Indole matrine derivative photoaffinity active molecular probe and application thereof

Abstract

The invention discloses an indole sophocarpidine derivative photoaffinity active molecular probe which is formed by connecting an active group and a reactive group, the active group is a sophocarpidine derivative, the reactive group is a substance containing a diaziridine group and an alkyne group, and the chemical formula of the sophocarpidine derivative is as shown in the formula I. The invention further discloses a preparation method of the indole sophocarpidine derivative photoaffinity active molecular probe. After Spodoptera frugiperda Sf9 cells are treated by the indole sophocarpidine derivative photoaffinity active molecular probe for 72 hours, the inhibition rate can reach 86.33%, potential target protein when the sophocarpidine derivative acts on the Sf9 cells can be determined by utilizing the indole sophocarpidine derivative photoaffinity active molecular probe, targeted modification of sophocarpidine is facilitated, and the application range of the indole sophocarpidine derivative photoaffinity active molecular probe is widened. Further, matrine pesticides with excellent insecticidal activity are efficiently synthesized, and agricultural pests are better prevented and controlled.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an indolematrine derivative photoaffinity active molecular probe and application thereof. Background Art

[0002] Spodoptera frugiperda belongs to the family Noctuidae of the order Lepidoptera, also known as the fall armyworm. It is native to tropical and subtropical regions of the Americas. It has an extremely fast migration speed and can migrate long distances with the help of wind. Spodoptera frugiperda is also a polyphagous agricultural pest with the characteristics of omnivorous diet, large appetite and wide host range. It can feed on about 353 plants in 76 families, such as corn, rice, sugarcane, and sorghum. At the same time, the adult Spodoptera frugiperda has a strong reproductive capacity. Female insects can mate and lay eggs many times, and can lay about 900 to 1,000 eggs in their lifetime, and up to 2,000 eggs, which greatly promotes the reproduction of the population.

[0003] During the outbreak period, chemical control is still the main method of prevention and control of fall armyworm. However, fall armyworm has now developed obvious resistance to organophosphorus, nicotine and pyrethroid chemical pesticides.

[0004] Matrine (Mat) is a water-soluble alkaloid extracted from Sophora flavescens, Sophora tonkinensis or Sophora alopecuroides, which has a wide range of medical and agricultural activities and is a general term for a variety of alkaloids. Studies have found that matrine and its derivatives show a wide range of biological properties, and they play an important role in anti-cancer, anti-cardiovascular and cerebrovascular diseases, anti-virus, regulating the immune system, regulating the digestive system and anti-tumor. In addition, matrine and its derivatives are pesticides with no residue and low toxicity in the agricultural field, which are of great significance to the development of green ecology, the control of environmental pollution and the improvement of agricultural product quality.

[0005] In order to improve the biological activity of matrine, more and more studies have been conducted on the transformation and modification of related structures of matrine. However, due to the fact that the target proteins of matrine and its derivatives have not been clearly identified and the related mechanism of action is not clear, the research efficiency of matrine and its derivatives is low, and the synthesis and application process is relatively difficult. Among them, the design and synthesis of new derivatives of matrine are mainly based on experimental experience, which leads to relatively blind synthesis and development design. The activities of the new derivatives obtained are uneven, and it is impossible to carry out targeted development and design according to the needs of the experimental purpose, which makes the screening and application of new derivatives with high activity more difficult in the later stage, greatly increasing the economic cost of matrine research and development.

[0006] At present, there is an urgent need for molecular probes that can identify potential target proteins in S. frugiperda Sf9 cells, and then specifically transform and modify the structure of matrine to synthesize matrine derivative pesticides with better insecticidal activity. Summary of the invention

[0007] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a photoaffinity active molecular probe of an indolematrine derivative and its application.

[0008] The first object of the present invention is to provide a photoaffinity-active molecular probe of an indolematrine derivative.

[0009] The second objective of the present invention is to provide the use of the above-mentioned indolematrine derivative photoaffinity active molecular probe in determining the target of matrine pesticides.

[0010] The third object of the present invention is to provide the use of the above-mentioned indolematrine derivative photoaffinity active molecular probe in guiding agricultural pest control.

[0011] The fourth object of the present invention is to provide a method for determining the target of matrine pesticides.

[0012] In order to achieve the above object, the present invention is implemented by the following scheme:

[0013] An indolematrine derivative photoaffinity active molecular probe, wherein the indolematrine derivative photoaffinity active molecular probe is obtained by connecting an active group and a reactive group;

[0014] The active group is a matrine derivative, and the reactive group is a substance containing a diaziridine group and an alkyne group;

[0015] The chemical formula of the matrine derivative is shown in Formula I,

[0016]

[0017] When R1 is NH2, R2 is H; when R2 is NH2, R1 is H.

[0018] Preferably, the chemical formula of the matrine derivative is as shown in Formula II,

[0019]

[0020] Preferably, the substance containing a diazirine group and an alkyne group is alkyne-diazirine-acid.

[0021] More preferably, the chemical formula of the indolematrine derivative active molecular probe is as shown in Formula III,

[0022]

[0023] The present invention also claims the use of any of the above-mentioned indolematrine derivative photoaffinity active molecular probes in determining the target of matrine pesticides.

[0024] Preferably, the target is actin muscle-type A2, histone H4, SFRICE_009909, unconvertional myosin-XV and / or sulfatase D.

[0025] The present invention also seeks to protect the use of any of the above-mentioned indolematrine derivative photoaffinity active molecular probes in guiding agricultural pest control.

[0026] Preferably, the guiding agricultural pest control is guiding the synthesis of pesticides targeting the action site.

[0027] More preferably, the target is actin muscle-type A2, histone H4, SFRICE_009909, unconvertional myosin-XV and / or sulfatase D.

[0028] More preferably, the pesticide is a matrine pesticide.

[0029] More preferably, the insecticide is fall armyworm.

[0030] The present invention also claims a method for preparing a photoaffinity-active molecular probe of an indolematrine derivative, comprising the following steps:

[0031] S11. introducing 4-nitroindole or 5-nitroindole at position 13 of sophocarpine by Michael addition reaction, followed by hydrogenation reaction to obtain a matrine derivative as shown in Formula I;

[0032] S12. performing an amidation reaction on the active group and the reactive group to obtain an affinity-active molecular probe of an indolematrine derivative;

[0033] The active group is the matrine derivative obtained in step S11, and the reactive group is a substance containing a diazirine group and an alkyne group;

[0034]

[0035]

[0036] When 4-nitroindole is introduced in step S11, R1 is NH2 and R2 is H; when 5-nitroindole is introduced in step S11, R1 is H and R2 is NH2.

[0037] Preferably, step S11 is: introducing 4-nitroindole at position 13 of sophocarpine by Michael addition reaction.

[0038] Preferably, the hydrogenation reaction in step S11 is a Pd / C hydrogenation reduction reaction.

[0039] Preferably, the reactive group in step S12 is alkyne-diaziridine-acid.

[0040] The present invention also claims a method for determining the target of action of matrine pesticides, comprising the following steps:

[0041] S1. Co-incubating any of the above-mentioned indolematrine derivative photoaffinity active molecular probes with target cells, irradiating with ultraviolet light during the co-incubation period, lysing and collecting the supernatant, and obtaining an indolematrine derivative photoaffinity active molecular probe that specifically binds to the target cell protein;

[0042] The target cells are cells that act on matrine pesticides;

[0043] S2. Connect a reporter group to the indolematrine derivative photoaffinity-active molecular probe that specifically binds to the target cell protein obtained in step S1 to obtain an indolematrine derivative photoaffinity-active molecular probe that is connected to a reporter gene and specifically binds to the target cell protein, and perform proteomics detection to determine the target.

[0044] Preferably, the co-incubation time in step S1 is 30 min.

[0045] Preferably, the target cells are Sf9 cells.

[0046] Preferably, the reporter group in step S2 is biotin, rhodamine and / or BODLPY.

[0047] More preferably, the reporter group in step S2 is biotin.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] The present invention provides an indolematrine derivative photoaffinity active molecular probe, which is formed by connecting an active group and a reactive group, wherein the active group is a matrine derivative, and the reactive group is a substance containing a diazirine group and an alkyne group, and the chemical formula of the matrine derivative is shown in Formula I. After the indolematrine derivative photoaffinity active molecular probe of the present invention treats fall armyworm Sf9 cells for 72 hours, the inhibition rate can reach 86.33%, and the indolematrine derivative photoaffinity active molecular probe can be used to determine the potential target protein when the matrine derivative acts on the Sf9 cell, which is helpful for targeted transformation and modification of matrine, and then efficiently synthesizing matrine pesticides with excellent insecticidal activity, so as to achieve better prevention and control of agricultural pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The synthetic route of 13-(4-aminoindole)-matrine in Example 1;

[0051] Figure 2 This is a graph showing the FTIR test results of the reddish brown solid powder in Example 1;

[0052] Figure 3 is the MS graph of the reddish brown solid powder in Example 1;

[0053] Figure 4 is the reddish brown solid powder in Example 1 1 H NMR test results;

[0054] Figure 5 is the reddish brown solid powder in Example 1 13 C Nuclear magnetic resonance detection results;

[0055] Figure 6 The synthetic route of the photoaffinity-active molecular probe of the indolematrine derivative in Example 2;

[0056] Figure 7 This is the FTIR test result of the light yellow powder in Example 2;

[0057] Figure 8 This is the MS diagram of the light yellow powder in Example 2;

[0058] Fig. 9 For the light yellow powder in Example 2 1 H NMR test results;

[0059] Fig.10 Light yellow powder 13 C Nuclear magnetic resonance detection results;

[0060] Fig.11This is a graph showing the results of determining the inhibition rate of matrine at different concentrations on Spodoptera frugiperda Sf9 cells in Example 3;

[0061] Fig.12 The morphological characteristics of Spodoptera frugiperda Sf9 cells after being cultured with different concentrations of matrine in Example 3 are observed; A to D are the cell morphologies at 0 h, 24 h, 48 h and 72 h after being treated with 2 mM Mat, E to H are the cell morphologies at 0 h, 24 h, 48 h and 72 h after being treated with 2.5 mM Mat, and I to L are the cell morphologies at 0 h, 24 h, 48 h and 72 h after being treated with 3 mM Mat.

[0062] Fig.13 These are observation diagrams of the morphological characteristics of fall armyworm Sf9 cells after being cultured with different concentrations of 4-NO2-Ind-Mat in Example 3; A to D are the cell morphologies at 0 h, 24 h, 48 h and 72 h after being treated with 1 mM 4-NO2-Ind-Mat, E to H are the cell morphologies at 0 h, 24 h, 48 h and 72 h after being treated with 1 mM 4-NO2-Ind-Mat, and I to L are the cell morphologies at 0 h, 24 h, 48 h and 72 h after being treated with 2 mM 4-NO2-Ind-Mat, respectively;

[0063] Fig.14 These are observation diagrams of the morphological characteristics of Spodoptera frugiperda Sf9 cells after being cultured with different concentrations of 4-NH2-Ind-Mat in Example 3; A to D are the cell morphologies when treated with 1 mM 4-NH2-Ind-Mat for 0 h, 24 h, 48 h and 72 h, respectively; E to H are the cell morphologies when treated with 1.5 mM 4-NH2-Ind-Mat for 0 h, 24 h, 48 h and 72 h, respectively; I to L are the cell morphologies when treated with 2 mM 4-NH2-Ind-Mat for 0 h, 24 h, 48 h and 72 h, respectively;

[0064] Fig.15These are observation diagrams of morphological characteristics of Spodoptera frugiperda Sf9 cells cultured with different concentrations of 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine in Example 3; A to D are cell morphologies at 0 h, 24 h, 48 h and 72 h after treatment with 1 mM 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine, respectively; E to H are cell morphologies at 0 h, 24 h, 48 h and 72 h after treatment with 1.5 mM 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine, respectively; I to L are cell morphologies at 0 h, 24 h, 48 h and 72 h after treatment with 2 mM 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine, respectively;

[0065] Fig.16 The silver staining results of different proteins in Example 4 are shown;

[0066] Fig.17 The figure is a search and identification result diagram of the actin protein in Example 4;

[0067] Fig.18 is the secondary mass spectrum of the unique peptide in the antin protein in Example 4;

[0068] Fig.19 This is a diagram showing the search and identification results of the Histone H4 protein in Example 4;

[0069] Fig. 20 The secondary mass spectrum of the unique peptide in the Histone H4 protein in Example 4;

[0070] Fig.21 This is the search and identification result diagram of SFRICE_009909 in Example 4;

[0071] Fig. 22 This is a search and identification result diagram of SFRICE_002323 (Fragment) in Example 4;

[0072] Fig.23 This is a search and identification result diagram of Sulfatase D in Example 4;

[0073] Fig.24 is the secondary mass spectrum of the unique peptide in the SFRICE_009909 protein in Example 4;

[0074] Fig.25is the secondary mass spectrum of the unique peptide in SFRICE_002323 (Fragment) in Example 4;

[0075] Fig.26 This is the secondary mass spectrum of the unique peptide in Sulfatase D in Example 4. DETAILED DESCRIPTION

[0076] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0077] Example 1 Preparation and testing of active groups

[0078] 1. Experimental Methods

[0079] The synthetic route of 13-(4-aminoindole)-matrine (i.e., matrine derivative, 4-NH2-Ind-Mat) is as follows Figure 1 As shown, the details are as follows:

[0080] 1. Synthesis of 13-(4-nitroindole)-matrine (4-NO2-Ind-Mat)

[0081] Into a 50mL three-necked flask, 0.3745g of sophoracarpine (1.520mmol), 0.3745g of 4-nitroindole (2.31mmol), 0.1453g of cesium carbonate (0.4460mmol) and 12mL of 1,4-dioxane (analytical grade) were added, and the reaction was condensed and refluxed at 120°C for 8h. After the reaction was completed, 10mL of dichloromethane was added after cooling to 25°C and filtered. The filtrate was collected and rotary evaporated to obtain a crude product. The crude product was purified by column chromatography (dichloromethane: ethyl acetate: anhydrous methanol = 15:4:1, V:V) to obtain a brown-yellow solid (4-NO2-Ind-Mat) with a yield of 40.15%.

[0082] 2. Synthesis of 4-NH2-Ind-Mat

[0083] Into a 25 mL branch flask, 15 mL of anhydrous methanol, 0.1000 g of 4-nitroindolematrine (0.2448 mmol), and 0.02 g of Pd / C (10%, w / w) were added in sequence. The gas in the branch flask was replaced with hydrogen using a hydrogen balloon. The replacement was repeated three times to fill the branch flask with hydrogen.

[0084] After stirring at 50° C. for 6 h, the Pd / C was removed by suction filtration to obtain a red-brown liquid. The red-brown liquid was purified by column chromatography (dichloromethane:methanol=10:1, V:V) to obtain a red-brown solid powder with a yield of 97.84%.

[0085] The reddish brown solid powder was subjected to infrared spectroscopy (FTIR), mass spectrometry (MS) and nuclear magnetic resonance (NMR) detection, and the corresponding test results were recorded; the nuclear magnetic resonance detection includes 1 H NMR detection and 13 C nuclear magnetic resonance detection.

[0086] 2. Experimental Results

[0087] The FTIR test results of reddish brown solid powder are shown in the figure Figure 2 As shown, the result shows: 3346cm -1 and 3347cm -1 The peak at 3044 cm is the H stretching vibration absorption peak on C-NH2. -1 The carbon-hydrogen stretching vibration absorption peak on the benzene ring is 1632 cm -1 The peak of lactam C=O stretching vibration is 1477 cm -1 The C=C vibration absorption peak of the benzene ring skeleton is 731 cm -1 It is the CN stretching vibration absorption peak.

[0088] The MS image of the reddish brown solid powder is as follows Figure 3 As shown, the results show that the relative molecular mass (M) of 13-(4-aminoindole)-matrine is 378.5, and the molecular ion peak m / z in the mass spectrum is 379.8, which is [M+H] + peak, which is consistent with the theoretical value; Figure 3 There is also a peak of m / z=757.2, which is a diploid peak [2M+H] caused by too high injection concentration. + , thus confirming that the nitro group on 13-(4-nitroindolyl)matrine was successfully reduced to an amino group.

[0089] Reddish brown solid powder 1 The results of H NMR detection are shown in Figure Figure 4 As shown, 1H NMR (500MHz, DMSO-d6) 7.06 (d, J=3.3Hz, 1H), 6.83 (t, J=7.8Hz, 1H), 6.69 (d, J=8.2Hz, 1H), 6.57 (d, J=3.3 Hz, 1H), 6.18 (d, J=7.4Hz, 1H), 5.21 (s, 2H), 4.85-4.74 (m, 1H), 4.20 (dd, J=12.3, 4.4Hz, 1H), 2.96 (d, J=1 2.4Hz, 1H), 3.80-3.71 (m, 1H), 2.76 (s, 1H), 2.69 (dd, J=12.0, 3.3Hz, 3H), 2.50 (p, J=1.8Hz, 1H), 2.37-2. 28 (m, 1H), 2.09 (t, J=3.0Hz, 1H), 2.04-1.95 (m, 1H), 1.83 (dq, J=13.9, 5.6, 5.0Hz, 3H), 1.62-1.19 (m, 8H).

[0090] Reddish brown solid powder 13 C NMR test results are shown in Figure Figure 5 As shown, 13 C NMR (126MHz, DMSO-d6) δ166.52, 142.16, 137.03, 123.18, 121.62, 117.31, 102.65, 100.04, 98.53, 63. 93, 57.04 (d, J=2.4Hz), 51.09, 46.47, 42.26, 40.79, 38.47, 35.83, 30.41, 27.91, 26.79, 21.20, 20.50.

[0091] Combination Figure 2 to Figure 5 As shown in the results, the reddish brown solid powder is recorded as 13-(4-aminoindole)-matrine (4-NH2-Ind-Mat), and the chemical formula of 4-NH2-Ind-Mat is shown in Formula II;

[0092]

[0093]

[0094] Example 2 Preparation and Detection of Photoaffinity-Active Molecular Probes of Indolematrine Derivatives

[0095] 1. Experimental Methods

[0096] The synthetic route of the photoaffinity active molecular probe of indolematrine derivative, namely 13-[(4-diaziridine-7-alkyne-1-octanamide)indole]matrine is as follows: Figure 6 As shown, the details are as follows:

[0097] Into a 50 mL flask, 1 equiv of alkyne-diaziridine-acid, 1.2 equiv of 4-NH2-Ind-Mat prepared in Example 1, 2.1 equiv of 1-methylimidazole, 1.1 equiv of N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate and 16 mL of acetonitrile were added in sequence, and the mixture was stirred at 25 ° C for 24 h, followed by rotary evaporation to obtain a crude product, which was purified by column chromatography (dichloromethane: anhydrous methanol = 10:1, v / v) to obtain a light yellow powder with a yield of 73.52%.

[0098] The light yellow powder was subjected to infrared spectroscopy (FTIR), mass spectrometry (MS) and nuclear magnetic resonance (NMR) detection, and the corresponding test results were recorded; the NMR detection includes 1 H NMR detection and 13 C nuclear magnetic resonance detection.

[0099] 2. Experimental Results

[0100] The FTIR test results of the light yellow powder are as follows Figure 7 As shown, the result shows: 3288cm -1 The amide NH stretching vibration absorption peak is at 3061 cm -1 The peak at 2925 cm is the CH stretching vibration absorption peak on the benzene ring. -1 The absorption peak of CH stretching vibration of alkanes is 2116 cm -1 The peak at 1632cm is the stretching vibration absorption peak of the carbon-carbon triple bond of alkyne; -1 The peak at 1540 cm is the C=O stretching vibration absorption peak of lactam. -1 The peaks are NH bending and CN stretching vibrations, 1431 cm -1 The C=C vibration absorption peak on the benzene ring skeleton is 1133cm -1 The CN stretching vibration peak is 742 cm -1 It is the CN stretching vibration absorption peak.

[0101] The MS image of the light yellow powder is as follows Figure 8 The results show that the molecular formula of 13-[(4-diaziridine-7-alkyne-1-octanamide)indole]matrine is C 31 H 38 N6O2, its theoretical relative molecular mass M is 526.69, [M+1] and m / z=527.4 in the figure, which is the [M+H]+ peak, are basically consistent with the theoretical value.

[0102] Light yellow powder 1 The results of H NMR detection are shown in Figure Fig. 9 As shown,1 H NMR (500MHz, DMSO-d6) δ7.46 (d, J=7.5Hz, 1H), 7.29 (d, J=8.3Hz, 1H), 7.17 (s, 2H), 7.15 (d, J=4.5Hz, 1H), 6.68 (d, J=3.4Hz, 1H) , 4.92 (d, J = 7.2Hz, 1H), 4.83 (s, 2H), 4.34 (dd, J = 12.6, 4.4Hz, 1H), 3.84 (dd, J = 11.2, 5.7Hz, 1H), 3.11 (s, 1H), 2.99-2.92 (m, 1H) , 2.85 (d, J = 7.2Hz, 1H), 2.81 (d, J = 2.5Hz, 4H), 2.79-2.72 (m, 2H), 2.48 (d, J = 5.8Hz, 1H), 2.36-2.23 (m, 5H), 2.15-2.09 (m, 1H), 2.06 (d, J=7.6Hz, 1H), 2.00 (dd, J=12.0, 3.3Hz, 1H), 1.93-1.85 (m, 3H), 1.67 (q, J=7.5, 6.8Hz, 3H), 1.33 (q, J=12.6, 9.9Hz, 3H).

[0103] Light yellow powder 13 C NMR test results are shown in Figure Fig.10 As shown, 13 C NMR (126MHz, DMSO-d6) δ173.04, 169.41, 138.15, 131.43, 124.53, 123.55, 122.98, 114.36, 107.76, 100.86, 83.68, 70.38, 65.15, 58.18, 52.42, 47.87, 43.44, 42.82, 38.91, 38.66, 37.06, 33.52, 31.60, 31.50, 29.86, 29.02, 28.70, 27.43, 22.08, 21.42, 13.92.

[0104] Combination Figure 7 to Figure 10 The results shown in the figure show that the light yellow powder is an indole matrine derivative photoaffinity active molecular probe (i.e., 13-[(4-Bisacridine-7-alkyne-1-octylamide) Indole] Matrine), and its chemical formula is shown in Formula III;

[0105]

[0106] Example 3 Effects of various groups and indolematrine derivative photoaffinity active molecular probes on the activity of Sf9 cells

[0107] 1. Experimental Methods

[0108] The effects of matrine (Mat), 13-(4-nitroindole)-matrine (4-NO2-Ind-Mat) prepared in Example 1, 13-(4-aminoindole)-matrine (4-NH2-Ind-Mat) prepared in Example 1, and the indolematrine derivative photoaffinity active molecular probe prepared in Example 2 on the activity of Spodoptera frugiperda Sf9 cells were detected respectively. Taking the detection of matrine (Mat) as an example, the specific results are as follows:

[0109] 1. Effect of Matrine on the Activity of Spodoptera frugiperda Sf9 Cells

[0110] Select healthy and well-shaped Spodoptera frugiperda Sf9 cells and prepare them to a cell density of 1×10 6 Then, 1.2×10 cells / mL of cell suspension were added to each well of a 96-well plate. 5 The cells were added to Spodoptera frugiperda Sf9 cells and pre-cultured at 27°C for 24 h.

[0111] Matrine (Mat) was then added to the 96-well plate to obtain cell wells with Mat concentrations of 1.0 mM, 2.0 mM, 3.0 mM, 4.0 mM and 5.0 mM, respectively. Three experimental time points (24 h, 48 h and 72 h) were set for the cell wells of each matrine concentration, and 4 wells were repeated at each time point, for a total of 3×4=12 wells.

[0112] After adding matrine to the 96-well plate, when the cell wells reach the corresponding experimental time point, 10 μL of methyl tetrazolium blue (MTT) is added to each well, and the wells are sealed and placed in a 27°C incubator away from light for 4 hours. After the treatment, the liquid in the wells is discarded, and 150 μL of dimethyl sulfoxide (DMSO) is added to each well to dissolve the formazan crystals produced in the cells. After the crystals are completely dissolved, the OD value corresponding to the cell wells at 492 nm is measured and recorded as OD treatment .

[0113] The blank control wells (Background) are: 1.2×10 5 After adding the Spodoptera frugiperda Sf9 cells, 0.5% (w / w) DMSO was added for culture;

[0114] Control wells: 1.2×10 5 After adding Spodoptera frugiperda Sf9 cells, PBS (phosphate buffer) was added at 150 μL / well.

[0115] Determine the OD value corresponding to the blank control well at 492nm and record it as OH background ; At the same time, the OD value corresponding to the control well at 492nm was measured and recorded as OD control ; The inhibition rate of matrine on Spodoptera frugiperda Sf9 cells was calculated according to formula I;

[0116]

[0117] 2. Inverted phase contrast microscopy (IPCM) morphological feature detection

[0118] Select Spodoptera frugiperda Sf9 cells with good morphology and prepare them to a cell density of 1×10 6 Cell suspension of cells / mL was added to each well of a 12-well plate at a rate of 0.5 mL. After culturing for 24 h, matrine (Mat) was then added to a 96-well plate to obtain cell wells with Mat concentrations of 2.0 mM, 2.5 mM, and 3.0 mM. The cell morphology in the cell wells was observed using an inverted phase contrast microscope (IPCM) at 0 h, 24 h, 48 h, and 72 h of culture, respectively.

[0119] Matrine (Mat) was replaced by 4-NO2-Ind-Mat, 4-NH2-Ind-Mat and 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine prepared in Example 2, and the same treatment was performed to detect the effects of 4-NO2-Ind-Mat, 4-NH2-Ind-Mat and 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine on the activity of Spodoptera frugiperda Sf9 cells and the activity of 4-NO2-Ind-Mat, 4-NH2-Ind-Mat and 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine. The morphological characteristics of Spodoptera frugiperda Sf9 cells after treatment with ne; when testing the effect on the activity of Spodoptera frugiperda Sf9 cells, the concentrations of 4-NO2-Ind-Mat, 4-NH2-Ind-Mat and 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine were 0.25mM, 0.50Mm, 1.00mM, 1.50mM and 2.00mM, and when detecting the morphological characteristics of Spodoptera frugiperda Sf9 cells after treatment, the concentrations of 4-NO2-Ind-Mat, 4-NH2-Ind-Mat and 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine were 1.0mM, 1.5mM and 2.00mM.

[0120] 2. Experimental Results

[0121] The results of the inhibition rate determination of different concentrations of matrine on Spodoptera frugiperda Sf9 cells are shown in Fig.11 And as shown in Table 1.

[0122] Table 1 Inhibition rate of different concentrations of matrine on Spodoptera frugiperda Sf9 cells

[0123]

[0124]

[0125] The results showed that after treating Sf9 cells with Mat for 24 hours, even when the Mat concentration was 3.0 mM, it only showed a weak inhibitory effect (14.27%); it was not until 48 hours after treating Sf9 cells that different concentrations of Mat showed a more obvious inhibitory effect on Sf9 cells, but the inhibitory activity was weak. When Sf9 cells were treated with 3.0 mM Mat for 48 hours, the inhibition rate was only 25.47%.

[0126] The morphological characteristics of Spodoptera frugiperda Sf9 cells cultured with different concentrations of matrine are shown in the figure below. Fig.12 As shown, A to D are the cell morphologies at 0 h, 24 h, 48 h and 72 h after treatment with 2 mM Mat, E to H are the cell morphologies at 0 h, 24 h, 48 h and 72 h after treatment with 2.5 mM Mat, and I to L are the cell morphologies at 0 h, 24 h, 48 h and 72 h after treatment with 3 mM Mat.

[0127] The results showed that after Sf9 cells were treated with low concentration (<3mM) of Mat for 24 hours, only a small number of cells showed vacuolation, with no significant difference from the untreated state. When the Mat concentration reached 3.0mM and the treatment lasted for more than 48 hours, the number of vacuolated cells increased significantly, and some cells began to swell and become round, and began to disintegrate.

[0128] The results of the inhibition rate determination of different concentrations of 4-NO2-Ind-Mat on Spodoptera frugiperda Sf9 cells are shown in Table 2.

[0129] Table 2 Inhibition rate of different concentrations of 4-NO2-Ind-Mat on Spodoptera frugiperda Sf9 cells The results showed that after the cells were treated with 4-NO2-Ind-Mat at concentrations of 0.25, 0.50, 1.00, 1.50, and 2.00 mmol / L for 24, 48, and 72 hours, the inhibitory activity of each treatment group on the growth of Sf9 cells was higher than that of the 2 mmol / L Mat treatment group for the same time. With the continuous increase of drug concentration and treatment time, the inhibitory activity of 4-NO2-Ind-Mat on the growth of Sf9 cells also continued to increase, indicating that the inhibition of cell proliferation by 4-NO2-Ind-Mat was time-concentration dependent. Among them, after 72 hours of treatment with 2.0 mmol / L 4-NO2-Ind-Mat, the inhibition rate reached 75.23%, which was the highest inhibition rate among the five treatment groups.

[0130] The morphological characteristics of Spodoptera frugiperda Sf9 cells cultured with different concentrations of 4-NO2-Ind-Mat are shown in the figure below. Fig.13 As shown, A to D are the cell morphologies when treated with 1 mM 4-NO2-Ind-Mat for 0 h, 24 h, 48 h and 72 h, respectively; E to H are the cell morphologies when treated with 1 mM 4-NO2-Ind-Mat for 0 h, 24 h, 48 h and 72 h, respectively; I to L are the cell morphologies when treated with 2 mM 4-NO2-Ind-Mat for 0 h, 24 h, 48 h and 72 h, respectively.

[0131] The results showed that after 24 hours of treatment with drugs at various concentrations, some cells in the 1.0mmol / L, 1.5mmol / L, and 2.0mmol / L treatment groups showed cavitation, and with the increase of concentration, the proportion of cavitation in each treatment group increased; among them, the 2.0mmol / L treatment group began to show cell shrinkage and cell disintegration. After 48 hours of treatment with drugs at various concentrations, more cavitation and a small number of cells in the 1.0mmol / L and 1.5mmol / L treatment groups showed shrinkage or disintegration, while most of the cells in the 2.0mmol / L treatment group had disintegrated; after 72 hours of treatment with drugs at various concentrations, some cells in the 1.0mmol / L and 1.5mmol / L treatment groups showed shrinkage or disintegration; the 2.0mmol / L treatment group still showed a large number of cell disintegration.

[0132] The results of the inhibition rate determination of different concentrations of 4-NH2-Ind-Mat on Spodoptera frugiperda Sf9 cells are shown in Table 3.

[0133] Table 3 Inhibition rate of different concentrations of 4-NH2-Ind-Mat on Spodoptera frugiperda Sf9 cells The results showed that after the cells were treated with 4-NH2-Ind-Mat at concentrations of 0.25, 0.50, 1.00, 1.50, and 2.00 mmol / L for 24, 48, and 72 hours, the inhibitory activity of each treatment group on the growth of Sf9 cells was higher than that of the 2 mmol / L Mat treatment group for the same time; with the continuous increase of drug concentration and treatment time, the inhibitory effect of 4-NH2-Ind-Mat at each concentration on the growth of Sf9 cells also increased, indicating that 4-NH2-Ind-Mat has a time-concentration dependence on cell proliferation inhibition. Among them, after 72 hours of treatment with 2.0 mmol / L 4-NH2-Ind-Mat, the inhibition rate reached 56.63%, which was the highest inhibition rate among the five treatment groups.

[0134] The morphological characteristics of Spodoptera frugiperda Sf9 cells cultured with 4-NH2-Ind-Mat at different concentrations are shown in the figure below. Fig.14 As shown, A to D are the cell morphologies when treated with 1 mM 4-NH2-Ind-Mat for 0 h, 24 h, 48 h and 72 h, respectively; E to H are the cell morphologies when treated with 1.5 mM 4-NH2-Ind-Mat for 0 h, 24 h, 48 h and 72 h, respectively; I to L are the cell morphologies when treated with 2 mM 4-NH2-Ind-Mat for 0 h, 24 h, 48 h and 72 h, respectively.

[0135] The results showed that after 24 hours of drug treatment at various concentrations, some Sf9 cells in the 1.0mmol / L, 1.5mmol / L, and 2.0mmol / L treatment groups showed vacuolation, and the proportion of vacuolated cells increased with the increase of drug concentration; after 48 hours of drug treatment at various concentrations, more vacuolated cells appeared in the 1.0mmol / L and 1.5mmol / L treatment groups, and a small number of cells in the 2.0mmol / L treatment group shrank or disintegrated. After 72 hours of drug treatment at various concentrations, cells in the 1.0mmol / L and 1.5mmol / L treatment groups showed shrinkage or disintegration, and a large number of cells in the 2.0mmol / L treatment group disintegrated.

[0136] The results of the inhibition rate determination of different concentrations of 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine on Spodoptera frugiperda Sf9 cells are shown in Table 4.

[0137] Table 4 Inhibition rate of different concentrations of 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine on Spodoptera frugiperda Sf9 cells

[0138]

[0139] The results showed that when Sf9 cells were treated with 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine, except for the 0.25mM experimental group, the inhibitory effect of this substance on cells was equivalent to that of the active group indolematrine derivative 4-NO-Ind-Mat; and as the cell treatment time of each concentration of 13-[(4-biaziridine-7-alkyne-1-octylamide)indole]matrine molecular probe increased, the corresponding cell inhibitory activity was similar to that of other indolematrine derivatives as active groups; after 72h of treatment with 2mmol / L 13-[(4-biaziridine-7-alkyne-1-octylamide)indole]matrine molecular probe, the inhibition rate reached 86.33%.

[0140] The morphological characteristics of Spodoptera frugiperda Sf9 cells cultured with different concentrations of 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine are shown in the figure below. Fig.15As shown, A to D are the cell morphologies at 0 h, 24 h, 48 h and 72 h after treatment with 1 mM 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine, respectively; E to H are the cell morphologies at 0 h, 24 h, 48 h and 72 h after treatment with 1.5 mM 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine, respectively; I to L are the cell morphologies at 0 h, 24 h, 48 h and 72 h after treatment with 2 mM 13-[(4-Bisacridine-7-alkyne-1-octylamide)Indole]Matrine, respectively.

[0141] The results showed that after 24 hours of treatment with 13-[(4-diaziridine-7-alkyne-1-octanamide) indole] matrine molecular probe, the cell morphology of Sf9 cells changed. Some cells in the three experimental groups showed vacuolization, and the proportion increased with the increase of concentration. After 48 hours of treatment, the cell morphology of the 1.0mmol / L and 1.5mmol / L experimental groups showed more vacuolization and some cells disintegrated and shrank; more cells disintegrated in the 2mmol / L experimental group. After 72 hours of treatment, the cell morphology of the three concentration experimental groups showed obvious abnormalities; after 72 hours of treatment with 2mmol / L 13-[(4-diaziridine-7-alkyne-1-octanamide) indole] matrine molecular probe, a large number of cells shrank or disintegrated, showing obvious apoptotic characteristics.

[0142] Example 4: Fishing and identification of proteins using photoaffinity-active molecular probes of indolematrine derivatives

[0143] 1. Identification of specific protein bands

[0144] 1. Experimental methods

[0145] (1) Sf9 cell culture dish setup

[0146] The cell density in 2 mL was 0.5 × 10 5 ~1.0×10 6 Well-grown Sf9 cells at a concentration of 1 mM were inoculated into a 25 mm culture dish at an inoculation area of ​​80%, and pre-cultured in a 27°C incubator for 24 h. The culture dish was then taken out and placed in a clean bench to discard the original solution. After washing with PBS, 2 mL of the indolematrine derivative photoaffinity-active molecular probe prepared in Example 2 was added to obtain an Sf9 cell culture dish co-incubated with the photoaffinity-active molecular probe.

[0147] The cell density in 2 mL was 0.5 × 105 ~1.0×10 6 Well-grown Sf9 cells at a concentration of 1 mM were inoculated into a 25 mm culture dish at an inoculation area of ​​80%, and pre-cultured in a 27°C incubator for 24 h. The culture dish was then taken out and placed in a clean bench to discard the original solution. After washing with PBS, 2 mL of 1 mM 4-NH2-Ind-Mat prepared in Example 1 was added to obtain a Sf9 cell culture dish co-incubated with 4-NH2-Ind-Mat.

[0148] The cell density in 2 mL was 0.5 × 10 5 ~1.0×10 6 Well-growing Sf9 cells of 100 / mL were inoculated into a 25 mm culture dish according to an inoculation area of ​​80%, and pre-cultured in a 27°C incubator for 24 hours. Then, the culture dish was taken out and placed in an ultra-clean workbench to discard the original solution. After washing with PBS, 2 mL of 1 mM biotin was added to obtain a biotin-co-incubated Sf9 cell culture dish.

[0149] (2) Indolematrine photoaffinity active molecular probe to fish for potential targets in Sf9 cells

[0150] The Sf9 cell culture dish co-incubated with the photoaffinity active molecular probe was placed in a 27°C incubator for 24 hours. After irradiation with ultraviolet light for 30 minutes during the culture period, the culture medium was discarded, the cells were washed with PBS, and 0.5 mL of RIPA lysis buffer (containing PMSF) was added to the culture dish. After shaking to fully contact the cells, it was incubated on ice for 30 minutes, followed by centrifugation at 10,000 g and 4°C for 8 minutes, and the supernatant (indolematrine photoaffinity active molecular probe specifically bound to Sf9 cell protein) was collected.

[0151] The protein concentration in the supernatant was determined using a BCA protein concentration assay kit (P0012S, purchased from Biotime) according to its instructions, and the supernatant was diluted with pre-cooled PBS to a concentration of 1 mg / mL of a pure protein solution (indolylmatrine photoaffinity active molecular probe specifically bound to Sf9 cell protein).

[0152] Then, a Click reaction reagent was added to the pure protein solution, wherein the Click reaction reagent contained 10 μL of THPTA (10 mM aqueous solution), 10 μL of TCEP (100 mM aqueous solution), 10 μL of copper sulfate (100 mM aqueous solution) and 10 μL of azide-biotin (5 mM dissolved in DMSO), and the mixture was placed at 25°C for a Click reaction for 3 hours to introduce biotin (reporter group) to the indolematrine photoaffinity active molecular probe to form an indolematrine photoaffinity active molecular probe connected with a reporter group as shown in Formula IV, and then 4 times the volume of the Click reaction reagent was added with acetone (precooled at -20°C), and the mixture was placed at -20°C for precipitation for 30 minutes, and then centrifuged at 11000 g and 4°C for 15 minutes using a refrigerated centrifuge, and the centrifugation was repeated twice, the precipitate was collected, washed and dried, and re-dissolved with PBS to obtain a protein solution to be tested;

[0153]

[0154] (3) BCA method for protein concentration determination

[0155] The total protein concentration extracted from the cells was determined using a BCA protein concentration assay kit (P0012S, purchased from Bio-Tech), and the specific operation was as follows:

[0156] According to the different concentrations of protein standards shown in Table 5, the OD values ​​of the different concentrations of protein standards in Table 5 at 562 nm were tested using a microplate reader, and △OD was calculated; △OD = OD n -OD0, where OD0 is the OD value of the protein standard with a concentration of 0 at 562 nm, OD n is the OD value of protein standard at each concentration at 562 nm.

[0157] Table 5 Protein standards of different concentrations

[0158] Serial number / n Protein standard concentration (mg / mL) <![CDATA[OD 562 ]]> <![CDATA[△OD(OD n -OD0)]]> 0 0 <![CDATA[OD0]]> <![CDATA[OD0-OD0]]> 1 0.025 <![CDATA[OD1]]> <![CDATA[OD1-OD0]]> 2 0.05 <![CDATA[OD2]]> <![CDATA[OD2-OD0]]> 3 0.1 <![CDATA[OD3]]> <![CDATA[OD3--OD0]]> 4 0.2 <![CDATA[OD4]]> <![CDATA[OD4-OD0 <!-- 12 -->]]> 5 0.3 <![CDATA[OD5]]> <![CDATA[OD5-OD0]]> 6 0.4 <![CDATA[OD6]]> <![CDATA[OD6-OD0]]> 7 0.5 <![CDATA[OD7]]> <![CDATA[OD7-OD0]]>

[0159] The concentrations of protein standards of different concentrations were combined with their △OD values ​​and substituted into the linear equation shown in Formula I to calculate the k value of Formula I, thereby obtaining the protein concentration linear standard equation.

[0160] Formula I: C 蛋白 (mg / mL) = 10 × {(OD n -OD0) / k}.

[0161] The OD value of the protein solution to be tested at 562 nm was measured using an enzyme marker, and the difference between the OD value and OD0 was calculated and substituted into the protein concentration linear standard equation to obtain the protein concentration of the protein solution to be tested.

[0162] (4) Protein enrichment using the biotin-streptavidin system

[0163] Place 100 μL of Streptavidin Magpoly Beads liquid on a magnetic separator, discard the liquid after magnetic separation until clear, then resuspend with 500 μL of balancing solution (0.1 M PBS) and 500 μL of washing solution (0.15 M NaCl, pH 7.0), mix and shake thoroughly to wash, then place on a magnetic separation rack for separation, discard the liquid to obtain the magnetic beads, and repeat the washing twice.

[0164] Add 50 μL of magnetic beads to the protein solution to be tested, place it on a rotating mixer for mixing and incubation for 60 minutes, and place it on a magnetic separation rack for magnetic separation after the incubation to obtain magnetic beads adsorbed with proteins and residual protein solution; use a washing solution (0.15 M NaCl, pH 7.0) to fully wash the magnetic beads adsorbed with proteins to obtain magnetic beads adsorbed with proteins; and collect the residual protein after the first washing, the third washing solution, the sixth washing solution and the ninth washing solution respectively.

[0165] After washing, add 100 μL of SDS (0.1%, v / v) to each tube and heat at 95°C for 3 min to elute to obtain an eluate containing protein. 中和液 :V 洗脱液 =1:10, the eluate containing protein and the neutralization solution (from the Tris-HCl kit) were mixed to obtain a protein sample; then V 蛋白样品 :V 蛋白上样缓冲液 The protein sample and protein loading buffer (5×Protein loading buffer) were mixed evenly in a ratio of 4:1, placed in a 100°C high-temperature water bath for inactivation for 6 min, cooled and centrifuged, and the electrophoresis loading solution was collected.

[0166] The total protein in the Sf9 cell culture dish co-incubated with the photoaffinity active molecular probe was extracted using a total protein extraction kit according to the kit instructions to obtain the matrine derivative-Sf9 cell total protein.

[0167] The total protein in the Sf9 cell culture dish co-incubated with 4-NH2-Ind-Mat in step (1) was extracted using a total protein extraction kit according to the kit instructions to obtain 4-NH2-Ind-Mat-Sf9 cell total protein.

[0168] As shown in step (2), the Sf9 cell culture dish co-incubated with biotin shown in step (1) is treated to obtain a biotin co-incubated Sf9 test protein solution; 50 μL of the above-mentioned magnetic beads are added to the biotin co-incubated Sf9 test protein solution, and the solution is placed on a rotating mixer for mixing and incubation for 60 minutes. After the incubation, the solution is placed on a magnetic separation rack for magnetic separation to obtain magnetic beads 1 adsorbed with proteins and residual protein solution 1, and the protein on the magnetic beads 1 adsorbed with proteins is eluted to obtain a biotin co-incubated Sf9 protein sample solution.

[0169] (5) Gel-based affinity proteomics verification

[0170] Prepare separation gel and concentrated gel according to the ingredients shown in Table 6, pour the prepared separation gel into the electrophoresis gel plate, let it stand for 30 minutes, then add the concentrated gel, let it stand for 30 minutes to obtain the electrophoresis gel plate, and install the electrophoresis gel plate into the electrophoresis tank.

[0171] Table 6 Separation gel and stacking gel composition

[0172] Components Volume of separation gel used / mL Volume of stacking gel used / mL Double distilled water 4.6 2.1 30% (w / w) acrylamide 2.7 0.5 Tris-HCl 1.5M (pH = 8.8) 2.5 / Tris-HCl 1.0M (pH = 6.8) / 0.38 10%SDS(w / w) 0.1 0.03 10%(w / w)AP 0.1 0.03 TEMED 0.006 0.003

[0173] A 10-hole electrophoresis comb was used to process the electrophoresis gel plate, and samples were loaded on the corresponding lanes of the electrophoresis gel plate according to the sample types and gel loading amounts shown in Table 7.

[0174] Table 7 Sample types and gel loading amounts

[0175] Lanes Sample Type Silver staining amount 1 <![CDATA[Marker BeyoColor TM Color Prestained Protein Molecular Weight Standard (6.5-270kDa) Set]]> 1μL 2 Total protein of Sf9 cells 2μg 3 <![CDATA[Total protein of 4-NH2-Ind-Mat-Sf9 cells]]> 2μg 4 Biotin co-incubated Sf9 protein sample solution 200 μg 5 Remaining protein liquid 1 200 μg 6 Electrophoresis loading solution 200 μg 7 Remaining protein liquid 200 μg 8 The third washing liquid 200 μg 9 6th washing solution 200 μg 10 9th washing liquid 200 μg

[0176] Then fill the inner and outer tanks of the electrophoresis instrument with electrophoresis buffer, and control the voltage of the electrophoresis instrument to 220V. When the samples of each group enter the separation gel, adjust the voltage to 120V. When it reaches the bottom of the separation gel, turn off the electrophoresis instrument and remove the electrophoresis gel plate. Use the rapid silver staining kit (BL620A) to perform protein silver staining on the electrophoresis gel plate according to the kit instructions, and record the silver staining results of different proteins.

[0177] 2. Experimental results

[0178] Silver staining results of different proteins are shown in the figure Fig.16 As shown, the results showed that in lane 6, after the protein sample of the photoaffinity probe co-incubation group adsorbed and eluted by streptavidin magnetic beads was separated by electrophoresis, a specific target protein band appeared at around 66Kda.

[0179] The electrophoresis results of total cell protein in lane 2 and the electrophoresis results of total protein extracted by 4-NH2-Ind-Mat co-incubation in lane 3 were used as references, proving that the target protein (66Kda) existed in the total protein before adsorption and separation; while in the electrophoresis results of the protein sample in lane 4 in which biotin was co-incubated with cells and eluted by adsorption, no protein band was found at 66Kda; in the remaining protein sample in lane 5 in which biotin was co-incubated with cells and adsorbed, a protein band appeared at 66Kda, indicating that the protein band at 66Kda was not a specific protein binding band of biotin.

[0180] Compared with lane 4, the electrophoresis results of lane 6 clearly show an additional protein band at 66 Kda; lane 7 is the electrophoresis band of the remaining protein sample after the photoaffinity probe was co-incubated with cells and adsorbed, and no protein band appeared at 66 Kda; the electrophoresis results of lanes 8, 9, and 10 show no protein band at 66 Kda, indicating that the protein at 66 Kda is a protein band that can specifically bind to the indolematrine derivative photoaffinity active molecular probe prepared in Example 2.

[0181] 2. Affinity proteomics detection based on high-resolution mass spectrometry

[0182] 1. Experimental methods

[0183] (1) Proteolysis

[0184] Will Fig.16 The protein band at the molecular weight of 66Kda in lane 6 was cut to obtain an electrophoresis gel strip containing the target protein.

[0185] Place the electrophoresis strip in an EP tube and wash it twice with ultrapure water, each time for 1 min. After washing, discard the liquid to obtain the washed electrophoresis strip. Mix 30mM K3Fe(CN)6 and 100mM NaS2O3 in a volume ratio of 1:1, add the washed electrophoresis strip until it is submerged, and after the silver color of the washed electrophoresis strip is removed, discard the liquid in the EP tube and wash it twice with ultrapure water to obtain a colorless electrophoresis strip.

[0186] Then, 100% (w / w) ACN was added to the EP tube, and after shaking for 30 seconds, the liquid was discarded to obtain a white strip after the colorless electrophoresis strip turned white, and the dry white strip was obtained after drying; 150 μL of DTT (25 mM) was added to the dry white strip to submerge the strip, and after reacting at 56°C for 30 minutes, the DTT was aspirated, and then 150 μL of IAA (55 mM) was added, and the dark reaction was carried out at 25°C for 30 minutes, and the IAA was aspirated, and the strip was washed with ultrapure water for 3 times, and then the strip was dehydrated with 100% (w / w) ACN. After dehydration, the liquid in the EP tube was discarded to obtain an EP tube containing a pretreated strip.

[0187] Use 25mM NH4HCO3 to adjust the concentration of trypsin to 20ng / μL, then add trypsin (20ng / μL) to the EP tube containing the pretreated strip to submerge the strip. After an ice bath for 30 minutes, add 25mM NH4HCO3 to submerge the pretreated strip and incubate the strip in a 37°C incubator for 12h.

[0188] After the enzyme digestion is completed, the EP tube is placed in an ultrasonic instrument for 15 to 20 minutes, and the peptide fragments are sucked out and placed in a new EP tube.

[0189] (2) ZipTip purification of peptides

[0190] The peptide obtained in step (1) was dissolved in 30 μL of 0.1% (v / v) FA as the sample to be processed.

[0191] Use the extraction pipette tip (Tip) to repeatedly pipette 10 μL of 100% (w / w) CAN to wet the chromatography column twice, and discard the ACN; use the Tip to repeatedly pipette 10 μL of 0.1% (v / v) FA to acidify the chromatography column twice, and discard the FA; use the Tip to blow and aspirate the sample 15 times, then use 10 μL of 0.1% (v / v) FA to wash twice, and then elute the Tip with 10 μL of 70% (w / w) ACN solution containing 0.1% FA (v / v), collect the eluate in a new EP tube, and vacuum dry to obtain a mass spectrometry sample.

[0192] (3) RPLC-MS protein spectrum detection

[0193] The mass spectrometry sample obtained in step (2) was dissolved in a sample dissolving solution (containing 0.1% formic acid and 5% acetonitrile, w / w), mixed evenly, and centrifuged at 13200 rpm, 4°C for 20 min, and the supernatant was collected as the mass spectrometry loading solution.

[0194] The column information of RPLC-MS is: 300um idx 5mm, Acclaim PepMap RSLC C18, 5um, (Thermo, 160454);

[0195] Acclaim PepMap 75um X 150mm, C18, 3um, 100A (Thermo, 160321).

[0196] The mobile phase information is: mobile phase A: 0.1% (w / w) formic acid; mobile phase B: 80% (w / w) ACN solution containing 0.1% (w / w) formic acid; flow rate: 300 nL / min; analysis time: 65 min; effective gradient: phase B increases from 5% to 90%.

[0197] According to the mobile phase parameter table shown in Table 8 and the mass spectrometry parameter setting table shown in Table 9, RPLC-MS protein spectrum was performed on the mass spectrometry loading liquid to obtain mass spectrometry data, and the mass spectrometry data was converted into an MGF format file using MM File Conversion software. Then, according to the retrieval parameters shown in Table 10, MASCOT (http: / / www.matrixscience.com / ) was used to search the uniprot database, and the screening condition pep_expect < 0.05 was set to obtain protein peptide information.

[0198] Table 8 Mobile phase parameters

[0199] Time (min) Phase B 0 5％ 5 5％ 45 50％ 50 90％ 55 90％ 65 5％

[0200] Table 9 Mass spectrometry parameter settings

[0201]

[0202] Table 10 Search parameters

[0203]

[0204]

[0205] 2. Experimental results

[0206] The peptide information (pep_expect<0.05) obtained based on the RPLC-MS mass spectrometry data is shown in Table 11, and the protein information is shown in Table 12.

[0207] Table 11 Peptide information

[0208] Table 12 Protein information

[0209]

[0210] The results showed that under the condition of pep_expect<0.05, 8 peptides and 5 proteins were screened and identified in the RPLC-MS mass spectrometry data; the two proteins with the highest scores were actin protein and Histone H4 protein, with scores of 71 and 43 respectively, the mass of actin protein was 42232, and the mass of Histone H4 protein was 11374.

[0211] In the peptide information shown in Table 11, among the 8 identified peptides, 4 polypeptide fragments matched the actin (muscle-type A2) protein sequence of Spodoptera frugiperda, accounting for 15.4% of the actin protein sequence; 1 polypeptide fragment matched the Histone H4 protein sequence of Spodoptera frugiperda, accounting for 9.7% of the Histone H4 protein sequence.

[0212] The search and identification results of actin protein are as follows Fig.17 As shown, the results show that there are 4 sequences in the protein sequence that are covered by 4 polypeptide fragments matching the actin (muscle-type A2) protein sequence in Table 11 ( Fig.17 The gray area in the middle of the protein sequence shows that the amino acid residue at position 307 is oxidatively modified (M).

[0213]

[0214] The protein matched in the NCBI database is actin (muscle-type A2) [Spodoptera frugiperda] protein, whose code in the gene library is XM_035583379.2 (gene name: LOC118268744), with a length of 377 amino acids. The secondary mass spectrum of the unique peptide in the antin protein is as follows Fig.18 shown.

[0215] The results of the retrieval and identification of Histone H4 protein are as follows Fig.19 As shown in the figure, the results show that: there is one sequence on the protein sequence that is covered by a polypeptide fragment matching the Histone H4 protein sequence in Table 11; the protein matched by the protein sequence in the NCBI database is the histone H4 [Spodoptera frugiperda] protein, which is encoded in the gene library as XM_035599717.2 (gene name: LOC118279879), with a length of 104 amino acids. The secondary mass spectrum of the unique peptide in the Histone H4 protein is shown in Fig. 20 shown.

[0216] Based on Tables 11 and 12, three proteins, SFRICE_009909, SFRICE_002323 (Fragment) and Sulfatase D, were also identified. The search and identification results of SFRICE_009909 are as follows: Fig.21 As shown in the figure, the search and identification results of SFRICE_002323 (Fragment) are as follows Fig. 22As shown in the figure, the search and identification results of Sulfatase D are as follows Fig.23 shown.

[0217] The results showed that there was one sequence in each of the three proteins that was covered by the corresponding peptide fragments in Table 11, and there was one oxidation modification (M) in the Sulfatase D protein sequence; the proteins that matched the three proteins in the NCBI database were SFRICE_009909, unconventional myosin-XV (SFRICE_002323), and Sulfatase D protein. The secondary mass spectrum of the unique peptide in the SFRICE_009909 protein is shown in Fig.24 As shown, the secondary mass spectrum of the unique peptide in SFRICE_002323 (Fragment) is as follows Fig.25 As shown, the secondary mass spectrum of the unique peptide in Sulfatase D is as follows Fig.26 shown.

[0218] In summary, the photoaffinity-active molecular probe of the indolematrine derivative prepared in Example 2 can specifically target and bind to actin muscle-type A2, histone H4, SFRICE_009909, unconvertional myosin-XV and sulfatase D proteins of the fall armyworm, thereby guiding the design and synthesis of indole matrine derivatives for killing the fall armyworm.

[0219] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A photoaffinity-active molecular probe of an indolematrine derivative, characterized in that: The indolematrine derivative photoaffinity active molecular probe is obtained by connecting an active group and a reactive group; The active group is a matrine derivative, and the reactive group is a substance containing a diaziridine group and an alkyne group; The chemical formula of the matrine derivative is shown in Formula I, When R1 is NH2, R2 is H; when R2 is NH2, R1 is H.

2. The photoaffinity-active molecular probe of indolematrine derivatives according to claim 1, characterized in that: The chemical formula of the matrine derivative is shown in Formula II, 3. The indolematrine derivative photoaffinity active molecular probe according to claim 1, characterized in that: The substance containing a diazirine group and an alkyne group is alkyne-diazirine-acid.

4. The indolematrine derivative photoaffinity active molecular probe according to claim 3, characterized in that: The chemical formula of the indolematine derivative active molecular probe is shown in Formula III:

5. Use of the photoaffinity-active molecular probe of the indolematrine derivative according to any one of claims 1 to 4 in determining the target of matrine pesticides.

6. The use according to claim 5, characterized in that: The targets are actin muscle-type A2, histone H4, SFRICE_009909, unconvertional myosin-XV and / or sulfatase D.

7. Use of the photoaffinity-active molecular probe of the indolematrine derivative according to any one of claims 1 to 4 in guiding agricultural pest control.

8. The use according to claim 7, characterized in that: The guiding agricultural pest control is guiding the synthesis of pesticides targeting the action sites.

9. A method for determining the target of a matrine pesticide, characterized in that: The following steps are involved: S1. Co-incubating the indolematrine derivative photoaffinity active molecular probe according to any one of claims 1 to 3 with target cells, irradiating with ultraviolet light during the co-incubation period, lysing and collecting the supernatant, and obtaining the indolematrine derivative photoaffinity active molecular probe that specifically binds to the target cell protein; The target cells are cells that act on matrine pesticides; S2. Connect a reporter group to the indolematrine derivative photoaffinity-active molecular probe that specifically binds to the target cell protein obtained in step S1 to obtain an indolematrine derivative photoaffinity-active molecular probe that is connected to a reporter gene and specifically binds to the target cell protein, and perform proteomics detection to determine the target.

10. The method according to claim 9, characterized in that The reporter group in step S2 is biotin, rhodamine and / or BODLPY.

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