CN115109814B

CN115109814B - Method for preparing 2 '-deoxy-2' -fluoro-beta-D-arabinoside adenosine analogue

Info

Publication number: CN115109814B
Application number: CN202211028953.XA
Authority: CN
Inventors: 张波琨; 谢磊; 李�杰; 黄月园; 孙丰来; 谈宏宇
Original assignee: Changzhou Hequan Pharmaceutical Co ltd; Changshu Yaomingkangde New Drug Development Co ltd
Current assignee: Changzhou Hequan Pharmaceutical Co ltd; Changshu Yaomingkangde New Drug Development Co ltd
Priority date: 2022-08-26
Filing date: 2022-08-26
Publication date: 2022-11-29
Anticipated expiration: 2042-08-26
Also published as: CN115109814A

Abstract

The invention discloses a method for preparing 2 '-deoxy-2' -fluoro-beta-D-arabinoside adenosine analogues, which comprises the following steps: step 1: in a liquid reaction system, taking a compound shown in a formula II and a compound shown in a formula III as substrates, and carrying out enzyme catalytic reaction under the catalysis of deoxyribotransferase NDT to form a compound shown in a formula I; and 2, step: separating the compound shown in the formula I from the reaction system after the reaction in the step 1; wherein the Genbank number of the deoxyribotransferase is WP _011253881.1. The invention also provides a reaction system. The invention has the advantages of chemical modification of nucleotide by adopting enzyme catalysis reaction, short reaction step, mild condition and high substrate conversion rate.

Description

Method for preparing 2 '-deoxy-2' -fluoro-beta-D-arabinosyl adenosine analogue

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a method for preparing a 2 '-deoxy-2' -fluoro-beta-D-arabinosyl adenosine analogue.

Background

The 2 '-deoxy-2' -fluoro-beta-D-arabinoside adenosine analog is a nucleoside analog which has good antiviral and antitumor activities and can be effectively embedded into a DNA chain and an RNA chain of a virus, thereby inhibiting division and replication of cells.

RNA interference, a technique for controlling normal gene expression, has recently been used for the treatment of tumors, infectious diseases, and metabolic diseases. However, RNA interference techniques still have certain development obstacles, such as 1) the degradation of oligonucleotides by nucleases; 2) Poor binding selectivity and stability to the target RNA; 3) Triggering an unwanted immune response; 4) Transport and binding proteins present certain obstacles. Chemically modified nucleotides may solve some of the above-mentioned obstacles. In the modified chemical group, the 2 '-deoxy-2' -fluoro-beta-D-arabinose modified nucleoside has unique RNA interference characteristics, the effect of the 2 '-deoxy-2' -fluoro-beta-D-arabinose modified nucleotide in the synthesized siRNA is more than 4 times that of the common siRNA, the half-life period in serum is about 6h, and the half-life period is more than 24 times that of the common siRNA. Therefore, the use of 2 '-deoxy-2' -fluoro- β -D-arabinose for chemical modification of nucleotides is a preferred option to overcome RNA interference techniques.

At present, 2 '-deoxy-2' -fluoro-beta-D-arabinosyl adenosine analogs are generally synthesized by chemical synthesis methods and enzymatic methods. The chemical synthesis method is a multi-step process, needs a process of protecting and deprotecting groups in order to realize regioselectivity and stereoselectivity, has complex operation steps and high cost, is easy to generate toxic and harmful substances, and causes pollution to the environment. The enzymatic synthesis is more and more emphasized by people due to factors such as high regioselectivity, stereoselectivity, mild reaction conditions, environmental protection and no pollution, but the enzymatic synthesis of the 2 '-deoxy-2' -fluoro-beta-D-arabinosyladenosine analogue has the defect of low substrate conversion rate, which seriously limits the application of the enzymatic synthesis in preparation and production.

In the prior art, 2 '-deoxy-2' -fluoro-beta-D-arabinosyladenosine and analogues thereof are synthesized by an enzymatic method, and 2 '-deoxy-2' -fluoro-beta-D-arabinosyluridine is used as a substrate to generate a product under the transglycosylation action of nucleoside phosphorylase. Coli purine nucleoside phosphorylase (PNP, ec.2.4.2.1) and pyrimidine nucleoside phosphorylase (PyNP, ec.2.4.2.2) have low conversion rates when 2 '-deoxy-2' -fluoro- β -D-arabinose modified nucleotides are used as substrates, limiting their large-scale application.

Therefore, there is a need in the art to develop a new method for preparing 2 '-deoxy-2' -fluoro- β -D-arabinoside adenosine analogs.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of a 2 '-deoxy-2' -fluoro-beta-D-arabinoside adenosine analogue, which comprises the following steps:

step 1: in a liquid reaction system, taking a compound of a formula II and a compound of a formula III as substrates, and removingCarrying out an enzyme catalytic reaction under the catalysis of an ribose transferase NDT to form a compound shown in a formula I; wherein R is selected from NH ₂ Cl, S or F;

and 2, step: separating the compound shown in the formula I from the reaction system after the reaction in the step 1;

the Genbank serial number of the deoxyribotransferase NDT is WP _011253881.1, the compound shown in the formula II is 2 '-deoxy-2' -fluoro-beta-D-arabinouridine, and the compound shown in the formula III is an adenine analogue.

In some embodiments, the compound of formula II is present at a concentration of 0.1 to 500g/L. Preferably, the compound of formula II is present at a concentration of 0.1g/L or more, 0.2g/L or more, 0.5g/L or more, 1g/L or more, 10g/L or more, 25g/L or more, 50g/L or more, 75g/L or more, 100g/L or more, 125g/L or more, 150g/L or more, 175g/L or more, 200g/L or more, 220g/L or more, 240g/L or more, 260g/L or more, 280g/L or more, 300g/L or more, 320g/L or more, 350g/L or more, 400g/L or more, or 450g/L or more.

Preferably, the concentration of the compound shown in the formula II is 1-500g/L; more preferably, the concentration of the compound shown in the formula II is 10-400g/L; most preferably, the compound of formula II is present at a concentration of 50 to 350g/L.

In some embodiments, the molar ratio of the compound of formula II to the compound of formula III is 1: (1 to 10). Preferably, the molar ratio of the compound of formula II to the compound of formula III is 1: (1.1 to 5). More preferably, the molar ratio of the compound of formula II to the compound of formula III is 1: (1.5 to 3).

In some embodiments, the mass ratio (w/w) of the deoxyribotransferase NDT to the compound of formula II is (0.1 to 10): 1. preferably, the mass ratio (w/w) of the deoxyribotransferase NDT to the compound of formula II is (0.1 to 5): 1. more preferably, the mass ratio (w/w) of the deoxyribotransferase NDT to the compound of the formula II is (0.5 to 2): 1.

in some embodiments, the liquid reaction system is an aqueous system. Preferably, the aqueous system contains a buffer.

In some embodiments, the buffer of the liquid reaction system is citric acid-sodium citrate buffer, na ₂ HPO ₄ -NaOH disodium hydrogen phosphate-sodium hydroxide buffer or tris buffer. Preferably, the buffer of the reaction system is a Tris (hydroxymethyl) methylamine salt buffer (Tris-HCl).

In some embodiments, in step 1, the temperature of the enzyme-catalyzed reaction is 20 ℃ to 90 ℃. Preferably, the temperature of the enzyme catalysis reaction is 40-80 ℃. More preferably, the temperature of the enzyme-catalyzed reaction is 55 ℃ to 70 ℃.

In some embodiments, the time for the enzyme-catalyzed reaction in step 1 is 1 to 72 hours. Preferably, the time of the enzyme-catalyzed reaction is 2 to 48 hours. More preferably, the time of the enzyme-catalyzed reaction is 4 to 36 hours. Most preferably, the time of the enzyme-catalyzed reaction is from 12 to 24 hours.

In some embodiments, in step 1, the pH of the system for the enzyme-catalyzed reaction is 6.0 to 9.0. Preferably, the pH value of the enzyme-catalyzed reaction is 6.5 to 8.5. More preferably, the pH value of the enzyme-catalyzed reaction is 7 to 8.

In some embodiments, the liquid reaction system, the deoxyribotransferase NDT is present in the form of: free form, immobilized enzyme or enzyme in the form of bacterial cells.

In some embodiments, in step 2, the method of separating comprises: after the reaction is terminated, the reaction solution is centrifuged or filtered, the filtrate is extracted with an extraction solvent, and the organic layer is concentrated.

In some embodiments, in step 2, the operation of terminating the reaction is adding methanol or a methanol-water mixed solution with a volume concentration of 50% or more.

In some embodiments, in step 2, in the reaction system after the reaction, not less than 40% of the compound of formula II is converted into the compound of formula I. Preferably, not less than 50% of the compound of formula II is converted into the compound of formula I.

The present invention also provides a reaction system comprising:

(1) An aqueous solvent;

(2) A substrate 1, said substrate 1 being a compound of formula II;

(3) A substrate 2, wherein the substrate 2 is a compound of formula III;

wherein R is selected from NH ₂ Cl, S or F;

(4) The Genbank number of the deoxyribotransferase NDT is WP _011253881.1.

In some embodiments, the concentration of the compound of formula II in the reaction system is from 0.1 to 500g/L.

Preferably, the concentration of the compound of formula II in the reaction system is 0.1g/L or more, 0.2g/L or more, 0.5g/L or more, 1g/L or more, 10g/L or more, 25g/L or more, 50g/L or more, 75g/L or more, 100g/L or more, 125g/L or more, 150g/L or more, 175g/L or more, 200g/L or more, 220g/L or more, 240g/L or more, 260g/L or more, 280g/L or more, 300g/L or more, 320g/L or more, 350g/L or more, 400g/L or more, or 450g/L or more.

Preferably, in the reaction system, the concentration of the compound shown in the formula II is 1-500g/L; more preferably, in the reaction system, the concentration of the compound shown in the formula II is 10-400g/L; most preferably, the concentration of the compound of formula II in the reaction system is 50-350g/L.

In some embodiments, the molar ratio of the compound of formula II to the compound of formula III in the reaction system is 1: (1 to 10). Preferably, the molar ratio of the compound of formula II to the compound of formula III is 1: (1.1 to 5). More preferably, the molar ratio of the compound of formula II to the compound of formula III is 1: (1.5 to 3).

In some embodiments, in the reaction system, the mass ratio (w/w) of the deoxyribotransferase to the compound of formula II is (0.01 to 10): 1. preferably, the mass ratio (w/w) of the deoxyribotransferase to the compound of formula II is (0.01 to 5): 1. more preferably, the mass ratio (w/w) of the deoxyribotransferase to the compound of formula II is (0.5 to 2): 1.

in some embodiments, the aqueous solvent is a buffer.

In some embodiments, the buffer of the reaction system is Phosphate Buffered Saline (PBS), tris-methylamine salt buffer (Tris-HCl), or Tris-methylamine sulfate buffer (Tris-H) ₂ SO ₄ ). Preferably, the buffer of the reaction system is Tris (hydroxymethyl) methylamine salt buffer (Tris-HCl).

In some embodiments, ≧ 40% of the compound of formula II is converted into the compound of formula I in the reaction system after the reaction. Preferably, not less than 50% of the compound of formula II is converted into the compound of formula I.

The reaction system provided by the invention can carry out enzymatic reaction and prepare the 2 '-deoxy-2' -fluoro-beta-D-arabinoside adenosine analogue with high conversion rate. In the above reaction system, the concentration of the compound of formula II, the molar ratio of the compound of formula II to the compound of formula III, and the mass ratio (w/w) of the oxyribotransferase to the compound of formula II all refer to the initial parameters and/or the feeding parameters of the reaction system.

The invention also provides a biocatalytic preparation method of the 2 '-deoxy-2' -fluoro-beta-D-arabino adenosine analogue based on the reaction system, which performs enzymatic reaction under the reaction system to prepare a compound shown in the formula I:

。

In some embodiments, the mass ratio (w/w) of the deoxyribotransferase NDT to the compound of formula II is (0.1 to 10): 1. preferably, the mass ratio (w/w) of the deoxyribotransferase NDT to the compound of formula II is (0.1 to 5): 1. more preferably, the mass ratio (w/w) of the deoxyribotransferase NDT to the compound of formula II is (0.5 to 2): 1.

in some embodiments, in step 2, in the reaction system after the reaction, ≧ 40% of the compound of formula II is converted into the compound of formula I. Preferably, not less than 50% of the compound of formula II is converted into the compound of formula I.

Terminology of the technology

Deoxyribotransferase NDT, nucleotide 2' -deoxyribosyltransferases, in the present invention, "deoxyribotransferase" is an enzyme capable of transferring the sugar group of deoxynucleosides to free purines or pyrimidines to synthesize different deoxynucleoside compounds. The deoxyribotransferase NDT of the present invention is derived from Lactobacillus helveticus (Lactobacillus helveticus), hereinafter referred to as LhNDT, and its Genbank number is WP _011253881.1. In the reaction system of the present invention, the deoxyribotransferase can be produced by constructing recombinant plasmids and recombinant engineering bacteria in the form of wet cells, crude enzyme solution, crude enzyme powder, pure enzyme, etc., which are commercially available products, or by referring to the molecular cloning laboratory Manual.

Chemical bond(s)

In the present application, a chemical bond represents a single bond or a double bond. In the case of CR in the structure of the compounds of the formula I, when the substituent R is a monovalent radical, for example F, cl, br, OH, NH ₂ When the C and the R are connected through a single bond; when the substituent R is a divalent group, such as O or S, C and R are connected by a double bond, i.e. C = O, C = S is formed.

Enzyme-catalyzed preparation method

The invention provides a method for preparing a compound shown in a formula I by catalyzing a compound shown in a formula II and a compound shown in a formula III to react through deoxyribotransferase NDT produced by recombinant engineering bacteria. The reaction formula is shown as follows:

a specific implementation process of the enzyme catalysis preparation method is as follows: adding the compound of the formula II, the compound of the formula III and deoxyribotransferase NDT into a Tris-HCl buffer solution, uniformly mixing, maintaining the temperature at 20-90 ℃, and reacting under stirring for 1-72 hours. After the reaction is finished, adding a methanol-water solution into the reaction solution, centrifuging and/or filtering by diatomite, taking supernatant or filtrate, and concentrating the collected solution to obtain the product.

The present inventors have made extensive and intensive studies and, as a result of extensive screening experiments, have unexpectedly developed for the first time a biological production method of a 2 '-deoxy-2' -fluoro- β -D-arabinoadenosyl analogue having a high conversion rate, in which deoxyribotransferase exhibits specificity and selectivity for a substrate of reaction.

The main advantages of the invention include:

1) Compared with the prior art, the deoxyribotransferase NDT screened by the method and the preparation method thereof are used for preparing the compound shown in the formula I, the conversion rate of the substrate compound shown in the formula II is improved, and the reaction cost is reduced.

2) The deoxyribotransferase NDT screened by the method has selectivity on different reaction substrates, and the compound of the formula II is used as the reaction substrate, so that the enzymatic reaction is carried out under the reaction conditions provided by the method, and the obtained conversion rate is high.

3) The deoxyribotransferase screened by the method can be used for preparing the compound shown in the formula I from the compound shown in the formula II and the compound shown in the formula III through one-step reaction, the reaction steps are short, the reaction conditions are mild, only centrifugal filtration is needed for post-treatment, the operation is simple and convenient, and the industrial production is easy.

Drawings

FIG. 1 is a graph showing the effect of pH on the catalytic efficiency of deoxyribotransferase NDT, where the abscissa represents pH and the ordinate represents conversion;

FIG. 2 is a graph showing the effect of temperature on the catalytic efficiency of deoxyribotransferase NDT, where the abscissa represents temperature and the ordinate represents conversion rate.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: cold Spring Harbor laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise specified, the test materials of the present invention are commercially available reagents and are commercially available.

Example 1: obtaining of deoxyribotransferase NDT enzyme powder

1) Construction and transformation of recombinant vectors

Coli BL21 (DE 3) pET-28a-LhNDT was constructed as an example

Activating E.coli DH5 alpha/pET-28 a (+) strain (purchased from Novodak Biotechnology Co., ltd.), adding 3-5. Mu.L of pET-28a-LhNDT (50 ng/. Mu.L) into 50-100. Mu.L of E.coli BL21 (DE 3) competent cells (purchased from Novodak Biotechnology Co., ltd.), placing on ice for 20 min, performing heat shock at 42 ℃ for 90 sec, rapidly returning to ice for 5 min, adding 800. Mu.L of non-resistant LB culture solution, culturing for 1 h at 37 ℃, spreading on LB plate culture medium containing resistance, and culturing at 37 ℃ overnight for 12 h to obtain recombinant strain E.coli BL21 (DE 3) containing pET-28 a-LhNDT.

The Genbank number of LhNDT is WP _011253881.1.

The formula of the non-resistant LB culture solution is as follows: 5g/L of yeast powder, 10g/L of sodium chloride and 10g/L of peptone;

the formula of LB agar plate culture medium containing kanamycin resistance is as follows: 5g/L of yeast powder, 10g/L of sodium chloride, 10g/L of peptone, 20g/L of agar powder and 25 mu g/mL of sodium bicarbonate.

2) Construction of expression vector and inducible expression

The recombinant strain E.coli BL21 (DE 3) containing pET-28a-LhNDT obtained in 1) was directly spread on a solid LB plate containing 25. Mu.g/mL kanamycin resistance, and cultured at 37 ℃ for 12-14 h to obtain a monoclonal colony. A single colony of E.coli BL21 (DE 3) containing the LhNDT recombinant vector was picked from a Carna plate, inoculated in 1mL of liquid LB medium containing 25. Mu.g/mL of Carna resistance, and cultured with shaking at 37 ℃ for 12 hours. Then inoculating into fresh 1L liquid LB culture medium containing 25 μ g/mL kanamycin resistance according to the inoculation amount of 2% (v/v), culturing at 37 ℃ until OD600 is about 0.6-0.8, adding IPTG (purchased from Shanghai Allandin Biotech Co., ltd.) to the final concentration of 1.0 mmol/L, inducing expression at 200 rpm and 25 ℃ for 20h, centrifuging (4 ℃,4000 rpm,30 min) to remove supernatant, and washing the obtained bacterial sludge with 30mL of 0.9% NaCl solution for re-suspension for later use.

3) Preparation of lyophilized enzyme powder

Washing the bacterial liquid obtained in the step 2) twice by using 50 mmol/L Tris-HCl, then suspending the thalli in 50 mmol/L Tris-HCl (pH8.0) buffer solution, carrying out ultrasonic cell disruption in ice bath (amplitude transformer 6, power 500W, switching on for 2s, switching off for 5s, and 30 minutes. And centrifuging the sample subjected to ultrasonic disruption at 12000 rpm at 4 ℃ for 30 min, taking the supernatant, putting the supernatant into a freeze dryer at-80 ℃ for freeze drying for 24h, and grinding the obtained freeze-dried sample to prepare the freeze-dried enzyme powder.

When the method provided by the application is used for preparing the 2 '-deoxy-2' -fluoro-beta-D-arabinosyladenosine analogue, the deoxyribotransferase prepared in the embodiment is used as an enzyme catalyst, the reaction steps only need one step, the operation is simple and convenient, and the conversion rate is high.

Example 2: effect of deoxyribotransferase NDT on the reaction of different substrates

The reaction formula is as follows:

reaction operation: to a 10 mL reaction flask was added 2 mL Tris-HCl buffer (pH 8.0, 50 mM), and then 10mg of the compound of formula II (8.12 mmol, 1 eq), 10mg of the compound of formula III (20.3 mmol, 2.5 eq) and 2mg of deoxyribosyltransferase (lyophilized enzyme powder) were added, mixed well, the temperature was adjusted to 60 ℃ and the reaction was stirred for 20 hours. After the reaction, 100. Mu.L of the reaction solution was taken and added to a 1.5 mL centrifuge tube, then 1ml of 50% methanol-water post-treatment solution was added to terminate the reaction, the centrifuge tube was centrifuged at 1000rpm, and the supernatant after centrifugation was subjected to result detection by HPLC. High Performance Liquid Chromatography (HPLC) is purchased from Agilent technologies, inc., and the HPLC detection conditions are as follows: the mobile phase was 10mM NH4OAc mobile phase A:5% acetonitrile, mobile phase B:95% acetonitrile, temperature 40 ℃, flow rate 1.3mL/min, column Xbridge C18 (from Waters/Watts, USA). In this application, the R substituent may be selected to be NH ₂ The results of the reaction for substrates with different R substituents are shown in Table 1.

TABLE 1

As shown by comparison of experiments 1-6, the effect of deoxyribotransferase (Genbank number WP _ 011253881.1) on the reaction of substrates with different R substituents is significantly different. When the R substituent is NH ₂ When Cl, S or F are involved, removingThe conversion of the enzyme-catalyzed reaction of the compounds of the formulae II and III by the enzyme oxidoreductase (Genbank number WP _ 011253881.1) is high, whereas the conversion is significantly lower when the R substituent is O or OH.

Example 3: effect of different deoxyribotransferases on the reaction

The reaction formula is as follows:

reaction operation: the same substrate was used, and the same reaction procedure as in example 2 was carried out using a different deoxyribotransferase. The reaction results are shown in table 2.

TABLE 2

As can be seen from the comparison of experiments 1, 7 and 8, the reaction effects of different deoxyribotransferases are significantly different.

Example 4: search for optimum conditions for enzymatic reactions

The optimum catalytic temperature and the optimum pH of the deoxyribotransferase LhNDT catalytic reaction are determined:

the reaction formula is as follows:

the R substituent is NH ₂ The compound in the formula II is 2 '-deoxy-2' -fluoro-beta-D-arabinoside uridine, the compound in the formula III is adenine, and the compound in the formula I is 2 '-deoxy-2' -fluoro-beta-D-arabinoside adenosine.

Optimum pH: 2mg of enzyme powder is subjected to enzyme activity reaction with 10mg of 2 '-deoxy-2' -fluoro-beta-D-arabinouridine and 10mg of adenine under the conditions of 60 ℃ and 1000rpm in different pH values (4.0-11.0), and the conversion rate of the 2 '-deoxy-2' -fluoro-beta-D-arabinoadenosine is measured after 20 hours. Three buffer systems were used to adjust the pH of the solution.

Wherein the three buffer systems are citric acid-sodium citrate buffer (50mmol, pH4-6), tris-HCl buffer (50mmol, pH6-9), and Na ₂ HPO ₄ NaOH buffer (50mmol, pH 9-11).

The reaction results are shown in FIG. 1, wherein the reaction conversion is up to 80.5% at pH7.0, the reaction conversion is increasing from pH4.0 to 7.0, the reaction conversion is substantially constant at pH7.0 to 8.0, and the reaction conversion is decreasing at pH8.0 to 11.0.

Optimum temperature: 2mg of enzyme powder is subjected to enzyme activity reaction with 10mg of 2 '-deoxy-2' -fluoro-beta-D-arabinouridine and 10mg of adenine in a Tris-HCl buffer solution (50 mM, pH 7.0) at 1000rpm at different temperatures (40-80 ℃), and the conversion rate of 2 '-deoxy-2' -fluoro-beta-D-arabinoadenosine is measured after 20 hours.

The reaction results are shown in FIG. 2, in which the conversion of the reaction is 81% at maximum at a temperature of 60 ℃ and increases continuously from 40 to 60 ℃ and decreases at a temperature above 60 ℃ and reaches 25% at 80 ℃.

According to the above examples, the optimal pH for the reaction catalyzed by LhNDT to produce 2 '-deoxy-2' -fluoro-. Beta. -D-arabinoadenosyl was 7.0 and the optimal reaction temperature was 60 ℃.

In summary, the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A method for preparing a 2 '-deoxy-2' -fluoro- β -D-arabinoadenosine analog comprising the steps of:

step 1: in a liquid reaction system, taking a compound shown in a formula II and a compound shown in a formula III as substrates, and carrying out enzyme catalytic reaction under the catalysis of deoxyribotransferase NDT to form a compound shown in a formula I; wherein R is selected from NH ₂ Cl or F;

step 2: separating the compound shown in the formula I from the reaction system after the reaction in the step 1;

wherein the Genbank number of the deoxyribotransferase NDT is WP _011253881.1, the compound shown in the formula II is 2 '-deoxy-2' -fluoro-beta-D-arabinouridine, and the compound shown in the formula III is an adenine analogue;

the concentration of the compound of the formula II is 50-350g/L, and the molar ratio of the compound of the formula II to the compound of the formula III is 1: (1.5 to 3), wherein the mass ratio of the deoxyribotransferase NDT to the compound of the formula II is (0.5 to 2): 1, in the step 1, the temperature of the enzyme catalysis reaction is 55-70 ℃, the time of the enzyme catalysis reaction is 12-24 hours, and the pH value of the system of the enzyme catalysis reaction is 7-8.

2. The method of claim 1, wherein the buffer of the liquid reaction system is citric acid-sodium citrate buffer, na ₂ HPO ₄ -NaOH disodium hydrogen phosphate-sodium hydroxide buffer or tris buffer.

3. A reaction system for preparing 2 '-deoxy-2' -fluoro- β -D-arabinoadenosyl analogues, the reaction system comprising:

(1) An aqueous solvent;

(2) A substrate 1, said substrate 1 being a compound of formula II;

(3) A substrate 2, wherein the substrate 2 is a compound of formula III;

wherein R is selected from NH ₂ Cl or F;

(4) The Genbank serial number of the deoxyribotransferase NDT is WP _011253881.1;

the pH value of the reaction system is 7 to 8.

4. A biocatalytic preparation method of 2 '-deoxy-2' -fluoro- β -D-arabino adenosine analogues, characterized in that said preparation method uses the reaction system of claim 3 to perform an enzymatic reaction to obtain the compound of formula I, said reaction system having a temperature of 55 ℃ to 70 ℃, said reaction formula being:

。