D-biotin-labeled crocin probe and its synthesis method

Abstract

The present invention discloses a D-biotin-labeled crocin probe and a synthesis method thereof, and relates to the field of chemical synthesis. The molecular formula of the D-biotin-labeled crocin probe is as shown in formula (I). The method includes: (1) mixing D-biotin and N-Boc-1,6-hexanediamine, adding a solvent, a condensing agent and an organic base to react to obtain compound 3-1; (2) in the presence of dichloromethane, compound 3-1 is reacted with trifluoroacetic acid to remove the Boc group to obtain compound 3-2; (3) compound 3-2 is dissolved in pyridine to obtain reaction solution a; under an ice bath, bromoacetyl bromide is dissolved in dichloromethane to obtain reaction solution b; then reaction solution a is added dropwise to reaction solution b, and the reaction is completely carried out under an ice bath to obtain compound 3-3; (4) under luminescence, column chromatography purification obtains a crocin probe. The synthesized probe provides technical support for the discovery of subsequent crocin target sites.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical synthesis, and in particular to a D-biotin-labeled crocin probe and a synthesis method thereof. Background Art

[0002] Crocin, also known as crocin or crocin, is a water-soluble carotenoid mainly composed of crocin I (crocin I). It is a major medicinal ingredient in the traditional Chinese medicine crocus and is also a quality evaluation indicator of crocus in the pharmacopoeia. It has a wide range of good pharmacological effects. Modern research shows that crocin has a good therapeutic effect on a variety of central nervous system and cardiovascular system diseases. It also has anti-cancer, anti-inflammatory, antioxidant, liver-protecting and choleretic, and anti-diabetic effects. Other studies have shown that crocin increases intracellular Ca by activating the ERK1 / 2 signaling pathway. 2+ concentration, can promote the proliferation of human umbilical vein endothelial cells in vitro.

[0003] However, the specific molecular targets of crocin in organisms are still unclear. Therefore, a technology that can track small molecule drugs to facilitate the discovery and identification of their direct targets is needed.

[0004] Currently, the most common method for finding targets for small molecule drugs is chemical proteomics technology, which uses D-biotin-labeled small molecule drug probes as tools and means to label their target proteins, utilizes the high affinity between D-biotin and avidin to separate and purify the target proteins, and identifies the target proteins through mass spectrometry technology.

[0005] However, there is currently no precedent for using D-biotin labeling technology for crocin labeling, and no specific probe synthesis method has been found. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a D-biotin-labeled crocin probe and a synthesis method thereof, thereby providing technical support for the subsequent discovery of crocin targets.

[0007] One aspect of the present invention is to provide a D-biotin-labeled crocin probe, the molecular formula of which is shown in formula (I):

[0008]

[0009] The molecular formula of the D-biotin-labeled crocin probe of formula (I) is as follows:

[0010]

[0011] In the above molecular formula, the crocin molecule is abbreviated as crocin, and the simplified formula of the D-biotin-labeled crocin probe is as follows:

[0012]

[0013] Another aspect of the present invention is to provide a method for synthesizing a D-biotin-labeled crocin probe, comprising the following steps:

[0014] (1) D-biotin and N-Boc-1,6-hexanediamine were first mixed, and then a solvent, a condensing agent, and an organic base were added, and the mixture was reacted at room temperature to obtain a first product mixture; the first product mixture was added to ultrapure water under stirring to precipitate a solid, and the solid was filtered and dried to obtain compound 3-1;

[0015] Wherein, the condensing agent is selected from any one of 1-ethyl-3-(3-dimethylpropylamine)carbodiimide hydrochloride (abbreviated as EDCI), N,N'-dicyclohexylcarbodiimide (DCC) or 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU);

[0016] The organic base is selected from any one of N,N-diisopropylethylamine (DIPEA) and triethylamine (TEA);

[0017] The solvent is a polar aprotic solvent, selected from any one of N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexamethylphosphoramide (HMPT) or N-methylpyrrolidone.

[0018] The synthetic route is:

[0019]

[0020] In the present invention, room temperature refers to a temperature of about 25°C.

[0021] In one embodiment of the method of the present invention, in step (1), the reaction time is 10-15 h, for example 12 h.

[0022] In one embodiment of the method of the present invention, in step (1), the volume ratio of the ultrapure water to the first product mixture is 5:1.

[0023] In one embodiment of the method of the present invention, in step (1), the drying temperature of the solid is 50° C. and the drying time is 3-5 hours.

[0024] In the method of the present invention, in step (1), there is no special requirement for the order of adding the solvent, the condensing agent and the organic base; preferably, the solvent is added first, and then the condensing agent and the organic base are added under stirring.

[0025] (2) In the presence of dichloromethane, compound 3-1 is reacted with trifluoroacetic acid at room temperature to remove the -Boc group to obtain a second product mixture; the second product mixture is evaporated under reduced pressure to remove the dichloromethane and the trifluoroacetic acid to obtain compound 3-2.

[0026] In the method of the present invention, in step (2), the reaction is a Boc removal reaction. Dichloromethane is the solvent of the reaction system; trifluoroacetic acid is used for the removal of the Boc group, and HCl can also be used instead of trifluoroacetic acid, which is related to the instability of the Boc protecting group to acid.

[0027] In the method of the present invention, in step (2), a rotary evaporator can be used for reduced pressure evaporation, which not only provides a reduced pressure environment to evaporate the solvent, but also provides an oxygen-free environment for the system, making the compound less likely to deteriorate.

[0028] The synthetic route is:

[0029]

[0030] In one embodiment of the method of the present invention, in step (2), dichloromethane must be added first, and then trifluoroacetic acid. If trifluoroacetic acid is added first, local exotherm may occur, causing the raw materials to deteriorate, resulting in the inability to obtain compound 3-2.

[0031] In one embodiment of the method of the present invention, in step (2), the second product mixture is dried using a rotary evaporator, which utilizes the function of dichloromethane to extract trifluoroacetic acid. Trifluoroacetic acid is not easy to be completely dried, and the dichloromethane can be added to the rotary evaporator multiple times and dried to remove as much residual trifluoroacetic acid as possible; for example, the dichloromethane can be added and dried 2-4 times, such as 3 times or 2 times. If the trifluoroacetic acid has been completely removed by two rotary evaporations, the compound 3-2 can be directly used in the next step after the second rotary evaporation without the third addition.

[0032] In one embodiment of the method of the present invention, in step (2), the temperature of the rotary evaporator for drying is 45°C.

[0033] (3) Compound 3-2 was dissolved in pyridine to obtain reaction solution a; bromoacetyl bromide was dissolved in dichloromethane under ice bath to obtain reaction solution b; reaction solution a was then added to reaction solution b under ice bath to react and obtain compound 3-3.

[0034] The synthetic route is:

[0035]

[0036] In one embodiment of the method of the present invention, in step (3), the "ice bath" refers to maintaining the temperature at 0-4° C. In step (3), the reaction of compound 3-2 with bromoacetyl bromide should be carried out at a temperature of 0-4° C. If the temperature is higher, for example, at room temperature, the reaction compound will deteriorate, and compound 3-3 will not be obtained.

[0037] (4) Under light-shielding conditions, crocin is dissolved in the same solvent as that in step (1) to obtain a reaction solution c, compound 3-3 is added to the reaction solution c at room temperature to react to obtain a final product mixture, the final product mixture is slowly dripped into ethyl acetate to precipitate a solid, the solid is filtered to obtain a crude product, and the crude product is subjected to column chromatography to obtain a purified D-biotin-labeled crocin probe.

[0038] The synthetic route is:

[0039]

[0040] In one embodiment of the method of the present invention, step (4) is carried out in the dark to prevent crocin from deteriorating during the synthesis process. In step (4), it is preferably carried out in the dark throughout the entire process.

[0041] In one embodiment of the method of the present invention, in step (4), the reaction time is 10-15 h, for example 12 h.

[0042] In one embodiment of the method of the present invention, in step (4), the crocin can be precipitated using ethyl acetate because of its good water solubility but poor solubility in ethyl acetate; while some unreacted raw materials can be dissolved in ethyl acetate, thereby removing some impurities.

[0043] In a further embodiment of the method of the present invention, in step (4), the crude product is dissolved in a methanol-water mixture with a volume ratio of 10:1 and 200-300 mesh silica gel is added thereto, and then the mixture is dried using a rotary evaporator to obtain a sample for column chromatography; the column chromatography process includes flushing the separation column twice, first flushing the separation column with a dichloromethane-methanol mixture as an eluent, and then flushing the separation column with an ethyl acetate-methanol-water mixture as an eluent to obtain a purified D-biotin-labeled crocin probe.

[0044] In a further embodiment of the method of the present invention, in step (4), crocin is first dissolved in a small amount of water (crocin has good water solubility), then diluted with 10 times the volume of methanol, and then silica gel is added to mix the sample, thereby obtaining a sample for dry column packing. Here, the volume ratio of methanol to water is 10:1, which is related to the good water solubility and poor fat solubility of crocin, and the low water content (<10%) of silica gel during sample preparation makes it easier to remove.

[0045] In a further embodiment of the method of the present invention, in step (4), the temperature of the rotary evaporator for drying is in the range of 45-50° C. The use of the rotary evaporator can evaporate the toxic methanol as airtightly as possible, ensuring the on-site safety of the preparation method.

[0046] In a further embodiment of the method of the present invention, in step (4), a mixture of dichloromethane and methanol is first used as an elution system to remove impurities related to the D-biotin intermediate; and then a mixture of ethyl acetate, methanol and water is used as an elution system to elute the target product crocin probe.

[0047] In a further embodiment of the method of the present invention, the column chromatography process includes flushing the separation column twice, first flushing the separation column with a mixture of dichloromethane and methanol in a volume ratio of 10:1 as an eluent, and then flushing the separation column with a mixture of ethyl acetate, methanol and water in a volume ratio of 10:3:1 as an eluent to obtain a purified D-biotin-labeled crocin probe.

[0048] In a further embodiment of the method of the present invention, in step (4), the proportion of water in the eluent used is relatively small, so as to protect the silica gel as much as possible and prevent it from being dissolved by water.

[0049] In yet a further embodiment of the method of the present invention, in step (1), the solvent is selected from N,N-dimethylformamide (DMF); the condensing agent is selected from 1-ethyl-3-(3-dimethylpropylamine)carbodiimide hydrochloride (EDCI); and the organic base is selected from N,N-diisopropylethylamine (DIPEA).

[0050] In a further embodiment of the method of the present invention, in step (1), the added condensing agent, solvent, and organic base are all high-purity substances. The molar ratio of D-biotin to N-Boc-1,6-hexanediamine is 1:1.2; the molar ratio of D-biotin to DIPEA is 1:3; the molar ratio of D-biotin to EDCI is 1:2; and the ratio of the mass of D-biotin to the volume of DMF is 1:20.

[0051] In a further embodiment of the method of the present invention, in step (1), the amount of D-Boc-1,6-hexanediamine component used is slightly excessive relative to the amount of D-biotin component used, so as to achieve as complete a reaction of the D-biotin raw material as possible; and D-Boc-1,6-hexanediamine is a liquid and can be removed by subsequent filtration.

[0052] In a further embodiment of the method of the present invention, in step (1), the molar ratio of D-biotin to DIPEA is controlled to be 1:3, and the molar ratio of D-biotin to EDCI is controlled to be 1:2, so as to facilitate complete reaction of the D-biotin. D-biotin has poor solubility in the reaction raw material system of the present invention and is difficult to remove. Therefore, allowing it to react completely in step (1) is beneficial for improving the purity of the target product, the D-biotin-labeled carthamin probe.

[0053] In a further embodiment of the method of the present invention, in step (2), the volume ratio of the dichloromethane to the trifluoroacetic acid is 5:1.

[0054] In a further embodiment of the method of the present invention, in step (2), the added dichloromethane and trifluoroacetic acid are both high-purity substances; in step (2), the molar ratio of the compound 3-1 to the dichloromethane to the trifluoroacetic acid is 1:156:27.

[0055] In a further embodiment of the method of the present invention, in step (3), the molar ratio of the compound 3-2 to the bromoacetyl bromide is 1:1; and / or in step (4), the molar ratio of the compound 3-3 to the crocin is 1:1.

[0056] In a further embodiment of the method of the present invention, in step (3), the volume ratio of bromoacetyl bromide to dichloromethane is 1:40.

[0057] The beneficial effects of the present invention are as follows:

[0058] 1. The D-biotin-labeled crocin probe of the present invention is a newly developed substance with good application prospects in clinical medicine.

[0059] 2. D-biotin was first connected to N-Boc-1,6-hexanediamine, then to bromoacetyl bromide under ice bath conditions, and finally crocin was used to undergo a nucleophilic substitution reaction, thereby successfully constructing the target probe.

[0060] 3. When finally purifying the target product, in the first step, impurities are removed using an eluent with a dichloromethane-methanol volume ratio of 10:1; in the second step, impurities are removed using an eluent with a volume ratio of ethyl acetate-methanol-water of 10:3:1, resulting in a two-step elution and purification. Without the first step, some impurities in the product cannot be removed; in the second step, conventional eluents are discarded, and the proportion of water in the added eluent is relatively low. This is a setting made considering the water solubility of crocin targeted by the method of the present invention and the purity of the target product D-biotin-labeled crocin probe: crocin has good water solubility and can be completely dissolved using less water; silica gel has a certain water solubility, and the eluent containing less water can avoid the dissolution of silica gel caused by a large proportion of water, which affects the separation effect and thus the purity of the target product. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Compound 3-1 prepared in Example 1 of the present invention 1 HNMR spectrum.

[0062] Figure 2 Compound 3-2 prepared in Example 1 of the present invention 1 HNMR spectrum.

[0063] Figure 3 The D-biotin labeled crocin probe prepared in Example 1 of the present invention 1 HNMR spectrum.

[0064] Figure 4 This is a gel imaging image of the probe targeting the binding protein prepared in Example 1 of the present invention.

[0065] Figure 5 This is the mass spectrometry base peak diagram of the probe targeting the binding protein prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0066] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0067] raw material:

[0068]

[0069]

[0070] Example 1 Synthesis of D-biotin-labeled crocin probe

[0071] (1) Add 4 mmol (1 g) of D-biotin and 4.8 mmol (1.098 mL) of N-Boc-1,6-hexanediamine to a single-necked flask, then add 20 mL of anhydrous N,N-dimethylformamide (DMF), and then add 12 mmol (2.028 mL) of N,N-diisopropylethylamine (DIPEA) and 8 mmol (1.5768 g) of 1-ethyl-3-(3-dimethylpropylamino)carbodiimide hydrochloride (EDCI) under stirring. The mixture is reacted at room temperature (about 25°C) for 12 hours. After the reaction is complete, a reaction solution is obtained. Whether the reaction is complete can be determined by observing whether the reaction solution is clear (because D-biotin has poor solubility in DMF solvent), which can be used as one of the judgment criteria; then thin layer chromatography (TLC) can be used to determine whether the reaction is complete.

[0072] Add 100 ml of ultrapure water to a beaker and slowly add the reaction solution dropwise while stirring to precipitate a solid. Filter the solid and dry it to obtain compound 3-1 (500 mg) with a yield of approximately 23%.

[0073] 1 H NMR(400MHz,DMSO-d6)δ7.73(t,J=5.5Hz,1H),6.80-6.73(m,1H),6.42(s,1H ),6.36(s,1H),4.34-4.27(m,1H),4.16-4.09(m,1H),3.10-3.07(m,1H),3.0 1(dd,J=12.7,6.6Hz,1H),2.89(dd,J=13.2,6.4Hz,1H),2.82(dd,J=12.4,5. 1Hz, 1H), 2.58 (d, J = 12.4Hz, 1H), 2.04 (t, J = 7.4Hz, 2H), 1.68-1.15 (m, 23H).

[0074] (2) Add 500 mg (1.13 mmol) of compound 3-1 to a single-necked flask, then add 10 mL of dichloromethane, and finally add 2 mL of trifluoroacetic acid with stirring. The mixture is allowed to react at room temperature for 3 h. After the reaction is complete, the reaction solution is dried using a rotary evaporator, and then 10 mL of dichloromethane is added and dried again (repeat 3 times) to obtain compound 3-2 (200 mg) without trifluoroacetic acid.

[0075] 1H NMR (400MHz, DMSO-d6) δ7.85-7.75(m,4H),6.43-6.35(m,2H),4.31(dd,J=7.6,5.0Hz,1H),4.13(dd,J=7.7,4.4Hz,1H),3.12-3 .05(m,1H),3.01(dd,J=12.8,6.7Hz,2H),2.85-2.72(m,3H),2.58(d,J=12.4Hz,1H),2.04(t,J=7.4Hz,2H),1.68-1.16(m,14H).

[0076] (3) 0.451 mmol (200 mg) of compound 3-2 was dissolved in 2 mL of pyridine to obtain reaction solution a. 0.451 mmol (41 μL) of bromoacetyl bromide was dissolved in dichloromethane (1.6 mL) under ice-bath conditions to obtain reaction solution b. Reaction solution a was slowly added dropwise to the stirred reaction solution b under ice-bath conditions. The mixture was reacted under ice-bath conditions for 10 min to obtain compound 3-3. Compound 3-3 was used directly in the next step without further treatment.

[0077] (4) 0.451 mmol (460 mg) of crocin was dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF). Compound 3-3 was slowly added dropwise while stirring at room temperature. The reaction was allowed to react for 12 h. After the reaction was complete, the reaction solution was slowly added dropwise to stirred ethyl acetate to precipitate a yellow solid. The yellow solid was filtered to obtain a crude product of the probe.

[0078] The synthetic route is as follows:

[0079]

[0080] The crude product was purified by column chromatography. Purification conditions included dissolving the crude product in a 10:1 volume ratio of methanol to water; then adding 200-300 mesh silica gel and drying using a rotary evaporator. The column chromatography purification process involved first flushing the column with a 10:1 volume ratio of dichloromethane to methanol to remove some impurities; then performing a secondary cleanup with a 10:3:1 volume ratio of ethyl acetate to methanol to water to obtain 10 mg of purified (95% purity) D-biotin-labeled crocin probe (Biotin-Crocin) in a 2% yield (protect from light during these operations).

[0081] Figure 2 In the figure, 1.25-1.75 are the hydrogens of the aliphatic chain in 3-2, and 2.7-3.2 are the peaks of the biotin S-ortho position and the amino-ortho position in 3-2.

[0082] Figure 3In the figure, 1.75-2.0 is the characteristic methyl peak of crocin, the triplet peak at 2.25 is the carbonyl peak of biotin in 3-2, and 3.25-4.0 is the carbonyl peak of crocin.

[0083] Target protein detection of the D-biotin-labeled crocin probe of the present invention

[0084] Purpose of detection: To obtain the target protein of crocin in vascular endothelial cells and provide support for further research on its biological effects.

[0085] Detection methods: Co-IP experiments and protein profiling.

[0086] Detection steps (CO-IP experiment):

[0087] (1) Prepare 6 culture dishes (10 cm) filled with primary human umbilical vein endothelial cells, wash twice with PBS, then add 1 ml of PBS to each culture dish to cover the cell surface, collect the cells with a scraper into an EP tube, centrifuge at 800 rpm for 15 min, collect the cell pellet, add 500 μl of lysis buffer (RIPA:PS:PSMF is 100:2:1) to the pellet, let it stand on ice for 30 min, shake it every 10 minutes, then centrifuge at 4°C and 12,000 rpm for 30 min, and collect the supernatant. Divide the supernatant into three parts, i.e., set up three groups (Input group, IgG group, IP group).

[0088] (2) The IgG group was the negative control, and 2 μl of IgG (10 μg / ml) was added; the IP group was the experimental group, and 100 μl of crocin probe solution labeled with D-biotin (29.5×10 3 umol / L) and incubated with rotation at 4°C overnight to form a D-biotin-labeled crocin probe-target protein complex (abbreviated as: biotin / crocin / target protein complex); the input group served as the positive control.

[0089] (3) On the second day, 20 μl of streptavidin magnetic beads were added to the IgG group and IP group respectively. The beads were rotated and incubated at 4°C for 2 hours. Streptavidin and biotin specifically bound to form a "magnetic bead / biotin / crocin / target protein complex". The magnetic bead complex was collected by magnetic adsorption and washed four times with 500 μl of RIPA. Then, loading buffer was added at a volume ratio of 1:2. The loading buffer was added to the supernatant of the Input group at the above ratio.

[0090] (4) The samples prepared above were subjected to polyacrylamide gel electrophoresis to obtain the target protein, and the electrophoresis gel was stained with Coomassie brilliant blue for 30 minutes, and then washed with clean water until obvious electrophoresis bands appeared at the corresponding positions. The gel was imaged. The results were as follows: Figure 4 shown.

[0091] (5) Protein spectrum: Collect IP group strips and perform protein spectrum detection.

[0092] The mass spectrum base peak of this sample is as follows Figure 5 As shown. Figure 5 The base peak diagram of mass spectrometry detection is shown. The horizontal axis is the peptide retention time and the vertical axis is the mass spectrometry signal intensity. Figure 5 If we see that there are many peaks eluting at different times and their relative abundance is high, it means that there are many types of peptides and they are highly complex.

[0093] The statistical results of the number of proteins and peptides identified in this sample are shown in Table 1 below.

[0094] Table 1 Statistics of protein identification results from combined search

[0095] Sample name Number of protein identifications Number of peptide identifications Number of PSM identifications crocin 546 2111 2541

[0096] After mass spectrometry data retrieval, PSM FDR ≤ 0.01 and protein FDR ≤ 0.01 were used as screening criteria for peptide, site, and protein identification, respectively. Table 1 shows the protein identification results, which show that the crocin probe targets 546 proteins and 2111 peptides. PSM represents the number of matches between the spectrum and peptides.

[0097] The above results show that the constructed biotin-crocin probe can be effectively used for the analysis of crocin target protein, providing experimental data basis and support for further research on the medicinal mechanism of saffron.

[0098] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the spirit of the present invention.

Claims

1. A D-biotin-labeled crocin probe, characterized in that: The molecular formula is shown in formula (I): Formula (I).

2. A method for synthesizing the D-biotin-labeled crocin probe according to claim 1, characterized in that: The following steps are involved: (1) D-biotin and N-Boc-1,6-hexanediamine are first mixed, and then a solvent, a condensing agent, and an organic base are added, and the mixture is reacted at room temperature to obtain a first product mixture; the first product mixture is added to ultrapure water to precipitate a solid, and the solid is filtered and dried to obtain compound 3-1; Wherein, the condensing agent is selected from any one of 1-ethyl-3-(3-dimethylpropylamine)carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate or O-benzotriazole-tetramethyluronium hexafluorophosphate; The organic base is selected from any one of N,N-diisopropylethylamine and triethylamine; The solvent is a polar aprotic solvent selected from any one of N,N-dimethylformamide, dimethyl sulfoxide, hexamethylphosphoramide or N-methylpyrrolidone; The synthetic route is: ； (2) in the presence of dichloromethane, reacting compound 3-1 with trifluoroacetic acid at room temperature to remove the -Boc group, thereby obtaining a second product mixture; and evaporating the second product mixture under reduced pressure to remove the dichloromethane and trifluoroacetic acid, thereby obtaining compound 3-2; ； (3) Compound 3-2 was dissolved in pyridine to obtain reaction solution a; bromoacetyl bromide was dissolved in dichloromethane under ice bath to obtain reaction solution b; reaction solution a was then added to reaction solution b under ice bath to react and obtain compound 3-3; ； (4) Under light-shielding conditions, crocin is dissolved in the same solvent as that in step (1) to obtain a reaction solution c, compound 3-3 is added to the reaction solution c at room temperature to react to obtain a final product mixture, the final product mixture is slowly dripped into ethyl acetate to precipitate a solid, the solid is filtered to obtain a crude product, and the crude product is subjected to column chromatography to obtain a purified D-biotin-labeled crocin probe.

3. The method for synthesizing a D-biotin-labeled crocin probe according to claim 2, wherein In step (4), a sample is prepared for column chromatography using methanol-water in a volume ratio of 10:1; the column chromatography process includes flushing the separation column twice, firstly flushing the separation column with a mixture of dichloromethane-methanol as an eluent, and then flushing the separation column with a mixture of ethyl acetate-methanol-water as an eluent, to obtain a purified D-biotin-labeled crocin probe.

4. The method for synthesizing a D-biotin-labeled crocin probe according to claim 3, wherein The column chromatography process includes two flushing of the separation column. First, the separation column is flushed with a mixture of dichloromethane and methanol in a volume ratio of 10:1 as an eluent, and then the separation column is flushed with a mixture of ethyl acetate, methanol and water in a volume ratio of 10:3:1 as an eluent to obtain a purified D-biotin-labeled crocin probe.

5. The method for synthesizing a D-biotin-labeled crocin probe according to any one of claims 2 to 4, wherein: In step (1), the solvent is selected from N,N-dimethylformamide; the condensing agent is selected from 1-ethyl-3-(3-dimethylpropylamine)carbodiimide hydrochloride; and the organic base is selected from N,N-diisopropylethylamine.

6. The method for synthesizing a D-biotin-labeled crocin probe according to any one of claims 2 to 4, wherein: In step (1), the molar ratio of D-biotin to N-Boc-1,6-hexanediamine is 1:1.2; The molar ratio of D-biotin to DIPEA is 1:3; The molar ratio of D-biotin to EDCI is 1:

2.

7. The method for synthesizing a D-biotin-labeled crocin probe according to any one of claims 2 to 4, wherein: In step (2), the volume ratio of the dichloromethane to the trifluoroacetic acid is 5:

1.

8. The method for synthesizing a D-biotin-labeled crocin probe according to any one of claims 2 to 4, wherein: In step (3), the molar ratio of the compound 3-2 to the bromoacetyl bromide is 1:1; and / or in step (4), the molar ratio of the compound 3-3 to the crocin is 1:1.

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