Near-infrared fluorescent probe for detecting cysteine as well as preparation and application of near-infrared fluorescent probe

Abstract

The invention discloses a near-infrared fluorescent probe DCIC as well as a preparation method and biological imaging application thereof. According to the structure of the probe, (E)-2-(3-(3, 5-difluoro-4-hydroxystyryl)-5, 5-methylcyclohex-2-alkenyl) malononitrile is used as a near-infrared fluorophore, a dynamic response unit is constructed through acryloyl modification, and specific recognition and lasting signal output of Cys can be achieved. After the probe reacts with Cys, near-infrared fluorescence enhancement is generated at 650 nm, and the probe has the advantages of being short in response time, high in sensitivity and good in selectivity. Compared with the prior art, the rapid imaging of plant cells and living body Cys metabolism can be synchronously realized. The invention provides a high-sensitivity and high-temporal-spatial-resolution molecular tool for plant stress physiological research and disease-related Cys metabolism monitoring.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and particularly relates to a near-infrared fluorescent probe for detecting cysteine in plants, a preparation method thereof, and an application thereof. Background Art

[0002] Cysteine (Cys), as an essential sulfur-containing amino acid, plays a key role in plant physiological processes, including redox homeostasis regulation, glutathione synthesis, metal ion chelation, and stress response. Although traditional detection methods such as high-performance liquid chromatography (HPLC), electrochemical analysis, and mass spectrometry have high sensitivity, they have inherent defects such as complex sample pretreatment, destruction of the integrity of plant tissues, and inability to achieve in-situ real-time monitoring, which severely restricts the study of the dynamic metabolic process of Cys in living plants.

[0003] In recent years, the fluorescent probe technology based on molecular recognition has received extensive attention due to its high spatio-temporal resolution and non-destructive detection characteristics. Most of the reported Cys fluorescent probes use excitation / emission wavelengths in the visible light region (400 - 650 nm). However, plant tissues show strong autofluorescence interference and light scattering effects in this wavelength band, resulting in a significant reduction in the signal-to-noise ratio. In addition, thiol substances such as glutathione (GSH) and homocysteine (Hcy) widely present in plant cells have similar reaction activities to Cys, and existing probes generally have insufficient selectivity and are difficult to achieve specific recognition. It is worth noting that the physical barrier effect of the plant cell wall and the vacuolar compartmentalization effect further reduce the transmembrane efficiency and targeting ability of the probe molecule, and the particularity of the plant cell microenvironment is rarely considered in the existing probe design.

[0004] Although near-infrared (NIR, 650 - 900 nm) fluorescent probes have made significant progress in animal in vivo imaging due to their advantages such as deep tissue penetration, low light damage, and weak background interference, their application in plant systems still faces the following challenges: (1) The absorption effect of complex pigment components (such as chlorophyll and carotenoids) in plants on near-infrared light has not been effectively avoided; (2) The probe response mechanism mostly depends on the thiol-mediated nucleophilic cyclization reaction, and the Cys concentration (in the μM level) in plant cells is significantly lower than that in the animal system (in the mM level), and the detection limit of existing probes is difficult to meet the detection requirements for low-abundance Cys; (3) There is a lack of stability regulation strategies for plant cell pH fluctuations (such as the acidic environment of vacuoles) and changes in ionic strength, resulting in insufficient reliability of probe signals.

[0005] Therefore, developing a new type of fluorescent probe with near-infrared emission characteristics, high selectivity, low detection limit, and excellent plant cell compatibility has important scientific value and application potential for revealing the regulation mechanism of plant sulfur metabolism networks, evaluating heavy metal stress responses, and optimizing crop stress resistance traits. Summary of the Invention

[0006] The object of the present invention is to provide a near-infrared fluorescence probe for detecting Cys in plants and a preparation method thereof. The fluorescence probe prepared by this method can be used for rapidly and highly selectively identifying Cys in plants and for bioimaging.

[0007] The object of the present invention can be achieved by the following technical solutions: A near-infrared fluorescence probe DCIC, whose structural formula is as follows: 。

[0008] The synthesis route of the near-infrared fluorescence probe DCIC described in the present invention is as Figure 2 shown.

[0009] The preparation method of the near-infrared fluorescence probe DCIC described in the present invention includes: Dissolve the compound (E)-2-(3-(3,5-difluoro-4-hydroxystyryl)-5,5-dimethylcyclohex-2-enylidene) malononitrile (DCI-OH), acryloyl chloride and triethylamine in dichloromethane, react under stirring. After the reaction is completed, remove dichloromethane, and obtain a yellow solid through purification, namely the near-infrared fluorescence probe DCIC.

[0010] In some embodiments, the molar ratio of the compound (E)-2-(3-(3,5-difluoro-4-hydroxystyryl)-5,5-dimethylcyclohex-2-enylidene) malononitrile to acryloyl chloride is 1:(1.2 - 1.5).

[0011] In some embodiments, the molar ratio of the compound (E)-2-(3-(3,5-difluoro-4-hydroxystyryl)-5,5-dimethylcyclohex-2-enylidene) malononitrile to triethylamine is 1:(1.2 - 1.5).

[0012] In some embodiments, the reaction temperature is 20 - 35 °C.

[0013] In some embodiments, the reaction time is 8 - 16 h.

[0014] In some embodiments, dichloromethane is removed by evaporation, preferably removed by a rotary evaporator.

[0015] In some embodiments, the purification is column chromatography separation, and the column chromatography conditions are: ethyl acetate and petroleum ether as eluents, with a volume ratio of 1:(3 - 15); silica gel is 200 - 300 mesh.

[0016] The near-infrared fluorescence probe described in the present invention can be used for rapidly and highly selectively identifying Cys, such as Figure 1As shown, the specific process is as follows: The fluorescence probe shows almost no fluorescence emission in a mixed solution of phosphate buffer and DMSO (volume ratio 6:4). After adding a solution containing Cys, since the acrylate functional group serves as a common specific detection site for Cys, Cys reacts with acrylate through Michael addition. Then, the amino group attacks the ester bond to form a seven-membered cyclic lactam byproduct through intramolecular cyclization and release the fluorophore, resulting in a significant enhancement of the probe's fluorescence at 650 nm.

[0017] The present invention also provides the application of the above-mentioned near-infrared fluorescence probe DCIC in detecting Cys in plants.

[0018] The present invention also provides the application of the above-mentioned near-infrared fluorescence probe DCIC in preparing a reagent for imaging detection of cysteine in plant cells or plant tissues.

[0019] The beneficial effects of the present invention are as follows: (1) The fluorescence probe of the present application has a novel structure, and the synthesis method is simple and easy to separate and purify.

[0020] (2) The probe of the present invention can exclude the interference of common reactive small molecules, has a highly specific selectivity for Cys, and has strong anti-interference ability.

[0021] (3) The fluorescence probe of the present invention has a fast response time (100 s) and high sensitivity (38.4 nM) for recognizing Cys, and is suitable for rapid real-time monitoring of Cys in the environment and organisms.

[0022] (4) The fluorescence probe of the present invention monitors and images Cys in plants through fluorescence at 650 nm, and has the advantages of low background interference and little light damage to biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 Shows the reaction principle of the fluorescence probe DCIC of the present invention for detecting Cys; Figure 2 Shows the synthesis route of the fluorescence probe DCIC of the present invention; Figure 3 Is the nuclear magnetic resonance hydrogen spectrum of the fluorescence probe DCIC of the present invention (the solvent is CDCl3); Figure 4 13C NMR spectrum of the fluorescence probe DCIC of the present invention (the solvent is CDCl3); Figure 5 High resolution mass spectrum of the fluorescence probe DCIC of the present invention; Figure 6 UV and fluorescence selectivity diagrams of the fluorescence probe DCIC of the present invention for recognizing Cys; Figure 7 Kinetic curve diagram of the fluorescence probe DCIC of the present invention for recognizing Cys; Figure 8 UV and fluorescence titration diagrams of the fluorescence probe DCIC of the present invention for recognizing Cys; Figure 9 Lowest detection limit diagram of the fluorescence probe DCIC of the present invention for recognizing Cys; Figure 10 Fluorescence imaging diagram of the fluorescence probe DCIC of the present invention for recognizing Cys in onion epidermal cells; Figure 11 Fluorescence imaging diagram of the fluorescence probe DCIC of the present invention for recognizing Cys in the root tips of tobacco seedlings.

[0026] Detailed Description of the Invention The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0027] Example 1 Synthesis of the near-infrared fluorescence probe DCIC: In a 50 mL round-bottom flask containing 10 mL of dichloromethane, (E)-2-(3-(3,5-difluoro-4-hydroxystyryl)-5,5-methylcyclohex-2-enylidene) malononitrile (326.3 mg, 1 mmol) and triethylamine (151.8 mg, 1.5 mmol) were added. Then, a dichloromethane solution (5 mL) of acryloyl chloride (135.8 mg, 1.5 mmol) was added under stirring. The above mixed solution was stirred at 25 °C for 12 hours.

[0028] After the reaction was completed, the excess dichloromethane solution was removed by rotary evaporation. The crude product was purified by silica gel column chromatography, and the eluent was ethyl acetate: petroleum ether = 1:6 to obtain the target yellow probe compound with a yield of 85%.

[0029] 1H NMR measurement: 1 1H NMR (400 MHz, Chloroform-d ) δ 7.15 (d, J = 8.3Hz, 2H), 6.93 (d, J =2.5 Hz, 2H), 6.90 (s, 1H), 6.72 (d, J = 17.3 Hz, 1H),6.40 (dd, J = 17.3, 10.5 Hz, 1H), 6.15 (d, J = 11.6 Hz, 1H), 2.64 (s, 2H),2.47 (s, 2H), 1.11 (s, 6H). 1H NMR spectrum determination: 13 C NMR (101 MHz, Chloroform- d ) δ 168.85, 162.34,156.69 (d, J = 4.8 Hz), 154.19 (d, J = 4.8 Hz), 152.32, 135.10 - 134.43 (m),133.55 (t, J = 2.8 Hz), 131.50, 127.61 (t, J = 16.4 Hz), 126.05, 124.92,113.14, 112.35, 110.98 - 110.51 (m), 80.24, 42.95, 39.19, 32.04, 28.00. High-resolution mass spectrometry determination: HR-MS (m / z) calcd for chemical formula C 22 H 18 F2NaO2 + [M] + :403.1234, Found: 403.1229. The 1H NMR spectrum is as shown in Figure 3 , the 13C NMR spectrum is as shown in Figure 4 , and the high-resolution mass spectrum is as shown in Figure 5 .

[0030] Example 2 Preparation of 1 mM probe solution: Accurately weigh the fluorescent probe DCIC prepared in Example 1 and dissolve it in dimethyl sulfoxide (DMSO) solution to prepare a 1 mM solution for standby.

[0031] In a phosphate buffer solution with pH = 7.4 and DMSO (volume ratio 6:4), in order to explore the selectivity of the probe DCIC, a selectivity test was carried out.

[0032] In the ultraviolet absorption spectrum ( Figure 6A), The absorption of the individual probe is at 390 nm, while after reacting with Cys, the absorption redshifts to 500 nm. Among other analytes, except for a partial enhancement of GSH, other interfering substances do not react with the probe DCIC, and the absorption remains unchanged. The solution of the individual probe (10 μM) has a very weak fluorescence emission at 650 nm (the excitation wavelength is 550 nm, Figure 6 B), After adding Cys (100 μM), the fluorescence intensity increases significantly. After adding other interfering substances, the fluorescence emission hardly changes.

[0033] The above experimental results show that under the excitation condition of 500 nm, the probe has good selectivity for Cys.

[0034] Example 3 In a phosphate buffer solution with pH = 7.4 and DMSO (volume ratio 6:4), the kinetic test of the reaction process between the probe DCIC and Cys was carried out.

[0035] When the excitation wavelength is 500 nm, a graph is plotted with the fluorescence intensity at 650 nm as the ordinate and time as the abscissa ( Figure 7 ). Within 20 minutes, the fluorescence intensity of the solution of the individual probe DCIC hardly changes, indicating that the probe has good stability. After adding Cys (0 - 100 μM), at 100 seconds, the fluorescence intensity tends to balance. Therefore, the probe can quickly achieve the purpose of detecting Cys and has the potential for detection in plants.

[0036] Example 4 An ultraviolet spectrophotometer and a fluorescence spectrum analyzer were used to conduct the ultraviolet absorption titration and fluorescence spectrum titration tests for the detection of Cys by the probe DCIC.

[0037] In a phosphate buffer solution with pH = 7.4 and DMSO (volume ratio 6:4), the concentration of the probe DCIC was fixed at 10 µM, and then different concentrations of Cys (0 - 30 µM) were added to measure the changes in the absorption spectrum and fluorescence emission of the probe DCIC (10 μM) for different concentrations of Cys (0 - 100 μM). As Figure 8 shown in A, as the concentration of Cys increases, the absorption redshifts to 500 nm and the absorption intensity increases. As Figure 8 shown in B. When excited with light at 500 nm, Cys (0 - 100 μM) was gradually added, and the fluorescence intensity at 650 nm increased significantly. Then the relationship between the fluorescence intensity value at 650 nm and the concentration of Cys was discussed ( Figure 9 ). When the concentration of Cys increased from 0 μM to 10 μM, the two showed an excellent linear fitting relationship (R 2= 0.9984), and the calculated detection limit was 38.4 nM.

[0038] Example 5 Study whether the probe DCIC can be used to detect Cys in plant cells.

[0039] As Figure 10 shown in A2, 10 μM probe DCIC was added to onion epidermal cells and incubated at room temperature for 30 minutes. No obvious fluorescence appeared under the red light channel. Then, after treating the cells with 100 μM Cys solution and incubating for 30 minutes, the fluorescence enhanced under the red channel scan ( Figure 10 B2). The white light channel ( Figure 10 A1 and Figure 10 B1) showed that the cell morphology was good after the cells were incubated with the probe and Cys. The above results indicate that the probe DCIC has good cell permeability and compatibility with cells. Therefore, the probe DCIC is applicable to Cys in plant cells.

[0040] Example 6 Further evaluate the effect of the probe DCIC in detecting Cys in plant living tissues.

[0041] As Figure 11 shown in A2, tobacco seedlings were incubated with 10 μM probe DCIC at room temperature for 30 minutes. No obvious fluorescence appeared under the red light channel. Then, the tobacco seedlings were treated with 100 μM Cys solution, and after incubating for 30 minutes, the fluorescence enhanced under the red channel scan ( Figure 11 B2). The white light channel ( Figure 11 A1 and Figure 11 B1) showed that the root tip tissue of tobacco was in good morphology after being incubated with the probe and Cys. Therefore, the fluorescence imaging results indicate that the probe DCIC is applicable to detecting Cys in plant tissues.

[0042] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A near-infrared fluorescent probe, the structural formula of which is as follows: 。 2. The method for preparing the near-infrared fluorescent probe according to claim 1, comprising: The compound (E)-2-(3-(3,5-difluoro-4-hydroxyphenylvinyl)-5,5-methylcyclohex-2-enylidene)malononitrile, acryloyl chloride and triethylamine are dissolved in dichloromethane and reacted under stirring. After the reaction is completed, the dichloromethane is removed and purified to obtain a yellow solid, namely the near-infrared fluorescent probe DCIC.

3. The preparation method according to claim 2, characterized in that: The molar ratio of the compound DCI-OH to acryloyl chloride is 1:(1.2-1.5).

4. The preparation method according to claim 2, characterized in that: The molar ratio of the compound DCI-OH to triethylamine is 1:(1.2-1.5).

5. The preparation method according to claim 2, characterized in that: The reaction temperature is 20-35°C.

6. The preparation method according to claim 2, characterized in that: The reaction time is 8-16h.

7. The preparation method according to claim 2, characterized in that: The purification is performed by column chromatography separation, and the column chromatography conditions are as follows: ethyl acetate and petroleum ether are used as eluents in a volume ratio of 1:(3-15); and the silica gel is 200-300 meshes.

8. Use of the near-infrared fluorescent probe according to claim 1 or the near-infrared fluorescent probe obtained by the preparation method according to any one of claims 2 to 7 in detecting cysteine.

9. Use of the near-infrared fluorescent probe according to claim 1 or the near-infrared fluorescent probe obtained by the preparation method according to any one of claims 2 to 7 in preparing a cysteine ​​imaging detection agent in plant cells or plant tissues.

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