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CN-122012644-A - Method for preparing chiral compound by resolving racemization lactam and application thereof

CN122012644ACN 122012644 ACN122012644 ACN 122012644ACN-122012644-A

Abstract

The invention relates to the technical field of enzyme engineering, and discloses a method for preparing chiral compounds by resolving racemization lactam and application thereof, wherein the method comprises the following steps: chiral resolution is carried out on a substrate by using racemized lactam as the substrate and amidase or amidase-producing microorganism to obtain chiral compounds. The method provided by the invention can be specifically applied to chiral resolution of raceme beta-lactam and raceme gamma-lactam, the optical purity of the obtained product reaches 99.0%, the development of a green and environment-friendly biocatalysis chemical process is facilitated, and meanwhile, the method provided by the invention can be widely applied to preparation of optical pure chiral medicine intermediates of taxol antitumor medicines and nucleoside antiviral medicines.

Inventors

  • ZHU SHAOZHOU
  • Sun Jiabei
  • YAO JING
  • HUANG HAIWEI
  • CHEN HUA

Assignees

  • 中国食品药品检定研究院

Dates

Publication Date
20260512
Application Date
20251215

Claims (10)

  1. 1. Use of amidase or amidase-producing microorganism for the preparation of chiral compounds by resolution of racemic lactams, characterized in that the amidase is a protein as shown in (a) or (b): (a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1; (b) A protein derived from (a) having an activity of resolving racemic lactam by substituting, deleting and/or adding one or more amino acids in the amino acid sequence of (a).
  2. 2. A method for preparing chiral compounds by resolution of racemic lactams is characterized by comprising the steps of taking racemic lactams as substrates, using amidases or amidase-producing microorganisms as catalysts, and carrying out chiral resolution on the substrates by using the catalysts to obtain chiral compounds, wherein the amidases are proteins shown in the following (a) or (b): (a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1; (b) A protein derived from (a) having an activity of resolving racemic lactam by substituting, deleting and/or adding one or more amino acids in the amino acid sequence of (a).
  3. 3. The method according to claim 2, wherein the racemic lactam comprises β -lactam and/or γ -lactam.
  4. 4. The method according to claim 2, wherein the chiral compound comprises (3 r,4 s) -HPO and/or (-) - γ -lactam; the (3R, 4S) -HPO is (3R, 4S) -3-hydroxy-4-phenyl-azetidin-2-one; the (-) -gamma-lactam is (1R, 4S) -2-azabicyclo [2.2.1] hept-5-en-3-one.
  5. 5. The method according to any one of claims 2 to 4, wherein the method comprises resuspending the substrate and the catalyst in a buffer to obtain a biocatalysis system, and subjecting the biocatalysis system to a chiral resolution reaction to obtain the chiral compound.
  6. 6. The method according to any one of claims 2 to 5, wherein the concentration of the substrate in the biocatalytic system is in the range of 5 to 10 mM.
  7. 7. The method according to any one of claims 2 to 6, wherein when the catalyst is amidase, the concentration of the catalyst in the biocatalysis system ranges from 10 to 50 μg/mL; When the catalyst is an amidase-producing microorganism, the concentration range of the catalyst in the biocatalysis system is 5-20 mg/mL.
  8. 8. The method according to any one of claims 2 to 7, wherein the chiral resolution has a reaction pH of 7.0 to 9.0.
  9. 9. The method according to any one of claims 2 to 8, wherein the reaction temperature of the chiral resolution is 40-60 ℃.
  10. 10. The method according to any one of claims 2 to 9, wherein the reaction time for chiral resolution is 1 to 4 hours.

Description

Method for preparing chiral compound by resolving racemization lactam and application thereof Technical Field The invention relates to the technical field of enzyme engineering, in particular to a method for preparing chiral compounds by resolving racemization lactam and application thereof. Background Beta-and gamma-lactams are key cyclic amide intermediates, the diverse cyclic backbones and the abundant functional group reactivities make them an integral part of pharmaceutical and fine chemical syntheses. For example, beta-lactams, gamma-lactams and delta-lactams form the core skeleton of many antibiotics, antiviral agents, antitumor drugs, and central nervous system active compounds, while epsilon-lactams are important precursors for the mass production of large-scale chemicals such as nylon-6. In order to meet the increasing demands for structurally diverse, highly bioactive drugs, lactam intermediates have made significant progress in asymmetric synthesis, site-selective functionalization and downstream applications thereof, in particular, innovations in enzymatic kinetic resolution and other biocatalytic strategies, greatly improving the efficiency and stereoselectivity of lactam construction, and simultaneously providing an easily amplified, environmentally friendly synthetic route. Among the β -lactams, optically pure (3R, 4S) -3-hydroxy-4-phenyl-azetidin-2-one ((3R, 4S) -HPO) has been of great interest as a key intermediate for the semisynthesis of paclitaxel. Paclitaxel is a representative taxane natural product originally isolated from the bark of Taxus brevifolia (Taxus brevelfolia), having a unique bicyclic diterpene skeleton and multiple polar substituents, wherein the C-13 side chain-3-phenylisoserine derivative is critical for the antimitotic activity of paclitaxel. The discovery of paclitaxel has opened up microtubule-targeted anticancer therapies and has induced the generation of a variety of subsequent drugs, such as docetaxel, cabazitaxel, and albumin-bound paclitaxel, as well as the next generation analogs, laroxib (larotaxel) and TPI-287, which are currently in preclinical evaluation. Because of extremely low natural abundance of taxol, industrial synthesis mainly relies on a semisynthetic route to extract 10-deacetylbaccatin III (10-deacetylbaccatin III) from taxol leaves, construct a 3-phenylisoserine side chain through beta-lactam or amino acid derivatives thereof, and then carry out nucleophilic ring-opening coupling at the C-13 position. Therefore, the development of a beta-lactam biocatalytic resolution process with high yield, high stereoselectivity and environmental protection is of great importance for the production of paclitaxel and analogues thereof. In recent years, the enzyme-catalyzed approach to paclitaxel side chain synthesis has also been significantly advanced. The most common strategies at present are lipase mediated kinetic resolution of enantiomers, with commonly used biocatalysts from shigella (archrobacter spp.) and saccharomyces cerevisiae (Saccharomyces cerevisiae). Cyclooxygenase enzymes also have potential for preparing (2R, 3S) -ethyl 3-phenylglycinate, but these processes still need to be further optimized to improve efficiency and selectivity. In contrast, enzymatic ring opening routes based on beta-lactams or esters are still rare, and only few methods of highly enantioselective hydrolysis of the (3 s,4 r) -enantiomer have been reported to date. In terms of gamma-lactams, 2-azabicyclo [2.2.1] hept-5-en-3-ones (Vince lactams, including (+) -gamma-lactam ((1S, 4R) -2-azabicyclo [2.2.1] hept-5-en-3-one) and (-) -gamma-lactam ((1R, 4S) -2-azabicyclo [2.2.1] hept-5-en-3-one) racemates are favored because of their ability to provide a stereodefining cyclopentane skeleton after ring opening, the double bond of which is also readily derivable. To date Vince lactams have been used to synthesize key carbocyclic nucleoside antiviral and therapeutic agents, such as peramivir (peramivir), abacavir (abacavir), MK-0812 and meloagliptin (melogliptin), which have been demanded in the pharmaceutical industry for many tons. At present, the production of optically pure Vince lactam mainly depends on biocatalytic kinetic resolution, and gamma-lactamase shows excellent selectivity under mild and environment-friendly conditions, so that the method is the first choice for industrial production. The prior art has mainly extracted and purified amidase from microorganism for resolving gamma-lactam, preparing (+) -gamma-lactam or (-) -gamma-lactam, lacks the application of catalyzing and resolving beta-lactam to prepare (3R, 4S) -HPO, and simultaneously, has not been reported biological enzyme capable of catalyzing and resolving gamma-lactam and beta-lactam at the same time. Disclosure of Invention The invention provides a method for preparing chiral compounds by resolving raceme lactam and application thereof, which aims to solve the problem of high-activity chiral resolution of r