Method for synthesizing 2,2',4,5,5'-pentachloro-4-biphenylcarbinol based on pig liver microsomal incubation system

Abstract

The application discloses a method for synthesizing 2,2',4,5,5'-pentachloro-4-biphenyl alcohol based on a pig liver microsome incubation system, which comprises the following steps: establishing a pig liver microsome incubation system, and adding a substrate 2,2',4,5,5'-pentachlorobiphenyl; the 2,2',4,5,5'-pentachlorobiphenyl is metabolically converted by pig liver microsomes to form 2,2',4,5,5'-pentachloro-4-biphenyl alcohol; after termination of the incubation, isopropyl alcohol, dichloromethane and n-hexane are added to the incubation solution, and after oscillation and centrifugation, the organic phase is transferred and volatilized under high-temperature and reduced-pressure conditions; the 2,2',4,5,5'-pentachloro-4-biphenyl alcohol is directly converted, the lipid impurities are removed through concentrated sulfuric acid sulfonation reaction, the residual 2,2',4,5,5'-pentachlorobiphenyl is separated by using a silica gel column, and thus the purified 2,2',4,5,5'-pentachloro-4-biphenyl alcohol is obtained, and the method has the advantages of simple operation, high conversion efficiency, no interference of isomers, and high purity of target products.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing, specifically relating to a method for the biosynthesis and purification of 2,2',4,5,5'-pentachloro-4-benzyl alcohol based on a porcine liver microsomal incubation system. Background Technology

[0002] Polychlorinated biphenyls (PCBs) are a class of biphenyl molecules with varying numbers of chlorine atoms substituted for each other. Historically, they were produced and used on a large scale as industrial products. PCBs have a very stable chemical structure, allowing them to persist in the environment for extended periods and accumulate through the food chain. They pose health hazards to animals and humans, including carcinogenicity, endocrine disruption, neurotoxicity, reproductive toxicity, and immunotoxicity. Therefore, PCBs are listed in the Stockholm Convention on Persistent Organic Pollutants and are strictly controlled hazardous substances in the environmental and food safety fields. Research has found that PCB homologues with specific molecular structures can be metabolized by animals, plants, and microorganisms to produce hydroxyPCBs. HydroxyPCBs are more toxic and have stronger endocrine disrupting effects than their parent PCBs, making them a current research focus. 2,2',4,5,5'-Pentachlorobiphenyl (IUPAC designation PCB101) is one of the most frequently found PCB homologues in food. It can be metabolized by different types of animals to form two hydroxypolychlorinated biphenyls: 2,2',4,5,5'-pentachloro-4-biphenylol (abbreviated 4'-OH-PCB101) and 2,2',4,5,5'-pentachloro-3-biphenylol (abbreviated 3'-OH-PCB101).

[0003] Based on the varying numbers and positions of chlorine and hydroxyl atoms, theoretically there are 837 homologues of hydroxypolychlorinated biphenyls (HPCBs), but only a few dozen have been discovered to date that can be produced through biological metabolism. Currently, HPCBs are only used for detection; their preparation is limited to serving as standards or standard solutions for detection. The preparation of HPCBs typically employs chemical synthesis, which offers advantages such as rapid preparation, low cost, and high yield. However, due to the large number of HPCB homologues, chemical synthesis cannot precisely control the number and positions of hydroxyl and chlorine atoms on the benzene ring, requiring the synthesis of a series of mixtures followed by separation. This results in significant drawbacks, including difficulty in controlling directional synthesis conditions, the generation of numerous byproducts and homologues, and extreme difficulty in separating and purifying individual homologues, leading to complex preparation processes and high separation and purification costs. Summary of the Invention

[0004] To address the shortcomings of existing technologies and solve the aforementioned technical problems, this application provides a method for synthesizing 2,2',4,5,5'-pentachloro-4-biphenylol based on a porcine liver microsomal incubation system.

[0005] This application leverages the advantages of bioenzymes, such as high specificity, high catalytic efficiency, and mild reaction conditions, to generate hydroxypolychlorinated biphenyls (PCBs) with specific structures from a particular PCB molecule. By screening suitable bioenzymes, the idea of ​​directionally catalyzing PCBs to produce hydroxypolychlorinated biphenyls with specific single structures can be realized, further eliminating interference from homologues and facilitating further separation and purification to obtain the target product.

[0006] Animal liver microsomes are rich in enzymes that metabolize polychlorinated biphenyls (PCBs). By incubating PCBs with animal liver microsomes in vitro and controlling the conditions, hydroxyPCBs with specific structures can be generated rapidly and accurately.

[0007] Therefore, developing a method for the preparation and purification of hydroxypolychlorinated biphenyls (PCBs) based on in vitro metabolism in animal liver microsomes is of great practical significance and application value for further developing single-structure PCB standards, reducing preparation costs, improving synthesis efficiency, and reducing resource waste.

[0008] Based on the characteristics of porcine liver microsomes specifically metabolizing PCB101 (a common polychlorinated biphenyl contaminant in food) to form 4'-OH-PCB101, mouse liver microsomes specifically metabolizing PCB101 to form 3'-OH-PCB101, and other animal liver microsomes simultaneously metabolizing PCB101 to produce both 4'-OH-PCB101 and 3'-OH-PCB101, this application proposes a method for biosynthesizing 4'-OH-PCB101 based on porcine liver microsomes.

[0009] Based on the above technical approach, the following technical solution was specifically designed in this case to solve the problems existing in the background technology:

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] Step 1: 2,2',4,5,5'-pentachlorobiphenyl is metabolized into 2,2',4,5,5'-pentachloro-4-biphenyl alcohol in a porcine liver microsomal incubation system;

[0012] The above steps specifically include the construction of a porcine liver microsomal incubation system, pretreatment of 2,2',4,5,5'-pentachlorobiphenyl, and initiation of the metabolic reaction of porcine liver microsomes. Step 101: Porcine liver microsomal incubation system

[0013] Pig liver microsomes were suspended in a buffer salt solution to form a pig liver microsome incubation system.

[0014] System establishment method: A porcine liver microsome incubation system was constructed to uniformly disperse the metabolic enzymes in porcine liver microsomes in a buffer salt solution and provide a suitable pH environment for the metabolic process. Maintaining an appropriate concentration of porcine liver microsomes (based on protein) in the buffer salt solution is beneficial for the smooth progress of metabolic reactions; excessively high or low microsome protein concentrations will lead to reduced conversion efficiency.

[0015] In one feasible approach, the specific system construction process involves using 0.01M phosphate buffer as a solvent, with the following component concentrations: microsomal protein 0.5 mg / mL, nicotinamide adenine dinucleotide phosphate (NADP+) 1.3 mM, glucose-6-phosphate 3.3 mM, magnesium chloride 3.3 mM, glucose-6-phosphate dehydrogenase 0.4 U / mL, sodium citrate 0.05 mM; and 2,2',4,5,5'-pentachlorobiphenyl added to the porcine liver microsomal incubation system at a concentration of 2 μg / mL.

[0016] In particular, the pH of the buffer salt solution should be controlled at 7.2 ± 0.2.

[0017] Specifically, the concentration of porcine liver microsomes (based on protein) in buffered saline solution should be controlled between 0.25 and 1.0 mg / mL, with 0.5 mg / mL being the preferred concentration.

[0018] Step 102: Pretreatment steps for 2,2',4,5,5'-pentachlorobiphenyl

[0019] A solution of 2,2',4,5,5'-pentachlorobiphenyl was added to the porcine liver microsome incubation system to ensure that the 2,2',4,5,5'-pentachlorobiphenyl came into full contact with the metabolic enzymes of the porcine liver microsomes.

[0020] It should be noted that 2,2',4,5,5'-pentachlorobiphenyl is a hydrophobic substance and insoluble in aqueous systems. It must first be dissolved in a suitable solvent before being introduced into the porcine liver microsome incubation system to ensure sufficient contact between 2,2',4,5,5'-pentachlorobiphenyl and the metabolic enzymes of the porcine liver microsomes, thus ensuring conversion efficiency. The concentration of 2,2',4,5,5'-pentachlorobiphenyl is also a key factor affecting conversion efficiency.

[0021] The designers of this case discovered that the concentration of 2,2',4,5,5'-pentachlorobiphenyl added to the porcine liver microsomal system exhibited a two-stage functional relationship with the formation rate of 2,2',4,5,5'-pentachloro-4-biphenyl alcohol, which conforms to the Michaelis-Menten equation of enzyme reaction kinetics.

[0022] v = 22.5 * S / (1.42 + S), where v is the reaction rate and S is the substrate concentration.

[0023] Initially, as the concentration of 2,2',4,5,5'-pentachlorobiphenyl increased, the formation rate of 2,2',4,5,5'-pentachloro-4-biphenyl alcohol increased accordingly; when the concentration of 2,2',4,5,5'-pentachlorobiphenyl increased to a certain extent, the formation rate of 2,2',4,5,5'-pentachloro-4-biphenyl alcohol tended to stabilize and no longer increased.

[0024] Therefore, in this case, 2,2',4,5,5'-pentachlorobiphenyl was first dissolved in dimethyl sulfoxide (DMSO) to prepare a 0.2 mg / mL 2,2',4,5,5'-pentachlorobiphenyl solution, which was then added to the porcine liver microsome incubation system at a volume ratio of 1:99. This ensured that 2,2',4,5,5'-pentachlorobiphenyl was uniformly dispersed in the porcine liver microsome incubation system, guaranteeing the smooth progress of the enzymatic reaction.

[0025] Preferably, the concentration of 2,2',4,5,5'-pentachlorobiphenyl in the porcine liver microsome incubation system is set at 2 μg / mL to obtain the maximum formation rate of 2,2',4,5,5'-pentachloro-4-biphenyl alcohol.

[0026] Step 103: Initiate the metabolic response of porcine liver microsomes

[0027] Under suitable temperature and stirring conditions, coenzymes were added to initiate the metabolic reaction of 2,2',4,5,5'-pentachlorobiphenyl by porcine liver microsomes.

[0028] It should be noted that the enzymatic reactions of metabolic enzymes in liver microsomes require suitable environmental conditions such as temperature, and also require the participation of coenzymes. To enhance the contact between the enzyme and 2,2',4,5,5'-pentachlorobiphenyl, the liver microsome incubation solution should be stirred. Too low a stirring rate will cause precipitation of pig liver microsomes, while vigorous stirring will cause denaturation of microsomal proteins, resulting in loss of their metabolic function.

[0029] Based on this, the temperature of the porcine liver microsome incubation system in this case is controlled between 37°C and 40°C, with 38°C being the preferred setting.

[0030] Furthermore, the incubation system is placed on a rotating shaker with the rotation speed controlled at 30–80 rpm, preferably 50 rpm.

[0031] In this case, the concentrations of the coenzyme and buffer salts added to the incubation system were as follows: nicotinamide adenine dinucleotide phosphate (NADP+) 1.3 mM, glucose-6-phosphate 3.3 mM, magnesium chloride 3.3 mM, glucose-6-phosphate dehydrogenase 0.4 U / mL, and sodium citrate 0.05 mM.

[0032] Step 2: Remove biomass impurities such as fats from the extraction solution using concentrated sulfuric acid;

[0033] 2,2',4,5,5'-pentachloro-4-biphenylol was extracted from a porcine liver microsomal incubation system using an organic solvent via liquid-liquid extraction. The 2,2',4,5,5'-pentachloro-4-biphenylol produced from the metabolism of 2,2',4,5,5'-pentachlorobiphenyl by porcine liver microsomes needs to be extracted from the incubation system solution.

[0034] It is worth noting that, since 2,2',4,5,5'-pentachloro-4-biphenylol is a weakly polar substance, a nonpolar-polar mixed solution can effectively extract weakly polar substances from complex biological matrix solutions. However, directly mixing the nonpolar-polar mixed solution with a protein-rich porcine liver microsome incubation system may lead to severe emulsification, resulting in extraction failure. Therefore, using a suitable demulsifier is an important condition for the extraction of 2,2',4,5,5'-pentachloro-4-biphenylol.

[0035] Therefore, in one feasible approach, isopropanol can be used as the demulsifier, and n-hexane-dichloromethane mixed solution can be used as the non-polar-polar mixed solution; preferably, the ratio of the three solvents, isopropanol, n-hexane, and dichloromethane, is 1:3:3 by volume.

[0036] The extraction solution is prepared by mixing isopropanol, n-hexane, and dichloromethane solutions in a specific ratio. The volume ratio of the extraction solution to the porcine liver microsome incubation system solution is 5:1.

[0037] Furthermore, the extraction method is as follows: after adding the extraction solution to the porcine liver microsome incubation system solution, extraction can be carried out by shaking.

[0038] Preferably, the oscillation rate is 200 rpm and the time is 20 min.

[0039] After extraction, the solution is separated into layers by standing or centrifugation.

[0040] Furthermore, the solution was allowed to stand for 1 hour. After the solution separated into layers, the upper organic phase was the crude extract of 2,2',4,5,5'-pentachloro-4-biphenylol.

[0041] Step 3: Remove unreacted 2,2',4,5,5'-pentachlorobiphenyl and lipids from biomass by rinsing with a nonpolar solvent through a silica gel column;

[0042] Step 301: Remove lipids

[0043] In this case, the crude extract was solvent-converted, and concentrated sulfuric acid was used to remove lipids and other biomass components. This included evaporating the solvent from the upper organic phase by vacuum evaporation, dissolving the residue with n-hexane, and then adding concentrated sulfuric acid to remove lipids and other biomass components from the solution.

[0044] Although the crude extract separates inorganic salts, proteins, and other substances from 2,2',4,5,5'-pentachloro-4-biphenylol, it still contains lipids and other biomass, as well as unreacted 2,2',4,5,5'-pentachlorobiphenyl. Concentrated sulfuric acid can chemically react with lipids and other biomass, decomposing them into inorganic substances, thus separating them from 2,2',4,5,5'-pentachloro-4-biphenylol dissolved in organic solvents. Dichloromethane in the crude extract can react with concentrated sulfuric acid, and the resulting product is soluble in organic solvents; therefore, the solvent for the crude extract needs to be converted to a solvent that is chemically inert to concentrated sulfuric acid.

[0045] In an optional embodiment, the crude extract is evaporated using a rotary evaporator and then redissolved with an organic solvent that is inert to concentrated sulfuric acid.

[0046] Those skilled in the art will know that rotary evaporators are used multiple times, including the upper layer solution described below. During the purification, separation, and concentration of the target substance, the solution needs to be evaporated to dryness using a rotary evaporator multiple times, then reconstituted with the solution for further processing, followed by evaporation, and then reconstitution. The specific operating methods are well-known and will not be elaborated upon in this document.

[0047] The inert solvent mentioned above is n-hexane, and the volume used is the same as the volume of the crude extract.

[0048] For impurity removal, add concentrated sulfuric acid at 5-10 times the volume of the microsomal protein to the solution. Then, rotate the container at 50 rpm for 20 minutes to allow the concentrated sulfuric acid to react with the organic matter in the solution. Finally, allow the solution to stand or centrifuge at 5000 rpm to separate the layers. The lower layer is the product of the sulfonation reaction between concentrated sulfuric acid and organic matter, and the upper layer is a hexane solution containing 2,2',4,5,5'-pentachloro-4-biphenylol and 2,2',4,5,5'-pentachlorobiphenyl. Evaporate the upper solution to 1-3 mL of concentrated solution using a rotary evaporator for further purification.

[0049] Preferably, the weight of concentrated sulfuric acid added is 10 times the amount of microsomal protein.

[0050] Step 302: Remove 2,2',4,5,5'-pentachlorobiphenyl and other persistent organic contaminants from the concentrate.

[0051] Approach: Use silica gel column chromatography to separate 2,2',4,5,5'-pentachlorobiphenyl and other persistent organic pollutants from the concentrate to obtain a purified 2,2',4,5,5'-pentachloro-4-biphenyl alcohol solution.

[0052] First, it should be noted that while the concentrated solution purified by concentrated sulfuric acid has removed all lipids and other biomass, it still contains nonpolar and weakly polar impurities that are chemically inert to concentrated sulfuric acid. These are mainly 2,2',4,5,5'-pentachlorobiphenyl and other persistent organic pollutants that may be present in the liver microsome incubation system. 2,2',4,5,5'-pentachloro-4-biphenyl alcohol is a weakly polar substance, while these impurities are all nonpolar. Therefore, based on the differences in retention characteristics of compounds with different polarities on a silica gel column, solvents of different polarities can be used to elute and separate the nonpolar and weakly polar substances.

[0053] As a further optimization step of the method in this case, the silica gel column is prepared by packing silica gel packing material into a glass chromatography column. The silica gel packing material has a particle diameter range of 0.063-0.100 mm and a moisture content of 5% by mass; the glass chromatography column has a diameter of 1 cm, and the amount of silica gel packing material is 3 g.

[0054] For solvents of different polarities, n-hexane can be used as a non-polar solvent, while a mixed solution of dichloromethane and n-hexane with a volume ratio of 2:3 can be used as a weakly polar solvent.

[0055] Before use, activate the silica gel column with 20 mL of n-hexane. Load the concentrate and rinse the silica gel column with 30 mL of n-hexane to remove 2,2',4,5,5'-pentachlorobiphenyl and other persistent organic contaminants.

[0056] Step 4: Elute 2,2',4,5,5'-pentachloro-4-biphenylol on the silica gel column using a weakly polar mixed solution;

[0057] The 2,2',4,5,5'-pentachloro-4-biphenyl alcohol adsorbed on the chromatographic column was eluted with 30 mL of a 2:3 dichloromethane-n-hexane mixed solution to obtain a purified 2,2',4,5,5'-pentachloro-4-biphenyl alcohol solution.

[0058] In an optional embodiment, 2,2',4,5,5'-pentachloro-4-biphenylol adsorbed on the silica gel column is eluted with 1.5 times the volume of dichloromethane / n-hexane (volume ratio 2:3) to obtain a 2,2',4,5,5'-pentachloro-4-biphenylol eluent.

[0059] Step 5: The eluent was dried under vacuum to obtain purified 2,2',4,5,5'-pentachloro-4-biphenyl alcohol.

[0060] The purified 2,2',4,5,5'-pentachloro-4-biphenyl alcohol solution was placed in a vacuum desiccator to evaporate the solvent, and then dissolved in an appropriate amount of low-volatility solvent to obtain a 2,2',4,5,5'-pentachloro-4-biphenyl alcohol solution that can be stored for a long time.

[0061] 2,2',4,5,5'-pentachloro-4-biphenylol crystals were obtained. Acetonitrile was added at a ratio of 1 mL per mg of crystals to obtain a 1 mg / mL 2,2',4,5,5'-pentachloro-4-biphenylol acetonitrile solution.

[0062] The purified 2,2',4,5,5'-pentachloro-4-biphenyl alcohol is stable and can be stored at room temperature for a long time. To reduce concentration errors caused by solvent evaporation, the highly volatile dichloromethane-n-hexane mixed solution should be replaced with a less volatile solvent.

[0063] In an optional embodiment, acetonitrile can be used as a low-volatility solvent. The purified 2,2',4,5,5'-pentachloro-4-biphenylol solution is placed in a vacuum desiccator to evaporate the solvent, yielding 2,2',4,5,5'-pentachloro-4-biphenylol crystals.

[0064] The mass of the obtained crystals was calculated using the mass difference method. Acetonitrile was added at a ratio of 1 mL per mg of crystals to obtain a 1 mg / mL solution of 2,2',4,5,5'-pentachloro-4-biphenylol acetonitrile. The 1 mg / mL solution of 2,2',4,5,5'-pentachloro-4-biphenylol acetonitrile was diluted with acetonitrile to 1 μg / mL and identified by mass spectrometry.

[0065] In summary, the present invention has the following main beneficial effects:

[0066] 1) This invention utilizes porcine liver microsomes as a bioreactor to convert 2,2',4,5,5'-pentachlorobiphenyl into 2,2',4,5,5'-pentachloro-4-biphenylol. It has the advantages of simple product structure, high conversion efficiency, and simple and controllable reaction conditions, and overcomes the disadvantages of chemical synthesis methods, such as difficult control of reaction conditions, low yield, many types of impurity compounds, and difficulty in separating and purifying the target analyte.

[0067] 2) This invention employs concentrated sulfuric acid sulfonation to remove biomass from the porcine liver microsomal reaction system. 2,2',4,5,5'-pentachloro-4-biphenyl alcohol is further separated and purified using a silica gel column. By utilizing the difference in molecular polarity between impurity compounds and 2,2',4,5,5'-pentachloro-4-biphenyl alcohol, residual inorganic salts, non-polar persistent organic pollutants, and unreacted 2,2',4,5,5'-pentachlorobiphenyl can be effectively removed from the porcine liver microsomal reaction system, thereby obtaining highly purified 2,2',4,5,5'-pentachloro-4-biphenyl alcohol. Compared with traditional size exclusion chromatography purification methods, this method has advantages such as simple operation, high speed, and thorough impurity separation.

[0068] Therefore, the present invention has the advantages of simple operation, short time consumption, high efficiency, low cost and good stability. Attached Figure Description

[0069] Figure 1 This is a synthetic route diagram for the present invention.

[0070] Figure 2 This diagram illustrates the possible metabolic pathways and metabolites of 2,2',4,5,5'-pentachlorobiphenyl of the present invention.

[0071] Figure 3 This invention relates to the metabolic pathway and metabolites of 2,2',4,5,5'-pentachlorobiphenyl via porcine liver microsomes.

[0072] Figure 4 Michaelis equation fitting plots for the metabolism of 2,2',4,5,5'-pentachlorobiphenyl to 2,2',4,5,5'-pentachloro-4-biphenylol in liver microsomes of different breeds of pigs; where (a) liver microsomes of Bama miniature pigs; (b) liver microsomes of Landrace pigs; (c) liver microsomes of Duroc × Landrace × Large White crossbred pigs. Detailed Implementation

[0073] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Note: Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the raw materials, reagents, consumables, etc. in the following embodiments are commercially available products.

[0074] See attached document Figure 1The purpose of this invention is to provide a method for producing 2,2',4,5,5'-pentachlorobiphenyl by metabolizing 2,2',4,5,5'-pentachloro-4-biphenylol via porcine liver microsomes, followed by further separation and purification of 2,2',4,5,5'-pentachloro-4-biphenylol. The method involves adding 2,2',4,5,5'-pentachlorobiphenyl to a porcine liver microsome incubation system and incubating at 37–40°C. The 2,2',4,5,5'-pentachlorobiphenyl is then catalyzed by enzymes in the porcine liver microsomes to produce 2,2',4,5,5'-pentachloro-4-biphenylol. The separation and purification method involves extracting 2,2',4,5,5'-pentachloro-4-biphenylol from the incubation solution using an organic solvent after termination of incubation, removing biomass impurities such as fats from the extraction solution with concentrated sulfuric acid, separating unreacted 2,2',4,5,5'-pentachlorobiphenyl using a silica gel column, eluting the 2,2',4,5,5'-pentachloro-4-biphenylol adsorbed on the silica gel column with a weakly polar mixed solution, and further drying the eluent under vacuum to obtain purified 2,2',4,5,5'-pentachloro-4-biphenylol.

[0075] It should be noted that the metabolism of 2,2',4,5,5'-pentachlorobiphenyl to hydroxylated polychlorinated biphenyls varies among animal species. Normally, 2,2',4,5,5'-pentachlorobiphenyl can be metabolized by animal liver microsomes, forming two metabolites: 2,2',4,5,5'-pentachloro-4-biphenylol and 2,2',4,5,5'-pentachloro-3-biphenylol. (Appendix) Figure 2 The possible metabolic pathways and metabolites of 2,2',4,5,5'-pentachlorobiphenyl are shown.

[0076] In this case, the inventors compared liver microsomes from different species of animals, including rats, mice, fish, pigs, cattle, and chickens (detailed in Experimental Example 2 below), and found that only pig liver microsomes could specifically metabolize 2,2',4,5,5'-pentachlorobiphenyl to 2,2',4,5,5'-pentachloro-4-biphenylol. The metabolic reactions of liver microsomes from other animals would simultaneously convert 2,2',4,5,5'-pentachlorobiphenyl to 2,2',4,5,5'-pentachloro-4-biphenylol and 2,2',4,5,5'-pentachloro-3-biphenylol, or only to 2,2',4,5,5'-pentachloro-3-biphenylol.

[0077] Appendix Figure 3This study demonstrates the process by which 2,2',4,5,5'-pentachlorobiphenyl undergoes metabolism via porcine liver microsomes to form an epoxidized transition state, subsequently producing 2,2',4,5,5'-pentachloro-4-biphenylol. Therefore, this application selects porcine liver microsomes as the bioreactor for 2,2',4,5,5'-pentachlorobiphenyl, which can directionally form the molecularly singular 2,2',4,5,5'-pentachloro-4-biphenylol, avoiding interference from its homologue, 2,2',4,5,5'-pentachloro-3-biphenylol.

[0078] In addition, throughout the research process, the research team found that the best synthesis effect of pig liver microsomes was obtained from the livers of healthy 3- to 10-month-old Bama miniature pigs raised in clean areas free from industrial pollution sources.

[0079] Because the quality of porcine liver microsomes has a significant impact on the conversion efficiency and purification of 2,2',4,5,5'-pentachloro-4-biphenylol: unhealthy pigs often have impaired liver function, leading to loss or weakening of liver microsomal metabolic capacity; pigs that are too young have underdeveloped livers, while pigs that are too old have declining liver function, and different types of persistent organic pollutants may accumulate in the liver, affecting the separation and purification of 2,2',4,5,5'-pentachloro-4-biphenylol; industrial pollution in the breeding area increases the intake of persistent organic pollutants from the environment by pigs, leading to impaired liver function and accumulation of persistent organic pollutants in the liver.

[0080] Therefore, the inventors of this case also investigated the ability of liver microsomes from different breeds of pigs to biotransform 2,2',4,5,5'-pentachloro-4-biphenylol. They found that the liver microsomes from the miniature pig Bama Miniature Pig had a significantly higher metabolic capacity than those from large pigs such as Landrace and Duroc × Landrace × Large White crossbred pigs. Therefore, liver microsomes from Bama Miniature Pigs were preferred.

[0081] As a feasible and preferred approach, this application uses healthy 3- to 10-month-old Bama miniature pigs raised in clean areas free from industrial pollution sources to obtain liver microsomes. This ensures metabolic activity against 2,2',4,5,5'-pentachlorobiphenyl while effectively avoiding the introduction of other potential persistent organic pollutants.

[0082] Based on the above research approach, the applicant proposes a feasible implementation method as follows:

[0083] Example 1

[0084] This embodiment provides a method for the biosynthesis and purification of 2,2',4,5,5'-pentachloro-4-biphenylol, as detailed below:

[0085] Step (1): Weigh 10 mg of 2,2',4,5,5'-pentachlorobiphenyl into a glass reagent bottle, add 50 mL of dimethyl sulfoxide, and stir with a glass rod for 2 min to dissolve it, which is a 0.2 mg / mL 2,2',4,5,5'-pentachlorobiphenyl solution.

[0086] Step (2): Weigh 8g of sodium chloride (NaCl), 0.2g of potassium chloride (KCl), 1.44g of disodium hydrogen phosphate (Na2HPO4) and 0.24g of potassium dihydrogen phosphate (KH2PO4) and dissolve them in 800mL of pure water. Adjust the pH of the solution to 7.4 with hydrochloric acid (HCl) and add pure water to make up to 1L. This is the 0.01M phosphate buffer solution.

[0087] Step (3): Take 452.5 mL of 0.01 M phosphate buffer solution into a glass bottle, add 12.5 mL of Bama miniature pig liver microsomal solution with a protein concentration of 20 mg / mL, add 5 mL of 0.2 mg / mL 2,2',4,5,5'-pentachlorobiphenyl solution, place in a 38±0.5℃ water bath shaker and shake at 50 rpm until the solution temperature stabilizes at 38±0.5℃, then add 24.9 mL of NADPH incubation system A solution (containing 26.1 mM NADP+, 66 mM glucose-6-phosphate, and 66 mM magnesium chloride aqueous solution) and 5 mL of NADPH incubation system B solution (containing 40 U / mL glucose-6-phosphate dehydrogenase and 5 mM sodium citrate aqueous solution) to start the reaction; incubate in a 38±0.5℃ water bath shaker at 50 rpm for 2 h.

[0088] Step (4): Stop the incubation. For ease of operation, divide the solution into 5 equal portions and place them in reagent bottles (100 mL / bottle). Add 500 mL of isopropanol-dichloromethane-n-hexane mixed solution (volume ratio 1:3:3) to the reagent bottle and extract for 20 min by shaking on a shaker (200 rpm). After extraction, let the solution stand for 1 h, and the solution will separate into two layers. Transfer the upper layer solution to a rotary evaporator flask and evaporate the solvent under reduced pressure until all the solvent has evaporated. Add 50 mL of n-hexane to the rotary flask to dissolve the residue, and then transfer the solution to a pear-shaped flask.

[0089] Step (5): Add 0.5g of concentrated sulfuric acid to the pear-shaped flask containing the hexane complex solution, place it on a rotary evaporator, and rotate it for 20 minutes at atmospheric pressure, 30℃, and 50 rpm. Remove the pear-shaped flask and place it on a reagent bottle rack to stand until the solution separates into two layers. Transfer the upper layer solution to a rotary evaporator flask and use a rotary evaporator to evaporate the solution to 1-3mL of concentrated solution for the next purification step.

[0090] Step (6): Place 95g of silica gel packing material in a muffle furnace and bake at 550℃ for 12h to activate it. After cooling, add 5g of pure water and shake vigorously to evenly disperse the added water into the silica gel packing material to prepare 5% water-silica gel. Take 3g of 5% water-silica gel and fill it into a glass chromatography column with a diameter of 1cm. Add 20mL of n-hexane and open the column outlet valve. When the liquid level drops to 1-3mm from the surface of the silica gel packing layer, close the outlet valve. Then, add the concentrated solution obtained in step (5) to the silica gel column, open the column outlet valve, and close the outlet valve when the liquid level drops to 1-3mm from the surface of the silica gel packing layer. Place a pear-shaped bottle at the outlet end of the glass chromatography column to collect the solution. Add 30mL of dichloromethane-n-hexane (volume ratio 2:3) to the silica gel column, open the outlet valve, and collect all the eluent.

[0091] Step (7): Place the collected eluent on a rotary evaporator and evaporate the solution to 1-2 mL under reduced pressure at 60℃ and 75 rpm. Take a reagent bottle and accurately weigh the empty bottle to 0.0001 g. Transfer the concentrated eluent to the reagent bottle and place it in a vacuum desiccator. After the solution in the reagent bottle has completely evaporated, weigh the reagent bottle (accurate to 0.0001 g) and subtract the weight of the empty bottle to obtain the weight of the obtained 2,2',4,5,5'-pentachloro-4-biphenylol. Add acetonitrile to it at a ratio of 1 mg / mL to obtain a 1 mg / mL 2,2',4,5,5'-pentachloro-4-biphenylol acetonitrile solution.

[0092] Step (8): Take 1 μL of the above solution and mix it with 999 μL of acetonitrile, and identify it using a mass spectrometer.

[0093] The mass spectrometry conditions were as follows: quadrupole tandem mass spectrometer (TQS, Waters Corporation, USA), electron spray ionization (ESI) source, negative ion mode, capillary voltage -3.0 kV; capillary temperature 450 °C; desolvation gas was high-purity nitrogen at a flow rate of 850 L / Hr, collision gas was high-purity argon, single-stage mass spectrometry scanning mode, and the mass-to-nuclear ratio (m / z) range was 50–800. 2,2',4,5,5'-pentachloro-4-biphenylol showed significant peak signals at mass-to-nuclear ratios of m / z = 399, 341, 343, and 345, and the response intensity was significantly higher than that at other mass-to-nuclear ratios, indicating that the purified 2,2',4,5,5'-pentachloro-4-biphenylol was successfully prepared.

[0094] The specifications of the materials and reagents used are as follows:

[0095] Acetonitrile: CAS No. 75-05-8, chromatographic grade.

[0096] Isopropanol: CAS No. 67-63-0, chromatographic grade.

[0097] Dichloromethane: CAS No. 75-09-2, analytical grade.

[0098] n-Hexane: CAS No. 110-54-3, analytical grade.

[0099] Concentrated sulfuric acid: CAS No. 7664-93-9, analytical grade.

[0100] Silica gel filler: particle size 0.063-0.100mm.

[0101] 2,2',4,5,5'-Pentachlorobiphenyl: CAS No. 37680-73-2, purity ≥99%.

[0102] The water used in the experiment was Milli-Q ultrapure water.

[0103] Taking the biosynthesis of 2,2',4,5,5'-pentachloro-4-biphenylol in Example 1 as an example, the metabolites of liver microsomes from different animals and the biotransformation efficiency of liver microsomes from different breeds of pigs were investigated.

[0104] Experimental Example 1: Investigation of microsomal metabolites in the livers of different animals

[0105] Referring to Example 1, the method of metabolizing 2,2',4,5,5'-pentachlorobiphenyl to 2,2',4,5,5'-pentachloro-4-biphenylol using a porcine liver microsomal incubation system was investigated. The metabolites of 2,2',4,5,5'-pentachlorobiphenyl in the liver microsomes of other animals, including rats, mice, carp, and chickens, were also examined. Pigs, rats, mice, carp, and chickens were all purchased from Beijing Huizhi Taikang Pharmaceutical Technology Co., Ltd.

[0106] The specific method is as follows:

[0107] (1) Metabolism of 2,2',4,5,5'-pentachlorobiphenyl by liver microsomes in different animals

[0108] A 0.5 mL incubation system for pig, rat, mouse, carp, and chicken liver microsomes was constructed. 452.5 μL of 0.01 M phosphate buffer (pH = 7.2), 5 μL of 0.2 mg / mL 2,2',4,5,5'-pentachlorobiphenyl-dimethyl sulfoxide solution, and 12.5 μL of liver microsomes with a protein concentration of 20 mg / mL were added sequentially to glass test tubes. The mixture was incubated at 38 °C for 5 min. Then, 24.9 μL of NADPH incubation system A (26.1 mM NADP+, 66 mM glucose-6-phosphate, 66 mM magnesium chloride aqueous solution) and 5 μL of solution B (40 U / mL glucose-6-phosphate dehydrogenase, 5 mM sodium citrate aqueous solution) were added to initiate the reaction. The mixture was incubated in a shaking incubator at 38 °C for 2 h. Each animal liver microsomal incubation system was tested in triplicate, with control experiments included (without substrate, without microsomal incubation, and without coenzyme factor) to ensure that no impurities or other reactions would affect the final identification of hydroxypolychlorinated biphenyls.

[0109] (2) Sample pretreatment

[0110] Transfer the incubation solution to a 10 mL screw-capped glass centrifuge tube, add 10 μL of 0.2 μg / mL 13C12-4-OH-2,3,3`,4`,5-hexachlorobiphenyl (13C-4-OH-PCB107) isotope internal standard solution, equilibrate for 30 min, then add 0.3 mL isopropanol, 1.0 mL dichloromethane, and 1.0 mL n-hexane sequentially, vortex for 1 min; then centrifuge at 5000 rpm for 2 min; transfer the supernatant to a 10 mL centrifuge tube using a glass pipette, then add 2 mL of dichloromethane-n-hexane (1:1 volume ratio) to the sample and repeat the extraction once, combining the two extraction solutions. The sample extract was dried under nitrogen, and 5 mL of n-hexane was added to dissolve the residue. 0.1 g of concentrated sulfuric acid was then added, and the mixture was shaken at 200 rpm for 20 min, centrifuged at 5000 rpm for 5 min, and the supernatant was collected and blown under nitrogen to obtain approximately 1 mL of concentrate. The concentrate was passed through a 5% water-silica gel column, eluted sequentially with 20 mL of n-hexane and then with 30 mL of dichloromethane-n-hexane (2:3 v / v). The eluent was concentrated to 50 μL by rotary evaporation and nitrogen blowing for LC-MS / MS analysis.

[0111] (3) LC-MS / MS determination

[0112] A. Chromatographic conditions

[0113] Chromatographic column: Waters ACQUITY UPL CEBEH Shield RP18 column, 100 mm in length, 2.1 mm in inner diameter, and 1.7 μm in particle size; column temperature: 40 °C; flow rate: 0.3 mL / min; injection volume: 5 L. Mobile phase A: 0.01% formic acid solution, mobile phase B: 0.01% formic acid in methanol solution, gradient elution program is shown in Table 1.

[0114] Table 1 Gradient elution program

[0115]

[0116]

[0117] B. Mass Spectrometry Conditions

[0118] Ionization method: electrospray ionization, negative ion mode (ESI-); Detection method: multiple reaction monitoring (MRM); Capillary voltage: -3.0kV; Ion source temperature: 150℃; Desolventization temperature: 450℃; Desolventization gas: nitrogen 850L / Hr; Multiple reaction monitoring (MRM) mode, monitoring ion pairs, cone voltage and collision energy are shown in Table 2.

[0119] Table 2 Retention time, MRM ion pairs, cone voltage, collision energy

[0120]

[0121] a These are quantitative ions.

[0122] C. Data Analysis

[0123] The concentrations of 2,2',4,5,5'-pentachloro-3-biphenylol and 2,2',4,5,5'-pentachloro-4-biphenylol produced in the liver microsomal incubation solution were calculated using the isotope internal standard method. The values ​​are expressed as mean ± standard deviation.

[0124] (4) Results Analysis

[0125] The concentrations of 2,2',4,5,5'-pentachloro-3-biphenylol and 2,2',4,5,5'-pentachloro-4-biphenylol produced by the metabolism of 2,2',4,5,5'-pentachlorobiphenyl by liver microsomes in different animals were determined, and the results are shown in Table 3.

[0126] Table 3. Concentrations of products from the microsomal metabolism of 2,2',4,5,5'-pentachlorobiphenyl in the livers of different animals.

[0127]

[0128]

[0129] As shown in Table 3, the product patterns of 2,2',4,5,5'-pentachlorobiphenyl metabolites in the liver microsomes of different animals exhibit significant differences. Specifically, porcine and carp liver microsomes specifically metabolize 2,2',4,5,5'-pentachloro-4-biphenylol, while liver microsomes from other animals simultaneously or exclusively produce 2,2',4,5,5'-pentachloro-3-biphenylol. Compared to carp liver microsomes, porcine liver microsomes, under the same incubation conditions, produce approximately 10 times higher concentrations of 2,2',4,5,5'-pentachloro-4-biphenylol. Therefore, porcine liver microsomes are preferably used in this application for the biosynthesis of 2,2',4,5,5'-pentachloro-4-biphenylol.

[0130] Experimental Example 2: Investigation of microsomes from different breeds of pig liver and optimal substrate addition levels

[0131] This experiment has two objectives: first, to investigate the metabolic capacity of different breeds of pigs for PCB101; and second, to optimize the optimal substrate (PCB101) concentration.

[0132] Referring to Example 1, a method for metabolizing 2,2',4,5,5'-pentachlorobiphenyl to 2,2',4,5,5'-pentachloro-4-biphenylol using a porcine liver microsomal incubation system was investigated. The enzymatic reaction kinetics of the metabolism of 2,2',4,5,5'-pentachlorobiphenyl to 2,2',4,5,5'-pentachloro-4-biphenylol by liver microsomes derived from Bama miniature pigs, Landrace pigs, and Duroc × Landrace × Large White crossbred pigs were examined. By comparing the enzymatic reaction kinetic parameters, the pig breed with the highest metabolic efficiency and the optimal substrate (2,2',4,5,5'-pentachlorobiphenyl) concentration in the incubation system were determined.

[0133] The specific method is as follows:

[0134] (1) Experiment on the metabolism of 2,2',4,5,5'-pentachlorobiphenyl in liver microsomes of different breeds of pigs

[0135] A 0.5 mL incubation system for liver microsomes of Bama miniature pigs, Landrace pigs, and Duroc × Landrace × Large White three-way crossbred pigs was constructed. Different volumes of 2,2',4,5,5'-pentachlorobiphenyl-dimethyl sulfoxide solution were added to the liver microsome incubation system to achieve concentrations of 0.1 μg / mL, 0.5 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL. The enzymatic kinetics of the metabolism of 2,2',4,5,5'-pentachlorobiphenyl to 2,2',4,5,5'-pentachloro-4-biphenylol in the liver microsomes of different pig breeds were investigated.

[0136] Add 452.5 μL of 0.01M phosphate buffer solution (pH = 7.2), an appropriate amount of 2,2',4,5,5'-pentachlorobiphenyl-dimethyl sulfoxide solution, and 12.5 μL of liver microsomes with a protein concentration of 20 mg / mL to glass test tubes sequentially. Incubate at 38°C for 5 min. Then add 24.9 μL of NADPH incubation system A (26.1 mM NADP+, 66 mM glucose-6-phosphate, 66 mM magnesium chloride aqueous solution) and 5 μL of solution B (40 U / mL glucose-6-phosphate dehydrogenase, 5 mM sodium citrate aqueous solution) to start the reaction. Incubate at 38°C in a shaking incubator for 2 h. Each animal liver microsome incubation system was set up in triplicate, and control experiments were set up (without adding reaction substrate, without adding microsomes, and without adding coenzyme factors) to ensure that there were no impurities or other reactions that would affect the final identification of hydroxypolychlorinated biphenyls.

[0137] (2) Sample pretreatment

[0138] Transfer the incubation solution to a 10 mL screw-capped glass centrifuge tube, add 10 μL of 0.2 μg / mL 13C12-4-OH-2,3,3',4',5-hexachlorobiphenyl (13C-4-OH-PCB107) isotope internal standard solution, equilibrate for 30 min, then add 0.3 mL isopropanol, 1.0 mL dichloromethane, and 1.0 mL n-hexane sequentially, vortex for 1 min; then centrifuge at 5000 rpm for 2 min; transfer the supernatant to a 10 mL centrifuge tube using a glass pipette, then add 2 mL of dichloromethane-n-hexane (1:1 volume ratio) to the sample and repeat the extraction once, combining the two extraction solutions. The sample extract was dried under nitrogen, and 5 mL of n-hexane was added to dissolve the residue. 0.1 g of concentrated sulfuric acid was then added, and the mixture was shaken at 200 rpm for 20 min, centrifuged at 5000 rpm for 5 min, and the supernatant was collected and blown under nitrogen to obtain approximately 1 mL of concentrate. The concentrate was passed through a 5% water-silica gel column, eluted sequentially with 20 mL of n-hexane and then with 30 mL of dichloromethane-n-hexane (2:3 v / v). The eluent was concentrated to 50 μL by rotary evaporation and nitrogen blowing for LC-MS / MS analysis.

[0139] (3) LC-MS / MS determination

[0140] A. Chromatographic conditions

[0141] Chromatographic column: Waters ACQUITY UPL CEBEH Shield RP18 column, 100 mm in length, 2.1 mm in inner diameter, and 1.7 μm in particle size; column temperature: 40 °C; flow rate: 0.3 mL / min; injection volume: 5 L. Mobile phase A: 0.01% formic acid solution, mobile phase B: 0.01% formic acid in methanol solution, gradient elution program is shown in Table 1.

[0142] B. Mass spectrometry conditions: Ionization mode: electrospray ionization, negative ion mode (ESI-); Detection mode: multiple reaction monitoring (MRM); Capillary voltage: -3.0kV; Ion source temperature: 150℃; Desolventizing temperature: 450℃; Desolventizing gas: nitrogen 850L / Hr; In multiple reaction monitoring (MRM) mode, the monitored ion pairs, cone voltage and collision energy are shown in Table 2.

[0143] C. Data Analysis

[0144] The concentrations of 2,2',4,5,5'-pentachloro-3-biphenylol and 2,2',4,5,5'-pentachloro-4-biphenylol produced in the liver microsomal incubation solution were calculated using the isotope internal standard method. The values ​​are expressed as mean ± standard deviation.

[0145] (4) Results Analysis

[0146] The concentrations of 2,2',4,5,5'-pentachloro-3-biphenylol and 2,2',4,5,5'-pentachloro-4-biphenylol produced by the metabolism of 2,2',4,5,5'-pentachlorobiphenyl by liver microsomes from different breeds were determined. The results showed that 2,2',4,5,5'-pentachloro-3-biphenylol was not detected in any of the treatment groups, but 2,2',4,5,5'-pentachloro-4-biphenylol was detected in all of them.

[0147] Based on the Michaelis-Menten equation for enzyme reaction kinetics, nonlinear fitting was performed using Origin 9.1 software to analyze the concentrations of substrate (2,2',4,5,5'-pentachlorobiphenyl) and product (2,2',4,5,5'-pentachloro-4-biphenylol). The Michaelis-Menten equation is as follows:

[0148]

[0149] Where V0 is the initial reaction rate, Vmax is the maximum reaction rate, [S] is the substrate concentration, and K is the maximum reaction rate. m is the Michaelis constant.

[0150] The Michaelis-Menten equation fitting plot of substrate concentration versus reaction rate is shown in the figure. Figure 4 , Figure 4Michaelis equation fitting plots of liver microsomes from different breeds of pigs to produce 2,2',4,5,5'-pentachlorobiphenyl and 2,2',4,5,5'-pentachloro-4-biphenylol. (a) Liver microsomes of Bama miniature pig; (b) Liver microsomes of Landrace pig; (c) Liver microsomes of Duroc × Landrace × Large White crossbred pig.

[0151] Depend on Figure 4 It is evident that the maximum reaction rate (Vmax) for the biotransformation of 2,2',4,5,5'-pentachloro-4-biphenylol by liver microsomes differed significantly among the three pig breeds. The Vmax values ​​for liver microsomes from Bama miniature pigs, Landrace pigs, and Duroc × Landrace × Large White hybrid pigs were 22.5±10.0, 13.1±3.1, and 14.4±2.6, respectively, indicating that the Vmax of Bama miniature pig liver microsomes was significantly higher than that of Landrace pigs and Duroc × Landrace × Large White hybrid pigs. This suggests that the biotransformation efficiency of 2,2',4,5,5'-pentachloro-4-biphenylol by Bama miniature pig liver microsomes is significantly higher than that of Landrace pigs and Duroc × Landrace × Large White hybrid pigs. This may be because Bama miniature pigs are a small breed with a vigorous metabolic capacity, resulting in a stronger ability to metabolize and transform exogenous compounds compared to large pig breeds. Therefore, the preferred pig liver microsomes in this invention are those from Bama miniature pigs. Furthermore, from... Figure 4 It can also be seen that when the x-axis is greater than 2, the y-axis almost no longer increases with the increase of the x-axis value, indicating that the saturation concentration of 2,2',4,5,5'-pentachlorobiphenyl in the microsomal metabolism of the liver of the three selected pig breeds is approximately 2 μg / mL. Therefore, the preferred concentration of 2,2',4,5,5'-pentachlorobiphenyl in the pig liver microsomal incubation system of this invention is 2 μg / mL.

[0152] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for synthesizing 2,2',4,5,5'-pentachloro-4-biphenylol based on a porcine liver microsomal incubation system, characterized in that, This includes establishing a porcine liver microsomal system and adding 2,2',4,5,5'-pentachlorobiphenyl dissolved in an aqueous phase to the system. 2,2',4,5,5'-pentachloro-4-biphenyl alcohol is obtained through extraction, impurity removal, concentration, and drying purification. The establishment of the porcine liver microsomal system specifically involves adding the following components in the following amounts using 0.01M phosphate buffer as a solvent: Microsomal protein 0.5 mg / mL; Nicotinamide adenine dinucleotide phosphate (NADP+) 1.3 mM; Glucose-6-phosphate 3.3 mM; Magnesium chloride 3.3 mM; Glucose-6-phosphate dehydrogenase 0.4 U / mL; Sodium citrate 0.05mM; The extraction steps include preparing an antiemulsifying extraction solution, adding it to a porcine liver microsome incubation system solution, and then extracting a crude extract of 2,2',4,5,5'-pentachloro-4-biphenylol by shaking, settling or centrifugation. The impurity removal step includes converting the crude extract to a different solvent, removing impurities with concentrated sulfuric acid, and then evaporating the solvent using a rotary evaporator to obtain a concentrated extract. The purification process also includes purification of the concentrate, specifically involving the use of a silica gel column to separate 2,2',4,5,5'-pentachlorobiphenyl and other persistent organic contaminants from the concentrate, thereby obtaining a purified 2,2',4,5,5'-pentachloro-4-biphenyl alcohol solution.

2. The method for synthesizing 2,2',4,5,5'-pentachloro-4-biphenylol based on the porcine liver microsomal incubation system according to claim 1, characterized in that, The concentration of 2,2',4,5,5'-pentachlorobiphenyl added to the porcine liver microsomal incubation system was 2 μg / mL.

3. The method for synthesizing 2,2',4,5,5'-pentachloro-4-biphenylol based on the porcine liver microsomal incubation system according to claim 2, characterized in that, The 2,2',4,5,5'-pentachlorobiphenyl was dissolved in dimethyl sulfoxide (DMSO) to prepare a 0.2 mg / mL 2,2',4,5,5'-pentachlorobiphenyl solution, which was then added to the porcine liver microsome incubation system at a volume ratio of 1:

99.

4. The method for synthesizing 2,2',4,5,5'-pentachloro-4-biphenylol based on the porcine liver microsomal incubation system according to claim 1, characterized in that, Specifically, the preparation of the antiemulsifying extraction solution involves mixing isopropanol, n-hexane, and dichloromethane solutions in a volume ratio of 1:3:3 to form the antiemulsifying extraction solution. The volume ratio of the antiemulsifying extraction solution to the volume of the pig liver microsome incubation system solution is 5:

1.

5. The method for synthesizing 2,2',4,5,5'-pentachloro-4-biphenylol based on the porcine liver microsomal incubation system according to claim 1, characterized in that, The conversion solvent is n-hexane, with the same volume as the crude extract, and the amount of concentrated sulfuric acid added is 10 times the amount of microsomal protein.

6. The method for synthesizing 2,2',4,5,5'-pentachloro-4-biphenylol based on the porcine liver microsomal incubation system according to claim 5, characterized in that, The drying and purification process involves placing the purified 2,2',4,5,5'-pentachloro-4-biphenylol solution in a vacuum desiccator to evaporate the solvent, followed by dissolution with a low-volatility solvent to obtain a 2,2',4,5,5'-pentachloro-4-biphenylol solution that can be stored for a long time.

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