A β-galactosidase-activated near-infrared fluorescent probe, its preparation method and application

Abstract

The application discloses a kind of β-galactoside enzyme activated near-infrared fluorescent probes, the structural formula of the fluorescent probe Gal-QCS is as follows: the response site of β-Gal is introduced on the good nature semi-florin fluorophore of the fluorescent probe, the fluorescence of probe itself is quenched due to intramolecular charge transfer effect, after reacting with β-Gal again, glycosidic bond is hydrolyzed, so that its fluorescence recovers. By the change of absorption wavelength and fluorescence wavelength before and after response, the quantitative detection of β-Gal can be realized;The emission wavelength of the fluorescent probe is longer, can quickly, specifically identify β-Gal, has higher signal-to-background ratio, reduces the error caused by background fluorescence.

Description

Technical Field

[0001] This invention belongs to the field of near-infrared small molecule fluorescent probes, specifically relating to a β-galactosidase activated near-infrared fluorescent probe, its preparation method, and its application. Background Technology

[0002] β-galactosidase (β-Gal) is a glycoside hydrolase with diverse physiological and pathological functions; its deficiency or overexpression can lead to a variety of rare diseases. The main function of β-Gal is to catalyze the hydrolysis of glycosides and the removal of galactose residues from gangliosides, sphingolipids, and glycoproteins. Numerous studies have demonstrated that β-Gal is an important biomarker for primary ovarian cancer and cellular senescence. Therefore, developing methods for the detection of β-Gal is essential.

[0003] Currently, colorimetric methods, electrochemical methods, single-photon emission computed tomography (SPECT), and positron emission tomography (PET) imaging can all be used to detect β-Gal. However, these methods have certain drawbacks, such as long experimental duration, complex operating conditions, and high experimental costs. Furthermore, these methods cannot non-invasively monitor β-Gal in real time at the cellular or in vivo level. In recent years, optical imaging has become an important tool in biological research due to its non-invasive nature, high sensitivity, rapid response, high spatiotemporal resolution, and real-time imaging capabilities. Activated molecular probes have been developed for the sensitive and specific detection of β-Gal. These probes are small in size, low in cost, easy to chemically modify, and have broad applicability, and can be used for cell staining and in vitro detection.

[0004] Several fluorescent probes for detecting β-Gal have been developed in the existing technology, but some limitations still need to be addressed. These include short emission wavelengths, relatively slow response rates, poor selectivity, and low signal-to-background ratios, which are crucial for applications in living cells or tissues. Summary of the Invention

[0005] To address the problems in the background art, the present invention aims to provide a β-galactosidase-activated near-infrared fluorescent probe, its preparation method, and its applications. This fluorescent probe introduces a β-Gal response site onto a suitable hemicyanine fluorophore. The probe itself undergoes fluorescence quenching due to intramolecular charge transfer. Upon reaction with β-Gal, the glycosidic bond is hydrolyzed, restoring its fluorescence. Quantitative detection of β-Gal can be achieved by observing the changes in absorption and fluorescence wavelengths before and after the response. This fluorescent probe has a relatively long emission wavelength, enabling rapid and specific identification of β-Gal, a high signal-to-background ratio, and reduced errors caused by background fluorescence.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a β-galactosidase-activated near-infrared fluorescent probe (Gal-QCS), the structural formula of which is as follows:

[0008]

[0009] The fluorescent probe, named Gal-QCS, features a longer wavelength, rapid response, high selectivity, and high signal-to-background ratio.

[0010] This invention also provides a method for preparing the β-galactosidase-activated near-infrared fluorescent probe, comprising the following steps:

[0011] (1) Compound 1 and Compound 2 were dissolved in acetonitrile, and an appropriate amount of sodium acetate was added. The mixture was heated and stirred at 80-85°C for 6-8 hours. After cooling, the mixture was extracted, and the solvent was evaporated by rotary chromatography to obtain Compound 3.

[0012] (2) Compound 3 was dissolved in methanol, and an appropriate amount of concentrated sulfuric acid was added dropwise under ice bath. The mixture was heated and stirred at 88-92°C for 6-8 hours. After cooling, the mixture was extracted, and the organic phase was dried by rotary evaporation and separated by column chromatography to obtain compound 4.

[0013] (3) Compound 4 and compound 5 were dissolved in acetonitrile, N,N-diisopropylethylamine was added, and the mixture was heated under reflux for 6-8 h. After the reaction was completed, the mixture was extracted, the solvent was evaporated, and column chromatography was performed to separate the compound 6.

[0014] (4) Dissolve compound 6 in methanol, add anhydrous potassium carbonate, stir at room temperature for 4-6 h, extract after the reaction, evaporate the solvent and perform column chromatography to obtain the fluorescent probe Gal-QCS.

[0015] The synthetic route is as follows:

[0016]

[0017] Furthermore, in step (1), the molar ratio of compound 1, compound 2, and sodium acetate is 1:(1-1.5):2, and the molar volume ratio of compound 1 to acetonitrile is 1:(8-10) mmol / ml.

[0018] Furthermore, in step (2), the volume ratio of methanol to concentrated sulfuric acid is 20:1.

[0019] Furthermore, in step (3), the molar ratio of compound 4, compound 5, and N,N-diisopropylethylamine is 1:(1-3):(1-3), and the molar volume ratio of compound 4 to acetonitrile is 1:(7-10) mmol / ml.

[0020] Furthermore, in step (4), the molar ratio of compound 6 to anhydrous potassium carbonate is 1:(4-6), and the molar volume ratio of compound 6 to methanol is 1:(4-6) mmol / ml.

[0021] Furthermore, the inventors characterized the fluorescent probe Gal-QCS using mass spectrometry, proton NMR, carbon NMR, ultraviolet light, and fluorescence spectrometry, respectively, proving that the probe was successfully synthesized.

[0022] The present invention also provides the application of the near-infrared fluorescent probe (Gal-QCS) activated by the β-galactosidase to detect β-galactosidase in live cells or tissues.

[0023] The response process of the near-infrared fluorescent probe Gal-QCS is as follows:

[0024]

[0025] Specifically, the detection of β-galactosidase using the aforementioned near-infrared fluorescent probe Gal-QCS is carried out as follows:

[0026] In this system, β-galactosidase is used as the fluorescent probe substrate. The incubation environment is in a 20% EtOH / H2O buffer system, with an incubation temperature between 25 and 45°C, a pH between 3 and 10°C, and an incubation time between 0 and 120 min. This ensures that the substrate can completely hydrolyze the glycosidic bond of the probe. The system can be detected simultaneously using a fluorescence detector and a UV spectrophotometer.

[0027] UV detection conditions: the maximum absorption wavelength of the probe is 630 nm, and the maximum absorption wavelength of the dye is 780 nm;

[0028] The fluorescence detection conditions are: excitation wavelength of 760 nm, no fluorescence before probe response, and maximum emission wavelength of response product of 830 nm.

[0029] The optimal incubation conditions selected through preliminary experiments are: a temperature of 37℃, an ambient pH of 6-8, and an incubation time of no more than 30 minutes.

[0030] The near-infrared fluorescent probe Gal-QCS described in this invention has a maximum absorption wavelength of 630 nm and almost no fluorescence before the addition of substrate β-Gal. When β-Gal is added, the absorption at 630 nm gradually weakens, the absorption at 780 nm gradually strengthens, and the emission at 830 nm also gradually strengthens.

[0031] The applications of the near-infrared fluorescent probe Gal-QCS described in this invention are as follows: This hemisensory fluorescent probe exhibits high fluorescence quantum yield, good photostability and chemical stability, and an emission wavelength between 650 nm and 900 nm, belonging to the near-infrared band. It can achieve fluorescence analysis and detection at the cellular and in vivo levels, reducing fluorescence interference caused by biological tissues themselves, improving the imaging signal-to-background ratio, enhancing tissue penetration depth, and increasing response sensitivity. The fluorescent probe of this invention can be used for the detection of ovarian cancer and the diagnosis of aging diseases at the cellular and in vivo levels. It is expected to bring new methods and breakthroughs to research fields such as the diagnosis of ovarian cancer and aging diseases, as well as the evaluation of drug treatment efficacy, and also provide a powerful tool for related biological fields.

[0032] The fluorescent probe provided by this invention allows Gal-QCS to be specifically recognized by β-galactosidase (β-Gal), causing the galactose residues to be hydrolyzed and released, releasing free hemicyanine dye QCS. The activity of β-galactosidase can be detected by the absorption at 780 nm and the fluorescence change at 830 nm of the probe and its reaction product. The probe of this invention has a rapid and sensitive response, and the emission wavelength of its reaction product is located in the near-infrared region, which can realize in vivo imaging analysis, reduce the interference caused by the self-fluorescence of biomolecules, enhance the tissue penetration depth, and has a high signal-to-noise ratio.

[0033] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0034] (1) The raw materials for synthesizing the fluorescent probe Gal-QCS described in this invention are inexpensive and readily available, with a high yield and easy to promote.

[0035] (2) The fluorescent probe Gal-QCS described in this invention has near-infrared fluorescence emission, which can reduce the tissue's own fluorescence background and enhance the tissue penetration depth.

[0036] (3) The fluorescent probe Gal-QCS described in this invention has a fast response speed and can saturate within 10 minutes.

[0037] (4) The fluorescent probe Gal-QCS described in this invention has good selectivity and is not affected by other biological factors. Attached Figure Description

[0038] Figure 1 This is the 1H NMR spectrum of compound 4.

[0039] Figure 2 This is the 1C NMR spectrum of compound 4.

[0040] Figure 3 This is the mass spectrum of compound 4.

[0041] Figure 4 This is the mass spectrometry of the fluorescent probe Gal-QCS.

[0042] Figure 5 This is the ultraviolet absorption spectrum of the fluorescent probe in solution in Example 2.

[0043] Figure 6 This is the fluorescence emission spectrum of the fluorescent probe in solution in Example 2.

[0044] Figure 7 This is a bar chart showing the selectivity test of the fluorescent probe for β-Gal in Example 3.

[0045] Figure 8 This is the UV absorption spectrum of the fluorescent probe in Example 4 responding to different concentrations of β-Gal.

[0046] Figure 9 This is the fluorescence emission spectrum of the fluorescent probe in Example 4 responding to different concentrations of β-Gal.

[0047] Figure 10 This is the fluorescence emission spectrum of the fluorescent probe in Example 5 responding to β-Gal. Detailed Implementation

[0048] The present invention will be further described below with reference to the specific embodiments described. It should be noted that the specific embodiments described below are only for explaining the present invention and are not intended to limit the scope of the present invention. The described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Unless otherwise specified, the test materials used in this invention are all conventional methods, and the raw materials are all obtainable from publicly available commercial sources unless otherwise specified.

[0050] The present invention will be further illustrated below with reference to the embodiments:

[0051] Example 1

[0052] The synthetic route of the fluorescent probe Gal-QCS is as follows, specifically including the following steps:

[0053] (1) Dissolve 1.5 mmol of compound 1 and 1 mmol of compound 2 in acetonitrile, add an appropriate amount of 2 mmol of sodium acetate, heat and stir at 85°C for 6-8 h, cool and extract with dichloromethane and ultrapure water, evaporate the solvent and separate by column chromatography to obtain compound 3.

[0054] (2) Dissolve 1 mmol of compound 3 in 10 ml of methanol, add 0.5 ml of concentrated sulfuric acid dropwise under ice bath, heat and stir at 90 °C for 6-8 h, cool and extract with dichloromethane and ultrapure water, and separate the organic phase by column chromatography to obtain compound 4.

[0055] (3) Dissolve 0.8 mmol of compound 4 and 1.5 mmol of compound 5 in 8 ml of acetonitrile, add 1.5 mmol of N,N-diisopropylethylamine, heat under reflux for 6-8 h, after the reaction is complete, extract with dichloromethane and ultrapure water, evaporate the solvent and separate by column chromatography to obtain compound 6;

[0056] (4) Dissolve 0.5 mmol of compound 6 in 3 ml of methanol, add 3 mmol of anhydrous potassium carbonate, stir at room temperature for 4-6 h, after the reaction is complete, extract with dichloromethane and ultrapure water, evaporate the solvent and perform column chromatography to obtain the fluorescent probe Gal-QCS.

[0057] Figure 1 It is compound 4. 1 HNMR spectra, from Figure 1 It can be seen that each hydrogen atom in compound 4 has a corresponding assigned peak.

[0058] Figure 2 It is compound 4. 1 CNMR spectra, from Figure 2 It can be seen that each carbon in compound 4 has a corresponding assigned peak.

[0059] Figure 3 This is the mass spectrum of compound 4, from Figure 3 It can be seen that the molecular weight of compound 4 is 612.

[0060] Figure 4 This is the mass spectrometry of the fluorescent probe Gal-QCS, from... Figure 4 It can be seen that the molecular weight of the fluorescent probe Gal-QCS is 880.

[0061] Example 2

[0062] The response of the fluorescent probe to β-Gal was detected using a UV-Vis absorption spectrophotometer and a fluorescence spectrometer.

[0063] The fluorophore probe Gal-QCS from Example 1 was prepared as a 1 mM dimethyl sulfoxide (DMSO) stock solution, and a 20% EtOH / H2O test system was prepared.

[0064] Five μl of the above DMSO stock solution was added to two separate 20% EtOH / H2O buffer systems and diluted to a final test concentration of 5 μM. One system served as a blank control, and the other was incubated with 1.6 U / ml β-Gal. After incubation at 37°C for 30 min, the maximum absorption wavelength was measured using a UV spectrophotometer. The maximum absorption wavelength was 780 nm. The UV absorption spectrum is shown below. Figure 5 The maximum emission wavelength was measured using a fluorescence spectrometer. The excitation wavelength was set to 760 nm, and the maximum emission wavelength was 830 nm. The fluorescence spectrum is shown below. Figure 6 This indicates that the glycosidic bond of the probe is hydrolyzed by β-Gal, releasing the fluorophore of hemicyanine.

[0065] Example 3

[0066] Selectivity test of fluorescent probe for β-Gal:

[0067] Five μl of the probe stock solution from Example 2 was added to 15 separate 20% EtOH / H2O buffer systems to make the final test system 5 μM. β-Gal and different interfering substances were added to each system, and the mixtures were incubated at 37°C for 30 min. The absorbance was measured using a UV spectrophotometer, and a bar chart was created based on the absorbance at 780 nm. The results are shown below. Figure 7 As shown in the figure. Where 0 is blank, 1 is ALP (0.05 U / ml), 2 is cys (100 μM), 3 is β-Gal (1.6 U / ml), 4 is GGT (0.05 U / ml), 5 is GSH (1 μM), 6 is H2O2 (100 μM), 7 is HClO (100 μM), 8 is Hcy (100 μM), 9 is LAP (0.05 U / ml), and 10 is Na+. + (500μM), 11 is ONOO - (10μM). According to Figure 7 It can be seen that the fluorescent probe Gal-QCS only responds to β-Gal. Only after the addition of β-Gal does the absorption at 780nm show a significant increase. However, the absorption at 780nm does not increase significantly after the addition of other interfering substances. This indicates that the fluorescent probe has high selectivity and specificity and is not affected by other interfering substances in the biological environment.

[0068] Example 4

[0069] Five μl of the probe stock solution from Example 2 was added to 15 separate 20% EtOH / H2O buffer systems to make the final test system 5 μM. Different gradient concentrations of β-Gal (0-1.6 U / ml) were added to each system, and the mixtures were incubated at 37°C for 30 min to ensure consistent incubation conditions for the same probe at different enzyme concentrations. The absorbance was measured using a UV spectrophotometer; the absorbance at 780 nm gradually increased, with a fold increase of approximately 36 times (results are shown in Figure 1). Figure 8 As shown in the figure). Fluorescence was measured using a fluorescence spectrometer; the fluorescence intensity at 830 nm gradually increased, with a fold increase of approximately 11 times (results are shown in the figure). Figure 9 (As shown).

[0070] Example 5

[0071] Reaction kinetics of fluorescent probes to β-Gal:

[0072] Five μl of the probe stock solution from Example 2 was added to two 20% EtOH / H2O buffer systems to make the final test system 5 μM. One system served as a blank control, while the other was incubated with β-Gal to achieve a final enzyme concentration of 1.6 U / ml. The mixture was incubated at 37°C for 1 hour, and fluorescence was measured using a fluorescence spectrometer. It was found that only the sample with added β-Gal showed a gradual increase in fluorescence intensity at 830 nm. Furthermore, the probe response reached saturation within 10 minutes, indicating that the fluorescent probe has a relatively fast response speed (results are shown in Figure 1). Figure 10 (As shown).

[0073] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. For those skilled in the art, improvements and modifications obtained without departing from the inventive concept should also be considered within the scope of protection of the present invention.

Claims

1. A β-galactoside-activated near-infrared fluorescent probe, characterized by, The structural formula of the fluorescent probe Gal-QCS is as follows:

2. The method for preparing the β-galactosidase-activated near-infrared fluorescent probe according to claim 1, characterized in that, The method comprises the following steps: (1) Compound 1 and compound 2 are dissolved in acetonitrile, an appropriate amount of sodium acetate is added, and stirring is carried out at 80-85 DEG C for 6-8 h; after cooling, extraction is carried out, and the solvent is spin-dried and subjected to column chromatography separation to obtain compound 3; (2) Compound 3 is dissolved in methanol, and an appropriate amount of concentrated sulfuric acid is added dropwise under ice bath; stirring is carried out at 88-92 DEG C for 6-8 h; after cooling, extraction is carried out, and the organic phase is spin-dried and subjected to column chromatography separation to obtain compound 4; (3) Compound 4 and compound 5 are dissolved in acetonitrile, and N,N-diisopropylethylamine is added; refluxing is carried out for 6-8 h; after the reaction is completed, extraction is carried out, and the solvent is spin-dried and subjected to column chromatography separation to obtain compound 6; (4) Compound 6 is dissolved in methanol, and anhydrous potassium carbonate is added; stirring is carried out at room temperature for 4-6 h; after the reaction is completed, extraction is carried out, and the solvent is spin-dried and subjected to column chromatography separation to obtain the fluorescent probe Gal-QCS, and the synthesis route is as follows:

3. The method for preparing the β-galactosidase-activated near-infrared fluorescent probe according to claim 2, characterized in that, In the step (1), the molar ratio of compound 1, compound 2 and sodium acetate is 1:(1-1.5):2, and the molar volume ratio of compound 1 and acetonitrile is 1:(8-10) mmol / ml.

4. The method for preparing the β-galactosidase-activated near-infrared fluorescent probe according to claim 2, characterized in that, In the step (2), the volume ratio of methanol and concentrated sulfuric acid is 20:

1.

5. The method for preparing the β-galactosidase-activated near-infrared fluorescent probe according to claim 2, characterized in that, In the step (3), the molar ratio of compound 4, compound 5 and N,N-diisopropylethylamine is 1:(1-3):(1-3), and the molar volume ratio of compound 4 and acetonitrile is 1:(7-10) mmol / ml.

6. The method for preparing the β-galactosidase-activated near-infrared fluorescent probe according to claim 2, characterized in that, In the step (4), the molar ratio of compound 6 and anhydrous potassium carbonate is 1:(4-6), and the molar volume ratio of compound 6 and methanol is 1:(4-6) mmol / ml.

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