A fluorescent probe for monitoring FAO levels in living cells and macrophage polarization phenotypes, and its preparation method and application

Abstract

The present invention relates to the field of detection technology, specifically a fluorescent probe for monitoring FAO levels in living cells and macrophage polarization phenotypes, as well as its preparation method and application. Combining the advantages of fluorescent probes themselves, the present invention designs a novel fluorescent probe from a metabolic perspective as a FAO metabolic substrate. After FAO metabolism, the probe directly releases a fluorophore, is easy to operate, and has a significant fluorescence "on" effect, enabling visual monitoring of changes in FAO levels in living cells. Furthermore, the probe can also serve as a novel selective M2 macrophage fluorescent probe to dynamically monitor the polarization process of macrophages M2 and the reprogramming process between M1 and M2.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, in particular to a fluorescent probe for monitoring FAO levels in living cells and polarization phenotypes of macrophages, and a preparation method and application thereof. Background Art

[0002] Fatty acid β-oxidation (FAO) is a major pathway in lipid metabolism, and FAO disturbances occur in pathological conditions such as inflammation, cancer, and diabetic nephropathy. Macrophages participate in processes such as inflammation, immunity, and maintaining homeostasis. FAO heterogeneity and functional heterogeneity exist between macrophages of different phenotypes (M1 / M2), differentially impacting the development, progression, and treatment of diseases (such as inflammation and tumors). Therefore, visual monitoring of FAO levels in living cells, the proportion of M1 and M2 phenotypes within macrophage populations, and macrophage reprogramming are of great significance for understanding disease pathology and conducting pharmacological studies. Currently, fluorescent probes for optical monitoring of cellular FAO are extremely scarce, and only a few have been reported for FAO monitoring. For example, the coumarin-based and quinone methide-based fluorescent probes developed by Akio Ojida's team (Uchinomiya et al., 2020) serve as FAO substrates, enabling monitoring of FAO levels in the mitochondria of living cells. In terms of macrophage monitoring, the Young-TaeChang team (Cho et al., 2023) developed a series of fluorescent probes, CDg16, CDr17, and CDg18, based on the transporter uptake preferences and expression differences of macrophages. These probes selectively fluorescently label macrophages of different phenotypes and are used to monitor macrophage reprogramming.

[0003] Therefore, in the search for FAO monitoring in living cells, the coumarin-based fluorescent probes currently developed have short emission wavelengths, weak fluorescence intensity, and poor on-response effects, limiting their application in disease models. Quinone methide-based fluorescent probes, as bioorthogonal molecular probes, must be used in combination with fluorescein, Cy-5 azide, and other agents to monitor FAO, resulting in a relatively complex analysis process. In the monitoring of macrophage reprogramming, due to differences in transporter expression in different cell types, currently developed fluorescent probes such as CDg16, CDr17, and CDg18 lack widespread applicability in practical applications. Furthermore, the relatively small number of transport proteins involved in transporting specific substances limits the development of such probes.

[0004] Therefore, finding a fluorescent probe that can realize visual monitoring of FAO levels in living cells and has good fluorescence turn-on effect and optical stability is an urgent problem that needs to be solved. Summary of the Invention

[0005] In order to solve the above technical problems existing in the prior art, the present invention provides a fluorescent probe for monitoring the FAO level of living cells and the polarization phenotype of macrophages, as well as a preparation method and application thereof.

[0006] A fluorescent probe for monitoring FAO levels in living cells and macrophage polarization phenotype, the specific structural formula of which is as follows:

[0007]

[0008] Furthermore, X is any one of heteroatoms such as O, S, Si, Se, and N.

[0009] Furthermore, the R is an alkane or a substituted alkane, and the final structure is an aliphatic carboxylic acid or an aliphatic carboxylic acid ester.

[0010] Furthermore, R2 is a saturated alkyl group, an unsaturated alkyl group or an alkyl group containing other branches with a carbon chain length of 1-20 (n=1-20).

[0011] Furthermore, R3 is any one of heteroatoms such as O, S, and N, or a group that can undergo a nucleophilic addition reaction, such as a dicyano group, a 4-methylquinoline substituent, a 1,4-dimethylquinoline iodide substituent, a 4-methylpyridine substituent, a 1,4-dimethylpyridine iodide substituent, a dicyanoisophorone substituent, a 2-methylbenzothiazole, a 1-2-dimethylbenzothiazole iodide substituent, a 2-methylindole substituent, a 1,2-dimethylindole iodide substituent, or a 1,1,2-trimethyl-1H-benzo[e]indole.

[0012] Furthermore, R4 is a five- or six-membered alkyl ring.

[0013] The method for preparing the fluorescent probe for monitoring FAO levels in living cells and macrophage polarization phenotypes has the following specific reaction scheme:

[0014]

[0015] The compound of formula (III) is reacted with potassium carbonate, and then the corresponding brominated fatty acid chain / fatty acid ester chain is added to react with sodium iodide to obtain the product of formula (II); the product of formula (II) is subjected to a thiolation reaction with Lawesson's reagent or reacted with dicyano, 4-methylquinoline, 1,4-dimethylquinoline iodide, 4-methylpyridine, 1,4-dimethylpyridine iodide, dicyanoisophorone, 2-methylbenzothiazole, 1-2-dimethylbenzothiazole iodide, etc. to obtain the product of formula (I).

[0016] Furthermore, in step (a), the solvent is dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF), the substitution reagent is iodopropane, and the base is sodium hydroxide or potassium hydroxide.

[0017] Specifically, formula (III) is dissolved in anhydrous N,N-dimethylformamide (DMF), 3 equivalents of potassium carbonate are added and reacted at room temperature for 30 minutes, followed by the addition of the corresponding brominated fatty acid chain / fatty acid ester chain and sodium iodide, and the reaction is carried out overnight in a 90°C oil bath. The reaction solution is extracted with ethyl acetate and water, the oil phase is obtained and spin-dried, and purified on a 100-200 mesh silica gel column with an eluent of PE:EA = 10:1 to obtain the product formula (II). Formula (I) can be obtained from formula (II) by thiolation reaction with Lawesson's reagent, or by reacting formula (II) with dicyano, 4-methylquinoline, 1,4-dimethylquinoline iodide, 4-methylpyridine, 1,4-dimethylpyridine iodide, dicyanoisophorone, 2-methylbenzothiazole, 1-2-dimethylbenzothiazole iodide, etc. in an environment of anhydrous ethanol or the like with piperidine (1.5 equivalents) as a catalyst to obtain the product formula (I).

[0018] Furthermore, in order to improve the reaction efficiency of the compound of formula (I), the acid acceptor preferably used in this reaction includes inorganic bases such as sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydride, etc., and organic bases such as triethylamine, diisopropylethylamine, etc. In the reaction of introducing a substituent in formula (II), 1.5 equivalents of hexahydropyridine are added as a catalyst to accelerate the reaction rate. As the solvent for the above reaction, any solvent that does not adversely affect the reaction can be used. Anhydrous dimethylformamide, anhydrous ethanol, acetonitrile, and methanol are preferably used.

[0019] The fluorescent probe of the present invention can monitor changes in FAO levels in living cells and can be used as an optical tool for batch screening of new drugs targeting FAO. It has the ability to selectively detect M2 macrophages, which can distinguish M2 macrophages from M0 / M1 macrophages, dynamically monitor the polarization process of macrophages into M2 types and the reprogramming process between M1 / M2 types, and quickly evaluate the effects of drugs on the phenotypic conversion of macrophages into M1 and M2 types.

[0020] The fluorescent probe for monitoring FAO levels in living cells and macrophage polarization phenotypes can be used to monitor changes in FAO levels in living cells, as a novel selective M2 macrophage fluorescent probe, and to distinguish M2 macrophages from M0 and M1 macrophages.

[0021] Compared with the prior art, the technical effects created by the present invention are embodied in:

[0022] (1) The fluorescent probe of the present invention can realize the visual monitoring of FAO levels in living cells, has good fluorescence onset effect and optical stability, and the maximum fluorescence emission wavelength can be located in the visible-near infrared band (360nm-980nm). It has the ability to be widely applied in disease models and can be used as an optical tool for batch screening of new drugs targeting FAO.

[0023] (2) The fluorescence intensity of the fluorescent probe of the present application in M2 macrophages is higher than that in M0 and M1 macrophages. It has the ability to selectively characterize M2 macrophages and can distinguish M2 macrophages from M0 / M1 macrophages. It can be used as a new type of selective M2 macrophage fluorescent probe to dynamically monitor the polarization process of macrophages M2 and the reprogramming process between M1 / M2.

[0024] (3) The fluorescent probe of the present application can also be used for batch screening of effective small molecule compounds, natural product monomers or extracts, traditional Chinese medicine compounds, and related drugs in different dosage forms that regulate FAO levels. It can not only be used to quickly evaluate the effects of drugs on the phenotypic transformation of macrophages M1 and M2, but also provide a practical tool for screening new drugs targeting FAO. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The following is a reaction scheme for synthesizing the compound of the present invention.

[0026] Figure 2 Schematic diagram of the metabolic process and metabolic products of the probe of the present invention.

[0027] Figure 3 It is the nuclear magnetic hydrogen spectrum of the probe of the present invention.

[0028] Figure 4 This is a graph showing the results of monitoring FAO levels in living cells using the probe of the present invention.

[0029] Figure 5 This is a graph showing the results of detecting FAO levels in living cells using a flow cytometer using the probe of the present invention.

[0030] Figure 6 This is a diagram showing the results of the probe of the present invention selectively labeling M2 macrophages.

[0031] Figure 7 This is a diagram of the probe of the present invention visually monitoring the macrophage reprogramming process

[0032] Figure 8 This is a diagram of the probe of the present invention being used for batch screening of drugs. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0034] Example 12: Synthesis of bromocyclohexane-1-enecarboxaldehyde

[0035] DMF (11.92 g, 163.02 mmol, 4 eq) was added to 15 mL of chloroform, and the mixture was cooled to 0°C in an ice bath. PBr3 (33.10 g, 122.27 mmol, 3 eq) was slowly added dropwise over 10 min. White flocs were generated in the reaction solution. A mixed solution of cyclohexanone and chloroform was then added. The reaction was allowed to react at room temperature for 12 h. The reaction was quenched with ice water, and the pH was adjusted to neutral using sodium bicarbonate. The organic phase was extracted with DCM and saturated brine, and concentrated to give a clear, transparent, dark yellow liquid product, namely compound Br-6 (6.2 g, 80.47%).

[0036] Example 2 Synthesis of 6-methoxy-2,3-dihydro-1H-xanthene-4-carbaldehyde

[0037] Compound Br-6 (3 g, 15.58 mmol, 1 eq) was added to 9 mL of DMF, followed by dihydroxytetramethoxybenzaldehyde (2-Hydroxy-4-methoxybenzaldehyde, 2.17 g, 14.28 mmol, 0.9 eq) and cesium carbonate (Cs2CO3, 15.51 g, 47.63 mmol, 3 eq), and the mixture was allowed to react overnight at room temperature. After the reaction, bright yellow spots were observed on TCL. The residue was filtered off using a sand core funnel and rinsed several times with EA. After extraction with EA and saturated brine, the organic phase was concentrated and purified by silica gel column chromatography with an eluent polarity of PE:EA=20:1. The final product was a yellow solid powder (3.2 g, 83.23%).

[0038] 1 H NMR (400MHz, CHLOROFORM-D) δ10.31 (s, 1H), 7.08 (d, J = 9.2Hz, 1H), 6.66 (d, J = 2.6Hz, 3H), 3.84 (s, 3H), 2.56 (t, J = 6.2Hz, 2H), 2.44 (t, J = 6.1Hz, 2H).

[0039] Example 3 Synthesis of 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde

[0040] Compound Meo-R (1 g, 4.13 mmol, 1 eq) was dissolved in 15 ml of DCM and placed in an ice bath until the reaction solution cooled to 0°C. BBr (3.98 mL, 41.28 mmol, 10 eq) was slowly added dropwise to the reaction solution over 10 minutes using a syringe under ventilation. The reaction solution gradually turned from yellow to vermilion. After reacting in an ice bath for 20 minutes, the ice bath was removed and the reaction was continued at room temperature for 4 hours. Upon completion of the reaction, the reaction solution was poured into ice water to quench the reaction. The residue was rinsed with DCM and the pH of the reaction solution was adjusted to neutral with NaHCO. The organic phase was concentrated after extraction with DCM and saturated brine and purified by silica gel column chromatography using a DCM:MeOH ratio of 40:1 as the eluent. The final product was a dark yellow solid powder (825 mg, 84.57%).

[0041] Example 4 Synthesis of methyl 9-((4-formyl-2,3,9,9a-tetrahydro-1H-xanthene-6-yl)oxy)nonanoate

[0042] Compound OH-R (100 mg, 1 eq) and KCO were reacted in ultra-dry DMF at room temperature for 20 min. Compound 9Z (220.08 mg, 2 eq) and NaI were then added. The DMF was heated to 90°C and allowed to react overnight. The organic phase was concentrated after extraction with EA and saturated brine and purified by silica gel column chromatography with an eluent ratio of PE:EA = 5:1 to yield a yellow solid powder (150 mg, 85.92%).

[0043] 1H NMR (400MHz, CHLOROFORM-D) δ10.29(s,1H),7.07(d,J=9.2Hz,1H),6.67–6.62(m,3H),3.96(t,J=6.5Hz,2H),3.66(s,3H),2.59–2.53(m,2H),2.43(t ,J=6.1Hz,2H),2.30(t,J=7.6Hz,2H),1.84–1.74(m,2H),1.70(q,J=6.1Hz ,2H),1.62(t,J=7.5Hz,2H),1.44(d,J=8.1Hz,2H),1.36–1.29(m,6H).13C NMR(101MHz,CHLOROFORM-D)δ187.62,174.45,161.18,161.06,153.48,127.55,127.32,126.47,114.58,112.5 0,111.52,100.99,68.51,51.62,34.16,33.68,30.01,29.27,29.24,29.14,26.02,25.56,25.00,21.60,20.49.

[0044] HRMS(ESI)m / z:calculated for C24H31O5 + [M+H] + ,399.21660,found399.21600.calculated for C24H30O5Na + [M+Na] + ,421.19855,found 421.19772.

[0045] Example 5 Flow cytometry analysis of FAO levels in probe cells

[0046] Conventional cell culture method was used. After the cells were in the logarithmic growth phase, the cells were cultured at a rate of 1.0*10 5 The cells were seeded at a density of cells / mL in a six-well plate, cultured at 37°C and 5% CO2 for 24 hours, and washed twice with PBS. The inhibitor group was pre-incubated with serum-free DMEM medium and 5 μM Etomoxir for 12 hours, while the normal group was pre-incubated with blank serum and the same volume of PBS as Etomoxir. After the pre-incubation, the probe obtained in Example 4 was used to incubate for 1 hour and 6 hours respectively, and then the cells in the cell culture plate were collected. The cells were resuspended in flow cytometry loading buffer and the fluorescence information of the corresponding light path was collected. The flow cytometry analysis results of the probe in Example 4 are shown in the figure. Figure 5 shown.

[0047] In other embodiments of the present invention, the fluorescence imaging analysis and flow cytometry analysis of the probe intracellular FAO level monitoring are consistent with the above-mentioned operation method, and the difference in intracellular FAO level is also visually monitored. The results are as follows Figure 4 ,as well as Figure 8 As shown in C and D.

[0048] Example 6 Probe Selectively Labels M2 Macrophages

[0049] An M1 / M2 RAW cell model was constructed, washed twice with PBS and replaced with serum-free medium. 10 μM of the probe obtained in Example 4 was added and incubated in a constant temperature cell culture incubator for 1 hour. Without washing, the cells were directly placed under a laser confocal microscope to observe fluorescence. At the same time, cells were collected and fluorescence differences were analyzed by flow cytometry. Figure 6 shown

[0050] Example 7 Probe Visualization Monitoring of Macrophage Reprogramming Process

[0051] After IL-4 was used to induce RAW cells into M2 macrophages, different concentrations of HS-1793 were added and treated for 6 h. Then, 10 μM of the probe obtained in Example 4 was added and incubated in a constant temperature cell culture incubator for 1 h. Without washing, the cells were directly placed under a laser confocal microscope to observe fluorescence. At the same time, cells were collected and fluorescence differences were analyzed by flow cytometry. Figure 7 shown.

[0052] Example 8 Application in Drug Batch Screening

[0053] After using IL-4 to induce RAW cells into M2 macrophages, the natural product and the active ingredients of the seedling medicine were added for an appropriate time, washed twice with PBS, and then 10 μM of the FAO probe obtained in Example 4 was added and incubated in a constant temperature cell culture incubator for 1 hour. Without washing, the cells were directly placed under a laser confocal microscope to observe fluorescence; and the cells were collected and the fluorescence difference was analyzed by flow cytometry. Figure 8 shown.

[0054] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solutions of the present invention are not limited to the above embodiments and are subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A fluorescent probe for monitoring FAO levels in living cells and macrophage polarization phenotype, characterized in that: It is methyl 9-((4-formyl-2,3,9,9a-tetrahydro-1H-xanthene-6-yl)oxy)nonanoate, and its specific structural formula is as follows:

2. Use of the fluorescent probe for monitoring FAO levels in living cells and macrophage polarization phenotype according to claim 1 in the preparation of a reagent for monitoring changes in FAO levels in living cells.

3. Use of the fluorescent probe for monitoring FAO levels in living cells and macrophage polarization phenotype according to claim 1 in the preparation of a reagent for a selective M2 macrophage fluorescent probe.

4. Use of the fluorescent probe for monitoring FAO levels in living cells and macrophage polarization phenotype according to claim 1 in the preparation of a reagent for distinguishing M2 macrophages from M0 and M1 macrophages.

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