Bacterial lipid droplet membrane rhodamine fluorescent dyes and synthesis and use thereof

Abstract

The application provides a rhodamine bacterial lipid droplet membrane fluorescent dye and synthesis and application thereof. The fluorescent dye is based on the affinity of a long carbon chain connected to a nitrogen atom of rhodamine to lipids, the affinity of a positively charged open-loop rhodamine to a negative potential lipid membrane, and the enhancement of a benzene ring to TICT to reduce the background of non-lipid droplet membrane sites. A dye (C4, C6, C8) capable of positioning on a bacterial lipid droplet membrane is designed and synthesized, and the structural formula is shown in formula (1), wherein n is 2, 4 or 6. The dye realizes the lipid droplet membrane imaging of Bacillus cereus, Rhodococcus opacus , Rhodococcus jostii RHA1.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence imaging and labeling, specifically relating to a class of rhodamine bacterial lipid droplet membrane fluorescent probes and their synthesis and application. Background Technology

[0002] Lipid droplets are organelles widely found in plants, animals, fungi, and some prokaryotes. Besides lipid storage, they also participate in physiological processes such as cell communication, metabolic diseases, and the immune system's defense against pathogen invasion. Lipid droplets from various oil-producing bacteria can serve as green biofuels, showing potential to replace fossil fuels in the future. The dynamic processes of lipid droplet formation, division, fusion, and contact with other organelles can be monitored and analyzed in situ using fluorescent dyes and fluorescence microscopy.

[0003] Lipid droplets consist of a neutral lipid core, a monolayer phospholipid membrane enclosing the neutral lipids, and some lipid droplet-related proteins. Current organic small-molecule lipid droplet dyes mainly use Nile Red, BODIPY, and naphthalimide as parent compounds. These dyes are highly lipid-soluble, have zero net charge, and possess excellent staining ability for the neutral lipid core of lipid droplets, but they cannot distinguish the monolayer phospholipid membrane. Some lipid droplet-related proteins are located within the phospholipid membrane to perform their functions. Currently, there is a lack of dyes for imaging the phospholipid membrane of lipid droplets, hindering the monitoring and study of more complex physiological processes. Therefore, the development and application of super-resolution lipid droplet membrane dyes are crucial for the study of lipid droplet-related physiological processes and have broad application prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a class of rhodamine bacterial lipid droplet membrane fluorescent dyes and their synthesis and application.

[0005] This invention provides a class of fluorescent dyes for labeling bacterial lipid droplet membranes. Using rhodamine as the fluorescent group, long carbon chains are introduced onto the nitrogen atoms on both sides to increase lipid solubility. A benzene ring is introduced to enhance TICT in non-lipid environments, thereby reducing background at non-target sites. Replacing the spirocyclic ring with a carboxyl group gives the dye a positive charge, providing targeting specificity to negatively charged bacterial lipid droplet membranes. This fluorescent dye can label bacterial lipid droplet membranes and features low staining concentration, fast staining speed, good biocompatibility, high fluorescence intensity, and strong photostability. A class of rhodamine fluorescent dyes for labeling bacterial lipid droplet membranes has the following structure:

[0006]

[0007] Where n = 2, 4, or 6 or more

[0008] The synthetic route for a class of rhodamine fluorescent dyes used for labeling bacterial lipid droplets is as follows:

[0009]

[0010] The specific synthesis steps are as follows:

[0011] (1) Synthesis of intermediate aniline compounds

[0012] Iodobenzene, aliphatic amine compounds, palladium acetate, 1,1'-binaphthyl-2,2'-bis(diphenylphosphine), and potassium tert-butoxide were dissolved in dry dioxane. The reaction solution was heated to 60-120 °C under nitrogen protection and stirred for 12-24 h. The solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography. Petroleum ether and dichloromethane (volume ratio 1-200:1) were used as eluents to remove the solvent, yielding intermediate aniline compounds.

[0013] The aliphatic amine compounds are butylamine, hexylamine, and octylamine.

[0014] (2) Synthesis of intermediate aniline rhodamine

[0015] Aniline compounds, tris(dibenzylacetone)dipalladium, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl, cesium carbonate, and fluorescein bis(trifluoromethanesulfonate) were dissolved in dry dioxane; the reaction solution was heated to 60-120 °C and stirred for 12-24 h under nitrogen protection; the solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography using dichloromethane and methanol in a volume ratio of 1-200:1 to remove the solvent, yielding the intermediate aniline rhodamine.

[0016] The aniline compounds are N-butylaniline, N-hexylaniline, and N-octylaniline.

[0017] (3) Synthesis of bacterial lipid droplet membrane probes

[0018] The intermediate aniline rhodamine was dissolved in anhydrous ethanol, and concentrated sulfuric acid was slowly added dropwise under ice bath conditions. The mixture was heated to 40-90 °C and stirred for 24-48 h. The pH was adjusted to alkaline by adding saturated sodium carbonate aqueous solution, and the mixture was extracted three times with ethyl acetate. The organic phase was collected. The solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography. The solvent was removed by using dichloromethane and methanol in a volume ratio of 1-200:1 to elute the lipid droplet membrane rhodamine fluorescent dye.

[0019] In step (1), the mass ratio of iodobenzene, fatty amine compounds, palladium acetate, 1,1'-binaphthyl-2,2'-bis(diphenylphosphine), and potassium tert-butoxide is 1:0.5-3:0.1-1:0.1-1:0.1-1; the mass ratio of iodobenzene to dioxane is 1:10-50 g / mL.

[0020] In step (2), the mass ratio of fluorescein bis(trifluoromethanesulfonate), aniline compounds, tris(dibenzylacetone)dipalladium, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl, and cesium carbonate is 1:0.5-3:0.1-1:0.1-1:0.1-1; the mass ratio of fluorescein bis(trifluoromethanesulfonate) to dioxane is 1:10-50 g / mL.

[0021] In step (3), the mass ratio of intermediate aniline rhodamine to concentrated sulfuric acid is 1:0.5-3; the mass ratio of intermediate aniline rhodamine to anhydrous ethanol is 1:200-1000 g / mL.

[0022] This invention provides a method for synthesizing rhodamine fluorescent dyes for labeling bacterial lipid droplets, which has the advantages of simple purification, easy operation, and high yield.

[0023] Application of a class of rhodamine fluorescent dyes for bacterial lipid droplet membranes in fluorescence imaging and labeling. These fluorescent dyes can specifically label lipid droplet membranes within live bacteria, enabling super-resolution fluorescence imaging.

[0024] This invention has the following characteristics:

[0025] The dye of the present invention has the advantages of low cost of raw materials, simple method, high yield and easy derivatization.

[0026] The dye of this invention exhibits high specificity and stability. The introduction of a benzene ring onto rhodamine nitrogen enhances the dye's TICT (transient inductively coupled plasma) effect. At non-target sites, the free torsion of the benzene ring leads to the TICT effect, causing the dye to be in a fluorescence-quenched state. After specific binding to the bacterial lipid droplet membrane, the torsion of the benzene ring is restricted, the TICT effect is suppressed, and the dye fluorescence intensity is significantly increased, achieving specific imaging of the bacterial lipid droplet membrane. The long carbon chain can improve the dye's affinity for the lipid membrane and enhance its retention capacity on the membrane. Therefore, introducing a long carbon chain onto rhodamine nitrogen can achieve stable labeling of the lipid droplet membrane, thus realizing super-resolution fluorescence imaging of the bacterial lipid droplet membrane.

[0027] The dye of this invention can precisely locate lipid droplet membranes in live bacteria in real time, and can be applied to the study of lipid droplet dynamics in bacteria and the study of the interaction between lipid droplets and other subcellular structures.

[0028] This invention's fluorescent dye is based on the affinity of the long carbon chain of rhodamine linked to the nitrogen atom for lipids, the affinity of positively charged open-ring rhodamine for negatively charged lipid membranes, and the benzene ring-enhanced TICT to reduce background at non-lipid droplet membrane sites. This dye achieves the desired effect on Bacillus cereus, ... Rhodococcusopacus , Rhodococcusjostii Lipid droplet imaging of RHA1. Attached Figure Description

[0029] Figure 1 The 1H NMR spectrum of C4 prepared in Example 1;

[0030] Figure 2 The carbon NMR spectrum of C4 prepared in Example 1;

[0031] Figure 3 The ultraviolet absorption spectra of C4 prepared in Example 1 in methanol and glycerol solvents with different ratios are shown. The horizontal axis represents wavelength and the vertical axis represents absorption intensity. The concentration of the fluorescent dye is 2 µM.

[0032] Figure 4 Fluorescence emission spectra of C4 prepared in Example 1 in methanol and glycerol solvents with different ratios. The horizontal axis represents wavelength and the vertical axis represents fluorescence intensity. The concentration of the fluorescent dye is 2 µM.

[0033] Figure 5 The 1H NMR spectrum of C6 prepared in Example 2;

[0034] Figure 6 C1NMR spectrum of C6 prepared in Example 2;

[0035] Figure 7 The ultraviolet absorption spectra of C6 prepared in Example 2 in methanol and glycerol solvents with different ratios are shown. The horizontal axis represents wavelength and the vertical axis represents absorption intensity. The concentration of the fluorescent dye is 2 µM.

[0036] Figure 8 Fluorescence emission spectra of C6 prepared in Example 2 in solvents with different ratios of methanol and glycerol. The horizontal axis represents wavelength, and the vertical axis represents fluorescence intensity. The concentration of the fluorescent dye is 2 µM.

[0037] Figure 9 The 1H NMR spectrum of C8 prepared in Example 3;

[0038] Figure 10 Carbon NMR spectrum of C8 prepared in Example 3;

[0039] Figure 11 The ultraviolet absorption spectra of C8 prepared in Example 3 in methanol and glycerol solvents with different ratios are shown. The horizontal axis represents wavelength and the vertical axis represents absorption intensity. The concentration of the fluorescent dye is 2 µM.

[0040] Figure 12 Fluorescence emission spectra of C8 prepared in Example 3 in methanol and glycerol solvents with different ratios. The horizontal axis represents wavelength and the vertical axis represents fluorescence intensity. The concentration of the fluorescent dye is 2 µM.

[0041] Figure 13 The lipid droplet film dye C4 in Example 4 in bacteria Rhodococcusopacus Super-resolution fluorescence imaging of lipid droplet membranes in China;

[0042] Figure 14 In Example 5, the lipid droplet film dye C4 was used in... Rhodococcusjostii Super-resolution fluorescence imaging of lipid droplet membranes in RHA1;

[0043] Figure 15 Super-resolution fluorescence imaging of lipid droplet membranes in Bacillus cereus using the lipid droplet dye C6 in Example 6;

[0044] Figure 16 Super-resolution fluorescence imaging of lipid droplet membranes in Bacillus cereus using the lipid droplet dye C8 in Example 7;

[0045] Figure 17 The super-resolution fluorescence image of lipid droplet film dye C6 in Example 8 after 100 min of stable staining of lipid droplet film in Bacillus cereus. Detailed Implementation

[0046] Example 1

[0047] C4 Synthesis

[0048] (1) Synthesis of intermediate N-butylaniline

[0049]

[0050] Iodobenzene (2 g, 9.85 mmol), butylamine (5 g, 68 mmol), palladium acetate (0.4 g, 1.78 mmol), 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (0.4 g, 0.64 mmol), and potassium tert-butoxide (2 g, 17.82 mmol) were dissolved in dry dioxane (30 mL). The reaction mixture was heated to 100 °C and stirred for 12 h under nitrogen protection. The solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography. The solvent was removed by eluent in a 20:1 volume ratio of petroleum ether and dichloromethane to give 440 mg of pale yellow liquid N-butylaniline, with a yield of 30%.

[0051] (2) Synthesis of intermediate butylaniline rhodamine

[0052]

[0053] N-Butylaniline (600 mg, 4.0 mmol), tris(dibenzylacetone)palladium (20 mg, 0.022 mmol), 2-dicyclohexylphosphine-2',4',6'-triisoylbiphenyl (20 mg, 0.042 mmol), cesium carbonate (300 mg, 0.92 mmol), and fluorescein bis(trifluoromethanesulfonate) (200 mg, 0.336 mmol) were dissolved in dry dioxane (5 mL). The reaction mixture was heated to 120 °C and stirred for 12 h under nitrogen protection. The solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography using dichloromethane and methanol in a volume ratio of 10:1 to remove the solvent, yielding 90 mg of the pale pink intermediate butylaniline rhodamine, with a yield of 45%. The specific 1H NMR data of the butylaniline rhodamine prepared in Example 1 are as follows:

[0054] 1 H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 7.6 Hz, 1H), 7.55 (t, J = 7.2 Hz, 1H), 7.48 (t, J = 7.4 Hz, 1H), 7.27 (dd, J = 8.4, 7.3 Hz, 4H), 7.14 (d, J = 7.6 Hz,1H), 7.07 (m, 6H), 6.47 (d, J = 2.4 Hz, 2H), 6.44 (d, J = 8.8 Hz, 2H), 6.32 (dd, J = 8.8, 2.4 Hz, 2H), 3.58 (m, 4H), 1.56 (m, 4H), 1.26 (dq, J = 14.7, 7.4 Hz, 4H), 0.84 (t, J = 7.4 Hz, 6H).

[0055] (3) Synthesis of bacterial lipid droplet membrane probe C4

[0056]

[0057] The intermediate butylaniline rhodamine (50 mg, 0.084 mmol) was dissolved in anhydrous ethanol (10 mL), and concentrated sulfuric acid (50 mg) with a mass concentration of 98% was slowly added dropwise under ice bath conditions. The mixture was heated to 60 °C and stirred for 48 h. The pH was adjusted to 10 by adding saturated sodium carbonate aqueous solution, and the mixture was extracted three times with ethyl acetate. The organic phase was collected, the solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography. The solvent was removed by using dichloromethane and methanol in a volume ratio of 10:1 to obtain 47 mg of purple lipid droplet rhodamine fluorescent dye, with a yield of 85%.

[0058] The C4 hydrogen NMR spectrum of the bacterial lipid droplet membrane probe prepared in Example 1 is as follows: Figure 1 As shown, the specific data is as follows:

[0059] 1 H NMR (400 MHz, MeOD) δ 8.29 (d, J = 7.7 Hz, 1H), 7.82 (dt, J = 24.1, 7.5 Hz, 2H), 7.57 (t, J = 7.6 Hz, 4H), 7.47 (t, J = 7.4 Hz, 2H), 7.41 (d, J = 7.4Hz, 1H), 7.33 (d, J = 7.8 Hz, 4H), 7.08 (d, J = 9.4 Hz, 2H), 6.84 (m, 4H), 4.04(q, J = 7.1 Hz, 4H), 3.95 (m, 4H), 1.74 (m, 4H), 1.43 (m, 4H), 1.03 (t, J = 7.1Hz, 3H), 0.95 (t, J = 7.3 Hz, 6H).

[0060] The C4 NMR spectrum of the bacterial lipid droplet membrane probe prepared in Example 1 is as follows: Figure 2 As shown, the specific data is as follows:

[0061] 13C NMR (176 MHz, CDCl3) δ 165.00, 160.40, 157.79, 157.03, 143.15,133.28, 131.20, 131.08, 130.79, 130.52, 130.27, 128.74, 127.46, 115.99, 114.64, 97.97, 62.74, 61.61, 53.82, 29.35, 20.06, 15.32, 13.82.

[0062] Its high-resolution mass spectrometry data are as follows: Theoretical value of high-resolution mass spectrometry C 40 H 35 N2O3[M] + 623.3268, measured value 623.3266.

[0063] Upon testing, its structure is shown in equation C4 above, and its spectral properties are as follows:

[0064] C4 was dissolved in DMSO solution to prepare a 2mM stock solution. Different concentration test solutions were prepared as needed to detect its fluorescence spectrum and ultraviolet absorption spectrum. Super-resolution imaging of the lipid droplet membrane of bacteria was performed for dynamic tracking.

[0065] The ultraviolet absorption and fluorescence emission spectra of C4 were measured in different ratios of methanol and glycerol solvents (volume ratio, methanol / glycerol = 0 / 10, 1 / 9, 2 / 8, 3 / 7, 4 / 6, 5 / 5, 6 / 4, 7 / 3, 8 / 2, 9 / 1, 10 / 0). Each time, 4 μL of C4 stock solution was added to 4 mL of solvent, and the mixture was sonicated to ensure homogeneity, preparing a 2 μM fluorescent dye test solution for ultraviolet absorption and fluorescence emission spectra measurement.

[0066] The UV absorption spectra of C4 in methanol and glycerol solvents with different ratios are as follows: Figure 3 As shown.

[0067] The fluorescence emission spectra of C4 in methanol and glycerol solvents with different ratios are as follows: Figure 4 As shown, with the increase of glycerol content in the solvent, the solvent viscosity increases, the TICT of C4 is suppressed, and the fluorescence increases significantly.

[0068] Example 2

[0069] C6 Synthesis

[0070] (1) Synthesis of intermediate N-hexylaniline

[0071]

[0072] Iodobenzene (1.5 g, 7.35 mmol), hexylamine (4 g, 39.6 mmol), palladium acetate (0.2 g, 0.89 mmol), 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (0.8 g, 1.28 mmol), and potassium tert-butoxide (1 g, 8.91 mmol) were dissolved in dry dioxane (50 mL). The reaction mixture was heated to 90 °C and stirred for 16 h under nitrogen protection. The solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography using petroleum ether and dichloromethane in a volume ratio of 25:1 to remove the solvent, yielding 495 mg of pale yellow liquid N-hexylaniline, with a yield of 38%. The specific 1H NMR data of the N-hexylaniline prepared in Example 2 are as follows:

[0073] 1 H NMR (700 MHz, CDCl3) δ 7.16 (t, J = 7.8 Hz, 2H), 6.68 (t, J = 7.3 Hz, 1H), 6.59 (d, J = 8.0 Hz, 2H), 3.58 (s, 1H), 3.09 (t, J = 7.2 Hz, 2H), 1.63 –1.58 (m, 2H), 1.39 (dt, J = 14.3, 7.2 Hz, 2H), 1.32 (dd, J = 11.5, 8.1 Hz, 4H), 0.90 (t, J = 6.8 Hz, 3H).

[0074] (2) Synthesis of intermediate hexylaniline rhodamine

[0075]

[0076] N-hexylaniline (300 mg, 1.69 mmol), tris(dibenzylacetone)palladium (200 mg, 0.22 mmol), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (200 mg, 0.42 mmol), cesium carbonate (300 mg, 0.92 mmol), and fluorescein bis(trifluoromethanesulfonate) (300 mg, 0.503 mmol) were dissolved in dry dioxane (5 mL). The reaction mixture was heated to 100 °C and stirred for 14 h under nitrogen protection. The solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography. The solvent was removed by eluent in a mixture of dichloromethane and methanol in a volume ratio of 8:1 to obtain 120 mg of the pale pink intermediate hexylaniline rhodamine, with a yield of 37%.

[0077] The specific 1H NMR data of the hexylaniline rhodamine prepared in Example 2 are as follows:

[0078] 1 H NMR (700 MHz, CDCl3) δ 8.01 (s, 1H), 7.67 (s, 1H), 7.60 (s, 1H), 7.38 (s, 4H), 7.19 (s, 7H), 6.60 (s, 2H), 6.56 (d, J = 7.3 Hz, 2H), 6.44 (d, J =5.5 Hz, 2H), 3.70 (s, 4H), 1.70 (s, 4H), 1.32 (s, 12H), 0.91 (s, 6H).

[0079] (3) Synthesis of bacterial lipid droplet membrane probe C6

[0080]

[0081] The intermediate hexylaniline rhodamine (100 mg, 0.153 mmol) was dissolved in anhydrous ethanol (30 mL), and concentrated sulfuric acid (100 mg) with a mass concentration of 80% was slowly added dropwise under ice bath conditions. The mixture was heated to 75 °C and stirred for 30 h. The pH was adjusted to 9 by adding saturated sodium carbonate aqueous solution, and the mixture was extracted three times with ethyl acetate. The organic phase was collected, the solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography. The solvent was removed by using dichloromethane and methanol in a volume ratio of 10:1 to obtain 88 mg of purple lipid droplet rhodamine fluorescent dye, with a yield of 80%.

[0082] The C6 NMR spectrum of the bacterial lipid droplet membrane probe prepared in Example 2 is as follows: Figure 5 As shown, the specific data is as follows:

[0083] 1 H NMR (700 MHz, CDCl3) δ 8.26 (d, J = 7.9 Hz, 1H), 7.83 (t, J = 7.5 Hz, 1H), 7.72 (t, J = 7.7 Hz, 1H), 7.54 (t, J = 7.6 Hz, 4H), 7.45 (t, J = 7.4 Hz, 2H), 7.34 (d, J = 7.5 Hz, 1H), 7.25 (d, J = 7.7 Hz, 4H), 7.04 (d, J= 9.3 Hz, 2H), 6.75(s, 4H), 4.10 (m, 2H), 3.91 – 3.87 (m, 4H), 1.78 – 1.73 (m, 4H), 1.38 (s,4H), 1.30 m, 8H), 1.12 (t, J = 7.1 Hz, 3H), 0.87 (t, J = 6.3 Hz, 6H).

[0084] The C6 NMR spectrum of the bacterial lipid droplet membrane probe prepared in Example 2 is as follows: Figure 6 As shown, the specific data is as follows:

[0085] 13 C NMR (176 MHz, CDCl3) δ 165.01, 160.43, 157.78, 157.02, 143.14,133.29, 131.29, 131.20, 130.80, 130.54, 130.29, 129.85, 128.76, 127.46,115.95, 114.65, 97.96, 62.70, 61.61, 54.03, 31.46, 27.29, 26.46, 22.53,15.33, 13.98, 13.88.

[0086] Its high-resolution mass spectrometry data are as follows: Theoretical value of high-resolution mass spectrometry C 46 H 51 N2O3 + [M] + 679.3894, measured value 679.3835.

[0087] Upon testing, its structure is shown in equation C6 above, and its spectral properties are as follows:

[0088] C6 was dissolved in DMSO solution to prepare a 2mM stock solution. Different concentration test solutions were prepared as needed to detect its fluorescence spectrum and ultraviolet absorption spectrum. Super-resolution imaging of the lipid droplet membrane of bacteria was performed for dynamic tracking.

[0089] The ultraviolet absorption and fluorescence emission spectra of C6 were measured in different ratios of methanol and glycerol solvents (volume ratio, methanol / glycerol = 0 / 10, 1 / 9, 2 / 8, 3 / 7, 4 / 6, 5 / 5, 6 / 4, 7 / 3, 8 / 2, 9 / 1, 10 / 0). For each test, 4 μL of C6 stock solution was added to 4 mL of solvent, and the mixture was sonicated to ensure homogeneity, preparing a 2 μM fluorescent dye test solution for ultraviolet absorption and fluorescence emission spectra measurement.

[0090] The UV absorption spectra of C6 in methanol and glycerol solvents with different ratios are as follows: Figure 7 As shown.

[0091] The fluorescence emission spectra of C6 in methanol and glycerol solvents with different ratios are as follows: Figure 8 As shown, with the increase of glycerol content in the solvent, the solvent viscosity increases, the TICT of C6 is suppressed, and the fluorescence increases significantly.

[0092] Example 3

[0093] C8 synthesis

[0094] (1) Synthesis of intermediate N-octylaniline

[0095]

[0096] Iodobenzene (1 g, 4.90 mmol), octylamine (2 g, 15.50 mmol), palladium acetate (0.3 g, 1.34 mmol), 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (0.6 g, 0.96 mmol), and potassium tert-butoxide (0.9 g, 8.02 mmol) were dissolved in dry dioxane (40 mL). The reaction mixture was heated to 110 °C and stirred for 13 h under nitrogen protection. The solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography. The solvent was removed by eluent in a 15:1 (v / v) mixture of petroleum ether and dichloromethane to obtain 140 mg of pale yellow liquid N-octylaniline, with a yield of 14%. The specific 1H NMR data of the N-octylaniline prepared in Example 3 are as follows:

[0097] 1 H NMR (400 MHz, CDCl3) δ 7.22 – 7.08 (m, 2H), 6.67 (t, J = 7.3 Hz, 1H), 6.63 – 6.47 (m, 2H), 3.57 (s, 1H), 3.09 (t, J = 7.1 Hz, 2H), 1.64 – 1.56 (m,2H), 1.42 – 1.26 (m, 10H), 0.88 (t, J = 6.8 Hz, 3H).

[0098] (2) Synthesis of intermediate octylaniline rhodamine

[0099]

[0100] N-Octylane (300 mg, 1.46 mmol), tris(dibenzylacetone)palladium (38 mg, 0.042 mmol), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (40 mg, 0.084 mmol), cesium carbonate (150 mg, 0.46 mmol), and fluorescein difluoromethanesulfonate (250 mg, 0.42 mmol) were dissolved in dry dioxane (8 mL). The reaction mixture was heated to 120 °C and stirred for 17 h under nitrogen protection. The solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography. The solvent was removed by eluent in a 9:1 ratio of dichloromethane and methanol to obtain 148 mg of the pale pink intermediate octylaniline rhodamine, with a yield of 50%.

[0101] The specific 1H NMR data of the octylaniline rhodamine prepared in Example 3 are as follows:

[0102] 1 H NMR (700 MHz, CDCl3) δ 7.97 (d, J = 7.4 Hz, 1H), 7.63 (t, J = 7.2 Hz, 1H), 7.56 (t, J = 7.3 Hz, 1H), 7.34 (t, J = 7.4 Hz, 4H), 7.21 (d, J = 7.4 Hz, 1H), 7.14 (t, J = 8.3 Hz, 6H), 6.55 (s, 2H), 6.51 (d, J = 8.7 Hz, 2H), 6.39 (d, J = 8.2Hz, 2H), 3.68 – 3.62 (m, 4H), 1.65 (s, 4H), 1.33 – 1.20 (m, 24H), 0.86 (t, J =6.7 Hz, 6H).

[0103] (3) Synthesis of bacterial lipid droplet membrane probe C8

[0104]

[0105] The intermediate octylaniline rhodamine (80 mg, 0.113 mmol) was dissolved in anhydrous ethanol (35 mL), and concentrated sulfuric acid (200 mg) with a mass concentration of 90% was slowly added dropwise under ice bath conditions. The mixture was heated to 75 °C and stirred for 28 h. The pH was adjusted to 11 by adding saturated sodium carbonate aqueous solution, and the mixture was extracted three times with ethyl acetate. The organic phase was collected, the solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography. The solvent was removed by using dichloromethane and methanol in a volume ratio of 12:1 to obtain 65 mg of purple lipid droplet rhodamine fluorescent dye, with a yield of 75%.

[0106] The C8 nuclear magnetic resonance (NMR) spectrum of the bacterial lipid droplet membrane probe prepared in Example 3 is as follows: Figure 9 As shown, the specific data is as follows:

[0107] 1 H NMR (700 MHz, CDCl3) δ 8.26 (d, J = 7.8 Hz, 1H), 7.83 (t, J = 7.4 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.54 (t, J = 7.4 Hz, 4H), 7.45 (t, J = 7.3 Hz, 2H), 7.34 (d, J = 7.4 Hz, 1H), 7.25 (d, J = 7.5 Hz, 4H), 7.04 (d, J = 9.1 Hz, 2H), 6.75(s, 4H), 4.10 (m, 2H), 3.90 – 3.85 (m, 4H), 1.78 – 1.73 (m, 4H), 1.37 (m,4H), 1.30 – 1.22 (m, 18H), 1.12 (t, J = 7.0 Hz, 3H), 0.86 (t, J = 6.7 Hz, 6H).

[0108] The C8 carbon NMR spectrum of the bacterial lipid droplet membrane probe prepared in Example 3 is as follows: Figure 10 As shown, the specific data is as follows:

[0109] 13C NMR (176 MHz, CDCl3) δ 165.02, 160.44, 157.79, 157.02, 143.14,133.31, 131.19, 131.11, 130.80, 130.54, 130.30, 129.84, 128.75, 127.46,115.97, 114.66, 97.95, 62.70, 61.61, 54.03, 31.71, 29.26, 29.14), 27.31,26.79, 22.58, 15.33, 14.07, 13.88.

[0110] Its high-resolution mass spectrometry data are as follows: Theoretical value of high-resolution mass spectrometry C 50 H 59 N2O3 + [M] + 735.4520, measured value 735.4562.

[0111] Upon testing, its structure is shown in formula C8 above.

[0112] C8 was dissolved in DMSO solution to prepare a 2mM stock solution. Different concentration test solutions were prepared as needed to detect its fluorescence spectrum and ultraviolet absorption spectrum. Super-resolution imaging of the lipid droplet membrane of bacteria was performed for dynamic tracking.

[0113] The ultraviolet absorption and fluorescence emission spectra of C8 were measured in different ratios of methanol and glycerol solvents (volume ratio, methanol / glycerol = 0 / 10, 1 / 9, 2 / 8, 3 / 7, 4 / 6, 5 / 5, 6 / 4, 7 / 3, 8 / 2, 9 / 1, 10 / 0). For each test, 4 μL of C8 stock solution was added to 4 mL of solvent, and the mixture was sonicated to ensure homogeneity, preparing a 2 μM fluorescent dye test solution for ultraviolet absorption and fluorescence emission spectra measurement.

[0114] The UV absorption spectra of C8 in methanol and glycerol solvents with different ratios are as follows: Figure 11 As shown.

[0115] The fluorescence emission spectra of C8 in methanol and glycerol solvents with different ratios are as follows: Figure 12 As shown, with the increase of glycerol content in the solvent, the solvent viscosity increases, the TICT of C8 is suppressed, and the fluorescence increases significantly.

[0116] Example 4

[0117] C4 pairs Rhodococcusopacus ( Beijing Beina Innovation Biotechnology Research Institute, BNCC337018 ）After staining, superresolution fluorescence was measured. 0.2 μL of C4 stock solution (C4 dissolved in DMSO solution to prepare a 2 mM stock solution) was dissolved in 200 μL of a solution with an OD600 value of 1. Rhodococcusopacus In the process, super-resolution fluorescence imaging was performed after incubation at room temperature for 10 minutes.

[0118] The final concentration of C4 is 1 μmol. Rhodococcusopacus Super-resolution fluorescence imaging of the culture medium is shown below. Figure 13 As shown: C4 staining Rhodococcusopacus The lipid droplet membrane has a distinct hollow structure, and C4 can specifically label the lipid droplet membrane of bacteria.

[0119] Example 5

[0120] C4 pairs Rhodococcusjostii RHA1 (accession number NBRC 108803) was subjected to super-resolution fluorescence testing after staining. 0.2 μL of C4 stock solution (C4 dissolved in DMSO solution to prepare a 2 mM stock solution) was dissolved in 200 μL of a solution with an OD600 value of 1. Rhodococcusjostii In RHA1, super-resolution fluorescence imaging was performed after incubation at room temperature for 10 minutes.

[0121] The final concentration of C4 is 1 μmol. Rhodococcusjostii Super-resolution fluorescence imaging of RHA1 culture medium is shown below. Figure 14 As shown: C4 staining Rhodococcusjostii RHA1 lipid droplet membranes have a distinct hollow structure, and C4 can specifically label the bacterial lipid droplet membranes.

[0122] Example 6

[0123] Super-resolution fluorescence testing was performed on Bacillus cereus (accession number CICC 20726) after staining with C6. 0.2 μL of C6 stock solution (C6 dissolved in DMSO solution to prepare a 2 mM stock solution) was dissolved in 200 μL of Bacillus cereus culture medium with an OD600 value of 1. After incubation at room temperature for 10 minutes, super-resolution fluorescence imaging was performed.

[0124] Super-resolution fluorescence imaging of Bacillus cereus culture medium with a final C6 concentration of 1 μmol is shown in the image below. Figure 15 As shown: C6 staining of the lipid droplet membrane of Bacillus cereus has a distinct hollow structure, and C6 can specifically label the lipid droplet membrane of Bacillus cereus.

[0125] Example 7

[0126] Super-resolution fluorescence testing was performed on Bacillus cereus (accession number CICC 20726) after staining with C8. 0.2 μL of C8 stock solution (C8 dissolved in DMSO solution to prepare a 2 mM stock solution) was dissolved in 200 μL of Bacillus cereus culture medium with an OD600 value of 1. After incubation at room temperature for 10 minutes, super-resolution fluorescence imaging was performed.

[0127] Super-resolution fluorescence imaging of Bacillus cereus culture medium with a final C8 concentration of 1 μmol is shown below. Figure 16 As shown: C8 staining of the lipid droplet membrane of Bacillus cereus has a distinct hollow structure, and C8 can specifically label the lipid droplet membrane of Bacillus cereus.

[0128] Example 8

[0129] Super-resolution fluorescence testing was performed on Bacillus cereus (accession number CICC 20726) after staining with C6. 0.2 μL of C6 stock solution (C6 dissolved in DMSO solution to prepare a 2 mM stock solution) was dissolved in 200 μL of Bacillus cereus culture medium with an OD600 value of 1. After incubation at room temperature for 10 minutes, super-resolution fluorescence imaging was performed. The same lipid droplet was imaged every 10 minutes for a total duration of 100 minutes.

[0130] Long-term super-resolution fluorescence imaging of Bacillus cereus culture medium with a final C6 concentration of 1 μmol is shown in the image below. Figure 17 As shown: C6 staining of Bacillus cereus lipid droplet membrane has a distinct hollow structure. C6 can specifically label the lipid droplet membrane of Bacillus cereus, and C6 can stably label the lipid droplet membrane within 100 min of imaging time.

Claims

1. A type of rhodamine fluorescent dye for bacterial lipid droplet membranes, with the following structure: ； Where n = 2, 4 or 6.

2. The method for synthesizing a type of bacterial lipid droplet membrane rhodamine fluorescent dye as described in claim 1, characterized in that, The steps are as follows: (1) Synthesis of intermediate aniline rhodamine: Aniline compounds, tris(dibenzylacetone)dipalladium, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl, cesium carbonate, and fluorescein bis(trifluoromethanesulfonate) were dissolved in a dry dioxane solvent. The reaction solution was heated to 60-120 °C and stirred for 12-24 h under nitrogen protection. The solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography. The eluent was dichloromethane and methanol in a volume ratio of 1-200:

1. After removing the eluent, the intermediate aniline rhodamine was obtained. The aniline compound is one of N-butylaniline, N-hexylaniline, and N-octylaniline; (2) Synthesis of rhodamine fluorescent dye for bacterial lipid droplet membranes: The intermediate aniline rhodamine was dissolved in anhydrous ethanol, and concentrated sulfuric acid was added dropwise under ice bath conditions. The mixture was heated to 40-90 °C and stirred for 24-48 h. The pH was adjusted to alkaline by adding saturated sodium carbonate aqueous solution, and the mixture was extracted 2-6 times with ethyl acetate. The organic phase was collected. The solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography using dichloromethane and methanol in a volume ratio of 1-200:1 as eluents. After removing the eluents, the lipid droplet membrane rhodamine fluorescent dye was obtained.

3. The synthesis method according to claim 2, characterized in that, The synthesis of the aniline compounds includes: Iodobenzene, aliphatic amine compounds, palladium acetate, 1,1'-binaphthyl-2,2'-bisdiphenylphosphine, and potassium tert-butoxide were dissolved in a dry dioxane solvent. The reaction solution was heated to 60-120 °C under nitrogen protection and stirred for 12-24 h. The solvent was removed under reduced pressure, and the mixture was separated by silica gel column chromatography. Petroleum ether and dichloromethane in a volume ratio of 1-200:1 were used as eluents. After removing the eluents, aniline compounds were obtained. The fatty amine compound is one of butylamine, hexylamine, or octylamine; The aniline compound is one of N-butylaniline, N-hexylaniline, and N-octylaniline.

4. The synthesis method according to claim 3, characterized in that: The mass ratio of iodobenzene, fatty amine compounds, palladium acetate, 1,1'-binaphthyl-2,2'-bis(diphenylphosphine), and potassium tert-butoxide is 1:0.5-3:0.1-1:0.1-1:0.1-1; the mass ratio of iodobenzene to dioxane is 1:10-50 g / mL.

5. The synthesis method according to claim 2, characterized in that: In step (1), the mass ratio of fluorescein bis(trifluoromethanesulfonate), aniline compounds, tris(dibenzylacetone)dipalladium, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl, and cesium carbonate is 1:0.5-3:0.1-1:0.1-1:0.1-1.5; the mass ratio of fluorescein bis(trifluoromethanesulfonate) to dioxane is 1:10-50 g / mL.

6. The method for synthesizing a type of bacterial lipid droplet membrane rhodamine fluorescent dye according to claim 2, characterized in that: In step (2), the mass ratio of intermediate aniline rhodamine to concentrated sulfuric acid is 1:0.5-3; the mass ratio of intermediate aniline rhodamine to anhydrous ethanol is 1:200-1000 g / mL. The mass concentration of concentrated sulfuric acid ranges from 70% to 98%; the pH range is from 9 to 14, which is alkaline.

7. The application of the bacterial lipid droplet membrane rhodamine fluorescent dye of claim 1 in fluorescence imaging and / or labeling processes.

8. The application according to claim 7, characterized in that: The fluorescent dye is used as a lipid droplet dye.

9. The application according to claim 7 or 8, characterized in that: The fluorescent dye can specifically label the lipid droplet membrane of bacteria.

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