A method for preparing 2'-methoxy-uridine triphosphate

Abstract

The application discloses a method for preparing 2'-methoxy-uridine triphosphate, which comprises the following steps: taking 2'-methoxy-uridine acid as a substrate, taking adenosine triphosphate as an initial phosphate donor, constructing a phosphate circulation system, adding an enzyme catalyst, and adopting one-pot enzyme method to perform enzyme catalysis reaction, so as to obtain the end product 2'-methoxy-uridine triphosphate. The phosphate circulation system is composed of adenosine triphosphate, adenosine diphosphate and phosphate compounds. The enzyme catalyst catalyzes the dephosphorylation of the phosphate compounds to provide inorganic phosphate to the adenosine diphosphate, and the adenosine triphosphate is regenerated to realize the circulation energy supply. The method adopts high-activity enzyme combination of specific sources, has high conversion rate of the substrate 2'-methoxy-uridine acid, and has high conversion rate of the final 2'-methoxy-uridine triphosphate up to 92.6%, and has the advantages of high synthesis efficiency, simple steps, green environmental protection and controllable cost.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to a method for preparing 2′-methoxy-uridine triphosphate. Background Technology

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

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

[0004] Approved nucleic acid drugs such as silencing RNA drugs (siRNA drugs) Patisiran and Givosiiran, ASO drugs Nusinersen, Inotersen and Volanesorsen, and aptamer drug Peg-aptanib have all demonstrated the pharmacological value and clinical efficacy of 2′-methoxy (2′OMe) modification, making 2′-methoxy modified nucleotides a key intermediate in drug development.

[0005] 2′-Methoxyuridine triphosphate (2′-OME-UTP), as a UTP analog, possesses enhanced resistance to hydrolysis and nuclease cleavage of the RNA chain due to methylation at the 2-position of the ribose, making it an important raw material for aptamer synthesis. However, this compound is unstable, and its commercial price is as high as 50 μL (100 mM) / £10,000 (CAS: 143028-99-3). Currently, there is no efficient and targeted synthetic method. In addition, existing nucleotide production methods also have various drawbacks, such as: chemical synthesis methods have long routes, poor stereoselectivity, expensive and toxic reagents, and high production costs; RNA enzymatic hydrolysis produces a mixture of four nucleotides, which is difficult to extract and has low purity; microbial fermentation methods are limited by cell permeability; and although traditional biocatalysis methods have solved the cell permeability problem, they are not efficient enough in the synthesis of complex modified nucleotides.

[0006] Therefore, there is an urgent need in this field to develop a simple, easy-to-operate, economical, environmentally friendly, and efficient method for preparing 2′-methoxy-uridine triphosphate, 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 of the Invention

[0007] To address the above technical problems, this invention provides a method for preparing 2′-methoxy-uridine triphosphate. 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 is constructed. An enzyme catalyst is added, and a one-pot enzymatic reaction is performed to obtain the final product 2′-methoxy-uridine triphosphate.

[0008] 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;

[0009] 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.

[0010] Specifically, the phosphate compound is acetylphosphoric acid.

[0011] Specifically, 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.

[0012] Specifically, the enzyme catalyst further includes a polyphosphoric acid kinase, which is derived from... Erysipelotrichaceae bacterium The Genbank serial number is UKS89443.1, and the phosphate compound is sodium hexametaphosphate.

[0013] 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.

[0014] Specifically, the concentration of adenosine triphosphate is 1-40 mmol / L; preferably, the concentration of adenosine triphosphate is 2-20 mmol / L; more preferably, the concentration of adenosine triphosphate is 2-10 mmol / L; and most preferably, the concentration of adenosine triphosphate is 4 mmol / L.

[0015] Specifically, the concentration of acetyl phosphate is 1~100 mmol / L; preferably, the concentration of acetyl phosphate is 10~100 mmol / L; more preferably, the concentration of acetyl phosphate is 40~100 mmol / L; even more preferably, the concentration of acetyl phosphate is 78.5 mmol / L.

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

[0017] Specifically, the concentration of sodium hexametaphosphate is 1~200 mmol / L, preferably 1~100 mmol / L; more preferably 1~50 mmol / L; and even more preferably 10 mmol / L.

[0018] Specifically, the concentrations of uridine kinase, acetate kinase, and polyphosphokinase are all 0.05–50 mg / mL; preferably, the concentrations of uridine kinase, acetate kinase, and polyphosphokinase are all 1–20 mg / mL; more preferably, the concentrations of uridine kinase, acetate kinase, and polyphosphokinase are all 1–10 mg / mL; and most preferably, the concentrations of uridine kinase, acetate kinase, and polyphosphokinase are all 5 mg / mL.

[0019] Specifically, 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, preferably 50 mmol / L; the liquid reaction system also includes a coenzyme and a magnesium salt, wherein the coenzyme is thiamine pyrophosphate with a concentration of 0.1%~2% of the substrate concentration, preferably 2% of the substrate concentration.

[0020] Specifically, the concentration of the magnesium salt is 1 mmol / L to 20 mmol / L, and the magnesium salt is any one or a combination of magnesium chloride, magnesium sulfate, and magnesium nitrate; preferably, the magnesium salt is magnesium chloride, and the concentration of magnesium chloride is 25 mmol / L.

[0021] Specifically, the temperature of the enzyme-catalyzed reaction is 30℃ to 60℃, the reaction time is 2 to 48 hours, and the pH of the enzyme-catalyzed reaction is 5.0 to 10.0.

[0022] Preferably, the temperature of the enzyme-catalyzed reaction is 30°C, the reaction time is 20 h, and the pH of the enzyme-catalyzed reaction is 7.0~8.0; more preferably, the pH of the enzyme-catalyzed reaction is 7.5.

[0023] Specifically, in the liquid reaction system, the uridine kinase, uridine kinase, and acetate kinase exist in the form of free enzymes, immobilized enzymes, or bacterial enzymes.

[0024] Another aspect of the present invention provides a reaction system comprising a buffer solution, 2′-methoxyuridine monophosphate, an enzyme catalyst, adenosine triphosphate, a phosphate compound, a coenzyme, and a kinase stabilizer.

[0025] The enzyme catalyst includes 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, and the phosphate compound is acetyl phosphate.

[0026] Preferably, the enzyme catalyst further includes pyruvate oxidase, which is derived from... Aerococcus vivridans The phosphate compound is sodium pyruvate.

[0027] Preferably, the enzyme catalyst further includes a polyphosphoric acid kinase, which is derived from... Erysipelotrichaceae bacterium The phosphate compound is sodium hexametaphosphate.

[0028] Specifically, the coenzyme is thiamine pyrophosphate, and the kinase stabilizer is any one or a combination of magnesium chloride, magnesium sulfate, and magnesium nitrate.

[0029] Specifically, the buffer solution is a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution.

[0030] The reaction system provided by this invention can carry out an enzymatic reaction to prepare 2′-methoxy-uridine triphosphate.

[0031] In the aforementioned reaction system, the concentrations of 2′-methoxyuridine acid, adenosine triphosphate, acetyl phosphate, sodium pyruvate, sodium hexametaphosphate, thiamine pyrophosphate, and kinase stabilizer all refer to the initial parameters and / or feeding parameters of the reaction system.

[0032] Technical terms

[0033] Uridine kinase (UMK) is a pyrimidine ribonucleotide kinase that is widely found in microorganisms, animals, and humans. It is an important catalyst in the nucleotide metabolic compensation pathway, catalyzing the transfer of γ-phosphate from adenosine triphosphate (ATP) to 2′-methoxy-uridine acid (2′-OME-UMP) to form 2′-methoxy-uridine diphosphate (2′-OME-UDP). The reaction requires ATP as a phosphate donor.

[0034] The uridine kinase used in this application is derived from Arabidopsis thaliana. Arabidopsis thaliana Its Genbank serial number is NP_850867.1. It was obtained by gene recombination, expression in a recombinant vector, cell disruption and purification, and freeze-drying.

[0035] Acetyl kinase (ACK) is a member of the phosphotransferase superfamily, widely distributed in microorganisms, and is responsible for catalyzing the reversible reaction of acetyl phosphate and adenosine diphosphate (ADP) to generate acetic acid and adenosine triphosphate (ATP). In this invention, it can also catalyze the transfer of γ-phosphate from adenosine triphosphate (ATP) to 2′-methoxy-uridine monophosphate (2′-OME-UMP) to form 2′-methoxy-uridine diphosphate (2′-OME-UDP).

[0036] In this application, the acetate kinase used is derived from *Thermophyton floccosum*. Thermotoga maritima Its Genbank serial number is AKE29949.1. It was obtained by gene recombination, expression in a recombinant vector, cell disruption and purification, and freeze-drying.

[0037] Pyruvate oxidase (POX) is a class of oxidases that catalyze the oxidative decarboxylation of pyruvate. It is widely found in microorganisms and can oxidize and decarboxylate pyruvate to produce hydrogen peroxide and the energy storage metabolite acetyl phosphate, providing phosphate donor precursors for the phosphate cycle system.

[0038] In this application, the pyruvate oxidase used is derived from *Bacillus viride*. Aerococcus vivridans It is obtained by gene recombination, expression in a recombinant vector, cell disruption and purification, and freeze-drying.

[0039] Polyphosphoric acid kinase (PPK) is one of the key kinases catalyzing the synthesis of polyphosphate (Poly P). It is widely present in microorganisms and can catalyze the synthesis of Poly P. n Generate polyP n+1 The reversible reaction can transfer phosphorus from polyphosphate to adenosine diphosphate (ADP) to generate adenosine triphosphate (ATP), providing a phosphate donor for the phosphate cycle system.

[0040] The polyphosphate kinase used in this application is derived from Erysipelothrix bacteria. Erysipelotrichaceae bacterium It is obtained by gene recombination, expression in a recombinant vector, cell disruption and purification, and freeze-drying.

[0041] In the reaction system of this application, the above-mentioned uridine kinase, acetate kinase, pyruvate oxidase and polyphosphokinase can be produced in the form of wet bacterial cells, crude enzyme solution, crude enzyme powder or pure enzyme, etc., which can be commercially available products, or can be produced by constructing recombinant plasmids and recombinant engineered bacteria in accordance with "Molecular Cloning: A Laboratory Manual".

[0042] Through extensive and in-depth research, and by conducting numerous screening experiments and optimizing conditions, the inventors of this application have unexpectedly developed an enzymatically catalytic preparation method for 2′-methoxy-uridine triphosphate for the first time. The uridine kinase, acetate kinase, pyruvate oxidase, and polyphosphate kinase in this method all exhibit high conversion rates for the substrates of the reaction.

[0043] The beneficial effects of this application include:

[0044] 1. The method for preparing 2′-methoxy-uridine triphosphate provided in this application uses 2′-methoxy-uridine acid with ribose 2-substitution as raw material, and a combination of uridine kinase, acetate kinase, pyruvate oxidase or polyphosphokinase as catalysts, combined with a phosphate cycle system constructed with adenosine triphosphate as the initial phosphate donor, to prepare 2′-methoxy-uridine triphosphate by one-pot enzymatic catalysis. The reaction conditions are mild, no high temperature and high pressure equipment is required, and the reaction cycle is only 2 to 48 hours, which is significantly shorter than chemical synthesis (usually several days). It has the advantages of high synthesis efficiency, simple steps, and single product. It overcomes the defects of long chemical synthesis routes and avoids the problem of difficult purification by enzymatic hydrolysis. In addition, it can also effectively avoid the problem of cell permeability inhibiting the product in microbial fermentation.

[0045] 2. This application uses a combination of highly active enzymes from specific sources. After screening and verification, uridine kinase achieves a conversion rate of 78.6% for the substrate 2′-methoxy-uridine acid 2′-OME-UMP, and the final conversion rate of 2′-methoxy-uridine triphosphate 2′-OME-UTP is as high as 92.6%, and the substrate can be completely consumed.

[0046] 3. The ATP-ADP phosphate cycling system constructed in this application regenerates ATP by supplying Pi with acetyl phosphate / sodium pyruvate / sodium hexametaphosphate, reducing ATP consumption by more than 5 times compared to the non-cycling system, and significantly reducing the consumption of expensive phosphate donors. In addition, the core enzyme of this method is expressed through genetic engineering recombination, which can be prepared on a large scale, and the lyophilized enzyme powder has strong stability and high reusability, reducing the production cost of the catalyst. Furthermore, this reaction does not require expensive and toxic chemical reagents, reducing the cost of environmental treatment. Finally, considering the fact that the product of this method, 2′-methoxy-uridine triphosphate, is expensive, the economic benefits of this method are further demonstrated, providing a key foundation for the development of industrial-scale production. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the catalytic reaction in Example 2.

[0048] Figure 2 The liquid phase diagram shown in Example 2 is used to screen uridine kinases from different sources to catalyze the reaction to generate 2′-methoxy-uridine diphosphate 2′-OME-UDP. The horizontal axis in the figure represents time (minutes) and the vertical axis represents absorbance (AU).

[0049] Figure 3 This is a schematic diagram of the catalytic reaction in Example 3.

[0050] Figure 4 The liquid phase diagram for screening the generation of 2′-methoxy-uridine triphosphate 2′-OME-UTP by acetate kinase from different sources in Example 3 is shown. The horizontal axis represents time (minutes) and the vertical axis represents absorbance (AU).

[0051] Figure 5 This is a schematic diagram of the catalytic reaction in Example 4.

[0052] Figure 6 The liquid phase diagram for the one-pot enzymatic preparation of 2′-methoxy-uridine triphosphate 2′-OME-UTP in Example 4 is shown. The horizontal axis represents time (minutes) and the vertical axis represents absorbance (AU).

[0053] Figure 7 This is a schematic diagram of the catalytic reaction in Example 5.

[0054] Figure 8 The liquid phase diagram for preparing 2′-methoxy-uridine triphosphate 2′-OME-UTP using uridine kinase UMK, pyruvate kinase and acetate kinase ACK in Example 5 is shown. The horizontal axis represents time (minutes) and the vertical axis represents absorbance (AU).

[0055] Figure 9 This is a schematic diagram of the catalytic reaction in Example 6.

[0056] Figure 10 The liquid phase diagram for preparing 2′-methoxy-uridine triphosphate 2′-OME-UTP using uridine kinase UMK, polyphosphate kinase and acetate kinase ACK in Example 6 is shown. The horizontal axis represents time (minutes) and the vertical axis represents absorbance (AU). Detailed Implementation

[0057] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. All experimental materials used in this invention are commercially available reagents unless otherwise specified. In the following examples, ATP is adenosine triphosphate, ADP is adenosine diphosphate, 2′-OME-UMP is 2′-methoxy-uridine monophosphate, 2′-OME-UDP is 2′-methoxy-uridine diphosphate, and 2′-OME-UTP is 2′-methoxy-uridine triphosphate.

[0059] Example 1: Obtaining freeze-dried enzyme powder

[0060] 1) Construction and transformation of recombinant vectors

[0061] Taking the construction of recombinant strain E. coli BL21(DE3) pET-28a-UMK as an example:

[0062] Urydidine kinase (UMK) genes from different sources were fully synthesized by Shanghai Genewise Biotech Co., Ltd., and ligated into the pET28a vector to obtain the recombinant vector pET28a-UMK. The Urydidine kinase (UMK) genes from different sources are shown in Table 1 below.

[0063] Table 1

[0064]

[0065] Activate E. coli DH5α / pET-28a(+) strain (purchased from Novizan Biotechnology Co., Ltd.), add 3-5 μL of pET28a-UMK (50 ng / μL) to 50-100 μL of E. coli BL21(DE3) competent cells (purchased from Novizan Biotechnology Co., Ltd.), place on ice for 20 min, heat shock at 42 ℃ for 90 sec, quickly return to ice for 5 min, add 800 μL of antibiotic-free LB medium, incubate at 37 ℃ and 200 rpm for 1 h, then spread on LB agar plates containing Kansas antibiotics, and incubate overnight at 37 ℃ for 12 h to obtain recombinant E. coli BL21(DE3) containing pET28a-UMK.

[0066] The formulation of antibiotic-free LB medium is as follows: yeast extract 5 g / L, sodium chloride 10 g / L, peptone 10 g / L;

[0067] The formulation of LB agar plate medium containing cannabinoid resistance is as follows: yeast extract 5 g / L, sodium chloride 10 g / L, peptone 10 g / L, agar powder 20 g / L, cannabinoid 25 μg / mL.

[0068] The construction and transformation of recombinant vectors for acetate kinase ACK, pyruvate oxidase POX, and polyphosphate kinase PPK were performed using the same methods as described above. Acetate kinase ACK genes from different sources are shown in Table 2.

[0069] Table 2

[0070]

[0071] 2) Induced expression of expression vectors

[0072] The recombinant E. coli BL21(DE3) containing pET28a-UMK obtained in step 1) was directly plated onto a solid LB agar plate containing 25 μg / mL kanamycin resistance and cultured at 37 °C for 12–14 h to obtain single colonies. A 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 medium containing 25 μg / mL kanamycin resistance, and cultured at 37 °C with shaking for 12 h. Then, at an inoculum rate of 2% (v / v), the culture was inoculated into 1 L of fresh liquid LB medium containing 25 μg / mL of Kanamycin resistance. The medium was cultured at 37 °C until the 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. The culture was induced at 200 rpm and 25 °C for 20 h. After induction, the culture was centrifuged (4 °C, 4000 rpm, 30 min) to remove the supernatant. The resulting bacterial sludge was washed and resuspended in 30 ml of 0.9% NaCl solution for later use to obtain a bacterial culture containing the UMK target protein.

[0073] Taking the uridine kinase UMK expression vector as an example, the induction expression of recombinant vectors for acetate kinase ACK, pyruvate oxidase POX, and polyphosphate kinase PPK in this invention is the same. Using the same method, bacterial cultures containing the UMK target protein, the ACK target protein, the POX target protein, and the PPK target protein can be obtained.

[0074] 3) Preparation of freeze-dried enzyme powder

[0075] In this invention, both bacterial cultures containing the UMK target protein and the ACK target protein were prepared into lyophilized enzyme powder using the following method: First, the bacterial culture was collected, washed twice with 50 mmol / L Tris-HCl, and then resuspended in 50 mmol / L Tris-HCl (pH 8.0) buffer. The cells were then sonicated in an ice bath (amplitude bar 6, power 500W, on for 2 seconds, off for 5 seconds, for 30 minutes). The sonicated sample was then centrifuged at 12000 rpm at 4°C for 30 minutes. Afterward, the supernatant was placed in a -80°C freeze dryer and lyophilized for 24 hours. The resulting lyophilized sample was then ground to prepare lyophilized crude enzyme powder.

[0076] Example 2: Screening of uridine kinase UMK from different sources

[0077] The lyophilized enzyme powders of uridine kinase UMK from different sources obtained in Example 1 were subjected to a catalytic reaction. 2 mL of phosphate buffer (phosphate concentration 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 were added to an 8 mL reaction flask. The pH of the reaction solution was controlled at 7.5 using 5 M NaOH. 10 mg of uridine kinase from different sources was added to initiate the reaction. The temperature of the reaction solution was controlled at 30°C, and the reaction speed was controlled at 1000 rpm. After 3 hours of reaction, 100 μL of the reaction solution was taken, an equal volume of methanol solution was added for inactivation, and the mixture was centrifuged and filtered. The conversion rate of the product 2′-OME-UDP was determined by high-performance liquid chromatography (HPLC). The reaction process is shown in the figure below. Figure 1 As shown in the figure, the HPLC liquid chromatography results are as follows: Figure 2 As shown in the figure. 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%.

[0078] The conversion rate of 2′-methoxy-uridine diphosphate (2′-OME-UDP) determined by liquid chromatography was calculated as the proportion of the detected product peak area (Area).

[0079] The conversion rate of 2′-methoxy-uridine diphosphate (2′-OME-UDP) Conv(2′-OME-UDP) % = Area[2′-OME-UDP / ( 2′-OME-UMP + 2′-OME-UDP)] *100%.

[0080] The high-performance liquid chromatograph (HPLC) was purchased from Agilent Technologies. The HPLC detection conditions were as follows: mobile phase was 10 mM NH4Oac, mobile phase A was 5% acetonitrile, mobile phase B was 95% acetonitrile, temperature was 40 ℃, flow rate was 1.3 mL / min, and the chromatographic column was Xbridge C18 (purchased from Waters, USA).

[0081] The conversion rates of uridine monophosphate kinase (UMK) from various sources are shown in Table 3 below:

[0082] Table 3

[0083]

[0084] The results show that Arabidopsis thaliana The uridine kinase UMK from this source had the highest catalytic conversion rate, while the conversion rate of 2′-OME-UDP was 71.4%.

[0085] Example 3: Screening for ACK acetate from different sources

[0086] The vector construction and expression of acetate kinase ACK, as well as the preparation of lyophilized crude enzyme powder, were the same as in Example 1. Acetyl kinase ACK enzyme powders from different sources obtained in Example 1 were subjected to a catalytic reaction. 2 mL of phosphate buffer (phosphate concentration 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 were added to the reaction flask (8 mL). The pH of the reaction solution was controlled at 7.5 using 5 M NaOH. Arabidopsis thaliana 10 mg of uridine kinase from a specific source and 10 mg of acetate kinase from different sources were used to initiate the reaction. The temperature of the reaction solution was controlled at 30℃, and the reaction speed was controlled at 1000 rpm. After 20 h of reaction, 100 μL of the reaction solution was taken, an equal volume of methanol solution was added for inactivation, and the mixture was centrifuged and filtered. The conversion rate of the product 2′-OME-UTP was determined by high-performance liquid chromatography (HPLC). The reaction process is shown in the figure below. Figure 3 As shown in the figure, the HPLC liquid chromatography results are as follows: Figure 4 As shown in the figure, the elution time of 2′-OME-UTP was 5.32 min, with a Conv(2′-OME-UTP) % of 30.1%; the elution time of 2′-OME-UDP was 4.91 min, with a Conv(2′-OME-UDP) % of 13.3%; and the elution time of 2′-OME-UMP was 4.33 min, with a Conv(2′-OME-UMP) % of 6.1%. Furthermore, at a reaction time of 20 h, a large amount of the hydrolysis byproduct 2′-methoxy-uridine (2′-OME-Urd) was generated, with a elution time of 0.501 min and a Conv(2′-OME-Urd) % of 50.5%.

[0087] The 2′-OME-UTP conversion rate measured by liquid chromatography is calculated as a percentage of the peak area of ​​the detected product:

[0088] 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%

[0089] The high-performance liquid chromatograph (HPLC) was purchased from Agilent Technologies. The HPLC detection conditions were as follows: mobile phase was 10 mM NH4OAc, mobile phase A was 5% acetonitrile, mobile phase B was 95% acetonitrile, temperature was 40 ℃, flow rate was 1.3 mL / min, and the chromatographic column was Xbridge C18 (purchased from Waters, USA).

[0090] The conversion rates of ACK from various sources are shown in Table 4 below:

[0091] Table 4

[0092]

[0093] Combining the liquid phase diagram with the table above, it can be seen that... Thermotoga maritima Acetyl kinase ACK from this source catalyzed a higher conversion rate, reaching 30.1%.

[0094] Example 4: Preparation of 2′-methoxy-uridine triphosphate using a one-pot enzymatic method with uridine kinase UMK and acetate kinase ACK

[0095] Based on the screening results of Examples 2 and 3, uridine kinase UMK and acetate kinase ACK with the best conversion rates were obtained. A one-pot enzymatic catalysis was then performed to prepare 2′-methoxy-uridine triphosphate. The synthetic route is as follows: Figure 5 As shown.

[0096] Add 2 mL of phosphate buffer (phosphate concentration controlled at 200 mmol / L), 10 mg of 2′-methoxyuridine monophosphate (2′-OME-UMP), 4.5 mg of ATP, 5 mg of magnesium chloride, 2 mg of thiamine pyrophosphate, and 22 mg of acetyl phosphate to the reaction vessel. Adjust the pH of the reaction solution to 7.5 using 5 M NaOH. Add 10 mg of... Arabidopsis thaliana 2mg of uridine diphosphate kinase (UMK) enzyme powder. Thermotoga maritima The purified acetylkinase ACK enzyme powder was used to start the reaction. The temperature of the above reaction solution was controlled at 30℃ and the reaction speed was controlled at 1000 rpm. After 3 h of reaction time, 100 μl of the reaction solution was taken, and an equal volume of methanol solution was added for inactivation. After centrifugation and filtration, the reaction conversion rate of the product 2′-OME-UTP was determined by HPLC.

[0097] The 2′-OME-UTP conversion rate measured by liquid chromatography is calculated as a percentage of the peak area of ​​the detected product:

[0098] Conversion rate of 2′-OME-UTP (2′-methoxy-uridine triphosphate) Conv(2′-OME-UTP) % = Area[2′-OME-UTP / (2′-OME-UMP + 2′-OME-UDP + 2′-OME-UTP)] * 100%

[0099] The high-performance liquid chromatograph (HPLC) was purchased from Agilent Technologies. The HPLC detection conditions were as follows: mobile phase was 10 mM NH4OAc, mobile phase A was 5% acetonitrile, mobile phase B was 95% acetonitrile, temperature was 40 ℃, flow rate was 1.3 mL / min, and the chromatographic column was Xbridge C18 (purchased from Waters, USA).

[0100] like Figure 6 The figure shows the liquid chromatography (LC) chromatogram for the production of 2′-methoxy-uridine triphosphate (2′-OME-UTP) using uridine kinase UMK and acetate kinase ACK, which exhibit the best conversion rates. LC analysis showed that the 2′-OME-UTP eluted at 4.61 min, with a Conv(2′-OME-UTP) % of 92.6%, indicating a high conversion rate of 92.6%. The 2′-OME-UDP eluted at 4.22 min, with a Conv(2′-OME-UDP) % of 1.0%, indicating complete consumption of 2′-OME-UMP. The 2′-OME-Urd eluted at 0.451 min, with a Conv(2′-OME-Urd) % of 6.5%. It can be seen that this method requires a short reaction time of only 3 h, reduces substrate hydrolysis side reactions, and achieves highly efficient synthesis of 2′-methoxy-uridine triphosphate (2′-OME-UTP).

[0101] Example 5: Preparation of 2′-methoxy-uridine triphosphate using a one-pot enzymatic method with uridine kinase UMK, pyruvate oxidase POX, and acetate kinase ACK

[0102] Based on the screening results of Examples 2 and 3, the uridine kinase UMK and acetate kinase ACK with the best conversion rates were obtained, combined with those derived from... Aerococcus vivridans The pyruvate oxidase POX was used in a one-pot enzyme-catalyzed synthesis to prepare 2′-methoxy-uridine triphosphate. The synthetic route is as follows: Figure 7 As shown.

[0103] Add 2 mL of phosphate buffer (phosphate concentration controlled at 200 mmol / L), 10 mg of 2′-methoxyuridine 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 to the reaction vessel. Adjust the pH of the reaction solution to 7.5 using 5 M NaOH. Add 10 mg of... Arabidopsis thalianaUrydidine kinase (UMK) enzyme powder, 2mg Thermotoga maritima Purified acetate kinase (ACK) powder from the source and 2 mg Aerococcus vivridans The pyruvate oxidase POX enzyme powder was used to start the reaction; the temperature of the above reaction solution was controlled at 30℃ and the reaction speed was controlled at 1000 rpm; after the reaction time was 3 h, 100 μl of the reaction solution was taken, an equal volume of methanol solution was added for inactivation, and then the mixture was centrifuged and filtered. The reaction conversion rate of the product 2′-OME-UTP was determined by HPLC.

[0104] The 2′-OME-UTP conversion rate measured by liquid chromatography is calculated as a percentage of the peak area of ​​the detected product:

[0105] Conversion rate of 2′-OME-UTP (2′-methoxy-uridine triphosphate) Conv(2′-OME-UTP) % = Area[2′-OME-UTP / (2′-OME-UMP + 2′-OME-UDP + 2′-OME-UTP)] * 100%

[0106] The high-performance liquid chromatograph (HPLC) was purchased from Agilent Technologies. The HPLC detection conditions were as follows: mobile phase was 10 mM NH4OAc, mobile phase A was 5% acetonitrile, mobile phase B was 95% acetonitrile, temperature was 40 ℃, flow rate was 1.3 mL / min, and the chromatographic column was Xbridge C18 (purchased from Waters, USA).

[0107] like Figure 8 The figure shows the liquid chromatography (LC) chromatogram for the production of 2′-methoxy-uridine triphosphate (2′-OME-UTP) using uridine kinase UMK, acetate kinase ACK, and pyruvate oxidase POX, which exhibit the best conversion rates. LC analysis showed that the peak time for 2′-OME-UTP was 5.265 min, with a Conv(2′-OME-UTP) % of 34.19%, indicating a 2′-OME-UTP conversion rate of 34.19%. The peak times for 2′-OME-UDP were 4.886 min, with a Conv(2′-OME-UDP) % of 9.97%; for 2′-OME-UMP, it was 4.321 min, with a Conv(2′-OME-UMP) % of 2.54%; and for 2′-OME-Urd, it was 0.473 min, with a Conv(2′-OME-Urd) % of 53.13%.

[0108] Example 6 Preparation of 2′-methoxy-uridine triphosphate using a one-pot enzymatic method with uridine kinase UMK, polyphosphate kinase PPK, and acetate kinase ACK

[0109] Based on the screening results of Example 2, uridine kinase UMK and acetate kinase ACK with the best conversion rates were obtained, combined with those derived from... Erysipelotrichaceae bacterium Polyphosphate kinase (PPK) was used in a one-pot enzymatic process to prepare 2′-methoxy-uridine triphosphate. The synthetic route is as follows: Figure 9 As shown.

[0110] Add 2 mL of phosphate buffer (phosphate concentration controlled at 200 mmol / L), 10 mg of 2′-methoxyuridine monophosphate (2′-OME-UMP), 4.5 mg of ATP, 5 mg of magnesium chloride, and 25 mg of sodium hexametaphosphate to the reaction vessel. Adjust the pH of the reaction solution to 7.5 using 5 M NaOH. Add 10 mg of... Arabidopsis thaliana Urydidine kinase (UMK) enzyme powder, 2mg Thermotoga maritima Purified acetate kinase (ACK) powder from the source and 2 mg Erysipelotrichaceae bacterium The polyphosphate kinase (PPK) enzyme powder was used to start the reaction. The temperature of the reaction solution was controlled at 30℃ and the reaction speed was controlled at 1000 rpm. After 3 h of reaction time, 100 μl of the reaction solution was taken, and an equal volume of methanol solution was added for inactivation. After centrifugation and filtration, the reaction conversion rate of the product 2′-OME-UTP was determined by HPLC.

[0111] The 2′-OME-UTP conversion rate measured by liquid chromatography is calculated as a percentage of the peak area of ​​the detected product:

[0112] Conversion rate of 2′-OME-UTP (2′-methoxy-uridine triphosphate) Conv(2′-OME-UTP) % = Area[2′-OME-UTP / (2′-OME-UMP + 2′-OME-UDP + 2′-OME-UTP)] * 100%

[0113] The high-performance liquid chromatograph (HPLC) was purchased from Agilent Technologies. The HPLC detection conditions were as follows: mobile phase was 10 mM NH4OAc, mobile phase A was 5% acetonitrile, mobile phase B was 95% acetonitrile, temperature was 40 ℃, flow rate was 1.3 mL / min, and the chromatographic column was Xbridge C18 (purchased from Waters, USA).

[0114] like Figure 10The figure shows the liquid chromatography (LC) chromatogram for the production of 2′-methoxy-uridine triphosphate (2′-OME-UTP) using uridine kinase UMK, acetate kinase ACK, and polyphosphate kinase PPK, which exhibit the best conversion rates. LC analysis showed that the 2′-OME-UTP eluted at 5.243 min, with a Conv(2′-OME-UTP) % of 48.19%, indicating a 2′-OME-UTP conversion rate of 48.19%. The 2′-OME-UDP eluted at 4.552 min, with a Conv(2′-OME-UDP) % of 33.79%, the 2′-OME-UDP eluted at 3.525 min, with a Conv(2′-OME-UMP) % of 15.7%, and the 2′-OME-Urd eluted at 0.456 min, with a Conv(2′-OME-Urd) % of 2.32%.

[0115] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

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 phosphate compounds to provide inorganic phosphate to adenosine diphosphate, and regenerates adenosine triphosphate to achieve cyclic energy supply. The phosphate compound is acetyl phosphate, and the concentration of acetyl phosphate is 78.5 mmol / L; The concentration of the 2′-methoxy-uridine acid is 15 mmol / L; The concentration of the adenosine triphosphate was 4 mmol / L; The concentration of the uridine kinase is 5 mg / mL; The concentration of the acetate kinase was 1 mg / mL. In the liquid reaction system, the buffer solution is a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer with a concentration of 200 mmol / L. The liquid reaction system also includes a coenzyme and a magnesium salt. The coenzyme is thiamine pyrophosphate with a concentration of 2% of the substrate concentration. The magnesium salt has a concentration of 25 mmol / L and is magnesium chloride. The enzyme-catalyzed reaction is carried out at a temperature of 30°C, for a reaction time of 3 hours, and at a pH of 7.5.

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