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CN-121975876-A - Enzymatic synthesis method of dorzolamide chiral intermediate

CN121975876ACN 121975876 ACN121975876 ACN 121975876ACN-121975876-A

Abstract

The invention discloses an enzyme catalytic synthesis method of a dorzolamide chiral intermediate, which comprises the steps of taking methyl acetoacetate as a substrate, adding a buffer solution, the substrate, alcohol dehydrogenase, formate dehydrogenase, ammonium formate and coenzyme into a reaction container, reacting for 2-24 hours at 20-40 ℃, extracting, separating and steaming, and then obtaining the dorzolamide chiral intermediate (R) -3-hydroxybutyrate, wherein the alcohol dehydrogenase is a mutant of ADH1 from Ralstonia, and the amino acid sequence of the ADH1 is shown as SEQ ID No. 1. The method has the advantages of mild reaction conditions, high stereoselectivity, more environment-friendly property, high conversion rate, high chiral purity, less enzyme consumption, high-concentration substrate tolerance and low preparation cost, is suitable for industrial production, avoids chiral resolution and heavy metal residues in products, and makes up the defects of a chemical method.

Inventors

  • LUO ZHIBO
  • WU YAN
  • CUI YONGYAN
  • CAO MIN
  • HAO XIN
  • MA MUQING
  • DU FENG
  • DENG YAPING
  • DENG YONGPIAO

Assignees

  • 杭州微远生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260409

Claims (9)

  1. 1. An enzyme catalysis synthesis method of a dorzolamide chiral intermediate is characterized in that methyl acetoacetate is used as a substrate, buffer solution, the substrate, alcohol dehydrogenase, formate dehydrogenase, ammonium formate and coenzyme are added into a reaction container to react for 2-24 hours at 20-40 ℃, and after extraction, separation and rotary evaporation, the dorzolamide chiral intermediate (R) -3-hydroxybutyrate is obtained; The alcohol dehydrogenase is a mutant of ADH1 from Ralstonia, and the amino acid sequence of the ADH1 is shown in SEQ ID No. 1.
  2. 2. The method of claim 1, wherein the alcohol dehydrogenase and the formate dehydrogenase are added as wet bacterial forms of recombinant genetically engineered bacteria.
  3. 3. The method for synthesizing the enzyme catalysis according to claim 2, wherein the dosage proportion of each raw material in a 1L reaction system is 5-100 g of recombinant escherichia coli wet bacterial cells expressing alcohol dehydrogenase, 10-100 g of recombinant escherichia coli wet bacterial cells expressing formate dehydrogenase, 20-100 g of substrate, 0.1-1.0M of ammonium formate, 0.1-2.0 mM of coenzyme and the balance of buffer solution.
  4. 4. The method for synthesizing the enzyme catalysis according to claim 3, wherein the dosage ratio of each raw material in the 1L reaction system is 5-100 g of recombinant escherichia coli wet bacterial cells expressing alcohol dehydrogenase, 50-75 g of recombinant escherichia coli wet bacterial cells expressing formate dehydrogenase, 20-100 g of substrate, 0.1-1.0M of ammonium formate, 0.1-2.0 mM of coenzyme and the balance of buffer solution.
  5. 5. The enzymatic synthesis process according to any one of claims 1-4, characterized in that the mutant of ADH1 is selected from one of ADH1-T55F/L96A/V110F/D82F,ADH1-T55F/L96A/V110F/G129P,ADH1-T55F/L96A/V110F/N176E,ADH1-T55F/L96A/V110F/Q191R,ADH1-T55F/L96A/V110F/V214P,ADH1-T55F/L96A/V110F/G129P/Q191R; ADH1-T55F/L96A/V110F/D82F is that the T at position 55 of the ADH1 amino acid sequence is changed to F, the L at position 96 is changed to A, the V at position 110 is changed to F, and the D at position 82 is changed to F; ADH1-T55F/L96A/V110F/G129P is that the T at position 55 of the ADH1 amino acid sequence is changed to F, the L at position 96 is changed to A, the V at position 110 is changed to F, and the G at position 129 is changed to P; ADH1-T55F/L96A/V110F/N176E is that the T at position 55 of the ADH1 amino acid sequence is changed to F, the L at position 96 is changed to A, the V at position 110 is changed to F, and the N at position 176 is changed to E; ADH1-T55F/L96A/V110F/Q191R is that the T at position 55 of the ADH1 amino acid sequence is changed to F, the L at position 96 is changed to A, the V at position 110 is changed to F, and the Q at position 191 is changed to R; ADH1-T55F/L96A/V110F/V214P is that the T at position 55 of the ADH1 amino acid sequence is changed into F, the L at position 96 is changed into A, the V at position 110 is changed into F, and the V at position 214 is changed into P; ADH1-T55F/L96A/V110F/G129P/Q191R is the ADH1 amino acid sequence 55 th T to F, 96 th L to A, 110 th V to F, 129 th G to P, and 191 th Q to R.
  6. 6. The method of enzyme-catalyzed synthesis according to any of claims 1 to 4, wherein the buffer is selected from the group consisting of TEA buffer, PB buffer, tris-HCl buffer, HEPES buffer.
  7. 7. The method for enzymatic synthesis according to claim 6, wherein the concentration of the TEA buffer is 0.05-0.20M, the pH value is 6.0-8.0, the concentration of the PB buffer is 0.05-0.10M, the pH value is 6.0-7.0, the concentration of the Tris-HCl buffer is 0.05-0.10M, the pH value is 7.0-8.0, and the concentration of the HEPES buffer is 0.01-0.10M, and the pH value is 6.0-8.0.
  8. 8. The method of claim 1, wherein the coenzyme is NADP + or NAD + .
  9. 9. An alcohol dehydrogenase, characterized in that the alcohol dehydrogenase is a mutant of ADH1 derived from ralstonia, and the amino acid sequence of ADH1 is shown in SEQ ID No. 1; the mutant of ADH1 is selected from one of ADH1-T55F/L96A/V110F/D82F,ADH1-T55F/L96A/V110F/G129P,ADH1-T55F/L96A/V110F/N176E,ADH1-T55F/L96A/V110F/Q191R,ADH1-T55F/L96A/V110F/V214P,ADH1-T55F/L96A/V110F/G129P/Q191R; ADH1-T55F/L96A/V110F/D82F is that the T at position 55 of the ADH1 amino acid sequence is changed to F, the L at position 96 is changed to A, the V at position 110 is changed to F, and the D at position 82 is changed to F; ADH1-T55F/L96A/V110F/G129P is that the T at position 55 of the ADH1 amino acid sequence is changed to F, the L at position 96 is changed to A, the V at position 110 is changed to F, and the G at position 129 is changed to P; ADH1-T55F/L96A/V110F/N176E is that the T at position 55 of the ADH1 amino acid sequence is changed to F, the L at position 96 is changed to A, the V at position 110 is changed to F, and the N at position 176 is changed to E; ADH1-T55F/L96A/V110F/Q191R is that the T at position 55 of the ADH1 amino acid sequence is changed to F, the L at position 96 is changed to A, the V at position 110 is changed to F, and the Q at position 191 is changed to R; ADH1-T55F/L96A/V110F/V214P is that the T at position 55 of the ADH1 amino acid sequence is changed into F, the L at position 96 is changed into A, the V at position 110 is changed into F, and the V at position 214 is changed into P; ADH1-T55F/L96A/V110F/G129P/Q191R is the ADH1 amino acid sequence 55 th T to F, 96 th L to A, 110 th V to F, 129 th G to P, and 191 th Q to R.

Description

Enzymatic synthesis method of dorzolamide chiral intermediate Technical Field The invention relates to the technical field of enzyme catalytic synthesis, in particular to an enzyme catalytic synthesis method of a dorzolamide chiral intermediate. Background Dorzolamide (trade name Trusopt) is a topical ocular carbonic anhydrase inhibitor, is a non-bacteriostatic sulfonamide, and can inhibit carbonic anhydrase activity. The external eye drop is an anti-glaucoma external eye drop, is mainly used for reducing intraocular pressure of patients with open angle glaucoma and ocular hypertension, is developed by merck company, and is approved by the FDA in 1995 in the United states. The pharmacological activity of dorzolamide is highly dependent on its enantiomer of S configuration, one of the key to its synthesis being the obtaining of methyl (R) -3-hydroxybutyrate of high optical purity. As an indispensable key chiral building block for the synthesis of dorzolamide, the construction of its chiral center must have extremely high enantiomeric excess values, usually requiring e.e. values > 99% to meet stringent drug regulatory requirements. Early, the synthesis of methyl (R) -3-hydroxybutyrate was largely dependent on chemical catalysis, which may be technically mature, but has inherent drawbacks that are difficult to overcome. The chemical method generally needs expensive chiral ligand or reagent, has harsh reaction conditions and generally uses organic solvent to generate a large amount of three wastes, which is contrary to the green pharmaceutical concept. In contrast, biocatalysis (in particular, ketoreductase-mediated asymmetric reduction) is becoming a research hotspot, and has the advantages of mild conditions, high stereoselectivity, environmental friendliness and the like. However, the early biocatalysis process still has the problems of low conversion rate, unstable optical selectivity, poor process compatibility and the like. A representative synthetic route for methyl (R) -3-hydroxybutyrate is as follows: The (R) -3-hydroxy methyl butyrate (CN 113416130A) is prepared by taking polyhydroxyalkanoate as a raw material and a mixed solution of methanesulfonic acid/methyl methanesulfonate and methanol as a solvent. The method relates to a strong corrosive reagent (methanesulfonic acid), has high requirements on materials of reaction equipment, brings the problems of safe operation and waste liquid treatment, has high raw material cost, and does not accord with the green chemistry principle. The 3-hydroxy methyl butyrate (CN 1181041C) is prepared by using octacarbonyl cobalt-pyridine compound-sodium salt as a catalyst and using propylene oxide, carbon monoxide and methanol as raw materials. The method produces by-products propylene glycol and dimethyl ether, the method has no chiral control, a raceme is obtained, an optical pure (R) -configuration can be obtained only by an additional chiral resolution step, the highest conversion rate of a resolution theory is only 50%, and the yield is low. The starch is used as a carbon source, and the (R) -3-hydroxy methyl butyrate (CN 101864458A) is prepared by virtue of microbial fermentation by virtue of an auxiliary substrate ethanol. The method has the advantages of complex and lengthy process route, lower yield compared with a chemical method, complex product separation and purification, and larger process optimization space. The existing method for preparing (R) -3-hydroxy-methyl butyrate has low efficiency, low concentration of catalytic substrate, long reaction time and high cost, and cannot be used for industrial production. For example, CN113416130B reports a reaction time of about 72: 72 h, and the conversion efficiency cannot meet the industrial requirements. Other problems of insufficient enzyme stability, high coenzyme regeneration cost, difficult product separation and the like exist, and the large-scale application of the novel recombinant enzyme is further limited. Disclosure of Invention The invention aims to provide an enzyme catalytic synthesis method of a dorzolamide chiral intermediate, which has the advantages of mild reaction conditions, high stereoselectivity, more environment friendliness, high conversion rate, high chiral purity, small enzyme dosage, high-concentration substrate tolerance and low preparation cost, is suitable for industrial production, avoids chiral resolution and heavy metal residues in products, and overcomes the defects of a chemical method. The technical scheme adopted for solving the technical problems is as follows: An enzyme catalysis synthesis method of a dorzolamide chiral intermediate, which takes methyl acetoacetate as a substrate, adds buffer solution, the substrate, alcohol dehydrogenase, formate dehydrogenase, ammonium formate and coenzyme into a reaction container, reacts for 2-24 hours at 20-40 ℃, and extracts, separates and rotationally evaporates the dorzolamide chiral intermediate (R) -3-hydroxybutyrate; The alco