Method for preparing alpha-lipoic acid through catalysis of hydrolase

Abstract

The invention relates to the technical field of bioengineering and medicine, in particular to a method for preparing alpha-lipoic acid through catalysis of hydrolase, and the method adopts a microfluidic channel reactor, the microfluidic channel reactor comprises a mixer, a first-stage reaction channel, a second-stage reaction channel and a product collector which are connected in sequence, and the method comprises the steps that the mixer, the first-stage reaction channel, the second-stage reaction channel and the product collector are sequentially connected; the method comprises the following steps: placing dimethyl sulfoxide and a mixture of ethyl 6, 8-dichloro caprylate and water in a molar mass ratio of 1: (1.1-1.5) in a mixer, placing an enzyme catalysis system in a first-stage reaction channel, pushing raw materials in the mixer to be continuously introduced into the first-stage reaction channel by a charging pump for reaction, controlling reaction conditions, and performing post-treatment on a product to obtain alpha-lipoic acid. According to the method for preparing alpha-lipoic acid through catalysis of the hydrolase, the reaction steps are few, the content of by-products is low, energy consumption and production cost are reduced, a continuous and controllable production process is achieved, toxic solvent emission is reduced, and the method conforms to the production concept of green chemistry.

Description

Technical Field

[0001] The present application relates to the field of bioengineering and pharmaceutical technology, and in particular to a method for preparing α-lipoic acid catalyzed by hydrolase. Background Art

[0002] α-lipoic acid is a vitamin-like compound that can eliminate free radicals that accelerate aging and cause disease. It is both water-soluble and fat-soluble, and can assist coenzymes in physiological metabolism that is beneficial to the body's immunity. It is a universal antioxidant drug. α-lipoic acid has a certain effect on the treatment of many diseases such as liver disease, diabetes, HIV virus, tumors, nervous system degeneration, radiation damage, arsenic, mercury, cadmium and other heavy metal poisoning.

[0003] The main method for industrial production is to synthesize α-lipoic acid from 6,8-disubstituted octanoic acid or octanoic acid esters through one-step or multi-step reactions. The synthesis route generally involves the reaction of 6,8-disubstituted octanoic acid esters with Na2S2 in DMF, followed by hydrolysis and acidification to obtain α-lipoic acid. The above process route inevitably has defects, mainly low yield, complicated operation steps, and inevitable by-products in the reaction that are not only difficult to remove but also prone to undesirable polymerization. The boiling point of DMF is relatively high, and the post-reaction treatment is relatively complicated, which is bound to greatly reduce the total yield and increase the cost, thereby affecting the quality and yield of the product.

[0004] In view of the problems of existing α-lipoic acid synthesis methods such as many by-products, low reaction yields, long saponification reaction time, and complex post-treatment, it is still of great significance to develop an α-lipoic acid synthesis process with fewer by-products, higher reaction yields, and more environmental protection. Summary of the invention

[0005] In order to effectively improve the above problems, the present application provides a method for preparing α-lipoic acid by hydrolase catalysis, which has fewer reaction steps, shorter reaction time, fewer by-products, higher reaction yield, milder conditions, and is more environmentally friendly and safer.

[0006] The present application provides a method for preparing α-lipoic acid catalyzed by hydrolase, which adopts the following technical scheme: A method for preparing alpha-lipoic acid by hydrolase catalysis comprises the following steps: using a microfluidic channel reactor, the microfluidic channel reactor comprising a mixer, a primary reaction channel, a secondary reaction channel and a product collector connected in sequence, the mixer being connected with a feeding pump, the inlet of the primary reaction channel being connected with a first interface, the outlet of the secondary hydrolysis channel being connected with a second interface, and the second interface being connected with the product collector; the method comprises: placing dimethyl sulfoxide and a mixture of ethyl 6,8-dichlorooctanoate and water in a molar mass ratio of 1:(1.1-1.5) in the mixer, placing 0.5-1.0 g of an enzyme catalytic system in the primary reaction channel, the feeding pump driving the raw materials of the mixer to continuously pass into the primary reaction channel for reaction, controlling the temperature to be 50-60°C for reaction for 20-35 min, collecting the reaction liquid in the product collector, and obtaining alpha-lipoic acid through post-treatment.

[0007] By adopting the above technical scheme, a microfluidic channel reactor is used to synthesize α-lipoic acid online using an enzyme catalytic system with 6,8-dichlorooctanoic acid ethyl ester as a raw material. This method not only greatly shortens the reaction time, but also has a high conversion rate and selectivity, fewer reaction steps, low by-product content, and reduced reaction costs, and has the advantages of being economical and efficient.

[0008] In the present application, the enzyme catalytic system catalyzes the hydrolysis of ester bonds, adopts a two-stage reaction channel, increases the hydrolysis channel, improves the reaction rate and greatly reduces the possibility of by-product generation; the applicant analyzes the enzyme catalytic mechanism by detecting the reaction process, and roughly includes: in the first step, the biological enzyme in the enzyme catalytic system catalyzes the rearrangement of DMSO oxidation molecules to form a sulfur-containing ester substrate intermediate, the enzyme active hydroxyl group specifically nucleophilically attacks the carbonyl group in the sulfur-containing ester group, forms a hydrogen bond, and gives a proton to form an "oxygen anion hole", the oxygen anion attacks the carbonyl group, a part of the substrate intermediate combines with the previous proton to form an alcohol, and the other part forms a sulfur-containing acyl complex intermediate, thereby reducing the activation energy of the catalytic reaction, reducing the reaction potential energy, and through the enzyme's specific selective catalysis, there are few by-products; in the second step, the alcohol formed by the combination of the enzyme catalytic system solvent or the proton of the previous step activates the water molecule, the water molecule is activated to nucleophilically attack the carbonyl group of the active intermediate of the sulfur-containing acyl complex to hydrolyze it, and at the same time provides a proton to the sulfur-containing acyl complex, releasing the sulfur-containing acyl part to form an acid, completing the enzyme's specific catalytic hydrolysis process. Therefore, a continuous two-stage reaction channel is set up to prevent the enzyme catalytic active site from being complexed or blocked by by-products. By increasing the reaction channel, the contact area is increased and the direction and position of the enzyme are adjusted to provide an ideal environment for the hydrolysis of the substrate, reduce the impurity hydrolysis reaction, increase the product generation rate, reduce the reaction time, improve the reaction efficiency, and have high selectivity and good reproducibility.

[0009] The enzyme-catalyzed hydrolysis reaction raw materials 6,8-dichlorooctanoic acid ethyl ester and water are used in a certain range of ratios to keep the enzyme-catalyzed reaction rate stable, thereby preventing the content of one party from being too low, causing the enzyme-catalyzed rate to decrease and become unstable, resulting in adverse reactions, separation difficulties, and reduced yields.

[0010] In a specific embodiment, the enzyme catalytic system includes one of lipase, pepsin and trypsin. Preferably, the enzyme catalytic system is lipase B.

[0011] By adopting the above technical scheme, lipase, protease and the like can participate in a variety of biochemical reactions. For the hydrolysis of the intermediates in the reaction of the present application, the intermediate products are converted into carboxylic acids with the participation of water molecules. Lipase B is selected for the specificity of the reaction of the present application, so that lipase B can catalyze hydrolysis at a high rate at the oil-water interface, and has an association effect with the oil-water interface, so that the active site of the enzyme is exposed, and the ability of the substrate to bind to the lipase is enhanced, which is conducive to the folding of the side chain of the substrate to the surface of the enzyme molecule, making the enzyme catalysis easy to carry out.

[0012] In a specific embodiment, the enzyme catalytic system includes lipase and crown ether.

[0013] Preferably, the mass ratio of the lipase to the crown ether is 1:(0.6-0.8).

[0014] The applicant discovered that crown ethers are highly compatible with lipase, and that crown ethers can activate lipase amino acid residues, thereby making the enzyme highly active and stable. The applicant optimized the reaction rate and reaction conversion rate with better reproducibility by adjusting the masses of lipase and crown ether participating in the reaction within a certain range. Analysis showed that crown ethers may act as enzyme-preserving solvents, and the cavity channels can maintain the activity of the enzyme to a certain extent, regulating the catalytic ability of the enzyme, so that different amino acid substitutions at the enzyme site promote different activities, thereby affecting the reaction rate and product.

[0015] The mass ratio of dimethyl sulfoxide to the enzyme catalysis system is (5-12):1.

[0016] The oxidation reaction of dimethyl sulfoxide is promoted by using an enzyme catalytic system and dimethyl sulfoxide in a certain range of proportions, so that the molecular rearrangement proceeds smoothly, the generation of reaction by-products is reduced, and the generation of trisulfide heterocyclic esters that are difficult to separate by-products is prevented.

[0017] The post-treatment operation is to add 1.5-3g of activated carbon to the reaction solution, filter it after cooling it slightly, and distill it under reduced pressure to obtain a crude product. The single eluents obtained by column chromatography separation are combined and evaporated to dryness to obtain α-lipoic acid.

[0018] Activated carbon is used to adsorb unreacted or inactivated enzymes and some reaction solid impurities, and the reaction solvent is removed by rotary evaporation and the target product is obtained by column chromatography separation.

[0019] In a specific embodiment, the inner diameter of the reaction channel is 2.8-3.6 mm, and the lengths of the primary reaction channel and the secondary reaction channel are both 1.0-1.5 m.

[0020] Preferably, the reaction channel material is one of stainless steel or polytetrafluoroethylene (PTFE).

[0021] By adopting the above technical solution, the reaction channel is designed so that the reactants are fully mixed in the inner diameter channel, the reaction distance is shortened, the substrate and the active center of the lipase are closer, and the association reaction is easier. The stainless steel or polytetrafluoroethylene material ensures the corrosion resistance and smoothness of the channel, further reducing the generation of by-product impurities.

[0022] In a specific embodiment, the microfluidic channel reactor comprises a thermostat, and the primary reaction channel is placed in the thermostat.

[0023] By adopting the above technical solution, a constant temperature box is set in the primary reaction channel to enable the catalytic reaction to proceed smoothly, reduce the increase of heating side reactions in the hydrolysis process, make separation difficult and reduce the yield.

[0024] In summary, the present application includes at least one of the following beneficial technical effects: 1. By adopting a microfluidic channel reactor, an enzyme catalysis system is used to synthesize alpha-lipoic acid online with 6,8-dichlorooctanoic acid ethyl ester as a raw material. This method not only greatly shortens the reaction time, but also has high conversion rate and selectivity, few reaction steps, low by-product content, reduces reaction cost, and has the advantage of economic efficiency. In the present application, the enzyme catalysis system catalyzes the hydrolysis of ester bonds, adopts a two-stage reaction channel, increases the hydrolysis channel, improves the reaction rate and greatly reduces the possibility of by-product generation; a continuous two-stage reaction channel is set to prevent the enzyme catalysis active site from being complexed or blocked by by-products, and by increasing the reaction channel, increasing the contact area and adjusting the direction and position of the enzyme to provide an ideal environment for the hydrolysis of the substrate, reducing the impurity hydrolysis reaction, increasing the product generation rate, reducing the reaction time, improving the reaction efficiency, high selectivity, and good reproducibility.

[0025] 2. Lipases and proteases can participate in a variety of biochemical reactions. For the hydrolysis of the intermediates in the reaction of this application, the intermediate products are converted into carboxylic acids with the participation of water molecules. Lipase B is selected for the specificity of the reaction of this application, so that lipase B can catalyze hydrolysis at a high rate at the oil-water interface, and has an association effect with the oil-water interface, so that the active site of the enzyme is exposed, and the ability of the substrate to bind to the lipase is enhanced, which is conducive to the folding of the side chain of the substrate to the surface of the enzyme molecule, making the enzyme catalysis easy to carry out.

[0026] 3. The enzyme-catalyzed hydrolysis reaction raw materials 6,8-dichlorooctanoic acid ethyl ester and water are used in a certain range of ratios to keep the enzyme-catalyzed reaction rate stable, thereby preventing one side from being too little, the enzyme-catalyzed rate from being reduced and unstable, causing adverse reactions, separation difficulties, and reduced yields. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a diagram of the reaction apparatus of the method for preparing α-lipoic acid catalyzed by hydrolase in Example 1.

[0028] Explanation of the reference numerals: 1. mixer; 2. primary reaction channel; 3. secondary reaction channel; 4. product collector; 5. first interface; 6. second interface; 7. feeding pump; 8. constant temperature box. DETAILED DESCRIPTION

[0029] In the present application, the lipase is Candida antarctica lipase B (5000LU / g) with the catalog number TXT21915; the trypsin with the catalog number T128770: purchased from Aladdin; the DMSO (≥99%) with the catalog number ST1276; all the raw materials in the present application can be obtained commercially.

[0030] Example 1 Reference Figure 1 , a method for preparing alpha-lipoic acid by hydrolase catalysis, using a microfluidic channel reactor, the microfluidic channel reactor comprising a mixer 1, a primary reaction channel 2, a secondary reaction channel 3 and a product collector 4 connected in sequence, and also comprising a thermostat, the primary reaction channel 2 is placed in the thermostat, the mixer 1 is connected to a feed pump 7, the inlet of the primary reaction channel 2 is connected to a first interface 5, the outlet of the secondary hydrolysis channel is connected to a second interface 6, and the second interface 6 is connected to the product collector 4; wherein the inner diameter of the reaction channel is 2.8 mm, the lengths of the primary reaction channel 2 and the secondary reaction channel 3 are both 1.0 m, and the material of the reaction channel is polytetrafluoroethylene; the preparation method is to mix 9.6 g of dimethyl sulfoxide and a mixture of 0.05 mol of 6,8-dichlorooctanoic acid ethyl ester and 0.058 mol of water The mixture was placed in a mixer 1, and 0.8 g of an enzyme catalytic system was placed in a primary reaction channel 2, wherein the enzyme catalytic system was antarctic Candida lipase B. The feed pump 7 was turned on to push the raw material of the mixer 1 to continuously pass into the primary reaction channel 2 for reaction. The flow rate of the reaction liquid was 10.4 μL / min, and the temperature was controlled to be 50°C for reaction for 35 minutes. The reaction liquid was collected in the product collector 4, and the feed pump 7 was turned off. Subsequently, 1.5 g of activated carbon was added to the reaction liquid, and the reaction liquid was filtered after being cooled slightly. The solvent was removed by vacuum distillation to obtain a crude product, and the crude product was separated by column chromatography (the elution reagent was petroleum ether: ethyl acetate volume ratio = 20:1). The crude product was dissolved with a small amount of elution reagent and then wet-coated. The obtained single product eluates were combined and evaporated at 60°C to obtain the target product, with a total yield of 93.2% and an HPLC purity of 95.5%.

[0031] Example 2 A method for preparing alpha-lipoic acid by hydrolase catalysis, using a microfluidic channel reactor, the microfluidic channel reactor comprising a mixer 1, a primary reaction channel 2, a secondary reaction channel 3 and a product collector 4 connected in sequence, and also comprising a thermostat, wherein the primary reaction channel 2 is placed in the thermostat, the mixer 1 is connected to a feed pump 7, the inlet of the primary reaction channel 2 is connected to a first interface 5, the outlet of the secondary hydrolysis channel is connected to a second interface 6, and the second interface 6 is connected to the product collector 4; wherein the inner diameter of the reaction channel is 2.8 mm, the lengths of the primary reaction channel 2 and the secondary reaction channel 3 are both 1.0 m, and the material of the reaction channel is polytetrafluoroethylene; the preparation method is as follows: 6 g of dimethyl sulfoxide, 0.05 mol of ethyl 6,8-dichlorooctanoate and 0.058 mol of water are mixed; The raw materials are placed in a mixer 1, and 1g of an enzyme catalytic system is placed in a primary reaction channel 2, wherein the enzyme catalytic system is Antarctic Candida lipase B. The feed pump 7 is turned on to push the raw materials in the mixer 1 to continuously pass into the primary reaction channel 2 for reaction. The flow rate of the reaction liquid is 10.4μL / min, and the temperature is controlled to be 60°C for reaction for 20min. The reaction liquid is collected in the product collector 4, and the feed pump 7 is turned off. Subsequently, 3g of activated carbon is added to the reaction liquid, and the reaction liquid is filtered after being cooled slightly. The solvent is removed by distillation under reduced pressure to obtain a crude product, and the crude product is separated by column chromatography (the elution reagent is petroleum ether: ethyl acetate volume ratio = 20:1). The crude product is dissolved with a small amount of elution reagent and then wet-coated, and the obtained single product eluates are combined and evaporated at 60°C to obtain the target product, with a total yield of 92.8% and an HPLC purity of 94.9%.

[0032] Example 3 A method for preparing alpha-lipoic acid by hydrolase catalysis, adopting a microfluidic channel reactor, wherein the microfluidic channel reactor comprises a mixer 1, a primary reaction channel 2, a secondary reaction channel 3 and a product collector 4 connected in sequence, and also comprises a thermostat, wherein the primary reaction channel 2 is placed in the thermostat, the mixer 1 is connected to a feeding pump 7, the inlet of the primary reaction channel 2 is connected to a first interface 5, the outlet of the secondary hydrolysis channel is connected to a second interface 6, and the second interface 6 is connected to the product collector 4; wherein the inner diameter of the reaction channel is 2.8 mm, the lengths of the primary reaction channel 2 and the secondary reaction channel 3 are both 1.0 m, and the material of the reaction channel is polytetrafluoroethylene; the preparation method comprises the following steps: mixing 9.6 g of dimethyl sulfoxide, 0.05 mol of ethyl 6,8-dichlorooctanoate and 0.075 mol of water into a mixture; The mixture was placed in a mixer 1, and 0.8 g of an enzyme catalytic system was placed in a primary reaction channel 2, wherein the enzyme catalytic system was antarctic Candida lipase B. The feed pump 7 was turned on to push the raw material of the mixer 1 to continuously pass into the primary reaction channel 2 for reaction. The flow rate of the reaction liquid was 10.4 μL / min, and the temperature was controlled to be 50°C for reaction for 35 minutes. The reaction liquid was collected in the product collector 4, and the feed pump 7 was turned off. Subsequently, 1.5 g of activated carbon was added to the reaction liquid, and the mixture was filtered after being cooled slightly. The solvent was removed by distillation under reduced pressure to obtain a crude product, and the crude product was separated by column chromatography (the elution reagent was petroleum ether: ethyl acetate volume ratio = 20:1). The crude product was dissolved with a small amount of elution reagent and then wet-coated. The obtained single product eluates were combined and evaporated at 60°C to obtain the target product, with a total yield of 93.1% and an HPLC purity of 95.1%.

[0033] Example 4 A method for preparing alpha-lipoic acid by hydrolase catalysis, using a microfluidic channel reactor, the microfluidic channel reactor comprising a mixer 1, a primary reaction channel 2, a secondary reaction channel 3 and a product collector 4 connected in sequence, and also comprising a thermostat, wherein the primary reaction channel 2 is placed in the thermostat, the mixer 1 is connected to a feeding pump 7, the inlet of the primary reaction channel 2 is connected to a first interface 5, the outlet of the secondary hydrolysis channel is connected to a second interface 6, and the second interface 6 is connected to the product collector 4; wherein the inner diameter of the reaction channel is 2.8 mm, the lengths of the primary reaction channel 2 and the secondary reaction channel 3 are both 1.0 m, and the material of the reaction channel is polytetrafluoroethylene; the preparation method is as follows: 9.6 g of dimethyl sulfoxide and 0.05 mol of ethyl 6,8-dichlorooctanoate and 0.058 mol of water are mixed. The mixture was placed in a mixer 1, and 0.8 g of an enzyme catalytic system was placed in a primary reaction channel 2, wherein the enzyme catalytic system was trypsin. The feed pump 7 was turned on to push the raw material of the mixer 1 to continuously pass into the primary reaction channel 2 for reaction. The flow rate of the reaction liquid was 10.4 μL / min, and the temperature was controlled to be 50° C. for reaction for 35 min. The reaction liquid was collected in the product collector 4, and the feed pump 7 was turned off. Subsequently, 1.5 g of activated carbon was added to the reaction liquid, and the reaction liquid was filtered after being cooled slightly. The solvent was removed by vacuum distillation to obtain a crude product, and the crude product was separated by column chromatography (the elution reagent was petroleum ether: ethyl acetate volume ratio = 20:1). The crude product was dissolved with a small amount of elution reagent and then wet-coated. The obtained single product eluates were combined and evaporated to dryness at 60° C. to obtain the target product, with a total yield of 91.7% and an HPLC purity of 92.3%.

[0034] Example 5 A method for preparing alpha-lipoic acid by hydrolase catalysis, adopting a microfluidic channel reactor, wherein the microfluidic channel reactor comprises a mixer 1, a primary reaction channel 2, a secondary reaction channel 3 and a product collector 4 connected in sequence, and also comprises a thermostat, wherein the primary reaction channel 2 is placed in the thermostat, the mixer 1 is connected to a feeding pump 7, the inlet of the primary reaction channel 2 is connected to a first interface 5, the outlet of the secondary hydrolysis channel is connected to a second interface 6, and the second interface 6 is connected to the product collector 4; wherein the inner diameter of the reaction channel is 2.8 mm, the lengths of the primary reaction channel 2 and the secondary reaction channel 3 are both 1.0 m, and the material of the reaction channel is polytetrafluoroethylene; the preparation method comprises the following steps: mixing 3.2 g of dimethyl sulfoxide, 0.05 mol of ethyl 6,8-dichlorooctanoate and 0.058 mol of water into a mixture; The mixture was placed in a mixer 1, and 0.8 g of an enzyme catalytic system was placed in a primary reaction channel 2, wherein the enzyme catalytic system was antarctic Candida lipase B. The feed pump 7 was turned on to push the raw material of the mixer 1 to continuously pass into the primary reaction channel 2 for reaction. The flow rate of the reaction liquid was 10.4 μL / min, and the temperature was controlled to be 50°C for reaction for 35 minutes. The reaction liquid was collected in the product collector 4, and the feed pump 7 was turned off. Subsequently, 1.5 g of activated carbon was added to the reaction liquid, and the mixture was filtered after being cooled slightly. The solvent was removed by distillation under reduced pressure to obtain a crude product, and the crude product was separated by column chromatography (the elution reagent was petroleum ether: ethyl acetate volume ratio = 20:1). The crude product was dissolved with a small amount of elution reagent and then wet-coated. The obtained single product eluates were combined and evaporated to dryness at 60°C to obtain the target product, with a total yield of 91.8% and an HPLC purity of 93.1%.

[0035] Example 6 A method for preparing alpha-lipoic acid by hydrolase catalysis, using a microfluidic channel reactor, the microfluidic channel reactor comprising a mixer 1, a primary reaction channel 2, a secondary reaction channel 3 and a product collector 4 connected in sequence, and also comprising a thermostat, wherein the primary reaction channel 2 is placed in the thermostat, the mixer 1 is connected to a feed pump 7, the inlet of the primary reaction channel 2 is connected to a first interface 5, the outlet of the secondary hydrolysis channel is connected to a second interface 6, and the second interface 6 is connected to the product collector 4; wherein the inner diameter of the reaction channel is 2.8 mm, the lengths of the primary reaction channel 2 and the secondary reaction channel 3 are both 1.0 m, and the material of the reaction channel is polytetrafluoroethylene; the preparation method is as follows: placing a mixture of 12 g of dimethyl sulfoxide, 0.05 mol of ethyl 6,8-dichlorooctanoate and 0.058 mol of water in a reaction chamber; In the mixer 1, 0.8 g of the enzyme catalytic system is placed in the primary reaction channel 2, wherein the enzyme catalytic system is Antarctic Candida lipase B, the feed pump 7 is turned on to push the raw material of the mixer 1 to continuously pass into the primary reaction channel 2 for reaction, the flow rate of the reaction liquid is 10.4 μL / min, the temperature is controlled to be 50°C for reaction for 35 minutes, the reaction liquid is collected in the product collector 4, the feed pump 7 is turned off, and then 1.5 g of activated carbon is added to the reaction liquid, and the reaction liquid is filtered after cooling slightly, and the solvent is removed by vacuum distillation to obtain a crude product, and the crude product is separated by column chromatography (the elution reagent is petroleum ether: ethyl acetate volume ratio = 20:1). The crude product is dissolved with a small amount of elution reagent and then wet-coated, and the obtained single product eluates are combined and evaporated at 60°C to obtain the target product, with a total yield of 92.4% and an HPLC purity of 93.4%.

[0036] Example 7 A method for preparing alpha-lipoic acid by hydrolase catalysis adopts a microfluidic channel reactor, wherein the microfluidic channel reactor comprises a mixer 1, a primary reaction channel 2, a secondary reaction channel 3 and a product collector 4 connected in sequence, and also comprises a thermostat, wherein the primary reaction channel 2 is placed in the thermostat, the mixer 1 is connected to a feeding pump 7, the inlet of the primary reaction channel 2 is connected to a first interface 5, the outlet of the secondary hydrolysis channel is connected to a second interface 6, and the second interface 6 is connected to the product collector 4; wherein the inner diameter of the reaction channel is 2.8 mm, the lengths of the primary reaction channel 2 and the secondary reaction channel 3 are both 1.0 m, and the material of the reaction channel is polytetrafluoroethylene; the preparation method comprises the following steps: placing a mixture of 9.6 g of dimethyl sulfoxide, 0.05 mol of ethyl 6,8-dichlorooctanoate and 0.058 mol of water in the mixer 1, and 0.8g of enzyme catalytic system was placed in the primary reaction channel 2, wherein the enzyme catalytic system was Antarctic Candida lipase B and 18-crown ether-6 in a mass ratio of 1:0.8, the feed pump 7 was turned on to push the raw materials of the mixer 1 to continuously pass into the primary reaction channel 2 for reaction, the flow rate of the reaction liquid was 10.4μL / min, the temperature was controlled to be 50°C for reaction for 35min, the reaction liquid was collected in the product collector 4, the feed pump 7 was turned off, and then 1.5g of activated carbon was added to the reaction liquid, and the reaction liquid was filtered after cooling slightly, and the solvent was removed by vacuum distillation to obtain a crude product, and the crude product was separated by column chromatography (the elution reagent was petroleum ether: ethyl acetate volume ratio = 20:1). The crude product was dissolved with a small amount of elution reagent and then wet-coated, and the obtained single product eluates were combined and evaporated at 60°C to obtain the target product, with a total yield of 95.6% and an HPLC purity of 94.8%.

[0037] Example 8 A method for preparing alpha-lipoic acid by hydrolase catalysis adopts a microfluidic channel reactor, wherein the microfluidic channel reactor comprises a mixer 1, a primary reaction channel 2, a secondary reaction channel 3 and a product collector 4 connected in sequence, and also comprises a thermostat, wherein the primary reaction channel 2 is placed in the thermostat, the mixer 1 is connected to a feeding pump 7, the inlet of the primary reaction channel 2 is connected to a first interface 5, the outlet of the secondary hydrolysis channel is connected to a second interface 6, and the second interface 6 is connected to the product collector 4; wherein the inner diameter of the reaction channel is 2.8 mm, the lengths of the primary reaction channel 2 and the secondary reaction channel 3 are both 1.0 m, and the material of the reaction channel is polytetrafluoroethylene; the preparation method comprises the following steps: placing a mixture of 9.6 g of dimethyl sulfoxide, 0.05 mol of ethyl 6,8-dichlorooctanoate and 0.058 mol of water in the mixer 1, and 0.8g of enzyme catalytic system was placed in the primary reaction channel 2, wherein the enzyme catalytic system was Antarctic Candida lipase B and 18-crown ether-6 in a mass ratio of 1:0.6, the feed pump 7 was turned on to push the raw materials of the mixer 1 to continuously pass into the primary reaction channel 2 for reaction, the flow rate of the reaction liquid was 10.4μL / min, the temperature was controlled to be 50°C for reaction for 35min, the reaction liquid was collected in the product collector 4, the feed pump 7 was turned off, and then 1.5g of activated carbon was added to the reaction liquid, and the reaction liquid was filtered after cooling slightly, and the solvent was removed by vacuum distillation to obtain a crude product, and the crude product was separated by column chromatography (the elution reagent was petroleum ether: ethyl acetate volume ratio = 20:1). The crude product was dissolved with a small amount of elution reagent and then wet-coated, and the obtained single product eluates were combined and evaporated at 60°C to obtain the target product, with a total yield of 95.1% and an HPLC purity of 95.9%.

[0038] Example 9 A method for preparing alpha-lipoic acid by hydrolase catalysis adopts a microfluidic channel reactor, wherein the microfluidic channel reactor comprises a mixer 1, a primary reaction channel 2, a secondary reaction channel 3 and a product collector 4 connected in sequence, and also comprises a thermostat, wherein the primary reaction channel 2 is placed in the thermostat, the mixer 1 is connected to a feeding pump 7, the inlet of the primary reaction channel 2 is connected to a first interface 5, the outlet of the secondary hydrolysis channel is connected to a second interface 6, and the second interface 6 is connected to the product collector 4; wherein the inner diameter of the reaction channel is 2.8 mm, the lengths of the primary reaction channel 2 and the secondary reaction channel 3 are both 1.0 m, and the material of the reaction channel is polytetrafluoroethylene; and the preparation method comprises the following steps: placing a mixture of 9.6 g of dimethyl sulfoxide, 0.05 mol of ethyl 6,8-dichlorooctanoate and 0.058 mol of water in the mixer 1, 0.8g of enzyme catalytic system was placed in the primary reaction channel 2, wherein the enzyme catalytic system was Antarctic Candida lipase B and 18-crown ether-6 in a mass ratio of 1:1, the feed pump 7 was turned on to push the raw materials of the mixer 1 to continuously pass into the primary reaction channel 2 for reaction, the flow rate of the reaction liquid was 10.4μL / min, the temperature was controlled to be 50°C for reaction for 35min, the reaction liquid was collected in the product collector 4, the feed pump 7 was turned off, and then 1.5g of activated carbon was added to the reaction liquid, and the reaction liquid was filtered after cooling slightly, and the solvent was removed by vacuum distillation to obtain a crude product, and the crude product was separated by column chromatography (elution reagent was petroleum ether: ethyl acetate volume ratio = 20:1). The crude product was dissolved with a small amount of elution reagent and then wet-coated, and the obtained single product eluates were combined and evaporated at 60°C to obtain the target product, with a total yield of 94.7% and an HPLC purity of 94.1%.

[0039] Example 10 A method for preparing alpha-lipoic acid by hydrolase catalysis adopts a microfluidic channel reactor, wherein the microfluidic channel reactor comprises a mixer 1, a primary reaction channel 2, a secondary reaction channel 3 and a product collector 4 connected in sequence, and also comprises a thermostat, wherein the primary reaction channel 2 is placed in the thermostat, the mixer 1 is connected to a feeding pump 7, the inlet of the primary reaction channel 2 is connected to a first interface 5, the outlet of the secondary hydrolysis channel is connected to a second interface 6, and the second interface 6 is connected to the product collector 4; wherein the inner diameter of the reaction channel is 2.8 mm, the lengths of the primary reaction channel 2 and the secondary reaction channel 3 are both 1.0 m, and the material of the reaction channel is polytetrafluoroethylene; the preparation method comprises the following steps: placing a mixture of 9.6 g of dimethyl sulfoxide, 0.05 mol of ethyl 6,8-dichlorooctanoate and 0.058 mol of water in the mixer 1, and 0.8g of enzyme catalytic system was placed in the primary reaction channel 2, wherein the enzyme catalytic system was Antarctic Candida lipase B and 18-crown ether-6 in a mass ratio of 1:0.4, the feed pump 7 was turned on to push the raw materials of the mixer 1 to continuously pass into the primary reaction channel 2 for reaction, the flow rate of the reaction liquid was 10.4μL / min, the temperature was controlled to be 50°C for reaction for 35min, the reaction liquid was collected in the product collector 4, the feed pump 7 was turned off, and then 1.5g of activated carbon was added to the reaction liquid, and the reaction liquid was filtered after cooling slightly, and the solvent was removed by vacuum distillation to obtain a crude product, and the crude product was separated by column chromatography (the elution reagent was petroleum ether: ethyl acetate volume ratio = 20:1). The crude product was dissolved with a small amount of elution reagent and then wet-coated, and the obtained single product eluates were combined and evaporated at 60°C to obtain the target product, with a total yield of 93.8% and an HPLC purity of 93.2%.

[0040] Embodiment 11 A method for preparing alpha-lipoic acid by hydrolase catalysis adopts a microfluidic channel reactor, wherein the microfluidic channel reactor comprises a mixer 1, a primary reaction channel 2, a secondary reaction channel 3 and a product collector 4 connected in sequence, and also comprises a thermostat, wherein the primary reaction channel 2 and the secondary reaction channel 3 are both placed in the thermostat, the mixer 1 is connected to a feeding pump 7, the inlet of the primary reaction channel 2 is connected to a first interface 5, the outlet of the secondary hydrolysis channel is connected to a second interface 6, and the second interface 6 is connected to the product collector 4; wherein the inner diameter of the reaction channel is 2.8 mm, the lengths of the primary reaction channel 2 and the secondary reaction channel 3 are both 1.0 m, and the material of the reaction channel is polytetrafluoroethylene; the preparation method comprises the following steps: 9.6 g of dimethyl sulfoxide and 0.05 mol of ethyl 6,8-dichlorooctanoate and 0.058 mol of water are mixed. The mixture is placed in a mixer 1, and 0.8g of an enzyme catalytic system is placed in a primary reaction channel 2, wherein the enzyme catalytic system is antarctic Candida lipase B. The feed pump 7 is turned on to push the raw material of the mixer 1 to continuously pass into the primary reaction channel 2 for reaction. The flow rate of the reaction liquid is 10.4μL / min, and the temperature is controlled to be 50°C for reaction for 35min. The reaction liquid is collected in the product collector 4, and the feed pump 7 is turned off. Subsequently, 1.5g of activated carbon is added to the reaction liquid, and the reaction liquid is filtered after cooling slightly. The solvent is removed by distillation under reduced pressure to obtain a crude product, and the crude product is separated by column chromatography (the elution reagent is petroleum ether: ethyl acetate volume ratio = 20:1). The crude product is dissolved with a small amount of elution reagent and then wet-coated. The obtained single product eluates are combined and evaporated at 60°C to obtain the target product, with a total yield of 93.0% and an HPLC purity of 93.8%.

[0041] Comparative Example 1 A method for preparing alpha-lipoic acid by hydrolase catalysis adopts a microfluidic channel reactor, wherein the microfluidic channel reactor comprises a mixer 1, a primary reaction channel 2, a secondary reaction channel 3 and a product collector 4 connected in sequence, and also comprises a thermostat, wherein the primary reaction channel 2 and the secondary reaction channel 3 are both placed in the thermostat, the mixer 1 is connected to a feeding pump 7, the inlet of the primary reaction channel 2 is connected to a first interface 5, the outlet of the secondary hydrolysis channel is connected to a second interface 6, and the second interface 6 is connected to the product collector 4; wherein the inner diameter of the reaction channel is 2.8 mm, the lengths of the primary reaction channel 2 and the secondary reaction channel 3 are both 1.0 m, and the material of the reaction channel is polytetrafluoroethylene; the preparation method comprises the following steps: 9.6 g of dimethyl sulfoxide and 0.05 mol of ethyl 6,8-dichlorooctanoate and 0.045 mol of water are mixed. The mixture is placed in a mixer 1, and 0.8g of an enzyme catalytic system is placed in a primary reaction channel 2, wherein the enzyme catalytic system is Antarctic Candida lipase B. The feed pump 7 is turned on to push the raw material of the mixer 1 to continuously pass into the primary reaction channel 2 for reaction. The flow rate of the reaction liquid is 10.4μL / min, and the temperature is controlled to be 50°C for reaction for 35min. The reaction liquid is collected in the product collector 4, and the feed pump 7 is turned off. Subsequently, 1.5g of activated carbon is added to the reaction liquid, and the reaction liquid is filtered after cooling slightly. The solvent is removed by vacuum distillation to obtain a crude product, and the crude product is separated by column chromatography (the elution reagent is petroleum ether: ethyl acetate volume ratio = 20:1). The crude product is dissolved with a small amount of elution reagent and then wet-coated. The obtained single product eluates are combined and evaporated at 60°C to obtain the target product, with a total yield of 89.2% and an HPLC purity of 92.1%.

[0042] Comparative Example 2 A method for preparing alpha-lipoic acid by hydrolase catalysis adopts a microfluidic channel reactor, wherein the microfluidic channel reactor comprises a mixer 1, a primary reaction channel 2, a secondary reaction channel 3 and a product collector 4 connected in sequence, and also comprises a thermostat, wherein the primary reaction channel 2 and the secondary reaction channel 3 are both placed in the thermostat, the mixer 1 is connected to a feeding pump 7, the inlet of the primary reaction channel 2 is connected to a first interface 5, the outlet of the secondary hydrolysis channel is connected to a second interface 6, and the second interface 6 is connected to the product collector 4; wherein the inner diameter of the reaction channel is 2.8 mm, the lengths of the primary reaction channel 2 and the secondary reaction channel 3 are both 1.0 m, and the material of the reaction channel is polytetrafluoroethylene; the preparation method comprises the following steps: 9.6 g of dimethyl sulfoxide and 0.05 mol of ethyl 6,8-dichlorooctanoate and 0.09 mol of water are mixed. The mixture is placed in a mixer 1, and 0.8g of an enzyme catalytic system is placed in a primary reaction channel 2, wherein the enzyme catalytic system is antarctic Candida lipase B. The feed pump 7 is turned on to push the raw material of the mixer 1 to continuously pass into the primary reaction channel 2 for reaction. The flow rate of the reaction liquid is 10.4μL / min, and the temperature is controlled to be 50°C for reaction for 35min. The reaction liquid is collected in the product collector 4, and the feed pump 7 is turned off. Subsequently, 1.5g of activated carbon is added to the reaction liquid, and the reaction liquid is filtered after cooling slightly. The solvent is removed by vacuum distillation to obtain a crude product, and the crude product is separated by column chromatography (the elution reagent is petroleum ether: ethyl acetate volume ratio = 20:1). The crude product is dissolved with a small amount of elution reagent and then wet-coated. The obtained single product eluates are combined and evaporated at 60°C to obtain the target product, with a total yield of 85.4% and an HPLC purity of 93.2%.

[0043] Comparative Example 3 A method for preparing alpha-lipoic acid by hydrolase catalysis, using a microfluidic channel reactor, the microfluidic channel reactor comprising a mixer 1, a primary reaction channel 2 and a product collector 4 connected in sequence, and also comprising a thermostat, the primary reaction channel 2 is placed in the thermostat, the mixer 1 is connected to a feed pump 7, the inlet of the primary reaction channel 2 is connected to a first interface 5, and the first interface 5 is connected to the product collector 4; wherein the inner diameter of the reaction channel is 2.8 mm, the length of the primary reaction channel 2 is 1.0 m, and the material of the reaction channel is polytetrafluoroethylene; the preparation method is as follows: 9.6 g of dimethyl sulfoxide, 0.05 mol of ethyl 6,8-dichlorooctanoate and 0.058 mol of water are placed in the mixer 1, 0.8 g of enzyme catalyst is added, The system is placed in the primary reaction channel 2, wherein the enzyme catalytic system is Antarctic Candida lipase B, the feed pump 7 is turned on to push the raw material of the mixer 1 to continuously pass into the primary reaction channel 2 for reaction, the flow rate of the reaction liquid is 10.4 μL / min, the temperature is controlled to be 50°C for reaction for 35 minutes, the reaction liquid is collected in the product collector 4, the feed pump 7 is turned off, and then 1.5 g of activated carbon is added to the reaction liquid, and the reaction liquid is filtered after cooling slightly, and the solvent is removed by vacuum distillation to obtain a crude product, and the crude product is separated by column chromatography (the elution reagent is petroleum ether: ethyl acetate volume ratio = 20:1). The crude product is dissolved with a small amount of elution reagent and then wet-coated, and the obtained single product eluate is combined and evaporated at 60°C to obtain the target product, with a total yield of 84.6% and an HPLC purity of 89.1%.

[0044] The present application adopts a microfluidic channel reactor and utilizes an enzyme catalytic system to synthesize α-lipoic acid online using 6,8-dichlorooctanoic acid ethyl ester as a raw material. The method not only greatly shortens the reaction time (from the traditional reaction time of 20-72h to 20-35min in the present application), but also has a high conversion rate and selectivity. The yield of the embodiments is more than 90%, the reaction steps are few, the by-product content is low, the energy consumption and production cost are reduced, a continuous and controllable production process is realized, and the emission of toxic solvents is reduced, which is in line with the production concept of green chemistry.

[0045] Comparative Example 1 and Example 4-6 show that the catalytic activity of lipase B depends on the protein structure, has certain stability and reproducibility, and lipase B catalyzes the hydrolysis of the present application's specific intermediates with higher selectivity, and a certain range of enzyme catalytic systems and dimethyl sulfoxide promote the smooth reaction and reduce the occurrence of by-products. Comparative Example 1 and Example 7-10 show that crown ethers can be highly compatible with lipase, and crown ethers can activate lipase amino acid residues, so that the enzyme has high activity and stability, the reaction conversion rate is optimal, and the reproducibility is better; Comparative Example 1 and Comparative Example 1-2 show that a certain range of enzyme catalytic hydrolysis reaction raw materials 6,8-dichlorooctanoic acid ethyl ester and water can be used to keep the enzyme catalytic reaction rate stable, one side content is too low, the enzyme catalytic rate is reduced and unstable, so that adverse reactions occur, separation is difficult, and the yield is reduced. By comparing Example 1 with Example 11 and Comparative Example 3, it can be seen that setting up a continuous two-stage reaction channel prevents the enzyme catalytic active site from being complexed or blocked by the by-products. By increasing the reaction channel, increasing the contact area and adjusting the direction and position of the enzyme, an ideal environment is provided for the hydrolysis of the substrate, reducing the impurity hydrolysis reaction and increasing the product generation rate.

[0046] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing α-lipoic acid by hydrolase catalysis, characterized in that: The method adopts a microfluidic channel reactor, which comprises a mixer (1), a primary reaction channel (2), a secondary reaction channel (3) and a product collector (4) connected in sequence, wherein the mixer (1) is connected to a feeding pump (7), the inlet of the primary reaction channel (2) is connected to a first interface (5), the outlet of the secondary hydrolysis channel is connected to a second interface (6), and the second interface (6) is connected to the product collector (4); the method comprises placing dimethyl sulfoxide and a mixture of ethyl 6,8-dichlorooctanoate and water in a molar mass ratio of 1:(1.1-1.5) in the mixer (1), placing 0.5-1.0 g of an enzyme catalytic system in the primary reaction channel (2), driving the raw materials in the mixer (1) to continuously pass into the primary reaction channel (2) by the feeding pump (7) for reaction, controlling the temperature to be 50-60°C for reaction for 20-35 minutes, collecting the reaction liquid in the product collector (4), and obtaining α-lipoic acid through post-treatment.

2. The method for preparing α-lipoic acid by hydrolase catalysis according to claim 1, characterized in that: The enzyme catalysis system includes one of lipase, pepsin and trypsin.

3. The method for preparing α-lipoic acid by hydrolase catalysis according to claim 1, characterized in that: The enzyme catalysis system comprises lipase and crown ether.

4. The method for preparing α-lipoic acid by hydrolase catalysis according to claim 3, characterized in that: The mass ratio of the lipase to the crown ether is 1:(0.6-0.8).

5. The method for preparing α-lipoic acid by hydrolase catalysis according to claim 1, characterized in that: The mass ratio of dimethyl sulfoxide to the enzyme catalysis system is (5-12):

1.

6. The method for preparing α-lipoic acid by hydrolase catalysis according to claim 1, characterized in that: The post-treatment operation is to add 1.5-3g of activated carbon to the reaction solution, filter it after cooling it slightly, and distill it under reduced pressure to obtain a crude product. The single eluents obtained by column chromatography separation are combined and evaporated to dryness to obtain α-lipoic acid.

7. The method for preparing α-lipoic acid by hydrolase catalysis according to claim 1, characterized in that: The microfluidic channel reactor comprises a thermostatic box (8), and the primary reaction channel (2) is placed in the thermostatic box (8).

8. The method for preparing α-lipoic acid by hydrolase catalysis according to claim 1, characterized in that: The inner diameter of the reaction channel is 2.8-3.6 mm, and the lengths of the primary reaction channel (2) and the secondary reaction channel (3) are both 1.0-1.5 m.

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