Near-infrared fluorescent probe for specifically recognizing D-lysine as well as preparation method and application of near-infrared fluorescent probe

Abstract

The invention discloses a near-infrared fluorescent probe for specifically recognizing D-lysine as well as a preparation method and application of the near-infrared fluorescent probe, and belongs to the technical field of biological fluorescence analysis. The probe can accurately identify D-lysine from various chiral amino acids in a short time, and qualitative and quantitative analysis can be performed in a water phase; the chemical selectivity and enantioselectivity of the D-lysine can be identified at low concentration, the influence of other interferents is avoided, and the detection efficiency is improved; the probe has near-infrared emission, ensures the accuracy and reliability of complex biological environment detection, and overcomes the defects of the existing chiral fluorescent probe; the probe is stable in structure and is not easy to dissociate or degrade in a living body, so that the reliability and repeatability of detection are ensured; the preparation method is simple, mild in reaction condition, simple in purification step, easily available in raw materials, low in cost and suitable for large-scale production; the fluorescent probe is used for real-time and rapid fluorescence imaging of cell exogenous D-lysine, and a powerful tool is provided for research and treatment of metabolic disorder related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biofluorescence analysis. Specifically, it relates to a near-infrared fluorescent probe for specifically recognizing D-lysine, and its preparation method and application. Background Art

[0002] Lysine is one of the essential amino acids for the human body, closely participating in the ornithine cycle and polyamine synthesis, and having positive significance in promoting human growth and metabolism, enhancing immunity, promoting nutrient absorption, antiviral, and treating depression. D-lysine is an optical isomer of lysine, belonging to basic amino acids, and having important biological functions in vivo. With the continuous formation and increase in the amount of D-amino acids during food processing, the development, pathophysiological processes, and roles in treatment of D-amino acids in the human body in diseases such as cancer and Alzheimer's disease are being gradually revealed. Different from L-lysine, D-lysine is related to cytotoxicity and metabolic abnormalities, such as uremia. Therefore, the chiral analysis of D-lysine is of great significance for ensuring the safety of food and drugs and the research on diseases related to metabolic abnormalities. Due to the high structural similarity among the 20 common amino acids in the human body, the mutual influence between different configurations of the same amino acid may also reduce the specific recognition ability of chiral amino acids. How to achieve the specific recognition of D-lysine has become a challenge.

[0003] Traditional methods for detecting lysine include luminol chemiluminescence method, gas chromatography-mass spectrometry, synchrotron radiation X-ray powder diffraction, and thermogravimetric analysis. Although they have high accuracy, they still have the disadvantages of expensive equipment and cumbersome detection, limiting their application in bioimaging. Fluorescence analysis technology has high sensitivity, simple action process, and low cost, and can be applied to chiral recognition. Traditional fluorescent probes have limited penetration depth in biological tissues and are easily interfered by background fluorescence, affecting their application effects in complex biological systems. Developing new fluorescent probes to improve their penetration depth and specificity in biological tissues has become an important direction for the development of fluorescent probe technology. Near-infrared fluorescent probes are molecular probes with fluorescence characteristics in the near-infrared spectral region, having a deeper penetration depth, lower background interference, and smaller light damage. Therefore, developing a near-infrared fluorescent probe capable of specifically recognizing D-lysine is of great significance for improving the accuracy and sensitivity of biomedical research and clinical diagnosis. Summary of the Invention

[0004] In view of the technical status quo that the existing technologies cannot achieve the dual recognition of chemical selectivity and enantioselectivity for D-lysine, the emission wavelength of the existing chiral fluorescent probes is relatively low (less than 550 nm), the selectivity and sensitivity need to be improved, and they cannot be applied to real-time imaging in complex biological environments, etc., the purpose of the present invention is to provide a near-infrared fluorescent probe for specifically recognizing D-lysine, its preparation method and application.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides a near-infrared fluorescent probe for specifically recognizing D-lysine, and the structure of the near-infrared fluorescent probe for specifically recognizing D-lysine is as follows: , wherein, R 1 is any one of -H, -OH, -OCH 3 , -X, -NO 2 , -SO 3 H, -COOH, -Ar, -NH 2 , CF 3和 and C1-C5 alkyl; R 2 is any one of -H, -OH, -OCH 3 , -X, -NO 2 , -SO 3 H, -COOH, -Ar, -NH 2 , -CF 3 and C1-C5 alkyl.

[0006] The present invention provides a preparation method of the above near-infrared fluorescent probe for specifically recognizing D-lysine, including: Step 1: React 2-(3,5,5-trimethylcyclohex-2-en-1-ylidene)malononitrile with Compound 1 to prepare Compound S 1 ; The structural formula of the Compound 1 is: , wherein, R 2 is any one of -H, -OH, -OCH 3 , -X, -NO 2 , -SO 3 H, -COOH, -Ar, -NH 2 , -CF 3 and C1-C5 alkyl; The structural formula of the Compound S 1 is: , wherein, R 2 is any one of -H, -OH, -OCH 3 , -X, -NO 2, -SO 3 H, -COOH, -Ar, -NH 2 , -CF 3 and any one of C1-C5 alkyl groups; Step 2: Under argon protection, mix and react compound S1 and hexamethylenetetramine, extract, dry, and purify by column chromatography to obtain compound S 2 ; The structure formula of the said compound S 2 is: , where R 2 is -H, -OH, -OCH 3 , -X, -NO 2 , -SO 3 H, -COOH, -Ar, -NH 2 , -CF 3 and any one of C1-C5 alkyl groups; Step 3: Under argon protection, dissolve compound S 2 , compound 2 and sodium triacetoxyborohydride, remove the solvent after reaction, and purify by column chromatography to obtain a near-infrared fluorescent probe that specifically recognizes D-lysine; The structure formula of the said compound 2 is: , where R 1 is -H, -OH, -OCH 3 , -X, -NO 2 , -SO 3 H, -COOH, -Ar, -NH 2 , -CF 3 and any one of C1-C5 alkyl groups.

[0007] In Step 1, the molar ratio of 2-(3,5,5-trimethylcyclohex-2-en-1-ylidene)malononitrile to compound 1 is 1:1-3, the reaction temperature is 80°C-90°C, and the reaction time is 8-14 h.

[0008] In Step 2, the molar ratio of compound S1 to hexamethylenetetramine is 1:1-5, the reaction temperature is 90°C-110°C, and the reaction time is 6-10 h.

[0009] In Step 3, the molar ratio of compound S 2 , compound 2 and sodium triacetoxyborohydride is 1:1-2:1-3, and the reaction condition is to react at room temperature for 20-30 h.

[0010] In Step 3, the eluent used in the column chromatography is a mixed solvent of dichloromethane and methanol with a volume ratio of 3:1.

[0011] The present invention provides the use of the above-mentioned near-infrared fluorescent probe for specifically recognizing D-lysine in the preparation of a reagent for measuring, screening or detecting D-lysine.

[0012] The present invention provides application of the above-mentioned near-infrared fluorescent probe that specifically recognizes D-lysine in cell fluorescence imaging.

[0013] The present invention provides a kit for detecting the presence of D-lysine in a sample or determining the content of D-lysine in a sample. The kit comprises the above-mentioned near-infrared fluorescent probe that specifically recognizes D-lysine.

[0014] The present invention provides a method for detecting the presence or content of D-lysine in a sample, comprising: (1) contacting the above kit with a sample to form a fluorescent compound; (2) Determining the fluorescence properties of the fluorescent compound.

[0015] Compared with the prior art, the present invention achieves the following technical effects: The present invention provides a near-infrared fluorescent probe for specifically recognizing D-lysine. The probe can specifically recognize D-lysine from 39 chiral amino acids in a short time, and can perform qualitative and quantitative analysis on D-lysine in an aqueous phase, thereby realizing chemical selectivity and enantioselectivity dual recognition of D-lysine at a relatively low concentration, and can avoid interference of other interferents on the recognition of D-lysine, thereby improving the detection efficiency. The probe has near-infrared emission, thereby ensuring the accuracy and reliability of the probe in a complex biological environment, and overcomes the shortcomings of existing chiral fluorescent probes, such as low emission wavelength, need to improve selectivity and sensitivity, and inability to be used for real-time imaging in a complex biological environment. The probe has a stable structure and is not easily dissociated or degraded in vivo, thereby ensuring the reliability and repeatability of the detection.

[0016] The preparation method of the near-infrared fluorescent probe for specifically recognizing D-lysine provided by the present invention has the advantages of simple preparation, mild reaction conditions, easy control, simple purification steps, easy-to-obtain raw materials, low cost, and suitability for large-scale production.

[0017] The application provided by the present invention, the near-infrared fluorescent probe that specifically recognizes D-lysine can realize real-time and rapid fluorescence imaging of exogenous D-lysine in cells, provides a powerful tool for in-depth understanding of the occurrence and treatment of metabolic abnormality-related diseases, helps to reveal the role of D-lysine in metabolic abnormality-related diseases, and provides a basis for the treatment of metabolic abnormality-related diseases, and has potential biomedical research and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The synthetic route of the near-infrared fluorescent probe L1 that specifically recognizes D-lysine; Figure 2 UV absorption spectra of the probe L1 of the present invention before and after reacting with D- and L-lysine; Figure 3 Fluorescence emission spectra of the probe L1 of the present invention before and after reacting with D- and L-lysine; Figure 4 Fluorescence emission spectra of the probe L1 of the present invention before and after reacting with (0 - 20 eq) D-lysine; Figure 5 Fluorescence intensity changes (665 nm) of the probe L1 of the present invention and the probe L1 after adding 3 equivalents of Mn 2+ at different times; Figure 6 Fluorescence intensity changes (665 nm) of the probe L1 of the present invention after reacting with 30 equivalents of D-lysine or L-lysine at different times; Figure 7 Fluorescence intensity comparison chart of the selective recognition of 39 different configurations of amino acids by the probe L1 of the present invention.

[0019] Figure 8 Cell survival rate after co-incubating the probe L1 of the present invention at different concentrations for 24 h; Figure 9 Laser confocal imaging map (HepG2 cells) of the recognition of exogenous D-lysine and L-lysine by the probe L1 of the present invention, scale bar 50 μm. Detailed implementation manners

[0020] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] For those experimental steps or conditions not specified in the examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments without indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0022] In the present invention, unless otherwise specified, all experimental raw materials used are commercially available products well-known to those skilled in the art Example 1 Refer to the appendix Figure 1 , this example provides a near-infrared fluorescent probe L1 for specifically recognizing D-lysine, and the specific preparation steps are as follows: (1) Preparation of Compound S1

[0023] Add compound 2-(3,5,5-trimethylcyclohex-2-en-1-ylidene) malononitrile (400 mg, 2 mmol) and p-hydroxybenzaldehyde (400 mg, 3 mmol) into a reaction flask, dissolve them in anhydrous ethanol (10 mL), add piperidine (60 µL), and reflux the mixture overnight. After the reaction is completed, concentrate the solvent under reduced pressure to obtain the crude product. The crude product is purified by flash column chromatography (petroleum ether: ethyl acetate = 5:1) to obtain 540.4 mg of orange solid compound S1 with a yield of 83%.

[0024] 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.99 (s, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.21(d, J = 4.0Hz, 2H), 6.85 – 6.75 (m, 3H), 2.60 (s, 2H), 2.53 (s, 2H), 1.01 (s,6H) The structural formula of compound S1 is:

[0025] (2) Preparation of Compound S2

[0026] Add compound S1 (290mg, 1mmol) and hexamethylenetetramine (140 mg, 1 mmol) into a reaction flask, add trifluoroacetic acid (5 mL) and reflux for 10 h. Then add 2M HCl (8 mL), stir for 2 h, let stand overnight, extract the reaction solution with dichloromethane, dry it with anhydrous sodium sulfate, and remove the solvent. Finally, purify the crude product by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain 138 mg of orange solid S2 with a yield of 43%.

[0027] 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.18 (s, 1H), 10.29 (s, 1H), 7.97 (s,1H), 7.89 (d, J= 8.8 Hz, 1H), 7.32 (s, 2H), 7.04 (d, J = 8.4 Hz, 1H), 6.87 (s,1H), 3.36 (s, 2H), 2.61 (s, 2H), 1.01 (s, 6H). The structural formula of compound S2 is as follows:

[0028] (3)Near-infrared fluorescent probe L1 that specifically recognizes D-lysine

[0029] Compound S2 (64 mg, 0.2 mmol), (1R, 2S)-aminoindanol (30 mg, 0.2 mmol) and sodium triacetoxyborohydride (59 mg, 0.28 mmol) were added to a reaction flask. Under argon protection, 2.5 mL of 1,2-dichloroethane was added, and the reaction was carried out at room temperature for 24 h. The crude product was purified by silica gel column chromatography (the eluent was dichloromethane and methanol with a volume ratio of 3:1) to obtain 63 mg of brown solid L1 with a yield of 70%.

[0030] 1 H NMR (600 MHz, DMSO- d 6 ) δ 7.63 (s, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.35(s, 1H), 7.21 (d, J = 12.0 Hz, 5H), 6.80 (d, J = 7.8 Hz, 2H), 4.52 (s, 1H), 4.11(d, J = 14.4 Hz, 1H), 3.99 (d, J = 4.8 Hz, 1H), 3.95 (d, J = 14.4 Hz, 1H), 2.97 (m,1H), 2.85 (d, J = 16.2 Hz, 1H), 2.58 (s, 2H), 2.52 (s, 2H), 1.01 (s, 6H). 13 C NMR (150 MHz, DMSO- d 6) δ 170.64, 160.05, 157.20, 143.12, 141.45, 139.02, 129.34, 129.18, 127.90, 127.26, 126.68, 126.61, 125.62, 125.44, 124.81, 121.74, 116.51, 114.62, 113.82, 75.15, 70.99, 64.73, 48.51, 42.81, 38.71, 32.12(2), 27.92(2). HRMS [M+H] + : C 29 H 30 N 3 O 2 + , Exact Mass 452.2333, found 452.2331.

[0031] The structural formula of the near-infrared fluorescent probe L1 that specifically recognizes D-lysine is as follows: .

[0032] Example 2 Based on Example 1, the performance of the near-infrared fluorescent probe L1 of the present invention was measured in this example.

[0033] (1) Ultraviolet absorption spectrum In this experiment, the ultraviolet absorption spectra of the near-infrared fluorescent probe L1 of Example 1 of the present invention before and after the response to chiral lysine were measured.

[0034] Accurately weigh the probe L1, dissolve it with dimethyl sulfoxide solution to prepare a probe stock solution with a molar concentration of 1.0 mM, and store it at 4-8 °C for later use; prepare a manganese ion standard solution with a molar concentration of 5 mM with anaerobic distilled water and store it at 4-8 °C for later use; prepare an amino acid standard solution with a molar concentration of 50 mM with anaerobic distilled water and store it at 4-8 °C for later use.

[0035] Add 50 μL of the probe L1 stock solution (final concentration 10 μM) to a 5 mL volumetric flask, add the solvent (EtOH:H 2 O = 2:8) and make up the volume, then add 30 μL of the manganese ion stock solution (final concentration 30 μM), mix well and then add 30 μL of the chiral lysine stock solution. After reacting for 10 minutes, use a UV spectrometer to scan the reacted solution and record its ultraviolet absorption spectrum. The specific results are shown in the appendix Figure 2 as shown.

[0036] From the appendix Figure 2As can be seen from the data, when only probe L1 was present, the ultraviolet absorption spectrum showed a maximum absorption peak at 417 nm; when manganese ions were added to the probe solution and L-lysine was further added and reacted for ten minutes, the maximum absorption peak of the probe did not change significantly; while when D-lysine was added, its maximum absorption wavelength underwent an obvious red shift to 458 nm, indicating that a new substance was formed after the reaction of probe L1 with manganese ions and D-lysine, thus affecting its absorption characteristics at 417 nm, and also verifying that probe L1 as a near-infrared fluorescent probe can respond to the presence of D-lysine and exhibit detectable ultraviolet spectral changes.

[0037] (2)Fluorescence emission spectra of near-infrared fluorescent probe L1 before and after responding to chiral lysine In this experiment, the fluorescence emission spectra of near-infrared fluorescent probe L1 of Example 1 of the present invention before and after responding to chiral lysine were measured.

[0038] Add 50 μL of the mother liquor of probe L1 (final concentration 10 μM) to a 5 mL volumetric flask, add the solvent (EtOH:H 2 O = 2:8) and make up the volume. Then add 30 μL of the mother liquor of manganese ions (final concentration 30 μM), mix well and add 30 μL of the mother liquor of chiral lysine. After reacting for 10 minutes, use a fluorescence spectrometer to scan the reacted solution and record its fluorescence emission spectrum. The specific results are shown in the appendix Figure 3 as follows.

[0039] As can be seen from the appendix Figure 3 data, with an excitation wavelength of 470 nm, when only probe L1 was present and manganese ions were added to the probe solution, the fluorescence emission spectra were basically the same. When L-lysine or D-lysine was further added and reacted for ten minutes, the change in the fluorescence intensity of the probe was measured. The fluorescence intensity of the solution at the emission wavelength of 665 nm increased significantly, and the fluorescence intensity after adding D-lysine was significantly higher than that after adding L-lysine, indicating that a new substance was formed after the reaction of probe L1 with manganese ions and chiral lysine, thus affecting its fluorescence spectral characteristics at 665 nm, and also verifying that probe L1 as a near-infrared fluorescent probe can respond to the presence of D-lysine and exhibit detectable fluorescence spectral changes.

[0040] (3)Fluorescence emission spectra of near-infrared fluorescent probe L1 before and after responding to different concentrations of D-lysine In this experiment, the fluorescence emission spectra of near-infrared fluorescent probe L1 of Example 1 of the present invention before and after responding to different concentrations of D-lysine were measured.

[0041] After adding 30 μL of the manganese ion stock solution to the test solution of probe L1 and mixing well, different concentrations of D-lysine solution (0 - 200 μM) were added. After reacting for 10 minutes, the reacted solution was scanned using a fluorescence spectrometer, and its fluorescence emission spectrum was recorded. For specific results, see the appendix Figure 4 as shown.

[0042] From the appendix Figure 4 data, it can be seen that as the concentration of D-lysine increases, the fluorescence intensity at 665 nm also increases, indicating that probe L1 has a good concentration response to D-lysine; there are also significant changes in fluorescence intensity at lower concentrations of D-lysine, showing the high sensitivity of probe L1.

[0043] (4)Response time dependence after the reaction of near-infrared fluorescent probe L1 with manganese ions In this experiment, the response time dependence after the reaction of the near-infrared fluorescent probe L1 of Example 1 of the present invention with manganese ions was measured to study the fluorescence stability of probe L1 in solution.

[0044] For the test solution of probe L1 and the test solution of probe L1 (10 μM) with 30 μL of the manganese ion stock solution added, the reacted solution was subjected to kinetic scanning using a fluorescence spectrometer, and the fluorescence intensities at different time points (changing with time) were recorded. For specific measurement results, see the appendix Figure 5 as shown.

[0045] From the appendix Figure 5 data, it can be seen that as time extends, the fluorescence intensity at 665 nm decreases slightly and levels off after 4 minutes, indicating that probe L1 has good fluorescence stability in solution; showing the good stability of probe L1.

[0046] (5)Response time dependence of near-infrared fluorescent probe L1 with the same concentration of D-lysine and L-lysine In this test, the response time dependence of the near-infrared fluorescent probe L1 of Example 1 of the present invention with the same concentration of D-lysine and L-lysine was measured to study the fluorescence stability of probe L1 in detecting chiral lysine in solution.

[0047] After adding 30 μL of the manganese ion stock solution to the test solution of probe L1 (10 μM), 30 eq of D-lysine and L-lysine were added respectively. The reacted solution was subjected to kinetic scanning using a fluorescence spectrometer, and the fluorescence intensities at different time points (changing with time) were recorded. For specific measurement results, see the appendix Figure 6 as shown.

[0048] From the appendix Figure 6As can be seen from the data, as time prolongs, the fluorescence intensity of the probe solution with D-lysine added slightly decreases at 665 nm. The fluorescence intensity of the probe solution with L-lysine added decreases rapidly within 10 minutes at 665 nm, and remains basically unchanged at 665 nm after 10 minutes. At this time, the probe L1 can best distinguish different configurations of lysine. This result indicates that the probe L1 can distinguish the configuration of chiral lysine in solution in a relatively short time, and shows that the probe L1 also has good photostability for the detection of chiral lysine.

[0049] (6)Specific recognition of D-lysine by near-infrared fluorescence probe L1 In this experiment, the fluorescence intensity changes of the near-infrared fluorescence probe L1 in Example 1 of the present invention with 39 amino acids of the same concentration (glycine, D-alanine, D-valine, D-leucine, D-isoleucine, D-methionine, D-proline, D-tryptophan, D-serine, D-tyrosine, D-cysteine, D-phenylalanine, D-asparagine, D-glutamine, D-threonine, D-aspartic acid, D-glutamic acid, D-lysine, D-arginine, D-histidine, L-alanine, L-valine, L-leucine, L-isoleucine, L-methionine, L-proline, L-tryptophan, L-serine, L-tyrosine, L-cysteine, L-phenylalanine, L-asparagine, L-glutamine, L-threonine, L-aspartic acid, L-glutamic acid, L-lysine, L-arginine, L-histidine) at 665 nm were measured to study the specific recognition of D-lysine by the probe L1 in solution.

[0050] After adding 30 μL of manganese ion mother liquor to the probe L1 test solution (10 μM), 39 amino acids of the same concentration were added respectively, and the solution was scanned spectroscopically using a fluorescence spectrometer. The test conditions were: λ ex =470 nm, λ em =665 nm, silt band: 3nm / 3nm. The specific measurement results are shown in the appendix Figure 7 as follows.

[0051] From the appendix Figure 7 data, it can be seen that except for D-lysine, the fluorescence intensity of other amino acids basically does not change significantly at 665 nm after reacting with the probe L1. Only the fluorescence intensity of the probe solution with D-lysine added increases significantly at 665 nm. This shows that the fluorescence probe can specifically recognize D-lysine among 39 chiral amino acids. This example shows that the fluorescence probe L1 has high selectivity for D-lysine and is suitable for the specific detection of D-lysine.

[0052] (7) Cytotoxicity experiment This experiment conducted the cytotoxicity experiment of the near-infrared fluorescent probe L1 in Example 1 of the present invention on human liver cancer HepG2 cells.

[0053] Disperse the cells (1.5×10 4 cells / well) in a 96-well plate and culture at 37 °C for 24 h. Subsequently, aspirate the culture medium, and then add 100 μL of the culture medium mixed with different concentrations of probe L1 (0 μM, 10 μM, 20 μM, 40 μM, 80 μM), and continue to culture for 24 h. Subsequently, aspirate the culture medium, and then add the culture medium mixed with 10% CCK-8 reagent (10 μL CCK-8, 90 μL RPMI1640 medium), and after culturing for another 2 h, measure the absorbance of the cell lysate at 450 nm with an enzyme-labeled instrument. The specific measurement results are shown in the appendix Figure 8 as shown

[0054] From the appendix Figure 8 data, it can be seen that after HepG2 cells are incubated with probe L1 (≤80 μM) at 37 °C for 24 h, the survival rate remains above 80%, reflecting the low cytotoxicity of probe L1 and indicating that the probe has good biocompatibility, laying a good foundation for its subsequent bioimaging.

[0055] (8) Laser confocal experiment This experiment conducted the laser confocal experiment on the exogenous chiral lysine recognition of the near-infrared fluorescent probe L1 in Example 1 of the present invention on human liver cancer HepG2 cells.

[0056] Seed HepG2 cells in a confocal dish. Probe L1 group: Incubate HepG2 cells with only 10 μM of the probe for 30 min; Mn 2+ group: Incubate HepG2 cells with 10 μM of the probe for 30 min, wash 3 times with PBS, and then incubate with manganese chloride solution (30 μM) for 30 min; D-lysine group: Incubate HepG2 cells with 10 μM of the probe for 30 min, wash 3 times with PBS, and then incubate with manganese chloride solution (30 μM) and D-lysine solution (300 μM) for 30 min; L-lysine group: Incubate HepG2 cells with 10 μM of the probe for 30 min, wash 3 times with PBS, and then incubate with manganese chloride solution (30 μM) and L-lysine solution (300 μM) for 30 min. After each group is treated, directly perform FV3000 laser confocal imaging (Ex / Em: 480 nm / 600 - 700 nm). The test results are shown in the appendix Figure 9 as shown

[0057] From the appendix Figure 9 data, it can be seen that compared with the probe group, Mn 2+The fluorescence intensity of the red channel group showed no obvious change. When exogenous D-lysine was present in the cells, the fluorescence signal was significantly enhanced by 2.2 times compared with the probe group and the Mn 2+ group; when exogenous L-lysine was present in the cells, the fluorescence signal decreased slightly after the probe bound to it compared with the probe group and the Mn 2+ group. This result indicates that the probe L1 can achieve rapid, specific, and real-time detection of D-lysine in cells. In this experiment, through laser confocal imaging technology, it was successfully verified that the probe L1 has good application potential in detecting D-lysine in cells, which provides an important tool for understanding the role of D-lysine in the development of metabolic disorder-related diseases.

[0058] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution according to the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A near-infrared fluorescent probe that specifically recognizes D-lysine, characterized in that: The structure of the near-infrared fluorescent probe that specifically recognizes D-lysine is as follows: ， Wherein, R1 is any one of -H, -OH, -OCH3, -X, -NO2, -SO3H, -COOH, -Ar, -NH2, -CF3 and C1~C5 alkyl; R2 is any one of -H, -OH, -OCH3, -X, -NO2, -SO3H, -COOH, -Ar, -NH2, -CF3 and C1~C5 alkyl.

2. The method for preparing a near-infrared fluorescent probe for specifically recognizing D-lysine according to claim 1, characterized in that: include: Step 1: using 2-(3,5,5-trimethylcyclohex-2-ene-1-ylidene)malononitrile to react with compound 1 to prepare compound S1; The structural formula of the compound 1 is: , wherein R2 is any one of -H, -OH, -OCH3, -X, -NO2, -SO3H, -COOH, -Ar, -NH2, -CF3 and C1~C5 alkyl; The structural formula of the compound S1 is: , wherein R2 is any one of -H, -OH, -OCH3, -X, -NO2, -SO3H, -COOH, -Ar, -NH2, -CF3 and C1~C5 alkyl; Step 2: Under argon protection, compound S1 and hexamethylenetetramine were mixed for reaction, extracted, dried, and purified by column chromatography to obtain compound S2; The structural formula of the compound S2 is: , wherein R2 is any one of -H, -OH, -OCH3, -X, -NO2, -SO3H, -COOH, -Ar, -NH2, -CF3 and C1~C5 alkyl; Step 3: Under argon protection, compound S2, compound 2 and sodium triacetoxyborohydride are mixed and dissolved, the solvent is removed after the reaction, and a near-infrared fluorescent probe that specifically recognizes D-lysine is obtained by purification by column chromatography; The structural formula of the compound 2 is: , wherein R1 is any one of -H, -OH, -OCH3, -X, -NO2, -SO3H, -COOH, -Ar, -NH2, -CF3 and C1~C5 alkyl.

3. The method for preparing a near-infrared fluorescent probe that specifically recognizes D-lysine according to claim 2, characterized in that: In step 1, the molar ratio of 2-(3,5,5-trimethylcyclohex-2-ene-1-ylidene)malononitrile to compound 1 is 1:1-3, the reaction temperature is 80°C-90°C, and the reaction time is 8-14h.

4. The method for preparing a near-infrared fluorescent probe that specifically recognizes D-lysine according to claim 2, characterized in that: In step 2, the molar ratio of the compound S1 to hexamethylenetetramine is 1:1-5, the reaction temperature is 90° C.-110° C., and the reaction time is 6-10 h.

5. The method for preparing a near-infrared fluorescent probe that specifically recognizes D-lysine according to claim 2, characterized in that: In step 3, the molar ratio of compound S2, compound 2 and sodium triacetoxyborohydride is 1:1-2:1-3, and the reaction conditions are room temperature for 20-30 hours.

6. The method for preparing a near-infrared fluorescent probe that specifically recognizes D-lysine according to claim 2, characterized in that: In step 3, the eluent used in the column chromatography is a mixed solvent of dichloromethane and methanol in a volume ratio of 3:

1.

7. Use of the near-infrared fluorescent probe for specifically recognizing D-lysine according to claim 1 in preparing a reagent for measuring, screening or detecting D-lysine.

8. Use of the near-infrared fluorescent probe for specifically recognizing D-lysine as claimed in claim 1 in cell fluorescence imaging.

9. A kit for detecting the presence or content of D-lysine in a sample, characterized in that: The kit comprises the near-infrared fluorescent probe for specifically recognizing D-lysine as described in claim 1.

10. A method for detecting the presence or content of D-lysine in a sample, characterized in that: include: (1) using the kit for detecting the presence or content of D-lysine in a sample as claimed in claim 9 to contact the sample to form a fluorescent compound; (2) Determining the fluorescence properties of the fluorescent compound.

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