Method for preparing chiral compound by splitting racemate lactam and application of chiral compound

Abstract

The invention relates to the technical field of enzyme engineering, and discloses a method for preparing a chiral compound through resolution of racemate lactam and application of the method, and the method comprises the following steps: taking racemate lactam as a substrate, and carrying out chiral resolution on the substrate by using amidase or amidase-producing microorganisms to obtain the chiral compound. The method provided by the invention can be specifically applied to chiral resolution of racemate beta-lactam and racemate gamma-lactam, the optical purity of the obtained product reaches 99.0%, and development of a green and environment-friendly biological catalysis chemical process is facilitated; meanwhile, the method provided by the invention can be widely applied to preparation of optical pure chiral drug intermediates of paclitaxel antitumor drugs and nucleoside antiviral drugs.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, specifically to a method for preparing chiral compounds by resolving racemic in vivo amides and its application. Background Technology

[0002] β- and γ-lactams are key cyclic amide intermediates, whose diverse cyclic skeletons and abundant functional group reactivity make them indispensable in the synthesis of pharmaceuticals and fine chemicals. For example, β-lactams, γ-lactams, and δ-lactams constitute the core skeletons of many antibiotics, antiviral agents, antitumor drugs, and central nervous system active compounds, while ε-lactams are important precursors for the large-scale production of bulk chemicals such as nylon-6. To meet the growing demand for structurally diverse and highly bioactive drugs, significant progress has been made in the asymmetric synthesis, site-selective functionalization, and downstream applications of lactam intermediates. In particular, innovations in enzyme-catalyzed kinetic resolution and other biocatalytic strategies have greatly improved the efficiency and stereoselectivity of lactam construction, while providing easily scalable and environmentally friendly synthetic routes.

[0003] Among β-lactams, optically pure (3R,4S)-3-hydroxy-4-phenyl-azacyclobutane-2-one ((3R,4S)-HPO) has attracted much attention as a key intermediate in the semi-synthesis of paclitaxel. Paclitaxel, the first representative taxane natural product isolated from the bark of the Pacific yew (Taxus brevifolia), possesses a unique bicyclic diterpenoid skeleton and multiple polar substituents, among which the C-13 side chain-3-phenylisoserine derivative is crucial for the antimitotic activity of paclitaxel. The discovery of paclitaxel pioneered microtubule-targeted anticancer therapy and spurred the development of numerous subsequent drugs, such as docetaxel, cabazitaxel, and albumin-bound paclitaxel, as well as next-generation analogs larotaxel and TPI-287, which are currently in the preclinical evaluation stage. Due to the extremely low natural abundance of paclitaxel, industrial synthesis mainly relies on semi-synthetic routes: 10-deacetylbaccatin III is extracted from yew leaves, a 3-phenylisoserine side chain is constructed via β-lactam or its amino acid derivatives, followed by nucleophilic ring-opening coupling at the C-13 position. Therefore, developing a high-yield, highly stereoselective, and environmentally friendly biocatalytic resolution process for β-lactams is crucial for the production of paclitaxel and its analogues.

[0004] In recent years, significant progress has been made in enzymatic catalytic methods for the synthesis of paclitaxel side chains. The most commonly used strategy is lipase-mediated enantiokinetic resolution, with commonly used biocatalysts derived from *Arthrobacter pp.* and *Saccharomyces cerevisiae*. Epoxyhydrolases have also shown potential in the preparation of (2R,3S)-ethyl-3-phenylglycine esters, but these processes still require further optimization to improve efficiency and selectivity. In contrast, enzymatic ring-opening routes based on β-lactams or esters are still rare, with only a few methods for the highly enantioselective hydrolysis of (3S,4R)-enantiomers reported to date.

[0005] In the field of γ-lactams, 2-azabicyclo[2.2.1]hepta-5-en-3-one (Vince lactam, comprising the racemic mixtures of (+)-γ-lactam ((1S,4R)-2-azabicyclo[2.2.1]hepta-5-en-3-one) and (-)-γ-lactam ((1R,4S)-2-azabicyclo[2.2.1]hepta-5-en-3-one)) is favored for its well-defined cyclopentane skeleton after ring-opening, and its double bonds are readily derivatized. To date, Vince lactam has been used to synthesize key carbocyclic nucleoside antiviral drugs and therapeutic agents, such as peramivir, abacavir, MK-0812, and melogliptin, with annual demand in the pharmaceutical industry reaching several tons. Currently, the production of optically pure Vince lactam mainly relies on biocatalytic kinetic resolution. γ-lactamases exhibit excellent selectivity under mild and environmentally friendly conditions, thus becoming the preferred method for industrial production.

[0006] Existing technologies mostly extract and purify amidases from microorganisms to cleave γ-lactams and prepare (+)-γ-lactams or (-)-γ-lactams. There is a lack of applications of enzymes that catalyze the cleavage of β-lactams to prepare (3R,4S)-HPO. At the same time, there are no reports of biological enzymes that can simultaneously catalyze the cleavage of γ-lactams and β-lactams. Summary of the Invention

[0007] This invention provides a method for preparing chiral compounds by resolving racemic β-lactams and its application, in order to solve the problem of highly active chiral resolution of racemic β-lactams and racemic γ-lactams.

[0008] In a first aspect, the present invention provides the use of amidases or amidase-producing microorganisms in the resolution of racemic amides to prepare chiral compounds, wherein the amidase is a protein as shown in (a) or (b) below: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (b) A protein derived from (a) that has one or more amino acids substituted, deleted and / or added in the amino acid sequence of (a) and has resolving racemic in vivo amide activity.

[0009] Secondly, the present invention also provides a method for preparing chiral compounds by resolving racemic amides in vivo, the method comprising: using a racemic amide in vivo as a substrate, using an amidase or an amidase-producing microorganism as a catalyst, and using the catalyst to chirally resolve the substrate to obtain a chiral compound, wherein the amidase is a protein as shown in (a) or (b) below: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (b) A protein derived from (a) that has one or more amino acids substituted, deleted and / or added in the amino acid sequence of (a) and has resolving racemic in vivo amide activity.

[0010] In one alternative embodiment, the racemic endomemamide comprises β-lactam and / or γ-lactam.

[0011] In one alternative embodiment, the chiral compound comprises (3R,4S)-HPO and / or (-)-γ-lactam; The (3R,4S)-HPO is (3R,4S)-3-hydroxy-4-phenyl-azacyclobutane-2-one; The (-)-γ-lactam is (1R,4S)-2-azabicyclo[2.2.1]hept-5-en-3-one.

[0012] In one alternative embodiment, the method includes: resuspending the substrate and catalyst in a buffer solution to obtain a biocatalytic system; and subjecting the biocatalytic system to a chiral resolution reaction to obtain a chiral compound.

[0013] In an alternative embodiment, when the substrate is a racemic β-lactam, the chiral compound obtained by chiral resolution is (3R,4S)-HPO.

[0014] In an alternative embodiment, when the substrate is a racemic γ-lactam, the chiral compound obtained by chiral resolution is (-)-γ-lactam.

[0015] In one alternative embodiment, the concentration of the substrate in the biocatalytic system ranges from 5 to 10 mM.

[0016] In one alternative embodiment, when the catalyst is an amidase, the concentration of the catalyst in the biocatalytic system ranges from 10 to 50 µg / mL.

[0017] In one alternative embodiment, when the catalyst is an amidase-producing microorganism, the concentration of the catalyst in the biocatalytic system ranges from 5 to 20 mg / mL.

[0018] In one alternative embodiment, the amidase-producing microorganism includes *Escherichia coli* expressing the amidase.

[0019] In one alternative embodiment, the amidase is codon-optimized to make it suitable for heterologous expression in Escherichia coli.

[0020] In one alternative embodiment, the reaction pH for the chiral resolution is 7.0 to 9.0.

[0021] In one optional embodiment, the reaction temperature for the chiral separation is 40~60°C.

[0022] In one alternative implementation, the reaction time for the chiral split is 1 to 4 hours.

[0023] In one alternative embodiment, the reaction buffer for chiral resolution comprises Tris, boric acid, citric acid, and / or Na2HPO4.

[0024] In one alternative embodiment, the reaction buffer comprises 50 mM Tris, 50 mM boric acid, 33 mM citric acid and / or 50 mM Na2HPO4.

[0025] The technical solution of this invention has the following advantages: 1. This invention provides a method for preparing chiral compounds by resolving racemic intramethylene amides. The method comprises: using racemic intramethylene amides as substrates, using an amidase or an amidase-producing microorganism as a catalyst, and using the catalyst to chirally resolve the substrates to obtain chiral compounds. The amidase is a protein as shown in (a) or (b) below: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO. 1; (b) a protein derived from (a) having one or more amino acids substituted, deleted, and / or added to the amino acid sequence in (a) and possessing the activity of resolving racemic intramethylene amides. The method provided by this invention can be specifically applied to the chiral resolution of racemic 3-hydroxy-4-phenyl-azacyclobutane-2-one (β-lactam) and racemic 2-azabicyclo[2.2.1]hept-5-en-3-one (γ-lactam) to obtain chiral compounds (3R,4S)-HPO or (-)-γ-lactams with an optical purity of 99.0%. The method provided by this invention is beneficial for developing green and environmentally friendly biocatalytic chemical processes, and can also be widely used in the preparation of optically pure chiral drug intermediates for paclitaxel-based antitumor drugs and nucleoside antiviral drugs.

[0026] 2. This invention also provides the application of amidases or amidase-producing microorganisms in the resolution of racemic in vivo amides to prepare chiral compounds, wherein the amidase is a protein as shown in (a) or (b) below: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO.1; (b) a protein derived from (a) having one or more amino acids substituted, deleted, and / or added to the amino acid sequence in (a) and possessing the activity of resolving racemic in vivo amides. The application described in this invention can be specifically applied to the chiral resolution of racemic 3-hydroxy-4-phenyl-azacyclobutane-2-one (β-lactam) and racemic 2-azabicyclo[2.2.1]hept-5-en-3-one (γ-lactam) to obtain chiral compounds (3R,4S)-HPO or (–)-γ-lactam with an optical purity of 99.0%, which is beneficial for developing green and environmentally friendly biocatalytic chemical processes. It can also be widely used in the preparation of optically pure chiral drug intermediates for paclitaxel-based antitumor drugs and nucleoside antiviral drugs. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 This is an SDS-PAGE image of amidase Rb-ami expression; Figure 2 This is a chiral resolution liquid chromatogram of racemic 2-azabicyclo[2.2.1]hept-5-en-3-one and racemic 3-hydroxy-4-phenyl-azacyclobutane-2-one catalyzed by amidase Rb-ami; Figure 2 Among them, (A) racemic 2-azabicyclo[2.2.1]hept-5-en-3-one; (B) racemic 3-hydroxy-4-phenyl-azabicyclobutane-2-one; Figure 3 This is a characterization diagram of the optimal reaction temperature and thermal stability of the amidase Rb-ami; Figure 4 This is a characterization diagram of the optimal reaction pH and pH stability of the amidase Rb-ami. Detailed Implementation

[0029] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0030] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0031] The materials and reagents used in the embodiments of this invention are as follows: IPTG was purchased from Sigma Aldirch; chiral compound reference standards were purchased from Biostar Pharmaceuticals. Sodium dihydrogen phosphate, disodium hydrogen phosphate, boric acid, and citric acid were purchased from Sinopharm Beijing Chemical Reagent Co., Ltd.; trifluoroacetic acid was purchased from Sigma Aldirch.

[0032] Example 1: A method for preparing chiral compounds by resolving racemic amides in vivo. This embodiment provides a method for preparing chiral compounds by resolving racemic amides in vivo, and the specific steps are as follows: 1. Preparation of amidase (1) Obtaining amidase Rb-ami Obtained from Genbank Rhodococcus baikonurensis The original gene sequence of the amidase Rb-ami (gene number: WP_298775653.1) was optimized for heterologous expression in E. coli through codon optimization, and a histidine tag was added to its N-terminus for protein purification to obtain the Rb-ami coding sequence. The Rb-ami coding sequence as shown in SEQ ID NO.2 was directly synthesized by solid-phase phosphoramide chemical synthesis, and then inserted into the NdeI and XhoI sites of the pET28a(+) vector using Gibson Assembly gene recombination to obtain the recombinant plasmid pET28a(+)-Rb-ami.

[0033] (2) Recombinant protein expression and purification The recombinant plasmid pET28a(+)-Rb-ami was introduced into *E. coli* BL21(DE3) via heat shock to obtain transformants, which were single colonies of recombinant *E. coli* BL-21(pET-28-Rb-ami). The transformed cells, i.e., *E. coli* BL21(DE3) cells carrying the Rb-ami plasmid, were cultured overnight (16 h) in 50 mL of LB medium containing 50 µg / mL kanamycin at 37°C and 220 rpm. Two mL of the overnight culture was inoculated into 200 mL of fresh LB medium (containing 50 µg / mL kanamycin) and cultured at 37°C and 220 rpm until the optical density (OD600) at 600 nm reached 0.7. The culture system was then cooled to 14°C, and protein expression was induced by adding IPTG to a final concentration of 0.05 mM. The expression was then induced for 16 hours in a shaker at 14°C and 150 rpm to obtain the fermentation broth. The fermentation broth was centrifuged (5000 rpm, 10 min) and the cells were collected. The bacterial cells were resuspended in lysis buffer containing 1 mg / mL lysozyme (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0) and lysed by sonication. Insoluble impurities were removed by centrifugation (8,000 × g, 30 min, 4°C), and the clarified lysis buffer was loaded into NiO2 pre-equilibrated with 30 mL of lysis buffer. 2+ The sample was loaded onto an NTA affinity column. After washing with 50 mL of wash buffer (50 mM Tris-HCl, 300 mM NaCl, 30 mM imidazole, pH 8.0), the sample was eluted with 10 mL of elution buffer (50 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, pH 8.0) to obtain the eluent containing recombinant amidase Rb-ami. The eluent was concentrated and further purified by molecular sieve (gel filtration, Cytiva). The chromatographic peaks corresponding to Rb-ami were collected, fractionated, combined, and concentrated to the desired volume using an Amicon Millipore centrifuge with a molecular weight cutoff of 10 kDa to obtain recombinant amidase Rb-ami. The amino acid sequence of recombinant amidase Rb-ami is shown in SEQ ID NO.1.

[0034] 2. Chiral splitting The recombinant amidase Rb-ami obtained in the above steps was added to 500 µL of reaction buffer at a dosage of 25 µg / mL to obtain a catalyst system. 5 mM (3R,4S)-HPO4 and 5 mM (3S,4R)-HPO4 were added to the catalyst system to obtain a biocatalytic system. The biocatalytic system was reacted at 50 °C for 4 hours. After the reaction was completed, 2 μL of trifluoroacetic acid was added to terminate the reaction, yielding a reaction mixture. 1 mL of ethyl acetate (chromatographic grade, purchased from Sigma) was added to the reaction mixture for extraction. After extraction, the mixture was centrifuged (1000 rpm, 10 min), and the upper organic phase was collected to obtain the product containing the chiral compound. The reaction buffer consisted of 50 mM Tris, 50 mM boric acid, 33 mM citric acid, and 50 mM Na2HPO4, with water as the solvent and pH 7.0.

[0035] Example 2: A method for preparing chiral compounds by resolving racemic amides in vivo. This embodiment provides a method for preparing chiral compounds by resolving racemic amides in vivo, and the specific steps are as follows: Based on the chiral resolution steps in Example 1, the substrate system was changed to 5mM (+)-γ-lactam and 5mM (-)-γ-lactam for chiral resolution, and other conditions were the same as in Example 1.

[0036] Example 3: A method for preparing chiral compounds by resolving racemic amides in vivo. This embodiment provides a method for preparing chiral compounds by resolving racemic amides in vivo, and the specific steps are as follows: Based on the chiral separation step in Example 1, the recombinant amidase Rb-ami was replaced with 10 mg of recombinant Escherichia coli used to extract the recombinant amidase, and other conditions were the same as in Example 1.

[0037] Example 4: A method for preparing chiral compounds by resolving racemic amides in vivo. This embodiment provides a method for preparing chiral compounds by resolving racemic amides in vivo, and the specific steps are as follows: Based on the chiral resolution steps in Example 1, the substrate system was changed to 5mM (+)-γ-lactam and 5mM (-)-γ-lactam for chiral resolution, and other conditions were the same as in Example 3.

[0038] Experimental Example 1: Purification and Identification of Amidease This experimental example demonstrates the purity analysis of the recombinant amidase Rb-ami prepared in Example 1. The specific steps are as follows: The purification effect of the recombinant amidase Rb-ami prepared in Example 1 was detected by SDS-PAGE.

[0039] Test results are shown Figure 1 , Figure 1 This is an SDS-PAGE image of the recombinant amidase Rb-ami expression, created by... Figure 1 It can be seen that the recombinant amidase Rb-ami prepared in Example 1 has high purity and most of the impurities have been removed.

[0040] Experimental Example 2: Analysis of amidase hydrolysis activity This experimental example analyzed the hydrolytic activity of the recombinant amidase Rb-ami prepared in Example 1. The specific steps are as follows: Chiral compounds were prepared using the methods described in Examples 1 and 2. After preparation, 10 µL of the upper organic phase was analyzed by HPLC. HPLC analysis was performed using a Shimadzu system and a Daicel Chiralpak AD-H chiral column. The HPLC analysis conditions were: flow rate 0.5 mL / min, gradient elution of 80:20 (volume fraction) bioacetonitrile:isopropanol for 30 min, and the product was detected at 220 nm.

[0041] The results are as follows Figure 2 As shown, Figure 2 The racemic 2-azabicyclo[2.2.1]hepta-5-en-3-one catalyzed by recombinant amidase Rb-ami ( Figure 2 A) and racemic 3-hydroxy-4-phenyl-azacyclobutane-2-one ( Figure 2 B) Chiral separation liquid chromatography chromatogram. (From...) Figure 2 It was found that the retention times of the two enantiomers could be determined using a reference standard (purchased from Bidex Pharmaceuticals). Compared to the blank control (reaction system without recombinant amidase Rb-ami), after incubation with recombinant amidase Rb-ami, one enantiomer ((3S,4R)-HPO or (+)-γ-lactam) was completely hydrolyzed, leaving optically pure enantiomers ((3R,4S)-HPO or (-)-γ-lactam). Figure 2 It is known that the recombinant amidase Rb-ami prepared in Example 1 of this invention has good chiral resolution activity for racemic 3-hydroxy-4-phenyl-azacyclobutane-2-one and racemic 2-azabicyclo[2.2.1]hept-5-en-3-one.

[0042] Experimental Example 3: Optimal Reaction Temperature and Thermal Stability Analysis of Amideases This experimental example analyzed the optimal reaction temperature and thermal stability of the recombinant amidase Rb-ami obtained in Example 1. The specific steps are as follows: (1) Optimal reaction temperature experiment: Based on the chiral resolution step in Example 1, the reaction pH was fixed at 7.0, and the reaction temperature was adjusted to 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃ and 90℃. Other reaction conditions were the same as in Example 1.

[0043] (2) Thermal stability experiment: Based on the chiral resolution step in Example 1, the recombinant amidase Rb-ami was heat-treated at different temperatures (30℃, 40℃, 50℃, 60℃, 70℃ and 80℃) for 1 hour before chiral resolution reaction was carried out, and the reaction pH was fixed at 7.0.

[0044] The results are as follows Figure 3 As shown, by Figure 3 It can be seen that the recombinant amidase Rb-ami obtained after purification has an optimal reaction temperature of 50℃ under the condition of pH 7.0, and the enzyme activity is close to 100%, and it still has more than 70% enzyme activity at 60℃. Depend on Figure 3 It can be seen that in the thermostability experiment, the hydrolytic activity of recombinant amidase Rb-ami was analyzed after heat treatment at different temperatures for 1 hour (method shown in Experiment Example 2). The results showed that the activity of recombinant amidase Rb-ami did not decrease after treatment at 30~40℃ for 1 hour; after treatment at 50℃ for 1 hour, it still retained more than 70% of the enzyme activity, indicating that recombinant amidase Rb-ami has high thermostability.

[0045] In summary, the recombinant amidase Rb-ami provided in Example 1 of this invention exhibits high lactam hydrolysis activity at 40~60℃ and good thermal stability.

[0046] Experimental Example 4: Optimal Reaction pH and pH Stability Analysis of Amideases This experimental example analyzed the optimal reaction pH and pH stability of the recombinant amidase Rb-ami obtained in Example 1. The specific steps are as follows: (1) Optimal reaction pH experiment: Based on the chiral resolution step in Example 1, the reaction temperature was fixed at 50℃, and the reaction pH was adjusted to 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. Other reaction conditions were the same as in Example 1.

[0047] (2) pH stability experiment: Based on the chiral separation step in Example 1, the recombinant amidase Rb-ami was treated at different pH values ​​(pH 3, pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, pH 10, pH 11 and pH 12) for 1 hour before chiral separation reaction was carried out, with the reaction temperature fixed at 50℃.

[0048] (3) The buffer solution used is formulated as follows: 50mM Tris, 50mM boric acid, 33mM citric acid and 50mM Na2HPO4, with pH adjusted by hydrochloric acid or sodium hydroxide.

[0049] The results are as follows Figure 4 As shown, by Figure 4 It can be seen that at a temperature of 50℃, the optimal reaction pH of recombinant amidase Rb-ami is 8.0, and the enzyme can maintain high enzyme activity in the pH range of 7.0~9.0. Depend on Figure 4 The pH stability experiment showed that the recombinant amidase Rb-ami retained more than 50% of its activity after being treated for 1 hour in the pH range of 6.0 to 9.0, indicating that it has high pH tolerance.

[0050] In summary, the recombinant amidase Rb-ami provided in Example 1 of this invention exhibits highly efficient lactam hydrolysis activity in the pH range of 7.0 to 9.0.

[0051] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of amidases or amidase-producing microorganisms in the resolution of racemic amides to prepare chiral compounds, characterized in that, The amidase is a protein as shown in (a) or (b) below: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (b) A protein derived from (a) that has one or more amino acids substituted, deleted and / or added in the amino acid sequence of (a) and has the resolving racemic in vivo amide activity.

2. A method for preparing chiral compounds by resolving racemic amides in vivo, characterized in that, The method includes: using a racemic amide as a substrate, and an amidase or an amidase-producing microorganism as a catalyst, chirally resolving the substrate using the catalyst to obtain a chiral compound, wherein the amidase is a protein as shown in (a) or (b) below: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (b) A protein derived from (a) that has one or more amino acids substituted, deleted and / or added in the amino acid sequence of (a) and has the resolving racemic in vivo amide activity.

3. The method according to claim 2, characterized in that, The racemic endomemamides include β-lactams and / or γ-lactams.

4. The method according to claim 2, characterized in that, The chiral compounds include (3R,4S)-HPO and / or (-)-γ-lactam; The (3R,4S)-HPO is (3R,4S)-3-hydroxy-4-phenyl-azacyclobutane-2-one; The (-)-γ-lactam is (1R,4S)-2-azabicyclo[2.2.1]hept-5-en-3-one.

5. The method according to any one of claims 2-4, characterized in that, The method includes: resuspending the substrate and catalyst in a buffer solution to obtain a biocatalytic system; and performing a chiral resolution reaction on the biocatalytic system to obtain a chiral compound.

6. The method according to any one of claims 2-5, characterized in that, The concentration range of the substrate in the biocatalytic system is 5–10 mM.

7. The method according to any one of claims 2-6, characterized in that, When the catalyst is an amidase, the concentration of the catalyst in the biocatalytic system ranges from 10 to 50 µg / mL; When the catalyst is an amidase-producing microorganism, the concentration of the catalyst in the biocatalytic system ranges from 5 to 20 mg / mL.

8. The method according to any one of claims 2-7, characterized in that, The reaction pH for the chiral resolution is 7.0 to 9.

0.

9. The method according to any one of claims 2-8, characterized in that, The reaction temperature for the chiral separation is 40~60℃.

10. The method according to any one of claims 2-9, characterized in that, The reaction time for the chiral separation is 1 to 4 hours.

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