CN116421721A - Beta-galactosidase activated photosensitizer, preparation method and application thereof - Google Patents
Beta-galactosidase activated photosensitizer, preparation method and application thereof Download PDFInfo
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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Abstract
The invention relates to the technical field of biological medicines, and provides a beta-galactosidase activated photosensitizer, a preparation method and application thereof, wherein the beta-galactosidase activated photosensitizer consists of a beta-galactosidase activated galactosyl substrate, a rearranged connector and BODIPY molecules containing positively charged pyridine rings, and the beta-galactosidase activated photosensitizer is a compound with the following structural formula:
before entering the aging 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, so thatThe photosensitizer activated by the beta-galactosidase has no fluorescence or no photodynamic activity; after entering the senescent cell, the beta-galactosidase can be activated by the beta-galactosidase highly expressed in the senescent cell, so that the photodynamic activity is shown, and the efficient elimination of the senescent cell is realized.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a photosensitizer activated by beta-galactosidase, a preparation method and application thereof.
Background
The photodynamic effect is to irradiate the photosensitizer absorbed by the tissue with laser light of a specific wavelength so as to make the photosensitizer reach an excited state, and the excited state photosensitizer transfers energy to oxygen molecules around cells to generate singlet oxygen with cytotoxicity, thereby causing the cells to be damaged and dead.
Cell aging is a permanent state of cell cycle arrest caused by various internal and external pressures, and research shows that cell aging is closely related to various age-related diseases, such as atherosclerosis, dyspnea syndrome, alzheimer's disease, etc., aging cells regulate functions of adjacent normal cells by promoting secretion of a series of inflammatory cytokines, chemokines, growth factors, etc., thereby interfering with physiological functions of tissues, resulting in development and poor prognosis of many diseases, thus eliminating aging cells or shutting down their secretion mechanisms, preventing or delaying age-related degeneration and prolonging life of organisms. Senescent cells share some common biological features, such as enlarged cell morphology, lipid and protein deposition, a significant increase in the number of lysosomes, high expression and activation of senescence-associated enzymes, especially β -galactosidase, an exoglycosidase that specifically hydrolyzes β -glycosidic linkages between galactose and its organic moiety; beta-galactosidase is the most common biomarker for senescent cells because it is easily detected in cells and tissues.
Although a series of compound molecules have been developed to date, they kill senescent cells by modulating related signaling pathways (e.g., anti-apoptotic, protein kinases, and transcription factors). However, such drug molecules are also subject to multiple aspects of cellular aging, dynamic and highly heterogeneous characteristics, especially when the cells are subjected to different stimuli or pressures, such molecules can fail off-target, with a significant risk in the clinic.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a photosensitizer activated by beta-galactosidase, a preparation method and application thereof.
The technical scheme adopted for solving the technical problems is as follows: in one aspect, the invention provides a beta-galactosidase-activated photosensitizer consisting of a beta-galactosidase-activated galactosyl substrate, a rearranged linker, and a BODIPY molecule comprising a positively charged pyridine ring, said beta-galactosidase-activated photosensitizer being a compound of the formula:
on the other hand, the invention also provides a preparation method of the beta-galactosidase activated photosensitizer, which comprises the following steps:
s1, dispersing a compound 1 in a solvent 1, adding N-iodinated succinimide, stirring, evaporating to dryness after the reaction is finished, and purifying to obtain a compound 2;
s2, dispersing the compound 3 and the compound 4 in a solvent 2, adding tetrabutylammonium bromide and potassium carbonate, stirring, evaporating to dryness after the reaction is finished, and purifying to obtain a compound 5;
s3, dispersing the compound 5 in a solvent 3, adding sodium borohydride, stirring, evaporating to dryness after the reaction is finished, and purifying to obtain a compound 6;
s4, dispersing the compound 6 in a solvent 4, adding phosphorus tribromide, stirring, evaporating to dryness after the reaction is finished, and purifying to obtain a compound 7;
s5, dispersing the compound 7 and potassium carbonate in the solvent 3, stirring, evaporating to dryness after the reaction is finished, and purifying to obtain a compound 8;
s6, dispersing the compound 8 and the compound 2 in a solvent 5, stirring, cooling, filtering, washing and drying after the reaction is finished to obtain a compound gal-PBDP;
wherein,,
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; the solvent 5 is acetonitrile.
Preferably, in the S1, the molar ratio of the compound 1 to the 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.
Preferably, in the S2, the molar ratio of the compound 3, the compound 4, the tetrabutylammonium bromide and the potassium carbonate is 1:1.2:0.5:3, the reaction temperature is room temperature, and the reaction time is 16-24 hours.
Preferably, in the S3, the molar ratio of the compound 5 to sodium borohydride is 1:2.5-1:4.0, the reaction temperature is room temperature, and the reaction time is 8-16 hours.
Preferably, in the S4, the molar ratio of the compound 6 to the phosphorus tribromide is 1:2.0-1:4.0, the reaction temperature is room temperature, and the reaction time is 6-12 hours.
Preferably, in the step S5, the molar ratio of the compound 7 to the 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.
Preferably, in the step S6, the molar ratio of the compound 8 to the compound 2 is 1:1.0-1:1.5, the reaction temperature is 80 ℃, and the reaction time is 20-30 hours.
In another aspect, the invention also provides the use of a beta-galactosidase-activated photosensitizer for the elimination of senescent cells, using a beta-galactosidase-activated photosensitizer as described above.
The invention has the beneficial effects that: unlike the prior art, the beta-galactosidase activated photosensitizer of the invention takes the highly expressed beta-galactosidase in the aging cells as a target point, and the beta-galactosidase activated photosensitizer with high selectivity for eliminating the aging cells is constructed by connecting a beta-galactosidase activated galactosyl substrate with a rearranged connector and a BODIPY molecule containing a positively charged pyridine ring; in addition, the photosensitizer activated by the beta-galactosidase can be directly added into a cell culture solution to be co-cultured with cells, so that the photodynamic effect on the aging cells is effectively exerted, and the aging cells are eliminated.
Drawings
FIG. 1 is a fluorescence spectrum of a β -galactosidase activated photosensitizer (gal-PBDP) before and after activation by β -galactosidase in an example of the invention;
FIG. 2 is a study of the specificity of a beta-galactosidase-activated photosensitizer (gal-PBDP) for beta-galactosidase in an example of the invention;
FIG. 3 is a confocal microscopy comparison of β -galactosidase activated photosensitizer (gal-PBDP) in normal and senescent cells according to an example of the present invention;
FIG. 4 shows the photodynamic effect of beta-galactosidase activated photosensitizer (gal-PBDP) on aging HeLa and HeLa in an example of the present invention;
FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of a beta-galactosidase activated photosensitizer (gal-PBDP) in an embodiment of the invention;
FIG. 6 is a nuclear magnetic resonance carbon spectrum of a beta-galactosidase activated photosensitizer (gal-PBDP) in an embodiment of the invention;
FIG. 7 is a mass spectrum characterization of the β -galactosidase activated photosensitizer (gal-PBDP) in an example of the invention.
Detailed Description
For the purpose of illustrating more clearly the objects, technical solutions and advantages of embodiments of the present invention, the present invention will be further described with reference to the accompanying drawings and embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by a person skilled in the art without any inventive effort, are intended to be within the scope of the present invention, based on the embodiments of the present invention.
Embodiment one:
the beta-galactosidase activated photosensitizer in the first embodiment of the invention consists of a beta-galactosidase activated galactosyl substrate, a rearranged connector and a BODIPY molecule containing a positively charged pyridine ring, wherein the beta-galactosidase activated galactosyl substrate, the rearranged connector and the BODIPY molecule containing the positively charged pyridine ring are all functional molecular groups, and the beta-galactosidase which is highly expressed in aging cells is taken as a target point, and the beta-galactosidase activated galactosyl substrate, the rearranged connector and the BODIPY molecule containing the positively charged pyridine ring are connected to construct the beta-galactosidase activated photosensitizer with high selectivity for eliminating aging cells, and the beta-galactosidase activated photosensitizer is a compound with the following structural formula:
before entering the senescent cells, the positive charge on the pyridine ring quenches the fluorescence of the photosensitizer BODIPY molecule and the generation of singlet oxygen through a photoelectric transfer effect (photo electron transfer), so that the photosensitizer activated by the beta-galactosidase has no fluorescence and no photodynamic activity.
Embodiment two:
the second embodiment of the invention provides a preparation method of a beta-galactosidase activated photosensitizer, which comprises the following reaction routes:
the method comprises the following specific steps:
s1, dispersing the compound 1 (0.72 g,2.21 mmol) in methylene chloride (40 ml), adding N-iodosuccinimide (1.27 g,5.53 mmol), stirring at room temperature for 8 hours, evaporating the methylene chloride after the reaction is finished, and purifying by silica gel column chromatography (petroleum ether: ethyl acetate=5:1) to obtain a red solid compound 2 (0.96 g, 75%);
in the step S1, the molar ratio of the compound 1 to the N-iodinated succinimide is 1:2.0-1:3.0, the reaction time is 6-10 hours, in the embodiment, 8 hours, and the reaction temperature is room temperature, namely 25 ℃ commonly referred to in chemical experiments;
in step S1, compound 1 is a compound of the following structural formula:
s2, dispersing compound 3 (1.23 g,3 mmol) and compound 4 (0.61 g,3.6 mmol) in a mixed solvent of water and dichloromethane (30 mL of water+30 mL of dichloromethane), adding tetrabutylammonium bromide (0.48 g,1.5 mmol) and potassium carbonate (1.24 g,9 mmol), stirring at room temperature for 20 hours, separating a dichloromethane phase after the reaction is finished, evaporating to dryness, and purifying by silica gel column chromatography (dichloromethane: methanol=10:1) to obtain a light yellow solid compound 5 (1.31 g, 88%);
in the step S2, the molar ratio of the compound 3 to the compound 4 to the tetrabutylammonium bromide to the potassium carbonate is 1:1.2:0.5:3, the reaction time is 16-24 hours, in the embodiment, 20 hours, and the reaction temperature is room temperature, namely 25 ℃ commonly referred to in chemical experiments;
in step S2, compound 3 is a compound of the following structural formula:
compound 4 is a compound of the formula:
compound 5 is a compound of the formula:
s3, dispersing the compound 5 (1.21 g,2.43 mmol) in a mixed solvent of methanol and dichloromethane (methanol 5 ml+dichloromethane 45 ml), adding sodium borohydride (275 mg,7.29 mmol), stirring at room temperature for 12 hours, evaporating the mixed solvent of methanol and dichloromethane after the reaction is finished, and purifying by silica gel column chromatography (dichloromethane: methanol=6:1) to obtain a white solid compound 6 (1.03 g, 85%);
in the step S3, the molar ratio of the compound 5 to the sodium borohydride is 1:2.5-1:4.0, the reaction time is 8-16 hours, in the embodiment, 12 hours, and the reaction temperature is room temperature, namely 25 ℃ commonly referred to in chemical experiments;
in step S3, compound 6 is a compound of the following structural formula:
s4, dispersing the compound 6 (0.98 g,1.96 mmol) in tetrahydrofuran (40 mL), adding phosphorus tribromide (1.59 g,5.88 mmol), stirring at room temperature for 8 hours, evaporating tetrahydrofuran after the reaction is finished, and purifying by silica gel column chromatography (dichloromethane: methanol=10:1) to obtain a white solid compound 7 (0.86 g, 78%);
in the step S4, the molar ratio of the compound 6 to the phosphorus tribromide is 1:2.0-1:4.0, the reaction time is 6-12 hours, in the embodiment, 8 hours, and the reaction temperature is room temperature, namely 25 ℃ commonly referred to in chemical experiments;
in step S4, compound 7 is a compound of the following structural formula:
s5, dispersing the compound 7 (0.72 g,1.28 mmol) and potassium carbonate (0.88 g,6.4 mmol) in a mixed solvent of methanol and dichloromethane (methanol 20 ml+dichloromethane 20 mL), stirring at room temperature for 12 hours, evaporating the mixed solvent of methanol and dichloromethane after the reaction is finished, and purifying by silica gel column chromatography (dichloromethane: methanol=4:1) to obtain a white solid compound 8 (0.46 g, 91%);
in the step S5, the molar ratio of the compound 7 to the potassium carbonate is 1:3.0-1:6.0, the reaction time is 8-16 hours, in the embodiment, 12 hours, and the reaction temperature is room temperature, namely 25 ℃ commonly referred to in chemical experiments;
in step S5, compound 8 is a compound of the following structural formula:
s6, dispersing a compound 8 (0.38 g,0.96 mmol) and a compound 2 (0.55 g,0.96 mmol) in acetonitrile (40 mL), stirring for 24 hours at 80 ℃, cooling the reaction solution to room temperature after the reaction is finished, filtering, washing the obtained solid with diethyl ether (20 mL) twice, and drying to obtain a dark red solid compound gal-PBDP (196 mg, 21%);
in the step S6, the molar ratio of the compound 8 to the compound 2 is 1:1.0-1:1.5, and the reaction time is 20-30 hours, in this example 24 hours;
in step S6, compound gal-PBDP is a compound of the formula:
the compound gal-PBDP is the final product: beta-galactosidase activated photosensitizers 。
Embodiment III:
the third embodiment of the present invention also provides an application of the β -galactosidase activated photosensitizer (gal-PBDP), referring to fig. 1 to 7, the β -galactosidase activated photosensitizer (gal-PBDP) prepared in the first embodiment or the second embodiment is used for activating β -galactosidase in aging cells, and the specific reaction principle is as follows:
first, the β -galactosidase-activated photosensitizer (gal-PBDP) of the present invention is in an "off" state due to the photoelectric transfer effect, resulting in non-fluorescent and non-photodynamic activity of the β -galactosidase-activated photosensitizer (gal-PBDP) before it is not activated by β -galactosidase; once the beta-galactosidase activated photosensitizer (gal-PBDP) enters the senescent cell, it can be activated by the beta-galactosidase in the senescent cell to release the photodynamic photosensitizer BODIPY molecule, thereby emitting fluorescence, as shown in fig. 1, 1 is before activation, 2 is after activation; further eliminating aging cells by photodynamic action; as can be seen from FIG. 2, the beta-galactosidase-activated photosensitizer (gal-PBDP) of the present invention is only responsive to beta-galactosidase and thus fluoresces.
Second, since normal cells lack or low expression of β -galactosidase, the β -galactosidase-activated photosensitizer (gal-PBDP) of the present invention is not activated efficiently by β -galactosidase and exhibits no fluorescence and photodynamic activity after entering normal cells, so that normal cells are not eliminated by photodynamic action, and as can be seen from fig. 3, the β -galactosidase-activated photosensitizer (gal-PBDP) of the present invention has a stronger fluorescence intensity in senescent HeLa cells than in HeLa cells; as can be seen from FIG. 4, the beta-galactosidase-activated photosensitizer (gal-PBDP) of the present invention can selectively kill senescent HeLa cells under light conditions, and thus, the beta-galactosidase-activated photosensitizer (gal-PBDP) of the present invention can act as a probe for senescent cells and selectively eliminate senescent cells.
FIG. 5 shows a nuclear magnetic resonance hydrogen spectrum of a beta-galactosidase activated photosensitizer (gal-PBDP) of the invention; hydrogen spectrum assignment: 1 H NMR(400MHz,DMSO-d 6 ):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,CH 3 )。
FIG. 6 shows a nuclear magnetic resonance carbon spectrum of a beta-galactosidase activated photosensitizer (gal-PBDP) of the invention; belonging to carbon spectrum: 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。
FIG. 7 shows a mass spectral characterization of the beta-galactosidase activated photosensitizer (gal-PBDP) of the present invention; HRMS (ESI) m/z calcd for C 31 H 32 BF 2 I 2 N 4 O 8 + [M] + :891.0370,found 891.0359。
It will be understood that modifications and variations will be apparent to those skilled in the art from the foregoing description, and it is intended that all such modifications and variations be included within the scope of the following claims.
Claims (9)
1. A β -galactosidase activated photosensitizer consisting of a β -galactosidase activated galactosyl substrate, a rearrangeable linker and a BODIPY molecule comprising a positively charged pyridine ring, characterized in that: the beta-galactosidase activated photosensitizer is a compound of the following structural formula:
2. a method for preparing a beta-galactosidase activated photosensitizer, which is characterized by comprising the following steps: the method comprises the following steps:
s1, dispersing a compound 1 in a solvent 1, adding N-iodinated succinimide, stirring, evaporating to dryness after the reaction is finished, and purifying to obtain a compound 2;
s2, dispersing the compound 3 and the compound 4 in a solvent 2, adding tetrabutylammonium bromide and potassium carbonate, stirring, evaporating to dryness after the reaction is finished, and purifying to obtain a compound 5;
s3, dispersing the compound 5 in a solvent 3, adding sodium borohydride, stirring, evaporating to dryness after the reaction is finished, and purifying to obtain a compound 6;
s4, dispersing the compound 6 in a solvent 4, adding phosphorus tribromide, stirring, evaporating to dryness after the reaction is finished, and purifying to obtain a compound 7;
s5, dispersing the compound 7 and potassium carbonate in the solvent 3, stirring, evaporating to dryness after the reaction is finished, and purifying to obtain a compound 8;
s6, dispersing the compound 8 and the compound 2 in a solvent 5, stirring, cooling, filtering, washing and drying after the reaction is finished to obtain a compound gal-PBDP;
wherein,,
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; the solvent 5 is acetonitrile.
3. A method of preparing a β -galactosidase activated photosensitizer as claimed in claim 2, wherein:
in the S1, the molar ratio of the compound 1 to the N-iodinated succinimide is 1:2.0-1:3.0, the reaction temperature is room temperature, and the reaction time is 6-10 hours.
4. A method of preparing a β -galactosidase activated photosensitizer as claimed in claim 2, wherein:
in the step S2, the molar ratio of the compound 3 to the compound 4 to the tetrabutylammonium bromide to the potassium carbonate is 1:1.2:0.5:3, the reaction temperature is room temperature, and the reaction time is 16-24 hours.
5. A method of preparing a β -galactosidase activated photosensitizer as claimed in claim 2, wherein:
in the step S3, the molar ratio of the compound 5 to the sodium borohydride is 1:2.5-1:4.0, the reaction temperature is room temperature, and the reaction time is 8-16 hours.
6. A method of preparing a β -galactosidase activated photosensitizer as claimed in claim 2, wherein:
in the S4, the molar ratio of the compound 6 to the phosphorus tribromide is 1:2.0-1:4.0, the reaction temperature is room temperature, and the reaction time is 6-12 hours.
7. A method of preparing a β -galactosidase activated photosensitizer as claimed in claim 2, wherein:
in the step S5, the molar ratio of the compound 7 to the potassium carbonate is 1:3.0-1:6.0, the reaction temperature is room temperature, and the reaction time is 8-16 hours.
8. A method of preparing a β -galactosidase activated photosensitizer as claimed in claim 2, wherein:
in the step S6, the molar ratio of the compound 8 to the compound 2 is 1:1.0-1:1.5, the reaction temperature is 80 ℃, and the reaction time is 20-30 hours.
9. Use of a β -galactosidase activated photosensitizer characterized by: use of a β -galactosidase-activated photosensitizer according to claim 1 for the elimination of senescent cells.
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