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CN-121975899-A - Method for synthesizing dehydroepiandrosterone by chemical-multienzyme coupling catalysis

CN121975899ACN 121975899 ACN121975899 ACN 121975899ACN-121975899-A

Abstract

The application provides a method for synthesizing dehydroepiandrosterone by chemical-multienzyme coupling catalysis, belonging to the technical field of pharmaceutical preparations with specific therapeutic activity. The method comprises the steps of taking 4-androstenedione as a substrate, esterifying and synthesizing 3-acetoxyandrostane-3, 5-diene-17-ketone, adding lipase for catalyzing 3-acetoxyandrostane-3, 5-diene-17-ketone to synthesize 5-androstenedione, adding ketoreductase and glucose dehydrogenase, and carrying out double-enzyme coupling for catalyzing 5-androstenedione to synthesize DHEA. The method can realize the efficient synthesis of DHEA.

Inventors

  • SHEN YONGMIAO
  • QIAN JIANWU
  • YING HUIFANG
  • TANG RENHE
  • PENG FENG
  • XI ZIWEI
  • LI CHAOYANG
  • Zhong Zonghai

Assignees

  • 江西百思康瑞药业有限公司
  • 浙江理工大学

Dates

Publication Date
20260505
Application Date
20260115

Claims (8)

  1. 1. A method for synthesizing dehydroepiandrosterone by chemical-multienzyme coupling catalysis, which is characterized by comprising the following steps: Step one, using 4-androstenedione as a substrate, adding p-toluenesulfonic acid and acetic anhydride, and esterifying to synthesize 3-acetoxyandrostane-3, 5-diene-17-ketone; step two, adding a lipohydrolase, catalyzing the hydrolysis of 3-acetoxyandrostane-3, 5-diene-17-ketone to synthesize 5-androstenedione, The lipase is a lipase Pa-lipase with nucleotide sequences of SEQ ID NO.1, a lipase Ps-lipase with nucleotide sequences of SEQ ID NO.3, a lipase Pl-lipase with nucleotide sequences of SEQ ID NO.5, a lipase Pp-lipase with nucleotide sequences of SEQ ID NO.7, a corresponding expression plasmid or genetic engineering bacterium wet cell, a cell disruption solution of the genetic engineering bacterium wet cell expressing the lipase Pp-lipase, or a pure enzyme solution separated after cell disruption and protein purification of the lipase Pp-lipase wet cell; step three, adding ketoreductase and glucose dehydrogenase, synthesizing dehydroepiandrosterone by double enzyme coupling catalysis of 5-androstenedione, The glucose dehydrogenase is glucose dehydrogenase Es-GDH with a nucleotide sequence of SEQ ID NO.11, a corresponding expression plasmid thereof, a cell disruption solution of genetically engineered bacteria thereof or a pure enzyme solution separated from wet thalli of the genetically engineered bacteria of the glucose dehydrogenase Es-GDH after cell disruption and protein purification; The ketoreductase is Rh-SDR with a nucleotide sequence of SEQ ID NO.13, sj-SDR with a nucleotide sequence of SEQ ID NO.15, ss-SDR with a nucleotide sequence of SEQ ID NO.17, sw-SDR with a nucleotide sequence of SEQ ID NO.19, mutants thereof, expression plasmids of the mutants or wet bacterial cells of genetically engineered bacteria, cell disruption liquid of the wet bacterial cells of the genetically engineered bacteria expressing the ketoreductase or pure enzyme liquid separated after cell disruption and protein purification of the wet bacterial cells of the genetically engineered bacteria of the ketoreductase.
  2. 2. The method for synthesizing dehydroepiandrosterone through coupling catalysis of chemical-multienzyme according to claim 1, wherein in the first step, the final concentration of 4-androstenedione in a conversion system of esterification synthesis is 10-100 g/L, the final concentration of toluene sulfonic acid is 10-100 g/L, and the concentration of acetic anhydride is 43.2-432 g/L.
  3. 3. The method for synthesizing dehydroepiandrosterone through coupling catalysis of chemical-multienzyme according to claim 1, wherein in the second step, the pH of a hydrolysis-catalyzed conversion system is 5-10, and the temperature is 15-60 ℃.
  4. 4. The method for synthesizing dehydroepiandrosterone according to claim 1 wherein in step two, any one of phosphate-citrate buffer, phosphate buffer, tris/HCI buffer and glycine-NaOH buffer is added to the hydrolysis-catalyzed conversion system as a reaction medium.
  5. 5. The method for synthesizing dehydroepiandrosterone by using chemical-multienzyme coupling catalysis as claimed in claim 1, wherein in the third step, the pH of a conversion system of the double-enzyme coupling synergistic catalysis is 6-7, and the temperature is 30-40 ℃.
  6. 6. The method for synthesizing dehydroepiandrosterone by using the chemical-multienzyme coupling catalysis as claimed in claim 1, wherein NADP + and glucose are further added into a conversion system of the double-enzyme coupling synergistic catalysis, the concentration of NADP + in the conversion system is 0.5-3.5 mM, and the addition molar ratio of glucose to 5-androstenedione is 0.5-2:1.
  7. 7. The method for synthesizing dehydroepiandrosterone by using chemical-multienzyme coupling catalysis as set forth in claim 1, wherein the solvent of the conversion system with the synergistic catalysis of the double-enzymatic coupling is any one of methanol, ethanol, isopropanol, n-hexane, dimethyl sulfoxide and 2-methyltetrahydrofuran.
  8. 8. The method for synthesizing dehydroepiandrosterone according to claim 1 wherein said mutant of Ss-SDR is a mutant obtained by mutating at least one of the following amino acid sequences: (1) phenylalanine at position 26 is substituted with alanine, (2) arginine at position 106 is substituted with serine, (3) tryptophan at position 157 is substituted with glycine, and (4) tyrosine at position 249 is substituted with serine.

Description

Method for synthesizing dehydroepiandrosterone by chemical-multienzyme coupling catalysis Technical Field The application relates to a method for synthesizing raw material Dehydroepiandrosterone (DHEA) by chemical-multienzyme coupling catalysis, belonging to the technical field of pharmaceutical preparations with specific therapeutic activity. Background As a raw material for synthesizing abiraterone, dehydroepiandrosterone (DHEA) mainly depends on semi-synthesis from plant raw materials such as diosgenin, and the route is lengthy, involves strong corrosive reagents and is accompanied by serious environmental pollution. With the deep concept of green synthesis, chemical-enzymatic synthetic routes have been developed and are the hot spot of current research. The research core of the route is to integrate the advantages of chemical synthesis and biocatalysis, namely, a mother nucleus is generally constructed through esterification reaction, and then, enzymes with high regioselectivity and stereoselectivity (lipase, ketoreductase, glucose dehydrogenase and the like) are utilized to catalyze key reactions such as hydroxylation, dehydrogenation or side chain modification of specific sites. The method aims at remarkably reducing the synthesis steps, avoiding the use of toxic reagents, improving the reaction efficiency and the optical purity of the product, thereby constructing a synthesis process with higher atom economy and more environment-friendly property. The deep research on the route of the method not only has important industrial value for realizing the green manufacture of DHEA, but also provides a technical reference for the biosynthesis of other complex medicaments. However, the current research is mainly on the synthesis route or application mode of abiraterone, and the synthesis of DHEA is less studied. Disclosure of Invention In view of this, the present application provides a method for the chemical-multienzyme coupling catalyzed synthesis of Dehydroepiandrosterone (DHEA), which synthesizes Dehydroepiandrosterone (DHEA) directly by chemical reaction-multienzyme coupling. Specifically, the application is realized by the following scheme: a method for synthesizing dehydroepiandrosterone by chemical-multienzyme coupling catalysis comprises the following steps: step one, using 4-androstenedione (4-AD) as an initial substrate, and esterifying to synthesize 3-acetoxyandrostane-3, 5-diene-17-ketone (3-acetyloxy-androsta-3, 5-dien-17-one); step two, adding a lipohydrolase, and hydrolyzing and catalyzing 3-acetoxyandrostane-3, 5-diene-17-ketone to synthesize 5-androstenedione (5-AD); And thirdly, adding ketoreductase and glucose dehydrogenase, and synthesizing Dehydroepiandrosterone (DHEA) by double-enzyme coupling synergistic catalysis of 5-androstenedione. Further, as preferable: In the first step, the first step is to perform, In the esterification and synthesis conversion system, the concentration of 4-androstenedione is 10-100 g/L. The conversion system of the esterification synthesis is added with p-toluenesulfonic acid and acetic anhydride for hydrolysis and esterification, the concentration of the toluenesulfonic acid in the conversion system is 10-100 g/L, the concentration of the acetic anhydride in the conversion system is 43.2-432 g/L, and the toluenesulfonic acid and the acetic anhydride simultaneously participate in the reaction and regulate the polarity of the system. The composite catalytic system formed by the two can realize the efficient activation of the hydroxyl site of the substrate through obvious synergistic effect, wherein p-toluenesulfonic acid promotes protonation to form an oxonium ion intermediate, acetic anhydride provides acetyl and captures generated water molecules, and the reaction balance is pushed to move towards the esterification direction. This catalytic strategy significantly increases the efficiency of selective acylation of the hydroxyl group at the 3-position and inhibits other competing side reactions such as rearrangement or over-acylation. Under the optimized condition, the reaction is directionally carried out, and the key intermediate 3-acetoxy androsta-3, 5-diene-17-ketone is finally successfully synthesized in high yield, and the product has important application value in the synthesis of steroid medicines. The method has the advantages of mild conditions, simple and convenient operation, obvious synergistic effect of the catalyst and high-efficiency and specific synthetic path for esterification modification of steroid with similar structure. The first step can be specifically set up in such a way that 4-AD, acetic anhydride and p-toluenesulfonic acid are added into a dry reaction flask under the protection of nitrogen. The reaction mixture is heated to a certain temperature (e.g., 40-50 ℃) and stirred and heated at that temperature for a number of hours. The reaction time needs to be monitored by TLC until the 4-AD starting material is substantially lost.