A lysosome-targeted, pH-activated so2 fluorescent probe and preparation and application thereof

Abstract

The application discloses a kind of targeting lysosome, pH activated SO2 fluorescent probe and its preparation and application.The probe presents good cell lysosome targeting ability, and under the action of acidic environment in lysosome, can release sulfur dioxide and derivative reaction site, SO2 And derivative can carry out Michael addition with unsaturated carbon-carbon double bond released by molecule, change the conjugated structure of molecule, cause the change of probe color and fluorescence, with strong fluorescence intensity, can realize the imaging and rapid detection of SO2 And derivative in lysosome, show good practical applicability.The preparation method of the probe of the application is simple, raw material is diversified, easy to mass production and application.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of SO2 and derivative detection, and particularly relates to a lysosome-targeted, pH-activated SO2 fluorescent probe and preparation and application thereof. BACKGROUND

[0002] Lysosomes are the main place for endogenous sulfur dioxide (SO2) production, and lysosomes, as scavengers of living cells, play a crucial role in the metabolic process. Abnormal SO2 will affect the number and activity of lysosomes in cells, thereby further damaging local immune function. Although fluorescent molecular probes have been used for the detection and fluorescence imaging of SO2 and its derivatives in lysosomes, in the prior art, the active sites of most probes are activated before entering cells or organelles, thereby resulting in low accuracy of detection results. Even though lysosome-targeted, photo-activated SO2 fluorescent probes have been developed and applied, the active sites of SO2 in the fluorescent probes need to be activated by ultraviolet irradiation, which will damage living cells and limit the application of SO2 fluorescent probes. SUMMARY

[0003] The present application aims to at least solve one of the above technical problems in the prior art. To this end, the purpose of the present application is to provide a lysosome-targeted, pH-activated SO2 fluorescent probe and preparation and application thereof, and particularly relates to a pH-induced molecular switch opening and closing ring, an acid-induced color change spiro compound. In the acidic environment in lysosomes, without applying other stimulation methods, the spiro carbon-heteroatom covalent bond of the spiro compound can be broken, accompanied by changes in color and fluorescence. At the same time, the electrophilicity of the carbon-carbon double bond connected thereto is enhanced, and SO2 and its derivatives can attack the unsaturated C=C bond by nucleophilic addition, change the electron cloud distribution in the spiro compound and interrupt its large conjugated structure, thereby changing the fluorescence wavelength and intensity of the molecular probe, and realizing the imaging and detection of SO2 and its derivatives in lysosomes.

[0004] To achieve the above purpose, the technical solution adopted by the present application is as follows:

[0005] The first aspect of the present application proposes a lysosome-targeted, pH-activated SO2 fluorescent probe.

[0006] The second aspect of the present application proposes a preparation method of the lysosome-targeted, pH-activated SO2 fluorescent probe.

[0007] The third aspect of the present application proposes an application of the lysosome-targeted, pH-activated SO2 fluorescent probe.

[0008] According to the first aspect of the present application, a lysosome-targeted, pH-activated SO2 fluorescent probe is proposed, which comprises a compound having the following formula I structure:

[0009]

[0010] wherein X is selected from O, S, Se, NH, a substituted N atom;

[0011] Ar1 is selected from an aromatic ring or a substituted aromatic ring having 6-14 carbon atoms;

[0012] R1 is a lysosome targeting group selected from one of hydrogen, morpholine, piperazine, pyridine, piperidine, 1,4,7-triazacyclononane, 1,4,7,10-tetraazacyclododecane;

[0013] R2, R3, R4, R5 are independently selected from one of H, alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, carbonyl, amido, halogen, nitro, cyano, aldehyde, hydroxylamino, sulfonyl, aryl or substituted aryl, heteroaryl;

[0014] In the compound of the above general structure, n is any integer between 1 and 5.

[0015] In the present application, the heterocycle of the spirocyclic fluorescent molecular probe undergoes bond cleavage under external stimulation such as pH change, thereby forming a π-extended large conjugated system, which has high molar absorption coefficient and long wavelength (near-infrared) fluorescence, and is easy to be captured and recognized by the naked eye or machine. The pH in lysosomes is 4.0-6.0, and under the stimulation of such an acid environment, the spirocyclic molecular probe designed in the present application can undergo cleavage of the spiro carbon-heteroatom covalent bond, and the negative ion and the heteroatom positive ion in the zwitterion are not in the same conjugated system, reducing the dispersion of the negative ion to the positive charge, greatly increasing the electrophilicity of the heteroatom positive ion part, so that the double bond adjacent thereto is more easily attacked by nucleophilic substances such as SO2 and SO2 derivatives. With the introduction and modification of the color group or the fluorescent group, the addition reaction produced can change the distribution of the electron cloud in the spirocyclic molecule and interrupt its large conjugated structure, affect the optical properties of the molecular probe, and then cause the color and fluorescence of the molecule to change obviously. The above process mechanism is shown as follows:

[0016]

[0017]

[0018] In some embodiments of the present application, the aromatic ring includes a monocyclic, bicyclic, or polycyclic aromatic ring.

[0019] In some embodiments of the present application, the aromatic ring is selected from one of a benzene ring, a naphthalene ring, a phenanthrene ring, and an anthracene ring.

[0020] In some embodiments of the present application, the substituent on the substituted aromatic ring is selected from F, Cl, Br, I, R, CX 1 3, CX 1 X 2 X 3 , NO2, OR, SR, SO2R, SOR, SO3R, NHR, NRR', CHO, CH2OR, CO2R, OCOR, OCOCH2R, CONHRNR'O; wherein X 1 , X 2 , X 3 are each independently selected from H, F, Cl, Br, I, NO2, OR, SR, NHR, NRR', (CH2) m CHO, (CH2) m CO2R, (CH2) m ; R and R' are each independently selected from H or a hydrocarbon substituent, and m is any integer between 0 and 4.

[0021] In some preferred embodiments of the present application, Ar1 is selected from an aromatic ring or a substituted aromatic ring having 6 to 10 carbon atoms.

[0022] In some preferred embodiments of the present application, X is O.

[0023] In some preferred embodiments of the present application, Ar1 is selected from one of a monocyclic, bicyclic aromatic ring, a substituted monocyclic aromatic ring.

[0024] In some preferred embodiments of the present application, R1 is selected from one of hydrogen, morpholine, piperazine, 1,4,7-triazacyclononane, 1,4,7,10-tetraazacyclododecane.

[0025] In some preferred embodiments of the present application, R2, R3, R4, R5 are each independently selected from one of H, alkylamino, or substituted alkylamino.

[0026] In some preferred embodiments of the present application, n = 1.

[0027] In some more preferred embodiments of the present application, the monocyclic aromatic ring is benzene.

[0028] In some more preferred embodiments of the present application, the bicyclic aromatic ring is naphthalene.

[0029] In some more preferred embodiments of the present application, the substituent on the substituted monocyclic aromatic ring is selected from one of NO2, CONHRNR'O, wherein R and R' are each independently selected from H or a hydrocarbon substituent.

[0030] In some more preferred embodiments of the application, the substituents on the substituted monocyclic aromatic ring are selected from the group consisting of NO2,

[0031] In some more preferred embodiments of the application, the compound of the structure of Formula I is selected from the group consisting of one of the following compounds:

[0032]

[0033] The names of the above compounds are, respectively, A-1 : 7-(diethylamino)-3-[(1 E)-2-(8,9-dihydro-11,11 -dimethylbenzoxazolo[3,2-a]indol-10a(11 H)-yl)vinyl]- 2H-chromen-2-one; A-2: 7-(diethylamino)-3-[(1 E)-2-(8,9-dihydro-11,11 - dimethylbenzoxazolo[3,2-a]indol-10a(11 H)-yl)vinyl]-4-morpholino-2H-1 - chromen-2-one; A-3: 7-(diethylamino)-3-[(1 E)-2-(8,9-dihydro-11,11 - dimethylbenzoxazolo[3,2-a]indol-10a(11 H)-yl)vinyl]-4-piperazin-1 -yl-2H-1 - chromen-2-one; A-4: 7-(diethylamino)-3-[(1 E)-2-(8,9-dihydro-11,11 - dimethylbenzoxazolo[3,2-a]indol-10a(11 H)-yl)vinyl]-4-[1,4,7-triazacyclononanyl]- 2H-1 -chromen-2-one; A-5: 7-(diethylamino)-3-[(1 E)-2-(8,9-dihydro-11,11 - dimethylbenzoxazolo[3,2-a]indol-10a(11 H)-yl)vinyl]-4-(1,4,7,10- tetraazacyclododecanyl)-2H-1 -chromen-2-one; A-6: 7-(diethylamino)-3-[(1 E)-2-(2,3-dihydro-9,9-dimethyl-6-nitrooxazolo[3,2-a]indol-9a(9H)- yl)vinyl]-4-morpholino-2H-1 -chromen-2-one; A-7: 7-(diethylamino)-3-[(1 E)-2-(2,3-dihydro-9,9-dimethyl-6-nitrooxazolo[3,2-a]indol-9a(9H)- yl)vinyl]-4-piperazin-1 -yl-2H-1 -chromen-2-one; A-8: 7-(diethylamino)-3-[(1 E)-2-(8,9-dihydro-11,11 -6-morpholinoethyl-oxazolo[3,2-a]indol-10a(11 H)- yl)vinyl]-2H-chromen-2-one.

[0034] ​In a second aspect of the present application, a preparation method of the lysosome-targeting, pH-activated SO2 fluorescent probe of the first aspect is provided, comprising the following steps: heating and reacting a compound shown in formula II with a compound shown in formula III in an organic solvent to obtain a compound shown in formula IV, then mixing the compound shown in formula IV with saturated sodium carbonate and ethyl acetate, stirring and reacting, separating, collecting the upper liquid, and obtaining the product.

[0035] Y is selected from one of F, Cl, Br, and I.

[0036] In some embodiments of the present application, the structural formula shown in formula II is selected from one of the following compounds: 1,1,2-trimethyl-1H-benzo[e]indole, 2,3,3-trimethyl-5-nitroindole, and 2,3,3-trimethyl-3H-indole-5-carboxylic acid.

[0037] In some embodiments of the present application, the structural formula shown in formula III is selected from one of the following compounds: 7-diethylamino-3-formyl-coumarin, 4-chloro-7-diethylamino-2-oxo-2H-chromen-3-carbaldehyde, and 2-oxo-2H-chromen-3-carbaldehyde.

[0038] In some embodiments of the present application, the heating reaction is performed at a temperature of 60-85°C for 12-36 hours.

[0039] In some embodiments of the present application, the molar ratio of the compound shown in formula II to the compound shown in formula III is 1:0.8-1.2.

[0040] In some embodiments of the present application, the organic solvent is a common organic solvent such as anhydrous ethanol, anhydrous acetonitrile, tetrahydrofuran, etc., and the molar volume ratio of the compound shown in formula II to the organic solvent is 1 mol:5-10 mL.

[0041] In some embodiments of the present application, the stirring reaction is performed at room temperature (20-30°C) for 0.5-3 hours.

[0042] In the present application, the purpose of the above stirring reaction step is to perform basic ring closure, and 3 hours is the time required to ensure complete ring closure.

[0043] In some preferred embodiments of the present application, after the upper liquid is collected, the product is concentrated by rotary evaporation and recrystallized in ethyl acetate.

[0044] According to a third aspect of the present application, the lysosome-targeting, pH-activated SO2 fluorescent probe of the first aspect is applied in imaging and / or detecting SO2 and its derivatives in lysosomes, and / or the lysosome-targeting, pH-activated SO2 fluorescent probe is applied in preparing a detection reagent for SO2 and its derivatives in lysosomes.

[0045] The present application has the following advantages:

[0046] The probe of the present application has good lysosome-targeting capability, and under the action of the acidic environment in lysosomes, can release the reaction site of sulfur dioxide and its derivatives, and the SO2 and its derivatives can undergo Michael addition with the unsaturated carbon-carbon double bond released by the molecule, change the conjugated structure of the molecule, cause the change of the color and fluorescence of the probe, have strong fluorescence intensity, and can realize the imaging and rapid detection of SO2 and its derivatives in lysosomes, and show good practical applicability.

[0047] The probe of the present application has simple preparation method, diversified raw material sources, and is easy to mass produce and apply. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 UV-Vis absorption spectra of A-1 in different pH solutions (ethanol-water) in Example 1;

[0049] Figure 2 Fluorescence emission spectra (490nm) of A-1 in different pH solutions (ethanol-water) in Example 1;

[0050] Figure 3 Fluorescence emission spectra (675nm) of A-1 in different pH solutions (ethanol-water) in Example 1;

[0051] Figure 4 UV-Vis absorption spectra of A-1 in different pH stimuli, A-1 to sulfite with a concentration of 1 μM in Example 1;

[0052] Figure 5 Fluorescence emission spectra (482nm) of A-1 in different pH stimuli, A-1 to sulfite with a concentration of 1 μM in Example 1;

[0053] Figure 6 Fluorescence emission spectra (675nm) of A-1 in different pH stimuli, A-1 to sulfite with a concentration of 1 μM in Example 1;

[0054] Figure 7 Fluorescence photos of A-1 in Example 1 under UV light irradiation in response to bisulfite under different pH solutions, "+" is to add sulfite, "-" is no sulfite;

[0055] Figure 8 Fluorescence emission spectra (482 nm, 675 nm) of A-1 in Example 1 for different molar concentrations of sulfite;

[0056] Figure 9 Variation plots of A-1 solution (ethanol-water) in Example 1 for different ions;

[0057] Figure 10 UV-Vis absorption spectra of A-1 in Example 1 for different ions;

[0058] Figure 11 Fluorescence emission spectra (475 nm) of A-1 in Example 1 for different ions;

[0059] Figure 12 Fluorescence emission spectra (667 nm) of A-1 in Example 1 for different ions;

[0060] Figure 13 UV-Vis absorption spectra of A-1 in Example 1 for different ions with simultaneous addition of HSO3 - ;

[0061] Figure 14 Selectivity analysis plots of A-1 in Example 1 for different ions;

[0062] Figure 15 Targeting imaging of fluorescent probe A-1 in Example 1 and commercial probe Lyso Green Tracker to cell lysosomes;

[0063] Figure 16 Targeting imaging of fluorescent probe A-2 in Example 2 and commercial probe Lyso Green Tracker to cell lysosomes;

[0064] Figure 17 Response of fluorescent probe A-1 in Example 1 to different concentrations of HSO3 - in cell lysosomes;

[0065] Figure 18 Response of fluorescent probe A-2 in Example 2 to different concentrations of HSO3 - in cell lysosomes. DETAILED DESCRIPTION

[0066] The present application is further described in detail by the following specific examples. The starting materials, reagents or apparatus used in the examples and comparative examples are commercially available or are obtained by methods known in the art unless otherwise specified. The test or test method is a conventional method in the art unless otherwise specified.

[0067] Example 1

[0068] This embodiment prepares a lysosome-targeting, pH-activated SO2 fluorescent probe A-1, and the specific process is as follows:

[0069] 2 mmol of 1,1,2-trimethyl-1H-benzo[e]indole (compound II) is reacted with 2.4 mmol of 2-bromoethanol in 5 mL of anhydrous acetonitrile at 82°C for 10 h, the reaction system is placed in a room temperature environment, the polarity is adjusted with ethyl acetate until a large amount of solid is precipitated, filtration is performed under reduced pressure, the filter cake is washed with ethyl acetate for 2-3 times, and drying is performed in a blast drying machine at 65°C for 3 h to obtain 3-(2-hydroxyethyl)-1,1,2-trimethyl-1H-benzo[e]indole bromide salt (compound III);

[0070]

[0071] 2 mmol of 7-diethylamino coumarin (compound IV) is dissolved in 5 mL of N,N-dimethylformamide (DMF), 200 μL of phosphorus oxychloride (POCl3) is used as a catalyst, stirring is performed at 65°C for 2 h, cooling is performed to room temperature, ice water is added to quench the reaction, 20 wt% NaOH is added dropwise to adjust the pH to a large amount of precipitate, the pH is measured to be pH≈7 with pH paper, filtration is performed under reduced pressure, and vacuum drying is performed to obtain a crude product, which is recrystallized with ethyl acetate, and the recrystallized precipitate is placed in a blast drying machine at 65°C for 2 h to obtain 7-diethylamino-3-formyl-coumarin (compound V);

[0072]

[0073] 1 mmol of compound III is reacted with 1.1 mmol of compound V in 5 mL of anhydrous ethanol (EtOH) at 79°C for 10 h, cooling is performed to room temperature, the solvent is spin-dried, recrystallization is performed with ethyl acetate, filtration is performed under reduced pressure, vacuum drying is performed, and then the filter cake is washed with ethyl acetate for 2-3 times and vacuum dried to obtain (E)-2-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)-3-(2-hydroxyethyl)-1,1-dimethyl-1H-benzoindol-3-ium (compound VI);

[0074]

[0075] The 1 mmol compound VI was mixed with 5 mL saturated sodium carbonate (Na2CO3) and 5 mL ethyl acetate (EA) at room temperature and stirred for 1 h. After the reaction, the solution was separated by a separatory funnel. The upper liquid was dried by a rotary evaporator, and then recrystallized with ethyl acetate. The precipitate was dried in a blast drier at 65 °C for 2 h to obtain A-1. It was yellow in pure organic solvent ethanol and dark blue in the ratio of 3:7 of organic solvent and water.

[0076]

[0077] Example 2

[0078] In this example, a lysosome-targeted, pH-activated SO2 fluorescent probe A-2 was prepared, and the specific process was as follows:

[0079] The 2 mmol 1,1,2-trimethyl-1H-benzo[e]indole (compound II) was heated and reacted with 2.4 mmol 2-bromoethanol in 5 mL anhydrous acetonitrile at 82 °C for 10 h. The reaction system was placed in a room temperature environment, and the polarity was adjusted with ethyl acetate until a large amount of solid precipitated. The filter cake was washed with ethyl acetate for 2-3 times, and dried in a blast drier at 65 °C for 3 h to obtain 3-(2-hydroxyethyl)-1,1,2-trimethyl-1H-benzo[e]indole bromide salt (compound III).

[0080]

[0081] The 5 mmol malonic acid (compound VII) was mixed with 10 mmol 2,4,6-trichlorophenol (compound VIII), 200 μL phosphorus oxychloride (POCl3) was added, and stirred at 100 °C for 4 h. After being placed at room temperature, 30 mL ice water was added to quench the reaction, and the pH was adjusted to neutral by adding saturated sodium carbonate solution drop by drop. Then, 20 mL dichloromethane was added for extraction, and the organic layer was reserved and dried with 2 g anhydrous magnesium sulfate. The solvent was dried by a rotary evaporator to obtain white bis(2,4,6-trichlorophenyl) malonate (compound IX).

[0082]

[0083] The 4.4 mmol bis(2,4,6-trichlorophenyl) malonate (compound IX) was mixed with 4 mmol 3-(N,N-diethyl) phenol (compound X) at a molar ratio of 1.1:1 in 15 mL organic solvent toluene (PhMe) at 110 °C for 2 h. After the reaction, the system was placed at room temperature, filtered, washed with toluene for 3 times, and dried in a blast drier at 65 °C for 3 h to obtain 3-hydroxy-7-diethylamino-coumarin (compound XI).

[0084]

[0085] To 3.5 mmol of 3-hydroxy-7-diethylamino-coumarin (Compound XI) and 7 mmol of phosphorus oxychloride (POCl3) in a 1:2 molar ratio, 10 mL of N,N-dimethylformamide (DMF) was used as solvent, and the mixture was stirred at 60°C for 6 h. The reaction was quenched by adding 20 mL of ice water, and the pH was adjusted to neutral by adding saturated sodium carbonate dropwise. The solid was filtered and dried in a blast drier at 65°C for 1 h to obtain orange 3-chloro-2-formyl-coumarin (Compound XII).

[0086]

[0087] To 3 mmol of 3-chloro-2-formyl-coumarin (Compound XII) and 3.3 mmol of morpholine (Compound XIII) in a 1:1.1 molar ratio, 10 mL of N,N-dimethylformamide (DMF) was used as organic solvent, and the mixture was stirred at 60°C for 1 h. The reaction was quenched by adding 10 mL of ice water, and the organic layer was extracted with 10 mL of dichloromethane (CH2Cl2). The organic layer was dried by rotary evaporation, and the solvent was removed by column chromatography to obtain pure 3-morpholino-2-formyl-coumarin (Compound XIV).

[0088]

[0089] To 2 mmol of Compound III and 2.4 mmol of Compound XIV in 10 mL of anhydrous ethanol (EtOH), 79°C was used for reaction for 10 h. The reaction solution was cooled to room temperature, dried by rotary evaporation, and recrystallized with ethyl acetate three times. The precipitate was dried in a blast drier at 65°C for 3 h to obtain pure (E)-7-diethylamino-3-(2-(11,11-dimethyl-8,9-dihydrobenzoxazolo[3,2-a]indol-10a(11H)-yl)vinyl)-4-morpholino-2H-thiophen-2-one (Compound XV).

[0090]

[0091] To 1 mmol of Compound XV in 5 mL of saturated sodium carbonate (Na2CO3) and 5 mL of ethyl acetate (EA), 30°C was used for stirring for 1 h. The solution was separated by a separatory funnel, and the upper liquid was dried by rotary evaporation and recrystallized with ethyl acetate. The precipitate obtained by recrystallization was dried in a blast drier at 65°C for 2 h to obtain A-2.

[0092]

[0093] Example 3

[0094] The embodiment prepares a lysosome-targeted, pH-activated SO2 fluorescent probe A-8, and the specific process is as follows:

[0095] 1 mmol of 5-nitro-2,3,3-trimethylindole (compound i) is reacted with 2.4 mmol of 2-bromoethanol in 5 mL of anhydrous acetonitrile at 82°C for 10 h, the reaction system is placed in a room temperature environment, the polarity is adjusted with ethyl acetate until a large amount of solid is precipitated, filtration is performed under reduced pressure, the filter cake is washed with ethyl acetate for 2-3 times, and drying is performed in a blast drying machine at 65°C for 3 h to obtain compound ii;

[0096]

[0097] 1 mmol of compound ii is reacted with 1.1 mmol of compound V in 5 mL of anhydrous ethanol (EtOH) at 79°C for 10 h, the solution is cooled to room temperature, the solvent is spin-dried, recrystallization is performed with ethyl acetate, filtration is performed under reduced pressure, vacuum drying is performed, and then the filter cake is washed with ethyl acetate for 2-3 times and vacuum dried to obtain compound iii;

[0098]

[0099] 0.5 mmol of compound iii is stirred in 5 mL of saturated sodium carbonate (Na2CO3) and 5 mL of ethyl acetate (EA) at 30°C for 1 h, the solution is separated by a separatory funnel after the reaction, the upper liquid is spin-dried by a rotary evaporator, and then recrystallization is performed with ethyl acetate; the precipitate obtained by recrystallization is placed in a blast drying machine and dried at 65°C for 2 h to obtain compound iv.

[0100]

[0101] 0.2 mmol of compound iv is reacted with 0.3 mmol of N-(2-aminoethyl)morpholine at a molar ratio of 1:1.5 in 5 mL of ethanol (EtOH) as an organic solvent, stirring is performed at 60°C for 1 h, 8 mL of ice water is added to quench the reaction, 10 mL of dichloromethane (CH2Cl2) is used for extraction, the organic layer is spin-dried, and column chromatography is used for separation to obtain 7-(diethylamino)-3-[(1E)-2-(8,9-dihydro-11,11-6-morpholinoethyl oxazolo[3,2-a]indol-10a(11H)-yl)vinyl]-2H-chromen-2-one, i.e., compound A-8.

[0102]

[0103] Test example

[0104] The probe A-1 prepared in Example 1 was weighed 2.4 mg in a 50 mL volumetric flask, and prepared into an ethanol solution (100 μmol / L) for standby. The aqueous solutions with different pH were prepared by using 1 mol / L dilute hydrochloric acid and 1 mol / L NaOH solution. 1 mL of the prepared solution was taken in a 10 mL volumetric flask, and 7 mL of the solution with different pH was taken in the volumetric flask, and ethanol was added to constant volume to 10 mL, at this time the concentration of the probe A-1 was 10 μmol / L, and the pH of the solution after constant volume was measured by a pH meter. 3 mL of the solution with different pH was taken in a cuvette, and the ultraviolet-visible absorption spectrum of A-1 in the ethanol-water solution with different pH was obtained by using a UV-visible spectrophotometer, and the results were shown in Figure 1 . Then the cuvette was placed in a fluorescence spectrophotometer, and the fluorescence emission spectrum was obtained by using 400 nm light excitation, and the results were shown in Figure 2 ; and the fluorescence emission spectrum in the wavelength range of 570-850 nm was obtained by using 550 nm light excitation, and the results were shown in Figure 3 . From the ultraviolet-visible absorption spectrum Figure 1 , it can be seen that with the increase of pH, the absorption peak at 592 nm gradually decreased, and the absorption peak at 421 nm gradually increased; and in the fluorescence emission spectrum Figure 2 and 3 , the emission peak at 490 nm gradually increased, and the emission peak at 675 nm gradually decreased, which collectively indicated that the probe A-1 was sensitive to the change of pH.

[0105] In the cuvettes of 3 mL of the solution with different pH, 30 μL of the prepared sodium sulfite (Na2SO3) solution with a concentration of 1 mM was added. The ultraviolet-visible absorption spectrum was tested, and the results were shown in Figure 4 ; the fluorescence emission spectrum under 400 nm wavelength excitation was shown in Figure 5 ; and the fluorescence emission spectrum under 550 nm wavelength excitation was shown in Figure 6 . When the pH was less than 4 and the pH was greater than 7, after the addition of Na2SO3, the ultraviolet-visible absorption spectrum and the fluorescence emission spectrum did not change obviously; only when the pH was between 4 and 7, after the addition of Na2SO3, the ultraviolet-visible absorption spectrum changed obviously, the absorption peak at 592 nm decreased obviously Figure 4 ; the fluorescence also changed obviously, the emission peak at 490 nm increased Figure 5 , and the emission peak at 675 nm decreased Figure 6 .

[0106] 2.4 mg of probe A-1 prepared in Example 1 was placed in a 50 mL volumetric flask and dissolved to prepare a 100 μmol / L solution for later use. Aqueous solutions of different pH values ​​were prepared using 1 mol / L dilute hydrochloric acid and a prepared 1 mol / L NaOH solution. 1 mL of the prepared probe A-1 solution was placed in a 10 mL volumetric flask, and 7 mL of each solution at different pH values ​​were placed in separate volumetric flasks. Ethanol was added to bring the volume to 10 mL, at which point the concentration of probe A-1 was 10 μmol / L. The pH of the solution after volume adjustment was measured using a pH meter. 6.3 mg of sodium sulfite (Na₂SO₃) was weighed and dissolved to prepare a 50 mL 1 mM solution. 3 mL of each solution at different pH values ​​were placed in cuvettes, resulting in four representative groups. In each group, one group had 30 μL of the prepared 1 mM sodium sulfite (Na₂SO₃) solution added, and the other group had no added solution. The results are shown in the figure. Figure 7 .from Figure 7 It can be seen that the fluorescence changes significantly from red to blue only when sulfite ions are added at pH levels between 4 and 7. Furthermore, the fluorescence spectrum changes significantly with increasing sulfite ion concentration, as shown in the results below. Figure 8 The blue fluorescence (492nm) becomes stronger, while the red fluorescence (674nm) weakens.

[0107] 2.4 mg of probe A-1 prepared in Example 1 was placed in a 50 mL volumetric flask and prepared as a 100 μmol / L solution for later use. 1 mL each of the following 1 mM solutions were prepared: sodium bicarbonate (NaHCO3), potassium thiocyanate (KSCN), sodium bisulfate (NaHSO4), potassium iodide (KI), glutathione (GSH), sodium chloride (NaCl), sodium hydrosulfite (NaHS), sodium acetate (CH3COONa), sodium sulfate (Na2SO4), cysteine ​​(CYS), sodium carbonate (Na2CO3), sodium sulfite (Na2SO3), and sodium bisulfite (NaHSO3). 1 mL of the prepared 100 μmol / L probe A-1 solution was placed in a 10 mL volumetric flask, 7 mL of ultrapure water was added, and then 2 mL of ethanol was added to bring the volume to 10 mL. 3 mL of the prepared A-1 solution was placed in 14 transparent vials, and 3 μL of each of the prepared ion solutions was added to each vial. The results are shown in the table below. Figure 9 .from Figure 9 It can be seen that A-1 reacts with various competing ions. The blue probe A-1 solution is reacted with various ions, including HSO3. - SO3 2- The color changed to pale yellow, while the color changes of other ion groups were not obvious, indicating that A-1 and HSO3... - SO3 2-The reaction is sensitive, but other competitive ions do not have this feature. Then the solution in the test tube was poured into the cuvette in turn, and the UV-Vis absorption spectra of the probe A-1 and different ions were obtained by scanning the wavelength range of 800-230 nm at a speed of 4000 nm / min using a UV-Vis spectrophotometer. The results are shown in Figure 10 , the absorption peak of the probe A-1 around 592 nm in the HSO3 - / SO3 2- group decreased significantly, and other ion groups did not have this trend. Then the above solutions were tested for fluorescence, and a fluorescence spectrophotometer was used to excite at 400 nm and 550 nm, respectively, Figure 11 and Figure 12 are the fluorescence emission spectra of the probe A-1 and different ions with HSO3 - excitation at 400 nm / 550 nm, respectively. It can be seen that the addition of HSO3 - / SO3 2- enhanced the fluorescence at 492 nm, while the fluorescence intensity at 675 nm was weakened, and other competitive ions did not have this trend. Then 30 μL of 1 mM Na2SO3 solution prepared in the previous step was added to the 14 cuvettes, and the UV-Vis absorption spectra of the probe A-1 and the mixed ions were obtained by scanning the wavelength range of 800-230 nm at a speed of 4000 nm / min using a UV-Vis spectrophotometer. The results are shown in Figure 13 . It can be seen that the absorption peak at 592 nm decreased after the addition of Na2SO3. The absorption peaks of Figure 10 and Figure 13 at 592 nm were compared with those of the pure A-1 probe, and the results are shown in Figure 14 . It can be concluded that the probe has a specific response to the SO2 derivative SO3 2- / HSO3 - .

[0108] The probe A-1 prepared in Example 1 was prepared into a mother liquor with a concentration of 500 μM using an ethanol-water solution (v / v, 3 / 7). (1) Plating: HepG2 cells were plated 24 h in advance into a 24-well plate and cultured in a cell culture medium containing 10% (v / v) FBS at 37°C in a culture box containing 5% CO2; (2) Preparation of a lysosome staining working solution: the mother liquor of A-1 with a concentration of 500 μM (dilution volume ratio 1:100) and Lyso-Tracker Green (dilution volume ratio 1:10000) were added into a cell culture medium to prepare a lysosome staining working solution with a concentration of 5 μM of probe A-1; (3) Staining: the culture medium of HepG2 cells was removed, washed with PBS solution for 3 times, and the prepared lysosome staining working solution was added, and incubated at 37°C for 30 min and 60 min, respectively; (4) Washing: the staining working solution was removed, washed with PBS solution for 3 times, and then fresh cell culture medium was added; (5) Image acquisition: observed and acquired images using an inverted automatic microscope, and the results are shown in Figure 15 . Figure 15 It is shown that probe A-1 is co-localized with commercial Lyso-Tracker Green in lysosomes, and the coincidence effect is good, and A-1 has good ability of targeting and positioning lysosomes, and can be used for quantitative detection of the content of SO2 derivative bisulfite in lysosomes.

[0109] The probe A-2 prepared in Example 2 was prepared into a mother liquor with a concentration of 500 μM using an ethanol-water solution (v / v, 3 / 7). (1) Plating: HepG2 cells were plated 24 h in advance into a 24-well plate and cultured in a cell culture medium containing 10% (v / v) FBS at 37°C in a culture box containing 5% CO2; (2) Preparation of a lysosome staining working solution: the mother liquor of A-2 with a concentration of 500 μM (dilution volume ratio 1:100) and Lyso-Tracker Green (dilution volume ratio 1:10000) were added into a cell culture medium to prepare a lysosome staining working solution with a concentration of 5 μM of probe A-2; (3) Staining: the culture medium of HepG2 cells was removed, washed with PBS solution for 3 times, and the prepared lysosome staining working solution was added, and incubated at 37°C for 30 min; (4) Washing: the staining working solution was removed, washed with PBS solution for 3 times, and then fresh cell culture medium was added; (5) Image acquisition: observed and acquired images using an inverted automatic microscope, and the results are shown in Figure 16 . Figure 16 It is shown that probe A-2 is co-localized with commercial Lyso-Tracker Green in lysosomes, and the coincidence effect is good, and A-2 has good ability of targeting and positioning lysosomes, and can be used for detection of the content of SO2 derivative bisulfite in lysosomes.

[0110] The probe A-1 prepared in Example 1 was prepared into a mother liquor with a concentration of 500 μM using an ethanol-water solution (v / v, 3 / 7). (1) Plating: HepG2 cells were plated 24 h in advance into a 24-well plate and cultured in a cell culture medium containing 10% (v / v) FBS at 37°C in a culture box containing 5% CO2; (2) Preparation of sodium bisulfite (NaHSO3) cell culture solutions with different concentrations: NaHSO3 cell culture solutions containing 10 μmol / L, 50 μmol / L and 100 μmol / L, respectively, were prepared; (3) Preparation of staining working solutions: the mother liquor of the probe A-1 with a concentration of 500 μM (dilution volume ratio 1:100) was added into the NaHSO3 cell culture solutions with different concentrations to prepare the staining working solutions; (4) Staining: the culture medium of the HepG2 cells was removed, washed with PBS solution for 3 times, and the prepared staining working solution was added to incubate at 37°C for 30 min; (5) Washing: the staining working solution was removed, washed with PBS solution for 3 times, and then fresh cell culture medium was added; (6) Image acquisition: observation and image acquisition were performed using an inverted automatic microscope, and the results are shown in Figure 17 The results show that the fluorescent probe A-1 has a good effect on detecting NaHSO3 in the cell lysosome, the blue fluorescence of the group without adding NaHSO3 is almost none, the red fluorescence is weakened and the blue fluorescence is enhanced with the increase of the concentration of NaHSO3, and good application performance is shown.

[0111] The probe A-2 prepared in Example 2 was prepared into a mother liquor with a concentration of 500 μM using an ethanol-water solution (v / v, 3 / 7). (1) Plating: HepG2 cells were plated 24 h in advance into a 24-well plate and cultured in a cell culture medium containing 10% (v / v) FBS at 37°C in a culture box containing 5% CO2; (2) Preparation of sodium bisulfite (NaHSO3) cell culture solutions with different concentrations: NaHSO3 cell culture solutions containing 10 μmol / L, 50 μmol / L and 100 μmol / L, respectively, were prepared; (3) Preparation of staining working solutions: the mother liquor of the probe A-2 with a concentration of 500 μM (dilution volume ratio 1:100) was added into the NaHSO3 cell culture solutions with different concentrations to prepare the staining working solutions; (4) Staining: the culture medium of the HepG2 cells was removed, washed with PBS solution for 3 times, and the prepared staining working solution was added to incubate at 37°C for 30 min; (5) Washing: the staining working solution was removed, washed with PBS solution for 3 times, and then fresh cell culture medium was added; (6) Image acquisition: observation and image acquisition were performed using an inverted automatic microscope, and the results are shown in Figure 18 The results show that the fluorescent probe A-2 has a good effect on detecting NaHSO3 in the cell lysosome, the blue fluorescence of the group without adding NaHSO3 is almost none, the red fluorescence is weakened and the blue fluorescence is enhanced with the increase of the concentration of NaHSO3, and good application performance is shown.

[0112] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A pH-activated SO2 fluorescent probe targeting lysosomes, characterized in that, Selected from one of the following compounds: and .

2. The method for preparing the fluorescent probe according to claim 1, characterized in that, The process includes the following steps: reacting the compound shown in Formula II with the compound shown in Formula III in anhydrous ethanol or anhydrous acetonitrile by heating to obtain the compound of Formula IV; then mixing the compound of Formula IV with saturated sodium carbonate and ethyl acetate; stirring the reaction; separating the liquids; and collecting the upper liquid to obtain the compound. Y is chosen from one of F, Cl, Br, and I.

3. The preparation method according to claim 2, characterized in that, The heating reaction is carried out at a temperature of 60 ℃ to 85 ℃ for a time of 12 h to 36 h; the stirring reaction is carried out at a temperature of 20 ℃ to 30 ℃ for a time of 1 h to 3 h.

4. The application of the targeted lysosome, pH-activated SO2 fluorescent probe of claim 1 in the preparation of imaging reagents for SO2 and its derivatives within lysosomes, wherein the SO2 derivative is selected from at least one of sulfite or bisulfite.

5. The application of the targeted lysosome, pH-activated SO2 fluorescent probe of claim 1 in the preparation of detection reagents for SO2 and its derivatives within lysosomes, wherein the SO2 derivative is selected from at least one of sulfite or bisulfite.

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