147.24,137.38,131.11,131.04,130.25,126.59,124.96,124.39,124.24,115.50,114.98,114.40,111.88,100.11。

Synthesis of Compound H:

dissolving compound A (0.15mmol) and compound D (0.17mmol) in 5mL of anhydrous ethanol, adding 10uL of piperidine, refluxing for 15min, pouring into water, extracting with EA, drying the organic phase with anhydrous sodium sulfate, rotary evaporating the organic solvent, and performing column chromatography to obtain a red solid compound H32 mg with a yield of 51%.¹H NMR(400MHz,CDCl₃)δ8.84(s,1H),7.88(d,J＝8.9Hz,1H),7.77(d,J＝2.2Hz,1H),7.65(d,J＝8.9Hz,1H),7.31–7.20(m,6H),7.08(dd,J＝13.4,7.5Hz,6H),2.65(s,3H)。¹³C NMR(101MHz,CDCl₃)δ194.74,158.60,155.95,148.01,145.86,142.41,134.91,130.63,129.16,128.68,124.83,124.27,123.40,122.12,120.60,112.91,111.12,110.92,52.41。HRMS(ESI):m/z calcd for C₂₇H₁₉NO₃[M+H]⁺406.1438,found 406.1446。

Synthesis of Compound I:

under the protection of argon, the compound A (50mg, 0.15mmol) and the methoxy formyl methylene triphenylphosphine (200mg, 0.15mmol) are subjected to a melt reaction at 180 ℃ for 2 hours, and column chromatography is carried out to obtain a yellow solid 30mg and the yield is 55%.¹H NMR(400MHz,DMSO)δ8.43(d,J＝9.8Hz,1H),8.09(d,J＝9.0Hz,1H),7.95(d,J＝8.9Hz,1H),7.88(d,J＝2.1Hz,1H),7.40(d,J＝8.9Hz,1H),7.37(t,J＝7.8Hz,4H),7.24(dd,J＝8.8,2.1Hz,1H),7.13(t,J＝8.4Hz,6H),6.48(d,J＝9.8Hz,1H)。¹³C NMR(101MHz,DMSO)δ160.46,154.49,147.93,147.28,140.51,133.36,130.74,130.61,130.22,126.39,125.02,124.31,123.35,115.21,113.48,112.25。HRMS(ESI):m/z calcd for C₂₅H₁₇NO₂[M+H]⁺364.1332,found 364.1323。

Example 4: cell co-localization assay of Compound F with BODIPY 493/503

Preparation of 10 of Compound F^-3M mother liquor, a 0.1mg/mL solution of BODIPY 493/503 was prepared. 980uL of culture medium was added to the confocal dish that was previously incubated with Hela cells for 24h, and 10uL of compound F stock solution and 10uL of BODIPY 493/503 in 0.1mg/mL solution were added to the confocal dish to stain the cells for 30 min. The PBS was rinsed three times and a clean 1mL PBS solution was added. Exciting light 400nm, 60.2% laser power laser confocal imaging, collection channel one: 500-550nm (BODIPY 493/503), channel two: 570-620nm (compound F).