A method for synthesizing a lactam compound

Abstract

The application discloses a synthesis method of a lactam compound, provides a brand-new green biosynthesis route, and obtains various lactams through dehydration condensation of carboxylic acid by using amino acid as raw material and by means of carboxylic acid reductase catalysis. Experiment proves that the method has the advantages of mild reaction condition, simple operation, environmental friendliness and high atom economy.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and specifically to a method for synthesizing lactams using carboxylic acid reductase catalysis. Background Technology

[0002] Lactam compounds are important pharmaceutical intermediates and are important backbones for drugs such as penicillin G (an antibiotic for treating bacterial infections), Zoladex (an antitumor drug), and brivaracetam (an antiepileptic drug).

[0003] Lactam compounds are mainly synthesized chemically. In 2013, William D. Wulff's team showed that aziridine-2-carboxylic acid reacts with oxalyl chloride to generate morpholino-2,3,5-trione, cyclic N-carboxylic anhydride, or β-lactam, depending on the nature of the substituent at the 3-position; after treatment with Wilsmeer's reagent, all substrates can be transferred to β-lactams (see Multifaceted interception of 2-chloro-2-oxoacetic anhydrides: a catalytic asymmetric synthesis of β-lactams[J]. Chemical Science, 2013, 4: 622.). Although this reaction has high yields, the lactam products are diastereoselective. In 2018, Seung Youn Hong et al. reported a method using an iridium(III) catalyst to synthesize β-lactams via sp... 3 and sp 2C–H amidation converts a wide range of 1,4,2-dioxazol-5-ones (carbonylnitrobenzene precursors readily available from carboxylic acids) into corresponding chiral five-membered ring lactams (see Selective formation of γ-lactams via CH amidation enabled by tailored iridium catalysts[J]. Science, 2018, 359(6379):1016.). These reactions typically require 40–80 °C for 12–48 h and hexafluoroisopropanol as the solvent. In 2019, Stephen J. Connon's team reported an anion-binding method for preparing enantiomeric enriched chiral five-membered ring lactams from enolized anhydrides and imines. The conversion rate of the chiral five-membered ring lactams prepared by this method was 84-99%, and the enantiomeric excess (ee) was 69-96% (see Catalytic Asymmetricγ-Lactam Synthesis from Enolisable Anhydrides and Imines[J]. Chemistry-A European Journal,2019,25(30):7275-7279.). In 2021, Buxing Han's team reported the synthesis of six-membered ring chiral lactams using ruthenium as a catalyst. The reaction required an organic solvent, and after 6 hours at 180°C, the yield reached 63% (see Production of Piperidine and δ-Lactam Chemicals from Biomass-Derived Triacetic Acid Lactone[J]. Angewandte Chemie, 2021, 60: 14405-14409.). This demonstrates that the chemical synthesis of lactams requires high temperatures, organic reagent treatment, complex steps, and a long reaction time.

[0004] Currently, enzymatic synthesis of β-lactams mainly uses amino acids and esters as substrates. In 1995, Baldwin's team first reported the synthesis of β-lactam rings using isopenicillin N synthase with non-natural amino acids (Ld-(α-aminohexyl)-L-cysteine-D-valine) as substrates (see Crystal structure of isopenicillin N synthase is the first from a new structural family of enzymes. [J]. Nature, 1995, 375: 700-704.). In 2001, Schofield's team reported the synthesis of β-lactam rings using natural amino acids (N...2 (2-Carboxyethyl)-l-arginine) was used as a substrate to synthesize the lactam ring via β-lactam synthase (see Enzymatic Synthesis of Monocyclic β-Lactams[J]. Bioorganic & Medicinal Chemistry Letters, 2002, 12(4): 597-599.). Enzymes from other fungal genera can also catalyze the synthesis of five- to nine-membered ring lactam products from C4-C8 straight-chain amino acids, such as esterase B from *Candida antarctica* (see Synthesis of lactams using enzyme-catalyzed aminolysis. Tetrahedron Letters, 2013, 54(5):370-372.), acyl-CoA ligase from *Streptomyces aizunensis* (see Application of an Acyl-CoALigase from *Streptomyces aizunensis* for Lactam Biosynthesiss[J]. ACS Synthetic Biology, 2017, 6(5):884-890), and *Citrobacter freundii* (see A synthesized microbial biosensor for high-throughput screening of lactambiocatalysts[J]. Nature). Communications, 2018, 9(1): 5053.) or 3-hydroxybutyrate dehydrogenase from Enterobacter kobei (see An Integrated Cofactor / Co-Product Recycling Cascade for the Biosynthesis of Nylon Monomers from Cycloalkylamines[J]. Angewandte Chemie International Edition, 2021, 60(7): 3481-3486.). In the above reactions using amino acids as substrates, the enzymes have a single substrate type, catalyze substrate conversion rates of <50%, and do not involve the synthesis of chiral lactams.

[0005] In 1992, Arie L. Gutman's research showed that pancreatic lipase from wild boar (Sus scrofa) can catalyze the synthesis of five-membered and six-membered ring lactam products from amino esters in organic solvents (see Enzymatic formation of lactamsin organic solvents[J].Tetrahedron Letters,1992,33(27):3943-3946.). In 2010, Turner's team reported that transaminases ATA-113 and ATA-117 from Chromobacterium violaceum use isopropylamine as an ammonia donor to amination ethyl 4-acetylbutyrate to form an amino ester. The amino ester undergoes self-cyclization to generate (S) and (R) types of 6-methyl-2-piperidinone (>99% ee) respectively (see Efficient Production of Enantiomerically Pure Chiral Amines at Concentrations of 50 g / L Using Transaminases[J]. Organic Process Research & Development, 2012, 14(1):234-237.). In 2019, Kyle et al. discovered that photoexcitation alters the catalytic function of flavoenzymes from *Gluconobacter oxydans*, enabling these enzymes to promote asymmetric radical cyclization. This reaction imparts a stereochemical preference (74:26er -> 99:1er) to the construction of penta, hexa, hepta, and octa-lactams by the enzyme's active site (see Photoexcitation of flavoenzymes enables a stereoselective radical cyclization[J]. Science, 2019, 364(6446):1166-1169.). Although the synthesis of lactams using esters as substrates exhibits high conversion rates (>80%) and a certain degree of stereochemical selectivity, problems such as byproducts and residual organic solvents exist during the reaction process.

[0006] In summary, the chemical synthesis of lactams requires high temperature, high pressure, and metal catalysts; the reaction conditions are harsh; the pollution is significant; the safety factor is low; and optically pure reaction products cannot be synthesized. Biological methods offer milder reaction conditions, but they require expensive cofactors and generate byproducts. Therefore, finding new green methods for synthesizing lactams has significant application value for industrial production. Summary of the Invention

[0007] To address the shortcomings of the existing technology, this invention provides a method for synthesizing lactams using carboxylic acid reductase catalysis. The method may include the following steps (reaction principle see...). Figure 1 ): Using amino acids with or without chiral centers as substrates, chiral or achiral lactams are generated through a reaction catalyzed by carboxylic acid reductase or its mutants.

[0008] This invention provides a method for synthesizing lactam compounds using carboxylic acid reductase or its mutant.

[0009] The carboxylic acid reductase is any one of the following: SrCAR (WP_007468889.1) carboxylic acid reductase derived from Segniliparus rugosus, a mutant of SrCAR, MmCAR (QQW35300.1) carboxylic acid reductase derived from Mycobacterium marinum; a mutant of MmCAR, MsCAR (VTP06723.1) carboxylic acid reductase derived from Mycolicibacterium smegmatis, NiCAR (AAR91681.1) carboxylic acid reductase derived from Nocardia iowensis, SeCAR (WP_013138593.1) carboxylic acid reductase derived from Segniliparus rotundus; a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins defined above; or an immobilized enzyme of any of the carboxylic acid reductases defined above.

[0010] The mutant of the carboxylic acid reductase SrCAR is any one of the following: SrCAR K524W SrCAR S702A SrCAR K524W / S702A , SrCAR-A or SrCAR K524W -A;

[0011] The SrCAR K524W The protein obtained by mutating position 524 of SrCAR to a tryptophan residue is a key site affecting its affinity for substrates. Mutating it to a tryptophan residue can significantly improve the catalytic activity of carboxylic acid reductase in converting amino acids to lactams.

[0012] The SrCAR S702AThe protein obtained by mutating position 702 of SrCAR to an alanine residue is a key site affecting its thioesterification activity. Mutating it to alanine only allows adenylate esterification activity (the step before the amino substrate is cyclized to form a lactam), but inhibits the subsequent thioesterification reaction (a competitive reaction of lactamation activity), which can significantly improve the catalytic activity of carboxylic acid reductase to convert amino acids to lactams.

[0013] The SrCAR K524W / S702A The protein obtained by simultaneously mutating the 524th and 702nd positions of SrCAR to tryptophan and alanine residues, respectively, is expected to have a positive synergistic effect, thereby improving the catalytic activity of carboxylic acid reductase in converting amino acids to lactams.

[0014] The SrCAR-A is a protein obtained by deleting amino acid residues 655-1188 of SrCAR and retaining amino acid residues 1-654. Amino acid residues 655-1188 are the structural domains responsible for reducing the substrate. Deleting amino acid residues 655-1188 can prevent the substrate from undergoing a reduction reaction, thereby promoting the substrate to undergo a cyclization reaction to generate a lactam.

[0015] SrCAR K524W -A is the protein obtained by mutating the 524th position of SrCAR to a tryptophan residue and simultaneously deleting amino acid residues 655-1188 of SrCAR, retaining amino acid residues 1-654. Simultaneous mutation at the 524th position and mutation truncation of amino acid residues 655-1188 are expected to obtain a positive synergistic effect, thereby further improving the catalytic activity of carboxylic acid reductase in converting amino acids to lactams.

[0016] Furthermore, the mutant of the carboxylic acid reductase MmCAR is: MmCAR-A;

[0017] MmCAR-A is a protein obtained by deleting amino acid residues 647-1174 of MmCAR, retaining amino acid residues 1-646. Amino acid residues 647-1174 are the domains responsible for reducing the substrate. Deleting amino acid residues 647-1174 can prevent the substrate from undergoing reduction, thereby promoting the substrate to undergo cyclization to generate lactam.

[0018] Furthermore, in the steps of the method, the carboxylic acid reductase and its mutants catalyze the reaction in the form of whole cells, crude enzyme solution, crude enzyme powder, or pure enzyme.

[0019] The carboxylic acid reductase was obtained through recombinant expression. Further, the whole cells, crude enzyme solution, crude enzyme powder, and pure enzyme were all prepared according to a method comprising the following steps: expressing the nucleic acid molecule of the carboxylic acid reductase or its mutant in host cells to obtain recombinant cells (i.e., whole cells); lysing the recombinant cells to obtain a crude enzyme solution; and purifying the crude enzyme using a column chromatography process to obtain the pure enzyme.

[0020] Furthermore, the nucleic acid molecule capable of expressing the carboxylic acid reductase or its mutant is introduced into the host cell via a recombinant vector. The recombinant vector can be a bacterial plasmid (such as an expression vector based on the T7 promoter in bacteria, specifically pET28a), a bacteriophage, a yeast plasmid (such as the YEp series vectors), or a retroviral packaging plasmid carrying the coding gene for the carboxylic acid reductase or its mutant.

[0021] The recombinant vector is specifically a recombinant plasmid obtained by replacing the small fragment between the restriction sites NdeⅠ and XhoⅠ of the pET28a vector with the coding gene of the carboxylic acid reductase or its mutant.

[0022] Furthermore, the host cell may be a prokaryotic cell or a lower eukaryotic cell.

[0023] Furthermore, the prokaryotic cell may specifically be a bacterium. The lower eukaryotic cell may specifically be a yeast cell.

[0024] In one embodiment of the present invention, the host cell is specifically *Escherichia coli*, more specifically *E. coli* BL21(DE3). Accordingly, the induction culture involves adding IPTG to the culture system to a final concentration of 0.1-0.5 mmol / L (specifically, 0.1 mmol / L) and inducing culture at 20-37°C (specifically, 20°C) for 12-24 h (specifically, 15 h).

[0025] During the catalytic reaction, the temperature can be 20–40°C (e.g., 25°C); the reaction time can be 8–28 h (e.g., 24 h).

[0026] When the carboxylic acid reductase carries out the catalytic reaction in the form of a pure enzyme, the catalytic reaction can be carried out in a buffer solution as shown below: a phosphate buffer solution with a concentration of 50 mmol / L and a pH of 6.5-9.5.

[0027] In this invention, the cofactor of the carboxylic acid reductase may specifically be ATP.

[0028] The concentration of the substrate in the reaction system can be 1-5 mmol / L (e.g., 5 mmol / L). The concentration of the purified enzyme in the reaction system can be 4-40 g / L (e.g., 20 g / L). The concentration of ATP in the reaction system can be 5-15 mmol / L (e.g., 15 mmol / L). The Mg... 2+ The concentration in the reaction system can be 5-10 mmol / L (e.g., 10 mmol / L).

[0029] The catalytic reaction system for the carboxylic acid reductase catalytic step consists of the following components: 50 mmol / L phosphate buffer (pH 6.5-9.5), amino acids at a final concentration of 1-5 mmol / L, ATP at a final concentration of 5-15 mmol / L, MgCl2 at a final concentration of 5-10 mmol / L, and the purified carboxylic acid reductase at a final concentration of 4-40 g / L.

[0030] The application of the carboxylic acid reductase or its carboxylic acid reductase mutant in the synthesis of chiral and achiral lactams falls within the scope of protection of this invention.

[0031] This invention provides a novel green biosynthesis method for lactams, which has the advantages of mild reaction conditions, simple operation, and environmental friendliness. Attached Figure Description

[0032] Figure 1 A schematic diagram of the reaction for preparing lactams from carboxylic acid reductase.

[0033] Figure 2-18 The detection results of carboxylate reductase in the preparation of lactams by liquid chromatography (HPLC) or gas chromatography (GC). Where: Standard: chromatographic detection result of the mixed lactam standard; Control: detection result of the reaction solution without carboxylate reductase (control). Detailed Implementation

[0034] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0036] Example 1: Preparation of engineered bacteria containing carboxylic acid reductase or its mutants

[0037] 1.1 Preparation of engineered carboxylic acid reductase bacteria

[0038] It has been reported that carboxylic acid reductases MmCAR from *Mycobacterium marinum*, NiCAR from *Nocardiaiowensis*, and SeCAR from *Segniliparus rotundus* can catalyze intermolecular amination reactions between carboxylic acid molecules and excess amino donors (see Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides. [J]. Angewandte Chemie International Edition, 2017, 56, 14498-14501.). Based on this, it is speculated that carboxylic acid reductases can catalyze intramolecular amination reactions between the carboxyl and amino groups of amino acid molecules to synthesize lactam products. SrCAR from *Segniliparus rugosus* is a carboxylic acid reductase whose crystal structure and mechanism have been extensively studied. MsCAR from *Mycolicibacterium smegmatis* is a carboxylic acid reductase that can be solublely expressed in *E. coli* and has a broad substrate spectrum.

[0039] Carboxylic acid reductases from the above five different bacterial genera were synthesized (Table 1) and ligated into the pET24a expression vector that was double-digested with NdeⅠ and XhoⅠ to obtain a recombinant expression vector containing the carboxylic acid reductase gene.

[0040] The recombinant expression vector was transformed into a suitable microbial host. The host microorganism can be any conventional microorganism in the art, as long as it can stably replicate on its own and effectively express the carboxylate reductase gene. In this embodiment, the recombinant expression plasmid was introduced into E. coli BL21(DE3) competent cells via electroporation and cultured upside down on LB agar plates containing kanamycin resistance for 12-16 hours. Positive transformants were selected for DNA sequencing verification; the correctly verified transformants were the carboxylate reductase gene-engineered strains.

[0041] 1.2 Preparation of carboxylic acid reductase mutant engineered bacteria

[0042] Using the carboxylic acid reductase gene obtained in 1.1 as a template, a carboxylic acid reductase mutant vector was constructed. Information on the mutant is shown in Table 1. Primers designed for constructing the mutant are shown in Table 2.

[0043] Among them, SrCAR K524WTo obtain the protein by mutating position 524 of SrCAR to a tryptophan residue, molecular dynamics simulations revealed that position 524 of SrCAR is a key site affecting its substrate affinity (see Computer-assisted engineering of the catalytic activity of a carboxylic acid reductase[J]. Journal of Biotechnology, 2019, 306: 97-104.). Mutating it to a tryptophan residue significantly enhanced the catalytic activity of carboxylic acid reductase in converting amino acids to lactams. S702A The protein obtained by mutating position 702 of SrCAR to an alanine residue is described in the literature. Position 702 of SrCAR is reportedly a key site affecting its thioesterification activity (see Structures of carboxylic acid reductase reveal domain dynamics underlying catalysis[J]. Nature Chemical Biology, 2017, 13(9):975-981.). Mutating it to alanine only allows adenylation activity (the step before the amino substrate is cyclized to form a lactam), but inhibits subsequent thioesterification (a competitive reaction for lactamation activity), significantly improving the catalytic activity of carboxylic acid reductase in converting amino acids to lactams. K524W / S702A The protein obtained by simultaneously mutating positions 524 and 702 of SrCAR to tryptophan and alanine residues, respectively, is expected to achieve a positive synergistic effect, thereby enhancing the catalytic activity of carboxylic acid reductase in converting amino acids to lactams. SrCAR-A is a protein obtained by deleting amino acid residues 655-1188 of SrCAR and retaining amino acid residues 1-654. According to literature reports, amino acid residues 655-1188 of SrCAR are the domains responsible for the thioesterification and subsequent reduction of the substrate (see Structures of carboxylic acid reductase reveal domain dynamics underlying catalysis[J]. Nature Chemical Biology,2017,13(9):975-981.). Deleting amino acid residues 655-1188 can prevent the substrate from undergoing reduction, thereby promoting the substrate to undergo cyclization to generate lactams. K524W-A is the protein obtained by mutating position 524 of SrCAR to tryptophan and simultaneously deleting amino acid residues 655-1188 of SrCAR, retaining amino acid residues 1-654. Simultaneous mutation at position 524 and truncation of amino acid residues 655-1188 are expected to achieve a positive synergistic effect, thereby further improving the catalytic activity of carboxylic acid reductase in converting amino acids to lactams. The mutant MmCAR-A of carboxylic acid reductase MmCAR is obtained by deleting amino acid residues 647-1174 of MmCAR, retaining amino acid residues 1-646. According to literature reports, amino acid residues 647-1174 of MmCAR are the domain responsible for the thioesterification and subsequent reduction of the substrate (see Structures of carboxylicacid reductase reveal domain dynamics underlying catalysis[J]. Nature). Chemical Biology, 2017, 13(9): 975-981.) Delete amino acid residues from position 647 to 1174 to prevent the substrate from undergoing reduction reaction, thereby promoting the substrate to undergo cyclization reaction to generate lactam.

[0044] Table 1 Information on carboxylic acid reductases or their mutants involved in this embodiment.

[0045]

[0046] Note: Naming conventions, such as replacing the original K with W in the 524th position of WP_007468889.1 and naming it "K524W"; multiple mutations are separated by forward slashes (" / "); △ represents deletion mutations.

[0047] Table 2. Mutants and primer sequences used

[0048]

[0049]

[0050] Note: Lowercase letters represent mutation sites.

[0051] To obtain a carboxylic acid reductase mutant, the following experiment was conducted:

[0052] First round of PCR: The reaction system is shown in Table 3, with a total volume of 25 μL. PCR conditions: 98℃: 2 min, (98℃: 10 s, 55℃: 15 s, 72℃: 20 s) 30 cycles, 72℃: 2 min.

[0053] Table 3 PCR reaction system 1

[0054]

[0055] Note: The specific primer sequences used for each mutant are shown in Table 2.

[0056] Second round of PCR: The reaction system is shown in Table 4, with a total volume of 50 μL. PCR conditions: 98℃: 2 min, (98℃: 10 s, 55℃: 15 s, 72℃: 4 min 20 s) 30 cycles, 72℃: 4 min.

[0057] Table 4 PCR reaction system 2

[0058]

[0059]

[0060] Truncated mutations (constructing SrCAR-A, SrCAR) K524W The PCR reaction system for MmCAR-A and MmCAR-A is shown in Table 5, with a total volume of 50 μL. PCR conditions: 98℃: 2 min, (98℃: 10 s, 55℃: 15 s, 72℃: 4 min) 30 cycles, 72℃: 4 min.

[0061] Table 5 PCR reaction system 3

[0062]

[0063] Take 2 μL of the above PCR product and transform it into E. coli BL21(DE3) electrocompetent cells. After recovery for 1 h (37℃, 220 rpm), centrifuge at 5,000 rpm for 30 s, and take 100 μL to spread on LB agar plates (containing 50 μg / mL kanamycin). Invert the plates and incubate at 37℃ for 15 h. Pick single colonies for sequencing verification. Transformants with correct sequencing verification are the engineered strains of the carboxylic acid reductase gene mutant.

[0064] Example 2: Expression and purification of carboxylic acid reductase or its mutant gene

[0065] The recombinant strain constructed in Example 1 was streaked onto LB agar plates (containing 50 μg / mL kanamycin). Single colonies were picked and transferred to 5 mL of LB liquid medium containing 50 μg / mL kanamycin. The culture was carried out at 37°C and 220 rpm for 12 h. The culture was then inoculated into TB liquid medium containing 50 μg / mL kanamycin at a 2% inoculum size and cultured at 37°C until OD500. 600When the concentration was 0.8-0.9, IPTG was added to a final concentration of 0.1 mmol / L, and expression was induced at 20℃ and 220 rpm for 16 h. The cells were then centrifuged at 4℃ and 7,000 rpm for 10 min to collect the cell pellet (i.e., whole cells). The cells were resuspended in phosphate buffer (50 mmol / L, pH 7.4), and the cells were autoclaved (4℃) to obtain whole cell lysate (i.e., crude enzyme solution). The crude enzyme solution was centrifuged at 10,000 rpm and 4℃ for 60 min to collect the supernatant. The supernatant was bound to a Ni column pre-equilibrated with solution A (20 mmol / L imidazole, 500 mmol / L NaCl dissolved in 50 mmol / L potassium phosphate buffer, pH 7.4). The column was washed with solution A, and the target protein was eluted with solution B (500 mmol / L imidazole dissolved in 50 mmol / L potassium phosphate buffer, pH 7.4). The eluted protein was concentrated, dialyzed to desalt, and then quantified.

[0066] Example 3: Carboxylic acid reductase catalyzes the formation of lactams from amino acids.

[0067] The carboxylic acid reductase prepared in Example 2 catalyzes the formation of lactams from amino acid substrates.

[0068] In the case of carboxylic acid reductase SrCAR, SrCAR K524W SrCAR S702A SrCAR K524W / S702A SrCAR-A, SrCAR K524W In reaction systems using MmCAR-A, MmCAR-A, and MmCAR-A as catalysts, 50 mmol / L phosphate buffer (pH 9.0), 5 mmol / L amino acid substrate, 10 mmol / L MgCl2, 10 mmol / L ATP, and 20 g / L purified enzyme were added. The reaction system was incubated at 25°C for 24 h, and the products were then analyzed by high-performance liquid chromatography (HPLC) or gas chromatography (GC).

[0069] In a reaction system using carboxylic acid reductase MsCAR as a catalyst, the following were added: 50 mmol / L phosphate buffer (pH 6.5), 1 mmol / L amino acid substrate, 5 mmol / L MgCl2, 5 mmol / L ATP, and 4 g / L purified enzyme. The reaction system was incubated at 30°C for 8 hours, and the product was then detected by high-performance liquid chromatography (HPLC) or gas chromatography (GC).

[0070] In a reaction system using carboxylic acid reductase NiCAR as a catalyst, the following were added: 50 mmol / L phosphate buffer (pH 8.0), 1 mmol / L amino acid substrate, 5 mmol / L MgCl2, 5 mmol / L ATP, and 4 g / L purified enzyme. The reaction system was incubated at 20°C for 28 h, and the product was detected by high-performance liquid chromatography (HPLC) or gas chromatography (GC).

[0071] In a reaction system using carboxylic acid reductase SeCAR as a catalyst, the following were added: 50 mmol / L phosphate buffer (pH 9.5), 5 mmol / L amino acid substrate, 10 mmol / L MgCl2, 15 mmol / L ATP, and 40 g / L purified enzyme. The reaction system was incubated at 40°C for 28 h, and the product was detected by high-performance liquid chromatography (HPLC) or gas chromatography (GC).

[0072] The detection methods for the target non-chiral lactams are as follows: Detection conditions for 1b-6b: chromatographic column: β-TBDAc (25m × 0.25mm); GC temperature program: initial temperature 110℃ (hold for 2.5min), increase to 135℃ at 25℃ / min (hold for 2min), increase to 210℃ at 30℃ / min (hold for 1.5min); Detection conditions for 7b: chromatographic column: SH-Rtx-1 (60m × 0.32mm); GC temperature program: initial temperature 80℃ (hold for 2.5min), increase to 135℃ at 5℃ / min (hold for 2min), increase to 210℃ at 5℃ / min (hold for 2.0min).

[0073] The detection conditions for the target chiral lactam product by GC / HPLC are shown in Table 6:

[0074] Table 6. Detection conditions for chiral lactams

[0075]

[0076] The detection results of carboxylic acid reductase catalyzing the formation of lactam from amino acids are shown in Tables 7 and 8.

[0077] The sample detection results of carboxylic acid reductase catalyzing the synthesis of lactams from amino acids are shown in the following chromatogram. Figure 2-18The results are shown in the table. Standard: Chromatographic detection results of the racemic lactam standard; Control: Detection results of the reaction solution without carboxylic acid reductase (control). The results show that carboxylic acid reductases from five different sources can catalyze the synthesis of lactam products from amino acid substrates. Some of these reductases achieved conversion rates exceeding 80%, especially the carboxylic acid reductase SrCAR from *Segniliparus rugosus* for 17a substrates and the mutant SrCARK. 524W The highest conversion rate for 12a and 14a substrates can reach 99%.

[0078] Table 7. Detection results of carboxylic acid reductase-catalyzed synthesis of non-chiral lactams from amino acids.

[0079]

[0080] [a] To the reaction system, add 50 mmol / L phosphate buffer (pH 9.0), 5 mmol / L amino acid substrate, 10 mmol / L MgCl2, 15 mmol / L ATP, and 20 g / L purified enzyme. Incubate the reaction system at 25°C for 24 h (the reaction endpoint was defined as when the product no longer increased), and determine the conversion rate by gas chromatography (GC).

[0081] [b]The reaction system was supplemented with 50 mmol / L phosphate buffer (pH 6.5), 1 mmol / L amino acid substrate, 5 mmol / L MgCl2, 5 mmol / L ATP, and 4 g / L purified enzyme. The reaction system was incubated at 30°C for 8 hours (the reaction endpoint was defined as when the product no longer increased), and the conversion rate was determined by gas chromatography (GC).

[0082] [c] The reaction system was supplemented with 50 mmol / L phosphate buffer (pH 8.0), 1 mmol / L amino acid substrate, 5 mmol / L MgCl2, 5 mmol / L ATP, and 4 g / L purified enzyme. The reaction system was incubated at 20°C for 28 h (the reaction endpoint was defined as when the product no longer increased), and the conversion rate was determined by gas chromatography (GC).

[0083] [d] The reaction system was supplemented with 50 mmol / L phosphate buffer (pH 9.5), 5 mmol / L amino acid substrate, 10 mmol / L MgCl2, 15 mmol / L ATP, and 40 g / L purified enzyme. The reaction system was incubated at 40 °C for 28 h (the reaction endpoint was defined as when the product no longer increased), and the conversion rate was determined by gas chromatography (GC).

[0084] nd: Target product not detected.

[0085] Table 8. Detection results of carboxylic acid reductase-catalyzed synthesis of chiral lactams from amino acids.

[0086]

[0087] [a] To the reaction system, add 50 mmol / L phosphate buffer (pH 9.0), 5 mmol / L amino acid substrate, 10 mmol / L MgCl2, 15 mmol / L ATP, and 20 g / L purified enzyme. Incubate the reaction system at 25°C for 24 h (ending when product production ceased). Determine the conversion and enantiomeric excess (ee) using GC or HPLC chiral column chromatography.

[0088] [b]The reaction system was supplemented with 50 mmol / L phosphate buffer (pH 6.5), 1 mmol / L amino acid substrate, 5 mmol / L MgCl2, 5 mmol / L ATP, and 4 g / L purified enzyme. The reaction system was incubated at 30°C for 8 hours (the reaction endpoint was defined as when the product no longer increased). The conversion and enantiomeric excess (ee) were determined by GC or HPLC chiral column chromatography.

[0089] [c] The reaction system was supplemented with 50 mmol / L phosphate buffer (pH 8.0), 1 mmol / L amino acid substrate, 5 mmol / L MgCl2, 5 mmol / L ATP, and 4 g / L purified enzyme. The reaction system was incubated at 20°C for 28 h (the reaction endpoint was defined as when the product no longer increased). The conversion and enantiomeric excess (ee) were determined by GC or HPLC chiral column chromatography.

[0090] [d] The reaction system was supplemented with 50 mmol / L phosphate buffer (pH 9.5), 5 mmol / L amino acid substrate, 10 mmol / L MgCl2, 15 mmol / L ATP, and 40 g / L purified enzyme. The reaction system was incubated at 40 °C for 28 h (the reaction endpoint was defined as when the product no longer increased). The conversion and enantiomeric excess (ee) were determined by GC or HPLC chiral column chromatography.

[0091] rac: racemic product.

[0092] nd: Target product not detected.

[0093] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for synthesizing lactam compounds using a carboxylic acid reductase, comprising the following steps: using a carboxylic acid reductase to catalyze the synthesis of lactams from an amino acid substrate; The carboxyl reductase is a mutant of SrCAR, derived from Segniliparus rugosus, with accession number WP_007468889.

1. K524W Alternatively, the carboxylic acid reductase may be a fusion protease obtained by attaching a tag to its N-terminus and / or C-terminus. The SrCAR mentioned K524W The protein obtained by mutating the 524th position of SrCAR to a tryptophan residue; The amino acid substrate is selected from any of the following: ； 。 2. The method according to claim 1, characterized in that: The carboxylic acid reductase is an immobilized enzyme obtained through immobilization.

3. The method according to claim 1, characterized in that: In the steps of the method, the carboxylic acid reductase catalyzes the reaction in the form of whole cells, crude enzyme solution, crude enzyme powder, or pure enzyme.

4. The method according to claim 1, characterized in that: In the reaction in which the carboxylic acid reductase catalyzes the formation of lactams from a substrate, the reaction system contains, in addition to the substrate and carboxylic acid reductase, Mg. 2+ And cofactor ATP.

5. The method according to claim 4, characterized in that: The reaction of producing lactam by using the carboxylic acid reductase as a biological enzyme substrate was carried out in a phosphate buffer solution with a concentration of 40 mmol / L-60 mmol / L and a pH of 6.5-9.5; the temperature of the catalytic reaction was 20-40°C; and the time of the catalytic reaction was 8-28 h.

6. The method according to claim 4, characterized in that: The substrate is present at a concentration of 1-5 mmol / L in the reaction system; the enzyme is present at a concentration of 4-40 g / L in the reaction system; the ATP is present at a concentration of 5-15 mmol / L in the reaction system; and the Mg... 2+ The concentration in the reaction system is 5-10 mmol / L.

7. The method according to claim 5, characterized in that: The catalytic reaction system for the carboxylic acid reductase catalytic step consists of the following components: phosphate buffer at a concentration of 50 mmol / L and a pH of 6.5-9.5; amino acids at a final concentration of 1-5 mmol / L; ATP at a final concentration of 5-15 mmol / L; MgCl2 at a final concentration of 5-10 mmol / L; and the purified carboxylic acid reductase at a final concentration of 4-40 g / L.

8. The method according to any one of claims 1 to 7, characterized in that: The carboxyl reductase was obtained through recombinant expression.

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