Method for preparing 2-amino-5-halogen benzamide based on enzyme catalysis
By catalyzing 2-amino-5-halogen benzamide in water, halogenase, coenzyme and coenzyme circulation systems, safety hazards and environmental pollution problems of chemical preparation are solved, and an efficient, safe and environmentally friendly preparation process is achieved. , suitable for large-scale production.
Patent Information
- Application Number
- CN202411313152.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-20
- Publication Date
- 2025-01-03
AI Technical Summary
The existing chemical method for preparing 2-amino-5-halogen benzamide has safety hazards, is complex in operation, is environmentally contaminated, is costly and is not suitable for large-scale production, and lacks catalytic preparation technology for biological enzyme method.
Using water, halogenase, coenzyme and coenzyme circulation systems, the enzyme catalytic reaction of 2-amino-benzamide and halogen salts is generated. The halogenase and coenzyme have high selectivity and reaction The conditions are mild, the operation is simple, and suitable for industrial production.
It improves the selectivity and conversion rate of halogen subsidies, reduces reaction costs, simplifies steps, improves safety and environmental protection, is suitable for industrial production, and has excellent industrial application prospects.
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Figure CN119242726A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medical technology, and particularly relates to a method for preparing 2-amino-5-halogen benzamide based on enzyme catalysis. Background Art
[0002] 2-Amino-5-chlorobenzamide, English name 2-Amino-5-chlorobenzamide, white crystals, melting point of 171-172℃, CAS 5202-85-7, molecular weight 170.6, widely used in the synthesis of chemical production such as medicine and fuel intermediates, is the basic raw material for the synthesis of quinoline compounds. 2-Amino-5-chlorobenzamide has good biological activity and is the starting material for some drugs with good therapeutic effects on human diseases. For example, 2,3-dihydroxyquinolinone compounds synthesized with 2-amino-5-chlorobenzamide as the starting material are a good class of antibiotics with analgesic and anti-vasodilatory effects; in addition, 2,3-quinazoline-4(1H)-one compounds also have good biological and pharmacological activities. 2-Amino-5-chlorobenzamide not only has good activity on humans, but also has good herbicidal and plant regulating effects on plants.
[0003] 2-Amino-5-bromobenzamide, English name: 2-Amino-5-bromobenzamide, white crystalline powder, has good solubility and stability, CAS number 16313-66-9, molecular weight 215.05. 2-Amino-5-bromobenzamide has certain applications in the field of medicine. It can be used as an intermediate for antibacterial drugs and plays an important role in the preparation of certain antibacterial drugs. In addition, it can also be used to treat diseases such as skin infections and respiratory infections. In addition, 2-amino-5-bromobenzamide can also be used in the field of cosmetics. It has antibacterial and antiseptic effects and is often added to skin care products, cosmetics and other products to extend their service life.
[0004] At present, the synthesis of 2-amino-5-halogen benzamide is mainly a chemical method. Taking 2-amino-5-chlorobenzamide as an example, Mahajan et al. adopt SO2, Cl2, SCl2, NH3 and other reagents to chemically synthesize 2-amino-5-chlorobenzamide. However, chlorination, acylation and ammonia solution reagents are all highly irritating to the human body, and severe patients can cause pulmonary edema and skin ulceration and burns, and even cause irreversible damage to the respiratory tract and eyes. Chinese patent application CN101575301B reports a two-step chemical method for synthesizing 2-amino-5-chlorobenzamide, first with anthranilate as starting material, sodium hypochlorite solution and glacial acetic acid as chlorination reagent, organic solvent and water as solvent, mixed at low temperatures for chlorination to obtain 2-amino-5-chlorobenzoic acid methyl ester; then add ammonia, react in a reactor heated and pressurized, finally carry out solvent, stir, and operations such as suction filtration obtain the final product. However, the chemical method for preparing 2-amino-5-halogenbenzamide is expensive, inconvenient to use, causes great damage to equipment, requires high qualifications for operators, and pollutes the environment. The entire production cost is high, the reaction is dangerous, and the steps are complicated, making it unsuitable for large-scale production in factories.
[0005] Halogenases are a class of flavin-dependent halogenases that have high substrate and regiospecificity and can partially halogenate indole units, including halogenation with 2-amino-benzamide as a substrate. However, there are few studies and reports on the preparation of 2-amino-5-halogenobenzamide using bioenzymatic methods.
[0006] Therefore, there is an urgent need in the art to develop a method for preparing 2-amino-5-halogenbenzamide based on enzyme catalysis, which has high safety, simple operation, good environmental protection, and can reduce costs while also having good synthesis efficiency and product yield. Summary of the invention
[0007] To solve the above technical problems, the present invention provides a method for preparing 2-amino-5-halogenobenzamide based on enzyme catalysis, comprising the following steps:
[0008] Step 1: using 2-amino-benzamide and halogen salt as substrates, reacting under the action of water, halogenase, coenzyme and coenzyme circulation system to generate a compound of formula I; wherein X is selected from F, Cl, Br or I;
[0009]
[0010] Step 2: isolating the compound of formula I from the reaction system after the reaction in step 1;
[0011] Wherein, the nucleotide sequence of the halogenase is as shown in SEQ ID NO:1 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto, and the amino acid sequence of the halogenase is as shown in SEQ ID NO:2 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto.
[0012] In a specific embodiment, in step 1, the halogen salt is one of sodium chloride, potassium chloride, sodium bromide or potassium bromide.
[0013] The "coenzyme" is a class of small organic molecules that can transfer chemical groups from one enzyme to another, are loosely bound to the enzyme, and are necessary for the activity of a specific enzyme.
[0014] In a specific embodiment, in step 1, the coenzymes are NAD and FAD.
[0015] In a specific embodiment, in step 1, the coenzymes are NADP and FAD.
[0016] The coenzyme circulation system is a chemical reaction system that occurs during the action of the coenzyme.
[0017] In a specific embodiment, in step 1, the coenzyme cycle system is a GDH-glucose coenzyme cycle system or an isopropanol coenzyme cycle system. The GDH-glucose coenzyme cycle system can transfer the H atoms required for the reaction substrate ketone to the product alcohol to glucose as the supply, which is dependent on the transfer of NAD (P), wherein GDH is a glucose dehydrogenase. The isopropanol coenzyme cycle system can transfer the H atoms required for the reaction substrate ketone to the product alcohol to isopropanol as the supply, which is dependent on the transfer of NAD (P).
[0018] In a specific embodiment, in step 1, the coenzyme circulation system includes flavin reductase, the nucleotide sequence of the flavin reductase is shown in SEQ ID NO:3 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto, and the amino acid sequence of the flavin reductase is shown in SEQ ID NO:4 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto. The flavin reductase (FRs enzyme) can reduce flavin adenine dinucleotide (FAD) to reduced flavin adenine dinucleotide (FADH2), which is then oxidized by molecular oxygen to generate a peroxyflavin intermediate, which then undergoes a series of reactions with halide ions to promote the halogenation reaction.
[0019] In a specific embodiment, in step 1, the reaction conditions are: pH value 6.0-8.0, reaction time 12-24h, reaction temperature 20-30°C.
[0020] In a specific embodiment, in step 1, in the reaction system, the concentration of 2-amino-benzamide is 0.1-20 mg / mL, the concentration of halogen salt is 1-100 mg / mL, the concentration of coenzyme is 0.01-1 mg / mL, the concentration of FAD is 0.01-10 mg / mL, and the concentration of halogenase is 0.1-10 mg / mL.
[0021] In a specific embodiment, in the GDH-glucose coenzyme circulation system, the concentration of glucose is 0.1-100 mg / mL.
[0022] In a specific embodiment, the concentration of the flavin reductase is 0.1-10 mg / mL.
[0023] In a specific embodiment, the halogenase is added in the form of crude halogenase powder, crude halogenase liquid or wet bacteria expressing halogenase.
[0024] The main advantages of the present invention include:
[0025] 1) The method for preparing 2-amino-5-halogenobenzamide based on enzyme catalysis disclosed in the present application is to carry out the reaction under the action of water, halogenase, coenzyme and coenzyme circulation system, the selectivity of the halogenation site is high, and the conversion rate of the substrate 2-amino-benzamide and the halogen salt is improved.
[0026] 2) The method for preparing 2-amino-5-halogenobenzamide based on enzyme catalysis disclosed in the present application uses 2-amino-benzamide and halogen salt as reaction substrates, reduces the reaction cost, and under the reaction conditions provided in the present application, has a short reaction step, mild reaction conditions and simple operation, high safety, low pollution to the environment, and is easy to industrialize. Therefore, the method has excellent industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 The schematic diagram of the synthetic route of the method for preparing 2-amino-5-halogenbenzamide based on enzyme catalysis disclosed in the present application;
[0028] Figure 2 This is the HPLC spectrum of the synthetic product 2-amino-5-chlorobenzamide in Example 2 of the present application;
[0029] Figure 3 This is the HPLC spectrum of the synthetic product 2-amino-5-bromobenzamide in Example 3 of the present application. DETAILED DESCRIPTION
[0030] The invention discloses a halogenase (Halogenase, Hal) derived from Streptomyces rugosporus and flavin reductases (Flavinreductases, FRs) derived from Escherichia coli, and provides a method for preparing 2-amino-5-halogenobenzamide by a halogenase method.
[0031] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Unless otherwise specified, the raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.
[0032] The present invention will be further described below in conjunction with embodiments:
[0033] Example 1
[0034] 1) Construction and transformation of recombinant vector
[0035] Taking the construction of recombinant strain E. coli BL21 (DE3) pET-28a-Hal as an example
[0036] Activate E. coli DH5α / pET-28a(+) strain (purchased from Novizan Biotechnology Co., Ltd.), add 3-5 μL pET-28a-Hal (50 ng / μL) to 50-100 μL E. coli BL21(DE3) competent cells (purchased from Novizan Biotechnology Co., Ltd.), place on ice for 20 min, heat shock at 42°C for 90 sec, quickly return to ice for 5 min, add 800 μL of resistance-free LB culture solution, incubate at 37°C, 200 rpm, culture for 1 h, then spread on LB agar plate medium containing kana resistance, and culture overnight at 37°C for 12 h to obtain recombinant E. coli BL21(DE3) containing pET-28a-Hal.
[0037] The nucleotide sequence of halogenase (Hal) is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.
[0038] The formula of the non-resistant LB culture medium is: 5g / L yeast powder, 10g / L sodium chloride and 10g / L peptone;
[0039] The formula of LB agar plate medium containing kana resistance is: yeast powder 5g / L, sodium chloride 10g / L, peptone 10g / L, agar powder 20g / L and kana 25μg / mL.
[0040] 2) Construction of expression vector and induced expression
[0041] The recombinant E. coli BL21 (DE3) containing pET-28a-Hal obtained in 1) was directly spread on a solid LB plate containing 25 μg / mL kanamycin resistance, and cultured at 37°C for 12-14 hours to obtain a monoclonal colony. A single colony of E. coli BL21 (DE3) containing the Hal recombinant vector was picked from the kana plate, inoculated into 1 mL of liquid LB medium containing 25 μg / mL kanamycin resistance, and cultured at 37°C for 12 hours with shaking. Then, the cells were inoculated at a 2% (v / v) inoculation rate into 1 L of fresh liquid LB medium containing 25 μg / mL kanamycin resistance, cultured at 37°C until OD600 was approximately 0.6-0.8, IPTG (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to a final concentration of 1.0 mmol / L, 200 rpm, 25°C to induce expression for 20 h, and then centrifuged (4°C, 4000 rpm, 30 min) to remove the supernatant, and the obtained bacterial sludge was washed with 30 ml of 0.9% NaCl solution and resuspended for standby use.
[0042] 3) Preparation of halogenated enzyme freeze-dried enzyme powder
[0043] The bacterial solution obtained in 2) was washed twice with 50 mmol / L Tris-HCl, and then resuspended in 50 mmol / L Tris-HCl (pH 8.0) buffer, and the cells were ultrasonically disrupted in an ice bath (amplifier 6, power 500 W, on 2 s, off 5 s, 30 minutes). The ultrasonically disrupted sample was centrifuged at 12000 rpm and 4°C for 30 min, and the supernatant was placed in a -80°C freeze dryer for lyophilization for 24 h, and the obtained lyophilized sample was ground to prepare lyophilized enzyme powder.
[0044] 4) Preparation of freeze-dried enzyme powder in the coenzyme circulation system
[0045] In this embodiment, the coenzyme cycle system is a GDH-glucose coenzyme cycle system, wherein the preparation method of flavin reductase (FRs) freeze-dried enzyme powder is as shown in 1) to 3), the nucleotide sequence of flavin reductase (FRs) is as shown in SEQ ID NO: 3, and the amino acid sequence is as shown in SEQ ID NO: 4.
[0046] When preparing 2-amino-5-halogenobenzamide by the method provided in the present application, the freeze-dried enzyme powder prepared in this example is used for catalysis.
[0047] Example 2 Preparation of 2-amino-5-chlorobenzamide
[0048] Please refer to Figure 1 The synthetic route shown in the figure is as follows: the freeze-dried enzyme powder prepared in Example 1 is taken, and 2-amino-benzamide, NaCl, NAD, FAD, GDH, glucose and buffer solution are added respectively. The corresponding reaction system is shown in Table 1.
[0049] Table 1
[0050]
[0051] The reaction solution was placed at 25° C. and 1000 rpm. After reacting for 20 h, 100 μL of the reaction solution was taken out and inactivated by adding 100 μL of acetonitrile solution. The reaction solution was then centrifuged and filtered. The product 2-amino-5-chlorobenzamide was determined by HPLC to obtain the reaction conversion rate.
[0052] The high performance liquid chromatograph (HPLC) was purchased from Agilent Technologies, and the HPLC detection conditions were as follows: the mobile phase was 10 mM NH4OAc, the mobile phase A was 5% acetonitrile, the mobile phase B was 95% acetonitrile, the temperature was 35°C, the flow rate was 1.5 mL / min, and the chromatographic column was Xbridge C18 (purchased from Waters, USA). Figure 2 The results showed that the reaction conversion rate was 92.1% after 20 h.
[0053] Example 3: Preparation of 2-amino-5-bromobenzamide
[0054] The freeze-dried enzyme powder prepared in Example 1 was taken, and 2-amino-benzamide, NaBr, NAD, FAD, GDH, glucose and buffer solution were added respectively. The corresponding reaction system is shown in Table 2.
[0055] Table 2
[0056]
[0057] The reaction solution was placed at 25° C. and 1000 rpm. After reacting for 20 h, 100 μL of the reaction solution was taken out and inactivated by adding 100 μL of acetonitrile solution. The reaction solution was then centrifuged and filtered. The product 2-amino-5-chlorobenzamide was measured by HPLC to obtain the reaction conversion rate.
[0058] The high performance liquid chromatograph (HPLC) was purchased from Agilent Technologies, and the HPLC detection conditions were as follows: the mobile phase was 10 mM NH4OAc, the mobile phase A was 5% acetonitrile, the mobile phase B was 95% acetonitrile, the temperature was 35°C, the flow rate was 1.5 mL / min, and the chromatographic column was Xbridge C18 (purchased from Waters, USA). Figure 3 The results showed that the reaction conversion rate was 75.3% after 20 h.
[0059] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for preparing 2-amino-5-halogen benzamide based on enzyme catalysis, characterized in that: The steps include: Step 1: using 2-amino-benzamide and halogen salt as substrates, reacting under the action of water, halogenase, coenzyme and coenzyme circulation system to generate a compound of formula I; wherein X is selected from F, Cl, Br or I; Step 2: isolating the compound of formula I from the reaction system after the reaction in step 1; Wherein, the nucleotide sequence of the halogenase is as shown in SEQ ID NO:1 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto, and the amino acid sequence of the halogenase is as shown in SEQ ID NO:2 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto.
2. The method for preparing 2-amino-5-halogen benzamide based on enzyme catalysis according to claim 1, characterized in that: In the step 1, the halogen salt is one of sodium chloride, potassium chloride, sodium bromide or potassium bromide.
3. The method for preparing 2-amino-5-halogen benzamide based on enzyme catalysis according to claim 1, characterized in that: In the step 1, the coenzymes are NAD and FAD.
4. The method for preparing 2-amino-5-halogen benzamide based on enzyme catalysis according to claim 1, characterized in that: In the step 1, the coenzymes are NADP and FAD.
5. The method for preparing 2-amino-5-halogenbenzamide based on enzyme catalysis according to claim 1, characterized in that: In the step 1, the coenzyme circulation system is a GDH-glucose coenzyme circulation system or an isopropanol coenzyme circulation system.
6. The method for preparing 2-amino-5-halogen benzamide based on enzyme catalysis according to claim 1, characterized in that: In the step 1, the coenzyme circulation system includes flavin reductase, the nucleotide sequence of the flavin reductase is as shown in SEQ ID NO:3 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto, and the amino acid sequence of the flavin reductase is as shown in SEQ ID NO:4 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto.
7. The method for preparing 2-amino-5-halogen benzamide based on enzyme catalysis according to claim 1, characterized in that: In the step 1, the reaction conditions are: pH value 6.0-8.0, reaction time 12-24h, reaction temperature 20-30°C.
8. The method for preparing 2-amino-5-halogen benzamide based on enzyme catalysis according to claim 1, characterized in that: In the step 1, in the reaction system, the concentration of 2-amino-benzamide is 0.1-20 mg / mL, the concentration of halogen salt is 1-100 mg / mL, the concentration of coenzyme is 0.01-1 mg / mL, the concentration of FAD is 0.01-10 mg / mL, and the concentration of halogenase is 0.1-10 mg / mL.
9. The method for preparing 2-amino-5-halogen benzamide based on enzyme catalysis according to claim 5, characterized in that: In the GDH-glucose coenzyme circulation system, the concentration of glucose is 0.1-100 mg / mL.
10. The method for preparing 2-amino-5-halogen benzamide based on enzyme catalysis according to claim 6, characterized in that: The concentration of the flavin reductase is 0.1-10 mg / mL.