A photosensitizer activated by beta-galactosidase and its preparation method

Abstract

The present invention relates to the field of biomedicine technology and provides a β-galactosidase-activated photosensitizer and a preparation method thereof. The β-galactosidase-activated photosensitizer comprises a β-galactosidase-activated galactosyl substrate, a rearrangeable linker, and a BODIPY molecule containing a positively charged pyridine ring. The β-galactosidase-activated photosensitizer is a compound having the following structural formula: before entering senescent cells, the positive charge on the pyridine ring quenches the fluorescence of the BODIPY molecule and the generation of singlet oxygen through the photoelectric transfer effect, rendering the β-galactosidase-activated photosensitizer non-fluorescent and non-photodynamically active; after entering senescent cells, the β-galactosidase-activated photosensitizer can be activated by the β-galactosidase highly expressed in the senescent cells, thereby exhibiting photodynamic activity and achieving efficient elimination of senescent cells.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a photosensitizer activated by beta-galactosidase and a preparation method thereof. Background Art

[0002] The photodynamic effect uses laser light of a specific wavelength to irradiate photosensitizers absorbed by tissues, causing the photosensitizers to reach an excited state. The excited photosensitizers transfer energy to oxygen molecules around the cells, generating singlet oxygen with cytotoxicity, thereby causing cell damage and death.

[0003] Cellular senescence is a permanent state of cell cycle arrest caused by various internal and external stressors. Studies have shown that cellular senescence is closely associated with a variety of age-related diseases, such as atherosclerosis, respiratory distress syndrome, and Alzheimer's disease. Senescent cells promote the secretion of a series of inflammatory cytokines, chemokines, and growth factors, which modulate the function of neighboring normal cells, thereby interfering with the physiological function of tissues and leading to the development of many diseases and poor prognosis. Therefore, eliminating senescent cells or shutting down their secretory mechanisms can prevent or delay age-related degeneration and prolong the lifespan of organisms. Senescent cells share several common biological characteristics, such as enlarged cell morphology, lipid and protein deposition, a significant increase in the number of lysosomes, and elevated expression and activation of senescence-related enzymes, particularly β-galactosidase. β-galactosidase is an exoglycosidase that specifically hydrolyzes the β-glycosidic bond between galactose and its organic moiety. Because β-galactosidase is easily detected in cells and tissues, it is the most commonly used biomarker for senescent cells.

[0004] Although a series of compound molecules have been developed to kill senescent cells by regulating related signaling pathways (such as anti-apoptosis, protein kinases, and transcription factors), such drug molecules are also affected by the multifaceted, dynamic, and highly heterogeneous characteristics of cellular senescence. In particular, when cells are subjected to different stimuli or stress, such molecules will fail to work on their targets, posing a greater risk in clinical practice. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a β-galactosidase activated photosensitizer and a preparation method thereof.

[0006] The technical solution adopted by the present invention to solve the technical problem is as follows: On the one hand, the present invention provides a β-galactosidase-activated photosensitizer, which is composed of a β-galactosidase-activated galactose substrate, a rearrangeable linker, and a BODIPY molecule containing a positively charged pyridine ring. The β-galactosidase-activated photosensitizer is a compound with the following structural formula:

[0007]

[0008] On the other hand, the present invention also provides a method for preparing a β-galactosidase activated photosensitizer, comprising the following steps:

[0009] S1, dispersing compound 1 in solvent 1, adding N-iodosuccinimide and stirring, evaporating to dryness and purifying after the reaction to obtain compound 2;

[0010] S2, dispersing compound 3 and compound 4 in solvent 2, adding tetrabutylammonium bromide and potassium carbonate and stirring, evaporating to dryness and purifying after the reaction to obtain compound 5;

[0011] S3, dispersing the compound 5 in the solvent 3, adding sodium borohydride and stirring, evaporating to dryness and purifying after the reaction to obtain compound 6;

[0012] S4, dispersing the compound 6 in the solvent 4, adding phosphorus tribromide and stirring, evaporating to dryness and purifying after the reaction to obtain compound 7;

[0013] S5, dispersing the compound 7 and potassium carbonate in the solvent 3 and stirring, evaporating to dryness and purifying after the reaction to obtain compound 8;

[0014] S6, dispersing the compound 8 and the compound 2 in the solvent 5 and stirring, cooling, filtering, washing, and drying after the reaction to obtain the compound gal-PBDP;

[0015] in,

[0016] The compound 1 is a compound of the following structural formula:

[0017] The compound 2 is a compound of the following structural formula:

[0018] The compound 3 is a compound of the following structural formula:

[0019] The compound 4 is a compound of the following structural formula:

[0020] The compound 5 is a compound of the following structural formula:

[0021] The compound 6 is a compound of the following structural formula:

[0022] The compound 7 is a compound of the following structural formula:

[0023] The compound 8 is a compound of the following structural formula:

[0024] The compound gal-PBDP is a compound of the following structural formula:

[0025]

[0026] The solvent 1 is dichloromethane; the solvent 2 is a mixed solvent of water and dichloromethane; the solvent 3 is a mixed solvent of methanol and dichloromethane; the solvent 4 is tetrahydrofuran; and the solvent 5 is acetonitrile.

[0027] Preferably, in S1, the molar ratio of compound 1 to N-iodosuccinimide is 1:2.0 to 1:3.0, the reaction temperature is room temperature, and the reaction time is 6 to 10 hours.

[0028] Preferably, in S2, the molar ratio of compound 3, compound 4, tetrabutylammonium bromide and potassium carbonate is 1:1.2:0.5:3, the reaction temperature is room temperature, and the reaction time is 16 to 24 hours.

[0029] Preferably, in S3, the molar ratio of compound 5 to sodium borohydride is 1:2.5 to 1:4.0, the reaction temperature is room temperature, and the reaction time is 8 to 16 hours.

[0030] Preferably, in S4, the molar ratio of compound 6 to phosphorus tribromide is 1:2.0 to 1:4.0, the reaction temperature is room temperature, and the reaction time is 6 to 12 hours.

[0031] Preferably, in S5, the molar ratio of compound 7 to potassium carbonate is 1:3.0 to 1:6.0, the reaction temperature is room temperature, and the reaction time is 8 to 16 hours.

[0032] Preferably, in S6, the molar ratio of compound 8 to compound 2 is 1:1.0 to 1:1.5, the reaction temperature is 80° C., and the reaction time is 20 to 30 hours.

[0033] The beneficial effects of the present invention are as follows: Different from the prior art, the β-galactosidase-activated photosensitizer of the present invention targets the β-galactosidase highly expressed in senescent cells, and constructs a β-galactosidase-activated photosensitizer with high selectivity for eliminating senescent cells by connecting the β-galactosidase-activated galactosyl substrate with a rearrangeable linker and a BODIPY molecule containing a positively charged pyridine ring; moreover, the β-galactosidase-activated photosensitizer of the present invention can be directly added to the cell culture medium and co-cultured with the cells to achieve efficient photodynamic effect on senescent cells, thereby eliminating senescent cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 11 is a fluorescence spectrum of the β-galactosidase-activated photosensitizer (gal-PBDP) before and after activation by β-galactosidase in an embodiment of the present invention;

[0035] Figure 2 This is a study on the specificity of the β-galactosidase-activated photosensitizer (gal-PBDP) for β-galactosidase in the examples of the present invention;

[0036] Figure 3 Confocal microscopy comparison of the photosensitizer (gal-PBDP) activated by β-galactosidase in normal and senescent cells in the embodiment of the present invention;

[0037] Figure 4 The photodynamic effect of the β-galactosidase-activated photosensitizer (gal-PBDP) on senescent HeLa and HeLa in the examples of the present invention;

[0038] Figure 5 1H NMR spectrum of the photosensitizer (gal-PBDP) activated by β-galactosidase in the embodiment of the present invention;

[0039] Figure 6 : is the carbon NMR spectrum of the photosensitizer (gal-PBDP) activated by β-galactosidase in the examples of the present invention;

[0040] Figure 7 This is the mass spectrometric characterization of the photosensitizer (gal-PBDP) activated by β-galactosidase in the examples of the present invention. DETAILED DESCRIPTION

[0041] In order to more clearly illustrate the purpose, technical solutions and advantages of the embodiments of the present invention, the present invention will be further described below with reference to the accompanying drawings and embodiments for a clear and complete description. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

[0042] Example 1:

[0043] The β-galactosidase-activated photosensitizer in Example 1 of the present invention is composed of a β-galactosidase-activated galactosyl substrate, a rearrangeable linker, and a BODIPY molecule containing a positively charged pyridine ring. The β-galactosidase-activated galactosyl substrate, the rearrangeable linker, and the BODIPY molecule containing a positively charged pyridine ring are all functional molecular groups. β-galactosidase, which is highly expressed in senescent cells, is targeted. By connecting the β-galactosidase-activated galactosyl substrate with the rearrangeable linker and the BODIPY molecule containing a positively charged pyridine ring, a β-galactosidase-activated photosensitizer with high selectivity for eliminating senescent cells is constructed. The β-galactosidase-activated photosensitizer is a compound with the following structural formula:

[0044]

[0045] Before entering senescent cells, the positive charge on the pyridine ring quenches the fluorescence of the photosensitizer BODIPY molecule and the generation of singlet oxygen through the photoelectron transfer effect, rendering the β-galactosidase-activated photosensitizer non-fluorescent and inactive in photodynamic activity.

[0046] Example 2:

[0047] Example 2 of the present invention provides a method for preparing a β-galactosidase-activated photosensitizer. The reaction scheme of the preparation method is as follows:

[0048]

[0049] The specific steps are as follows:

[0050] S1, compound 1 (0.72 g, 2.21 mmol) was dispersed in dichloromethane (40 ml), and N-iodosuccinimide (1.27 g, 5.53 mmol) was added. The mixture was stirred at room temperature for 8 hours. After the reaction, the dichloromethane was evaporated to dryness, and the product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain compound 2 (0.96 g, 75%) as a red solid.

[0051] In step S1, the molar ratio of compound 1 to N-iodosuccinimide is 1:2.0 to 1:3.0, the reaction time is 6 to 10 hours, 8 hours in this embodiment, and the reaction temperature is room temperature, i.e., 25° C., which is generally referred to in chemical experiments;

[0052] In step S1, compound 1 is a compound having the following structural formula:

[0053] Compound 2 is a compound with the following structural formula:

[0054] S2, compound 3 (1.23 g, 3 mmol) and compound 4 (0.61 g, 3.6 mmol) were dispersed in a mixed solvent of water and dichloromethane (30 mL of water + 30 mL of dichloromethane), tetrabutylammonium bromide (0.48 g, 1.5 mmol) and potassium carbonate (1.24 g, 9 mmol) were added, and the mixture was stirred at room temperature for 20 hours. After the reaction, the dichloromethane phase was separated, evaporated to dryness, and purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain compound 5 (1.31 g, 88%) as a light yellow solid;

[0055] In step S2, the molar ratio of compound 3, compound 4, tetrabutylammonium bromide and potassium carbonate is 1:1.2:0.5:3, the reaction time is 16 to 24 hours, 20 hours in this embodiment, and the reaction temperature is room temperature, i.e., 25° C., which is generally referred to in chemical experiments;

[0056] In step S2, compound 3 is a compound having the following structural formula:

[0057] Compound 4 is a compound with the following structural formula:

[0058] Compound 5 is a compound with the following structural formula:

[0059] S3, Compound 5 (1.21 g, 2.43 mmol) was dispersed in a mixed solvent of methanol and dichloromethane (5 ml of methanol + 45 ml of dichloromethane), and sodium borohydride (275 mg, 7.29 mmol) was added. The mixture was stirred at room temperature for 12 hours. After the reaction, the mixed solvent of methanol and dichloromethane was evaporated to dryness and purified by silica gel column chromatography (dichloromethane:methanol = 6:1) to obtain Compound 6 (1.03 g, 85%) as a white solid.

[0060] In step S3, the molar ratio of compound 5 to sodium borohydride is 1:2.5 to 1:4.0, the reaction time is 8 to 16 hours, 12 hours in this embodiment, and the reaction temperature is room temperature, i.e., 25° C., which is usually referred to in chemical experiments;

[0061] In step S3, compound 6 is a compound having the following structural formula:

[0062] S4, compound 6 (0.98 g, 1.96 mmol) was dispersed in tetrahydrofuran (40 mL), phosphorus tribromide (1.59 g, 5.88 mmol) was added, and the mixture was stirred at room temperature for 8 hours. After the reaction, the tetrahydrofuran was evaporated to dryness and purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain compound 7 (0.86 g, 78%) as a white solid;

[0063] In step S4, the molar ratio of compound 6 to phosphorus tribromide is 1:2.0 to 1:4.0, the reaction time is 6 to 12 hours, 8 hours in this embodiment, and the reaction temperature is room temperature, i.e., 25° C., which is usually referred to in chemical experiments;

[0064] In step S4, compound 7 is a compound having the following structural formula:

[0065] S5, compound 7 (0.72 g, 1.28 mmol) and potassium carbonate (0.88 g, 6.4 mmol) were dispersed in a mixed solvent of methanol and dichloromethane (20 mL of methanol + 20 mL of dichloromethane) and stirred at room temperature for 12 hours. After the reaction, the mixed solvent of methanol and dichloromethane was evaporated to dryness and purified by silica gel column chromatography (dichloromethane:methanol = 4:1) to obtain compound 8 (0.46 g, 91%) as a white solid;

[0066] In step S5, the molar ratio of compound 7 to potassium carbonate is 1:3.0 to 1:6.0, the reaction time is 8 to 16 hours, 12 hours in this embodiment, and the reaction temperature is room temperature, i.e., 25° C., which is usually referred to in chemical experiments;

[0067] In step S5, compound 8 is a compound having the following structural formula:

[0068] S6, compound 8 (0.38 g, 0.96 mmol) and compound 2 (0.55 g, 0.96 mmol) were dispersed in acetonitrile (40 mL) and stirred at 80°C for 24 hours. After the reaction, the reaction solution was cooled to room temperature and filtered. The filtered solid was washed twice with diethyl ether (20 ml) and dried to obtain a dark red solid compound gal-PBDP (196 mg, 21%).

[0069] In step S6, the molar ratio of compound 8 to compound 2 is 1:1.0 to 1:1.5, and the reaction time is 20 to 30 hours, and in this embodiment, 24 hours;

[0070] In step S6, the compound gal-PBDP is a compound having the following structural formula:

[0071]

[0072] The compound gal-PBDP is the final product: a photosensitizer activated by β-galactosidase.

[0073] Example 3:

[0074] The third embodiment of the present invention also provides an application of a β-galactosidase activated photosensitizer (gal-PBDP), please refer to Figures 1 to 7The β-galactosidase-activated photosensitizer (gal-PBDP) prepared in Example 1 or Example 2 is used to activate β-galactosidase in senescent cells. The specific reaction principle is as follows:

[0075]

[0076] First, the β-galactosidase-activated photosensitizer (gal-PBDP) of the present invention is in an "off" state due to the photoelectric transfer effect before being activated by β-galactosidase. Once the β-galactosidase-activated photosensitizer (gal-PBDP) enters senescent cells, it can be activated by the β-galactosidase in the senescent cells, releasing photosensitizer BODIPY molecules with photodynamic activity, thereby emitting fluorescence, such as Figure 1 As shown, 1 is before activation, 2 is after activation; then, senescent cells are eliminated through photodynamic therapy; Figure 2 As can be seen in the figure, the β-galactosidase activated photosensitizer (gal-PBDP) of the present invention can only respond to β-galactosidase, thereby emitting fluorescence.

[0077] Secondly, since normal cells lack or low-express β-galactosidase, the photosensitizer (gal-PBDP) activated by β-galactosidase in the present invention cannot be effectively activated by β-galactosidase after entering normal cells, and cannot show fluorescence and photodynamic activity. Therefore, normal cells will not be eliminated by photodynamic action. Figure 3 As can be seen from the figure, the fluorescence intensity of the photosensitizer (gal-PBDP) activated by β-galactosidase of the present invention in senescent HeLa cells is stronger than that in HeLa cells; Figure 4 It can be seen that the β-galactosidase-activated photosensitizer (gal-PBDP) of the present invention can selectively kill senescent HeLa cells under light conditions. Therefore, the β-galactosidase-activated photosensitizer (gal-PBDP) of the present invention can be used as a probe of senescent cells and selectively eliminate senescent cells.

[0078] Figure 5 The 1H NMR spectrum of the β-galactosidase activated photosensitizer (gal-PBDP) of the present invention is shown; the 1H NMR spectrum is assigned as follows: 1H NMR (400MHz, DMSO-d6): δ9.44 (d, J=6.8Hz, 2H, Ar-H), 8.48 (d, J=6.8Hz, 2H, Ar-H), 8.09 (d, J=2. 0Hz,1H,Ar-H),7.86(dd,J=8.8,2.0Hz,1H),7.53(dd,J=8.8Hz,1H),6.01(s,2H,PhCH2),5.22(br s,1H,OH),5.12(d,J=8.0Hz,1H,H-1),4.94(br s,1H,OH),4.67 4.94(br s,2H,OH),3.72(d,J=2.4Hz,1H,H-6a),3.68(d,J=8.0Hz,1H,H-2),3.61-3.54(m,2H,H-4,H-6b ),3.50-3.46(m,1H,H-5),3.41(dd,J=8.0,2.4Hz,1H,H-3),2.58(s,6H,CH3),1.38(s,6H,CH3).

[0079] Figure 6 The carbon NMR spectrum of the β-galactosidase activated photosensitizer (gal-PBDP) of the present invention is shown; carbon spectrum attribution: 13 C{ 1 H}NMR (100.6MHz, DMSO-d6): δ158.3,151.4,150.7,147.0,144.9,140.3,135.1,134.7,129. 6,129.2,127.8,126.0,118.0,101.2,88.8,76.2,73.8,70.4,68.4,62.7,60.7,17.9,16.5.

[0080] Figure 7 The mass spectrometric characterization of the β-galactosidase activated photosensitizer (gal-PBDP) of the present invention is shown; HRMS (ESI): m / z calcd for C 31 H 32 BF2I2N4O8 + [M] + :891.0370,found 891.0359.

[0081] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all such improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. A β-galactosidase-activated photosensitizer, comprising a β-galactosidase-activated galactose substrate, a rearrangeable linker, and a BODIPY molecule containing a positively charged pyridine ring, characterized in that: The β-galactosidase activated photosensitizer is a compound of the following structural formula:

2. A method for preparing a β-galactosidase-activated photosensitizer, characterized in that: Here are the steps: S1, dispersing compound 1 in solvent 1, adding N-iodosuccinimide and stirring, evaporating to dryness and purifying after the reaction to obtain compound 2; S2, dispersing compound 3 and compound 4 in solvent 2, adding tetrabutylammonium bromide and potassium carbonate and stirring, evaporating to dryness and purifying after the reaction to obtain compound 5; S3, dispersing the compound 5 in the solvent 3, adding sodium borohydride and stirring, evaporating to dryness and purifying after the reaction to obtain compound 6; S4, dispersing the compound 6 in the solvent 4, adding phosphorus tribromide and stirring, evaporating to dryness and purifying after the reaction to obtain compound 7; S5, dispersing the compound 7 and potassium carbonate in the solvent 3 and stirring, evaporating to dryness and purifying after the reaction to obtain compound 8; S6, dispersing the compound 8 and the compound 2 in the solvent 5 and stirring, cooling, filtering, washing, and drying after the reaction to obtain the compound gal-PBDP; in, The compound 1 is a compound of the following structural formula: The compound 2 is a compound of the following structural formula: The compound 3 is a compound of the following structural formula: The compound 4 is a compound of the following structural formula: The compound 5 is a compound of the following structural formula: The compound 6 is a compound of the following structural formula: The compound 7 is a compound of the following structural formula: The compound 8 is a compound of the following structural formula: The compound gal-PBDP is a compound of the following structural formula: The solvent 1 is dichloromethane; the solvent 2 is a mixed solvent of water and dichloromethane; the solvent 3 is a mixed solvent of methanol and dichloromethane; the solvent 4 is tetrahydrofuran; and the solvent 5 is acetonitrile.

3. The method for preparing a β-galactosidase-activated photosensitizer according to claim 2, wherein: In S1, the molar ratio of the compound 1 to N-iodosuccinimide is 1:2.0 to 1:3.0, the reaction temperature is room temperature, and the reaction time is 6 to 10 hours.

4. The method for preparing a β-galactosidase-activated photosensitizer according to claim 2, wherein: In S2, the molar ratio of compound 3, compound 4, tetrabutylammonium bromide and potassium carbonate is 1:1.2:0.5:3, the reaction temperature is room temperature, and the reaction time is 16 to 24 hours.

5. The method for preparing a β-galactosidase-activated photosensitizer according to claim 2, wherein: In S3, the molar ratio of compound 5 to sodium borohydride is 1:2.5 to 1:4.0, the reaction temperature is room temperature, and the reaction time is 8 to 16 hours.

6. The method for preparing a β-galactosidase-activated photosensitizer according to claim 2, wherein: In the step S4, the molar ratio of compound 6 to phosphorus tribromide is 1:2.0 to 1:4.0, the reaction temperature is room temperature, and the reaction time is 6 to 12 hours.

7. The method for preparing a β-galactosidase-activated photosensitizer according to claim 2, wherein: In S5, the molar ratio of compound 7 to potassium carbonate is 1:3.0 to 1:6.0, the reaction temperature is room temperature, and the reaction time is 8 to 16 hours.

8. The method for preparing a β-galactosidase-activated photosensitizer according to claim 2, wherein: In the S6, the molar ratio of the compound 8 to the compound 2 is 1:1.0 to 1:1.5, the reaction temperature is 80° C., and the reaction time is 20 to 30 hours.

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