Preparation method of near-infrared two-zone BODIPY dye for marking biological membrane

Abstract

The invention discloses a preparation method of a near-infrared two-region BODIPY dye for marking a biological membrane, and relates to the technical field of biological medicines. The invention aims to solve the technical problems of difficult synthesis, more steps and slower cell membrane labeling speed of the existing cell membrane labeling fluorescent molecules. The method comprises the following steps: by taking a near-infrared two-region aza-BODIPY dye as a main body structure, introducing an alkyl chain and hydrophilic sulfonate or quaternary ammonium salt on a molecular structure, and modifying and coupling to obtain BMP1, BMP2 and BMP3. According to the near-infrared two-region aza-Bodipy dye and the preparation method thereof, a series of BMP1, BMP2 and BMP3 are synthesized, and the near-infrared two-region aza-Bodipy dye is excellent in water solubility. The fluorescence brightness of the three materials before and after being combined with the lipidosome reaches more than 10 times, and the BMP3 material has more excellent hydrophilic performance, does not need DMSO to assist dissolution, can be better combined with the lipidosome, has larger change in fluorescence intensity before and after, and can better become a marking dye of a biological membrane structure material. The near-infrared two-region BODIPY dye prepared by the preparation method disclosed by the invention is used for marking a biomass film.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a method for preparing a near-infrared second-zone BODIPY dye for marking biofilms. Background Art

[0002] Biomembranes are ubiquitous lipid structures in living cells. They constitute the plasma membrane (PM) and delimit most organelles. Sensing and imaging of biomembranes have revealed remarkable heterogeneity in lipid organization. Thus, most sphingomyelin and cholesterol are present in the PM, where they are preferentially localized in the outer leaflet and constitute microdomains responsible for a plethora of cellular functions. 5,6 The development of biomembrane chemical tools is an important research direction, stimulated by the high demand of cell biologists and biophysicists. In particular, it concerns fluorescent probes, which have been driven by the remarkable development of fluorescence microscopy because of the high sensitivity of fluorescence technology. The large number of developed membrane fluorescent probes has prompted us to classify them according to their targeting principles, sensing profiles, and optical responses. In terms of targeting, there are probes targeting both cell membranes and organelles, with the cell membrane being perhaps the most discovered target. Typically, cell biologists require powerful tools to label the cell plasma membrane alone. The traditional tool is fluorescently labeled wheat germ agglutinin (WGA) (absorption 317 nm, emission fluorescence 553 nm), which targets membrane glycoproteins. However, its large size does not always allow for efficient staining of cells and tissues. Currently, the most commonly used commercial small molecule fluorescent probes are lipophilic cyanine derivatives, such as DiI (λex 550nm, λem 565nmin methanol), DiD (646 / 663nM), DIO (λex 484nm, λem 501nmin methanol), and DIR (λex 748 nm, em 780nmin DMSO). They are generally poorly soluble in water, so excess concentrations are required and provide heterogeneous labeling. In addition, these traditional cell membrane markers usually rely on the use of fluorophores to provide fluorescence emission in the visible and NIR-I spectral regions (400-700nm and 700-900nm, respectively). These fluorophores are mainly oriented towards in vitro cell imaging, and their use in in vivo cell tracking is limited due to the presence of a large amount of tissue autofluorescence and light scattering in such a short wavelength region. Imaging cell membrane markers in optical windows with less interference (such as the NIR-II region) may make a significant contribution to this important field. In addition, the excitation and emission of these cell membrane-labeled fluorescent molecules are usually in the visible light and near-infrared region I (400-1000nm), and there are still few cell membrane-labeled fluorescent small molecules reported in the near-infrared region II fluorescence imaging region (1000-1700nm). Currently, Professor Zhu Shoujun of the First Hospital of Jilin University has proposed a NIR-II region molecule FE-4ZW that introduces four zwitterionic sulfonyl ammonium terminal groups for in vivo detection of transplanted cells and brain tumor cells migrating to cervical lymph nodes. However, they are difficult to synthesize, have many steps, and label cell membranes relatively slowly. Therefore, it is also urgent to develop a near-infrared region II dye that can quickly stain membranes. Summary of the invention

[0003] The present invention provides a method for preparing a near-infrared second-zone BODIPY dye for marking biological membranes in order to solve the technical problems that the current cell membrane marking fluorescent molecules are difficult to synthesize, have many steps, and have a slow cell membrane marking speed.

[0004] A method for preparing a near-infrared second-region BODIPY dye for marking biofilms is specifically carried out in the following steps:

[0005] 1. Dissolve 4-diethylaminobenzaldehyde and 4-hydroxyacetophenone in anhydrous ethanol, stir at 0°C, then add NaOH solution, react at room temperature for 24 hours, monitor the reaction using TLC spot plate, and after the reaction is completed, obtain reaction solution 1, vacuum concentrate reaction solution 1, and separate by silica gel column chromatography to obtain compound 3;

[0006] The dosage ratio of the 4-diethylaminobenzaldehyde, 4-hydroxyacetophenone and anhydrous ethanol is 0.11 mol: 0.073 mol: 150 mL; the volume ratio of petroleum ether and ethyl acetate in the silica gel column chromatography purification is (10: 1) to (3: 1);

[0007] 2. Dissolve the compound 3 obtained in step 1, nitromethane and diethylamine in anhydrous ethanol, stir at 110° C., react for 8 to 12 hours, and monitor the reaction using a TLC spot plate. After the reaction is completed, obtain a reaction solution 2, vacuum concentrate the reaction solution 2, and separate it by silica gel column chromatography to obtain compound 4;

[0008] The usage ratio of the compound 3, nitromethane, diethylamine and anhydrous ethanol is 0.051 mol: 0.754 mol: 1.012 mol: 150 mL; the volume ratio of petroleum ether and dichloromethane used in the silica gel column chromatography purification is 1:1 to pure dichloromethane;

[0009] 3. Dissolve the compound 4 obtained in step 2 and ammonium acetate in n-butanol, stir at 130° C., react for 8 to 12 hours, and monitor the reaction using a TLC spot plate. After the reaction is completed, obtain reaction solution 3, concentrate the reaction solution 3 in vacuo to a quarter of the volume of the reaction solution 3, and filter to obtain compound 5;

[0010] The usage ratio of the compound 4, ammonium acetate and n-butanol is 0.03369 mol:1.168 mol:150 mL;

[0011] 4. Dissolve the compound 5 obtained in step 3 and N,N-diisopropylethylamine in anhydrous dichloromethane, stir at 0°C, add boron trifluoride etherate, react at room temperature for 8-12 hours, and monitor the reaction using a TLC spot plate. After the reaction is completed, a reaction solution 4 is obtained, and the reaction solution 4 is vacuum concentrated and separated by silica gel column chromatography to obtain a compound 6;

[0012] The usage ratio of the compound 5, N,N-diisopropylethylamine, anhydrous dichloromethane and boron trifluoride ether is 0.016 mol: 0.251 mol: 100 mL: 0.24 mol; the volume ratio of petroleum ether to ethyl acetate in silica gel column chromatography purification is 2: 1;

[0013] 5. Dissolve the compound 6 obtained in step 4, potassium carbonate and dodecane bromide in acetone, and react at 63° C. for 5 h. After the reaction is completed, a reaction solution 5 is obtained. The reaction solution 5 is vacuum concentrated and separated by silica gel column chromatography to obtain a compound 7;

[0014] The dosage ratio of the compound 6, potassium carbonate, dodecane bromide and acetone is 0.149 mol: 0.449 mol: 0.445 mol: 10 mL; the volume ratio of petroleum ether to ethyl acetate in silica gel column chromatography purification is 4: 1;

[0015] 6. Dissolve the compound 7 obtained in step 5, propyl sultone and potassium carbonate in anhydrous tetrahydrofuran, stir and react at 80°C for 8 to 12 hours, and monitor the reaction by TLC spot plate. After the reaction is completed, a reaction solution 6 is obtained. The reaction solution 6 is vacuum concentrated and separated by silica gel column chromatography to obtain near-infrared zone II BODIPY dye BMP1.

[0016] Furthermore, in step 6, the amount ratio of compound 7, propyl sultone, potassium carbonate and anhydrous tetrahydrofuran is 0.0477 mmol:0.0982 mmol:0.232 mmol:5 mL; and the volume ratio of dichloromethane to methanol in the silica gel column chromatography purification is 5:1.

[0017] Furthermore, the structural formula of the near-infrared second region BODIPY dye BMP1 is:

[0018]

[0019] Furthermore, replace step six with:

[0020] The compound 7 obtained in step five, 3-bromopropyltrimethylammonium bromide and potassium carbonate are dissolved in acetonitrile, stirred at 85°C, and refluxed for 8-12 hours. The reaction is monitored by TLC spot plate. After the reaction is completed, a reaction solution 7 is obtained. The reaction solution 7 is vacuum concentrated and separated by silica gel column chromatography to obtain near-infrared zone II BODIPY dye BMP2.

[0021] Furthermore, the dosage ratio of the compound 7, 3-bromopropyltrimethylammonium bromide, potassium carbonate and acetonitrile is 0.0441 mmol:0.0881 mmol:0.232 mmol:5 mL; and the volume ratio of dichloromethane to methanol in the silica gel column chromatography purification is 5:1.

[0022] Furthermore, the structural formula of the near-infrared second region BODIPY dye BMP2 is:

[0023]

[0024] Furthermore, replace step six with:

[0025] Dissolve the compound 7 obtained in step 5, N,N-dimethylchloropropane hydrochloride and cesium carbonate in anhydrous DMF, stir and react at 60°C for 8-12 hours, and monitor the reaction using a TLC spot plate. After the reaction is completed, add pure water, extract with dichloromethane to obtain a dichloromethane phase, and then concentrate in vacuo to obtain compound 8;

[0026] Compound 8 and (3-bromopropyl)trimethylammonium bromide were dissolved in anhydrous methanol, stirred and reacted at 80°C for 8-12 hours, and the reaction was monitored by TLC spot plate. After the reaction was completed, reaction solution 8 was obtained, which was vacuum concentrated and separated by reverse silica gel column chromatography to obtain near-infrared zone II BODIPY dye BMP3.

[0027] Furthermore, the usage ratio of compound 7, N,N-dimethylchloropropane hydrochloride, cesium carbonate, anhydrous DMF and pure water is 0.0357 mmol: 0.0506 mmol: 0.0984 mmol: 5 mL: 5 mL.

[0028] Furthermore, the dosage ratio of the compound 8, (3-bromopropyl)trimethylammonium bromide and anhydrous methanol is 0.0477mmol:0.2385mmol:5mL; the volume ratio of water to methanol in the reverse silica gel column chromatography purification is (95:5) to (5:95).

[0029] Furthermore, the structural formula of the near-infrared second region BODIPY dye BMP3 is:

[0030]

[0031] The present invention adopts near-infrared zone II aza-BODIPY dye as the main structure, which has a relatively stable DAD conjugated structure. Alkyl chains and hydrophilic sulfonates or quaternary ammonium salts are further introduced into the molecular structure to improve the hydrophilic properties of the molecule. After modification and coupling, a series of molecules BMP1, BMP2 and BMP3 molecules are obtained.

[0032] Beneficial effects of the present invention:

[0033] The present invention synthesizes three near-infrared second region aza-BODIPY dyes, namely BMP1, BMP2 and BMP3. Compared with BMP1 and BMP2, BMP3 has better water solubility, does not need to add 1% DMSO to promote solubility, and significantly improves the speed and brightness of marking cell membranes. The dye and liposomes only need to be mixed for 5 minutes and do not need centrifugal washing, and can be used as a material for marking biomass membrane structure.

[0034] The present invention synthesizes a series of excellent water-soluble near-infrared zone II aza-Bodipy dyes of BMP1, BMP2 and BMP3. The fluorescence brightness of these three materials before and after combining with liposomes is more than 10 times, and the BMP3 material has more excellent hydrophilicity, does not require DMSO to assist in dissolution, and can be better combined with liposomes. The fluorescence intensity changes more before and after, and can better become a marker dye for biological membrane structure materials.

[0035] The near-infrared second-zone BODIPY dye prepared by the invention is used for marking biomass membranes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The compound 3 described in Example 1 in methanol-d4 1 H NMR spectrum;

[0037] Figure 2 The compound 4 described in Example 4 in methanol-d4 1 H NMR spectrum;

[0038] Figure 3 The compound 6 described in Example 1 in CDCl3 1 H NMR spectrum;

[0039] Figure 4 is the compound 7 described in Example 7 in CDCl3 1 H NMR spectrum;

[0040] Figure 5 The BMP1 in DMSO-d6 1 H NMR spectrum;

[0041] Figure 6 The high-resolution mass spectrum of BMP1 described in the examples;

[0042] Figure 7 The BMP2 in DMSO-d6 described in the example 1 H NMR spectrum;

[0043] Figure 8 The high-resolution mass spectrum of BMP2 described in the example;

[0044] Fig. 9 The BMP3 in DMSO-d6 described in the examples 1 H NMR spectrum;

[0045] Fig.10 The high-resolution mass spectrum of BMP3 described in the embodiment;

[0046] Fig.11 The UV-Vis-NIR absorption spectra of BMP1, BMP2, BMP3, BMP1@Lipo, BMP2@Lipo and BMP3@Lipo in pure water described in the examples;

[0047] Fig.12 The fluorescence emission spectra of BMP1, BMP2, BMP3, BMP1@Lipo, BMP2@Lipo and BMP3@Lipo in pure water described in the examples;

[0048] Fig.13 The excitation was 808 nm (1000 nm filter) and the power was 40 mW cm -2 Under the same conditions, near-infrared second-zone fluorescence images of BMP1, BMP2, BMP3, BMP1@Lipo, BMP2@Lipo and BMP3@Lipo described in the examples are shown. DETAILED DESCRIPTION

[0049] Specific implementation method 1: This implementation method is a method for preparing a near-infrared second-region BODIPY dye for marking biofilms, which is specifically carried out in the following steps:

[0050] 1. Dissolve 4-diethylaminobenzaldehyde and 4-hydroxyacetophenone in anhydrous ethanol, stir at 0°C, then add NaOH solution, react at room temperature for 24 hours, monitor the reaction using TLC spot plate, and after the reaction is completed, obtain reaction solution 1, vacuum concentrate reaction solution 1, and separate by silica gel column chromatography to obtain compound 3;

[0051] The dosage ratio of the 4-diethylaminobenzaldehyde, 4-hydroxyacetophenone and anhydrous ethanol is 0.11 mol: 0.073 mol: 150 mL; the volume ratio of petroleum ether and ethyl acetate in the silica gel column chromatography purification is (10: 1) to (3: 1);

[0052] 2. Dissolve the compound 3 obtained in step 1, nitromethane and diethylamine in anhydrous ethanol, stir at 110° C., react for 8 to 12 hours, and monitor the reaction using a TLC spot plate. After the reaction is completed, obtain a reaction solution 2, vacuum concentrate the reaction solution 2, and separate it by silica gel column chromatography to obtain compound 4;

[0053] The usage ratio of the compound 3, nitromethane, diethylamine and anhydrous ethanol is 0.051 mol: 0.754 mol: 1.012 mol: 150 mL; the volume ratio of petroleum ether and dichloromethane used in the silica gel column chromatography purification is 1:1 to pure dichloromethane;

[0054] 3. Dissolve the compound 4 obtained in step 2 and ammonium acetate in n-butanol, stir at 130° C., react for 8 to 12 hours, and monitor the reaction using a TLC spot plate. After the reaction is completed, obtain reaction solution 3, concentrate the reaction solution 3 in vacuo to a quarter of the volume of the reaction solution 3, and filter to obtain compound 5;

[0055] The usage ratio of the compound 4, ammonium acetate and n-butanol is 0.03369 mol:1.168 mol:150 mL;

[0056] 4. Dissolve the compound 5 obtained in step 3 and N,N-diisopropylethylamine in anhydrous dichloromethane, stir at 0°C, add boron trifluoride etherate, react at room temperature for 8-12 hours, and monitor the reaction using a TLC spot plate. After the reaction is completed, a reaction solution 4 is obtained, and the reaction solution 4 is vacuum concentrated and separated by silica gel column chromatography to obtain a compound 6;

[0057] The usage ratio of the compound 5, N,N-diisopropylethylamine, anhydrous dichloromethane and boron trifluoride ether is 0.016 mol: 0.251 mol: 100 mL: 0.24 mol; the volume ratio of petroleum ether to ethyl acetate in silica gel column chromatography purification is 2: 1;

[0058] 5. Dissolve the compound 6 obtained in step 4, potassium carbonate and dodecane bromide in acetone, and react at 63° C. for 5 h. After the reaction is completed, a reaction solution 5 is obtained. The reaction solution 5 is vacuum concentrated and separated by silica gel column chromatography to obtain a compound 7;

[0059] The dosage ratio of the compound 6, potassium carbonate, dodecane bromide and acetone is 0.149 mol: 0.449 mol: 0.445 mol: 10 mL; the volume ratio of petroleum ether to ethyl acetate in silica gel column chromatography purification is 4: 1;

[0060] 6. Dissolve the compound 7 obtained in step 5, propyl sultone and potassium carbonate in anhydrous tetrahydrofuran, stir and react at 80°C for 8 to 12 hours, and monitor the reaction by TLC spot plate. After the reaction is completed, a reaction solution 6 is obtained. The reaction solution 6 is vacuum concentrated and separated by silica gel column chromatography to obtain near-infrared zone II BODIPY dye BMP1.

[0061] Specific embodiment 2: This embodiment differs from specific embodiment 1 in that: the usage ratio of compound 7, propyl sultone, potassium carbonate and anhydrous tetrahydrofuran in step 6 is 0.0477mmol:0.0982mmol:0.232mmol:5mL; the volume ratio of dichloromethane to methanol in the silica gel column chromatography purification is 5:1. The rest is the same as specific embodiment 1.

[0062] Specific embodiment 3: This embodiment is different from specific embodiment 1 or 2 in that the structural formula of the near-infrared second region BODIPY dye BMP1 is: The rest is the same as the first or second specific implementation.

[0063] Specific implementation method 4: This implementation method is different from any one of the specific implementation methods 1 to 3 in that: step 6 is replaced by:

[0064] The compound 7 obtained in step 5, 3-bromopropyltrimethylammonium bromide and potassium carbonate were dissolved in acetonitrile, stirred at 85°C, and refluxed for 8 to 12 hours, and the reaction was monitored by TLC spot plate. After the reaction was completed, a reaction solution 7 was obtained, and the reaction solution 7 was vacuum concentrated and separated by silica gel column chromatography to obtain near-infrared second-zone BODIPY dye BMP2. The rest is the same as that of the first to third embodiments.

[0065] Specific embodiment 5: This embodiment differs from specific embodiments 1 to 4 in that the usage ratio of compound 7, 3-bromopropyltrimethylammonium bromide, potassium carbonate and acetonitrile is 0.0441mmol:0.0881mmol:0.232mmol:5mL; the volume ratio of dichloromethane to methanol in the silica gel column chromatography purification is 5:1. The rest is the same as specific embodiments 1 to 4.

[0066] Specific embodiment 6: This embodiment is different from any one of the specific embodiments 1 to 5 in that the structural formula of the near-infrared second region BODIPY dye BMP2 is:

[0067] The rest is the same as that of Specific Implementation Methods 1 to 5.

[0068] Specific implementation method 7: This implementation method is different from any one of the specific implementation methods 1 to 6 in that: step 6 is replaced by:

[0069] Dissolve the compound 7 obtained in step 5, N,N-dimethylchloropropane hydrochloride and cesium carbonate in anhydrous DMF, stir and react at 60°C for 8-12 hours, and monitor the reaction using a TLC spot plate. After the reaction is completed, add pure water, extract with dichloromethane to obtain a dichloromethane phase, and then concentrate in vacuo to obtain compound 8;

[0070] Compound 8 and (3-bromopropyl)trimethylammonium bromide were dissolved in anhydrous methanol, stirred at 80°C for 8-12 hours, and the reaction was monitored by TLC spot plate. After the reaction was completed, a reaction solution 8 was obtained, which was vacuum concentrated and separated by reverse silica gel column chromatography to obtain near-infrared second-zone BODIPY dye BMP3. The rest is the same as the first to sixth embodiments.

[0071] Specific embodiment eight: This embodiment differs from specific embodiments one to seven in that the usage ratio of compound 7, N,N-dimethylchloropropane hydrochloride, cesium carbonate, anhydrous DMF and pure water is 0.0357mmol:0.0506mmol:0.0984mmol:5mL:5mL. The rest is the same as specific embodiments one to seven.

[0072] Specific embodiment 9: This embodiment differs from specific embodiments 1 to 8 in that the usage ratio of compound 8, (3-bromopropyl)trimethylammonium bromide and anhydrous methanol is 0.0477mmol:0.2385mmol:5mL; the volume ratio of water to methanol in the reverse silica gel column chromatography purification is (95:5) to (5:95). The rest is the same as specific embodiments 1 to 8.

[0073] Specific embodiment 10: This embodiment differs from any one of specific embodiments 1 to 9 in that the structural formula of the near-infrared second region BODIPY dye BMP3 is: The rest is the same as that of Specific Embodiments 1 to 9.

[0074] The content of the present invention is not limited to the content of the above-mentioned embodiments. The combination of one or several specific embodiments can also achieve the purpose of the invention.

[0075] Example:

[0076] This embodiment provides a method for preparing a near-infrared second-region BODIPY dye for marking biofilms, which is specifically carried out in the following steps:

[0077] 1. Dissolve 0.11 mol 4-diethylaminobenzaldehyde and 0.073 mol 4-hydroxyacetophenone in 150 mL anhydrous ethanol, stir at 0°C, then add 50% NaOH solution (41.17 g NaOH and 41 mL water), react at room temperature for 24 hours, monitor the reaction by TLC spot plate, and after the reaction is completed, obtain reaction solution 1, vacuum concentrate reaction solution 1, separate by silica gel column chromatography, and obtain red solid crystalline compound 3 (15 g, 0.051 mol, 69.5%); 1H NMR (400MHz, Methanol-d4) δ8.04–7.99(m,2H),7.74(d,J=15.3Hz,1H),7.61(d,J=8.9Hz,2H),7.51(d, J=15.3Hz,1H),6.94–6.88(m,2H),6.76(d,J=8.9Hz,2H),3.50(q,J=7.0Hz,4H),1.23(t,J=7.0Hz,6H);

[0078] The volume ratio of petroleum ether to ethyl acetate in the silica gel column chromatography purification is (10:1) to (3:1);

[0079]

[0080] 2. Dissolve 0.051 mol of compound 3 obtained in step 1, 0.754 mol of nitromethane and 1.012 mol of diethylamine in 150 mL of anhydrous ethanol, stir at 110° C., react for 8 to 12 h, and monitor the reaction using a TLC spot plate. After the reaction is completed, obtain reaction solution 2, vacuum concentrate reaction solution 2, and separate by silica gel column chromatography to obtain red-yellow solid crystalline compound 4 (12 g, 0.03369 mol, yield 66.05%); 1 H NMR (400MHz, Methanol-d4) δ7.88–7.83(m,2H),7.15–7.08(m,2H),6.87–6.81(m,2H),6.67–6.61(m,2H),4.83(ddd,J=12.4,6.3,1.4Hz,1H ),4.69(ddd,J=12.4,8.9,1.4Hz,1H),4.08–3.98(m,1H),3.45–3.39(m,1H),3.33(qd,J=6.9,6.5,3.3Hz,6H),1.12(td,J=7.1,1.4Hz,6H);

[0081] The silica gel column chromatography purifies petroleum ether and dichloromethane in a volume ratio of 1:1 to pure dichloromethane;

[0082]

[0083] 3. Dissolve 0.03369 mol of compound 4 obtained in step 2 and 1.168 mol of ammonium acetate in 150 mL of n-butanol, stir at 130°C, react for 8-12 h, and monitor the reaction using TLC spot plate. After the reaction is completed, obtain reaction solution 3, vacuum concentrate reaction solution 3 to one-fourth of the volume of reaction solution 3, and filter to obtain blue crude product crystal compound 5 (10 g, 0.016 mol, yield 47.6%);

[0084]

[0085] 4. Dissolve 0.016 mol of compound 5 obtained in step 3 and 0.251 mol of N,N-diisopropylethylamine in 100 mL of anhydrous dichloromethane, stir at 0°C, add 0.24 mol of boron trifluoride etherate, react at room temperature for 8-12 h, monitor the reaction using TLC spot plate, and after the reaction is completed, obtain reaction solution 4, concentrate reaction solution 4 in vacuo, and separate by silica gel column chromatography to obtain blue-purple crystalline compound 6 (1 g, 1.49 mmol, yield 9.31%); 1 H NMR(400MHz,DMSO-d6)δ8.07–8.00(m,4H),7.66(d,J=8.7Hz,4H),7.60(d,J=3.6Hz,2H),6.90 (dd,J=8.8,2.3Hz,4H),6.71(d,J=8.7Hz,4H),3.43(q,J=7.0Hz,8H),1.14(t,J=7.0Hz,12H);

[0086] The volume ratio of petroleum ether to ethyl acetate in silica gel column chromatography purification was 2:1;

[0087]

[0088] 5. Dissolve 0.149 mol of compound 6 obtained in step 4, 0.449 mol of potassium carbonate and 0.445 mol of dodecane bromide in 10 mL of acetone, and react at 63° C. for 5 h. After the reaction is completed, a reaction solution 5 is obtained. The reaction solution 5 is vacuum concentrated and separated by silica gel column chromatography to obtain a blue-purple crystalline compound 7 (40 mg, 0.0477 mmol, yield 32.01%). 1 HNMR(400MHz,Chloroform-d)δ8.05(dd,J=36.2,9.0Hz,8H),7.04–6.68(m,10H),4.04(t,J=6.5Hz,2H),3. 49(s,8H),1.85–1.79(m,2H),1.48(q,J=7.1Hz,2H),1.29(dt,J=16.0,6.2Hz,30H),0.92(t,J=6.7Hz,3H).

[0089] The volume ratio of petroleum ether to ethyl acetate in silica gel column chromatography purification was 4:1;

[0090]

[0091] 6. Dissolve 0.0477 mmol of compound 7 obtained in step 5, 0.0982 mmol of propyl sultone and 0.232 mmol of potassium carbonate in 5 mL of anhydrous tetrahydrofuran, stir and react at 80° C. for 8 to 12 h, monitor the reaction using a TLC spot plate, and after the reaction is completed, obtain reaction solution 6. Concentrate the reaction solution 6 in vacuo and separate it by silica gel column chromatography. The volume ratio of dichloromethane to methanol in the silica gel column chromatography is 5:1 to obtain a blue solid near-infrared second-zone BODIPY dye BMP1 (30 mg, 0.0312 mmol, yield 65.4%). 1 H NMR(400MHz,MeOD)δ8.09,8.06,8.04,8.02,8.00,7.65,7.65,7.38,6.98,6.96,6.85,6.83,6 .77,6.75,6.73,5.34,4.76,4.40,4.19,4.17,4.16,4.05,4.03,4.02,3.50,3.48,3.46,3.35 ,3.35,3.34,3.34,3.34,3.10,3.08,3.06,2.32,2.23,2.21,2.19,2.03,1.81,1.79,1.62,1.48,1.40,1.38,1.35,1.32,1.30,1.29,1.28,1.26,1.24,1.22,0.91,0.89,0.88,0.10,0.00. 13 C NMR (151MHz, DMSO) δ161.03,160.99,148.88,144.65,142.40,131.53,131.4 5,124.42,124.39,119.73,114.99,114.96,111.78,68.20,67.44,49.07,48. 28,44.48,40.44,40.32,40.18,40.04,39.90,39.76,39.63,39.49,31.80,2 9.56,29.52,29.25,29.08,25.98,25.67,22.61,14.46,13.00.HRMS(ESI)m / z calcd for C55H70BF2N5O5S + (M+H) + 961.51533, found 961.51587.

[0092]

[0093] 7. Dissolve 0.0441 mmol of compound 7 obtained in step 5, 0.0881 mmol of 3-bromopropyltrimethylammonium bromide and 0.232 mmol of potassium carbonate in 5 mL of acetonitrile, stir at 85°C, and reflux for 8 to 12 hours. Monitor the reaction by TLC spot plate. After the reaction is completed, obtain reaction solution 7. Concentrate the reaction solution 7 in vacuo and separate it by silica gel column chromatography. The volume ratio of dichloromethane to methanol in the silica gel column chromatography is 5:1. Obtain blue crystalline near-infrared second zone BODIPY dye BMP2 (20 mg, 0.0198 mmol, yield 44.9%); 1H NMR(400MHz,Chloroform-d)δ8.08(d,J=8.7Hz,4H),7.98(t,J=9.3Hz,4H),6.94(dd ,J=8.9,3.1Hz,4H),6.80–6.71(m,6H),4.07(t,J=5.2Hz,2H),3.98(t,J=6.5Hz,2H) ,3.72(t,J=8.3Hz,2H),3.46(qd,J=7.1,2.6Hz,8H),3.19(s,9H),2.13(s,2H),1.78 (d,J=7.7Hz,2H),1.48–1.42(m,2H),1.32–1.23(m,28H),0.90(t,J=6.8Hz,3H).13C NMR(151MHz,Chloroform-d)δ160.93,159.71,155.96,148.54,148.48,145.25,144. 98,143.24,142.88,131.26,131.23,125.67,124.77,120.48,120.40,114.51,114.4 5,114.17,111.38,111.36,68.28,64.36,63.99,53.36,44.65,31.97,29.74,29.70, 29.65,29.50,29.41,29.26,26.09,23.25,22.74,14.19,12.76,1.06.HRMS(ESI)m / z calcd for C58H78BF2N6O2+(M)+939.62419,found939.62488.

[0094]

[0095] 8. Dissolve 0.0357 mmol of compound 7 obtained in step 5, 0.0506 mmol of N,N-dimethylchloropropane hydrochloride and 0.0984 mmol of cesium carbonate in 5 mL of anhydrous DMF, stir and react at 60 ° C for 8 to 12 h, and monitor the reaction by TLC spot plate. After the reaction is completed, add 5 mL of pure water, extract twice with dichloromethane to obtain a dichloromethane phase, and then concentrate in vacuo to obtain compound 8 (18 mg, 0.0194 mmol, yield 54.3%); 1 H NMR(600MHz,Chloroform-d)δ8.08(d,J=8.8Hz,4H),7.99(dd,J=14.7,8.5Hz,4H),6 .95(dd,J=8.7,4.9Hz,4H),6.81–6.72(m,6H),4.07(t,J=5.5Hz,2H),3.98(t,J=6.5H z,2H),3.73–3.67(m,2H),3.47(qd,J=7.2,3.2Hz,8H),3.18(s,9H),2.17–2.08(m,2 H),1.82–1.77(m,2H),1.49–1.36(m,2H),1.32–1.24(m,28H),0.91(t,J=6.9Hz,3H). 1 H NMR(600MHz,Chloroform-d)δ8.08(d,J=8.8Hz,4H),7.99(dd,J=14.7,8.5Hz,4H),6.95 (dd,J=8.7,4.9Hz,4H),6.81–6.72(m,6H),4.07(t,J=5.5Hz,2H),3.98(t,J=6.5Hz,2H), 3.73–3.67(m,2H),3.47(qd,J=7.2,3.2Hz,8H),3.18(s,9H),2.17–2.08(m,2H),1.82–1 .77(m,2H),1.49–1.36(m,2H),1.32–1.24(m,28H),0.91(t,J=6.9Hz,3H).HRMS(ESI)m / z calcd for C58H78BF2N6O2 + (M) + 939.62419, found 939.62488.

[0096]

[0097] IX. Dissolve 0.0477 mmol of compound 8 and 0.2385 mmol of (3-bromopropyl)trimethylammonium bromide in 5 mL of anhydrous methanol, stir and react at 80°C for 8 to 12 hours, monitor the reaction using a TLC spot plate, and after the reaction is completed, obtain a reaction solution 8. Concentrate the reaction solution 8 in vacuo and separate it by reverse silica gel column chromatography. The volume ratio of water to methanol in the reverse silica gel column chromatography is (95:5) to (5:95); obtain near-infrared second zone BODIPY dye BMP3 (40 mg, 0.039 mmol, yield 36.1%); 1 H NMR (600MHz, DMSO-d6) δ8.13–8.05(m,8H),7.20(d,J=19.7Hz,2H),7.09(dd,J=29.7,8.4Hz,4H),6.84(d,J= 8.6Hz,4H),4.20(d,J=6.2Hz,2H),4.07(d,J=6.7Hz,2H),3.49(d,J=7.3Hz,8H),3.34(dt,J=15.8,7.8Hz,6H ),3.13(d,J=13.1Hz,15H),2.23(ddt,J=27.4,11.9,5.9Hz,4H),1.77–1.71(m,2H),1.43(t,J=7.6Hz,2H),1 .34(t,J=7.3Hz,3H),1.25(d,J=10.8Hz,17H),1.18(t,J=7.2Hz,12H),0.85(d,J=7.2Hz,3H).HRMS(ESI)m / z calcd for C 63 H 90 BF2N7O2 2+ (M) 2+ 512.86031, found 512.86041.

[0098]

[0099] C 12 Synthesis of -TPAE:

[0100] First, DMPC (6 mg), DOPE (4 mg), DSPE-PEG 2000(0.5 mg) was dissolved in a mixed solution of chloroform and methanol in a volume ratio of 1:1; the mixed solution was evaporated at 30°C in a vacuum environment to form a uniform lipid film; then, the lipid film was dispersed in 1.0 mL of phosphate buffered saline (PBS) and ultrasonicated in an ice bath for 2.5 min to obtain a liposome preparation (named Lipo); then, three freeze-thaw cycles were performed between -50 and 60°C; a small extruder was used to extrude it back and forth 10 times through a polycarbonate membrane with a pore size of 400 nm and a polycarbonate membrane with a pore size of 200 nm in turn; and blank liposomes were obtained.

[0101] BMP1 (1 mM, DMSO: pure water = 1:99), BMP2 (1 mM, DMSO: pure water = 1:99) and BMP3 (1 mM, pure water) prepared in the example were mixed with blank liposomes respectively, and stirred for 5 minutes to obtain samples BMP1@Lipo, BMP2@Lipo and BMP3@Lipo, which were stored in a refrigerator at 4°C after synthesis.

[0102] The BMP1, BMP2 and BMP3 prepared in the examples, as well as the samples BMP1@Lipo, BMP2@Lipo and BMP3@Lipo mixed with liposomes were tested and analyzed.

[0103] Figure 1 UV spectra of BMP1, BMP2, BMP3, BMP1@Lipo, BMP2@Lipo and BMP3@Lipo in pure water;

[0104] Figure 2 Fluorescence emission spectra of BMP1, BMP2, BMP3, BMP1@Lipo, BMP2@Lipo and BMP3@Lipo in pure water;

[0105] Figure 3 The excitation was 808 nm (1000 nm filter) and the power was 40 mW cm -2 Near-infrared second-zone fluorescence images of BMP1, BMP2, BMP3, BMP1@Lipo, BMP2@Lipo and BMP3@Lipo under the following conditions.

[0106] The Aza-BODIPY dye prepared by the present invention has unique advantages such as good optical properties, light and heat stability, low biological cell toxicity and simple synthesis method. It is analyzed by UV-visible-near infrared spectroscopy. As shown in the figure, BMP1, BMP2, and BMP3 molecules show a strong absorption peak (~808nm) in the near-infrared region in pure water solvent, and an emission peak at about 1000nm. It can be found that BMP1, BMP2 and BMP3 dyes have almost no fluorescence in water, but have strong fluorescence on biological membranes, with a difference of more than 10 times, and can be used as near-infrared second zone biological membrane fluorescence imaging materials.

Claims

1. A method for preparing a near-infrared second-region BODIPY dye for marking biofilms, characterized in that The method is specifically carried out in the following steps:

1. Dissolve 4-diethylaminobenzaldehyde and 4-hydroxyacetophenone in anhydrous ethanol, stir at 0°C, then add NaOH solution, react at room temperature for 24 hours, monitor the reaction using TLC spot plate, and after the reaction is completed, obtain reaction solution 1, vacuum concentrate reaction solution 1, and separate by silica gel column chromatography to obtain compound 3; The dosage ratio of the 4-diethylaminobenzaldehyde, 4-hydroxyacetophenone and anhydrous ethanol is 0.11 mol: 0.073 mol: 150 mL; the volume ratio of petroleum ether and ethyl acetate in the silica gel column chromatography purification is (10: 1) to (3: 1); 2. Dissolve the compound 3 obtained in step 1, nitromethane and diethylamine in anhydrous ethanol, stir at 110° C., react for 8 to 12 hours, and monitor the reaction using a TLC spot plate. After the reaction is completed, obtain a reaction solution 2, vacuum concentrate the reaction solution 2, and separate it by silica gel column chromatography to obtain compound 4; The usage ratio of the compound 3, nitromethane, diethylamine and anhydrous ethanol is 0.051 mol: 0.754 mol: 1.012 mol: 150 mL; the volume ratio of petroleum ether and dichloromethane used in the silica gel column chromatography purification is 1:1 to pure dichloromethane; 3. Dissolve the compound 4 obtained in step 2 and ammonium acetate in n-butanol, stir at 130° C., react for 8 to 12 hours, and monitor the reaction using a TLC spot plate. After the reaction is completed, obtain reaction solution 3, concentrate the reaction solution 3 in vacuo to a quarter of the volume of the reaction solution 3, and filter to obtain compound 5; The usage ratio of the compound 4, ammonium acetate and n-butanol is 0.03369 mol:1.168 mol:150 mL; 4. Dissolve the compound 5 obtained in step 3 and N,N-diisopropylethylamine in anhydrous dichloromethane, stir at 0°C, add boron trifluoride etherate, react at room temperature for 8-12 hours, and monitor the reaction using a TLC spot plate. After the reaction is completed, a reaction solution 4 is obtained, and the reaction solution 4 is vacuum concentrated and separated by silica gel column chromatography to obtain a compound 6; The usage ratio of the compound 5, N,N-diisopropylethylamine, anhydrous dichloromethane and boron trifluoride ether is 0.016 mol: 0.251 mol: 100 mL: 0.24 mol; the volume ratio of petroleum ether to ethyl acetate in silica gel column chromatography purification is 2: 1; 5. Dissolve the compound 6 obtained in step 4, potassium carbonate and dodecane bromide in acetone, and react at 63° C. for 5 h. After the reaction is completed, a reaction solution 5 is obtained. The reaction solution 5 is vacuum concentrated and separated by silica gel column chromatography to obtain a compound 7; The dosage ratio of the compound 6, potassium carbonate, dodecane bromide and acetone is 0.149 mol: 0.449 mol: 0.445 mol: 10 mL; the volume ratio of petroleum ether to ethyl acetate in silica gel column chromatography purification is 4: 1; 6. Dissolve the compound 7 obtained in step 5, propyl sultone and potassium carbonate in anhydrous tetrahydrofuran, stir and react at 80°C for 8 to 12 hours, and monitor the reaction by TLC spot plate. After the reaction is completed, a reaction solution 6 is obtained. The reaction solution 6 is vacuum concentrated and separated by silica gel column chromatography to obtain near-infrared zone II BODIPY dye BMP1.

2. The method for preparing a near-infrared second-region BODIPY dye for marking biofilm according to claim 1, characterized in that In step 6, the amount ratio of compound 7, propyl sultone, potassium carbonate and anhydrous tetrahydrofuran is 0.0477 mmol:0.0982 mmol:0.232 mmol:5 mL; the volume ratio of dichloromethane to methanol in the silica gel column chromatography purification is 5:

1.

3. The method for preparing a near-infrared second-region BODIPY dye for marking biofilm according to claim 1, characterized in that The structural formula of the near-infrared second region BODIPY dye BMP1 is:

4. The method for preparing a near-infrared second-region BODIPY dye for marking biofilm according to claim 1, characterized in that Replace step six with: The compound 7 obtained in step five, 3-bromopropyltrimethylammonium bromide and potassium carbonate are dissolved in acetonitrile, stirred at 85°C, and refluxed for 8-12 hours. The reaction is monitored by TLC spot plate. After the reaction is completed, a reaction solution 7 is obtained. The reaction solution 7 is vacuum concentrated and separated by silica gel column chromatography to obtain near-infrared zone II BODIPY dye BMP2.

5. The method for preparing a near-infrared second-region BODIPY dye for marking biofilm according to claim 4, characterized in that The dosage ratio of the compound 7, 3-bromopropyltrimethylammonium bromide, potassium carbonate and acetonitrile is 0.0441 mmol:0.0881 mmol:0.232 mmol:5 mL; the volume ratio of dichloromethane to methanol in the silica gel column chromatography purification is 5:

1.

6. The method for preparing a near-infrared second-region BODIPY dye for marking biofilm according to claim 4, characterized in that The structural formula of the near-infrared second region BODIPY dye BMP2 is:

7. The method for preparing a near-infrared second-region BODIPY dye for marking biofilm according to claim 1, characterized in that Replace step six with: Dissolve the compound 7 obtained in step 5, N,N-dimethylchloropropane hydrochloride and cesium carbonate in anhydrous DMF, stir and react at 60°C for 8-12 hours, and monitor the reaction using a TLC spot plate. After the reaction is completed, add pure water, extract with dichloromethane to obtain a dichloromethane phase, and then concentrate in vacuo to obtain compound 8; Compound 8 and (3-bromopropyl)trimethylammonium bromide were dissolved in anhydrous methanol, stirred and reacted at 80°C for 8-12 hours, and the reaction was monitored by TLC spot plate. After the reaction was completed, reaction solution 8 was obtained, which was vacuum concentrated and separated by reverse silica gel column chromatography to obtain near-infrared zone II BODIPY dye BMP3.

8. The method for preparing a near-infrared second-region BODIPY dye for marking biofilm according to claim 5, characterized in that The usage ratio of the compound 7, N,N-dimethylchloropropane hydrochloride, cesium carbonate, anhydrous DMF and pure water is 0.0357 mmol: 0.0506 mmol: 0.0984 mmol: 5 mL: 5 mL.

9. The method for preparing a near-infrared second-region BODIPY dye for marking biofilm according to claim 5, characterized in that The dosage ratio of the compound 8, (3-bromopropyl)trimethylammonium bromide and anhydrous methanol is 0.0477mmol:0.2385mmol:5mL; the volume ratio of water to methanol in the reverse silica gel column chromatography purification is (95:5) to (5:95).

10. The method for preparing a near-infrared second-region BODIPY dye for marking biofilm according to claim 5, characterized in that The structural formula of the near-infrared second-region BODIPY dye BMP3 is:

Images