Fluorescent probe for monitoring FAO level of living cells and polarization phenotype of macrophages as well as preparation method and application of fluorescent probe

Abstract

The invention relates to the technical field of detection, in particular to 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 of the fluorescent probe. The advantages of the fluorescent probe are combined, the novel fluorescent probe is designed as an FAO metabolism substrate from the metabolism angle, fluorophores are directly released after FAO metabolism, operation is convenient and fast, the obvious fluorescence opening effect is achieved, and the FAO level change in living cells is visually monitored. In addition, the fluorescent probe can also be used as a novel selective M2 type macrophage fluorescent probe for dynamically monitoring a macrophage M2 type polarization process and a reprogramming process between M1 and M2 types.

Description

Technical Field

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

[0002] Fatty acid β-oxidation (FAO) is one of the main pathways of lipid metabolism. FAO disorders exist in pathological conditions such as inflammation, cancer, and diabetic nephropathy. Macrophages are involved in inflammation, immunity, and maintaining the stability of the internal environment. There is FAO heterogeneity and functional heterogeneity between macrophages of different phenotypes (M1 / M2), which differentially affect the occurrence, development, and treatment of diseases (inflammation, tumors, etc.). Therefore, visual monitoring of FAO levels in living cells, the proportion of M1 and M2 phenotypes in macrophage populations, and macrophage reprogramming are of great significance for understanding the pathological mechanisms and pharmacological studies of diseases. At present, fluorescent probes based on optical monitoring of cell FAO are very scarce, and only a few probes have been reported for FAO monitoring. For example, the coumarin fluorescent probes and quinone methide fluorescent probes developed by Akio Ojida's team (Uchinomiya et al., 2020) are FAO substrates, which realize the monitoring of FAO levels in 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, to selectively fluorescently label macrophages of different phenotypes and monitor macrophage reprogramming.

[0003] Therefore, in the search for FAO monitoring in living cells, the coumarin fluorescent probes currently developed have short emission wavelengths, weak fluorescence intensity, and poor turn-on effects, which limit their application in disease models. Quinone methide fluorescent probes, as bioorthogonal molecular probes, need to be used in combination with fluorescein, Cy-5 azide, etc. to achieve FAO monitoring, and the analysis process is relatively complicated. In the monitoring of macrophage reprogramming, due to the differences in the expression of transporters on different types of cells, the currently developed fluorescent probes such as CDg16, CDr17, and CDg18 lack wide applicability in practical applications. In addition, the number of transport proteins related to the transport of specific types of substances is small, which 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, and 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, 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, the R4 is a five- or six-membered alkyl ring.

[0013] The specific reaction route of the method for preparing the fluorescent probe for monitoring the FAO level of living cells and the polarization phenotype of macrophages is as follows:

[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 Lawesson reagent thiolation reaction 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, and then the corresponding brominated fatty acid chain / fatty acid ester chain and sodium iodide are added, and the reaction is carried out overnight in an oil bath environment at 90°C. The reaction solution is extracted with ethyl acetate and water, the oil phase is obtained and spin-dried, and purified with a 100-200 mesh silica gel column, and the eluent is PE:EA=10:1, and the product formula (II) can be obtained. Formula (I) can be obtained by Lawesson's reagent thioreaction of formula (II), 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 and other solvents and piperidine (1.5 equivalents) as a catalyst to obtain the product formula (I).

[0018] Further, 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 substituents 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 has no adverse effect on 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 the 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, can distinguish M2 macrophages from M0 / M1 macrophages, dynamically monitor the polarization process of M2 macrophages and the reprogramming process between M1 / M2 types, and quickly evaluate the effect of drugs on the phenotypic transformation of macrophages M1 and M2.

[0020] The fluorescent probe for monitoring the FAO level in living cells and the polarization phenotype of macrophages can be used to monitor the changes in FAO levels in living cells, to be used 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 turn-on 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 used 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 compounds of the present invention.

[0026] Figure 2 It is a 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 the probe of the present invention monitoring the FAO level in living cells.

[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 within 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. Sodium bicarbonate was used to adjust the pH to neutral. The organic phase was concentrated after extraction with DCM and saturated brine to obtain a clear, transparent dark yellow liquid product, which was compound Br-6 (6.2 g, 80.47%).

[0036] Example 26 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, and 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) were added, 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 out using a sand core funnel, and the residue was rinsed with EA several times. The organic phase was concentrated after extraction with EA and saturated brine, and purified by silica gel column chromatography, with the 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 DCM and placed in an ice bath until the reaction solution cooled to 0°C. In a ventilated environment, BBr3 (3.98 mL, 41.28 mmol, 10 eq) was slowly added dropwise to the reaction solution using a syringe within 10 min. The reaction solution gradually changed from yellow to vermilion. After reacting in an ice bath for 20 min, the ice bath was removed and the reaction was carried out at room temperature for 4 h. After the reaction was completed, the reaction solution was poured into ice water to quench the reaction, and the residue was washed with DCM. The pH of the reaction solution was adjusted to neutral using NaHCO3. The organic phase was concentrated after extraction with DCM and saturated brine, and purified by silica gel column chromatography. The eluent polarity was DCM: MeOH = 40: 1. 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 K2CO3 were placed in ultra-dry DMF and reacted at room temperature for 20 min, then compound (9Z, 220.08 mg, 2 eq) and NaI were added, and the temperature of DMF was raised to 90°C, and the reaction was allowed to proceed overnight. The organic phase was concentrated after extraction with EA and saturated brine, and purified by silica gel column chromatography, with the eluent polarity of PE:EA=5:1, and the product was 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] Using conventional cell culture methods, after the cells are in the logarithmic growth phase, the cells are cultured at a rate of 1.0*10 5 The cells were seeded at a density of cells / mL in a six-well plate and cultured at 37°C and 5% CO2 for 24 hours. The cells were 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 incubated for 1 hour and 6 hours respectively, and then the cells in the cell culture plate were collected. The cells were resuspended with 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] The fluorescence imaging analysis and flow cytometry analysis of the FAO level in the probe cells of other embodiments of the present invention are consistent with the above-mentioned operation method, and the difference of FAO level in the cells 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] The 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 placed in a constant temperature cell culture incubator for 1 hour, and then placed directly under a laser confocal microscope to observe fluorescence without washing. At the same time, cells were collected and the fluorescence difference was analyzed by flow cytometry. The results are shown in Figure 6 Shown

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

[0051] After using IL-4 to induce RAW cells into M2 macrophages, different concentrations of HS-1793 were added for 6 h, and then 10 μM of the probe obtained in Example 4 was added and placed 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 the 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, natural products and active ingredients of seedling medicine were added for a suitable time, washed twice with PBS, and then 10 μM of the FAO probe obtained in Example 4 was added and placed 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 analyzed for fluorescence differences by flow cytometry. Figure 8 shown.

[0054] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A fluorescent probe for monitoring FAO levels in living cells and macrophage polarization phenotype, characterized in that: Its specific structural formula is as follows:

2. The fluorescent probe according to claim 1, characterized in that The X is any one of heteroatoms such as O, S, Si, Se, and N.

3. The fluorescent probe according to claim 1, characterized in that The R is an alkane or a substituted alkane.

4. The fluorescent probe according to claim 1, characterized in that The R2 is a saturated alkyl group, an unsaturated alkyl group or an alkyl group containing other branches with a carbon chain length of 1 to 20.

5. The fluorescent probe according to claim 1, characterized in that The R3 is any one of heteroatoms such as O, S, N, etc., or a group that can undergo a nucleophilic addition reaction.

6. The fluorescent probe according to claim 1, characterized in that The R4 is a five- or six-membered alkyl ring.

7. The method for preparing the fluorescent probe for monitoring the FAO level of living cells and the polarization phenotype of macrophages according to claim 1, characterized in that: The specific reaction route is as follows: 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 Lawesson reagent thiolation reaction 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).

8. Use of the fluorescent probe for monitoring FAO levels in living cells and macrophage polarization phenotypes as claimed in claim 1 in monitoring changes in FAO levels in living cells.

9. Use of the fluorescent probe for monitoring FAO levels in living cells and polarization phenotype of macrophages as claimed in claim 1 as a novel selective M2 macrophage fluorescent probe.

10. Use of the fluorescent probe for monitoring FAO levels in living cells and polarization phenotype of macrophages as claimed in claim 1 in distinguishing M2 macrophages from M0 and M1 macrophages.

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