Method for preparing 2 '-methoxy-uridine triphosphate

Abstract

The invention discloses a method for preparing 2 '-methoxy-uridine triphosphate, which comprises the following steps: in a liquid reaction system, constructing a phosphoric acid circulating system by taking 2'-methoxy-uridine monophosphate as a substrate and adenosine triphosphate as an initial phosphoric acid donor, adding an enzyme catalyst, and carrying out an enzyme catalysis reaction by adopting a one-pot enzyme method to obtain the 2 '-methoxy-uridine triphosphate. The phosphoric acid circulating system is composed of adenosine triphosphate, adenosine diphosphate and a phosphate compound, inorganic phosphoric acid is provided for adenosine diphosphate by catalyzing dephosphorizing of the phosphate compound through the enzyme catalyst, and adenosine triphosphate is regenerated to achieve circulating energy supply. According to the method, a high-activity enzyme combination with a specific source is adopted, the conversion rate of the substrate 2 '-methoxyl-uridine monophosphate is high, the final conversion rate of 2'-methoxyl-uridine triphosphate reaches up to 92.6%, and the method has the advantages of being high in synthesis efficiency, simple in step, environmentally friendly and controllable in cost.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a method for preparing 2'-methoxy-uridine triphosphate. BACKGROUND

[0002] Nucleotides, as the basic structural units of DNA and RNA, have multiple key functions in life activities. They are not only carriers of genetic information, but also participate in energy metabolism, cell signal transduction in the form of nucleoside triphosphates (ATP, GTP, CTP, UTP), and play a role as enzyme reaction cofactors.

[0003] RNA interference technology is widely used in the treatment of tumors, infectious diseases and metabolic diseases, but it faces problems such as easy degradation of oligonucleotides by nucleases, poor selectivity and stability of binding to target RNA, easy triggering of immune response, and transport barrier. Chemically modified nucleotides can effectively solve some of the above defects. Among them, ribose 2'-hydroxyl modification is a research hotspot. 2'-methyl modification can enhance the binding of drugs to target mRNA, inhibit nuclease hydrolysis, reduce immunogenicity, and improve lipid solubility. 2'-methoxy (2'OMe) modified nucleotides, as their analogs, have better affinity and anti-enzymatic ability for target mRNA.

[0004] Approved nucleic acid drugs such as silencing RNA drugs (siRNA drugs) Patisiran and Givosiran, ASO drugs Nusinersen, Inotersen and Volanesorsen, and aptamer drugs Peg-aptanib have verified the pharmacological value and clinical utility of 2'-methoxy (2'OMe) modification. 2'-methoxy modified nucleotides have thus become a key intermediate in drug research and development.

[0005] 2'-methoxy-uridine triphosphate (2'-OME-UTP), as a UTP analog, has stronger anti-hydrolysis and anti-nuclease cutting stability due to the methylation of ribose 2, and is an important raw material for aptamer synthesis. However, this compound is unstable in nature, and the market price is as high as 50 μL (100 mM) / 100,000 pounds (CAS: 143028-99-3). Currently, there is no efficient and targeted synthesis method. In addition, the existing nucleotide production methods also have various defects, such as: chemical synthesis method has long route, poor stereoselectivity, expensive and toxic reagents, and high production cost; the product of RNA enzymatic method is a mixture of 4 nucleotides, which is difficult to extract and has low purity; microbial fermentation method is limited by cell permeability; traditional biological catalysis method solves the problem of cell permeability, but has low efficiency in the synthesis of complex modified nucleotides.

[0006] Therefore, there is an urgent need in the art to develop a simple, easy-to-operate, economical and environmentally friendly method for preparing 2'-methoxy-uridine triphosphate, which is single and efficient, so as to adapt to the large-scale production of 2'-methoxy-uridine triphosphate and help the research and development and industrialization of nucleic acid drugs. SUMMARY

[0007] To solve the above technical problems, the present application provides a method for preparing 2'-methoxy-uridine triphosphate, which uses 2'-methoxy-uridine acid as a substrate, adenosine triphosphate as an initial phosphate donor, constructs a phosphate cycle system, adds an enzyme catalyst, and performs enzyme catalytic reaction by one-pot enzyme method to obtain the end product 2'-methoxy-uridine triphosphate, wherein: The enzyme catalyst comprises uridine acid kinase and acetate kinase, the uridine acid kinase is derived from Arabidopsis thaliana, Thermotoga maritima Genbank No. NP_850867.1, the acetate kinase is derived from Aerococcus vivridans , Genbank No. AKE29949.1; The phosphate cycle system is composed of adenosine triphosphate, adenosine diphosphate and phosphate compound, and the enzyme catalyst catalyzes the dephosphorylation of the phosphate compound to provide inorganic phosphate to adenosine diphosphate, and regenerates adenosine triphosphate to realize the cycle energy supply.

[0008] Specifically, the phosphate compound is acetyl phosphate.

[0009] Specifically, the enzyme catalyst further comprises pyruvate oxidase, the pyruvate oxidase is derived from Erysipelotrichaceae bacterium , Genbank No. WP_026465373.1, and the phosphate compound is sodium pyruvate.

[0010] Specifically, the enzyme catalyst further comprises polyphosphate kinase, the polyphosphate kinase is derived from Arabidopsis thaliana, , Genbank No. UKS89443.1, and the phosphate compound is sodium hexametaphosphate.

[0011] Specifically, the concentration of 2'-methoxy-uridine acid is 10-80 mmol / L; preferably, the concentration of 2'-methoxy-uridine acid is 12-60 mmol / L; more preferably, the concentration of 2'-methoxy-uridine acid is 14-40 mmol / L; and most preferably, the concentration of 2'-methoxy-uridine acid is 15 mmol / L.

[0012] Specifically, the concentration of the adenosine triphosphate is 1-40 mmol / L; preferably, the concentration of the adenosine triphosphate is 2-20 mmol / L; more preferably, the concentration of the adenosine triphosphate is 2-10 mmol / L; most preferably, the concentration of the adenosine triphosphate is 4 mmol / L.

[0013] Specifically, the concentration of the acetyl phosphate is 1-100 mmol / L; preferably, the concentration of the acetyl phosphate is 10-100 mmol / L; more preferably, the concentration of the acetyl phosphate is 40-100 mmol / L; more preferably, the concentration of the acetyl phosphate is 78.5 mmol / L.

[0014] Specifically, the concentration of the sodium pyruvate is 1-200 mmol / L, preferably, the concentration of the sodium pyruvate is 50-150 mmol / L; more preferably, the concentration of the sodium pyruvate is 60-120 mmol / L; more preferably, the concentration of the sodium pyruvate is 80 mmol / L.

[0015] Specifically, the concentration of the sodium pyruvate is 1-200 mmol / L, preferably, the concentration of the sodium pyruvate is 50-150 mmol / L; more preferably, the concentration of the sodium pyruvate is 60-120 mmol / L; more preferably, the concentration of the sodium pyruvate is 80 mmol / L.

[0016] Specifically, the concentration of the sodium pyruvate is 1-200 mmol / L, preferably, the concentration of the sodium pyruvate is 50-150 mmol / L; more preferably, the concentration of the sodium pyruvate is 60-120 mmol / L; more preferably, the concentration of the sodium pyruvate is 80 mmol / L.

[0017] Specifically, the concentration of the sodium pyruvate is 1-200 mmol / L, preferably, the concentration of the sodium pyruvate is 50-150 mmol / L; more preferably, the concentration of the sodium pyruvate is 60-120 mmol / L; more preferably, the concentration of the sodium pyruvate is 80 mmol / L.

[0018] Specifically, the concentration of the sodium pyruvate is 1-200 mmol / L, preferably, the concentration of the sodium pyruvate is 50-150 mmol / L; more preferably, the concentration of the sodium pyruvate is 60-120 mmol / L; more preferably, the concentration of the sodium pyruvate is 80 mmol / L.

[0019] Specifically, the temperature of the enzyme catalysis reaction is 30-60℃, the reaction time of the enzyme catalysis reaction is 2-48h, and the pH of the enzyme catalysis reaction is 5.0-10.0.

[0020] Preferably, the temperature of the enzyme catalysis reaction is 30℃, the reaction time of the enzyme catalysis reaction is 20h, and the pH of the enzyme catalysis reaction is 7.0-8.0; more preferably, the pH of the enzyme catalysis reaction is 7.5.

[0021] Specifically, in the liquid reaction system, the uridine kinase, the uridine acid kinase and the acetic acid kinase are in the form of free enzyme, immobilized enzyme or bacterial enzyme.

[0022] Another aspect of the present application also provides a reaction system, which comprises a buffer, 2'-methoxy-uridine acid, an enzyme catalyst, adenosine triphosphate, a phosphate compound, a coenzyme and a kinase stabilizer. The enzyme catalyst comprises uridine acid kinase and acetic acid kinase, the uridine acid kinase is derived from Thermotoga Genbank sequence number is NP_850867.1, the acetic acid kinase is derived from maritima Aerococcus vivridans Genbank sequence number is AKE29949.1, and the phosphate compound is acetyl phosphate.

[0023] Preferably, the enzyme catalyst further comprises pyruvate oxidase, the pyruvate oxidase is derived from Erysipelotrichaceae bacterium The phosphate compound is sodium pyruvate.

[0024] Preferably, the enzyme catalyst further comprises polyphosphate kinase, the polyphosphate kinase is derived from Arabidopsis thaliana The phosphate compound is sodium hexametaphosphate.

[0025] Specifically, the coenzyme is thiamine pyrophosphate, and the kinase stabilizer is a combination of any one or more of magnesium chloride, magnesium sulfate and magnesium nitrate.

[0026] Specifically, the buffer is potassium phosphate buffer.

[0027] The reaction system provided by the present application can perform enzyme reaction to prepare 2'-methoxy-uridine triphosphate.

[0028] In the foregoing reaction system, the concentration of 2'-methoxy-uridine acid, the concentration of adenosine triphosphate, the concentration of acetyl phosphate, the concentration of sodium pyruvate, the concentration of sodium hexametaphosphate, the concentration of thiamine pyrophosphate and the concentration of the kinase stabilizer are all the starting parameters and / or feeding parameters of the reaction system.

[0029] Technical terms Uridylate kinase (UMK) is a pyrimidine ribonucleotide kinase, which is widely present in microorganisms, animals and human bodies, is an important catalyst in nucleotide metabolic compensation pathway, catalyzes the γ-phosphoryl of adenosine triphosphate (ATP) to be transferred to 2'-methoxy-uridylate (2'-OME-UMP) to form 2'-methoxy-uridine diphosphate (2'-OME-UDP), and the reaction needs adenosine triphosphate (ATP) as a phosphate donor.

[0030] In the present application, the used uridylate kinase is derived from Arabidopsis thaliana Thermotoga maritima , the Genbank sequence number of which is NP_850867.1, and is obtained by gene recombination, expression in a recombination vector, cell disruption and purification and then freeze-drying.

[0031] Acetate kinase (ACK) is a member of the phosphate transferase superfamily, which is widely present in microorganisms, is responsible for catalyzing the reversible reaction of acetyl phosphate and adenosine diphosphate (ADP) to generate acetic acid and adenosine triphosphate (ATP). In the present application, the γ-phosphoryl of adenosine triphosphate (ATP) can also be transferred to 2'-methoxy-uridylate (2'-OME-UMP) to form 2'-methoxy-uridine diphosphate (2'-OME-UDP).

[0032] In the present application, the used acetate kinase is derived from Thermotoga maritima Aerococcus vivridans , the Genbank sequence number of which is AKE29949.1, and is obtained by gene recombination, expression in a recombination vector, cell disruption and purification and then freeze-drying.

[0033] Pyruvate oxidase (POX) is a kind of oxidase which catalyzes the oxidative decarboxylation of pyruvate, is widely present in microorganisms, can produce hydrogen peroxide and energy storage metabolite acetyl phosphate by oxidative decarboxylation of pyruvate, and provides phosphate donor precursors for the phosphate cycle system.

[0034] In the present application, the used pyruvate oxidase is derived from Aerococcus viridans Erysipelotrichaceae , and is obtained by gene recombination, expression in a recombination vector, cell disruption and purification and then freeze-drying.

[0035] Polyphosphate kinase (PPK) is one of the key kinases for catalyzing the synthesis of polyphosphate (Poly P), which is widely present in microorganisms, can catalyze the reversible reaction of polyP n to polyP n+1 , and can also transfer the phosphorus of polyphosphate to adenosine diphosphate (ADP) to generate adenosine triphosphate (ATP), thereby providing phosphate donors for the phosphate cycle system.

[0036] In the present application, the used polyphosphate kinase is derived from the bacteria of the genus Erysiphalesbacterium Figure 1 , after gene recombination, expression in a recombinant vector, cell disruption and purification, and then lyophilization.

[0037] In the reaction system of the present application, the wet bacteria bodies, crude enzyme solution, crude enzyme powder or pure enzyme of the above uridine phosphorylase, acetate kinase, pyruvate oxidase and polyphosphate kinase can be used, which can be commercially available products or produced by constructing recombinant plasmids and recombinant engineering bacteria according to the Molecular Cloning Laboratory Guide.

[0038] The inventors of the present application have developed, for the first time, an enzyme-catalyzed preparation method of 2'-methoxy-uridine triphosphate through extensive and in-depth research, a large number of screening tests and condition optimization, and the uridine phosphorylase, acetate kinase, pyruvate oxidase and polyphosphate kinase in the method all show high conversion rate to the substrate of the reaction.

[0039] The beneficial effects of the present application include: 1. The preparation method of 2'-methoxy-uridine triphosphate provided by the present application uses 2'-methoxy-uridine acid substituted at the 2 position of ribose as a raw material, uses a variety of combinations of uridine phosphorylase, acetate kinase, pyruvate oxidase or polyphosphate kinase as a catalyst, combines with a phosphate recycling system constructed with adenosine triphosphate as an initial phosphate donor, and adopts one-pot enzyme catalysis to prepare 2'-methoxy-uridine triphosphate, which has the advantages of high synthesis efficiency, simple steps, single product and the like, overcomes the defects of long chemical synthesis route, avoids the problem of difficult purification in enzymatic hydrolysis method, and effectively avoids the problem of cell permeability inhibition of products in microbial fermentation method.

[0040] 2. The present application uses a high-activity enzyme combination from a specific source, and the conversion rate of uridine phosphorylase to the substrate 2'-methoxy-uridine acid 2'-OME-UMP is 78.6%, and the conversion rate of the final 2'-methoxy-uridine triphosphate 2'-OME-UTP is as high as 92.6%, and the substrate can be completely consumed.

[0041] 3、The ATP-ADP phosphate cycle system constructed in the application regenerates ATP by acetyl phosphate / sodium pyruvate / sodium hexametaphosphate, which reduces the ATP consumption by more than 5 times compared with the non-circulation system, greatly reduces the consumption of expensive phosphoric acid donors, in addition, the core enzyme of the method is expressed by genetic engineering, which can be prepared on a large scale, and the freeze-dried enzyme powder has strong stability and high reusability, which reduces the production cost of the catalyst; furthermore, the reaction does not require expensive and toxic chemical reagents, which reduces the cost of environmental protection treatment, finally, combined with the fact that the product 2'-methoxy-uridine triphosphate of the method is already expensive, the economy of the method is further embodied, which provides a key basis for industrialized production development. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 2 A schematic diagram of the catalytic reaction in Example 2.

[0043] Figure 3 A liquid chromatogram of Example 2, screening different sources of uridine acid kinase catalytic reaction to generate 2'-methoxy-uridine diphosphate 2'-OME-UDP, in which the horizontal coordinate represents time (minutes), and the vertical coordinate represents absorbance (AU).

[0044] Figure 4 A schematic diagram of the catalytic reaction in Example 3.

[0045] Figure 5 A liquid chromatogram of Example 3, screening different sources of acetic acid kinase catalytic reaction to generate 2'-methoxy-uridine triphosphate 2'-OME-UTP, in which the horizontal coordinate represents time (minutes), and the vertical coordinate represents absorbance (AU).

[0046] Figure 6 A schematic diagram of the catalytic reaction in Example 4.

[0047] Figure 7 A liquid chromatogram of Example 4, one-pot enzyme method for preparing 2'-methoxy-uridine triphosphate 2'-OME-UTP, in which the horizontal coordinate represents time (minutes), and the vertical coordinate represents absorbance (AU).

[0048] Figure 8 A schematic diagram of the catalytic reaction in Example 5.

[0049] Figure 9 A liquid chromatogram of Example 5, using uridine acid kinase UMK, pyruvate kinase and acetic acid kinase ACK to prepare 2'-methoxy-uridine triphosphate 2'-OME-UTP, in which the horizontal coordinate represents time (minutes), and the vertical coordinate represents absorbance (AU).

[0050] Figure 10 A schematic diagram of the catalytic reaction in Example 6.

[0051] Figure 1 For Example 6, a liquid phase diagram for the preparation of 2'-methoxy-uridine triphosphate 2'-OME-UTP using uridine kinase UMK, polyphosphate kinase and acetate kinase ACK was prepared, in which the abscissa represents time (min) and the ordinate represents absorbance (AU). DETAILED DESCRIPTION

[0052] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0053] The experimental methods in the following examples, unless otherwise specified, are generally carried out according to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. The experimental materials involved in the present application, unless otherwise specified, are commercial reagents, which can be obtained from commercial channels. In the following examples, ATP is adenosine triphosphate, ADP is adenosine diphosphate, 2'-OME-UMP is 2'-methoxy-uridine acid, 2'-OME-UDP is 2'-methoxy-uridine diphosphate, and 2'-OME-UTP is 2'-methoxy-uridine triphosphate.

[0054] Example 1 Obtaining of lyophilized enzyme powder 1) Construction and transformation of recombinant vector For example, the recombinant strain E. coli BL21(DE3) pET-28a-UMK was constructed as follows: UMK genes from different sources were synthesized by Shanghai Jinweizhi Biological Engineering Co., Ltd., and were connected to the pET28a vector to obtain the recombinant vector pET28a-UMK. The UMK genes from different sources are shown in Table 1 below: Table 1

[0055] The activated E. coli DH5a / pET-28a(+) strain (purchased from Novozyme Biotech Co., Ltd.) was prepared by adding 3-5 pL of pET28a-UMK (50 ng / pL) into 50-100 pL of E. coli BL21(DE3) competent cells (purchased from Novozyme Biotech Co., Ltd.), placing on ice for 20 min, heating at 42 °C for 90 sec, quickly returning to ice bath for 5 min, adding 800 pL of LB culture solution without resistance, culturing at 37 °C and 200 rpm for 1 h, and then coating on LB agar plate culture medium containing kanamycin resistance and culturing at 37 °C for 12 h to obtain recombinant bacteria E. coli BL21(DE3) containing pET28a-UMK.

[0056] The formula of the LB culture solution without resistance is as follows: 5 g / L of yeast powder, 10 g / L of sodium chloride, and 10 g / L of proteose peptone. The formula of the LB agar plate culture medium containing kanamycin resistance is as follows: 5 g / L of yeast powder, 10 g / L of sodium chloride, 10 g / L of proteose peptone, 20 g / L of agar powder, and 25 pg / mL of kanamycin.

[0057] The construction of recombinant vectors of acetic acid kinase ACK, pyruvate oxidase POX, and polyphosphate kinase PPK and the operation of transformation are the same as the above method. The acetic acid kinase ACK genes from different sources are shown in Table 2: Table 2

[0058] 2) Induced expression of the expression vector The recombinant bacteria E. coli BL21(DE3) containing pET28a-UMK obtained in 1) was directly coated on a solid LB plate containing 25 pg / mL of kanamycin resistance and cultured at 37 °C for 12-14 h to obtain a single colony. The single colony of E. coli BL21(DE3) containing the UMK recombinant vector was picked from the kanamycin plate and inoculated into 1 mL of liquid LB culture medium containing 25 pg / mL of kanamycin resistance and cultured at 37 °C for 12 h. Then, 2% (v / v) of the inoculum was inoculated into 1 L of fresh liquid LB culture medium containing 25 pg / mL of kanamycin resistance, and when the OD600 was about 0.6-0.8, IPTG (purchased from Shanghai Aladdin Biochem Technology Co., Ltd.) was added to a final concentration of 1.0 mmol / L, and the culture was induced at 200 rpm and 25 °C for 20 h. After centrifugation (4 °C, 4000 rpm, 30 min), the supernatant was removed, and the obtained bacterial slurry was washed with 30 ml of 0.9% NaCl solution and resuspended for use to obtain a bacterial solution containing the UMK target protein.

[0059] With the Uridylate kinase UMK expression vector as an example, the induced expression of the recombinant vectors of acetate kinase ACK, pyruvate oxidase POX and polyphosphate kinase PPK in the present application is the same as this. By using the same method, the bacterial liquid containing the UMK target protein, the bacterial liquid containing the ACK target protein, the bacterial liquid containing the POX target protein and the bacterial liquid containing the PPK target protein can be obtained.

[0060] 3) Preparation of freeze-dried enzyme powder In the present application, the bacterial liquid containing the UMK target protein and the bacterial liquid containing the ACK target protein are prepared into freeze-dried enzyme powder by the following method: first, the bacterial liquid is collected and washed twice with 50 mmol / L Tris-HCl, then the bacterial body is resuspended in 50 mmol / L Tris-HCl (pH 8.0) buffer, and the cells are broken by ultrasonic in an ice bath (amplitude rod 6, power 500W, on 2s, off 5s, 30min, the sample after ultrasonic breaking is centrifuged at 12000 rpm, 4℃ for 30min. Then, the supernatant is placed in a freeze-drying machine at -80℃ and freeze-dried for 24h, and the freeze-dried sample is ground to prepare freeze-dried crude enzyme powder.

[0061] Example 2: Screening of uridylate kinases UMK from different sources The freeze-dried enzyme powder of the uridylate kinases UMK from different sources obtained in Example 1 is subjected to catalytic reaction, 2 mL of phosphate buffer is added to the reaction bottle (8 mL), the phosphate concentration is 200 mmol / L, 10 mg of 2'-OME-UMP, 15 mg of ATP, 2 mg of magnesium chloride and 0.2 mg of thiamine pyrophosphate are added, the pH of the reaction solution is controlled at 7.5 with 5 M NaOH; 10 mg of uridylate kinase from different sources is added to start the reaction; the temperature of the above reaction solution is controlled at 30℃, and the rotation speed of the reaction is controlled at 1000 rpm; 100 μl of the reaction solution is taken after 3h of reaction, an equal volume of methanol solution is added for inactivation, then centrifugal filtration is performed, and the reaction conversion rate of the product 2'-OME-UDP is determined by high performance liquid chromatography (HPLC). The reaction process diagram is shown in Figure 2 , and the HPLC liquid chromatography result diagram is shown in Arabidopsis thaliana . Among them, the peak time of 2'-OME-UDP is 1.251 min, Conv(2'-OME-UDP) % = 78.6%, and the peak time of 2'-OME-UMP is 3.651 min, Conv(2'-OME-UMP) % = 21.4%.

[0062] Among them, the conversion rate of 2'-methoxy-uridine diphosphate (2'-OME-UDP) measured by liquid chromatography is calculated based on the proportion of the peak area Area of the detected product: 2'-methoxy-uridine diphosphate (2'-OME-UDP) conversion rate Conv(2'-OME-UDP) % = Area[2'-OME-UDP / (2'-OME-UMP + 2'-OME-UDP)] * 100%.

[0063] The high performance liquid chromatograph (HPLC) is purchased from Agilent Technologies Co., Ltd. The HPLC detection conditions are as follows: the mobile phase is 10 mM NH4Oac, the mobile phase A is 5% acetonitrile, the mobile phase B is 95% acetonitrile, the temperature is 40°C, the flow rate is 1.3 mL / min, and the chromatographic column is Xbridge C18 (purchased from the United States Waters / Waters).

[0064] The conversion rates of the uridine monophosphate kinases UMK from different sources are as shown in Table 3. Table 3

[0065] The results show that, Arabidopsis thaliana The uridine monophosphate kinase UMK from different sources has the highest catalytic conversion rate, and the conversion rate of 2'-OME-UDP is 71.4%.

[0066] Example 3: Screening of acetic acid kinases ACK from different sources The vector construction, expression of acetic acid kinases ACK, and preparation of freeze-dried crude enzyme powder are the same as in Example 1. The acetic acid kinases ACK enzyme powder from different sources obtained in Example 1 is subjected to catalytic reaction. 2 mL of phosphate buffer is added to the reaction bottle (8 mL), the phosphate concentration is 200 mmol / L, 10 mg of 2'-OME-UMP, 4.5 mg of ATP, 5 mg of magnesium chloride, 2 mg of thiamine pyrophosphate, and 22 mg of acetyl phosphate are added, and the pH of the reaction solution is controlled at 7.5 with 5 M NaOH; 10 mg of uridine monophosphate kinase from different sources and 10 mg of acetic acid kinase from different sources are added, and the reaction is started. Figure 3 The temperature of the above reaction solution is controlled at 30°C, and the reaction speed is controlled at 1000 rpm. After 20 h of reaction, 100 μL of the reaction solution is taken, an equal volume of methanol solution is added for inactivation, centrifuged and filtered, and the reaction conversion rate of the product 2'-OME-UTP is determined by high performance liquid chromatography HPLC. The reaction process diagram is shown in Figure 4 The HPLC liquid chromatogram is shown in Thermotoga maritimaThe peak time of 2'-OME-UTP is 5.32 min, Conv(2'-OME-UTP) %=30.1%, the peak time of 2'-OME-UDP is 4.91 min, Conv(2'-OME-UDP) %=13.3%, and the peak time of 2'-OME-UMP is 4.33 min, Conv(2'-OME-UMP) %=6.1%. In addition, the reaction time is 20 h, and a large amount of hydrolysis by-product 2'-methoxy-uridine (2'-OME-Urd) is generated, the peak time of 2'-OME-Urd is 0.501 min, and Conv(2'-OME-Urd) %=50.5%.

[0067] The conversion rate of 2'-OME-UTP detected by liquid chromatography is calculated based on the proportion of the detected product peak area Area: The conversion rate of 2'-methoxy-uridine triphosphate (2'-OME-UTP) Conv(2'-OME-UTP) %=Area[2'-OME-UTP / (2'-OME-UMP+2'-OME-UDP+2'-OME-UTP)]*100% The high-performance liquid chromatograph (HPLC) is purchased from Agilent Technologies Co., Ltd. The HPLC detection conditions are as follows: the mobile phase is 10 mM NH4OAc mobile phase A: 5% acetonitrile, and the mobile phase B: 95% acetonitrile, the temperature is 40 ℃, the flow rate is 1.3 mL / min, and the chromatographic column is Xbridge C18 (purchased from the United States Waters / Waters).

[0068] The conversion rate of the acetic acid kinase ACK from each source is as shown in Table 4: Table 4

[0069] Combining the liquid chromatogram with the above table, it can be seen that Figure 5 The acetic acid kinase ACK from the source has a higher catalytic conversion rate, and the conversion rate is 30.1%.

[0070] Example 4: One-pot enzyme method for preparing 2'-methoxy-uridine triphosphate using uridine acid kinase UMK and acetic acid kinase ACK According to the screening results of Examples 2 and 3, the uridine acid kinase UMK and the acetic acid kinase ACK with the optimal conversion rate are obtained, and a one-pot enzyme catalysis is performed to prepare 2'-methoxy-uridine triphosphate. The synthesis route is as shown in Arabidopsis thaliana .

[0071] Into the reactor, 2 mL of phosphate buffer was added, the phosphate concentration was controlled at 200 mmol / L, 10 mg of 2'-methoxy-uridine acid (2'-OME-UMP), 4.5 mg of ATP, 5 mg of magnesium chloride, 2 mg of thiamine pyrophosphate, and 22 mg of acetyl phosphate were added, and the pH of the reaction solution was controlled at 7.5 by using 5 M NaOH; 10 mg of UMK enzyme powder from U. maydis was added, and the reaction was started. Thermotoga maritima 2 mg of purified ACK enzyme powder from U. maydis was added, and the reaction was started. Figure 6 The temperature of the above reaction solution was controlled at 30°C, and the rotation speed of the reaction was controlled at 1000 rpm; 100 μl of the reaction solution was taken after 3 h of reaction, inactivated after adding an equal volume of methanol solution, centrifuged and filtered, and the reaction conversion rate of the product 2'-OME-UTP was determined by HPLC.

[0072] The 2'-OME-UTP conversion rate determined by liquid chromatography was calculated as the proportion of the detected product peak area Area: 2'-methoxy-uridine triphosphate 2'-OME-UTP conversion rate Conv(2'-OME-UTP) % = Area[2'-OME-UTP / (2'-OME-UMP + 2'-OME-UDP + 2'-OME-UTP)] *100% 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 mobile phase A: 5% acetonitrile, mobile phase B: 95% acetonitrile, temperature 40°C, flow rate 1.3 mL / min, and the chromatographic column was Xbridge C18 (purchased from Waters, USA).

[0073] As shown in Aerococcus vivridans The liquid phase diagram for producing 2'-methoxy-uridine triphosphate 2'-OME-UTP using the optimal conversion rate of uridine acid kinase UMK and acetic acid kinase ACK is shown. The liquid phase detection showed that the 2'-OME-UTP peak appeared at 4.61 min, the Conv(2'-OME-UTP) % = 92.6%, i.e., the 2'-OME-UTP conversion rate was as high as 92.6%. The 2'-OME-UDP peak appeared at 4.22 min, the Conv(2'-OME-UDP) % = 1.0%, the 2'-OME-UMP was completely consumed, the 2'-OME-Urd peak appeared at 0.451 min, and the Conv(2'-OME-Urd) % = 6.5%. It can be seen that the reaction time required by the present method is short, only 3 h, the substrate hydrolysis side reaction is reduced, and the efficient synthesis of 2'-methoxy-uridine triphosphate 2'-OME-UTP is realized.

[0074] Example 5 One-pot enzymatic preparation of 2'-methoxy-uridine triphosphate using uridine monophosphate kinase UMK, pyruvate oxidase POX and acetate kinase ACK According to the screening results of Examples 2 and 3, the uridine monophosphate kinase UMK and the acetate kinase ACK with the optimal conversion rate were obtained, and combined with the pyruvate oxidase POX derived from Figure 7 , one-pot enzymatic catalysis was performed to prepare 2'-methoxy-uridine triphosphate, and the synthesis route is shown in Arabidopsis thaliana .

[0075] 2 mL of phosphate buffer was added to the reaction kettle, the phosphate concentration was controlled at 200 mmol / L, 10 mg of 2'-methoxy-uridine monophosphate (2'-OME-UMP), 4.5 mg of ATP, 5 mg of magnesium chloride, 2 mg of thiamine pyrophosphate and 17.6 mg of sodium pyruvate were added, and the pH of the reaction solution was controlled at 7.5 with 5 M NaOH; 10 mg of uridine monophosphate kinase UMK enzyme powder derived from Thermotoga maritima , 2 mg of purified acetate kinase ACK enzyme powder derived from Aerococcus and 2 mg of pyruvate oxidase POX enzyme powder derived from vivridans Figure 8 were added to start the reaction; the temperature of the above reaction solution was controlled at 30°C, and the rotation speed was controlled at 1000 rpm; 100 μl of the reaction solution was taken after 3 h of reaction, and an equal volume of methanol solution was added for inactivation, followed by centrifugal filtration, and the reaction conversion rate of the product 2'-OME-UTP was determined by HPLC.

[0076] Among them, the conversion rate of 2'-OME-UTP detected by liquid chromatography is calculated as the proportion of the peak area Area: 2'-methoxy-uridine triphosphate 2'-OME-UTP conversion rate Conv(2'-OME-UTP) % = Area[2'-OME-UTP / (2'-OME-UMP + 2'-OME-UDP + 2'-OME-UTP)] *100% Among them, the high-performance liquid chromatograph (HPLC) is purchased from Agilent Technology Co., Ltd., the HPLC detection conditions are as follows: the mobile phase is 10 mM NH4OAc mobile phase A: 5% acetonitrile, mobile phase B: 95% acetonitrile, temperature 40°C, flow rate 1.3 mL / min, and the chromatographic column is Xbridge C18 (purchased from the United States Waters / Waters).

[0077] As Erysipelotrichaceae bacteriumThe liquid phase diagram for producing 2'-methoxy-uridine triphosphate 2'-OME-UTP using the optimal conversion rate of uridine acid kinase UMK, acetate kinase ACK, and pyruvate oxidase POX is shown. The liquid phase detection showed that the 2'-OME-UTP peak time was 5.265 min, Conv(2'-OME-UTP) % = 34.19%, i.e., the conversion rate of 2'-OME-UTP was 34.19%. The 2'-OME-UDP peak time was 4.886 min, Conv(2'-OME-UDP) % = 9.97%, the 2'-OME-UMP peak time was 4.321 min, Conv(2'-OME-UMP) % = 2.54%, and the 2'-OME-Urd peak time was 0.473 min, Conv(2'-OME-Urd) % = 53.13%.

[0078] Example 6 One-pot enzymatic preparation of 2'-methoxy-uridine triphosphate using uridine acid kinase UMK and polyphosphate kinase PPK and acetate kinase ACK According to the screening results of Example 2, the optimal conversion rate of uridine acid kinase UMK and acetate kinase ACK was obtained, and combined with the polyphosphate kinase PPK derived from Figure 9 , one-pot enzymatic catalysis was performed to prepare 2'-methoxy-uridine triphosphate, and the synthesis route is shown as Arabidopsis thaliana .

[0079] 2 mL of phosphate buffer was added to the reaction kettle, the phosphate concentration was controlled at 200 mmol / L, 10 mg of 2'-methoxy-uridine acid (2'-OME-UMP), 4.5 mg of ATP, 5 mg of magnesium chloride, and 25 mg of sodium hexametaphosphate were added, and the pH of the reaction solution was controlled at 7.5 with 5 M NaOH; 10 mg of Thermotoga maritima uridine acid kinase UMK enzyme powder derived from E. coli, 2 mg of Erysipelotrichaceae purified acetate kinase ACK enzyme powder derived from E. coli, and 2 mg of bacterium Figure 10 polyphosphate kinase PPK enzyme powder derived from E. coli were added, and the reaction was started; the temperature of the above reaction solution was controlled at 30°C, and the reaction speed was controlled at 1000 rpm; after 3 h of reaction, 100 μl of reaction solution was taken, inactivated after adding an equal volume of methanol solution, centrifuged and filtered, and the reaction conversion rate of the product 2'-OME-UTP was determined by HPLC.

[0080] Among them, the conversion rate of 2'-OME-UTP detected by liquid chromatography is calculated based on the proportion of the peak area Area of the detected product: 2'-methoxy-uridine triphosphate 2'-OME-UTP conversion rate Conv(2'-OME-UTP) %=Area[2'-OME-UTP / (2'-OME-UMP + 2'-OME-UDP+2'-OME-UTP)]*100% Wherein, the high performance liquid chromatograph (HPLC) is purchased from Agilent Technologies Co., Ltd., the HPLC detection condition is that the mobile phase is 10 mM NH4OAc mobile phase A: 5% acetonitrile, mobile phase B: 95% acetonitrile, temperature 40 ℃, flow rate 1.3 mL / min, and the chromatographic column is Xbridge C18 (purchased from the United States Waters / Waters).

[0081] As shown in ​ The liquid phase diagram for producing 2'-methoxy-uridine triphosphate 2'-OME-UTP using the optimal conversion rate of uridine acid kinase UMK, acetic acid kinase ACK and polyphosphate kinase PPK is shown. The liquid phase detection shows that the 2'-OME-UTP peak time is 5.243 min, Conv(2'-OME-UTP) %=48.19%, that is, the conversion rate of 2'-OME-UTP is 48.19%. The 2'-OME-UDP peak time is 4.552 min, Conv(2'-OME-UDP) %=33.79%, the 2'-OME-UMP peak time is 3.525 min, Conv(2'-OME-UMP) %=15.7%, and the 2'-OME-Urd peak time is 0.456 min, Conv(2'-OME-Urd) %=2.32%.

[0082] To sum up, the above-mentioned embodiments are only the preferred embodiments of the present application, and do not limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing 2′-methoxy-uridine triphosphate, characterized in that, In a liquid reaction system, using 2′-methoxy-uridine acid as a substrate and adenosine triphosphate as the initial phosphate donor, a phosphate cycling system was constructed. An enzyme catalyst was added, and a one-pot enzymatic reaction was performed to obtain the final product 2′-methoxy-uridine triphosphate, wherein: The enzyme catalyst comprises uridine kinase and acetate kinase, wherein the uridine kinase is derived from... Arabidopsis thaliana, The Genbank serial number is NP_850867.1, and the acetate kinase is derived from... Thermotoga maritima The Genbank serial number is AKE29949.1; The phosphoric acid cycle system consists of adenosine triphosphate, adenosine diphosphate, and phosphate compounds. The enzyme catalyst catalyzes the dephosphorylation of the phosphate compounds to provide inorganic phosphate to adenosine diphosphate, and regenerates adenosine triphosphate to achieve cyclic energy supply.

2. The method for preparing 2′-methoxy-uridine triphosphate according to claim 1, characterized in that, The phosphate compound is acetylphosphoric acid.

3. The method for preparing 2′-methoxy-uridine triphosphate according to claim 1, characterized in that, The enzyme catalyst further includes pyruvate oxidase, which is derived from... Aerococcus vivridans The Genbank serial number is WP_026465373.1, and the phosphate compound is sodium pyruvate.

4. The method for preparing 2′-methoxy-uridine triphosphate according to claim 1, characterized in that, The enzyme catalyst also includes polyphosphokinase, which is derived from... Erysipelotrichaceae bacterium The Genbank serial number is UKS89443.1, and the phosphate compound is sodium hexametaphosphate.

5. The method for preparing 2′-methoxy-uridine triphosphate according to claim 1, characterized in that, The concentration of the 2′-methoxy-uridine acid is 10~80 mmol / L.

6. The method for preparing 2′-methoxy-uridine triphosphate according to claim 2, characterized in that, The concentration of adenosine triphosphate is 2-40 mmol / L, the concentration of acetyl phosphate is 1-100 mmol / L, and the concentrations of uridine kinase and acetate kinase are both 0.05-50 mg / mL.

7. The method for preparing 2′-methoxy-uridine triphosphate according to claim 3, characterized in that, The concentration of adenosine triphosphate is 1-20 mmol / L, the concentration of sodium pyruvate is 1-200 mmol / L, and the concentrations of uridine kinase, acetate kinase, and pyruvate oxidase are all 0.05-50 mg / mL.

8. The method for preparing 2′-methoxy-uridine triphosphate according to claim 4, characterized in that, The concentration of adenosine triphosphate is 1-20 mmol / L, the concentration of sodium hexametaphosphate is 1-200 mmol / L, and the concentrations of uridine kinase, acetate kinase, and polyphosphokinase are all 0.05-50 mg / mL.

9. The method for preparing 2′-methoxy-uridine triphosphate according to claim 1, characterized in that, In the liquid reaction system, the buffer solution is a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution with a concentration of 50-500 mmol / L. The liquid reaction system also includes a coenzyme and a magnesium salt. The coenzyme is thiamine pyrophosphate with a concentration of 0.1%-2% of the substrate concentration. The magnesium salt has a concentration of 1 mmol / L-20 mmol / L and is any one or a combination of magnesium chloride, magnesium sulfate, and magnesium nitrate.

10. The method for preparing 2′-methoxy-uridine triphosphate according to claim 1, characterized in that, The enzyme-catalyzed reaction is carried out at a temperature of 30°C to 60°C, for a reaction time of 2 to 48 hours, and at a pH of 5.0 to 10.0.

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