Method for preparing cinepazide by enzyme method

Abstract

The invention discloses a method for preparing cinepazide by using an enzyme method. The enzyme is an amido bond synthetase derived from Azospiracil lipoferum B.510 and / or a mutant of the amido bond synthetase. The invention further discloses a method for preparing the cinepazide by using the enzyme method, wherein the amido bond synthetase is an amido bond synthetase derived from Azospiracil lipoferum B.510. The amino acid sequence of the enzyme is shown as SEQ ID NO: 1; the mutant is a hydrophobic and small-volume amino acid (A) obtained by mutating P at the 328th site and / or V at the 329th site of an enzyme. According to the enzyme, (E)-3, 4, 5-trimethoxy cinnamic acid is used as an initial substrate, cinepazide is produced through one-step catalysis of a single enzyme, and the final conversion rate can reach 99% or above. Compared with a chemical synthesis method, the production cost can be greatly reduced, the process synthesis route is simplified, and the production efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the application of an amide bond synthase mutant in the preparation of guipizide. Background Technology

[0002] Cinepazide, chemically named (E)-1-(pyrrolidone-1-ylformylmethyl)-4-(3,4,5-trimethoxycinnamoyl)piperazine (CAS No.: 23887-46-9), is a multi-target vasodilator belonging to the piperazine derivative class. Clinically, it is mainly used to treat cardiovascular and cerebrovascular diseases and peripheral circulatory disorders. It plays a crucial role in inhibiting adenosine deaminase activity and adenosine reabsorption, as well as increasing glucose uptake in brain tissue, improving cellular nutrition and metabolism, increasing cerebral blood flow, and weakly blocking calcium ions. Cinepazide is the active ingredient of cinpazide maleate (trade name: Clinozide), and the maleate ion can improve the drug's water solubility and stability. Guipizide maleate was first developed by Sanofi-Aventis in France and launched there in 1974. It was subsequently launched in many other countries, including my country in 2002. Confirmatory clinical trials were completed in 2019, and in 2020, the National Medical Products Administration (NMPA) approved guipizide maleate for improving neurological symptoms, activities of daily living, and functional impairments caused by acute ischemic stroke. This drug can be used for patients with multiple coexisting conditions, relieving angina, cerebral ischemia, and limb numbness by preferentially improving microcirculation (such as coronary arteries, cerebral vessels, and peripheral vessels). It has few side effects, strong drug compatibility, and robust market demand.

[0003] The existing synthetic methods for guipizide are mainly chemical synthesis methods. The current optimal synthetic route (patent number: CN201910475471.0) is as follows: starting with (E)-3,4,5-trimethoxycinnamoyl chloride and diethanolamine, they react under alkaline conditions to generate the intermediate (E)-N,N-(2-hydroxyethyl)-3,4,5-trimethoxycinnamoamide. Then, the hydroxyl group is chlorinated under the action of sulfoxide to obtain the intermediate (E)-N,N-(2-chloroethyl)-3,4,5-trimethoxycinnamoamide, which then reacts with α-aminoacetylpyrrolidone to generate guipizide. This route requires the use of large amounts of toxic reagents (such as sulfoxide) and multiple separation and purification processes of the intermediates. From the perspective of green chemistry and industrial feasibility, this synthetic route still needs further improvement. Summary of the Invention

[0004] The purpose of this invention is to provide an enzymatic method for preparing cinnarizine, which mainly solves the following technical problems: (1) simplifying the synthesis process route; (2) reducing the use of toxic reagents; and (3) reducing process costs while maintaining a high product conversion rate.

[0005] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0006] The enzyme of the present invention is an amide bond synthase Azo (Uniprot:D3NZ98) derived from Azospirillum ipoferum B.510, whose amino acid sequence is shown in SEQ ID NO:1. Using this enzyme as a template, by mutating the substrate-binding pocket amino acid residues, a mutant with high catalytic activity for producing guipizide was finally obtained.

[0007] Preferably, the mutant is one in which the proline (P) at position 328 and / or the valine (V) at position 329 of the enzyme are both mutated into small side chains, hydrophobic amino acids.

[0008] Preferably, the mutant is one in which both P at position 328 and V at position 329 of the enzyme are mutated to A.

[0009] Specifically, in this invention, referring to the complete protein sequence of *Azospirillum lipogenes* Azo (Uniprot: D3NZ98), its amino acid sequence is shown in SEQ ID NO:1, and its nucleotide sequence is shown in SEQ ID NO:4. A 6×His tag and a thrombin protease cleavage site are introduced upstream of the gene encoding this protein sequence. Then, based on the parental sequence with the amino acid sequence of SEQ ID NO:1, amino acid position 328 of the parental sequence is mutated to alanine (A), resulting in the amino acid sequence of SEQ ID NO:2, and its nucleotide sequence is shown in SEQ ID NO:6. Amino acid position 329 of SEQ ID NO:2 is mutated to alanine (A), resulting in the amino acid sequence of SEQ ID NO:3, and its nucleotide sequence is shown in SEQ ID NO:6.

[0010] The present invention also provides a method for preparing guipizide, which can provide suitable enzymatic reaction conditions in vitro to obtain the target product.

[0011] A method for preparing cinnarizine by enzymatic means, wherein the enzyme is an amide bond synthase derived from Azospirillum lipoferum B.510 and / or its mutants.

[0012] Preferably, the enzyme is prepared by a one-step catalytic reaction using (E)-3,4,5-trimethoxycinnamic acid and 1-(2-piperazin-1-ylacetyl)pyrrolidine as substrates to obtain cinnamic acid-piperazine.

[0013] Preferably, the above reaction system further includes ATP and / or substances that can generate ATP, as well as Mg. 2+ and Tris buffer.

[0014] Preferably, the reaction system comprises: (E)-3,4,5-trimethoxycinnamic acid 0.5–4 mM; 1-(2-piperazin-1-ylacetyl)pyrrolidine 1–10 mM; ATP 1–10 mM; Mg 2+ 2–16 mM; Tris-HCl 30–200 mM; enzyme 5–55 μM.

[0015] Preferably, the reaction system comprises: (E)-3,4,5-trimethoxycinnamic acid 1±0.5mM; 1-(2-piperazin-1-ylacetyl)pyrrolidine 5±2mM; ATP 5±2mM; Mg 2+ 10±2mM; Tris-HCl 50~100mM; enzyme 25±10μM.

[0016] Preferably, the reaction conditions are 20–40°C, pH = 7.0–9.0, and the reaction is carried out with stirring in a water bath.

[0017] Preferably, the reaction conditions are 30±5℃, stirring speed is 100-500rpm, and reaction time is 24±6h.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The enzyme of the present invention can use (E)-3,4,5-trimethoxycinnamic acid as a substrate to catalyze the production of cinnamic acid, which can greatly reduce the production cost.

[0020] (2) This invention uses the protein “Azo” (Uniprot: D3NZ98) from Azospirillum lipoferum B.510 as a template for modification. By mutating the amino acid residues in its substrate binding pocket, a mutant with higher catalytic conversion activity is finally obtained. The conversion rate of guipizide can reach more than 99% in 24 hours, which is conducive to further reducing production costs.

[0021] (3) The method of the present invention is superior to the chemical synthesis preparation process, and shows outstanding advantages in terms of production cost, energy consumption, product quality and green index. Attached Figure Description

[0022] Figure 1SDS-PAG electrophoresis images of purified Azo and its mutants; M: Protein Marker; Lane 1: Azo wild-type purified enzyme; Lane 2: Azo-P328A mutant purified enzyme; Lane 3: Azo-P328A-V329A purified enzyme.

[0023] Figure 2 The UV peak diagrams are for cinnamic acid standard (a), (E)-3,4,5-trimethoxycinnamic acid standard (b), and actual reaction sample (c).

[0024] Figure 3 The conversion efficiency versus reaction time curves of Azo wild-type and its mutants (P328A, P328A-V329A) against guipizide. Detailed Implementation

[0025] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0026] The terms “comprising,” “including,” and “having” are used interchangeably in this document to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” in this document also provides for schemes “consisting of…”.

[0027] When used herein, the term “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0028] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids.

[0029] The term "nucleic acid" in this document includes any compound and / or substance comprising a polymer containing nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5′ to 3′. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers containing mixtures of two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derived sugar or phosphate backbones bonded or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as carriers for the direct expression of the enzymes of the present invention in vitro and / or in vivo, such as in a host or patient.

[0030] Enzyme-related information: The template protein for the amide bond synthase in this invention is the amide bond synthase Azo (Uniprot: D3NZ98) from *Azospirillum lipoferum* B.510, whose amino acid sequence is SEQ ID NO:1. Using this as a template, Azo-P328A and Azo-P328A-V329A were obtained, with their amino acid sequences being SEQ ID NO:2 and SEQ ID NO:3, respectively.

[0031] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. For process parameters not specifically specified, conventional techniques can be referred to. The test materials used in the present invention are all common commercially available products.

[0032] Example 1

[0033] Construction of wild-type Azo and mutant recombinant expression vectors and expression strains

[0034] Includes the following steps:

[0035] 1.1 The synthesized Azo gene was seamlessly cloned and ligated with the purified gene fragment and plasmid pET28a(+) using primers, and transformed into *E. coli*-Top10 competent cells. The clones were plated on LB agar plates (containing 100 μg / mL kanamycin). Positive clones were picked and subjected to colony PCR and gene sequencing. The pET28a-Azo recombinant plasmid of wild-type Azo was extracted using a plasmid extraction kit.

[0036] 1.2 Design the mutant primers, as shown in the table below.

[0037] Table 1

[0038]

[0039] Note: Underlined sequences represent mutation sites.

[0040] 1.3 Using the plasmid pET28a(+)-Azo containing Azo as a template, the plasmid containing the mutant gene was amplified by PCR using the primers in Table 1. The PCR amplification system is shown in Table 2 below, and the PCR amplification program is shown in Table 3. The mutant described in this invention was constructed using the overlap PCR method.

[0041] Table 2 PCR amplification system

[0042]

[0043] Table 3 PCR amplification program

[0044]

[0045] After the PCR amplification products were confirmed by 1% agarose gel electrophoresis, the PCR products were digested and purified using the restriction endonuclease DpnI to remove the original template contained in the system. The digestion system is shown in Table 4 below.

[0046] Table 4 Digestion reaction system

[0047]

[0048] The above system was placed at a constant temperature of 37℃ for 30-60 minutes.

[0049] 1.4 The enzyme digestion products were purified by PCR, and the purified products were confirmed by nucleic acid electrophoresis.

[0050] 1.5 The purified product was mixed with TOP10 competent E. coli cells, placed on ice for 30 min, heat-shocked at 42 °C for 90 s, and then quickly placed on ice for 2 min.

[0051] 1.6 Add the competent cells obtained in the previous step to 1 ml of LB medium and activate them at 37°C and 200 rpm for 45 min. After activation, centrifuge the competent cells at 8000 rpm for 2 min, remove most of the medium, resuspend the cells, and spread the resulting transformation solution evenly on LB plates containing 100 μg / mL kanamycin. Incubate overnight at 37°C. Pick a single colony from the plate and inoculate it into 5 ml of LB liquid medium (containing 100 μg / mL Kanamycin). Incubate overnight at 37°C and 200 rpm. After confirming the amplification results by gene sequencing, extract the plasmid for later use.

[0052] Example 2

[0053] Fermentation and purification of recombinant proteins from wild-type Azo and its mutant recombinant expression strains

[0054] 2.1 Expression of mutants

[0055] The plasmid of the Azo mutant obtained in Example 1 was transformed into Escherichia coli competent cells BL21(DE3). The transformation solution was plated on LB plates containing 100 μg / mL kanamycin and incubated overnight at 37°C. The single colony that grew was the expressed strain.

[0056] Single colonies of the Azo mutant were picked and inoculated into 5 ml of LB medium and incubated overnight at 37°C and 220 rpm until OD500 was reached. 600 After approximately 0.8-0.9%, inoculate at a rate of 2% (v / v) into 500 mL Erlenmeyer flasks containing 100 mL of kanamycin-resistant LB liquid medium. Incubate at 37°C and 220 rpm on a constant temperature shaker until OD reaches zero. 600 After reaching approximately 0.8-0.9, the culture was scaled up to 500 ml of self-induction medium (1% (w / v) peptone, 0.5% (w / v) yeast extract, 0.05% (w / v) glucose, 0.2% (w / v) lactose, 0.5% (w / v) glycerol, 25 mM Na₂HPO₄, 25 mM KH₂PO₄, 50 mM NH₄Cl, 5 mM Na₂SO₄, 2 mM MgSO₄) at a 5% (v / v) ratio for further expansion. The OD was then calculated. 600 Once the culture temperature reaches approximately 1.0, the culture temperature is lowered from 37℃ to 16℃, and the rotation speed is adjusted to 200 rpm for induction, which lasts for 20 hours. After induction, the bacteria are harvested. The fermentation broth is centrifuged at 4℃ and 8000 rpm for 20 minutes, then the culture medium is discarded, and the bacterial cells are collected. The centrifuged bacterial cells are washed twice with pre-cooled Tris-HCl buffer (pH 9.0) to remove residual culture medium components.

[0057] 2.2 Purification of mutants

[0058] The bacterial cells obtained above were resuspended in Tris-HCl buffer (pH 9.0) and the cells were sonicated to disrupt the cell disruption. The cell disruption solution was centrifuged (10000 r / min, 30 min), and the supernatant was collected. This supernatant is the prepared amide bond synthase Azo or mutant crude enzyme solution.

[0059] The bacterial cells were resuspended in Tris-HCl buffer (pH 9.0) at a ratio of 1:10 (m / v) and sonicated on ice for 20 min. The disruption was complete when the bacterial suspension was clear and homogeneous. The disruption solution was centrifuged at 11,000 rpm for 30 min at 4°C. The supernatant crude enzyme solution was collected and filtered through a 0.45 μm aqueous microporous membrane to remove insoluble impurities and small bacterial fragments. The amide bond synthase was purified using an AKTA Purifier protein purification system with His Prep affinity chromatography columns. TM FF16 / 10 and HiPrep salt-changing chromatography column TM On October 26th, all solutions entering the AKTA protein purifier were filtered through a 0.45 μm aqueous microporous membrane. The specific operating steps are as follows: First, the alcohol-protected liquid in the protein purifier was drained with deionized water. Then, the HisPrep affinity chromatography column was equilibrated with loading buffer at a flow rate of 5 mL / min. TM FF16 / 10, to OD 280 After the conductivity reaches equilibrium, the crude enzyme solution is pumped into the protein purifier at a flow rate of 3 mL / min. Once the binding sites on the Ni column are fully occupied by the target protein, the affinity chromatography column is equilibrated with Tris-HCl buffer (pH 9.0) containing 50 mM imidazole at a flow rate of 5 mL / min to remove unbound contaminating proteins, until the OD value reaches equilibrium. 280 The electrolyte and conductivity were re-equilibrated; then the target protein was eluted with Tris-HCl buffer (pH 9.0) containing 300 mM imidazole at a rate of 3 mL / min, and collected in centrifuge tubes. The collected target protein was then subjected to a salt-change chromatography column, HiPrep. TM On October 26th, for salt replacement, the system was first equilibrated with salt replacement buffer at a flow rate of 5 mL / min. Then, the collected target protein was pumped into the protein purifier at an appropriate volume, and the salt replacement operation was repeated with salt replacement buffer until completion. The final target protease solution was concentrated to an appropriate concentration using a 10 kDa ultrafiltration tube, aliquoted, pre-frozen in liquid nitrogen, and stored at -80°C. The target protein bands are shown in the appendix. Figure 1 , 2 .

[0060] 2.3 Detection of mutant protein concentration

[0061] The concentration of mutant protein was determined using the Brandford Protein Assay Kit. A series of bovine serum albumin (BSA) concentrations were prepared as protein standard solutions to construct a standard curve. 10 μl of standard protein at different concentrations was added to each well of a 96-well plate, followed by 200 μl of Brandford reagent. After reacting at room temperature for 5 min, the absorbance was measured at 595 nm. The protein concentration standard curve was obtained based on the known protein concentrations and their corresponding absorbance values. The mutant protein was measured at 595 nm under the same reaction conditions. The experiment was repeated in triplicate, and the mutant protein concentration was determined by comparing the results with the protein concentration standard curve.

[0062] Example 3

[0063] Guipizide was prepared by enzyme catalysis using Azo wild-type and mutants (Azo-P328A, Azo-P328A-V329A).

[0064] The reaction system for the catalytic synthesis of guipizide is shown in Table 5 below. The solutions in this table were added to 2 mL centrifuge tubes and then placed in a constant-temperature metal bath. The reaction was carried out at 30°C and 500 rpm for 24 h. After the reaction was complete, an equal volume of acetonitrile was added to the reaction system as a co-solvent, and the mixture was vortexed for 1-2 min, followed by heating at 60°C for 5 min. The enzyme protein was then removed by centrifugation at 15,000 rpm for 5 min. The supernatant organic phase was filtered through a syringe filter and then transferred to a chromatographic vial for subsequent HPLC analysis. The blank control group used the corresponding buffer solution instead of the enzyme solution, with all other conditions remaining the same. Each experiment was conducted in triplicate.

[0065] Table 5. Catalytic synthesis reaction system of Azo and its mutants for guipizide.

[0066]

[0067] After 24 hours, HPLC analysis showed that the conversion rates for Azo-WT were 10.98%, Azo-P328A were 73.01%, and Azo-P328A-V329A were >99%. (See attached data.) Figure 3 .

[0068] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing cinnarizine using an enzymatic process, characterized in that, The enzyme is an amide bond synthase derived from Azospirillum lipoferum B.510 and / or its mutants.

2. The method according to claim 1, characterized in that, The amino acid sequence of the enzyme is shown in SEQ ID NO:1; the mutant is one in which the P at position 328 and / or the V at position 329 of the enzyme are both mutated to hydrophobic and small-volume amino acids.

3. The method according to claim 2, characterized in that, The mutant is one in which both P at position 328 and / or V at position 329 of the enzyme are mutated to A.

4. The method according to claim 1, 2, or 3, characterized in that, The enzyme is used as a substrate of (E)-3,4,5-trimethoxycinnamic acid and 1-(2-piperazin-1-ylacetyl)pyrrolidine to obtain cinnamic acid via a one-step catalytic reaction.

5. The method according to claim 4, characterized in that, The reaction system also includes ATP and / or substances that can generate ATP, as well as Mg. 2+ and Tris buffer.

6. The method according to claim 5, characterized in that, The reaction system includes: (E)-3,4,5-trimethoxycinnamic acid 0.5–4 mM; 1-(2-piperazin-1-ylacetyl)pyrrolidine 1–10 mM; ATP 1–10 mM; Mg 2+ 2–16 mM; Tris-HCl 30–200 mM; enzyme 5–55 μM.

7. The method according to claim 6, characterized in that, The reaction system includes: (E)-3,4,5-trimethoxycinnamic acid 1±0.5mM; 1-(2-piperazin-1-ylacetyl)pyrrolidine 5±2mM; ATP 5±2mM; Mg 2+ 10±2mM; Tris-HCl 50~100mM; enzyme 25±10μM.

8. The method according to claim 5, characterized in that, The reaction conditions are 20–40℃, pH = 7.0–9.0, and the reaction is carried out with stirring in a water bath.

9. The method according to claim 5, characterized in that, The reaction conditions were 30±5℃, stirring speed of 100-500rpm, and reaction time of 24±6h.

10. The method according to claim 6, characterized in that, The reaction conditions were 30±5℃, pH=7.0-9.0, and the reaction was carried out by stirring at 100-500rpm for 24±6h.

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