Reaction type two-photon fluorescent probe for rapidly detecting in-vivo and in-vitro monoamine oxidase as well as preparation method and application of reaction type two-photon fluorescent probe

Abstract

The invention discloses a two-photon fluorescent probe capable of quickly responding to monoamine oxidase and a synthesis method of the two-photon fluorescent probe, and aims to detect the activity of monoamine oxidase in an animal body. According to the method, 2 '-hydroxyacetophenone and p-dimethylaminobenzaldehyde are used as raw materials to synthesize a compound HTC, then the compound HTC reacts with N-Boc-3-aminopropyl bromide, and finally the fluorescent probe HTBA is obtained in a system of trifluoroacetic acid and dichloromethane. According to the invention, the defect of long detection time of a traditional probe is effectively overcome, and after the synthesized fluorescent probe responds to monoamine oxidase, an exposed parent shows a two-photon characteristic, so that not only can interference of background fluorescence signals be weakened, but also high-resolution fluorescence microscopic imaging can be realized, and the penetration depth of tissues is enhanced. Besides, the probe provided by the invention not only can detect the activity of endogenous monoamine oxidase in cells, but also can rapidly detect monoamine oxidase in zebrafish bodies, and the application of the technology provides a more efficient and accurate tool for biomedical research.

Description

Technical Field

[0001] The present invention relates to a reactive two-photon fluorescence probe for rapid response to monoamine oxidase, its synthesis method and application in detecting monoamine oxidase in animals, belonging to the technical field of fluorescence probes. Background Art

[0002] Monoamine oxidase (MAO) is an enzyme dependent on flavin adenine dinucleotide (FAD), mainly located in the outer mitochondrial membrane, and is responsible for participating in amine metabolism in the human body. It can oxidize monoamine substances produced during life metabolism and externally ingested into corresponding aldehydes, while generating hydrogen peroxide (H2O2). In the human body, MAO plays a key role in maintaining the homeostasis of neurotransmitters. Currently, there are two known subtypes of human MAO: MAO-A and MAO-B. These two enzymes are encoded by different genes. Although their amino acid sequence homology is as high as 70%, there are significant differences in substrate specificity, tissue distribution and selective inhibitors. Abnormal expression of MAO-A and MAO-B is closely related to various mental diseases and neurodegenerative diseases. For example, studies have shown that high expression of MAO-A is significantly associated with schizophrenia and depression, while MAO-B shows a significant increase in expression in the brains of patients with Parkinson's disease (PD) and Alzheimer's disease (AD). Specifically, MAO-A has a strong affinity for serotonin and norepinephrine, while MAO-B mainly metabolizes benzylamine and phenethylamine. As a widely used detection method, fluorescence probes have attracted attention due to their high sensitivity, low detection limit and good non-destructive penetration ability. Based on the structure of monoamine oxidase, researchers have developed a variety of fluorescence probes for detecting MAO. However, the reported MAO reactive fluorescence probes generally have a long response time, usually several hours, even up to dozens of hours, and this long response time limits the real-time monitoring of endogenous MAO. Therefore, there is an urgent need to develop new rapid-response MAO fluorescence probes.

[0003] Two-photon microscopy (TPM) has become a highly promising in situ, real-time biological imaging system due to its robustness, high sensitivity, and non-invasive nature. Using a two-photon light source with a wavelength range of 700-1450 nm, TPM enables deeper light penetration and excellent spatial selectivity. The signal intensity of TP fluorescent probes exhibits a quadratic dependence on the intensity of the incident laser beam. This allows fluorescence emission to be precisely controlled within the focal plane of the incident laser beam, avoiding interference from out-of-focus light. This results in the generation of high-quality optical sectioning images with submicron lateral resolution and micron-level temporal resolution. Furthermore, small-molecule enzyme probes can be structurally designed based on their targets and are easily synthesized, enabling the localization of enzyme activity in its native environment with high sensitivity and specificity. Therefore, the development of fluorescent probes suitable for TPM to enable three-dimensional resolution of enzyme activity in deep scattering tissues, particularly for in vivo enzyme activity studies, is of great scientific significance. Such technological advancements will facilitate the acquisition of real-time data on in vivo enzyme activity with minimal interference, driving research in related fields. Summary of the Invention

[0004] The purpose of the present invention is to provide a reactive two-photon fluorescent probe that rapidly responds to monoamine oxidase, a synthesis method thereof, and an application in detecting monoamine oxidase in animals, thereby providing an important rapid detection technology for monitoring the activity of MAOs in cells and detecting MAOs in zebrafish.

[0005] The implementation process of the present invention is as follows: A reactive two-photon fluorescent probe for rapid detection of monoamine oxidase in vivo and in vitro, the structural formula of which is as follows: .

[0006] The above compound is abbreviated as HTBA.

[0007] The method for synthesizing the reactive two-photon fluorescent probe for rapid detection of monoamine oxidase in vivo and in vitro comprises the following steps: (1)

[0008] In the presence of sodium hydroxide, 2'-hydroxyacetophenone reacts with p-dimethylaminobenzaldehyde to obtain compound HTC; (2)

[0009] In the presence of potassium carbonate, compound HTC reacts with N-Boc-3-aminopropyl bromide to obtain compound A; (3)

[0010] In trifluoroacetic acid and CH2Cl2 system, compound A is converted into compound HTBA.

[0011] In the above step (1), the molar ratio of 2'-hydroxyacetophenone to p-dimethylaminobenzaldehyde is 1:(0.8 - 1.2).

[0012] In the above step (2), the molar ratio of compound HTC, N-Boc-3-aminopropyl bromide to potassium carbonate is 1:(0.8 - 1.2):(1.5 - 3).

[0013] In the above step (3), the volume ratio of trifluoroacetic acid to CH2Cl2 is 1:(1.5 - 3).

[0014] The compound of the present invention is used in the preparation of a preparation for detecting the activity of monoamine oxidase. Specifically, the two-photon fluorescence probe for rapidly responding to monoamine oxidase provided by the present invention can be used to detect the activity of monoamine oxidase in an in vitro buffer solution, detect the activity of monoamine oxidase in living cells, and detect the activity of monoamine oxidase in zebrafish.

[0015] Since the present invention adopts a two-photon fluorescence dye probe, the probe can specifically recognize MAOs in vivo and in vitro, and has rapid detection, high selectivity and good biocompatibility. The probe has low toxicity to normal cells NIH-3T3 (mouse embryonic fibroblasts) with low expression of MAOs. The probe is excited by 808 nm of a two-photon confocal microscope for two-photon imaging of zebrafish, and shows excellent two-photon imaging performance in in vivo experiments.

[0016] Compared with the prior art, the present invention has the following remarkable technical advantages: the two-photon fluorescence probe of the present invention shows high selectivity for monoamine oxidase. After the probe responds to monoamine oxidase, its naked parent body exhibits two-photon characteristics, effectively resisting the interference of background fluorescence signals, thereby improving the anti-interference ability of the probe; the two-photon fluorescence probe of the present invention has good targeting to intracellular organelles, and the probe can rapidly detect endogenous monoamine oxidase in cells and perform fluorescence imaging; in addition, the two-photon fluorescence probe of the present invention can also rapidly detect and perform fluorescence imaging on monoamine oxidase in zebrafish. These technical effects provide a more efficient and accurate tool for related biomedical research. Brief Description of the Drawings

[0017] Figure 1 It is the change curve of the fluorescence spectrum of the two-photon fluorescence probe responding to monoamine oxidase A with time increasing. The abscissa is wavelength (nm), and the ordinate is fluorescence emission intensity; Figure 2 It is the change curve of the fluorescence spectrum of the two-photon fluorescence probe responding to monoamine oxidase B with time increasing. The abscissa is wavelength (nm), and the ordinate is fluorescence emission intensity; Figure 3It is the change curve of the fluorescence spectrum of the two-photon fluorescence probe with the increase in the concentration of monoamine oxidase A. The abscissa is the wavelength (nm), and the ordinate is the fluorescence emission intensity; Figure 4 It is the change curve of the fluorescence spectrum of the two-photon fluorescence probe with the increase in the concentration of monoamine oxidase B. The abscissa is the wavelength (nm), and the ordinate is the fluorescence emission intensity; Figure 5 It is the confocal fluorescence imaging detection diagram of the two-photon fluorescence probe for living cells; Figure 6 It is the confocal fluorescence imaging diagram of the co-localization of the two-photon fluorescence probe for cells; Figure 7 It is the two-photon fluorescence imaging of the two-photon fluorescence probe for zebrafish. Specific embodiments

[0018] The present invention will be further described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present application. The methods are all conventional methods unless otherwise specified, and the raw materials can be obtained from public commercial channels unless otherwise stated.

[0019] The synthetic route of the present invention is as follows:

[0020] A synthetic method for the two-photon fluorescence probe HTBA, comprising the following steps: (1) After fully dissolving sodium hydroxide in deionized water, add absolute ethanol. Under room temperature and nitrogen protection, slowly drip a mixed solution of 2'-hydroxyacetophenone, p-dimethylaminobenzaldehyde and absolute ethanol into a round-bottom flask using a constant-pressure dropping funnel. Continue stirring at room temperature for 48 h. After tracking the reaction by TLC until the reaction is complete, slowly drip 2 mL of 30% H2O2 into the mixed solution using a constant-pressure dropping funnel, and continue stirring for 12 h. Track the reaction by TLC. After the reaction is completed, adjust the pH to neutral with 2 M hydrochloric acid, pour it into ice water and continue stirring. A solid will precipitate. Filter, take the filter residue for recrystallization to obtain an orange solid, denoted as compound HTC; (2) After completely dissolving compound HTC and N-Boc-3-aminopropyl bromide in anhydrous DMF, quickly add potassium carbonate. The mixture is stirred in an oil bath at 45 °C - 55 °C for 5 h, and the reaction is tracked by TLC. After the reaction is completed, cool to room temperature. Dilute the reaction solution with ethyl acetate, wash it three times with saturated brine, dry the organic phase with anhydrous sodium sulfate, remove the solvent under reduced pressure, mix the sample, purify it by column chromatography, and dry it to obtain a yellow solid, denoted as compound A.

[0021] (3) Dissolve compound A in dry CH2Cl2. Under ice bath conditions and nitrogen protection, slowly drip the mixed solution of trifluoroacetic acid and dry CH2Cl2 into the reaction system, stir for 1 h, and monitor the reaction by TLC until compound a reacts completely. After the reaction is completed, remove the solvent under reduced pressure, mix the sample, purify by column chromatography, and dry to obtain a yellow solid, denoted as HTBA.

[0022] Synthesis of the two-photon fluorescence probe HTBA for rapid response to monoamine oxidase in Example 1.

[0023] (1) Synthesis of compound HTC: Add 2.02 g of NaOH and 5 mL of deionized water to a 100 mL round-bottom flask. After complete dissolution, add 30 mL of absolute ethanol. Under room temperature and nitrogen protection, slowly drip the mixed solution of 2'-hydroxyacetophenone (0.713 mL, 6.02 mmoL), p-dimethylaminobenzaldehyde (895.1 mg, 6.02 mmol), and 20 mL of absolute ethanol into the round-bottom flask using a constant pressure dropping funnel. Continue to stir at room temperature for 48 h. After monitoring the reaction by TLC until it is complete, slowly drip 2 mL of 30% H2O2 into the mixed solution using a constant pressure dropping funnel, and continue to stir for 12 h, monitoring the reaction by TLC. After the reaction is completed, adjust the pH to neutral with 2 M hydrochloric acid, pour it into 200 mL of H2O and continue to stir. A solid will precipitate. Filter and take the filter residue for recrystallization to obtain compound HTC as an orange solid (638.6 mg, yield 37.7%). 1H NMR (400 MHz, DMSO- d 6) δ 8.15 - 8.10 (m, 2H), 8.08 (dd, J = 7.9, 1.6 Hz, 1H), 7.75 (ddd, J =8.2, 6.5, 1.6 Hz, 1H), 7.71 (dd, J = 8.5, 1.6 Hz, 1H), 7.43 (ddd, J = 8.1,6.5, 1.6 Hz, 1H), 6.86 - 6.81 (m, 2H), 3.01 (s, 6H). HRMS (ESI) m / z calcd forC 17 H 16 NO3 + (M+H) + : 282.1125, Found: 282.1109.

[0024] (2) Synthesis of compound a: Compound HTC (281 mg, 1 mmol) and N-Boc-3-aminopropyl bromide (238 mg, 1 mmol) were added into a 50 mL round-bottom flask. 10 mL of anhydrous DMF was added to dissolve them completely. Potassium carbonate (636 mg, 2 mmol) was added rapidly. The mixture was stirred for 5 h under nitrogen protection at 45 °C - 55 °C, and the reaction was monitored by TLC. After the reaction was completed, it was cooled to room temperature. The reaction solution was diluted with 80 mL of ethyl acetate and washed 3 times with saturated brine. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The sample was triturated and purified by column chromatography (PE:EtOAc = 10:1), and then dried to obtain compound a as a yellow solid (284.8 mg, yield 65%). 1 H NMR (400 MHz, DMSO- d 6) δ 8.08 - 8.00 (m, 3H), 7.77 (ddd, J = 8.6,6.9, 1.6 Hz, 1H), 7.71 (dd, J = 8.5, 1.2 Hz, 1H), 7.45 (ddd, J = 8.1, 6.9,1.2 Hz, 1H), 6.87 - 6.80 (m, 3H), 3.96 (t, J = 6.5 Hz, 2H), 3.07 (q, J = 6.6Hz, 2H), 3.03 (s, 6H), 1.79 (p, J = 6.7 Hz, 2H), 1.36 (s, 9H). HRMS (ESI) m / zcalcd for C 25 H 30 N2NaO5 + (M+Na) + : 461.2047, Found: 461.2028。

[0025] (3) Synthesis of HTBA: Compound a (87.6 mg, 0.2 mmol) and 20 mL of dry CH2Cl2 were added into a 50 mL two-neck round-bottom flask. Under ice bath conditions and nitrogen protection, a mixed solution of 1.5 mL of trifluoroacetic acid and 3 mL of dry CH2Cl2 was slowly added dropwise into the reaction system. The mixture was stirred for 1 h, and the reaction was monitored by TLC until compound a was completely reacted. After the reaction was completed, the solvent was removed under reduced pressure. The sample was triturated and purified by column chromatography (CH2Cl2:CH3OH = 20:1), and then dried to obtain compound HTBA as a yellow solid (46 mg, yield 68%).1 H NMR (400 MHz, DMSO- d 6) δ 8.08 (dd, J J = 8.0, 1.7 Hz,1H), 8.06 - 8.01 (m, 2H), 7.81 (ddd, J J = 8.6, 6.9, 1.7 Hz, 1H), 7.75 (dd, J J =8.5, 1.2 Hz, 1H), 7.48 (ddd, J J = 8.0, 6.9, 1.2 Hz, 1H), 6.87 (dq, J J = 10.1,2.3 Hz, 2H), 4.01 (t, J J = 5.8 Hz, 2H), 3.08 - 3.00 (m, 8H), 2.04 (p, J J = 6.4Hz, 2H). HRMS (ESI) m / z calcd for C 20 H 22 N2O3 + (M+Na) + : 339.1664, Found:339.1684。

[0026] Example 2 Detection of Monoamine Oxidase by HTBA Probe in Vitro Take a certain volume of MAOs with a concentration of 2.5 mg / 0.5 mL and dilute them with PBS buffer solution (pH = 7.4) to a test solution of 1 μg / μL respectively. Dilute the probe stock solution (10 mM) with DMSO to a test solution of 10 μM. The test system is PBS buffer solution (pH = 7.4) containing 10% DMSO. Set the excitation wavelength of the fluorometer to 420 nm, and the emission wavelength receiving range to 440 - 800 nm. The results are as Figure 1 and Figure 2 shown. The HTBA probe has a good response to both monoamine oxidase A and monoamine oxidase B, and the response time is fast. Especially for MAO - B, the response is extremely fast, and the response time is only 2 min.

[0027] Example 3 Response of Probe HTBA and Different Concentrations of MAOs The preparation method of the spectral test solution is the same as that in Example 2. The response results of probe HTBA and different concentrations of MAO - A and MAO - B are as Figure 3 and Figure 4As shown, the fluorescence intensity increases with the increase in the concentration of MAOs, indicating that the HTBA probe has a certain concentration dependence on both MAO-A and MAO-B.

[0028] Example 4 Fluorescence Imaging Detection of Monoamine Oxidase in Living Cells by Probe HTBA HepG2 cells were seeded in a confocal microscope dish and cultured in an incubator for 12 hours. Subsequently, the probe HTBA (at a concentration of 3 μM) was co-incubated with HepG2 cells for 2 minutes. Then, the cells were washed three times with sterile PBS to remove the unabsorbed probe. Imaging of the cells was performed using a TCS SP8 DIVE confocal microscope, selecting 488 nm as the excitation wavelength and collecting fluorescence signals in the range of 498 - 598 nm. As Figure 5 shown, untreated cells themselves had no obvious fluorescence signal. However, after incubation with the probe HTBA (3 μM), obvious green fluorescence signals appeared in the green fluorescence channel. Notably, when we first treated the cells with the MAOs inhibitor isoniazid, the green fluorescence signal almost completely disappeared. This indicates that the probe HTBA can effectively detect endogenous monoamine oxidase (MAOs) and has good cell permeability.

[0029] Example 5 Targeting Ability of Probe HTBA to Organelles in Cells HepG2 cells were seeded in a confocal microscope dish and cultured in an incubator for 12 hours. Subsequently, the probe HTBA at a concentration of 3 μM was co-incubated with HepG2 cells for 2 minutes. Then, two commercial organelle fluorescent dyes (Mito-Tracker Red CMXRos and Lyso-Tracker Red) were added respectively. After 30 minutes of treatment, the cells were washed three times with sterile PBS to remove the unabsorbed probe. Then, imaging of the cells was performed using a TCS SP8 DIVE confocal microscope. As Figure 6 shown, the green fluorescence of the probe HTBA overlapped very highly with the red fluorescence of the lysosome dye, and the Pearson's correlation coefficient was P = 0.90. This indicates that after the probe HTBA reacts rapidly with MAO-B in the cells, most of the HTC catalyzed and hydrolyzed by MAO-B enters the lysosome. This result further supports the effective detection of MAO-B by the probe HTBA in cells and its good targeting to organelles.

[0030] Example 6 Detection of Monoamine Oxidase in Zebrafish by Probe HTBA in Two-Photon Imaging Normal five-day-old zebrafish larvae were divided into three groups. The first group was not treated with anything. The second group was incubated with the probe HTBA for 10 minutes and then washed three times with sterile PBS to remove the excess unabsorbed probe. The third group was first treated with the MAOs inhibitor isoniazid for 1 hour, then the probe HTBA was added for a 10-minute incubation, and then the washing process was repeated. Next, two-photon imaging of the zebrafish was performed using a TCS SP8 DIVE confocal microscope, 808 nm was selected as the excitation wavelength, and the fluorescence signal in the range of 500 - 600 nm was collected. As Figure 7 shown, there was no obvious fluorescence signal in the untreated zebrafish itself. However, after incubation with the probe HTBA (10 μM), obvious green fluorescence signals appeared in the green fluorescence channel. Notably, when the zebrafish were first treated with the MAOs inhibitor isoniazid, the green fluorescence signals almost completely disappeared. This indicates that the probe HTBA can effectively detect endogenous monoamine oxidases (MAOs) in zebrafish.

Claims

1. A reactive two-photon fluorescent probe for rapid detection of monoamine oxidase in vivo and in vitro, characterized in that: The structural formula is as follows: 。 2. The method for synthesizing a reactive two-photon fluorescent probe for rapid detection of monoamine oxidase in vivo and in vitro as claimed in claim 1, characterized in that The following steps are involved: （1） ， In the presence of sodium hydroxide, 2'-hydroxyacetophenone reacts with p-dimethylaminobenzaldehyde to obtain compound HTC; （2） ， In the presence of potassium carbonate, compound HTC reacts with N-Boc-3-aminopropyl bromide to obtain compound A; （3） ， In trifluoroacetic acid and CH2Cl2 system, compound A is converted into compound HTBA.

3. The synthesis method according to claim 2, wherein: In the above step (1), the molar ratio of 2'-hydroxyacetophenone to p-dimethylaminobenzaldehyde is 1:(0.8-1.2).

4. The synthesis method according to claim 2, wherein: In the above step (2), the molar ratio of the compound HTC, N-Boc-3-aminopropyl bromide and potassium carbonate is 1: (0.8-1.2): (1.5-3).

5. The synthesis method according to claim 2, wherein: In the above step (3), the volume ratio of trifluoroacetic acid to CH2Cl2 is 1: (1.5~3).

6. Use of the compound according to claim 1 in preparing a preparation for detecting monoamine oxidase activity in a buffer solution in vitro.

7. Use of the compound according to claim 1 in preparing a preparation for detecting monoamine oxidase activity in living cells.

8. Use of the compound according to claim 1 in preparing a preparation for detecting monoamine oxidase activity in zebrafish.

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