Method for preparing cytidine compound

Abstract

The invention discloses a method for preparing a cytidine compound. The method has the characteristics of high conversion rate, high product purity, simple production process, reasonable cost and environmental friendliness. The invention also provides a separated nucleic acid for coding the N-deoxyribose transferase, a recombinant expression vector, a transformant, an enzyme reaction system and application of the N-deoxyribose transferase or the enzyme reaction system in preparation of a compound with a general formula a as shown in the specification.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical intermediate synthesis, specifically relating to a method for preparing cytidine compounds. Background Technology

[0002] 5-Methyl-2'-deoxycytidine (5MedC) is a pyrimidine nucleoside formed by deoxycytidine enzyme-catalyzed methylation. Methylation can affect the structure and function of DNA, thereby regulating cell development, differentiation and function (Rodrigo Hasbún, Luís Valledor, José L. Rodríguez, et al. HPCE quantification of 5-methyl-2'-deoxycytidine in genomic DNA:methodological optimization for chestnut and other woody species.[J]. Plant Physiol Biochem, 2008, 46(8-9):815-822.DOI:10.1016 / j.plaphy.2008.04.009.). In the human body, 3% of all deoxycytidine residues are methylated. Methylation occurs after DNA synthesis, mainly in the CpG sequence (Fraga MF, Uriol E, Diego LB, et al. High-performance capillary electrophoretic method for the quantification of 5-methyl 2'-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues.[J]. Electrophoresis, 2015, 23(11):1677-1681.DOI:10.1002 / 1522-2683(200206)23:11<1677::AID-ELPS1677>3.0.CO;2-Z.). 5-methyl-2'-deoxycytidine is involved in the regulation of gene expression and the silencing of invading viral genomes in the CpG sequence. It is the only nucleoside that is majorly modified in eukaryotic DNA. After incorporation into single-stranded DNA, it can signal DNA methylation through cis-action. Abnormal methylation of 5-methyl-2'-deoxycytidine is associated with the occurrence and development of some diseases.For example, abnormal DNA methylation in certain tumors can lead to gene misregulation and cellular dysfunction (Application of N-Halogeno-N-sodiobenzenesulfonamide Reagents to the Selective Detection of 5-Methylcytosine in DNA Sequences[J]. Journal of the American Chemical Society, 2013, 135(4):1240-1243.DOI:10.1021 / ja311229n.). Furthermore, 5-methyl-2'-deoxycytidine and its derivatives have been used in epigenetic studies to investigate the dynamics of DNA methylation patterns in gene expression control. It is also speculated that it is a key element controlling vertebrate gene function and cell differentiation (Razin A, Riggs A D. DNA methylation and gene function. Science[J]. 1980.DOI:10.1126 / science.6254144.).

[0003] WING L. et al. reported a chemical synthesis method for 5-methyl-2'-deoxycytidine in the literature. The method uses thymidine as the starting material and obtains the product through two steps. First, thymidine undergoes a substitution reaction with triazole to generate the intermediate 5-methyl-4-(1,2,4-triazolyl)-1-(β-D-3,5-di-O-acetyl-2-deoxyfuran-O syl)pyrimidin-2(1H)-one. Then, it is treated with ammonia water to finally obtain 5-methyl-2'-deoxycytidine. (Sung W L. ChemInform Abstract: CHEMICAL CONVERSION OF THYMIDINE INTO 5-METHYL-2′-DEOXYCYTIDINE[J]. ChemischerInformationsdienst, 1982, 13(7). DOI: 10.1002 / chin.198207315.) Although the reaction system of this method is relatively mild, the first step of the reaction takes up to three days, and a more efficient and convenient synthetic route still needs to be explored.

[0004] In recent years, N4-benzoyl-2'-deoxycytidine has attracted widespread attention as a low-polyester nucleotide inducer and manufacturing intermediate for various antiviral, anticancer, anticlonal disease, and antirheumatic agents, and as an anti-DNA raw material (JP2004210681A). Cytidine compounds with similar structures to N4-benzoyl-2'-deoxycytidine (general formula as shown in formula a, where R is, for example, NH2, Cl, SH, F, or CH3) can also serve as important pre-synthetic intermediates for drug development, showing broad application prospects.

[0005]

[0006] Existing technologies all use chemical synthesis methods to prepare N4-benzoyl-2'-deoxy-5-methylcytidine (as shown in Formula 1). These methods often suffer from drawbacks such as generating significant amounts of waste, which is detrimental to the environment; producing numerous byproducts; and complex production processes due to the synthetic routes. Currently, there is an urgent need for methods to prepare cytidine compounds that offer high conversion rates, high product purity, simple production processes, reasonable costs, and environmental friendliness. Summary of the Invention

[0007] To address the shortcomings of existing technologies that use chemical synthesis methods to prepare compounds with the general formula shown in formula a, such as generating a large amount of waste that is detrimental to the environment, producing numerous byproducts, and having complex production processes due to the frequent use of synthetic routes, this invention provides a method for preparing cytidine compounds, which features high conversion rate, high product purity, simple production process, reasonable cost, and environmental friendliness.

[0008] The reaction route of this invention is shown below:

[0009]

[0010] Where R can be NH2, Cl, thio group, F, CH3, etc.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0012] The first aspect of the present invention provides a method for preparing cytidine compounds, wherein the general formula of the cytidine compounds is shown in Formula a, and the method includes the step of reacting an N-deoxyribotransferase with a substrate.

[0013] The reaction route is shown below:

[0014]

[0015] Wherein, R is an alkyl, halogen, amino, or thio group; the N-deoxyribotransferase is derived from the genus Lactobacillus, preferably from Lactobacillus fermentum, Lactobacillus sakei, or Lactobacillus helveticus.

[0016] In some embodiments of the present invention, the alkyl group is C1 to C6, preferably methyl; the halogen is Cl or F; and / or, the N-deoxyribotransferase comprises an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

[0017] In some preferred embodiments of the present invention, the reaction system of the reaction comprises a compound as shown in formula b, deoxythymidine, N-deoxyribotransferase, and a buffer solution.

[0018] In some preferred embodiments of the present invention, the reaction system further satisfies at least one of the following:

[0019] (i) The molar ratio of the compound shown in formula b to deoxythymidine is 1:(0.5-10), preferably 1:(1-5), for example 1:4;

[0020] (ii) The mass ratio of the N-deoxyribotransferase to deoxythymidine is 1:(5-50), preferably 1:(10-30), for example 1:21;

[0021] (iii) The buffer solution is Tris-HCl with a pH of 6-9 and a concentration of 20-100 mmol, for example, 50 mmol;

[0022] (iv) The reaction system of the reaction also contains a co-solvent, which is selected from one or more of DMSO, Triton-X100, Tween 80 and PEG-200.

[0023] In some embodiments of the present invention, the N-deoxyribonuclease is used as a liquid enzyme, such as a crude enzyme solution, a solid enzyme, such as enzyme powder, an immobilized enzyme, or cells expressing the N-deoxyribonuclease.

[0024] In some preferred embodiments of the present invention, the N-deoxyribotransferase is used in the form of enzyme powder.

[0025] In some embodiments of the present invention, the pH of the reaction is 7-8; and / or the temperature of the reaction is 40°C-60°C, preferably 50°C-60°C.

[0026] A second aspect of the present invention provides an isolated nucleic acid encoding an N-deoxyribotransferase, said nucleic acid comprising a nucleotide sequence as shown in SEQ ID NO:7.

[0027] A third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the second aspect.

[0028] In some embodiments of the present invention, the backbone plasmid of the recombinant expression vector is pET-28a(+).

[0029] A fourth aspect of the present invention provides a transformant comprising a nucleic acid as described in the second aspect or a recombinant expression vector as described in the third aspect.

[0030] In some embodiments of the present invention, the substrate bacteria of the transformant are Escherichia coli, such as E. coli BL21(DE3) or E. coli MG1655.

[0031] The fifth aspect of the present invention provides a method for preparing N-deoxyribotransferase, the method comprising the steps of growing the transformant as described in the fourth aspect in a culture medium and expressing the N-deoxyribotransferase.

[0032] In some embodiments of the present invention, the method includes:

[0033] (1) The seed culture of the transformant was inoculated into a culture medium and cultured.

[0034] (2) Add an inducing agent and culture with shaking;

[0035] (3) The bacterial cells obtained in step (2) are broken up and the supernatant is collected;

[0036] In some optional embodiments of the present invention, the method further includes the step of freeze-drying the supernatant obtained in step (3) to prepare enzyme powder.

[0037] In some embodiments of the present invention, the method satisfies one or more of the following conditions:

[0038] In step (1), the culture medium is LB liquid culture medium, the inoculation amount of the seed liquid is 1-5%, the % is a volume percentage, and / or the culture temperature is 30-37℃;

[0039] In step (2), the inducing agent is isopropyl-β-D-thiogalactoside, the temperature of the shaking culture is 20-25℃, the shaking speed of the shaking culture is 120-200rpm, and / or the shaking culture time is 16-22h;

[0040] In step (3), the crushing is ultrasonic crushing, and the crushing is carried out under ice bath conditions.

[0041] The sixth aspect of the present invention provides an enzyme reaction system, which is a reaction system as defined in the method described in the first aspect.

[0042] The seventh aspect of the present invention provides the use of N-deoxyribonuclease or the enzyme reaction system as described in the sixth aspect in the preparation of compounds of general formula a;

[0043]

[0044] Wherein, R is an alkyl, halogen, amino, or thio group.

[0045] In some embodiments of the present invention, the alkyl group is C1 to C6, preferably methyl; the halogen is Cl or F; and / or, the N-deoxyribonuclease is derived from the genus Lactobacillus, preferably from Lactobacillus fermentum, Lactobacillus sakei, or Lactobacillus helveticus.

[0046] In some specific embodiments of the present invention, the N-deoxyribotransferase comprises an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

[0047] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0048] The reagents and raw materials used in this invention are all commercially available.

[0049] The significant advantages of this invention are as follows: It provides a method for preparing cytidine compounds, and also provides an isolated nucleic acid encoding N-deoxyribonuclease, a recombinant expression vector, a transformant, a method for preparing N-deoxyribonuclease, an enzyme reaction system, and the application of the N-deoxyribonuclease or enzyme reaction system in the preparation of compounds of formula a. The method for preparing cytidine compounds provided by this invention utilizes a one-step enzymatic catalysis of nucleoside 2'-deoxyribonuclease (NDT) to replace several protecting-deprotecting steps in the original chemical glycosylation reaction, resulting in high conversion rate, high product purity, a simplified production process, and environmental friendliness, significantly reducing production costs. After reacting at 50°C, 1000 rpm for 20 hours, the conversion rate reaches 87.7%. Attached Figure Description

[0050] Figure 1The reaction route for the enzymatic preparation of compounds with the general formula shown in formula a is given.

[0051] Figure 2 The HPLC results of N4-benzoyl-2'-deoxy-5-methylcytidine obtained by NDT lyophilized enzyme powder in Example 4 are shown.

[0052] Figure 3 The reaction conversion rate of NDT in Example 6 was measured at different pH values.

[0053] Figure 4 The reaction conversion rate of NDT in Example 6 was measured at different temperatures. Detailed Implementation

[0054] This invention discloses a nucleoside 2′-deoxyribosyltransferase (NDT) derived from *Limosilactobacillus fermentum*, *Latilactobacillus sakei*, or *Lactobacillus shelveticus*, and provides a method for preparing N4-benzoyl-2′-deoxy-5-methylcytidine and its analogues using this NDT. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments; those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0055] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0056] Example 1: Construction and transformation of recombinant vectors

[0057] Taking the construction of recombinant strain E.coli BL21(DE3)pET-28a-NDT as an example.

[0058] Activate E. coli DH5α / pET-28a(+) strain (E. coli DH5α purchased from Novizan Biotechnology Co., Ltd.), add 3-5 μL of pET-28a-NDT (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 s, quickly return to ice water 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 kanamycin resistance, and incubate overnight at 37℃ for 12 h to obtain recombinant E. coli BL21(DE3) containing pET-28a-NDT.

[0059] Among them, NDT is derived from Limosilactobacillus fermentum, with the amino acid sequence shown in SEQ ID NO:1 and the nucleotide sequence shown in SEQ ID NO:7.

[0060] The formula for antibiotic-free LB medium is as follows: yeast extract 5g / L, sodium chloride 10g / L, peptone 10g / L;

[0061] The formulation of LB agar plate medium containing kanamycin resistance is as follows: yeast extract 5 g / L, sodium chloride 10 g / L, peptone 10 g / L, agar powder 20 g / L, kanamycin 25 μg / mL.

[0062] Example 2: Construction and Expression of Expression Vectors

[0063] The construction and expression of recombinant E. coli BL21(DE3) containing pET28a-NDT are taken as an example.

[0064] The transformed recombinant strain *E. coli* BL21(DE3) containing pET28a-NDT was directly plated onto solid LB agar plates containing 25 μg / mL kanamycin resistance and cultured at 37°C for 12–14 h to obtain single colonies. Single colonies of *E. coli* BL21(DE3) containing the NDT recombinant vector were picked from the kanamycin plates 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 a 2% (v / v) inoculation rate, the culture was separately inoculated into 1 L of fresh liquid LB medium containing 25 μg / mL kanamycin resistance and cultured at 37°C until OD (dose elapsed). 600When the concentration was approximately 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG; purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to a final concentration of 1.0 mmol / L. Expression was induced at 200 rpm and 25℃ for 20 h. After centrifugation (4℃, 4000 rpm, 30 min), the supernatant was removed, and the resulting bacterial sludge was washed and resuspended in 30 mL of 0.9% NaCl solution for later use.

[0065] Example 3: Preparation of freeze-dried enzyme powder

[0066] Take the preparation of NDT enzyme powder as an example.

[0067] The collected bacterial suspension was 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, 30 min). The sonicated sample was centrifuged at 12000 rpm at 4℃ for 30 min, and the supernatant was placed in a -80℃ freeze dryer for 24 h. The resulting freeze-dried sample was then ground to prepare freeze-dried enzyme powder.

[0068] Example 4: Preparation of NDT from Other Sources

[0069] The vector construction and expression of NDT derived from Lactiplantibacillus plantarum WCFS1, and the enzyme powder preparation were the same as in Examples 1-3.

[0070] The vector construction and expression of NDT derived from Latilactobacillus sakei, and the preparation of enzyme powder were the same as in Examples 1-3.

[0071] The vector construction and expression of NDT derived from Thiomonas arsenitoxydans, and the enzyme powder preparation were the same as in Examples 1-3.

[0072] The vector construction and expression of NDT derived from Lactobacillus helveticus, and the enzyme powder preparation were the same as in Examples 1-3.

[0073] The vector construction and expression of Lactobacillus johnsonii NCC 533-derived NDT, and the enzyme powder preparation were the same as in Examples 1-3.

[0074] Example 5: Synthesis of N4-benzoyl-2'-deoxy-5-methylcytidine (CAS: 104579-02-4) by NDT enzymes from different sources.

[0075] The reaction route is shown below:

[0076]

[0077] R = CH3

[0078] Take 2 mg of the enzyme powder obtained in Examples 1-4, add 10 mg of N4-benzoyl-5-methylcytosine as shown in Formula 2, 42 mg of deoxythymidine as shown in Formula 3, 80 μL of DMSO, and 1.92 mL of buffer solution. Place the above reaction solution at 50 °C and 1000 rpm for 20 h. Take 50 μL of the reaction solution, add 50% methanol solution to inactivate it, centrifuge and filter, and determine the reaction conversion rate of the product N4-benzoyl-2'-deoxy-5-methylcytosine by HPLC.

[0079] 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 35℃, flow rate was 1.5 mL / min, and the chromatographic column was Xbridge C18 (purchased from Waters, USA).

[0080] The experimental results are shown in Table 1:

[0081] Table 1. Synthesis of N4-benzoyl-2'-deoxy-5-methylcytidine by NDT enzymes from different sources.

[0082]

[0083] The results showed that the NDT enzyme derived from Limosilactobacillus fermentum had the highest conversion rate, so it was chosen for subsequent screening.

[0084] Example 6: Synthesis of deoxyribotransferases (compounds shown in Formula 1)

[0085]

[0086] Take 4 mg of the lyophilized enzyme powder obtained in Examples 1-3, and add N4-benzoyl-5-methylcytosine as shown in Formula 2, deoxythymidine as shown in Formula 3, and buffer solution respectively. The corresponding reaction systems are shown in Table 2.

[0087] Table 2 NDT Catalytic Reaction System

[0088]

[0089] The above reaction solution was placed at 50℃ and 1000rpm for 20h. After that, 50μL of the reaction solution was taken, and 50% methanol solution was added to inactivate it. After centrifugation and filtration, the reaction conversion rate was obtained by determining the product N4-benzoyl-2'-deoxy-5-methylcytidine by HPLC.

[0090] The high-performance liquid chromatograph (HPLC) was purchased from Agilent Technologies. HPLC detection conditions were as follows: mobile phase 10 mM NH4OAc, mobile phase A: 5% acetonitrile, mobile phase B: 95% acetonitrile, temperature 35℃, flow rate 1.5 mL / min, and Xbridge C18 column (purchased from Waters, USA). Results showed that NDT conversion was highest at 87.1% after 20 h. The reaction results are shown in [Figure number missing]. Figure 2 .

[0091] Example 7: Synthesis of N4-benzoyl-2'-deoxy-5-aminocytidine

[0092] The implementation was the same as in Example 6, and the reaction route was the same as in Example 5, except that the cytosine R group in the raw materials was replaced with NH2.

[0093] Take 4 mg of the enzyme powder obtained in Examples 1-3, add 20 mg of N4-benzoyl-5-aminocytosine, 84 mg of deoxythymidine, 160 μL of DMSO, and 1.84 mL of buffer solution. Place the above reaction solution at 50 °C and 1000 rpm for 20 h. Take 50 μL of the reaction solution, add 50% methanol solution to inactivate it, centrifuge and filter, and determine the reaction conversion rate of the product N4-benzoyl-2'-deoxy-5-methylcytidine by HPLC.

[0094] The detection method was the same as in Example 5. The results showed that the conversion rate of NDT to N4-benzoyl-2'-deoxy-5-aminocytidine was 75.5%.

[0095] Example 8: Synthesis of N4-benzoyl-2'-deoxy-5-chloro-cytidine

[0096] The implementation was the same as in Example 6, and the reaction route was the same as in Example 5, except that the cytosine R group in the raw materials was replaced with Cl.

[0097] Take 4 mg of the enzyme powder obtained in Examples 1-3, add 22 mg of N4-benzoyl-5-chloro-cytosine, 84 mg of deoxythymidine, 160 μL of DMSO, and 1.84 mL of buffer solution. Place the above reaction solution at 50 °C and 1000 rpm for 20 h. Take 50 μL of the reaction solution, add 50% methanol solution to inactivate it, centrifuge and filter, and determine the reaction conversion rate of the product N4-benzoyl-2'-deoxy-5-chloro-cytosine by HPLC.

[0098] The detection method was the same as in Example 5. The results showed that the conversion rate of NDT to N4-benzoyl-2'-deoxy-5-chloro-cytidine was 65.1%.

[0099] Example 9: Synthesis of N4-benzoyl-2'-deoxy-5-thiocytidine

[0100] The implementation was the same as in Example 6, and the reaction route was the same as in Example 5, except that the cytosine R group in the raw materials was replaced with a thio group.

[0101] Take 4 mg of the enzyme powder obtained in Examples 1-3, add 22 mg of N4-benzoyl-5-thiocytosine, 84 mg of deoxythymidine, 160 μL of DMSO, and 1.84 mL of buffer solution. Place the above reaction solution at 50 °C and 1000 rpm for 20 h. Take 50 μL of the reaction solution, add 50% methanol solution to inactivate it, centrifuge and filter. Measure the product N4-benzoyl-2'-deoxy-5-thiocytosine by HPLC to obtain the reaction conversion rate.

[0102] The detection method was the same as in Example 5. The results showed that the conversion rate of NDT to N4-benzoyl-2'-deoxy-5-thio-cytidine was 62.0%.

[0103] Example 10: Synthesis of 4-benzoyl-2'-deoxy-5-fluorocytidine

[0104] The implementation was the same as in Example 6, and the reaction route was the same as in Example 5, except that the cytosine R group in the raw materials was replaced with F.

[0105] Take 4 mg of the enzyme powder obtained in Examples 1-3, add 20 mg of N4-benzoyl-5-fluoro-cytosine, 84 mg of deoxythymidine, 160 μL of DMSO, and 1.84 mL of buffer solution. Place the above reaction solution at 50 °C and 1000 rpm for 20 h. Take 50 μL of the reaction solution, add 50% methanol solution to inactivate it, centrifuge and filter. Measure the product N4-benzoyl-2'-deoxy-5-fluoro-cytosine by HPLC to obtain the reaction conversion rate.

[0106] The detection method was the same as in Example 5. The results showed that the conversion rate of NDT to N4-benzoyl-2'-deoxy-5-fluoro-cytidine was 34.0%.

[0107] In conjunction with Examples 6-10, NDT showed the highest conversion rate for N4-benzoyl-2'-deoxy-5-methylcytidine.

[0108] Example 11: Determination of Optimal Catalytic Temperature and pH for NDT

[0109] Optimal pH: 4 mg of enzyme powder obtained in Examples 1-3 was reacted with 20 mg N4-benzoyl-5-methylcytosine and 84 mg deoxythymidine at different pH values ​​(4.0-11.0) at 50°C and 1000 rpm for enzyme activity. After 20 h, the conversion rate of N4-benzoyl-2'-deoxy-5-methylcytosine was measured. The pH of the solution was adjusted using three buffer systems.

[0110] The three buffer systems are: citrate-sodium citrate buffer (50 mmol, pH 4-6), Tris-HCl buffer (50 mmol, pH 6-9), and Na2HPO4-NaOH buffer (50 mmol, pH 9-11).

[0111] The reaction results are shown in Figure 3 As shown in Table 3, the conversion rate of the reaction was the highest at pH 7.0 (87.2%). The conversion rate increased continuously from pH 4.0 to 7.0, remained basically unchanged from pH 7.0 to 8.0, and decreased from pH 8.0 to 11.0.

[0112] Table 3 pH optimization

[0113]

[0114] Optimal temperature: 4 mg of enzyme powder was reacted with 20 mg N4-benzoyl-5-methylcytosine and 84 mg deoxythymidine at different temperatures (30-70℃) in Tris-HCl buffer (50 mM, pH 7.0) at 1000 rpm for enzyme activity. The conversion rate of N4-benzoyl-2'-deoxy-5-methylcytosine was measured after 20 h.

[0115] Table 4 Temperature Optimization

[0116] Temperature (°C) Conversion rate (%) 30 51 40 63 50 87.7 60 77 70 20

[0117] The reaction results are shown in Figure 4 According to Table 4, the reaction conversion rate is the highest at 50℃ (87.7%). The reaction conversion rate increases continuously from 30℃ to 50℃. When the temperature exceeds 50℃, the reaction conversion rate decreases, and the reaction conversion rate is 20% at 70℃.

[0118] According to the above embodiments, the optimal pH for the NDT-catalyzed reaction to generate N4-benzoyl-2'-deoxy-5-methylcytidine is 7.0, and the optimal reaction temperature is 50°C.

[0119] sequence list

[0120] amino acid sequence

[0121] SEQ ID NO:1

[0122] Limosilactobacillus fermentum

[0123] MKNTDPVANTKIYLATSFFNEEQRARIPQALAQLEANPTVGVVHQPFDFQYKDARVDSDPAGVFGSLEWQIATYNNDLNAVGTSDVCVALYDMDQIDEGICMEIGMFVALHKPIVLLPFTKKDKSAYEANLMLARGVTTWLEPNDFSPPLKFPNFFPFPNFFFQVFFQV

[0124] SEQ ID NO:2

[0125] Lactiplantibacillus plantarum WCFS1

[0126] MNNVYLAAPFFDEAQQQVKSALLANPTINPDGIFIPEEHQFEEEPFGSRAWQQYVYASDMRQVHRADVVVAILDFDMTSATNEPDSG™FEIGAAVAEKTPVIIVQFDANKELNLMIAQGLTAYFDASKDGLKELSAYNCDDLRSKPAHRPVF

[0127] SEQ ID NO:3

[0128] Latilactobacillus sakei

[0129] MSTNKKIYLAGPFFSPEQIERLDKVAALLAQNPTVVETESIFRPNQHSYSEAEFGSFEWQTATFGFDIRQIDQADLVVAVLDYQTESGQFEPDSG™WECGYAFAHNKPVVLARYKDDLPINLMLSGSATAVFNGDADLTNLATYDFNALQTKYVATKIY

[0130] SEQ ID NO:4

[0131] Thiomonas arsenitoxydans

[0132] MTKPRVYLAGPDLFFEDRDARYARLRAACANAGLEAVAPTDGLEVHTERPLSVAEQIYQHNLRLLRDCDGVLVNLSPFRGVEPDSGTVFEAAFATAIGKPVAAWIGDHWNTQERSAVLRKVWRDADGRVRDSHDGGLVEDFGLPANLMLACSFSVSPAPWQAIERLLMALPVMEGV

[0133] SEQ ID NO:5

[0134] Lactobacillus helveticus

[0135] MNKKKTLYFGAGWFNEKQNKAYKEAMAALKENPTVDLENSYVPLENQYKGIRIDEHPEYLHNIEWASATYHNDLVGIKTSDVMLGVYLPEEEDVGLGMELGYALSQGKYILLVIPDEDYGKPINLMSWGVCDNAIKISELKDFDFNKPRYNFYDGAVY

[0136] SEQ ID NO:6

[0137] Lactobacillus johnsonii NCC 533

[0138] MAGWFTETQNKAYKDAMSALNANPTIDLENSYVPLQNQYKDIRVDEHPEYLHDKEWAQATYNGDLVGIKTSDVMLGVYVPKEEDVGLGMELGYAMSQGKYVLLVIPDELYGESINLMSWGVADNVIKMSELATFDFNRPRYNFYDGAVY

[0139] nucleotide sequence

[0140] SEQ ID NO:7

[0141] Escherichia coli BL21(DE3) / pEt-28a(+)

[0142] ATGAAAAACACCGATCCGGTGGCGAACACCAAAATTTATCTGGCGACGAGCTTTTTTAACGAAGAACAGCGCGCGCGCATTCCGCAAGCGCTGGCGCAGCTGGAAGCGAACCCGACCGTGGGCGTGGTGCATCAGCCGTTTGATTTTCAGTATAAAGATGCGCGCGTGGATAGCGATCCGGCGGGCGTGTTTGGCAGCCTGGAATGGCAGATTGCGACCTATAACAACGATCTGAACGCGGTGGGCACGAGCGATGTGTGCGTGGCGCTGTATGATATGGATCAGATTGATGAAGGCATTTGCATGGAAATTGGCATGTTTGTGGCGCTGCATAAACCGATTGTGCTGCTGCCGTTTACCAAAAAAGATAAAAGCGCGTATGAAGCGAACCTGATGCTGGCGCGCGGCGTGACCACCTGGCTGGAACCGAACGATTTTAGCCCGCTGAAAGATTTTAACTTTAACCATCCGATGGCGCAGCCGTTTCCGCCGTTTAAAGTGTTTTAA。

Claims

1. A method for preparing cytidine compounds, wherein the general formula of the cytidine compounds is shown in formula a, characterized in that, The method includes the step of reacting N-deoxyribonuclease with a substrate; The reaction route is shown below: Wherein, R is an alkyl, halogen, amino, or thiol group; the N-deoxyribotransferase is derived from the genus *Lactobacillus*, preferably from *Limosilactobacillus fermentum*, *Latilactobacillus sakei*, or *Lactobacillus helveticus*.

2. The method as described in claim 1, characterized in that, The alkyl group is C1 to C6, preferably methyl; the halogen is Cl or F; and / or, the N-deoxyribotransferase contains an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; Preferably, the reaction system comprises a compound as shown in Formula b, deoxythymidine, N-deoxyribotransferase, and a buffer solution; More preferably, the reaction system also satisfies at least one of the following: (i) The molar ratio of the compound shown in formula b to deoxythymidine is 1:(0.5-10), preferably 1:(1-5); (ii) The mass ratio of the N-deoxyribotransferase to deoxythymidine is 1:(5-50), preferably 1:(10-30); (iii) The buffer solution is Tris-HCl with a pH of 6-9 and a concentration of 20-100 mmol; (iv) The reaction system of the reaction also contains a co-solvent, which is selected from one or more of DMSO, Triton-X100, Tween 80 and PEG-200.

3. The method as described in claim 1 or 2, characterized in that, The N-deoxyribonuclease is used in the form of a liquid enzyme, such as a crude enzyme solution, a solid enzyme, such as enzyme powder, an immobilized enzyme, or cells expressing the N-deoxyribonuclease; Preferably, the N-deoxyribonuclease is used in the form of enzyme powder.

4. The method according to any one of claims 1-3, characterized in that, The pH of the reaction is 7-8; and / or the temperature of the reaction is 40℃-60℃, preferably 50℃-60℃.

5. An isolated nucleic acid encoding N-deoxyribotransferase, characterized in that, The nucleic acid contains a nucleotide sequence as shown in SEQ ID NO:

7.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 5; Preferably, the backbone plasmid of the recombinant expression vector is pET-28a(+).

7. A transformant, characterized in that, The transformant comprises the nucleic acid as described in claim 5 or the recombinant expression vector as described in claim 6; Preferably, the substrate bacteria of the transformant is Escherichia coli, such as E. coli BL21(DE3) or E. coli MG1655.

8. A method for preparing N-deoxyribotransferase, characterized in that, The method includes the steps of growing the transformant as described in claim 7 in a culture medium and expressing the N-deoxyribonuclease; Preferably, the method includes: (1) The seed culture of the transformant was inoculated into a culture medium and cultured. (2) Add an inducing agent and culture with shaking; (3) The bacterial cells obtained in step (2) are broken up and the supernatant is collected; Optionally, the method further includes the step of freeze-drying the supernatant obtained in step (3) to prepare enzyme powder; Preferably, the method satisfies one or more of the following conditions: In step (1), the culture medium is LB liquid culture medium, the inoculation amount of the seed liquid is 1-5%, the % is a volume percentage, and / or the culture temperature is 30-37℃; In step (2), the inducing agent is isopropyl-β-D-thiogalactoside, the temperature of the shaking culture is 20-25℃, the shaking speed of the shaking culture is 120-200rpm, and / or the shaking culture time is 16-22h; In step (3), the crushing is ultrasonic crushing, and the crushing is carried out under ice bath conditions.

9. An enzyme reaction system, characterized in that, The enzyme reaction system is the reaction system defined in any one of claims 2-4.

10. The use of an N-deoxyribotransferase or the enzyme reaction system as described in claim 9 in the preparation of compounds of general formula a; in, R is an alkyl, halogen, amino, or mercapto group; Preferably, the alkyl group is C1 to C6, preferably methyl; the halogen is Cl or F; and / or, the N-deoxyribonuclease is derived from the genus Lactobacillus, preferably from Lactobacillus fermentum, Lactobacillus sakei, or Lactobacillus helveticus. More preferably, the N-deoxyribotransferase comprises an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

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