CN109912612B

CN109912612B - Synthesis, preparation and application of fluorescent probe for identifying Cys, GSH and HOCl based on molecular logic gate

Info

Publication number: CN109912612B
Application number: CN201910269455.6A
Authority: CN
Inventors: 叶勇; 牛华伟; 彭淑新; 张永茹; 赵玉芬
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2019-04-04
Filing date: 2019-04-04
Publication date: 2021-05-11
Anticipated expiration: 2039-04-04
Also published as: CN109912612A

Abstract

A synthesis, preparation and application of fluorescent probes for identifying Cys, GSH and HOCl based on a molecular logic gate belong to the field of small molecular fluorescent probes and relate to the technical field of molecular computers, and the multi-identification fluorescent probe has a structural formula shown in a formula (I):

the novel fluorescent probe for identifying Cys, GSH and HOCl based on the molecular logic gate is reported for the first time, has the advantages of low detection limit, high sensitivity and good selectivity, and can realize visual detection; the probe has the advantages of cheap and easily-obtained raw materials, high yield and low preparation cost, and is suitable for industrial production; the solution to be tested is 100% buffer solution, and the buffer capacity of the solution to be tested is consistent with the buffer capacity of the physiological environment in the organism; the invention constructs a molecular logic gate by taking Cys, GSH and HOCl as input signals and maximum fluorescence emission peak fluorescence intensities at 514 nm and 412nm as output signals, and is based on molecular logic gate identification reported for the first timeThe fluorescent probes for Cys, GSH and HOCl can solve the limitation caused by single identification fluorescent probe.

Description

Synthesis, preparation and application of fluorescent probe for identifying Cys, GSH and HOCl based on molecular logic gate

Technical Field

The invention belongs to the field of small molecular fluorescent probes, relates to the technical field of molecular computers, and particularly relates to a synthesis method of a multi-recognition fluorescent probe, a construction method of a molecular logic gate and application of the multi-recognition fluorescent probe in detection of Cys, GSH and HOCl.

Background

The traditional semiconductor silicon material realizes logic operation through voltage change; the supermolecule chemistry corresponds to the input and output in the logic calculation process one by one through the interaction process of the object and the host and the change of the corresponding spectrum signal. The logical operation of the fluorescent probe is to identify two or more target molecules by the probe molecules and to change the fluorescence generated in the identification process. The target molecules are used as input signals, fluorescence emission is used as output signals, and the target molecules and the fluorescence emission are used for constructing a molecular logic gate together, so that different analytes can be detected in the same biological sample in a rapid and continuous mode, and the target molecules can be used for detecting certain diseases. Typical fluorescent probes are uniquely identifiable. However, single recognition of fluorescent probes has certain limitations. In biological applications, there may be certain associations between active substances, and changes in one active substance may result in changes in the concentration of the relevant substance, which cannot be tracked by a single identification. The dual-recognition or multi-recognition fluorescent probe can solve the limitation caused by a single recognition fluorescent probe.

Typical fluorescent probes are uniquely identifiable. However, single recognition of fluorescent probes has certain limitations. In biological applications, there may be certain associations between active substances, and changes in one active substance may result in changes in the concentration of the relevant substance, which cannot be tracked by a single identification. The dual-recognition or multi-recognition fluorescent probe can solve the limitation caused by a single recognition fluorescent probe.

Hypochlorous acid (HOCl), which is generated in vivo by hydrogen peroxide and chloride ions under myeloperoxidase catalysis, is one of typical Reactive Oxygen Species (ROS) and has strong oxidative properties and antibacterial activity in biology. However, in vivo hypochlorous acid abnormalities are associated with a number of diseases, such as cardiovascular disease, injury to red blood cells in humans, lung injury, rheumatic disease and cancer. In addition, excess HOCl can cause oxidative stress, which plays a very important role in cell death signaling.

Glutathione (GSH) is the most abundant non-protein thiol in normal cells. GSH plays an important role in redox activity and gene regulation. However, once the GSH level is abnormal, many diseases such as malignant tumor, heart failure, renal failure, etc. may be caused. In addition, intracellular GSH also acts as a reservoir for cysteine (Cys). As one of the biological thiols, Cys is involved in biological redox homeostasis, biocatalysis, detoxification of xenobiotics and other important physiological processes. However, lack of Cys can lead to slow growth, liver damage, muscle and fat loss, pathology and other diseases.

Excessive HOCl can cause oxidative stress, and intracellular Cys and GSH are important small molecule antioxidants that can directly consume the excess HOCl in the organism. However, the exact changes in HOCl and Cys/GSH under oxidative stress are not well understood due to the lack of high sensitivity, specificity and rapid detection methods. Therefore, the development of molecular logic gate-based fluorescent probes for detecting Cys, GSH and HOCl is a problem that needs to be solved by those skilled in the art.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention aims to provide a novel fluorescent probe which is good in selectivity and high in sensitivity and can visually detect Cys, GSH and HOCl, and a preparation method of the novel fluorescent probe is correspondingly provided, and raw materials are cheap and easy to obtain, and the preparation cost is low.

Another objective of the invention is to construct molecular logic gates to recognize Cys, GSH and HOCl, avoiding the drawback of single recognition.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a fluorescent probe for identifying Cys, GSH and HOCl based on molecular logic gate has molecular formula of C₃₃H₂₉N₃O₄The probe compound is abbreviated as probe NPCC and has a structural formula shown as a formula (I):

formula (I).

A synthetic method of the fluorescent probe comprises the following steps:

step (1): dissolving a known compound 10-acetyl-9-hydroxy-2, 3,6, 7-tetrahydro-1H-pyrano [2,3-f ] pyrido [3,2,1-ij ] quinolin-11 (5H) -one and benzaldehyde into ethanol, adding piperadine, and heating and refluxing to obtain a red solid, namely the compound 1, wherein the structural formula is as follows:

；

step (2): adding phenylhydrazine and glacial acetic acid into an ethanol solution containing the compound 1, and heating and refluxing to obtain a yellow solid, namely a compound 2, wherein the structural formula of the yellow solid is as follows:

；

and (3): and dropwise adding the dichloromethane solution of acryloyl chloride into the dichloromethane mixed solution containing the compound 2 and triethylamine, reacting at room temperature, and performing silica gel chromatography to obtain a probe compound NPCC.

In the step (1), the 10-acetyl-9-hydroxy-2, 3,6, 7-tetrahydro-1H-pyrano [2,3-f ] pyrido [3,2,1-ij ] quinolin-11 (5H) -one: the molar ratio of benzaldehyde is 1: (1-1.05).

In the step (1), the addition amount of the piperadine is 6-9 drops.

In the step (1), the reaction time is preferably 12 to 16 hours.

In the step (1), the separation and purification step comprises: cooling the reaction liquid to room temperature, separating out solid, filtering, washing the filter cake with frozen ethanol, and vacuum drying.

In step (2), the compound 1: the molar ratio of phenylhydrazine is 1: (1-3).

In the step (2), the adding amount of the glacial acetic acid is 0.5 mL-1 mL.

In the step (2), the reaction time is preferably 3 to 6 hours.

In the step (2), the separation and purification step is as follows: cooling the reaction liquid to room temperature, separating out solid, filtering, washing the filter cake with frozen ethanol, and vacuum drying.

In step (3), the compound 2: acryloyl chloride: the molar ratio of triethylamine is 1: (1-1.2): (1-1.4).

In the step (3), the temperature for adding the acryloyl chloride is 0-5 ℃ and the time is 0.5 hour.

In the step (3), the reaction temperature after the dropwise addition of the acryloyl chloride is room temperature, the reaction temperature is 25-28 ℃, and the reaction time is preferably 2-4 hours.

In the step (3), the elution machine used for the silica gel column chromatography is preferably composed of petroleum ether and dichloromethane in a volume ratio of 1: 1.

The synthetic route of the probe of the invention is as follows:

。

the compound NPCC is short for the fluorescent probe compound provided by the invention.

One application of the fluorescent probe prepared by the invention is to detect Cys, GSH and HOCl in water environment.

The second application of the fluorescent probe prepared by the invention is to construct a molecular logic gate.

The above application, in particular, includes:

dissolving the probe in dimethyl sulfoxide (DMSO) to prepare a probe stock solution (1 mM); adding the probe solution into the solution to be detected; the test solution is preferably Tris-HCl (10 mM, 1 mM CTAB) buffer, and the pH value of the buffer is 7.4.

Preferably, when the solution to be detected does not contain Cys or GSH, the fluorescence intensity of the maximum fluorescence emission peak at 514 nm of the solution does not obviously change; when Cys or GSH exists in the solution to be detected, the fluorescence intensity of the maximum fluorescence emission peak at 514 nm of the mixed solution is obviously increased, and green fluorescence is emitted under a 365 nm fluorescent lamp.

Preferably, when Cys is added into the liquid to be detected, the time required for the maximum fluorescence emission peak fluorescence intensity at 514 nm to reach the response platform is 60 minutes, and when GSH is added into the liquid to be detected, the time required for the maximum fluorescence emission peak fluorescence intensity at 514 nm to reach the response platform is 15 minutes.

Preferably, the difference in response time of the probe to Cys and GSH allows kinetic discrimination between Cys and GSH.

Preferably, when Cys is added to the solution to be detected, HOCl is added after 60 minutes, the green fluorescence of the mixed solution is weakened under a 365 nm fluorescent lamp, and the mixed solution does not have obvious blue fluorescence.

Preferably, when GSH is added into the solution to be detected, HOCl is added after 15 minutes, green fluorescence of the mixed solution disappears under a 365 nm fluorescent lamp, and the mixed solution emits blue fluorescence.

Preferably, Cys and GSH are added into the solution to be detected, HOCl is added, and the change conditions of green fluorescence and blue fluorescence of the mixed solution under a 365 nm fluorescent lamp are different.

Preferably, the mixed solution has different changes of green fluorescence and blue fluorescence under 365 nm fluorescent lamp, and Cys, GSH and HOCl can be visually detected.

Preferably, the lower limit of the detection concentration of Cys, GSH and HOCl detected by the probe is respectively as follows: cys was 8.95 nM, GSH 4.26 nM, HOCl 0.43. mu.M.

The molecular logic gate is constructed based on the action of a fluorescent probe on Cys, GSH and HOCl and the change of fluorescence intensity, and takes the maximum fluorescence emission peak fluorescence intensity at 514 nm and 412nm as a judgment basis.

The molecular logic gate comprises an OR type logic gate AND an AND type logic gate.

The construction methods of the molecular logic gate including an OR type logic gate AND an AND type logic gate are respectively as follows:

(1) taking a fluorescent probe compound NPCC solution as a template, taking one of Cys OR GSH as an input signal, enabling the interaction between the Cys OR GSH and the probe compound NPCC to enhance the green fluorescence of the probe compound NPCC under a 365 nm fluorescent lamp, and taking the maximum fluorescence emission peak at 514 nm as an output signal to construct an OR type logic gate;

(2) taking a fluorescent probe compound NPCC solution as a template, taking GSH AND HOCl which exist in sequence as input signals, enabling blue fluorescence of the probe compound NPCC to be enhanced under a 365 nm fluorescent lamp through sequential interaction of the GSH AND the probe compound NPCC, AND taking a maximum fluorescence emission peak at 412nm as an output signal to construct an AND type logic gate.

Compared with the prior art, the beneficial effects of the invention are embodied in the following aspects:

1. the novel fluorescent probe for identifying Cys, GSH and HOCl is reported for the first time, has the advantages of low detection limit, high sensitivity and good selectivity, and has the capability of resisting the interference of other small molecules in organisms and wide potential application value;

2. the probe has the advantages of cheap and easily-obtained raw materials, high yield and low preparation cost, and is suitable for industrial production;

3. the solution to be tested is 100% buffer solution, and the buffer capacity of the solution is consistent with the buffer capacity of the physiological environment in the organism;

4. the fluorescent probe is used for detecting Cys, GSH and HOCl, the color change of the fluorescent probe can be seen by naked eyes, and the visual detection can be realized;

5. the invention takes Cys, GSH and HOCl as input signals, and fluorescence emission at 514 nm and 412nm as output signals to construct a molecular logic gate, and is a fluorescent probe for identifying Cys, GSH and HOCl based on the molecular logic gate reported for the first time.

Drawings

FIG. 1 is the variation of the UV absorption spectrum and fluorescence emission spectrum of the probe with Cys, GSH and HOCl in example 2; after adding 10 mu mol/L Cys, adding 300 mu mol/L HOCl after 60 minutes; after addition of 10. mu. mol/L GSH, after 15 minutes, 300. mu. mol/L HOCl was added.

FIG. 2 shows the change of fluorescence spectrum of the probe in example 3 with increasing addition of Cys; FIG. 2 is an inset of a dotted plot of the maximum fluorescence emission peak fluorescence intensity at 514 nm as a function of Cys concentration and green fluorescence emitted by a 365 nm fluorescent lamp after identifying Cys.

FIG. 3 is a graph showing the change of fluorescence spectrum of the probe in example 4 with an increase in the amount of GSH added; FIG. 3 is an inset of a dot plot showing the change in maximum fluorescence emission peak fluorescence intensity at 514 nm with GSH concentration, and green fluorescence is emitted in a 365 nm fluorescent lamp after GSH is recognized.

FIG. 4 shows the change of fluorescence spectrum of probe obtained by adding GSH to the probe mixture of example 5 and adding HOCl at different concentrations after 15 minutes; FIG. 4 inset is a plot of the maximum fluorescence emission peak fluorescence intensity at 412nm as the HOCl concentration increases and blue fluorescence is emitted at 365 nm fluorescent lamp after HOCl is identified.

FIG. 5 shows the addition of Cys, GSH and other various interfering substances to the probe mixture of example 6; in the figure, other different interfering substances are Hcy, His, Glu, Asp, Val, Phe, Tyr, Ala, Ser, Leu, Arg, Pro, Thy, Lys, Gly, Na⁺，K⁺，F^–，Cl^–，Br^–，I^–，CO₃ ^2–，HCO₃ ^–，Ac^–，SO₄ ^2–，PO₄ ^3–，NO₃ ^–，NO₂ ^–，•OH，O₂•^–HOCl and H₂O₂。

FIG. 6 shows GSH is added to the probe mixture in example 7, and HOCl and other various interfering substances are added after 15 minutes; in the figure, other different interfering objectsIs Na in nature⁺，K⁺，F^–，Cl^–，Br^–，I^–，CO₃ ^2–，HCO₃ ^–，Ac^–，SO₄ ^2–，S₂O₈ ^2–，PO₄ ^3–，ClO₄ ^–，HSO₃ ^–，HS^–，NO₃ ^–，•OH，NO₂ ^–，¹O₂，O₂•^–And H₂O₂。

FIG. 7a is a kinetic experiment of the probe response to Cys and GSH in example 8; FIG. 7b is a kinetic experiment of the probe response to HOCl in example 9.

FIG. 8 is a schematic diagram of an OR molecular logic gate in example 10 AND an AND molecular logic gate in example 11.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, but it should not be understood that the scope of the above-described subject matter of the present invention is limited to the following embodiments, and all other embodiments obtained by a person of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

Example 1

Synthesis of fluorescent Probe Compound NPCC.

(1) Synthesis of Compound 1

The known compound 10-acetyl-9-hydroxy-2, 3,6, 7-tetrahydro-1H-pyrano [2,3-f ] pyrido [3,2,1-ij ] quinolin-11 (5H) -one (897 mg, 3 mmol) and benzaldehyde (334 mg, 3.15 mmol) were dissolved in 25mL of ethanol, 6-9 drops of piperazine were added during stirring and the mixture was refluxed for 14 hours. After completion of the reaction, it was cooled to room temperature, filtered with suction, and the filter cake was washed with frozen ethanol and dried under vacuum to give Compound 1 (896.4 mg, yield 77.2%) as a red solid.

Hydrogen nuclear magnetic resonance spectroscopy:¹H NMR (400 MHz, CDCl₃, ppm):1.92-1.99 (m, 4H), 2.76-2.85 (m, 4H), 3.29-3.32 (m, 4H),7.42 (m, 4H), 7.70 (m, 2H), 7.94 (d, 1 H, J = 15.8 Hz), 8.46 (d, 1 H, J = 15.8 Hz)。

nuclear magnetic resonance carbon spectrum measurement:¹³C NMR (100 MHz, CDCl₃, ppm): δ = 20.14, 20.25, 21.22, 27.48, 49.82, 50.25, 98.06, 103.11, 105.36, 118.70, 122.75, 124.07, 128.88, 128.96, 130.55, 135.27, 144.41, 149.38, 152.26, 161.51, 179.36, 191.56。

high-resolution mass spectrometry: [ M + H ]]⁺ calcd for [C₂₄H₂₁NO₄+H ]⁺: 388.1543. Found: 388.1545。

(2) Synthesis of Compound 2

Phenylhydrazine (0.162 g, 1.5 mmol) and 0.5 mL of acetic acid were added to a solution of compound 2 (194 mg, 0.5 mmol) in 25mL of ethanol. The reaction mixture was refluxed for 4 hours. After completion of the reaction, a precipitate separated out, separated by filtration, washed with chilled ethanol, and dried under vacuum. Compound 3 was obtained as a yellow solid (233 mg, 97.7% yield).

Hydrogen nuclear magnetic resonance spectroscopy:¹H NMR (400 MHz, CDCl₃, ppm): 2.00 (m, 4H), 2.82-2.90 (m, 4H), 3.30-3.31 (m, 4H), 3.53-3.60 (m, 1H), 4.21-4.29 (m, 1H), 5.09-5.15 (m, 1H), 6.85 (t, 1H, J = 31.4 Hz), 6.93 (d, 2H, J = 17.4 Hz), 7.23 (t, 2 H, J = 21.4 Hz), 7.30 (s, 1 H), 7.36-7.42 (m, 5 H), 13.85 (s, 1 H)。

nuclear magnetic resonance carbon spectrum measurement:¹³C NMR (100 MHz, CDCl₃, ppm): δ = 20.38, 20.51, 21.48, 27.67, 46.89, 49.65, 50.09, 63.43, 91.85, 103.15, 105.84, 113.15, 118.51, 119.56, 121.05, 125.99, 127.59, 129.10, 129.47, 142.27, 144.65, 147.12, 150.76, 151.57, 162.10, 167.36。

high-resolution mass spectrometry: HR-MS M/z [ M + H ]]⁺ calcd for [C₃₀H₂₁N₃O₃+H ]⁺: 478.2125. Found: 478.2115。

(3) Synthesis of fluorescent Probe Compound NPCC

A solution of acryloyl chloride (90.5 mg, 0.225 mmol) in dichloromethane (5 mL) was added dropwise to a mixed solution of compound 2 (83.5 mg, 0.175 mmol) and triethylamine (25 mg, 0.25 mmol) in dichloromethane (10 mL) at 0-5 deg.C. After the dropwise addition was completed within half an hour, the temperature was slowly raised to room temperature. After completion of the reaction for 2.5 hours at room temperature, the mixture was washed with brine (10 mL. times.3) and dried over anhydrous sodium sulfate. The combined organic layers were filtered and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent petroleum ether: dichloromethane = 1: 1) to give probe NPCC as a red-brown solid (60 mg, yield 65%).

high-resolution mass spectrometry: HR-MS M/z [ M + H ]]⁺ calcd for [C₃₃H₂₉N₃O₄+H ]⁺: 532.2231. Found: 532.2222。

Example 2

The ultraviolet visible spectrum and fluorescence emission spectrum of the probe of the invention change when the probe acts with Cys, GSH and HOCl.

The probe prepared in example 1 was dissolved in DMSO to prepare a probe stock solution having a probe concentration of 1 mM. The test solution was Tris-HCl (10 mM, 1 mM CTAB) buffer, pH 7.4. And adding 30 mu L of the probe mother solution into 3 mL of the solution to be detected to obtain a probe mixed solution, wherein the final concentration of the probe in the mixed solution is 10 mu mol/L. And testing the change conditions of the ultraviolet-visible spectrum and the fluorescence spectrum when the probe acts on Cys, GSH and HOCl by using an ultraviolet-visible spectrophotometer and a fluorescence spectrophotometer. As can be seen from FIG. 1, after Cys (10 μ M) or GSH (10 μ M) is added, the maximum ultraviolet absorption peak of the probe is blue-shifted from 471 nm to 441 nm, the fluorescence intensity of the probe at 514 nm is enhanced, in the process, the color of the solution is changed from orange yellow to yellow, green fluorescence appears under a 365 nm fluorescent lamp, and visual detection of Cys and GSH is realized. After the addition of HOCl (30 μ M) to the above solutions with Cys and GSH, respectively, a distinct experimental phenomenon was seen: probe solutions respectively added with GSH and HOCl change color from yellow to colorless, emit blue fluorescence under a 365 nm fluorescent lamp, the maximum ultraviolet absorption peak is blue-shifted from 441 nm to 380 nm, the maximum fluorescence emission peak is blue-shifted from 514 nm to 412nm, and the probe solutions have the characteristics of ratio type detection of ultraviolet absorption spectrum and fluorescence emission spectrum for HOCl; the color of the probe solution added with Cys and HOCl respectively changes from yellow to light yellow, the green fluorescence becomes light under 365 nm fluorescent lamp, no blue line fluorescence appears, and the ultraviolet absorption peak at 441 nm and the fluorescence emission peak at 514 nm do not completely disappear.

Example 3

The invention relates to a fluorescence titration experiment for identifying Cys by using a probe.

Example 2. mu.L of the prepared probe mother solution was added to 3 mL of the solution to be detected, the final concentration of the probe was 10. mu. mol/L, and Cys mother solutions of different equivalents (0-4 equivalents) were added, as shown in FIG. 2, the maximum fluorescence emission peak fluorescence intensity at 514 nM gradually increased with the increase of Cys concentration, and the detection limit of Cys recognized by the probe was 8.95 nM.

Example 4

The invention relates to a fluorescence titration experiment for identifying GSH by a probe.

Example 2. mu.L of the prepared probe mother solution was added to 3 mL of the solution to be tested, the final concentration of the probe was 10. mu. mol/L, and different equivalents (0-5 equivalents) of GSH mother solutions were added, as shown in FIG. 3, the maximum fluorescence emission peak fluorescence intensity at 514 nM gradually increased with the increase of the GSH concentration, and the detection limit for the probe to recognize GSH was 4.26 nM.

Example 5

The invention relates to a fluorescent titration experiment for identifying HOCl by a probe.

In example 2, 30. mu.L of the prepared probe mother solution is added into 3 mL of solution to be detected, the final concentration of the probe is 10. mu. mol/L, 10 equivalents of GSH mother solution are added, after 15 minutes, HOCl mother solutions with different equivalents (0-30 equivalents) are added into the solution to be detected, to which the probe and GSH are added, as shown in FIG. 4, the fluorescence intensity at 514 nm gradually decreases and the fluorescence intensity at 412nm gradually increases with the increase of the HOCl concentration, and the probe has the characteristic of detecting HOCl in a fluorescence emission spectrum ratio type, and the detection limit of HOCl identified by the probe is 0.43. mu.M.

Example 6

The invention relates to fluorescence selectivity experiments for identifying Cys and GSH.

Example 2 preparation of probe mother liquor 30. mu.L was added to 3 mL of solution to be tested, the final concentration of probe was 10. mu. mol/L, and 10 equivalents of the following different interfering substances were added: hcy, His, Glu, Asp, Val, Phe, Tyr, Ala, Ser, Leu, Arg, Pro, Thy, Lys, Gly, Na⁺，K⁺，F^–，Cl^–，Br^–，I^–，CO₃ ^2–，HCO₃ ^–，Ac^–，SO₄ ^2–，PO₄ ^3–，NO₃ ^–，NO₂ ^–，•OH，O₂•^–HOCl and H₂O₂. And detecting the ultraviolet and fluorescence spectrum changes of the test solution by using an ultraviolet-visible spectrophotometer and a fluorescence spectrophotometer. As can be seen from FIG. 5, when various interfering substances were added to the test solution, the ultraviolet absorption peak at 471 nm did not change significantly, and the fluorescence emission peak at 514 nm did not increase significantly. Hcy is the lowest thiol-containing amino acid content in normal organisms, so the interference caused by the addition of Hcy is not significant. However, after addition of Cys or GSH, the maximum UV absorption peak of the test solution blueshifted from 471 nm to 441 nm, and the fluorescence intensity increased at 514 nm. The experimental results show that the probe NPCC has good selectivity on Cys and GSH.

Example 7

The invention relates to a fluorescence selectivity experiment for identifying HOCl.

EXAMPLE 2 preparation of Probe mother liquorAdding 30 mu L of the probe into 3 mL of solution to be detected, wherein the final concentration of the probe is 10 mu mol/L, firstly adding 10 equivalents of GSH mother liquor, and after 15 min, respectively adding 30 equivalents of the following different interfering substances: na (Na)⁺，K⁺，F^–，Cl^–，Br^–，I^–，CO₃ ^2–，HCO₃ ^–，Ac^–，SO₄ ^2–，S₂O₈ ^2–，PO₄ ^3–，ClO₄ ^–，HSO₃ ^–，HS^–，NO₃ ^–，•OH，NO₂ ^–，¹O₂，O₂•^–And H₂O₂. And detecting the ultraviolet and fluorescence spectrum changes of the test solution by using an ultraviolet-visible spectrophotometer and a fluorescence spectrophotometer. As can be seen from FIG. 6, when various interfering substances were added to the test solution, the ultraviolet absorption peak at 441 nm did not change significantly, the fluorescence emission peak at 514 nm did not decrease significantly, and the fluorescence emission peak at 412nm did not increase significantly. However, after HOCl is added, the maximum ultraviolet absorption peak of the test solution is blue-shifted from 441 nm to 380 nm, the fluorescence intensity of the fluorescence emission peak at 514 nm is weakened, the fluorescence intensity of the fluorescence emission peak at 412nm is increased, and the ratio type detection of HOCl by an ultraviolet absorption spectrum and a fluorescence emission spectrum is realized. The experimental results show that the probe NPCC has good selectivity to HOCl.

Example 8

The invention relates to a dynamic experiment of a probe and Cys and GSH, namely the relationship between the fluorescence intensity of the probe and time after adding Cys or GSH.

Example 2 preparation of Probe mother solution 30. mu.L was added to 3 mL of the solution to be tested, the final concentration of probe was 10. mu. mol/L, and 10 equivalents of Cys and GSH mother solutions were added, respectively, and the concentration of Cys and GSH was 100. mu. mol/L. The fluorescence spectrum of the probe was measured as a change in fluorescence intensity with time upon excitation with an excitation wavelength of 440 nm. As shown in FIG. 7a, the fluorescence intensity at 512 nm gradually increased with time, the solution to be measured to which the probe and Cys were added reached a stable value within 60 minutes, and the solution to be measured to which the probe and GSH were added reached a stable value within 15 minutes. The experimental results demonstrate that the probe can distinguish Cys from GSH by kinetics.

Example 9

The probe of the invention is used for HOCl kinetic experiments, namely, after GSH is added to reach a response platform, HOCl is added, and the relationship between the fluorescence intensity of the probe and time is obtained.

Example 2 mu.L of the prepared probe mother solution was added to 3 mL of the solution to be tested, the final concentration of the probe was 10. mu. mol/L, 10 equivalents of Cys and GSH mother solutions were added, 30 equivalents of HOCl were added, the concentration of Cys and GSH was 100. mu. mol/L, and the concentration of HOCl was 300. mu. mol/L. The ratio of fluorescence intensity (I) at 412nm and 514 nm of the probe is tested by excitation with an excitation wavelength of 380 nm₄₁₂/I₅₁₄) Fluorescence spectra as a function of time. As shown in FIG. 7b, over time, 10 equivalents of Cys were added first, followed by 30 equivalents of HOCl, I₄₁₂/I₅₁₄The fluorescence intensity ratio of (A) does not change obviously; adding 10 equivalents of GSH, then 30 equivalents of HOCl, I₄₁₂/I₅₁₄The ratio of fluorescence intensities of (a) and (b) increased significantly and reached a stable value within 15 minutes. The experimental results show that the probe can effectively distinguish Cys, GSH and HOCl through dynamics.

Example 10

And (4) constructing an OR type logic gate.

The principle of the construction of the OR type logic gate is shown in FIG. 8, Cys OR GSH is used as an output signal, the fluorescence intensity of the maximum fluorescence emission peak of the probe NPCC at 514 nm is used as an output signal, the fluorescence intensity of the maximum fluorescence emission peak at 514 nm is enhanced, the corresponding true value is 1, and the corresponding true value table is 0 when no obvious fluorescence emission peak exists at 514 nm. When the input is (0, 0), 30 μ L of the probe mother liquor prepared in example 2 is added into 3 mL of the solution to be tested, the final concentration of the probe is 10 μmol/L, the fluorescence intensity is tested under the excitation of 441 nm, and the output value is 0 because the probe NPCC has intramolecular charge transfer and the probe has no obvious fluorescence; when the input is (1, 0), 30 μ L of the probe mother liquor prepared in example 2 is added into 3 mL of the solution to be tested, the final concentration of the probe is 10 μmol/L, 10 equivalents of Cys mother liquor are added, the final concentration of Cys is 100 μmol/L, the fluorescence intensity of the probe is tested under the excitation of 441 nm, and the maximum fluorescence emission peak fluorescence intensity of the probe at 514 nm is remarkably increased due to the inhibition of charge transfer in the probe NPCC molecule, so the output value is 1; when the input is (0, 1), 30. mu.L of the probe mother liquor prepared in example 2 is added into 3 mL of the solution to be tested, the final concentration of the probe is 10. mu. mol/L, 10 equivalents of GSH mother liquor are added, the final concentration of GSH is 100. mu. mol/L, the fluorescence intensity of the probe is tested under the excitation of 441 nm, and the maximum fluorescence emission peak fluorescence intensity of the probe at 514 nm is remarkably increased due to the inhibition of charge transfer in the NPCC molecule of the probe, so that the output value is 1; when the input was (1, 1), 30. mu.L of the probe mother liquor prepared in example 2 was added to 3 mL of the solution to be tested, the final concentration of the probe was 10. mu. mol/L, 10 equivalents of Cys and 10 equivalents of GSH mother liquor were added, respectively, and the final concentrations of Cys and GSH were 100. mu. mol/L, and the fluorescence intensity thereof was measured under excitation at 441 nm, and since the intramolecular charge transfer of the probe NPCC was suppressed, the fluorescence intensity of the maximum fluorescence emission peak of the probe at 514 nm was significantly increased, so that the output was 1.

The structure of the OR-type logic gate, different input signals, and corresponding truth table of output signals are shown in table 1.

OR type logic gate truth table

Input 1 (Cys)	Input 2 (GSH)	Output (514 nm fluorescence intensity)
			0	0	0
1	0	1
			0	1	1
1	1	1

Example 11

AND (5) constructing an AND type logic gate.

The principle of the AND-type logic gate is shown in fig. 8, with GSH AND HOCl as output signals, the fluorescence intensity of the maximum fluorescence emission peak at 412nm of the probe NPCC as output signals, the fluorescence intensity of the maximum fluorescence emission peak at 412nm is enhanced, the corresponding true value is 1, AND when no significant fluorescence emission peak exists at 412nm, the corresponding truth table is 0. When the input is (0, 0), 30 μ L of the probe mother liquor prepared in example 2 is added into 3 mL of the solution to be tested, the final concentration of the probe is 10 μmol/L, the fluorescence intensity is tested under the excitation of 380 nm, the probe has no obvious fluorescence, so the output value is 0; when the input is (1, 0), 30. mu.L of the probe mother liquor prepared in example 2 is added into 3 mL of the solution to be tested, the final concentration of the probe is 10. mu. mol/L, 10 equivalents of GSH mother liquor are added, the final concentration of GSH is 100. mu. mol/L, the fluorescence intensity is tested under the excitation of 380 nm, the fluorescence intensity of the probe at 412nm is not obviously increased, so the output value is 0; when the input is (0, 1), 30 μ L of the probe mother liquor prepared in example 2 is added into 3 mL of the solution to be tested, the final concentration of the probe is 10 μmol/L, 30 equivalents of HOCl mother liquor are added, the final concentration of HOCl is 300 μmol/L, the fluorescence intensity of the probe is tested under the excitation of 380 nm, and the output value is 0 because the NPCC probe cannot be directly contacted with HOCl and the fluorescence intensity of the probe at 412nm is not obviously increased; when the input was (1, 1), 30. mu.L of the probe stock solution prepared in example 2 was added to 3 mL of the solution to be tested, the final concentration of the probe was 10. mu. mol/L, 10 equivalents of GSH were added, 30 equivalents of the GSH stock solution were added after 15 minutes, the final concentration of GSH was 100. mu. mol/L, and the final concentration of HOCl was 300. mu. mol/L, and the fluorescence intensity was measured under excitation at 380 nm, and the maximum fluorescence emission peak fluorescence intensity at 412nm of the probe significantly increased, so the output was 1.

AND type logic gate truth table

Input 1 (GSH)	Input 2 (HOCl)	Output (412 nm fluorescence intensity)
			0	0	0
1	0	0
			0	1	0
1	1	1

Claims

1. A fluorescent probe compound for identifying Cys, GSH and HOCl based on a molecular logic gate is abbreviated as a probe NPCC, and is characterized in that the chemical structure of the fluorescent probe compound is shown as a formula (I):

formula (I).

2. The method for synthesizing the fluorescent probe compound for detecting Cys, GSH and HOCl according to claim 1, which comprises the following steps:

(1) refluxing 10-acetyl-9-hydroxy-2, 3,6, 7-tetrahydro-1H-pyrano [2,3-f ] pyrido [3,2,1-ij ] quinolin-11 (5H) -one, benzaldehyde and piperidine in ethanol, and filtering the product to obtain compound 1:

(2) refluxing the compound 1, phenylhydrazine and glacial acetic acid in ethanol, and filtering a product to obtain a compound 2:

(3) and dropwise adding the dichloromethane solution of acryloyl chloride into the dichloromethane mixed solution containing the compound 2 and triethylamine, slowly heating to room temperature, reacting at room temperature, and separating and purifying the product to obtain the fluorescent probe NPCC.

3. The method of claim 2, wherein in step (1), the 10-acetyl-9-hydroxy-2, 3,6, 7-tetrahydro-1H-pyrano [2,3-f ] pyrido [3,2,1-ij ] quinolin-11 (5H) -one: the molar ratio of benzaldehyde is 1: (1-1.05), wherein the addition amount of the piperidine is 6-9 drops; in step (2), the compound 1: the molar ratio of phenylhydrazine is 1: (1-3), wherein the adding amount of the glacial acetic acid is 0.5-1 mL; in step (3), the compound 2: acryloyl chloride: the molar ratio of triethylamine is 1: (1-1.2): (1-1.4).

4. The synthesis method according to claim 2, wherein in the step (1), the reflux temperature is 80 ℃, and the reaction time is 12-16 hours; in the step (2), the reflux temperature is 80 ℃, and the reaction time is 3-6 hours; in the step (3), the adding temperature of the acryloyl chloride is 0-5 ℃, the reaction time is 0.5 h, after the acryloyl chloride is added, the temperature is slowly increased to the room temperature, the reaction temperature is 25-28 ℃, and the reaction time is 2-4 h.

5. The synthesis method according to claim 2, wherein in the step (1), the filtration is cooling to room temperature, then filtering, washing with frozen ethanol, and vacuum drying; in the step (2), the filtration is carried out after cooling to room temperature, and the filtration is washed by frozen ethanol and dried in vacuum; in the step (3), the separation and purification step is that the crude product reaction solution is washed with brine and dried over anhydrous sodium sulfate, the combined organic layers are filtered, the solvent is evaporated under reduced pressure, and the crude product is purified by silica gel column chromatography.