Asymmetric BODIPY-based lipid droplet fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a lipid droplet fluorescent probe based on asymmetric BODIPY as well as a preparation method and application of the lipid droplet fluorescent probe. The fluorescent probe has a structure as shown in a formula I; wherein R1 is selected from any one of aryl, aralkyl and heteroaryl; r2 is selected from at least one of aryl, aralkyl and heterocyclic radical; the aryl is unsubstituted aryl or aryl substituted by at least one substituent; the heteroaryl is unsubstituted heteroaryl or heteroaryl substituted by at least one substituent; the aralkyl is unsubstituted aralkyl or aralkyl substituted by at least one substituent; the heterocyclic group is an unsubstituted heterocyclic group or a heterocyclic group substituted by at least one substituent; the substituent group is selected from any one of alkyl, methoxyl, halogen and halogenated alkyl. According to the fluorescent probe provided by the invention, the advantages of large Stokes displacement, small background interference and the like of the asymmetric BODIPY are utilized, and the targeted lipid droplet fluorescent probe with high sensitivity and high stability is obtained.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a lipid droplet fluorescent probe based on asymmetric BODIPY, its preparation method, and its application. Background Technology

[0002] Lipid droplets (LDs) are highly dynamic organelles that play a crucial role in regulating cellular lipid metabolism. Firstly, structurally, LDs consist of a neutral lipid core (i.e., triacylglycerol and sterol esters) encased in a phospholipid monolayer and modified with specific proteins. Their diameters range from 20 nm to 100 μm, allowing them to grow in different cells and perform different functions. Secondly, LDs participate in various cellular processes, such as membrane formation, transport, and protein interactions, thus playing a vital role in regulating lipid storage and energy supply. Studies have found that LD metabolic disorders and functional impairments are associated with various diseases, including obesity, hepatic steatosis, diabetes, cardiac dysfunction, atherosclerosis, and fatty liver. Therefore, understanding the influencing factors of LD formation and its interactions with other organelles is crucial. This will help elucidate the relationship between LD generation and disease pathogenesis, and to develop targeted treatment methods.

[0003] Traditional methods for visualizing lipid droplets (LDs) include electron microscopy, cryo-electron microscopy, Raman imaging, immunoelectron microscopy, direct organelle mass spectrometry, and optical diffraction tomography. These advanced techniques require cell fixation or extraction of LDs for detection, but they neglect the real-world environment of living cells, significantly reducing their relevance in practical research. In this context, fluorescence imaging (FLI) offers a non-invasive method for visualizing cellular components. FLI boasts advantages such as high sensitivity, high temporal resolution, ease of operation, and real-time tracking, making it an important tool for disease diagnosis and monitoring, as well as image-guided surgery, greatly promoting the development of medical treatment. Although many fluorescent probes based on different mechanisms have been reported for lipid droplet imaging, they suffer from low imaging sensitivity and significant background interference, hindering lipid droplet imaging. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a fluorescent probe based on asymmetric BODIPY. The asymmetric BODIPY structure in the molecular structure of this fluorescent probe enables the probe to have a large Stokes shift, effectively avoiding the limitation of self-absorption phenomenon of traditional organic fluorescent probes, thereby enabling clearer and more stable lipid droplet imaging.

[0005] The present invention also proposes a method for preparing the above-mentioned fluorescent probe.

[0006] The present invention also proposes an application.

[0007] According to a first aspect of the present invention, an asymmetric BODIPY-based fluorescent probe is provided, the fluorescent probe having a structure as shown in Formula I:

[0008]

[0009] Wherein, R1 is a substituted or unsubstituted aromatic group;

[0010] R2 is selected from substituted or unsubstituted aromatic groups and substituted or unsubstituted C. 1～6 alkyl;

[0011] The aromatic group is selected from: aryl, heteroaryl, and arylamino groups containing 1 to 6 phenyl groups;

[0012] The phenyl groups in the aryl group containing 1 to 6 phenyl groups are connected by a hydrocarbon group or a heteroatom selected from N, O and S;

[0013] The heteroaryl group is a 3- to 7-membered monocyclic, 6- to 10-membered bicyclic, or 13- to 16-membered polycyclic system having one, two, or three independent heteroatom ring members selected from N, O, S, and B;

[0014] The substituent is selected from C. 1～6 Alkyl, C 1～6 Alkoxy, heteroaryl, heterocyclic, C 1～6 Any one of alkyl-amino, halogen, and haloalkyl.

[0015] In some embodiments of the present invention, R1 is selected from any of the following structures:

[0016]

[0017] In some embodiments of the present invention, R2 is selected from any of the following structures:

[0018]

[0019] According to a second aspect of the present invention, a method for preparing the asymmetric BODIPY-based fluorescent probe described in the first aspect of the present invention is provided, the method specifically comprising the following steps:

[0020] S1: A haloaromatic hydrocarbon with N-Boc-pyrrole group and an arylboronic acid with substituent R1 are coupled by a Suzuki reaction to obtain intermediate a;

[0021] S2: A halogen substituent is introduced into intermediate a through a substitution reaction to obtain intermediate b;

[0022] S3: Sodium methoxide is used to react with intermediate b to remove the Boc group, and then a cyclization reaction is carried out under the catalysis of boron trifluoride diethyl ether and triethylamine to obtain intermediate c;

[0023] S4: In the presence of a catalyst and a base, arylboronic acid with substituent R2 is reacted with intermediate c to obtain the fluorescent probe based on asymmetric BODIPY.

[0024] In some embodiments of the present invention, the reaction system of the Suzuki coupling reaction in step S1 further includes a catalyst, a ligand, and a base.

[0025] In some embodiments of the present invention, the catalyst in step S1 includes a palladium catalyst.

[0026] In some embodiments of the present invention, the palladium catalyst comprises palladium acetate or tetra(triphenylphosphine)palladium.

[0027] In some embodiments of the present invention, the ligand comprises at least one selected from 2-biscyclohexylphosphine-2',4',6'-triisopropylbiphenyl, 2-dicyclohexylphosphine-2',6'-diisopropoxy-1,1'-biphenyl, 2-biscyclohexylphosphine-2',6'-dimethoxybiphenyl, triphenylphosphine, and tricyclohexylphosphine.

[0028] In some embodiments of the present invention, the alkali includes at least one selected from sodium bicarbonate, sodium carbonate, potassium carbonate, and cesium carbonate.

[0029] In some embodiments of the present invention, the molar ratio of the haloaromatic hydrocarbon with N-Boc-pyrrole group and the arylboronic acid with substituent R1 in step S1 is 1:(1-3).

[0030] In some embodiments of the present invention, the molar ratio of the haloaromatic hydrocarbon with N-Boc-pyrrole group and the arylboronic acid with substituent R1 in step S1 is 2:(3-5).

[0031] In some embodiments of the present invention, the molar ratio of the N-Boc-pyrrole group-containing haloaromatic hydrocarbon to the catalyst in step S1 is (15-25):1.

[0032] In some embodiments of the present invention, the molar ratio of the N-Boc-pyrrole group-containing haloaromatic hydrocarbon to the catalyst in step S1 is (18-22):1.

[0033] In some embodiments of the present invention, the molar ratio of the N-Boc-pyrrole haloalkane to the ligand in step S1 is (5-15):1.

[0034] In some embodiments of the present invention, the molar ratio of the N-Boc-pyrrole haloalkane to the ligand in step S1 is (8-12):1.

[0035] In some embodiments of the present invention, the solvent of the reaction system in step S1 includes tetrahydrofuran.

[0036] In some embodiments of the present invention, the reaction temperature of the Suzuki coupling reaction in step S1 is 70°C to 90°C.

[0037] In some embodiments of the present invention, the reaction time of the Suzuki coupling reaction in step S1 is 10 to 20 hours.

[0038] In some embodiments of the present invention, the halogen substituent in step S2 includes any one of fluorine, chlorine, bromine and iodine.

[0039] In some embodiments of the present invention, the halogen donor in the substitution reaction in step S2 includes N-bromosuccinimide.

[0040] In some embodiments of the present invention, the molar ratio of intermediate a to halogen substituent in step S2 is 1:(1-3).

[0041] In some embodiments of the present invention, the molar ratio of intermediate a to halogen substituent in step S2 is 2:(3-5).

[0042] In some embodiments of the present invention, the solvent of the reaction system in step S2 includes tetrahydrofuran.

[0043] In some embodiments of the present invention, the reaction temperature of the substitution reaction in step S2 is room temperature (25°C to 30°C).

[0044] In some embodiments of the present invention, the reaction time of the substitution reaction in step S2 is 3 to 5 hours.

[0045] In some embodiments of the present invention, the molar ratio of sodium methoxide to intermediate b in step S3 is (5-15):1.

[0046] In some embodiments of the present invention, the molar ratio of sodium methoxide to intermediate b in step S3 is (8-12):1.

[0047] In some embodiments of the present invention, the molar ratio of intermediate b to triethylamine in step S3 is 1:(3-8).

[0048] In some embodiments of the present invention, the molar ratio of intermediate b to triethylamine in step S3 is 1:(3-5).

[0049] In some embodiments of the present invention, the molar ratio of intermediate b to boron trifluoride ethyl ether in step S3 is 1:(1-5).

[0050] In some embodiments of the present invention, the molar ratio of intermediate b to boron trifluoride ethyl ether in step S3 is 1:(2-3).

[0051] In some embodiments of the present invention, the solvent of the reaction system in step S3 includes tetrahydrofuran and / or methanol.

[0052] In some embodiments of the present invention, the temperature at which the Boc group is removed in step S3 is 40°C to 60°C.

[0053] In some embodiments of the present invention, the reaction in step S3 to remove the Boc group takes 0.5 to 2 hours.

[0054] In some embodiments of the present invention, the temperature of the cyclization reaction in step S3 is 70°C to 90°C.

[0055] In some embodiments of the present invention, the cyclization reaction in step S3 takes 15 to 20 hours.

[0056] In some embodiments of the present invention, the catalyst in step S4 includes a palladium catalyst.

[0057] In some embodiments of the present invention, the palladium catalyst comprises palladium acetate or tetra(triphenylphosphine)palladium.

[0058] In some embodiments of the present invention, the alkali in step S4 includes at least one of sodium bicarbonate, sodium carbonate, and potassium carbonate.

[0059] In some embodiments of the present invention, the molar ratio of the arylboronic acid with substituent R2 in step S4 to the intermediate c is (2-6):1.

[0060] In some embodiments of the present invention, the molar ratio of the arylboronic acid with substituent R2 in step S4 to the intermediate c is (3-5):1.

[0061] In some embodiments of the present invention, the molar ratio of intermediate c to catalyst in step S4 is (25-40):1.

[0062] In some embodiments of the present invention, the molar ratio of intermediate c to catalyst in step S4 is (30-35):1.

[0063] In some embodiments of the present invention, the solvent of the reaction system in step S4 includes tetrahydrofuran.

[0064] In some embodiments of the present invention, the temperature of the reaction in step S4 is 70°C to 90°C.

[0065] In some embodiments of the present invention, the reaction time in step S4 is 20 to 30 hours.

[0066] According to a third aspect of the invention, the application of the asymmetric BODIPY-based fluorescent probe described in the first aspect of the invention is proposed in the preparation of products for lipid droplet imaging.

[0067] The present invention has at least the following beneficial effects:

[0068] 1) This invention introduces electron-withdrawing groups such as tetraphenylethylene and triphenylamine groups into the asymmetric BODIPY molecule to construct a fluorescent probe based on the ICT effect, which has a large Stokes shift, high fluorescence quantum yield, high sensitivity, high lipophilicity, and can perform lipid droplet imaging. This invention overcomes the shortcomings of existing lipid droplet fluorescent probes, such as small Stokes shift, large background interference, and weak solid-state emission fluorescence. It effectively avoids the limitation of self-absorption phenomenon of traditional organic fluorescent probes, and thus enables clearer and more stable lipid droplet imaging.

[0069] 2) The fluorescent probe provided by this invention has practical application value in the field of lipid droplet imaging research and can be used for imaging lipid droplets including those in obesity, hepatic steatosis, diabetes, cardiac dysfunction, atherosclerosis, fatty liver, etc.

[0070] 3) The fluorescent probe provided by this invention has a simple synthesis process, uses inexpensive and readily available raw materials, and can be mass-produced. Attached Figure Description

[0071] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0072] Figure 1 This is the high-resolution mass spectrum of Probe-1 in Embodiment 1 of the present invention;

[0073] Figure 2 The images show the fluorescence spectra of Probe-1 in CHCl3 / Hexane solutions with different ratios in the experimental examples of this invention.

[0074] Figure 3 This is the ultraviolet absorption spectrum of Probe-2 in DMSO in the experimental example of this invention;

[0075] Figure 4 This is the fluorescence spectrum of Probe-2 in DMSO in the experimental example of this invention. Detailed Implementation

[0076] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0077] Terminology Definition

[0078] In this invention, unless otherwise stated, the terms alkyl, aryl, aralkyl, heteroaryl, heterocyclic, halogen, and haloalkyl should be understood as follows: "alkyl" generally refers to a saturated hydrocarbon group having 1 to 6 carbon atoms in a straight-chain or branched configuration, including but not limited to methyl, ethyl, n-propyl (also known as propyl or propalkyl), isopropyl, n-butyl (also known as butyl or butalkyl), isobutyl, sec-butyl, tert-butyl, n-pentyl (also known as pentyl or pentalkyl), n-hexyl (also known as hexyl or hexalkyl), etc.

[0079] "Aryl" generally refers to a monocyclic, bicyclic, or polycyclic aromatic carbon ring structure group, including but not limited to phenyl, naphthyl, anthracene, fluorenyl, azulenyl, phenanthrene, etc. Where permissible chemical valence, the aryl group may optionally be substituted with the substituents described in this invention.

[0080] "Heteroaryl" usually refers to a monocyclic, bicyclic, or polycyclic aromatic carbon atom ring structure group in which one or more carbon atom ring members have been replaced with one or more heteroatoms, such as O, S, or N atoms, provided that structural stability allows.

[0081] "Heterocyclic group" generally refers to a monocyclic, bicyclic, or polycyclic carbon atom ring structure group that is saturated or partially unsaturated, in which one or more carbon atom ring members have been replaced with heteroatoms, such as O, S, or N atoms, where structural stability allows.

[0082] "Halogen" usually refers to halogen atomic groups, including fluorine, chlorine, bromine and iodine.

[0083] "Halogenated alkyl" generally refers to an alkyl-halogenated group in which the alkyl group is partially or completely replaced by one or more halogen atoms, where the available chemical valence allows.

[0084] Example 1

[0085] In this embodiment, a fluorescent probe Probe-1 based on asymmetric BODIPY was prepared, the structure of which is shown below:

[0086]

[0087] The synthetic route is shown below:

[0088]

[0089] Specifically, the following steps are included:

[0090] (1) Synthesis of Compound 1

[0091] 1,4-Dichloroisoquinoline (2.0 g, 10.1 mmol), 1-Boc-2-pyrroloboronic acid (2.5 g, 12.1 mmol), and tetrakis(triphenylphosphine)palladium (2 g, 10.1 mmol) were dissolved in tetrahydrofuran (20 mL), followed by the addition of 2M sodium bicarbonate solution (0.5 mL). The mixture was stirred and refluxed in an oil bath at 80 °C for 15 hours under nitrogen protection, and the reaction was monitored by thin-layer chromatography (TLC). After the reaction was completed, the solvent was evaporated to obtain the crude product, which was then extracted three times with saturated NaCl and CH2Cl2, dried over anhydrous magnesium sulfate, concentrated and dried, and separated by column chromatography to obtain compound 1 (2.7 g, 81%) as a pale yellow powder.

[0092] NMR data for compound 1: 1 H NMR(500MHz,Chloroform-d)δ8.61(s,1H),8.24(d,J=7.9Hz,1H),7.82(s,1H),7.80(d,J=6.7Hz,1H),7.60(t ,J=8.3Hz,1H),7.50(dd,J=3.3,1.7Hz,1H),6.48(dd,J=3.3,1.8Hz,1H),6.38(t,J=3.3Hz,1H),0.98(s,9H).

[0093] (2) Synthesis of Compound 2

[0094] Compound 1 (2.0 g, 6.08 mmol), (4-(1,2,2-triphenylvinyl)phenyl)boronic acid (4.58 g, 12.16 mmol), palladium acetate (0.068 g, 0.3 mmol), and 2-bicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (0.29 g, 0.608 mmol) were dissolved in tetrahydrofuran (20 mL), followed by the addition of 2M sodium bicarbonate solution (0.5 mL). The mixture was stirred and refluxed in an oil bath at 80 °C for 15 hours under nitrogen protection, and the reaction was monitored by TLC. After the reaction was completed, the solvent was evaporated to obtain the crude product, which was then extracted three times with saturated NaCl and CH2Cl2, dried over anhydrous magnesium sulfate, concentrated and dried, and separated by column chromatography to obtain compound 2 (3 g, 79%) as a yellow powder.

[0095] NMR data for compound 2:1 H NMR(500MHz,Chloroform-d)δ8.47(s,1H),7.82(dd,J=12.8,8.4Hz,2H),7.66–7.60(m,1H),7.55–7.50(m,2H),7. 24(s,2H),7.21–7.10(m,15H),7.10–7.06(m,2H),6.51(dd,J=3.3,1.7Hz,1H),6.39(t,J=3.3Hz,1H),0.93(s,9H).

[0096] (3) Synthesis of compound 3

[0097] Compound 2 (2.0 g, 3.2 mmol) and N-bromosuccinimide (0.68 g, 3.84 mmol) were dissolved in tetrahydrofuran (15 mL). The mixture was stirred at -78 °C for 30 minutes under nitrogen protection. The reaction mixture was then heated to room temperature and reacted at room temperature for 3.5 hours, monitored by TLC. After the reaction was complete, the solvent was evaporated to obtain the crude product, which was then extracted three times with saturated NaCl and CH₂Cl₂, dried over anhydrous magnesium sulfate, concentrated, dried, and separated by column chromatography to obtain compound 3 (1.9 g, 86%) as a yellow-green powder.

[0098] NMR data for compound 3: 1 H NMR(500MHz,Chloroform-d)δ8.44(s,1H),8.10(d,J=8.4Hz,1H),7.85(d,J=8.4Hz,1H),7.65(t,J=7.7Hz,1H),7.57(t,J=7.6Hz,1H ),7.24(d,J=8.1Hz,2H),7.21–7.11(m,15H),7.08(dd,J=7.3,2.4Hz,2H),6.53(d,J=3.6Hz,1H),6.46(d,J=3.6Hz,1H),1.05(s,9H).

[0099] (4) Synthesis of compound 4

[0100] Compound 3 (1.5 g, 2.13 mmol) was dissolved in tetrahydrofuran (15 mL), and sodium methoxide (1.15 g, 21.3 mmol) was dissolved in methanol (5 mL). The two solutions were mixed and stirred at 50 °C for 1 h under nitrogen protection. The reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated to obtain the crude product, which was then extracted three times with saturated Na₂CO₃ and CH₂Cl₂, dried over anhydrous magnesium sulfate, concentrated and dried, and the resulting compound was dissolved in toluene (10 mL). Triethylamine (0.71 mL, 9.4 mmol) was added, and after nitrogen protection, boron trifluoride diethyl ether (1.3 mL, 5.64 mmol) was added. The reaction was carried out at 80 °C for 18 h, and the reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated to obtain the crude product, which was then extracted three times with saturated Na₂CO₃ and CH₂Cl₂, dried over anhydrous magnesium sulfate, concentrated and dried, and the mixture was separated by column chromatography to obtain an orange-yellow powder (0.93 g, 67%).

[0101] NMR data for compound 4: 1 H NMR(500MHz,Chloroform-d)δ8.49(d,J=8.3Hz,1H),7.98(s,1H),7.92–7.83(m,2H),7 .78(t,J=7.6Hz,1H),7.23–7.07(m,19H),7.03(d,J=3.8Hz,1H),6.39(d,J=3.7Hz,1H).

[0102] (5) Synthesis of Probe-1

[0103] Compound 4 (0.5 g, 0.87 mmol), 4-(diphenylamino)phenylboronic acid (1.0 g, 3.48 mmol), and tetra(triphenylphosphine)palladium (0.03 g, 0.026 mmol) were dissolved in tetrahydrofuran (15 mL), followed by the addition of 2M sodium bicarbonate solution (0.2 mL). The mixture was stirred and refluxed in an oil bath at 80 °C for 24 h under nitrogen protection, and the reaction was monitored by TLC. After the reaction was completed, the solvent was evaporated to obtain the crude product, which was then extracted three times with saturated NaCl and CH2Cl2, dried over anhydrous magnesium sulfate, concentrated and dried, and separated by column chromatography to obtain Probe-1 (0.71 g, 83%) as a red powder.

[0104] NMR data from Probe-1: 1H NMR(500MHz,Chloroform-d)δ8.55(d,J=8.2Hz,1H),7.92(s,1H),7.87–7.84(m,2H),7.83–7.7 7(m,2H),7.76–7.72(m,1H),7.29(d,J=8.6Hz,3H),7.26–7.01(m,29H),6.72(d,J=3.8Hz,1H).

[0105] High-resolution mass spectra of Probe-1, such as Figure 1 As shown.

[0106] Example 2

[0107] In this embodiment, a fluorescent probe Probe-2 based on asymmetric BODIPY was prepared, the structure of which is shown below:

[0108]

[0109] The synthetic route is shown below:

[0110]

[0111] Specifically, the following steps are included:

[0112] The synthesis steps of compounds 1 to 4 are consistent with the synthesis steps described in Example 1.

[0113] Compound 4 (0.5 g, 0.87 mmol), (4-(1,2,2-triphenylvinyl)phenyl)boronic acid (1.3 g, 3.48 mmol), and tetra(triphenylphosphine)palladium (0.03 g, 0.026 mmol) were dissolved in tetrahydrofuran (15 mL), followed by the addition of 2M sodium bicarbonate solution (0.5 mL). The mixture was stirred and refluxed in an oil bath at 80 °C for 24 hours under nitrogen protection, and the reaction was monitored by TLC. After the reaction was completed, the solvent was evaporated to obtain the crude product, which was then extracted three times with saturated NaCl and CH2Cl2, dried over anhydrous magnesium sulfate, concentrated and dried, and separated by column chromatography to obtain Probe-2 (0.67 g, 85%) as an orange powder.

[0114] NMR data from Probe-2: 1H NMR(500MHz,Chloroform-d)δ8.55(d,J=8.3Hz,1H),7.92(s,1H),7.86(d,J=8.4Hz,1H),7.81(t,J=7.6Hz,1H ),7.75(d,J=8.7Hz,1H),7.71(d,J=8.1Hz,2H),7.25–7.03(m,36H),6.91–6.87(m,1H),6.73(d,J=3.8Hz,1H).

[0115] Test case

[0116] This experiment tested the fluorescence properties of the fluorescent probes prepared in Examples 1 and 2. The specific experimental methods and results are as follows:

[0117] 1. The Probe-1 obtained in Example 1 was dissolved in a PBS solution containing 80% DMSO to obtain 10 -3 A mol / L Probe-1 solution was prepared, and then diluted with PBS to obtain 10 mol / L. -5 A mol / L Probe-1 solution was prepared. Because the polarity of CHCl3 is close to that of lipid droplets, different proportions of CHCl3 / Hexane were added, and the changes in Probe-1 fluorescence were tested. The results are as follows: Figure 2 As shown.

[0118] from Figure 2 It can be seen that as the proportion of CHCl3 increases, the wavelength gradually red-shifts and the fluorescence intensity decreases, indicating that the probe has strong red fluorescence in lipid droplets.

[0119] 2. The Probe-2 obtained in Example 2 was dissolved in a PBS solution containing 80% DMSO to obtain 10 -3 A mol / L Probe-2 solution was prepared, and then diluted with PBS to obtain 10 mol / L. -5 A 20 μL solution of Probe-2 (mol / L) was added to 1.98 mL of DMSO solution, and its UV and fluorescence spectra were measured. The results are as follows: Figure 3 and Figure 4 As shown.

[0120] Depend on Figure 3 and Figure 4 It can be seen that Probe-2 has strong fluorescence at 557 nm and a Stokes shift of 74, indicating that the fluorescent probe provided by this invention has a large Stokes shift after modification.

[0121] The fluorescent probes provided by this invention are prepared by the Suzuki reaction using 2-(isoquinoline-1-acyl)pyrrole-boron complexes with different substitution structures and various typical electron-withdrawing groups. The lipid-water partition coefficients of the fluorescent probes provided by this invention are all greater than 10, exhibiting strong lipophilicity and enabling excellent targeting of lipid droplets. Furthermore, due to the presence of electron donors and acceptors in the fluorescent probes, intermolecular charges are significantly separated, forming a DAD structure, which enhances intramolecular charge transfer, causing a redshift in wavelength. This results in high sensitivity, high temporal resolution, and high lipophilicity, thus enabling precise lipid droplet imaging.

[0122] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A fluorescent probe based on asymmetric BODIPY, characterized in that, The fluorescent probe has the structure shown in Formula I: Wherein, R1 is a substituted or unsubstituted aromatic group; R2 is selected from substituted or unsubstituted aromatic groups and substituted or unsubstituted C. 1～6 alkyl; The aromatic group is selected from: aryl, heteroaryl, and arylamino groups containing 1 to 6 phenyl groups; The phenyl groups in the aryl group containing 1 to 6 phenyl groups are connected by a hydrocarbon group or a heteroatom selected from N, O and S; The heteroaryl group is a 3- to 7-membered monocyclic, 6- to 10-membered bicyclic, or 13- to 16-membered polycyclic system having one, two, or three independent heteroatom ring members selected from N, O, S, and B; The substituent is selected from C. 1～6 Alkyl, C 1～6 Alkoxy, heteroaryl, heterocyclic, C 1～6 Any one of alkyl-amino, halogen, and haloalkyl.

2. The fluorescent probe according to claim 1, characterized in that, R1 is selected from any of the following structures:

3. The fluorescent probe according to claim 1, characterized in that, R2 is selected from any of the following structures:

4. The method for preparing the asymmetric BODIPY-based fluorescent probe according to any one of claims 1 to 3, characterized in that, The preparation method specifically includes the following steps: S1: A haloaromatic hydrocarbon with N-Boc-pyrrole group and an arylboronic acid with substituent R1 are coupled by a Suzuki reaction to obtain intermediate a; S2: A halogen substituent is introduced into intermediate a through a substitution reaction to obtain intermediate b; S3: Sodium methoxide is used to react with intermediate b to remove the Boc group, and then a cyclization reaction is carried out under the catalysis of boron trifluoride diethyl ether and triethylamine to obtain intermediate c; S4: In the presence of a catalyst and a base, arylboronic acid with substituent R2 is reacted with intermediate c to obtain the fluorescent probe based on asymmetric BODIPY.

5. The preparation method according to claim 4, characterized in that, The reaction system for the Suzuki coupling reaction described in step S1 also includes a catalyst, a ligand, and a base; Preferably, the catalyst comprises a palladium catalyst; Preferably, the ligand comprises at least one selected from 2-bicyclohexylphosphine-2',4',6'-triisopropylbiphenyl, 2-dicyclohexylphosphine-2',6'-diisopropoxy-1,1'-biphenyl, 2-bicyclohexylphosphine-2',6'-dimethoxybiphenyl, triphenylphosphine, and tricyclohexylphosphine; Preferably, the alkali includes at least one selected from sodium bicarbonate, sodium carbonate, potassium carbonate, and cesium carbonate.

6. The preparation method according to claim 5, characterized in that, The molar ratio of the haloaromatic hydrocarbon with N-Boc-pyrrole group and the arylboronic acid with substituent R1 in step S1 is 1:(1-3). Preferably, the molar ratio of the N-Boc-pyrrole group to the catalyst is (15-25):1; Preferably, the molar ratio of the N-Boc-pyrrole group to the ligand is (5-15):

1.

7. The preparation method according to claim 4, characterized in that, The halogen substituents mentioned in step S2 include any one of fluorine, chlorine, bromine, and iodine; Preferably, the halogen donor in the substitution reaction in step S2 includes N-bromosuccinimide; Preferably, the molar ratio of intermediate a to the halogen substituent is 1:(1-3).

8. The preparation method according to claim 4, characterized in that, The molar ratio of sodium methoxide to intermediate b in step S3 is (5-15):1; Preferably, the molar ratio of intermediate b to triethylamine is 1:(3-8); Preferably, the molar ratio of intermediate b to boron trifluoride ethyl ether is 1:(1-5).

9. The preparation method according to claim 4, characterized in that, The catalyst mentioned in step S4 includes a palladium catalyst; Preferably, the alkali in step S4 includes at least one of sodium bicarbonate, sodium carbonate, and potassium carbonate; Preferably, the molar ratio of the arylboronic acid with substituent R2 to the intermediate c is (2-6):1; Preferably, the molar ratio of the intermediate c to the catalyst is (25-40):

1.

10. The use of the asymmetric BODIPY-based fluorescent probe as described in any one of claims 1 to 3 in the preparation of products for lipid droplet imaging.

Images