A trifunctional probe based on photocrosslinking groups and its preparation method and application

Abstract

The present invention belongs to the field of probe technology, and provides a trifunctional probe based on a photocrosslinking group, a preparation method thereof, and an application thereof. The method comprises the following steps: under a protective atmosphere, compound 1, compound 2, 1-hydroxybenzotriazole, dimethylformamide, carbodiimide, and N,N-diisopropylethylamine are mixed, and a condensation reaction is performed to obtain a trifunctional probe based on a photocrosslinking group. The probe provided by the present invention can be bonded with baicalein, TFAE, and scutellaria baicalensis medicated serum to form a corresponding probe, and compete with its original compound. Under excitation of different ultraviolet wavelengths, the probe can be cross-linked with small molecules and target proteins in traditional Chinese medicine, and accurately reflect the level of target protein labeling of the reactive group in the proteome through a "reporter group", and can also selectively enrich the labeled target protein, and achieve the identification of single compounds, compound combinations, and natural drug extract targets through subsequent experiments.

Description

Technical Field

[0001] The present invention relates to the field of probe technology, and in particular to a trifunctional probe based on a photocrosslinking group, and a preparation method and application thereof. Background Art

[0002] Traditional Chinese medicine (TCM) is an effective treatment approach, proven through long-term medical trials. Its hallmark is the combined use of multiple drugs. Existing research is often guided by a reductionist approach, primarily deconstructing the complex systems of TCM into simple individual chemical components. These components are then used to identify their pharmacological targets. Targets are the foundation for the active ingredients of TCM to exert their effects. TCM active ingredients target biomacromolecules in the body, modulating their biological activity. This in turn regulates cellular signaling pathways downstream of the target molecules, perturbing disease networks and thereby preventing and treating diseases. Therefore, identifying the targets of TCM active ingredients is crucial for TCM research and development. Currently, the most prominent and practical technology for identifying the targets of TCM active ingredients is to transform them into active probes. However, this approach also has limitations. For example, the probes may enrich nonspecific proteins while targeting the target protein, which can introduce errors in experimental results. Therefore, developing probes that can efficiently identify the targets of TCM compounds is of great significance. Summary of the Invention

[0003] The purpose of the present invention is to overcome the problems existing in the prior art and provide a trifunctional probe based on a photocrosslinking group and a preparation method and application thereof.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a trifunctional probe based on a photocrosslinking group, wherein the structure of the trifunctional probe is:

[0006] ,

[0007] The n is any natural number.

[0008] Preferably, the structure of the trifunctional probe is:

[0009] 、 、 、 、 or .

[0010] The present invention also provides a method for preparing the trifunctional probe based on photocrosslinking groups, comprising the following steps:

[0011] Under a protective atmosphere, compound 1, compound 2, 1-hydroxybenzotriazole, dimethylformamide, carbodiimide and N,N-diisopropylethylamine are mixed and subjected to a condensation reaction to obtain the trifunctional probe based on the photocrosslinking group;

[0012] The compound 1 is ,

[0013] The compound 2 is ,

[0014] The n is any natural number.

[0015] Preferably, the mass ratio of the compound 1, 1-hydroxybenzotriazole, carbodiimide and N,N-diisopropylethylamine is 100-200:10-100:50-200:100-300.

[0016] Preferably, the mass volume ratio of the compound 1 to dimethylformamide is 100-300 mg: 1-10 mL.

[0017] Preferably, the molar ratio of 1-hydroxybenzotriazole to compound 2 is 0.2-3.5:0.1-2.

[0018] Preferably, the temperature of the condensation reaction is 10-35° C., and the time of the condensation reaction is 1-5 hours.

[0019] The present invention also provides the use of the trifunctional probe based on photocrosslinking groups in identifying the target sites of single compounds, compound combinations and natural drug extracts.

[0020] The beneficial effects of the present invention are:

[0021] The present invention provides a trifunctional probe based on a photocrosslinking group, which can be used as a trifunctional probe to realize the target identification of single compounds, compound combinations and natural drug extracts. It bonds with baicalein, TFAE (baicalein, wogonin, melaleuca alternifolia), and scutellaria baicalensis drug-containing serum to form corresponding probes and compete with their original compounds.

[0022] The probe provided by the present invention can achieve cross-linking with small molecules and target proteins in traditional Chinese medicine under the excitation of different ultraviolet light wavelengths, and accurately reflect the level of labeling of the target protein by the reactive group in the proteome through the "reporter group". It can also selectively enrich the target protein labeled by the probe and analyze and identify it through subsequent experimental methods, thereby realizing the identification of single compounds, compound combinations and targets of natural drug extracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1When n=0 of compound 2 in Example 1, the trifunctional probe based on the photocrosslinking group

[0024] hydrogen spectrum;

[0025] Figure 2 1 is the hydrogen spectrum of the trifunctional probe based on the photocrosslinking group when n=1 of compound 2 in Example 1;

[0026] Figure 3 1 is the hydrogen spectrum of the trifunctional probe based on the photocrosslinking group when n=2 of compound 2 in Example 1;

[0027] Figure 4 1 is the hydrogen spectrum of the trifunctional probe based on the photocrosslinking group when n=3 of compound 2 in Example 1;

[0028] Figure 5 1 is the hydrogen spectrum of the trifunctional probe based on the photocrosslinking group when n=4 of compound 2 in Example 1;

[0029] Figure 6 1 is the hydrogen spectrum of the trifunctional probe based on the photocrosslinking group when n=5 for compound 2 in Example 1;

[0030] Figure 7 This is a schematic diagram of the protein molecular weight and input amount results in Example 1;

[0031] Figure 8 The electrophoresis results of different hook protein systems in Example 1 are shown;

[0032] Figure 9 This is a schematic diagram of the protein molecular weight and input amount results in Example 2;

[0033] Figure 10 The electrophoresis results of different hook protein systems in Example 2 are shown;

[0034] Figure 11 This is a schematic diagram of the protein molecular weight and input amount results in Example 3;

[0035] Figure 12 This is the electrophoresis result diagram of the protein fishing system in different systems in Example 3. DETAILED DESCRIPTION

[0036] The present invention provides a trifunctional probe based on a photocrosslinking group, wherein the structure of the trifunctional probe is:

[0037] ,

[0038] The n is any natural number.

[0039] In the present invention, the structure of the trifunctional probe is preferably:

[0040] 、 、 、 、 or .

[0041] The present invention also provides a method for preparing the trifunctional probe based on photocrosslinking groups, comprising the following steps:

[0042] Under a protective atmosphere, compound 1, compound 2, 1-hydroxybenzotriazole, dimethylformamide, carbodiimide and N,N-diisopropylethylamine are mixed and subjected to a condensation reaction to obtain the trifunctional probe based on the photocrosslinking group.

[0043] In the present invention, the protective atmosphere is preferably nitrogen, neon or argon.

[0044] In the present invention, the compound 1 is

[0045] .

[0046] In the present invention, the compound 2 is

[0047] ,

[0048] The n is any natural number.

[0049] In the present invention, the mass ratio of the compound 1, 1-hydroxybenzotriazole, carbodiimide and N,N-diisopropylethylamine is preferably 100-200:10-100:50-200:100-300, more preferably 110-190:20-90:70-195:120-290, and more preferably 120-180:25-85:75-190:150-250.

[0050] In the present invention, the mass volume ratio of the compound 1 to dimethylformamide is preferably 100-300 mg:1-10 mL, more preferably 120-200 mg:2-9 mL, and even more preferably 130-190 mg:3-8 mL.

[0051] In the present invention, the molar ratio of 1-hydroxybenzotriazole to compound 2 is preferably 0.2-3.5:0.1-2, more preferably 0.3-2.5:0.2-1.9, and even more preferably 0.5-2.3:0.3-1.8.

[0052] In the present invention, the extent of the condensation reaction is monitored by thin layer chromatography. The temperature of the condensation reaction is preferably 10-35°C, more preferably 15-30°C, and more preferably 17-28°C. The time of the condensation reaction is preferably 1-5 h, more preferably 1.5-4.5 h, and more preferably 2-3 h.

[0053] In the present invention, after the condensation reaction is completed, a large amount of solid is precipitated in the system, which is filtered and purified by column (purification conditions are shown in Table 1) to obtain a trifunctional probe product; the column purification is performed using conventional technical means in the art.

[0054] In the present invention, the upper column purification is performed by a reverse phase column, the filler of the upper column purification is silica gel, and the flow rate of the upper column purification is preferably 1.3~1.7mL / min, more preferably 1.4~1.6mL / min, and more preferably 1.45~1.55mL / min.

[0055] Table 1 Column purification conditions

[0056] Time (min) 0.05% formic acid water 0.05% formic acid in acetonitrile 0 95% 5% 1.5 5% 95% 3 5% 95% 3.01 95% 5% 3.2 95% 5%

[0057] In the present invention, as the chain length of compound 2 increases to n≥4, the product obtained by the condensation reaction is an oily substance without solid precipitation. In this case, the steps of filtration and column purification can be omitted.

[0058] The present invention also provides the use of the trifunctional probe based on photocrosslinking groups in identifying the target sites of single compounds, compound combinations and natural drug extracts.

[0059] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0060] Example 1

[0061] Under a nitrogen atmosphere, 179 mg of compound 1, compound 2 (wherein n=1 in compound 2, and the molar ratio of compound 2 to 1-hydroxybenzotriazole is 1:1.2), 91 mg of 1-hydroxybenzotriazole, 2.8 mL of dimethylformamide, 121 mg of carbodiimide, and 181 mg of N,N-diisopropylethylamine were mixed and condensed at 25°C for 2.5 hours. After the reaction, a large amount of solid precipitated in the system, which was filtered and purified by column chromatography (filler: silica gel, flow rate: 1.5 mL / min, other conditions are shown in Table 1) to obtain the trifunctional probe based on photocrosslinking groups (abbreviated as ABP).

[0062] While keeping other conditions unchanged, the value of n in compound 2 was replaced with 0, 2, 3, 4, and 5, respectively (when n was 4 or 5, no subsequent filtration and purification steps were required), to obtain trifunctional probes based on photocrosslinking groups of different chain lengths.

[0063] When n=0, the hydrogen spectrum of the trifunctional probe is as follows Figure 1 shown; from Figure 1 It can be obtained that when n=0, the hydrogen spectrum data of the trifunctional probe is 1 HNMR(400MHz,DMSO-d6)δ8.86(m,2H),8.27(d,J=8.8Hz,2H),8.15(dd,J=8.9,3.0Hz,4H),8.02(d,J=8.4Hz,2H),7.81(d,J=8.3Hz,2H), 7.78-7.67(m,3H),7.58(dd,J=8.3,7.0Hz,2H),7.23(d,J=8.9Hz,2H),4.92(d,J=2.4Hz,2H),3.64(t,J=2.4Hz,1H),3.55-3.48(m,4H).

[0064] When n=1, the hydrogen spectrum of the trifunctional probe is as follows Figure 2 shown; from Figure 2 It can be obtained that when n=1, the hydrogen spectrum data of the trifunctional probe is 1 HNMR(400MHz,DMSO-d6)δ8.72(t,J=5.5Hz,1H),8.68(t,J=5.4Hz,1H),8.20(d,J=8.7Hz,2H),8.15-8.06(m,2H),7.97(d,J=8.4Hz,2H),7 .83-7.62(m,5H),7.53(t,J=7.7Hz,2H),7.29-7.16(m,2H),4.93(d,J=2.4Hz,2H),3.63(ddd,J=11.9,5.2,2.0Hz,5H),3.56-3.39(m,4H).

[0065] When n=2, the hydrogen spectrum of the trifunctional probe is as follows Figure 3 shown; from Figure 3 It can be obtained that when n=2, the hydrogen spectrum data of the trifunctional probe is 1HNMR(400MHz,DMSO-d6)δ8.76(t,J=5.6Hz,1H),8.71(t,J=5.5Hz,1H),8.24(d,J=8.8Hz,2H),8.18-8.07(m,4H),7.98(d,J=8.4Hz,2H),7.7 7(d,J=8.4Hz,2H),7.85-7.54(m,5H),7.33-7.14(m,2H),4.92(d,J=2.4Hz,2H),3.64(t,J=2.4Hz,1H),3.57(d,J=5.8Hz,8H),3.45(m,4H).

[0066] When n=3, the hydrogen spectrum of the trifunctional probe is as follows Figure 4 shown; from Figure 4 It can be obtained that when n=3, the hydrogen spectrum data of the trifunctional probe is 1 HNMR(400MHz,DMSO-d6)δ8.76(t,J=5.6Hz,1H),8.71(t,J=5.6Hz,1H),8.24(d,J=8.8Hz,2H),8.13(d,J=8.8Hz,4H),7.98(d,J=8.4Hz,2H),7.90-7 .64(m,5H),7.57(t,J=7.7Hz,2H),7.22(d,J=8.9Hz,2H),4.92(d,J=2.4H z,2H),3.64(t,J=2.4Hz,1H),3.60-3.49(m,12H),3.44(p,J=5.8Hz,4H).

[0067] When n=4, the hydrogen spectrum of the trifunctional probe is as follows Figure 5 shown; from Figure 5 It can be obtained that when n=4, the hydrogen spectrum data of the trifunctional probe is 1 HNMR(400MHz,DMSO-d6)δ8.76(t,J=5.6Hz,1H),8.71(t,J=5.5Hz,1H),8.25(d,J=8.8Hz,2H),8.20-8.04(m,4H),7.99(d,J=8.4Hz,2H), 7.88-7.64(m,5H),7.57(dd,J=8.4,6.9Hz,2H),7.22(d,J=8.9Hz,2H),4.92(d,J=2.4Hz,2H),3.64(t,J=2.4Hz,1H),3.59-3.40(m,20H).

[0068] When n=5, the hydrogen spectrum of the trifunctional probe is as follows Figure 6 shown; from Figure 6It can be obtained that when n=5, the hydrogen spectrum data of the trifunctional probe is 1 HNMR(400MHz,DMSO-d6)δ8.76(t,J=5.6Hz,1H),8.71(t,J=5.6Hz,1H),8.35-8.20(m,2H),8.19-8.08(m,4H),8.05-7.94(m,2H),7 .88-7.65(m,5H),7.57(t,J=7.7Hz,2H),7.22(d,J=8.9Hz,2H),4.92(d,J=2.4Hz,2H),3.64(t,J=2.4Hz,1H),3.62-3.37(m,24H).

[0069] The protein solutions of rat heart, liver, spleen, lung, pancreas, intestine, stomach, brain, kidney, bladder, testis, and prostate tissues were diluted to 2 mg / mL respectively, and 1 mL of each solution was evenly mixed together to obtain a protein system; ABP was prepared at a concentration of 100 mmol / L. n=5 The mother solution and 1mmol / L baicalein solution were used to separate ABP n=5 The stock solution was diluted to 1, 1.5, 2, 2.5 and 3 mmol / L; 20 μL of ABP at concentrations of 1, 1.5, 2, 2.5 and 3 mmol / L was taken. n=5 The solution was mixed evenly with 20 μL of 1 mmol / L baicalein solution to obtain ABP in different proportions. n=5 - baicalein solution; irradiate each of them under light of wavelength 365nm for 20min, take 12.5μL of the above-mentioned ABP of different proportions respectively n=5 - Baicalein solution was mixed with 50 μL protein system, and the mixed solution was shaken at 25°C and 800 rpm for 20 min. They were transferred to 96-well plates, irradiated under 302 nm UV light for 10 min, and then transferred back to EP tubes. 5 μL of orthogonal mixture of tris(2-carbonylethyl)phosphine hydrochloride (TCEP), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), and copper sulfate (CuSO4) (volume ratio of 1:3:1) and 0.3 μL of 20 mmol / L rhodamine solution were added to 5 EP tubes respectively (the whole process was carried out under light-proof conditions). After the addition, the mixture was shaken at 25°C and 800 rpm for 1 h in the dark, and then 12.5 μL of Solebol 5X Loading was added to each EP tube. Buffer, shake and mix, boil at 95℃ for 5min, and then centrifuge for 20s to obtain sample solutions 1, 2, 3, 4, and 5. The ABP content in each sample solution was n=5 and baicalein concentrations, as shown in Table 2 .

[0070] Table 2 ABP in each sample solution n=5 and baicalein concentration results

[0071] Sample solution 1 2 3 4 5 <![CDATA[ABP before mixing n=5 Concentration (mmol / L)]]> 1 1.5 2 2.5 3 Baicalein concentration before mixing (mmol / L) 1 1 1 1 1 <![CDATA[ABP after mixing n=5 Concentration (mmol / L)]]> 0.5 0.75 1 1.25 1.5 Baicalein concentration after mixing (mmol / L) 0.5 0.5 0.5 0.5 0.5

[0072] Then the protein molecular weight and input amount in the system are tested to obtain a schematic diagram of the protein molecular weight and input amount in the system, as shown in Figure 7 As shown in the figure, it can be seen that the amount of protein input in each sample is equal, and the molecular weight distribution of the protein ranges from 1kD to 300kD. That is, the probe of this embodiment is applicable to a wide range of protein molecular weights and has excellent practical value.

[0073] Finally, perform polypropylene gel electrophoresis experiments, install glass plates and check for leaks, prepare 10% concentration separation gel, and pour separation gel between the glass plates (preparation method: 2.08 mL of pure water, 1.67 mL of 30% Acr-Bis (29:1), 1.25 mL of SDS-PAGE Separating Gel Buffer, 0.05 mL of 10% APS, 0.002 mL of TEMED), immediately cover with a layer of anhydrous ethanol to remove bubbles, wait 40 minutes, and prepare stacking gel (preparation method: 1.14 mL of pure water, 0.34 mL of 30% Acr-Bis (29:1), 0.5 mL of SDS-PAGE Stacking Gel). Buffer, 0.02mL of 10% APS, 0.002mL of TEMED), remove anhydrous ethanol and pour concentrated gel between the two plates and insert the sample comb, wait 30min; install the electrophoresis system, add electrode buffer, load the sample, add a group of 10uL sample solution to each channel (sample solution 1, 2, 3, 4, 5), add 5uL of Loading Buffer to the front and back of the sample, and add 2uL of Marking to another channel. After completion, start running the gel, first modulate the voltage to 130V and run for 10min, then modulate to 190V and run for 40min, that is, until bromophenol blue just runs out of the separation gel, remove the gel, use Taton fluorescence imaging to observe the labeling effect, and then use Coomassie Brilliant Blue staining to test the protein quality of each lane, and obtain the electrophoresis result diagram of sample 1~5 hook protein system, as shown below. Figure 8 As shown in the figure, the ABP of each ratio n=5 -Baicalein can achieve the hooking of protein, also known as ABP n=5 In the subsequent experiments, we selected the ratio of sample solution 1 (i.e. ABP n=5 and baicalein in a ratio of 1:1) for testing and verification.

[0074] Prepare 25mmol / L and 75mmol / L baicalein solutions, respectively. Take 1μL of each solution and add it to 49μL of protein system to obtain mixed solutions. The concentrations of baicalein in the mixed solutions are 0.5mmol / L and 1.5mmol / L, respectively. Oscillate the mixed solutions at 25℃ and 800rpm for 1h, then take 12.5μL ABP n=5 -Baicalein (ABP n=5 The mixture was mixed with the two protein systems, and then shaken at 25°C and 800 rpm for 20 min. They were transferred to 96-well plates, irradiated under 302 nm UV light for 10 min, and then transferred back to EP tubes. 5 μL of orthogonal mixture (tris(2-carbonylethyl)phosphine hydrochloride (TCEP), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), copper sulfate (CuSO4) in a volume ratio of 1:3:1) and 0.3 μL of 20 mmol / L rhodamine solution were added to two EP tubes respectively (the whole process was carried out in the dark). After the addition, the mixture was shaken at 25°C and 800 rpm for 1 h in the dark. Then, 12.5 μL of 5X Loading Buffer, shake and mix, cook at 95℃ for 5min, then centrifuge for 20s, and perform electrophoresis polyacrylamide gel experiment according to the above method. n=5 - Electrophoresis results of baicalein hook protein system, such as Figure 8 As shown. Figure 8 It can be obtained by adding baicalein first, baicalein combines with protein, and then adding ABP n=5 -baicalein, which significantly reduced the amount of protein captured, and the binding of baicalein to proteins could be competed with the ABP-baicalein probe. This also demonstrated that sample solutions 1-5 effectively identified the protein system.

[0075] Example 2

[0076] Under a helium atmosphere, 175 mg of compound 1, compound 2 (wherein n in compound 2 is 5, and the molar ratio of compound 2 to 1-hydroxybenzotriazole is 1:1), 85 mg of 1-hydroxybenzotriazole, 2.6 mL of dimethylformamide, 116 mg of carbodiimide and 175 mg of N,N-diisopropylethylamine were mixed and condensed at 22° C. for 2.7 hours to obtain the trifunctional probe based on the photocrosslinking group.

[0077] The protein solutions of rat heart, liver, spleen, lung, pancreas, intestine, stomach, brain, kidney, bladder, testis, and prostate tissues were diluted to 2 mg / mL, and 1 mL of each solution was evenly mixed to obtain a protein system. A 100 mmol / L ABP stock solution and a 1 mmol / L TFAE solution (TFAE average molecular weight: 270.24×65.8%+284.26×21.2%+284.26×13%=275.03) were prepared. The ABP stock solution was diluted to 1, 1.5, 2, 2.5, and 3 mmol / L using dimethyl sulfoxide (DMSO). 20 μL of the 1, 1.5, 2, 2.5, and 3 mmol / L ABP solutions were taken and evenly mixed with 20 μL of the 1 mmol / L TFAE solution to obtain ABP-TFAE solutions of different ratios. These solutions were irradiated at a wavelength of 365 nm for 2 min. At 0 min, 12.5 μL of the above-mentioned ABP-TFAE solution with different ratios was mixed with 50 μL of the protein system. The mixed solutions were shaken at 25°C and 800 rpm for 20 min, transferred to 96-well plates, irradiated under 302 nm UV light for 10 min, and then transferred back to EP tubes. 5 μL of an orthogonal mixture (tris(2-carbonylethyl)phosphine hydrochloride (TCEP), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), and copper sulfate (CuSO4) in a volume ratio of 1:3:1) and 0.3 μL of 20 mmol / L rhodamine solution were added to each of the five EP tubes (the entire process was performed in the dark). After the addition, the mixture was shaken at 25°C and 800 rpm for 1 h in the dark, and then 12.5 μL of Solebol 5X was added to each EP tube. Loading Buffer was added, mixed by vortexing, boiled at 95°C for 5 min, and then centrifuged for 20 s to obtain sample solutions 6, 7, 8, 9, and 10. The concentrations of ABP and TFAE in each sample solution are shown in Table 3.

[0078] Table 3 Concentration results of ABP and TFAE in each sample solution

[0079] Sample solution 6 7 8 9 10 ABP concentration before mixing (mmol / L) 1 1.5 2 2.5 3 TFAE concentration before mixing (mmol / L) 1 1 1 1 1 ABP concentration after mixing (mmol / L) 0.5 0.75 1 1.25 1.5 TFAE concentration after mixing (mmol / L) 0.5 0.5 0.5 0.5 0.5

[0080] Then the protein molecular weight and input amount in the system are tested to obtain a schematic diagram of the protein molecular weight and input amount in the system, as shown in Figure 9 As shown in the figure, it can be seen that the amount of protein input in each sample is equal, and the molecular weight distribution of the protein ranges from 1kD to 300kD. That is, the probe of this embodiment is applicable to a wide range of protein molecular weights and has excellent practical value.

[0081] Finally, perform polypropylene gel electrophoresis experiments, install glass plates and check for leaks, prepare 10% concentration separation gel, and pour 5 mL of separation gel between the glass plates (preparation method: 2.08 mL of pure water, 1.67 mL of 30% Acr-Bis (29:1), 1.25 mL of SDS-PAGE Separating Gel Buffer, 0.05 mL of 10% APS, 0.002 mL of TEMED). Immediately cover with a layer of anhydrous ethanol to remove bubbles, wait 40 minutes, and prepare 2 mL of stacking gel (preparation method: 1.14 mL of pure water, 0.34 mL of 30% Acr-Bis (29:1), 0.5 mL of SDS-PAGE Stacking Gel). Buffer, 0.02mL of 10% APS, 0.002mL of TEMED), remove anhydrous ethanol and pour concentrated gel between the two plates and insert the sample comb, wait 30min; install the electrophoresis system, add electrode buffer, load the sample, a group of 10uL sample solution in each channel (sample solution 6, 7, 8, 9, 10), add 5uL of Loading Buffer to the front and back of the sample, and add 2uL of Marking to one channel. After completion, start running the gel, first modulate the voltage to 130V and run for 10min, then modulate it to 190V and run for 40min, that is, until bromophenol blue just runs out of the separation gel, remove the gel, use Taton fluorescence imaging to observe the labeling effect, and then use Coomassie Brilliant Blue staining to test the protein quality of each lane, and obtain the electrophoresis result diagram of sample 6~10 hook protein system, as shown below. Figure 10 As shown in the figure, all ABP-TFAE ratios were able to capture the protein, indicating that ABP and TFAE successfully bonded. In subsequent experiments, we used the ratio of sample solution 6 (i.e., a 1:1 ratio of ABP and TFAE) for testing and verification.

[0082] TFAE solutions with concentrations of 25 mmol / L and 75 mmol / L were prepared respectively, 1 μL of each solution was drawn and added to 49 μL of the protein system to obtain mixed solutions. At this time, the concentrations of TFAE in the mixed solutions were 0.5 mmol / L and 1.5 mmol / L respectively; the mixed solutions were shaken at a temperature of 25°C and a speed of 800 rpm for 1 h, and then 12.5 μL ABP-TFAE (ABP and TFAE ratio was 1:1) was taken and mixed with the above two protein systems respectively, and then shaken at a temperature of 25°C and a speed of 800 rpm for 20 min, and they were transferred to 96-well plates respectively. Irradiate under 302nm UV light for 10 minutes, then transfer back to the EP tube. Add 5μL of an orthogonal mixture of tris(2-carbonylethyl)phosphine hydrochloride (TCEP), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), and copper sulfate (CuSO4) in a volume ratio of 1:3:1) and 0.3μL of 20mmol / L rhodamine solution to each of the two EP tubes (the entire process was performed under light-proof conditions). After the addition, shake at 25°C and 800rpm for 1 hour in the dark. Then, add 12.5μL of 5X Loading Buffer to each EP tube, shake and mix, boil at 95°C for 5 minutes, and then centrifuge for 20 seconds. Perform the polyacrylamide gel electrophoresis experiment according to the above method. The electrophoresis results of different concentrations of TFAE and ABP-TFAE hook protein system are as shown below. Figure 10 As shown. Figure 10 As can be seen from the results, after TFAE is first added, TFAE binds to the protein. When ABP-TFAE is then added, the amount of protein captured is significantly reduced, and the binding of baicalein to the protein can be competed for by the ABP-baicalein probe. This also demonstrates that the aforementioned sample solutions 6-10 do effectively identify the protein system.

[0083] Example 3

[0084] Under a nitrogen atmosphere, 183 mg of compound 1, compound 2 (wherein n=3 in compound 2, and the molar ratio of compound 2 to 1-hydroxybenzotriazole is 1:1.3), 96 mg of 1-hydroxybenzotriazole, 3.8 mL of dimethylformamide, 127 mg of carbodiimide, and 186 mg of N,N-diisopropylethylamine were mixed and condensed at 28°C for 2.1 h. After the reaction, a large amount of solid precipitated in the system, which was filtered and purified by column chromatography (filler: silica gel, flow rate: 1.6 mL / min, other conditions are shown in Table 1) to obtain the trifunctional probe based on the photocrosslinking group.

[0085] The experiment was carried out in accordance with the same test method as in Example 1 to obtain the schematic diagram of the protein molecular weight and input amount results in Example 1, as shown in FIG. Figure 11 As shown in the figure, it can be seen that the amount of protein input in each sample is equal, and the molecular weight distribution of the protein ranges from 1kD to 300kD, that is, the probe of this embodiment is applicable to a wide range of protein molecular weights and has excellent practical value; at the same time, the electrophoresis results of different system hook protein systems are obtained, as shown in Figure 12 As shown in the figure, it can be seen that the ABP of each ratio n=3 -Baicalein can achieve the hooking of protein, also known as ABP n=3 The protein was successfully bonded to baicalein. After baicalein was added first, baicalein combined with the protein, and then ABP was added. n=3 - Baicalein, the amount of protein hooked by it was significantly reduced, and the binding of baicalein to protein could be competed by ABP-baicalein probe. n=3 -Baicalein does achieve effective identification of protein systems.

[0086] As can be seen from the above examples, the present invention provides a trifunctional probe based on a photocrosslinking group, which can be used as a trifunctional probe to identify single compounds, compound combinations, and natural drug extract targets. It can bond with baicalein, TFAE (baicalein, wogonin, melaleuca alternifolia), and scutellaria baicalensis medicated serum to form corresponding probes and compete with their original compounds. The probe provided by the present invention can achieve crosslinking with small molecules in traditional Chinese medicine and target proteins under the excitation of different ultraviolet light wavelengths, and accurately reflect the level of labeling of the target protein by the reactive group in the proteome through the "reporter group". The target protein labeled by the probe can also be selectively enriched and analyzed and identified through subsequent experimental methods, thereby achieving the identification of single compounds, compound combinations, and natural drug extract targets.

[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A trifunctional probe based on a photocrosslinking group, characterized in that: The structure of the trifunctional probe is:

2. The method for preparing a trifunctional probe based on a photocrosslinking group according to claim 1, characterized in that: It includes the following steps: Under a protective atmosphere, compound 1, compound 2, 1-hydroxybenzotriazole, dimethylformamide, carbodiimide and N,N-diisopropylethylamine are mixed and subjected to a condensation reaction to obtain the trifunctional probe based on the photocrosslinking group; The compound 1 is The compound 2 is The n is 2, 3, 4 or 5.

3. The preparation method according to claim 2, wherein The mass ratio of the compound 1, 1-hydroxybenzotriazole, carbodiimide and N,N-diisopropylethylamine is 100-200:10-100:50-200:100-300.

4. The preparation method according to claim 3, wherein The mass volume ratio of the compound 1 to dimethylformamide is 100-300 mg:1-10 mL.

5. The preparation method according to claim 4, wherein The molar ratio of the 1-hydroxybenzotriazole to the compound 2 is 0.2-3.5:0.1-2.

6. The preparation method according to claim 4 or 5, characterized in that The temperature of the condensation reaction is 10 to 35° C., and the time of the condensation reaction is 1 to 5 hours.

7. Use of the trifunctional probe based on photocrosslinking groups according to claim 1 in the preparation of drugs for identifying target points of baicalein, a composition of baicalein, wogonin and melaleuca alternifolia, and serum containing baicalein.

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