CN114478384A - Lysosome targeted pH fluorescent probe and preparation and application thereof - Google Patents

Lysosome targeted pH fluorescent probe and preparation and application thereof Download PDF

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CN114478384A
CN114478384A CN202210179172.4A CN202210179172A CN114478384A CN 114478384 A CN114478384 A CN 114478384A CN 202210179172 A CN202210179172 A CN 202210179172A CN 114478384 A CN114478384 A CN 114478384A
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fluorescent probe
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吴柏旭
程翎
卫严冰
王哲
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Luoxi Medical Technology Hangzhou Co ltd
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    • C—CHEMISTRY; METALLURGY
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    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
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Abstract

The application discloses a lysosome targeting pH fluorescent probe and preparation and application thereof, wherein the lysosome targeting pH fluorescent probe has a structural formula shown as (I):

Description

Lysosome targeted pH fluorescent probe and preparation and application thereof
Technical Field
The application relates to the technical field of pH detection, in particular to a lysosome targeted pH fluorescent probe and preparation and application thereof.
Background
Intracellular pH plays a crucial role in many cellular processes, such as cellular growth, apoptosis, ion transport, autophagy, enzymatic activity, homeostasis, etc. Subcellular organelles of eukaryotic cells have different pH values, e.g., the endosomes and lysosomes are slightly acidic (pH 4.0-6.0) internally and the mitochondria are basic (pH-8.0). As an important indicator of cellular health, small changes in intracellular pH can lead to cellular dysfunction and serious diseases such as cancer, stroke, alzheimer's disease, etc. Because the pH value in the cell has important physiological significance, the monitoring of the change of the pH value in the organelle has important significance for exploring cell metabolism and understanding physiological and pathological processes. Fluorescent probes are receiving attention because of their advantages such as high sensitivity and real-time detection.
Disclosure of Invention
The application provides a novel high sensitivity pH fluorescence probe, can accurate target lysosome to show linear relation between fluorescence probe and pH concentration in certain pH within range, be favorable to improving the detection precision.
A lysosome targeting pH fluorescent probe having the structural formula shown in (I):
Figure BDA0003521698530000011
the application also provides a preparation method of the lysosome targeting pH fluorescent probe, which comprises the following steps:
taking a compound (II) and a compound (III) as reactants, adding glacial acetic acid and sodium triacetoxyborohydride to react in a solvent, and separating and purifying reaction liquid after the reaction is finished to obtain the lysosome targeted pH fluorescent probe with the structural formula shown in (I):
Figure BDA0003521698530000021
the synthetic route is as follows:
Figure BDA0003521698530000022
optionally, the amount ratio of the compound (II), the compound (III), the glacial acetic acid and the sodium triacetoxyborohydride is 1: 1-1.2: 10-12: 4-5. Further, the ratio of the amounts of the compound (II), the compound (III), glacial acetic acid and sodium triacetoxyborohydride substances is 1:1.2:10: 4.
Optionally, the solvent is anhydrous tetrahydrofuran.
Optionally, the reaction conditions are as follows: stirring and reacting for 10-14 h at 25-30 ℃.
Alternatively, the separation and purification method may be as follows: concentrating the reaction solution under reduced pressure, and separating and purifying by column chromatography, wherein the eluent is dichloromethane/methanol with volume ratio of 40:1 to obtain compound (I).
The compound (II) of the present application is a compound disclosed in the following references W.Shen, P.Wang, Z.Xie, H.Zhou, Y.Hu, M.Fu, Q.Zhu, Abifungical probe reactions involved in diagnosis and hydrogen sulfite in zebra fish model of Parkinson's disease. Talanta2021, 122621; the compound (III) is obtained by purchase.
The application finds that the fluorescent probe has lysosome targeting property, can detect the change of the pH value in cells, and the pH value of lysosome in the cells is 4.0-6.0.
Therefore, the application also provides a use of the lysosome targeting pH fluorescent probe in cell imaging or in preparation of a cell imaging agent.
Optionally, the cell is a tumor cell. Further, the cell is human cervical carcinoma cell Hela cell.
The application also provides application of the lysosome targeted pH fluorescent probe in preparation of a pH fluorescent detection product.
Optionally, the pH fluorescence detection product is a test strip or a kit.
The application also provides a lysosome targeted pH detection kit, which comprises the lysosome targeted pH fluorescent probe. The lysosome-targeted pH fluorescent probe is prepared into a kit according to methods well established in the art.
The fluorescent probe can be used for quantitatively detecting the concentration of pH in an aqueous solution by a fluorescence ratio method and is used for the fluorescent quantitative detection of the pH. The principle of fluorescence detection for quantifying pH concentration is as follows: hydroxyl and morpholine groups of the compound (I) are taken as pH sensitive groups, and a proton is obtained/lost to form NH under the acidic or alkaline environment+/O-A group. Measuring the fluorescence intensity of the probe at an emission wavelength of 470nm and 560nm at an excitation of 380nmAnd varied to obtain a pH value. The principle of pH concentration detection using the novel lysosome-targeted pH fluorescent probe of the present application is as follows:
Figure BDA0003521698530000031
the lysosome-targeted pH fluorescent probe is weak in blue fluorescence, namely the fluorophore 1, 8-naphthalimide in the probe is quenched, and the principle of detecting the pH is that after the compound (I) reacts with the pH, a strong electron-donating group O is generated-The fluorescence of the 1, 8-naphthalimide is recovered, and strong yellow fluorescence is emitted, so that the effect of ratio detection of pH is realized.
Based on this, the present application also provides a method for quantitative detection of pH for non-diagnostic purposes, comprising:
adding a compound with a structural formula shown in (I) into a solution to be detected, and measuring fluorescence intensity values at 560nm and 470nm respectively by taking 380nm as an excitation wavelength after the reaction is finished; then dividing the fluorescence intensity value at 560nm by the fluorescence intensity value at 470nm, and substituting the obtained result into a linear standard curve to calculate the pH value of the solution to be measured;
Figure BDA0003521698530000041
optionally, the final concentration of the lysosome targeting pH fluorescent probe in the solution to be detected is 5 μ M, and the pH value of the solution to be detected is 4.5-7. When the detection method is adopted, the final concentration of the compound added into the solution to be detected is 5 mu M, and the compound has a good linear relation with the pH value of the solution to be detected at 4.5-7, so that the pH concentration of the solution to be detected can be detected more accurately.
In the linear standard curve, the ordinate is the absorption intensity value of the reaction solution at 560nm divided by the absorption intensity value at 470 nm; the abscissa is the pH value.
The preparation method of the linear standard curve comprises the following steps:
accurately weighing a certain amount of compound (I), preparing a probe mother solution with the concentration of 1mM by using dimethyl sulfoxide, sucking 2 mu L of the probe mother solution by using a pipette gun, adding the probe mother solution into 0.398mL of PBS buffer solutions with different pH values (the final pH value in water is 3.5-10), reacting for 3 hours at 37 ℃, adding the probe mother solution into a 96-well plate, and measuring the fluorescence emission spectrum of the compound (I) by using a multifunctional microplate reader. And taking 380nm as an excitation wavelength, and drawing a value obtained by dividing the fluorescence intensity value at 560nm by the fluorescence intensity value at 470nm as a vertical coordinate and taking the pH concentration as a horizontal coordinate to obtain a linear standard curve.
Optionally, the linear equation of the linear standard curve is: y-2.31 x +7.15 (R)20.9946); wherein y is the absorption intensity value of the reaction solution at 560nm divided by the absorption intensity value at 470nm, and x is the pH concentration.
Compared with the prior art, the application has at least one of the following beneficial effects:
(1) the compound (I) can be used as a fluorescent probe for detecting pH, the pH in an aqueous solution is detected by a fluorescence ratio method, the selectivity to the pH is good, the target effect on lysosomes is good, and an effective research tool is provided for further accurately researching the lysosome pH.
(2) The compound (I) can be used for cell imaging and detecting pH change in cells.
Drawings
FIG. 1 is a nuclear magnetic hydrogen spectrum of compound (I) prepared in example 1 of the present application.
FIG. 2 shows the nuclear magnetic carbon spectrum of compound (I) prepared in example 1 of the present application.
Fig. 3 is a graph showing fluorescence absorption spectra of compound (I) prepared in example 1 of the present application added to DMSO/PBS buffer (v/v-1/199) at different pH.
Fig. 4 shows fluorescence emission spectra (excitation wavelength 380nm) of compound (I) prepared in example 1 of the present application added to DMSO/PBS buffer (v/v-1/199) at different pH.
Fig. 5 is a plot of the ratio of the fluorescence intensity of compound (I) at 560nm/470nm when compound (I) prepared in example 1 of the present application was added to DMSO/PBS buffer (v/v-1/199) at different pH.
FIG. 6 is a plot of the fluorescence intensity ratio at 560nm/470nm as a point plot (excitation wavelength 380nm) of compound (I) prepared in example 1 of the present application added to DMSO/PBS buffer (v/v: 1/199) at varying pH from 4.5-7.
Fig. 7 is a fluorescence plot of the selectivity results of compound (I) prepared in example 1 of the present application in DMSO/PBS buffer (pH 4 and 7.4, v/v 1/199), respectively. Wherein, 1-18 are PBS, acetaldehyde, benzaldehyde, p-nitrobenzaldehyde, p-hydroxybenzaldehyde, acetone, formic acid, glucose, glutathione, homocysteine, cysteine, sodium bisulfate, hydrogen peroxide, tert-butyl hydroperoxide, hypochlorous acid, sodium ions, potassium ions and magnesium ions respectively.
FIG. 8 is a graph of the results of the reproducibility of compound (I) prepared in example 1 of the present application at different pH values (excitation wavelength 380 nm).
FIG. 9 is a photograph of an image of cells of compound (I) prepared in application example 1 and a commercially available Lyso-Tracker Red.
FIG. 10 is a graph showing the results of cellular imaging of Compound (I) prepared in application example 1 under different pH environments.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
Example 1: preparation of Compound (I)
In a dry 25mL round bottom flask, compound II (59.4mg, 0.2mmol) was dissolved in 5mL anhydrous tetrahydrofuran and cooled to 0 deg.C, followed by the sequential addition of compound III (34.6mg, 0.24mmol), glacial acetic acid (120mg, 2mmol), and finally the addition of sodium triacetoxyborohydride (168.8mg, 0.8mmol) in portions. The mixture was brought to room temperature and reacted overnight. After the reaction of the starting materials was completed, the organic solvent was spin-dried under reduced pressure and separated and purified by column chromatography using dichloromethane/methanol 40:1(v/v) as an eluent to give 40.3mg of a yellow solid in 49.2% yield.
Figure BDA0003521698530000071
1H NMR(500MHz,Chloroform-d)δ8.18(dd,J＝8.0,1.2Hz,1H),8.01–7.99(m,1H),7.70(s,1H),7.54(d,J＝8.2Hz,1H),6.76(t,J＝6.0Hz,1H),3.69(t,J＝9.4Hz,2H),3.65–3.59(m,5H),3.39–3.33(m,2H),2.51(t,J＝7.7Hz,2H),2.44–2.41(m,4H),1.84(t,J＝7.3Hz,2H),1.73–1.65(m,2H),1.42–1.34(m,2H),0.97(t,J＝5.6Hz,3H).
13C NMR(125MHz,Chloroform-d)δ167.44,166.38,145.27,136.46,128.41,126.67,126.44,125.90,125.84,123.01,122.14,120.77,111.18,66.32,54.37,54.18,43.43,40.95,30.24,26.14,19.56.
The nuclear magnetic hydrogen spectrum is shown in figure 1, and the nuclear magnetic carbon spectrum is shown in figure 2.
Example 2: fluorescence absorption spectroscopy of compound (I) (5 μ M) in DMSO/PBS buffer (v/v-1/199) at different pH.
An amount of the compound (I) prepared in example 1 was accurately weighed, prepared into a probe stock solution with a concentration of 0.1mM using dimethyl sulfoxide, and 2. mu.L of the solution was pipetted into 0.398mL of PBS buffers with different pH values (final pH value in water is 3.5 to 10), reacted at 37 ℃ for 3 hours, added to a 96-well plate, and then the fluorescence emission spectrum of the compound (I) was measured.
The fluorescence absorption spectrum is shown in FIG. 3. The experimental result shows that when the pH value is lower, the fluorescence absorption peak value of the compound (I) at 380nm is higher; the fluorescence absorption peak of compound (I) at 450nm gradually increased as the pH was gradually neutral and basic. Indicating that the probe is sensitive to pH.
Example 3: fluorescence emission spectra of compound (I) (5 μ M) at different pH. The excitation wavelength was 380 nm.
An amount of the compound (I) prepared in example 1 was accurately weighed, prepared into a probe stock solution with a concentration of 0.1mM using dimethyl sulfoxide, and 2. mu.L of the solution was pipetted into 0.398mL of PBS buffers with different pH values (final pH value in water is 3.5 to 10), reacted at 37 ℃ for 3 hours, added to a 96-well plate, and then the fluorescence emission spectrum of the compound (I) was measured.
The fluorescence spectrum is shown in FIG. 4. The experimental result shows that when the compound is excited at the wavelength of 380nm and the pH value is lower, the fluorescence intensity of the compound (I) at 470nm is stronger; when the pH is neutral and basic, the fluorescence intensity of compound (I) at 560nm gradually increases, while the fluorescence intensity at 470nm gradually decreases. The probe is shown to exhibit a ratio change for pH. And the ratio of fluorescence intensity at 560nm/470nm of the compound (I) prepared in example 1 of the present application is plotted as FIG. 5. The pKa value of compound (I) was calculated to be 5.2 and was suitable as a lysosomal pH fluorescent probe.
And further dividing the fluorescence intensity value at 560nm by the fluorescence intensity value at 470nm to obtain a value on the ordinate and pH concentration on the abscissa, and plotting a linear relationship, as shown in FIG. 6. It can be found that the pH concentration is in the range of 4.5-7, the pH concentration and the pH value have a good linear relation, namely a linear equation: y-2.31 x +7.15 (R)20.9946) demonstrated that the probe can be quantitatively detected in acidic solutions approximating lysosomal pH by fluorescence ratio detection mode.
Example 4: study of the specificity of Compound (I) (5. mu.M) for pH.
An amount of the compound (I) prepared in example 1 was accurately weighed, prepared into a probe stock solution with a concentration of 0.1mM using dimethyl sulfoxide, and 2. mu.L of the solution was pipetted into 0.398mL of the solution, to which 18 chemicals possibly existing in the living system were added, respectively. The fluorescence profiles of the selectivity results of compound (I) in DMSO/PBS buffer (pH 4 and 7.4, v/v 1/199) are shown in fig. 7, which shows the ratio of fluorescence I after the addition of 18 chemicals possibly present in the living system, respectively560nm/I470nmThe changes were all minor, indicating that compound (I) (5 μ M) has good specificity for pH and is not interfered by other chemicals.
Example 5: repeated studies (excitation wavelength 380nm) of compound (I) (5. mu.M) at different pH values were carried out.
An amount of the compound (I) prepared in example 1 was accurately weighed, a probe stock solution having a concentration of 0.1mM was prepared using dimethyl sulfoxide, 2. mu.L of the solution was aspirated by a pipette and added to 0.398mL of a PBS buffer solution having a pH of 4, and after reaction at 37 ℃ for 1 hour, the solution was added to a 96-well plate, and then the fluorescence emission value of the compound (I) at an excitation wavelength of 380nm was measured. And the solvent is adjusted at pH 4 and 7.4, and the pH repeatability of the compound (I) is repeatedly tested.
From FIG. 8, it is found that the fluorescence intensity can be changed continuously, which proves that the probe has certain repeatability in detecting pH.
Example 4 cellular imaging of Compound (I) with a commercial Lyso-Tracker Red.
A certain amount of probe (I) was accurately weighed, prepared into a 10mM stock solution with dimethyl sulfoxide, and 2. mu.L of the stock solution was pipetted into 1.998mL of DMEM medium. 1mL of the culture medium containing compound (I) was added to Hela cells, incubated at 37 ℃ for 0.5h, washed twice with DMEM medium, incubated with commercial Lyso-Tracker Red at 37 ℃ for 20min, washed twice with PBS, and finally subjected to fluorescence imaging using an Olympus Fluoview FV 1200 confocal microscope.
FIG. 9 is a diagram of the effect of confocal fluorescence imaging of cells: (a) the compound (I) has an excitation wavelength of 405nm and an acceptance wavelength range of 500-600 nm, (b) a Lyso-Tracker Red, an excitation wavelength of 543nm and an acceptance wavelength range of 590-640 nm; (c) a mixing channel; (d) reference bar, 20 μm.
The experimental results show that the compound (I) can detect the pH of lysosomes in cells (Pearson's correlation coefficient of 0.95).
Example 5 cytographic imaging of Compound (I) at different pH environments.
A certain amount of probe (I) was accurately weighed, prepared into a 10mM stock solution with dimethyl sulfoxide, and 2. mu.L of the solution was pipetted into 1.998mL of DMEM medium with different pH. 1mL of the culture containing compound (I) was added to Hela cells, and 1. mu.L of a stock solution of nigericin prepared in dimethyl sulfoxide was added to make the final concentration of nigericin 5. mu.M, incubated at 37 ℃ for 0.5h, washed twice with DMEM medium, and finally fluorescence-imaged with Olympus Fluoview FV 1200 confocal microscope.
FIG. 10 is a diagram showing the effect of confocal fluorescence imaging of cells. Green channel: the excitation wavelength of the compound (I) is 405nm, and the receiving wavelength range is 450-500 nm; yellow channel: the excitation wavelength of the compound (I) is 405nm, and the receiving wavelength range is 500-600 nm; reference bar, 20 μm.
The experimental results show that when the pH value of the compound (I) is increased, the fluorescence intensity of a yellow channel is gradually increased, and the fluorescence in a green channel is reduced, so that the change of the intracellular pH value can be further detected.
The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. A lysosomal targeting pH fluorescent probe having the structural formula shown in (I):
Figure FDA0003521698520000011
2. the method of making the lysosomal targeting pH fluorescent probe of claim 1, comprising:
taking a compound (II) and a compound (III) as reactants, adding glacial acetic acid and sodium triacetoxyborohydride, reacting in a solvent, and separating and purifying reaction liquid after the reaction is finished to obtain the lysosome targeted pH fluorescent probe with the structural formula shown as (I):
Figure FDA0003521698520000012
3. the preparation method according to claim 3, wherein the amount ratio of the compound (II) to the compound (III) to the glacial acetic acid to the sodium triacetoxyborohydride is 1:1 to 1.2:10 to 12:4 to 5; the reaction conditions are as follows: stirring and reacting for 10-14 h at 25-30 ℃.
4. Use of the lysosomal targeted pH fluorescent probe of claim 1 in the manufacture of a pH fluorescent test product.
5. The use of claim 4, wherein the pH fluorescence detection product is a test strip or a kit.
6. A lysosomal targeted pH detection kit comprising the lysosomal targeted pH fluorescent probe of claim 1.
7. Use of the lysosomal targeted pH fluorescent probe of claim 1 for cell imaging or in the preparation of a cell imaging agent.
8. Use according to claim 7, wherein the cell is a tumor cell.
9. A method for quantitative pH determination for non-diagnostic purposes, comprising:
adding a compound with a structural formula shown in (I) into a solution to be detected, and measuring fluorescence intensity values at 560nm and 470nm respectively by taking 380nm as an excitation wavelength after the reaction is finished; then dividing the fluorescence intensity value at 560nm by the fluorescence intensity value at 470nm, and substituting the obtained result into a linear standard curve to calculate the pH value of the solution to be measured;
Figure FDA0003521698520000021
10. the method for quantitative detection of pH according to claim 9, characterized in that the final concentration of the lysosome targeting pH fluorescent probe in the solution to be tested is 5 μ M, and the pH value of the solution to be tested is 4.5-7.
CN202210179172.4A 2022-02-25 2022-02-25 Lysosome targeted pH fluorescent probe and preparation and application thereof Withdrawn CN114478384A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116283771A (en) * 2023-01-12 2023-06-23 常熟理工学院 Fluorescent compound, application thereof in measuring pH value of solution and pH detection device

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116283771A (en) * 2023-01-12 2023-06-23 常熟理工学院 Fluorescent compound, application thereof in measuring pH value of solution and pH detection device
CN116283771B (en) * 2023-01-12 2024-05-28 常熟理工学院 Fluorescent compound, application thereof in measuring pH value of solution and pH detection device

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Application publication date: 20220513