Method for synthesizing nitrogen-containing heterocyclic ring compound through multi-enzyme-stage combination

Abstract

The invention belongs to the technical field of biological catalysis, and discloses a method for synthesizing a nitrogen-containing heterocyclic ring compound through multi-enzyme-stage combination, which comprises the following steps: by taking alpha, omega-diamine as a substrate, catalyzing one amino group into aldehyde (carbonyl) through one-step enzyme catalysis, then carrying out ammonia-aldehyde condensation on the intermediate in an aqueous solution, and spontaneously cyclizing to generate cyclic imine, thereby obtaining the nitrogen-containing heterocyclic ring compound. And finally, further catalyzing imine by utilizing imine reductase to obtain the nitrogen-containing heterocyclic ring compound. The nitrogen-containing heterocyclic ring compound is synthesized from alpha, omega-diamine by utilizing two-step enzyme catalysis and constructing different amine receptor and cofactor circulation systems, the method belongs to a biosynthesis method, the raw materials are easy to obtain, the operation is simple and convenient, an efficient method is provided for synthesis of the nitrogen-containing heterocyclic ring compound, and the method is suitable for industrialization.

Description

Technical Field

[0001] The invention belongs to the technical field of biosynthesis, and particularly relates to a method for synthesizing nitrogen-containing heterocyclic compounds by multi-enzyme cascade. Background Art

[0002] Nitrogen-containing heterocyclic compounds (such as piperidine and pyrrolidine) are a class of organic compounds with important biological activities and industrial applications. They are widely used in pharmaceutical synthesis, pesticide manufacturing, and fine chemical production. For example, the piperidine structure is the core skeleton of many bioactive molecules (such as anti-tumor drugs, central nervous system drugs, and antibacterial drugs) and also has important applications in the preparation of functional materials, dyes, and fragrances. Therefore, the development of efficient, green, and sustainable methods for the synthesis of nitrogen-containing heterocyclic compounds is not only of great scientific significance but also meets the urgent needs of industrial production.

[0003] Currently, the synthesis of nitrogen-containing heterocyclic compounds mainly relies on traditional chemical catalytic methods, such as transition metal-catalyzed cyclization reactions, acid-catalyzed condensation reactions, and reductive amination reactions. However, these methods have significant limitations: First, chemical catalytic methods usually require high temperature, high pressure, or strong acid and base environments, resulting in high energy consumption, complex equipment requirements, and low operational safety; second, chemical catalytic methods often have difficulty achieving high regioselectivity and stereoselectivity, resulting in a large number of byproducts and low product purity, increasing the difficulty and cost of separation and purification; finally, chemical catalysis processes often use toxic reagents (such as heavy metal catalysts) or produce harmful byproducts, causing environmental pollution and not meeting the requirements of green chemistry and sustainable development.

[0004] In recent years, biocatalytic methods have become a research hotspot for the synthesis of nitrogen-containing heterocyclic compounds due to their high efficiency, high selectivity, and environmental friendliness. Multienzyme cascade reactions, by integrating multiple enzymatic steps, enable efficient conversion of simple substrates to complex products, offering advantages such as high selectivity, mild reaction conditions, and environmental friendliness. However, existing biocatalytic methods for the synthesis of nitrogen-containing heterocyclic compounds still face numerous challenges. First, many key enzymes (e.g., imine reductase and amine dehydrogenase) rely on cofactors (e.g., NADPH and NADH), the high cost of which restricts their large-scale industrial application. Second, in cascade reactions, enzyme activity and stability can be limited by reaction conditions (e.g., pH, temperature, and substrate concentration), impacting catalytic efficiency. Finally, existing enzymatic methods have poor substrate adaptability, making them difficult to meet diverse synthetic needs and limiting their application. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention provides a multi-enzyme cascade method for synthesizing nitrogen-containing heterocyclic compounds. Specifically, this method utilizes a multi-step enzymatic cascade reaction to convert α,ω-diamines into nitrogen-containing heterocycles. By constructing an amine receptor and cofactor recycling system, the reaction efficiency can be significantly improved and byproduct formation can be reduced. This reaction is simple to operate, environmentally friendly, and economically viable, with mild reaction conditions and a wide range of substrate compatibility, enabling the efficient synthesis of a variety of nitrogen-containing heterocycles.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows: A multi-enzyme cascade synthesis method for nitrogen-containing heterocyclic compounds comprises: using α,ω-diamine as a raw material, an amine receptor and an NADPH circulatory system, and reacting at 10-60°C and pH 5.0-10.0 for 1-48 hours under the action of a transaminase, an imine reductase, and PLP to obtain nitrogen-containing heterocyclic compounds. The nitrogen-containing heterocyclic compounds include but are not limited to azetidine, pyrrolidine, piperidine, azepane, azacyclooctane, and derivatives thereof; the amine receptor and the NADPH circulatory system comprise one or a combination of glucose dehydrogenase (GDH), L-alanine dehydrogenase (AlaDH), L-amino acid deaminase (LAAD), D-amino acid oxidase (DAAO), alanine racemase (ALR), and catalase (CAT), a cosubstrate, pyruvate, and a cofactor.

[0007] As an improvement, the α,ω-diamine includes but is not limited to straight-chain α,ω-diamines such as 1,3-propylenediamine, 1,4-butylenediamine, 1,5-pentanediamine, 1,6-hexanediamine, and 1,7-heptanediamine, as well as cyclohexane-1,3-diamine, cyclohexane-1,4-diamine, cyclohexane-1,3-dimethylamine, or cyclohexane-1,4-dimethylamine.

[0008] As an improvement, the amino acid sequence of the transaminase includes but is not limited to AKK18527, Q9I6J2, RBP85366, 4CE5_A, WP_011781668, WP_003255866, HBB1607555, KUN50029, WP_010975253, WP_011049154, WP_011749320; the amino acid sequence of the imine reductase includes but is not limited to XP_045278909, XP_001827659, XP_748217, WP_071905000, OAQ82145, WP_020017817, 3ZGY_A, WP_015347361, WP_077989474.

[0009] As an improvement, the added amount of the transaminase is 2-10 U / mL, and the added amount of the imine reductase is 2-10 U / mL.

[0010] As an improvement, the co-substrate includes but is not limited to one or both of glucose and pyruvate.

[0011] As an improvement, the cofactor includes but is not limited to NAD + , NADH, NADP + , or NADPH.

[0012] As an improvement, the amino acid sequence of glucose dehydrogenase includes but is not limited to 1GEE_A, AFQ56330; the amino acid sequence of L-alanine dehydrogenase includes but is not limited to WP_257713275, AGE64789, ABI71643, WP_233079927, CVN26252; the amino acid sequence of L-amino acid deaminase includes but is not limited to AVK70349, WP_004248621; the amino acid sequence of D-amino acid oxidase includes but is not limited to ONH69498, XP_057061749, Q99042; the amino acid sequence of alanine racemase includes but is not limited to QRQ54233, QRQ52941; the amino acid sequence of catalase includes but is not limited to CAK38326, O28050, AMW80800.

[0013] As an improvement, the added amount of the glucose dehydrogenase is 1~10 U / mL; the added amount of the L-alanine dehydrogenase is 1~10 U / mL; the added amount of the L-amino acid deaminase is 1~10 U / mL; the added amount of the D-amino acid oxidase is 1~10 U / mL; the added amount of the alanine racemase is 1~10 U / mL; and the added amount of the catalase is 1~10 U / mL.

[0014] Beneficial effects: Compared to existing technologies, this invention provides a multi-enzyme cascade method for synthesizing nitrogen-containing heterocyclic compounds. By designing an efficient cofactor regeneration system and optimizing enzyme combinations and reaction conditions, it uses a multi-enzyme cascade to catalyze the synthesis of nitrogen-containing heterocycles from α,ω-diamines. This system constructs a multi-amine receptor and cofactor circulation system, reduces the reaction's dependence on high concentrations of amine receptors and cofactors, and broadens the biosynthetic pathway for nitrogen-containing heterocycles. Specific advantages are as follows: First, the use of cofactor regeneration enzymes such as L-alanine dehydrogenase significantly reduces the cost of NADPH and solves the problem of strong cofactor dependence; Secondly, by rationally designing the enzyme combination and reaction sequence, efficient conversion from diamines to nitrogen-containing heterocycles was achieved, improving the reaction efficiency and yield. Finally, the present invention utilizes readily available raw materials, mild reaction conditions, and an economical, environmentally friendly synthesis process. It eliminates the need for toxic reagents and produces few byproducts, meeting the requirements of green chemistry and sustainable development. This method addresses the challenges of harsh reaction conditions, poor selectivity, and severe environmental pollution encountered in existing technologies, providing a highly efficient, green, and sustainable new method for the synthesis of nitrogen-containing heterocyclic compounds, with significant scientific significance and industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The reaction flow chart of the synthesis of nitrogen-containing heterocycles from α,ω-diamines catalyzed by multi-enzyme cascade.

[0016] Figure 2 The reaction flow chart of the multi-enzyme cascade catalyzed synthesis of nitrogen-containing heterocycles from α,ω-diamines with the addition of an amine acceptor and cofactor co-circulation system.

[0017] Figure 3 Liquid chromatogram of azetidine synthesis.

[0018] Figure 4 This is a liquid chromatogram of the synthesis of pyrrolidine.

[0019] Figure 5 This is the liquid chromatogram of piperidine synthesis.

[0020] Figure 6 This is the liquid chromatogram of the synthesis of azepane.

[0021] Figure 7 This is the liquid chromatogram of the synthesis of azacyclooctane.

[0022] Figure 8 This is the liquid chromatogram of the synthesis of 3-azabicyclo[3.3.1]nonane. DETAILED DESCRIPTION

[0023] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The liquid phase assay method used in the examples of the present invention for piperidine and hydroxypiperidine involved derivatization of the samples with Fmoc-Cl or dansyl chloride and detection using a Shimadzu LC-20A HPLC (Shimadzu, Janpan). The chromatographic column used was an Agilent 5 HC-C18 column (4.6 mm × 250 mm; Agilent); the column temperature was 35°C; the detection wavelengths were 254 nm / 263 nm; and the mobile phase was CH3CN / H2O (65:35, v / v).

[0025] Unless otherwise specified, the enzymes used in the following examples are transaminase (AKK18527), imine reductase (WP_077989474), glucose dehydrogenase (AFQ56330), L-alanine dehydrogenase (WP_257713275), L-amino acid deaminase (AVK70349), D-amino acid oxidase (Q99042), alanine racemase (QRQ54233), and catalase (AMW80800).

[0026] Example 1 A 1 mL reaction system (5 U / mL transaminase, 2 U / mL imine reductase, 2 U / mL glucose dehydrogenase, 10 mM 1,4-butanediamine, 20 mM pyruvate, 20 mM glucose, 0.1 mM PLP, 1 mM NADP+, Tris-HCl buffer, pH 9) was incubated at 30 °C, 800 rpm for 48 h and then inactivated by boiling.

[0027] Liquid phase analysis revealed a pyrrolidine concentration of 4.68 mM and a yield of 46.8%. Adding the cofactor NADPH resulted in a higher yield of pyrrolidine.

[0028] Comparative Example 1 A 1 mL reaction system (5 U / mL transaminase, 2 U / mL imine reductase, 10 mM 1,4-butanediamine, 20 mM pyruvate, 0.1 mM PLP, 10 mM NADPH, pH 9 Tris-HCl buffer) was incubated at 30 °C, 800 rpm for 48 h and then inactivated by boiling.

[0029] Liquid phase analysis revealed that the pyrrolidine concentration in the reaction solution was 1.62 mM, and the yield was 16.2%.

[0030] Example 2 A 1 mL reaction system (5 U / mL transaminase, 2 U / mL imine reductase, 2 U / mL glucose dehydrogenase, 5 U / mL D-amino acid oxidase, 2 U / mL alanine racemase, 2 U / mL catalase, 10 mM 1,4-butanediamine, 1 mM pyruvate, 20 mM glucose, 0.1 mM PLP, 1 mM NADP+, Tris-HCl buffer, pH 9) was incubated at 800 rpm at 30 °C for 48 h and then inactivated by boiling.

[0031] Liquid phase analysis revealed that the pyrrolidine concentration in the reaction solution was 5.63 mM, and the yield was 56.3%.

[0032] Example 3 A 1 mL reaction system (5 U / mL transaminase, 2 U / mL imine reductase, 2 U / mL glucose dehydrogenase, 2 U / mL L-amino acid deaminase, 10 mM 1,4-butanediamine, 1 mM pyruvate, 20 mM glucose, 0.1 mM PLP, 1 mM NADP+, Tris-HCl buffer, pH 9) was incubated at 800 rpm at 30 °C for 48 h and then inactivated by boiling.

[0033] The pyrrolidine concentration in the reaction solution was measured by liquid chromatography-mass spectrometry to be 4.89 mM, and the yield was 48.9%.

[0034] Example 4 A 1 mL reaction system (5 U / mL transaminase, 2 U / mL imine reductase, 5 U / mL L-alanine dehydrogenase, 2 U / mL glucose dehydrogenase, 10 mM 1,4-butanediamine, 1 mM pyruvate, 20 mM glucose, 0.1 mM PLP, 15 mM NAD+, 1 mM NADP+, Tris-HCl buffer, pH 9) was incubated at 30 °C, 800 rpm for 48 h and then inactivated by boiling.

[0035] Liquid phase analysis revealed a pyrrolidine concentration of 6.34 mM and a yield of 63.4%. The addition of a dual amine donor and cofactor circulation system dominated by L-alanine dehydrogenase and glucose dehydrogenase increased the pyrrolidine yield compared to Examples 4 and 5.

[0036] Comparative Example 2 A 1 mL reaction system (5 U / mL transaminase, 2 U / mL imine reductase, 5 U / mL L-alanine dehydrogenase, 10 mM 1,4-butanediamine, 1 mM pyruvate, 0.1 mM PLP, 1 mM NADP+, pH 9 Tris-HCl buffer) was incubated at 30 °C, 800 rpm for 48 h and then inactivated by boiling.

[0037] Liquid chromatography revealed a pyrrolidine concentration of 0.52 mM in the reaction solution, yielding 5.2%. Testing the ability of L-alanine dehydrogenase to catalyze the conversion of alanine and NADP+ to pyruvate and NADPH revealed a significant decrease in pyrrolidine yield, suggesting that L-alanine dehydrogenase is unable to utilize NADP+, or has difficulty utilizing it.

[0038] Example 5 A 1 mL reaction system (5 U / mL transaminase, 2 U / mL imine reductase, 5 U / mL L-alanine dehydrogenase (WP_233079927) D198A mutant, 10 mM 1,4-butanediamine, 1 mM pyruvate, 0.1 mM PLP, 1 mM NADP+, pH 9 Tris-HCl buffer) was incubated at 30 °C, 800 rpm for 48 h and then inactivated by boiling.

[0039] Liquid phase analysis revealed an azetidine concentration of 9.63 mM in the reaction solution, yielding 96.3%. The modified L-alanine dehydrogenase (WP_233079927) mutant D198A catalyzes the conversion of alanine and NADP+ to pyruvate and NADPH. The amine donor and cofactor co-circulation system, dominated by the L-alanine dehydrogenase (WP_233079927) mutant D198A, exhibited the highest product yield among all the recycling systems and is therefore preferred.

[0040] Example 6 The reaction system was the same as Example 5 except that the substrate 1,4-butanediamine was replaced by 1,3-propylenediamine.

[0041] The concentration of azetidine in the reaction solution was determined to be 1.65 mM by liquid phase analysis, and the yield was 16.5%.

[0042] Example 7 The reaction system was the same as Example 5 except that the substrate 1,4-butanediamine was replaced by 1,5-pentanediamine.

[0043] The concentration of piperidine in the reaction solution was determined to be 9.98 mM by liquid chromatography, and the yield was 99.8%.

[0044] Example 8 The reaction system was the same as Example 5 except that the substrate 1,4-butanediamine was replaced by 1,6-hexanediamine.

[0045] Liquid phase analysis revealed that the concentration of azepane in the reaction solution was 9.66 mM, and the yield was 96.6%.

[0046] Example 9 The reaction system was the same as Example 5 except that the substrate 1,4-butanediamine was replaced by 1,7-heptanediamine.

[0047] Liquid phase analysis showed that the concentration of azocyclooctane in the reaction solution was 0.35 mM, and the yield was 3.5%.

[0048] Example 10 The reaction system was the same as Example 5 except that the substrate 1,4-butanediamine was replaced by cyclohexane-1,3-dimethylamine.

[0049] Liquid phase analysis showed that the concentration of 3-azabicyclo[3.3.1]nonane in the reaction solution was 3.62 mM, and the yield was 36.2%.

[0050] Example 11 Except that the substrate 1,4-butanediamine in the reaction system was replaced by cyclohexane-1,4-diamine, other conditions were the same as those in Example 5.

[0051] Liquid phase analysis revealed that the concentration of 7-azabicyclo[2.2.1]heptane in the reaction solution was 3.76 mM, and the yield was 37.6%.

[0052] Example 12 The reaction system was the same as Example 5 except that the substrate 1,4-butanediamine was replaced by cyclohexane-1,3-diamine.

[0053] Liquid phase analysis revealed that the concentration of 6-azabicyclo[3.1.1]heptane in the reaction solution was 2.87 mM, and the yield was 28.7%.

[0054] Example 13 The reaction system was the same as Example 5 except that the substrate 1,4-butanediamine was replaced by spermidine.

[0055] Liquid chromatography revealed that the concentration of 1-(3-aminopropyl)pyrrolidine in the reaction solution was 2.36 mM, with a yield of 23.6%.

[0056] In summary, the present invention provides a novel multi-enzyme cascade method for catalyzing the conversion of diamines to nitrogen-containing heterocyclic compounds. By constructing a diverse amine receptor and cofactor circulation system, the reaction requires fewer high concentrations of these compounds, thus avoiding the formation of significant byproducts. The ability of the multi-enzyme cascade system to synthesize different nitrogen-containing heterocyclic compounds from diamines of varying carbon chain lengths was also investigated. The results demonstrate that carbon chain lengths between 4 and 6 carbon chains are more likely to form the corresponding products, while carbon chain lengths of 3 or less or 7 or greater make it difficult to form the corresponding nitrogen-containing heterocyclic compounds.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for synthesizing nitrogen-containing heterocyclic compounds by multi-enzyme cascade, characterized in that: Using α,ω-diamine as a raw material, an amine receptor and an NADPH circulation system, and under the action of transaminase, imine reductase and PLP, the reaction is carried out at 10-60°C and pH 5.0-10.0 for 1-48 hours to obtain a nitrogen-containing heterocyclic compound, and the nitrogen-containing heterocyclic compound includes but is not limited to azetidine, pyrrolidine, piperidine, azepane, azacyclooctane and their derivatives; the amine receptor and the NADPH circulation system are composed of one or a combination of glucose dehydrogenase (GDH), L-alanine dehydrogenase (AlaDH), L-amino acid deaminase (LAAD), D-amino acid oxidase (DAAO), alanine racemase (ALR), catalase (CAT), a cosubstrate, pyruvate, and a cofactor.

2. The method for synthesizing nitrogen-containing heterocyclic compounds by multi-enzyme cascade according to claim 1, characterized in that: The α,ω-diamine includes but is not limited to linear α,ω-diamines such as 1,3-propylenediamine, 1,4-butylenediamine, 1,5-pentanediamine, 1,6-hexanediamine, and 1,7-heptanediamine, as well as cyclohexane-1,3-diamine, cyclohexane-1,4-diamine, cyclohexane-1,3-dimethylamine, or cyclohexane-1,4-dimethylamine.

3. The method for synthesizing nitrogen-containing heterocyclic compounds by multi-enzyme cascade according to claim 1, characterized in that: The amino acid sequence of the transaminase includes but is not limited to AKK18527, Q9I6J2, RBP85366, 4CE5_A, WP_011781668, WP_003255866, HBB1607555, KUN50029, WP_010975253, WP_011049154, and WP_011749320; the amino acid sequence of the imine reductase includes but is not limited to XP_045278909, XP_001827659, XP_748217, WP_071905000, OAQ82145, WP_020017817, 3ZGY_A, WP_015347361, and WP_077989474.

4. The method for synthesizing nitrogen-containing heterocyclic compounds by multi-enzyme cascade according to claim 1, characterized in that: The added amount of the transaminase is 2-10 U / mL, and the added amount of the imine reductase is 2-10 U / mL.

5. The method for synthesizing nitrogen-containing heterocyclic compounds by multi-enzyme cascade according to claim 1, characterized in that: The co-substrate includes but is not limited to one or both of glucose and pyruvate.

6. The method for synthesizing nitrogen-containing heterocyclic compounds by multi-enzyme cascade according to claim 1, characterized in that: Such cofactors include but are not limited to NAD + , NADH, NADP + , or NADPH.

7. The method for synthesizing nitrogen-containing heterocyclic compounds by multi-enzyme cascade according to claim 1, characterized in that: The amino acid sequence of glucose dehydrogenase includes but is not limited to 1GEE_A, AFQ56330; the amino acid sequence of L-alanine dehydrogenase includes but is not limited to WP_257713275, AGE64789, ABI71643, WP_233079927, CVN26252; the amino acid sequence of L-amino acid deaminase includes but is not limited to AVK70349, WP_004248621; the amino acid sequence of D-amino acid oxidase includes but is not limited to ONH69498, XP_057061749, Q99042; the amino acid sequence of alanine racemase includes but is not limited to QRQ54233, QRQ52941; the amino acid sequence of catalase includes but is not limited to CAK38326, O28050, AMW80800.

8. The method for synthesizing nitrogen-containing heterocyclic compounds by multi-enzyme cascade according to claim 1, characterized in that: The added amount of the glucose dehydrogenase is 1~10 U / mL; the added amount of the L-alanine dehydrogenase is 1~10 U / mL; the added amount of the L-amino acid deaminase is 1~10 U / mL; the added amount of the D-amino acid oxidase is 1~10 U / mL; the added amount of the alanine racemase is 1~10 U / mL; and the added amount of the catalase is 1~10 U / mL.

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